RESUMEN
To form tissues with unique functions and structures, it is important that the cells that comprise them maintain physical contact. On the other hand, with each mitosis, drastic changes in cell shapes, cell adhesion, and cytoskeletal architecture may cause such contacts to be temporarily weakened, risking improper development and maintenance of tissues. Despite such risks, tissues form properly during normal development. However, it is not well understood whether mitotic abnormalities affect tissue formation. Here, analysis of zebrafish embryos with aberrant mitosis shows that proper progression of mitosis is important to maintain cell contact in developing tissues. By screening mutants with abnormal trunk and tail development, we obtained a mutant with perturbed expression of some tissue-specific genes in embryonic caudal regions. The responsible gene is mastl/gwl, which is involved in progression of mitosis. Analysis focusing on the chordo-neural hinge (CNH), the primordium of axial tissues, shows that cell detachment from the CNH is increased in mastl mutant embryos. Time-lapse imaging reveals that this cell detachment occurs during mitosis. These results suggest that cells are unable to maintain contact due to abnormalities in progression of mitosis in mastl mutants.
RESUMEN
The metameric pattern of somites is created based on oscillatory expression of clock genes in presomitic mesoderm. However, the mechanism for converting the dynamic oscillation to a static pattern of somites is still unclear. Here, we provide evidence that Ripply/Tbx6 machinery is a key regulator of this conversion. Ripply1/Ripply2-mediated removal of Tbx6 protein defines somite boundary and also leads to cessation of clock gene expression in zebrafish embryos. On the other hand, activation of ripply1/ripply2 mRNA and protein expression is periodically regulated by clock oscillation in conjunction with an Erk signaling gradient. Whereas Ripply protein decreases rapidly in embryos, Ripply-triggered Tbx6 suppression persists long enough to complete somite boundary formation. Mathematical modeling shows that a molecular network based on results of this study can reproduce dynamic-to-static conversion in somitogenesis. Furthermore, simulations with this model suggest that sustained suppression of Tbx6 caused by Ripply is crucial in this conversion.
Asunto(s)
Somitos , Pez Cebra , Animales , Somitos/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Mesodermo/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
The presumptive somite boundary in the presomitic mesoderm (PSM) is defined by the anterior border of the expression domain of Tbx6 protein. During somite segmentation, the expression domain of Tbx6 is regressed by Ripply-meditated degradation of Tbx6 protein. Although the expression of zebrafish tbx6 remains restricted to the PSM, the transcriptional regulation of tbx6 remains poorly understood. Here, we show that the expression of zebrafish tbx6 is maintained by transcriptional autoregulation. We find that a proximal-located cis-regulatory module, TR1, which contains two putative T-box sites, is required for somite segmentation in the intermediate body and for proper expression of segmentation genes. Embryos with deletion of TR1 exhibit significant reduction of tbx6 expression at the 12-somite stage, although its expression is initially observed. Additionally, Tbx6 is associated with TR1 and activates its own expression in the anterior PSM. Furthermore, the anterior expansion of tbx6 expression in ripply gene mutants is suppressed in a TR1-dependent manner. The results suggest that the autoregulatory loop of zebrafish tbx6 facilitates immediate removal of Tbx6 protein through termination of its own transcription at the anterior PSM.
Asunto(s)
Tipificación del Cuerpo/genética , Homeostasis/genética , Somitos/embriología , Proteínas de Dominio T Box/metabolismo , Transcripción Genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Sitios de Unión/genética , Embrión no Mamífero/metabolismo , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Homocigoto , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Somitos/metabolismo , Proteínas de Dominio T Box/química , Proteínas de Dominio T Box/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Somites sequentially form with a regular interval by the segmentation from the anterior region of the presomitic mesoderm (PSM). The expression of several genes involved in the somite segmentation is switched off at the transition from the anterior PSM to somites. Zebrafish Ripply1, which down-regulates a T-box transcription factor Tbx6, is required for the suppression of segmentation gene expression. However, the functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior PSM remain elusive. In this study, we generated ripply1 mutants and examined genetic interaction between ripply1/2 and tbx6. Zebrafish ripply1-/- embryos failed to form the somite boundaries as was observed in knockdown embryos. We found that somite segmentation defects in ripply1 mutants were suppressed by heterozygous mutation of tbx6 or partial translational inhibition of tbx6 by antisense morpholino. We further showed that somite boundaries that were recovered in tbx6+/-; ripply1-/- embryos were dependent on the function of ripply2, indicating that relative gene dosage between ripply1/2 and tbx6 plays a critical role in the somite formation. Interestingly, the expression of segmentation genes such mesp as was still not fully suppressed at the anterior PSM of tbx6+/-; ripply1-/- embryos although the somite formation and rostral-caudal polarity of somites were properly established. Furthermore, impaired myogenesis was observed in the segmented somites in tbx6+/-; ripply1-/- embryos. These results revealed that partial suppression of the segmentation gene expression by Ripply is sufficient to establish the rostral-caudal polarity of somites, and that stronger suppression of the segmentation gene expression by Ripply is required for proper myogenesis in zebrafish embryos.
