RESUMO
The primary cilium is a hair-like projection that controls cell development and tissue homeostasis. Although accumulated studies identify the molecular link between cilia and cilia-related diseases, the underlying etiology of ciliopathies has not been fully understood. In this paper, we determine the function of Rab34, a small GTPase, as a key regulator for controlling ciliogenesis and type I collagen trafficking in craniofacial development. Mechanistically, Rab34 is required to form cilia that control osteogenic proliferation, survival, and differentiation via cilia-mediated Hedgehog signaling. In addition, Rab34 is indispensable for regulating type I collagen trafficking from the ER to the Golgi. These results demonstrate that Rab34 has both ciliary and non-ciliary functions to regulate osteogenesis. Our study highlights the critical function of Rab34, which may contribute to understanding the novel etiology of ciliopathies that are associated with the dysfunction of RAB34 in humans.
Assuntos
Cílios , Osteogênese , Proteínas rab de Ligação ao GTP , Cílios/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Animais , Camundongos , Humanos , Crânio/metabolismo , Proteínas Hedgehog/metabolismo , Diferenciação Celular , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Transdução de Sinais , Desenvolvimento Ósseo , Ossos Faciais/metabolismo , Ossos Faciais/crescimento & desenvolvimento , Ossos Faciais/embriologia , Proliferação de Células , Transporte Proteico , Complexo de Golgi/metabolismoRESUMO
BACKGROUND: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling. RESULTS: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis. CONCLUSIONS: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown.
Assuntos
Proteínas Aviárias/biossíntese , Galinhas , Ossos Faciais/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/biossíntese , MicroRNAs/biossíntese , Transdução de Sinais , Animais , Padronização Corporal , Embrião de Galinha , Ectoderma/embriologiaRESUMO
In the amniote embryo, the upper jaw and nasal cavities form through coordinated outgrowth and fusion of craniofacial prominences. Adjacent to the embryonic prominences are the developing eyes, which abut the maxillary and lateral nasal prominences. The embryos of extant sauropsids (birds and nonavian reptiles) develop particularly large eyes in comparison to mammals, leading researchers to propose that the developing eye may facilitate outgrowth of prominences towards the midline in order to aid prominence fusion. To test this hypothesis, we performed unilateral and bilateral ablation of the developing eyes in chicken embryos, with the aim of evaluating subsequent prominence formation and fusion. Our analyses revealed minor interaction between the developing craniofacial prominences and the eyes, inconsequential to the fusion of the upper beak. At later developmental stages, the skull exhibited only localized effects from missing eyes, while geometric morphometrics revealed minimal effect on overall shape of the upper jaw when it develops without eyes. Our results indicate that the substantial size of the developing eyes in the chicken embryo exert little influence over the fusion of the craniofacial prominences, despite their effect on the size and shape of maxillary prominences and components of the skull.
Assuntos
Embrião de Galinha/embriologia , Galinhas/fisiologia , Olho/embriologia , Ossos Faciais/embriologia , Crânio/embriologia , Animais , Embrião de Galinha/fisiologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/fisiologia , Ossos Faciais/fisiologia , Mamíferos/embriologia , Mamíferos/fisiologia , Maxila/embriologia , Maxila/fisiologia , Crânio/fisiologiaRESUMO
The formation of the craniofacial skeleton is a highly dynamic process that requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Alterations that occur during cNCC development result in congenital birth defects and craniofacial abnormalities such as cleft lip with or without cleft palate. While the gene regulatory networks facilitating neural crest development have been extensively studied, the epigenetic mechanisms by which these pathways are activated or repressed in a temporal and spatially regulated manner remain largely unknown. Chromatin modifiers can precisely modify gene expression through a variety of mechanisms including histone modifications such as methylation. Here, we investigated the role of two members of the PRDM (Positive regulatory domain) histone methyltransferase family, Prdm3 and Prdm16 in craniofacial development using genetic models in zebrafish and mice. Loss of prdm3 or prdm16 in zebrafish causes craniofacial defects including hypoplasia of the craniofacial cartilage elements, undefined posterior ceratobranchials, and decreased mineralization of the parasphenoid. In mice, while conditional loss of Prdm3 in the early embryo proper causes mid-gestation lethality, loss of Prdm16 caused craniofacial defects including anterior mandibular hypoplasia, clefting in the secondary palate and severe middle ear defects. In zebrafish, prdm3 and prdm16 compensate for each other as well as a third Prdm family member, prdm1a. Combinatorial loss of prdm1a, prdm3, and prdm16 alleles results in severe hypoplasia of the anterior cartilage elements, abnormal formation of the jaw joint, complete loss of the posterior ceratobranchials, and clefting of the ethmoid plate. We further determined that loss of prdm3 and prdm16 reduces methylation of histone 3 lysine 9 (repression) and histone 3 lysine 4 (activation) in zebrafish. In mice, loss of Prdm16 significantly decreased histone 3 lysine 9 methylation in the palatal shelves but surprisingly did not change histone 3 lysine 4 methylation. Taken together, Prdm3 and Prdm16 play an important role in craniofacial development by maintaining temporal and spatial regulation of gene regulatory networks necessary for proper cNCC development and these functions are both conserved and divergent across vertebrates.
