RESUMO
Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Dysfunction of apoptosis is involved in many fatal diseases such as cancer. Visualization of apoptosis in living animals is necessary to understand the mechanism of apoptosis-related diseases. Here, we describe a genetically encoded fluorescent probe for imaging apoptosis in living multicellular organisms, based on spontaneous complementation of two fragments of a green fluorescent protein (GFP) variant (GFP OPT). The probe is designed for detection of mitochondria-mediated apoptosis during which a mitochondrial protein of Smac is released into cytosol. The Smac is connected with a carboxy-terminal fragment of GFP OPT (GFP11), whereas the remainder of GFP OPT (GFP(1-10)) is located in the cytosol. Under an apoptotic condition, the Smac is released from mitochondria into cytosol, allowing complementation of the GFP-OPT fragments and the emission of fluorescence. Live-cell imaging demonstrates that the probe enables detection of apoptosis in living cells with a high signal-to-background ratio. We applied the probe to living zebrafish, in which apoptotic cells were visualized with fluorescence. The technique provides a useful tool for the study of apoptosis in living animals, facilitating elucidation of the mechanisms of apoptosis-related diseases.
Assuntos
Apoptose/genética , Corantes Fluorescentes/metabolismo , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Animais , Sobrevivência Celular , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
The precise government of the left-right (LR) specification of an organ is an essential aspect of its morphogenesis. Multiple signaling cascades have been implicated in the establishment of vertebrate LR asymmetry. Recently, mTOR signaling was found to critically regulate the development of LR asymmetry in zebrafish. However, the upstream factor(s) that activate mTOR signaling in the context of LR specification are as yet unknown. In this study, we identify the SLC7 amino acid transporters Slc7a7 and Slc7a8 as novel regulators of LR asymmetry development in the small fish medaka. Knockdown of Slc7a7 and/or Slc7a8 in medaka embryos disrupted LR organ asymmetries. Depletion of Slc7a7 hindered left-sided expression of the southpaw (spaw) gene, which is responsible for LR axis determination. Work at the cellular level revealed that Slc7a7 coordinates ciliogenesis in the epithelium of Kupffer's vesicle and thereby the generation of the nodal fluid flow required for LR asymmetry. Interestingly, knockdown of Slc7a7 depressed mTOR signaling activity in medaka embryos. Treatment with rapamycin, an inhibitor of mTOR signaling, together with Slc7a7 knockdown synergistically perturbed spaw expression, indicating an interaction between Slc7a7 and mTOR signaling affecting gene expression required for LR specification. Taken together, our results demonstrate that Slc7a7 governs the regulation of LR asymmetry development via the activation of mTOR signaling.
Assuntos
Padronização Corporal/fisiologia , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Organogênese/fisiologia , Oryzias/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Sistema y+L de Transporte de Aminoácidos , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinais/fisiologiaRESUMO
UNLABELLED: During vertebrate embryogenesis, the liver develops at a precise location along the endodermal primitive gut tube because of signaling delivered by adjacent mesodermal tissues. Although several signaling molecules have been associated with liver formation, the molecular mechanism that regulates liver specification is still unclear. We previously performed a screen in medaka to isolate mutants with impaired liver development. The medaka hio mutants exhibit a profound (but transient) defect in liver specification that resembles the liver formation defect found in zebrafish prometheus (prt) mutants, whose mutation occurs in the wnt2bb gene. In addition to their liver abnormality, hio mutants lack pectoral fins and die after hatching. Positional cloning indicated that the hio mutation affects the raldh2 gene encoding retinaldehyde dehydrogenase type2 (RALDH2), the enzyme principally responsible for retinoic acid (RA) biosynthesis. Mutations of raldh2 in zebrafish preclude the development of pectoral fins. Interestingly, in hio mutants, expression of wnt2bb in the lateral plate mesoderm (LPM) directly adjacent to the liver-forming endoderm was completely lost. CONCLUSION: Our data reveal the unexpected finding that RA signaling positively regulates the wnt2bb gene expression required for liver specification in medaka. These results suggest that a common molecular mechanism may underlie liver and pectoral fin specification during piscine embryogenesis.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Fígado/embriologia , Oryzias/genética , Tretinoína/fisiologia , Proteína Wnt2/genética , Animais , Transdução de SinaisRESUMO
Stress-induced Sapk/Jnk signaling is involved in cell survival and apoptosis. Recent studies have increased our understanding of the physiological roles of Jnk signaling in embryonic development. However, still unclear is the precise function of Jnk signaling during gastrulation, a critical step in the establishment of the vertebrate body plan. Here we use morpholino-mediated knockdown of the zebrafish orthologs of the Jnk activators Mkk4 and Mkk7 to examine the effect of Jnk signaling abrogation on early vertebrate embryogenesis. Depletion of zebrafish Mkk4b led to abnormal convergent extension (CE) during gastrulation, whereas Mkk7 morphants exhibited defective somitogenesis. Surprisingly, Mkk4b morphants displayed marked upregulation of wnt11, which is the triggering ligand of CE and stimulates Jnk activation via the non-canonical Wnt pathway. Conversely, ectopic activation of Jnk signaling by overexpression of an active form of Mkk4b led to wnt11 downregulation. Mosaic lineage tracing studies revealed that Mkk4b-Jnk signaling suppressed wnt11 expression in a non-cell-autonomous manner. These findings provide the first evidence that wnt11 itself is a downstream target of the Jnk cascade in the non-canonical Wnt pathway. Our work demonstrates that Jnk activation is indispensable for multiple steps during vertebrate body plan formation. Furthermore, non-canonical Wnt signaling may coordinate vertebrate CE movements by triggering Jnk activation that represses the expression of the CE-triggering ligand wnt11.
