RESUMEN
Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.
Asunto(s)
Quinasas DyrK , Proteínas de Xenopus , Xenopus laevis , Animales , Región Branquial/embriología , Región Branquial/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/metabolismo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/embriología , Cresta Neural/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
The dynamics of multipotent neural crest cell differentiation and invasion as cells travel throughout the vertebrate embryo remain unclear. Here, we preserve spatial information to derive the transcriptional states of migrating neural crest cells and the cellular landscape of the first four chick cranial to cardiac branchial arches (BA1-4) using label-free, unsorted single-cell RNA sequencing. The faithful capture of branchial arch-specific genes led to identification of novel markers of migrating neural crest cells and 266 invasion genes common to all BA1-4 streams. Perturbation analysis of a small subset of invasion genes and time-lapse imaging identified their functional role to regulate neural crest cell behaviors. Comparison of the neural crest invasion signature to other cell invasion phenomena revealed a shared set of 45 genes, a subset of which showed direct relevance to human neuroblastoma cell lines analyzed after exposure to the in vivo chick embryonic neural crest microenvironment. Our data define an important spatio-temporal reference resource to address patterning of the vertebrate head and neck, and previously unidentified cell invasion genes with the potential for broad impact.
Asunto(s)
Región Branquial/crecimiento & desarrollo , Cabeza/crecimiento & desarrollo , Cuello/crecimiento & desarrollo , Cresta Neural/crecimiento & desarrollo , Animales , Tipificación del Cuerpo/genética , Región Branquial/embriología , Diferenciación Celular/genética , Movimiento Celular/genética , Microambiente Celular/genética , Embrión de Pollo , Embrión de Mamíferos , Embrión no Mamífero , Desarrollo Embrionario/genética , Cabeza/embriología , Humanos , Mesodermo/crecimiento & desarrollo , Células Madre Multipotentes/citología , Cuello/embriología , Cresta Neural/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Organogénesis/genética , Microambiente Tumoral/genética , Vertebrados/genética , Vertebrados/crecimiento & desarrolloRESUMEN
Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.
Asunto(s)
Tipificación del Cuerpo , Cara/embriología , Factor de Transcripción GATA3/metabolismo , Animales , Región Branquial/citología , Región Branquial/embriología , Región Branquial/metabolismo , Muerte Celular , Proliferación Celular , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Embrión de Mamíferos , Factor de Transcripción GATA3/genética , Regulación del Desarrollo de la Expresión Génica , Mandíbula/citología , Mandíbula/embriología , Maxilar/citología , Maxilar/embriología , Ratones , Morfogénesis , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismoRESUMEN
OBJECTIVE: To elucidate the branchial origin of the articular and the square (homology of the malleus and the incus of mammals), we used immunohistochemistry to analyse the expression of the Hox-A2 protein during cephalogenesis in chickens. MATERIALS AND METHODS: Immunohistochemistry on paraffin sections of embryos from stage HH16 to HH40. RESULTS: In addition to the columella (equivalent to the mammalian stapes), the joint between the articular and the quadrate bones, and the retro-articular process of the articular (homologous to the short process of the malleus) express Hox-A2, suggesting an intervention of the 2nd arch in their formation. However, we fortuitously observed very intense expression within the early muscle plate of the second arch, which then generalized to all cephalic muscles, and extended to the trunk's myotomes. In the cartilage, the presence of the protein disappeared at stage 35. DISCUSSION AND CONCLUSION: The present results, while confirming the contribution of the second arch to the development of avian equivalents of the mammalian ear ossicles, strongly suggest that the Hox-A2 gene plays a role in muscle development, which remains to be elucidated by more sophisticated techniques.
Asunto(s)
Cartílago , Proteínas de Homeodominio , Animales , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Embrión de Pollo , Cartílago/metabolismo , Cartílago/embriología , Pollos/metabolismo , Pollos/genética , Maxilares/embriología , Maxilares/metabolismo , Región Branquial/metabolismo , Región Branquial/embriología , Desarrollo de Músculos , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Músculo Esquelético/metabolismo , Músculo Esquelético/embriologíaRESUMEN
Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.
Asunto(s)
Región Branquial/embriología , Nervios Craneales/embriología , Ectodermo/embriología , Receptores Notch/fisiología , Animales , Tipificación del Cuerpo , Diferenciación Celular , Linaje de la Célula , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genotipo , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Fenotipo , Dominios Proteicos , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.
