RESUMO
The vertebrate nervous system develops from embryonic neural plate and neural crest. Although genetic mechanisms governing vertebrate neural development have been investigated in depth, epigenetic regulation of this process remains less understood. Redundancy of epigenetic factors and early lethality of animals deficient in critical epigenetic components pose major challenges in characterization of epigenetic factors in vertebrate neural development. In this study, we use the amphibian model Xenopus laevis to investigate the roles of non-redundant, obligatory components of all histone H3K4 activating methylation complexes (COMPASS, also known as SET1/MLL complexes) in early neural development. The two genes that we focus on, Ash2l and Dpy30, regulate mesendodermal differentiation in mouse embryonic stem cells and cause early embryonic lethality when removed from mouse embryos. Using targeted knockdown of the genes in dorsal ectoderm of Xenopus that gives rise to future nervous system, we show here that ash2l and dpy30 are required for neural and neural crest marker expression in Xenopus late neurula embryos but are dispensable for early neural and neural plate border gene expression. Co-immunoprecipitation assays reveal that Dpy30 and Ash2L associate with the neural plate border transcription factors, such as Msx1 and Tfap2a. Chromatin immunoprecipitation (ChIP) assay further demonstrates that Ash2L and the H3K4me3 active histone mark accumulate at the promoter regions of the neural crest gene sox10 in a Tfap2a-dependent manner. Collectively, our data suggest that Ash2l and Dpy30 interact with specific transcription factors to recruit COMPASS complexes to the regulatory regions of neural crest specification genes to control their expression and influence development of the nervous system during vertebrate embryogenesis.
Assuntos
Epigênese Genética , Crista Neural , Animais , Camundongos , Regulação da Expressão Gênica no Desenvolvimento/genética , Metilação , Placa Neural/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
VAMP/synaptobrevin-associated protein B (VAP-B) is a type II ER membrane protein, but its N-terminal MSP domain (MSPd) can be cleaved and secreted. Mutations preventing the cleavage and secretion of MSPd have been implicated in cases of human neurodegenerative diseases. The site of VAP cleavage and the tissues capable in releasing the processed MSPd are not understood. In this study, we analyze the C. elegans VAP-B homolog, VPR-1, for its processing and secretion from the intestine. We show that intestine-specific expression of an N-terminally FLAG-tagged VPR-1 rescues underdeveloped gonad and sterility defects in vpr-1 null hermaphrodites. Immunofluorescence studies reveal that the tagged intestinal expressed VPR-1 is present at the distal gonad. Mass spectrometry analysis of a smaller product of the N-terminally tagged VPR-1 identifies a specific cleavage site at Leu156. Mutation of the leucine results in loss of gonadal MSPd signal and reduced activity of the mutant VPR-1. Thus, we report for the first time the cleavage site of VPR-1 and provide direct evidence that intestinally expressed VPR-1 can be released and signal in the distal gonad. These results establish the foundation for further exploration of VAP cleavage, MSPd secretion, and non-cell-autonomous signaling in development and diseases.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Membrana/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Genes de Helmintos , Gônadas/química , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Proteínas de Helminto/química , Organismos Hermafroditas/genética , Organismos Hermafroditas/metabolismo , Organismos Hermafroditas/fisiologia , Infertilidade , Intestinos/citologia , Intestinos/fisiologia , Leucina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fenótipo , Mutação Puntual , Domínios Proteicos , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Heterochromatin protein 1 (HP1) is associated with and plays a role in compact chromatin conformation, but the function of HP1 in vertebrate embryogenesis is not understood completely. RESULTS: Here, we explore the activity of HP1 in early neural development in the frog Xenopus laevis. We show that the three isoforms of HP1, HP1α, ß, and γ, are expressed in similar patterns in the neural and neural crest derivatives in early embryos. Despite this, knockdown of HP1ß and HP1γ, but not HP1α, in presumptive neural tissues leads to head defects. Late pan-neural markers and neural crest specifier genes are reduced, but early neural and neural plate border genes are less affected in the morphant embryos. Further investigation reveals that neuronal differentiation is impaired and a pluripotency-associated gene, pou5f3.2/oct25, is expanded in HP1ß morphants. Ectopic expression of pou5f3.2/oct25 mimics the effect of HP1ß knockdown on marker expression, whereas simultaneous knockdown of HP1ß and pou5f3.2/oct25 partially rescues expression of these genes. CONCLUSION: Taken together, the data suggest that HP1ß regulates transition from precursor to more differentiated cell types during neural and neural crest development in Xenopus, and it does so at least partially via repression of the pluripotency-associated transcription regulator pou5f3.2/oct25.
