The spinal cord contains a diverse array of physiologically distinct interneuron cell types that subserve specialized roles in somatosensory perception and motor control. The mechanisms that generate these specialized interneuronal cell types from multipotential spinal progenitors are not known. In this study, we describe a temporally regulated transcriptional program that controls the differentiation of Renshaw cells (RCs), an anatomically and functionally discrete spinal interneuron subtype. We show that the selective activation of the Onecut transcription factors Oc1 and Oc2 during the first wave of V1 interneuron neurogenesis is a key step in the RC differentiation program. The development of RCs is additionally dependent on the forkhead transcription factor Foxd3, which is more broadly expressed in postmitotic V1 interneurons. Our demonstration that RCs are born, and activate Oc1 and Oc2 expression, in a narrow temporal window leads us to posit that neuronal diversity in the developing spinal cord is established by the composite actions of early spatial and temporal determinants.

The transcription factors Foxd3 and Pax3 are important early regulators of neural crest (NC) progenitor cell properties. Homozygous mutations of Pax3 or a homozygous NC-specific deletion of Foxd3 cause marked defects in most NC derivatives, but neither loss of both Foxd3 alleles nor loss of one Pax3 allele alone greatly affects overall development of cardiac NC derivatives. In contrast, compound mutant embryos homozygous for a NC-specific Foxd3 mutation and heterozygous for Pax3 have fully penetrant persistent truncus arteriosus, severe thymus hypoplasia, and midgestation lethality. Foxd3; Pax3 compound mutant embryos have increased cell death in the neural folds and a drastic early reduction of NC cells, with an almost complete absence of NC caudal to the first pharyngeal arch. The genetic interaction between these genes implicates gene dosage-sensitive roles for Foxd3 and Pax3 in cardiac NC progenitors. Foxd3 and Pax3 act together to affect survival and maintenance of cardiac NC progenitors, and loss of these progenitors catastrophically affects key aspects of later cardiovascular development. genesis, 2011. © 2010 Wiley-Liss, Inc.

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation.

Modifications to the core histones are thought to contribute to ESC pluripotency by priming tissue-specific promoters and enhancers for later activation. However, it is unclear how these marks are targeted in ESCs and maintained during differentiation. Here, we show that the ESC factor Sox2 targets H3K4 methylation to monovalent and bivalent domains. In ESCs, Sox2 contributes to the formation of a monovalent mark at an enhancer in the pro/pre-B cell-specific lambda5-VpreB1 locus. Binding of Foxd3 suppresses intergenic transcription of the enhancer and surrounding sequences. In pro-B cells, enhancer activity is dependent on the Sox and Fox binding sites, and the enhancer is bound by Sox4, which is required for efficient expression of lambda5. Our results lead us to propose a factor relay model whereby ESC factors establish active epigenetic marks at tissue specific elements before being replaced by cell type-specific factors as cells differentiate.

Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6(CRE-IRES-GFP)(Dlx5/6(CIG)) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx(-/y);Dlx5/6(CIG) (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx(-/+);Dlx5/6(CIG)) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.

Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.

Understanding the molecular mechanisms of stem cell maintenance is crucial for the ultimate goal of manipulating stem cells for the treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3(-/-) embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue-specific deletion of Foxd3 in the neural crest, we show that Foxd3(flox/-); Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo.