RESUMO
The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.
Assuntos
Crista Neural , Peixe-Zebra , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Crista Neural/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Autofagia/genética , Morte Celular , Mutação/genéticaRESUMO
We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Fator de Transcrição GATA3/genética , Proteínas Hedgehog/metabolismo , Fenótipo , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Fator de Transcrição GATA3/metabolismo , Haploinsuficiência , Mutação com Perda de Função , Mutação , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Organogênese , Crânio/citologia , Crânio/embriologia , Peixe-Zebra/embriologiaRESUMO
BACKGROUND: Fetal alcohol spectrum disorders (FASD) collectively refer to all deleterious outcomes due to prenatal alcohol exposures. Alterations to the face are common phenotypes in FASD. While alcohol exposure is the underlying cause of FASD, many variables modify the outcomes of such exposures. Genetic risk is one such variable, yet we still have a limited understanding of the nature of the genetic loci mediating susceptibility to FASD. METHODS: We employed ENU-based random mutagenesis in zebrafish to identify mutations that enhanced the teratogenicity of ethanol (EtOH). F3 embryos obtained from 126 inbred F2 families were exposed to 1% EtOH in the medium (approximately 41 mM tissue levels). Zebrafish stained with Alcian Blue and Alizarin Red were screened for qualitative alterations to the craniofacial skeleton between 4 and 7 days postfertilization (dpf). RESULTS: In all, we recovered 6 EtOH-sensitive mutants, 5 from the genetic screen itself and one as a background mutation in one of our wild-type lines. Each mutant has a unique EtOH-induced phenotype relative to the other mutant lines. All but 1 mutation appears to be recessive in nature, and only 1 mutant, au29, has apparent craniofacial defects in the absence of EtOH. To validate the genetic screen, we genetically mapped au29 and found that it carries a mutation in a previously uncharacterized gene, si:dkey-88l16.3. CONCLUSIONS: The phenotypes of these EtOH-sensitive mutants differ from those in previous characterizations of gene-EtOH interactions. Thus, each mutant is likely to provide novel insights into EtOH teratogenesis. Given that most of these mutants only have craniofacial defects in the presence of EtOH and our mapping of au29, it is also likely that many of the mutants will be previously uncharacterized. Collectively, our findings point to the importance of unbiased genetic screens in the identification, and eventual characterization, of risk alleles for FASD.
Assuntos
Modelos Animais de Doenças , Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/genética , Testes Genéticos/métodos , Mutação/efeitos dos fármacos , Mutação/genética , Animais , Anormalidades Craniofaciais/induzido quimicamente , Anormalidades Craniofaciais/genética , Feminino , Transtornos do Espectro Alcoólico Fetal/patologia , Predisposição Genética para Doença/genética , Gravidez , Peixe-ZebraRESUMO
Integrated development of diverse tissues gives rise to a functional, mobile vertebrate musculoskeletal system. However, the genetics and cellular interactions that drive the integration of muscle, tendon, and skeleton are poorly understood. In the vertebrate head, neural crest cells, from which cranial tendons derive, pattern developing muscles just as tendons have been shown to in limb and trunk tissue, yet the mechanisms of this patterning are unknown. From a forward genetic screen, we determined that cyp26b1 is critical for musculoskeletal integration in the ventral pharyngeal arches, particularly in the mandibulohyoid junction where first and second arch muscles interconnect. Using time-lapse confocal analyses, we detail musculoskeletal integration in wild-type and cyp26b1 mutant zebrafish. In wild-type fish, tenoblasts are present in apposition to elongating muscles and condense in discrete muscle attachment sites. In the absence of cyp26b1, tenoblasts are generated in normal numbers but fail to condense into nascent tendons within the ventral arches and, subsequently, muscles project into ectopic locales. These ectopic muscle fibers eventually associate with ectopic tendon marker expression. Genetic mosaic analysis demonstrates that neural crest cells require Cyp26b1 function for proper musculoskeletal development. Using an inhibitor, we find that Cyp26 function is required in a short time window that overlaps the dynamic window of tenoblast condensation. However, cyp26b1 expression is largely restricted to regions between tenoblast condensations during this time. Our results suggest that degradation of RA by this previously undescribed population of neural crest cells is critical to promote condensation of adjacent scxa-expressing tenoblasts and that these condensations are subsequently required for proper musculoskeletal integration.