Asunto(s)
Tipificación del Cuerpo/genética , Desarrollo Embrionario/genética , Proteínas Nucleares/genética , Proteínas de Dominio T Box/genética , Proteínas de Pez Cebra/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Mesodermo/crecimiento & desarrollo , Morfolinos/genética , Desarrollo de Músculos/genética , Somitos/crecimiento & desarrollo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
The segmental pattern of somites is generated by sequential conversion of the temporal periodicity provided by the molecular clock. Whereas the basic structure of this clock is conserved among different species, diversity also exists, especially in terms of the molecular network. The temporal periodicity is subsequently converted into the spatial pattern of somites, and Mesp2 plays crucial roles in this conversion in the mouse. However, it remains unclear whether Mesp genes play similar roles in other vertebrates. In this study, we generated zebrafish mutants lacking all four zebrafish Mesp genes by using TALEN-mediated genome editing. Contrary to the situation in the mouse Mesp2 mutant, in the zebrafish Mesp quadruple mutant embryos the positions of somite boundaries were clearly determined and morphological boundaries were formed, although their formation was not completely normal. However, each somite was caudalized in a similar manner to the mouse Mesp2 mutant, and the superficial horizontal myoseptum and lateral line primordia were not properly formed in the quadruple mutants. These results clarify the conserved and species-specific roles of Mesp in the link between the molecular clock and somite morphogenesis.
Asunto(s)
Somitos/metabolismo , Pez Cebra/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Morfogénesis/genética , Morfogénesis/fisiología , Mutación/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The somite is the most prominent metameric structure observed during vertebrate embryogenesis, and its metamerism preserves the characteristic structures of the vertebrae and muscles in the adult body. During vertebrate somitogenesis, sequential formation of epithelialized cell boundaries generates the somites. According to the "clock and wavefront model," the periodical and sequential generation of somites is achieved by the integration of spatiotemporal information provided by the segmentation clock and wavefront. In the anterior region of the presomitic mesoderm, which is the somite precursor, the orchestration between the segmentation clock and the wavefront achieves morphogenesis of somites through multiple processes such as determination of somite boundary position, generation of morophological boundary, and establishment of the rostrocaudal polarity within a somite. Recently, numerous studies using various model animals including mouse, zebrafish, and chick have gradually revealed the molecular aspect of the "clock and wavefront" model and the molecular mechanism connecting the segmentation clock and the wavefront to the multiple processes of somite morphogenesis. In this review, we first summarize the current knowledge about the molecular mechanisms underlying the clock and the wavefront and then describe those of the three processes of somite morphogenesis. Especially, we will discuss the conservation and diversification in the molecular network of the somitigenesis among vertebrates, focusing on two typical model animals used for genetic analyses, i.e., the mouse and zebrafish. In this review, we described molecular mechanism for the generation of somites based on the spatiotemporal information provided by "segmentation clock" and "wavefront" focusing on the evidences obtained from mouse and zebrafish.
