Abstract
Purpose :
The choroid fissure is a crucial transient structure through which retinal axons exit and vasculature enters the eye. Disruption of choroid fissure development results in uveal coloboma. Loss-of-function mutations in the Hedgehog (Hh) receptor patched2 (ptch2) lead to overactive Hh signaling and result in coloboma in humans and zebrafish. We previously determined the cellular basis of wild type choroid fissure formation and identified specific early cell movements disrupted by overactive Hh signaling. Despite this, the downstream molecular signaling mechanisms by which overactive Hh signaling disrupts cell motility remain unknown. Is Gli-dependent transcription, or non-canonical Hh signaling via Src-family kinases (SFKs), required for the coloboma phenotype? In which cells is overactive Hh signaling acting to disrupt cell movements?
Methods :
Zebrafish mutant lines were used: ptch2 (blwtc294z) and gli1 (dtrts269). We established a transgenic line (Tg(ptch2:H2A-mCherry)), and performed antibody staining for pax2a. The SFK inhibitors PP2 and SU6656 were used. To determine whether overactive Hh signaling acts in a cell autonomous or non-autonomous manner to disrupt movements, we utilized blastula cell transplantation.
Results :
In the ptch2 mutant, we find that both our transgenic reporter and endogenous pax2a are expanded and upregulated, concurrent with disrupted early cell movements. Loss of gli1 partially rescues the ptch2 mutant coloboma phenotype. Treatment of ptch2 mutant embryos with two structurally unrelated SFK inhibitors yields no rescue of the coloboma phenotype. Finally, using blastula cell transplantation, we find that ptch2 mutant cells behave normally when transplanted into a wild type host; conversely, wild type cells take on a mutant phenotype when transplanted into a ptch2 mutant host.
Conclusions :
Our results indicate that in the ptch2 mutant, Gli1-dependent transcription is required to cause coloboma, however, non-canonical Hh signaling via SFK activation is not. Additionally, overactive Hh signaling acts in a non-autonomous manner to misregulate cell migration. Together, our data suggest a model in which overactive Hh signaling upregulates expression of a currently unidentified cell surface or secreted molecule that disrupts cell motility during choroid fissure formation. We are currently working to identify this downstream molecule.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.