Assuntos
Homólogo 5 da Proteína Cromobox/genética , Crista Neural/embriologia , Isoformas de Proteínas/genética , Proteínas de Xenopus/genética , Animais , Homólogo 5 da Proteína Cromobox/metabolismo , Regulação da Expressão Gênica , Crista Neural/metabolismo , Neurogênese/genética , Isoformas de Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The transcription factor Hypermethylated in Cancer 1 (HIC1) is associated with both tumorigenesis and the complex human developmental disorder Miller-Dieker Syndrome. While many studies have characterized HIC1 as a tumor suppressor, HIC1 function in development is less understood. Loss-of-function mouse alleles show embryonic lethality accompanied with developmental defects, including craniofacial abnormalities that are reminiscent of human Miller-Dieker Syndrome patients. However, the tissue origin of the defects has not been reported. In this study, we use the power of the Xenopus laevis model system to explore Hic1 function in early development. We show that hic1 mRNA is expressed throughout early Xenopus development and has a spatial distribution within the neural plate border and in migrating neural crest cells in branchial arches. Targeted manipulation of hic1 levels in the dorsal ectoderm that gives rise to neural and neural crest tissues reveals that both overexpression and knockdown of hic1 result in craniofacial defects with malformations of the craniofacial cartilages. Neural crest specification is not affected by altered hic1 levels, but migration of the cranial neural crest is impaired both in vivo and in tissue explants. Mechanistically, we find that Hic1 regulates cadherin expression profiles and canonical Wnt signaling. Taken together, these results identify Hic1 as a novel regulator of the canonical Wnt pathway during neural crest migration.
Assuntos
Movimento Celular , Crista Neural/embriologia , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Animais , Crista Neural/citologia , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Apical constriction regulates epithelial morphogenesis during embryonic development, but how this process is controlled is not understood completely. Here, we identify a Rho guanine nucleotide exchange factor (GEF) gene plekhg5 as an essential regulator of apical constriction of bottle cells during Xenopus gastrulation. plekhg5 is expressed in the blastopore lip and its expression is sufficient to induce ectopic bottle cells in epithelia of different germ layers in a Rho-dependent manner. This activity is not shared by arhgef3, which encodes another organizer-specific RhoGEF. Plekhg5 protein is localized in the apical cell cortex via its pleckstrin homology domain, and the GEF activity enhances its apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation. Knockdown of plekhg5 inhibits activin-induced bottle cell formation and endogenous blastopore lip formation in gastrulating frog embryos. Apical accumulation of actomyosin, apical constriction and bottle cell formation fail to occur in these embryos. Taken together, our data indicate that transcriptional regulation of plekhg5 expression at the blastopore lip determines bottle cell morphology via local polarized activation of Rho by Plekhg5, which stimulates apical actomyosin activity to induce apical constriction.
Assuntos
Polaridade Celular , Gastrulação , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Ativinas/metabolismo , Actomiosina/metabolismo , Animais , Citoesqueleto/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Movimento , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.
