Role of Zic genes during neurulation
Neurulation is the embryonic event that results in formation of the neural tube, the primordium of the central nervous system. Impairment of this process leads to neural tube defects (NTDs), which are among the commonest congenital malformations in humans. Zic1, 2, 3 and 4 encode a family of 2C2H-like zinc finger transcription factors of which two members, Zic2 and Zic3, have been implicated in the causation of NTDs. The aim of this thesis was to investigate the function of Zic genes during neural tube closure in the mouse embryo. The expression of Zic genes was examined at the time of neurulation by in situ hybridisation. All 4 Zic genes have partially overlapping but distinct expression domains, with Zic2 and Zic3 mRNA detected at the posterior neuropore region, consistent with the occurrence of spina bifida and sacral agenesis in Zic2 and Zic3 mutants. Expression of Zic2 was not altered during abnormal neurulation in the mouse mutants ct, Lp and Sp2H, indicating that Zic2 does not act downstream of these mutant genes in the production of NTDs. Zic3 expression was also unaffected in the ct and Sp2H mutants but showed downregulation in Lp homozygous embryos, suggesting that Zic3 may be regulated downstream of the Lp gene. A novel ENU-generated mouse model of spina bifida, the Kumba mouse (Zic2Ku), carries a mutation in the zinc finger domain region of Zic2. Morphological analysis of neurulation in Zic2Ku/Ku embryos showed that Zic2 is required for normal bending of the neural plate. Absence of dorsolateral bending during neural tube closure can explain the subsequent development of spina bifida in Zic2Ku/Ku embryos. In order to understand the molecular pathway by which Zic2 mediates its function, the yeast two-hybrid system was used to identify protein-binding partners. Capicua, p53 binding protein 1, Glis2 and Krox20 were among a series of genes whose protein products were found to interact with Zic2. These protein-protein interactions were confirmed by co-immunolocalisation studies of cultured transfected cells and by glutathione-S-transferase pull-down assays. In situ hybridisation studies demonstrated that Capicua is expressed in the posterior neuropore region of E9.5 embryos consistent with a role of these proteins in Zic2-dependent spina bifida. In conclusion, this thesis has established an early embryonic role for Zic2 in low spinal neurulation and has identified several binding proteins that may participate with Zic2 in the regulation of neural tube closure.