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1.
Cold Spring Harb Protoc ; 2019(5)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30885966

RESUMO

Hi-C is a sequencing-based method that captures three-dimensional (3-D) genome interactions by counting the interaction frequencies of pairs of genomic loci. This protocol describes the application of in situ Hi-C to the Xenopus embryo. Briefly, after fixing embryos with formaldehyde, nuclei are isolated and chromatin is digested with a restriction enzyme. Restriction sites are filled in with a biotinylated nucleotide and the blunted ends are re-ligated in place, all while still contained in the nuclei (i.e., in situ). Subsequently, the re-ligated genomic DNA is isolated and fragmented by sonication. Biotinylated ligation junctions are captured with streptavidin-coated beads, and DNA fragments are amplified by ligation-mediated polymerase chain reaction (LM-PCR). The PCR product is isolated and sequenced from both ends (paired-end), and informatics methods are then applied to align the two sides of the ligation junctions to the reference genome. Because ligation occurs much more frequently intra- than interchromosomally, and with generally decreasing frequency the further away DNA loci are from each other on the linear chromosome, interaction frequency information can be used to assist in assembling genomes and to phase haplotypes, which is especially useful in the case of a tetraploid organism such as X. laevis Our streamlined version of in situ Hi-C was optimized for high throughput and low cost, and enables generation of high-quality Hi-C libraries from small cell numbers (down to ∼10,000 cells) in 2 d.


Assuntos
Cromatina/genética , Técnicas Genéticas , Genoma , Xenopus laevis/genética , Animais , Biotinilação , Reagentes de Ligações Cruzadas/química , DNA/isolamento & purificação , Biblioteca Gênica , Microesferas , Estreptavidina/metabolismo , Xenopus laevis/embriologia
3.
Genesis ; 55(1-2)2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28095645

RESUMO

Over the past years, the Xenopus embryo has emerged as an incredibly useful model organism for studying the formation and function of cilia and ciliated epithelia in vivo. This has led to a variety of findings elucidating the molecular mechanisms of ciliated cell specification, basal body biogenesis, cilia assembly, and ciliary motility. These findings also revealed the deep functional conservation of signaling, transcriptional, post-transcriptional, and protein networks employed in the formation and function of vertebrate ciliated cells. Therefore, Xenopus research can contribute crucial insights not only into developmental and cell biology, but also into the molecular mechanisms underlying cilia related diseases (ciliopathies) as well as diseases affecting the ciliated epithelium of the respiratory tract in humans (e.g., chronic lung diseases). Additionally, systems biology approaches including transcriptomics, genomics, and proteomics have been rapidly adapted for use in Xenopus, and broaden the applications for current and future translational biomedical research. This review aims to present the advantages of using Xenopus for cilia research, highlight some of the evolutionarily conserved key concepts and mechanisms of ciliated cell biology that were elucidated using the Xenopus model, and describe the potential for Xenopus research to address unresolved questions regarding the molecular mechanisms of ciliopathies and airway diseases.


Assuntos
Ciliopatias/genética , Larva/crescimento & desenvolvimento , Pneumopatias/genética , Depuração Mucociliar/genética , Animais , Cílios/genética , Cílios/patologia , Ciliopatias/patologia , Modelos Animais de Doenças , Humanos , Pneumopatias/patologia , Transdução de Sinais/genética , Biologia de Sistemas , Xenopus laevis/genética
4.
PLoS Genet ; 13(1): e1006538, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28103240

RESUMO

Cooperative transcription factor binding at cis-regulatory sites in the genome drives robust eukaryotic gene expression, and many such sites must be coordinated to produce coherent transcriptional programs. The transcriptional program leading to motile cilia formation requires members of the DNA-binding forkhead (Fox) and Rfx transcription factor families and these factors co-localize to cilia gene promoters, but it is not clear how many cilia genes are regulated by these two factors, whether these factors act directly or indirectly, or how these factors act with specificity in the context of a 3-dimensional genome. Here, we use genome-wide approaches to show that cilia genes reside at the boundaries of topological domains and that these areas have low enhancer density. We show that the transcription factors Foxj1 and Rfx2 binding occurs in the promoters of more cilia genes than other known cilia transcription factors and that while Rfx2 binds directly to promoters and enhancers equally, Foxj1 prefers direct binding to enhancers and is stabilized at promoters by Rfx2. Finally, we show that Rfx2 and Foxj1 lie at the anchor endpoints of chromatin loops, suggesting that target genes are activated when Foxj1 bound at distal sites is recruited via a loop created by Rfx2 binding at both sites. We speculate that the primary function of Rfx2 is to stabilize distal enhancers with proximal promoters by operating as a scaffolding factor, bringing key regulatory domains bound by Foxj1 into close physical proximity and enabling coordinated cilia gene expression.


