Project description:The evolutionary origin of powered flight was fundamental to the establishment and radiation of the avian clade, and it remains a salient feature of modern birds. However, flight has been lost multiple times throughout the evolution of the avian lineage. Ratites (flightless palaeognaths, including the emu, ostrich and other well-known groups) are perhaps the most notable flightless birds, and their convergent losses of flight often coincide with adaptations to a cursorial lifestyle, including robust legs, digit loss, and reduced wings. Although there is a wealth of comparative anatomical knowledge for several ratites, the underlying genetic mechanisms producing these changes remain debated. Here we use a multidisciplinary approach employing embryological, genetic, and genomic techniques to interrogate the mechanisms underlying the delay in forelimb development contributing to the diminution of the forelimb in emu embryos. We show that the epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) and muscle precursor migration, the initiating events of limb formation, occur at equivalent stages in the emu and chick. However, the early emu forelimb fails to proliferate at HH18. The unique emu forelimb expression of Nkx2.5, previously associated with diminished wing development, does not initiate until after this stage, concomitant with migration of myoblasts into the limb bud, and hence would not appear to be the proximal cause of limb reduction in this species. In contrast, RNA-sequencing of HH18 limb tissues reveals significantly lower Fgf10 expression in the emu forelimb. Artificially increasing mesenchymal Fgf10 expression to the nascent emu wing induces ectodermal Fgf8 expression and results in a proliferative limb bud. Analyzing open chromatin reveals differentially active regulatory elements near Fgf10 and Sall-1 in the emu wing compared to emu hindlimb and both chicken limbs. Additionally, we show that the Sall-1 enhancer activity is dependent on an Ets transcription factor-binding site likely mediated by Fgf-signaling. Taken together, our results support a model where regulatory changes result in lower expression of Fgf10 and a concomitant failure to induce the genes required for limb proliferation in the early emu wing bud at HH18.

Project description:The analysis of differentially expressed genes is a powerful approach to elucidate the genetic mechanisms underlying the morphological and evolutionary diversity among serially homologous structures, both within the same organism (e.g., hand vs. foot) and between different species (e.g., hand vs. wing). In the developing embryo, limb-specific expression of Pitx1, Tbx4, and Tbx5 regulates the determination of limb identity. However, numerous lines of evidence, including the fact that these three genes encode transcription factors, indicate that additional genes are involved in the Pitx1-Tbx hierarchy. To examine the molecular distinctions coded for by these factors, and to identify novel genes involved in the determination of limb identity, we have used Serial Analysis of Gene Expression (SAGE) to generate comprehensive gene expression profiles from intact, developing mouse forelimbs and hindlimbs. To minimize the extraction of erroneous SAGE tags from low-quality sequence data, we used a new algorithm to extract tags from -analyzed sequence data and obtained 68,406 and 68,450 SAGE tags from forelimb and hindlimb SAGE libraries, respectively. We also developed an improved method for determining the identity of SAGE tags that increases the specificity of and provides additional information about the confidence of the tag-UniGene cluster match. The most differentially expressed gene between our SAGE libraries was Pitx1. The differential expression of Tbx4, Tbx5, and several limb-specific Hox genes was also detected; however, their abundances in the SAGE libraries were low. Because numerous other tags were differentially expressed at this low level, we performed a 'virtual' subtraction with 362,344 tags from six additional nonlimb SAGE libraries to further refine this set of candidate genes. This subtraction reduced the number of candidate genes by 74%, yet preserved the previously identified regulators of limb identity. This study presents the gene expression complexity of the developing limb and identifies candidate genes involved in the regulation of limb identity. We propose that our computational tools and the overall strategy used here are broadly applicable to other SAGE-based studies in a variety of organisms. Keywords: other

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Developmental and transcriptional programs that define the initiation of the forelimb II

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