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During positive selection at the transition from CD4+CD8+ doublepositive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing. [ABSTRACT FROM AUTHOR]

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail-chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca2+ homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin;bothFoxa2 andE-cadherinwere re-expressed atmetastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification. The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development. [ABSTRACT FROM AUTHOR]

Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the fork head/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons. [ABSTRACT FROM AUTHOR]

The transcription factors Foxa1 and Foxa2 promote the specification of midbrain dopaminergic (mDA) neurons and the floor plate. Whether their role is direct has remained unclear as they also regulate the expression of Shh, which has similar roles. We characterized the Foxa2 cis-regulatory network by chromatin immunoprecipitation followed by high-throughput sequencing of mDA progenitors. This identified 9160 high-quality Foxa2 binding sites associated with 5409 genes, providing mechanistic insights into Foxa2-mediated positive and negative regulatory events. Foxa2 regulates directly and positively key determinants of mDA neurons, including Lmx1a, Lmx1b, Msx1 and Ferd3l, while negatively inhibiting transcription factors expressed in ventrolateral midbrain such as Helt, Tle4, Otx1, Sox1 and Tal2. Furthermore, Foxa2 negatively regulates extrinsic and intrinsic components of the Shh signaling pathway, possibly by binding to the same enhancer regions of co-regulated genes as Gli1. Foxa2 also regulates the expression of floor plate factors that control axon trajectories around the midline of the embryo, thereby contributing to the axon guidance function of the floor plate. Finally, this study identified multiple Foxa2-regulated enhancers that are active in the floor plate of the midbrain or along the length of the embryo in mouse and chick. This work represents the first comprehensive characterization of Foxa2 targets in mDA progenitors and provides a framework for elaborating gene regulatory networks in a functionally important progenitor population. [ABSTRACT FROM AUTHOR]

We have previously shown that a forkhead transcription factor Foxal interacts with androgen signaling and controls prostate differentiated response. Here, we show the mouse Foxal expression marks the entire embryonic urogenital sinus epithelium (UGE), contrasting with Shh and Foxa2, which are restricted to the basally located cells during prostate budding. The Foxal-deficient mouse prostate shows a severely altered ductal pattern that resembles primitive epithelial cords surrounded by thick stromal layers. Characterization of these mutant cells indicates a population of basal-like cells similar to those found in the embryonic UGE, whereas no differentiated or mature luminal epithelial cells are found in Foxal-deficient epithelium. These phenotypic changes are accompanied with molecular aberrations, including focal epithelial activation of Shh and elevated Foxa2 and Notchl in the null epithelium. Perturbed epithelial-stromal interactions induced by Foxal-deficient epithelium is evident, as demonstrated by the expansion of surrounding smooth muscle and elevated levels of stromal factors (Bmp4, Fgf7, Fgf10 and Gli). The prostatic homeobox protein Nkx3.1, a known proliferation inhibitor, was downregulated in Foxal-deficient epithelial cells, while several prostate-specific androgen-regulated markers, including a novel Foxal target, are absent in the null prostate. These data indicate that Foxal plays a pivotal role in controlling prostate morphogenesis and cell differentiation. [ABSTRACT FROM AUTHOR]

Birth defects, which occur in one out of 20 live births, often affect multiple organs that have common developmental origins. Human and mouse studies indicate that haploinsufficiency of the transcription factor TBX1 disrupts pharyngeal arch development, resulting in the cardiac and craniofacial features associated with microdeletion of 22q11 (del22q11), the most frequent human deletion syndrome. Here, we have generated an allelic series of Tbx1 deficiency that reveals a lower critical threshold for Tbx1 activity in the cardiac outflow tract compared with other pharyngeal arch derivatives, including the palatal bones. Mice hypomorphic for Tbx1 failed to activate expression of the forkhead transcription factor Foxa2 in the pharyngeal mesoderm, which contains cardiac outflow precursors derived from the anterior heart field. We identified a Fox-binding site upstream of Tbx1 that interacted with Foxa2 and was necessary for pharyngeal mesoderm expression of Tbx1, revealing an autoregulatory loop that may explain the increased cardiac sensitivity to Tbx1 dose. Downstream of Tbx1, we found a fibroblast growth factor 8 (Fgf8) enhancer that was dependent on Tbx1 in vivo for regulating expression in the cardiac outflow tract, but not in pharyngeal arches. Consistent with its role in regulating cardiac outflow tract cells Tbx1 gain of function resulted in expansion of the cardiac outflow tract segment derived from the anterior heart field as marked by Fgfl0. These findings reveal a Tbx1-dependent transcriptional and signaling network in the cardiac outflow tract that renders mouse cardiovascular development more susceptible than craniofacial development to a reduction in Tbx1 dose, similar to humans with del22q11. [ABSTRACT FROM AUTHOR]

