RESUMO
Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Fosfatidilinositóis/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/genética , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Lisina/metabolismo , Metilação , Camundongos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Interferência de RNA , Homologia de Sequência de Aminoácidos , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Polycomb group (PcG) proteins are transcriptional repressors that mediate epigenetic gene silencing by chromatin modification. PcG-mediated gene repression is implicated in development, cell differentiation, stem-cell fate maintenance and cancer. However, analysis of the roles of PcG proteins in orchestrating vertebrate developmental programs in vivo has been hampered by the early embryonic lethality of several PcG gene knockouts in mice. Here, we demonstrate that zebrafish Ring1b, the E3 ligase in Polycomb Repressive Complex 1 (PRC1), is essential for pectoral fin development. We show that differentiation of lateral plate mesoderm (LPM) cells into presumptive pectoral fin precursors is initiated normally in ring1b mutants, but fin bud outgrowth is impaired. Fgf signaling, which is essential for migration, proliferation and cell-fate maintenance during fin development, is not sufficiently activated in ring1b mutants. Exogenous application of FGF4, as well as enhanced stimulation of Fgf signaling by overactivated Wnt signaling in apc mutants, partially restores the fin developmental program. These results reveal that, in the absence of functional Ring1b, fin bud cells fail to execute the pectoral fin developmental program. Together, our results demonstrate that PcG-mediated gene regulation is essential for sustained Fgf signaling in vertebrate limb development.
Assuntos
Nadadeiras de Animais/embriologia , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Nadadeiras de Animais/efeitos dos fármacos , Nadadeiras de Animais/metabolismo , Animais , Sequência de Bases , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Proteínas do Grupo Polycomb , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tretinoína/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel-Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells.
Assuntos
Metabolismo Energético , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Peixe-Zebra/metabolismo , Animais , Metabolismo Basal , Polaridade Celular , Embrião não Mamífero/enzimologia , Embrião não Mamífero/patologia , Privação de Alimentos , Fator de Crescimento Insulin-Like I/metabolismo , Intestinos/patologia , Larva/citologia , Larva/metabolismo , Mutação/genética , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Valvas Cardíacas/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Divisão Celular , Linhagem da Célula , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes APC , Genótipo , Valvas Cardíacas/anormalidades , Valvas Cardíacas/citologia , Valvas Cardíacas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Mutação/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Transativadores/genética , Proteínas Wnt , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , beta CateninaRESUMO
Plastic nanoparticles originating from weathering plastic waste are emerging contaminants in aquatic environments, with unknown modes of action in aquatic organisms. Recent studies suggest that internalised nanoplastics may disrupt processes related to energy metabolism. Such disruption can be crucial for organisms during development and may ultimately lead to changes in behaviour. Here, we investigated the link between polystyrene nanoplastic (PSNP)-induced signalling events and behavioural changes. Larval zebrafish exhibited PSNP accumulation in the pancreas, which coincided with a decreased glucose level. By using hyperglycemic and glucocorticoid receptor (Gr) mutant larvae, we demonstrate that the PSNP-induced disruption in glucose homoeostasis coincided with increased cortisol secretion and hyperactivity in challenge phases. Our work sheds new light on a potential mechanism underlying nanoplastics toxicity in fish, suggesting that the adverse effect of PSNPs are at least in part mediated by Gr activation in response to disrupted glucose homeostasis, ultimately leading to aberrant locomotor activity.
Assuntos
Nanopartículas/toxicidade , Poliestirenos/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Hidrocortisona/metabolismo , Larva/efeitos dos fármacos , Larva/fisiologia , Atividade Motora/efeitos dos fármacos , Mutação , Plásticos/toxicidade , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Distribuição Tecidual , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Notch signaling plays an essential role in the proliferation, differentiation and cell fate determination of various tissues, including the developing pancreas. One regulator of the Notch pathway is GDE2 (or GDPD5), a transmembrane ecto-phosphodiesterase that cleaves GPI-anchored proteins at the plasma membrane, including a Notch ligand regulator. Here we report that Gdpd5-knockdown in zebrafish embryos leads to developmental defects, particularly, impaired motility and reduced pancreas differentiation, as shown by decreased expression of insulin and other pancreatic markers. Exogenous expression of human GDE2, but not catalytically dead GDE2, similarly leads to developmental defects. Human GDE2 restores insulin expression in Gdpd5a-depleted zebrafish embryos. Importantly, zebrafish Gdpd5 orthologues localize to the plasma membrane where they show catalytic activity against GPI-anchored GPC6. Thus, our data reveal functional conservation between zebrafish Gdpd5 and human GDE2, and suggest that strict regulation of GDE2 expression and catalytic activity is critical for correct embryonic patterning. In particular, our data uncover a role for GDE2 in regulating pancreas differentiation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Organogênese , Pâncreas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/anormalidades , Embrião não Mamífero/diagnóstico por imagem , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Morfolinos/metabolismo , Pâncreas/diagnóstico por imagem , Pâncreas/embriologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Filogenia , Domínios Proteicos , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Autophagy is an evolutionarily conserved process that degrades cellular components to restore energy homeostasis under limited nutrient conditions. How this starvation-induced autophagy is regulated at the whole-body level is not fully understood. Here, we show that the tumor suppressor Lkb1, which activates the key energy sensor AMPK, also regulates starvation-induced autophagy at the organismal level. Lkb1-deficient zebrafish larvae fail to activate autophagy in response to nutrient restriction upon yolk termination, shown by reduced levels of the autophagy-activating proteins Atg5, Lc3-II and Becn1, and aberrant accumulation of the cargo receptor and autophagy substrate p62. We demonstrate that the autophagy defect in lkb1 mutants can be partially rescued by inhibiting mTOR signaling but not by inhibiting the PI3K pathway. Interestingly, mTOR-independent activation of autophagy restores degradation of the aberrantly accumulated p62 in lkb1 mutants and prolongs their survival. Our data uncover a novel critical role for Lkb1 in regulating starvation-induced autophagy at the organismal level, providing mechanistic insight into metabolic adaptation during development.
