We thank Dr. Clark Riley, Ms. Carol Davenport, and Ms. Jani ne Ptak for synthetic oligonucleotides; Dr. Keith Fry for the gift of mAb ABS; Ms. Cathy Blizzard for cat eyes; Dr. Mark Molliver for donating fixed monkey eyes; Drs. Masafumi Tanaka and Wi nshi p Herr for Oct.1 cDNA; Mr. Hao Zhou for the GST-Ott-1 POU domain protein; Dr. Y.-W. Peng for rabbit retinas and Dr. Hua-Shun Li for chicken retinas; Dr. Elio Ravi ol a for advice on histologic techniques; and Drs. Robert Rodieck, Jen-Chi h Hsieh, and King-wai Yau for hel pful comments on the manuscript.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Immunohistochemistry. Immunostaining of dorsal root and trigeminal ganglia, retinal sections, and retinal flat mounts was performed as previously described (6, 7). Antibodies to Brn3a, Brn-3b, and Brn-3c are described in refs. 6 and 7. Additional antibodies were obtained from the following sources: antiAbbreviations: ES, embryonic stem; mAb, monoclonal antibody; CNS, central nervous system; en, embryonic day n; DAPI, 4',6-diamidino-2-phenylindole; TUNEL, terminal dUTP nick end labeling.

During vertebrate retinogenesis, seven classes of cells are specified from multipotent progenitors. To date, the mechanisms underlying multipotent cell fate determination by retinal progenitors remain poorly understood. Here, we show that the Foxn4 winged helix/forkhead transcription factor is expressed in a subset of mitotic progenitors during mouse retinogenesis. Targeted disruption of Foxn4 largely eliminates amacrine neurons and completely abolishes horizontal cells, while overexpression of Foxn4 strongly promotes an amacrine cell fate. These results indicate that Foxn4 is both necessary and sufficient for commitment to the amacrine cell fate and is nonredundantly required for the genesis of horizontal cells. Furthermore, we provide evidence that Foxn4 controls the formation of amacrine and horizontal cells by activating the expression of the retinogenic factors Math3, NeuroD1, and Prox1. Our data suggest a model in which Foxn4 cooperates with other key retinogenic factors to mediate the multipotent differentiation of retinal progenitors.

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This misspecification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.