RESUMO
The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/ß-catenin/TCF- and IL-1ß/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and ß-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.
RESUMO
The homeodomain transcription factor Otx2 has essential roles in head and eye formation via the negative and positive regulation of its target genes, but it remains elusive how this dual activity of Otx2 affects cellular functions. In the current study, we first demonstrated that both exogenous and endogenous Otx2 are phosphorylated at multiple sites. Using Xenopus embryos, we identified three possible cyclin-dependent kinase (Cdk) sites and one Akt site, and analyzed the biological activities of phosphomimetic (4E) and nonphosphorylatable (4A) mutants for those sites. In the neuroectoderm, the 4E but not the 4A mutant downregulated the Cdk inhibitor gene p27xic1 (cdknx) and posterior genes, and promoted cell proliferation, possibly forming a positive-feedback loop consisting of Cdk, Otx2 and p27xic1 for cell proliferation, together with anteriorization. Conversely, the 4A mutant functioned as an activator on its own and upregulated the expression of eye marker genes, resulting in enlarged eyes. Consistent with these results, the interaction of Otx2 with the corepressor Tle1 is suggested to be phosphorylation dependent. These data suggest that Otx2 orchestrates cell proliferation, anteroposterior patterning and eye formation via its phosphorylation state.
Assuntos
Padronização Corporal , Proliferação de Células , Fatores de Transcrição Otx/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Quinases Ciclina-Dependentes/metabolismo , Embrião não Mamífero , Olho/embriologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Xenopus laevis/genéticaRESUMO
Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-ß-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.
Assuntos
Autoantígenos/genética , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/crescimento & desenvolvimento , Análise Espaço-Temporal , Animais , Autoantígenos/análise , Biomarcadores , Conexinas , Embrião de Mamíferos , Folículo Piloso/embriologia , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Proteínas de Peixe-ZebraRESUMO
Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.
Assuntos
Padronização Corporal , Endoderma/fisiologia , Proteínas de Homeodomínio/fisiologia , RNA Mensageiro/metabolismo , Proteínas com Domínio T/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Alfa-Amanitina/farmacologia , Animais , Proteínas de Homeodomínio/análise , Proteínas com Domínio T/análise , Proteínas com Domínio T/genética , Fatores de Transcrição , Proteínas de Xenopus/análise , Proteínas de Xenopus/genéticaRESUMO
Cell proliferation is strictly regulated by the dosage balance among cell-cycle regulators such as CDK/cyclin complexes and CDK-Inhibitors. Even in the allotetraploid genome of Xenopus laevis, the dosage balance must be maintained for animals to stay alive, and the duplicated homeologous genes seem to have gradually changed, through evolution, resulting in the best genes for them to thrive. In the Xenopus laevis genome, while homeologous gene pairs of CDKs are fundamentally maintained and a few cyclin genes are amplified, homeologous gene pairs of the important CDK-Inhibitors, CDKn1c and CDKn2a, are deleted from chromosomes L and S. Although losses of CDKn1c and CDKn2a can lead to diseases in humans, their loss in X. laevis does not affect the animals' health. Also, another gene coding CDKn1b is lost besides CDKn1c and CDKn2a in the genome of Xenopus tropicalis. These findings suggest a high resistance of Xenopus to diseases. We also found that CDKn2c.S expression is higher than that of CDKn2c.L, and a conserved noncoding sequence (CNS) of CDKn2c genomic loci on X. laevis chromosome S and X. tropicalis has an enhancement activity in regulating the different expression. These findings together indicate a surprising fragility of CDK inhibitor gene loci in the Xenopus genome in spite of their importance, and may suggest that factors other than CDK-inhibitors decelerate cell-cycling in Xenopus.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas de Xenopus/genética , Xenopus/genética , Sequência de Aminoácidos , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/genética , Diploide , Embrião não Mamífero/fisiologia , Evolução Molecular , Deleção de Genes , Especiação Genética , Instabilidade Genômica , Família Multigênica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Tetraploidia , Xenopus/embriologia , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.
Assuntos
Genes , Cromossomos Sexuais/genética , Diferenciação Sexual/genética , Xenopus laevis/genética , Animais , Evolução Biológica , Inversão Cromossômica , Elementos de DNA Transponíveis/genética , Diploide , Evolução Molecular , Feminino , Duplicação Gênica , Haploidia , Hibridização in Situ Fluorescente , Masculino , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Processos de Determinação Sexual/genéticaRESUMO
Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species.
