RESUMO
Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRß, in all vertebrates, and the expression of TRα but not TRß is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.
Assuntos
Células-Tronco Adultas/metabolismo , Ciclo Celular , Proliferação de Células , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Receptores alfa dos Hormônios Tireóideos/deficiência , Tri-Iodotironina/metabolismo , Proteínas de Xenopus/deficiência , Xenopus/metabolismo , Células-Tronco Adultas/patologia , Animais , Apoptose , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/patologia , Larva/genética , Larva/metabolismo , Metamorfose Biológica , Transdução de Sinais , Receptores alfa dos Hormônios Tireóideos/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMO
Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.
Assuntos
Cílios/patologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/fisiopatologia , Proteínas dos Microfilamentos/deficiência , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas de Xenopus/deficiência , Animais , Transtornos da Motilidade Ciliar/patologia , Modelos Animais de Doenças , Éxons/genética , Feminino , Deleção de Genes , Genes Letais , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Rotação , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMO
Animal cells divide by a process called cytokinesis which relies on the constriction of a contractile actomyosin ring leading to the production of two daughter cells. Cytokinesis is an intrinsic property of cells which occurs even for artificially isolated cells. During division, isolated cells undergo dramatic changes in shape such as rounding and membrane deformation as the division furrow ingresses. However, cells are often embedded in tissues and thus are surrounded by neighbouring cells. How these neighbours might influence, or might themselves be influenced by, the shape changes of cytokinesis is poorly understood in vertebrates. Here, we show that during cytokinesis of epithelial cells in the Xenopus embryo, lateral cell-cell contacts remain almost perpendicular to the epithelial plane. Depletion of the tight junction-associated protein GEF-H1 leads to a transient and stereotyped deformation of cell-cell contacts. Although, this deformation occurs only during cytokinesis, we show that it originates from immediate neighbours of the dividing cell. Moreover, we show that exocyst and recycling endosome regulation by GEF-H1 are involved in adaptation of cell-cell contacts to deformation. Our results highlight the crucial role of tight junctions and GEF-H1 in cell-cell contact adaptation when cells are exposed to a mechanical stress such as cytokinesis.
Assuntos
Citocinese/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Junções Íntimas/metabolismo , Proteínas de Xenopus/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Junções Aderentes/metabolismo , Junções Aderentes/ultraestrutura , Amidas/farmacologia , Animais , Comunicação Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Forma Celular , Embrião não Mamífero , Células Epiteliais/ultraestrutura , Morfolinos/genética , Morfolinos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/deficiência , Junções Íntimas/ultraestrutura , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/deficiência , Xenopus laevis , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.
Assuntos
Axonema/genética , Axonema/metabolismo , Proteínas de Transporte/genética , Cílios/patologia , Dineínas/química , Dineínas/metabolismo , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Mutação , Animais , Axonema/patologia , Axonema/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/genética , Dineínas/ultraestrutura , Exoma/genética , Éxons/genética , Imunofluorescência , Genes Recessivos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Xenopus , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genéticaRESUMO
Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.
Assuntos
Ciclina T/genética , Quinase 9 Dependente de Ciclina/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes myc , Crista Neural/embriologia , Fator B de Elongação Transcricional Positiva/genética , Elongação da Transcrição Genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Animais , Ciclina T/deficiência , Quinase 9 Dependente de Ciclina/deficiência , Isoxazóis/farmacologia , Leflunomida , Morfolinos/farmacologia , Fator B de Elongação Transcricional Positiva/deficiência , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA Polimerase II/metabolismo , Fatores de Transcrição SOXE/biossíntese , Fatores de Transcrição SOXE/genética , Elongação da Transcrição Genética/efeitos dos fármacos , Transcrição Gênica , Proteínas de Xenopus/deficiência , Xenopus laevis/genéticaRESUMO
The NF2 gene product Merlin is a FERM-domain protein possessing a broad tumor-suppressing function. NF2/Merlin has been implicated in regulating multiple signaling pathways critical for cell growth and survival. However, it remains unknown whether NF2/Merlin regulates Wnt/ß-catenin signaling during vertebrate embryogenesis. Here we demonstrate that NF2/Merlin is required for body pattern formation in the Xenopus laevis embryo. Depletion of the maternal NF2/Merlin enhances organizer gene expression dependent on the presence of ß-catenin, and causes dorsanteriorized development; Morpholino antisense oligo-mediated knockdown of the zygotic NF2/Merlin shifts posterior genes anteriorwards and reduces the anterior development. We further demonstrate that targeted depletion of NF2 in the presumptive dorsal tissues increases the levels of nuclear ß-catenin in the neural epithelial cells. Biochemical analyses reveal that NF2 depletion promotes the production of active ß-catenin and concurrently decreases the level of N-terminally phosphorylated ß-catenin under the stimulation of the endogenous Wnt signaling. Our findings suggest that NF2/Merlin negatively regulates the Wnt/ß-catenin signaling activity during the pattern formation in early X. laevis embryos.
Assuntos
Neurofibromina 2/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genes da Neurofibromatose 2 , Hibridização In Situ , Masculino , Neurofibromina 2/deficiência , Neurofibromina 2/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Zigoto/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis. RESULTS: Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2. CONCLUSIONS: Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.
Assuntos
Actinas/fisiologia , Cones de Crescimento/metabolismo , Microtúbulos/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Elongação Traducional da Cadeia Peptídica/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas de Xenopus/fisiologia , Actinas/antagonistas & inibidores , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Transporte/metabolismo , Colchicina/farmacologia , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Quinase do Fator 2 de Elongação/metabolismo , Regulação da Expressão Gênica , Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/genética , Netrina-1 , Nocodazol/farmacologia , Oócitos , Fator 2 de Elongação de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais/fisiologia , Tiazolidinas/farmacologia , Moduladores de Tubulina/farmacologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Morfolinos/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularização Fisiológica/genética , Oligorribonucleotídeos Antissenso/genética , Oócitos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Heterotaxy is a disorder of left-right body patterning, or laterality, that is associated with major congenital heart disease. The aetiology and mechanisms underlying most cases of human heterotaxy are poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer, where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals. The mechanism that specifies these two cilia types remains unknown. Here we show that the N-acetylgalactosamine-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking the ciliopathy primary ciliary dyskinesia. Together our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the left-right organizer to determine laterality, and reveal a novel mechanism for human heterotaxy.
Assuntos
Padronização Corporal , Cílios/fisiologia , Síndrome de Heterotaxia/genética , N-Acetilgalactosaminiltransferases/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Cílios/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Glicosilação , Humanos , Camundongos , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/deficiência , N-Acetilgalactosaminiltransferases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptor Notch1/química , Receptor Notch1/deficiência , Receptor Notch1/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genéticaRESUMO
The formation of the dorsal-ventral (DV) and anterior-posterior (AP) axes, fundamental to the body plan of animals, is regulated by several groups of polypeptide growth factors including the TGF-ß, FGF, and Wnt families. In order to ensure the establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated. However, the molecular mechanisms responsible for these interactions remain unclear. Here, we demonstrate that the forkhead box transcription factor FoxB1, which is upregulated by the neuralizing factor Oct-25, plays an important role in the formation of the DV and AP axes. Overexpression of FoxB1 promoted neural induction and inhibited BMP-dependent epidermal differentiation in ectodermal explants, thereby regulating the DV patterning of the ectoderm. In addition, FoxB1 was also found to promote the formation of posterior neural tissue in both ectodermal explants and whole embryos, suggesting its involvement in embryonic AP patterning. Using knockdown analysis, we found that FoxB1 is required for the formation of posterior neural tissues, acting in concert with the Wnt and FGF pathways. Consistent with this, FoxB1 suppressed the formation of anterior structures via a process requiring the function of XWnt-8 and eFGF. Interestingly, while downregulation of FoxB1 had little effect on neural induction, we found that it functionally interacted with its upstream factor Oct-25 and plays a supportive role in the induction and/or maintenance of neural tissue. Our results suggest that FoxB1 is part of a mechanism that fine-tunes, and leads to the coordinated formation of, the DV and AP axes during early development.
Assuntos
Padronização Corporal/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Animais , Sequência de Bases , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Morfolinos/genética , Neurogênese/genética , Neurogênese/fisiologia , Oligonucleotídeos Antissenso/genética , Fatores do Domínio POU/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Regulação para Cima , Proteínas Wnt/fisiologia , Via de Sinalização Wnt , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Biologia Computacional , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Anormalidades do Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microinjeções , Dados de Sequência Molecular , Filogenia , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Proteínas de Xenopus/química , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/metabolismoRESUMO
Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.
Assuntos
Hipotricose/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação Puntual/genética , Proteínas Wnt/antagonistas & inibidores , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Embrião de Galinha , Mapeamento Cromossômico , Cromossomos Humanos Par 18/genética , Genes Dominantes/genética , Genes Reporter/genética , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Humanos , Hipotricose/metabolismo , Hipotricose/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiência , Proteínas de Membrana , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Couro Cabeludo , Transdução de Sinais , Pele , Medula Espinal/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMO
Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.
Assuntos
Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Actinas/metabolismo , Animais , Polaridade Celular , Sobrevivência Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Humanos , Mitose , Modelos Biológicos , Miosinas/química , Miosinas/deficiência , Transporte Proteico , Proteínas de Xenopus/química , Proteínas de Xenopus/deficiênciaRESUMO
Pontin (Tip49) and Reptin (Tip48) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. Here we show that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. We conclude that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. We propose a novel function of xPontin and xReptin as co-repressors in the c-Myc/Miz-1 pathway.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Biomarcadores , Proteínas de Transporte/genética , Proliferação de Células , DNA Helicases/genética , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Mutação/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Tempo , Fatores de Transcrição/genética , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, since Nkx2.5-null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed subtractive hybridization by using RNA isolated from wild-type and Nkx2.5-null hearts at embryonic day 8.5. We isolated a mouse cDNA encoding myocardin A, which is an alternative spliced isoform of myocardin and the most abundant isoform in the heart from embryo to adult. The expression of myocardin A and myocardin was markedly downregulated in Nkx2.5-null mouse hearts. Transient-cotransfection analysis showed that Nkx2.5 transactivates the myocardin promoter. Inhibition of myocardin function in the teratocarcinoma cell line P19CL6 prevented differentiation into cardiac myocytes after dimethyl sulfoxide treatment. Myocardin A transactivated the promoter of the atrial natriuretic factor gene through the serum response element, which was augmented by bone morphogenetic protein 2 and transforming growth factor beta-activated kinase 1. These results suggest that myocardin expression is regulated by Nkx2.5 and that its function is required for cardiomyogenesis.
Assuntos
Coração Fetal/embriologia , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição , Proteínas de Xenopus/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Regulação para Baixo , Coração Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ativação Transcricional , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genéticaRESUMO
In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes.