RESUMO
Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.
Assuntos
Pigmentação , Peixe-Zebra , Animais , Forma Celular , Melanóforos/fisiologia , Pigmentação/genética , Pele , Peixe-Zebra/genéticaRESUMO
In Darwin's and Mendel's times, researchers investigated a wealth of organisms, chosen to solve particular problems for which they seemed especially well suited. Later, a focus on a few organisms, which are accessible to systematic genetic investigations, resulted in larger repertoires of methods and applications in these few species. Genetic animal model organisms with large research communities are the nematode Caenorhabditis elegans, the fly Drosophila melanogaster, the zebrafish Danio rerio, and the mouse Mus musculus. Due to their specific strengths, these model organisms have their strongest impacts in rather different areas of biology. C. elegans is unbeatable in the analysis of cell-to-cell contacts by saturation mutagenesis, as worms can be grown very fast in very high numbers. In Drosophila, a rich pattern is generated in the embryo as well as in adults that is used to unravel the underlying mechanisms of morphogenesis. The transparent larvae of zebrafish are uniquely suited to study organ development in a vertebrate, and the superb versatility of reverse genetics in the mouse made it the model organism to study human physiology and diseases. The combination of these models allows the in-depth genetic analysis of many fundamental biological processes using a plethora of different methods, finally providing many specific approaches to combat human diseases. The plant model Arabidopsis thaliana provides an understanding of many aspects of plant biology that might ultimately be useful for breeding crops.
Assuntos
Arabidopsis , Crescimento e Desenvolvimento , Modelos Animais , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Pesquisa em Genética , Crescimento e Desenvolvimento/genética , Humanos , Camundongos , Melhoramento Vegetal , Peixe-Zebra/genéticaRESUMO
The autonomic portion of the peripheral nervous system orchestrates tissue homeostasis through direct innervation of internal organs, and via release of adrenalin and noradrenalin into the blood flow. The developmental mechanisms behind the formation of autonomic neurons and chromaffin cells are not fully understood. Using genetic tracing, we discovered that a significant proportion of sympathetic neurons in zebrafish originates from Schwann cell precursors (SCPs) during a defined period of embryonic development. Moreover, SCPs give rise to the main portion of the chromaffin cells, as well as to a significant proportion of enteric and other autonomic neurons associated with internal organs. The conversion of SCPs into neuronal and chromaffin cells is ErbB receptor dependent, as the pharmacological inhibition of the ErbB pathway effectively perturbed this transition. Finally, using genetic ablations, we revealed that SCPs producing neurons and chromaffin cells migrate along spinal motor axons to reach appropriate target locations. This study reveals the evolutionary conservation of SCP-to-neuron and SCP-to-chromaffin cell transitions over significant growth periods in fish and highlights relevant cellular-genetic mechanisms. Based on this, we anticipate that multipotent SCPs might be present in postnatal vertebrate tissues, retaining the capacity to regenerate autonomic neurons and chromaffin cells.
Assuntos
Movimento Celular/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Células de Schwann/fisiologia , Sistema Simpático-Suprarrenal/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sistema Simpático-Suprarrenal/citologia , Peixe-ZebraRESUMO
Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.
Assuntos
Citocinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Drosophila , Olho/metabolismo , Humanos , Linfoma/enzimologia , Crista Neural/enzimologia , Células PC12 , Pigmentação , Ratos , Peixe-ZebraRESUMO
The development of the pigmentation pattern in zebrafish is a tightly regulated process that depends on both the self-organizing properties of pigment cells and extrinsic cues from other tissues. Many of the known mutations that alter the pattern act cell-autonomously in pigment cells, and our knowledge about external regulators is limited. Here, we describe novel zebrafish mau mutants, which encompass several dominant missense mutations in Aquaporin 3a (Aqp3a) that lead to broken stripes and short fins. A loss-of-function aqp3a allele, generated by CRISPR-Cas9, has no phenotypic consequences, demonstrating that Aqp3a is dispensable for normal development. Strikingly, the pigment cells from dominant mau mutants are capable of forming a wild-type pattern when developing in a wild-type environment, but the surrounding tissues in the mutants influence pigment cell behaviour and interfere with the patterning process. The mutated amino acid residues in the dominant alleles line the pore surface of Aqp3a and influence pore permeability. These results demonstrate an important effect of the tissue environment on pigment cell behaviour and, thereby, on pattern formation.
