Limb patterning genes and heterochronic development of the emu wing bud

Abstract

BackgroundThe forelimb of the flightless emu is a vestigial structure, with greatly reduced wing elements and digit loss. To explore the molecular and cellular mechanisms associated with the evolution of vestigial wings and loss of flight in the emu, key limb patterning genes were examined in developing embryos.MethodsLimb development was compared in emu versus chicken embryos. Immunostaining for cell proliferation markers was used to analyze growth of the emu forelimb and hindlimb buds. Expression patterns of limb patterning genes were studied, using whole-mount in situ hybridization (for mRNA localization) and RNA-seq (for mRNA expression levels).ResultsThe forelimb of the emu embryo showed heterochronic development compared to that in the chicken, with the forelimb bud being retarded in its development. Early outgrowth of the emu forelimb bud is characterized by a lower level of cell proliferation compared the hindlimb bud, as assessed by PH3 immunostaining. In contrast, there were no obvious differences in apoptosis in forelimb versus hindlimb buds (cleaved caspase 3 staining). Most key patterning genes were expressed in emu forelimb buds similarly to that observed in the chicken, but with smaller expression domains. However, expression of Sonic Hedgehog (Shh) mRNA, which is central to anterior–posterior axis development, was delayed in the emu forelimb bud relative to other patterning genes. Regulators of Shh expression, Gli3 and HoxD13, also showed altered expression levels in the emu forelimb bud.ConclusionsThese data reveal heterochronic but otherwise normal expression of most patterning genes in the emu vestigial forelimb. Delayed Shh expression may be related to the small and vestigial structure of the emu forelimb bud. However, the genetic mechanism driving retarded emu wing development is likely to rest within the forelimb field of the lateral plate mesoderm, predating the expression of patterning genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-016-0063-5) contains supplementary material, which is available to authorized users.

Co-option of the cardiac transcription factor Nkx2.5 during development of the emu wing

“…However, there was a strong reduction in cellular proliferation within Nkx2.5 -expressing limb buds, suggesting that the major influence on limb morphology may be through growth restriction. This is consistent with the reduced cell proliferation we recently reported in the developing emu wing 19 . Furthermore, in mouse embryos, ectopic expression of Nkx2.5 in the prospective sinoatrial node within the developing heart, where it is normally repressed, results in a marked reduction in cellular proliferation 50 , 51 , supporting the ability of Nkx2.5 to repress tissue growth.…”

“…It has previously been reported that there is a delay in the initiation of Tbx5 expression during the stages preceding limb bud outgrowth in the emu 18 . Similarly, our previous work has revealed alterations in the expression of a number of patterning factors such as Sonic Hedgehog and Patched1 , in embryonic emu limb buds 19 . These altered expression patterns may simply be the result of delayed development and reduced size of the emu forelimb bud, or they could be causative.…”

“…6 ), and it is not clear whether delayed expression of Tbx5 in the lateral plate mesoderm contributes to the diminutive size of the wing or is a consequence of a reduced wing bud field. Similarly, the reduced wing bud relative to the leg bud expression of Shh we have observed in emu 19 may contribute to reduction in the anterioposterior growth of the forelimb autopod or may reflect a reduced requirement for Shh within the smaller wing bud field. In addition, our expression profiling was performed at a single time point, soon after initiation of wing bud outgrowth.…”

“…4l, m ), indicating that Nkx2.5 is already expressed in resident (lateral-plate-derived) mesenchymal cells. In addition, we have recently demonstrated reduced cell proliferation associated with retarded emu forelimb bud development associated with resident mesenchyme but not Pax7 + immigrating cells 19 , implicating resident mesenchymal cells as the cell type likely involved in development of the reduced emu wing bud.…”

Evolution of the avian digital pattern

“…A posterior expression domain of Alx4 appears at stage 26 in the forelimb bud of all three birds, suggesting that it is not unique to emu. In addition, the emu forelimb bud has been proposed to develop heterochronically based on delayed SHH expression during the initiation of limb outgrowth 24 . The conserved expression pattern of Alx4 observed in this study is inconsistent with this model at least at stages 25 and 26.…”