The Hedgehog (Hh) pathway has indispensable functions in development and tissue homeostasis. Hh ligand acts as a morphogen, signaling across developmental fields creating a gradient that leads to patterned cell differentiation. Proper differentiation depends on slight differences in ligand concentration; therefore, small fluctuations in ligand or modulations of signaling efficiency have significant impacts on development and homeostasis. This work focuses on characterization of a new and unexpected modulator of this pathway, the intracellular mobilization of calcium via Ryanodine Receptors.
Ryanodine receptors (RyRs) mediate release of calcium from intracellular stores contained in the endoplasmic/sarcoplasmic reticulum and the nuclear envelope. We have found that disruption of RyR function in the zebrafish embryo causes loss or reduction of Hh-dependent muscle, neurons, spinal cord precursors and fins in a manner consistent with reduced Hh signaling. Furthermore, blocking RyR function reduces Hh-dependent gene expression. My hypothesis is that intracellular calcium release from RyRs is necessary for proper Hh signal transduction.
In future experiments, we will visualize ciliary and cytoplasmic calcium dynamics during periods of Hh signaling in vivo in zebrafish. Additionally, we are currently using novel gene targeting in zebrafish to generate a Smo-mCherry targeted knock-in fusion protein that will allow us to visualize Hh component trafficking in real-time. We will visualize Smo trafficking defects upon depletion of RyR function. Our ultimate goal is to determine where in the Hh pathway RyR function is required.