Carkett, MD; (2016) Comparative analysis of Sonic Hedgehog signalling and the response to Sonic Hedgehog signalling in vertebrate forelimbs and hindlimbs. Doctoral thesis , UCL (University College London).

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Abstract

The Sonic Hedgehog (Shh) morphogen is required to establish anteroposterior (AP) pattern in vertebrate limbs. Limb progenitors exposed to increasing levels or durations of Shh signalling ultimately give rise to progressively more posterior structures. However, how Shh specifies different digit identities at a molecular level is poorly understood and molecular markers of individual digits are yet to be determined. Shh also patterns the dorsoventral axis of the vertebrate neural tube, where desensitisation to Shh signalling via Patched-­‐mediated negative feedback – termed temporal adaptation -­‐ is required for correct interpretation of the Shh morphogen gradient. To investigate how limb progenitors respond to, and integrate, different levels and durations of Shh signalling at a molecular level I have developed an ex vivo assay and used RNA-­‐sequencing to examine the immediate transcriptional responses of chick limb progenitors exposed to defined concentrations of Shh over fixed periods of time. I observe that limb progenitors initially respond equivalently to different concentrations of Shh but establish a graded response over time through a variation of a temporal adaptation mechanism in which both signal desensitisation and signal accumulation are required to generate distinct transcriptional outputs. I demonstrate that signal desensitisation is mediated, at least in part, by Patched-­‐mediated negative feedback, but that additional cell-­‐autonomous and noncell-­‐autonomous feedback mechanisms operating through Sufu/Gli and Disp1 also exist. I further use in silico analyses to identify candidate markers of digit identities that are induced by different levels of Shh signalling. I show a subset of candidate markers are expressed in intermediate AP domains, consistent with predictions, and may mark or specify middle digit identities. Finally, I have investigate d differences in Shh signalling dynamics and the response to Shh signalling in chick forelimbs and hindlimbs and provide evidence that hindlimbs are patterned by Shh over a shorter period of time.

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