Asunto(s)
Acuaporina 1/fisiología , Movimiento Celular/fisiología , Cresta Neural/citología , Seudópodos/fisiología , Animales , Región Branquial/citología , Región Branquial/embriología , Membrana Celular/fisiología , Microambiente Celular , Embrión de Pollo , Biología Computacional , Adhesiones Focales , Cresta Neural/embriología , Receptor EphB1/metabolismo , Receptor EphB3/metabolismoRESUMEN
Vertebrate heart development requires the integration of temporally distinct differentiating progenitors. However, few signals are understood that restrict the size of the later-differentiating outflow tract (OFT). We show that improper specification and proliferation of second heart field (SHF) progenitors in zebrafish lazarus (lzr) mutants, which lack the transcription factor Pbx4, produces enlarged hearts owing to an increase in ventricular and smooth muscle cells. Specifically, Pbx4 initially promotes the partitioning of the SHF into anterior progenitors, which contribute to the OFT, and adjacent endothelial cell progenitors, which contribute to posterior pharyngeal arches. Subsequently, Pbx4 limits SHF progenitor (SHFP) proliferation. Single cell RNA sequencing of nkx2.5+ cells revealed previously unappreciated distinct differentiation states and progenitor subpopulations that normally reside within the SHF and arterial pole of the heart. Specifically, the transcriptional profiles of Pbx4-deficient nkx2.5+ SHFPs are less distinct and display characteristics of normally discrete proliferative progenitor and anterior, differentiated cardiomyocyte populations. Therefore, our data indicate that the generation of proper OFT size and arch arteries requires Pbx-dependent stratification of unique differentiation states to facilitate both homeotic-like transformations and limit progenitor production within the SHF.
Asunto(s)
Aorta Torácica/embriología , Región Branquial/embriología , Cardiomegalia/genética , Proteínas de Unión al ADN/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Región Branquial/citología , Proliferación Celular/fisiología , Corazón/embriología , Corazón/fisiología , Miocitos Cardíacos/citología , Células Madre/citología , Pez Cebra/genéticaRESUMEN
During neurulation, cranial neural crest cells (CNCCs) migrate long distances from the neural tube to their terminal site of differentiation. The pathway traveled by the CNCCs defines the blueprint for craniofacial construction, abnormalities of which contribute to three-quarters of human birth defects. Biophysical cues like naturally occurring electric fields (EFs) have been proposed to be one of the guiding mechanisms for CNCC migration from the neural tube to identified position in the branchial arches. Such endogenous EFs can be mimicked by applied EFs of physiological strength that has been reported to guide the migration of amphibian and avian neural crest cells (NCCs), namely galvanotaxis or electrotaxis. However, the behavior of mammalian NCCs in external EFs has not been reported. We show here that mammalian CNCCs migrate towards the anode in direct current (dc) EFs. Reversal of the field polarity reverses the directedness. The response threshold was below 30 âmV/mm and the migration directedness and displacement speed increased with increase in field strength. Both CNCC line (O9-1) and primary mouse CNCCs show similar galvanotaxis behavior. Our results demonstrate for the first time that the mammalian CNCCs respond to physiological EFs by robust directional migration towards the anode in a voltage-dependent manner.
Asunto(s)
Región Branquial/embriología , Diferenciación Celular , Movimiento Celular , Electricidad , Transducción de Señal , Animales , Región Branquial/citología , Línea Celular , Ratones , Cresta Neural/citologíaRESUMEN
We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.
