Analysis of the requirement for the Chx10 homeobox gene and FGF signalling for normal retinal development

Mutation in the human CHX10 homeobox gene causes microphthalmia, and the ocular retardation (Chx10^{orJ/orJ}) mouse models this phenotype. Lack of Chx10 causes reduced proliferation of retinal progenitor cells, resulting in an abnormally small neural retina, as well as loss of the optic nerve and failure of bipolar cells to differentiate. This study aimed to characterise the molecular mechanisms through which microphthalmia arises, to assess the importance of FGF signalling in Chx10-mediated control of early retinal development, and to assess whether Chx10 acts cell autonomously or regulates intercellular signalling. Real time RT-PCR and RNA in situ hybridisation was used to compare the expression levels and patterns of a set of genes potentially regulated by Chx10 in wild type and Chx10^{orJ/orJ} embryonic neural retinas. Increased expression of a number of RPE-specific genes was observed in the Chx10^{orJ/orJ} retina, indicating a role for Chx10 in maintenance of the neural retina/RPE boundary, whereas expression of several genes involved in cell cycle progression, cell differentiation and FGF signalling was decreased. Blocking FGF signalling in wild type embryonic retinal explants, cultured ex vivo for 24 hours, resulted in Chx10^{orJ/orJ}-like gene expression, implying FGF signalling is required for mediation of Chx10 activity. Addition of exogenous FGF to Chx10^{orJ/orJ} explants did not rescue the gene expression defect, indicating Chx10 is required to potentiate the FGF signalling mechanism. Finally, chimaeric embryos were generated combining wild type and Chx10^{orJ/orJ} cells, to assess the cell-intrinsic requirement for Chx10 expression. Defects in the timing of the onset of neuronal differentiation were not rescued in Chx10^{orJ/orJ} cells. However the presence of wild type cells was sufficient to reduce abnormal expansion of the ciliary body and to increase the number of Chx10^{orJ/orJ} cells in the neural retina, indicating a role for Chx10 in regulating extrinsic factors critical for normal eye development. This study shows that Chx10 plays an early role in maintaining the boundary between the neural retina and the RPE in the embryonic optic cup. It suggests a Chx10/FGF regulatory loop is important for maintaining retinal progenitor cell identity and regulating the onset of retinal histogenesis