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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03696</article-id><article-id pub-id-type="doi">10.7554/eLife.03696</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="author-15375"><name><surname>Isern</surname><given-names>Joan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-10"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-15376"><name><surname>García-García</surname><given-names>Andrés</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-8"/><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15377"><name><surname>Martín</surname><given-names>Ana M</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15378"><name><surname>Arranz</surname><given-names>Lorena</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15379"><name><surname>Martín-Pérez</surname><given-names>Daniel</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15380"><name><surname>Torroja</surname><given-names>Carlos</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15381"><name><surname>Sánchez-Cabo</surname><given-names>Fátima</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15171"><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-9805-9988</contrib-id><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-5"/><xref ref-type="other" rid="par-6"/><xref ref-type="other" rid="par-7"/><xref ref-type="other" rid="par-11"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Stem Cell Niche Pathophysiology Group</institution>, <institution>Centro Nacional de Investigaciones Cardiovasculares</institution>, <addr-line><named-content content-type="city">Madrid</named-content></addr-line>, <country>Spain</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Cossu</surname><given-names>Giulio</given-names></name><role>Reviewing editor</role><aff><institution>University of Manchester</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>joan.isern@cnic.es</email> (JI);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>smendez@cnic.es</email> (SM-F)</corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>25</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03696</elocation-id><history><date date-type="received"><day>16</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>24</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Isern et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Isern et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03696.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03696.001</object-id><p>Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin<sup>−</sup> MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin<sup>+</sup> cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP<sup>+</sup> Pdgfrα<sup>−</sup> cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.001">http://dx.doi.org/10.7554/eLife.03696.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03696.002</object-id><title>eLife digest</title><p>During the earliest phases of development, the embryo is formed by groups of stem cells that can develop into all the different types of tissue in the body—from bones to brain tissue. Later in life, small stockpiles of adult stem cells are found in various tissues and provide a reservoir of new cells available for replacing old or damaged cells. The most important source of blood stem cells is the bone marrow, which produces and stores cells that are capable of developing into blood and immune system cells. These processes are assisted by different bone marrow cells called stromal cells, which create a specialized local environment or ‘niche’.</p><p>But are the stromal stem cells that form the skeleton the same ones that form this niche during development? Or do the various types of stromal stem cells develop from distinct groups of cells in the embryo? Furthermore, it is unclear which cells guide blood stem cells towards the forming bones.</p><p>Other types of cells, including some of the cells of the nervous system, can communicate with the stem cells in the adult marrow and influence their behavior. This led scientists to wonder whether the stem cells in the bone marrow niche and the cells that communicate with them developed from the same type of embryonic stem cell.</p><p>Isern et al. tracked down the developmental origins of different types of bone marrow stromal stem cells by examining the bone marrow from the long bones (for example, the bones in the leg) of unborn and infant mice. It turns out that not all stromal stem cells in the developing bone marrow are alike. In fact, one pool of stromal stem cells forms the skeleton and loses stem cell activity in the process. In contrast, a different population of stromal stem cells develops from the same group of embryonic cells that gives rise to the cells of the nervous system. The stromal stem cells in this second group function as a niche to recruit and store the incoming blood stem cells and retain their stem cell activity throughout life.</p><p>The findings of Isern et al. help to explain why the nervous system is able to communicate with stem cells in the adult marrow, and provide a model for understanding how stem cell niches in organs that contain nerve tissue are established.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.002">http://dx.doi.org/10.7554/eLife.03696.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>mesenchymal stem cells</kwd><kwd>hematopoietic stem cells</kwd><kwd>stem cell niche</kwd><kwd>bone marrow</kwd><kwd>neural crest</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><award-id>55007426</award-id><principal-award-recipient><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100003329</institution-id><institution>Ministerio de Economía y Competitividad</institution></institution-wrap></funding-source><award-id>SAF-2011-30308</award-id><principal-award-recipient><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100003329</institution-id><institution>Ministerio de Economía y Competitividad</institution></institution-wrap></funding-source><award-id>BFU2012-35892</award-id><principal-award-recipient><name><surname>Isern</surname><given-names>Joan</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Ramon y Cajal Program</institution></institution-wrap></funding-source><award-id>RYC-2011-09209</award-id><principal-award-recipient><name><surname>Isern</surname><given-names>Joan</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Ramon y Cajal Program</institution></institution-wrap></funding-source><award-id>RYC-2009-04703</award-id><principal-award-recipient><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution>Marie Curie Actions</institution></institution-wrap></funding-source><award-id>FP7-PEOPLE-2011-RG-294262</award-id><principal-award-recipient><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution>ConsEPOC-Comunidad de Madrid</institution></institution-wrap></funding-source><award-id>S2010/BMD-2542</award-id><principal-award-recipient><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution>Fundación Ramón Areces</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>García-García</surname><given-names>Andrés</given-names></name></principal-award-recipient></award-group><award-group id="par-9"><funding-source><institution-wrap><institution>Fundación La Caixa</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>García-García</surname><given-names>Andrés</given-names></name></principal-award-recipient></award-group><award-group id="par-10"><funding-source><institution-wrap><institution>Ministerio de Educatión</institution></institution-wrap></funding-source><award-id>SB2010-0023</award-id><principal-award-recipient><name><surname>Isern</surname><given-names>Joan</given-names></name></principal-award-recipient></award-group><award-group id="par-11"><funding-source><institution-wrap><institution>Spanish Cell Therapy Network</institution></institution-wrap></funding-source><award-id>TerCel</award-id><principal-award-recipient><name><surname>Méndez-Ferrer</surname><given-names>Simón</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Developing long bones contain distinct mesenchymal stem-cell populations derived from mesoderm and neural crest, which have specialized functions in skeleton formation and the establishment of the hematopoietic stem-cell niche, respectively.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Bone marrow stromal cells (BMSCs) are a heterogeneous population. The different mesenchymal cell types might either arise from a variety of resident progenitors or might ultimately be derived from a single population of rare MSCs (<xref ref-type="bibr" rid="bib11">Caplan, 1991</xref>). In adult mammals, a multiple origin of skeletal MSCs is suggested by the distinct germ layer derivation of different bone structures, with craniofacial bones generated by the neuroectoderm, whereas the axial and appendicular bones are respectively derived from paraxial and lateral mesoderm. Mesoderm generates chondrocytes, which are progressively replaced by osteoblasts through the process of endochondral ossification (<xref ref-type="bibr" rid="bib57">Olsen et al., 2000</xref>). MSCs share cell-surface markers and localization with pericytes, suggesting that some pericytes might be MSCs (<xref ref-type="bibr" rid="bib16">Crisan et al., 2008</xref>). However, it remains unclear whether the bone marrow hosts ontogenically distinct MSCs in the same bones and whether they are endowed with specific functions.</p><p>In adult bone marrow, a variety of mesenchymal cells regulate HSCs (<xref ref-type="bibr" rid="bib10">Calvi et al., 2003</xref>; <xref ref-type="bibr" rid="bib81">Zhang et al., 2003</xref>; <xref ref-type="bibr" rid="bib3">Arai et al., 2004</xref>; <xref ref-type="bibr" rid="bib64">Sacchetti et al., 2007</xref>; <xref ref-type="bibr" rid="bib13">Chan et al., 2008</xref>; <xref ref-type="bibr" rid="bib54">Naveiras et al., 2009</xref>; <xref ref-type="bibr" rid="bib47">Mendez-Ferrer et al., 2010</xref>; <xref ref-type="bibr" rid="bib58">Omatsu et al., 2010</xref>; <xref ref-type="bibr" rid="bib62">Raaijmakers et al., 2010</xref>). Nevertheless, the specialized functions and developmental origin of these cells are largely unknown. Adult HSCs are also regulated by certain other non-hematopoietic lineages, including endothelial cells (<xref ref-type="bibr" rid="bib5">Avecilla et al., 2004</xref>; <xref ref-type="bibr" rid="bib36">Kiel et al., 2005</xref>; <xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>), sympathetic neurons and associated non-myelinating Schwann cells (<xref ref-type="bibr" rid="bib35">Katayama et al., 2006</xref>; <xref ref-type="bibr" rid="bib72">Spiegel et al., 2007</xref>; <xref ref-type="bibr" rid="bib46">Mendez-Ferrer et al., 2008</xref>; <xref ref-type="bibr" rid="bib78">Yamazaki et al., 2011</xref>), perivascular cells expressing the leptin receptor (<xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>) and mesodermal derivatives (<xref ref-type="bibr" rid="bib26">Greenbaum et al., 2013</xref>). However, the relationships and potential overlap among these populations remain unclear. It is also not known whether MSCs that form the HSC niche also generate other stromal cells or are a specialized population that arises earlier in embryogenesis and persists into adulthood.</p><p>In this study, we investigated the developmental origin and functions of MSCs in the primordial marrow of long bones. We show that, like peripheral neural and glial cells, HSC niche-forming MSCs in perinatal bone marrow arise from the trunk neural crest and make only a modest contribution to endochondrogenesis. Thus, whereas mesoderm-derived MSCs are mostly involved in endochondral ossification, neural crest-derived cells have a specialized function in establishing the HSC niche in the developing marrow of the same bones. These results provide compelling evidence for functional segregation of MSCs derived from different germ layers. The data also show that three HSC niche components—peripheral sympathetic neurons, Schwann cells, and MSCs—share a common origin.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Fetal bone marrow nestin<sup>+</sup> cells are quiescent and distinct from osteochondral cells</title><p>In adult mouse bone marrow, stromal cells expressing the green fluorescent protein (GFP) driven by the regulatory elements of nestin promoter (Nes-GFP<sup>+</sup>) display features of both MSCs and HSC niche cells (<xref ref-type="bibr" rid="bib47">Mendez-Ferrer et al., 2010</xref>). This finding prompted us to characterize Nes-GFP<sup>+</sup> cells during marrow development in limb bones. GFP<sup>+</sup> cells were already present in E16.5 bone marrow, associated preferentially with blood vessels infiltrating the cartilage scaffold (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A–C</xref>). At E18.5 Nes-GFP<sup>+</sup> cells were frequently associated with arterioles and sprouting endothelial cells within the osteochondral junction (<xref ref-type="fig" rid="fig1">Figure 1A–C</xref>). Fetal bone marrow Nes-GFP<sup>+</sup> cells were heterogeneous, composed of a majority of BMSCs but also including a small subset of CD31<sup>+</sup> putative endothelial cells that increased during the postnatal period (<xref ref-type="fig" rid="fig1">Figure 1D,E</xref> and <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D</xref>). Compared with Nes-GFP<sup>-</sup> BMSCs, the Nes-GFP<sup>+</sup> cell population was enriched in endogenous <italic>Nestin</italic> mRNA expression (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Arterioles were associated with an intense fluorescence microscopy signal, due to the presence of several concentric GFP<sup>+</sup> cells, including an outer layer that expressed smooth muscle actin and an inner layer of endothelial cells (<xref ref-type="fig" rid="fig1">Figure 1G</xref> and <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1E,F</xref>). Fetal bone marrow Nes-GFP<sup>+</sup> cells were distinct from S100-expressing chondrocytes and osteoblastic cells genetically labeled with the 2.3-kilobase proximal fragment of the α1(I)-collagen promoter (<xref ref-type="bibr" rid="bib17">Dacquin et al., 2002</xref>) (<xref ref-type="fig" rid="fig1">Figure 1H–J</xref>). Contrasting the marked proliferation of Nes-GFP<sup>-</sup> BMSCs in perinatal life, Nes-GFP<sup>+</sup> cells remained mostly quiescent (<xref ref-type="fig" rid="fig1">Figure 1K</xref> and <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1G</xref>). As a result, whereas Nes-GFP<sup>-</sup> BMSCs steadily expanded, Nes-GFP<sup>+</sup> BMSC number did not change significantly (<xref ref-type="fig" rid="fig1">Figure 1L</xref>). Fetal bone marrow Nes-GFP<sup>+</sup> cells thus include a small subset (<10%) of endothelial cells and a large population of non-endothelial stromal cells (>90%). Unlike Nes-GFP<sup>-</sup> stromal cells, Nes-GFP<sup>+</sup> cells proliferate slowly and do not express osteochondral protein cell markers.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.003</object-id><label>Figure 1.</label><caption><title>Fetal bone marrow nestin<sup>+</sup> cells proliferate slowly and are distinct from osteochondral cells.</title><p>(<bold>A</bold>–<bold>C</bold>) Nes-GFP<sup>+</sup> cells in fetal bones undergoing endochondral ossification. Whole-mount confocal projection of E18.5 <italic>Nes-Gfp</italic> femoral bone marrow stained with CD31 (magenta) to mark endothelium. Note the perivascular distribution of GFP<sup>+</sup> cells (green) in arterioles (<bold>B</bold>–<bold>B′′</bold>) and small vessels invading the primary spongiosa (<bold>C</bold>–<bold>C′′</bold>). (<bold>D</bold>–<bold>E</bold>) <italic>Nes-Gfp</italic> transgene is expressed by a subset of bone marrow endothelial cells. Flow cytometry histograms show the frequency of CD45<sup>−</sup> Nes-GFP<sup>+</sup> cells expressing CD31. (<bold>F</bold>) Endogenous <italic>Nestin</italic> mRNA expression measured by qPCR in stromal populations isolated from <italic>Nes-Gfp</italic> mice at the indicated stages (mean ± SD, <italic>n</italic> = 3–5). (<bold>G</bold>) <italic>Nes-Gfp</italic> bone marrow section stained with smooth muscle actin antibodies (αSma, red; asterisks) to reveal arterioles. (<bold>H</bold>) Limb section from an E17.5 <italic>Nes-Gfp</italic>;<italic>Col2.3-Cre</italic>;<italic>KFP</italic> embryo showing <italic>Nes-</italic>GFP<sup>+</sup> (green) and osteoblasts identified with antibodies to Katushka (KFP) protein (red), driven by the 2.3-kb proximal fragment of the α1(I)-collagen promoter. Arrowheads, endosteal surface. (<bold>I</bold>) Metaphysis of E17.5 <italic>Nes-Gfp</italic> embryo showing S100<sup>+</sup> chondrocytes (red). (<bold>J</bold>) Magnified view of boxed area in (<bold>I</bold>). (<bold>K</bold>) Representative cell cycle profiles of bone marrow stromal Nes-GFP<sup>+/-</sup> cells at early postnatal stages. Frequencies of cells in G<sub>2</sub>/S-M (%) are indicated. (<bold>L</bold>) Number of stromal Nes-GFP<sup>+/−</sup> cells in postnatal bone marrow (mean ± SEM, <italic>n</italic> = 3–4). Scale bars: 200 μm (<bold>A</bold>, <bold>A′</bold>, <bold>B′′</bold>, <bold>C</bold>, <bold>H</bold>), 100 μm (<bold>G</bold>, <bold>I</bold> and <bold>J</bold>); (<bold>A′</bold>, <bold>G</bold>–<bold>J</bold>) dashed line indicates bone contour. <italic>BM</italic>, bone marrow; <italic>C</italic>, cartilage; <italic>PS</italic>, primary spongiosa.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.003">http://dx.doi.org/10.7554/eLife.03696.003</ext-link></p></caption><graphic xlink:href="elife03696f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Perivascular and endothelial Nes-GFP<sup>+</sup> cells invade the incipient bone marrow associated with blood vessels.</title><p>(<bold>A</bold>) Longitudinal section of E16.5 <italic>Nes-Gfp</italic> forelimb. A profuse perivascular network of GFP<sup>+</sup> cells can be observed, mostly associated with arterial blood vessels. (<bold>B</bold>) High magnification detail (inset B’) of arterioles containing Nes-GFP<sup>+</sup> cells during invasion of the distal shaft of fetal bone. (<bold>C</bold>) Section through an E16.5 <italic>Nes-Gfp</italic> metaphyseal region undergoing vascularization, revealed by CD31 immunostaining of endothelial cells (red). (<bold>D</bold>) Representative FACS histograms of anti-CD31-stained CD45<sup>-</sup> bone marrow cells from 2-week old <italic>Nes-Gfp</italic> mice. The frequency of GFP<sup>+</sup> putative endothelial cells is indicated. (<bold>E</bold> and <bold>F</bold>) Confocal high magnification detail is showing several layers of perivascular GFP<sup>+</sup> cells (arrows) encircling an arteriole. Innermost endothelial cells immunostained with CD31 (red) also expressed GFP (yellow overlay, asterisks). (<bold>G</bold>) Femoral bone marrow section from newborn <italic>Nes-Gfp</italic> mouse stained with Ki67 (red) to label proliferative cells. Arrows indicate GFP<sup>+</sup> Ki67<sup>+</sup> cells; arrowheads depict GFP<sup>+</sup> Ki67<sup>−</sup> cells; dashed line marks bone contour. (A-C,E,G) Nuclei were counterstained with DAPI (gray). Scale bars: 500 μm (<bold>A</bold>); 200 μm (<bold>B</bold>); 100 μm (C,G); 50 μm (<bold>E</bold> and <bold>F</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.004">http://dx.doi.org/10.7554/eLife.03696.004</ext-link></p></caption><graphic xlink:href="elife03696fs001"/></fig></fig-group></p></sec><sec id="s2-2"><title>Bone marrow nestin<sup>+</sup> cells do not contribute to fetal endochondrogenesis</title><p>We next studied whether Nes-GFP<sup>+</sup> cells displayed osteoprogenitor activity in fetal bone marrow. The axial and appendicular skeleton is thought to originate solely from mesoderm. During endochondral ossification, cartilage is progressively replaced by osteoblast precursors that express the transcription factor osterix and infiltrate the perichondrium along the invading blood vessels (<xref ref-type="bibr" rid="bib44">Maes et al., 2010</xref>). To identify mesodermal derivatives, we performed lineage-tracing studies by crossing mice expressing the <italic>RCE</italic> reporter—a sensitive reporter that drives stronger GFP expression than other reporter lines (<xref ref-type="bibr" rid="bib71">Sousa et al., 2009</xref>)—with mice expressing inducible <italic>Cre</italic> recombinase under the regulatory elements of the <italic>Hoxb6</italic> gene, which is expressed in the lateral plate mesoderm (<xref ref-type="bibr" rid="bib55">Nguyen et al., 2009</xref>). The resulting double-transgenic mice were administered tamoxifen at E10.5, a stage when the <italic>Hoxb6</italic> gene is still expressed. These mice and newborn <italic>Nes-gfp</italic> embryos were analyzed for osterix protein expression, which marks cells committed to the osteoblast lineage. Unlike osteoblast precursors derived from lateral plate mesoderm, Nes-GFP<sup>+</sup> cells in fetal-limb bone marrow did not express highly osterix protein (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.005</object-id><label>Figure 2.