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Cranial placodes are transient ectodermal thickenings that contribute to a diverse array of organs in the vertebrate head. They develop from a common territory, the pre-placodal region that over time segregates along the antero-posterior axis into individual placodal domains: the adenohypophyseal, olfactory, lens, trigeminal, otic, and epibranchial placodes. These placodes terminally differentiate into the anterior pituitary, the lens, and contribute to sensory organs including the olfactory epithelium, and inner ear, as well as several cranial ganglia. To study cranial placodes and their derivatives and generate cells for therapeutic purposes, several groups have turned to in vitro derivation of placodal cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). In this review, we summarize the signaling cues and mechanisms involved in cranial placode induction, specification, and differentiation in vivo, and discuss how this knowledge has informed protocols to derive cranial placodes in vitro. We also discuss the benefits and limitations of these protocols, and the potential of in vitro cranial placode modeling in regenerative medicine to treat cranial placode-related pathologies., Competing Interests: Declaration of competing interest The authors report no competing interests. The authors alone are responsible for the content and writing of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi., (© 2023. The Author(s).)

Epithelial-mesenchymal transition (EMT) is an early event in cell dissemination from epithelial tissues. EMT endows cells with migratory, and sometimes invasive, capabilities and is thus a key process in embryo morphogenesis and cancer progression. So far, matrix metalloproteinases (MMPs) have not been considered as key players in EMT but rather studied for their role in matrix remodelling in later events such as cell migration per se. Here, we used Xenopus neural crest cells to assess the role of MMP28 in EMT and migration in vivo. We show that a catalytically active MMP28, expressed by neighbouring placodal cells, is required for neural crest EMT and cell migration. We provide strong evidence indicating that MMP28 is imported in the nucleus of neural crest cells where it is required for normal Twist expression. Our data demonstrate that MMP28 can act as an upstream regulator of EMT in vivo raising the possibility that other MMPs might have similar early roles in various EMT-related contexts such as cancer, fibrosis, and wound healing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gouignard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 45th Annual Meeting at the Sanford Consortium for Regenerative Medicine at the University of California, San Diego on October 20th-21st, 2022. The meeting included presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Drs. Ralph Marcucio and Loydie Jerome-Majewska and four scientific sessions that highlighted new discoveries in signaling in craniofacial development, genomics of craniofacial development, human genetics of craniofacial development and translational and regenerative approaches in craniofacial biology. The meeting also included workshops on analysis of single cell RNA sequencing datasets and using human sequencing data from the Gabriella Miller Kids First Pediatric Research Program. There were 110 faculty and trainees in attendance that represent a diverse group of researchers from all career stages in the fields of developmental biology and genetics. The meeting, which also included outdoor poster presentations, provided opportunities for participant interactions and discussions, thus strengthening the SCGDB community., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: AD is affiliated with Regeneron Pharmaceuticals. The author has no financial interests to declare, HJ, CG, JS No competing interests declared, (© 2023, Devotta, Juraver-Geslin et al.)

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.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 44th Annual Meeting in a virtual format on October 18-19, 2021. The SCGDB meeting included presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Drs. Paul Trainor and Jeff Bush and four scientific sessions on the genomics of craniofacial development, craniofacial morphogenesis and regeneration, translational craniofacial biology and signaling during craniofacial development. The meeting also included workshops on professional development for faculty and trainees, National Institutes of Health (NIH)/National Institute of Craniofacial and Dental Research funding and usage of Genomics Software, as well as two poster sessions. An exhibitor booth run by FaceBase was also present to facilitate the upload and download of datasets relevant to the craniofacial community. Over 200 attendees from 12 countries and 23 states, representing over 80 different scientific institutions, participated. This diverse group of scientists included cell biologists, developmental biologists, and clinical geneticists. Although the continuing COVID-19 pandemic forced a virtual meeting format for a second year in a row, the meeting platform provided ample opportunities for participant interactions and discussions, thus strengthening the community., (© 2022 Wiley Periodicals LLC.)

