RESUMO
The vertebrate planar cell polarity (PCP) pathway shares molecular components with the ß-catenin-mediated canonical Wnt pathway but acts through membrane complexes containing Vang or Frizzled to orient neighboring cells coordinately. The molecular interactions underlying the action of Vang in PCP signaling and specification, however, are yet to be delineated. Here, we report the identification of Rack1 as an interacting protein of a vertebrate Vang protein, Vangl2. We demonstrate that Rack1 is required in zebrafish for PCP-regulated processes, including oriented cell division, cellular polarization, and convergent extension during gastrulation. We further show that the knockdown of Rack1 affects membrane localization of Vangl2 and that the Vangl2-interacting domain of Rack1 has a dominant-negative effect on Vangl2 localization and gastrulation. Moreover, Rack1 antagonizes canonical Wnt signaling. Together, our data suggest that Rack1 regulates the localization of an essential PCP protein and acts as a molecular switch to promote PCP signaling.
Assuntos
Polaridade Celular/fisiologia , Gástrula/metabolismo , Gastrulação/fisiologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Divisão Celular/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Gástrula/citologia , Proteínas de Membrana/genética , Camundongos , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Proteínas Wnt/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Eritropoese/genética , Mitose/genética , Mutação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Anemia Diseritropoética Congênita/genética , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Peixe-Zebra/sangueRESUMO
Endothelial tubular morphogenesis relies on an exquisite interplay of microtubule dynamics and actin remodeling to propel directed cell migration. Recently, the dynamicity and integrity of microtubules have been implicated in the trafficking and efficient translation of the mRNA for HIF-1α (hypoxia-inducible factor), the master regulator of tumor angiogenesis. Thus, microtubule-disrupting agents that perturb the HIF-1α axis and neovascularization cascade are attractive anticancer drug candidates. Here we show that EM011 (9-bromonoscapine), a microtubule-modulating agent, inhibits a spectrum of angiogenic events by interfering with endothelial cell invasion, migration and proliferation. Employing green-fluorescent transgenic zebrafish, we found that EM011 not only inhibited vasculogenesis but also disrupted preexisting vasculature. Mechanistically, EM011 caused proteasome-dependent, VHL-independent HIF-1α degradation and repressed expression of HIF-1α downstream targets, namely VEGF and survivin. Furthermore, EM011 inhibited membrane ruffling and impeded formation of filopodia, lamellipodia and stress fibers, which are critical for cell migration. These events were associated with a drug-mediated decrease in activation of Rho GTPases- RhoA, Cdc42 and Rac1, and correlated with a loss in the geometric precision of centrosome reorientation in the direction of movement. This is the first report to describe a previously unrecognized, antiangiogenic property of a noscapinoid, EM011, and provides evidence for novel anticancer strategies recruited by microtubule-modulating drugs.
Assuntos
Inibidores da Angiogênese/farmacologia , Dioxóis/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Isoquinolinas/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Centrossomo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , Masculino , Camundongos , Microtúbulos/efeitos dos fármacos , Paxilina/fisiologia , Ativação Transcricional , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Complex carbohydrates represent one of the most polymorphic classes of macromolecules, but their functions during embryonic development remain poorly defined. Herein, we show that knockdown of FucT8, the fucosyltransferase responsible for adding an α1,6 fucosyl residue to the core region of N-linked oligosaccharides, results in defective midline patterning during zebrafish development. Reduced FucT8 expression leads to mild cyclopia, small forebrains, U-shaped somites, among other midline patterning defects. One of the principal FucT8 substrates was identified as Apolipoprotein B (ApoB), the major scaffold protein that is responsible for assembly and secretion of lipoprotein particles in vertebrates. In Drosophila, lipoprotein particles are thought to facilitate cell signaling by serving as a transport vehicle for lipid-modified cell signaling proteins, such as hedgehog. In this regard, knockdown of ApoB expression in zebrafish embryos leads to similar midline patterning defects as those seen in FucT8 morphant embryos. Furthermore, preliminary studies suggest that ApoB facilitates Sonic hedgehog signaling during zebrafish development, analogous to the function of lipoprotein particles during hedgehog signaling in Drosophila.
