Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors.

ONECUT transcription factors induce neuronal characteristics and remodel chromatin accessibility.

Remodeling of chromatin accessibility is necessary for successful reprogramming of fibroblasts to neurons. However, it is still not fully known which transcription factors can induce a neuronal chromatin accessibility profile when overexpressed in fibroblasts. To identify such transcription factors, we used ATAC-sequencing to generate differential chromatin accessibility profiles between human fibroblasts and iNeurons, an in vitro neuronal model system obtained by overexpression of Neurog2 in induced pluripotent stem cells (iPSCs). We found that the ONECUT transcription factor sequence motif was strongly associated with differential chromatin accessibility between iNeurons and fibroblasts. All three ONECUT transcription factors associated with this motif (ONECUT1, ONECUT2 and ONECUT3) induced a neuron-like morphology and expression of neuronal genes within two days of overexpression in fibroblasts. We observed widespread remodeling of chromatin accessibility; in particular, we found that chromatin regions that contain the ONECUT motif were in- or lowly accessible in fibroblasts and became accessible after the overexpression of ONECUT1, ONECUT2 or ONECUT3. There was substantial overlap with iNeurons, still, many regions that gained accessibility following ONECUT overexpression were not accessible in iNeurons. Our study highlights both the potential and challenges of ONECUT-based direct neuronal reprogramming.

An empirical model of Onecut binding activity at the sea urchin SM50 C-element gene regulatory region.

Studying the formation of endoskeleton in many species is complex and difficult. The sea urchin embryo offers an unparalleled platform for understanding this process because of the ease with which its skeletogenic mesenchyme cells can be manipulated. In this study, preliminary evidence from biochemical studies towards understanding the role of the Onecut transcription factor during sea urchin skeletogenic mesenchyme cell specification is presented. Based on the evidence, an empirical model is proposed showing how Onecut, together with associated co-factors, may be using the C-element of the SM50 gene regulatory region in advance of the sea urchin Strongylocentrotus purpuratus spicule development. In the model, Onecut recognizes and binds the DNA sequence CATCGATCTC in the C-element without temporal restriction. Onecut then utilizes different sets of co-factors to switch from its unknown function early in development (four cell stage to the mesenchyme blastula stage), to its known role in the oral-aboral boundary thereafter. At the writing of this report, definitive evidence as to whether the "early" factors are expressed in all cells except the micromere lineages, or whether the "late" factors are expressed in micromere descendants or ectodermal precursors only are lacking. The former would suggest a possible Onecut repression function for the early co-factors outside the micromere lineages; the latter scenario would suggest a Onecut activation function for the late co-factors in the presumptive ciliary band.

cis-Regulatory control of the initial neurogenic pattern of onecut gene expression in the sea urchin embryo.

Specification of the ciliated band (CB) of echinoid embryos executes three spatial functions essential for postgastrular organization. These are establishment of a band about 5 cells wide which delimits and bounds other embryonic territories; definition of a neurogenic domain within this band; and generation within it of arrays of ciliary cells that bear the special long cilia from which the structure derives its name. In Strongylocentrotus purpuratus the spatial coordinates of the future ciliated band are initially and exactly determined by the disposition of a ring of cells that transcriptionally activate the onecut homeodomain regulatory gene, beginning in blastula stage, long before the appearance of the CB per se. Thus the cis-regulatory apparatus that governs onecut expression in the blastula directly reveals the genomic sequence code by which these aspects of the spatial organization of the embryo are initially determined. We screened the entire onecut locus and its flanking region for transcriptionally active cis-regulatory elements, and by means of BAC recombineered deletions identified three separated and required cis-regulatory modules that execute different functions. The operating logic of the crucial spatial control module accounting for the spectacularly precise and beautiful early onecut expression domain depends on spatial repression. Previously predicted oral ectoderm and aboral ectoderm repressors were identified by cis-regulatory mutation as the products of goosecoid and irxa genes respectively, while the pan-ectodermal activator SoxB1 supplies a transcriptional driver function.

Auto and cross regulatory elements control Onecut expression in the ascidian nervous system.

The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.

MicroRNA-9 promotes the switch from early-born to late-born motor neuron populations by regulating Onecut transcription factor expression.