Background: Development of paired sensory organs is a highly complex and coordinated process. These organs arise from ectodermal thickenings in the cephalic region known as cranial placodes. We have previously shown that Zic1 is a critical regulator for the formation of the pre-placodal region (PPR), the common territory for the development of all cranial placodes in Xenopus laevis., Results: In this study, we have analyzed a number of Zic1 targets for their expression during PPR patterning, as well as their regulation by retinoic acid (RA) and one of its major metabolites, 4-oxo-RA. Our findings show that anteriorly Zic1 regulates several transcription factors, Crx, Fezf2, Nkx3-1, and Xanf1 as well as a serine/threonine/tyrosine kinase, Pkdcc.2. These factors are all expressed in the vicinity of the PPR and as such are candidate regulators of placode formation downstream of Zic1. In addition to their differential regulation by RA, we find that 4-oxo-RA is also capable of modulating the expression of these genes, as well as a broad array of RA-regulated genes., Conclusion: Our data highlight the complexity of retinoid-mediated regulation required for Zic1-activated anterior structure specification in Xenopus, and the potential physiological role of 4-oxo-RA in cranial placode development., (© 2021 American Association for Anatomy.)

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., Competing Interests: YW, AD, DJ, JK, LN, KB, JA, JS, AD No competing interests declared, (© 2022, Wu et al.)

The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a network of transcription factors working in concert with chromatin modifiers. The high mobility group nucleosome binding protein 1 (Hmgn1) is a nonhistone chromatin architectural protein, associated with transcriptionally active chromatin. Here we report the expression and function of Hmgn1 during Xenopus neural crest and craniofacial development. Hmgn1 is broadly expressed at the gastrula and neurula stages, and is enriched in the head region at the tailbud stage, especially in the eyes and the pharyngeal arches. Hmgn1 knockdown affected the expression of several neural crest specifiers, including sox8, sox10, foxd3, and twist1, while other genes (sox9 and snai2) were only marginally affected. The specificity of this phenotype was confirmed by rescue, where injection of Hmgn1 mRNA was able to restore sox10 expression in morphant embryos. The reduction in neural crest gene expression at the neurula stage in Hmgn1 morphant embryos correlated with a decreased number of sox10- and twist1-positive cells in the pharyngeal arches at the tailbud stage, and hypoplastic craniofacial cartilages at the tadpole stage. These results point to a novel role for Hmgn1 in the control of gene expression essential for neural crest and craniofacial development. Future work will investigate the precise mode of action of Hmgn1 in this context., (© 2021 Wiley Periodicals LLC.)

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10 -10 ), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases., (© 2021. The Author(s).)

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In Xenopus , Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here, we have identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as being crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain. Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that pitx2c is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a program of RA containment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Matrix metalloproteinases (MMPs) are a large family of proteases comprising 24 members in vertebrates. They are well known for their extracellular matrix remodelling activity. MMP28 is the latest member of the family to be discovered. It is a secreted MMP involved in wound healing, immune system maturation, cell survival and migration. MMP28 is also expressed during embryogenesis in human and mouse. Here, we describe the detailed expression profile of MMP28 in Xenopus laevis embryos. We show that MMP28 is expressed maternally and accumulates at neurula and tail bud stages specifically in the cranial placode territories adjacent to migrating neural crest cells. As a secreted MMP, MMP28 may be required in neural crest-placode interactions. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

The spliceosome is a complex of RNA and proteins that function together to identify intron-exon junctions in precursor messenger-RNAs, splice out the introns, and join the flanking exons. Mutations in any one of the genes encoding the proteins that make up the spliceosome may result in diseases known as spliceosomopathies. While the spliceosome is active in all cell types, with the majority of the proteins presumably expressed ubiquitously, spliceosomopathies tend to be tissue-specific as a result of germ line or somatic mutations, with phenotypes affecting primarily the retina in retinitis pigmentosa, hematopoietic lineages in myelodysplastic syndromes, or the craniofacial skeleton in mandibulofacial dysostosis. Here we describe the major spliceosomopathies, review the proposed mechanisms underlying retinitis pigmentosa and myelodysplastic syndromes, and discuss how this knowledge may inform our understanding of craniofacial spliceosomopathies., (© 2020 Wiley Periodicals LLC.)