Assuntos
Anormalidades Craniofaciais/genética , Proteínas de Ligação a DNA/fisiologia , Histona Metiltransferases/fisiologia , Proteína do Locus do Complexo MDS1 e EVI1/fisiologia , Crânio/embriologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Cromatina/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Orelha Média/anormalidades , Orelha Média/embriologia , Ossos Faciais/embriologia , Feminino , Genes Letais , Código das Histonas/genética , Histona Metiltransferases/deficiência , Histona Metiltransferases/genética , Histonas/metabolismo , Arcada Osseodentária/embriologia , Proteína do Locus do Complexo MDS1 e EVI1/deficiência , Proteína do Locus do Complexo MDS1 e EVI1/genética , Masculino , Metilação , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional/genética , Especificidade da Espécie , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
3D imaging approaches based on X-ray microcomputed tomography (microCT) have become increasingly accessible with advancements in methods, instruments and expertise. The synergy of material and life sciences has impacted biomedical research by proposing new tools for investigation. However, data sharing remains challenging as microCT files are usually in the range of gigabytes and require specific and expensive software for rendering and interpretation. Here, we provide an advanced method for visualisation and interpretation of microCT data with small file formats, readable on all operating systems, using freely available Portable Document Format (PDF) software. Our method is based on the conversion of volumetric data into interactive 3D PDF, allowing rotation, movement, magnification and setting modifications of objects, thus providing an intuitive approach to analyse structures in a 3D context. We describe the complete pipeline from data acquisition, data processing and compression, to 3D PDF formatting on an example of craniofacial anatomical morphology in the mouse embryo. Our procedure is widely applicable in biological research and can be used as a framework to analyse volumetric data from any research field relying on 3D rendering and CT-biomedical imaging.
Assuntos
Imageamento Tridimensional/estatística & dados numéricos , Software , Microtomografia por Raio-X/estatística & dados numéricos , Animais , Compressão de Dados/estatística & dados numéricos , Processamento Eletrônico de Dados , Ossos Faciais/anatomia & histologia , Ossos Faciais/embriologia , Disseminação de Informação/métodos , Camundongos , Modelos Anatômicos , Interpretação de Imagem Radiográfica Assistida por Computador , Crânio/anatomia & histologia , Crânio/embriologiaRESUMO
BACKGROUND: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process. RESULTS: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression. CONCLUSION: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Ossos Faciais/embriologia , Proteínas de Homeodomínio/genética , Crista Neural/embriologia , Crânio/embriologia , Animais , Animais Geneticamente Modificados , Embrião de Galinha , Anormalidades Craniofaciais/genética , Desenvolvimento Embrionário/genética , Ossos Faciais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mandíbula/embriologia , Mandíbula/metabolismo , Maxila/embriologia , Maxila/metabolismo , Crista Neural/metabolismo , Crânio/metabolismoRESUMO
Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.
