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To contribute to devise successful beta-cell differentiation strategies for the cure of Type 1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Orthodenticle-related (Otx) proteins are a highly conserved class of homeobox-containing transcription factors found in a wide range of organisms. They function in numerous developmental events, most prominently, anterior head patterning in insects and vertebrates. In the sea urchin, Strongylocentrotus purpuratus, an orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specific Spec2a gene and more generally the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from other species, we isolated cDNA and genomic clones containing SpOtx sequences. Here, we report that SpOtx exists in two forms (alpha and beta) that are generated by alternative RNA splicing from a single SpOtx gene. SpOtx(alpha) and SpOtx(beta) had identical C-termini and homeoboxes but were entirely different in their N-terminal domains. SpOtx(alpha) mRNAs were transcribed from a single start site and accumulated in all cells during cleavage, but were gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contrast, three distinct SpOtx(beta) mRNAs resulted from two separate transcriptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later were largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx(beta) appeared later in development than SpOtx(alpha). Overexpression of SpOtx(alpha) and SpOtx(beta) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx(alpha) was able to repress the accumulation of SpOtx(beta) transcripts, whereas SpOtx(beta) had no effect on the accumulation of SpOtx(alpha) transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus. The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

The control region of the aboral ectoderm-specific Spec2a gene of Strongylocentrotus purpuratus contains a 188-bp enhancer element, the RSR enhancer, required for temporal activation and aboral ectoderm/mesenchyme cell expression. Within the enhancer is a positive cis-regulatory element with the core consensus sequence TAATCC, which is capable of binding the sea urchin orthodenticle-related homeobox protein SpOtx. In this report, we extend our analysis of the RSR enhancer by dissecting it into smaller pieces and testing these pieces in an enhancer activation assay. The 188-bp enhancer region could not be divided without partial loss of activity, and two of the three pieces tested exhibited some activity. Using site-directed mutagenesis, we showed that three Otx consensus binding sites were responsible for the activity of the enhancer, acting in a non-cooperative manner to yield full activity. Mutagenizing the three Otx sites and a fourth one just upstream abolished all activity in the context of the complete Spec2a control region. Bandshift analysis revealed that the Otx sites were able to bind SpOtx, suggesting that this transcription factor mediates positive control at these sites. Non-SpOtx binding sites overlapping two of the Otx sites may also play a role in Spec2a expression. Using a lacZ reporter gene, we showed that a 76-bp DNA fragment containing two of the Otx sites was sufficient for aboral ectoderm/mesenchyme cell expression. These results suggest that the RSR enhancer plus an upstream DNA element required for mesenchyme cell repression are necessary and sufficient for the proper temporal activation and aboral ectoderm expression of the Spec2a gene and that the Otx elements play a positive role in this process.