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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">01440</article-id><article-id pub-id-type="doi">10.7554/eLife.01440</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><subj-group subj-group-type="heading"><subject>Genomics and evolutionary biology</subject></subj-group></article-categories><title-group><article-title>Dual mode of embryonic development is highlighted by expression and function of <italic>Nasonia</italic> pair-rule genes</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="author-7354"><name><surname>Rosenberg</surname><given-names>Miriam I</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7863"><name><surname>Brent</surname><given-names>Ava E</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa2">‡</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7804"><name><surname>Payre</surname><given-names>François</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-3168"><name><surname>Desplan</surname><given-names>Claude</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Center for Developmental Genetics, Department of Biology</institution>, <institution>New York University</institution>, <addr-line><named-content content-type="city">New York</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Centre de Biologie du Développement</institution>, <institution>UMR5547 CNRS/ Université de Toulouse</institution>, <addr-line><named-content content-type="city">Toulouse</named-content></addr-line>, <country>France</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pan</surname><given-names>Duojia</given-names></name><role>Reviewing editor</role><aff><institution>HHMI, Johns Hopkins University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>cd38@nyu.edu</email> (CD);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>miriamr1@tx.technion.ac.il</email> (MIR)</corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel</p></fn><fn fn-type="present-address" id="pa2"><label>‡</label><p>Department of Biological Sciences, Columbia University, New York, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>05</day><month>03</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01440</elocation-id><history><date date-type="received"><day>13</day><month>09</month><year>2013</year></date><date date-type="accepted"><day>24</day><month>01</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Rosenberg et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Rosenberg et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">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="elife01440.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01440.001</object-id><p>Embryonic anterior–posterior patterning is well understood in <italic>Drosophila</italic>, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp <italic>Nasonia</italic> (<italic>Nv</italic>) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in <italic>Nasonia</italic> suggest long germ embryogenesis. However, the <italic>Nasonia</italic> pair-rule genes <italic>even-skipped</italic>, <italic>odd-skipped</italic>, <italic>runt</italic> and <italic>hairy</italic> are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of <italic>Nv eve</italic>, <italic>odd</italic> or <italic>h</italic> causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that <italic>Nasonia</italic> uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling <italic>Drosophila</italic> at the anterior and ancestral <italic>Tribolium</italic> at the posterior.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.001">http://dx.doi.org/10.7554/eLife.01440.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01440.002</object-id><title>eLife digest</title><p>Networks of genes that work together are widespread in nature. The conservation of individual genes across species and the tendency of their networks to stick together is a sign that they are working efficiently. Furthermore, it is common for existing gene networks to be adapted to perform new tasks, instead of new networks being invented every time a similar but distinct demand arises. One important question is: how can evolution use the same building blocks—such as the genes in a functioning network—in different ways to achieve new outcomes?</p><p>The gene network that sets up the ‘body plan’ of insects during development has been well studied, most deeply in the fruit fly, <italic>Drosophila</italic>. Like all insects, the body of a fruit fly is divided into three main parts—the head, the thorax and the abdomen—and each of these parts is made up of several smaller segments. There is a remarkable diversity of insect body plans in nature, and yet, these seem to arise from the same gene networks in the embryo.</p><p>When a <italic>Drosophila</italic> embryo is growing into a larva, all the different body segments develop at the same time. In most other insects, however, segments of the abdomen emerge later and sequentially during the development process. The ancestors of most insects are also thought to have developed in this way, which is known as ‘short germ embryogenesis’. So how did the so-called ‘long germ embryogenesis’, as observed in <italic>Drosophila</italic>, evolve from the short germ embryogenesis that is observed in most other insects?</p><p>The gene network that controls development includes the ‘pair-rule genes’ that are expressed in a pattern of alternating stripes that wrap around, top to bottom, along most of the length of the embryo. These stripes mark where the edges of each body segment will eventually develop. In fruit flies, this pattern extends along the entire length of the embryo and the stripes all appear at one time. However, in the abdominal region of short germ insects, the pair-rule genes are expressed in waves that pass through the posterior region as it grows, with new segments being added one behind the other.</p><p>Now, Rosenberg et al. have attempted to explain how the same genes can be used to direct the segmentation process in such different ways by studying another long germ insect species, the jewel wasp. Analysis of the expression of pair-rule genes in the jewel wasp shows that it uses a mixed strategy to control segmentation. The development of segments at the front of its body is directed in the same way as the fruit fly, with all these segments laid down together. However, the segments at the rear of the body are only patterned later, one after the other, like most other insects.</p><p>The work of Rosenberg et al. suggests that the jewel wasp represents an intermediate step between ancestral insects and <italic>Drosophila</italic> in the evolution of the gene network that patterns the ‘body plan’<italic>.</italic> Identifying and studying these intermediate forms allows us to understand the ways in which evolution can innovate by building upon what has come before.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.002">http://dx.doi.org/10.7554/eLife.01440.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd><italic>Nasonia vitripennis</italic></kwd><kwd><italic>Tribolium</italic></kwd><kwd>embryonic patterning</kwd><kwd>evolution</kwd><kwd>segmentation</kwd><kwd>pair-rule genes</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>F32GM084563</award-id><principal-award-recipient><name><surname>Rosenberg</surname><given-names>Miriam I</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>Damon Runyon Cancer Research Foundation</institution></institution-wrap></funding-source><award-id>DRG-1870-05</award-id><principal-award-recipient><name><surname>Brent</surname><given-names>Ava E</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>American Cancer Society</institution></institution-wrap></funding-source><award-id>120323-PF-11-242-01-DDC</award-id><principal-award-recipient><name><surname>Rosenberg</surname><given-names>Miriam I</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>5R01GM064864</award-id><principal-award-recipient><name><surname>Desplan</surname><given-names>Claude</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>Pair-rule genes in the wasp <italic>Nasonia</italic> function as in <italic>Drosophila</italic> in patterning anterior segments, and similar to ancestral insects in patterning posterior segments, illustrating a mixed-mode transition state between short and long germ embryogenesis.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Control of axial patterning and embryonic development is well understood in <italic>Drosophila</italic> (reviewed in <xref ref-type="bibr" rid="bib42">Liu and Kaufman, 2005b</xref>; <xref ref-type="bibr" rid="bib59">Peel et al., 2005</xref>; <xref ref-type="bibr" rid="bib65">Rosenberg et al., 2009</xref>; <xref ref-type="bibr" rid="bib51">Pankratz and Jaj, 1993</xref>). Extensive work has elucidated the genetic basis of establishment of the anterior–posterior (A–P) and dorsal–ventral (D–V) axes of the fly embryo. For the A–P axis, maternally loaded mRNAs generate localized signaling centers at each pole of the egg to establish morphogenetic gradients. These gradients instruct, in a concentration dependent manner, broad domains of expression of early zygotic genes, the ‘gap genes’ (<xref ref-type="bibr" rid="bib11">Chen et al., 2012</xref>). This is made possible in part by the syncytial environment of the early blastoderm embryo where nuclei are not bounded by membranes, allowing diffusion of morphogen transcription factors through a shared cytoplasm without the need for cell–cell signaling. In this environment, broad activation by maternal factors coupled with repressive activities by the gap genes leads to the expression of the pair-rule genes in two-segment periodicity, as pair-rule stripes. The overlapping registers of different pair-rule genes ultimately establish segment polarity through activation of the segment polarity genes, each expressed in stripes with single segmental register. This mode of development is termed ‘long germ’ embryogenesis because the embryo occupies all of the blastoderm apart from a dorsal region representing the extraembryonic ammnioserosa. A striking feature of long germ embryogenesis is that virtually all of segment patterning is completed synchronously in the syncytial environment. However, forays into other insect models have revealed that the <italic>Drosophila</italic> paradigm is an evolutionarily derived state, and that insects generally undergo a very different type of embryogenesis and segmentation (reviewed in <xref ref-type="bibr" rid="bib42">Liu and Kaufman, 2005b</xref>; <xref ref-type="bibr" rid="bib59">Peel et al., 2005</xref>; <xref ref-type="bibr" rid="bib65">Rosenberg et al., 2009</xref>).</p><p>Unlike flies, most insect embryonic primordia occupy only a small portion of the blastoderm and only few anterior segments (head and thorax) are patterned in a syncytial environment. The remainder of the embryo is generated after cellularization via a ‘growth zone’, at the posterior region of the embryo. This mode is termed ‘short germ’ embryogenesis. Recently, the mechanisms governing posterior segment patterning and growth in the <italic>Tribolium</italic> embryo were characterized in elegant detail (<xref ref-type="bibr" rid="bib16">Choe et al., 2006</xref>; <xref ref-type="bibr" rid="bib15">Choe and Brown, 2009</xref>; <xref ref-type="bibr" rid="bib26">El-Sherif et al., 2012</xref>; <xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>): Oscillations of the pair-rule gene <italic>Tc’odd-skipped</italic> (<italic>Tc’odd)</italic> in the growth zone are in turn linked to a circuit of two other pair-rule genes, <italic>Tc’runt</italic> and <italic>Tc’even-skipped (Tc’eve</italic>), such that each new pair of segments experiences a pulse of <italic>Tc’odd</italic> and requires both <italic>Tc’eve</italic> and <italic>Tc’runt</italic> expression in order to progress; the driver of these oscillations is still unknown. The waves of expression of <italic>Tc’odd-skipped</italic> pass through the growth zone rhythmically, generating segments and new stripes of stable expression with each periodic pulse (<xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>). RNAi of <italic>Tc’odd</italic>, <italic>Tc’runt</italic>, or <italic>Tc’eve</italic> results in asegmental embryos, underscoring their requirement in both growth zone-derived segments and earlier blastoderm anterior segments (<xref ref-type="bibr" rid="bib16">Choe et al., 2006</xref>). In contrast, the pair-rule genes <italic>Tc’sloppy paired (Tc’slp)</italic> and <italic>Tc’paired</italic> (<italic>Tc’prd</italic>) appear in two-segment periodicity in head stripes and in stripes that emerge from the growth zone, and RNAi of those genes produce classical pair-rule phenotypes, in which alternating segments are lost (<xref ref-type="bibr" rid="bib14">Choe and Brown, 2007</xref>). Live imaging revealed that formation of posterior segments results primarily from convergent extension and short-range cell movements and not strictly from cell division within the ‘growth zone’. This mechanism appears similar to both segmentation of vertebrate presomitic mesoderm (reviewed in [<xref ref-type="bibr" rid="bib22">Dubrulle and Pourquie, 2004</xref>] and [<xref ref-type="bibr" rid="bib61">Pourquie, 2011</xref>]) and to segment formation in more basal arthropods, including the centipede <italic>Strigamia maritima</italic> (<xref ref-type="bibr" rid="bib13">Chipman et al., 2004</xref>; <xref ref-type="bibr" rid="bib12">Chipman and Akam, 2008</xref>) and the spider <italic>Cupiennius salei</italic> (<xref ref-type="bibr" rid="bib76">Stollewerk et al., 2003</xref>), suggesting it as an ancient mechanism inherited from the last common ancestor of all segmented animals (though this interpretation is still debated; reviewed in [<xref ref-type="bibr" rid="bib20">Davis and Patel, 1999</xref>]).</p><p>As <italic>Drosophila</italic> is only one example of a derived long germ strategy, one outstanding question is how transitions from short germ to long germ embryogenesis occurred, such that the same set of segmentation genes possesses different functions. The careful study of additional long germ insects should shed light on what aspects of <italic>Drosophila</italic> development are essential facets of long germ embryogenesis and which aspects are more evolutionarily labile. Other model species have been studied, including long germ beetles (e.g., <italic>Callosobruchus</italic> order: <italic>Coleoptera</italic>; (<xref ref-type="bibr" rid="bib58">Patel et al., 1994</xref>)), and several members of the order <italic>Hymenoptera</italic>, including the honeybee, <italic>Apis mellifera</italic> (<xref ref-type="bibr" rid="bib21">Dearden et al., 2006</xref>; <xref ref-type="bibr" rid="bib83">Wilson et al., 2010</xref>; <xref ref-type="bibr" rid="bib81">Wilson and Dearden, 2011</xref>, <xref ref-type="bibr" rid="bib82">2012</xref>) and the jewel wasp, <italic>Nasonia vitripennis</italic> (<italic>Nv</italic>) (<xref ref-type="bibr" rid="bib62">Pultz et al., 1999</xref>; <xref ref-type="bibr" rid="bib80">Werren et al., 2010</xref>). However, systematic characterization of their pair-rule genes and segmentation mechanisms is still incomplete.</p><p>We use the wasp <italic>Nasonia vitripennis</italic> as a model for the study of A–P patterning, as a species that appears to have evolved, independently of <italic>Drosophila,</italic> a similar mode of long germ embryogenesis. We have previously characterized the early patterns of <italic>Nasonia</italic> segmentation genes and found that maternal and gap gene expression confirms a long germ mode of embryogenesis. This conclusion was based on the existence of two polar signaling centers, each utilizing localized maternal <italic>Nv orthodenticle (otd)</italic> mRNA that encodes a morphogen. <italic>Nv otd</italic> acts in combination with <italic>Nv hunchback</italic> (<italic>hb</italic>) and localized maternal <italic>Nv giant (gt)</italic> at the anterior, and with localized maternal <italic>Nv caudal (cad)</italic> at the posterior, to specify positional identity. The domains of zygotic expression of <italic>Nv hb</italic>, <italic>Nv gt</italic>, <italic>Nv cad, Nv Krüppel</italic> (<italic>Kr</italic>), <italic>Nv tailless</italic> (<italic>tll</italic>), and <italic>Nv knirps</italic> (<italic>kni</italic>) closely resemble their <italic>Drosophila</italic> counterparts, consistent with a similar mode of blastoderm allocation (<xref ref-type="bibr" rid="bib64">Pultz et al., 2005</xref>; <xref ref-type="bibr" rid="bib45">Lynch et al., 2006</xref>; <xref ref-type="bibr" rid="bib55">Olesnicky et al., 2006</xref>; <xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>). Although these data support <italic>Drosophila-</italic>like early regulatory interactions and a long germ mode of embryogenesis, little was known about later stages of <italic>Nasonia</italic> embryonic patterning.</p><p>We analyzed the expression and function of the pair-rule genes <italic>Nv eve, Nv odd</italic>, <italic>Nv runt</italic> and <italic>Nv h</italic> during embryogenesis. We found that each gene is expressed in both a canonical long-germ pair-rule stripe pattern at the anterior, as well as late-forming posterior stripes, indicating a dual mode of regulation. Strikingly, <italic>Nv eve</italic> is ultimately expressed in a total of 16 segmental stripes, of which six are derived from a single posterior stripe in the cellularized blastoderm. We also observe waves of <italic>Nv odd</italic> expression that resemble the waves of <italic>Tribolium odd</italic> expression<italic>,</italic> suggesting the residual activity of a segmentation clock in <italic>Nasonia.