Investigation of the SHH gradient during limb development through quantitation of transcriptional regulation, expression, and protein distribution.

Correct number and pattern of digits is determined in a time and concentration-dependent manner by a gradient of Sonic Hedgehog (SHH) across the anterior-posterior axis of the embryonic limb bud. Owing to the potent morphogenic/mitogenic capabilities of SHH, transcription of the SHH gene in the limb is tightly regulated by feedback loops with other signalling pathways and by the Zone of Polarising Activity regulatory sequence (ZRS). The ZRS is a long-range, cis-regulatory limb-specific enhancer of SHH, and is essential for correct limb SHH expression. The Silkie, a polydactylous breed of chicken, possesses a C>A mutation in the ZRS, resulting in ectopic SHH expression in the anterior limb and hindlimb-specific polydactyly. We employ the Silkie mutant to investigate how SHH is regulated by the ZRS, and how Hedgehog signalling can modulate SHH expression in an autoregulatory manner. We further characterise the effects that the Silkie mutation has on subsequent limb development; investigating the dependence of increased posterior SHH, increased Hedgehog-dependent growth and necessary genotype in both the posterior and anterior limb bud. Several fundamental questions regarding SHH during limb development have yet to be fully addressed: how much SHH protein is present, and does it form a gradient as hypothesised by Wolpert’s Morphogen Gradient Model? By developing a standard curve-based method to assess absolute quantities of processed SHH protein, N-SHH, we find that the quantity of N-SHH protein increases through limb development, and does indeed form a quantifiable gradient across the posterior limb. By comparing quantity of N-SHH protein in equivalently staged mouse, rat, emu and chicken limbs, we find that there is no significant link between N-SHH protein quantity and digit number between mammalian and avian species, and investigate how digit number is modulated in the late limb. A number of species exhibit reduced numbers of digits, including the wings of the emu, cassowary and kiwi. Unlike in mammalian examples of digit loss (i.e. cow, pig) the emu wing has delayed and significantly reduced SHH expression. Through sequencing and functional in vivo testing of ZRS sequences of ratite bird species, we investigate whether the ZRS has a role in evolutionary digit loss. We also demonstrate the aspects of digit loss and Hh signalling are shared with examples of mammalian digit loss. This thesis presents novel research into multiple aspects of genetic regulation, limb development, and evolutionary developmental biology; elucidating both long held dogmas and upcoming areas of limb development