Assuntos
Desenvolvimento Embrionário/genética , Desenvolvimento Maxilofacial/genética , Morfogênese/genética , Ácido Retinoico 4 Hidroxilase/genética , Animais , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Arcada Osseodentária/embriologia , Desenvolvimento Muscular/genética , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Tendões/embriologia , Tendões/crescimento & desenvolvimento , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18â h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Endoderma/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Morfogênese , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Contagem de Células , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Face/embriologia , Morfogênese/efeitos dos fármacos , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Crista Neural/patologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Crânio/efeitos dos fármacos , Crânio/patologia , Fatores de TempoRESUMO
Human birth defects are highly variable and this phenotypic variability can be influenced by both the environment and genetics. However, the synergistic interactions between these two variables are not well understood. Fetal alcohol spectrum disorders (FASD) is the umbrella term used to describe the wide range of deleterious outcomes following prenatal alcohol exposure. Although FASD are caused by prenatal ethanol exposure, FASD are thought to be genetically modulated, although the genes regulating sensitivity to ethanol teratogenesis are largely unknown. To identify potential ethanol-sensitive genes, we tested five known craniofacial mutants for ethanol sensitivity: cyp26b1, gata3, pdgfra, smad5 and smoothened. We found that only platelet-derived growth factor receptor alpha (pdgfra) interacted with ethanol during zebrafish craniofacial development. Analysis of the PDGF family in a human FASD genome-wide dataset links PDGFRA to craniofacial phenotypes in FASD, prompting a mechanistic understanding of this interaction. In zebrafish, untreated pdgfra mutants have cleft palate due to defective neural crest cell migration, whereas pdgfra heterozygotes develop normally. Ethanol-exposed pdgfra mutants have profound craniofacial defects that include the loss of the palatal skeleton and hypoplasia of the pharyngeal skeleton. Furthermore, ethanol treatment revealed latent haploinsufficiency, causing palatal defects in â¼62% of pdgfra heterozygotes. Neural crest apoptosis partially underlies these ethanol-induced defects in pdgfra mutants, demonstrating a protective role for Pdgfra. This protective role is mediated by the PI3K/mTOR pathway. Collectively, our results suggest a model where combined genetic and environmental inhibition of PI3K/mTOR signaling leads to variability within FASD.
Assuntos
Anormalidades Craniofaciais/prevenção & controle , Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/prevenção & controle , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/anormalidades , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fissura Palatina/etiologia , Fissura Palatina/genética , Anormalidades Craniofaciais/etiologia , Anormalidades Craniofaciais/genética , Modelos Animais de Doenças , Feminino , Transtornos do Espectro Alcoólico Fetal/etiologia , Transtornos do Espectro Alcoólico Fetal/genética , Interação Gene-Ambiente , Heterozigoto , Humanos , Mutação , Crista Neural/anormalidades , Crista Neural/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca(b1086) loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal-ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.
Assuntos
Desenvolvimento Ósseo/genética , Larva/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Genes Homeobox , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a highly variable set of phenotypes caused by fetal alcohol exposure. Numerous factors influence FASD phenotypes, including genetics. The zebrafish is a powerful vertebrate model system with which to identify these genetic factors. Many zebrafish mutants are housed at the Zebrafish International Resource Center (ZIRC). These mutants are readily accessible and an excellent source to screen for ethanol (EtOH)-sensitive developmental structural mutants. METHODS: We screened mutants obtained from ZIRC for sensitivity to EtOH teratogenesis. Embryos were treated with 1% EtOH (41 mM tissue levels) from 6 hours postfertilization onward. Levels of apoptosis were evaluated at 24 hours postfertilization. At 4 days postfertilization, the craniofacial skeleton, peripheral axon projections, and sensory neurons of neuromasts were examined. Fish were genotyped to determine whether there were phenotype/genotype correlations. RESULTS: Five of 20 loci interacted with EtOH. Notable among these was that vangl2, involved in convergent extension movements of the embryonic axis, interacted strongly with EtOH. Untreated vangl2 mutants had normal craniofacial morphology, while severe midfacial defects including synophthalmia and narrowing of the palatal skeleton were found in all EtOH-treated mutants and a low percentage of heterozygotes. The cell cycle gene, plk1, also interacted strongly with EtOH. Untreated mutants have slightly elevated levels of apoptosis and loss of ventral craniofacial elements. Exposure to EtOH results in extensive apoptosis along with loss of neural tissue and the entire craniofacial skeleton. Phenotypes of hinfp, mars, and foxi1 mutants were also exacerbated by EtOH. CONCLUSIONS: Our results provide insight into the gene-EtOH interactions that may underlie EtOH teratogenesis. They support previous findings that EtOH disrupts elongation of the embryonic axis. Importantly, these results show that the zebrafish is an efficient model with which to test for gene-EtOH interactions. Understanding these interactions will be crucial to understanding of the FASD variation.
Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Anormalidades Craniofaciais/induzido quimicamente , Etanol/efeitos adversos , Transtornos do Espectro Alcoólico Fetal/genética , Peixe-Zebra/genética , Animais , Anormalidades Craniofaciais/genética , Genes cdc , Proteínas de Membrana/genética , Fenótipo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed "maturation," when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.
Assuntos
Condrócitos/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Osteogênese/genética , Pentosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Cartilagem/crescimento & desenvolvimento , Cartilagem/ultraestrutura , Células Cultivadas , Condrócitos/ultraestrutura , Proteoglicanas de Sulfatos de Condroitina/genética , Colágeno/genética , Proteínas Hedgehog/metabolismo , Mutação , Peixe-Zebra/metabolismo , UDP Xilose-Proteína XilosiltransferaseRESUMO
The proper function of the craniofacial skeleton requires the proper shaping of many individual skeletal elements. Neural crest cells generate much of the craniofacial skeleton and morphogenesis of skeletal elements occurs in transient, reiterated structures termed pharyngeal arches. The shape of individual elements depends upon intrinsic patterning within the neural crest as well as extrinsic signals to the neural crest from adjacent tissues within the arches. Hedgehog (Hh) signaling is known to play roles in craniofacial development, yet its involvement in intrinsic and extrinsic patterning of the craniofacial skeleton is still not well understood. Here, we show that morphogenetic movements of the pharyngeal arches and patterning of the neural crest require Hh signaling. Loss of Hh signaling, in smoothened (smo) mutants, disrupts the expression of some Dlx genes as well as other markers of dorsal/ventral patterning of the neural crest. Transplantation of wild-type neural crest cells into smo mutants rescues this defect, demonstrating that the neural crest requires reception of Hh signals for proper patterning. Despite the rescue, morphogenesis of the facial skeleton is not fully recovered. Through transplant analyses, we find two additional requirements for Hh signaling. The endoderm requires the reception of Hh signals for proper morphogenetic movements of the pharyngeal arches and the neural crest require the reception of Hh signaling for the activity of a reverse signal that maintains sonic hedgehog expression in the endoderm. Collectively, these results demonstrate that Hh signaling is essential to establish intrinsic and extrinsic patterning information for the craniofacial skeleton.
Assuntos
Padronização Corporal , Osso e Ossos/embriologia , Região Branquial/embriologia , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Animais , Biomarcadores/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Região Branquial/citologia , Região Branquial/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Face , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Crista Neural/citologia , Crista Neural/metabolismo , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Cristalino/embriologia , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Alelos , Animais , Apoptose , Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética , Epitélio/embriologia , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Cristalino/citologia , Cristalino/metabolismo , Mutação , Retina/citologia , Retina/embriologia , Retina/metabolismo , Transativadores/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Human palatal clefting is debilitating and difficult to rectify surgically. Animal models enhance our understanding of palatogenesis and are essential in strategies designed to ameliorate palatal malformations in humans. Recent studies have shown that the zebrafish palate, or anterior neurocranium, is under similar genetic control to the amniote palatal skeleton. We extensively analyzed palatogenesis in zebrafish to determine the similarity of gene expression and function across vertebrates. By 36 hours postfertilization (hpf) palatogenic cranial neural crest cells reside in homologous regions of the developing face compared with amniote species. Transcription factors and signaling molecules regulating mouse palatogenesis are expressed in similar domains during palatogenesis in zebrafish. Functional investigation of a subset of these genes, fgf10a, tgfb2, pax9, and smad5 revealed their necessity in zebrafish palatogenesis. Collectively, these results suggest that the gene regulatory networks regulating palatogenesis may be conserved across vertebrate species, demonstrating the utility of zebrafish as a model for palatogenesis.
