title: Limb patterning genes and heterochronic development of the emu wing bud.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Limb 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).
  
  
  RESULTS
  The 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 ofSonic 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,Gli3andHoxD13, also showed altered expression levels in the emu forelimb bud.
  
  
  CONCLUSIONS
  These data reveal heterochronic but otherwise normal expression of most patterning genes in the emu vestigial forelimb. DelayedShhexpression 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.
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800 Australia.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Smith
authors.affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, VIC 3052 Australia.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Farlie
authors.affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, VIC 3052 Australia.
authors.forename: Nadia M
authors.initials: NM
authors.lastname: Davidson
authors.affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, VIC 3052 Australia.
authors.forename: Kelly N
authors.initials: KN
authors.lastname: Roeszler
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800 Australia.
authors.forename: Claire
authors.initials: C
authors.lastname: Hirst
authors.affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, VIC 3052 Australia.
authors.forename: Alicia
authors.initials: A
authors.lastname: Oshlack
authors.affiliation: Environmental Futures Research Institute, Griffith University, Nathan, QLD 4111 Australia.
authors.forename: David M
authors.initials: DM
authors.lastname: Lambert
keywords: Emu embryo; Heterochronic; Limb bud; Limb development; Limb patterning; Sonic Hedgehog