RESUMO
Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.
Assuntos
Desenvolvimento Embrionário/genética , Proteínas de Ligação a RNA/genética , Animais , Diferenciação Celular/genética , Autorrenovação Celular/genética , Regulação da Expressão Gênica , Mamíferos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.
Assuntos
Diferenciação Celular , Células Epidérmicas/citologia , Quinase I-kappa B , Queratinócitos , Proteínas/metabolismo , Animais , Epiderme , Quinase I-kappa B/genética , Camundongos , UbiquitinaçãoRESUMO
Cilia in most vertebrate left-right organizers are involved in the original break in left-right (L-R) symmetry, however, less is known about their roles in subsequent steps of the cascade - relaying the signaling and maintaining the established asymmetry. Here we describe the L-R patterning cascades in two mutants of a ciliary transition zone protein TMEM107, revealing that near-complete loss of cilia in Tmem107null leads to left pulmonary isomerism due to the failure of the midline barrier. Contrary, partially retained cilia in the node and the midline of a hypomorphic Tmem107schlei mutant appear sufficient for the formation of the midline barrier and establishment and maintenance of the L-R asymmetry. Despite misregulation of Shh signaling in both mutants, the presence of normal Lefty1 expression and midline barrier formation in Tmem107schlei mutants, suggests a requirement for cilia, but not necessarily Shh signaling for Lefty1 expression and midline barrier formation.
Assuntos
Cílios , Síndrome de Heterotaxia , Pulmão , Proteínas de Membrana/deficiência , Transdução de Sinais , Animais , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Síndrome de Heterotaxia/embriologia , Síndrome de Heterotaxia/genética , Síndrome de Heterotaxia/patologia , Pulmão/embriologia , Pulmão/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of Pitx1, which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between Pitx1 and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in Pitx1 regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, Pitx1 expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the Pitx1 promoter and potentially redundant enhancers. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1-/- mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.
Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , Fatores de Transcrição Box Pareados/metabolismo , Animais , Elementos Facilitadores Genéticos/genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Fenótipo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cilia are key regulators of animal development and depend on intraflagellar transport (IFT) proteins for their formation and function, yet the roles of individual IFT proteins remain unclear. We examined the Ift56hop mouse mutant and reveal novel insight into the function of IFT56, a poorly understood IFTB protein. Ift56hop mice have normal cilia distribution but display defective cilia structure, including abnormal positioning and number of ciliary microtubule doublets. We show that Ift56hop cilia are unable to accumulate Gli proteins efficiently, resulting in developmental patterning defects in Shh signaling-dependent tissues such as the limb and neural tube. Strikingly, core IFTB proteins are unable to accumulate normally within Ift56hop cilia, including IFT88, IFT81 and IFT27, which are crucial for key processes such as tubulin transport and Shh signaling. IFT56 is required specifically for the IFTB complex, as IFTA components and proteins that rely on IFTA function are unaffected in Ift56hop cilia. These studies define a distinct and novel role for IFT56 in IFTB complex integrity that is crucial for cilia structure and function and, ultimately, animal development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Padronização Corporal , Cílios/metabolismo , Microtúbulos/metabolismo , Vertebrados/embriologia , Vertebrados/metabolismo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Cílios/ultraestrutura , Códon sem Sentido/genética , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/ultraestrutura , Tubo Neural/metabolismo , Fenótipo , Polidactilia/patologia , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
During development and homeostasis, precise control of Wnt/ß-catenin signaling is in part achieved by secreted and membrane proteins that negatively control activity of the Wnt co-receptors Lrp5 and Lrp6. Lrp4 is related to Lrp5/6 and is implicated in modulation of Wnt/ß-catenin signaling, presumably through its ability to bind to the Wise (Sostdc1)/sclerostin (Sost) family of Wnt antagonists. To gain insights into the molecular mechanisms of Lrp4 function in modulating Wnt signaling, we performed an array of genetic analyses in murine tooth development, where Lrp4 and Wise play important roles. We provide genetic evidence that Lrp4 mediates the Wnt inhibitory function of Wise and also modulates Wnt/ß-catenin signaling independently of Wise. Chimeric receptor analyses raise the possibility that the Lrp4 extracellular domain interacts with Wnt ligands, as well as the Wnt antagonists. Diverse modes of Lrp4 function are supported by severe tooth phenotypes of mice carrying a human mutation known to abolish Lrp4 binding to Sost. Our data suggest a model whereby Lrp4 modulates Wnt/ß-catenin signaling via interaction with Wnt ligands and antagonists in a context-dependent manner.
