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1.
Proc Natl Acad Sci U S A ; 121(19): e2311685121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38683994

RESUMO

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.


Assuntos
Crista Neural , Análise de Célula Única , Xenopus laevis , Animais , Crista Neural/citologia , Crista Neural/metabolismo , Análise de Célula Única/métodos , Xenopus laevis/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Movimento Celular , Redes Reguladoras de Genes , Transcriptoma , Gastrulação , Placa Neural/metabolismo , Placa Neural/embriologia , Placa Neural/citologia , Transição Epitelial-Mesenquimal/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/citologia , Neurulação/genética , Neurulação/fisiologia , Diferenciação Celular
2.
Cells Dev ; : 203910, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38452855

RESUMO

One hundred years ago, Hilde Mangold and Hans Spemann published their seminal paper on what came to be known as The Organizer, but seven decades would pass before the molecular basis of this remarkable phenomenon was revealed. Richard Harland and his laboratory played a key role in that discovery, and in this interview he discusses not just the science and the people but also other important factors like mental health and luck.

3.
Nat Commun ; 15(1): 579, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233380

RESUMO

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.


Assuntos
Cromatina , Evolução Molecular , Animais , Cromatina/genética , Genoma/genética , Anuros/genética , Xenopus/genética , Centrômero/genética
4.
Cold Spring Harb Protoc ; 2023(5): pdb.top106195, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-36283836

RESUMO

Collecting eggs from adult Xenopus laevis and Xenopus tropicalis to raise healthy embryos and tadpoles is relatively simple but requires careful handling of the frog. Eggs can be fertilized through natural matings or by in vitro fertilization and examined visually. Here we review how eggs are obtained and how to recognize healthy eggs that will develop into high-quality embryos.


Assuntos
Fertilização in vitro , Fertilização , Animais , Xenopus laevis , Xenopus , Larva
5.
Cold Spring Harb Protoc ; 2023(1): 19-21, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36167670

RESUMO

Adult frogs that are well-cared-for will give high-quality eggs and embryos for use in every Xenopus protocol. Thoughtful frog husbandry is thus pivotal to successful research using these organisms. Protocols for successfully raising tadpoles, establishing and maintaining water quality, and detecting specific pathogens are key to maintaining healthy frog populations.


Assuntos
Xenopus laevis , Animais , Larva
6.
J Evol Biol ; 35(12): 1777-1790, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36054077

RESUMO

In many groups, sex chromosomes change frequently but the drivers of their rapid evolution are varied and often poorly characterized. With an aim of further understanding sex chromosome turnover, we investigated the polymorphic sex chromosomes of the Marsabit clawed frog, Xenopus borealis, using genomic data and a new chromosome-scale genome assembly. We confirmed previous findings that 54.1 Mb of chromosome 8L is sex-linked in animals from east Kenya and a laboratory strain, but most (or all) of this region is not sex-linked in natural populations from west Kenya. Previous work suggests possible degeneration of the Z chromosomes in the east population because many sex-linked transcripts of this female heterogametic population have female-biased expression, and we therefore expected this chromosome to not be present in the west population. In contrast, our simulations support a model where most or all of the sex-linked portion of the Z chromosome from the east acquired autosomal segregation in the west, and where much genetic variation specific to the large sex-linked portion of the W chromosome from the east is not present in the west. These recent changes are consistent with the hot-potato model, wherein sex chromosome turnover is favoured by natural selection if it purges a (minimally) degenerate sex-specific sex chromosome, but counterintuitively suggest natural selection failed to purge a Z chromosome that has signs of more advanced and possibly more ancient regulatory degeneration. These findings highlight complex evolutionary dynamics of young, rapidly evolving Xenopus sex chromosomes and set the stage for mechanistic work aimed at pinpointing additional sex-determining genes in this group.


Assuntos
Genômica , Cromossomos Sexuais , Masculino , Animais , Feminino , Xenopus laevis/genética , Cromossomos Sexuais/genética , Genoma , Evolução Molecular , Cromossomo X
7.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34345915

RESUMO

The FET family of atypical RNA-binding proteins includes Fused in sarcoma (FUS), Ewing's sarcoma (EWS) and the TATA-binding protein-associate factor 15 (TAF15). FET proteins are highly conserved, suggesting specialized requirements for each protein. Fus regulates splicing of transcripts required for mesoderm differentiation and cell adhesion in Xenopus, but the roles of Ews and Taf15 remain unknown. Here, we analyze the roles of maternally deposited and zygotically transcribed Taf15, which is essential for the correct development of dorsoanterior neural tissues. By measuring changes in exon usage and transcript abundance from Taf15-depleted embryos, we found that Taf15 may regulate dorsoanterior neural development through fgfr4 and ventx2.1. Taf15 uses distinct mechanisms to downregulate Fgfr4 expression, namely retention of a single intron within fgfr4 when maternal and zygotic Taf15 is depleted, and reduction in the total fgfr4 transcript when zygotic Taf15 alone is depleted. The two mechanisms of gene regulation (post-transcriptional versus transcriptional) suggest that Taf15-mediated gene regulation is target and co-factor dependent, contingent on the milieu of factors that are present at different stages of development.


Assuntos
Encéfalo/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Xenopus/metabolismo , Animais , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Éxons/fisiologia , Feminino , Masculino , Neurônios/fisiologia , Xenopus/fisiologia
9.
Neuron ; 109(5): 788-804.e8, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33497602

RESUMO

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.


Assuntos
Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Córtex Cerebral/crescimento & desenvolvimento , Estrogênios/fisiologia , Neurogênese , Animais , Transtorno do Espectro Autista/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Avaliação Pré-Clínica de Medicamentos , Estrogênios/administração & dosagem , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Fatores de Risco , Transdução de Sinais , Xenopus
11.
Development ; 147(21)2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32467234

RESUMO

DYRK1A [dual specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 A] is a high-confidence autism risk gene that encodes a conserved kinase. In addition to autism, individuals with putative loss-of-function variants in DYRK1A exhibit microcephaly, intellectual disability, developmental delay and/or congenital anomalies of the kidney and urinary tract. DYRK1A is also located within the critical region for Down syndrome; therefore, understanding the role of DYRK1A in brain development is crucial for understanding the pathobiology of multiple developmental disorders. To characterize the function of this gene, we used the diploid frog Xenopus tropicalis We discover that Dyrk1a is expressed in ciliated tissues, localizes to ciliary axonemes and basal bodies, and is required for ciliogenesis. We also demonstrate that Dyrk1a localizes to mitotic spindles and that its inhibition leads to decreased forebrain size, abnormal cell cycle progression and cell death during brain development. These findings provide hypotheses about potential mechanisms of pathobiology and underscore the utility of X. tropicalis as a model system for understanding neurodevelopmental disorders.


Assuntos
Encéfalo/anatomia & histologia , Cílios/metabolismo , Embrião não Mamífero/anatomia & histologia , Transtornos do Neurodesenvolvimento/genética , Organogênese/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Xenopus/genética , Animais , Encéfalo/embriologia , Ciclo Celular/genética , Sobrevivência Celular , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Tamanho do Órgão , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fatores de Risco , Fuso Acromático/metabolismo , Telencéfalo/anatomia & histologia , Proteínas de Xenopus/metabolismo
12.
Development ; 146(15)2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31285353

RESUMO

During Xenopus gastrulation, Wnt and FGF signaling pathways cooperate to induce posterior structures. Wnt target expression around the blastopore falls into two main categories: a horseshoe shape with a dorsal gap, as in Wnt8 expression; or a ring, as in FGF8 expression. Using ChIP-seq, we show, surprisingly, that the FGF signaling mediator Ets2 binds near all Wnt target genes. However, ß-catenin preferentially binds at the promoters of genes with horseshoe patterns, but further from the promoters of genes with ring patterns. Manipulation of FGF or Wnt signaling demonstrated that 'ring' genes are responsive to FGF signaling at the dorsal midline, whereas 'horseshoe' genes are predominantly regulated by Wnt signaling. We suggest that, in the absence of active ß-catenin at the dorsal midline, the DNA-binding protein TCF binds and actively represses gene activity only when close to the promoter. In contrast, genes without functional TCF sites at the promoter may be predominantly regulated by Ets at the dorsal midline and are expressed in a ring. These results suggest recruitment of only short-range repressors to potential Wnt targets in the Xenopus gastrula.


Assuntos
Gástrula/embriologia , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-ets-2/metabolismo , Fatores de Transcrição TCF/metabolismo , Xenopus laevis/embriologia , Animais , Sítios de Ligação/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Ligação Proteica/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismo
13.
Dev Biol ; 452(1): 8-20, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980799

RESUMO

The Western clawed frog Xenopus tropicalis is a diploid model system for both frog genetics and developmental biology, complementary to the paleotetraploid X. laevis. Here we report a chromosome-scale assembly of the X. tropicalis genome, improving the previously published draft genome assembly through the use of new assembly algorithms, additional sequence data, and the addition of a dense genetic map. The improved genome enables the mapping of specific traits (e.g., the sex locus or Mendelian mutants) and the characterization of chromosome-scale synteny with other tetrapods. We also report an improved annotation of the genome that integrates deep transcriptome sequence from diverse tissues and stages. The exon-intron structures of these genes are highly conserved relative to both X. laevis and human, as are chromosomal linkages ("synteny") and local gene order. A network analysis of developmental gene expression will aid future studies.