The neural crest is a multipotent population of cells that originates a variety of cell types. Many animal models are used to study neural crest induction, migration and differentiation, with amphibians and birds being the most widely used systems. A major technological advance to study neural crest development in mouse, chick and zebrafish has been the generation of transgenic animals in which neural crest specific enhancers/promoters drive the expression of either fluorescent proteins for use as lineage tracers, or modified genes for use in functional studies. Unfortunately, no such transgenic animals currently exist for the amphibians Xenopus laevis and tropicalis, key model systems for studying neural crest development. Here we describe the generation and characterization of two transgenic Xenopus laevis lines, Pax3-GFP and Sox10-GFP, in which GFP is expressed in the pre-migratory and migratory neural crest, respectively. We show that Pax3-GFP could be a powerful tool to study neural crest induction, whereas Sox10-GFP could be used in the study of neural crest migration in living embryos., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Hes3 belongs to the Hes basic helix-loop-helix family of transcriptional repressors that play central roles in maintaining progenitor cells and regulating binary cell fate decisions in the embryo. During Xenopus laevis development, hes3 is expressed in the embryonic ectoderm in a horseshoe shape domain at the edge of the developing neural pate. Hes3 mis-expression at early neurula stage blocks neural crest (snai2, sox8, sox9 and sox10) and cranial placode (six1 and dmrta1) gene expression, and promotes neural plate (sox2 and sox3) fate. At tailbud stage, these embryos exhibited a massive up-regulation of both sox8 and sox10 expression, associated with an increase in genes important for melanocytes differentiation (mitf and dct). Using a hormone inducible construct we show that Hes3 does not induce a pigment cell differentiation program de novo, rather it maintains progenitor cells in an undifferentiated state, and as Hes3 expression subsides overtime these cells adopt a pigment cell fate. We demonstrate that mechanistically Hes3 mediates its activity through inhibition of Wnt/β-catenin signaling, a molecular pathway critical for neural crest specification and pigment cell lineage differentiation. We propose that Hes3 at the edge of the neural plate spatially restricts the response to mesoderm-derived Wnt ligands, thereby contributing to the establishment of sharp boundaries of gene expression at the neural plate border., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/β-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/β-catenin signaling. Dkk2 depletion in Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or β-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and β-catenin, however unlike Wnt8, in a GSK3β independent manner. These findings suggest that Wnt8 and Dkk2 converge on β-catenin using distinct transduction pathways both independently required to activate Wnt/β-catenin signaling and induce neural crest cells., Competing Interests: AD, CH, JS No competing interests declared, (© 2018, Devotta et al.)

Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca 2+ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca 2+ safely remain unclear. Our previous work contradicted the dogma that Ca 2+ is ferried through the cytosol of Ca 2+ -transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca 2+ stores and their concomitant refilling by store-operated Ca 2+ entry (SOCE) mediated by Ca 2+ release activated Ca 2+ (CRAC) channels. Given that Ca 2+ 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 Ca 2+ concentration ([Ca 2+ ] 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 Ca 2+ imaging showed that CCK stimulation increased [Ca 2+ ] 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 Ca 2+ transcytosis paradigm for ER-based transport of bulk Ca 2+ . Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca 2+ transport.

The neural crest (NC) multipotent progenitor cells form at the neural plate border and migrate to diverse locations in the embryo to differentiate into many cell types. NC is specified by several embryonic pathways, however the role of noncanonical Wnt signaling in this process remains poorly defined. Daam1 is a formin family protein that is present in embryonic ectoderm at the time of NC formation and can mediate noncanonical Wnt signaling. Our interference experiments indicated that Daam1 is required for NC gene activation. To further study the function of Daam1 in NC development we used a transgenic reporter Xenopus line, in which GFP transcription is driven by sox10 upstream regulatory sequences. The activation of the sox10:GFP reporter in a subset of NC cells was suppressed after Daam1 depletion and in embryos expressing N-Daam1, a dominant interfering construct. Moreover, N-Daam1 blocked reporter activation in neuralized ectodermal explants in response to Wnt11, but not Wnt8 or Wnt3a, confirming that the downstream pathways are different. In complementary experiments, a constitutively active Daam1 fragment expanded the NC territory, but this gain-of-function activity was eliminated in a construct with a point mutation in the FH2 domain that is critical for actin polymerization. These observations suggest a new role of Daam1 and actin remodeling in NC specification., (© 2018 Wiley Periodicals, Inc.)