Asunto(s)
Síndrome de DiGeorge/patología , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Dominio T Box/metabolismo , Animales , Región Branquial/embriología , Síndrome de DiGeorge/genética , Modelos Animales de Enfermedad , Embrión de Mamíferos , Endodermo/embriología , Femenino , Factores de Transcripción Forkhead/genética , Corazón/embriología , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación , Miocardio/patología , Proteínas de Dominio T Box/genéticaRESUMEN
BACKGROUND: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. RESULTS: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap. CONCLUSIONS: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cíclidos/embriología , Desarrollo Embrionario/genética , Aletas de Animales/embriología , Aletas de Animales/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Región Branquial/embriología , Región Branquial/metabolismo , Cíclidos/genética , Cíclidos/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Intestinos/embriología , Intestinos/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Miocardio/metabolismo , Sistema Nervioso Periférico/embriología , Sistema Nervioso Periférico/metabolismo , Homología de Secuencia , Cráneo/embriología , Cráneo/metabolismo , Diente/embriología , Diente/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Pharyngeal arches (PA) are sequentially generated in an anterior-to-posterior order. Ripply3 is essential for posterior PA development in mouse embryos and its expression is sequentially activated in ectoderm and endoderm prior to formation of each PA. Since the PA phenotype of Ripply3 knockout (KO) mice is similar to that of retinoic acid (RA) signal-deficient embryos, we investigated the relationship between RA signaling and Ripply3 in mouse embryos. RESULTS: In BMS493 (pan-RAR antagonist) treated embryos, which are defective in third and fourth PA development, Ripply3 expression is decreased in the region posterior to PA2 at E9.0. This expression remains and its distribution is expanded posteriorly at E9.5. Conversely, high dose RA exposure does not apparently change its expression at E9.0 and 9.5. Knockout of retinaldehyde dehydrogenase 2 (Raldh2), which causes more severe PA defect, attenuates sequential Ripply3 expression at PA1 and reduces its expression level. EGFP reporter expression driven by a 6 kb Ripply3 promoter fragment recapitulates the endogenous Ripply3 mRNA expression during PA development in wild-type, but its distribution is expanded posteriorly in BMS493-treated and Raldh2 KO embryos. CONCLUSION: Spatio-temporal regulation of Ripply3 expression by RA signaling is indispensable for the posterior PA development in mouse.
Asunto(s)
Región Branquial/embriología , Proteínas Represoras/genética , Tretinoina/metabolismo , Animales , Benzoatos/farmacología , Región Branquial/efectos de los fármacos , Región Branquial/metabolismo , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Embarazo , Proteínas Represoras/metabolismo , Receptor alfa de Ácido Retinoico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estilbenos/farmacología , Tretinoina/farmacología , Tretinoina/fisiologíaRESUMEN
Growth and remodeling of the primitive pharyngeal arch artery (PAA) network into the extracardiac great vessels is poorly understood but a major source of clinically serious malformations. Undisrupted blood flow is required for normal PAA development, yet specific relationships between hemodynamics and remodeling remain largely unknown. Meeting this challenge is hindered by the common reductionist analysis of morphology to single idealized models, where in fact structural morphology varies substantially. Quantitative technical tools that allow tracking of morphological and hemodynamic changes in a population-based setting are essential to advancing our understanding of morphogenesis. Here, we have developed a methodological pipeline from high-resolution nano-computed tomography imaging and live-imaging flow measurements to multiscale pulsatile computational models. We combine experimental-based computational models of multiple PAAs to quantify hemodynamic forces in the rapidly morphing Hamburger Hamilton (HH) stage HH18, HH24 and HH26 embryos. We identify local morphological variation along the PAAs and their association with specific hemodynamic changes. Population-level mechano-morphogenic variability analysis is a powerful strategy for identifying stage-specific regions of well and poorly tolerated morphological and/or hemodynamic variation that may protect or initiate cardiovascular malformations.
Asunto(s)
Aorta Torácica/embriología , Aorta Torácica/fisiología , Región Branquial/embriología , Región Branquial/fisiología , Hemodinámica/fisiología , Remodelación Vascular , Puntos Anatómicos de Referencia , Animales , Embrión de Pollo , Simulación por Computador , Hidrodinámica , Imagenología Tridimensional , Análisis de la Onda del Pulso , Reproducibilidad de los ResultadosRESUMEN
The evolution of a hinged moveable jaw with variable morphology is considered a major factor behind the successful expansion of the vertebrates. DLX homeobox transcription factors are crucial for establishing the positional code that patterns the mandible, maxilla and intervening hinge domain, but how the genes encoding these proteins are regulated remains unclear. Herein, we demonstrate that the concerted action of the AP-2α and AP-2ß transcription factors within the mouse neural crest is essential for jaw patterning. In the absence of these two proteins, the hinge domain is lost and there are alterations in the size and patterning of the jaws correlating with dysregulation of homeobox gene expression, with reduced levels of Emx, Msx and Dlx paralogs accompanied by an expansion of Six1 expression. Moreover, detailed analysis of morphological features and gene expression changes indicate significant overlap with various compound Dlx gene mutants. Together, these findings reveal that the AP-2 genes have a major function in mammalian neural crest development, influencing patterning of the craniofacial skeleton via the DLX code, an effect that has implications for vertebrate facial evolution, as well as for human craniofacial disorders.