Assuntos
Ossos Faciais/embriologia , Proteínas de Homeodomínio/genética , Desenvolvimento Maxilofacial/genética , Crânio/embriologia , Fatores de Transcrição/genética , Animais , Desenvolvimento Ósseo/genética , Células Cultivadas , Embrião de Mamíferos , Ossos Faciais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Histona Acetiltransferases , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Crânio/metabolismoRESUMO
BACKGROUND: The platelet-derived growth factor (PDGF) family consists of four ligands (PDGF-A, PDGF-B, PDGF-C, PDGF-D) and two tyrosine kinase receptors (PDGFR-α and PDGFR-ß). In vertebrates, PDGF signaling influences cell proliferation, migration, and matrix deposition, and its up-regulation is implicated in cancer progression. Despite this evidence, the role of each family member during embryogenesis is still incomplete and partially controversial. In particular, study of the role of pdgf signaling during craniofacial development has been focused on pdgf-a, while the role of pdgf-b is almost unknown due to the lethal phenotypes of pdgf-b-null mice. RESULTS: By using a pdgf-b splice-blocking morpholino approach, we highlighted impairment of neural crest cell (NCC) migration in Xenopus laevis morphants, leading to alteration of NCC derivatives formation, such as cranial nerves and cartilages. We also uncovered a possible link between pdgf-b and the expression of cadherin superfamily members cdh6 and cdh11, which mediate cell-cell adhesion promoting NCC migration. CONCLUSIONS: Our results suggested that pdgf-b signaling is involved in cranial NCC migration and it is required for proper formation of craniofacial NCC derivatives. Taken together, these data unveiled a new role for pdgf-b during vertebrate development, contributing to complete the picture of pdgf signaling role in craniofacial development.
Assuntos
Ossos Faciais/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-sis/fisiologia , Crânio/crescimento & desenvolvimento , Animais , Caderinas/metabolismo , Adesão Celular , Movimento Celular , Embrião não Mamífero , Ossos Faciais/embriologia , Camundongos , Crista Neural/citologia , Transdução de Sinais , Crânio/embriologia , Xenopus laevis/embriologia , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Human CLCN7 encodes voltage-gated chloride channel 7 (ClC-7); mutations of CLCN7 lead to osteopetrosis which is characterized by increased bone mass and impaired osteoclast function. In our previous clinical practice, we noticed that osteopetrosis patients with CLCN7 mutations had some special deformities in craniofacial morphology and tooth dysplasia. It is unclear whether these phenotypes are the typical features of CLCN7 involved osteopetrosis and whether ClC-7 could regulate the development of craniofacial bone and tooth in some signaling pathways. Methods: First, we collected 80 osteopetrosis cases from the literature and compared their craniofacial and dental phenotypes. Second, four osteopetrosis pedigrees with CLCN7 mutations were recruited from our clinic for gene testing and clinical analysis of their craniofacial and dental phenotypes. Third, we used a zebrafish model with clcn7 morpholino treatment to detect the effects of ClC-7 deficiency on the development of craniofacial and dental phenotypes. General observation, whole mount alcian blue and alizarin red staining, whole mount in situ hybridization, scanning electron microscope observation, lysoSensor staining, Q-PCR and western blotting were performed to observe the in vivo characteristics of craniofacial bone and tooth changes. Fourth, mouse marrow stromal cells were further primarily cultured to detect ClC-7 related mRNA and protein changes using siRNA, Q-PCR and western blotting. Results: Over 84% of osteopetrosis patients in the literature had some typical craniofacial and tooth phenotypes, including macrocephaly, frontal bossing, and changes in shape and proportions of facial skeleton, and these unique features are more severe and frequent in autosomal recessive osteopetrosis than in autosomal dominant osteopetrosis patients. Our four pedigrees with CLCN7 mutations confirmed the aforementioned clinical features. clcn7 knockdown in zebrafish reproduced the craniofacial cartilage defects and various dental malformations combined the decreased levels of col10a1, sp7, dlx2b, eve1, and cx43. Loss of clcn7 function resulted in lysosomal storage in the brain and jaw as well as downregulated cathepsin K (CTSK). The craniofacial phenotype severity also presented a dose-dependent relationship with the levels of ClC-7 and CTSK. ClC-7/CTSK further altered the balance of TGF-ß/BMP signaling pathway, causing elevated TGF-ß-like Smad2 signals and reduced BMP-like Smad1/5/8 signals in clcn7 morphants. SB431542 inhibitor of TGF-ß pathway partially rescued the aforementioned craniofacial bone and tooth defects of clcn7 morphants. The ClC-7 involved CTSK/BMP and SMAD changes were also confirmed in mouse bone marrow stromal cells. Conclusion: These findings highlighted the vital role of clcn7 in zebrafish craniofacial bone and tooth development and mineralization, revealing novel insights for the causation of osteopetrosis with CLCN7 mutations. The mechanism chain of ClC-7/CTSK/ TGF-ß/BMP/SMAD might explain the typical craniofacial bone and tooth changes in osteopetrosis as well as pycnodysostosis patients.
Assuntos
Canais de Cloreto/metabolismo , Ossos Faciais/embriologia , Proteínas Mutantes/metabolismo , Osteopetrose/fisiopatologia , Crânio/embriologia , Dente/embriologia , Animais , Canais de Cloreto/genética , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Teóricos , Proteínas Mutantes/genética , Osteopetrose/patologia , Peixe-Zebra/embriologiaRESUMO
Epigenetic modifier lysine demethylase 3a (Kdm3a) specifically demethylates mono and dimethylated ninth lysine of histone 3 and belongs to the Jumonji domaincontaining group of demethylases. Kdm3a serves roles during various biological and pathophysiological processes, including spermatogenesis and metabolism, determination of sex, androgen receptormediated transcription and embryonic carcinoma cell differentiation. In the present study, physiological functions of Kdm3a were evaluated during embryogenesis of Xenopus laevis. Spatiotemporal expression pattern indicated that kdm3a exhibited its expression from early embryonic stages until tadpole stage, however considerable increase of kdm3a expression was observed during the neurula stage of Xenopus development. Depleting kdm3a using kdm3a antisense morpholino oligonucleotides induced anomalies, including head deformities, smallsized eyes and abnormal pigmentation. Wholemount in situ hybridization results demonstrated that kdm3a knockdown was associated with defects in neural crest migration. Further, quantitative polymerase chain reaction revealed abnormal expression of neural markers in kdm3a morphants. RNA sequencing of kdm3a morphants indicated that kdm3a was implicated in mesoderm formation, cell adhesion and metabolic processes of embryonic development. In conclusion, the results of the present study indicated that Kdm3a may serve a role in neural development during Xenopus embryogenesis and may be targeted for treatment of developmental disorders. Further investigation is required to elucidate the molecular mechanism underlying the regulation of neural development by Kdm3a.
Assuntos
Desenvolvimento Embrionário/genética , Ossos Faciais/embriologia , Histona Desmetilases com o Domínio Jumonji/genética , Neurogênese/genética , Organogênese/genética , Crânio/embriologia , Proteínas de Xenopus/genética , Animais , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Masculino , Xenopus laevisRESUMO
The skull is a vertebrate novelty. Morphological adaptations of the skull are associated with major evolutionary transitions, including the shift to a predatory lifestyle and the ability to masticate while breathing. These adaptations include the chondrocranium, dermatocranium, articulated jaws, primary and secondary palates, internal choanae, the middle ear, and temporomandibular joint. The incredible adaptive diversity of the vertebrate skull indicates an underlying bauplan that promotes evolvability. Comparative studies in craniofacial development suggest that the craniofacial bauplan includes three secondary organizers, two that are bilaterally placed at the Hinge of the developing jaw, and one situated in the midline of the developing face (the FEZ). These organizers regulate tissue interactions between the cranial neural crest, the neuroepithelium, and facial and pharyngeal epithelia that regulate the development and evolvability of the craniofacial skeleton.
Assuntos
Evolução Biológica , Ossos Faciais/embriologia , Crista Neural/embriologia , Crânio/embriologia , Animais , Padronização Corporal/genética , Ossos Faciais/anatomia & histologia , Ossos Faciais/metabolismo , Peixes/anatomia & histologia , Peixes/embriologia , Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/anatomia & histologia , Crista Neural/metabolismo , Crânio/anatomia & histologia , Crânio/metabolismoRESUMO
Chrondrocranium, the cartilaginous skull, is one of the major innovations that underlie evolution of the vertebrate head. Control of the induction and shaping of the cartilage is a key for the formation of the facial bones and largely defines facial shape. The appearance of cartilage in the head enabled many new functions such as protection of central nervous system and sensory structures, support of the feeding apparatus and formation of muscle attachment points ensuring faster and coordinated jaw movements. Here we review the evolution of cartilage in the cranial region and discuss shaping of the chondrocranium in different groups of vertebrates.