Assuntos
Proteínas de Homeodomínio/genética , Família Multigênica , Proteínas de Xenopus/genética , Xenopus/genética , Sequência de Aminoácidos , Animais , Blástula/metabolismo , Padronização Corporal/genética , Mapeamento Cromossômico , Sequência Conservada , Diploide , Embrião não Mamífero/metabolismo , Gástrula/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Organizadores Embrionários , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Tetraploidia , Via de Sinalização Wnt , Xenopus/embriologia , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
Inductive interactions mediated by the TGF-ß and FGF-MAPK pathways are essential for specification of the germ layers and embryonic body axes during early vertebrate embryogenesis. TGF-ß and FGF ligands signal through receptor Ser/Thr and Tyr kinases, respectively, and these signaling pathways cross-talk to regulate transcription and cell behavior. The allotetraploid Xenopus laevis and its ancestral diploid Xenopus tropicalis are versatile model organisms with which to study the inductive interactions and mechanisms of these signal transduction pathways. Here we have analyzed the draft genome of X. laevis with respect to the genomic organization and differential expression of genes in the TGF-ß and FGF pathways. Genomic structure and gene expression analyses of pathway components in X. laevis revealed that genetic modulations, including deletions resulting in singletons and differential expression of homeologs, have occurred frequently among extracellular regulatory factors of the TGF-ß pathway after allotetraploidization. Moreover, differential gene expression was found for factors regulating various cellular responses including co-receptors, decoy receptors, and intracellular negative regulators in both the TGF-ß and FGF-MAPK pathways. We summarize the patterns of genetic alterations in the allotetraploid frog X. laevis and discuss the importance of these changes with regard to developmental processes.
Assuntos
Indução Embrionária/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Diploide , Embrião não Mamífero/metabolismo , Epigênese Genética , Fatores de Crescimento de Fibroblastos/genética , Especiação Genética , Genômica , Ligantes , Sistema de Sinalização das MAP Quinases/genética , Anotação de Sequência Molecular , Receptores de Fatores de Crescimento/fisiologia , Proteínas Smad/metabolismo , Tetraploidia , Fator de Crescimento Transformador beta/genética , Xenopus/genética , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.
Assuntos
Perfilação da Expressão Gênica , Proteínas Hedgehog/genética , Receptores Notch/genética , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Receptores Frizzled/biossíntese , Receptores Frizzled/genética , Expressão Gênica , Genoma , Proteínas Hedgehog/biossíntese , Anotação de Sequência Molecular , Receptores Notch/biossíntese , Frações Subcelulares/metabolismo , Sintenia , Tetraploidia , Transcriptoma , Proteínas Wnt/biossíntese , Via de Sinalização Wnt/genética , Proteínas de Xenopus/biossínteseRESUMO
Xenopus laevis has an allotetraploid genome of 3.1Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otx1 suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.
Assuntos
Perfilação da Expressão Gênica , Fatores de Transcrição/genética , Xenopus laevis/genética , AnimaisRESUMO
Extracellular factors belonging to the TGF-ß family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-ß family genes in response to allotetraploidization.
Assuntos
Genoma , Família Multigênica , Fator de Crescimento Transformador beta/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Evolução Biológica , Mapeamento Cromossômico , Evolução Molecular , Deleção de Genes , Duplicação Gênica , Hibridização in Situ Fluorescente , Filogenia , Pseudogenes , Especificidade da Espécie , Sintenia , Tetraploidia , Xenopus/genéticaRESUMO
Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Queratinas/genética , Família Multigênica/genética , Proteínas de Xenopus/genética , Xenopus/genética , Animais , Diploide , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Evolução Molecular , Perfilação da Expressão Gênica , Genoma , Genômica , Filogenia , Proteômica/métodos , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Tetraploidia , Transcriptoma , Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.
Assuntos
Proteínas de Homeodomínio/metabolismo , Animais , Análise por Conglomerados , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox/genética , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/genética , Hibridização In Situ , Xenopus laevisRESUMO
BACKGROUND: Primary cilia are essential for Hedgehog (Hh) signal transduction in vertebrates. Although the core components of the Hh pathway are highly conserved, the dependency on cilia in Hh signaling is considered to be lower in fish than in mice, suggesting the presence of species-specific mechanisms for Hh signal transduction. RESULTS: To precisely understand the role of cilia in Hh signaling in fish and explore the evolution of Hh signaling, we have generated a maternal-zygotic medaka (Oryzias latipes) mutant that lacks cytoplasmic dynein heavy chain 2 (dhc2; MZdhc2), a component required for retrograde intraflagellar transport. We found that MZdhc2 exhibited the shortened cilia and partial defects in Hh signaling, although the Hh defects were milder than zebrafish mutants which completely lack cilia. This result suggests that Hh activity in fish depends on the length of cilium. However, the activity of Hh signaling in MZdhc2 appeared to be higher than that in mouse Dnchc2 mutants, suggesting a lower requirement for cilia in Hh signaling in fish. We confirmed that Ptch1 receptor is exclusively localized on the cilium in fish as in mammals. Subsequent analyses revealed that Fused, an essential mediator for Hh signaling in Drosophila and fish but not in mammals, augments the activity of Hh signaling in fish as a transcriptional target of Hh signaling. CONCLUSIONS: Ciliary requirement for Hh signaling in fish is lower than that in mammals, possibly due to fused-mediated positive feedback in Hh signaling. The finding of this fish-specific augmentation provides a novel insight into the evolution of Hh signaling.
Assuntos
Dineínas/genética , Proteínas Hedgehog/metabolismo , Mutação , Oryzias/embriologia , Transdução de Sinais , Animais , Padronização Corporal , Oryzias/genética , Medula Espinal/embriologiaRESUMO
How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann-Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.