Assuntos
Padronização Corporal/fisiologia , Fucosiltransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Western Blotting , Padronização Corporal/genética , Fucosiltransferases/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Células Hep G2 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Peixe-Zebra/genéticaRESUMO
The vertebrate inner ear arises from the otic placode, a transient thickening of ectodermal epithelium adjacent to neural crest domains in the presumptive head. During late gastrulation, cells fated to comprise the inner ear are part of a domain in cranial ectoderm that contain precursors of all sensory placodes, termed the preplacodal region (PPR). The combination of low levels of BMP activity coupled with high levels of FGF signaling are required to establish the PPR through induction of members of the six/eya/dach, iro, and dlx families of transcription factors. The zebrafish dlx3b/4b transcription factors are expressed at the neural plate border where they play partially redundant roles in the specification of the PPR, otic and olfactory placodes. We demonstrate that dlx3b/4b assist in establishing the PPR through the transcriptional regulation of the BMP antagonist cv2. Morpholino-mediated knockdown of Dlx3b/4b results in loss of cv2 expression in the PPR and a transient increase in Bmp4 activity that lasts throughout early somitogenesis. Through the cv2-mediated inhibition of BMP activity, dlx3b/4b create an environment where FGF activity is favorable for PPR and otic marker expression. Our results provide insight into the mechanisms of PPR specification as well as the role of dlx3b/4b function in PPR and otic placode induction.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Orelha Interna/embriologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Biomarcadores/metabolismo , Padronização Corporal , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Região Branquial/citologia , Região Branquial/metabolismo , Orelha Interna/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fenótipo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Somitos/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaAssuntos
Transplante de Pulmão/efeitos adversos , Disfunção Primária do Enxerto/diagnóstico , Insuficiência Respiratória/terapia , Algoritmos , Aloenxertos , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/mortalidade , Doença Crônica , Simulação por Computador , Rejeição de Enxerto , Humanos , Transplante de Pulmão/métodos , Fenótipo , Disfunção Primária do Enxerto/mortalidade , Pneumologia/normas , Teoria de Sistemas , Resultado do TratamentoRESUMO
BACKGROUND: Chronic lung allograft dysfunction and its main phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), are major causes of mortality after lung transplantation (LT). RAS and early-onset BOS, developing within 3 years after LT, are associated with particularly inferior clinical outcomes. Prediction models for early-onset BOS and RAS have not been previously described. METHODS: LT recipients of the French and Swiss transplant cohorts were eligible for inclusion in the SysCLAD cohort if they were alive with at least 2 years of follow-up but less than 3 years, or if they died or were retransplanted at any time less than 3 years. These patients were assessed for early-onset BOS, RAS, or stable allograft function by an adjudication committee. Baseline characteristics, data on surgery, immunosuppression, and year-1 follow-up were collected. Prediction models for BOS and RAS were developed using multivariate logistic regression and multivariate multinomial analysis. RESULTS: Among patients fulfilling the eligibility criteria, we identified 149 stable, 51 BOS, and 30 RAS subjects. The best prediction model for early-onset BOS and RAS included the underlying diagnosis, induction treatment, immunosuppression, and year-1 class II donor-specific antibodies (DSAs). Within this model, class II DSAs were associated with BOS and RAS, whereas pre-LT diagnoses of interstitial lung disease and chronic obstructive pulmonary disease were associated with RAS. CONCLUSION: Although these findings need further validation, results indicate that specific baseline and year-1 parameters may serve as predictors of BOS or RAS by 3 years post-LT. Their identification may allow intervention or guide risk stratification, aiming for an individualized patient management approach.