Assuntos
Autofagia , Proteínas Serina-Treonina Quinases/metabolismo , Inanição , Estresse Fisiológico , Proteínas Supressoras de Tumor/metabolismo , Animais , Autofagia/genética , Biomarcadores , Imunofluorescência , Imuno-Histoquímica , Larva , Mutação , Proteínas Serina-Treonina Quinases/genética , Estresse Fisiológico/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/genética , Peixe-ZebraRESUMO
Stem cells have the unique ability to both maintain the stem cell population via self-renewal and give rise to differentiated cells. The balance between these options is very delicate and important for the short- and long-term maintenance of tissue homeostasis in an organism. Pathways involved in integrating environmental cues and in directing energy metabolism play an important role in the fate decisions of stem cells. In this review, we give an overview of the effects of cellular and systemic metabolic states on stem-cell fate in both embryonic and in adult stem cell populations, with a particular emphasis on cell-cycle regulation. We discuss the major pathways implicated in sensing energetic status and regulating metabolism, including: the mTOR pathway, Forkhead-box-O transcription factors (FoxOs), Sirtuins, reactive oxygen species (ROS), AMP-activated kinase (AMPK) and LKB1, the mTOR pathway and hypoxia inducible factors (HIFs). Given the importance of a correct balance between self-renewal and differentiation, understanding the mechanisms that drive stem-cell fate in different metabolic conditions will provide more insight in stem cell biology in both health and disease.
Assuntos
Células-Tronco Adultas/fisiologia , Ciclo Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Metabolismo Energético/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco Adultas/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Humanos , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Polycomb group (PcG) genes are chromatin modifiers that mediate epigenetic silencing of target genes. PcG-mediated epigenetic silencing is implicated in embryonic development, stem cell plasticity, cell fate maintenance, cellular differentiation and cancer. However, analysis of the roles of PcG proteins in maintaining differentiation programs during vertebrate embryogenesis has been hampered due to the early embryonic lethality of several PcG knock-outs in the mouse. Here, we show that zebrafish Ring1b/Rnf2, the single E3 ubiquitin ligase in the Polycomb Repressive Complex 1, critically regulates the developmental program of craniofacial cell lineages. Zebrafish ring1b mutants display a severe craniofacial phenotype, which includes an almost complete absence of all cranial cartilage, bone and musculature. We show that Cranial Neural Crest (CNC)-derived cartilage precursors migrate correctly into the pharyngeal arches, but fail to differentiate into chondrocytes. This phenotype is specific for cartilage precursors, since other neural crest-derived cell lineages, including glia, neurons and chromatophores, are formed normally in ring1b mutants. Our results therefore reveal a critical and specific role for Ring1b in promoting the differentiation of cranial neural crest cells into chondrocytes. The molecular mechanisms underlying the pathogenesis of craniofacial abnormalities, which are among the most common genetic birth defects in humans, remain poorly understood. The zebrafish ring1b mutant provides a molecular model for investigating these mechanisms and may lead to the discovery of new treatments or preventions of craniofacial abnormalities.
Assuntos
Padronização Corporal/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Condrócitos/citologia , Condrogênese/genética , Arcada Osseodentária/embriologia , Músculos/embriologia , Músculos/metabolismo , Mutação , Crista Neural/embriologia , Crista Neural/metabolismo , Osteogênese/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Phosphatidylinositol 5-phosphate 4-kinases (PIP4Ks) phosphorylate phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5-bisphosphate; their most likely function is the regulation of the levels of PI5P, a putative signalling intermediate. There are three mammalian PIP4Ks isoforms (α, ß and γ), but their physiological roles remain poorly understood. In the present study, we identified the zebrafish orthologue (zPIP4Kα) of the high-activity human PIP4K α isoform and analyzed its role in embryonic development. RT-PCR analysis and whole-mount in situ hybridization experiments showed that zPIP4Kα is maternally expressed. At later embryonic stages, high PIP4Kα expression was detected in the head and the pectoral fins. Knockdown of zPIP4Kα by antisense morpholino oligonucleotides led to severe morphological abnormalities, including midbody winding defects at 48hpf. The abnormal phenotype could be rescued, at least in large part, by injection of human PIP4Kα mRNA. Our results reveal a key role for PIP4Kα and its activity in vertebrate tissue homeostasis and organ development.
