RESUMO
We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).
Assuntos
Sistemas CRISPR-Cas , DNA de Cadeia Simples/genética , Técnicas de Introdução de Genes/métodos , Proteínas de Membrana Transportadoras/genética , Reparo de DNA por Recombinação , Animais , DNA de Cadeia Simples/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/metabolismo , Melanócitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/genética , Pigmentação da Pele , Xenopus laevis , Zigoto/metabolismoRESUMO
The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.
Assuntos
Sistemas CRISPR-Cas/genética , Mutação INDEL/genética , Xenopus/genética , Animais , Edição de Genes , Marcação de Genes , Heterozigoto , Larva/genéticaRESUMO
The Xenopus oocyte contains components of both the planar cell polarity and apical-basal polarity pathways, but their roles are not known. Here, we examine the distribution, interactions and functions of the maternal planar cell polarity core protein Vangl2 and the apical-basal complex component aPKC. We show that Vangl2 is distributed in animally enriched islands in the subcortical cytoplasm in full-grown oocytes, where it interacts with a post-Golgi v-SNARE protein, VAMP1, and acetylated microtubules. We find that Vangl2 is required for the stability of VAMP1 as well as for the maintenance of the stable microtubule architecture of the oocyte. We show that Vangl2 interacts with atypical PKC, and that both the acetylated microtubule cytoskeleton and the Vangl2-VAMP1 distribution are dependent on the presence of aPKC. We also demonstrate that aPKC and Vangl2 are required for the cell membrane asymmetry that is established during oocyte maturation, and for the asymmetrical distribution of maternal transcripts for the germ layer and dorsal/ventral determinants VegT and Wnt11. This study demonstrates the interaction and interdependence of Vangl2, VAMP1, aPKC and the stable microtubule cytoskeleton in the oocyte, shows that maternal Vangl2 and aPKC are required for specific oocyte asymmetries and vertebrate embryonic patterning, and points to the usefulness of the oocyte as a model to study the polarity problem.
Assuntos
Padronização Corporal/genética , Proteínas de Membrana/fisiologia , Oócitos/metabolismo , Proteína Quinase C/fisiologia , RNA Mensageiro Estocado/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Feminino , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Oócitos/fisiologia , Ligação Proteica , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Distribuição Tecidual , Proteína 1 Associada à Membrana da Vesícula/metabolismo , Xenopus/embriologia , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.
Assuntos
Padronização Corporal , Fatores de Crescimento de Fibroblastos/metabolismo , Mesoderma/embriologia , Xenopus laevis/embriologia , Ativinas/metabolismo , Ativinas/farmacologia , Animais , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
Of the three Dishevelled (Dvl) genes, only Dvl2 and Dvl3 are maternally encoded in the frog, Xenopus laevis. We show here by loss of function analysis that single depletion of either Dvl2 or Dvl3 from the oocyte causes the same embryonic phenotype. We find that the effects of loss of function of Dvl2 and 3 together are additive, and that the proteins physically interact, suggesting that both are required in the same complex. We show that maternal Dvl2 and 3 are required for convergence extension movements downstream of the dorsally localized signaling pathway activated by Xnr3, but not downstream of the pathway activated by activin. Also, depletion of maternal Dvl2 and 3 mRNAs causes the up-regulation of a subset of zygotic ectodermal genes, including Foxi1e, with surprisingly no significant effect on the canonical Wnt direct target genes Siamois and Xnr3. We suggest that the likely reason for continued expression of the Wnt target genes in Dvl2/3-depleted embryos is that maternal Dvl mRNA depletion is insufficient to deplete stored punctae of Dvl protein in the oocyte cortex, which may transduce dorsal signaling after fertilization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Células Cultivadas , Proteínas Desgrenhadas , Feminino , Imunofluorescência , Imunoprecipitação , Oligonucleotídeos Antissenso , Fosfoproteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas de Xenopus/genéticaRESUMO
In CRISPR-Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR-Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR-Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR-Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR-Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73-104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , DNA , Edição de Genes/métodos , Oócitos , Xenopus laevis/genéticaRESUMO
We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing in Xenopus embryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR-Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilized Xenopus eggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F1 generation. Therefore, we can avoid the more challenging "oocyte host transfer" technique, which is particularly difficult for Xenopus tropicalis, that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5'-end phosphorothioate-modification to obtain higher-efficiency HDR.