RESUMO
We describe a novel hereditary cancer syndrome in the rat that is transmitted by a recessive gene mutation. Animals exhibiting the mutant phenotype develop multiple neuroendocrine malignancies within the first year of life. The endocrine neoplasia is characterized by bilateral adrenal pheochromocytoma, multiple extra-adrenal pheochromocytoma, bilateral medullary thyroid cell neoplasia, bilateral parathyroid hyperplasia, and pituitary adenoma. The appearance of neoplastic disease is preceded by the development of bilateral juvenile cataracts. Although the spectrum of affected tissues is reminiscent of human forms of multiple endocrine neoplasia (MEN), no germ-line mutations were detected in the Ret or Menin genes that are responsible for the dominantly inherited MEN syndromes in humans. Segregation studies in F1 and F2 crosses yielded frequencies of affected animals entirely consistent with a recessive autosomal mode of inheritance. The lack of the phenotype in F1 animals effectively excludes a germ-line tumor suppressor gene mutation as the causal event. The absence of mutation of known MEN genes and the unique constellation of affected tissues, plus the recessive mode of inheritance, lead us to conclude that the mutation of an as yet unknown gene is responsible for this syndrome of inherited neuroendocrine cancer.
Assuntos
Proteínas de Drosophila , Genes Recessivos/genética , Neoplasia Endócrina Múltipla/genética , Animais , Feminino , Mutação em Linhagem Germinativa , Masculino , Neoplasia Endócrina Múltipla/patologia , Proteínas de Neoplasias/genética , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Over 2000 proteins in the Ensembl human genome database have been linked with disease information from OMIM. In comparison with all human proteins, we find that disease-associated proteins tend to have less designable folds in terms of their SCOP family counts, suggesting that they are intrinsically less robust to mutation and environmental stress. Disease proteins also tend to have isoelectric points closer to neutrality and more alternating hydrophilic-hydrophobic amino acid stretches compared with the average human protein. These results suggest that protein aggregation is a significant phenomenon associated with diseases. Another finding in this work is that many disease proteins are highly sequence similar to other disease proteins, suggesting that gene duplication has contributed to the expansion of disease-prone protein families.
Assuntos
Doença , Desenho de Fármacos , Duplicação Gênica , Genoma Humano , Proteínas/química , Proteínas/genética , Substituição de Aminoácidos , Bases de Dados Genéticas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ponto Isoelétrico , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Radical oesophageal resection has until now been regarded as the gold standard for treatment in intraepithelial high-grade neoplasia or early adenocarcinoma of the oesophagus. However, the mortality and morbidity rates are substantial. DESIGN: A new therapeutic approach involving low-risk endoscopic therapy modalities was examined in the framework of a prospective study. PATIENTS: A total of 115 patients with intraepithelial high-grade neoplasia (19) and early adenocarcinoma (96) in Barrett's oesophagus. METHODS: Endoscopic mucosal resection (EMR) was used in 70 patients, and photodynamic therapy (PDT) was used in 32 patients. The two procedures were combined in ten patients. Three patients underwent primary treatment with argon plasma coagulation (APC). The average follow-up was 34 +/- 10 months (range 24-60 months). RESULTS: Complete local remission was achieved in 98%. The overall complication rate was 9.5%. Major complications, such as perforation and severe bleeding, did not occur. Minor complications included not haemoglobin relevant bleeding (drop of haemoglobin less than 2 g/dl) (5) and stenosis (3) after EMR, and long-lasting odynophagia (1) and sunburn (2) after PDT. In all, 13 patients have died so far, but in only one case due to the underlying disease. The calculated overall 3-year survival rate is 88%. During the follow-up period, a 30% rate of metachronous lesions was observed; endoscopic therapy was performed successfully in all but one of these patients. CONCLUSIONS: These good acute-phase and intermediate results, along with low morbidity rates and no mortality, suggest that the organ-preserving local endoscopic procedure including EMR and PDT is an attractive alternative to oesophageal resection. Therefore, endoscopic therapy might replace radical oesophageal resection in future in cases of intraepithelial high-grade neoplasia and early mucosal adenocarcinoma in Barrett's oesophagus.