Assuntos
Palato/embriologia , Palato/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Fator de Transcrição PAX9/genética , Fator de Transcrição PAX9/metabolismo , Transdução de Sinais , Proteína Smad5/genética , Proteína Smad5/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
South American and African weakly electric fish independently evolved electric organs from muscle. In both groups, a voltage-gated sodium channel gene independently lost expression from muscle and gained it in the electric organ, allowing the channel to become specialized for generating electric signals. It is unknown how this voltage-gated sodium channel gene is targeted to muscle in any vertebrate. We describe an enhancer that selectively targets sodium channel expression to muscle. Next, we demonstrate how the loss of this enhancer, but not trans-activating factors, drove the loss of sodium channel gene expression from muscle in South American electric fish. While this enhancer is also altered in African electric fish, key transcription factor binding sites and enhancer activity are retained, suggesting that the convergent loss of sodium channel expression from muscle in these two electric fish lineages occurred via different processes.
RESUMO
We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into one cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton.
Assuntos
Regeneração Óssea/genética , Organogênese/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Nadadeiras de Animais/fisiologia , Nadadeiras de Animais/cirurgia , Animais , Animais Geneticamente Modificados , Condrócitos/metabolismo , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular , Orelha/embriologia , Orelha/crescimento & desenvolvimento , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Larva/genética , Larva/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
Pharyngeal endoderm is essential for and can reprogram development of the head skeleton. Here we investigate the roles of specific endodermal structures in regulating craniofacial development. We have isolated an integrinalpha5 mutant in zebrafish that has region-specific losses of facial cartilages derived from hyoid neural crest cells. In addition, the cranial muscles that normally attach to the affected cartilage region and their associated nerve are secondarily reduced in integrinalpha5- animals. Earlier in development, integrinalpha5 mutants also have specific defects in the formation of the first pouch, an outpocketing of the pharyngeal endoderm. By fate mapping, we show that the cartilage regions that are lost in integrinalpha5 mutants develop from neural crest cells directly adjacent to the first pouch in wild-type animals. Furthermore, we demonstrate that Integrinalpha5 functions in the endoderm to control pouch formation and cartilage development. Time-lapse recordings suggest that the first pouch promotes region-specific cartilage development by regulating the local compaction and survival of skeletogenic neural crest cells. Thus, our results reveal a hierarchy of tissue interactions, at the top of which is the first endodermal pouch, which locally coordinates the development of multiple tissues in a specific region of the vertebrate face. Lastly, we discuss the implications of a mosaic assembly of the facial skeleton for the evolution of ray-finned fish.
Assuntos
Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osso Hioide/crescimento & desenvolvimento , Integrina alfa5/genética , Integrinas/metabolismo , Mutação , Animais , Padronização Corporal , Cartilagem/embriologia , Cartilagem/metabolismo , Cartilagem/patologia , Linhagem da Célula , Eletroporação , Evolução Molecular , Proteínas de Fluorescência Verde/metabolismo , Osso Hioide/embriologia , Microscopia Confocal , Modelos Biológicos , Dados de Sequência Molecular , Crista Neural/metabolismo , Neurônios/metabolismo , Fenótipo , Fatores de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/químicaRESUMO
Eph receptor tyrosine kinases and ephrins are required for axon patterning and plasticity in the developing nervous system. Typically, Eph-ephrin interactions promote inhibitory events; for example, prohibiting the entry of neural cells into certain embryonic territories. Here, we show that distinct subsets of motor neurons that express EphA4 respond differently to ephrin-A5. EphA4-positive LMC(l) axons avoid entering ephrin-A5-positive hindlimb mesoderm. In contrast, EphA4-positive MMC(m) axons extend through ephrin-A5-positive rostral half-sclerotome. Blocking EphA4 activation in MMC(m) neurons or expanding the domain of ephrin-A5 expression in the somite results in the aberrant growth of MMC(m) axons into the caudal half-sclerotome. Moreover, premature expression of EphA4 in MMC(m) neurons leads to a portion of their axons growing into novel ephrin-A5-positive territories. Together, these results indicate that EphA4-ephrin-A5 signaling acts in a positive manner to constrain MMC(m) axons to the rostral half-sclerotome. Furthermore, we show that Eph activation localizes to distinct subcellular compartments of LMC(l) and MMC(m) neurons, consistent with distinct EphA4 signaling cascades in these neuronal subpopulations.