</label><caption><title>Bone marrow nestin<sup>+</sup> cells are different from mesodermal osteo-chondroprogenitors.</title><p>(<bold>A</bold> and <bold>B</bold>) Bone marrow sections from <italic>Nes-Gfp</italic> (<bold>A</bold>–<bold>A′′</bold>) and <italic>Hoxb6-CreER</italic><sup><italic>T2</italic></sup>;<italic>RCE</italic> (<bold>B</bold>–<bold>B′′</bold>) E18.5 embryos (tamoxifen-induced at E10.5) immunostained with Osterix antibodies (Osx, red) to label osteoprogenitor cells. GFP<sup>+</sup> Osx<sup>+</sup> mesodermal-derived osteoprogenitors are marked with asterisks (insets 2–3). (<bold>C</bold>–<bold>C′′′</bold>) Perinatal recombination in <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> mice efficiently targets bone marrow stromal Nes-GFP<sup>+</sup> cells. Bone marrow section of a P7 <italic>Nes-Gfp;Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> mouse that received tamoxifen at birth, showing Nes-GFP<sup>+</sup> cells (green), <italic>Nes</italic>-derived progeny (red), and double-positive cells (arrowheads). (<bold>D</bold>–<bold>F</bold>) Fate mapping of the progeny of nestin<sup>+</sup> cells and limb mesoderm in E18.5/19.5 femoral bone marrow from <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;RCE</italic> (<bold>D</bold>–<bold>D′′</bold>, <bold>F</bold>–<bold>F′</bold>) and <italic>Hoxb6-CreER;RCE</italic> fetuses (<bold>E</bold>). (<bold>D</bold>) GFP (green) and nuclei counterstained with DAPI (gray) in bone of E18.5 fetus induced with tamoxifen at E13.5. Neither proliferating (*) nor hypertrophic (**) chondrocytes showed GFP fluorescence (inset 1). (<bold>D′</bold>–<bold>D′′</bold>) <italic>Nes</italic>-derived cells with a similar morphology and distribution to Nes-GFP<sup><italic>+</italic></sup> cells were detected near the cartilage–perichondrium interface (arrows) and within the chondro–osseous junction (arrowheads). (<bold>E</bold> and <bold>F</bold>) Bone marrow sections of (<bold>E</bold>) <italic>Hoxb6-CreER;RCE</italic> and (<bold>F</bold>) <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;RCE</italic> E18.5 embryos induced with tamoxifen at E10.5 and E8.5, respectively, stained with S100 antibodies to label chondrocytes (red). High magnification views of cartilage (inset 2) showing abundant double-positive chondrocytes (arrowheads). (<bold>F</bold>) <italic>Nes</italic>-traced cells (green) were not chondrocytes (red, *) but infiltrated the chondro–osseous junction and trabecular bone (arrowheads). Scale bars: 200 μm (<bold>A</bold>–<bold>A′</bold>, <bold>B</bold>–<bold>B′</bold>), 100 μm (<bold>A′′</bold>, <bold>B′′</bold>), 50 μm (<bold>B′′</bold>3, <bold>C</bold>). <italic>BM</italic>, bone marrow<italic>; C,</italic> cartilage<italic>; GIFM</italic>, genetic inducible fate mapping.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.005">http://dx.doi.org/10.7554/eLife.03696.005</ext-link></p></caption><graphic xlink:href="elife03696f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Sub-fractionation of fetal bone marrow mesenchymal progenitors.</title><p>(<bold>A</bold>) Neonatal <italic>Nes-Gfp</italic> bone marrow section immunostained with anti-osterix antibodies (red). Right panels, perichondrium detail showing inner rim of GFP<sup>+</sup> cells (green) adjacent to distinctive perichondrial osterix<sup>+</sup> cells. Nuclei were counterstained with DAPI (gray). Scale bar, 100 μm. <italic>PC</italic>, perichondrium. (<bold>B</bold>) Scheme showing FACS isolation of bone marrow stromal populations from <italic>Nes-Gfp</italic> mice and MSC assays. (<bold>C</bold>) Representative FACS plots and gating strategy to isolate stromal GFP<sup>+/-</sup> cells from <italic>Nes-Gfp</italic> bone marrow.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.006">http://dx.doi.org/10.7554/eLife.03696.006</ext-link></p></caption><graphic xlink:href="elife03696fs002"/></fig></fig-group></p><p>We next performed genetically inducible fate mapping using Nes-<italic>CreER</italic><sup><italic>T2</italic></sup> mice (<xref ref-type="bibr" rid="bib6">Balordi and Fishell, 2007</xref>). In these mice, tamoxifen administration triggers labeling of Nes-GFP<sup>+</sup> cells and their progeny (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>). Tamoxifen was administered at E13.5 (when primary ossification centers start forming) (<xref ref-type="bibr" rid="bib44">Maes et al., 2010</xref>), and at E8.5, to mark earlier nestin<sup>+</sup> embryonic precursors. Unlike <italic>Hoxb6</italic>-traced mesodermal derivatives, nestin<sup>+</sup> cells did not contribute to cartilage formation during this period. In contrast, <italic>Nes</italic>-traced cells with a similar morphology and distribution to Nes-GFP<sup>+</sup> cells were observed in the chondro–osseous junction (<xref ref-type="fig" rid="fig2">Figure 2E,F</xref>). Similarly, Nes-GFP<sup>+</sup> cells were not present inside the cartilage but were found in the innermost part of the perichondrium (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A</xref>), a region enriched in MSCs (<xref ref-type="bibr" rid="bib44">Maes et al., 2010</xref>; <xref ref-type="bibr" rid="bib79">Yang et al., 2013</xref>; <xref ref-type="bibr" rid="bib80">Zaidi and Mendez-Ferrer, 2013</xref>). The results thus show that, unlike mesodermal derivatives, Nes-GFP<sup>+</sup> cells do not exhibit osteochondral progenitor activity in the fetal bone marrow.</p></sec><sec id="s2-3"><title>MSC activity is progressively enriched in nestin<sup>+</sup> cells</title><p>The lack of a contribution by nestin<sup>+</sup> cells to fetal endochondrogenesis raised questions regarding their putative MSC properties in fetal bone marrow. We therefore measured mesenchymal progenitor activity in purified bone-marrow stromal subsets using the fibroblastic colony-forming unit (CFU-F) assay (<xref ref-type="bibr" rid="bib24">Friedenstein et al., 1970</xref>) and the multipotent self-renewing sphere-forming assay (<xref ref-type="bibr" rid="bib47">Mendez-Ferrer et al., 2010</xref>) (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>). BMSCs were isolated according to Nes-GFP expression (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C</xref>). CFU-F efficiency was nearly three times higher in Nes-GFP<sup>-</sup> cells than in Nes-GFP<sup>+</sup> cells at E17.5 (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Conversely, non-adherent sphere formation was markedly enriched in GFP<sup>+</sup> cells, whereas most spheres derived from GFP<sup>−</sup> cells rapidly attached to plastic and spontaneously differentiated into adipocytes (<xref ref-type="fig" rid="fig3">Figure 3B–G</xref>), suggesting that Nes-GFP<sup>-</sup> BMSCs are in a more committed state. Spheres formed by Nes-GFP<sup>+</sup> bone marrow cells contained mesenchyme-like spindle-shaped GFP<sup>+</sup> cells (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). At E18.5 and during the first postnatal week, CFU-F frequency was 6-fold higher in the GFP<sup>−</sup> stromal population than in GFP<sup>+</sup> cells (<xref ref-type="fig" rid="fig3">Figure 3H</xref>). However, at later postnatal stages, CFU-F activity was progressively restricted to Nes-GFP<sup>+</sup> cells due to a sharp drop in activity in GFP<sup>−</sup> BMSCs (>100-fold reduction between E18.5 and P14, compared with a 0.5-fold reduction in Nes-GFP<sup>+</sup> cells). At P7, CFU-Fs derived from Nes-GFP<sup>-</sup> cells contained mostly preosteoblasts (<xref ref-type="fig" rid="fig3">Figure 3I–K</xref>). The expression of genes associated with chondrocyte development was higher in Nes-GFP<sup>-</sup> than in Nes-GFP<sup>+</sup> BMSCs at E18.5; in contrast, the expression of master regulators of chondrogenesis, osteogenesis, and adipogenesis was progressively enriched in postnatal Nes-GFP<sup>+</sup> BMSCs (<xref ref-type="fig" rid="fig3">Figure 3L</xref>), consistent with the increasing MSC enrichment in this population. Together, these results suggest that most fetal BMSCs do not express nestin and quickly differentiate towards committed skeletal precursors, losing most MSC activity by the second week after birth. In contrast, nestin<sup>+</sup> cells conserve MSC activity throughout life.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.007</object-id><label>Figure 3.</label><caption><title>Perinatal enrichment of MSC activity in bone marrow nestin<sup>+</sup> cells.</title><p>(<bold>A</bold> and <bold>B</bold>) Fibroblast colony-forming units (CFU-F) and mesensphere-forming activities segregate in Nes-GFP<sup>-</sup> and Nes-GFP<sup>+</sup> fetal bone marrow cells, respectively. Frequencies of CFU-F and mesensphere-forming efficiency in E17.5 <italic>Nes-Gfp</italic> embryos. (<bold>C</bold>–<bold>E</bold>) Representative sphere cultures from both mesenchymal subpopulations sorted from bone marrow of <italic>Nes-Gfp</italic> fetuses. Note the presence of GFP<sup>+</sup> fibroblast-like cells (<bold>E′</bold> and insets1–3). (<bold>F</bold>–<bold>G</bold>) Adherent colonies derived from GFP<sup>-</sup> population stained with Oil Red O (red), to reveal mature adipocytes. (<bold>H</bold>) MSC activity is progressively restricted to bone marrow Nes-GFP<sup>+</sup> cells. Frequency of CFU-Fs in cultures of stromal (CD45<sup>−</sup> CD31<sup>−</sup> Ter119<sup>−</sup>) GFP<sup>+/−</sup> cells, isolated from the bone marrow of <italic>Nes-Gfp</italic> mice of the indicated age. Right panels show representative CFU-Fs in cell populations from adult mice. (<bold>I</bold>) Frequency of osteoblastic colony-forming units (CFU-OB) in bone marrow stromal GFP<sup>+/−</sup> cells of the indicated age. (<bold>J</bold>) Representative Giemsa-stained CFU-F from 3- and 10-day old bone marrow subpopulations. (<bold>K</bold>) Stained CFU-OB from E18.5 (alkaline phosphatase staining, left panels) and 1-week old (alizarin red staining, right panels) bone marrow subpopulations. (<bold>L</bold>) qPCR analysis of mesenchymal genes in bone marrow stromal populations isolated from fetal (E18.5) or 1-week old (P7) <italic>Nes-Gfp</italic> mice, as depicted (Figure 3—figure supplement 1). (<bold>A</bold>–<bold>B</bold>, <bold>H</bold>–<bold>L</bold>) Mean ± SD, n = 3–6; *p < 0.05, unpaired two-tailed <italic>t</italic> test. Scale bars: 200 μm (<bold>D</bold>, <bold>E′</bold>, <bold>G</bold>), 100 μm (G), 50 μm (<bold>E′</bold>1–3).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.007">http://dx.doi.org/10.7554/eLife.03696.007</ext-link></p></caption><graphic xlink:href="elife03696f003"/></fig></p></sec><sec id="s2-4"><title>The trunk neural crest contributes to bone marrow nestin<sup>+</sup> MSCs</title><p>Neural crest cells are characterized by nestin expression and sphere-forming ability. Although cells traced to neural crest origin have been reported in adult murine bone marrow (<xref ref-type="bibr" rid="bib52">Nagoshi et al., 2008</xref>; <xref ref-type="bibr" rid="bib51">Morikawa et al., 2009b</xref>; <xref ref-type="bibr" rid="bib25">Glejzer et al., 2011</xref>; <xref ref-type="bibr" rid="bib37">Komada et al., 2012</xref>), their precise identity, developmental dynamics, and function have remained elusive. Moreover, the neural crest-specific <italic>Wnt1-Cre</italic> line used in these studies displays ectopic <italic>Wnt1</italic> activation. To trace neural crest derivatives, we performed genetic fate-mapping studies with a recent <italic>Wnt1-Cre2</italic> line that does not induce ectopic Wnt1 activity (<xref ref-type="bibr" rid="bib41">Lewis et al., 2013</xref>). Unexpectedly, limb bones from <italic>Wnt1-Cre2</italic>;<italic>R26-Tomato</italic> double-transgenic neonates showed some neural crest-derived osteoblasts and osteocytes aligning the most recent layers of bone deposition, as well as similarly distributed chondrocytes in the outermost layers of the femur head (<xref ref-type="fig" rid="fig4">Figure 4A,B</xref>). As expected, neural crest-traced Schwann cells expressing glial fibrillary acidic protein (GFAP) were also detected in the bone marrow of one-week old mice (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A,B</xref>). Intriguingly, GFAP<sup>−</sup> perivascular cells with a similar morphology and distribution to Nes-GFP<sup>+</sup> cells were also derived from Wnt1<sup>+</sup> cells (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1C,D</xref>). The number of neural crest-traced osteochondral cells increased in the first postnatal week (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). By P28, CFU-F activity was much higher in <italic>Wnt1-Cre2</italic>-traced cells than in non-neural crest-traced bone marrow stromal cells (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). These results show that the neural crest contributes to limb bones late in development.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.008</object-id><label>Figure 4.</label><caption><title>Contribution of trunk neural crest cells to mesenchymal lineages in long bones.</title><p>(<bold>A</bold> and <bold>B</bold>) Fate mapping of neural crest derivatives in femoral bone marrow of neonatal <italic>Wnt1-Cre</italic>2;<italic>R26-Tomato</italic> mice. (<bold>A</bold>) Section through femoral distal epiphysis showing cortical neural-crest-derived chondrocytes (arrowheads, red); green signal corresponds to phalloidin staining. Nuclei were counterstained with DAPI (gray). (<bold>B</bold>) Bone marrow section showing <italic>Wnt1-Cre2</italic>-derived Tomato+ (red) osteocytes. (Inset 1) Neural-crest-derived osteocytes (asterisks) in endosteal region, showing their typical morphology revealed by phalloidin staining (green). (<bold>C</bold>–<bold>E</bold>) The neural crest contributes to Pdgfrα<sup>+</sup> BMSCs in long bones. (<bold>C</bold>–<bold>C</bold>′′) Fluorescent signals of GFP, Tomato, and DAPI in bone marrow sections from 1-week old <italic>Wnt1-Cre2;R26-Tomato;Nes-Gfp</italic> mice. (<bold>D</bold>) Frequency of fibroblastic colony-forming units (CFU-F) in CD31<sup>-</sup> CD45<sup>-</sup> Ter119<sup>-</sup> Tomato<sup>+/-</sup> bone marrow cells sorted from 1-week old <italic>Wnt1-Cre2;R26-Tomato</italic> mice (<italic>n</italic> = 3); N.D., not detectable. (<bold>D′</bold>) Examples of Giemsa staining (top panel) and Tomato fluorescence in neural crest-derived CFU-Fs. (<bold>E</bold>) Representative flow cytometry analysis of bone marrow stromal cells from 4-week old <italic>Wnt1-Cre2;R26-Tomato;Nes-Gfp</italic> mice. (<bold>F</bold>) Flow cytometry analysis of bone marrow stromal cells from <italic>Nes-Gfp</italic>;<italic>Sox10-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> triple-transgenic mice stained with Pdgfrα antibody. (<bold>E</bold>, <bold>F</bold>) Frequencies of neural crest-traced BMSCs are indicated. Scale bars: 200 μm (<bold>A</bold>–<bold>A′′</bold>, <bold>C</bold>, <bold>D</bold>–<bold>D′</bold>), 100 μm (<bold>B</bold>), 20 μm (<bold>B1</bold>, <bold>C′</bold>–<bold>C′′′</bold>). Dashed line depicts the bone and cartilage contour (<bold>A</bold>–<bold>C</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.008">http://dx.doi.org/10.7554/eLife.03696.008</ext-link></p></caption><graphic xlink:href="elife03696f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.009</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Bone marrow Nes-GFP<sup>+</sup> cells are distinct from mature Schwann cells.</title><p>(<bold>A</bold>–<bold>C</bold>) Diaphysis section stained with antibodies against glial fibrillary acidic protein (Gfap, cyan). Neural crest-traced cells included Gfap<sup>+</sup> Schwann cells (inset 1, arrowheads) and Gfap<sup>−</sup> putative BMSCs (inset 2, asterisks) (<bold>B-B</bold>′′, <bold>C-C</bold>′′). (<bold>D-D</bold>′′′) Staining of basal lamina with collagen IV antibodies (blue) showing close association of neural crest-derived cells (red) with blood vessels. (<bold>E</bold>) Surface expression of Pdgfrα enriches for fetal bone marrow mesenchymal progenitors. Fibroblast colony-forming units (CFU-F) in bone marrow stromal cells (CD45<sup>-</sup> CD31<sup>-</sup> Ter119<sup>-</sup>) isolated from E17.5 <italic>Nes-Gfp</italic> mice according to GFP and Pdgfrα expression. (<bold>F</bold>) Frequency of CFU-F colonies obtained from sorted populations of <italic>Wnt1-Cre2;R26-Tomato;Nes-Gfp</italic> 1-week old pups. (<bold>G</bold>) Schwann cells are closely associated with distinctive Nes-GFP<sup>+</sup> perivascular cells (arrowheads). Immunofluorescence of P9 <italic>Nes-Gfp</italic> tibial bone marrow showing GFP<sup>+</sup> cells (green), Gfap<sup>+</sup> Schwann cells (red), and CD31<sup>+</sup> endothelial cells (pink); nuclei were counterstained with DAPI (gray). (<bold>G′</bold>–<bold>G′′</bold>) Details of central diaphyseal region (insets) at high magnification. (<bold>G′</bold>) Note the long Gfap<sup>+</sup> Schwann cells extending through the arteriole, surrounded by distinctive Nes-GFP<sup>+</sup> cells (arrowhead). (<bold>G</bold>′′) Magnified view of a long arteriole. Asterisks indicate Gfap<sup>-</sup> perivascular Nes-GFP<sup>+</sup> cells. Scale bars: 200 μm (<bold>A</bold>, <bold>D</bold>, <bold>G</bold>), 50 μm (<bold>B</bold>, <bold>C</bold>, <bold>G′</bold>–<bold>G′′</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.009">http://dx.doi.org/10.7554/eLife.03696.009</ext-link></p></caption><graphic xlink:href="elife03696fs003"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.010</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Contribution of trunk neural crest to bone marrow stromal lineages.