Asunto(s)
Embrión de Mamíferos/embriología , Embrión no Mamífero/embriología , Morfogénesis/fisiología , Somitos/embriología , Pez Cebra/embriología , Animales , RatonesRESUMEN
Somitogenesis in vertebrates is a complex and dynamic process involving many sequences of events generated from the segmentation clock. Previous studies with mouse embryos revealed that the presumptive somite boundary is periodically created at the anterior border of the expression domain of Tbx6 protein. Ripply1 and Ripply2 are required for the determination of the Tbx6 protein border, but the mechanism by which this Tbx6 domain is regulated remains unclear. Furthermore, since zebrafish and frog Ripplys are known to be able to suppress Tbx6 function at the transcription level, it is also unclear whether Ripply-mediated mechanism of Tbx6 regulation is conserved among different species. Here, we tested the generality of Tbx6 protein-mediated process in somite segmentation by using zebrafish and further examined the mechanism of regulation of Tbx6 protein. By utilizing an antibody against zebrafish Tbx6/Fss, previously referred to as Tbx24, we found that the anterior border of Tbx6 domain coincided with the presumptive intersomitic boundary even in the zebrafish and it shifted dynamically during 1 cycle of segmentation. Consistent with the findings in mice, the tbx6 mRNA domain was located far anterior to its protein domain, indicating the possibility of posttranscriptional regulation. When both ripply1/2 were knockdown, the Tbx6 domain was anteriorly expanded. We further directly demonstrated that Ripply could reduce the expression level of Tbx6 protein depending on physical interaction between Ripply and Tbx6. Moreover, the onset of ripply1 and ripply2 expression occurred after reduction of FGF signaling at the anterior PSM, but this expression initiated much earlier on treatment with SU5402, a chemical inhibitor of FGF signaling. These results strongly suggest that Ripply is a direct regulator of the Tbx6 protein level for the establishment of intersomitic boundaries and mediates a reduction in FGF signaling for the positioning of the presumptive intersomitic boundary in the PSM.
Asunto(s)
Somitos/embriología , Somitos/metabolismo , Proteínas de Dominio T Box/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas de Dominio T Box/química , Proteínas de Dominio T Box/genética , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas Portadoras/genética , Desarrollo Embrionario/genética , Proteínas de Xenopus/genética , Pez Cebra/genética , Alelos , Animales , Proteínas Portadoras/biosíntesis , Proteínas del Citoesqueleto/genética , Citoesqueleto/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Dominios PDZ/genética , Fenotipo , ARN Mensajero/biosíntesis , Proteínas Wnt/genética , Xenopus , Proteínas de Xenopus/biosíntesis , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
Animal body color is generated primarily by neural crest-derived pigment cells in the skin. Mammals and birds have only melanocytes on the surface of their bodies; however, fish have a variety of pigment cell types or chromatophores, including melanophores, xanthophores, and iridophores. The medaka has a unique chromatophore type called the leucophore. The genetic basis of chromatophore diversity remains poorly understood. Here, we report that three loci in medaka, namely, leucophore free (lf), lf-2, and white leucophore (wl), which affect leucophore and xanthophore differentiation, encode solute carrier family 2, member 15b (slc2a15b), paired box gene 7a (pax7a), and solute carrier family 2 facilitated glucose transporter, member 11b (slc2a11b), respectively. Because lf-2, a loss-of-function mutant for pax7a, causes defects in the formation of xanthophore and leucophore precursor cells, pax7a is critical for the development of the chromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, as these chromatophores have purine-dependent light reflection. Our identification of slc2a15b and slc2a11b as genes critical for the differentiation of leucophores and xanthophores in medaka led to a further finding that the existence of these two genes in the genome coincides with the presence of xanthophores in nonmammalian vertebrates: birds have yellow-pigmented irises with xanthophore-like intracellular organelles. Our findings provide clues for revealing diverse evolutionary mechanisms of pigment cell formation in animals.
Asunto(s)
Cromatóforos/fisiología , Regulación del Desarrollo de la Expresión Génica , Oryzias/embriología , Animales , Tipificación del Cuerpo , Diferenciación Celular , Embrión de Pollo , Cromatóforos/metabolismo , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos/metabolismo , Genoma , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Melanóforos/metabolismo , Datos de Secuencia Molecular , Mutación , Cresta Neural/citología , Cresta Neural/patología , Oryzias/fisiología , Factor de Transcripción PAX7/metabolismo , Fenotipo , Filogenia , Pigmentación , VertebradosRESUMEN
