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1.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502237

RESUMO

Neural crest (NC) cells are highly migratory cells that contribute to various vertebrate tissues, and whose migratory behaviors resemble cancer cell migration and invasion. Information exchange via dynamic NC cell-cell contact is one mechanism by which the directionality of migrating NC cells is controlled. One transmembrane protein that is most likely involved in this process is protein tyrosine kinase 7 (PTK7), an evolutionary conserved Wnt co-receptor that is expressed in cranial NC cells and several tumor cells. In Xenopus, Ptk7 is required for NC migration. In this study, we show that the Ptk7 protein is dynamically localized at cell-cell contact zones of migrating Xenopus NC cells and required for contact inhibition of locomotion (CIL). Using deletion constructs of Ptk7, we determined that the extracellular immunoglobulin domains of Ptk7 are important for its transient accumulation and that they mediate homophilic binding. Conversely, we found that ectopic expression of Ptk7 in non-NC cells was able to prevent NC cell invasion. However, deletion of the extracellular domains of Ptk7 abolished this effect. Thus, Ptk7 is sufficient at protecting non-NC tissue from NC cell invasion, suggesting a common role of PTK7 in contact inhibition, cell invasion, and tissue integrity.


Assuntos
Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Movimento Celular , Inibição de Contato , Neoplasias Pulmonares/metabolismo , Crista Neural/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Polaridade Celular , Humanos , Neoplasias Pulmonares/patologia , Xenopus laevis
3.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445520

RESUMO

Endocytic trafficking is an under-appreciated pathway in cardiac development. Several genes related to endocytic trafficking have been uncovered in a mutagenic ENU screen, in which mutations led to congenital heart defects (CHDs). In this article, we review the relationship between these genes (including LRP1 and LRP2) and cardiac neural crest cells (CNCCs) during cardiac development. Mice with an ENU-induced Lrp1 mutation exhibit a spectrum of CHDs. Conditional deletion using a floxed Lrp1 allele with different Cre drivers showed that targeting neural crest cells with Wnt1-Cre expression replicated the full cardiac phenotypes of the ENU-induced Lrp1 mutation. In addition, LRP1 function in CNCCs is required for normal OFT lengthening and survival/expansion of the cushion mesenchyme, with other cell lineages along the NCC migratory path playing an additional role. Mice with an ENU-induced and targeted Lrp2 mutation demonstrated the cardiac phenotype of common arterial trunk (CAT). Although there is no impact on CNCCs in Lrp2 mutants, the loss of LRP2 results in the depletion of sonic hedgehog (SHH)-dependent cells in the second heart field. SHH is known to be crucial for CNCC survival and proliferation, which suggests LRP2 has a non-autonomous role in CNCCs. In this article, other endocytic trafficking proteins that are associated with CHDs that may play roles in the NCC pathway during development, such as AP1B1, AP2B1, FUZ, MYH10, and HECTD1, are reviewed.


Assuntos
Etilnitrosoureia/efeitos adversos , Cardiopatias Congênitas/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Animais , Linhagem da Célula , Modelos Animais de Doenças , Endocitose , Cardiopatias Congênitas/induzido quimicamente , Camundongos , Mutação , Crista Neural/metabolismo , Transdução de Sinais
4.
Biomolecules ; 11(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34439783

RESUMO

Neuroblastoma is a pediatric cancer, onset with localized as well as metastatic disease. Localized tumors usually show a high content of aneuploid cells. It is suggested that aneuploid cells with numerical copy number variation (CNV) are generated by chromosome instability (CIN). Patients with a localized tumor respond well to the therapy and show a good outcome. On the contrary, patients with a metastatic tumor have worse outcomes and the cells with structural CNV show high levels of CIN. It is proposed that a favorable outcome in patients with localized disease is associated to the grade of CIN.


Assuntos
Aneuploidia , Genoma Humano , Neoplasias do Sistema Nervoso/genética , Neuroblastoma/genética , Fatores de Proteção , Antineoplásicos/uso terapêutico , Instabilidade Cromossômica , Variações do Número de Cópias de DNA , Humanos , Lactente , Metástase Neoplásica , Neoplasias do Sistema Nervoso/tratamento farmacológico , Neoplasias do Sistema Nervoso/mortalidade , Neoplasias do Sistema Nervoso/patologia , Crista Neural/metabolismo , Crista Neural/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Análise de Sobrevida , Resultado do Tratamento
5.
Biomolecules ; 11(7)2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34356679

RESUMO

Schwann cell development and peripheral nerve myelination are finely orchestrated multistep processes; some of the underlying mechanisms are well described and others remain unknown. Many posttranslational modifications (PTMs) like phosphorylation and ubiquitination have been reported to play a role during the normal development of the peripheral nervous system (PNS) and in demyelinating neuropathies. However, a relatively novel PTM, SUMOylation, has not been studied in these contexts. SUMOylation involves the covalent attachment of one or more small ubiquitin-like modifier (SUMO) proteins to a substrate, which affects the function, cellular localization, and further PTMs of the conjugated protein. SUMOylation also regulates other proteins indirectly by facilitating non-covalent protein-protein interaction via SUMO interaction motifs (SIM). This pathway has important consequences on diverse cellular processes, and dysregulation of this pathway has been reported in several diseases including neurological and degenerative conditions. In this article, we revise the scarce literature on SUMOylation in Schwann cells and the PNS, we propose putative substrate proteins, and we speculate on potential mechanisms underlying the possible involvement of this PTM in peripheral myelination and neuropathies.


Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Proteína SUMO-1/metabolismo , Células de Schwann/metabolismo , Animais , Proliferação de Células , Doença de Charcot-Marie-Tooth/patologia , Epigênese Genética , Humanos , Bainha de Mielina/metabolismo , Crista Neural/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/genética , Sumoilação
6.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445433

RESUMO

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2ß (AP-2ß) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2ß is conditionally deleted in the NCCs (AP-2ß NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2ß deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2ß NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/ß-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Regulação para Baixo , Epitélio Corneano/anormalidades , Deleção de Genes , Fator de Transcrição AP-2/genética , Animais , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Epitélio Corneano/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Queratina-12/metabolismo , Queratina-15/metabolismo , Masculino , Camundongos , Crista Neural/metabolismo , Fenótipo , Fator de Transcrição AP-2/metabolismo , Via de Sinalização Wnt
7.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202161

RESUMO

The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.


Assuntos
Crista Neural/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Transdução de Sinais , Sistema Urinário/inervação , Sistema Urinário/metabolismo , Animais , Sistema Nervoso Autônomo , Diferenciação Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Camundongos , Crista Neural/embriologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuritos/metabolismo , Neurogênese , Crescimento Neuronal , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Receptores 5-HT3 de Serotonina/genética , Transcriptoma , Sistema Urinário/embriologia
8.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299157

RESUMO

Curcumin, a yellow polyphenol extracted from the turmeric root is used as a diet supplement. It exhibits anti-inflammatory, antioxidant, and antitumor properties by modulating different intracellular mechanisms. Due to their low solubility in water, the curcumin molecules must be encapsulated into liposomes to improve the bioavailability and biomedical potential. For the periodontal tissue and systemic health, it is essential to regulate the local inflammatory response. In this study, the possible beneficial effect of liposomes loaded with curcumin (CurLIP) in neural crest-derived human periodontal ligament stem cells (hPDLSCs) and in endothelial-differentiated hPDLSCs (e-hPDLSCs) induced with an inflammatory stimulus (lipopolysaccharide obtained from Porphyromonas gingivalis, LPS-G) was evaluated. The CurLIP formulation exhibited a significant anti-inflammatory effect by the downregulation of Toll-like receptor-4 (TLR4)/Myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa light chain enhancer of activated B cells (NFkB)/NLR Family Pyrin Domain Containing 3 (NLRP3)/Caspase-1/Interleukin (IL)-1ß inflammation cascade and reactive oxygen species (ROS) formation. Moreover, the exposure to LPS-G caused significant alterations in the expression of epigenetic modifiers, such as DNA Methyltransferase 1 (DNMT1) and P300, while the CurLIP treatment showed physiological expression. Overall, our in vitro study provides novel mechanistic insights into the intracellular pathway exert by CurLIP in the regulation of inflammation and epigenetic modifications.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Curcumina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Crista Neural/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Diferenciação Celular , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lipossomos/administração & dosagem , Lipossomos/química , Crista Neural/citologia , Crista Neural/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/química , Espécies Reativas de Oxigênio , Células-Tronco/citologia , Células-Tronco/metabolismo
9.
Dev Biol ; 477: 251-261, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34102166