Assuntos
Inativação Gênica/fisiologia , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Transativadores/metabolismo , Ubiquitinação/fisiologia , Animais , Células HeLa , Histonas/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Nucleossomos/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transativadores/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl â Daam1 cascade during CE. Our finding uncovers Vangl2-Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Polaridade Celular/fisiologia , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Defeitos do Tubo Neural/metabolismo , Neurulação , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Vertebrate embryos undergo dramatic shape changes at gastrulation that require locally produced and anisotropically applied forces, yet how these forces are produced and transmitted across tissues remains unclear. We show that depletion of myosin regulatory light chain (RLC) levels in the embryo blocks force generation at gastrulation through two distinct mechanisms: destabilizing the myosin II (MII) hexameric complex and inhibiting MII contractility. Molecular dissection of these two mechanisms demonstrates that normal convergence force generation requires MII contractility and we identify a set of molecular phenotypes correlated with both this failure of convergence force generation in explants and of blastopore closure in whole embryos. These include reduced rates of actin movement, alterations in C-cadherin dynamics and a reduction in the number of polarized lamellipodia on intercalating cells. By examining the spatial relationship between C-cadherin and actomyosin we also find evidence for formation of transcellular linear arrays incorporating these proteins that could transmit mediolaterally oriented tensional forces. These data combine to suggest a multistep model to explain how cell intercalation can occur against a force gradient to generate axial extension forces. First, polarized lamellipodia extend mediolaterally and make new C-cadherin-based contacts with neighboring mesodermal cell bodies. Second, lamellipodial flow of actin coalesces into a tension-bearing, MII-contractility-dependent node-and-cable actin network in the cell body cortex. And third, this actomyosin network contracts to generate mediolateral convergence forces in the context of these transcellular arrays.
Assuntos
Gastrulação , Modelos Moleculares , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos/fisiologia , Caderinas/metabolismo , Polaridade Celular , Embrião não Mamífero/metabolismo , Modelos Biológicos , Morfogênese , Miosina Tipo II/metabolismo , Notocorda/citologia , Fenótipo , Fosforilação , Pseudópodes/metabolismo , Xenopus laevis/metabolismoRESUMO
Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Células-Tronco Embrionárias Murinas/metabolismo , Ácido Retinoico 4 Hidroxilase/metabolismo , Tretinoína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Células Cultivadas , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/enzimologia , Ácido Retinoico 4 Hidroxilase/genética , Transdução de Sinais , Xenopus laevisRESUMO
During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation.
Assuntos
Padronização Corporal/genética , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Análise de Sequência de RNA , Xenopus/genética , Ativinas/fisiologia , Animais , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas da Matriz Extracelular/fisiologia , Gástrula/ultraestrutura , Camadas Germinativas/metabolismo , Morfogênese/genética , Organizadores Embrionários , Proteínas Tirosina Quinases/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Xenopus/embriologia , Proteínas de Xenopus/fisiologiaRESUMO
Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.
Assuntos
Astrócitos/metabolismo , Diferenciação Celular/fisiologia , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Células Cultivadas , Imunofluorescência , Immunoblotting , Hibridização In Situ , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Xenopus laevisRESUMO
Neural crest cells arise from the border of the neural plate and epidermal ectoderm, migrate extensively and differentiate into diverse cell types during vertebrate embryogenesis. Although much has been learnt about growth factor signals and gene regulatory networks that regulate neural crest development, limited information is available on how epigenetic mechanisms control this process. In this study, we show that Polycomb repressive complex 2 (PRC2) cooperates with the transcription factor Snail2/Slug to modulate neural crest development in Xenopus. The PRC2 core components Eed, Ezh2 and Suz12 are expressed in the neural crest cells and are required for neural crest marker expression. Knockdown of Ezh2, the catalytic subunit of PRC2 for histone H3K27 methylation, results in defects in neural crest specification, migration and craniofacial cartilage formation. EZH2 interacts directly with Snail2, and Snail2 fails to expand the neural crest domains in the absence of Ezh2. Chromatin immunoprecipitation analysis shows that Snail2 regulates EZH2 occupancy and histone H3K27 trimethylation levels at the promoter region of the Snail2 target E-cadherin. Our results indicate that Snail2 cooperates with EZH2 and PRC2 to control expression of the genes important for neural crest specification and migration during neural crest development.