The molecular mechanism underlying somite development differs along the embryonic antero-posterior axis. In zebrafish, cell lineage tracing and genetic analysis have revealed a difference in somite development between the trunk and tail. For instance, spadetail/tbx16 (spt) mutant embryos lack trunk somites but not tail ones. Trunk and tail somites are developed from mesodermal progenitor cells (MPCs) located in the tailbud. While the undifferentiated state of MPCs is maintained by mutual activation between Wnt and Brachyury/Ntl, the mechanism by which the MPCs differentiate into presomitic mesoderm (PSM) cells remains largely unclear. Especially, the molecules that promote PSM differentiation during tail development should be clarified. Here, we show that zebrafish embryos defective in mesogenin1 (msgn1) and spt failed to differentiate into PSM cells in tail development and show increased expression of wnt8 and ntl. Msgn1 acted in a cell-autonomous manner and as a transcriptional activator in PSM differentiation. The expression of msgn1 initially overlapped with that of ntl in the ventral tailbud, as previously reported; and its mis-expression caused ectopic expression of tbx24, a PSM marker gene, only in the tailbud and posterior notochord, both of which expressed ntl in zebrafish embryos. Furthermore, the PSM-inducing activity of misexpressed msgn1 was enhanced by co-expression with ntl. Thus, Msgn1 exercised its PSM-inducing activity in cells expressing ntl. Based on these results, we speculate that msgn1 expression in association with that of ntl may allow the differentiation of progenitor cells to proceed during development of somites in the tail.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Mesodermo/metabolismo , Cola (estructura animal)/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Embrión no Mamífero/metabolismo , Proteínas Fetales , Mesodermo/citología , Somitos/citología , Somitos/metabolismo , Células Madre/metabolismo , Proteínas de Dominio T Box/metabolismo , Cola (estructura animal)/citologíaRESUMEN
Females homozygous for a mutation in cellular island (cei) produce embryos with defects in cytokinesis during early development. Analysis of the cytoskeletal events associated with furrow formation reveal that these defects include a general delay in furrow initiation as well as a complete failure to form furrow-associated structures in distal regions of the blastodisc. A linkage mapping-based candidate gene approach, including transgenic rescue, shows that cei encodes the zebrafish Aurora B kinase homologue. Genetic complementation analysis between the cei mutation and aurB zygotic lethal mutations corroborate gene assignment and reveal a complex nature of the maternal-effect cei allele, which appears to preferentially affect a function important for cytokinesis in the early blastomeres. Surprisingly, in cei mutant embryos a short yet otherwise normal furrow forms in the center of the blastodisc. Furrow formation is absent throughout the width of the blastodisc in cei mutant embryos additionally mutant for futile cycle, which lack a spindle apparatus, showing that the residual furrow signal present in cei mutants is derived from the mitotic spindle. Our analysis suggests that partially redundant signals derived from the spindle and astral apparatus mediate furrow formation in medial and distal regions of the early embryonic blastomeres, respectively, possibly as a spatial specialization to achieve furrow formation in these large cells. In addition, our data also suggest a role for Cei/AurB function in the reorganization of the furrow-associated microtubules in both early cleavage- and somite-stage embryos. In accordance with the requirement for cei/aurB in furrow induction in the early cleavage embryo, germ plasm recruitment to the forming furrow is also affected in embryos lacking normal cei/aurB function.
Asunto(s)
Citocinesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Aurora Quinasas , Tipificación del Cuerpo , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Proteínas Serina-Treonina Quinasas/genética , Especificidad de la Especie , Huso Acromático/enzimología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
A female-sterile zebrafish maternal-effect mutation in cellular atoll (cea) results in defects in the initiation of cell division starting at the second cell division cycle. This phenomenon is caused by defects in centrosome duplication, which in turn affect the formation of a bipolar spindle. We show that cea encodes the centriolar coiled-coil protein Sas-6, and that zebrafish Cea/Sas-6 protein localizes to centrosomes. cea also has a genetic paternal contribution, which when mutated results in an arrested first cell division followed by normal cleavage. Our data supports the idea that, in zebrafish, paternally inherited centrosomes are required for the first cell division while maternally derived factors are required for centrosomal duplication and cell divisions in subsequent cell cycles. DNA synthesis ensues in the absence of centrosome duplication, and the one-cycle delay in the first cell division caused by cea mutant sperm leads to whole genome duplication. We discuss the potential implications of these findings with regards to the origin of polyploidization in animal species. In addition, the uncoupling of developmental time and cell division count caused by the cea mutation suggests the presence of a time window, normally corresponding to the first two cell cycles, which is permissive for germ plasm recruitment.