Retinoic acid (RA) is a vital morphogen for early patterning and organogenesis in the developing embryo. RA is a diffusible, lipophilic molecule that signals via nuclear RA receptor heterodimeric units that regulate gene expression by interacting with RA response elements in promoters of a significant number of genes. For precise RA signaling, a robust gradient of the morphogen is required. The developing embryo contains regions that produce RA, and specific intracellular concentrations of RA are created through local degradation mediated by Cyp26 enzymes. In order to elucidate the mechanisms by which RA executes precise developmental programs, the kinetics of RA metabolism must be clearly understood. Recent advances in techniques for endogenous RA detection and quantification have paved the way for mechanistic studies to shed light on downstream gene expression regulation coordinated by RA. It is increasingly coming to light that RA signaling operates not only at precise concentrations but also employs mechanisms of degradation and feedback inhibition to self-regulate its levels. A global gradient of RA throughout the embryo is often found concurrently with several local gradients, created by juxtaposed domains of RA synthesis and degradation. The existence of such local gradients has been found especially critical for the proper development of craniofacial structures that arise from the neural crest and the cranial placode populations. In this review, we summarize the current understanding of how local gradients of RA are established in the embryo and their impact on craniofacial development., (© 2018 Wiley Periodicals, Inc.)

During embryogenesis vertebrates develop a complex craniofacial skeleton associated with sensory organs. These structures are primarily derived from two embryonic cell populations the neural crest and cranial placodes, respectively. Neural crest cells and cranial placodes are specified through the integrated action of several families of signaling molecules, and the subsequent activation of a complex network of transcription factors. Here we describe the expression and function of Anosmin-1 (Anos1), an extracellular matrix protein, during neural crest and cranial placodes development in Xenopus laevis. Anos1 was identified as a target of Pax3 and Zic1, two transcription factors necessary and sufficient to generate neural crest and cranial placodes. Anos1 is expressed in cranial neural crest progenitors at early neurula stage and in cranial placode derivatives later in development. We show that Anos1 function is required for neural crest and sensory organs development in Xenopus, consistent with the defects observed in Kallmann syndrome patients carrying a mutation in ANOS1. These findings indicate that anos1 has a conserved function in the development of craniofacial structures, and indicate that anos1-depleted Xenopus embryos represent a useful model to analyze the pathogenesis of Kallmann syndrome., (Copyright © 2017. Published by Elsevier Inc.)

The transcription factors Pax3 and Zic1 are critical to specify the neural plate border and to promote neural crest formation. In a microarray screen designed to identify genes regulated by Pax3 and Zic1 in Xenopus we isolated Znf703/Nlz1 a transcriptional repressor member of the NET (NocA/Nlz, Elbow, and TLP-1) protein family. At early neurula stage znf703 is expressed in the dorsal ectoderm, spanning the neural plate and neural plate border, with an anterior boundary of expression corresponding to rhombomeres 3 and 4 (r3/r4) in the prospective hindbrain. As a bonafide target of Pax3 and Zic1, znf703 is activated by neural plate border inducing signals, and its expression depends on Pax3 and Zic1 function in the embryo. Znf703 morpholino-mediated knockdown expanded several posterior hindbrain genes, while Znf703 overexpression completely obliterated the expression of these segmental genes, signifying that the transcriptional repressor activity of Znf703 is critical to pattern the hindbrain. Furthermore, snai2 and sox10 expression was severely impaired upon manipulation of Znf703 expression levels in the embryo suggesting that Znf703 participates in neural crest formation downstream of Pax3 and Zic1 in Xenopus., (© 2017 Wiley Periodicals, Inc.)

Historically, techniques to analyze the localized distribution of mRNAs during development were performed on sectioned embryos using radioactively labeled riboprobes. The processing of the tissues and the use of emulsion autoradiography were laborious and time-consuming, leading to the development of more direct approaches. The nonradioactive whole-mount in situ hybridization method was first introduced in Drosophila embryos, and later adapted to Xenopus embryos for abundant transcripts such as muscle actin. Since then, the technique has been improved and is now broadly used for the spatial detection of even less abundant transcripts in Xenopus The technique has been especially powerful in the analysis of changes in gene expression in embryos manipulated by mRNA or antisense oligonucleotides microinjection, and in animal cap explants exposed to exogenous factors. The protocol described here provides an excellent signal-to-noise ratio for most labeled probes. It also is relatively high-throughput: With a little practice, approximately 50 samples can easily be processed simultaneously., (© 2017 Cold Spring Harbor Laboratory Press.)

Purpose of Review: Craniofacial disorders are among the most common human birth defects and present an enormous health care and social burden. The development of animal models has been instrumental to investigate fundamental questions in craniofacial biology and this knowledge is critical to understand the etiology and pathogenesis of these disorders., Recent Findings: The vast majority of craniofacial disorders arise from abnormal development of the neural crest, a multipotent and migratory cell population. Therefore, defining the pathogenesis of these conditions starts with a deep understanding of the mechanisms that preside over neural crest formation and its role in craniofacial development., Summary: This review discusses several studies using Xenopus embryos to model human craniofacial conditions, and emphasizes the strength of this system to inform important biological processes as they relate to human craniofacial development and disease., Competing Interests: Conflict of interest Aditi Dubey and Jean-Pierre Saint-Jeannet declare that they have no conflict of interest.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

Segmentation of the vertebrate hindbrain is an evolutionarily conserved process. Here, we identify the transcription factor early growth response 4 (egr4) as a novel regulator of posterior hindbrain development in Xenopus. egr4 is specifically and transiently expressed in rhombomeres 5 and 6 (r5/r6), and Egr4 knockdown causes a loss of mafb/kreisler and krox20/egr2 expression in r5/r6 and r5, respectively. This phenotype can be fully rescued by injection of frog or mouse Egr4 mRNA. Moreover Egr4-depleted embryos exhibit a specific loss of the neural crest stream adjacent to r5, and have inner ear defects. While the homeodomain protein vHnf1/Hnf1b directly activates Mafb and Krox20 expression in the mouse hindbrain to specify r5, we show that in Xenopus this process is indirect through the activation of Egr4. We provide evidence that rearrangements in the regulatory sequences around egr4 and mafb genes may account for this difference.

Transcription factors Pax3 and Zic1 are among the earliest genes activated at the neural plate border. In Xenopus, they are necessary and sufficient to promote the formation of multiple neural plate border cell types, including the neural crest, cranial placodes, and hatching gland. Pax3 is especially critical for the formation of the hatching gland, a group of cells that produce proteolytic enzymes essential to digest the egg vitelline envelope and jelly coat in order to release the tadpole into the environment. In a screen designed to identify downstream targets of Pax3, we isolated a member of the astacin family of metalloproteases, related to Xenopus hatching enzyme (Xhe), that we named Xhe2. Xhe2 is exclusively expressed in hatching gland cells as they first emerge at the lateral edge of the anterior neural plate, and persists in this tissue up to the tadpole stage. Knockdown experiments show that Xhe2 expression depends entirely on Pax3 function. Gain-of-function studies demonstrate that Pax3 can induce premature hatching through the upregulation of several proteolytic enzymes including Xhe2. Interestingly, Xhe2 overexpression is sufficient to induce early hatching, indicating that Xhe2 is one of the key components of the degradation mechanism responsible for breaking down the vitelline membrane., (© 2014 Wiley Periodicals, Inc.)

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., (© 2014 Wiley Periodicals, Inc.)

Specialized sensory organs in the vertebrate head originate from thickenings in the embryonic ectoderm called cranial sensory placodes. These placodes, as well as the neural crest, arise from a zone of ectoderm that borders the neural plate. This zone separates into a precursor field for the neural crest that lies adjacent to the neural plate, and a precursor field for the placodes, called the pre-placodal region (PPR), that lies lateral to the neural crest. The neural crest domain and the PPR are established in response to signaling events mediated by BMPs, FGFs and Wnts, which differentially activate transcription factors in these territories. In the PPR, members of the Six and Eya families, act in part to repress neural crest specific transcription factors, thus solidifying a placode developmental program. Subsequently, in response to environmental cues the PPR is further subdivided into placodal territories with distinct characteristics, each expressing a specific repertoire of transcription factors that provide the necessary information for their progression to mature sensory organs. In this review we summarize recent advances in the characterization of the signaling molecules and transcriptional effectors that regulate PPR specification and its subdivision into placodal domains with distinct identities., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The neural crest (NC) is a multipotent population of migratory cells unique to the vertebrate embryo, contributing to the development of multiple organ systems. Transcription factors pax3 and zic1 are among the earliest genes activated in NC progenitors, and they are both necessary and sufficient to promote NC fate. In order to further characterize the function of these transcription factors during NC development we have used hormone inducible fusion proteins in a Xenopus animal cap assay, and DNA microarray to identify downstream targets of Pax3 and Zic1. Here we present the results of this screen and the initial validation of these targets using quantitative RT-PCR, in situ hybridization and morpholinos-mediated knockdown. Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. We also obtained several factors with no known function in Xenopus NC, which represent novel regulators of NC fate. The comprehensive characterization of Pax3 and Zic1 targets function in the NC gene regulatory network, are essential to understanding the mechanisms regulating the emergence of this important cell population., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cranial placodes are thickenings of the embryonic head ectoderm that contribute to the paired sense organs and to the cephalic peripheral nervous system. Here we report the spatiotemporal expression pattern of transcription factor Pitx2c during Xenopus laevis cranial placode formation, focusing more specifically on key stages of trigeminal and profundal placode development. We also compare its expression to five genes that have been associated with development of these sensory placodes, namely Foxi1c, Islet1, NeuroD, Pax3, and Six1. We show that while initially expressed in both the trigeminal and profundal placodes, Pitx2c is later restricted to the prospective profundal ganglion, where it is co-expressed with Islet1, NeuroD and Pax3. This combination of factors defines a molecular signature for the characterization of the profundal versus trigeminal ganglia in Xenopus.

Background: Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3, and foxn1, and evaluated the neural crest contribution to thymus development., Results: In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T-cell differentiation in frogs., Conclusions: These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms., (Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.)

To illustrate the impact developmental biology and genetics have already had on the clinical management of the million infants born worldwide each year with CHD, we have chosen three stories which have had particular relevance for pediatric cardiologists, cardiothoracic surgeons, cardiac anesthesiologists, and cardiac nurses. First, we show how Margaret Kirby's finding of the unexpected contribution of an ectodermal cell population - the cranial neural crest - to the aortic arch arteries and arterial pole of the embryonic avian heart provided a key impetus to the field of cardiovascular patterning. Recognition that a majority of patients affected by the neurocristopathy DiGeorge syndrome have a chromosome 22q11 deletion, have also spurred tremendous efforts to characterize the molecular mechanisms contributing to this pathology, assigning a major role to the transcription factor Tbx1. Second, synthesizing the work of the last two decades by many laboratories on a wide gamut of metazoans (invertebrates, tunicates, agnathans, teleosts, lungfish, amphibians, and amniotes), we review the >20 major modifications and additions to the ancient circulatory arrangement composed solely of a unicameral (one-chambered), contractile myocardial tube and a short proximal aorta. Two changes will be discussed in detail - the interposition of a second cardiac chamber in the circulation and the septation of the cardiac ventricle. By comparing the developmental genetic data of several model organisms, we can better understand the origin of the various components of the multicameral (multi-chambered) heart seen in humans. Third, Martina Brueckner's discovery that a faulty axonemal dynein was responsible for the phenotype of the iv/iv mouse (the first mammalian model of human heterotaxy) focused attention on the biology of cilia. We discuss how even the care of the complex cardiac and non-cardiac anomalies seen in heterotaxy syndrome, which have long seemed impervious to advancements in surgical and medical intensive care, may yet yield to strategies grounded in a better understanding of the cilium. The fact that all cardiac defects seen in patients with full-blown heterotaxy can also be seen in patients without obvious laterality defects hints at important roles for ciliary function not only in left-right axis specification but also in cardiovascular morphogenesis. These three developmental biology stories illustrate how the remaining unexplained mortality and morbidity of congenital heart disease can be solved., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Lower vertebrates develop a unique set of primary sensory neurons located in the dorsal spinal cord. These cells, known as Rohon-Beard (RB) sensory neurons, innervate the skin and mediate the response to touch during larval stages. Here we report the expression and function of the transcription factor Xaml1/Runx1 during RB sensory neurons formation. In Xenopus embryos Runx1 is specifically expressed in RB progenitors at the end of gastrulation. Runx1 expression is positively regulated by Fgf and canonical Wnt signaling and negatively regulated by Notch signaling, the same set of factors that control the development of other neural plate border cell types, i.e. the neural crest and cranial placodes. Embryos lacking Runx1 function fail to differentiate RB sensory neurons and lose the mechanosensory response to touch. At early stages Runx1 knockdown results in a RB progenitor-specific loss of expression of Pak3, a p21-activated kinase that promotes cell cycle withdrawal, and of N-tub, a neuronal-specific tubulin. Interestingly, the pro-neural gene Ngnr1, an upstream regulator of Pak3 and N-tub, is either unaffected or expanded in these embryos, suggesting the existence of two distinct regulatory pathways controlling sensory neuron formation in Xenopus. Consistent with this possibility Ngnr1 is not sufficient to activate Runx1 expression in the ectoderm. We propose that Runx1 function is critically required for the generation of RB sensory neurons, an activity reminiscent of that of Runx1 in the development of the mammalian dorsal root ganglion nociceptive sensory neurons., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In vertebrate embryos, cardiac precursor cells of the primary heart field are specified in the lateral mesoderm. These cells converge at the ventral midline to form the linear heart tube, and give rise to the atria and the left ventricle. The right ventricle and the outflow tract are derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart has a single ventricle and an outflow tract with an incomplete spiral septum; however, it is unknown whether the cardiac neural crest is also involved in outflow tract septation, as in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to that described in birds and mammals. However, in contrast to what we see in amniotes, it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus, cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.

