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B.S. Tsinghua University – 2007
Thesis Advisor: James H. Millonig, Ph.D.
Graduate Program in Neuroscience
CABM, Room 010
Thursday, September 26, 2013
Neural tube defects (NTDs) and congenital cataract are human birth defects that have a multi-factorial basis. Vacuolated lens (vl) is a spontaneous mutation that arose on the C3H/HeSnJ background. Vl affects the apposition/fusion of neural folds and secondary lens fiber differentiation, causing NTDs and congenital cataract. These phenotypes are due to a deletion/frameshift mutation in the orphan G protein-coupled receptor, Gpr161, which is expressed on the tips of neural folds and lens fiber cells. Previously Gpr161vl-C3H was crossed to different inbred backgrounds including MOLF/EiJ, and the Gpr161vl mutant phenotypes were rescued. QTL analysis mapped five modifiers (Modvl1-5: Modifier of vl). Two QTL are investigated here: Modvl4 (LOD=4.4; Chr15) and Modvl5 (LOD=5.0; Chr18).
For Modvl5, we demonstrate that Modvl5MOLF congenic rescues the Gpr161vl-associated lethality and NTDs but not cataract. Bioinformatics determined the transcription factor, Cdx1, as one QTG that contains a functional poly-Q repeats polymorphism. Using Cdx1 as an entry point, we identified retinoid acid (RA), canonical Wnt and cell adhesion pathways as downstream targets of Gpr161. QRT-PCR, ISH and IHC determined that expression of RA and Wnt genes, as well as cell adhesion molecules (E-/N-cadherins) are down-regulated in Gpr161vl/vl but rescued by the Modvl5MOLF congenic during neurulation. Intraperitoneal RA injection restores the expression of canonical Wnt markers and rescues Gpr161vl/vl NTDs. These results indicate that Modvl5 and Cdx1 bypass Gpr161vl mutation by regulating these downstream targets.
For Modvl4, the Modvl4MOLF congenic partially rescues the lens fiber defect and cataract in Gpr161vl. Analyzing three subcongenic lines narrowed down the candidate modifiers from 381 genes/ESTs to only 14 protein-coding genes that are situated in a 15 Mb interval in proximal Modvl4. QRT-PCR and online gene expression database identified 6 genes to be candidate cataract modifiers. Three of them encode type II cadherins (Cdh6, 10 & 12). We hypothesize that these three genes co-regulate cell adhesion and cell shape change during lens fiber differentiation.
Lastly, an additional mating crossed Gpr161vl-C3H to PWK/PhJ. PWK displays similar modifying effects as MOLF and QTL analysis mapped four additional QTL (Modvl6, 7, 8 and 9). Future analysis on these 4 QTL will identify additional genes and pathways regulating Gpr161vl-associated NTDs and cataract.