</italic> As in <italic>Tribolium</italic>, we found that mitoses do not occur exclusively at the site of late forming segments, but mitotic figures are not randomly distributed throughout the embryo. Instead, coordinated mitoses resembling the later mitotic domains of <italic>Drosophila</italic> (<xref ref-type="bibr" rid="bib28">Foe, 1989</xref>) appear and progress in waves from anterior to posterior, and are largely excluded from stripes of <italic>eve</italic> expression, suggesting a coordination of mitoses by segmentation genes. Using morpholinos to knock down gene function, we found that <italic>Nv eve, Nv odd</italic> and <italic>Nv h</italic> phenotypes do not affect alternating segments at the posterior, unlike what is observed in <italic>Drosophila</italic>. Instead, these ‘pair-rule’ genes are required for the formation of a continuous posterior region comprising abdominal segments A5–A10. Phenotypes in the anterior of the embryo are gene-specific; each gene exhibits a partial pair-rule phenotype in the allelic series. We suggest that <italic>Nasonia</italic> uses ‘pair-rule’ genes to pattern the embryo in a manner that resembles both <italic>Drosophila</italic> and <italic>Tribolium</italic>. We present a model for how this mixed mode of segmentation is achieved.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title><italic>Nasonia</italic> even-skipped exhibits character of both long and short germ patterning</title><p>The expression of eve has been studied in many insects, owing to a widely cross-reacting antibody. Its promoter has also been well studied in <italic>Drosophila</italic> and has become a classic example of modular gene control (<xref ref-type="bibr" rid="bib57">Patel et al., 1992</xref>; <xref ref-type="bibr" rid="bib71">Small et al., 1992</xref>, <xref ref-type="bibr" rid="bib72">1996</xref>). We used circular RACE from total embryo RNA (<xref ref-type="bibr" rid="bib48">McGrath, 2011</xref>) to generate a fragment of approximately 1 kb corresponding to the coding region of <italic>Nv eve</italic>, including the highly conserved homeodomain (Genbank Accession# KC168090). Several minor transcript variants were captured and sequenced, but not studied further (see ‘Materials and methods’ for GenBank accession numbers). The homeodomain of <italic>Nv</italic> Eve shares 81.7% amino acid identity with its <italic>Drosophila</italic> counterpart.</p><p>We used in situ hybridization to look at the expression pattern of <italic>Nv eve</italic> during <italic>Nasonia</italic> embryogenesis (<xref ref-type="fig" rid="fig1">Figure 1</xref>). <italic>Nv eve</italic> expression becomes detectable as a broad early domain in the blastoderm embryo at around 3 hr after egg laying (AEL) (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). This domain broadens and its boundaries sharpen between 3 and 4 hr, by which time a faint posterior stripe (hereafter referred to as ‘stripe 6’, see below) becomes evident (<xref ref-type="fig" rid="fig1">Figure 1B,C</xref>). As embryogenesis progresses toward cellularization, the anterior domain splits into three distinct double-segment periodicity pair-rule stripes, stripes 1, 2 and 3 (<xref ref-type="fig" rid="fig1">Figure 1D–F</xref>). By cellularization, around 6 hr, a faint 4/5 stripe appears between the anterior stripes and stripe 6, which has become more intense (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Between 6 and 8 hr AEL, stripe 4/5 splits into distinct double-segment periodicity stripes 4 and 5, whereas stripes 1 and 2 split into single-segment periodicity segmental stripes (<xref ref-type="fig" rid="fig1">Figure 1G–I</xref>). In <italic>Drosophila</italic>, eve secondary stripes form de novo, between primary pair-rule stripes, in contrast to secondary <italic>paired</italic> stripes that later split from primary stripes, forming segmental stripes that affect all segments (<xref ref-type="bibr" rid="bib47">Macdonald et al., 1986</xref>; <xref ref-type="bibr" rid="bib36b">Kilchherr et al., 1986</xref>). Splitting of <italic>Nasonia eve</italic> double-segment stripes into single-segment stripes may occur by a similar mechanism (see below). As gastrulation progresses between 8 and 10 hr AEL, double-segment pair-rule stripes 3–5 also split to give rise to two distinct single-segment stripes each (<xref ref-type="fig" rid="fig1">Figure 1J–L</xref>). This anterior to posterior progression of <italic>Nv</italic> eve stripes is consistent with the sequential appearance of the segment polarity genes <italic>Nv wg</italic> (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>) and <italic>Nv en</italic> (<xref ref-type="bibr" rid="bib62">Pultz et al., 1999</xref>), which are first detected around cellularization in a few anterior segments and then appear in stripes progressively, in an anterior to posterior manner.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.003</object-id><label>Figure 1.</label><caption><title>Summary of <italic>Nasonia eve</italic> mRNA expression.</title><p>Embryos are shown with anterior left and dorsal up. <italic>Nv eve</italic> is initially expressed in a broad domain (<bold>A</bold> and <bold>B</bold>), which sharpens as a posterior stripe becomes visible at around 4 hr after embryo laying (AEL) (<bold>C</bold> and <bold>D</bold>). The broad domain retracts anteriorly and gives rise to three apparently double-segment stripes (<bold>E</bold> and <bold>F</bold>). Between stripes 3 and posterior stripe 6, an additional double stripe precursor comes up at around 6 hr AEL (stripe 4/5; panels <bold>F</bold> and <bold>G</bold>) and this splits to form two double-segment stripes, ‘4’ and ‘5’ as double-segment stripes 1–3 split into two single-segment stripes each between 6 and 8 hr AEL (<bold>F</bold>–<bold>J</bold>). Stripes 4 and 5 also split to form single-segment stripes during early gastrulation, and stripe 6 broadens (<bold>K</bold> and <bold>L</bold>), giving rise to stripes that are visibly distinct during germ band extension in non-fluorescent staining by 10–12 hr AEL (<bold>M</bold>–<bold>R</bold>, arrowheads). There are a total of 16 single-segment stripes of <italic>Nv eve</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.003">http://dx.doi.org/10.7554/eLife.01440.003</ext-link></p></caption><graphic xlink:href="elife01440f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title><italic>Nv wingless (Nv wg)</italic> mRNA expression in the embryo.</title><p>All embryos are shown with anterior to the left and dorsal up except as indicated. (<bold>A</bold>) Precellular blasoderm with no expression of <italic>Nv wg</italic>. (<bold>B</bold>) Cellular blastoderm embryo exhibiting head expression and one stripe of <italic>Nv wg</italic>. (<bold>C</bold>) Early gastrula embryo exhibiting <italic>Nv wg</italic> staining in three segmental stripes comprising thoracic segments. (<bold>D</bold>) Ventral view of early gastrula embryo with four segmental stripes, including one abdominal segment. (<bold>E</bold>) Germ band extension embryo with six segmental stripes of <italic>Nv wg</italic> expression. (<bold>F</bold>) Germ band extension embryo exhibiting eight segmental stripes, including three thoracic and five abdominal stripes, as well as additional head segmental stripes. (<bold>G</bold>) Ventral view of a fully extended germ band with the full complement of 16 segmental <italic>Nv wg</italic> stripes. (<bold>H</bold>) Lateral view of germ band retracting embryo with 16 segmental stripes, including three clear segmental stripes in the head. (<bold>I</bold>) Lateral view of dorsal closure embryo exhibiting fading segmental <italic>Nv wg</italic> staining in all segments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.004">http://dx.doi.org/10.7554/eLife.01440.004</ext-link></p></caption><graphic xlink:href="elife01440fs001"/></fig></fig-group></p><p>A remarkable feature of <italic>Nv eve</italic> expression is that the posterior stripe 6 broadens significantly at about 10 hr AEL (<xref ref-type="fig" rid="fig1">Figure 1L–N</xref>), before generating six additional stripes with single-segment periodicity, allowing the embryo to reach 16 individual segmental stripes by the time the germ band is fully extended. This division of stripe 6 initiates with an anterior band that will give rise to four stripes (segmental stripes 11–14; <xref ref-type="fig" rid="fig1">Figure 1P,Q</xref>, arrowheads; see below), and two later-appearing segmental stripes 15 and 16 (<xref ref-type="fig" rid="fig1">Figure 1Q</xref>, arrowheads). The last stripe, <italic>Nv eve</italic> 16, appears only at full germ band extension (<xref ref-type="fig" rid="fig1">Figure 1R</xref>) completing the 16 stripes observed at this stage.</p><p>The <italic>Nasonia</italic> embryo has 16 segments, whereas <italic>Drosophila</italic> has only 14 (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). In <italic>Drosophila</italic>, <italic>eve</italic> and other pair-rule genes are expressed with double-segment periodicity: seven transverse ‘pair-rule’ stripes are evident as a full complement in the blastoderm embryo at cellularization. If the <italic>Nasonia</italic> embryo were patterned using the same mechanisms as <italic>Drosophila</italic>, then eight pair-rule stripes would be predicted. However, only five truly pair-rule (double segment) stripes are apparent at cellularization, while stripe 6 gives rise later to four, then six single-segment stripes and six segments and is therefore not pair-rule (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). This delayed sequential posterior segmentation is therefore more reminiscent of the segmentation described in short germ insects.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.005</object-id><label>Figure 2.</label><caption><title><italic>Nv eve</italic> epistasis with maternal and gap genes.</title><p>(<bold>A</bold>) Schematic representation of the germ-band-extended embryo, showing 16 single-segment stripes of <italic>Nv eve</italic> expression, and their segment counterparts in the patterned larval cuticle. Colored boxes cover the segments of the larval cuticle that are lost or fused in each RNAi background. All embryos are shown anterior left, dorsal up (except where indicated). <italic>Nv eve</italic> mRNA expression is shown in each embryo (<bold>B</bold>–<bold>G</bold>). Wild-type (WT) embryos are shown as staged controls for RNAi embryos. (<bold>B</bold>) WT early blastoderm embryo. (<bold>C</bold>) WT cellular blastoderm embryo. (<bold>D</bold>) WT early gastrula extension embryo. (<bold>E</bold>) WT germ-band-retracted embryo. (<bold>F</bold>–<bold>H</bold>) <italic>gt</italic> RNAi embryos stained for <italic>Nv eve</italic> mRNA expression. (<bold>F</bold>) Cellular blastoderm embryo with reduced <italic>Nv gt</italic> exhibits loss of anterior <italic>Nv eve</italic> stripes (x). (<bold>G</bold>) <italic>Nv gt</italic> RNAi embryo in early germ-band-extension exhibits loss of anterior <italic>Nv eve</italic> stripes and improper splitting of <italic>Nv eve</italic> stripe 5, as well as aberrant dorsal anterior expression of <italic>Nv eve</italic>. (<bold>H</bold>) <italic>Nv gt</italic> RNAi embryo at dorsal closure exhibits a stripe of <italic>Nv eve</italic> at the anterior, as well as a reduced number of posterior segmental <italic>Nv eve</italic> stripes. (<bold>I</bold>–<bold>L</bold>) <italic>Nv hb</italic> mutant embryos stained for <italic>Nv eve</italic> mRNA expression. (<bold>I</bold>) Early blastoderm <italic>Nv hb</italic> mutant embryos have a reduced central <italic>Nv eve</italic> domain (bounded by black arrowheads), and an ectopic anterior <italic>Nv eve</italic> stripe (white arrowhead). (<bold>J</bold>) <italic>Nv hb</italic> mutant cellular blastoderm embryo with a single anterior domain of <italic>Nv eve</italic> that has failed to resolve, and a single stripe 4 which exhibits delayed splitting. (<bold>K</bold>) <italic>Nv Hb</italic> mutant germ-band extension embryo with fused anterior domain (line) and 6 segmental stripes, representing derivatives of <italic>Nv eve</italic> stripes 4 and 5 and two derivatives of stripe 6; additional stripe 6 derivatives are absent (x). (<bold>L</bold>) <italic>hb</italic> mutant dorsal closure embryo exhibiting fused anterior domain (line) and the same number of derivatives as in (<bold>M</bold>), with more posterior segments missing (x). (<bold>M</bold>–<bold>O</bold>) <italic>Nv cad</italic> RNAi embryos stained for <italic>Nv eve</italic> mRNA expression. (<bold>M</bold>) <italic>Nv cad</italic> RNAi early blastoderm with reduced central <italic>Nv eve</italic> domain that is also posteriorly shifted (anterior boundary indicated by black arrowhead). (<bold>N</bold>) <italic>Nv cad</italic> RNAi cellular blastoderm embryo with posteriorly shifted (arrowhead), reduced <italic>Nv eve</italic> central domain, whose splitting is delayed. (<bold>O</bold>) <italic>Nv cad</italic> RNAi early gastrula embryo with posterior shift in <italic>Nv eve</italic> expression (black arrowhead). Four double-segment periodicity stripes are split into single-segment stripes and stripe 5 remains intact. (<bold>P</bold>–<bold>S</bold>) <italic>Nv Kr</italic> RNAi embryos stained for <italic>Nv eve</italic> mRNA expression. (<bold>P</bold>) <italic>Nv Kr</italic> RNAi precellular blastoderm embryo with aberrant <italic>Nv eve</italic> central domain resolution, where stripes 2–3 appear posteriorly shifted. (<bold>Q</bold>) Dorsolateral view of a <italic>Nv Kr</italic> RNAi embryo where stripes 2 and 3 are less refined than WT and 3 is posteriorly shifted. No stripe 4/5 expression is detected (X). (<bold>R</bold>) <italic>Nv Kr</italic> RNAi early gastrula embryo with aberrant stripe 2 splitting and aberrant resolution of stripes 3–5. (<bold>S</bold>) Moderately affected <italic>Nv Kr</italic> RNAi germ-band retraction embryo with fused segments in the middle of the embryo (line). (<bold>T</bold>–<bold>V</bold>) <italic>Nv tll</italic> RNAi embryos stained for <italic>Nv eve</italic> mRNA expression. (<bold>T</bold>) <italic>Nv tll</italic> RNAi early blastoderm embryo with expanded <italic>Nv eve</italic> expression domains toward both poles (arrowheads). (<bold>U</bold>) <italic>Nv tll</italic> RNAi precellular blastoderm embryo showing delayed resolution of <italic>Nv eve</italic> stripes 1–3 and <italic>Nv eve</italic> stripe 6 shifted to the extreme posterior pole of the embryo (arrowhead). (<bold>V</bold>) <italic>Nv tll</italic> RNAi dorsal closure embryo showing abnormal posterior <italic>Nv eve</italic> stripe formation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.005">http://dx.doi.org/10.7554/eLife.01440.005</ext-link></p></caption><graphic xlink:href="elife01440f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title><italic>Nv eve/Nv gt</italic> double FISH in the embryo.</title><p>Lateral view of early <italic>Nasonia</italic> gastrula embryo double stained for <italic>Nv eve</italic> and <italic>Nv gt</italic> mRNA. (<bold>A</bold>) <italic>Nv eve</italic> mRNA. (<bold>B</bold>) <italic>Nv gt</italic> mRNA. (<bold>C</bold>) Merge of <italic>Nv eve</italic> and <italic>Nv gt</italic> channels. Note the position of the late posterior stripe of <italic>Nv gt</italic> relative to <italic>Nv eve</italic> stripe 6.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.006">http://dx.doi.org/10.7554/eLife.01440.006</ext-link></p></caption><graphic xlink:href="elife01440fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.007</object-id><label>Figure 2—figure supplement 2.</label><caption><p>(<bold>A</bold>) Summary of maternal and gap gene loss of function phenotypes in <italic>Nasonia</italic>. On the vertical axis, genes affected by RNAi or genetic lesion are listed, and indicated as either maternal or zygotic phenotypes. Segments lost in each background are indicated as gray bars. Segments that remain in a given background are annotated alphanumerically.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.007">http://dx.doi.org/10.7554/eLife.01440.007</ext-link></p></caption><graphic xlink:href="elife01440fs003"/></fig></fig-group></p></sec><sec id="s2-2"><title>Control of Nv eve by gap genes</title><p>To explore this apparent combination of short and long germ characters, we determined how <italic>Nv eve</italic> expression is controlled by upstream genes in the known <italic>Nasonia</italic> A–P patterning network. Early embryonic expression of <italic>Drosophila eve</italic> is controlled by maternal and gap genes, including <italic>bicoid (bcd), hb, Kr, gt, kni</italic> and <italic>torso</italic> (<xref ref-type="bibr" rid="bib51">MJaJ & H, 1993</xref>; <xref ref-type="bibr" rid="bib71">Small et al., 1992</xref>, <xref ref-type="bibr" rid="bib72">1996</xref>; <xref ref-type="bibr" rid="bib67">Schroeder et al., 2004</xref>; <xref ref-type="bibr" rid="bib70">Small and Levine, 1991</xref>), whereas later maintenance is achieved via autoregulation (<xref ref-type="bibr" rid="bib35">Jiang et al., 1991</xref>). Some <italic>Tribolium eve</italic> pair-rule stripes are also under the control of gap genes although some of the segments themselves are born much later than gap gene expression (<xref ref-type="bibr" rid="bib78">Sulston and Anderson, 1996</xref>; <xref ref-type="bibr" rid="bib10">Cerny et al., 2005</xref>). For example, in <italic>Tc’Kr</italic> mutant embryos<italic>,</italic> segments anterior to the normal <italic>Kr</italic> expression domain (T1–T3) appear wild type, but expression of both <italic>Tc’eve</italic> and <italic>Tc’en</italic> is lost in posterior segments and no segments are formed posterior to A4 (<xref ref-type="bibr" rid="bib10">Cerny et al., 2005</xref>). Therefore, <italic>Tribolium</italic> gap genes can affect the specification of segments that are not yet formed, presumably because of interactions with the growth zone. In other short germ insects, like <italic>Oncopeltus fasciatus, eve</italic> acts as a gap gene, regulating expression of <italic>hunchback</italic> and <italic>Krüppel</italic> (<xref ref-type="bibr" rid="bib41">Liu and Kaufman, 2005a</xref>).</p><p>To determine how the known maternal and gap genes regulate <italic>Nv eve</italic> expression in the early embryo, we used parental RNAi injections in pupal <italic>Nasonia</italic> females to knockdown <italic>Nv gt, Nv Kr, Nv tll</italic> and <italic>Nv cad</italic> mRNA, as well as a null mutation in <italic>Nv hb</italic> (<xref ref-type="bibr" rid="bib63">Pultz et al., 2000</xref>, <xref ref-type="bibr" rid="bib64">2005</xref>).</p></sec><sec id="s2-3"><title>Nv gt</title><p>As we previously reported, <italic>Nv giant</italic> knockdown results in the loss of all segments anterior to A1 and fusion of segments A6 and A7 (<xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>). <italic>Nv gt</italic> RNAi blastoderm embryos exhibit the loss of <italic>Nv eve</italic> double-segment stripes 1–3 (<xref ref-type="fig" rid="fig2">Figure 2F</xref>), as well as aberrant resolution of the first splitting events of stripe 6 (<xref ref-type="fig" rid="fig2">Figure 2G</xref>, arrowheads). Double in situ hybridization shows that, in the wild type, a late posterior stripe of <italic>Nv gt</italic> forms after <italic>Nv eve</italic> stripe 6 and appears to be within the <italic>Nv eve</italic> stripe 6 domain (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). These data suggest that a posterior <italic>Nv gt</italic> domain may partially affect stripe 6 splitting. Late <italic>Nv gt</italic> RNAi embryos exhibit strong anterior defects after dorsal closure, but the other posterior single-segment stripes of <italic>Nv eve</italic> appear unaffected (<xref ref-type="fig" rid="fig2">Figure 2H</xref>).</p></sec><sec id="s2-4"><title>Nv hb</title><p>In <italic>Nv hb</italic> mutant (<italic>headless</italic>) embryos, both head and thoracic fates and abdominal fates posterior to A6 are lost (<xref ref-type="bibr" rid="bib64">Pultz et al., 2005</xref>). Consistent with this phenotype, <italic>Nv eve</italic> double-segment stripes 1–3 (that give rise to head and thoracic fates) form but never resolve (<xref ref-type="fig" rid="fig2">Figure 2K</xref>, black arrowheads). An ectopic stripe of <italic>eve</italic> can be seen in the anterior of some embryos (<xref ref-type="fig" rid="fig2">Figure 2I</xref>, white arrowhead), consistent with the ectopic stripe of <italic>Nv cad</italic> (an activator of <italic>eve</italic>) in the head of some <italic>Nv hb</italic> mutant embryos (<xref ref-type="bibr" rid="bib55">Olesnicky et al., 2006</xref>). Gastrula and later germ band embryos exhibit normal <italic>Nv eve</italic> double-segment stripe formation and splitting of stripes 4 and 5, which give rise to segments A1–A4. However, only the two anteriormost segments are formed from <italic>Nv eve</italic> stripe 6 (6a and 6b) in <italic>Nv hb</italic> mutant embryos (<xref ref-type="fig" rid="fig2">Figure 2K,L</xref>).</p></sec><sec id="s2-5"><title>Nv caudal</title><p><italic>Nv caudal</italic> (<italic>cad</italic>) is expressed maternally as a mRNA gradient with a localized posterior source (<xref ref-type="bibr" rid="bib55">Olesnicky et al., 2006</xref>). <italic>Nv cad</italic> RNAi results in loss of all segments posterior to A1. Moderately affected <italic>Nv cad</italic> RNAi embryos exhibit a reduced early broad domain of <italic>Nv eve</italic> that is slightly shifted posteriorly (<xref ref-type="fig" rid="fig2">Figure 2M</xref>). This domain resolves poorly, with only weak activation of anterior <italic>Nv eve</italic> stripes and no posterior abdominal expression of <italic>Nv eve</italic> (<xref ref-type="fig" rid="fig2">Figure 2N–O</xref>).</p></sec><sec id="s2-6"><title>Nv Kr</title><p>As in <italic>Drosophila</italic>, <italic>Nv Krüppel</italic> (Kr) is expressed in a central domain, and <italic>Nv Kr</italic> is required for formation of segments T3 to A4 (<xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>). In <italic>Nv Kr</italic> RNAi embryos, both anterior and posterior domains of <italic>Nv hb</italic> expression expand towards the center of the embryo (<xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>). Consistent with expansion of <italic>Nv hb</italic>, we observed that <italic>Nv eve</italic> stripe 2 and 3 exhibit aberrant resolution and <italic>Nv eve</italic> stripes 4 and 5 fail to resolve in embryos with knocked-down <italic>Nv Kr</italic> (<xref ref-type="fig" rid="fig2">Figure 2P–S</xref>). Posterior segments are unaffected, as reflected by normal expression of <italic>Nv eve</italic> posterior to stripe 5 (segment A4; <xref ref-type="fig" rid="fig2">Figure 2R,S</xref>). This phenotype is dramatically different from <italic>Tc’Kr</italic> knockdown where all posterior segments are deleted, likely because <italic>Nv Kr</italic> is expressed anterior to the growth zone while <italic>Tc’Kr</italic> abuts it.</p></sec><sec id="s2-7"><title>Nv tll</title><p><italic>tailless</italic> mRNA is expressed in both an anterior and a posterior domain, though only posterior segments are affected by <italic>Nv tll</italic> RNAi (<xref ref-type="bibr" rid="bib46">Lynch et al., 2006</xref>). The most severely affected embryos are missing the six posterior abdominal segments. These embryos also exhibit an apparent slight anterior shift of the broad early domain of <italic>Nv eve</italic> expression and of stripe 6 (<xref ref-type="fig" rid="fig2">Figure 2T,U</xref>). Stripe 6 does not appear to resolve, resulting in an enduring ring of <italic>Nv eve</italic> expression and no <italic>Nv eve</italic> single-segment stripes posterior to this ring are apparent (<xref ref-type="fig" rid="fig2">Figure 2V</xref>).</p><p>Taken together, and consistent with previously described cuticular phenotypes for maternal and gap genes in <italic>Nasonia</italic> (<xref ref-type="bibr" rid="bib64">Pultz et al., 2005</xref>; <xref ref-type="bibr" rid="bib46">Lynch et al., 2006</xref>; <xref ref-type="bibr" rid="bib55">Olesnicky et al., 2006</xref>; <xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>), these data show that early <italic>Nv eve</italic> expression in blastoderm embryos involves regulatory interactions reminiscent of those underlying <italic>Drosophila</italic> long germ embryogenesis. However, since severe RNAi phenotypes of several genes, such as <italic>Nv cad</italic> and <italic>Nv tll</italic> results in total loss of posterior segments, these did not provide additional information for understanding the establishment of posterior <italic>Nv eve</italic> expression.</p></sec><sec id="s2-8"><title><italic>Nasonia</italic> embryos have mitotic domains but Nv eve posterior stripe resolution does not require localized cell division</title><p>To determine whether cell division plays a role in subdivision of the <italic>Nv eve</italic> posterior domain into single-segment stripes, we used in situ hybridization to visualize <italic>Nv eve</italic> mRNA in embryos where mitotic cells were labeled with antibodies against phosphorylated histone H3. We found that there is no cell division that is consistent with a role in pair-rule stripe splitting (<xref ref-type="fig" rid="fig3">Figure 3A–A″</xref>), or in stripe 6 resolution (<xref ref-type="fig" rid="fig3">Figure 3B–B″</xref>), suggesting that the dynamics of the <italic>Nv eve</italic> mRNA pattern mostly involves transcriptional regulation. Nevertheless, later mitoses occur in restricted spatial domains, reminiscent of later <italic>Drosophila</italic> mitotic domains within segments of the expanding germ band (<xref ref-type="fig" rid="fig3">Figure 3B–D″</xref>). A relationship between gap gene function and regulation of mitotic domains via regulation of <italic>string</italic> has been suggested in <italic>Drosophila</italic> (<xref ref-type="bibr" rid="bib25">Edgar et al., 1994</xref>) but not demonstrated. <italic>Nasonia</italic> mitotic domains appear in an anterior to posterior progression, allowing for progressive expansion of segments along the A–P axis via concerted cell divisions within domains. Strikingly, mitotic figures appear to be largely excluded from <italic>Nv eve</italic> stripes in these early stages of embryogenesis (<xref ref-type="fig" rid="fig3">Figure 3B″,C″</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Later embryos in which anterior <italic>Nv eve</italic> stripes are beginning to fade exhibit overlap of mitotic figures with weakened <italic>Nv eve</italic> stripe expression (<xref ref-type="fig" rid="fig3">Figure 3D″</xref>). Ultimately, embryos exhibit widespread mitotic figures that do not correspond to any apparent concerted domains or pattern, more like the pattern of mitoses described in several short germ insects (<xref ref-type="bibr" rid="bib31">Handel et al., 2005</xref>; <xref ref-type="bibr" rid="bib43">Liu and Kaufman, 2009</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.008</object-id><label>Figure 3.</label><caption><title><italic>Nv eve</italic> expression and cell division appear to be coordinated.</title><p>Embryos co-stained for <italic>Nv eve</italic> mRNA using in situ hybridization and fluorescent detection, as well as for mitotic figures, using an antibody against phospho Histone H3. Embryos are shown with anterior left and dorsal up, except columns <bold>B</bold> and <bold>C</bold>, which are ventral views. (<bold>A</bold>–<bold>A″</bold>) An early gastrula embryo exhibiting 15 stripes of <italic>Nv eve</italic>, including five derivatives of stripe 6 (<bold>A</bold>), has no evident mitotic figures in the posterior domain of <italic>Nv eve</italic> stripe 6 differentiation (<bold>A′</bold>). (<bold>A″</bold>) Merge of panels <bold>A</bold> and <bold>A′</bold>. (<bold>B</bold>–<bold>D″</bold>) Timecourse series of wild-type embryos stained for <italic>Nv eve</italic> mRNA and phospho-Histone H3. (<bold>B</bold>–<bold>D</bold>). Top panels are <italic>Nv eve</italic> in situ alone, middle panels (<bold>B′</bold>–<bold>D′</bold>) are phospho-Histone H3 antibody staining, and bottom panels (<bold>B″</bold>–<bold>D″</bold>) are merge images of upper panels, showing localization of mitotic figures relative to <italic>Nv eve</italic> stripes.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.008">http://dx.doi.org/10.7554/eLife.01440.008</ext-link></p></caption><graphic xlink:href="elife01440f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.009</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Quantification of PH3 positive cells relative to <italic>Nv eve</italic> stripes in the embryo.</title><p>Embryos stained for phosphorylated histone H3 (to label mitotic figures) and for <italic>Nv eve</italic> mRNA (as shown in <xref ref-type="fig" rid="fig3">Figure 3</xref>) were analyzed to quantify the number of mitotic cells occurring between stripes as compared to within <italic>eve</italic> stripes. Cell number counted is plotted on the Y axis as a function of position along the A–P axis of the embryo (as indicated by <italic>Nv eve</italic> stripe position), which is indicated on the X axis. Mitotic cells that fall partially on a stripe are counted as occurring in the stripe. Each unique color indicates one embryo quantified in this manner, and embryos corresponding to those shown in <xref ref-type="fig" rid="fig3">Figure 3</xref> are also labeled according to their position in the figure.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.009">http://dx.doi.org/10.7554/eLife.01440.009</ext-link></p></caption><graphic xlink:href="elife01440fs004"/></fig></fig-group></p></sec><sec id="s2-9"><title>Knockdown of <italic>Nv eve</italic> results in gap and segment polarity defects and posterior truncation</title><p>The expression of <italic>Nv eve</italic> suggests a combination of long germ and short germ character. To further explore this possibility, we knocked down <italic>Nv eve</italic> gene function. Although parental RNAi in <italic>Nasonia</italic> is effective for maternal and early zygotic genes (<xref ref-type="bibr" rid="bib44">Lynch and Desplan, 2006</xref>; <xref ref-type="bibr" rid="bib45">Lynch et al., 2006</xref>; <xref ref-type="bibr" rid="bib55">Olesnicky et al., 2006</xref>; <xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>), it often does not provide significant knockdown of later-acting genes. To overcome this limitation, we designed an <italic>Nv eve</italic> morpholino overlapping the translation start site, as well as one directed at the exon–intron junction in the homeobox. These two independent morpholinos are expected to disrupt <italic>Nv eve</italic> activity and indeed result in comparable phenotypes.</p><p>The <italic>Nasonia</italic> larval cuticle has relatively few landmarks to allow for interpretation of segmentation phenotypes. Beyond the denticle belts present on each of the three thoracic segments and ten abdominal segments, large spiracles are found on segments T2, A1, A2 and A3 (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, yellow arrowheads). In the head, two bright structures indicate the positions of antennal papillae. Morpholino block of <italic>Nv eve</italic> causes a range of phenotypes (<xref ref-type="fig" rid="fig4">Figure 4B–E</xref>), resulting in severe truncation of the embryo with loss of posterior-derived segments as well as a partial pair-rule phenotype for more anterior segments. The phenotypic series includes progressive truncation at the posterior, causing fusion of segments A9–10 in the least affected cuticles, and then A8–10 (<xref ref-type="fig" rid="fig4">Figure 4C</xref>) with segment A6 eventually lost, whereas A7 remains virtually intact. In the most severely affected embryos, the entire posterior of the embryo is truncated with A5–A10 missing. (The approximate percentage of embryos in each phenotypic class shown in <xref ref-type="fig" rid="fig4">Figure 4</xref> is indicated in the ‘Materials and methods’.)<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.010</object-id><label>Figure 4.</label><caption><title>Morpholino knockdown of <italic>Nv eve</italic>, <italic>Nv hairy</italic>, and <italic>Nv odd</italic> results in embryo patterning defects.</title><p>First instar larval cuticles are shown with anterior left and generally ventral denticle patterns are shown. (<bold>A</bold>, <bold>F</bold>, <bold>K</bold>) Wild-type larval cuticles. Yellow arrows indicate spiracles present on segments T2, A1, A2 and A3. Bright anterior labral appendages are apparent at the extreme anterior of the larva. (<bold>B</bold>–<bold>E</bold>) Unhatched larvae from <italic>Nv eve</italic> morpholino (MO)-injected embryos, in order of increasing phenotype severity. Red arrowheads indicate loss of midline cuticle. Blue dot indicates head open defect. Yellow arrowheads indicate position of spiracles. (<bold>G</bold>–<bold>J</bold>) Unhatched larvae from <italic>Nv odd</italic> morpholino (MO)-injected embryos, in order of increasing phenotype severity. Yellow arrows indicate position of spiracles, red arrows indicate A3/A4 fusion. X indicates naked cuticle from segment loss. Yellow line indicates multi-segment fusion. (<bold>L</bold>–<bold>O</bold>) Unhatched larvae from <italic>Nv hairy</italic> morpholino (MO)-injected embryos, in order of increasing phenotype severity. Yellow arrowheads indicate position of spiracles. Red or orange arrowheads indicate aberrantly positioned or missing spiracles. Yellow line indicates segment fusion.