Motor neurons in the vertebrate spinal cord are stereotypically organized along the rostro-caudal axis in discrete columns that specifically innervate peripheral muscle domains. Originating from the same progenitor domain, the generation of spinal motor neurons is orchestrated by a spatially and temporally tightly regulated set of secreted molecules and transcription factors such as retinoic acid and the Lim homeodomain transcription factors Isl1 and Lhx1. However, the molecular interactions between these factors remained unclear. In this study we examined the role of the microRNA 9 (miR-9) in the specification of spinal motor neurons and identified Onecut1 (OC1) as one of its targets. miR-9 and OC1 are expressed in mutually exclusive patterns in the developing chick spinal cord, with high OC1 levels in early-born motor neurons and high miR-9 levels in late-born motor neurons. miR-9 efficiently represses OC1 expression in vitro and in vivo. Overexpression of miR-9 leads to an increase in late-born neurons, while miR-9 loss-of-function induces additional OC1(+) motor neurons that display a transcriptional profile typical of early-born neurons. These results demonstrate that regulation of OC1 by miR-9 is a crucial step in the specification of spinal motor neurons and support a model in which miR-9 expression in late-born LMCl neurons downregulates Isl1 expression through inhibition of OC1. In conclusion, our study contributes essential factors to the molecular network specifying spinal motor neurons and emphasizes the importance of microRNAs as key players in the generation of neuronal diversity.

MiR-495 and miR-218 regulate the expression of the Onecut transcription factors HNF-6 and OC-2.

MicroRNAs are small, non-coding RNAs that posttranscriptionally regulate gene expression mainly by binding to the 3'UTR of their target mRNAs. Recent data revealed that microRNAs have an important role in pancreas and liver development and physiology. Using cloning and microarray profiling approaches, we show that a unique repertoire of microRNAs is expressed at the onset of liver and pancreas organogenesis, and in pancreas and liver at key stages of cell fate determination. Among the microRNAs that are expressed at these stages, miR-495 and miR-218 were predicted to, respectively, target the Onecut (OC) transcription factors Hepatocyte Nuclear Factor-6 (HNF-6/OC-1) and OC-2, two important regulators of liver and pancreas development. MiR-495 and miR-218 are dynamically expressed in developing liver and pancreas, and by transient transfection, we show that they target HNF-6 and OC-2 3'UTRs. Moreover, when overexpressed in cultured cells, miR-495 and miR-218 decrease the endogenous levels of HNF-6 and OC-2 mRNA. These results indicate that the expression of regulators of liver and pancreas development is modulated by microRNAs. They also suggest a developmental role for miR-495 and miR-218.

A ONECUT homeodomain protein communicates X chromosome dose to specify Caenorhabditis elegans sexual fate by repressing a sex switch gene.

Sex is determined in Caenorhabditis elegans through a dose-dependent signal that communicates the number of X chromosomes relative to the ploidy, the number of sets of autosomes. The sex switch gene xol-1 is the direct molecular target of this X:A signal and integrates both X and autosomal components to determine sexual fate. X chromosome number is relayed by X signal elements (XSEs) that act cumulatively to repress xol-1 in XX animals, thereby inducing hermaphrodite fate. Ploidy is relayed by autosomal signal elements (ASEs), which counteract the single dose of XSEs in XO animals to activate xol-1 and induce the male fate. Our goal was to identify and characterize new XSEs and further analyze known XSEs to understand the principles by which a small difference in the concentration of an intracellular signal is amplified to induce dramatically different developmental fates. We identified a new XSE, the ONECUT homeodomain protein CEH-39, and showed that it acts as a dose-dependent repressor of xol-1 transcript levels. Unexpectedly, most other XSEs also repress xol-1 predominantly, but not exclusively, at the transcript level. The twofold difference in X dose between XO and XX animals is translated into the male vs. hermaphrodite fate by the synergistic action of multiple, independent XSEs that render xol-1 active or inactive, primarily through transcriptional regulation.

Role of the Onecut transcription factors in pancreas morphogenesis and in pancreatic and enteric endocrine differentiation.

The Onecut (OC) transcription factor HNF-6 (OC-1) is required during embryogenesis for pancreatic specification, morphogenesis and endocrine differentiation. In mammals, HNF-6 has two paralogs, OC-2 and OC-3, which share DNA-binding and transcriptional activation properties and have expression patterns that overlap with that of HNF-6. This suggested that OC-2 and OC-3 play redundant roles with HNF-6 in pancreas development. Here, we have addressed this hypothesis by analyzing the phenotype of mice knockout for the Onecut factors. We found that neither OC-2 nor OC-3 is required for pancreas specification. However, OC-2 plays partially redundant roles with HNF-6 in pancreas morphogenesis and in the differentiation of endocrine precursors. As similar molecular events drive endocrine differentiation in the pancreas and gastrointestinal tract, we also investigated if Onecut factors are involved in enteroendocrine differentiation. OC-2 and OC-3 were found to delineate specific antero-posterior regions of the gut around embryonic day 12.5. Later on, OC2 was expressed in several gut cell types, whereas OC-3 behaved as a specific marker of the enteroendocrine lineage. However, OC-2 and OC-3, alone or in combination, were dispensable for gut development and enteroendocrine differentiation. In conclusion, our data reveal partially redundant roles for HNF-6 and OC-2 in developing pancreas and identify new markers for antero-posterior patterning of the gut and for enteroendocrine differentiation.

Temporal regulation of enhancer function in intestinal epithelium: a role for Onecut factors.

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.