Assuntos
Receptores de LDL/metabolismo , Dente/embriologia , Dente/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/deficiência , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Mutantes , Receptores de LDL/deficiência , Receptores de LDL/genética , Dente/patologia , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genéticaRESUMO
The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.
Assuntos
Agrina/fisiologia , Junção Neuromuscular/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Agrina/deficiência , Agrina/genética , Processamento Alternativo , Animais , Diafragma/embriologia , Diafragma/crescimento & desenvolvimento , Ativação Enzimática , Feminino , Proteínas Relacionadas a Receptor de LDL , Longevidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/fisiologia , Junção Neuromuscular/embriologia , Junção Neuromuscular/crescimento & desenvolvimento , Doenças da Junção Neuromuscular/enzimologia , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/fisiopatologia , Fosforilação , Densidade Pós-Sináptica/fisiologia , Processamento de Proteína Pós-Traducional , Receptores Colinérgicos/fisiologia , Receptores de LDL/deficiência , Receptores de LDL/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Teste de Desempenho do Rota-RodRESUMO
The proximate causes of multiple human genetic syndromes (ciliopathies) are disruptions in the formation or function of the cilium, an organelle required for a multitude of developmental processes. We previously identified Tmem107 as a critical regulator of cilia formation and embryonic organ development in the mouse. Here, we describe a patient with a mutation in TMEM107 that developed atypical Orofaciodigital syndrome (OFD), and show that the OFD patient shares several morphological features with the Tmem107 mutant mouse including polydactyly and reduced numbers of ciliated cells. We show that TMEM107 appears to function within cilia to regulate protein content, as key ciliary proteins do not localize normally in cilia derived from the Tmem107 mouse mutant and the human patient. These data indicate that TMEM107 plays a key, conserved role in regulating ciliary protein composition, and is a novel candidate for ciliopathies of unknown etiology.
Assuntos
Cílios/genética , Proteínas de Membrana/genética , Mutação , Síndromes Orofaciodigitais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Pré-Escolar , Cílios/patologia , Exoma , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Síndromes Orofaciodigitais/diagnóstico , Síndromes Orofaciodigitais/mortalidade , Cultura Primária de Células , Alinhamento de SequênciaRESUMO
The future site of skin appendage development is marked by a placode during embryogenesis. Although Wnt/ß-catenin signaling is known to be essential for skin appendage development, it is unclear which cellular processes are controlled by the signaling and how the precise level of the signaling activity is achieved during placode formation. We have investigated roles for Lrp4 and its potential ligand Wise (Sostdc1) in mammary and other skin appendage placodes. Lrp4 mutant mice displayed a delay in placode initiation and changes in distribution and number of mammary precursor cells leading to abnormal morphology, number and position of mammary placodes. These Lrp4 mammary defects, as well as limb defects, were associated with elevated Wnt/ß-catenin signaling and were rescued by reducing the dose of the Wnt co-receptor genes Lrp5 and Lrp6, or by inactivating the gene encoding ß-catenin. Wise-null mice phenocopied a subset of the Lrp4 mammary defects and Wise overexpression reduced the number of mammary precursor cells. Genetic epistasis analyses suggest that Wise requires Lrp4 to exert its function and that, together, they have a role in limiting mammary fate, but Lrp4 has an early Wise-independent role in facilitating placode formation. Lrp4 and Wise mutants also share defects in vibrissa and hair follicle development, suggesting that the roles played by Lrp4 and Wise are common to skin appendages. Our study presents genetic evidence for interplay between Lrp4 and Wise in inhibiting Wnt/ß-catenin signaling and provides an insight into how modulation of Wnt/ß-catenin signaling controls cellular processes important for skin placode formation.
Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Glândulas Mamárias Animais/embriologia , Receptores de LDL/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Epistasia Genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Mapeamento de Interação de Proteínas , Receptores de LDL/genética , Pele/embriologia , Pele/metabolismo , Pele/patologia , Vibrissas/metabolismo , Vibrissas/patologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The long bones of the vertebrate body are built by the initial formation of a cartilage template that is later replaced by mineralized bone. The proliferation and maturation of the skeletal precursor cells (chondrocytes) within the cartilage template and their replacement by bone is a highly coordinated process which, if misregulated, can lead to a number of defects including dwarfism and other skeletal deformities. This is exemplified by the fact that abnormal bone development is one of the most common types of human birth defects. Yet, many of the factors that initiate and regulate chondrocyte maturation are not known. We identified a recessive dwarf mouse mutant (pug) from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. pug mutant skeletal elements are patterned normally during development, but display a ~20% length reduction compared to wild-type embryos. We show that the pug mutation does not lead to changes in chondrocyte proliferation but instead promotes premature maturation and early ossification, which ultimately leads to disproportionate dwarfism. Using sequence capture and high-throughput sequencing, we identified a missense mutation in the Xylosyltransferase 1 (Xylt1) gene in pug mutants. Xylosyltransferases catalyze the initial step in glycosaminoglycan (GAG) chain addition to proteoglycan core proteins, and these modifications are essential for normal proteoglycan function. We show that the pug mutation disrupts Xylt1 activity and subcellular localization, leading to a reduction in GAG chains in pug mutants. The pug mutant serves as a novel model for mammalian dwarfism and identifies a key role for proteoglycan modification in the initiation of chondrocyte maturation.