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.010">http://dx.doi.org/10.7554/eLife.01440.010</ext-link></p></caption><graphic xlink:href="elife01440f004"/></fig></p><p>At the anterior, T1 is lost with fusion of T3 and A1 (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Segments A2 and A3 are also fused, and there is a continuous lawn of denticles from A4 to the truncated posterior. This is accompanied by a disruption of the remaining denticle belts, leaving naked cuticle along the midline (<xref ref-type="fig" rid="fig4">Figure 4</xref>, red arrows). In the most severely affected embryos, segments anterior to A1 are lost and are accompanied by head closure defects. This phenotype represents a partial pair-rule phenotype, accompanied by posterior truncation of the embryo. It also does not exhibit the lawn of denticles phenotype of strong <italic>eve</italic> alleles in flies (e.g., <italic>eve</italic><sup><italic>R13</italic></sup> [<xref ref-type="bibr" rid="bib47">Macdonald et al., 1986</xref>; <xref ref-type="bibr" rid="bib30">Fujioka et al., 1999</xref>]), although severely affected <italic>Nasonia</italic> embryos also exhibit cuticle defects beyond a pair-rule phenotype. Hence, these results support a mixed mode of embryogenesis in <italic>Nasonia</italic>, with characteristic features resembling both long germ and short germ insects. To further examine this possibility, we then investigated the expression patterns in <italic>Nasonia</italic> of other genes acting as pair-rule in <italic>Drosophila,</italic> and undertook functional characterization of their activity during embryonic development.</p></sec><sec id="s2-10"><title><italic>Nasonia odd-skipped</italic> expression and function</title><p>In the long germ <italic>Drosophila</italic> embryo, <italic>odd</italic> is expressed with a double-segment periodicity complementary to that of <italic>eve</italic>, and its inactivation causes the absence of odd segments (<xref ref-type="bibr" rid="bib54">Nusslein-Volhard and Wieschaus, 1980</xref>; <xref ref-type="bibr" rid="bib18">Coulter et al., 1990</xref>). Its critical function as a mediator of the segmentation clock in the short germ beetle was recently elegantly described (<xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>). <italic>Tc’odd</italic> begins with blastoderm expression in double-segment periodicity stripes alternating with <italic>Tc’eve</italic> expression. Then, new double-segment stripes emanate from the growth zone to generate the entire complement of <italic>odd</italic> stripes. Secondary single-segment stripes arise later (<xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>).</p><p>There are three <italic>odd</italic> paralogs in <italic>Nasonia</italic>, as in flies, where they are named <italic>odd, bowl</italic>, and <italic>sister of bowl</italic> (or <italic>sob;</italic> (<xref ref-type="bibr" rid="bib32">Hart et al., 1996</xref>)). We used sequence alignment (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>) and phylogenetic analysis (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>) to identify the <italic>Nasonia</italic> paralog that is closest to <italic>Drosophila odd-skipped</italic>, and refer to it hereafter as <italic>Nv odd</italic>. An <italic>Nv odd</italic> cDNA fragment comprising the region encoding the conserved DNA binding domain was used as a probe for in situ hybridization (GenBank Accession # KC142194). As observed above for <italic>Nv eve,</italic> the embryonic expression of <italic>Nv odd</italic> begins as a broad early domain in syncytial blasoderm embryos (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). As this broad domain strengthens and sharpens, a ventral head patch and a posterior cap appear (<xref ref-type="fig" rid="fig5">Figure 5B,C</xref>). The broad domain resolves into two clear apparent double-segment stripes (Stripes 1 and 2, <xref ref-type="fig" rid="fig5">Figure 5D</xref>). A third double-segment stripe arises from the second stripe, expanding posteriorly (<xref ref-type="fig" rid="fig5">Figure 5D–F</xref>). At the same time, a stronger posterior domain apparently advances anteriorly. Pair-rule stripe 4 (double-segment periodicity) arises at the anterior of the first advancing ‘wave’ at cellularization (<xref ref-type="fig" rid="fig5">Figure 5G–H</xref>) before the posterior domain recedes again (<xref ref-type="fig" rid="fig5">Figure 5I–J</xref>). The fifth double-segment stripe arises during a second ‘wave’ (<xref ref-type="fig" rid="fig5">Figure 5K–M</xref>) at the onset of gastrulation. A sixth stripe arises in an apparently similar manner, though it is much fainter and appears while more posterior stripes are already differentiated (<xref ref-type="fig" rid="fig5">Figure 5N</xref>, arrowhead); the posterior cap generates two thin pair-rule stripes (<xref ref-type="fig" rid="fig5">Figure 5O</xref>) during early germ band extension. At full germ band extension, a total of eight stripes are visible (<xref ref-type="fig" rid="fig5">Figure 5P</xref>) before these fade from anterior to posterior. This dynamic expression of <italic>Nv odd</italic> in the posterior of the embryo is reminiscent of the waves of growth zone expression of <italic>Tribolium odd</italic>, where blastoderm-derived stripes initially have double-segment periodicity and later single-segment periodicity (<xref ref-type="bibr" rid="bib16">Choe et al., 2006</xref>; <xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.011</object-id><label>Figure 5.</label><caption><title>Summary of <italic>Nv odd-skipped</italic> mRNA expression.</title><p>Embryos are shown with anterior left and dorsal up, except where indicated. (<bold>A</bold>) Precellular blastoderm embryo showing early expression of <italic>Nv odd</italic> in a broad domain and a posterior cap with a slight clearing in between. (<bold>B</bold>) Precellular blastoderm embryo showing ventral head patch and darkened central broad domain and distinct posterior cap. (<bold>C</bold>) Precellular blastoderm embryo with sharpening pair-rule stripes and expanding posterior cap. (<bold>D</bold>) Precellular blastoderm embryo with dark ventral head patch and posterior cap, and expansion of expression between broad central domain and posterior domain. (<bold>E</bold> and <bold>F</bold>) Cellularizing blastoderm embryos with three double-segment periodicity stripes, and a continuous posterior domain of variable staining intensity. Arrowhead indicates boundary of faint expression, which prefigures position of double-segment stripe 4. (<bold>G</bold>) Ventral view of cellularizing embryo with three strong double-segment stripes, and a fourth stripe forming at the anterior boundary of a more uniformly staining posterior cap (arrowhead). (<bold>H</bold>) Cellularized blastoderm embryo with four distinct double-segment stripes and a receding posterior cap domain (arrowhead). (<bold>I</bold>) Ventral view of cellular blastoderm showing four strong double-segment stripes and receding posterior cap (arrowhead), whose anterior boundary prefigures the position of stripe 5. (<bold>J</bold>) Ventrolateral view of cellular blasoderm embryo showing early appearance of stripe 5 at the anterior boundary of receding posterior domain, whose staining intensity is now less uniform. (<bold>K</bold>) Cellular blastoderm embryo with five double-segment stripes of expression, a strong ventral head spot, and a reduced, uniform posterior cap. (<bold>L</bold>) Same as <bold>K</bold>, with five equivalently strong double-segment stripes. Arrowhead indicates slightly expanded posterior cap. (<bold>M</bold>) Early germ-band extension embryo with five double-segment periodicity stripes and two stripes becoming evident within the posterior cap. (<bold>N</bold>) Slightly later embryo than <bold>M</bold>, with 2 posterior cap stripes more clearly differentiated. (<bold>O</bold>) Slightly later embryo than <bold>N</bold>, with anterior stripes fading and posterior segments expanding. (<bold>P</bold>) Dorsal view, dorsal closure embryo exhibiting eight single-segment periodicity stripes.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.011">http://dx.doi.org/10.7554/eLife.01440.011</ext-link></p></caption><graphic xlink:href="elife01440f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.012</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Phylogenetic analysis of odd-skipped.</title><p>Phylogenetic analysis of odd-skipped protein was performed using Maximum likelihood analysis with 1000-fold bootstrap support, using RAxML via the CIPRES portal (<xref ref-type="bibr" rid="bib17">Copf et al., 2003</xref>; <xref ref-type="bibr" rid="bib53">Nagy et al., 1994</xref>). The best scoring maximum likelihood tree is shown with bootstrap support values indicated adjacent to branch.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.012">http://dx.doi.org/10.7554/eLife.01440.012</ext-link></p></caption><graphic xlink:href="elife01440fs005"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.013</object-id><label>Figure 5—figure supplement 2.</label><caption><title>odd-skipped protein sequence alignment.</title><p>Predicted odd-skipped protein sequences from <italic>Nasonia</italic> were aligned to odd-skipped related proteins sequences from <italic>Drosophila melanogaster, Anopheles gambiae, Apis mellifera</italic>, and <italic>Tribolium castaneum</italic> using ClustalW using standard parameters. Full-length sequences were used for alignment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.013">http://dx.doi.org/10.7554/eLife.01440.013</ext-link></p></caption><graphic xlink:href="elife01440fs006"/></fig></fig-group></p><p>Using double fluorescent in situ hybridization, we confirmed that the pair-rule stripes of <italic>Nv odd</italic> and <italic>Nv eve</italic> are indeed complementary to each other, although their mode of appearance is totally different. <italic>Nv odd</italic> double-segment stripes are posterior to, and abut each posterior single-segment stripe (i.e., 1b, 2b) from each <italic>eve</italic> pair-rule doublet (<xref ref-type="fig" rid="fig6">Figure 6A–C</xref>), that is, the even-numbered segmental stripes. Late forming <italic>Nv eve</italic> stripe 15/16 intercalates between the two <italic>Nv odd</italic> stripes 7 and 8 that derive from the cap, with <italic>Nv odd</italic> stripe 8 remaining posterior to all <italic>Nv eve</italic> stripes (excepting the last stripe, <italic>eve 16</italic>, which is the last to appear), a relationship that may have ancestral origins (see ‘Discussion’).<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.014</object-id><label>Figure 6.</label><caption><title>Phasing of Nasonia pair-rule genes in embryos using double fluorescent in situ hybridization.</title><p>(<bold>A</bold>) Lateral view of <italic>Nv eve</italic> expression in early gastrula embryo. (<bold>B</bold>) <italic>Nv odd</italic> expression alone in the same embryo. (<bold>C</bold>) Merge of <italic>Nv eve</italic> and <italic>Nv odd</italic> channels, illustrating their relative phasing. <italic>Nv eve</italic> mRNA is pseudo-colored pink, <italic>Nv odd</italic> is in green. Arrowheads indicate position of a posterior doublet of <italic>odd</italic> stripes. (<bold>D</bold>) Dorsolateral view of <italic>Nv eve</italic> in later gastrula embryo. (<bold>E</bold>) <italic>Nv odd</italic> expression alone in the same embryo. Arrowheads indicate position of posterior <italic>Nv odd</italic> stripes 6, 7 and 8. (<bold>F</bold>) Merge of <italic>Nv eve</italic> and <italic>Nv odd</italic> channels, illustrating their relative phasing. (<bold>G</bold>) Lateral view of <italic>Nv eve</italic> expression in blastoderm embryo. Arrowhead indicates position of <italic>Nv eve</italic> stripe 5. (<bold>H</bold>) <italic>Nv runt</italic> expression in the same blastoderm embryo. (<bold>I</bold>) Merge of <italic>Nv eve</italic> (green) and <italic>Nv runt</italic> (pink) channels, indicating relative phasing. (<bold>J</bold>) Lateral view of <italic>Nv eve</italic> expression in germ-band-extended embryo. Numbers indicate identity of <italic>Nv eve</italic> stripe. (<bold>K</bold>) <italic>Nv runt</italic> expression alone in the same embryo. Arrowheads indicate position of posterior primary <italic>Nv runt</italic> stripes. (<bold>L</bold>) Merge of <italic>Nv eve</italic> (green) and <italic>Nv runt</italic> (pink) channels, indicating relative phasing. Note that posterior <italic>Nv runt</italic> stripes, though faint, appear to be positioned posterior to odd-numbered <italic>Nv eve</italic> segmental stripes. (<bold>M</bold>) Lateral view of <italic>Nv eve</italic> expression in early gastrula embryo. Line indicates broadening stripe 6. (<bold>N</bold>) <italic>Nv hairy</italic> expression in the same gastrula embryo. Arrowheads indicate positions of three late forming posterior double-segment stripes. (<bold>O</bold>) Merge of <italic>Nv eve</italic> (pink) and <italic>Nv hairy</italic> (green) channels, indicating relative phasing. (<bold>P</bold>) Ventral view of gastrula embryo showing <italic>Nv eve</italic> expression alone. Arrowheads indicate positions of single-segment stripes derived from <italic>eve</italic> stripe 6. (<bold>Q</bold>) <italic>Nv hairy</italic> expression alone in the same gastrula embryo. Line indicates extended anterior domain continuous with stripe 1. (<bold>R</bold>) Merge of <italic>Nv eve</italic> (green) and <italic>Nv hairy</italic> (pink) channels, illustrating relative phasing.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.014">http://dx.doi.org/10.7554/eLife.01440.014</ext-link></p></caption><graphic xlink:href="elife01440f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.015</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Summary of <italic>Nv runt</italic> mRNA expression.</title><p>Embryos are shown anterior left and ventral up unless indicated. (<bold>A</bold>) Early energid stage embryo with faint staining evident in posterior. (<bold>B</bold>) Early precellular blastoderm embryo exhibiting stronger, mostly ubiquitous <italic>Nv runt</italic> staining biased towards the posterior. (<bold>C</bold>) Precellular blastoderm embryo with strong stripe of <italic>Nv runt</italic> expression within larger, gap-type domain. Expression is absent from the rest of the embryo. (<bold>D</bold>) Precellular blastoderm embryo exhibiting three clear pair-rule stripes of <italic>Nv runt</italic> expression. (<bold>E</bold>) Cellular blastoderm embryo with four pair-rule stripes of <italic>Nv runt</italic> expression. (<bold>F</bold>) Early gastrula embryo exhibiting three pair-rule stripes, an expanding stripe 4/5 stripe domain and faint posterior stripe 6. (<bold>G</bold>) Early gastrula embryo exhibiting 6 pair-rule stripes of <italic>Nv runt</italic> expression. (<bold>H</bold>) Gastrula embryo exhibiting splitting of anterior stripes 1 and 2 (and possibly 3), and stronger expression of stripes 4 and 5, with expanded interstripe distance between stripes 5 and 6. (<bold>I</bold>) Dorsal view of gastrula embryo at approximately the same stage as in (<bold>H</bold>). (<bold>J</bold>) Dorsolateral view of germ-band extension embryo with fading anterior stripes, and an additional pair-rule stripe appearing between stripes 5 and 6 (arrowhead). (<bold>K</bold>) Germ-band extension embryo exhibiting seven pair-rule stripes of expression with segmental stripes appearing in posterior interstripes (arrowheads). (<bold>L</bold>) Ventral view of fully extended germ band exhibiting segmental expression in all segments, in addition to continuous expression in the ventral anterior of the embryo.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.015">http://dx.doi.org/10.7554/eLife.01440.015</ext-link></p></caption><graphic xlink:href="elife01440fs007"/></fig></fig-group></p><p>To examine the function of <italic>Nv odd</italic> in the embryo, we used one translation blocking and one splice blocking morpholino to knock down its expression in embryos. Inactivating <italic>Nv odd</italic> function leads to loss of the most posterior germ band-derived segments A5–A10 with additional anterior defects. The most sensitive phenotypes are the fusion of segments A3 and A4, and loss of segment A5 (<xref ref-type="fig" rid="fig4">Figure 4G</xref>, red arrowheads and x). More severely affected embryos exhibit loss of most segments posterior to A3/A4 and naked cuticle anterior to T2, though larval head structures are still present (<xref ref-type="fig" rid="fig4">Figure 4I–J</xref>). In many severely affected embryos, <italic>Nv odd</italic> knockdown causes additional loss/fusion of A1, and of T2. Thus, phenotypes comprise loss of segments T2, A1, A3 and A5 and resemble a pair-rule phenotype. A small percentage of embryos are nearly asegmental, with only small patches of denticle bands of unknown identity (not shown). Therefore, as also observed for <italic>Nv eve</italic> knockdown, the segmentation defects resulting from <italic>Nv odd</italic> inactivation only corresponds to a partial <italic>Drosophila</italic>-like pair-rule phenotype, and rather, in the most severe cases, resemble the phenotype of <italic>Tc’odd</italic> loss of function. It is of note that <italic>Nv odd</italic> stripes 4, 5 and 6 appear to emerge from waves of expression at the posterior of the embryo that likely specify segments A3–A5, which are most sensitive to loss of <italic>Nv odd</italic> function (<xref ref-type="fig" rid="fig5">Figure 5E–L</xref>, <xref ref-type="fig" rid="fig4">Figure 4G</xref>).</p><p>In summary, <italic>Nv odd</italic> is expressed initially in three sequentially forming anterior double-segment periodicity stripes, which appear to have <italic>Drosophila</italic>-like pair-rule character. Three more posterior double-segment stripes (PR stripes 4–6) then form sequentially, apparently as ‘waves’ of <italic>Nv odd</italic> expression, resembling the clock-driven stripes of <italic>Tc’odd</italic>. Finally, two <italic>Nv odd</italic> stripes form from a posterior cap<italic>. Nv odd</italic> knockdown affects anterior thoracic segments with a partial pair-rule phenotype; it also leads to the loss of posterior segments A5–A10.