This repository contains scripts used in the following Ph.D. disseration:
Limbs, Genitals, and Gene Regulatory Networks: Identifying Conserved Targets of TBX5 in Embryonic Forelimbs and Genitalia - Link
Abstract
Although the morphology of the amniote limb and phallus differ dramatically, these appendage types express a similar suite of transcription factors and signaling molecules during development. This observation led to the hypothesis that the amniote phallus may have evolved, in part, through co-option of components of an ancient appendage gene regulatory network. Consistent with this, previous work from our lab has shown that many enhancers active in developing limbs are also active in the genital tubercle (GT). However, it remains unknown whether transcription factors expressed in the limbs and phallus interact with the same enhancers to regulate similar suites of target genes. In this dissertation, I address this question by investigating the regulatory targets of the TBX5 transcription factor. TBX5 plays a critical role in the growth and development of the vertebrate forelimb and is also known to be expressed in the developing genitalia of several amniote species. Using TBX5 ChIP-seq in mouse embryonic forelimbs and GTs, I have identified thousands of binding sites in each of these appendage types. Approximately 32% of forelimb peaks are shared with the GT and are significantly enriched near genes involved in limb development. Thus, despite the high overlap of active enhancers in embryonic limbs and genitalia, there are differences in the set of TBX5-bound cis-regulatory targets in these tissues. To investigate the degree to which TBX5 binding events are conserved in amniote appendages, I also performed parallel TBX5 ChIP-seq experiments in embryonic appendages of Anolis lizards, turtles, and alligators. Furthermore, I conditionally knocked out the Tbx5 gene during the early development of mouse forelimbs and genitalia and used RNA-seq to uncover genes that exhibit TBX5-dependent expression. Using an integrative ChIP-seq and RNA-seq approach, I have identified putative direct target genes regulated by TBX5 in embryonic appendages. Overall, the large genomic and transcriptomic datasets I have generated: 1) enhance our understanding of how TBX5 governs appendage development and 2) serve as valuable resources to form testable hypotheses of the mechanisms underlying the growth of these structures.
by Aaron Alcala
Genomic and transcriptomic data for this manuscript will be uploaded to the Gene Expression Omnibus database and updated accession numbers will be posted here.
For a list of all tools used in this manuscript, click here.
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If you are a Windows user (like me) and need to run Linux within a virtual machine to perform analyses, check out this guide or this one. I setup a shared folder (to transfer files between my host Windows PC and Virtual Machine Linux) using this guide.
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After generation of figures, vector graphics were edited in Adobe Illustrator and assembled for publication in Adobe InDesign. Other photo edits were made in either Adobe Photoshop or Lightroom.
- Aligning reads from RNA-seq experiment using HISAT2
- RNA-seq analysis pipeline to detect differentially expressed genes between Tbx5 cKO mutant forelimbs and controls
- Using bedtools to find peak coordinate intersections between ChIP-seq datasets
- Performing motif analysis of ChIP-seq peaks using HOMER
- Genome browser tracks of ChIP-seq peaks of interest (and the tracks file used). Mouse embryo photo is from the VISTA Enhancer Browser. Example:
- Volcano plots of differentially expressed genes found via RNA-seq - ggVolcanoR. Example:
- Gene-set enrichment analysis of differentially expressed genes from RNA-seq and generation of lollipop plots using ShinyGO. Example:
- Assign biological meaning to a set of ChIP-seq binding sites by analyzing the annotations of nearby genes using GREAT (Genomic Regions Enrichment of Annotations Tool). Example:
- Creating venn diagrams using eulerr. Example showing overlapping binding regions of TBX5, HOXA13, and HOXD13 in mouse forelimbs:
