The non-methylated DNA-binding function of Kaiso is not required in early Xenopus laevis development.
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ABSTRACT:
SUBMITTER: Ruzov A
PROVIDER: S-EPMC2685941 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and water absorption and waste excretion. The embryonic kidney of Xenopus laevis consists of a single functional nephron composed of regions that are analogous to those found in the human nephron, making it a simple model for the study of nephrogenesis. The exocyst complex, which traffics proteins to the cell membrane in vesicles via CDC42, is essential for normal kidney development. Here, we show that the CDC42-GEF, dynamin binding protein (Dnmbp/Tuba), is essential for nephrogenesis in Xenopus. dnmbp is expressed in Xenopus embryo kidneys during development, and knockdown of Dnmbp using two separate morpholino antisense oligonucleotides results in reduced expression of late pronephric markers, whereas the expression of early markers of nephrogenesis remains unchanged. A greater reduction in expression of markers of differentiated distal and connecting tubules was seen in comparison to proximal tubule markers, indicating that Dnmbp reduction may have a greater impact on distal and connecting tubule differentiation. Additionally, Dnmbp reduction results in glomus and ciliary defects. dnmbp knockout using CRISPR results in a similar reduction of late markers of pronephric tubulogenesis and also results in edema formation in later stage embryos. Overexpression of dnmbp in the kidney also resulted in disrupted pronephric tubules, suggesting that dnmbp levels in the developing kidney are tightly regulated, with either increased or decreased levels leading to developmental defects. Together, these data suggest that Dnmbp is required for nephrogenesis.
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Project description:In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.
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Project description:HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) regulates a wide variety of cellular processes. It has been shown that one of the targets of HACE1 is the GTP-bound form of the small GTPase Rac1. However, the role of HACE1 in early development remains unknown.In situ hybridization revealed that Xenopus laevis hace1 is specifically expressed in the ectoderm at the blastula and gastrula stages and in the epidermis, branchial arch, kidney, and central nervous system at the tailbud stage. Knockdown of hace1 in Xenopus laevis embryos via antisense morpholino oligonucleotides led to defects in body axis elongation, pigment formation, and eye formation at the tadpole stage. Experiments with Keller sandwich explants showed that hace1 knockdown inhibited convergent extension, a morphogenetic movement known to be crucial for body axis elongation. In addition, time lapse imaging of whole embryos during the neurula stage indicated that hace1 knockdown also delayed neural tube closure. The defects caused by hace1 knockdown were partly rescued by knockdown of rac1. Moreover, embryos expressing a constitutively active form of Rac1 displayed phenotypes similar to those of embryos with hace1 knocked down.Our results suggest that Xenopus laevis hace1 plays an important role in early embryonic development, possibly via regulation of Rac1 activity.
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Project description:Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5x10(4) cells) per experimental condition. Finally, we demonstrate the predicted binding of endogenous beta-catenin to the nodal-related 6 promoter, binding of tagged Fast-1/FoxH1 to the goosecoid promoter, and binding of tagged Tcf3 to the siamois and nodal-related 6 promoters as examples of the potential application of ChIP to embryological investigations. Developmental Dynamics 238:1422-1432, 2009. (c) 2009 Wiley-Liss, Inc.
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Project description:Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3, and foxn1, and evaluated the neural crest contribution to thymus development.In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T-cell differentiation in frogs.These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms.
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Project description:The novel adaptor protein Kazrin associates with multifunctional entities including p120-subfamily members (ARVCF-, delta-, and p0071-catenin). Critical contributions of Kazrin to development or homeostasis are indicated with respect to ectoderm formation, integrity and keratinocyte differentiation, whereas its presence in varied tissues suggests broader roles. We find that Kazrin is maternally loaded, is expressed across development and becomes enriched in the forming head. Kazrin's potential contributions to craniofacial development were probed by means of knockdown in the prospective anterior neural region. Cartilaginous head structures as well as eyes on injected sides were reduced in size, with molecular markers suggesting an impact upon neural crest cell establishment and migration. Similar effects followed the depletion of ARVCF (or delta-catenin), with Kazrin:ARVCF functional interplay supported upon ARVCF partial rescue of Kazrin knockdown phenotypes. Thus, Kazrin and its associating ARVCF- and delta-catenins, are required to form craniofacial tissues originating from cranial neural crest and precordal plate.
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Project description:In species of the frog genus Xenopus, lens regeneration occurs through a process of transdifferentiation, in which cornea epithelial cells presumably undergo dedifferentiation and subsequently redifferentiate to form a new lens. Experimental studies have shown that the retina provides the key signal required to trigger this process once the original lens is removed. A previous study showed that addition of an exogenous fibroblast growth factor (i.e., FGF1 protein) could initiate transdifferentiation of cornea epithelial cells in culture. To determine the role of FGF signaling in X. laevis lens regeneration, we have examined the presence of specific FGFs and their receptors (FGFRs) during this process and evaluated the necessity of FGFR signaling. Reverse transcriptase-polymerase chain reaction analyses reveal that a number of FGF family members are expressed in cornea epithelium and retinal tissues both before and during the process of lens regeneration. Of these, FGF1, FGF8, and FGF9 are expressed principally in retinal tissue and not in the cornea epithelium. Hence, these ligands could represent key signaling factors originating from the retina that trigger regeneration. The results of experiments using an in vitro eye culture system and an FGFR inhibitor (SU5402) suggest that FGFR signaling is required for lens regeneration in Xenopus.
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Project description:SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development.
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Project description:The neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression.To uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue. Ectodermal and blastula animal pole (blastula) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells.We have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm, neural or NC tissue. This has identified novel tissue specific miRNAs and highlighted differential expression of miR-427 isoforms.
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Project description:The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis.Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip(®) Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization.We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the impediment imposed by insufficient gene annotation. These findings heighten the relevance of Xenopus as a model organism for genetic investigations of inner ear organogenesis, morphogenesis, and regeneration.
| S-EPMC3532188 | biostudies-literature