RESUMO

BMP signaling plays iterative roles during vertebrate neural crest development from induction through craniofacial morphogenesis. However, far less is known about the role of BMP activity in cranial neural crest epithelial-to-mesenchymal transition and delamination. By measuring canonical BMP signaling activity as a function of time from specification through early migration of avian midbrain neural crest cells, we found elevated BMP signaling during delamination stages. Moreover, inhibition of canonical BMP activity via a dominant negative mutant Type I BMP receptor showed that BMP signaling is required for neural crest migration from the midbrain, independent from an effect on EMT and delamination. Transcriptome profiling on control compared to BMP-inhibited cranial neural crest cells identified novel BMP targets during neural crest delamination and early migration including targets of the Notch pathway that are upregulated following BMP inhibition. These results suggest potential crosstalk between the BMP and Notch pathways in early migrating cranial neural crest and provide novel insight into mechanisms regulated by BMP signaling during early craniofacial development.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Mesencéfalo/embriologia , Crista Neural/metabolismo , Transdução de Sinais , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião de Galinha , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/metabolismo , Crista Neural/embriologia , Crânio/embriologia , Crânio/metabolismo , Técnicas de Cultura de Tecidos
10.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34184034

RESUMO

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) is crucial for mammalian craniofacial development, although the mechanisms by which the activity of downstream intracellular effectors is regulated to mediate gene expression changes have not been defined. We find that the RNA-binding protein Srsf3 is phosphorylated at Akt consensus sites downstream of PI3K-mediated PDGFRα signaling in mouse palatal mesenchyme cells, leading to its nuclear translocation. We further demonstrate that ablation of Srsf3 in the mouse neural crest lineage leads to facial clefting due to defective cranial neural crest cell proliferation and survival. Finally, we show that Srsf3 regulates the alternative RNA splicing of transcripts encoding protein kinases in the mouse facial process mesenchyme to regulate PDGFRα-dependent intracellular signaling. Collectively, our findings reveal that alternative RNA splicing is an important mechanism of gene expression regulation downstream of PI3K/Akt-mediated PDGFRα signaling in the facial mesenchyme and identify Srsf3 as a critical regulator of craniofacial development.


Assuntos
Processamento Alternativo , Mesoderma/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ligantes , Masculino , Camundongos , Camundongos Knockout , Crista Neural/embriologia , Crista Neural/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética
11.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071193

RESUMO

Cutaneous melanoma represents one of the deadliest types of skin cancer. The prognosis strongly depends on the disease stage, thus early detection is crucial. New therapies, including BRAF and MEK inhibitors and immunotherapies, have significantly improved the survival of patients in the last decade. However, intrinsic and acquired resistance is still a challenge. In this review, we discuss two major aspects that contribute to the aggressiveness of melanoma, namely, the embryonic origin of melanocytes and melanoma cells and cellular plasticity. First, we summarize the physiological function of epidermal melanocytes and their development from precursor cells that originate from the neural crest (NC). Next, we discuss the concepts of intratumoral heterogeneity, cellular plasticity, and phenotype switching that enable melanoma to adapt to changes in the tumor microenvironment and promote disease progression and drug resistance. Finally, we further dissect the connection of these two aspects by focusing on the transcriptional regulators MSX1, MITF, SOX10, PAX3, and FOXD3. These factors play a key role in NC initiation, NC cell migration, and melanocyte formation, and we discuss how they contribute to cellular plasticity and drug resistance in melanoma.


Assuntos
Plasticidade Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Melanoma/metabolismo , Crista Neural/metabolismo , Neoplasias Cutâneas/metabolismo , Acrilonitrila/análogos & derivados , Acrilonitrila/farmacologia , Compostos de Anilina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular , Movimento Celular , Resistencia a Medicamentos Antineoplásicos/genética , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Transcrição MSX1/genética , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição PAX3/genética , Fenótipo , Pirimidinonas/farmacologia , Fatores de Transcrição SOXE/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia
12.
Commun Biol ; 4(1): 695, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099848

RESUMO

The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.


Assuntos
Melanoma/genética , Crista Neural/embriologia , Fatores de Transcrição SOXE/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Crista Neural/metabolismo
13.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34128978

RESUMO

Intramembranous ossification, which consists of direct conversion of mesenchymal cells to osteoblasts, is a characteristic process in skull development. One crucial role of these osteoblasts is to secrete collagen-containing bone matrix. However, it remains unclear how the dynamics of collagen trafficking is regulated during skull development. Here, we reveal the regulatory mechanisms of ciliary and golgin proteins required for intramembranous ossification. During normal skull formation, osteoblasts residing on the osteogenic front actively secreted collagen. Mass spectrometry and proteomic analysis determined endogenous binding between ciliary protein IFT20 and golgin protein GMAP210 in these osteoblasts. As seen in Ift20 mutant mice, disruption of neural crest-specific GMAP210 in mice caused osteopenia-like phenotypes due to dysfunctional collagen trafficking. Mice lacking both IFT20 and GMAP210 displayed more severe skull defects compared with either IFT20 or GMAP210 mutants. These results demonstrate that the molecular complex of IFT20 and GMAP210 is essential for the intramembranous ossification during skull development.