Assuntos
Fosfatidilinositol 4,5-Difosfato/biossíntese , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Morfolinos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas , Proteínas de Peixe-Zebra/genéticaRESUMO
The tumor suppressor LKB1 has emerged as a critical regulator of cell polarity and energymetabolism. Studies in diverse model organisms continue to unravel the pathways downstream of LKB1; the emerging picture is that the outcomes of LKB1 signaling are mediated by a plethora of tissuespecific and contextdependent effectors.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Polaridade Celular , Metabolismo Energético , Humanos , Síndrome de Peutz-Jeghers/patologia , Síndrome de Peutz-Jeghers/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Previous studies have shown that Wnt signals, relayed through beta-catenin and T-cell factor 4 (Tcf4), are essential for the induction and maintenance of crypts in mice. We have now generated a tcf4 (tcf7l2) mutant zebrafish by reverse genetics. We first observe a phenotypic defect at 4 weeks post-fertilization (wpf), leading to death at about 6 wpf. The phenotype comprises a loss of proliferation at the base of the intestinal folds of the middle and distal parts of the intestine. The proximal intestine represents an independent compartment, as it expresses sox2 in the epithelium and barx1 in the surrounding mesenchyme, which are early stomach markers in higher vertebrates. Zebrafish are functionally stomach-less, but the proximal intestine might share its ontogeny with the mammalian stomach. Rare adult homozygous tcf4(-/-) 'escapers' show proliferation defects in the gut epithelium, but have no other obvious abnormalities. This study underscores the involvement of Tcf4 in maintaining proliferative self-renewal in the intestine throughout life.
Assuntos
Proliferação de Células , Mucosa Intestinal/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Imuno-Histoquímica , Hibridização In Situ , Intestinos/citologia , Masculino , Mutação , Fenótipo , Fatores de Tempo , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Truncation of the tumour suppressor adenomatous polyposis coli (APC) constitutively activates the Wnt/beta-catenin signalling pathway. This event constitutes the primary transforming event in sporadic colorectal cancer in humans. Moreover, humans or mice carrying germline truncating mutations in APC develop large numbers of intestinal adenomas. Here, we report that zebrafish that are heterozygous for a truncating APC mutation spontaneously develop intestinal, hepatic and pancreatic neoplasias that are highly proliferative, accumulate beta-catenin and express Wnt target genes. Treatment with the chemical carcinogen 7,12-dimethylbenz[a]anthracene accelerates the induction of these lesions. These observations establish apc-mutant zebrafish as a bona fide model for the study of digestive tract cancer.
Assuntos
Adenoma/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/deficiência , Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias do Sistema Digestório/metabolismo , Neoplasias do Sistema Digestório/patologia , Peixe-Zebra/metabolismo , Adenoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Animais Geneticamente Modificados , Neoplasias do Sistema Digestório/genética , Neoplasias do Sistema Digestório/veterinária , Regulação Neoplásica da Expressão Gênica , Peixe-Zebra/genéticaRESUMO
Little is known about the signaling mechanisms that determine the highly regular patterning of the intestinal epithelium into crypts and villi. With the use of mouse models, we show that bone morphogenetic protein (BMP)-4 expression occurs exclusively in the intravillus mesenchyme. Villus epithelial cells respond to the BMP signal. Inhibition of BMP signaling by transgenic expression of noggin results in the formation of numerous ectopic crypt units perpendicular to the crypt-villus axis. These changes phenocopy the intestinal histopathology of patients with the cancer predisposition syndrome juvenile polyposis (JP), including the frequent occurrence of intraepithelial neoplasia. Many JP cases are known to harbor mutations in BMP pathway genes. These data indicate that intestinal BMP signaling represses de novo crypt formation and polyp growth.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Mucosa Intestinal/crescimento & desenvolvimento , Polipose Intestinal/patologia , Intestino Delgado/crescimento & desenvolvimento , Transdução de Sinais , Adenoma/patologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte , Diferenciação Celular , Proteínas do Citoesqueleto/metabolismo , Mucosa Intestinal/embriologia , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/patologia , Polipose Intestinal/metabolismo , Intestino Delgado/embriologia , Intestino Delgado/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas/genética , Proteínas/metabolismo , Transativadores/metabolismo , Xenopus , Proteínas de Xenopus , beta CateninaRESUMO
The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.