</title><p>(<bold>A-A</bold>′′′) Bone marrow section from newborn <italic>Wnt1-Cre</italic>2;<italic>R26-Tomato</italic> pup stained with calcein to mark calcium deposition (green), showing <italic>Wnt1-Cre2</italic>-traced Tomato<sup>+</sup> osteoblasts (red) in calcifying areas (asterisks). Scale bar = 100 μm. (<bold>B</bold>) Representative bone section of an E18.5 <italic>Sox10-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> mouse showing Tomato<sup>+</sup> osteocytes (asterisks) and bone-lining osteoblasts (red). Dashed line depicts the bone contour. Nuclei were counterstained with DAPI (gray). (<bold>C</bold> and <bold>D</bold>) Representative flow cytometry plots of bone marrow stained with Pdgfrα from neonatal (<bold>D</bold>) and 4-week old (<bold>C</bold>) <italic>Wnt1-Cre</italic>2;<italic>R26-Tomato;Nes-Gfp</italic> mice, after gating on the stromal (CD45<sup>-</sup> CD31<sup>-</sup> Ter119<sup>-</sup>) population. (<bold>E</bold>) Representative flow cytometry histogram of bone marrow cells from E18.5 <italic>Sox10-CreER;R26-Tomato;Nes-Gfp</italic> embryos, induced with tamoxifen at E9.5, after gating on the tomato-bright population. Scale bars: 200 μm (<bold>B</bold>), 100 μm (<bold>A</bold>, <bold>B′</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.010">http://dx.doi.org/10.7554/eLife.03696.010</ext-link></p></caption><graphic xlink:href="elife03696fs004"/></fig></fig-group></p><p>We further characterized cell surface marker expression by neural crest-derived MSCs. Platelet-derived growth factor receptor alpha (Pdgfrα) is required in mesodermal and neural crest-derived mesenchyme during development (<xref ref-type="bibr" rid="bib65">Schatteman et al., 1992</xref>; <xref ref-type="bibr" rid="bib70">Soriano, 1997</xref>). Mouse Pdgfrα<sup>+</sup> BMSCs are highly enriched in CFU-F activity (<xref ref-type="bibr" rid="bib74">Takashima et al., 2007</xref>; <xref ref-type="bibr" rid="bib50">Morikawa et al., 2009a</xref>) and most adult mouse bone marrow nestin<sup>+</sup> cells are also Pdgfrα<sup>+</sup> (<xref ref-type="bibr" rid="bib78">Yamazaki et al., 2011</xref>; <xref ref-type="bibr" rid="bib61">Pinho et al., 2013</xref>). We found that Pdgfrα<sup>+</sup> BMSCs were also enriched in CFU-F activity at fetal stages (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1E</xref>). In the bone marrow of 4-week old <italic>Nes</italic>-<italic>Gfp</italic>;<italic>Wnt1-Cre2;R26-Tomato</italic> mice, most neural crest-traced cells were also Pdgfrα<sup>+</sup> and Nes-GFP<sup>+</sup> (<xref ref-type="fig" rid="fig4">Figure 4E</xref>). For confirmation, we intercrossed <italic>Nes</italic>-<italic>Gfp</italic>;<italic>R26-Tomato</italic> mice with a line expressing tamoxifen inducible Cre recombinase under the regulatory elements of the gene encoding the neural crest transcription factor Sox10. <italic>Nes</italic>-<italic>Gfp</italic>;<italic>Sox10-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> mice were administered tamoxifen at E9.5 to label migratory neural crest-derived cells. Similar to the situation in stage-matched <italic>Nes-Gfp</italic> mice—and also consistent with <italic>Wnt1-Cre2</italic>-traced cells—most <italic>Sox10-CreER</italic><sup><italic>T2</italic></sup>-traced bone marrow stromal cells were Pdgfrα<sup>+</sup> and Nes-GFP<sup>+</sup> cells (<xref ref-type="fig" rid="fig4">Figure 4F</xref> and <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). These results thus demonstrate definitively that the neural crest contributes to nestin<sup>+</sup> BMSCs.</p></sec><sec id="s2-5"><title>The Nes-GFP<sup>+</sup> bone marrow population comprises Pdgfrα<sup>+</sup> MSCs and Pdgfrα<sup>−</sup> Schwann cell precursors</title><p>The finding that some fetal bone marrow Nes-GFP<sup>+</sup> cells expressed Pdgfrα while others did not prompted us to study the possible functional heterogeneity of this population. Recent work showed that most adult bone marrow nestin<sup>+</sup> cells are Pdgfrα<sup>+</sup> and also that nestin<sup>+</sup> Pdgfrα<sup>+</sup> Schwann cells contribute to HSC maintenance (<xref ref-type="bibr" rid="bib78">Yamazaki et al., 2011</xref>). We found that bone marrow Nes-GFP<sup>+</sup> cells were closely associated with distinctive Gfap<sup>+</sup> Schwann cells (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1G</xref>). After sorting of neonatal GFP<sup>+/−</sup> Pdgfrα<sup>+/−</sup> BMSCs (<xref ref-type="fig" rid="fig5">Figure 5A</xref>), the two populations were analyzed by next-generation sequencing. Detection of endogenous <italic>Pdgfrα</italic> and <italic>Nes</italic> transcripts verified the isolation strategy. Interestingly, whereas <italic>Ly6a</italic>/<italic>Sca1</italic> expression was higher in GFP<sup>−</sup>Pdgfrα<sup>+</sup> cells (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A,B</xref>), the expression levels of HSC maintenance genes (<italic>Cxcl12</italic>, <italic>Kitl</italic> and <italic>Angpt1</italic>) and the <italic>Leptin receptor</italic>, which marks HSC niche-forming mesenchymal cells (<xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>), was highly enriched in GFP<sup>+</sup> Pdgfrα<sup>+</sup> cells (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). This population also abundantly expressed other genes enriched in MSCs (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1C</xref>). In contrast, Nes-GFP<sup>+</sup> Pdgfrα<sup>−</sup> cells expressed genes characteristic of Schwann cell precursors (<italic>Sox10</italic>, <italic>Plp1</italic>, <italic>Erbb3</italic>, <italic>Dhh</italic>) but did not express mature Schwann cell genes, such as <italic>Gfap</italic> (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Gene ontology analysis of differentially expressed genes between the two Pdgfrα<sup>+</sup> subpopulations revealed enrichment of categories related to ossification, bone and blood vessel development, axon guidance, and Schwann cell differentiation (<xref ref-type="fig" rid="fig5s1 fig5s2">Figure 5—figure supplement 1D and supplement 2</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.011</object-id><label>Figure 5.</label><caption><title>The neonatal bone marrow Nestin-GFP<sup>+</sup> population contains Pdgfrα<sup>+</sup> MSCs and Pdgfrα<sup>−</sup> Schwann cell precursors.</title><p>(<bold>A</bold>) Representative flow cytometry profiles showing Nes-GFP and Pdgfrα expression in postnatal BMSCs. (<bold>B</bold> and <bold>C</bold>) Relative mRNA expression levels of (<bold>B</bold>) HSC niche-related genes and (<bold>C</bold>) Schwann cell progenitor genes by GFP<sup>+</sup> Pdgfrα<sup>+</sup> (black) or GFP<sup>+</sup> Pdgfrα<sup>−</sup> BMSCs. RNAseq data are expressed as fragments per kilobase of exon per million fragments mapped (FPKM; <italic>n=2</italic> independent samples from pooled newborns). Note that the neonatal GFP<sup>+</sup> Pdgfrα<sup>−</sup> subpopulation has a Schwann cell progenitor signature (<bold>C</bold>), whereas GFP<sup>+</sup> Pdgfrα<sup>+</sup> cells are enriched in HSC maintenance genes. (<bold>D</bold>) Principal component analysis comparing the transcriptome of neonatal <italic>Nes-Gfp</italic> bone marrow stromal subsets with available microarray expression data sets from neural crest-derived populations and primary adult mouse BMSCs (<xref ref-type="table" rid="tbl1">Table 1</xref>). (<bold>E</bold> and <bold>F</bold>) In vitro differentiation of neonatal subpopulations isolated as in (<bold>A</bold>) and cultured in mesenchymal (mesenchymal) and Schwann cell (glial) differentiation medium. Adipocytes were stained with Oil Red O (red) and counterstained with hematoxylin (left panels); Schwann cells were stained with antibodies against glial fibrillary acidic protein (Gfap, red) and overlaid with endogenous GFP fluorescence (right panels). Scale bars: 200 μm (top right insets: 50 μm).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.011">http://dx.doi.org/10.7554/eLife.03696.011</ext-link></p></caption><graphic xlink:href="elife03696f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.012</object-id><label>Figure 5—figure supplement 1.</label><caption><title>The neonatal Nes-GFP<sup>+</sup> bone marrow population is enriched in primitive mesenchymal progenitors.</title><p>(<bold>A</bold>) Representative FACS profile of GFP and Pdgfrα expression in CD45<sup>−</sup> CD31<sup>−</sup> Ter119<sup>−</sup> BMSCs from neonatal <italic>Nes-Gfp</italic> long bones. Four different stromal subpopulations identified based on expression levels of Pdgfrα and GFP were isolated and analyzed by RNAseq. (<bold>B</bold>) Endogenous transcript expression levels of <italic>Nestin</italic>, <italic>Pdgfra</italic>, <italic>Cspg4 (NG2)</italic>, and <italic>Sca1 (Ly6a)</italic> in isolated stromal populations depicted in (<bold>A</bold>), expressed in fragments per kilobase of exon per million fragments mapped (FPKM). (<bold>C</bold>) Expression profiles of characteristic mesenchymal genes in the stromal subpopulations. (<bold>D</bold>) Functional gene ontology enrichment analysis (DAVID software) of genes differentially expressed (p ≤ 0.10) between the GFP<sup>+</sup> and GFP<sup>−</sup> subsets of P1 Pdgfrα<sup>+</sup> bone marrow stromal cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.012">http://dx.doi.org/10.7554/eLife.03696.012</ext-link></p></caption><graphic xlink:href="elife03696fs005"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.013</object-id><label>Figure 5—figure supplement 2.</label><caption><title>RNA-seq data analysis.</title><p>Top 50 downregulated (<bold>A</bold>) and upregulated (<bold>B</bold>) genes in Nes-GFP<sup>+</sup> Pdgfrα<sup>+</sup> and Nes-GFP<sup>+</sup> Pdgfrα<sup>−</sup> cells from P1 bone marrow stroma.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.013">http://dx.doi.org/10.7554/eLife.03696.013</ext-link></p></caption><graphic xlink:href="elife03696fs006"/></fig></fig-group></p><p>To further characterize nestin<sup>+</sup> subpopulations, we compared the transcriptome-wide profile of neonatal Nes-GFP<sup>+/−</sup> Pdgfrα<sup>+/−</sup> BMSCs with publicly available microarray expression data sets from primary adult BMSCs or neural crest derivatives (<xref ref-type="table" rid="tbl1">Table 1</xref>). Unbiased hierarchical clustering and principal component analysis (<xref ref-type="fig" rid="fig5">Figure 5D</xref>) revealed that Nes-GFP<sup>+</sup> Pdgfrα<sup>+</sup> cells were more similar to adult primitive BMSCs and distinct from more differentiated osteoblastic cells (<xref ref-type="bibr" rid="bib53">Nakamura et al., 2010</xref>). Pdgfrα<sup>+</sup> Nes-GFP<sup>+/−</sup> cells clustered nearby, consistent with the increasing restriction of Pdgfrα to Nes-GFP<sup>+</sup> cells in postnatal bone marrow. In addition, Nes-GFP<sup>+</sup> Pdgfrα <sup>+</sup> cells clustered far from Nes-GFP<sup>+</sup> Pdgfrα<sup>−</sup> cells, whose genomic profile was closest to that of E12.5 Schwann cell precursors. MSC-like and neural crest stem-cell-like derived clones (<xref ref-type="bibr" rid="bib76">Wislet-Gendebien et al., 2012</xref>) were markedly different, probably because these were cultured cells. Intriguingly, we noted a maturation hierarchy of Schwann and osteolineage cells, from undifferentiated cells (<xref ref-type="fig" rid="fig5">Figure 5D</xref>, lower corners) to more mature lineages (<xref ref-type="fig" rid="fig5">Figure 5D</xref>, contralateral upper corners). At the intersection of these differentiation waves, adult bone marrow CD45<sup>−</sup> Nes-GFP<sup>+</sup> cells (<xref ref-type="bibr" rid="bib47">Mendez-Ferrer et al., 2010</xref>) converged with bone marrow HSC niche cells identified by the expression of stem cell factor (<xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>). These results suggest the existence of two nestin<sup>+</sup> populations with non-overlapping MSC and Schwann cell precursor features. To test this hypothesis functionally, we cultured neonatal Nes-GFP<sup>+/−</sup> Pdgfrα<sup>+/−</sup> BMSCs in differentiation medium, finding that mesenchymal and glial differentiation was segregated in Pdgfrα<sup>+</sup> and Pdgfrα<sup>−</sup> cells, respectively (<xref ref-type="fig" rid="fig5">Figure 5E,F</xref>). Thus two Nes-GFP<sup>+</sup> neural crest derivatives occur in postnatal bone marrow: Pdgfrα<sup>+</sup> MSCs enriched in HSC-supporting genes and Pdgfrα<sup>−</sup> Schwann cell precursors.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.014</object-id><label>Table 1.</label><caption><p>Description of publicly available data sets used for principal component analyses</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.014">http://dx.doi.org/10.7554/eLife.03696.014</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Plot ID</th><th>Cell Population</th><th>GEO<xref ref-type="table-fn" rid="tblfn1">*</xref> samples</th><th>Sample description</th><th>Ref</th></tr></thead><tbody><tr><td><bold>1</bold></td><td>BM osteoblastic cells (Alcam<sup>-</sup> Sca1<sup>-</sup>)</td><td>GSM437794</td><td>BM (adult) primary stromal<xref ref-type="table-fn" rid="tblfn2">†</xref> Alcam<sup>-</sup> Sca1<sup>-</sup></td><td rowspan="6"><xref ref-type="table-fn" rid="tblfn3">‡</xref></td></tr><tr><td><bold>2</bold></td><td>BM osteoblastic cells (Alcam<sup>+</sup> Sca1<sup>-</sup>)</td><td>GSM437795</td><td>BM (adult) primary stromal<xref ref-type="table-fn" rid="tblfn2">†</xref> Alcam<sup>+</sup> Sca1<sup>-</sup></td></tr><tr><td><bold>3</bold></td><td>BM osteoblastic cells (Pdgfrα<sup>-</sup> Sca1<sup>-</sup>)</td><td>GSM437797</td><td>BM (adult) primary stromal<xref ref-type="table-fn" rid="tblfn2">†</xref> Pdgfrα<sup>-</sup> Sca1<sup>-</sup></td></tr><tr><td><bold>4</bold></td><td>BM osteoblastic cells ( Pdgfrα<sup>+</sup> Sca1<sup>-</sup>)</td><td>GSM437798</td><td>BM (adult) primary stromal<xref ref-type="table-fn" rid="tblfn2">†</xref> Pdgfrα<sup>+</sup> Sca1<sup>-</sup></td></tr><tr><td><bold>5</bold></td><td>BM MPC’s (Pdgfrα<sup>+</sup> Sca1<sup>+</sup>)</td><td>GSM437799</td><td>BM (adult) primary stromal<xref ref-type="table-fn" rid="tblfn2">†</xref> Mesenchymal progenitor cells (MPC) (Pdgfrα<sup>+</sup> Sca1<sup>+</sup>)</td></tr><tr><td><bold>6</bold></td><td>BM MPC’s (Alcam<sup>+</sup> Sca1<sup>+</sup>)</td><td>GSM437796</td><td>BM (adult) primary stromal<xref ref-type="table-fn" rid="tblfn2">†</xref> Mesenchymal progenitor cells (MPC) (Alcam<sup>+</sup> Sca1<sup>+</sup>)</td></tr><tr><td><bold>7</bold></td><td>BM MSC-like derived clones</td><td>GSM795638-40</td><td>BM MSC-derived cell line, Wnt1Cre/R26R β-gal<sup>-</sup> selected clone passage>10</td><td rowspan="2"><xref ref-type="table-fn" rid="tblfn4">§</xref></td></tr><tr><td><bold>8</bold></td><td>BM NCSC-derived clones</td><td>GSM795641-43</td><td>BM NCSC-derived cell line, Wnt1Cre/R26R β-gal<sup>-</sup> selected clone passage>10</td></tr><tr><td><bold>9</bold></td><td>E13.5 vascular segment (mesoderm-derived)</td><td>GSM261911</td><td>E13.5 internal carotid artery vascular segment (smooth muscle mesoderm-derived)</td><td rowspan="2"><xref ref-type="table-fn" rid="tblfn5">¶</xref></td></tr><tr><td><bold>10</bold></td><td>E13.5 vascular segment (NC derived)</td><td>GSM261912</td><td>E13.5 external carotid artery vascular segment (smooth muscle NC-derived)</td></tr><tr><td><bold>11</bold></td><td>BM (adult) Nes-GFP<sup>+</sup></td><td>GSM545815-17</td><td>BM (adult) primary (CD45<sup>-</sup>) Nes-GFP<sup>+</sup> cells</td><td><xref ref-type="table-fn" rid="tblfn6">**</xref></td></tr><tr><td><bold>12</bold></td><td>BM (adult) Scf-GFP<sup>+</sup></td><td>GSM821066-68</td><td>BM (adult) primary Scf-GFP+ cells</td><td><xref ref-type="table-fn" rid="tblfn7">††</xref></td></tr><tr><td><bold>13</bold></td><td>P0 Schwann cells</td><td>GSM15386-88</td><td>P0 primary Schwann cells (Plp-GFP<sup>+</sup>) from sciatic nerve</td><td rowspan="5"><xref ref-type="table-fn" rid="tblfn8">‡‡</xref></td></tr><tr><td><bold>14</bold></td><td>E12.5 Schwann cell precursors (SCPs)</td><td>GSM15373-75</td><td>E12.5 primary Schwann cell precursors (Plp-GFP<sup>+</sup>) from sciatic nerve</td></tr><tr><td><bold>15</bold></td><td>E14.5 Enteric Neural crest cells (ENCC)</td><td>GSM844492-94</td><td>E14.5 primary ENCCs (Wnt1Cre/R26-YFP<sup>+</sup>) from gut</td></tr><tr><td><bold>16</bold></td><td>E9.5 Neural crest stem cells (NCSC)</td><td>GSM15370-72</td><td>E9.5 trunk primary Plp-GFP+ cells (migrating NCSCs)</td></tr><tr><td><bold>17</bold></td><td>E18.5 Schwann cells</td><td>GSM15383-85</td><td>E18.5 primary Schwann cells (Plp-GFP<sup>+</sup>) cells from sciatic nerve</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>Gene Expression Omnibus database (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>)</p></fn><fn id="tblfn2"><label>†</label><p>CD45<sup>-</sup>CD31<sup>-</sup>Ter119<sup>-</sup></p></fn><fn><p><bold>References:</bold></p></fn><fn id="tblfn3"><label>‡</label><p>Nakamura <italic>et al.</italic> Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells. Blood (2010) vol. 116 (9) pp. 1422-32.</p></fn><fn id="tblfn4"><label>§</label><p>Wislet-Gendebien <italic>et al.</italic> Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences. Cell Mol Life Sci (2012)vol. 69 (15) pp. 2593-608.</p></fn><fn id="tblfn5"><label>¶</label><p>Zhang <italic>et al.</italic> Origin-specific epigenetic program correlates with vascular bed-specific differences in Rgs5 expression. FASEB J (2012) vol. 26 (1) pp. 181-91.</p></fn><fn id="tblfn6"><label>**</label><p>Méndez-Ferrer <italic>et al.</italic> Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature (2010) vol. 466 (7308) pp. 829-34.</p></fn><fn id="tblfn7"><label>††</label><p>Ding and Morrison. Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. Nature (2013) pp. 1-6.</p></fn><fn id="tblfn8"><label>‡‡</label><p>Buchstaller <italic>et al.</italic> Efficient isolation and gene expression profiling of small numbers of neural crest stem cells and developing Schwann cells. J Neurosci (2004) vol. 24 (10) pp. 2357-65.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s2-6"><title>Deficient migration of neural crest-derived cells along nerves reduces the bone marrow MSC and HSC populations</title><p>Under similar culture conditions used to grow neural crest cells, adult mouse bone marrow Nes-GFP<sup>+</sup> cells can form self-renewing and multipotent mesenchymal spheres with the capacity to transfer hematopoietic activity to ectopic sites during serial transplantations (<xref ref-type="bibr" rid="bib47">Mendez-Ferrer et al., 2010</xref>). In addition, human bone marrow-derived mesenspheres secrete factors that can expand human cord blood HSCs through secreted factors (<xref ref-type="bibr" rid="bib29">Isern et al., 2013</xref>). These findings and the results presented so far together suggest that neural crest-derived MSCs might have a specialized function in establishing the HSC niche in the developing bone marrow. We further studied the role of neural crest-derived cells in this process using a loss-of-function model. Perineural migration of neural crest-derived cells requires the interaction of the receptor tyrosine-protein kinase ErbB3 with the ligand neuregulin-1, produced by developing nerves (<xref ref-type="bibr" rid="bib31">Jessen and Mirsky, 2005</xref>). <italic>Erbb3</italic>-deficient mice initially show normal development of peripheral nerves but later display impaired perineural migration of neural crest-derived cells and die at perinatal stage (<xref ref-type="bibr" rid="bib63">Riethmacher et al., 1997</xref>). Hematopoietic progenitors were increased in fetal liver of KO mice (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). In contrast, expression of the MSC marker CD90, enriched in Nes-GFP<sup>+</sup> cells (<xref ref-type="fig" rid="fig6">Figure 6B,C</xref>), was reduced two-fold in <italic>Erbb3</italic><sup>−/−</sup> limb bone marrow, associated with 5-fold drop in the number of bone marrow hematopoietic progenitors (<xref ref-type="fig" rid="fig6">Figure 6D,G</xref>). To further dissect the contribution of neural crest to fetal hematopoiesis, we performed a similar analysis in mice conditionally lacking ErbB3 in Schwann-committed cells. To label Schwann cells, we intercrossed <italic>R26-Tomato</italic> reporter mice intercrossed with a line expressing Cre recombinase under the regulatory elements of desert hedgehog (<italic>Dhh</italic>) promoter (<xref ref-type="bibr" rid="bib30">Jaegle et al., 2003</xref>) (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A–C</xref>). These <italic>Dhh-Cre</italic> mice were then intercrossed with the ErbB3 conditional KO. Similar to the constitutive KO, <italic>Dhh-Cre</italic>;<italic>Erbb3</italic><sup><italic>fl/fl</italic></sup> mice are virtually devoid of Schwann cells (<xref ref-type="bibr" rid="bib67">Sheean et al., 2014</xref>); however, unlike the constitutive KO, <italic>Dhh-Cre</italic>;<italic>Erbb3</italic><sup><italic>fl/fl</italic></sup> mice had a normal frequency of bone marrow hematopoietic progenitors (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1D</xref>). Together, these results suggest that neural crest cells not yet committed to the Schwann cell lineage migrate along developing nerves to the bone marrow, giving rise to HSC niche-forming MSCs.