Assuntos
Osso e Ossos/embriologia , Condrócitos/metabolismo , Osteogênese/genética , Pentosiltransferases/fisiologia , Animais , Sequência de Bases , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Nanismo/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Pentosiltransferases/genética , Análise de Sequência de DNA , Transdução de Sinais/genética , UDP Xilose-Proteína XilosiltransferaseRESUMO
Cell fate decisions during embryogenesis and adult life govern tissue formation, homeostasis and repair. Two key decisions that must be tightly coordinated are proliferation and differentiation. Overproliferation can lead to hyperplasia or tumor formation while premature differentiation can result in a depletion of proliferating cells and organ failure. Maintaining this balance is especially important in tissues that undergo rapid turnover like skin however, despite recent advances, the genetic mechanisms that balance cell differentiation and proliferation are still unclear. In an unbiased genetic screen to identify genes affecting early development, we identified an essential regulator of the proliferation-differentiation balance in epidermal progenitor cells, the Keratinocyte differentiation factor 1 (Kdf1; 1810019J16Rik) gene. Kdf1 is expressed in epidermal cells from early stages of epidermis formation through adulthood. Specifically, Kdf1 is expressed both in epidermal progenitor cells where it acts to curb the rate of proliferation as well as in their progeny where it is required to block proliferation and promote differentiation. Consequently, Kdf1 mutants display both uncontrolled cell proliferation in the epidermis and failure to develop terminal fates. Our findings reveal a dual role for the novel gene Kdf1 both as a repressive signal for progenitor cell proliferation through its inhibition of p63 and a strong inductive signal for terminal differentiation through its interaction with the cell cycle regulator Stratifin.
Assuntos
Diferenciação Celular , Epiderme/patologia , Proteínas/metabolismo , Células-Tronco/metabolismo , Proteínas 14-3-3/metabolismo , Alelos , Animais , Sequência de Bases , Diferenciação Celular/genética , Proliferação de Células , Fissura Palatina/embriologia , Fissura Palatina/genética , Fissura Palatina/patologia , Perda do Embrião/genética , Perda do Embrião/patologia , Epiderme/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Recessivos/genética , Teste de Complementação Genética , Heterozigoto , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Fosfoproteínas/metabolismo , Mutação Puntual/genética , Ligação Proteica , Proteínas/genética , Splicing de RNA/genética , Transativadores/metabolismoRESUMO
Cilia are dynamic organelles that are essential for a vast array of developmental patterning events, including left-right specification, skeletal formation, neural development, and organogenesis. Despite recent advances in understanding cilia form and function, many key ciliogenesis components have yet to be identified. By using a forward genetics approach, we isolated a novel mutant allele (schlei) of the mouse Transmembrane protein 107 (Tmem107) gene, which we show here is critical for cilia formation and embryonic patterning. Tmem107 is required for normal Sonic hedgehog (Shh) signaling in the neural tube and acts in combination with Gli2 and Gli3 to pattern ventral and intermediate neuronal cell types. schlei mutants also form extra digits, and we demonstrate that Tmem107 acts in the Shh pathway to determine digit number, but not identity, by regulating a subset of Shh target genes. Phenotypically, schlei mutants share several features with other cilia mutants; however, spatial restriction of mutant phenotypes and lack of left-right patterning defects in schlei animals suggest differential requirements for Tmem107 in cilia formation in distinct tissues. Also, in contrast to mutants with complete loss of cilia, schlei mutants retain some function of both Gli activator and repressor forms. Together, these studies identify a previously unknown regulator of ciliogenesis and provide insight into how ciliary factors affect Shh signaling and cilia biogenesis in distinct tissues.
Assuntos
Cílios/genética , Embrião de Mamíferos/metabolismo , Proteínas Hedgehog/genética , Proteínas de Membrana/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Extremidades/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Tubo Neural/embriologia , Tubo Neural/metabolismo , Tubo Neural/ultraestrutura , Homologia de Sequência de Aminoácidos , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Tetrapod limbs display a great variety of forms and functions. In an attempt to understand factors influencing this diversity, Cretekos and colleagues, in a recent issue of Genes & Development, provide molecular insight into the evolution of wing morphology in bats.
