Sf3b4-depleted Xenopus embryos: A model to study the pathogenesis of craniofacial defects in Nager syndrome.

Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause of Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4.

Developmental roles of natriuretic peptides and their receptors.

Natriuretic peptides and their receptors are implicated in the physiological control of blood pressure, bone growth, and cardiovascular and renal homeostasis. They mediate their action through the modulation of intracellular levels of cGMP and cAMP, two second-messengers that have broad biological roles. In this review, we briefly describe the major players of this signaling pathway and their physiological roles in the adult, and discuss several reports describing their activity in the control of various aspects of embryonic development in several species. While the core components of this signaling pathway are well conserved, their functions have diverged in the embryo and the adult to control a diverse array of biological processes.

Npr3 regulates neural crest and cranial placode progenitors formation through its dual function as clearance and signaling receptor.

Natriuretic peptide signaling has been implicated in a broad range of physiological processes, regulating blood volume and pressure, ventricular hypertrophy, fat metabolism, and long bone growth. Here, we describe a completely novel role for natriuretic peptide signaling in the control of neural crest (NC) and cranial placode (CP) progenitors formation. Among the components of this signaling pathway, we show that natriuretic peptide receptor 3 (Npr3) plays a pivotal role by differentially regulating two developmental programs through its dual function as clearance and signaling receptor. Using a combination of MO-based knockdowns, pharmacological inhibitors and rescue assays we demonstrate that Npr3 cooperate with guanylate cyclase natriuretic peptide receptor 1 (Npr1) and natriuretic peptides (Nppa/Nppc) to regulate NC and CP formation, pointing at a broad requirement of this signaling pathway in early embryogenesis. We propose that Npr3 acts as a clearance receptor to regulate local concentrations of natriuretic peptides for optimal cGMP production through Npr1 activation, and as a signaling receptor to control cAMP levels through inhibition of adenylyl cyclase. The intracellular modulation of these second messengers therefore participates in the segregation of NC and CP cell populations.

The Core Splicing Factors EFTUD2, SNRPB and TXNL4A Are Essential for Neural Crest and Craniofacial Development.

Mandibulofacial dysostosis (MFD) is a human congenital disorder characterized by hypoplastic neural-crest-derived craniofacial bones often associated with outer and middle ear defects. There is growing evidence that mutations in components of the spliceosome are a major cause for MFD. Genetic variants affecting the function of several core splicing factors, namely SF3B4, SF3B2, EFTUD2, SNRPB and TXNL4A, are responsible for MFD in five related but distinct syndromes known as Nager and Rodriguez syndromes (NRS), craniofacial microsomia (CFM), mandibulofacial dysostosis with microcephaly (MFDM), cerebro-costo-mandibular syndrome (CCMS) and Burn-McKeown syndrome (BMKS), respectively. Animal models of NRS and MFDM indicate that MFD results from an early depletion of neural crest progenitors through a mechanism that involves apoptosis. Here we characterize the knockdown phenotype of Eftud2, Snrpb and Txnl4a in Xenopus embryos at different stages of neural crest and craniofacial development. Our results point to defects in cranial neural crest cell formation as the likely culprit for MFD associated with EFTUD2, SNRPB and TXNL4A haploinsufficiency, and suggest a commonality in the etiology of these craniofacial spliceosomopathies.

Xbp1 and Brachyury establish an evolutionarily conserved subcircuit of the notochord gene regulatory network.

Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.

Dkk2 promotes neural crest specification by activating Wnt/ß-catenin signaling in a GSK3ß independent manner.

Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/ß-catenin signaling by Wnt8 is especially critical. Glycoproteins of the Dickkopf (Dkk) family are important modulators of Wnt signaling acting primarily as Wnt antagonists. Here we report that Dkk2 is required for neural crest specification functioning as a positive regulator of Wnt/ß-catenin signaling. Dkk2 depletion in Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or ß-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and ß-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and ß-catenin, however unlike Wnt8, in a GSK3ß independent manner. These findings suggest that Wnt8 and Dkk2 converge on ß-catenin using distinct transduction pathways both independently required to activate Wnt/ß-catenin signaling and induce neural crest cells.

Evidence That Calcium Entry Into Calcium-Transporting Dental Enamel Cells Is Regulated by Cholecystokinin, Acetylcholine and ATP.

Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca2+ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca2+ safely remain unclear. Our previous work contradicted the dogma that Ca2+ is ferried through the cytosol of Ca2+-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca2+ stores and their concomitant refilling by store-operated Ca2+ entry (SOCE) mediated by Ca2+ release activated Ca2+ (CRAC) channels. Given that Ca2+ handling is maximal during the enamel-mineralization stage (maturation), we anticipated that SOCE would also be elevated then. Confirmation was obtained here using single-cell recordings of cytosolic Ca2+ concentration ([Ca2+]cyt) in rat ameloblasts. A candidate SOCE agonist, cholecystokinin (CCK), was found to be upregulated during maturation, with Cck transcript abundance reaching 30% of that in brain. CCK-receptor transcripts were also detected and Ca2+ imaging showed that CCK stimulation increased [Ca2+]cyt in a dose-responsive manner that was sensitive to CRAC-channel inhibitors. Similar effects were observed with two other SOCE activators, acetylcholine and ATP, whose receptors were also found in enamel cells. These results provide the first evidence of a potential regulatory system for SOCE in enamel cells and so strengthen the Ca2+ transcytosis paradigm for ER-based transport of bulk Ca2+. Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca2+ transport.

Zic1 controls placode progenitor formation non-cell autonomously by regulating retinoic acid production and transport.

All cranial placode progenitors arise from a common precursor field anterior to the neural plate, the pre-placodal region (PPR). We showed that transcription factor Zic1, expressed at the anterior neural plate, is necessary and sufficient to promote placode fate. Here we reveal the non-cell autonomous activity of Zic1 and implicate retinoic acid (RA) signalling as a key player in cranial placode progenitor specification. In a screen for genes activated by Zic1, we identify several factors involved in RA metabolism and function. Among them we show that retinaldehyde dehydrogenase 2 (RALDH2) and lipocalin-type prostaglandin D2 synthase (LPGDS), which, respectively, regulate the synthesis and transport of RA, directly participate in the establishment of the PPR. We propose that RALDH2 and LPGDS induction by Zic1 at the anterior neural plate allows for the localized production and transport of RA, which in turn activates a cranial placode developmental programme in neighbouring cells.

Transcription factor AP2 epsilon (Tfap2e) regulates neural crest specification in Xenopus.

Transcription factors Pax3 and Zic1 are two important regulators of cell fate decision at the neural plate border, where they act synergistically to promote neural crest (NC) formation. To understand the role of these factors in NC development, we performed a microarray analysis to identify downstream targets of Pax3 and Zic1 in Xenopus embryos. Among the genes identified was a member of transcription factor activator protein 2 (Tfap2) family, Tfap2 epsilon (Tfap2e). Tfap2e is first expressed at early neurula stage in NC progenitors and Rohon-Beard sensory neurons, and persists in a subset of migrating cranial NC cells as they populate the pharyngeal arches. This is in contrast to other species in which Tfap2e is not detected in the early NC lineage. Tfap2e morpholino-mediated knockdown results in a loss of NC progenitors and an expansion of the neural plate. Tfap2e is also sufficient to activate NC-specific genes in animal cap explants, and gain-of-function experiments in the whole embryo indicate that Tfap2e can promote NC formation. We propose that Tfap2e is a novel player in the gene regulatory network controlling NC specification in Xenopus downstream of Pax3 and Zic1.