Nkx2.5 triggers vascular remodeling in chicken but not in emu wing mesoderm

Nkx2.5 is a key NK-2 homeobox transcription factor with well-established roles in cardiogenesis and the development of other splanchnopleure-derived structures in vertebrates. Unexpectedly, Nkx2.5 is also expressed in the developing wings of emus, a flightless birds with vestigial wings, and its misexpression in chicken wing fields was reported to cause limb reduction. To further investigate the cellular effects of Nkx2.5 in the limb field, we overexpressed Nkx2.5 in the somatic layer of the lateral plate mesoderm at the wing level of chicken embryos via localized electroporation. This induced ectopic expression of the hemangiogenic gene Scl, accompanied by disorganization of the somatopleure and changes in the ventral vasculature, suggesting a transient activation of hemangioblast-like programs in this tissue. In contrast, analysis of single-cell RNA sequencing datasets from emu embryos revealed that Nkx2.5 -positive cells were predominantly found within the muscle clusters, including a subset of co-expressing lateral mesoderm markers such as Hand2 and Prrx1 . However, these cells did not show a hemangiogenic transcriptional profile, and expression of Scl was not detected. These findings suggest that although localized Nkx2.5 expression can promote vascular changes under experimental conditions in chicken, it does not induce comparable effects during normal emu limb development. Our results underscore the context-dependent effect of Nkx2.5 , which reflect the broader transcriptional environment and lineage history of the cells in which it is expressed. This study helps refine our understanding of Nkx2.5 function in limb mesoderm and provide further insights into the unique developmental trajectory of emu wing formation. • Ectopic Nkx2.5 expression in the chicken limb field induced Scl expression. • Nkx2.5 induced a localized vascular remodeling and transient tissue disorganization. • ScRNAseq showed emu Nkx2.5-positive cells had limited hemangiogenic gene expression. • Emu Nkx2.5-positive limb cells belonged to muscle, not hematoendothelial clusters. • The developmental context is key to interpreting novel gene function in evolution.