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.015</object-id><label>Figure 6.</label><caption><title>Perineural migration of neural crest-derived cells to long bones generates nestin<sup>+</sup> MSCs with specialized HSC niche function.</title><p>(<bold>A</bold>) E17.5-E18.5 fetal liver (FL) sections from wild-type (wt) and <italic>Erbb3</italic>-null embryos stained with antibodies for mature hematopoietic lineage (blue) and c-Kit (red). Quantification of fetal liver Lin<sup>−</sup> c-Kit<sup>+</sup> hematopoietic progenitors (per 0.41 mm<sup>2</sup>). (<bold>B</bold>) Representative FACS profile of CD45<sup>-</sup> CD31<sup>-</sup>Ter119<sup>-</sup> bone marrow cells from 2-week old <italic>Nes-Gfp</italic> mice stained with the mesenchymal marker CD90, showing the expression enrichment in Nes-GFP<sup>+</sup> cells. (<bold>C</bold>) Neonatal bone marrow section stained with anti-CD90 (red), which labeled Nes-GFP<sup>+</sup> (green) cells. Scale bar: 50 μm. (<bold>D</bold>) Representative bone marrow sections from wt and <italic>Erbb3</italic>-null E17.5/18.5 mice immunostained with anti-CD90 (red). (<bold>E</bold>) Quantification of CD90 immunostaining of samples in (<bold>D</bold>); <italic>n</italic> = 3. (<bold>F</bold>) Staining of bone marrow sections from wt and <italic>Erbb3</italic>-null embryos with antibodies for mature hematopoietic lineage (blue) and c-Kit (red). (<bold>G</bold>) Quantification of bone marrow Lineage<sup>−</sup> c-Kit<sup>+</sup> hematopoietic progenitors in E17.5/18.5 wt and <italic>Erbb3</italic>-null mice (<italic>n</italic> = 3). (<bold>E</bold>, <bold>G</bold>) Mean ± SEM; *p < 0.05, unpaired two-tailed <italic>t</italic> test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.015">http://dx.doi.org/10.7554/eLife.03696.015</ext-link></p></caption><graphic xlink:href="elife03696f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.016</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Conditional <italic>Erbb3</italic> deletion after glial specification of Schwann cell precursors does not affect bone marrow HSCs.</title><p>(<bold>A-A</bold>′) Whole-mount view of ventral ribcage of a <italic>Dhh-Cre</italic>;<italic>R26-Tomato</italic> neonate, showing labeling of glial cells along the intercostal peripheral nerves (red). (<bold>B-B</bold>′) Surface detail of Tomato<sup>+</sup> Schwann cells between 2 ribs. (<bold>C</bold>) High magnification of ventral skull showing an intricate network of Tomato<sup>+</sup> Schwann cells. (<bold>D</bold>) Frequency of hematopoietic lineage<sup>−</sup> c-Kit<sup>+</sup> Sca-1<sup>+</sup> (LSK) cells in neonatal bone marrow of <italic>Dhh-Cre;Erbb3</italic><sup><italic>f/f</italic></sup> and control littermate mice (mean ± SD, <italic>n</italic> = 4–5).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.016">http://dx.doi.org/10.7554/eLife.03696.016</ext-link></p></caption><graphic xlink:href="elife03696fs007"/></fig></fig-group></p></sec><sec id="s2-7"><title>Neural crest-derived nestin<sup>+</sup> cells direct developmental HSC migration to the bone marrow</title><p>Despite the low frequency of HSCs and Nes-GFP<sup>+</sup> cells, detailed immunofluorescence analysis showed significant proximity of HSCs to Nes-GFP<sup>+</sup> cells in neonatal bone marrow (<xref ref-type="fig" rid="fig7">Figure 7A,B</xref>), suggesting that nestin<sup>+</sup> cells might attract circulating HSCs toward their final developmental destination in the bone marrow. To study the contribution of nestin<sup>+</sup> cells to HSC migration from fetal liver to bone marrow, we used mice expressing the diphtheria toxin (iDTA) or its receptor (iDTR) in nestin<sup>+</sup> cells. Depletion of nestin<sup>+</sup> cells at E15.5 in <italic>Nes-CreER</italic><sup><italic>T2</italic></sup>;<italic>iDTR</italic> mice caused an ∼4-fold reduction in fetal bone marrow HSC activity within 48 hr, inversely correlating with an ∼8-fold increase in fetal liver HSC activity (<xref ref-type="fig" rid="fig7">Figure 7C,D</xref>). The cell cycle profile and apoptosis in hematopoietic progenitors were unchanged (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1A</xref>), but their numbers increased in fetal liver by 40% (<xref ref-type="fig" rid="fig7">Figure 7E</xref>). Similar results were obtained by depleting nestin<sup>+</sup> cells during the first postnatal week in <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;iDTA</italic> mice, which otherwise showed normal bone marrow histology (<xref ref-type="fig" rid="fig7">Figure 7F</xref> and <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1B</xref>). Developmental HSC migration to bone marrow proceeds until the second week after birth (<xref ref-type="bibr" rid="bib22">Dzierzak and Speck, 2008</xref>), suggesting that the bone marrow environment might still mature during this period to accommodate HSCs. We therefore analyzed GFP<sup>+/−</sup> BMSCs from E18.5 and P7 <italic>Nes-Gfp</italic> bone marrow. The expression of HSC-supporting genes was markedly higher and progressively upregulated in GFP<sup>+</sup> cells during the first postnatal week (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). These results suggest that perinatal maturation of nestin<sup>+</sup> cells allows the colonization of bone marrow by circulating HSCs.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03696.017</object-id><label>Figure 7.</label><caption><title>CXCL12 produced by nestin<sup>+</sup> MSCs contributes to the establishment of the HSC niche in the bone marrow.</title><p>(<bold>A</bold> and <bold>B</bold>) HSCs are localized near Nes-GFP<sup>+</sup> cells in neonatal bone marrow. Neonatal femoral sections from <italic>Nes-Gfp</italic> mice were immunostained with antibodies for mature hematopoietic lineages, CD48 (blue) and CD150 (red). (<bold>A</bold>) Quantification of the distance of Lin<sup>−</sup> CD48<sup>−</sup> CD150<sup>+</sup> HSC-enriched cells from Nes-GFP<sup>+</sup> cells (mean ± SEM, <italic>n</italic> = 41). (<bold>B</bold>) Representative image of a putative HSC (asterisk) near a Nes-GFP<sup>+</sup> cell. (<bold>C</bold>–<bold>F</bold>) Depletion of nestin<sup>+</sup> cells compromises developmental HSC migration to bone marrow. (<bold>C</bold> and <bold>D</bold>) Long-term culture-initiating cell (LTCIC) assay from nestin-depleted fetal liver and bone-marrow cells. <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;iDTR</italic> (red dots) and control <italic>iDTR</italic> (black dots) mice were exposed to tamoxifen at E14.5 and diphtheria toxin at E15.5, and liver cells (<bold>C</bold>) and bone marrow cells (<bold>D</bold>) were isolated at E17.5 (<italic>n</italic> = 5-6). The percentage of culture dishes that failed to generate hematopoietic colony-forming units in culture (CFU-C) is plotted against five serial dilutions of (<bold>C</bold>) fetal liver Lin<sup>−</sup> Sca-1<sup>+</sup> cells and (<bold>D</bold>) nucleated bone marrow cells. HSC frequencies and p values are indicated (Pearson’s chi-squared test). (<bold>E</bold>) Frequency of Lin<sup>−</sup> Sca-1<sup>+</sup> E17.5 liver cells in mice in (<bold>C</bold>). (<bold>F</bold>) Bone marrow CFU-C content in 1-week old <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-DTA</italic> and control littermates treated with tamoxifen at birth (<italic>n</italic> = 3–7). (<bold>G</bold>) Expression of core HSC maintenance genes increases in perinatal Nes-GFP<sup>+</sup> BMSCs. qPCR analysis of <italic>Cxcl12</italic>, stem cell factor/kit ligand (<italic>Kitl</italic>), angiopoietin-1 (<italic>Angpt1</italic>), and vascular cell adhesion molecule-1 (<italic>Vcam1</italic>) mRNA in CD45<sup>-</sup> CD31<sup>-</sup> Ter119<sup>-</sup> GFP<sup>+/−</sup> cells isolated from E18.5 and P7 <italic>Nes-Gfp</italic> bone marrow. (<bold>H</bold>) Relative <italic>Cxcl12</italic> mRNA expression levels in endothelial cells and Nes-GFP<sup>+/-</sup> BMSCs isolated from 1-week old mice (qPCR; <italic>n</italic> = 2). (<bold>I</bold> and <bold>J</bold>) Relative enrichment of <italic>Cxcl12</italic> (I) and <italic>Nestin</italic> (J) mRNA expression in populations sorted from the bone marrow of P7 <italic>Nes</italic>-<italic>Gfp</italic>;<italic>Wnt1-Cre2;R26-Tomato</italic> compound transgenic mice. (<bold>K</bold>) Representative confocal image of a bone marrow section from a 1-week old (P7) <italic>Nes-Gfp;Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> mouse treated with tamoxifen at birth. Both sinusoidal (asterisk) and arteriolar GFP<sup>+</sup> cells express the <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>-derived</italic> Tomato (red) reporter (yellow in overlaid picture, <bold>K′</bold>). (<bold>L</bold> and <bold>M</bold>) Efficiency of perinatal <italic>Cxcl12</italic> excision by the <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> driver in CD45<sup>-</sup>Ter119<sup>−</sup>CD31<sup>-</sup> cells (L) and endothelial (M) cells isolated from P7 bone marrow; qPCR in CD45<sup>-</sup>Ter119<sup>−</sup>CD31<sup>-</sup> cells isolated from <italic>Cxcl12</italic><sup><italic>f/f</italic></sup>;<italic>Nes-CreER</italic><sup><italic>T2</italic></sup> (<bold>E</bold>) and control (<bold>C</bold>) littermates treated with tamoxifen at birth (<italic>n</italic> = 2-3). (<bold>N</bold> and <bold>O</bold>) Bone marrow CFU-C (N) and long-term HSC (O) content in P7 <italic>Cxcl12</italic><sup><italic>f/f</italic></sup><italic>;Nes-CreER</italic><sup><italic>T2</italic></sup> and control littermates treated with tamoxifen at birth. (<bold>O</bold>) Lethally-irradiated mice (CD45.1) were transplanted with 1 ×10<sup>6</sup> bone marrow cells from P7 <italic>Cxcl12</italic><sup><italic>f/f</italic></sup><italic>;Nes-CreER</italic> or <italic>Cxcl12</italic><sup><italic>f/f</italic></sup> mice (CD45.2), together with 1 × 10<sup>6</sup> recipient bone marrow cells (CD45.1). Peripheral donor-derived blood chimerism after 16 weeks is shown (<italic>n</italic> = 4 per group). (<bold>E</bold>, <bold>L</bold>) Each dot represents an individual mouse. (<bold>F</bold>, <bold>H</bold>–<bold>J</bold>) Mean ± SD. *p < 0.05, unpaired two-tailed <italic>t</italic> test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.017">http://dx.doi.org/10.7554/eLife.03696.017</ext-link></p></caption><graphic xlink:href="elife03696f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03696.018</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Neural crest-derived cells direct developmental HSC migration to the bone marrow.</title><p>(<bold>A</bold>) Cell cycle profile of Lin<sup>−</sup> Sca-1<sup>+</sup> E17.5 liver cells isolated from <italic>Nes-CreER</italic><sup><italic>T2</italic></sup>;<italic>iDTR</italic> embryos and control <italic>iDTR</italic> littermates 24 hr after tamoxifen administration and 48 hr after diphtheria toxin treatment (mean ± SEM, <italic>n</italic> = 6). (<bold>B</bold>) Representative femoral bone marrow section from a tamoxifen-treated <italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-DTA</italic> mouse immunostained with collagen IV antibody (red) to reveal blood vessels (<bold>B′</bold>). Nuclei were counterstained with DAPI (gray). Scale bar, 100 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03696.018">http://dx.doi.org/10.7554/eLife.03696.018</ext-link></p></caption><graphic xlink:href="elife03696fs008"/></fig></fig-group></p></sec><sec id="s2-8"><title>Nestin<sup>+</sup> MSC-derived Cxcl12 contributes to the establishment of the HSC niche in the bone marrow</title><p>HSC migration to fetal bone marrow is enhanced by Cxcl12 and stem cell factor (<xref ref-type="bibr" rid="bib15">Christensen et al., 2004</xref>), both of which are highly expressed and progressively upregulated in bone marrow nestin<sup>+</sup> cells at perinatal stages (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). Cxcl12 is produced by several stromal cells and is required for developmental bone marrow colonization by HSCs (<xref ref-type="bibr" rid="bib2">Ara et al., 2003</xref>). It has been argued that Cxcl12 produced by endothelial cells and nestin<sup>−</sup> mesenchymal progenitors—but not by nestin<sup>+</sup> cells—is necessary for adult HSC maintenance (<xref ref-type="bibr" rid="bib20">Ding and Morrison, 2013</xref>; <xref ref-type="bibr" rid="bib26">Greenbaum et al., 2013</xref>). We found that, one week after birth, <italic>Cxcl12</italic> mRNA levels in Nes-GFP<sup>+</sup> BMSCs were >20-fold higher than in bone marrow endothelial cells and 80-fold higher than in Nes-GFP<sup>-</sup> BMSCs (<xref ref-type="fig" rid="fig7">Figure 7H</xref>). Among neural crest-traced cells, Nes-GFP<sup>+</sup> BMSCs were particularly enriched in the expression of <italic>Cxcl12</italic> and endogenous <italic>Nestin</italic> (<xref ref-type="fig" rid="fig7">Figure 7I,J</xref>). To conditionally delete Cxcl12 in nestin<sup>+</sup> cells in the first postnatal week, we intercrossed <italic>Cxcl12</italic> <sup><italic>fl</italic></sup> mice (<xref ref-type="bibr" rid="bib75">Tzeng et al., 2010</xref>) with <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> mice, which mostly label Nes-GFP<sup>+</sup> cells during this period (<xref ref-type="fig" rid="fig7">Figure 7K</xref>). Tamoxifen administration did not significantly alter <italic>Cxcl12</italic> mRNA levels in bone marrow endothelial cells but decreased these levels by 5-fold in BMSCs (<xref ref-type="fig" rid="fig7">Figure 7L–M</xref>). This was associated with an ∼30% reduction of bone marrow hematopoietic progenitors and HSCs measured by long-term competitive repopulation assays (<xref ref-type="fig" rid="fig7">Figure 7N,O</xref>). These results demonstrate that Cxcl12 production by nestin<sup>+</sup> MSCs contributes to the HSC niche formation in the developing bone marrow.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The aim of this study was to investigate the ontogeny and specific functions of mesenchymal progenitors in the fetal bone marrow. We show that the developing bone marrow in axial and appendicular skeleton harbors different MSC populations with distinct origins and specialized roles. While mesoderm-derived nestin<sup>−</sup> MSCs give rise to bone and cartilage, the neuroectoderm provides an additional source of MSCs, marked by nestin expression, that are endowed with specific HSC niche functions. We therefore conclude that osteochondroprogenitor and stem cell niche functions are separate and non-overlapping during bone marrow ontogenesis. The neural crest thus gives rise to three regulators of adult HSC activity: sympathetic neurons, associated Schwann cells, and nestin<sup>+</sup> MSCs.</p><p>Although it is accepted that mammalian connective tissues, such as bone or skeletal muscle, are derived mainly from mesoderm, the precise origin of BMSCs has remained unclear. While the neural crest contributes to the craniofacial skeleton, the trunk neural crest is thought to generate mostly non-ectomesenchymal derivatives, including melanocytes, neurons, and glia of the peripheral nervous system. In the trunk skeleton, mesenchymal cells have thus been considered to be derived mostly from the mesoderm (<xref ref-type="bibr" rid="bib57">Olsen et al., 2000</xref>), but the neural crest is also a source of pericytes, mural cells, and fibroblasts (<xref ref-type="bibr" rid="bib7">Bergwerff et al., 1998</xref>; <xref ref-type="bibr" rid="bib23">Etchevers et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Joseph et al., 2004</xref>) that can differentiate into mesenchymal lineages in vitro (<xref ref-type="bibr" rid="bib51">Morikawa et al., 2009b</xref>; <xref ref-type="bibr" rid="bib25">Glejzer et al., 2011</xref>; <xref ref-type="bibr" rid="bib32">John et al., 2011</xref>; <xref ref-type="bibr" rid="bib37">Komada et al., 2012</xref>). In addition, mature endothelial cells can generate mesenchymal cells through endothelial-to-mesenchymal transition. Genetic fate-mapping studies using the neuroepithelial marker <italic>Sox1</italic> identified a neuroectodermal origin of the earliest trunk MSCs, but other MSCs are recruited from undefined sources at later stages (<xref ref-type="bibr" rid="bib74">Takashima et al., 2007</xref>). This picture raised the question of whether several MSCs might transiently coexist in the developing bone marrow and whether neural-crest-derived MSCs with specific functions might persist in the postnatal bone marrow.</p><p>In this study, we show that most mesenchymal activity and chondrogenic capacity in the fetal bone marrow is associated with nestin<sup>−</sup> MSCs, but that these rapidly differentiate towards committed osteochondral lineages early in postnatal life. In contrast, slow-proliferating neural crest-derived nestin<sup>+</sup> MSCs do not contribute to fetal endochondrogenesis but are instead required to establish the HSC niche in the same bones. Interestingly, we also found that nestin<sup>+</sup> cells retained most of their steady-state MSC activity after the second postnatal week. Future studies will determine the potential contribution of this MSC population to adult skeletal turnover.</p><p>Very recent data showed that <italic>Osterix-Cre</italic>-labeled cells in neonatal bone give rise to Nes-GFP<sup>+</sup> MSCs (<xref ref-type="bibr" rid="bib49">Mizoguchi et al., 2014</xref>; <xref ref-type="bibr" rid="bib59">Ono et al., 2014</xref>); however, the <italic>Osterix-Cre</italic> lines used in these studies have been shown previously to mark not only osteolineage cells but also a large variety of non-osteolineage cells, including adventitial reticular cells, vascular smooth muscle cells, adipocytes, and perineural cells (<xref ref-type="bibr" rid="bib43">Liu et al., 2013</xref>). It therefore remains possible that some Nes-GFP<sup>+</sup> cells, which highly express <italic>Osterix</italic> mRNA (but not the protein, as we show here), also express the <italic>Osterix-Cre</italic> transgene; however, this might not necessarily reflect a lineage relationship or osteoblastic commitment. Moreover, it is unclear whether this <italic>Osterix-Cre</italic>-traced population is uniformly mesodermal or neural crest-derived. We did not find Nes-GFP<sup>+</sup> cells within cartilage, but some GFP<sup>+</sup> cells were associated with the innermost part of the perichondrium, a region that contains MSCs (<xref ref-type="bibr" rid="bib44">Maes et al., 2010</xref>; <xref ref-type="bibr" rid="bib79">Yang et al., 2013</xref>; <xref ref-type="bibr" rid="bib80">Zaidi and Mendez-Ferrer, 2013</xref>). Moreover, we found perichondrial <italic>Wnt1-Cre2</italic>-traced cells and neural crest-derived chondrocytes in the most superficial layers of articular cartilage, suggesting that the neural crest also contributes to these mesenchymal cells outside the marrow. Neural crest-derived skeletal embryonic precursors might be Pdgfrα<sup>+</sup> Nes-GFP<sup>-</sup> BMSCs, since we have shown that most neural crest cells traced by <italic>Wnt1-Cre2</italic> are Pdgfrα<sup>+</sup> Nes-GFP<sup>+</sup> cells, which do not seem to contribute to fetal osteochondral lineages.