Assuntos
Mapeamento Cromossômico , Cromossomos/genética , Perfilação da Expressão Gênica , Genoma , Anotação de Sequência Molecular , Animais , Humanos , Xenopus
14.
Cell Mol Life Sci ; 76(11): 2185-2198, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30729254

RESUMO

RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.


Assuntos
Proteínas de Anfíbios/genética , Genoma , RNA Mensageiro/genética , Caracteres Sexuais , Transcriptoma , Xenopus/genética , Proteínas de Anfíbios/metabolismo , Animais , Embrião não Mamífero , Desenvolvimento Embrionário , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Masculino , Anotação de Sequência Molecular , Poliadenilação , RNA Mensageiro/metabolismo , Fatores Sexuais , Sequenciamento do Exoma , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
15.
Cells Tissues Organs ; 205(5-6): 279-292, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30300884

RESUMO

Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients. Here, we used the Xenopus embryonic mucociliary epidermis as well as human airway epithelial cells (HAECs) as models to investigate the functional roles of NHEs in mucociliary development and regeneration. In Xenopus embryos, NHEs 1-3 were expressed during epidermal development, and loss of NHE function impaired mucociliary clearance in tadpoles. Clearance defects were caused by reduced cilia formation, disrupted alignment of basal bodies in multiciliated cells (MCCs), and dysregulated mucociliary gene expression. These data also suggested that NHEs may contribute to the activation of Wnt signaling in mucociliary epithelia. In HAECs, pharmacological inhibition of NHE function also caused defective ciliation and regeneration in airway MCCs. Collectively, our data revealed a requirement for NHEs in vertebrate mucociliary epithelia and linked NHE activity to cilia formation and function in differentiating MCCs. Our results provide an entry point for the understanding of the contribution of NHEs to signaling, development, and pathogenesis in the human respiratory tract.


Assuntos
Epitélio/embriologia , Epitélio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Células Cultivadas , Epitélio/ultraestrutura , Humanos , Trocador 3 de Sódio-Hidrogênio/metabolismo , Via de Sinalização Wnt , Xenopus/embriologia , Xenopus/metabolismo
16.
Dev Biol ; 442(2): 276-287, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30096282

RESUMO

Microtubule remodeling is critical for cellular and developmental processes underlying morphogenetic changes and for the formation of many subcellular structures. Katanins are conserved microtubule severing enzymes that are essential for spindle assembly, ciliogenesis, cell division, and cellular motility. We have recently shown that a related protein, Katanin-like 2 (KATNAL2), is similarly required for cytokinesis, cell cycle progression, and ciliogenesis in cultured mouse cells. However, its developmental expression pattern, localization, and in vivo role during organogenesis have yet to be characterized. Here, we used Xenopus embryos to reveal that Katnal2 (1) is expressed broadly in ciliated and neurogenic tissues throughout embryonic development; (2) is localized to basal bodies, ciliary axonemes, centrioles, and mitotic spindles; and (3) is required for ciliogenesis and brain development. Since human KATNAL2 is a risk gene for autism spectrum disorders, our functional data suggest that Xenopus may be a relevant system for understanding the relationship of mutations in this gene to autism and the underlying molecular mechanisms of pathogenesis.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Katanina/metabolismo , Animais , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Cílios/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
17.
Science ; 360(6392): 967-968, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29853675
18.
PLoS Biol ; 15(10): e2004045, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29049289

RESUMO

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.


Assuntos
Ectoderma/embriologia , Crista Neural/embriologia , Neurônios/citologia , Células-Tronco/metabolismo , Xenopus laevis/embriologia , Algoritmos , Animais , Análise por Conglomerados , Bases de Dados Genéticas , Ectoderma/metabolismo , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Internet , Microdissecção , Neoplasias/genética , Crista Neural/metabolismo , Neurulação/genética , Análise de Componente Principal , Fatores de Tempo , Transcriptoma/genética , Proteínas Wnt/metabolismo , Xenopus laevis/genética
19.
Science ; 357(6353): 811-815, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28705989

RESUMO

The spacing of hair in mammals and feathers in birds is one of the most apparent morphological features of the skin. This pattern arises when uniform fields of progenitor cells diversify their molecular fate while adopting higher-order structure. Using the nascent skin of the developing chicken embryo as a model system, we find that morphological and molecular symmetries are simultaneously broken by an emergent process of cellular self-organization. The key initiators of heterogeneity are dermal progenitors, which spontaneously aggregate through contractility-driven cellular pulling. Concurrently, this dermal cell aggregation triggers the mechanosensitive activation of ß-catenin in adjacent epidermal cells, initiating the follicle gene expression program. Taken together, this mechanism provides a means of integrating mechanical and molecular perspectives of organ formation.


Assuntos
Células Epidérmicas , Epiderme/embriologia , Plumas/citologia , Plumas/embriologia , Mecanotransdução Celular , Organogênese/fisiologia , Animais , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , beta Catenina/metabolismo
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