Assuntos
Evolução Biológica , Extremidades/anatomia & histologia , Voo Animal , Mamíferos/anatomia & histologia , Animais , Quirópteros/anatomia & histologia , Membro Anterior/anatomia & histologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Camundongos , Sequências Reguladoras de Ácido NucleicoRESUMO
Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.
Assuntos
Transtornos da Motilidade Ciliar , Doenças Renais Policísticas , Retinose Pigmentar , Feminino , Gravidez , Humanos , Camundongos , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Retina/metabolismo , Doenças Renais Policísticas/genética , Retinose Pigmentar/metabolismo , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismoRESUMO
Meckel syndrome (MKS) is a rare autosomal recessive disease causing perinatal lethality associated with a complex syndrome that includes occipital meningoencephalocele, hepatic biliary ductal plate malformation, postaxial polydactyly and polycystic kidneys. The gene mutated in type 1 MKS encodes a protein associated with the base of the cilium in vertebrates and nematodes. However, shRNA knockdown studies in cell culture have reported conflicting results on the role of Mks1 in ciliogenesis. Here we show that loss of function of mouse Mks1 results in an accurate model of human MKS, with structural abnormalities in the neural tube, biliary duct, limb patterning, bone development and the kidney that mirror the human syndrome. In contrast to cell culture studies, loss of Mks1 in vivo does not interfere with apical localization of epithelial basal bodies but rather leads to defective cilia formation in most, but not all, tissues. Analysis of patterning in the neural tube and the limb demonstrates altered Hedgehog (Hh) pathway signaling underlies some MKS defects, although both tissues show an expansion of the domain of response to Shh signaling, unlike the phenotypes seen in other mutants with cilia loss. Other defects in the skull, lung, rib cage and long bones are likely to be the result of the disruption of Hh signaling, and the basis of defects in the liver and kidney require further analysis. Thus the disruption of Hh signaling can explain many, but not all, of the defects caused by loss of Mks1.
Assuntos
Anormalidades Múltiplas/metabolismo , Cílios/fisiologia , Modelos Animais de Doenças , Proteínas Hedgehog/metabolismo , Camundongos , Proteínas/metabolismo , Transdução de Sinais , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/fisiopatologia , Animais , Cílios/genética , Feminino , Técnicas de Silenciamento de Genes , Proteínas Hedgehog/genética , Humanos , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas/genéticaRESUMO
The murine embryonic blood-brain barrier (BBB) consists of endothelial cells (ECs), pericytes (PCs), and basement membrane. Although PCs are critical for inducing vascular stability, signaling pathways in PCs that regulate EC morphogenesis during BBB development remain unexplored. Herein, we find that murine embryos lacking the transforming growth factor ß (TGF-ß) receptor activin receptor-like kinase 5 (Alk5) in brain PCs (mutants) develop gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH). The germinal matrix (GM) is a highly vascularized structure rich in neuronal and glial precursors. We show that GM microvessels of mutants display abnormal dilation, reduced PC coverage, EC hyperproliferation, reduced basement membrane collagen, and enhanced perivascular matrix metalloproteinase activity. Furthermore, ALK5-depleted PCs downregulate tissue inhibitor of matrix metalloproteinase 3 (TIMP3), and TIMP3 administration to mutants improves endothelial morphogenesis and attenuates GMH-IVH. Overall, our findings reveal a key role for PC ALK5 in regulating brain endothelial morphogenesis and a substantial therapeutic potential for TIMP3 during GMH-IVH.
Assuntos
Encéfalo/patologia , Embrião de Mamíferos/patologia , Endotélio Vascular/patologia , Hemorragias Intracranianas/patologia , Pericitos/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Barreira Hematoencefálica , Encéfalo/metabolismo , Embrião de Mamíferos/metabolismo , Endotélio Vascular/metabolismo , Feminino , Humanos , Hemorragias Intracranianas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese/fisiologia , Pericitos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
The vertebrate axial skeleton comprises regions of specialized vertebrae, which vary in length between lineages. Aires et al. (2016) uncover a key role for Oct4 in determining trunk length in mice. Additionally, a heterochronic shift in Oct4 expression may underlie the extreme elongation of the trunk in snakes.
Assuntos
Evolução Biológica , Osso e Ossos/metabolismo , Camundongos/anatomia & histologia , Fatores de Transcrição de Octâmero/metabolismo , Serpentes/anatomia & histologia , Tronco/anatomia & histologia , Animais , Camundongos/metabolismo , Serpentes/metabolismoRESUMO
Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes.