Genetic analyses of the cilia-dependent hedgehog signal transduction in the mouse

Open Access
Author:
Liu, Jinling
Graduate Program:
Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 28, 2014
Committee Members:
  • Aimin Liu, Dissertation Advisor
  • Zhi Chun Lai, Committee Chair
  • Douglas Cavener, Committee Member
  • Robert Paulson, Committee Member
Keywords:
  • hedgehog
  • spinal cord patterning
  • Gli
  • Sufu
  • cilia
Abstract:
The Hedgehog (Hh) signaling is crucial for vertebrate development and physiology. The morphogen Sonic hedgehog (Shh), one of the Hh family of secreted proteins, plays a key role in patterning the mammalian spinal cord along its dorsoventral (D/V) axis through the activation of Glioma-associated oncogene (Gli) family of transcription factors. Suppressor of Fused (Sufu), a Gli-interacting protein, modulates the D/V patterning of the spinal cord by antagonizing Hh signaling. The molecular mechanisms underlying the function of Sufu in Hh pathway activation and spinal cord D/V patterning remains controversial, particularly in light of recent findings that Sufu protects the Gli2 and Gli3 proteins from proteasomal degradation. In this thesis, I show that Hh pathway activation and dorsal expansion of ventral spinal cord cell types in the absence of Sufu depends on the activator activities of all three Gli family proteins. I also show that Sufu plays a positive role in the maximal activation of Hh signaling that defines the ventral-most cell fate in the mammalian spinal cord, likely through protecting Gli2 and Gli3 proteins from degradation. Finally, by altering the level of Gli3 repressor on a background of reduced Gli activator activities, I reveal an important contribution of Gli3 repressor activity to the Hh pathway activation and the D/V patterning of the spinal cord. The primary cilium, a hair-like structure projecting from the cell surface, is involved in Hh signaling in vertebrates. Gli2, the primary effector of Hh signaling, localizes to the tip of the primary cilium, but the importance of its ciliary localization remains unclear. Here, I address the roles of Gli2 ciliary localization by replacing endogenous Gli2 with Gli2∆CLR, a Gli2 variant not localizing to the cilium. The resulting Gli2∆CLRKI and Gli2∆CLRKI;Gli3 double mutants resemble Gli2null and Gli2null;Gli3 double mutants, respectively, suggesting the lack of Gli2∆CLR activation in development. Significantly, Gli2∆CLR cannot be activated by either pharmacochemical activation of Smo in vitro or loss of Ptch1 in vivo. Finally, Gli2∆CLR exhibits strong transcriptional activator activity in the absence of Sufu, suggesting that the lack of its activation in vivo results from a specific failure in relieving the inhibitory function of Sufu. My results provide strong evidence that the ciliary localization of Gli2 is critical for cilium-dependent activation of Hh signaling. Through this work, I established a better understanding on the genetic interactions between Gli and Sufu proteins as well as the role of cilia in Gli activation and Hh signal transduction.