</p><p>Previous studies suggested that only endochondral cells can form a hematopoietic microenvironment when implanted beneath the kidney capsule (<xref ref-type="bibr" rid="bib13">Chan et al., 2008</xref>, <xref ref-type="bibr" rid="bib14">2013</xref>), although bones formed by intramembranous ossification, such as the skull, can also form hematopoietic marrow without progressing through a cartilage intermediate. Notably, two surface markers used to isolate osteochondrogenic precursors in these studies, CD105 and CD90/Thy1, are particularly enriched in bone marrow Nes-GFP<sup>+</sup> cells. Our genetic studies clearly show that neural crest-derived cells that are not yet committed to the Schwann cell lineage migrate to bone in association with developing nerve fibers and give rise to bone marrow nestin<sup>+</sup> MSCs with specialized HSC niche functions. Constitutive deletion of the neuregulin-1 receptor ErbB3<italic>,</italic> which does not directly affect the peripheral nerves but impairs perineural migration of neural crest-derived cells (<xref ref-type="bibr" rid="bib63">Riethmacher et al., 1997</xref>), reduces BMSC number and impairs developmental HSC migration from liver to bone marrow. In contrast, conditional deletion of <italic>ErbB3</italic> in committed glial precursors severely reduced Schwann cell numbers (<xref ref-type="bibr" rid="bib67">Sheean et al., 2014</xref>) but did not affect bone marrow HSCs in our study. HSC maintenance genes are highly enriched and progressively upregulated in Nes-GFP<sup>+</sup> Pdgfrα<sup>+</sup> MSCs, coincident with HSC bone marrow colonization (<xref ref-type="bibr" rid="bib15">Christensen et al., 2004</xref>). Perinatal deletion of nestin<sup>+</sup> cells or blockade of their Cxcl12 production prevented HSC bone marrow seeding. Therefore, we are confident that neural crest-derived nestin<sup>+</sup> MSCs are indeed required for developmental HSC migration and formation of the bone marrow HSC niche.</p><p>Uncertainties remain about the possible overlap among different adult bone marrow mesenchymal cells with proposed HSC niche functions, which might also differ in fetal and adult bone marrow. Two populations of adult bone marrow Nes-GFP<sup>+</sup> cells can be separated by fluorescence intensity by microscopy. Nes-GFP<sup>bright</sup> peri-arteriolar cells, unlike Nes-GFP<sup>dim</sup> peri-sinusoidal cells, were found highly enriched in CFU-F activity and expression of HSC maintenance genes (<xref ref-type="bibr" rid="bib38">Kunisaki et al., 2013</xref>). However, these features have been previously attributed to peri-sinusoidal cells (<xref ref-type="bibr" rid="bib36">Kiel et al., 2005</xref>; <xref ref-type="bibr" rid="bib64">Sacchetti et al., 2007</xref>), which, unlike arteriolar cells, express both Nes-GFP<sup>+</sup> and leptin receptor (<xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>). Alternatively, an intermediate type of vessel that connects arterioles with sinusoids near the bone surface might be highly enriched in nestin<sup>+</sup> MSCs. These vessels, different from arterioles and sinusoids, contain CD31<sup>hi</sup> Endomucin<sup>hi</sup> endothelial cells and have been associated with osteoprogenitor activity (<xref ref-type="bibr" rid="bib39">Kusumbe et al., 2014</xref>). We initially reported that adult bone marrow Nes-GFP<sup>+</sup> cells were CD31<sup>−</sup> after using an intensive enzymatic digestion protocol to isolate the cells (<xref ref-type="bibr" rid="bib47">Mendez-Ferrer et al., 2010</xref>). In the present study, we have used milder conditions that better preserve antigen expression. In these conditions, we detect Nes-GFP<sup>+</sup> CD31<sup>+</sup> putative bone marrow endothelial cells progressively increasing in number with age, as reported by others (<xref ref-type="bibr" rid="bib59">Ono et al., 2014</xref>). We also find that multiple layers of Nes-GFP<sup>+</sup> endothelial and perivascular cells likely make the arteriolar GFP signal appear brighter for GFP under the microscope. This raises the possibility that arteriolar Nes-GFP<sup>+</sup> cells (<xref ref-type="bibr" rid="bib38">Kunisaki et al., 2013</xref>) might not completely coincide with the brightest cells isolated by FACS. However, we also found that <italic>Cxcl12</italic> is more abundantly produced by Nes-GFP<sup>+</sup> BMSCs than by endothelial cells, and that <italic>Cxcl12</italic> deletion in <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> mice impairs developmental HSC migration by preferentially targeting BMSCs.</p><p>Different bone marrow cells have been proposed as the main producers of Cxcl12 for HSCs. One report has argued that the only relevant source of Cxcl12 for adult HSC maintenance is nestin<sup>–</sup>leptin receptor<sup>–</sup>mesenchymal progenitors targeted by the <italic>Prx1-Cre</italic> driver (<xref ref-type="bibr" rid="bib26">Greenbaum et al., 2013</xref>). This conclusion is based on the stronger HSC depletion when <italic>Cxcl12</italic> was deleted using the <italic>Prx1-Cre</italic> line—which targets somatic lateral plate mesoderm and its derivatives, including chondrogenic and osteogenic lineages—compared to deletion in more specific stromal populations. Also, the HSC defect was specifically attributed to CD45<sup>-</sup> Lin<sup>−</sup> Pdgfrα<sup>+</sup> Sca-1<sup>+</sup> <italic>Prx1-cre</italic>-tdtomato<sup>hi</sup> cells which were not enriched in the expression of nestin or leptin receptor but also showed no marked enrichment in the expression of <italic>Cxcl12</italic>, <italic>Kitl</italic> or other mesenchymal markers. An alternate model is that endothelial cells are needed for Cxcl12-mediated adult HSC maintenance (<xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>; <xref ref-type="bibr" rid="bib20">Ding and Morrison, 2013</xref>). Nonetheless, the <italic>Tek</italic>-<italic>Cre</italic> system used would also target endothelial cells in fetal hematopoietic organs, so some of the effects might have been exported to the bone marrow during development. The study, in essence, proposed that nestin-negative leptin receptor (<italic>Lepr</italic>)-<italic>Cre</italic>-traced mesenchymal progenitors are another key source of Cxcl12 for HSC maintenance. These studies, however, analyzed the adult bone marrow, whereas the focus of our study has been the fetal and perinatal period. Differences may also have arisen from distinct experimental settings, Cre drivers (constitutive/inducible), Cre induction regimes and reporters used. It is also likely that constitutive <italic>Prx1-Cre</italic> and <italic>Lepr-Cre</italic> lines target multiple mesenchymal derivatives and that combined deletion of <italic>Cxcl12</italic> in these populations would have a more pronounced effect than deletion in specific cell types; however, the responsible cell populations might not be clear yet. Conversely, the lower excision in <italic>Nes-Cre</italic> mice and inefficient bone marrow recombination in adult <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> mice might not target all MSCs, or might result in compensatory actions by other Cxcl12-producing cells. In the present study, recombination efficiency in Nes-GFP<sup>+</sup> MSCs was higher when tamoxifen was administered in neonatal <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> mice than in adults. It has also been proposed that <italic>Nes</italic>-G<italic>fp</italic> and <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> lines might target different populations (<xref ref-type="bibr" rid="bib21">Ding et al., 2012</xref>). To directly address this, we generated <italic>Nes</italic>-G<italic>fp</italic>;<italic>Nes-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> triple transgenic mice that demonstrate consistent labeling using a different induction protocol. Bone marrow endothelial <italic>Cxcl12</italic> expression levels are significantly lower in this model, and seem unaffected by <italic>Nes-CreER</italic><sup><italic>T2</italic></sup>-driven excision, contrasting with the reduction in BMSCs, which was associated with decreased HSPC numbers in perinatal bone marrow. Our results thus clearly show that Cxcl12 produced by nestin<sup>+</sup> MSCs is required for developmental HSC migration to bone marrow.</p><p>We recently reported that sympathetic neuropathy of the HSC niche is required for the manifestation of myeloproliferative neoplasms, disorders previously considered to be autonomously driven by mutated HSCs and typically associated with excessive fibroblasts and osteoblasts in the bone marrow. During this pathogenesis, bone marrow nestin<sup>+</sup> cells do not seem to differentiate into fibroblasts or osteoblasts, but instead activate the Schwann cell program as a consequence of the neuroglial damage caused in the bone marrow by mutated HSCs (<xref ref-type="bibr" rid="bib4"><italic>Arranz et al., 2014</italic></xref>). These changes could be explained by a neural crest contribution found for HSC niche-forming MSCs and suggest the possible re-programming of these cells towards the closest ontogenically-related linages during the pathogenesis of these disorders.</p><p>In summary, this study designates separate biologic functions to ontogenically distinct populations of MSCs, and demonstrates that not all MSCs are alike. In the appendicular skeleton, nestin<sup>−</sup> MSCs derived from the mesoderm have a primarily osteochondroprogenitor function. In contrast, a distinct population of neural crest-derived nestin<sup>+</sup> MSCs contributes to directed HSC migration through the secretion of the chemokine Cxcl12 to ultimately establish the HSC niche in the neonatal bone marrow. These niche-forming MSCs share a common origin with sympathetic neurons and Schwann cells, an ontogenic relationship that underscores our earlier observations on the sympathetic control of HSC niche function (<xref ref-type="bibr" rid="bib46">Mendez-Ferrer et al., 2008</xref>, <xref ref-type="bibr" rid="bib47">2010</xref>; <xref ref-type="bibr" rid="bib4">Arranz et al., 2014</xref>). Future studies will also determine whether tight regulation of other peripheral adult stem cell niches by the nervous system also builds upon an ontogenic relationship of their components.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Animals</title><p>Mouse lines used in this study (please see <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> for a detailed list of mouse strains used in this study) included <italic>Nes-Gfp</italic> (<xref ref-type="bibr" rid="bib48">Mignone et al., 2004</xref>), <italic>Nes-CreER</italic><sup><italic>T2</italic></sup> (<xref ref-type="bibr" rid="bib6">Balordi and Fishell, 2007</xref>), <italic>Sox10-CreER</italic><sup><italic>T2</italic></sup> (<xref ref-type="bibr" rid="bib45">Matsuoka et al., 2005</xref>), <italic>Col2.3-Cre</italic> (<xref ref-type="bibr" rid="bib17">Dacquin et al., 2002</xref>), <italic>Dhh-Cre</italic> (<xref ref-type="bibr" rid="bib30">Jaegle et al., 2003</xref>), <italic>RCE-loxP</italic> (<xref ref-type="bibr" rid="bib71">Sousa et al., 2009</xref>), <italic>LSL-KFP</italic> (<xref ref-type="bibr" rid="bib19">Dieguez-Hurtado et al., 2011</xref>), <italic>R26-DTA</italic> (<xref ref-type="bibr" rid="bib9">Brockschnieder et al., 2006</xref>), <italic>Cxcl12</italic><sup>floxed</sup> (<xref ref-type="bibr" rid="bib75">Tzeng et al., 2010</xref>), <italic>Erbb3</italic><sup>floxed</sup> (<xref ref-type="bibr" rid="bib67">Sheean et al., 2014</xref>), <italic>Erbb3</italic>-null (<xref ref-type="bibr" rid="bib63">Riethmacher et al., 1997</xref>), and <italic>129S4.Cg-Tg(Wnt1-cre)2Sor/J</italic>, <italic>C57BL/6-Gt(ROSA)26Sor</italic><sup><italic>tm1(HBEGF)Awai</italic></sup><italic>/J</italic>, <italic>B6.Cg-Gt(ROSA)26Sor</italic><sup><italic>tm14(CAG-tdTomato)Hze</italic></sup><italic>/J</italic>, wild-type CD1 and wild-type C57BL/6J (Jackson Laboratories). Material and methods were approved by the Animal Care and Use Committees of the Spanish National Cardiovascular Research Center and Comunidad Autónoma de Madrid (PA-47/11 and ES280790000176).</p></sec><sec id="s4-2"><title>Embryo analysis and genetic inducible fate mapping</title><p>Embryos were dissected as previously described (<xref ref-type="bibr" rid="bib28">Isern et al., 2008</xref>). Briefly, selected intercrosses between mice carrying the alleles of interest were set and the morning of detection of the vaginal plug was considered as day 0.5 of gestation. We preferentially used paternal transgene transmission, by mating compound or simple transgenic males with females of wild-type background (C57BL/6 or CD1). Inducible lineage tracing studies were conducted as follows. Tamoxifen (T5648; Sigma, St. Louis, MO) was dissolved in corn oil at a final concentration of 20 mg/mL and given to pregnant dams by oral gavage (100-150 mg/kg) on the morning of the indicated stages. For neonatal induction, mothers of newborn pups were given tamoxifen (by oral gavage, 4 mg) on days 1 and 3 after delivery.</p></sec><sec id="s4-3"><title>Histology</title><p>Dissected tissues for histology were fixed in 2% paraformaldehyde at 4°C, cryopreserved by consecutive equilibration in 15% and 30% sucrose, and snap frozen and embedded in OCT compound (Tissue-Tek). In some cases, fixed frozen limbs or sterna were trimmed sequentially from both sides to expose the central medullar cavity and processed further for whole-mount fluorescence staining. Cryostat sections (15 μm) were prepared and processed for immunostaining or regular hematoxylin–eosin staining. Oil red O staining was performed as described (<xref ref-type="bibr" rid="bib29">Isern et al., 2013</xref>).</p></sec><sec id="s4-4"><title>Immunohistochemistry</title><p>Cryostat sections were stained using standard procedures. Briefly, tissues were permeabilized for 5-10 min at room temperature (RT) with 0.1% Triton X-100 and blocked for 1 h at RT with TNB buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent, Perkin Elmer, Waltham, MA). Primary antibody incubations were conducted either for 1-2 h at RT or overnight (o/n) at 4°C. Secondary antibody incubations were conducted for 1 h at RT. Repetitive washes were performed with PBS + 0.05% Tween-20. Stained tissue sections were counterstained for 5 min with 5 μM DAPI and rinsed with PBS. Slides were mounted in Vectashield Hardset mounting medium (Vector Labs, Burlingame, CA) and sealed with nail polish.</p><p>For whole-mount staining of thick-sectioned tissue pieces, all the incubations, including permeabilization and blocking, were performed o/n at 4°C with gently agitation, and washing steps were extended. Specimens were mounted in glass bottom dishes (Mat-Tek, Ashland, MA).</p></sec><sec id="s4-5"><title>Immunofluorescence</title><p>Fetal bone marrow and fetal liver sections were stained following standard procedures. Antibodies used are indicated in the table below. SLAM staining was performed in bone marrow sections from neonate mice. Slides were first blocked in 20% goat serum in PBS for 45 min. Endogenous avidin and biotin were blocked with an Avidin/Biotin Blocking Kit (Vector Labs) for 30 min with each reagent, washing 3 times with PBS in between. Slides were then incubated with rat anti-mouse CD150 antibody (Biolegend, San Diego, CA) at 1:50 dilution in goat blocking buffer for 2 h, and after washes with goat anti-rat IgG conjugated to Alexa555 (Molecular Probes, Eugene, OR) at 1:200 dilution in 20% goat serum in PBS for 1 h. Slides were then blocked in 20% rat serum in PBS for 10 min and incubated for 1 h with hamster anti-mouse biotin-conjugated CD48 (Abcam, UK) and the Biotin Mouse Lineage Panel (BD Pharmingen, San Jose, CA), which includes rat anti-mouse B220, rat anti-mouse CD3, rat anti-mouse Gr1, rat anti-mouse Mac-1, and rat anti-mouse Ter119 antibodies, each at 1:200 dilution in rat blocking buffer. Cy5-conjugated streptavidin (Molecular Probes) was added at 1:200 in rat blocking buffer for 30 min. Finally, slides were incubated with DAPI (1:1000 dilution of 5 mg/ml stock) for 10 min at room temperature and mounted in Vectashield Mounting Medium (Vector Labs). Antibodies used for Immunohistochemistry:<table-wrap id="tblu1" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Name</th><th>Type</th><th>Company</th></tr></thead><tbody><tr><td>TH</td><td>Rabbit pAb</td><td>Millipore</td></tr><tr><td>GFAP</td><td>Rabbit pAb</td><td>Dako</td></tr><tr><td>CD31</td><td>Rat mAb</td><td>BD Pharmingen</td></tr><tr><td>S100</td><td>Rabbit pAb</td><td>Dako</td></tr><tr><td>Collagen type IV</td><td>Rabbit pAb</td><td>Millipore</td></tr><tr><td>Ki67</td><td>Rabbit pAb</td><td>Abcam</td></tr><tr><td>Anti-KFP</td><td>Rabbit pAb</td><td>Evrogen</td></tr><tr><td>CD150</td><td>Rat mAb</td><td>Biolegend</td></tr><tr><td>CD48</td><td>ArHm mAb</td><td>Abcam</td></tr><tr><td>Lineage-biotin</td><td>Rat mAb</td><td>BD Biosciences</td></tr><tr><td>Tuj1</td><td>Mouse mAb</td><td>Promega</td></tr><tr><td>Anti-GFP</td><td>Rabbit pAb</td><td>Abcam</td></tr><tr><td>c-kit</td><td>goat pAb</td><td>R&D</td></tr><tr><td>Nestin</td><td>Rabbit pAb</td><td>Abcam</td></tr><tr><td>α-SMA</td><td>Mouse mAb</td><td>Sigma</td></tr></tbody></table></table-wrap></p></sec><sec id="s4-6"><title>Imaging</title><p>Confocal images of fluorescent staining were acquired with a laser scanning confocal microscope (Zeiss LSM 700, 10×/0.45, 25×/0.85) or with a multi-photon Zeiss LSM 780 microscope (10×/0.7, 20×/1.0). Optical z-stack projections were generated with the Zen2011 software package (Zeiss, Germany) using a maximal intensity algorithm. Wide-field views of whole-mount specimens were imaged with a Leica MZFLIII stereomicroscope equipped with an Olympus DP71 color camera. Images were post-processed and quantified using ImageJ (<xref ref-type="bibr" rid="bib66">Schneider et al., 2012</xref>) and Photoshop (Adobe, San Jose, CA).</p></sec><sec id="s4-7"><title>Preparation of fetal and neonatal bone marrow cell suspensions</title><p>Fetal skeletal elements were sub-dissected from fetuses, homogenized by cutting, and digested for 15-30 min at 37°C with shaking in 0.25% collagenase (StemCell Technologies, Canada). Postnatal bone specimens were cleaned from surrounding tissue, crushed in a mortar with a pestle, and collagenase-digested for 45-60 min at 37°C, with constant agitation. After enzymatic treatment, skeletal preparations were filtered through a 40-μm cell strainer and undigested bone material was discarded. The resulting bone marrow-enriched cell suspensions were pelleted, washed twice, and resuspended in FACS staining buffer (2% FCS in PBS) for further analysis.</p></sec><sec id="s4-8"><title>Flow cytometry</title><p>Dispersed bone marrow cell preparations were stained in FACS buffer for 15-30 min on ice with selected multicolor antibody cocktails (see below), washed, and resuspended with streptavidin conjugates when necessary. Stained cells were pelleted and resuspended in buffer containing DAPI to exclude dead cells. Cell cycle was analyzed by first isolating defined stromal populations by FACS, and then acquiring the cell cycle profile after staining the sorted populations with Hoechst 33342. Flow cytometry analysis and FACS were done in FACS CantoII or LSRFortessa machines (BD Biosciences) equipped with Diva Software (BD Biosciences) or in a FACS AriaII cell sorter (BD Biosciences). Data were analyzed using Diva and FlowJo (Tree Star, Inc, Eugene, OR). Antibodies used for cytometry:<table-wrap id="tblu2" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Name</th><th>Clone</th><th>Company</th></tr></thead><tbody><tr><td>CD45-APC/Cy7</td><td>104</td><td>BD Biosciences</td></tr><tr><td>CD45-APC</td><td>104</td><td>BD Biosciences</td></tr><tr><td>CD31-APC</td><td>MEC 13.3</td><td>BD Biosciences</td></tr><tr><td>Ter119-APC</td><td>Ter119</td><td>BD Biosciences</td></tr><tr><td>CD140a-biotin</td><td>APA5</td><td>eBioscience</td></tr><tr><td>CD140a-APC</td><td>APA5</td><td>Biolegend</td></tr><tr><td>CD90.2-APC</td><td>53-2.1</td><td>eBioscience</td></tr><tr><td>Ly6a-PE</td><td>E13–161.7</td><td>BD Biosciences</td></tr><tr><td>Vcam1-PE</td><td>429 (MVCAM.A)</td><td>Biolegend</td></tr><tr><td>Streptavidin-PE</td><td>--</td><td>BD Biosciences</td></tr><tr><td>Lineage cocktail-biotin</td><td/><td>BD Biosciences</td></tr></tbody></table></table-wrap></p></sec><sec id="s4-9"><title>CFU-F and CFU-OB assays</title><p>For fibroblast colony-forming unit (CFU-F) assays, bone marrow cell suspensions were FACS sorted directly into 6-well plates at a cell density of 100–500 cells/cm<sup>2</sup> and cultured in maintenance medium (α-MEM/15% FCS with antibiotics). After 10-12 days in culture, adherent cells were fixed with 100% methanol and stained with Giemsa stain (Sigma) to reveal fibroblast clusters. Colonies with more than 50 cells were scored as CFU-Fs. For osteoblast colony-forming unit (CFU-OB) assays, plated cells were cultured in maintenance medium in the presence of 1 mM L-ascorbate-2-phosphate. All cultures were maintained with 5% CO<sub>2</sub> in a water-jacketed incubator at 37°C, and medium was changed weekly. After 25 days in culture, cells were fixed and stained with alizarin red or alkaline phosphatase (<xref ref-type="bibr" rid="bib29">Isern et al., 2013</xref>).