</p></sec><sec id="s2-11"><title><italic>Nasonia runt</italic> expression</title><p>The complementarity between <italic>Nv eve</italic> and <italic>Nv odd</italic> is suggestive of cross interaction between the two genes but it is only partial and only affects half of the <italic>Nv eve</italic> segmental stripes since <italic>Nv odd</italic> does not have single-segment periodicity stripes. We sought to determine whether the remaining single segment stripes where <italic>odd</italic> is not interdigitated with <italic>Nv eve</italic> may alternate with stripes of <italic>Nv runt</italic>, as is observed in <italic>Drosophila</italic>. We studied the expression of <italic>Nv runt</italic> throughout embryogenesis (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>) and then used double fluorescent in situ hybridization to visualize its register with <italic>Nv eve</italic> stripes at both early and late stages. <italic>Nv runt</italic> stripes appear cleanly in an anterior to posterior progression, with six double-segment periodicity stripes visible before cellularization; two additional double-segment stripes are added at the posterior during gastrulation. Single-segment periodicity stripes only appear much later at full germ band extension when the expression of the other pair-rule genes is already well established (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). In the early embryo, <italic>Nv runt</italic> double-segment stripes appear posterior to, and partly overlapping with, each <italic>Nv eve</italic> primary double-segment stripe (<xref ref-type="fig" rid="fig6">Figure 6G–I</xref>). Splitting of anterior <italic>Nv eve</italic> stripes moves the posterior of each doublet (i.e., even-numbered <italic>Nv eve</italic> single-segment stripes) more posteriorly beyond each <italic>Nv runt</italic> primary double-segment stripe (<xref ref-type="fig" rid="fig6">Figure 6G–I</xref>). The appearance of <italic>Nv eve</italic> stripe 5 between <italic>Nv runt</italic> double-segment stripes 4 and 5 as they split (<xref ref-type="fig" rid="fig6">Figure 6G–I</xref>, arrowhead; <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1F,G</xref>) suggests that <italic>eve</italic> may help to repress <italic>Nv runt</italic>, though this remains to be tested.</p><p>Late expression of <italic>Nv runt</italic> in the extending germ band is considerably weaker than that of other genes, making its detection more challenging. Still, at the posterior of the embryo, <italic>Nv runt</italic> double-segment stripes appear between <italic>Nv eve</italic> single-segment stripes arising from splitting of double-segment stripes (<xref ref-type="fig" rid="fig6">Figure 6J–L</xref>). Altogether, our data support a model in which <italic>Nv eve</italic> single-segment periodicity stripes are established through the complementary action of <italic>Nv odd</italic> for odd numbered single-segment stripes and <italic>Nv runt</italic> for even-numbered stripes, as summarized in our model below. However, these interactions are still speculative since we have been technically unable to complete the epistasis experiments needed to test this model. A predicted role for <italic>Nv h</italic> is also described below.</p></sec><sec id="s2-12"><title>Expression and function of <italic>Nasonia hairy</italic></title><p><italic>hairy</italic> is a primary pair-rule gene in <italic>Drosophila</italic>, but in <italic>Tribolium</italic>, its function is restricted to head segment differentiation (<xref ref-type="bibr" rid="bib9">Carroll et al., 1988</xref>; <xref ref-type="bibr" rid="bib8">Carroll and Vavra, 1989</xref>; <xref ref-type="bibr" rid="bib24">Edgar et al., 1989</xref>; <xref ref-type="bibr" rid="bib16">Choe et al., 2006</xref>). There are two <italic>hairy-</italic>like genes in <italic>Nasonia</italic>, and we identified the likely <italic>hairy</italic> (<italic>h</italic>) ortholog using phylogenetic analysis (<xref ref-type="fig" rid="fig7s1 fig7s2">Figure 7—figure supplements 1 and 2</xref>). We examined expression of <italic>Nv h</italic> using a probe directed against the full-length coding region (Genbank Accession # KC190514).</p><p><italic>Nv h</italic> expression begins as a single broad anterior double segment stripe 1 that incompletely spans the dorso–ventral axis. It is soon followed by a second broad double-segment stripe 2 just anterior to the middle of the embryo (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Double-segment stripes 3, 4 and 5 are then added sequentially before cellularization (<xref ref-type="fig" rid="fig7">Figure 7B–E</xref>); a faint stripe at the extreme posterior of the embryo is also visible. By gastrulation, an anterior cap becomes more visible with continuous low expression between the anterior pole and the strong stripe 1 (<xref ref-type="fig" rid="fig7">Figure 7H–J</xref>). As gastrulation progresses, this domain becomes stronger and more uniform, whereas double-segment stripes 6 and 7 appear sequentially (<xref ref-type="fig" rid="fig7">Figure 7H–K</xref>). Stripe 8 broadens, becoming a posterior cap whose intensity increases during germ band extension (<xref ref-type="fig" rid="fig7">Figure 7J–N</xref>). The anterior of the embryo exhibits diffuse staining that expands from the anterior ventral side posteriorly, until germ band retracted embryos are faintly but uniformly stained with dark segmental stripes on top (<xref ref-type="fig" rid="fig7">Figure 7O</xref>). <italic>Nv h</italic> double-segment stripes appear cleanly, and the timing and presentation of expression of posterior stripes suggest that they may respond to waves of <italic>Nv odd</italic>. Like <italic>Nv odd</italic> and early <italic>Nv runt</italic>, <italic>Nv h</italic> does not have stripes with single-segment periodicity.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.016</object-id><label>Figure 7.</label><caption><title>Summary of <italic>Nv hairy</italic> mRNA expression.</title><p>(<bold>A</bold>) Blastoderm embryo with two double-segment periodicity stripes of <italic>Nv hairy</italic> expression. Note that stripe 2 is broader and stronger than stripe 1. (<bold>B</bold>) Blastoderm embryo showing four double-segment periodicity stripes of expression plus an anterior accumulation of <italic>Nv hairy</italic> transcripts (arrowhead). (<bold>C</bold>) Dorsal view of embryo as in (<bold>B</bold>), illustrating the dorsal anterior expression (arrowhead) that is activated in the same pattern as the anterior domain of <italic>Nv tailless</italic> (<xref ref-type="bibr" rid="bib46">Lynch et al., 2006</xref>). (<bold>D</bold>) Blastoderm embryo with strong anterior and dorsal anterior expression of <italic>Nv hairy</italic> and five pair-rule stripes. (<bold>E</bold>) Dorsal view of embryo as in (<bold>D</bold>) with increased dorsal anterior expression of <italic>Nv hairy</italic>, and the anterior spreading of expression from the anterior of double-segment pair-rule stripe 1. (<bold>F</bold>) Blastoderm embryo with expanding anterior domain (line), five double-segment ‘pair-rule’ stripes, and two additional stripes coming up. Note that the anterior domain between stripe 1 and the anterior pole is becoming more continuous in expression. (<bold>G</bold>) Dorsolateral view of embryo as in (<bold>F</bold>) highlighting the dorsal anterior expression. Stripe 2 is still wider than other stripes. Stripe 6 appears to be of single-segment periodicity. (<bold>H</bold>) Early gastrula embryo exhibiting a non-homogenous but largely continuous anterior cap of <italic>Nv hairy</italic> expression (that includes stripe 1). Four additional double-segment stripes and three single-segment stripes (two derived from stripe 6) are now evident. (<bold>I</bold>) Dorsal view of embryo slightly older than embryo in (<bold>H</bold>) showing the nearly continuous head domain, and the apparent splitting of stripe 1 within that domain. Double-segment stripes are thinning. (<bold>J</bold>) Dorsolateral view of extending germ-band embryo. Head domain is continuous (line). Stripes 1–7 have single-segment periodicity, are of non-uniform strength; stripe eight appears darker and broader. (<bold>K</bold>) Germ-band extending embryo with a continuous head domain (line) and eight discrete stripes. (<bold>L</bold>) Dorsolateral view of germ-band extending embryo. Stripe 8 is expanded into a wedge abutting the pole cells, and the anterior domain is expanding to include stripe 2. (<bold>M</bold>) Germ-band extension embryo with expanding anterior domain, that extends to include stripe 3 (arrowhead). Posterior domain is expanded. (<bold>N</bold>) Dorsolateral view of embryo as in (<bold>M</bold>) showing further expansion of posterior stripe 8 domain (line). (<bold>O</bold>) Germ-band-retracted embryo exhibiting ubiquitous staining with striated expression evident.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.016">http://dx.doi.org/10.7554/eLife.01440.016</ext-link></p></caption><graphic xlink:href="elife01440f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.017</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Phylogenetic analysis of hairy.</title><p>Phylogenetic analysis of hairy-related protein sequences was performed using Maximum likelihood analysis with 1000-fold bootstrap support, using RAxML via the CIPRES portal (<xref ref-type="bibr" rid="bib17">Copf et al., 2003</xref>; <xref ref-type="bibr" rid="bib53">Nagy et al., 1994</xref>). The best scoring maximum likelihood tree is shown with bootstrap support values indicated adjacent to branch.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.017">http://dx.doi.org/10.7554/eLife.01440.017</ext-link></p></caption><graphic xlink:href="elife01440fs008"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01440.018</object-id><label>Figure 7—figure supplement 2.</label><caption><title>Hairy protein sequence alignment.</title><p>Predicted hairy-like protein sequences from <italic>Nasonia</italic> were aligned to hairy-like proteins from <italic>Drosophila melanogaster, Anopheles gambiae, Apis mellifera</italic>, and <italic>Tribolium castaneum</italic> using ClustalW. Full-length sequences were used for the alignment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.018">http://dx.doi.org/10.7554/eLife.01440.018</ext-link></p></caption><graphic xlink:href="elife01440fs009"/></fig></fig-group></p><p>Double fluorescent in situ hybridization with <italic>Nv eve</italic> reveals that early <italic>Nv h</italic> overlaps the anterior of <italic>Nv eve</italic> double-segment stripes (<xref ref-type="fig" rid="fig6">Figure 6M–O</xref>). Later, these stripes appear thinner, and they overlap the anterior <italic>Nv eve</italic> single-segment odd-numbered stripes in each double-segment doublet. <italic>Nv h</italic> double-segment stripe 6 anticipates the position of the most anterior derivative of the <italic>Nv eve</italic> stripe 6 quartet (segmental stripes 11–14). <italic>Nv h</italic> stripe 7 (double segment), which is thin, appears within the <italic>Nv eve</italic> early broad stripe 6 domain, coinciding with <italic>Nv eve</italic> single-segment stripe 13 (<xref ref-type="fig" rid="fig6">Figure 6P–R</xref>, <xref ref-type="fig" rid="fig8">Figure 8</xref>). A more posterior stripe, <italic>Nv h</italic> 8, anticipates, albeit more broadly, the site of <italic>Nv eve</italic> single-segment stripe 15 (<xref ref-type="fig" rid="fig6">Figure 6M–O,P–R</xref>, <xref ref-type="fig" rid="fig8">Figure 8</xref>). Thus, <italic>Nv h</italic> and <italic>Nv eve</italic> are co-expressed at the anterior of <italic>eve</italic> pair-rule stripes and in the first of each pair of <italic>eve</italic> (odd-numbered) single-segment stripes, similar to the relationship described for <italic>Drosophila eve</italic> and <italic>hairy</italic> as they initiate segment polarity (<xref ref-type="bibr" rid="bib79">Warrior and Levine, 1990</xref>).<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01440.019</object-id><label>Figure 8.</label><caption><title>Summary model of pair-rule gene expression in the Nasonia embryo.</title><p>(<bold>A</bold>) Model of register of pair-rule gene expression in the early embryo. <italic>Nv eve</italic> and <italic>Nv odd</italic> stripes are totally complementary, whereas <italic>Nv runt</italic> stripes partly overlap each of these genes at their interface. <italic>Nv hairy</italic> stripes overlap <italic>Nv eve</italic> stripes toward the anterior of each double-segment periodicity stripe. Towards the posterior of the embryo, an extended domain of low-level <italic>Nv odd</italic> expression exhibits dynamic behavior over several nuclear cycles, and stripes 4/5 of <italic>Nv eve, Nv odd</italic>, and <italic>Nv runt</italic> each differentiate during this interval. Even more posteriorly, <italic>Nv eve</italic> stripe 6 lay anterior to a continuous <italic>Nv odd</italic> cap that extends to the posterior pole of the embryo. This region is set aside for segment specification and differentiation during germ-band extension. (<bold>B</bold>) Model of register of pair-rule gene expression in the germ-band extension (late) embryo. Single-segment periodicity stripes in the germ-band-extended embryo exhibit a variation upon early gene expression patterns. <italic>Nv eve</italic> single-segment stripes are interrupted by <italic>Nv runt</italic> and then <italic>Nv odd</italic> such that <italic>Nv runt</italic> stripes follow odd-numbered <italic>Nv eve</italic> stripes, and <italic>Nv odd</italic> stripes follow even-numbered <italic>Nv eve</italic> stripes. Each of 8 <italic>Nv hairy</italic> stripes overlaps odd-numbered <italic>Nv eve</italic> stripes that derive from the anterior of <italic>Nv eve</italic> pair-rule stripes. Additional expression of several of these genes in the ventral and head domains, which appears to rely on different regulatory logic, is not shown.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01440.019">http://dx.doi.org/10.7554/eLife.01440.019</ext-link></p></caption><graphic xlink:href="elife01440f008"/></fig></p><p>We knocked down <italic>Nv hairy</italic> function using two independent morpholinos directed at two different splice junctions. Both resulted in a range of cuticle defects that indicate that <italic>Nv h</italic> is required for the formation of all posterior-derived segments and blastoderm-derived segments in the thorax and anterior abdomen (<xref ref-type="fig" rid="fig4">Figure 4L–O</xref>). At the posterior, mildly affected cuticles exhibit fusion of segments A9–10 (<xref ref-type="fig" rid="fig4">Figure 4L,N</xref>), along with partial loss of alternating abdominal segments posterior to A4. In more affected cuticles, alternating segments posterior to A2 are fused (<xref ref-type="fig" rid="fig4">Figure 4L–N</xref>), resembling a pair-rule phenotype. In severely affected embryos, all segments from A4–A10 are fused with a continuous lawn of denticles that covers the posterior of a severely reduced cuticle (<xref ref-type="fig" rid="fig4">Figure 4O</xref>, yellow line). These phenotypes suggest a requirement for <italic>Nv hairy</italic> in specification of posterior segments. The late <italic>Nv h</italic> stripes 6–8 are positioned to affect the late forming segments as supported by double <italic>in situs</italic> (<xref ref-type="fig" rid="fig6">Figure 6</xref>). In spite of <italic>Nv hairy</italic> expression in the head and extreme anterior of the embryo, labral structures in <italic>Nv hairy</italic> morpholino cuticles appear to be unaffected. The expression pattern of <italic>Nv hairy</italic> is thus strikingly similar to <italic>Tc’hairy</italic> (<xref ref-type="bibr" rid="bib73">Sommer and Tautz, 1993</xref>; <xref ref-type="bibr" rid="bib2">Aranda et al., 2008</xref>), though functionally quite different, since <italic>Tc’h</italic> seems to act exclusively in patterning head segments (<xref ref-type="bibr" rid="bib16">Choe et al., 2006</xref>; <xref ref-type="bibr" rid="bib2">Aranda et al., 2008</xref>).</p><p>At the anterior, A2 is also nearly always affected, exhibiting loss of denticles and displacement or loss of the associated spiracle (<xref ref-type="fig" rid="fig4">Figure 4L</xref>, orange arrowhead). Segment T1 appears to be lost and fused to T2. Finally, more affected embryos show a loss of T3 (<xref ref-type="fig" rid="fig4">Figure 4M–O</xref>). Therefore, segments T1, T3, and A2 are missing, which resembles an anterior pair-rule phenotype.</p><p>In summary, <italic>Nv h</italic> expression is highly dynamic and proceeds in an anterior to posterior progression. It is distinct from <italic>Nv eve</italic> and does not exhibit single-segment periodicity stripes. At the end of embryogenesis, its expression becomes nearly ubiquitous (<xref ref-type="fig" rid="fig7">Figure 7O</xref>).</p><p>Taken together, these data support a model wherein ‘pair-rule’ genes have weak fly-type ‘pair-rule’ phenotypes in the anterior, and are required for the formation of a suite of posterior segments. Their interdigitated expression suggests extensive interactions during patterning of the posterior region after cellularization, although this has not yet been tested due to current experimental limitations. Our summary model of the phasing of ‘pair-rule’ stripes in the embryo is given in <xref ref-type="fig" rid="fig8">Figure 8</xref>.