Assuntos
Sistemas CRISPR-Cas , DNA de Cadeia Simples , Animais , DNA de Cadeia Simples/genética , Xenopus laevis/genética , Xenopus/genética , Microinjeções , Edição de Genes/métodos , MutagêneseRESUMO
Xenopus oocytes have been widely used as a simple protein expression system particularly for the characterization of ion channels and membrane receptors. However, less attention has been given to their use as a means of synthesizing and analyzing secreted signaling molecules. In this review, we describe two assays that address this use of Xenopus oocytes. In the first, the paracrine assay, the oocytes secrete the signal and juxtaposed animal cap explants receive it. This provides an easy and efficient way to manipulate the signaling source since different doses of mRNA for the secreted ligand can be injected into the oocyte. Also the signaling response in the receiving cells can be read in several ways: in vivo by monitoring the localization of GFP-tagged signaling mediators, after fixation by immunostaining, or by monitoring changes in the transcriptional readout by RT-PCR. In a second approach, the oocyte is used to secrete a ligand, here the Wnt family members Wnt5a and 11, into the surrounding medium. This conditioned medium is then used to treat other cell lines to monitor their physiological changes in response to various combinations of Wnt proteins. Only a few recombinant Wnt proteins are commercially available and these are predominantly of mouse origin. Since Xenopus oocytes translate foreign RNA efficiently, this method provides an alternative source of Wnt protein derived from other model species.
Assuntos
Oócitos/metabolismo , Proteínas Wnt/metabolismo , Algoritmos , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Camundongos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Xenopus laevisRESUMO
The Wnt/beta-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machinery to beta-catenin, by its association with legless (Lgs)/ B-cell lymphoma-9 (Bcl9). Pygo was shown to be required for development in vertebrates, but the role of Lgs/Bcl9 is unknown. We identified an amphibian orthologue of Lgs/Bcl9, XBcl9, which interacted biochemically with Xbeta-catenin and XPygo2. The body axis promoting ability of Xbeta-catenin was diminished when residues required for its interaction with XBcl9 were mutated. In blastula embryos, XBcl9 was transiently preferentially expressed in nuclei of dorsoanterior cells and ectopically expressed XBcl9 required XPygo2 to localize to nuclei. Furthermore, while neither XBcl9 nor XPygo2 alone affected development when ectopically expressed, both were required to induce supernumerary axis and dorsal gene activation. Like XPygo2, depletion of maternal XBcl9 alone caused dorsal defects. These results indicated an essential role of the Pygo-Bcl9 duet in vertebrate body axis formation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Padronização Corporal/fisiologia , Análise por Conglomerados , Biologia Computacional , Sequência Conservada/genética , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imunoprecipitação , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Xenopus/genéticaRESUMO
Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.
Assuntos
Gastrulação/genética , Proteínas de Membrana/fisiologia , Junções Íntimas/metabolismo , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Animais , Claudinas , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Proteínas de Xenopus/genética , Xenopus laevis/anormalidades , Xenopus laevis/genéticaRESUMO
Clostridium difficile impairs Paneth cells, driving intestinal inflammation that exaggerates colitis. Besides secreting bactericidal products to restrain C. difficile, Paneth cells act as guardians that constitute a niche for intestinal epithelial stem cell (IESC) regeneration. However, how IESCs are sustained to specify Paneth-like cells as their niche remains unclear. Cytokine-JAK-STATs are required for IESC regeneration. We investigated how constitutive STAT5 activation (Ca-pYSTAT5) restricts IESC differentiation towards niche cells to restrain C. difficile infection. We generated inducible transgenic mice and organoids to determine the effects of Ca-pYSTAT5-induced IESC lineages on C. difficile colitis. We found that STAT5 absence reduced Paneth cells and predisposed mice to C. difficile ileocolitis. In contrast, Ca-pYSTAT5 enhanced Paneth cell lineage tracing and restricted Lgr5 IESC differentiation towards pYSTAT5+Lgr5-CD24+Lyso+ or cKit+ niche cells, which imprinted Lgr5hiKi67+ IESCs. Mechanistically, pYSTAT5 activated Wnt/ß-catenin signaling to determine Paneth cell fate. In conclusion, Ca-pYSTAT5 gradients control niche differentiation. Lack of pYSTAT5 reduces the niche cells to sustain IESC regeneration and induces C. difficile ileocolitis. STAT5 may be a transcription factor that regulates Paneth cells to maintain niche regeneration.