Assuntos
Cromatina/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição de Fator Regulador X/metabolismo , Ativação Transcricional , Proteínas de Xenopus/metabolismo , Animais , Cromatina/química , Cílios/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Pele/citologia , Pele/metabolismo , Especificidade da Espécie , Xenopus
5.
Development ; 143(24): 4654-4664, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27864379

RESUMO

Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators known to drive the gene expression required for this organelle biogenesis are activated by the related coiled-coil proteins Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those seen in mutants of Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 co-activates some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that whereas Foxn4 and Foxj1 frequently bind to different targets at distal enhancers, they largely bind together at MCC gene promoters. Consistent with this co-regulation, cilia extension by MCCs is more severely compromised in foxn4 and foxj1 double mutants than in single mutants. In contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. These results indicate that Foxn4 complements Foxj1 transcriptionally during MCC differentiation, thereby shaping the levels of gene expression required for the timely and complete biogenesis of multiple motile cilia.


Assuntos
Cílios/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Pele/embriologia , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Animais , Corpos Basais/fisiologia , Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Morfolinos/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo
6.
Elife ; 52016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27623009

RESUMO

Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.

7.
Development ; 138(4): 705-14, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21266406

RESUMO

Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of a `kidney-specific' form of the H(+)v-ATPase that localizes to the plasma membrane, orthologs of the Cl(-)/HCO(-)(3) antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H(+)v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H(+)v-ATPase cytosolically or basolaterally. These two subtypes are specified during early PSC differentiation by a binary switch that can be regulated by Notch signaling and by the expression of ubp1, a transcription factor of the grainyhead family. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous.


Assuntos
Bombas de Íon/metabolismo , Pele/metabolismo , Xenopus laevis/metabolismo , Animais , Comunicação Celular , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Bombas de Íon/genética , Receptores Notch/metabolismo , Transdução de Sinais , Pele/citologia , Pele/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética
8.
Dev Biol ; 344(1): 319-30, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20493833

RESUMO

The lateral ectoderm of the leech embryo arises from the o and p bandlets, two parallel columns of blast cells that collectively constitute the O/P equivalence group. Individual blast cells within this equivalence group become committed to alternative O or P developmental pathways in accordance with their respectively ventrolateral or dorsolateral position (Weisblat and Blair, 1984). We here describe a novel member of the Six gene transcription factor family, Hau-Six1/2A, which contributes to the patterning of these cell fates in the leech Helobdella sp. (Austin). During embryogenesis Hau-Six1/2A expression is restricted to the dorsolateral column of p blast cells, and thus correlates with P cell fate over most of the body's length. Experimental manipulations showed that Hau-Six1/2A expression is induced in p blast cells by the interaction with the adjoining q bandlet. In addition, misexpression of Hau-Six1/2A in the ventrolateral o blast cells by injection of an expression plasmid elicited the dorsolateral P cell fates ectopically. These data imply that Hau-Six1/2A is a component of the molecular pathway that normally distinguishes O and P cell fates within this equivalence group. Genomic analysis revealed that the Six1/2 subfamily has expanded to a total of six genes in Helobdella. The pattern of Hau-Six1/2A expression during later embryogenesis suggested that this gene may have lost ancestral function(s) and/or acquired novel roles in association with the gene duplications that produced this expansion.


Assuntos
Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Sanguessugas/genética , Sanguessugas/fisiologia , Sequência de Aminoácidos , Animais , Biologia do Desenvolvimento , Ectoderma/metabolismo , Embrião não Mamífero , Hibridização In Situ , Modelos Anatômicos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de Aminoácidos
9.
Dev Genes Evol ; 217(6): 459-68, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17508218

RESUMO

The leech Helobdella sp. (Austin) has two genes of the Pax6 subfamily, one of which is characterized in detail. Hau-Pax6A was expressed during embryonic development in a pattern similar to other bilaterian animals. RNA was detected in cellular precursors of the central nervous system (CNS) and in peripheral cells including a population associated with the developing eye. The CNS of the mature leech is a ventral nerve cord composed of segmental ganglia, and embryonic Hau-Pax6A expression was primarily localized to the N teloblast lineage that generates the majority of ganglionic neurons. Expression began when the ganglion primordia were four cells in length and was initially restricted to a single cell, n(s).a, whose descendants will form the ganglion's anterior edge. At later stages, the Hau-Pax6A expression pattern expanded to include additional CNS precursors, including some descendants of the O teloblast. Expression persisted through the early stages of ganglion morphogenesis but disappeared from the segmented body trunk at the time of neuronal differentiation. The timing and iterated pattern of Hau-Pax6A expression in the leech embryo suggests that this gene may play a role in the segmental patterning of CNS morphogenesis.


Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sanguessugas/embriologia , Sanguessugas/genética , Fatores de Transcrição Box Pareados/metabolismo , Sequência de Aminoácidos , Animais , Linhagem da Célula , Embrião não Mamífero/citologia , Hibridização In Situ , Dados de Sequência Molecular , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/genética , Filogenia , Fatores de Tempo
10.
Development ; 132(1): 89-104, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15563521

RESUMO

The developmental bases for species differences in adult phenotypes remain largely unknown. An emerging system for studying such variation is the adult pigment pattern expressed by Danio fishes. These patterns result from several classes of pigment cells including black melanophores and yellow xanthophores, which differentiate during metamorphosis from latent stem cells of presumptive neural crest origin. In the zebrafish D. rerio, alternating light and dark horizontal stripes develop, in part, owing to interactions between melanophores and cells of the xanthophore lineage that depend on the fms receptor tyrosine kinase; zebrafish fms mutants lack xanthophores and have disrupted melanophore stripes. By contrast, the closely related species D. albolineatus exhibits a uniform pattern of melanophores, and previous interspecific complementation tests identified fms as a potential contributor to this difference between species. Here, we survey additional species and demonstrate marked variation in the fms-dependence of hybrid pigment patterns, suggesting interspecific variation in the fms pathway or fms requirements during pigment pattern formation. We next examine the cellular bases for the evolutionary loss of stripes in D. albolineatus and test the simplest model to explain this transformation, a loss of fms activity in D. albolineatus relative to D. rerio. Within D. albolineatus, we demonstrate increased rates of melanophore death and decreased melanophore migration, different from wild-type D. rerio but similar to fms mutant D. rerio. Yet, we also find persistent fms expression in D. albolineatus and enhanced xanthophore development compared with wild-type D. rerio, and in stark contrast to fms mutant D. rerio. These findings exclude the simplest model in which stripe loss in D. albolineatus results from a loss of fms-dependent xanthophores and their interactions with melanophores. Rather, our results suggest an alternative model in which evolutionary changes in pigment cell interactions themselves have contributed to stripe loss, and we test this model by manipulating melanophore numbers in interspecific hybrids. Together, these data suggest evolutionary changes in the fms pathway or fms requirements, and identify changes in cellular interactions as a likely mechanism of evolutionary change in Danio pigment patterns.


Assuntos
Amidoidrolases/fisiologia , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/genética , Alelos , Animais , Linhagem da Célula , Cruzamentos Genéticos , Embrião não Mamífero , Peixes/genética , Peixes/metabolismo , Genótipo , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Melanóforos/metabolismo , Mutação , Fenótipo , Filogenia , Pigmentação , Pigmentos Biológicos , Especificidade da Espécie , Temperatura , Fatores de Tempo
11.
Development ; 131(24): 6053-69, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15537688

RESUMO

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.


Assuntos
Melanóforos/citologia , Metamorfose Biológica/fisiologia , Crista Neural/citologia , Pigmentação/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Linhagem da Célula/fisiologia , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Crista Neural/crescimento & desenvolvimento , Fenótipo , Filogenia , Pigmentos Biológicos/metabolismo , Peixe-Zebra/anatomia & histologia
12.
Microsc Res Tech ; 58(6): 442-55, 2002 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12242701

RESUMO

The zebrafish Danio rerio is an emerging model organism for understanding vertebrate development and genetics. One trait of both historical and recent interest is the pattern formed by neural crest-derived pigment cells, or chromatophores, which include black melanophores, yellow xanthophores, and iridescent iridophores. In zebrafish, an embryonic and early larval pigment pattern consists of several stripes of melanophores and iridophores, whereas xanthophores are scattered widely over the flank. During metamorphosis, however, this pattern is transformed into that of the adult, which comprises several dark stripes of melanophores and iridophores that alternate with light stripes of xanthophores and iridophores. In this review, we place zebrafish relative to other model and non-model species; we review what is known about the processes of chromatophore specification, differentiation, and morphogenesis during the development of embryonic and adult pigment patterns, and we address how future studies of zebrafish will likely aid our understanding of human disease and the evolution of form.


Assuntos
Evolução Biológica , Padronização Corporal , Cromatóforos , Modelos Genéticos , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Morfogênese , Peixe-Zebra/genética
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