Assuntos
Efrina-A4/biossíntese , Efrina-A5/fisiologia , Neurônios Motores/metabolismo , Inibição Neural/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Eletroporação , Efrina-A4/genética , Efrina-A5/genética , Efrina-A5/farmacologia , Membro Posterior/embriologia , Membro Posterior/inervação , Ligantes , Mesoderma/metabolismo , Neurônios Motores/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The severity of most human birth defects is highly variable. Our ability to diagnose, treat and prevent defects relies on our understanding of this variability. Mutation of the transcription factor GATA3 in humans causes the highly variable hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. Although named for a triad of defects, individuals with HDR can also exhibit craniofacial defects. Through a forward genetic screen for craniofacial mutants, we isolated a zebrafish mutant in which the first cysteine of the second zinc finger of Gata3 is mutated. Because mutation of the homologous cysteine causes HDR in humans, these zebrafish mutants could be a quick and effective animal model for understanding the role of gata3 in the HDR disease spectrum. We demonstrate that, unexpectedly, the chaperone proteins Ahsa1 and Hsp90 promote severe craniofacial phenotypes in our zebrafish model of HDR syndrome. The strengths of the zebrafish system, including rapid development, genetic tractability and live imaging, make this an important model for variability.
Assuntos
Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Hipoparatireoidismo/metabolismo , Hipoparatireoidismo/patologia , Chaperonas Moleculares/metabolismo , Nefrose/metabolismo , Nefrose/patologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Modelos Animais de Doenças , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Injeções , Chaperonas Moleculares/antagonistas & inibidores , Morfolinos/administração & dosagem , Morfolinos/farmacologia , Mutação/genética , Fenótipo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
In human, mutation of the transcription factor SATB2 causes severe defects to the palate and jaw. The expression and sequence of SATB2 is highly conserved across vertebrate species, including zebrafish. We sought to understand the regulation of satb2 using the zebrafish model system. Due to the normal expression domains of satb2, we analyzed satb2 expression in mutants with disrupted Hh signaling or defective ventral patterning. While satb2 expression appears independent of Edn1 signaling, appropriate expression requires Shha, Smo, Smad5 and Hand2 function. Transplantation experiments show that neural crest cells receive both Bmp and Hh signaling to induce satb2 expression. Dorsomorphin- and cyclopamine-mediated inhibition of Bmp and Hh signaling, respectively, suggests that proper satb2 expression requires a relatively earlier Bmp signal and a later Hh signal. We propose that Bmp signaling establishes competence for the neural crest to respond to Hh signaling, thus inducing satb2 expression.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Região Branquial/citologia , Região Branquial/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Região Branquial/embriologia , Endoderma/citologia , Endoderma/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Peixe-Zebra/embriologiaRESUMO
Disruption of signaling pathways such as those mediated by sonic hedgehog (Shh) or platelet-derived growth factor (Pdgf) causes craniofacial abnormalities, including cleft palate. The role that microRNAs play in modulating palatogenesis, however, is completely unknown. We show that, in zebrafish, the microRNA Mirn140 negatively regulates Pdgf signaling during palatal development, and we provide a mechanism for how disruption of Pdgf signaling causes palatal clefting. The pdgf receptor alpha (pdgfra) 3' UTR contained a Mirn140 binding site functioning in the negative regulation of Pdgfra protein levels in vivo. pdgfra mutants and Mirn140-injected embryos shared a range of facial defects, including clefting of the crest-derived cartilages that develop in the roof of the larval mouth. Concomitantly, the oral ectoderm beneath where these cartilages develop lost pitx2 and shha expression. Mirn140 modulated Pdgf-mediated attraction of cranial neural crest cells to the oral ectoderm, where crest-derived signals were necessary for oral ectodermal gene expression. Mirn140 loss of function elevated Pdgfra protein levels, altered palatal shape and caused neural crest cells to accumulate around the optic stalk, a source of the ligand Pdgfaa. These results suggest that the conserved regulatory interactions of mirn140 and pdgfra define an ancient mechanism of palatogenesis, and they provide candidate genes for cleft palate.