Final Report Summary - EYEDEVELOPMENT (Molecular genetic regulation of eye field development) Anophthalmia, microphthalmia and coloboma are birth defects that account for many cases of childhood blindness. These abnormalities could result from abnormal development of eye progenitors in the eye field during early embryogenesis. Six3 is a homeoprotein essential for normal forebrain and eye development. Loss of human SIX3 function can lead to a wide spectrum of eye malformations including anophthalmia or microphthalmia and coloboma. Zebrafish have three six3-related genes, six3a, six3b and six7. The combined loss of six3b and six7 leads to microphthalmia or anophthalmia whereas the role of six3a was unknown when this project started. In the six3b;six7-deficient embryos, eye progenitors form yet the eyes fail to develop. This raises the question of what happens to these progenitors – do they die? change their fate? fail to migrate to their final position? Another important question is what are the molecular mechanisms regulating the changes in eye progenitor cell behavior downstream of Six3. Answering these questions could provide important new insights into the biology of eye progenitors, which might contribute to development of cell replacement therapies in the future. In the first two years of the project we focused on identifying molecular and cellular mechanisms that underlie the lack of eye tissues in six3b;six7-deficient embryos. We generated a transgenic line, rx3:nls-eGFP, in which GFP is strongly expressed in eye progenitors from early eye field stage. We planned to use this transgene in the background of six3b;six7-deficient embryos to follow, in live embryos, the fate of eye progenitors. Furthermore, we planned to isolate the progenitors from normal and six3b;six7-deficient embryos and compare gene expression profiles in order to identify the molecular mechanisms downstream of Six3 in regulating progenitor development. Surprisingly, when placing rx3:nls-eGFP transgene in the six3b;six7-deficient background no EGFP could be detected even at early stages. This unexpected result precluded the use of this tool in accomplishing the original Aims of the project. Nevertheless, using genetic methods we continued to test potential mechanisms underlying loss of eye progenitors in Six3-deficient embryos and found that it was not due to P53-mediated cell death or to increased Wnt/beta-catenin signaling. Current work focuses on blocking cell death pathways to find whether cell death plays a role in loss of eye progenitors, as well as testing the roles of several candidate genes known to function downstream of Six3 in early eye development. To generate an additional way to identify the roles of Six3 in early eye development, we have generated a model in which the activity of both six3b and six3a is abrogated. We used a new, strong hypomorphic allele of the six3a gene, six3a L183S, which was identified by TILLING. Zebrafish embryos homozygous for the six3a L183S allele appear normal, as do embryos homozygous for the null allele of six3b. We therefore generated embryos mutant for both six3a and six3b. The double mutants appeared normal until two days post-fertilization, at which time abnormal light passage through the lens of the eye was observed. Cross sections through the eyes and histological staining revealed large optic disc colobomas in the double mutants, and suggested abnormalities in optic nerve axon pathfinding. Labeling the optic nerve by immunohistochemistry demonstrated that the optic nerve appeared reduced in size and defasciculated. The retinal axon pathfinding defects could stem from cell-autonomous defects in the retinal ganglion cells (RGCs), which send the axons of the optic nerve or from abnormalities in the molecular guidance cues in their environment. Given that six3a and six3b are expressed both in RGCs and in brain tissues, both mechanisms could be relevant. Initial analyses of expression of axon guidance molecules in the double mutants identified strong reduction in the expression of the chemokine cxcl12a, which is expressed in the optic disc region and its receptor cxcr4b, which is expressed in RGCs. Current work focuses on further understanding the molecular mechanisms that underlie axon pathfinding abnormalities when Six3 function is abrogated. The described results for the combined loss of function of six3a and six3b identify new roles for Six3 in optic nerve development and open new avenues for research. Given that optic nerve malformations are causes for blindness, studies that will elucidate the roles of Six3 in optic nerve development will contribute to the understanding of the molecular mechanisms that control this process, and could help in developing therapies in diseases that damage the optic nerve such as glaucoma.