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Decades of study of a variety of insects have yielded a deep understanding of the genes controlling anterior–posterior patterning of the embryo. The best-characterized model species are the extreme long germ <italic>Drosophila</italic> embryo and the short-germ <italic>Tribolium</italic> embryo. Additional insects, such as <italic>Gryllus bimaculatus</italic> (<xref ref-type="bibr" rid="bib50">Mito et al., 2007</xref>)<italic>, Bombyx mori</italic> (<xref ref-type="bibr" rid="bib84">Xu et al., 1997</xref>), <italic>Oncopeltus fasciatus</italic> (<xref ref-type="bibr" rid="bib39">Liu and Kaufman, 2004a</xref>, <xref ref-type="bibr" rid="bib40">2004b</xref>, <xref ref-type="bibr" rid="bib41">2005a</xref>), <italic>Schistocerca americana</italic> (<xref ref-type="bibr" rid="bib57">Patel et al., 1992</xref>) and others, represent informative intermediates, but many of these species are on the short end of this wide spectrum, consistent with ancestral insects being short germ band. <italic>Hymenoptera</italic>, including <italic>Apis</italic> and <italic>Nasonia</italic>, have evolved a long germ embryogenesis independently from flies and therefore provide an excellent context for addressing the question of how the transition from short to long germ occurred. Other well-characterized arthropod species, such as <italic>Cupiennius salei</italic> (<xref ref-type="bibr" rid="bib19">Damen et al., 2000</xref>; <xref ref-type="bibr" rid="bib76">Stollewerk et al., 2003</xref>) and <italic>Strigamia maritima</italic> (<xref ref-type="bibr" rid="bib13">Chipman et al., 2004</xref>; <xref ref-type="bibr" rid="bib12">Chipman and Akam, 2008</xref>), provide additional models of interest for understanding more ancient evolutionary history.</p><p><italic>Nasonia</italic> has been characterized as long germ because of the presence of two morphogenetic centers and the expression patterns and RNAi phenotypes of the gap genes (<xref ref-type="bibr" rid="bib34">Ingham et al., 1985</xref>; <xref ref-type="bibr" rid="bib64">Pultz et al., 2005</xref>; <xref ref-type="bibr" rid="bib45">Lynch et al., 2006</xref>; <xref ref-type="bibr" rid="bib55">Olesnicky et al., 2006</xref>; <xref ref-type="bibr" rid="bib5">Brent et al., 2007</xref>). In this study, we describe the expression and loss of function phenotypes of <italic>Nasonia eve, hairy</italic> and <italic>odd</italic>, three genes that act as pair-rule genes in <italic>Drosophila.</italic> While the expression of <italic>Nv eve</italic> is controlled by maternal and gap gene factors that are largely similar to their <italic>Drosophila</italic> counterparts, we find critical deviations from the <italic>Drosophila</italic> long germ paradigm. <italic>Nv</italic> ‘pair-rule’ genes display similarity to both <italic>Drosophila</italic> and <italic>Tribolium,</italic> suggesting that <italic>Nasonia</italic> has features of both short and long germ band development. Unlike the long germ embryo of the honeybee, <italic>Apis</italic>, <italic>Nasonia</italic> pair-rule genes do not seem to be maternally expressed, and so far, regulation of <italic>Nv</italic> gap genes by pair-rule genes (as reported for <italic>Apis</italic> [<xref ref-type="bibr" rid="bib82">Wilson and Dearden, 2012</xref>]) has yet to be studied systematically.</p><sec id="s3-1"><title>Expression and knockdown of <italic>Nasonia eve</italic>, <italic>hairy</italic> and <italic>odd</italic></title><p>In contrast to <italic>Drosophila,</italic> whose pair-rule genes are expressed in the blastoderm in seven double-segment periodicity stripes to determine the formation of 14 segments, their orthologs in <italic>Nasonia</italic> are expressed in diverse and more intricate patterns. In no case do we observe simply eight precellularization double-segment stripes, confirming that pair ‘rule’ does not represent the regulatory dynamics of these genes across insects. We observe wave-like behavior of <italic>Nv odd</italic> stripe 4–6, which underscores that cycling control may remain from the ancestral segmentation clock. <italic>Nv runt</italic> and, to a large degree, <italic>Nv h</italic>, also exhibit a sequential progression of sharp stripe appearance that may be responsive to the waves of <italic>Nv odd</italic> (<xref ref-type="fig" rid="fig7">Figure 7</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>).</p><p>It is clear that <italic>Nv eve, Nv odd</italic>, and <italic>Nv h</italic> exhibit multiple modes of regulation during embryogenesis. In each case, their anterior stripes formed in the syncytial blastoderm have double-segment periodicity and arise in a manner that could be explained by the type of enhancer logic exemplified by <italic>Drosophila eve</italic> (<xref ref-type="bibr" rid="bib70">Small and Levine, 1991</xref>; <xref ref-type="bibr" rid="bib71">Small et al., 1992</xref>, <xref ref-type="bibr" rid="bib72">1996</xref>; <xref ref-type="bibr" rid="bib67">Schroeder et al., 2004</xref>; <xref ref-type="bibr" rid="bib68">Schroeder et al., 2011</xref>). Anterior double-segment ‘pair-rule’ stripes of <italic>Nv eve</italic> appear to be regulated by maternal and gap genes as in <italic>Drosophila,</italic> and embryos knocked down for <italic>Nv eve, Nv odd</italic> and <italic>Nv h</italic> exhibit a pair-rule phenotype in the blastoderm-derived segments, although this phenotype is most often limited. It is worth noting that the severe <italic>Nv eve</italic> anterior defects are more regional, as observed for <italic>Oncopeltus eve</italic> that behaves as a gap gene, and is attributed to the broad early domain of expression (<xref ref-type="bibr" rid="bib41">Liu and Kaufman, 2005a</xref>). Perhaps, as in <italic>Oncopeltus</italic>, <italic>Nv eve</italic> is required in combination with <italic>Nv hb</italic> or <italic>Nv gt</italic> for activation of their targets, which are in turn required for the proper formation of head and thoracic segments.</p><p>Another mode must control the formation of stripes of <italic>Nv eve, Nv odd</italic>, and <italic>Nv h</italic> that arise later, in a cellular environment, from a posterior domain (whether at the posterior pole of the embryo, as in the case of <italic>Nv odd</italic> and <italic>Nv hairy</italic>, or from a broad posterior stripe, as in the case of <italic>Nv eve</italic>). Knockdown of each of the three genes produces a severe posterior truncation of the embryo, deleting all six posterior segments, indicating that each gene is required for the formation of posterior-derived segments. This phenotype is unlike flies, and resembles more the short germ pair-rule gene circuit of <italic>Tribolium</italic> (<xref ref-type="bibr" rid="bib16">Choe et al., 2006</xref>; <xref ref-type="bibr" rid="bib15">Choe and Brown, 2009</xref>; <xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>). Together with co-expression data, their phenotypes suggest that each of these genes is required for refinement or maintenance of each other’s activity or expression (<xref ref-type="fig" rid="fig6">Figure 6</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>).</p><p>We propose a model in which interactions among ‘pair-rule’ genes dominate in patterning the long germ <italic>Nasonia</italic> embryo. Unlike flies, posterior stripes of ‘pair-rule’ genes like <italic>Nv hairy</italic> and <italic>Nv runt</italic> appear sequentially. Indeed, the gene circuit involving interactions among <italic>Tc’odd</italic>, <italic>Tc’eve</italic> and <italic>Tc’runt</italic> in each round of posterior segment formation underscores the likely ancestral nature of this network, which might have been brought under the control of the gap and maternal genes in flies, and in the anterior segments of <italic>Nasonia.</italic> The ‘waves’ of <italic>Nv odd</italic> pair-rule stripe expression that give rise to blastoderm stripes 4, 5, and 6 suggest residual activity of a segmentation clock. The presumptive domain of six posterior segments indicated by early posterior expression of <italic>Nv eve</italic> and <italic>Nv odd</italic> may be similar to the ‘growth zone’ of short germ insects.</p><p>Waves of <italic>Nv odd</italic> 4, 5, and 6, and the sequential formation of <italic>Nv runt</italic> stripes both interrupt and likely pattern the continuous <italic>Nv eve</italic> stripe 6 domain (<xref ref-type="fig" rid="fig6">Figure 6A–L</xref>; <xref ref-type="fig" rid="fig8">Figure 8</xref>). <italic>Nv h</italic> expression anticipates the final position of several late forming <italic>Nv eve</italic> stripes, and in combination with the phenotype of <italic>Nv h</italic> knockdown and co-expression data, suggests that it is required for the formation of the posterior <italic>Nv eve</italic> stripes.</p><p>Thus, <italic>Nasonia</italic> represents a variation on embryo allocation and patterning, but the contribution of ‘pair-rule’ gene function is enduring. The use of a clock-like mechanism is not incompatible with long germ embryogenesis, and rather, retaining this character might allow for sampling transitional states between the short- and long germ strategies, which likely occurred multiple times within holometabola. Further, it may be that the absence of significant posterior elongation is the transition state that tips the balance toward elaboration of anterior segmentation control mechanisms and loss of late forming segments. Our characterization of the <italic>Nasonia</italic> pair-rule genes illustrates one way that these two strategies can co-exist.</p><p>It is also of note that although <italic>hairy</italic>-related genes are the oscillating components of vertebrate segmentation clocks (<xref ref-type="bibr" rid="bib56">Palmeirim et al., 1997</xref>), it is generally <italic>odd-skipped</italic>-related genes that oscillate in arthropods (<xref ref-type="bibr" rid="bib13">Chipman et al., 2004</xref>; <xref ref-type="bibr" rid="bib12">Chipman and Akam, 2008</xref>; <xref ref-type="bibr" rid="bib26">El-Sherif et al., 2012</xref>; <xref ref-type="bibr" rid="bib66">Sarrazin et al., 2012</xref>). Notch-signaling has been described for its involvement in regulating <italic>hairy-</italic>related oscillations in the vertebrate clock (e.g., <xref ref-type="bibr" rid="bib36">Jouve et al., 2000</xref>), and it may also be involved in the context of the arthropod segmentation clock (<xref ref-type="bibr" rid="bib76">Stollewerk et al., 2003</xref>; <xref ref-type="bibr" rid="bib27">Eriksson et al., 2013</xref>; <xref ref-type="bibr" rid="bib36a">Kainz et al., 2011</xref>); in all cases, the driver of the clock is yet to be elucidated (reviewed in <xref ref-type="bibr" rid="bib60">Pourquie, 2003</xref>).</p></sec><sec id="s3-2"><title>An ancestral role for eve in specifying posterior identity may be linked to growth zone behavior in short germ insects</title><p><italic>eve</italic> has been suggested to have its most ancestral function as a specifier of posteriorness (<xref ref-type="bibr" rid="bib1">Ahringer, 1996</xref>; <xref ref-type="bibr" rid="bib7">Brown et al., 1997</xref>). Indeed, the two mammalian <italic>eve</italic> genes are located at the most ‘posterior’ end of two of the Hox clusters (<xref ref-type="bibr" rid="bib3">Bastian et al., 1992</xref>), although <italic>eve</italic> is not a part of the Hox cluster in <italic>Nasonia</italic> or <italic>Tribolium</italic> or any other insects that have been studied (<xref ref-type="bibr" rid="bib69">Shippy et al., 2008</xref>; <xref ref-type="bibr" rid="bib80">Werren et al., 2010</xref>; <xref ref-type="bibr" rid="bib52">Munoz-Torres, 2009</xref>; <xref ref-type="bibr" rid="bib77">Suen et al., 2011</xref>). Yet, even in <italic>Drosophila,</italic> where there is no apparent sequential segmentation, a delayed pair-rule stripe (stripe 8) appears early in gastrulation (<xref ref-type="bibr" rid="bib47">Macdonald et al., 1986</xref>; <xref ref-type="bibr" rid="bib29">Frasch et al., 1987</xref>; <xref ref-type="bibr" rid="bib37">Kim et al., 2000</xref>). In <italic>Schistocerca, eve</italic> is expressed in a posterior mesodermal domain and no pair-rule stripes arise from this region, indicating that <italic>eve</italic> plays a role in posteriorness and not segmentation in basal insects (<xref ref-type="bibr" rid="bib57">Patel et al., 1992</xref>). <italic>Nasonia eve</italic> sets aside stripe 6 relatively early, at about the same time as <italic>Nv odd</italic> that is expressed even more posteriorly. This feature of <italic>Nv eve</italic> in posterior segmentation is not shared by the other pair-rule genes we studied, therefore supporting the notion of an ancestral role for <italic>eve</italic> in posteriorness in <italic>Nasonia</italic>. That its expression is complementary to that of <italic>odd</italic> in both <italic>Tribolium</italic> and <italic>Nasonia</italic> in a late-differentiating posterior region may hint at how this role in posteriorness evolved into a role in posterior growth. In non-insect arthropods, there is evidence for a role for <italic>eve</italic> in both posterior identity and segmentation. The centipede <italic>Lithobius atkinsoni</italic> expresses <italic>eve</italic> in a posterior domain and between segments (<xref ref-type="bibr" rid="bib33">Hughes and Kaufman, 2002</xref>), and the crustacean <italic>Artemia franciscana</italic> exhibits growth zone <italic>eve</italic> expression that precedes expression in stripes in emerging segments (<xref ref-type="bibr" rid="bib17">Copf et al., 2003</xref>). In other basal arthropods, like spiders (<xref ref-type="bibr" rid="bib19">Damen et al., 2000</xref>) and the centipede <italic>Strigamia maritima</italic> (<xref ref-type="bibr" rid="bib12">Chipman and Akam, 2008</xref>), <italic>eve</italic> expression in stripes suggests that its ancestral role is segmental.</p></sec><sec id="s3-3"><title>Mitotic domains are coordinated with pair-rule gene expression</title><p>The broad stripe 6 domain of <italic>eve</italic> appears to be subdivided by transcription control, likely through interactions with <italic>Nv odd</italic> and <italic>Nv runt</italic> (<xref ref-type="fig" rid="fig6">Figure 6</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). Although we observed apparent mitotic domains in the early gastrula that proceed from anterior to posterior, they do not match the pattern of initial differentiation of germ band-derived segments or the splitting of anterior pair-rule stripes. Cell division patterns in mitotic domains have not been described in most insects, apart from <italic>Drosophila</italic> and the precellular blastoderm of <italic>Bombyx</italic> (order: Lepidoptera; <xref ref-type="bibr" rid="bib53">Nagy et al., 1994</xref>). In the short germ <italic>Tribolium</italic> and <italic>Oncopeltus</italic> embryos, cell divisions during gastrulation and elongation occur throughout the germ band, with no evidence for mitotic domains (<xref ref-type="bibr" rid="bib6">Brown et al., 1994</xref>; <xref ref-type="bibr" rid="bib43">Liu and Kaufman, 2009</xref>).</p><p>The relationship between <italic>Nv eve</italic> and cell division suggests coordination of cell divisions by segmentation genes, a phenomenon that has been suggested for <italic>Drosophila</italic> (<xref ref-type="bibr" rid="bib28">Foe, 1989</xref>; <xref ref-type="bibr" rid="bib23">Edgar and O’Farrell, 1989</xref>; <xref ref-type="bibr" rid="bib4">Bianchi-Frias et al., 2004</xref>). Use of coordinated mitotic domains is a strategy that seems to have evolved multiple times (e.g., in flies and wasps). We propose that the apparent coordination of mitotic domains and segmentation gene expression of both <italic>Nasonia</italic> and <italic>Drosophila</italic> development may constitute a step in the transition to long germ embryogenesis.</p></sec><sec id="s3-4"><title>Conclusion</title><p>In summary, despite obvious differences in their expression patterns, <italic>Nasonia eve</italic>, <italic>odd</italic>, and <italic>hairy</italic> function in both blastoderm- and germ band-derived segment formation. While <italic>Nasonia</italic> exhibits fly-type expression of maternal and gap genes in the precellular blastoderm, dynamic expression patterns and extensive interactions among ‘pair-rule’ genes appear to pattern a suite of late forming posterior segments. Indeed, their relative expression patterns suggest that the regulation of posterior segments may be through the type of mutual regulation described for the pair-rule gene circuit of <italic>Tribolium</italic>. This is unlike the long germ embryo of <italic>Drosophila,</italic> whose segmentation utilizes pair-rule interactions only during the late blastoderm stage. We propose that late-forming segments are set aside using remnants of ancestral control of posteriorness and the segmentation clock. Thus, <italic>Nasonia</italic> relies on a dynamic, dual mode of segmentation that has characteristics of both ancestral short germ and derived long germ embryogenesis.