Assuntos
Clostridioides difficile , Colite/metabolismo , Colite/microbiologia , Celulas de Paneth/metabolismo , Celulas de Paneth/microbiologia , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organoides/metabolismo , Organoides/microbiologia , Nicho de Células-Tronco/fisiologia , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
The trachea and esophagus arise from the separation of a common foregut tube during early fetal development. Mutations in key signaling pathways such as Hedgehog (HH)/Gli can disrupt tracheoesophageal (TE) morphogenesis and cause life-threatening birth defects (TEDs); however, the underlying cellular mechanisms are unknown. Here, we use mouse and Xenopus to define the HH/Gli-dependent processes orchestrating TE morphogenesis. We show that downstream of Gli the Foxf1+ splanchnic mesenchyme promotes medial constriction of the foregut at the boundary between the presumptive Sox2+ esophageal and Nkx2-1+ tracheal epithelium. We identify a unique boundary epithelium co-expressing Sox2 and Nkx2-1 that fuses to form a transient septum. Septum formation and resolution into distinct trachea and esophagus requires endosome-mediated epithelial remodeling involving the small GTPase Rab11 and localized extracellular matrix degradation. These are disrupted in Gli-deficient embryos. This work provides a new mechanistic framework for TE morphogenesis and informs the cellular basis of human TEDs.
Assuntos
Endossomos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Morfogênese/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Sistema Digestório/metabolismo , Endoderma/metabolismo , Endossomos/genética , Esôfago/embriologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Mesoderma/metabolismo , Mutação/genética , XenopusRESUMO
We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.
Assuntos
Endoderma/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/fisiologia , Animais , Endoderma/efeitos dos fármacos , Endoderma/embriologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hibridização In Situ , Pirróis/administração & dosagem , Pirróis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
We have taken advantage of the well-established oocyte host transfer technique to optimize a method for CRISPR editing of Xenopus that provides an efficient non-mosaic targeted insertion of small DNA fragment through homology-directed repair mechanism.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Introdução de Genes/métodos , Mutação/genética , Xenopus/genética , Animais , Proteína 9 Associada à CRISPR/metabolismo , Fertilização , Técnicas de Genotipagem , Humanos , Injeções , Mutagênese/genética , Oócitos/metabolismo , RNA Guia de Cinetoplastídeos/genética , Análise de Sequência de DNARESUMO
Xenopus embryo yolk proteins present a serious barrier to fluorescence microscopy. Previously, in situ assays of gene transcripts in whole embryos was limited to the use of chromogenic alkaline phosphatase substrates, which restricted researchers' ability to gauge coexpression of transcripts. Here, we describe a modified in situ hybridization (ISH) protocol that uses fluorescent substrates and a novel yolk-clearing technique for simultaneous visualization of the expression of two genes in whole Xenopus embryos with high resolution. This protocol employs two well-known fluorescent substrates, nitro blue tetrazolium/5-bromo- 4-chloro-3-indolyl-phosphate (NBT/BCIP) and Vector Red, in a sequential dual in situ hybridization procedure. Subsequent clearing of the samples with refractive-index-matching solution (RIMS) renders the samples amenable to confocal microscopy, allowing imaging at sufficiently high resolution to discern coexpression of transcripts within individual cells.
Assuntos
Compostos Cromogênicos/metabolismo , Hibridização in Situ Fluorescente/métodos , Xenopus/metabolismo , Animais , Compostos Cromogênicos/análise , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Nodal class TGF-ß signalling molecules play essential roles in establishing the vertebrate body plan. In all vertebrates, nodal family members have specific waves of expression required for tissue specification and axis formation. In Xenopus laevis, six nodal genes are expressed before gastrulation, raising the question of whether they have specific roles or act redundantly with each other. Here, we examine the role of Xnr5. We find it acts at the late blastula stage as a mesoderm inducer and repressor of ectodermal gene expression, a role it shares with Vg1. However, unlike Vg1, Xnr5 depletion reduces the expression of the nodal family member xnr1 at the gastrula stage. It is also required for left/right laterality by controlling the expression of the laterality genes xnr1, antivin (lefty) and pitx2 at the tailbud stage. In Xnr5-depleted embryos, the heart field is established normally, but symmetrical reduction in Xnr5 levels causes a severely stunted midline heart, first evidenced by a reduction in cardiac troponin mRNA levels, while left-sided reduction leads to randomization of the left/right axis. This work identifies Xnr5 as the earliest step in the signalling pathway establishing normal heart laterality in Xenopus.