Assuntos
Adenocarcinoma/terapia , Esôfago de Barrett/terapia , Carcinoma in Situ/terapia , Neoplasias Esofágicas/terapia , Esofagoscopia/métodos , Fotocoagulação a Laser/métodos , Fotoquimioterapia/métodos , Idoso , Terapia Combinada/métodos , Feminino , Humanos , Masculino , Estudos Prospectivos , Resultado do TratamentoRESUMO
In zebrafish, BMP signaling establishes cell identity along the dorsoventral (DV) axis during gastrulation. Owing to the early requirements of BMP activity in DV patterning, it has been difficult to assign later roles in cell fate specification to specific BMP ligands. In this study, we have taken advantage of two follistatin-like genes (fstl1 and fstl2), as well as a transgenic zebrafish line carrying an inducible truncated form of the BMP-type 1 receptor to study the role of Bmp4 outside of the context of DV specification. Characterization of fstl1/2 suggests that they exert a redundant role as BMP antagonists during late gastrulation, regulating BMP activity in axial mesoderm. Maintenance of appropriate levels of BMP signaling is crucial for the proper development of chordamesoderm, a subset of axial mesoderm that gives rise to the notochord, but not prechordal mesoderm, which gives rise to the prechordal plate. Bmp4 activity in particular is required during a crucial window beginning at late gastrulation and lasting through early somitogenesis to promote chordamesoderm proliferation. In the absence of Bmp4, the notochord precursor pool is depleted, and the notochord differentiates prematurely. Our results illustrate a role for Bmp4 in the proliferation and timely differentiation of axial tissue after DV axis specification.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Notocorda/citologia , Notocorda/embriologia , Cauda/embriologia , Peixe-Zebra/embriologia , Animais , Padronização Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Gastrulação/efeitos dos fármacos , Ligantes , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Notocorda/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , N-Acetil-Lactosamina Sintase/fisiologia , Sequência de Aminoácidos , Animais , Matriz Extracelular/metabolismo , Galactose/metabolismo , Humanos , Laminina/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , N-Acetil-Lactosamina Sintase/química , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-ZebraRESUMO
Complex carbohydrates are highly polymorphic macromolecules that are involved in diverse biological processes; however, a detailed understanding of their function remains obscure. To better define the roles of complex carbohydrates during vertebrate embryogenesis, we have initiated an analysis of glycosyltransferase function using the zebrafish system. In this study, we report the characterization of a zebrafish beta1,4-galactosyltransferase (GalT), which has substantial homology with mammalian beta4GalT5 and is expressed zygotically throughout the zebrafish embryo. Downregulating the expression of beta4GalT5 by injection of specific morpholino oligonucleotides results in dorsalized zebrafish embryos, suggesting a role of beta4GalT5 in Bmp2-mediated specification of the dorsoventral axis. Consistent with this, morpholino-injected embryos have ventrally expanded chordin expression and reduced activation of the Bmp-dependent transcription factors Smad1/5/8. Because other growth factors, such as Egf and Fgf, require binding to extracellular proteoglycans for delivery and/or binding to their cognate receptors, we examined whether proteoglycans isolated from control and morpholino-injected embryos show differential binding affinities for Bmp2. In this regard, proteoglycans isolated from beta4GalT5 morphant embryos are underglycosylated and are unable to bind recombinant Bmp2 as efficiently as proteoglycans from control-injected embryos, whereas the binding of Bmp7 is relatively unaffected. These results suggest that beta4GalT5 is a previously unidentified zebrafish galactosyltransferase that is essential for proper patterning of the dorsoventral axis by regulating Bmp2 signaling. Furthermore, this work demonstrates that a relatively simple carbohydrate modification to endogenous proteoglycans can modulate the specificity of cytokine signaling.
Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/enzimologia , Galactosiltransferases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Clonagem Molecular , Embrião não Mamífero/metabolismo , Galactosiltransferases/química , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/metabolismo , Glicosilação , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fenótipo , Filogenia , Ligação Proteica , Proteoglicanas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Smad , Fator de Crescimento Transformador beta/genética , Peixe-Zebra/genéticaRESUMO
The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.
Assuntos
Estruturas Embrionárias/fisiologia , Proteínas de Homeodomínio/metabolismo , Crista Neural/embriologia , Neurônios/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proliferação de Células , Sobrevivência Celular , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Estruturas Embrionárias/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Hibridização In Situ , Crista Neural/citologia , Neurônios/citologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Fatores de Transcrição/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8, we completed sequencing of the entire gene, including the 5' and 3' UTRs. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8, or another early regulator, dlx3b, which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1, which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8. Disrupting pax8, pax2a and pax2b did not further impair otic induction relative to loss of pax8 alone. However, the amount of otic tissue gradually decreased in pax8-pax2a-pax2b-deficient embryos such that no otic tissue was detectable by 24 hours post-fertilization. Loss of otic tissue did not correlate with increased cell death, suggesting that otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain otic fate.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Orelha/embriologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Transativadores/genética , Transativadores/fisiologia , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Morte Celular , Linhagem da Célula , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Éxons , Fator 3 de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição Forkhead , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/química , Fator de Transcrição PAX2 , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Fenótipo , Isoformas de Proteínas , Análise de Sequência de DNA , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Sensory placodes are ectodermal thickenings that give rise to elements of the vertebrate cranial sensory nervous system, including the inner ear and nose. Although mutations have been described in humans, mice and zebrafish that perturb ear and nose development, no mutation is known to prevent sensory placode formation. Thus, it has been postulated that a functional redundancy exists in the genetic mechanisms that govern sensory placode development. We describe a zebrafish deletion mutation, b380, which results in a lack of both otic and olfactory placodes. The b380 deletion removes several known genes and expressed sequence tags, including dlx3 and dlx7, two transcription factors that share a homoeobox domain similar in sequence to the Drosophila Distal-less gene. dlx3 and dlx7 are expressed in an overlapping pattern in the regions that produce the otic and olfactory placodes in zebrafish. We present evidence suggesting that it is specifically the removal of these two genes that leads to the otic and olfactory phenotype of b380 mutants. Using morpholinos, antisense oligonucleotides that effectively block translation of target genes, we find that functional reduction of both dlx genes contributes to placode loss. Expression patterns of the otic marker pax2.1, olfactory marker anxV and eya1, a marker of both placodes, in morpholino-injected embryos recapitulate the reduced expression of these genes seen in b380 mutants. We also examine expression of dlx3 and dlx7 in the morpholino-injected embryos and present evidence for existence of auto- and cross-regulatory control of expression among these genes. We demonstrate that dlx3 is necessary and sufficient for proper otic and olfactory placode development. However, our results indicate that dlx3 and dlx7 act in concert and their importance in placode formation is only revealed by inactivating both paralogs.