Assuntos
Evolução Biológica , Cartilagem/embriologia , Ossos Faciais/embriologia , Anfioxos/embriologia , Crânio/embriologia , Vertebrados/embriologia , Animais , Cartilagem/anatomia & histologia , Cartilagem/crescimento & desenvolvimento , Ossos Faciais/anatomia & histologia , Ossos Faciais/crescimento & desenvolvimento , Humanos , Anfioxos/anatomia & histologia , Anfioxos/crescimento & desenvolvimento , Modelos Biológicos , Crânio/anatomia & histologia , Crânio/crescimento & desenvolvimento , Vertebrados/anatomia & histologia , Vertebrados/crescimento & desenvolvimentoAssuntos
Biologia do Desenvolvimento , Ossos Faciais/embriologia , Crânio/embriologia , Animais , Padronização Corporal/genética , Anormalidades Craniofaciais/diagnóstico , Anormalidades Craniofaciais/etiologia , Anormalidades Craniofaciais/terapia , Ossos Faciais/crescimento & desenvolvimento , Humanos , Crista Neural/embriologia , Crista Neural/metabolismo , Organogênese/genética , Crânio/crescimento & desenvolvimentoRESUMO
In order to elucidate the effect of chorioallantoic and yolk sac placenta on the embryonic/fetal toxicity in dibutyltin dichloride (DBTCl)-exposed rats, we examined the histopathological changes and the tissue distribution of dibutyltin in the placentas and embryos. DBTCl was orally administered to the groups at doses of 0 mg/kg during gestation days (GD)s 7-9 (control group) and 20 mg/kg during GDs 7-9 (GD7-9 treated group), and GDs 10-12 (GD10-12 treated group). The total fetal mortality was increased, and malformations characterized by craniofacial dysmorphism were detected in the GD7-9 treated group. The embryonic/fetal weight and placental weight showed a decrease in both DBTCl-treated groups. Histologically, some embryos on GD 9.5 in the GD7-9 treated group underwent apoptosis without any changes of yolk sac. In the laser ablation-inductively coupled plasma-mass spectrometry analysis (LA-ICP-MS), tin was detected in the embryo, allantois, yolk sac, ectoplacental cone and decidual mass surrounding the conceptus on GD 9.5 in the GD7-9 treated group. Thus, it is considered that the embryo in this period is specifically sensitive to DBTCl-induced apoptosis, compared with other parts. The chorioallantoic placentas in both DBTCl-treated groups showed the developmental delay and hypoplasia in the fetal parts of placenta, resulting from apoptosis and mitotic inhibition. Thus, it was speculated that the DBTCl-induced malformations and fetal resorption resulted from the apoptosis in the embryo caused by the direct effect of DBTCl. The DBTCl-induced lesions in the chorioallantoic placenta were a non-specific transient developmental retardation in the fetal parts of placenta, leading to intrauterine growth retardation.
Assuntos
Retardo do Crescimento Fetal/induzido quimicamente , Troca Materno-Fetal/efeitos dos fármacos , Compostos Orgânicos de Estanho/toxicidade , Placenta/efeitos dos fármacos , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Ossos Faciais/anormalidades , Ossos Faciais/embriologia , Feminino , Mortalidade Fetal , Peso Fetal/efeitos dos fármacos , Idade Gestacional , Masculino , Tamanho do Órgão/efeitos dos fármacos , Compostos Orgânicos de Estanho/administração & dosagem , Compostos Orgânicos de Estanho/farmacocinética , Placenta/anatomia & histologia , Placenta/metabolismo , Gravidez , Ratos Wistar , Crânio/anormalidades , Crânio/embriologia , Distribuição TecidualRESUMO
BACKGROUND: Craniofacial anomalies involve defective pharyngeal arch development and neural crest function. Copy number variation at 1p35, containing histone deacetylase 1 (Hdac1), or 6q21-22, containing Hdac2, are implicated in patients with craniofacial defects, suggesting an important role in guiding neural crest development. However, the roles of Hdac1 and Hdac2 within neural crest cells remain unknown. RESULTS: The neural crest and its derivatives express both Hdac1 and Hdac2 during early murine development. Ablation of Hdac1 and Hdac2 within murine neural crest progenitor cells cause severe hemorrhage, atrophic pharyngeal arches, defective head morphogenesis, and complete embryonic lethality. Embryos lacking Hdac1 and Hdac2 in the neural crest exhibit decreased proliferation and increased apoptosis in both the neural tube and the first pharyngeal arch. Mechanistically, loss of Hdac1 and Hdac2 upregulates cyclin-dependent kinase inhibitors Cdkn1a, Cdkn1b, Cdkn1c, Cdkn2b, Cdkn2c, and Tp53 within the first pharyngeal arch. CONCLUSIONS: Our results show that Hdac1 and Hdac2 function redundantly within the neural crest to regulate proliferation and the development of the pharyngeal arches by means of repression of cyclin-dependent kinase inhibitors. Developmental Dynamics 246:1015-1026, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Região Branquial/embriologia , Proliferação de Células/fisiologia , Ossos Faciais/embriologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Crista Neural/embriologia , Animais , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Camundongos , Camundongos TransgênicosRESUMO
Interferon Regulatory Factor 6 (IRF6) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos (Irf6 +/- ; Twist1 +/- ) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel's cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53. Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.
Assuntos
Epistasia Genética , Ossos Faciais/embriologia , Fatores Reguladores de Interferon/genética , Proteínas Nucleares/genética , Organogênese/genética , Crânio/embriologia , Proteína 1 Relacionada a Twist/genética , Alelos , Animais , Apoptose/genética , Morte Celular , Linhagem Celular , Proliferação de Células , Anormalidades Craniofaciais/diagnóstico , Anormalidades Craniofaciais/genética , Endotelina-1/genética , Endotelina-1/metabolismo , Elementos Facilitadores Genéticos , Feminino , Imunofluorescência , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Fatores Reguladores de Interferon/metabolismo , Masculino , Mandíbula/embriologia , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Fenótipo , Ligação Proteica , Proteína 1 Relacionada a Twist/metabolismoRESUMO
The ex vivo culture of embryonic tissue explants permits the continuous monitoring of growth and morphogenesis at specific embryonic stages. The functions of soluble regulatory molecules can be analyzed by introducing them into culture medium or locally with beads to the tissue. Gene expression in the manipulated tissue explants can be analyzed using in situ hybridization, quantitative PCR, and reporter constructs combined to organ culture to examine the functions of the signaling molecules.
Assuntos
Desenvolvimento Embrionário/genética , Ossos Faciais/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese/genética , Organogênese/genética , Crânio/embriologia , Animais , Hibridização In Situ , Camundongos , Técnicas de Cultura de Órgãos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The incisive canal of the incisive bone or premaxilla is a narrow bony canal through which pass the nasopalatine nerve and its concomitant vessels. However, its fetal development remains obscure. To assess its development, serial frontal sections of the heads of 26 human fetuses, of gestational age 9-20 weeks (crown-rump length, 46-183 mm), were examined. The nerve initially passed through a wide loose tissue space, but after ossification of the upper part of the incisive bone at 12-15 weeks, the canal became narrow and filled with tight fibrous tissue. Canals in seven fetuses were dilated and open unilaterally or bilaterally. In two of these seven fetuses, a nasopalatine duct passed through the canal and connected the nasal cavity to a central lumen of the paramedian epithelial pearl in the incisive fossa (not to an oral cavity). Even if the canal was closed, the duct was likely to remain above and below the closed part. Paramedian pearls were present in all specimens larger than 110 mm (15 weeks), with or without association of midline pearls. These paramedian pearls usually protruded toward and/or extended into the dilated or open canal, suggesting that these pearls, not any primitive oronasal communication pathway, contributed to keeping the canal open. The dilated canal, located on the superomedial side of the second and third teeth buds, seemed to be usually closed by further ossification. Even in fetuses, the nasopalatine duct seemed to be a variant or unusual phase of development temporally occurring after normal palate fusion. Anat Rec, 300:1093-1103, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Ossos Faciais/embriologia , Feto/anatomia & histologia , Idade Gestacional , HumanosRESUMO
Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular 'Fraser protein complex' that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton.
Assuntos
Região Branquial/embriologia , Epitélio/embriologia , Proteínas da Matriz Extracelular/fisiologia , Integrinas/fisiologia , Mesoderma/embriologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Indução Embrionária , Epitélio/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Ossos Faciais/embriologia , Fibrilina-2/metabolismo , Integrinas/genética , Mesoderma/metabolismo , Morfogênese , Mutação , RNA Mensageiro , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Abigail Tucker is a professor at King's College London, UK and her lab works on various aspects of craniofacial development - from basic, evolutionary and clinical biology perspectives. This year, Abigail will be awarded the first Cheryll Tickle Medal by the British Society for Developmental Biology (BSDB). We chatted with Abigail about her research, her commitment to public engagement and the challenges and rewards of working with emerging model systems.