</p></sec><sec id="s4-10"><title>Hematopoietic progenitor assays</title><p>Single cell suspensions were prepared from bone marrow and mixed with methylcellulose-containing medium with cytokines (<xref ref-type="bibr" rid="bib12">Casanova-Acebes et al., 2013</xref>). Cells (5-7.5 × 10<sup>4</sup>) were plated in duplicate 35 mm dishes (Falcon, BD) and incubated under 20% O<sub>2</sub> and 5% CO<sub>2</sub> in a water-jacketed incubator. Hematopoietic colonies (CFU-Cs) were scored after 6-7 days in culture.</p></sec><sec id="s4-11"><title>Long-term culture-initiating cell assay</title><p>Long-term culture-initiating cell assay was performed as described (<xref ref-type="bibr" rid="bib77">Woehrer et al., 2013</xref>). Briefly, the feeder fetal stromal cell line AFT024 (kindly provided by Dr. K. Moore) was maintained as previously described (<xref ref-type="bibr" rid="bib56">Nolta et al., 2002</xref>). One week before use, the feeders were irradiated (15 Gy) with a <sup>137</sup>Cs irradiator and seeded in 96-well plates at confluency. After 7-10 days, five serial dilutions (each with 16 replicates) of sorted fetal liver Lin<sup>−</sup> Sca1<sup>+</sup> cells and bone marrow nucleated cells were seeded on the irradiated feeders and cultured with Myelocult M5300 supplemented with 10<sup>−6</sup> M hydrocortisone (StemCell Technologies) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). Cultures were maintained for four weeks at 33°C under 20% O<sub>2</sub> and 5% CO<sub>2</sub> in a water-jacketed incubator. Medium was half-changed weekly. Each well was then trypsinized for 10 min, washed with PBS, and plated for the hematopoietic progenitor assay. Twelve days after plating, the percentage of culture dishes in each experimental group that failed to generate CFU-Cs was plotted against the number of test cells. The frequencies of long-term culture-initiating cells were calculated by the Newton–Raphson method of maximum likelihood and Poisson statistics (using L-CalcTM software; StemCell Technologies) as the reciprocal of the number of test wells that yielded a 37% negative response.</p></sec><sec id="s4-12"><title>Cell culture and in vitro differentiation</title><p>Primary bone marrow cells were obtained from dissected bones using a mortar. All cultures were maintained at 37°C with 20% O<sub>2</sub>, 5% CO<sub>2</sub> in a water-jacketed incubator.</p><p>To obtain CFU-Fs and CFU-OBs, 0.5 × 10<sup>6</sup> bone marrow nucleated cells were seeded in each well of a 12-well plate with α-MEM supplemented with 1% penicillin-streptomycin, 15% FBS (Invitrogen), and 1 mM L-ascorbic acid 2-phosphate (Sigma). Half medium was replaced every 5 days. The numbers of CFU-Fs and CFU-OBs were scored after 10 and 28 days in culture, respectively.</p><p>CFU-F cultures were fixed in methanol for 10 min at room temperature. Cultures were stained with Giemsa diluted 1:10 in phosphate buffer, pH 6.8, for 10 min at 37°C. CFU-F colonies (those with more than 50 cells) were counted the next day.</p><p>CFU-OB cultures were fixed with 4% paraformaldehyde (PFA) for 5 min at room temperature. von Kossa staining was performed by adding 5% AgNO<sub>3</sub> to the culture and exposing plates to UV radiation for 20 min. Cells were then incubated with 5% (NH<sub>4</sub>)<sub>2</sub>S<sub>2</sub>O<sub>3</sub> in distilled water for 5 min and counterstained with 2% eosin. For Alizarin Red staining, cells were incubated with 2% alizarin red reagent (Sigma) in distilled H<sub>2</sub>O for 15 min. For alkaline phosphatase staining, Sigma Fast BCIP/NBT substrate (Sigma) was added to cell cultures followed by incubation in the dark for 15 min.</p></sec><sec id="s4-13"><title>Nucleic acid purification and qPCR</title><p>RNA from CFU-Fs and osteoblast cultures was extracted with Trizol reagent (Sigma-Aldrich) and purified on RNeasy mini columns (Qiagen, Netherlands). An on-column DNase digest (Qiagen) was performed before the clean-up step to eliminate residual genomic DNA. For osteoblast cultures, mRNA was extracted with the Dynabead mRNA DIRECT kit (Invitrogen). cDNA was generated using High Capacity cDNA Reverse Transcription reagents (Applied Biosystems, Waltham, MA). qPCR was performed in triplicate with SYBRgreen Universal PCR Master Mix (Applied Biosystems), using primers optimized for each target gene. The expression level of each gene was determined by using the relative standard curve method. Briefly, a standard curve was performed by doing serial dilutions of a mouse reference total RNA (Clontech, Palo Alto, CA). The expression level of each gene was calculated by interpolation from the standard curve. Relative quantifications of each transcript were obtained by normalizing against <italic>Gapdh</italic> transcript abundance, using the standard curve method. The sequences of oligonucleotides for qPCR are detailed below.<table-wrap id="tblu3" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Target gene</th><th>Symbol</th><th>Forward</th><th>Reverse</th></tr></thead><tbody><tr><td><italic>Alkal.Phosphat.</italic></td><td><italic>Alpl</italic></td><td>CACAATATCAAGGATATCGACGTGA</td><td>ACATCAGTTCTGTTCTTCGGGTACA</td></tr><tr><td><italic>Osterix</italic></td><td><italic>Sp7</italic></td><td>ATGGCGTCCTCTCTGCTTGA</td><td>GAAGGGTGGGTAGTCATTTG</td></tr><tr><td><italic>Runx2</italic></td><td><italic>Runx2</italic></td><td>TTACCTACACCCCGCCAGTC</td><td>TGCTGGTCTGGAAGGGTCC</td></tr><tr><td><italic>Rank ligand</italic></td><td><italic>Rankl</italic></td><td>CAGCATCGCTCTGTTCCTGTA</td><td>CTGCGTTTTCATGGAGTCTCA</td></tr><tr><td><italic>Gpnmb</italic></td><td><italic>Gpnmb</italic></td><td>CCCCAAGCACAGACTTTTGAG</td><td>GCTTTCTGCATCTCCAGCCT</td></tr><tr><td><italic>Osteocalcin</italic></td><td><italic>Bglap</italic></td><td>GGGCAATAAGGTAGTGAACAG</td><td>GCAGCACAGGTCCTAAATAGT</td></tr><tr><td><italic>Osteoglycin</italic></td><td><italic>Ogn</italic></td><td>ACCATAACGACCTGGAATCTGT</td><td>AACGAGTGTCATTAGCCTTGC</td></tr><tr><td><italic>Rank</italic></td><td><italic>Rank</italic></td><td>TGCAGCTCAACAAGGATACG</td><td>GAGCTGCAGACCACATCTGA</td></tr><tr><td><italic>TRAP</italic></td><td><italic>Acp5</italic></td><td>CAGCAGCCAAGGAGGACTAC</td><td>ACATAGCCCACACCGTTCTC</td></tr><tr><td><italic>Cathepsin k</italic></td><td><italic>Ctsk</italic></td><td>GGCCTCTCTTGGCCATA</td><td>CCTTCCCACTCTGGGTAG</td></tr><tr><td><italic>Mmp-9</italic></td><td><italic>Mmp9</italic></td><td>CGTCGTGATCCCCACTTACT</td><td>AACACACAGGGTTTGCCTTC</td></tr><tr><td><italic>Ppar gamma</italic></td><td><italic>Pparg</italic></td><td>ACCACTCGCATTCCTTTGAC</td><td>TGGGTCAGCTCTTGTGAATG</td></tr><tr><td><italic>Adiponectin</italic></td><td><italic>Adipoq</italic></td><td>TGTTCCTCTTAATCCTGCCCA</td><td>CCAACCTGCACAAGTTCCCTT</td></tr><tr><td><italic>Adipsin</italic></td><td><italic>Cfd</italic></td><td>TGCATCAACTCAGAGTGTCAATCA</td><td>TGCGCAGATTGCAGGTTGT</td></tr><tr><td><italic>Sox9</italic></td><td><italic>Sox9</italic></td><td>GAACAGACTCACATCTCT</td><td>GTGGCAAGTATTGGTCAA</td></tr><tr><td><italic>Col2a1</italic></td><td><italic>Col2a1</italic></td><td>GTGGAGCAGCAAGAGCAAGGA</td><td>CTTGCCCCACTTACCAGTGTG</td></tr><tr><td><italic>Aggrecan</italic></td><td><italic>Acan</italic></td><td>CACGCTACACCCTGGACTTTG</td><td>CCATCTCCTCAGCGAAGCAGT</td></tr><tr><td><italic>Cxcl12</italic></td><td><italic>Cxcl12</italic></td><td>CGCCAAGGTCGTCGCCG</td><td>TTGGCTCTGGCGATGTGGC</td></tr><tr><td><italic>Kit ligand</italic></td><td><italic>Kitl</italic></td><td>CCCTGAAGACTCGGGCCTA</td><td>CAATTACAAGCGAAATGAGAGCC</td></tr><tr><td><italic>Angiopoietin 1</italic></td><td><italic>Angpt1</italic></td><td>CTCGTCAGACATTCATCATCCAG</td><td>CACCTTCTTTAGTGCAAAGGCT</td></tr><tr><td><italic>Nestin</italic></td><td><italic>Nes</italic></td><td>GCTGGAACAGAGATTGGAAGG</td><td>CCAGGATCTGAGCGATCTGAC</td></tr></tbody></table></table-wrap></p></sec><sec id="s4-14"><title>Primary sphere-forming cultures</title><p>For sphere formation, cells were plated at clonal density (<1000 cells/cm<sup>2</sup>) in ultra-low adherent 35 mm dishes (StemCell Technologies). The growth medium consisted of DMEM/F12 (1:1) mixed 1:2 with human endothelial serum-free medium (Invitrogen) and contained 15% chicken embryo extract, prepared as described (<xref ref-type="bibr" rid="bib73">Stemple and Anderson, 1992</xref>; <xref ref-type="bibr" rid="bib60">Pajtler et al., 2010</xref>); 0.1 mM ß-mercaptoethanol; 1% non-essential aminoacids (Sigma); 1% N2 and 2% B27 supplements (Invitrogen); recombinant human fibroblast growth factor (FGF)-basic; recombinant human epidermal growth factor (EGF); recombinant human platelet-derived growth factor (PDGF-AB); recombinant human oncostatin M (227 a.a. OSM) (20 ng/ml); and recombinant human insulin-like growth factor-1 (IGF-1; 40 ng/ml) (Peprotech, Rocky Hill, NJ). The cultures were kept at 37°C under 5% CO<sub>2</sub>, 20% O<sub>2</sub> in a water-jacketed incubator, and were left untouched for one week to prevent cell aggregation in low density cultures. Medium was half-changed weekly. Mesenspheres were scored on days 10–14.</p></sec><sec id="s4-15"><title>In vitro differentiation of Schwann cells from bone marrow precursors</title><p>We used an adaptation of the original method (<xref ref-type="bibr" rid="bib8">Biernaskie et al. 2006</xref>). Defined stromal populations were isolated based on GFP and Pdgfrα expression from collagenase-treated bone marrow of <italic>Nes-Gfp</italic> neonates. Sorted cells were plated onto laminin/polylysine-coated chamber slide dishes (Labtek) and allowed to attach and expand in SKP medium I. After 3 days, cells were changed to SKP medium II (containing neuregulin-1 at 50 ng/mL) and allowed to differentiate further for >10 days. In vitro-generated Schwann cells were defined by morphology as thin and elongated cells. After differentiation, cells were fixed in 4% PFA, gently permeabilized with Triton X-100, and stained for immunofluorescence with anti-glial fibrillary acidic protein (Gfap) antibody (Dako, Carpinteria, CA).</p></sec><sec id="s4-16"><title>In vitro differentiation of bone marrow mesenchymal cells</title><p>Defined stromal populations were isolated based on GFP and Pdgfrα expression from collagenase-treated bone marrow of <italic>Nes-Gfp</italic> neonates and plated directly onto plastic dishes to allow attachment of fibroblasts. Adherent cells were cultured for 7-14 days in regular α-MEM supplemented with 15% FBS. In some cases, recombinant human PDGF was added at 20 ng/mL. At the end of the culture period, cells were fixed and stained with Oil red O to reveal adipocytes and counterstained with hematoxylin.</p></sec><sec id="s4-17"><title>RNA-Seq</title><p>For next-generation sequencing, total RNA was isolated using the Arcturus Picopure RNA isolation kit (Life Technologies, Carlsbad, CA) from small numbers of sorted cells (15,000-80,000), obtained from neonatal <italic>Nes-Gfp</italic> bone marrow preparations (two biological replicates). Each independent set of samples was obtained from pooled skeletal elements (long bones and sterna) from multiple littermates.</p><sec id="s4-17-1"><title>RNA-Seq library production</title><p>Amplified cDNA was prepared with the Ovation RNA-Seq System V2 Kit (NuGEN Technologies Inc., San Carlos, CA) followed by sonication and TrueSeq DNA Library Preparation Kit from Illumina. The quality, quantity, and the size distribution of the Illumina libraries were determined using the DNA-1000 Kit (Agilent Bioanalyzer). Libraries were sequenced on a Genome Analyzer II× (Illumina Inc., San Diego, CA) following the standard RNA sequencing protocol with the TruSeq SBS Kit v5. Fastq files containing reads for each library were extracted and demultiplexed using the Casava v1.8.2 pipeline.</p></sec><sec id="s4-17-2"><title>RNA-Seq analysis</title><p>Sequencing adaptor contaminations were removed from reads using the cutadapt software tool (MIT), and the resulting reads were mapped and quantified on the transcriptome (NCBIM37 Ensembl gene-build 65) using RSEM v1.17 (<xref ref-type="bibr" rid="bib42">Li and Dewey, 2011</xref>). Only genes with >2 counts per million in ≥2 samples were considered for statistical analysis. Data were then normalized and differential expression assessed using the function Voom from the bioconductor package Limma (<xref ref-type="bibr" rid="bib40">Law et al., 2014</xref>; Smyth, 2005). We considered those genes as differentially expressed with a Benjamini–Hochberg adjusted p-value ≤0.05.</p></sec><sec id="s4-17-3"><title>Principal component analysis (PCA) comparison with previously published data</title><p>Normalized RNA-Seq data were compared via principal component analysis (PCA) with previously published array expression data (<xref ref-type="table" rid="tbl1">Table 1</xref>). GEO data sets were downloaded and pre-processed using the GEOquery Bioconductor package (<xref ref-type="bibr" rid="bib18">Davis and Meltzer, 2007</xref>). Data sets were adjusted to the same intensity range, as previously described (<xref ref-type="bibr" rid="bib27">Heider and Alt, 2013</xref>). Batch correction was applied to the data sets using ComBat (<xref ref-type="bibr" rid="bib33">Johnson et al., 2007</xref>) on the log2-normalized GEO data sets together with the log2-normalized counts from each RNA-Seq experiment.</p></sec></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The authors express sincere thanks to the following investigators for their generous donation of mice: G.E. Enikolopov (<italic>Nes-Gfp</italic>), G. Fishell (<italic>Nes-CreER</italic><sup><italic>T2</italic></sup>, <italic>RCE-loxP</italic>), S. Ortega (<italic>LSL-KFP)</italic>, D. Riethmacher (<italic>iDTA</italic>), V. Pachnis (<italic>Sox10-CreER</italic><sup><italic>T2</italic></sup>), S. MacKem (<italic>Hoxb6-CreER</italic><sup><italic>T2</italic></sup>), T. Müller and C. Birchmeier (<italic>Erbb3</italic><sup><italic>−/−</italic></sup>), D.N. Meijer (<italic>Dhh-Cre</italic>), S. Rocha and A. García-Arroyo (<italic>R26-Tomato</italic>). We are grateful to S. González-Hernández, O. Pérez-Howell, J.M. Ligos, A.B. Ricote, and the CNIC Genomics Unit for technical assistance, to members of SMF lab for helpful discussions, to M. Zaidi, J.B. Aquino, I. Adameyko, P. Ernfors, M. Torres, I. Delgado, L. Carramolino, and M. García-Fernández for helpful advice and support, and S. Bartlett for editing the manuscript. This work was supported by the Spanish Ministry of Economy and Competitiveness through the Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, SB2010-0023 grant to J.I, Plan Nacional grants BFU2012-35892 to J.I. and SAF-2011-30308 to S.M.-F., Ramón y Cajal Program grants RYC-2011-09209 to J.I. and RYC-2009-04703 to S.M.-F. The Marie Curie Career Integration Program (FP7-PEOPLE-2011-RG-294262), ConSEPOC-Comunidad de Madrid grant S2010/BMD-2542 to S.M.-F., and the Spanish Network of Cell Therapy (TerCel). A.G.-G. received a fellowship from Fundación Ramón Areces and is currently supported by Fundación La Caixa. S.M.-F. is supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>JI, Designed and performed most experiments, analyzed data and wrote the manuscript, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>AG-G, Performed experiments, Acquisition of data</p></fn><fn fn-type="con" id="con3"><p>AMM, Performed experiments, Acquisition of data</p></fn><fn fn-type="con" id="con4"><p>LA, Performed experiments, Acquisition of data</p></fn><fn fn-type="con" id="con5"><p>DM-P, Performed experiments, Acquisition of data</p></fn><fn fn-type="con" id="con6"><p>CT, Performed experiments, Acquisition of data</p></fn><fn fn-type="con" id="con7"><p>FS-C, Performed experiments, Acquisition of data</p></fn><fn fn-type="con" id="con8"><p>SM-F, Conceived the overall study. Contributed most reagents, designed experiments, analyzed data and wrote the manuscript, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: Experimental procedures were approved by the Animal Care and Use Committees of the Spanish National Cardiovascular Research Center and Comunidad Autónoma de Madrid (PA-47/11 and ES280790000176).</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03696.019</object-id><label>Supplementary file 1.</label><caption><p>Summary of mouse strains used in this study.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" 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Expression Omnibus.</comment></related-object></p><p>The following previously published datasets were used</p><p><related-object content-type="existing-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Fumio</surname><given-names>Arai</given-names></name> and <name><surname>Toshio</surname><given-names>Suda</given-names></name>, <year>2010</year><x>, </x><source>Gene expression profile of murine bone marrow endosteal populations</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17597">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17597</ext-link><x>, </x><comment>Publicly available at NCBI Gene Expression Omnibus.</comment></related-object></p><p><related-object content-type="existing-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" 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xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE10360">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE10360</ext-link><x>, </x><comment>Publicly available at NCBI Gene Expression Omnibus.</comment></related-object></p><p><related-object content-type="existing-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro5"><name><surname>Simon</surname><given-names>Mendez-Ferrer</given-names></name> and <name><surname>Paul S</surname><given-names>Frenette</given-names></name>, <year>2010</year><x>, </x><source>Expression profile in bone marrow Nestin-GFP cells</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21941">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21941</ext-link><x>, </x><comment>Publicly available at NCBI Gene Expression Omnibus.</comment></related-object></p><p><related-object content-type="existing-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro6"><name><surname>Ding</surname><given-names>L</given-names></name>, <name><surname>Morrison</surname><given-names>SJ</given-names></name>, <year>2012</year><x>, </x><source>Scf-GFP + cells from the bone marrow and whole bone marrow microarray</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33158">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33158</ext-link><x>, </x><comment>Publicly available at NCBI Gene Expression Omnibus.</comment></related-object></p><p><related-object content-type="existing-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro7"><name><surname>Buchstaller</surname><given-names>J</given-names></name>, <name><surname>Sommer</surname><given-names>L</given-names></name>, <name><surname>Bodmer</surname><given-names>M</given-names></name>, 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An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by Janet Rossant (Senior editor) and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>While all reviewers concur on the importance of your work and the potential impact in the field, two out of three are not satisfied by the quality of the images that support your conclusions. In a lineage tracing study this is a crucial requirement since, at low magnification, as all the images are, it is very difficult if not impossible to demonstrate co-localisation of the tracer with the tissue specific antigen under observation. Confocal, high magnification analysis would be required to substantiate the results in virtually all the figures presented. Moreover, even at low magnification there are clear inconsistencies: for example expression of CD31 is not known to occur in non-endothelial cells and thus a double staining should have been performed. Also, while Osterix appears not to be expressed in lateral plate derived cells (but very few cells are shown), the co-expression with Wnt1 labeled neural crest is partial and doubtful, making the main conclusion of the whole work particularly weak.