An analysis of the regulation of dorso-ventral patterning and T-box gene function in early eye development
Structural eye defects are often caused by aberrant mechanisms during the early stages of eye development. The underlying mechanisms of these defects are currently not fully known. The optic vesicle, which gives rise to the mature eye, is patterned by asymmetrical gene expression. The T-box transcription factors, Tbx2, Tbx3, and Tbx5y which are members of the evolutionarily conserved family of T-box genes, are expressed in the dorsal region of the developing eye. The expression of these genes overlaps with the expression of the secreted signalling factor Bone morphogenetic protein (Bmp) 4. Together, they represent the earliest dorsally expressed genes in the developing mouse eye and are co-expressed in several other vertebrate species. This thesis investigates the regulation of T-box gene expression by Bmp4 in the developing mouse eye, and the importance of the regional expression of these genes for early eye development. Bmp4 and Noggin coated bead implantation in combination with whole mouse embryo culture was performed to manipulate Bmp4 signalling during eye morphogenesis. Examination of gene expression revealed that manipulation of Bmp4 signalling alters the expression of the T-box genes as well as the ventrally expressed Vax2 transcription factor, thus disrupting dorso-ventral (D-V) patterning. These changes resulted in reduced eye size and growth. Cell division was decreased globally and cell death induced in the dorsal neural retina. To directly investigate the role of Tbx2 in eye development, mice with a targeted mutation of Tbx2 were analysed, and gain of function experiments were carried out using in ovo retroviral gene delivery in the chick embryo. Tbx2 was selected for further analysis, as it showed the largest change in expression in Bmp4 manipulated eyes. Both strategies resulted in reduced eye size. In the loss of function approach, establishment of the expression of D-V patterning genes occurred in the absence of Tbx2, but the maintenance of the expression of several of these was affected. Within the normal dorsal site of Tbx2 expression, an increased trend in cell division was detected. Several abnormalities were also found outside of the Tbx2 expression domain, including delayed formation of the ventral region of the eye, and abnormal lens and hyaloid vasculature defects. These findings suggest that Tbx2 regulates eye development both intrinsically and outside of its site of expression and that a Bmp- Tbx2 pathway regulates early growth and coordination of morphogenesis of the developing eye.