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.

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Shifts in embryonic oxygen levels cue heterochrony in limb initiation [scRNA-seq]

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