Assuntos
Crista Neural/citologia , Crista Neural/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Complexo Repressor Polycomb 2/genética , Ligação Proteica , Fatores de Transcrição/genética , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Epigenetic mechanisms, including histone modifications, have emerged as important factors influencing cell fate determination. The functional role of H3K4 methylation, however, remains largely unclear in the maintenance and differentiation of hematopoietic stem cells (HSCs)/hematopoietic progenitor cells (HPCs). Here we show that DPY30, a shared core subunit of the SET1/MLL family methyltransferase complexes and a facilitator of their H3K4 methylation activity, is important for ex vivo proliferation and differentiation of human CD34(+) HPCs. DPY30 promotes HPC proliferation by directly regulating the expression of genes critical for cell proliferation. Interestingly, while DPY30 knockdown in HPCs impaired their differentiation into the myelomonocytic lineage, it potently promoted hemoglobin production and affected the kinetics of their differentiation into the erythroid lineage. In an in vivo model, we show that morpholino-mediated dpy30 knockdown resulted in severe defects in the development of the zebrafish hematopoietic system, which could be partially rescued by coinjection of dpy30 messenger RNA. Taken together, our results establish a critical role of DPY30 in the proliferation and appropriate differentiation of hematopoietic progenitor cells and in animal hematopoiesis. Finally, we also demonstrate a crucial role of DPY30 in the growth of several MLL1-fusion-mediated leukemia cell lines.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Complexos Multiproteicos/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Hematopoese , Histona-Lisina N-Metiltransferase/química , Humanos , Leucemia/genética , Leucemia/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/química , Proteína de Leucina Linfoide-Mieloide/química , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Alinhamento de Sequência , Fatores de Transcrição , Peixe-ZebraRESUMO
Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation.
Assuntos
Actinas/metabolismo , Citoplasma/enzimologia , Embrião não Mamífero/enzimologia , Gastrulação , Proteínas Tirosina Quinases/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Caderinas/metabolismo , Adesão Celular , Linhagem da Célula , Movimento Celular , Embrião não Mamífero/citologia , Humanos , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/genética , Xenopus laevis/genéticaRESUMO
The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Oxirredutases do Álcool/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Alinhamento de Sequência , Tretinoína/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
Post-translational histone modifications have important regulatory roles in chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on histone H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X-chromosome inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. Here we report the identification and functional characterization of the major deubiquitinase for histone H2A, Ubp-M (also called USP16). Ubp-M prefers nucleosomal substrates in vitro, and specifically deubiquitinates histone H2A but not H2B in vitro and in vivo. Notably, knockdown of Ubp-M in HeLa cells results in slow cell growth rates owing to defects in the mitotic phase of the cell cycle. Further studies reveal that H2A deubiquitination by Ubp-M is a prerequisite for subsequent phosphorylation of Ser 10 of H3 and chromosome segregation when cells enter mitosis. Furthermore, we demonstrate that Ubp-M regulates Hox gene expression through H2A deubiquitination and that blocking the function of Ubp-M results in defective posterior development in Xenopus laevis. This study identifies the major deubiquitinase for histone H2A and demonstrates that H2A deubiquitination is critically involved in cell cycle progression and gene expression.