Asunto(s)
Centriolos/metabolismo , Proteínas Cromosómicas no Histona/genética , Duplicación de Gen , Genoma , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Secuencia de Aminoácidos , Animales , Proteínas Cromosómicas no Histona/química , Fase de Segmentación del Huevo/citología , Embrión no Mamífero/citología , Desarrollo Embrionario , Padre , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Mitosis , Modelos Genéticos , Datos de Secuencia Molecular , Madres , Proteínas Mutantes/metabolismo , Mutación/genética , Ploidias , Transporte de Proteínas , Espermatozoides/citología , Huso Acromático/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/químicaRESUMEN
Cytokinesis in early zebrafish embryos involves coordinated changes in the f-actin- and microtubule-based cytoskeleton, and the recruitment of adhesion junction components to the furrow. We show that exposure to inhibitors of non-muscle myosin II function does not affect furrow ingression during the early cleavage cycles but interferes with the recruitment of pericleavage f-actin and cortical beta-catenin aggregates to the furrow, as well as the remodeling of the furrow microtubule array. This remodeling is in turn required for the distal aggregation of the zebrafish germ plasm. Embryos with reduced myosin activity also exhibit at late stages of cytokinesis a stabilized contractile ring apparatus that appears as a ladder-like pattern of short f-actin cables, supporting a role for myosin function in the disassembly of the contractile ring after furrow formation. Our studies support a role for myosin function in furrow maturation that is independent of furrow ingression and which is essential for the recruitment of furrow components and the remodeling of the cytoskeleton during cytokinesis.
Asunto(s)
Miosina Tipo II/fisiología , Pez Cebra/embriología , Actinas/metabolismo , Animales , Azepinas/farmacología , Agregación Celular/efectos de los fármacos , Citocinesis/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , ARN Helicasas DEAD-box/genética , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Hibridación in Situ , Miosina Tipo II/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Naftalenos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , beta Catenina/metabolismoRESUMEN
The Bone morphogenetic protein (Bmp) signalling gradient has a major function in the formation of the dorso-ventral axis. The zebrafish ventralized mutant, ogon, encodes Secreted Frizzled (Sizzled). sizzled is ventrally expressed in a Bmp-dependent manner and is required for the suppression of Bmp signalling on the ventral side of zebrafish embryos. However, it remains unclear how Sizzled inhibits Bmp signalling and controls ventro-lateral cell fate. We found that Sizzled stabilizes Chordin, a Bmp antagonist, by binding and inhibiting the Tolloid-family metalloproteinase, Bmp1a, which cleaves and inactivates Chordin. The cysteine-rich domain of Sizzled is required for inhibition of Bmp1a activity. Loss of both Bmp1a and Tolloid-like1 (Tll1; another Tolloid-family metalloproteinase) function leads to a complete suppression and reversal of the ogon mutant phenotype. These results indicate that Sizzled represses the activities of Tolloid-family proteins, thereby creating the Chordin-Bmp activity gradient along the dorso-ventral axis. Here, we describe a previously unrecognized role for a secreted Frizzled-related protein.
Asunto(s)
Tipificación del Cuerpo/fisiología , Polaridad Celular , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Pez Cebra/fisiología , Pez Cebra , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Cisteína/química , Cisteína/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/antagonistas & inhibidores , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Morfogénesis/genética , Oligonucleótidos/farmacología , Fenotipo , Metaloproteinasas Similares a Tolloid , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.
Asunto(s)
Cadherinas/fisiología , Gástrula/fisiología , Regulación del Desarrollo de la Expresión Génica , Mutación , Alelos , Animales , Secuencia de Bases , Compuestos de Boro/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular , Movimiento Celular , Exones , Inmunohistoquímica , Hibridación in Situ , Indoles/farmacología , Microscopía Electrónica , Datos de Secuencia Molecular , Movimiento , Neuronas/metabolismo , Oligonucleótidos/farmacología , Fenotipo , Homología de Secuencia de Ácido Nucleico , Temperatura , Pez Cebra , CigotoRESUMEN
sax1/nkx1.2 and sax2/nkx1.1 are members of the evolutionally conserved NK-1 homeobox gene family. sax1/nkx1.2 is reported to be expressed in the central nervous system during early and late neurogenesis in the chick and mouse, but the expression of sax2/nkx1.1 has not been reported. We isolated zebrafish cDNAs for sax1/nkx1.2 and sax2/nkx1.1 and examined their expression. In zebrafish, unlike chick and mouse, sax1/nkx1.2 was expressed in the prospective medial floor plate from the mid-gastrula period and was dependent on Nodal signaling. From the early segmentation period, sax1/nkx1.2 was also expressed in the posterior neuroectoderm. sax2/nkx1.1 was expressed in the prospective extraocular muscles, mesencephalic neurons residing along the tract of the posterior commissure, ventral neurons in the hindbrain, and interneurons in the spinal cord.