Assuntos
Blástula/metabolismo , Padronização Corporal , Coração/crescimento & desenvolvimento , Ligantes da Sinalização Nodal/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Determinação Direita-Esquerda/metabolismo , Ligantes da Sinalização Nodal/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Embryo homing and implantation occur within a crypt (implantation chamber) at the antimesometrial (AM) pole along the uterus. The mechanism by which this is achieved is not known. Here, we show that villi-like epithelial projections from the main uterine lumen toward the AM pole at regularly spaced intervals that form crypts for embryo implantation were disrupted in mice with uterine loss or gain of function of Wnt5a, or loss of function of both Ror1 and Ror2. This disruption of Wnt5a-ROR signaling resulted in disorderly epithelial projections, crypt formation, embryo spacing, and impaired implantation. These early disturbances under abnormal Wnt5a-ROR signaling were reflected in adverse late pregnancy events, including defective decidualization and placentation, ultimately leading to compromised pregnancy outcomes. This study presents deeper insight regarding the formation of organized epithelial projections for crypt formation and embryo implantation for pregnancy success.
Assuntos
Decídua/metabolismo , Implantação do Embrião , Células Epiteliais/citologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Decídua/citologia , Decídua/fisiologia , Células Epiteliais/metabolismo , Feminino , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the "basolateral" state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Polaridade Celular/genética , Células Epiteliais/citologia , Epitélio/embriologia , Gastrulação/genética , Gastrulação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Cinética , Microscopia Confocal , Morfolinos/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Alpha (α)-E-catenin is a component of the cadherin complex, and has long been thought to provide a link between cell surface cadherins and the actin skeleton. More recently, it has also been implicated in mechano-sensing, and in the control of tissue size. Here we use the early Xenopus embryos to explore functional differences between two α-catenin family members, α-E- and α-N-catenin, and their interactions with the different classical cadherins that appear as tissues of the embryo become segregated from each other. We show that they play both cadherin-specific and context-specific roles in the emerging tissues of the embryo. α-E-catenin interacts with both C- and E-cadherin. It is specifically required for junctional localization of C-cadherin, but not of E-cadherin or N-cadherin at the neurula stage. α-N-cadherin interacts only with, and is specifically required for junctional localization of, N-cadherin. In addition, α -E-catenin is essential for normal tissue size control in the non-neural ectoderm, but not in the neural ectoderm or the blastula. We also show context specificity in cadherin/ α-catenin interactions. E-cadherin requires α-E-catenin for junctional localization in some tissues, but not in others, during early development. These specific functional cadherin/alpha-catenin interactions may explain the basis of cadherin specificity of actin assembly and morphogenetic movements seen previously in the neural and non-neural ectoderm.
Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Proteínas de Membrana/metabolismo , Xenopus/embriologia , Animais , Caderinas/genética , Desenvolvimento EmbrionárioRESUMO
Foxi1e is a zygotic transcription factor that is essential for the expression of early ectodermal genes. It is expressed in a highly specific pattern, only in the deep cell layers of the animal hemisphere, and in a mosaic pattern in which expressing cells are interspersed with non-expressing cells. Previous work has shown that several signals in the blastula control this expression pattern, including nodals, the TGFß family member Vg1, and Notch. However, these are all inhibitory, which raises the question of what activates Foxi1e. In this work, we show that a related Forkhead family protein, Foxi2, is a maternal activator of Foxi1e. Foxi2 mRNA is maternally encoded, and highly enriched in animal hemisphere cells of the blastula. ChIP assays show that it acts directly on upstream regulatory elements of Foxi1e. Its effect is specific, since animal cells depleted of Foxi2 are able to respond normally to mesoderm inducing signals from vegetal cells. Foxi2 thus acts as a link between the oocyte and the early pathway to ectoderm, in a similar fashion to the vegetally localized VegT acts to initiate endoderm and mesoderm formation.