Assuntos
Aquaporina 3/genética , Mutação/genética , Pigmentação , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Nadadeiras de Animais/anatomia & histologia , Nadadeiras de Animais/citologia , Animais , Aquaporina 3/química , Aquaporina 3/metabolismo , Cromatóforos/metabolismo , Genes Dominantes , Proteínas de Fluorescência Verde/metabolismo , Mutação de Sentido Incorreto/genética , Permeabilidade , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
The introduction of engineered site-specific DNA endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. In zebrafish, loss-of-function alleles have been successfully produced; however, germ line transmission of functional targeted knock-ins of protein tags or of SNP exchanges have not been reported. Here we show by phenotypic rescue that the CRISPR/Cas system can be used to target and repair a premature stop codon at the albino (alb) locus in zebrafish with high efficiency and precision. Using circular donor DNA containing CRISPR target sites we obtain close to 50% of larvae with precise homology-directed repair of the alb(b4) mutation, a small fraction of which transmitted the repaired allele in the germ line to the next generation (3/28 adult fish). The in vivo demonstration of germ line transmission of a precise SNP exchange in zebrafish underscores its suitability as a model for genetic research.
Assuntos
Códon sem Sentido/genética , Reparo do DNA/genética , Engenharia Genética/métodos , Genoma/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Primers do DNA/genética , DNA Circular/genética , Genótipo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The zebrafish (Danio rerio) is an important model organism for basic as well as applied bio-medical research. One main advantage is its genetic tractability, which was greatly enhanced by the introduction of the CRISPR/Cas method a decade ago. The generation of loss-of-function alleles via the production of small insertions or deletions in the coding sequences of genes with CRISPR/Cas systems is now routinely achieved with high efficiency. The method is based on the error prone repair of precisely targeted DNA double strand breaks by non-homologous end joining (NHEJ) in the cell nucleus. However, editing the genome with base pair precision, by homology-directed repair (HDR), is by far less efficient and therefore often requires large-scale screening of potential carriers by labour intensive genotyping. Here we confirm that the Cas9 protein variant SpRY, with relaxed PAM requirement, can be used to target some sites in the zebrafish genome. In addition, we demonstrate that the incorporation of an artificial nuclear localisation signal (aNLS) into the Cas9 protein variants not only enhances the efficiency of gene knockout but also the frequency of HDR, thereby facilitating the efficient modification of single base pairs in the genome. Our protocols provide a guide for a cost-effective generation of versatile and potent Cas9 protein variants and efficient gene editing in zebrafish.
Assuntos
Proteína 9 Associada à CRISPR , Edição de Genes , Animais , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Quebras de DNA de Cadeia DuplaRESUMO
bicoid messenger RNA localizes to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first localized cytoplasmic determinant to be identified, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that the mRNA is recognized by a complex of many redundant proteins, each of which binds to the localization element in the 3' untranslated region (UTR) with little or no specificity. Indeed, the only known RNA-binding protein that co-localizes with bicoid mRNA is Staufen, which binds non-specifically to double-stranded RNA in vitro. Here we show that mutants in all subunits of the ESCRT-II complex (VPS22, VPS25 and VPS36) abolish the final Staufen-dependent step in bicoid mRNA localization. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm. However, the role of ESCRT-II in bicoid localization seems to be independent of endosomal sorting, because mutations in ESCRT-I and III components do not affect the targeting of bicoid mRNA. Instead, VPS36 functions by binding directly and specifically to stem-loop V of the bicoid 3' UTR through its amino-terminal GLUE domain, making it the first example of a sequence-specific RNA-binding protein that recognizes the bicoid localization signal. Furthermore, VPS36 localizes to the anterior of the oocyte in a bicoid-mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This function of ESCRT-II as an RNA-binding complex is conserved in vertebrates and may clarify some of its roles that are independent of endosomal sorting.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Endossomos/química , Endossomos/metabolismo , Proteínas de Homeodomínio/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Transativadores/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Polaridade Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Conformação de Ácido Nucleico , Oócitos/citologia , Oócitos/metabolismo , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. The CSF1R pathway is a promising therapeutic target in many human diseases, including neurological disorders and cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands (Csf1a, Csf1b, and Il34) in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors, Csf1ra and Csf1rb, however there is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Furthermore, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation, resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rbΔ4bp-deficient and il34Δ5bp-deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors, and the expression of csf1ra and csf1rb is nonoverlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.