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Embryo collection, non-fluorescent in situ hybridization, two-color FISH, and immunohisochemistry</title><p><italic>Nasonia</italic> embryos were collected and fixed in 5% formaldehyde/1X PBS/Heptane for 28 min, affixed to double-sided tape, and hand peeled under 1X PBS +0.1% tween, as described previously (<xref ref-type="bibr" rid="bib64">Pultz et al., 2005</xref>), except that the embryos were collected from host-fed, mated females. The embryos were stored under methanol at −20°C between fixation and hybridization.</p><p>In situ hybridizations were carried out as described previously (<xref ref-type="bibr" rid="bib64">Pultz et al., 2005</xref>). Briefly, the embryos stored under methanol were gradually brought up to 1X PBT and washed three times in 1x PBS +0.1% tween-20 (PBT) before a 30-min post-fix step in 5% formaldehyde/1XPBT. The embryos were then washed three times and subjected to proteinase K treatment (final concentration of 4 μg/ml) before three PBT washes. The embryos were blocked for 1 hr in hybridization buffer before probe preparation and addition for overnight incubation at 65°C. The next day, the embryos were washed in formamide wash buffer and then 1X MABT buffer before blocking in 2% Blocking Reagent (BBR; Roche Applied Science, Germany) in 1X MABT for 1 hr, then in 10% horse serum/2% BBR/1XMABT for 2 hr. The embryos were incubated overnight with primary antibody in the second blocking solution at 4°C. Anti-DIG-AP Fab fragments (Roche Diagnostics) were used at 1:2000 for non-fluorescent in situs. On the third day, the embryos were washed in 1X MABT for ten, 20 min washes before equilibrating the embryos in AP staining buffer and developing in AP buffer with NBT/BCIP solution (Roche Diagnostics). After staining, the embryos were washed in 1× PBT three times for 5 min each before a single 25 min post-fix step in 5% formaldehyde/1XPBT. The embryos were then washed briefly and allowed to sink in 50% and then 70% glycerol/1XPBS, which were subsequently used for mounting.</p><p>For fluorescent in situs, DIG probes were detected using anti-DIG-POD Fab fragments (Roche Diagnostics) at 1:50 dilution, followed by FastRed HNPP detection system (Roche Diagnostics), according to manufacturer’s instructions. Fluorescein probes were detected using anti-Fluorescein-AP Fab fragments (Roche Diagnostics) at 1:500 dilution.</p><p>For antibody staining of mitotic cells, we used a rabbit anti-phosphorylated histone H3 serine 10 antibody (Millipore, Billerica, Massachusetts) at 1:200, and then a donkey anti-rabbit secondary conjugated to Alexa-647 (Invitrogen, Carlsbad, California) at 1:200. In combination with FastRed in situ detection using anti-DIG-AP Fab fragments (Roche Diagnostics) at 1:500, primary antibodies were added to blocking buffer together and incubated according to the in situ protocol, and secondary antibody detection was carried out after the FastRed staining was completed and following three 1X PBT washes.</p></sec><sec id="s4-2"><title>Cloning of <italic>Nasonia</italic> pair-rule gene cDNA fragments</title><p><italic>Nasonia</italic> pair-rule gene cDNAs were cloned from embryo cDNA pools generated from reverse transcription of total embryo RNA from mixed age embryos using Superscript First Strand Synthesis kit (with Superscript II; Invitrogen) according to manufacturer’s specifications. For cases in which long cDNA sequences could not be amplified with oligos designed according to automated genome annotation and prediction models, we used circular RACE to simultaneously amplify sequences 5′ and 3′ to smaller cloned cDNA fragments, as previously described (<xref ref-type="bibr" rid="bib48">McGrath, 2011</xref>).</p></sec><sec id="s4-3"><title>Phylogenetic analysis of <italic>Nasonia</italic> pair-rule gene paralogs</title><p><italic>Nasonia</italic> paralogs of fly pair-rule genes were identified by TBLASTN and aligned against predicted or experimentally validated (virtually translated) protein sequences of the same proteins from <italic>Tribolium castaneum</italic> (<italic>Tcas</italic>), <italic>Anopheles gambiae</italic> (<italic>Agam</italic>), <italic>Apis mellifera</italic> (<italic>Amel</italic>). Protein sequences were aligned using CLUSTALW (<xref ref-type="bibr" rid="bib38">Larkin et al., 2007</xref>) and rendered using Dendroscope (<xref ref-type="bibr" rid="bib33a">Huson et al., 2007</xref>). Evolutionary relationships were inferred using a maximum likelihood analysis with 1000-fold bootstrap support, via RaXML hosted online at CIPRES science gateway (<ext-link ext-link-type="uri" xlink:href="http://www.phylo.org/index.php/portal/">http://www.phylo.org/index.php/portal/</ext-link>) (<xref ref-type="bibr" rid="bib74">Stamatakis et al., 2008</xref>; <xref ref-type="bibr" rid="bib75">Stamatakis, 2006</xref>; <xref ref-type="bibr" rid="bib49">Miller et al., 2010</xref>).</p></sec><sec id="s4-4"><title>Morpholino injection and larval cuticle preparations</title><p>Antisense morpholinos targeting splice junctions or transcription initiation sites were designed and ordered from GeneTools, LLC (<ext-link ext-link-type="uri" xlink:href="http://www.gene-tools.com/">www.gene-tools.com</ext-link>, Philomath, Oregon). Lyophilized morpholinos were resuspended in sterile nuclease-free water to a final concentration of 5 mM. For <italic>Nv odd</italic> splice block morpholino, which yielded a high percentage of dead embryos with no cuticle, injections were also carried out at 1 mM, 0.5 mM, and 0.05 mM dilutions. <italic>Nasonia</italic> embryos were collected for 35 min at 28°C and dehydrated for 30 min before injection with morpholinos (approximate volume injected = 0.001 μl per embryo). The embryos were allowed to develop on the injection membrane at 28°C on a 1X PBS/1% agarose plate for approximately 30 hr, to ensure complete development. Unhatched larvae were peeled and transferred to a slide for cuticle preparations in Lacto–Hoyer’s media.</p><p>Morpholino sequences used are as follows:<list list-type="simple"><list-item><p>Eve translation block 5′ CAAAGCTCCTCTGGAATCCTTGCAT 3′</p></list-item><list-item><p>Eve E2I2 splice block: 5′ AAACGATAGTTACCTTGATGGTCGA 3′</p></list-item><list-item><p>Hairy E2I2 splice block: 5′ CTGAATCTGTCAAGATACTTACGTC 3′</p></list-item><list-item><p>Hairy E1I1 splice block: 5′ GAGCAAGTCGAGATACTAACCCGTC</p></list-item><list-item><p>Odd splice block: 5′ AGAGAGTGTACTAAC TTGTGGTCCC 3′</p></list-item><list-item><p>Odd translation block: 5′ GCTCCATCGCAAGCTGGGTAAACGT 3′</p></list-item></list></p></sec><sec id="s4-5"><title>cDNA sequence accession numbers</title><p><list list-type="simple"><list-item><p><italic>Nv odd</italic> cDNA GenBank Accession # KC142194</p></list-item><list-item><p><italic>Nv eve</italic> cDNA, isoform 1 GenBank Accession# KC168090</p></list-item><list-item><p><italic>Nv eve</italic> cDNA, isoform 2 GenBank Accession# KC168091</p></list-item><list-item><p><italic>Nv eve</italic> cDNA, isoform 3 GenBank Accession# KC168092</p></list-item><list-item><p><italic>Nv hairy</italic> cDNA GenBank Accession # KC190514</p></list-item></list></p><p>Accession numbers for sequences used in sequence alignments and trees:<list list-type="simple"><list-item><p>NvitH1: NP_001267498 XP_001601817 (Nvit Hairy)</p></list-item><list-item><p>NvitH2: Uniprot K7J0X7_NASVI (hairy-like/Nvit Dpn)</p></list-item><list-item><p>NvitH3: XP_001601600.2 GI:345484850 (hairy-like/HES like?)</p></list-item><list-item><p>DmelH: NP_523977.2 GI:24661088 (Dmel hairy)</p></list-item><list-item><p>DmelDpn: NP_476923.1 GI:17136808 (Dmel deadpan)</p></list-item><list-item><p>AmelH1: XP_001120814.2 GI:328784100 (hypothetical protein)</p></list-item><list-item><p>AmelH2: XP_393948.3 GI:110762302 (hairy-like)</p></list-item><list-item><p>AgambH1: XP_316733.3 GI:158296333 (corrected seq; Agam hairy)</p></list-item><list-item><p>AgambH2: XP_320206.4 GI:158300226</p></list-item><list-item><p>TcasH1: NP_001107765.1 GI:166796106 (Tcas Hairy)</p></list-item><list-item><p>TcasH2: XP_967694.1 GI:91092620 (Tcas similar to GA21268-PA)</p></list-item><list-item><p>TcasH3: XP_975187.1 GI:91083981 (Tcas HES1)</p></list-item><list-item><p>DmelOddsk: NP_722922.1 GI:24581484 (Dmel Odd skipped)</p></list-item><list-item><p>DmelSobow: NP_476882.1 GI:17136746 (Dmel Sister of odd and bowl)</p></list-item><list-item><p>DmelBowl: NP_476883.1 GI:17136748 (Dmel Brother of odd with entrails limited)</p></list-item><list-item><p>NvitOddbowlA: XP_001603713.1 GI:156545195 (predicted protein)</p></list-item><list-item><p>NvitOddbowlB: XP_001603827.2 GI:345481739(Nv bowel-like)</p></list-item><list-item><p>NvitOddbowlC: XP_001603660.1 GI:156545193(Nv odd-skipped like)</p></list-item><list-item><p>AmelOddbowlA: XP_001120949.1 GI:110762343(Amel odd-skipped like)</p></list-item><list-item><p>AmelOddbowlB: XP_393879.3 GI:110762378(Amel bowel-like)</p></list-item><list-item><p>AmelOddbowlC: XP_001120905.1 GI:110762341</p></list-item><list-item><p>TcasBowl: XP_972138.2 GI:189240088(Tcas bowl-like)</p></list-item><list-item><p>TcasOdd: XP_972086.2 GI:189240086 (Tcas odd-skipped)</p></list-item><list-item><p>TcasSob: XP_972035.1 GI:91088523(Tcas: predicted sister of odd and bowl)</p></list-item><list-item><p>Agam7972_PA: XP_306979.3 GI:118776890</p></list-item><list-item><p>Agam7973_PA: XP_317495.3 GI:118789549</p></list-item><list-item><p>Agam8222: XP_555242.1 GI:57914799</p></list-item></list></p><p>For <xref ref-type="fig" rid="fig4">Figure 4</xref>, the phenotypic classes are approximately as follows:<list list-type="simple"><list-item><p>Eve: B 20.4% C 24% D 30.1% E 25%.</p></list-item><list-item><p>Odd: G 27.7% H 22.3% I 29.2% J 20.8%.</p></list-item><list-item><p>Hairy: L 13.4% M 25.0% N 36.6% O 25.0%.</p></list-item></list></p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The authors would like to acknowledge Karin Kiontke and David Fitch for help with phylogenetic analyses, Jeremy Lynch for technical advice about multiple FISH, and Bruce Edgar and Pat O’Farrell for thoughtful comments and helpful advice on cell cycle data. Filipe Pinto Teixeira Sousa and Claire Bertet provided invaluable expertise and assistance with confocal imaging, and Jeremy Lynch and David Loehlin provided helpful comments on the manuscript. Terry Blackman and Cleo Tsanis provided immeasurable support, carrying out all of the injections described in the paper. The authors would also like to thank Bob Johnston, Leatt Gilboa, Zhenqing Chen, Michael Perry and Brent Wells for support and discussion during the course of this project. MIR would like to dedicate this paper in loving memory of Allen Rosenberg (1931-2013).</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>MIR, 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 fn-type="con" id="con2"><p>AEB, 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 fn-type="con" id="con3"><p>FP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>CD, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><sec sec-type="datasets"><title>Major datasets</title><p>The following previously published datasets were used:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><year>2013</year><x>, </x><source>UniprotKB data set for Nasonia vitripennis</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/?query=author:%22EnsemblMetazoa%22">http://www.uniprot.org/uniprot/?query=author:%22EnsemblMetazoa%22</ext-link><x>, </x><comment>Publicly available at UniProt (<ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/">http://www.uniprot.org/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Werren</surname><given-names>JH</given-names></name>, <etal/>, <year>2010</year><x>, </x><source>Nasonia vitripennis Official Gene set</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.hymenopteragenome.org/nasonia/?q=sequencing_and_analysis_consortium_datasets">http://www.hymenopteragenome.org/nasonia/?q=sequencing_and_analysis_consortium_datasets</ext-link><x>, </x><comment>Publicly available at NasoniaBase (<ext-link ext-link-type="uri" xlink:href="http://www.hymenopteragenome.org/nasonia/">http://www.hymenopteragenome.org/nasonia/</ext-link>).</comment></related-object></p></sec></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group 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pub-id-type="doi">10.7554/eLife.01440.020</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pan</surname><given-names>Duojia</given-names></name><role>Reviewing editor</role><aff><institution>HHMI, Johns Hopkins University</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). 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://elife.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 “Dual mode of embryonic development is highlighted by expression and function of <italic>Nasonia</italic> pair-rule genes” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 2 reviewers, one of whom, Duojia Pan, is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewer 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>Rosenberg et al. describe the expression patterns and phenotypes that result from knocking down several pair-rule genes in the wasp <italic>Nasonia</italic>. Previous studies, focused mostly on maternal and gap genes, had suggested that <italic>Nasonia</italic> uses a long-germ mode of embryogenesis, which evolved independently of long-germ development in flies. The results presented here lead the authors to re-evaluate that premise: pair-rule expression and phenotypes in <italic>Nasonia</italic> suggest both a 'long-germ' mode of action in anterior segments (patterning by subdivision in a syncytial blastoderm) and a 'short-germ' mode in posterior segments (sequential patterning in a cellularized environment, coupled with posterior elongation). The data are of excellent quality and largely support this interesting conclusion.</p><p>The reviewers suggested several major points for improvement:</p><p>1) The paper contains a number of imprecise statements that should be corrected by careful editing of the manuscript. On several occasions the authors refer to cross-regulatory interactions among the pair-rule genes, based entirely on expression patterns (e.g., several points in the Discussion section). I think that is a weak basis for proposing such interactions. I recommend testing these hypotheses by combining morpholino treatments with the relevant in situs. Alternatively, the authors should make it clear that the proposed interactions are hypothetical. The remaining suggestions are highlighted in the minor comments below.</p><p>2) The reviewers recommend separating, where possible, the description of data from the interpretations and the discussion that derives from it. Currently, much of the discussion is scattered within the Results section; in the Discussion the authors reiterate many of the same points.</p><p>Minor comments:</p><p>1) Introduction, 2<sup>nd</sup> paragraph: I am not aware of evidence suggesting that <italic>odd</italic> controls the oscillations, rather than being a read-out?</p><p>2) Introduction, 2<sup>nd</sup> paragraph: Whether the common ancestors of bilaterians was segmented is still controversial.</p><p>3) When describing the cuticular phenotypes induced by morpholinos (<xref ref-type="fig" rid="fig4">Figure 4</xref> and Results section), it is not always clear how the authors identify which of the segments are missing or affected? Are there distinctive cuticular features that identify each segment? These should be described.</p><p>4) Section entitled ‘<italic>Nasonia runt</italic> expression’: “Altogether, our data support a model in which <italic>Nv eve</italic> segmental stripes are established through the complementary action of <italic>Nv odd</italic> for odd numbered segmental stripes and <italic>Nv runt</italic> for even-numbered segmental stripes, as summarized in <xref ref-type="fig" rid="fig8">Figure 8</xref>.”</p><p>It is not clear how the data presented justify this statement. The authors could combine morpholino treatments with <italic>eve</italic> stainings to test that hypothesis.</p><p>5) Section entitled ‘Expression and knockdown of <italic>Nasonia eve</italic>, <italic>hairy</italic> and <italic>odd</italic>’, last paragraph: It is difficult to understand what these sentences mean. How is a clock-like mechanism compatible with long-germ embryogenesis?</p><p>6) Section entitled ‘An ancestral role for eve in specifying posterior identity may be linked to growth zone behavior in short germ insects’: “In <italic>Schistocerca</italic>, <italic>eve</italic> is expressed in a posterior mesodermal domain and pair-rule stripes arise from this region, indicating that eve plays a role in posteriorness and not segmentation in basal insects.”</p><p><italic>Eve</italic> is expressed in segmental patterns in spiders, myriapods and crustaceans, suggesting that the data from <italic>Schistocerca</italic> are not representative of the ancestral state.</p><p>7) 'Segmental' and 'pair-rule' stripes could be renamed stripes with single- and double-segment periodicity, for clarity, especially since pair-rule genes are sometimes expressed in stripes that have single-segment periodicity.</p><p>8) The authors point out the lack of 'segmental' stripes of odd (meaning stripes with single-segment periodicity). Have they checked whether one of the other paralogues of odd is expressed in the 'missing' stripes?</p><p>9) It would be useful if the authors could indicate the frequency of each phenotypic class in <xref ref-type="fig" rid="fig2 fig4">Figures 2 and 4</xref>.