Assuntos
Orelha Interna/embriologia , Proteínas de Homeodomínio/genética , Proteínas Nucleares , Condutos Olfatórios/embriologia , Fatores de Transcrição/genética , Peixe-Zebra/genética , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Orelha Interna/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/efeitos da radiação , Feminino , Raios gama , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Proteínas de Homeodomínio/metabolismo , Homozigoto , Mutação , Condutos Olfatórios/anormalidades , Oligonucleotídeos Antissenso/farmacologia , Fator de Transcrição PAX2 , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Gravidez , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-ZebraRESUMO
We have identified three novel members of the zebrafish forkhead class I gene family, which we have named foxi2, foxi3a, and foxi3b. We have reported previously the identification of zebrafish foxi1, which is required for otic placode and jaw development. Expression analysis shows that foxi2 is expressed within the chordamesoderm during early somitogenesis and the retina and pharyngeal arches during later stages. The foxi3a and foxi3b genes likely represent a recently duplicated pair, and they are similarly expressed in epidermal mucous cells throughout embryogenesis and early larval stages. None of these newly identified FoxI genes are expressed in otic precursor cells and, therefore, are unlikely to share functional overlap with foxi1 in the development of the inner ear. In addition to these zebrafish FoxI paralogs, we have identified 16 new FoxI sequences in species ranging from Ciona intestinalis to Homo sapiens. We present an extensive phylogenetic analysis of the FoxI class that includes these new sequences together with those previously reported. This analysis supports the existence of three subfamilies within the FoxI class, each containing at least one zebrafish member.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Filogenia , Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Embrião não Mamífero/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Morfogênese , Família Multigênica , Proteínas de Ligação a RNA/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/classificação , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/químicaRESUMO
The formation of the otic placode is a complex process requiring multiple inductive signals. In zebrafish, fgf3 and fgf8, dlx3b and dlx4b, and foxi1 have been identified as the earliest-acting genes in this process. fgf3 and fgf8 are required as inductive signals, whereas dlx3b, dlx4b, and foxi1 appear to act directly within otic primordia. We have investigated potential interactions among these genes. Depletion of either dlx3b and dlx4b or foxi1 leads to a delay of pax2a expression in the otic primordia and reduction of the otic vesicle. Depletion of both foxi1 and dlx3b results in a complete ablation of otic placode formation. A strong synergistic interaction is also observed among foxi1, fgf3, and fgf8, and a weaker interaction among dlx3b, fgf3, and fgf8. Misexpression of foxi1 can induce expression of pax8, an early marker for the otic primordia, in embryos treated with an inhibitor of fibroblast growth factor (FGF) signaling. Conversely, morpholino knockdown of foxi1 blocks ectopic pax8 expression and otic vesicle formation induced by misexpression of fgf3 and/or fgf8. The observed genetic interactions suggest a model in which foxi1 and dlx3b/dlx4b act in independent pathways together with distinct phases of FGF signaling to promote otic placode induction and development.
Assuntos
Orelha/embriologia , Indução Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero , Indução Embrionária/efeitos dos fármacos , Epistasia Genética , Modelos Biológicos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Transdução de SinaisRESUMO
Multiple endocrine neoplasia-like syndrome (MENX) is a hereditary cancer syndrome in the rat characterized by inborn cataract and multiple tumors affecting the neuroendocrine system developed within the first year of life. The spectrum of affected organs is intermediate between MEN type 1 (MEN1) and MEN type 2 (MEN2) syndromes in human, but, in contrast to them, MENX is inherited in a recessive fashion. Here we report the mapping of the MENX locus to rat Chromosome (Chr) 4 by a genome-wide linkage analysis. This analysis was done in 41 animals obtained from a (Wistar/Nhg x SDwe) x SDwe interstrain backcross, where SDwe (Sprague-Dawley white eye) indicates the affected animals. The MENX disease locus was ultimately mapped to a approximately 22-cM interval on Chr 4 that includes the rat homolog of the human RET proto-oncogene. As activating point mutations of RET are known to be responsible for MEN2 in human, we analyzed several markers located in the proximity of Ret for linkage to the disease phenotype. Our data exclude Ret involvement in MENX and establish that a second gene, playing a role in endocrine tumor formation, lies within the distal part of rat Chr 4. Although heritable human endocrine tumors are quite rare, sporadic tumors of MEN-affected tissues occur at a much higher frequency, and their pathogenesis is poorly understood. The identification of the MENX gene should contribute to our understanding of the genetic mechanisms of neuroendocrine tissue tumorigenesis and may assist in developing new and more appropriate therapeutic strategies for these diseases.