Assuntos
Receptor de Fator Estimulador de Colônias de Macrófagos , Peixe-Zebra , Animais , Proteínas de Transporte/metabolismo , Hematopoese , Ligantes , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Peixe-Zebra/genéticaRESUMO
Color patterns are prominent features of many animals and are of high evolutionary relevance. In basal vertebrates, color patterns are composed of specialized pigment cells that arrange in multilayered mosaics in the skin. Zebrafish (Danio rerio), the preeminent model system for vertebrate color pattern formation, allows genetic screens as powerful approaches to identify novel functions in a complex biological system. Adult zebrafish display a series of blue and golden horizontal stripes, composed of black melanophores, silvery or blue iridophores, and yellow xanthophores. This stereotyped pattern is generated by self-organization involving direct cell contacts between all three types of pigment cells mediated by integral membrane proteins [1-5]. Here, we show that neuropeptide signaling impairs the striped pattern in a global manner. Mutations in the genes coding either for galanin receptor 1A (npm/galr1A) or for its ligand galanin (galn) result in fewer stripes, a pale appearance, and the mixing of cell types, thus resembling mutants with thyroid hypertrophy [6]. Zebrafish chimeras obtained by transplantations of npm/galr1A mutant blastula cells indicate that mutant pigment cells of all three types can contribute to a normal striped pattern in the appropriate host. However, loss of galr1A expression in a specific region of the brain is sufficient to cause the mutant phenotype in an otherwise wild-type fish. Increased thyroid hormone levels in mutant fish suggest that galanin signaling through Galr1A in the pituitary is an upstream regulator of the thyroid hormone pathway, which in turn promotes precise interactions of pigment cells during color pattern formation.
Assuntos
Padronização Corporal/fisiologia , Galanina/genética , Receptor Tipo 1 de Galanina/genética , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Encéfalo/metabolismo , Cor , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Feminino , Galanina/metabolismo , Morfogênese , Mutação , Pigmentação/genética , Receptor Tipo 1 de Galanina/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell-contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.
Assuntos
Cor , Pigmentação/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Cromatóforos/metabolismo , Evolução Molecular , Hibridização Genética , Fenótipo , Especificidade da Espécie , Peixe-Zebra/classificaçãoRESUMO
The genetic basis of morphological variation, both within and between species, provides a major topic in evolutionary biology. Teleost fish produce most elaborate color patterns, and among the more than 20000 species a number have been chosen for more detailed analyses because they are suitable to study particular aspects of color pattern evolution. In several fish species, color variants and pattern variants have been collected, transcriptome analyses have been carried out, and the recent advent of gene editing tools, such as CRISPR/Cas9, has allowed the production of mutants. Covering mostly the literature from the last three years, we discuss the cellular basis of coloration and the identification of loci involved in color pattern differences between sister species in cichlids and Danio species, in which cis-regulatory changes seem to prevail.
Assuntos
Ciclídeos/genética , Evolução Molecular , Pigmentação/genética , Transcriptoma/genética , Animais , Sistemas CRISPR-Cas/genética , Ciclídeos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Pigmentação/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in vertebrate pigmentation. Mutations in this gene cause variations in coat coloration in amniotes. Additionally, in mammals MC1R is the main receptor for agouti-signaling protein (ASIP), making it the critical receptor for the establishment of dorsal-ventral countershading. In fish, Mc1r is also involved in pigmentation, but it has been almost exclusively studied in relation to melanosome dispersion activity and as a putative genetic factor involved in dark/light adaptation. However, its role as the crucial component for the Asip1-dependent control of dorsal-ventral pigmentation remains unexplored. Using CRISPR/Cas9, we created mc1r homozygous knockout zebrafish and found that loss-of-function of mc1r causes a reduction of countershading and a general paling of the animals. We find ectopic development of melanophores and xanthophores, accompanied by a decrease in iridophore numbers in the ventral region of mc1r mutants. We also reveal subtle differences in the role of mc1r in repressing pigment cell development between the skin and scale niches in ventral regions.
Assuntos
Padronização Corporal/genética , Mutação com Perda de Função/genética , Pigmentação/genética , Receptor Tipo 1 de Melanocortina/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Melanóforos/metabolismo , Modelos Biológicos , Fenótipo , Receptor Tipo 1 de Melanocortina/agonistas , Receptor Tipo 1 de Melanocortina/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar-phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.
RESUMO
Asymmetric cell division generates cell diversity in bacteria, yeast, and higher eukaryotes. In Drosophila, both neural and muscle progenitors divide asymmetrically. In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation. Abstrakt (Abs) is a DEAD-box protein that regulates aspects of cell polarity in oocytes and embryos. We use a conditional allele of abs to investigate its role in neural and muscle progenitor cell polarity. In neuroblasts we observe loss of apical Insc crescents, failure in basal protein targeting, and defects in spindle orientation. In the GMC4-2a cell we observe loss of apical Insc crescents, defects in basal protein targeting, and equalization of sibling neuron fates; muscle precursors show a similar equalization of sibling cell fates. These phenotypes resemble those of insc mutants; indeed, abs mutants show a striking loss of Insc protein levels but no change of insc RNA levels. Furthermore, we find that the Abs protein physically interacts with insc RNA. Our results demonstrate a novel role for Abs in the posttranscriptional regulation of insc expression, which is essential for proper cell polarity, spindle orientation, and the establishment of distinct sibling cell fates within embryonic neural and muscle progenitors.