</p><p>10) It would be useful to know the identity of sequences (conventional gene names and, ideally, accession numbers) in the Figures showing sequence alignments and trees. For example, do the hairy-related genes included in the analysis include E(spl) orthologues?</p><p>11) Are the phylogenetic trees based on the entire aligned sequence, or on specific sequence blocks (domains) that could be aligned reliably?</p><p>12) Given the interest in comparing the arthropod and vertebrate segmentation clocks, the authors could comment on the absence of <italic>hairy</italic> oscillations in the Discussion. Have they looked at the expression of E(spl) orthologues?</p><p>13) First paragraph of Discussion: The authors reiterate the view that <italic>Nasonia</italic> develops using a long-germ mode, when in fact their paper argues that this is not the case (next paragraph). It would be interesting if the authors could step back and discuss in more general terms what their data suggests in terms of short- and long-germ mechanisms (see reviews by Davis and Patel, Ann Rev Entomol 2002, and Peel and Akam, Curr Biol 2003).</p><p>14) Section entitled ‘An ancestral role for eve in specifying posterior identity may be linked to growth zone behavior in short germ insects’: The concept of “posteriorness” is vague and should be explained.</p><p>15) The formation of posterior segments seems to be accompanied by a mild elongation the posterior part of embryo. This is another hallmark of short-germ development, which the authors could discuss.</p><p>16) Wilson and Dearden described the expression patterns and phenotypes for some of the same pair-rule genes in another hymenopteran, the honeybee. In the Discussion, it would be useful if the authors briefly compared the results from the honeybee with those from <italic>Nasonia</italic>.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01440.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The paper contains a number of imprecise statements that should be corrected by careful editing of the manuscript. On several occasions the authors refer to cross-regulatory interactions among the pair-rule genes, based entirely on expression patterns (e.g., several points in the Discussion section). I think that is a weak basis for proposing such interactions. I recommend testing these hypotheses by combining morpholino treatments with the relevant in situs. Alternatively, the authors should make it clear that the proposed interactions are hypothetical. The remaining suggestions are highlighted in the minor comments below</italic>.</p><p>We agree that the best experiments would be morpholino knockdown of each gene followed by in situ for each of the others, to really delineate the cross interactions that we propose. However, these experiments faced insurmountable technical limitations, and it was not possible to perform these studies. We have now made it more clear in the text that the proposed interactions represent a model that is hypothetical and based only on the suggestive expression patterns that we describe.</p><p><italic>2) The reviewers recommend separating, where possible, the description of data from the interpretations and the discussion that derives from it. Currently, much of the discussion is scattered within the Results section; in the Discussion the authors reiterate many of the same points</italic>.</p><p>We have removed discussion from the Results section where possible (although in several places, we found it more appropriate to comment briefly on a result that will not be part of a general discussion), and have tried to ensure that there is no redundancy between the Results and Discussion. The Discussion is now shorter and smoother.</p><p><italic>Minor comments</italic>:</p><p><italic>1) Introduction, 2nd paragraph: I am not aware of evidence suggesting that </italic>odd<italic> controls the oscillations, rather than being a read-out</italic>?</p><p>We agree that the wording of our description of <italic>odd-skipped</italic> oscillations in <italic>Tribolium</italic> was not clear. This has been corrected and we now indicate that the expression of <italic>odd</italic> is indeed oscillating, but that the control of these oscillations is still not understood.</p><p><italic>2) Introduction, 2nd paragraph: Whether the common ancestors of bilaterians was segmented is still controversial</italic>.</p><p>We are now more cautious and have added a reference to an excellent review discussing the competing models about the origin of segmentation, since it is difficult to concisely present these models in this context of the manuscript.</p><p><italic>3) When describing the cuticular phenotypes induced by morpholinos (</italic><xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref> <italic>and Results section), it is not always clear how the authors identify which of the segments are missing or affected? Are there distinctive cuticular features that identify each segment? These should be described</italic>.</p><p>There are significant landmarks on the <italic>Nasonia</italic> cuticle that give clear indications of anterior segment identity. Spiracles are easy to detect on T2, A1, A2 and A3 and their position and order on critical segments allow most of the time their identification. Our interpretation of the phenotypes is based on the phenotypic series that we obtain through morpholino knockdown; we can often observe the reduction of structures or segments stepwise in this fashion. However, there are sometimes ambiguities. We now describe these landmarks in the text.</p><p><italic>4) Section entitled ‘</italic>Nasonia runt<italic> expression’: “Altogether, our data support a model in which </italic>Nv eve<italic> segmental stripes are established through the complementary action of </italic>Nv odd<italic> for odd numbered segmental stripes and </italic>Nv runt<italic> for even-numbered segmental stripes, as summarized in</italic> <xref ref-type="fig" rid="fig8"><italic>Figure 8</italic></xref><italic>.</italic>”</p><p><italic>It is not clear how the data presented justify this statement. The authors could combine morpholino treatments with eve stainings to test that hypothesis</italic>.</p><p>We are unable to carry out this experiment with the very small number of injected embryos that are both non-uniformly developmentally delayed by the injection and also cannot be reliably recovered for subsequent staining without losing their structure. We have added a qualifying statement to indicate that this model is still theoretical.</p><p><italic>5) Section entitled ‘Expression and knockdown of</italic> Nasonia eve, hairy <italic>and</italic> odd<italic>’, last paragraph: It is difficult to understand what these sentences mean. How is a clock-like mechanism compatible with long-germ embryogenesis</italic>?</p><p>It is difficult to imagine a clock-like mechanism as co-existing with an extreme long-germ insect, like <italic>Drosophila</italic>, where there is very little (or no) material that remains to be patterned/segmented after gastrulation. However, only <italic>Drosophila</italic> fits with a very restrictive definition of long germ.</p><p>Insect embryogenesis comprises a spectrum of short-germ to long-germ, and, although insects like <italic>Nasonia</italic> and <italic>Apis</italic> can be called long germ, they are less completely so than <italic>Drosophila</italic>, indicating that these two mechanisms do appear to co-exist, which is the major conclusion of our paper. It seems reasonable to imagine that evolution sampled more derived, long-germ embryogenesis types (with more of the germ segmented before cellularization) multiple times, while still relying on the ancestral mechanisms of segment patterning to deal with the remaining late-forming segments, even as the number of remaining segments varied. These two mechanisms would have had to co-exist at some point in the transition from the most extreme short germ embryo to the most extreme long-germ embryo. Our interpretation is that embryos like <italic>Nasonia</italic> represent a snapshot of that transition.</p><p><italic>6) Section entitled ‘An ancestral role for eve in specifying posterior identity may be linked to growth zone behavior in short germ insects’: “In</italic> Schistocerca<italic>, eve is expressed in a posterior mesodermal domain and pair-rule stripes arise from this region, indicating that eve plays a role in posteriorness and not segmentation in basal insects.</italic>”</p><p>Eve <italic>is expressed in segmental patterns in spiders, myriapods and crustaceans, suggesting that the data from</italic> Schistocerca <italic>are not representative of the ancestral state.</italic></p><p>A discussion of this point has been added to the text. While it is certainly true that several other arthropods express <italic>eve</italic> in segmental stripes, it is also true that other organisms, including vertebrates, have a posterior domain of <italic>eve</italic> expression like <italic>Nasonia</italic>. We discuss both of these points now in the text and leave open any conclusion about which is the ancestral state.</p><p><italic>7) 'Segmental' and 'pair-rule' stripes could be renamed stripes with single- and double-segment periodicity, for clarity, especially since pair-rule genes are sometimes expressed in stripes that have single-segment periodicity</italic>.</p><p>We had struggled with this and clearly the reviewers also noted this. We have changed the name of the stripes to double-segment and single-segment throughout the descriptions of each gene.</p><p><italic>8) The authors point out the lack of 'segmental' stripes of odd (meaning stripes with single-segment periodicity). Have they checked whether one of the other paralogues of odd is expressed in the 'missing' stripes</italic>?</p><p>We attempted to amplify and clone each of the three <italic>odd</italic> paralogs from <italic>Nasonia</italic> cDNA to look at this, using cDNA from staged animals (embryo, larva, adult). We were able to clone only the one described in the paper from embryo cDNA and one other (“<italic>oddbowl A”</italic> in the tree) from adult cDNA. The third was either expressed at levels too low to efficiently amplified, or may not be expressed. The expression of <italic>oddbowl</italic> A was not tested systematically in embryos, though a small-scale experiment revealed no embryonic expression.</p><p><italic>9) It would be useful if the authors could indicate the frequency of each phenotypic class in</italic> <xref ref-type="fig" rid="fig2 fig4"><italic>Figures 2 and 4</italic></xref>.</p><p><xref ref-type="fig" rid="fig2">Figure 2</xref> does not show phenotypic classes but representative stainings of embryos at different stages of development. The pRNAi experiments with gap genes have been extensively described in several previous publications (Pultz et al., 1999, 2005; Olesnicky et al., 2006, Brent et al., 2007).</p><p>We now describe the frequency of the different phenotypic classes presented in <xref ref-type="fig" rid="fig4">Figure 4</xref> obtained with morpholinos, which are approximately as follows:<table-wrap id="tbl1" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Eve:</th><th>B 20.4%</th><th>C 24%</th><th>D 30.1%</th><th>E 25%</th></tr></thead><tbody><tr><td>Odd:</td><td>G 27.7%</td><td>H 22.3%</td><td>I 29.2%</td><td>J 20.8%</td></tr><tr><td>Hairy:</td><td>L 13.4%</td><td>M 25.0%</td><td>N 36.6%</td><td>O 25.0%</td></tr></tbody></table></table-wrap></p><p>These values also now appear in the Materials and methods section of the paper.</p><p><italic>10) It would be useful to know the identity of sequences (conventional gene names and, ideally, accession numbers) in the Figures showing sequence alignments and trees. For example, do the hairy-related genes included in the analysis include E(spl) orthologues</italic>?</p><p>The hairy-related genes include one possible E(spl) ortholog. The accession numbers for the sequences used in the alignments and trees are now given in the Materials and methods. Sequences in the <italic>hairy</italic> group have been updated, including longer sequences that have been released, in the database since the original submission, and their accession numbers are provided. The resulting alignment and tree now replace the original figures – the assignment of <italic>Nvit H1</italic> as the true <italic>Nasonia hairy</italic> is unaffected.</p><p><italic>11) Are the phylogenetic trees based on the entire aligned sequence, or on specific sequence blocks (domains) that could be aligned reliably</italic>?</p><p>The phylogenetic trees were based on the entire aligned sequence; this is now mentioned in the figure legend.</p><p><italic>12) Given the interest in comparing the arthropod and vertebrate segmentation clocks, the authors could comment on the absence of </italic>hairy<italic> oscillations in the Discussion. Have they looked at the expression of E(spl) orthologues</italic>?</p><p>We have not looked at the expression of E(spl) orthologues in <italic>Nasonia.</italic> We have added a brief comparison of arthropod and vertebrate clocks in the Discussion.</p><p><italic>13) First paragraph of Discussion: The authors reiterate the view that Nasonia develops using a long-germ mode, when in fact their paper argues that this is not the case (next paragraph). It would be interesting if the authors could step back and discuss in more general terms what their data suggests in terms of short- and long-germ mechanisms (see reviews by Davis and Patel, Ann Rev Entomol 2002, and Peel and Akam, Curr Biol 2003)</italic>.</p><p>As discussed above, insects like <italic>Nasonia</italic> and <italic>Apis</italic> whose embryogenesis comprises a spectrum of short-germ to long-germ can be called long germ, though they are less completely so than <italic>Drosophila (</italic>which appears to be the only species that fulfill the strict definition of long germ). We agree with the reviewers that our work demonstrates that, unlike what we previously believed, <italic>Nasonia</italic> is not a strict long germ, indicating that these two mechanisms do appear to co-exist, which is the major conclusion of our paper. It seems reasonable to imagine that evolution sampled more derived, long-germ embryogenesis types (with more of the germ segmented before cellularization) multiple times, while still relying on the ancestral mechanisms of segment patterning to deal with the remaining late-forming segments, even as the number of remaining segments varied. These two mechanisms would have had to co-exist at some point in the transition from the most extreme short germ embryo to the most extreme long-germ embryo. Our interpretation is that embryos like <italic>Nasonia</italic> represent a snapshot of that transition.</p><p><italic>14) Section entitled ‘An ancestral role for eve in specifying posterior identity may be linked to growth zone behavior in short germ insects’: The concept of “posteriorness” is vague and should be explained</italic>.</p><p>This concept, though vague, has been raised previously in the literature (see e.g., Ahringer J, <italic>Genes Dev</italic> 1996; Brown SJ et al., <italic>Mech Dev</italic> 1997). The significance of the posterior expression domains of <italic>eve</italic> in <italic>Nasonia</italic> and other arthropods is still not entirely clear, but it remains a possibility that it is in some way distinct from its expression in stripes and function in segmentation. The fact that its expression delineates a posterior growth zone in animals whose posterior segmentation occurs post-embryonically (e.g., Platynereis-de Rosa et al., <italic>Evolution and Development</italic> 2005) suggests that its ability to delineate a region of growth or patterning can be achieved independently of its striped expression and function (and that it may be an ancient feature). We wanted only to highlight that feature and with it, the possibility that it provides some additional, more general identity/information in the embryo posterior, though this explanation and characterization are still vague.</p><p><italic>15) The formation of posterior segments seems to be accompanied by a mild elongation the posterior part of embryo. This is another hallmark of short-germ development, which the authors could discuss</italic>.</p><p>We didn’t emphasize the mild elongation of the posterior part of the embryo, since compared to the significant posterior growth in more short-germ insects, this elongation is modest in <italic>Nasonia</italic>. It is also difficult to distinguish what may be modest elongation in a short-germ sense vs simply the elongated appearance of a germband elongated (and retracted) embryo. We don’t feel that the data are sufficient to support further discussion.</p><p><italic>16) Wilson and Dearden described the expression patterns and phenotypes for some of the same pair-rule genes in another hymenopteran, the honeybee. In the Discussion, it would be useful if the authors briefly compared the results from the honeybee with those from</italic> Nasonia.</p><p>An additional comparison of <italic>Nasonia</italic> pair rule genes to those of <italic>Apis</italic> has been added in the second paragraph of the Discussion.</p></body></sub-article></article> |