</p><p>In addition all reviewers found the manuscript difficult to read and practically inaccessible to a non-specialised audience. The reviews are included below to assist with your revisions.</p><p><italic>Reviewer #1</italic>:</p><p>This manuscript reports an interesting and potentially important finding on functional heterogeneity and developmental origin of bone marrow stromal cells.</p><p>However the main and potentially ground breaking finding, i.e. that Nestin+ MSC are derived from neural crest and support hematopoiesis rather than building the bone itself, is not supported by the data presented, that are of poor quality and do not allow to draw final conclusions on the developmental origin of these cells.</p><p>Moreover, the manuscript is poorly written, difficult to follow and would greatly benefit from careful editing and, at least in some parts, re-writing.</p><p>1) Authors report that a small but increasing fraction of GFP+ also express CD31 and that this fraction increases with age. Since Nestin is not known to be expressed in endothelial cells, a co-staining with also anti-nestin antibody should be performed and co-localization investigated at confocal level, since this bears important implications on the fidelity of the GFP transgene.</p><p>2) The text describing data presented in <xref ref-type="fig" rid="fig2">Figure 2B</xref> is particularly confusing and almost impossible to interpret: “These mice and newborn Nes-gfp embryos were analyzed for osterix protein expression, which marks cells committed to the osteoblastic lineage. In contrast, cells genetically traced by the regulatory elements of Osterix gene do not only comprise osteoblastic cells, but also adventitial reticular cells, vascular smooth muscle cells, adipocytes and perineural cells (<xref ref-type="bibr" rid="bib43">Liu et al., 2013</xref>). Unlike cells derived from lateral plate mesoderm, fetal limb bone marrow Nes-GFP+ cells did not express osterix protein (<xref ref-type="fig" rid="fig2">Figure 2A-B</xref>) and therefore could not be considered osteoblast precursors.”</p><p>After describing the Hoxb6-CreERT mice (labelling lateral mesoderm derived cells) the Authors show no co-localization of Nes-GFP and Osterix, and then “presumed” co-localization of Hoxb6 (i.e. lateral plated) derived cells and Osterix (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). They conclude that because Nestin-GFP+ cells do not co-localize with Hoxb6 labelled cells they cannot be of lateral plate origin. This is a crucial point and particularly weak. First of all, at the magnification shown it is impossible to draw any conclusion. In A', very few GFP+ cells are visible and in an area where they are quite distant from Osx+ cells.</p><p>On the other hand, in B' only a very small minority of HoxB6 derived cells express Osx (and at that magnification it is hard to be sure): in most case the colour overlap seems partial, as it may originate from two cells one above the other. Even assuming that there is co-localization in few cells, what about all the others Osx+ cells? Even though recombination is never 100% efficient, one would conclude that the very large majority of Osx expressing cells do not derive from lateral mesoderm. Maybe they originate from paraxial mesoderm, but then another Cre would be required or the origin of Osx expressing cells remains largely unidentified in this work.</p><p>3) The overlap between Nestin-GFP and Nestin-Cre derived cells is only minor and, again, at that magnification it is impossible to draw solid conclusions. It is possible that Nestin-Cre derived cells have turned off the expression of Nestin gene, but then what have they become? Conversely, many Nestin-GFP+ do not seem to have originated from cells previously expressing Nestin. Even the example at “high” magnification (<xref ref-type="fig" rid="fig2">Figure 2D</xref>) shows one cell (**) that is clearly double labelled and another example (*) where there are two neighbouring but clearly distinct cells. These data are not convincing at all of a reliable lineage tracing.</p><p>4) Data on Wnt1-Cre appear more convincing, but again, a high magnification of a double-labelled cell at the confocal level would be necessary, especially for osteocytes (Cartilage staining is quite convincing even at low mag). However, FACS analysis shown in <xref ref-type="fig" rid="fig4">Figure 4E</xref> has problems. The Wnt1-Cre/Tomato clearly shows a dim (continuous with background) and a very bright population, both gated together to show that 62% of these are Nes-GFP+. I would have liked to see whether the Nes-GFP+ cells are equally represented in the bright and the dim populations. This is critical because the results are highly dependent on the specific gating. Most importantly, the IF shown in <xref ref-type="fig" rid="fig4">Figure 4D</xref> is highly unconvincing and does not support the claim that Nes-GFP+ are derived from Wnt1 expressing cells. The very few Tomato+ cells do not really co-localize with GFP+ cells and the examples shown in D look like at most closely located cells. Finally, the FACS analysis for Sox10-Cre also shows that gating has included both bright cells, clearly separated from the negative peak and the shoulder of this. I would like to see whether the bright cells only are GFP+.</p><p>5) The final sections of the Results, though also suffering of the poor quality of the images, are more convincing but less original. Furthermore DTR experiments are tricky, first because killing Nestin expressing cells may have also consequences in other tissue and possibly systemic effects. In addition, and this criticism applies to all of these studies, developmental biologists seem to be unaware of the “bystander effect”, well studied by gene therapists, where killing a specific cell, often causes the death of the nearest cells, thus mudding the results.</p><p><italic>Reviewer #2</italic>:</p><p>The manuscript by Isern et al describes the results of experiments examining the developmental origins (fetal, neonatal and adult) of the mesenchymal cells that are osteogenic and that support the hematopoietic system in the bone marrow. The experiments mainly utilize the nestin-gfp reporter mouse model in addition to other mouse models in which induced recombination mediated reporter expression defines specific neural crest derived populations. The manuscript is filled with information but it is sometimes difficult to understand how it is related to the main question. For example, it is not explained why CD31 immunostaining is performed on nestin gfp tissues; what cell types does it mark? Convention is the endothelial and hematopoietic cells express CD31, not MSC. The Discussion provides some information about levels of CD31 expression but this comes rather late in the manuscript.</p><p>In the Results section that describes BM nestin+ cells do not contribute to fetal endochondrogenesis, the RCE reporter is used, what is this? No definition is provided. Also, later in this paragraph, the sentence beginning with “In contrast, cells…” seems misplaced and confusing, making this text and results hard to follow. Similarly, it is not understood why a Wnt Cre line was used. What is the rationale? I seem to miss the connection. It would be helpful to reorganize and perhaps move some information from the main text to the Discussion or leave it out if it is not directly related to the question. It would be helpful to the reader if the manuscript could be more direct in describing the experimental rationale.</p><p>Altogether, the authors have done a large amount of work to show that there are different component populations of mesenchymal cells that contribute to the development of the long bones, bone marrow and the supportive hematopoietic microenvironment. This is an important set of results that supports a new conceptual model for the development of the hematopoietic supportive microenvironment.</p><p><italic>Reviewer #3</italic>:</p><p>This manuscript identifies the ontological source of an important bone marrow (BM) niche cells previously identified by the senior author. These cells are the Nestin-GFP+ (Nes-GFP+) cells in the adult BM that are a source of MSC and niche support. The Mendez-Ferrer group has extended this work to the developing fetal and early postnatal bone and observed exciting and enlightening results. They demonstrate that trunk neural crest cells are the developmental source for Nes-GFP+ MSC in the BM, in addition to their known contributions to peripheral sympathetic neurons and Schwann cells. Interestingly, all three of these cells types are important BM niche components. As such they share an ontological relationship that could theoretically extend to other niche components elsewhere in the body. Nes-GFP+ cells were characterized in the developing bone (E17.5), at P0 and at P7 with a variety of lineage tracing and Cre deletion transgenics in addition to cell surface IHC and flow cytometry. From a wide array of models they show:</p><p>1) Nes-GFP+ cells do not exhibit osteochondral progenitor activity. Nes-GFP- cells that derive from lateral plate mesoderm possess this activity in fetal bone.</p><p>2) Neural crest contribution to postnatal BM-MSCs was definitively demonstrated by genetic fate mapping with Wnt1-Cre2 and Sox10-CreERT2.</p><p>3) Cell surface and mRNA-seq studies of PDGFRa+/- and Nes-GFP+/- cells revealed that there are two Nes-GFP+ neural crest derived cells in postnatal BM. Schwann cell precursors are in the PDGFRa- fraction while PDGFRa+ cells contain the MSCs.</p><p>4) Neural crest cells migrate along developing nerves to the fetal bone before commitment to the Schwann cell lineage and give rise to niche forming MSCs.</p><p>5) Cxcl12 expression was shown to be 80 and 20-fold higher in Nes-GFP+ cells compared to Nes-GFP- and endothelial cells respectively.</p><p>6) Conditional deletion of Cxcl12 in Nes-GFP+ cells led to a significant 30% reduction in CFU and a non-significant decrease in repopulating HSC.</p><p>The experiments are well performed and described. With minor exceptions the figures are clear and well presented. I have only minor suggestions and comments about the paper. The reviewer appreciates the very thoughtful and extensive discussion that attempts to resolve some apparent controversies in the field. I believe the manuscript is a significant contribution the HSC niche field. So many different transgenic strains of mice, here at least 12 gets confusing with all the acronyms and the lack of good explanations, here a table would have helped.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03696.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>Reviewer #1:</p><p><italic>This manuscript reports an interesting and potentially important finding on functional heterogeneity and developmental origin of bone marrow stromal cells</italic>.</p><p>We thank the reviewer for finding our study interesting and potentially important, and for the suggestions that have helped to improve the paper.</p><p><italic>However the main and potentially ground breaking finding, i.e. that Nestin+ MSC are derived from neural crest and support hematopoiesis rather than building the bone itself, is not supported by the data presented, that are of poor quality and do not allow to draw final conclusions on the developmental origin of these cells</italic>.</p><p>We apologize if the quality of the images examined was suboptimal. Although we provided images covering wide bone marrow areas to convincingly illustrate our findings, these images also had good resolution and might have lost it during the compression and conversion to pdf. Nevertheless, we now provide insets from low magnification pictures with increased resolution. We have also shown high magnification details to facilitate viewing.</p><p>We have provided new data that further and fully support the main conclusion of our study already stated in the title that the neural crest is a source of MSCs with a specialized HSC niche function:</p><p>1) Higher detail images from triple transgenics showing neural-crest-derived cells co-labeled with Nestin-GFP, providing additional proof of a neural crest origin for a subset of Nestin-GFP+ cells (although other populations marked by Nestin-GFP+ might contain cells from other origins).</p><p>2) Functional data showing the enrichment in mesenchymal stem cell activity (CFU-F) and HSC niche features (Cxcl12 mRNA expression) within sorted bone marrow neural-crest-derived stromal cells, which are also enriched in endogenous Nestin mRNA expression.</p><p>3) Flow cytometry data showing that the majority of bone marrow cells derived from the neural crest are indeed stromal Nestin-GFP+ cells.</p><p><italic>Moreover, the manuscript is poorly written, difficult to follow and would greatly benefit from careful editing and, at least in some parts, re-writing</italic>.</p><p>We apologize for this. We have re-written some parts and have revised the whole manuscript to make it more accessible to the broad readership of <italic>eLife</italic>. Following the suggestion by Reviewer #3, we have also included a table indicating the numerous mouse strains used, to help the reader.</p><p><italic>1) Authors report that a small but increasing fraction of GFP+ also express CD31 and that this fraction increases with age. Since Nestin is not known to be expressed in endothelial cells, a co-staining with also anti-nestin antibody should be performed and co-localization investigated at confocal level, since this bears important implications on the fidelity of the GFP transgene</italic>.</p><p>Although nestin was originally identified as a neural stem cell marker in the developing central nervous system (Lendahl U et al. 1990 Cell), later studies have detected nestin expression in many other cell lineages, most pronouncedly during development (for a review, please</p><p>see Wiese et al. Nestin expression, a property of multi-lineage progenitor cells? Cell Mol Life Sci (2004) vol. 61 (19-20) pp. 2510-22). Many studies have reported nestin expression in endothelial cells, especially in proliferating vascular endothelial cells. Please see (among others):</p><p>Ono N et al. Dev Cell. 2014 May 12;29(3):330-9.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/pubmed?term=Wroblewski%20J%5BAuthor%5D%26cauthor=true%26cauthor_uid=9084133">Wroblewski J</ext-link> et al. Differentiation. 1997 Feb;61(3):151-9.</p><p>Sugawara K et al. Lab Invest. 2002 Mar;82(3):345-51.</p><p>Mokrý J et al. Stem Cells Dev. 2004 Dec;13(6):658-64.</p><p>Kim E et al. Invest Ophthalmol Vis Sci. 2014 Jul 17;55(8):5099-108</p><p>In fact, nestin has been recently proposed as a marker for newly formed blood vessels (Matsuda et al. World J Gastroenterol (2013) 19 (1) pp. 42-8). In light of this emerging literature, we have carefully analyzed the nestin<sup>+</sup> endothelial subset to avoid some potential confusion, although the population of interest was the stromal CD31<sup>-</sup> <italic>Nestin</italic>-GFP<sup>+</sup> population, which is highly enriched in mesenchymal progenitors. The other reason for using this marker to visualize blood vessels was to substantiate the perivascular or sub-endothelial localization of these mesenchymal progenitors.</p><p>Regarding the fidelity of the GFP transgene, we already showed in <xref ref-type="fig" rid="fig1">Figure 1F</xref> of the manuscript that <italic>Nestin-</italic>GFP+ cells are clearly enriched in endogenous <italic>Nestin</italic> mRNA expression. In the new <xref ref-type="fig" rid="fig7">Figure 7J</xref> we also show that bone marrow CD45<sup>-</sup> CD31<sup>-</sup> Ter119<sup>-</sup> Tomato+ cells sorted from 1-week old <italic>Wnt1-Cre2;R26-tomato;Nes-Gfp</italic> triple-transgenic mice are highly enriched in endogenous <italic>Nestin</italic> expression, compared with bone marrow CD45<sup>-</sup> CD31<sup>-</sup> Ter119<sup>-</sup> Tomato- cells. In the new data provided in new <xref ref-type="fig" rid="fig4">Figure 4D-F</xref> we show that the majority of neural crest cells tracked using two different drivers, <italic>Wnt1-Cre2</italic> and <italic>Sox10-CreER</italic><sup><italic>T2</italic></sup>, are in fact <italic>Nestin</italic>-GFP+ PDGFRa+ MSC-enriched cells which contain most bone marrow mesenchymal activity (CFU-F) at early postnatal stages. All this data convincingly demonstrate endogenous nestin expression in the cell population of interest in this study.</p><p><italic>2) The text describing data presented in</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2B</italic></xref> <italic>is particularly confusing and almost impossible to interpret: “These mice and newborn Nes-gfp embryos were analyzed for osterix protein expression, which marks cells committed to the osteoblastic lineage. In contrast, cells genetically traced by the regulatory elements of Osterix gene do not only comprise osteoblastic cells, but also adventitial reticular cells, vascular smooth muscle cells, adipocytes and perineural cells (</italic><xref ref-type="bibr" rid="bib43"><italic>Liu et al., 2013</italic></xref><italic>). Unlike cells derived from lateral plate mesoderm, fetal limb bone marrow Nes-GFP+ cells did not express osterix protein (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2A-B</italic></xref><italic>) and therefore could not be considered osteoblast precursors</italic>.<italic>”</italic></p><p>Whereas osterix protein marks osteolineage cells, <italic>Osterix</italic> mRNA or cells marked by the regulatory elements of <italic>Osterix</italic> promoter do not only contain osteoblastic-committed cells, but also a variety of other cells in the bone marrow, including adventitial reticular cells, vascular smooth muscle cells, adipocytes and perineural cells. This has been clearly shown in a study cited in our manuscript (<xref ref-type="bibr" rid="bib43">Liu et al., 2013</xref>). This is likely explained by the fact that different stem cells (including MSCs) actively transcribe differentiation factors which are not translated into proteins, which that does not imply lineage commitment but allows them to rapidly differentiate in response to stimuli. This explains some contradictions in the field. We have reworded the corresponding section to reduce confusion. Unlike mesodermal derivatives, <italic>Nes-</italic>GFP+ did not express highly osterix protein and they did not contribute significantly to fetal osteocytes. We therefore conclude that these cells were not osteoblast precursors (even though they might show <italic>Osterix</italic> mRNA expression).</p><p><italic>After describing the Hoxb6-CreERT mice (labelling lateral mesoderm derived cells) the Authors show no co-localization of Nes-GFP and Osterix, and then “presumed” co-localization of Hoxb6 (i.e. lateral plated) derived cells and Osterix (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2B</italic></xref><italic>). They conclude that because Nestin-GFP+ cells do not co-localize with Hoxb6 labelled cells they cannot be of lateral plate origin. This is a crucial point and particularly weak. First of all, at the magnification shown it is impossible to draw any conclusion. In A', very few GFP+ cells are visible and in an area where they are quite distant from Osx+ cells</italic>.</p><p><italic>Hoxb6-CreERT</italic> line would only label cells that express this transgene at the time that tamoxifen were administered. Together with limited recombination efficiency, this might explain why few GFP+ cells were observed using this mouse line and induction regimen. However, most of these cells labeled with cytoplasmic GFP also had nuclear osterix protein (please be aware of the different location of the fluorescent signal; new <xref ref-type="fig" rid="fig2">Figure 2B</xref> in the revised manuscript, also <xref ref-type="fig" rid="fig8">Author response image 1</xref> below). This is in sharp contrast with the virtual absence of osterix protein in stage-matched <italic>Nestin-</italic>GFP+ cells (new <xref ref-type="fig" rid="fig2">Figure 2A</xref>).<fig id="fig8" position="float"><label>Author response image 1.</label><caption><p>Expression of osterix by bone marrow cells of the <italic>Hoxb6</italic>-derived lineage. High magnification detail of E18.5 <italic>Hoxb6-CreERT2;RCE</italic> bone marrow (BM) stained with anti-osterix (red). Embryos were induced with tamoxifen at E10.5 to trace limb mesoderm with <italic>Hoxb6-Cre driver</italic>. Asterisks indicate nuclear osterix-positive cells also marked with GFP. Dashed line indicated the bone contour. <italic>Scale bar:</italic> 50μm.</p></caption><graphic xlink:href="elife03696f008"/></fig></p><p><italic>On the other hand, in B' only a very small minority of HoxB6 derived cells express Osx (and at that magnification it is hard to be sure): in most case the colour overlap seems partial, as it may originate from two cells one above the other. Even assuming that there is co-localization in few cells, what about all the others Osx+ cells? Even though recombination is never 100% efficient, one would conclude that the very large majority of Osx expressing cells do not derive from lateral mesoderm. Maybe they originate from paraxial mesoderm, but then another Cre would be required or the origin of Osx expressing cells remains largely unidentified in this work</italic>.</p><p>The degree of recombination using <italic>HoxB6-CreER</italic> driver line is only partial, which might be due both to the time window when tamoxifen was administered and to limited recombination efficiency by this Cre line. This might underestimate the real contribution of lateral mesoderm to osterix+ cells. Although it is possible, as suggested by the reviewer, that other mesodermal sources contribute to osteoprogenitor cells, this possibility would not affect the conclusions of our paper and also falls outside its current and already wide scope. We have taken the advantage of this genetic system to visualize mesodermal derivatives (in this case, lateral plate) and have demonstrated that this population, unlike <italic>Nestin-creER</italic><sup><italic>T2</italic></sup>-traced cells, overlaps with embryonic skeletal precursors expressing osterix protein (an accepted marker of osteoprogenitor cells). We have also shown an abundant contribution of mesoderm to mature skeletal cells in long bones. This is in sharp contrast with the virtual absence of osterix protein in the <italic>Nestin-GFP+</italic> population and the absence of <italic>Nestin-creER</italic><sup><italic>T2</italic></sup>-traced fetal osteoblasts or chondrocytes. Therefore the main conclusion of this part, that nestin+ cells do not significantly contribute to fetal osteochondral cells, is fully supported by our data.</p><p><italic>3) The overlap between Nestin-GFP and Nestin-Cre derived cells is only minor and, again, at that magnification it is impossible to draw solid conclusions. It is possible that Nestin-Cre derived cells have turned off the expression of Nestin gene, but then what have they become? Conversely, many Nestin-GFP+ do not seem to have originated from cells previously expressing Nestin. Even the example at “high” magnification (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2D</italic></xref><italic>) shows one cell (**) that is clearly double labelled and another example (*) where there are two neighbouring but clearly distinct cells. These data are not convincing at all of a reliable lineage tracing</italic>.</p><p>The overlap between <italic>Nes-</italic>GFP+ cells and <italic>Nes-creER</italic><sup><italic>T2</italic></sup><italic>-</italic>traced cells depends on the developmental stage, tamoxifen dose and stage of administration, the time between tamoxifen administration and analysis, and the strength of the reporter mouse. We have replaced former panel 2D, based on <italic>KFP</italic> mice, a weak reporter line, by the Rosa26-Tomato reporter which provides a much stronger signal. <italic>Nes-gfp;Nes-creER</italic><sup><italic>T2</italic></sup><italic>;Rosa26-Tomato</italic> triple-transgenic mice show a consistent labeling and significant overlap of GFP+ and Tomato+ cells when tamoxifen is administered at perinatal stages and the analysis is performed within few days/weeks. We have provided additional images at higher magnification to better support our conclusions (new <xref ref-type="fig" rid="fig2">Figure 2C-C</xref> and <xref ref-type="fig" rid="fig9">Author response image 2</xref>).<fig id="fig9" position="float"><label>Author response image 2.</label><caption><p>Overlap of <italic>Nestin-GFP</italic>+ cells with <italic>Nestin-CreER</italic><sup><italic>T2</italic></sup>-labelled cells in the bone marrow. Representative confocal projections of skull bone marrow, showing the endogenous fluorescent signals of Tomato in red (upper right) and GFP in green (lower right); their corresponding overlay is indicated in orange/yellow. <italic>Nestin</italic>-<italic>gfp</italic>;<italic>Nestin-CreER</italic><sup><italic>T2</italic></sup><italic>;R26-Tomato</italic> triple-transgenic mice were treated with tamoxifen (4 mg, oral gavage to mother) at neonatal stage (P0) and were analyzed after 2 weeks. Asterisks indicate double-positive cells.</p></caption><graphic xlink:href="elife03696f009"/></fig></p><p><italic>4) Data on Wnt1-Cre appear more convincing, but again, a high magnification of a double-labelled cell at the confocal level would be necessary, especially for osteocytes (Cartilage staining is quite convincing even at low mag)</italic>.</p><p>We have stained osteocytes with phalloidin in bone marrow sections from <italic>Wnt1-Cre2;R26-tomato</italic> mice. The pictures shown with high magnification clearly confirm the presence of phalloidin+ neural-crest-traced osteocytes in the neonatal long bones (new <xref ref-type="fig" rid="fig4">Figure 4B</xref>). We have also included a complete section of a long bone from <italic>Nes-Gfp;Wnt1-Cre2;R26-tomato</italic> triple-transgenic mice that does not only illustrate neural-crest-derived chondrocytes in the outermost layer of both diaphyses, but also abundant neural-crest-derived cells in the endosteal bone marrow region (new <xref ref-type="fig" rid="fig4">Figure 4C</xref>).</p><p><italic>However, FACS analysis shown in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4E</italic></xref> <italic>has problems. The Wnt1-Cre/Tomato clearly shows a dim (continuous with background) and a very bright population, both gated together to show that 62% of these are Nes-GFP+. I would have liked to see whether the Nes-GFP+ cells are equally represented in the bright and the dim populations. This is critical because the results are highly dependent on the specific gating</italic>.</p><p>We have provided the negative control to substantiate the gating strategy (new <xref ref-type="fig" rid="fig4">Figure 4E</xref>). We have also reanalyzed the data and have performed additional experiments in <italic>Wnt1-Cre2;R26-Tomato;Nes-Gfp</italic> triple-transgenic mice analyzed at neonatal stage. As the reviewer states, two stromal populations can be distinguished based on Tomato fluorescence intensity. Although both Tomato+ stromal populations contain <italic>Nes-</italic>GFP+ PDGFRα+ MSC-enriched cells, these cells are progressively enriched in the Tomato<sup>dim</sup> population with age (new <xref ref-type="fig" rid="fig4s2">Figure 4–figure supplement 2C-D</xref>).</p><p><italic>Most importantly, the IF shown in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4D</italic></xref> <italic>is highly unconvincing and does not support the claim that Nes-GFP+ are derived from Wnt1 expressing cells. The very few Tomato+ cells do not really co-localize with GFP+ cells and the examples shown in D look like at most closely located cells</italic>.</p><p>We admitted in our manuscript the heterogeneity of the <italic>Nes</italic>-GFP+ population, which does not only contain neural-crest-derived cells but also endothelial cells. In the revised manuscript we have provided more clear images showing <italic>Nes-</italic>GFP+ Tomato+ cells (new <xref ref-type="fig" rid="fig4">Figure 4C-C</xref> and <xref ref-type="fig" rid="fig10">Author response image 3</xref>).<fig id="fig10" position="float"><label>Author response image 3.</label><caption><p>High magnification confocal image from 1-week old <italic>Wnt1-Cre2;Tomato;Nes-Gfp</italic> BM section, showing co-localization of NC-derived Tomato+ cell with Nes-GFP (arrowhead).</p></caption><graphic xlink:href="elife03696f010"/></fig></p><p>We have performed additional experiments that fully support our main message that neural-crest-derived nestin+ MSCs have a specialized function in forming the HSC niche in the bone marrow. We have analyzed <italic>Cxcl12</italic> and <italic>Nestin</italic> mRNA expression in Tomato<sup>+/-</sup> GFP<sup>+/-</sup> bone marrow stromal cells sorted from P7 <italic>Wnt1-Cre2;R26-Tomato;Nes-Gfp</italic> triple-transgenic mice. Among neural-crest-traced cells, <italic>Nes</italic>-GFP<sup>+</sup> BMSCs were particularly enriched in the expression of <italic>Cxcl12</italic> and endogenous <italic>Nestin</italic> (<xref ref-type="fig" rid="fig7">Figure 7I-J</xref>). We have measured MSC activity (CFU-F) in Tomato<sup>+/-</sup> GFP<sup>+/-</sup> bone marrow stromal cells sorted from P7 <italic>Wnt1-Cre2;R26-Tomato</italic> double-transgenic and P7 <italic>Wnt1-Cre2;R26-Tomato;Nes-Gfp</italic> triple-transgenic mice. When plated at equal cell density, colonies were only detected in the neural-crest-derived cells (new <xref ref-type="fig" rid="fig4">Figure 4D</xref> and <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1F</xref>). These results fully support our original contention.</p><p><italic>Finally, the FACS analysis for Sox10-Cre also shows that gating has included both bright cells, clearly separated from the negative peak and the shoulder of this. I would like to see whether the bright cells only are GFP+</italic>.</p><p>The reviewer is correct that different levels of Tomato intensity were also detected in <italic>Sox10-creERT2</italic> mice, but both Tomato<sup>bright</sup> and Tomato<sup>dim</sup> bone marrow stromal populations stromal contained <italic>Nes-</italic>GFP+ PDGFRα+ MSC-enriched cells. We have reanalyzed our data gating on the Tomato<sup>bright</sup> population, as requested. The Tomato<sup>bright</sup> population contains a high percentage of <italic>Nes-</italic>GFP+ cells, also including PDGFRα+ cells (new <xref ref-type="fig" rid="fig4s2">Figure 4–figure supplement 2E</xref>).</p><p><italic>5) The final sections of the Results, though also suffering of the poor quality of the images, are more convincing but less original. Furthermore DTR experiments are tricky, first because killing Nestin expressing cells may have also consequences in other tissue and possibly systemic effects. In addition, and this criticism applies to all of these studies, developmental biologists seem to be unaware of the “bystander effect”, well studied by gene therapists, where killing a specific cell, often causes the death of the nearest cells, thus mudding the results</italic>.</p><p>We hope that the revised images show the quality level expected by the reviewer and us. We agree that selective cell depletion experiments might have some bystander effects; for this reason we already showed in our manuscript the normal bone marrow histology and blood vessel architecture of experimental <italic>Nes-creERT2;iDTA</italic> mice (<xref ref-type="fig" rid="fig7s1">Figure 7–figure supplement 1B</xref>) as a proof of the absence of noticeable bystander effects. To further substantiate this, we have included in the response to the reviewer bone marrow sections of <italic>Nestin-CreER</italic><sup><italic>T2</italic></sup>;<italic>iDTR</italic> and control <italic>iDTR</italic> mice injected with tamoxifen and diphtheria toxin to demonstrate that cell deletion using this inducible Cre line is not associated with noticeable abnormalities in bone marrow histology or vascular leakage of FITC-dextran (green, please see below) previously intravenously injected in the mice (<xref ref-type="fig" rid="fig11">Author response image 4</xref>).<fig id="fig11" position="float"><label>Author response image 4.</label><caption><p>Intravital microscopy of mice previously injected with FITC-dextran (green) to demonstrate the absence of vascular leakage (upper panels). H & E staining of bone marrow sections showing a normal histology of the bone marrow (lower panels).</p></caption><graphic xlink:href="elife03696f011"/></fig></p><p>Reviewer #2:</p><p><italic>The manuscript by Isern et al describes the results of experiments examining the developmental origins (fetal, neonatal and adult) of the mesenchymal cells that are osteogenic and that support the hematopoietic system in the bone marrow. The experiments mainly utilize the nestin-gfp reporter mouse model in addition to other mouse models in which induced recombination mediated reporter expression defines specific neural crest derived populations. The manuscript is filled with information but it is sometimes difficult to understand how it is related to the main question. For example, it is not explained why CD31 immunostaining is performed on nestin gfp tissues; what cell types does it mark? Convention is the endothelial and hematopoietic cells express CD31, not MSC. The Discussion provides some information about levels of CD31 expression but this comes rather late in the manuscript</italic>.</p><p>We are currently lacking robust and specific MSC markers for immunofluorescence. We have used CD31 marker for two reasons: 1) To mark blood vessels, since <italic>Nes-</italic>GFP+ cells are closely associated with them. 2) To distinguish the small fraction of perinatal bone marrow <italic>Nes-</italic>GFP+ CD31+ cells from the majority of <italic>Nes-</italic>GFP+ CD31- cells, which contain the population of interest in our study. Please see also Response to Major Comment #1 by Reviewer 1.</p><p><italic>In the Results section that describes BM nestin+ cells do not contribute to fetal endochondrogenesis, the RCE reporter is used, what is this? No definition is provided</italic>.</p><p>The RCE acronym stands for <italic><underline>R</underline>osa26 <underline>C</underline>AG <underline>E</underline>GFP</italic>, an <italic>EGFP</italic> reporter allele which is expressed upon <italic>Cre</italic>-mediated recombination from the hybrid CAG promoter in the <italic>Rosa26</italic> locus (Sousa et al. Characterization of Nkx6-2-Derived Neocortical Interneuron Lineages. Cerebral Cortex (2009) vol. 19 (Supplement 1) pp. i1-i10). We have defined it as “…a sensitive reporter that drives stronger GFP expression than other reporter lines (<xref ref-type="bibr" rid="bib71"><italic>Sousa et al., 2009</italic></xref>)…” .</p><p><italic>Also, later in this paragraph, the sentence beginning with “In contrast, cells…” seems misplaced and confusing, making this text and results hard to follow</italic>.</p><p>We have moved this to the Discussion and have re-written this paragraph. Please see response to the similar Major Comment #2 by Reviewer 1.</p><p><italic>Similarly, it is not understood why a Wnt Cre line was used. What is the rationale? I seem to miss the connection. It would be helpful to reorganize and perhaps move some information from the main text to the Discussion or leave it out if it is not directly related to the question. It would be helpful to the reader if the manuscript could be more direct in describing the experimental rationale</italic>.</p><p>We apologize for not being sufficiently clear. We have rewritten the manuscript to better explain the rationale of each single experiment and have made an effort to better link contiguous sections.</p><p><italic>Altogether, the authors have done a large amount of work to show that there are different component populations of mesenchymal cells that contribute to the development of the long bones, bone marrow and the supportive hematopoietic microenvironment. This is an important set of results that supports a new conceptual model for the development of the hematopoietic supportive microenvironment</italic>.</p><p>We thank the reviewer for considering that our results importantly support a new conceptual model for the establishment of stem cell niches.</p><p>Reviewer #3:</p><p><italic>This manuscript identifies the ontological source of an important bone marrow (BM) niche cells previously identified by the senior author. These cells are the Nestin-GFP+ (Nes-GFP+) cells in the adult BM that are a source of MSC and niche support. The Mendez-Ferrer group has extended this work to the developing fetal and early postnatal bone and observed exciting and enlightening results. They demonstrate that trunk neural crest cells are the developmental source for Nes-GFP+ MSC in the BM, in addition to their known contributions to peripheral sympathetic neurons and Schwann cells. Interestingly, all three of these cells types are important BM niche components. As such they share an ontological relationship that could theoretically extend to other niche components elsewhere in the body. Nes-GFP+ cells were characterized in the developing bone (E17.5), at P0 and at P7 with a variety of lineage tracing and Cre deletion transgenics in addition to cell surface IHC and flow cytometry. From a wide array of models they show</italic>:</p><p><italic>1) Nes-GFP+ cells do not exhibit osteochondral progenitor activity. Nes-GFP- cells that derive from lateral plate mesoderm possess this activity in fetal bone</italic>.</p><p><italic>2) Neural crest contribution to postnatal BM-MSCs was definitively demonstrated by genetic fate mapping with Wnt1-Cre2 and Sox10-CreERT2</italic>.</p><p><italic>3) Cell surface and mRNA-seq studies of PDGFRa+/- and Nes-GFP+/- cells revealed that there are two Nes-GFP+ neural crest derived cells in postnatal BM. Schwann cell precursors are in the PDGFRa- fraction while PDGFRa+ cells contain the MSCs</italic>.</p><p><italic>4) Neural crest cells migrate along developing nerves to the fetal bone before commitment to the Schwann cell lineage and give rise to niche forming MSCs</italic>.</p><p><italic>5) Cxcl12 expression was shown to be 80 and 20-fold higher in Nes-GFP+ cells compared to Nes-GFP- and endothelial cells respectively</italic>.</p><p><italic>6) Conditional deletion of Cxcl12 in Nes-GFP+ cells led to a significant 30% reduction in CFU and a non-significant decrease in repopulating HSC</italic>.</p><p><italic>The experiments are well performed and described. With minor exceptions the figures are clear and well presented. I have only minor suggestions and comments about the paper. The reviewer appreciates the very thoughtful and extensive discussion that attempts to resolve some apparent controversies in the field. I believe the manuscript is a significant contribution the HSC niche field. So many different transgenic strains of mice, here at least 12 gets confusing with all the acronyms and the lack of good explanations, here a table would have helped</italic>.</p><p>We thank very much the reviewer for appreciating the relevance of our study and for the very positive criticism. We have included a table containing a detailed summary of all the mouse strains used in supplementary procedures (Supplementary file 3).</p></body></sub-article></article> |