Asunto(s)
Tipificación del Cuerpo/fisiología , Región Branquial/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Cresta Neural/metabolismo , Factor de Transcripción AP-2/metabolismo , Animales , Región Branquial/citología , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Cresta Neural/citología , Factor de Transcripción AP-2/genéticaRESUMEN
It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts - both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.
Asunto(s)
Evolución Biológica , Región Branquial/embriología , Cabeza/anatomía & histología , Cabeza/embriología , Corazón/anatomía & histología , Corazón/embriología , Vertebrados/anatomía & histología , Vertebrados/embriología , Animales , Región Branquial/anatomía & histología , Región Branquial/citología , Mesodermo/citología , Modelos Biológicos , Músculos/anatomía & histología , Músculos/citología , Músculos/embriología , Cresta Neural/citologíaRESUMEN
Knowledge of variations of the internal carotid artery is significant to surgeons and radiologists. The internal carotid artery normally runs a straight course in the neck. Its anomalies can lead to its iatrogenic injuries. We report a case of a large loop of the internal carotid artery in a male cadaver aged about 75 years. The common carotid artery terminated by dividing it into the external carotid artery and internal carotid arteries at the level of the upper border of the thyroid cartilage. From the level of origin, the internal carotid artery coursed upwards, backwards and laterally, and formed a large loop behind the internal jugular vein. The variation was found on the left side of the neck and was unilateral. The uncommon looping of the internal carotid artery might result in altered blood flow to the brain and may lead to misperceptions in surgical, imaging, and invasive procedures.
Asunto(s)
Variación Anatómica , Arteria Carótida Interna/anomalías , Anciano , Angiografía/métodos , Región Branquial/embriología , Cadáver , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/prevención & control , Arteria Carótida Interna/embriología , Desarrollo Embrionario , Humanos , Venas Yugulares/anatomía & histología , Masculino , Disección del Cuello/efectos adversos , Disección del Cuello/métodosRESUMEN
Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.
Asunto(s)
Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Región Branquial/embriología , Región Branquial/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Proteínas de Pez Cebra/genéticaRESUMEN
We have proposed that independent origins of the tympanic membrane (TM), consisting of the external auditory meatus (EAM) and first pharyngeal pouch, are linked with distinctive middle ear structures in terms of dorsal-ventral patterning of the pharyngeal arches during amniote evolution. However, previous studies have suggested that the first pharyngeal arch (PA1) is crucial for TM formation in both mouse and chick. In this study, we compare TM formation along the anterior-posterior axis in these animals using Hoxa2 expression as a marker of the second pharyngeal arch (PA2). In chick, the EAM begins to invaginate at the surface ectoderm of PA2, not at the first pharyngeal cleft, and the entire TM forms in PA2. Chick-quail chimera that have lost PA2 and duplicated PA1 suggest that TM formation is achieved by developmental interaction between a portion of the EAM and the columella auris in PA2, and that PA1 also contributes to formation of the remaining part of the EAM. By contrast, in mouse, TM formation is highly associated with an interdependent relationship between the EAM and tympanic ring in PA1.
Asunto(s)
Región Branquial/embriología , Membrana Timpánica/embriología , Animales , Región Branquial/metabolismo , Embrión de Pollo , Pollos , Conducto Auditivo Externo/embriología , Oído Medio/embriología , Embrión de Mamíferos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Fenotipo , Codorniz/embriología , Membrana Timpánica/metabolismoRESUMEN
Craniofacial development depends on cell-cell interactions, coordinated cellular movement and differentiation under the control of regulatory gene networks, which include the distal-less (Dlx) gene family. However, the functional significance of Dlx5 in patterning the oropharyngeal region has remained unknown. Here, we show that loss of Dlx5 leads to a shortened soft palate and an absence of the levator veli palatini, palatopharyngeus and palatoglossus muscles that are derived from the 4th pharyngeal arch (PA); however, the tensor veli palatini, derived from the 1st PA, is unaffected. Dlx5-positive cranial neural crest (CNC) cells are in direct contact with myoblasts derived from the pharyngeal mesoderm, and Dlx5 disruption leads to altered proliferation and apoptosis of CNC and muscle progenitor cells. Moreover, the FGF10 pathway is downregulated in Dlx5-/- mice, and activation of FGF10 signaling rescues CNC cell proliferation and myogenic differentiation in these mutant mice. Collectively, our results indicate that Dlx5 plays crucial roles in the patterning of the oropharyngeal region and development of muscles derived from the 4th PA mesoderm in the soft palate, likely via interactions between CNC-derived and myogenic progenitor cells.
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Tipificación del Cuerpo , Región Branquial/embriología , Comunicación Celular , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Boca/embriología , Mioblastos/citología , Cresta Neural/citología , Cráneo/embriología , Animales , Región Branquial/metabolismo , Diferenciación Celular , Proliferación Celular , Regulación hacia Abajo/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones Noqueados , Desarrollo de Músculos , Mioblastos/metabolismo , Cresta Neural/metabolismo , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Transducción de Señal , Cráneo/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
In the present study, the morphological development of the Brycon amazonicus digestive tract is described to provide basic knowledge for nutritional studies and, therefore, increase the survival of this species during larviculture. Samples were collected from hatching up to 25 days of age, measured, processed and observed under a stereomicroscope and light microscopy. Newly hatched larvae presented their digestive tract as a straight tube, dorsal to the yolk sac, lined with a single layer of undifferentiated cells. At 24 h post-hatching (hPH), the buccopharyngeal cavity was open, but the posterior region of the digestive tube remained closed. At 25 hPH, the digestive tube was completely open and could be divided into buccopharyngeal cavity, oesophagus and intestine. At 35 hPH, the intestine presented a dilatation in the proximal region, which had the function of storing food. Differentiation of the stomach started at 83 hPH, and mucous cells were observed in the epithelium. These cells are important in the production of mucus, whose function is to protect the organ against acidity, although the gastric glands began developing only from 171 hPH, when three stomach regions were observed: cardiac, fundic and pyloric. The gastric glands were observed in the cardiac region, indicating that this organ already had digestive functionality. From 243 hPH, the absorption and assimilation of nutrients were already possible but, only from 412 hPH, the digestive tract was completely developed and functional.
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Characiformes/crecimiento & desarrollo , Tracto Gastrointestinal/crecimiento & desarrollo , Animales , Región Branquial/citología , Región Branquial/embriología , Región Branquial/crecimiento & desarrollo , Characiformes/anatomía & histología , Characiformes/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Desarrollo Embrionario/fisiología , Mucosa Gástrica/citología , Mucosa Gástrica/embriología , Mucosa Gástrica/crecimiento & desarrollo , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/embriología , Larva/citología , Larva/crecimiento & desarrollo , Mucosa Bucal/citología , Mucosa Bucal/embriología , Mucosa Bucal/crecimiento & desarrollo , Factores de TiempoRESUMEN
BACKGROUND: Congenital cardiovascular malformations are the most common birth defects affecting children. Several of these defects occur in structures developing from neural crest cells. One of the key signaling pathways regulating cardiac neural crest cell (CNCC) development involves the endothelin-A receptor (Ednra). However, the exact function of Ednra signaling in CNCC is unknown. RESULTS: The fate mapping of CNCC in Ednra embryos indicated that the migration of these cells is aberrant in the cardiac outflow tract (OFT), but not in the pharyngeal arches. This premature arrest of CNCC migration occurs independently of CNCC proliferation and apoptosis changes and major gene expression changes. Analysis of the Rho family of small GTPases in the mutant embryos revealed that Cdc42 failed to localize normally in the CNCC migrating in the OFT. The inhibition of Cdc42 activity in cultured embryos recapitulated the migratory phenotype observed in Ednra mice. Further analyses revealed that Cdc42 is part of the signaling pathway activated by endothelin specifically in OFT CNCC to control their migration. CONCLUSIONS: These results indicated that the activation of Cdc42 by endothelin signaling is important for CNCC migration in the OFT but this pathway is not involved in mandibular or pharyngeal arch artery patterning.