Assuntos
Ciclo Celular/fisiologia , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Proteínas de Xenopus/metabolismo , Animais , Divisão Celular , Endopeptidases/deficiência , Endopeptidases/genética , Genes Homeobox/genética , Células HeLa , Histonas/química , Proteínas de Homeodomínio/genética , Humanos , Fosfosserina/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
BACKGROUND: Smad ubiquitination regulatory factor (Smurf) 1 and 2 are E3 ubiquitin ligases originally identified as inhibitors of transforming growth factor beta signaling and are shown to modulate multiple cellular activities. The roles of Smurfs in vertebrate embryogenesis, however, are not completely understood. RESULTS: Here we investigate the function of Smurf2 during early Xenopus development. We show that distinctly from Smurf1, overexpression of Smurf2 in presumptive mesoderm interfered with mesoderm induction and caused axial defects, whereas knockdown of Smurf2 with antisense morpholino oligonucleotides resulted in expansion of the mesoderm. These results imply that Smurf2 may modulate nodal-mediated mesodermal induction. Consistently, ventral expression of Smurf2 induced a partial secondary axis with head structures. In the ectoderm, Smurf2 resembled Smurf1 in controlling neural and epidermal marker expression and influencing head formation. Smurf1, but not Smurf2, additionally affected neural tube closure. Interestingly, both Smurfs could enhance as well as repress neural crest markers, implying that they modulate their targets dynamically during neural plate border specification. CONCLUSION: Our data demonstrate that Smurf1 and Smurf2 have overlapping and distinct functionalities during early frog embryogenesis; collectively, they regulate ectodermal and mesodermal induction and patterning to ensure normal development of Xenopus embryos.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Animais , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Ubiquitina-Proteína Ligases/genética , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMO
Apical constriction results in apical surface reduction in epithelial cells and is a widely used mechanism for epithelial morphogenesis. Both medioapical and junctional actomyosin remodeling are involved in apical constriction, but the deployment of medial versus junctional actomyosin and their genetic regulation in vertebrate embryonic development have not been fully described. In this study, we investigate actomyosin dynamics and their regulation by the RhoGEF protein Plekhg5 in Xenopus bottle cells. Using live imaging and quantitative image analysis, we show that bottle cells assume different shapes, with rounding bottle cells constricting earlier in small clusters followed by fusiform bottle cells forming between the clusters. Both medioapical and junctional actomyosin signals increase as surface area decreases, though correlation of apical constriction with medioapical actomyosin localization appears to be stronger. F-actin bundles perpendicular to the apical surface form in constricted cells, which may correspond to microvilli previously observed in the apical membrane. Knockdown of plekhg5 disrupts medioapical and junctional actomyosin activity and apical constriction but does not affect initial F-actin dynamics. Taking the results together, our study reveals distinct cell morphologies, uncovers actomyosin behaviors, and demonstrates the crucial role of a RhoGEF protein in controlling actomyosin dynamics during apical constriction of bottle cells in Xenopus gastrulation.
Assuntos
Actomiosina , Gastrulação , Animais , Actomiosina/metabolismo , Xenopus laevis/metabolismo , Actinas/metabolismo , Constrição , Morfogênese , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
Polycomb group (PcG) proteins are key regulators of gene expression and developmental programs via covalent modification of histones, but the factors that interpret histone modification marks to regulate embryogenesis are less studied. We previously identified Remodeling and Spacing Factor 1 (RSF1) as a reader of histone H2A lysine 119 ubiquitination (H2AK119ub), the histone mark deposited by Polycomb Repressive Complex 1 (PRC1). In the current study, we used Xenopus laevis as a model to investigate how RSF1 affects early embryonic development and whether recognition of H2AK119ub is important for the function of RSF1. We showed that knockdown of Xenopus RSF1, rsf1, not only induced gastrulation defects as reported previously, but specific targeted knockdown in prospective neural precursors induced neural and neural crest defects, with reductions of marker genes. In addition, similar to knockdown of PRC1 components in Xenopus, the anterior-posterior neural patterning was affected in rsf1 knockdown embryos. Binding of H2AK119ub appeared to be crucial for rsf1 function, as a construct with deletion of the UAB domain, which is required for RSF1 to recognize the H2AK119ub nucleosomes, failed to rescue rsf1 morphant embryos and was less effective in interfering with early Xenopus development when ectopically expressed. Furthermore, ectopic deposition of H2AK119ub on the Smad2 target gene gsc using a ring1a-smad2 fusion protein led to ectopic recruitment of RSF1. The fusion protein was inefficient in inducing mesodermal markers in the animal region or a secondary axis when expressed in the ventral tissues. Taken together, our results reveal that rsf1 modulates similar developmental processes in early Xenopus embryos as components of PRC1 do, and that RSF1 acts at least partially through binding to the H2AK119ub mark via the UAB domain during development.
RESUMO
Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and "core members" of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.