Assuntos
Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fuso Acromático/fisiologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Drosophila , Imuno-Histoquímica , Neuropeptídeos , Fuso Acromático/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Color patterns are prominent features of many animals; they are highly variable and evolve rapidly leading to large diversities even within a single genus. As targets for natural as well as sexual selection, they are of high evolutionary significance. The zebrafish (Danio rerio) has become an important model organism for developmental biology and biomedical research in general, and it is the model organism to study color pattern formation in vertebrates. The fish display a conspicuous pattern of alternating blue and golden stripes on the body and on the anal and tail fins. This pattern is produced by three different types of pigment cells (chromatophores) arranged in precise layers in the hypodermis of the fish. In this essay, we will summarize the recent advances in understanding the developmental and genetic basis for stripe formation in the zebrafish. We will describe the cellular events leading to the formation of stripes during metamorphosis based on long-term lineage imaging. Mutant analysis has revealed that a number of signaling pathways are involved in the establishment and maintenance of the individual pigment cells. However, the striped pattern itself is generated by self-organizing mechanisms requiring interactions between all three pigment cell types. The involvement of integral membrane proteins, including connexins and potassium channels, suggests that direct physical contacts between chromatophores are involved, and that the directed transport of small molecules or bioelectrical coupling is important for these interactions. This mode of patterning by transmitting spatial information between adjacent tissues within three superimposed cell layers is unprecedented in other developmental systems. We propose that variations in the patterns among Danio species are caused by allelic differences in the genes responsible for these interactions.
Assuntos
Padronização Corporal/genética , Cromatóforos/metabolismo , Metamorfose Biológica/genética , Morfogênese/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , FenótipoRESUMO
The conspicuous striped coloration of zebrafish is produced by cell-cell interactions among three different types of chromatophores: black melanophores, orange/yellow xanthophores and silvery/blue iridophores. During color pattern formation xanthophores undergo dramatic cell shape transitions and acquire different densities, leading to compact and orange xanthophores at high density in the light stripes, and stellate, faintly pigmented xanthophores at low density in the dark stripes. Here, we investigate the mechanistic basis of these cell behaviors in vivo, and show that local, heterotypic interactions with dense iridophores regulate xanthophore cell shape transition and density. Genetic analysis reveals a cell-autonomous requirement of gap junctions composed of Cx41.8 and Cx39.4 in xanthophores for their iridophore-dependent cell shape transition and increase in density in light-stripe regions. Initial melanophore-xanthophore interactions are independent of these gap junctions; however, subsequently they are also required to induce the acquisition of stellate shapes in xanthophores of the dark stripes. In summary, we conclude that, whereas homotypic interactions regulate xanthophore coverage in the skin, their cell shape transitions and density is regulated by gap junction-mediated, heterotypic interactions with iridophores and melanophores.
RESUMO
Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. We present the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3'-5' EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3' untranslated region with high affinity. Through structure-guided mutagenesis, we show that Exu dimerization is essential for bcd localization. Our data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.
Assuntos
Proteínas de Drosophila/química , Drosophila melanogaster/enzimologia , Proteínas do Ovo/química , Exonucleases/química , Proteínas de Ligação a RNA/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Drosophila/fisiologia , Proteínas do Ovo/fisiologia , Exonucleases/fisiologia , Feminino , Proteínas de Homeodomínio/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Transporte Proteico , RNA/química , Proteínas de Ligação a RNA/fisiologia , Transativadores/metabolismoRESUMO
The neural crest is a transient, multipotent embryonic cell population in vertebrates giving rise to diverse cell types in adults via intermediate progenitors. The in vivo cell-fate potential and lineage segregation of these postembryonic progenitors is poorly understood, and it is unknown if and when the progenitors become fate restricted. We investigate the fate restriction in the neural crest-derived stem cells and intermediate progenitors in zebrafish, which give rise to three distinct adult pigment cell types: melanophores, iridophores, and xanthophores. By inducing clones in sox10-expressing cells, we trace and quantitatively compare the pigment cell progenitors at four stages, from embryogenesis to metamorphosis. At all stages, a large fraction of the progenitors are multipotent. These multipotent progenitors have a high proliferation ability, which diminishes with fate restriction. We suggest that multipotency of the nerve-associated progenitors lasting into metamorphosis may have facilitated the evolution of adult-specific traits in vertebrates.