Assuntos
Proteínas da Matriz do Complexo de Golgi/metabolismo , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Animais , Calcificação Fisiológica , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células , Colágeno/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Camundongos , Camundongos Knockout , Crista Neural/metabolismo , Osteoblastos , Osteogênese , Proteômica
14.
Dev Biol ; 477: 241-250, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34052210

RESUMO

A critical cell type participating in cardiac outflow tract development is a subpopulation of the neural crest cells, the cardiac neural crest cells (NCCs), whose defect causes a spectrum of cardiovascular abnormalities. Accumulating evidence indicates that mTOR, which belongs to the PI3K-related kinase family and impacts multiple signaling pathways in a variety of contexts, plays a pivotal role for NCC development. Here, we investigated functional roles of mTOR for cardiac neural crest development using several lines of mouse genetic models. We found that disruption of mTOR caused NCC defects and failure of cardiac outflow tract separation, which resulted in a spectrum of cardiac defects including persistent truncus arteriosus, ventricular septal defect and ventricular wall defect. Specifically, mutant neural crest cells showed reduced migration into the cardiac OFT and prematurely exited the cell cycle. A number of critical factors and fundamental signaling pathways, which are important for neural crest and cardiomyocyte development, were impaired. Moreover, actin dynamics was disrupted by mTOR deletion. Finally, by phenotyping the neural crest Rptor and Rictor knockout mice respectively, we demonstrate that mTOR acts principally through the mTORC1 pathway for cardiac neural crest cells. Altogether, these data established essential roles of mTOR for cardiac NCC development and imply that dysregulation of mTOR in NCCs may underline a spectrum of cardiac defects.


Assuntos
Anormalidades Cardiovasculares/genética , Coração/embriologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Miocárdio/metabolismo , Crista Neural/embriologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Células Cultivadas , Deleção de Genes , Redes e Vias Metabólicas , Camundongos , Crista Neural/metabolismo , Serina-Treonina Quinases TOR/genética
15.
PLoS Genet ; 17(5): e1009579, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34033651

RESUMO

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.


Assuntos
Proteínas Morfogenéticas Ósseas/genética , Fator de Transcrição GATA3/genética , Proteínas Hedgehog/metabolismo , Fenótipo , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Fator de Transcrição GATA3/metabolismo , Haploinsuficiência , Mutação com Perda de Função , Mutação , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Organogênese , Crânio/citologia , Crânio/embriologia , Peixe-Zebra/embriologia
16.
Life Sci ; 278: 119577, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961858

RESUMO

OBJECTIVES: This study aims to investigate the role of demethylase ALKBH5 mediated demethylation of TAGLN mRNA in the occurrence of Hirschsprung's disease (HSCR), and to clarify how ALKBH5 reduces the m6A level of TAGLN mRNA and inhibits its degradation, thereby inhibiting the proliferation and migration of neural crest cells, and potentially contributing to the occurrence of HSCR. MATERIAL AND METHODS: Quantitative real-time PCR (qRT-PCR) and Western-Blot (WB) were conducted to test the expression level of ALKBH5 and TAGLN genes. Cell function assays were adopted to detect cell phenotypes. The qRT-PCR and methylated RNA immunoprecipitation (MeRIP-qPCR) were used to test the regulation of TAGLN by ALKBH5. RESULTS: 1. Compared with control intestinal tissue, the expression level of TAGLN and ALKBH5 in the aganglionic intestinal tissue of HSCR is increased. 2. The MeRIP-PCR and dualluciferase report confirmed that ALKBH5 could bind to m6A sites of TAGLN mRNA and reduce the m6A level of TAGLN mRNA. 3. In vitro cell experiments confirmed that overexpression of ALKBH5 can inhibit the degradation of TAGLN mRNA, increase the expression of TAGLN, thereby inhibiting cell proliferation and migration. 4. A zebrafish model of ALKBH5 overexpression was constructed. Studies have shown that ALKBH5 could inhibit the proliferation and migration of zebrafish enteric neurons. CONCLUSIONS: ALKBH5 could demethylate TAGLN mRNA and up-regulate TAGLN expression, leading to the inhibition of proliferation and migration of enteric neural crest cells and contributing to the occurrence of HSCR.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Doença de Hirschsprung/metabolismo , Intestinos/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Crista Neural/metabolismo , Peixe-Zebra/genética , Animais , Sítios de Ligação , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Doença de Hirschsprung/genética , Humanos , Lactente , Masculino , Metiltransferases/metabolismo
17.
Dev Cell ; 56(9): 1268-1282.e6, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33852891

