RESUMO
All vertebrates display a characteristic asymmetry of internal organs with the cardiac apex, stomach and spleen towards the left, and the liver and gall bladder on the right. Left-right (L-R) axis abnormalities or laterality defects are common in humans (1 in 8,500 live births). Several genes (such as Nodal, Ebaf and Pitx2) have been implicated in L-R organ positioning in model organisms. In humans, relatively few genes have been associated with a small percentage of human situs defects. These include ZIC3 (ref. 5), LEFTB (formerly LEFTY2; ref. 6) and ACVR2B (encoding activin receptor IIB; ref. 7). The EGF-CFC genes, mouse Cfc1 (encoding the Cryptic protein; ref. 9) and zebrafish one-eyed pinhead (oep; refs 10, 11) are essential for the establishment of the L-R axis. EGF-CFC proteins act as co-factors for Nodal-related signals, which have also been implicated in L-R axis development. Here we identify loss-of-function mutations in human CFC1 (encoding the CRYPTIC protein) in patients with heterotaxic phenotypes (randomized organ positioning). The mutant proteins have aberrant cellular localization in transfected cells and are functionally defective in a zebrafish oep-mutant rescue assay. Our findings indicate that the essential role of EGF-CFC genes and Nodal signalling in left-right axis formation is conserved from fish to humans. Moreover, our results support a role for environmental and/or genetic modifiers in determining the ultimate phenotype in humans.
Assuntos
Anormalidades Múltiplas/genética , Desenvolvimento Embrionário e Fetal/genética , Substâncias de Crescimento/genética , Cabeça/anormalidades , Holoprosencefalia/genética , Peptídeos e Proteínas de Sinalização Intercelular , Morfogênese/genética , Vísceras/anormalidades , Anormalidades Múltiplas/embriologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Códon/genética , Análise Mutacional de DNA , DNA Complementar/genética , Dextrocardia/embriologia , Dextrocardia/genética , Embrião não Mamífero/anormalidades , Etiquetas de Sequências Expressas , Proteínas Fetais/genética , Mutação da Fase de Leitura , Genótipo , Substâncias de Crescimento/deficiência , Cabeça/embriologia , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Fenótipo , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Situs Inversus/genética , Especificidade da Espécie , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Animals show behavioral asymmetries that are mediated by differences between the left and right sides of the brain. We report that the laterality of asymmetric development of the diencephalic habenular nuclei and the photoreceptive pineal complex is regulated by the Nodal signaling pathway and by midline tissue. Analysis of zebrafish embryos with compromised Nodal signaling reveals an early role for this pathway in the repression of asymmetrically expressed genes in the diencephalon. Later signaling mediated by the EGF-CFC protein One-eyed pinhead and the forkhead transcription factor Schmalspur is required to overcome this repression. When expression of Nodal pathway genes is either absent or symmetrical, neuroanatomical asymmetries are still established but are randomized. This indicates that Nodal signaling is not required for asymmetric development per se but is essential to determine the laterality of the asymmetry.
Assuntos
Padronização Corporal/genética , Lateralidade Funcional/genética , Proteínas Nucleares , Prosencéfalo/anatomia & histologia , Prosencéfalo/embriologia , Transdução de Sinais/genética , Proteínas de Peixe-Zebra , Animais , Diencéfalo/anatomia & histologia , Diencéfalo/embriologia , Proteínas Fetais , Regulação da Expressão Gênica no Desenvolvimento , Habenula/anatomia & histologia , Habenula/embriologia , Habenula/metabolismo , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mutagênese Sítio-Dirigida , Proteína Nodal , Fatores de Transcrição Box Pareados , Glândula Pineal/anatomia & histologia , Glândula Pineal/embriologia , Glândula Pineal/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Peixe-Zebra , Proteína Homeobox PITX2RESUMO
Idiopathic scoliosis (IS) affects 3% of children worldwide, yet the mechanisms underlying this spinal deformity remain unknown. Here we show that ptk7 mutant zebrafish, a faithful developmental model of IS, exhibit defects in ependymal cell cilia development and cerebrospinal fluid (CSF) flow. Transgenic reintroduction of Ptk7 in motile ciliated lineages prevents scoliosis in ptk7 mutants, and mutation of multiple independent cilia motility genes yields IS phenotypes. We define a finite developmental window for motile cilia in zebrafish spine morphogenesis. Notably, restoration of cilia motility after the onset of scoliosis blocks spinal curve progression. Together, our results indicate a critical role for cilia-driven CSF flow in spine development, implicate irregularities in CSF flow as an underlying biological cause of IS, and suggest that noninvasive therapeutic intervention may prevent severe scoliosis.
