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The specification of the seven retinal cell types from a common pool of retina progenitor cells (RPCs) involves complex interactions between the intrinsic program and the environment. The proneural basic helix-loop-helix (bHLH) transcriptional regulators are key components for the intrinsic programming of RPCs and are essential for the formation of the diverse retinal cell types. However, the extent to which an RPC can re-adjust its inherent program and the mechanisms through which the expression of a particular bHLH factor influences RPC fate is unclear. Previously, we have shown that Neurod1 inserted into the Atoh7 locus activates the retinal ganglion cell (RGC) program in Atoh7-expressing RPCs but not in Neurod1-expressing RPCs, suggesting that Atoh7-expressing RPCs are not able to adopt the cell fate determined by Neurod1, but rather are pre-programmed to produce RGCs. Here, we show that Neurod1-expressing RPCs, which are destined to produce amacrine and photoreceptor cells, can be re-programmed into RGCs when Atoh7 is inserted into the Neurod1 locus. These results suggest that Atoh7 acts dominantly to convert a RPC subpopulation not destined for an RGC fate to adopt that fate. Thus, Atoh7-expressing and Neurod1-expressing RPCs are intrinsically different in their behavior. Additionally, ChIP-Seq analysis identified an Atoh7-dependent enhancer within the intronic region of Nrxn3. The enhancer recognized and used Atoh7 in the developing retina to regulate expression of Nrxn3, but could be forced to use Neurod1 when placed in a different regulatory context. The results indicate that Atoh7 and Neurod1 activate distinct sets of genes in vivo, despite their common DNA-binding element.

Retinal progenitor cells (RPCs) express basic helix-loop-helix (bHLH) factors in a strikingly mosaic spatiotemporal pattern, which is thought to contribute to the establishment of individual retinal cell identity. Here, we ask whether this tightly regulated pattern is essential for the orderly differentiation of the early retinal cell types and whether different bHLH genes have distinct functions that are adapted for each RPC. To address these issues, we replaced one bHLH gene with another. Math5 is a bHLH gene that is essential for establishing retinal ganglion cell (RGC) fate. We analyzed the retinas of mice in which Math5 was replaced with Neurod1 or Math3, bHLH genes that are expressed in another RPC and are required to establish amacrine cell fate. In the absence of Math5, Math5Neurod1-KI was able to specify RGCs, activate RGC genes and restore the optic nerve, although not as effectively as Math5. By contrast, Math5Math3-KI was much less effective than Math5Neurod1-KI in replacing Math5. In addition, expression of Neurod1 and Math3 from the Math5Neurod1-KI/Math3-KI allele did not result in enhanced amacrine cell production. These results were unexpected because they indicated that bHLH genes, which are currently thought to have evolved highly specialized functions, are nonetheless able to adjust their functions by interpreting the local positional information that is programmed into the RPC lineages. We conclude that, although Neurod1 and Math3 have evolved specialized functions for establishing amacrine cell fate, they are nevertheless capable of alternative functions when expressed in foreign environments.

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that occupies a key node in the RGC gene regulatory network and that is essential for RGC differentiation. However, little is known about the genes that connect upstream regulatory genes, such as Pou4f2 with downstream effector genes responsible for RGC differentiation. The purpose of this study was to characterize the retinal function of eomesodermin (Eomes), a T-box transcription factor with previously unsuspected roles in retinogenesis. We show that Eomes is expressed in developing RGCs and is a mediator of Pou4f2 function. Pou4f2 directly regulates Eomes expression through a cis-regulatory element within a conserved retinal enhancer. Deleting Eomes in the developing retina causes defects reminiscent of those in Pou4f2(-/-) retinas. Moreover, myelin ensheathment in the optic nerves of Eomes(-/-) embryos is severely impaired, suggesting that Eomes regulates this process. We conclude that Eomes is a crucial regulator positioned immediately downstream of Pou4f2 and is required for RGC differentiation and optic nerve development.

Blastocyst formation marks the segregation of the first two cell lineages in the mammalian preimplantation embryo: the inner cell mass (ICM) that will form the embryo proper and the trophectoderm (TE) that gives rise to the trophoblast lineage. Commitment to ICM lineage is attributed to the function of the two transcription factors, Oct4 (encoded by Pou5f1) and Nanog. However, a positive regulator of TE cell fate has not been described. The T-box protein eomesodermin (Eomes) and the caudal-type homeodomain protein Cdx2 are expressed in the TE, and both Eomes and Cdx2 homozygous mutant embryos die around the time of implantation. A block in early TE differentiation occurs in Eomes mutant blastocysts. However, Eomes mutant blastocysts implant, and Cdx2 and Oct4 expression are correctly restricted to the ICM TE. Blastocoel formation initiates in Cdx2 mutants but epithelial integrity is not maintained and embryos fail to implant. Loss of Cdx2 results in failure to downregulate Oct4 and Nanog in outer cells of the blastocyst and subsequent death of those cells. Thus, Cdx2 is essential for segregation of the ICM and TE lineages at the blastocyst stage by ensuring repression of Oct4 and Nanog in the TE.

While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.