The regenerative capacity of many placode-derived epithelial structures makes them of interest for understanding the molecular control of epithelial stem cells and their niches. Here, we investigate the interaction between the developing epithelium and its surrounding mesenchyme in one such system, the taste papillae and sensory taste buds of the mouse tongue. We identify follistatin (FST) as a mesenchymal factor that controls size, patterning and gustatory cell differentiation in developing taste papillae. FST limits expansion and differentiation of Sox2-expressing taste progenitor cells and negatively regulates the development of taste papillae in the lingual epithelium: in Fst-/- tongue, there is both ectopic development of Sox2-expressing taste progenitors and accelerated differentiation of gustatory cells. Loss of Fst leads to elevated activity and increased expression of epithelial Bmp7; the latter effect is consistent with BMP7 positive autoregulation, a phenomenon we demonstrate directly. We show that FST and BMP7 influence the activity and expression of other signaling systems that play important roles in the development of taste papillae and taste buds. In addition, using computational modeling, we show how aberrations in taste papillae patterning in Fst-/- mice could result from disruption of an FST-BMP7 regulatory circuit that normally suppresses noise in a process based on diffusion-driven instability. Because inactivation of Bmp7 rescues many of the defects observed in Fst-/- tongue, we conclude that interactions between mesenchyme-derived FST and epithelial BMP7 play a central role in the morphogenesis, innervation and maintenance of taste buds and their stem/progenitor cells. [ABSTRACT FROM AUTHOR]

In the mouse, one of the earliest events in the determination of cell fate is the segregation of cells into germ layers during gastrulation; however, the cellular and molecular details are not well defined due to intrauterine development. We were able to visualize a clear sequence of events occurring in the process of germ-layer formation, using immunohistochemistry and time-lapse confocal imaging. The T-box transcription factor brachyury (T) and the Forkhead transcription factor Foxa2 specify mesoderm and endoderm in the posterior epiblast. Fate-specified epiblast cells lose their polarity and undergo epithelial-mesenchymal transition to invade into the primitive streak region, where these cell populations quickly separate and differentiate into morphologically and molecularly distinct Foxa2-positive endoderm and T-positive mesoderm populations. The endoderm cells flatten and acquire apical-basal polarity during intercalation into the outside epithelium in order to establish proper intracellular junctions with pre-existing cells. By contrast, the mesodermal cells become spherical during migration and acquire a mesenchymal fate. Interestingly, axial mesodermal cells are descended from Foxa2-positive epiblast cells that upregulate T protein in the anterior primitive streak region. These cells, as well as Foxa2-positive endoderm cells, are highly polarized and epithelialized, suggesting that Foxa2 promotes an epithelial fate and suppresses a mesenchymal fate. This observation is supported by the fact that Foxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium. We propose that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype. [ABSTRACT FROM AUTHOR]

The liver and pancreas are specified from the foregut endoderm through an interaction with the adjacent mesoderm. However, the earlier molecular mechanisms that establish the foregut precursors are largely unknown. In this study, we have identified a molecular pathway linking gastrula-stage endoderm patterning to organ specification. We show that in gastrula and early-somite stage Xenopus embryos, Wnt/ß-catenin activity must be repressed in the anterior endoderm to maintain foregut identity and to allow liver and pancreas development. By contrast, high ß-catenin activity in the posterior endoderm inhibits foregut fate while promoting intestinal development. Experimentally repressing ß-catenin activity in the posterior endoderm was sufficient to induce ectopic organ buds that express early liver and pancreas markers. ß-catenin acts in part by inhibiting expression of the homeobox gene hhex, which is one of the earliest foregut markers and is essential for liver and pancreas development. Promoter analysis indicates that ß-catenin represses hhex transcription indirectly via the homeodomain repressor Vent2. Later in development, ß-catenin activity has the opposite effect and enhances liver development. These results illustrate that turning Wnt signaling off and on in the correct temporal sequence is essential for organ formation, a finding that might directly impact efforts to differentiate liver and pancreas tissue from stem cells. [ABSTRACT FROM AUTHOR]

Epithelial cells lining the peripheral lung synthesize pulmonary surfactant that reduces surface tension at the air-liquid interface. Lack of surfactant lipids and proteins in the lungs causes respiratory distress syndrome, a common cause of morbidity and mortality in preterm infants. We show that C/EBPα plays a crucial role in the maturation of the respiratory epithelium in late gestation, being required for the production of surfactant lipids and proteins necessary for lung function. Deletion of the Cebpa gene in respiratory epithelial cells in fetal mice caused respiratory failure at birth. Structural and biochemical maturation of the lung was delayed. Normal synthesis of surfactant lipids and proteins, including SP-A, SP-B, SP-C, SP-D, ABCA3 (a lamellar body associated protein) and FAS (precursor of fatty acid synthesis) were dependent upon expression of the C/EBPα in respiratory epithelial cells. Deletion of the Cebpa gene caused increased expression of Tgfb2, a growth factor that inhibits lung epithelial cell proliferation and differentiation. Normal expression of C/EBPα required Titf1 and Foxa2, transcription factors that also play an important role in perinatal lung differentiation. C/EBPα participates in a transcriptional network that is required for the regulation of genes mediating perinatal lung maturation and surfactant homeostasis that is necessary for adaptation to air breathing at birth. [ABSTRACT FROM AUTHOR]