The Sox family of transcriptional regulators has been implicated in the control of a broad array of developmental processes. One member of this family SOX9 was first identified as a candidate gene for campomelic dysplasia (CD), a human syndrome affecting skeletal, and testis development. In these patients most endochondral bones of the face fail to develop resulting in multiple defects such as micrognathia, cleft palate, and facial dysmorphia. In this review we describe Sox9 expression during embryonic development and summarize loss of function experiments in frog, fish, and mouse embryos highlighting the role of Sox9 in regulating morphogenesis of the face. We also discuss the mutations in and around SOX9 responsible for craniofacial defects in CD patients., (Copyright © 2011 Wiley-Liss, Inc.)

Runx genes encode a family of proteins defined by the highly conserved runt DNA-binding domain. Studies in several organisms have shown that these transcription factors regulate multiple aspects of embryonic development and are responsible for the pathogenesis of several human diseases. Here we report the cloning and expression of Runx3 during Xenopus development and compare its expression pattern to other Runx family members, Runx1 and Runx2, and to Cbfbeta, the obligatory binding partner of Runx proteins. Using in situ hybridization in the whole embryo and on sections we show that Runx3 is co-expressed with Runx1 in the hematopoietic lineage and in Rohon-Beard sensory neurons. In contrast Runx3 and Runx2 are co-expressed in craniofacial cartilage elements. Runx3 shows also unique expression domains in a number of derivatives of the neurogenic placodes, including the ganglia of the anteroposterior and middle lateral line nerves, and ganglia of the trigeminal, glossopharyngeal, facial and vagal nerves. These observations suggest a critical role for Runx3 in the development of cranial sensory neurons, while in other tissues its co-expression with Runx1 or Runx2 may signify functional redundancy between these family members., (Copyright 2010 Elsevier B.V. All rights reserved.)

The transcription factor Sox9 has been implicated in inner ear formation in several species. To investigate the long-term consequences of Sox9 depletion on inner ear development we analyzed the inner ear architecture of Sox9-depleted Xenopus tadpoles generated by injection of increasing amounts of Sox9 morpholino antisense oligonucleotides. We found that Sox9-depletion resulted in major defects in the development of vestibular structures, semicircular canals and utricle, while the ventrally located saccule was less severely affected in these embryos. Consistent with this phenotype, we observed a specific loss of the dorsal expression of Wnt3a expression in the otic vesicle of Sox9 morphants, associated with an increase in cell death and a reduction in cell proliferation in the region of the presumptive otic epithelium. We propose that, in addition to its early role in placode specification, Sox9 is also required for the maintenance of progenitors in the otic epithelium.

The valves and septa of the mature heart are derived from the cardiac cushions, which develop from discrete swellings in two regions of developing heart tube: the atrioventricular (AV) canal and the ventricular outflow tract (OFT). In higher vertebrates, three distinct lineages contribute to the heart valves and septa, the endocardium, the myocardium, and the cardiac neural crest that will populate the cardiac jelly of the OFT. Very little is known about cardiac cushions development in amphibians. Here, we describe the expression of eight genes during key stages of cardiac cushion development in Xenopus. Among these genes, the Wnt antagonist Frzb1 and the transcription factors Xl-Fli, Sox8, Sox9, and Sox10 are differentially expressed in the mesenchyme of the OFT and AV cushions. These genes can be used in combination with lineage-tracing experiments to determine the embryonic origin of the cardiac cushions mesenchyme in Xenopus., ((c) 2009 Wiley-Liss, Inc.)