Asunto(s)
Sistema Nervioso Central/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Factores de Transcripción/biosíntesis , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Proteína Nodal , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. We identified a novel maternal-effect recessive mutation, tokkaebi (tkk), that affects formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5-100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos displayed defects in the nuclear accumulation of beta-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly affected. Injection of RNAs for activated beta-catenin, dominant-negative forms of Axin1 and GSK3beta, and wild-type Dvl3, into the tkk embryos suppressed the ventralized phenotypes and/or dorsalized the embryos, and restored or induced an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs was affected in the tkk embryos. Inhibition of cytoplasmic calcium release elicited an ectopic and expanded expression of chordin in the wild-type, but did not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the beta-catenin-degradation machinery to control the stability of beta-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of beta-catenin is involved in dorsal axis formation in zebrafish.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/fisiología , Señalización del Calcio/fisiología , Núcleo Celular/metabolismo , Mapeo Cromosómico , Cruzamientos Genéticos , Proteínas del Citoesqueleto/metabolismo , Ligandos , Organizadores Embrionarios/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Proteínas Wnt , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra , beta CateninaRESUMEN
The zebrafish mutant ogon (also called mercedes and short tail) displays ventralized phenotypes similar to the chordino (dino) mutant, in which the gene for the Bmp antagonist Chordin is mutated. We isolated the gene responsible for ogon by a positional cloning strategy and found that the ogon locus encodes a zebrafish homolog of Secreted Frizzled (Sizzled), which has sequence similarity to a Wnt receptor, Frizzled. Unlike other secreted Frizzled-related proteins (sFrps) and the Wnt inhibitor Dickkopf1, the misexpression of Ogon/Sizzled dorsalized, but did not anteriorize, the embryos, suggesting a role for Ogon/Sizzled in Bmp inhibition. Ogon/Sizzled did not inhibit a Wnt8-dependent transcription in the zebrafish embryo. ogon/sizzled was expressed on the ventral side from the late blastula through the gastrula stages. The ventral ogon/sizzled expression in the gastrula stage was reduced or absent in the swirl/bmp2b mutants but expanded in the chordino mutants. Misexpression of ogon/sizzled did not dorsalize the chordino mutants, suggesting that Ogon/Sizzled required Chordin protein for dorsalization and Bmp inhibition. These data indicate that Ogon/Sizzled functions as a negative regulator of Bmp signaling and reveal a novel role for a sFrp in dorsoventral patterning.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Proteínas/metabolismo , Proteínas de Xenopus , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/fisiología , Retroalimentación Fisiológica , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
A homeobox gene, pnx, is expressed in prospective posterior neurogenic regions and later in primary neurons. pnx expression was regulated by a signal from the non-axial mesendoderm and by Notch signaling. Pnx contains an Eh1 repressor domain, which interacted with Groucho and acted as a transcriptional repressor. Misexpression of pnx increased neural precursor cells and postmitotic neurons, which express neurogenin1 and elavl3/HuC, respectively. Expression of an antimorphic Pnx (VP16Pnx) or inhibition of Pnx by antisense morpholino oligonucleotide led to the reduction in the number of a subset of primary neurons. Misexpression of pnx promoted neurogenesis independent of Notch signaling. Epistatic analyses showed that Pnx also functions downstream of the Notch signal. These data indicate that pnx is a novel repressor-type homeobox gene that regulates posterior neurogenesis.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio/genética , Sistema Nervioso/embriología , Proteínas Nucleares , Factores de Transcripción , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Filogenia , Receptores Notch , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
Dharma/Bozozok (Dha/Boz) is a homeodomain protein containing an Engrailed homology (Eh) 1 repressor motif. It is important in zebrafish dorsal organizer formation. Dha/Boz interacted with a co-repressor Groucho through the Eh1 motif. Expression of a Dha/Boz fused to the transcriptional activator VP16 repressed dorsal axis formation and the expression of organizer genes but led to the dorsal expansion of expression of the homeobox gene vox/vega1, indicating that Dha/Boz functions as a transcriptional repressor for dorsal axis formation. We also isolated a novel homeobox gene, ved, whose expression was negatively regulated by dha/boz. ved's sequence and expression profile were similar to those of vox/vega1 and vent/vega2. Like Vox/Vega1 and Vent/Vega2, Ved acted as a transcriptional repressor. The combined inhibition of ved, vox/vega1, and vent/vega2, by antisense morpholino injection, strongly dorsalized the embryos and elicited ventral expansion of organizer gene expression, compared with the effect of inhibiting each of these genes alone. These results suggest that ved is a target for the repressor Dha/Boz. Ved functions redundantly with vox/vega1 and vent/vega2 to restrict the organizer domain.