RESUMEN
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.
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Transición Epitelial-Mesenquimal , Cresta Neural , Movimiento Celular , Núcleo Celular , EpitelioRESUMEN
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.
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Ectodermo , Cráneo , Animales , Humanos , Vertebrados , Diferenciación Celular , Transducción de Señal , Regulación del Desarrollo de la Expresión GénicaRESUMEN
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.
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Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Homeodominio/metabolismo , Organogénesis , Factores de Transcripción/metabolismo , Tretinoina/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Sistema Enzimático del Citocromo P-450/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Inmunohistoquímica , Modelos Biológicos , Organogénesis/genética , Fenotipo , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 46th Annual Meeting at Cincinnati Children's Hospital Medical Center in Cincinnati, Ohio on October 10th-12th, 2023. On the first day of the meeting, Drs. Sally Moody and Justin Cotney were each honored with the SCGDB Distinguished Scientist Awards for their exceptional contributions to the field of craniofacial biology. The following two days of the meeting featured five sessions that highlighted new discoveries in signaling and genomic mechanisms regulating craniofacial development, human genetics, translational and regenerative approaches, and clinical management of craniofacial differences. Interactive workshops on spatial transcriptomics and scientific communication, as well as a poster session facilitated meaningful interactions among the 122 attendees representing diverse career stages and research backgrounds in developmental biology and genetics, strengthened the SCGDB community.
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Anomalías Craneofaciales , Biología Evolutiva , Humanos , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patologíaRESUMEN
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.
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Distinciones y Premios , Genómica , Niño , Humanos , Biología Evolutiva , Congresos como AsuntoRESUMEN
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.
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Regulación del Desarrollo de la Expresión Génica , Tretinoina , Animales , Ectodermo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacología , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
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.
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Pérdida Auditiva/genética , Disostosis Mandibulofacial/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Sordera/genética , Modelos Animales de Enfermedad , Oído Interno/metabolismo , Ectodermo/metabolismo , Desarrollo Embrionario/genética , Ganglios Parasimpáticos/embriología , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Pérdida Auditiva/fisiopatología , Disostosis Mandibulofacial/metabolismo , Disostosis Mandibulofacial/fisiopatología , Cresta Neural/embriología , Factores de Empalme de ARN/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
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.
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COVID-19 , Pandemias , Biología Evolutiva , Genómica , Humanos , Programas Informáticos , Estados UnidosRESUMEN
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.
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Desarrollo Embrionario/genética , Proteína HMGN1/genética , Cresta Neural/crecimiento & desarrollo , Factores de Transcripción SOXE/genética , Proteína 1 Relacionada con Twist/genética , Proteínas de Xenopus/genética , Animales , Cromatina/genética , Embrión no Mamífero , Factores de Transcripción Forkhead/genética , Gástrula/crecimiento & desarrollo , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Proteína HMGN1/antagonistas & inhibidores , Cresta Neural/metabolismo , Factor de Transcripción SOX9/genética , Factores de Transcripción/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 43rd annual meeting in a virtual format on October 19-20, 2020. The SCGDB meeting included the presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Marilyn Jones and Kerstin Ludwig and four scientific sessions on the molecular regulation of craniofacial development, craniofacial morphogenesis, translational craniofacial biology, and signaling during craniofacial development. The meeting also included workshops on career development, NIH/NIDCR funding, and the utility of the FaceBase database, as well as two poster sessions. Over 190 attendees from 21 states, representing over 50 different scientific institutions, participated. This diverse group of scientists included cell biologists, developmental biologists, and clinical geneticists. While in-person interactions were missed due to the virtual meeting format imposed by the COVID-19 pandemic, the meeting platform provided ample opportunities for participant interactions and discussions, thus strengthening the community.
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Anomalías Craneofaciales/genética , Biología Evolutiva , Animales , COVID-19 , Congresos como Asunto/organización & administración , Anomalías Craneofaciales/embriología , Genética Médica , Humanos , Pandemias , Sociedades Médicas/organización & administración , Sociedades Científicas/organización & administración , Comunicación por VideoconferenciaRESUMEN
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.
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Disostosis Mandibulofacial , Mutación , Síndromes Mielodisplásicos , Retinitis Pigmentosa , Empalmosomas , Animales , Humanos , Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/metabolismo , Disostosis Mandibulofacial/patología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Empalmosomas/genética , Empalmosomas/metabolismo , Empalmosomas/patologíaRESUMEN
The Society for Craniofacial Genetics and Developmental Biology (SCGDB) 42nd Annual Meeting was held at the MD Anderson Cancer Center in Houston, Texas from October 14-15, 2019. The SCGDB meeting included scientific sessions on the molecular regulation of craniofacial development, cell biology of craniofacial development, signaling during craniofacial development, translational craniofacial biology, and for the first time, a career development workshop. Over a one hundred attendees from 21 states, and representing over 50 different scientific institutions, participated. The diverse group of scientists included cell and developmental biologists and clinical geneticists, promoting excellent discussions about molecular pathways guiding abnormal cell behaviors and the resultant morphological changes to craniofacial development. The results were high-quality science and a welcoming environment for trainees interested in craniofacial biology.
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Anomalías Craneofaciales/genética , Biología Evolutiva , Animales , Distinciones y Premios , Selección de Profesión , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Cresta Neural/patología , Cresta Neural/fisiología , Sociedades Científicas , Xenopus/genética , Xenopus/crecimiento & desarrolloRESUMEN
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/ß-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.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Desarrollo Embrionario , Secuencias Hélice-Asa-Hélice , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Placa Neural/citología , Filogenia , Factores de Transcripción SOXB1/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
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.
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Cresta Neural/metabolismo , Factor de Transcripción PAX3/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica/genética , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes , Humanos , Cresta Neural/embriología , Cresta Neural/fisiología , Neurogénesis , Factor de Transcripción PAX3/fisiología , Factores de Transcripción SOXE/fisiología , Xenopus laevis/embriologíaRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Cresta Neural/embriología , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/fisiología , Animales , Ectodermo/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Placa Neural/metabolismo , Neurogénesis , Factores de Transcripción SOXE/genética , Transducción de Señal , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
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.
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Comunicación Celular , Desarrollo Fetal , Organogénesis , Cráneo/embriología , Cráneo/metabolismo , Tretinoina/metabolismo , Animales , Biomarcadores , Catálisis , Comunicación Celular/genética , Familia 26 del Citocromo P450/genética , Familia 26 del Citocromo P450/metabolismo , Desarrollo Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Cinética , Morfogénesis , Cresta Neural/embriología , Cresta Neural/metabolismo , Organogénesis/genética , Transducción de Señal , Tretinoina/químicaRESUMEN
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.
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Desarrollo Embrionario/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismo , Neurogénesis/fisiología , Cráneo/embriología , Cráneo/metabolismo , Proteínas de Xenopus/metabolismo , Animales , XenopusRESUMEN
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.
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Cresta Neural/crecimiento & desarrollo , Neurogénesis/genética , Proteínas Nucleares/genética , Factor de Transcripción PAX3/genética , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Morfolinos/genética , Cresta Neural/metabolismo , Rombencéfalo/crecimiento & desarrollo , Rombencéfalo/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción de la Familia Snail/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
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.