RESUMO

Cell fate commitment is controlled by cis-regulatory elements often located in remote regions of the genome. To examine the role of long-range DNA interactions in early development, we generated a high-resolution contact map of active enhancers in avian neural crest cells. This analysis uncovered a diverse repertoire of enhancers that are part of the gene regulatory network underlying specification. We found that neural crest identity is largely regulated by cis-regulatory elements that propagate signaling inputs to network components. These genomic sensors display a combination of optimal and suboptimal TCF/LEF-binding sites, which allow cells to respond to Wnt signaling in a position-dependent manner. We propose that, rather than acting as upstream activators, signaling systems feed into regulatory circuits in a hub-and-spoke architecture. These results shed light on the tridimensional organization of the neural crest genome and define how signaling systems provide progenitors with spatial cues that transform their molecular identity.


Assuntos
Conectoma , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/metabolismo , Transdução de Sinais/genética , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Embrião de Galinha , Cromatina/metabolismo , Redes Reguladoras de Genes , Modelos Biológicos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Via de Sinalização Wnt
18.
Int J Mol Sci ; 22(6)2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804711

RESUMO

The primary cilium plays a pivotal role during the embryonic development of vertebrates. It acts as a somatic signaling hub for specific pathways, such as Sonic Hedgehog signaling. In humans, mutations in genes that cause dysregulation of ciliogenesis or ciliary function lead to severe developmental disorders called ciliopathies. Beyond its role in early morphogenesis, growing evidence points towards an essential function of the primary cilium in neural circuit formation in the central nervous system. However, very little is known about a potential role in the formation of the peripheral nervous system. Here, we investigate the presence of the primary cilium in neural crest cells and their derivatives in the trunk of developing chicken embryos in vivo. We found that neural crest cells, sensory neurons, and boundary cap cells all bear a primary cilium during key stages of early peripheral nervous system formation. Moreover, we describe differences in the ciliation of neuronal cultures of different populations from the peripheral and central nervous systems. Our results offer a framework for further in vivo and in vitro investigations on specific roles that the primary cilium might play during peripheral nervous system formation.


Assuntos
Cílios/fisiologia , Sistema Nervoso Periférico/fisiologia , Biomarcadores , Movimento Celular , Imunofluorescência , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Crista Neural/metabolismo , Neurônios/metabolismo , Organogênese/genética
19.
Cell Mol Life Sci ; 78(9): 4435-4450, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33796894

RESUMO

During early neural development, changes in signalling inform the expression of transcription factors that in turn instruct changes in cell identity. At the same time, switches in adhesion molecule expression result in cellular rearrangements that define the morphology of the emerging neural tube. It is becoming increasingly clear that these two processes influence each other; adhesion molecules do not simply operate downstream of or in parallel with changes in cell identity but rather actively feed into cell fate decisions. Why are differentiation and adhesion so tightly linked? It is now over 60 years since Conrad Waddington noted the remarkable "Constancy of the Wild Type" (Waddington in Nature 183: 1654-1655, 1959) yet we still do not fully understand the mechanisms that make development so reproducible. Conversely, we do not understand why directed differentiation of cells in a dish is sometimes unpredictable and difficult to control. It has long been suggested that cells make decisions as 'local cooperatives' rather than as individuals (Gurdon in Nature 336: 772-774, 1988; Lander in Cell 144: 955-969, 2011). Given that the cadherin family of adhesion molecules can simultaneously influence morphogenesis and signalling, it is tempting to speculate that they may help coordinate cell fate decisions between neighbouring cells in the embryo to ensure fidelity of patterning, and that the uncoupling of these processes in a culture dish might underlie some of the problems with controlling cell fate decisions ex-vivo. Here we review the expression and function of cadherins during early neural development and discuss how and why they might modulate signalling and differentiation as neural tissues are formed.


Assuntos
Caderinas/metabolismo , Crista Neural/metabolismo , Animais , Caderinas/química , Caderinas/classificação , Adesão Celular , Diferenciação Celular , Evolução Molecular , Humanos , Crista Neural/citologia , Crista Neural/crescimento & desenvolvimento , Neurogênese , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Biochem Biophys Res Commun ; 554: 173-178, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33798944

RESUMO

Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.


Assuntos
Células-Tronco Adultas/citologia , Regeneração Óssea/fisiologia , Crista Neural/citologia , Transplante de Células-Tronco/métodos , Conchas Nasais/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Crista Neural/metabolismo , Conchas Nasais/metabolismo
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