Assuntos
Líquido Cefalorraquidiano/fisiologia , Modelos Animais de Doenças , Escoliose/líquido cefalorraquidiano , Escoliose/fisiopatologia , Coluna Vertebral/anormalidades , Peixe-Zebra/anormalidades , Animais , Animais Geneticamente Modificados , Cílios/fisiologia , Epêndima/anormalidades , Hidrocefalia/genética , Hidrocefalia/patologia , Mutação , Receptores Proteína Tirosina Quinases/genética , Escoliose/genética , Peixe-Zebra/líquido cefalorraquidiano , Peixe-Zebra/genética , Proteínas de Peixe-ZebraRESUMO
During the development of the egg-laying system in Caenorhabditis elegans hermaphrodites, central gonadal cells organize the alignment of the vulva with the sex myoblasts, the progenitors of the egg-laying muscles. A fibroblast growth factor [EGL-17(FGF)] and an FGF receptor [EGL-15(FGFR)] are involved in the gonadal signals that guide the migrations of the sex myoblasts. Here we show that EGL-17(FGF) can act as an instructive guidance cue to direct the sex myoblasts to their final destinations. We find that egl-17 reporter constructs are expressed in the primary vulval cell and that EGL-17(FGF) expression in this cell correlates with the precise positioning of the sex myoblasts. We postulate that EGL-17(FGF) helps to coordinate the development of a functional egg-laying system, linking vulval induction with proper sex myoblast migration.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Substâncias de Crescimento/genética , Proteínas de Helminto/genética , Peptídeos e Proteínas de Sinalização Intercelular , Vulva/crescimento & desenvolvimento , Animais , Caenorhabditis elegans/citologia , Movimento Celular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , Genes Reporter , Genitália Feminina/citologia , Genitália Feminina/crescimento & desenvolvimento , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Masculino , Modelos Biológicos , Desenvolvimento Muscular , Músculos/citologia , Mutação , Células-Tronco/citologia , Vulva/citologiaRESUMO
The proper guidance of the Caenorhabditis elegans hermaphrodite sex myoblasts (SMs) requires the genes egl-15 and egl-17. egl-15 has been shown to encode the C. elegans orthologue of the fibroblast growth factor receptor (FGFR). Here we clone egl-17 and show it to be a member of the fibroblast growth factor (FGF) family, one of the first functional invertebrate FGFs known. egl-17 shares homology with other FGF members, conserving the key residues required to form the distinctive tertiary structure common to FGFs. Genetic and molecular evidence demonstrates that the SM migration defect seen in egl-17 mutant animals represents complete loss of egl-17 function. While mutations in egl-17 affect only SM migration, mutations in egl-15 can result in larval arrest, scrawny body morphology, and the ability to suppress mutations in clr-1. We propose that EGL-17 (FGF) acts as a ligand for EGL-15 (FGFR) specifically during SM migration and that another ligand(s) activates EGL-15 for its other functions.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiologia , Fatores de Crescimento de Fibroblastos/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Sequência Conservada , Primers do DNA , Transtornos do Desenvolvimento Sexual/genética , Feminino , Fatores de Crescimento de Fibroblastos/química , Teste de Complementação Genética , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/química , Dados de Sequência Molecular , Família Multigênica , Músculos/fisiologia , Oviposição , Fenótipo , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Specification of the left-right (L-R) axis in the vertebrate embryo requires transfer of positional information from the node to the periphery, resulting in asymmetric gene expression in the lateral plate mesoderm. We show that this activation of L-R lateral asymmetry requires the evolutionarily conserved activity of members of the EGF-CFC family of extracellular factors. Targeted disruption of murine Cryptic results in L-R laterality defects including randomization of abdominal situs, hyposplenia, and pulmonary right isomerism, as well as randomized embryo turning and cardiac looping. Similarly, zebrafish one-eyed pinhead (oep) mutants that have been rescued partially by mRNA injection display heterotaxia, including randomization of heart looping and pancreas location. In both Cryptic and oep mutant embryos, L-R asymmetric expression of Nodal/cyclops, Lefty2/antivin, and Pitx2 does not occur in the lateral plate mesoderm, while in Cryptic mutants Lefty1 expression is absent from the prospective floor plate. Notably, L-R asymmetric expression of Nodal at the lateral edges of the node is still observed in Cryptic mutants, indicating that L-R specification has occurred in the node but not the lateral plate. Combined with the previous finding that oep is required for nodal signaling in zebrafish, we propose that a signaling pathway mediated by Nodal and EGF-CFC activities is essential for transfer of L-R positional information from the node.