Assuntos
Gástrula , Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase 4/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer's vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.
Assuntos
Proteínas de Homeodomínio/metabolismo , Microtúbulos/fisiologia , Oryzias/embriologia , Animais , Cílios/genética , Cílios/metabolismo , Cílios/fisiologia , Clonagem Molecular , Técnicas de Silenciamento de Genes , Coração/embriologia , Proteínas de Homeodomínio/genética , Fígado/anormalidades , Microtúbulos/genética , Microtúbulos/metabolismo , Oryzias/genética , Oryzias/metabolismo , Baço/anormalidadesRESUMO
The Komeda miniature rat Ishikawa (KMI) is a spontaneous animal model of dwarfism caused by a mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII). This strain has been maintained as a segregating inbred strain for the mutated allele mri. In this study, we characterized the phenotype of the KMI strain, particularly growth traits, craniofacial measurements, and organ weights. The homozygous mutant (mri/mri) animals were approximately 70% to 80% of the size of normal, heterozygous (mri/+) animals in regard to body length, weight, and naso-occipital length of the calvarium, and the retroperitoneal fat of mri/mri rats was reduced greatly. In addition, among progeny of the (BNxKMI-mri/mri)F1xKMI-mri/mri backcross, animals with the KMI phenotype (mri/mri) were easily distinguished from those showing the wild-type phenotype (mri/+) by using growth traits such as body length and weight. Genetic analysis revealed that all of the backcrossed progeny exhibiting the KMI phenotype were homozygous for the KMI allele in the 1.2-cM region between D14Rat5 and D14Rat80 on chromosome 14, suggesting strongly that mri acts in a completely recessive manner. The KMI strain is the first and only rat model with a confirmed mutation in Prkg2 and is a valuable model for studying dwarfism and longitudinal growth traits in humans and for functional studies of cGKII.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/genética , Nanismo/genética , Mutação , Animais , Peso Corporal/genética , Mapeamento Cromossômico , Proteína Quinase Dependente de GMP Cíclico Tipo II , Nanismo/enzimologia , Nanismo/patologia , Feminino , Masculino , Tamanho do Órgão/genética , Fenótipo , Ratos , Ratos Endogâmicos BN , Ratos MutantesRESUMO
Reelin (Reln) is an extracellular matrix protein secreted from distinct neuronal populations and controls neural cell positioning during brain development. Alterations of human RELN have been reported in two pedigrees with an autosomal recessive lissencephaly. Although several alleles of the mouse reeler mutation were identified as disruptions of Reln, there is no other animal model with a confirmed mutation in Reln. We recently established the Komeda Zucker creeping (KZC) rat strain with an autosomal recessive mutation creeping (cre), showing a reeler-like phenotype. We also found that creeping was located in the genomic segment on rat chromosome 4 containing Reln and that the expression level of Reln mRNA was markedly reduced in cre/cre homozygous mutant animals. Here we report positional candidate cloning of creeping, and identify a nucleotide insertion mutation in Reln. This mutation leads to a translational frameshift and results in truncation of the predicted protein in the fourth reelin-specific repeat, removing the C-terminal region required for secretion and function of the protein. We conclude that the mutation detected here is causative and is probably a null allele. The KZC rat is the first rat model with a confirmed Reln mutation and would therefore contribute to the understanding of the Reln function.
Assuntos
Encéfalo/anormalidades , Moléculas de Adesão Celular Neuronais/deficiência , Proteínas da Matriz Extracelular/deficiência , Transtornos dos Movimentos/genética , Mutação/genética , Neurônios/metabolismo , Animais , Sequência de Bases/genética , Encéfalo/metabolismo , Encéfalo/patologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/isolamento & purificação , Mapeamento Cromossômico , Clonagem Molecular , Códon sem Sentido/genética , Análise Mutacional de DNA , DNA Complementar/análise , DNA Complementar/genética , Modelos Animais de Doenças , Éxons/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/isolamento & purificação , Genótipo , Dados de Sequência Molecular , Transtornos dos Movimentos/metabolismo , Proteínas do Tecido Nervoso , Neurônios/patologia , Fenótipo , Ratos , Ratos Endogâmicos , Proteína Reelina , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Serina EndopeptidasesRESUMO
The Komeda diabetes-prone (KDP) rat is a spontaneous animal model of human autoimmune type 1 diabetes. By positional cloning of the non-MHC major susceptibility locus lddm/kdp1, we recently identified a nonsense mutation in Cblb and also found that lymphocytes of KDP rats infiltrate into various tissues, indicating autoimmunity. The maintenance and production of KDP rats has been a critical problem owing to the poor reproductive ability of diabetic animals. To solve the problem, we here established the KDP rat as a segregating inbred strain. We first identified animals that were heterozygous at the lddm/kdp1 region in a breeding colony of KDP rats. The heterozygous region spans at least from D11Yok1 to Cblb on rat chromosome 11. By mating between the heterozygous rats, we obtained homozygotes, heterozygotes and wild-types with the expected ratio of 1:2:1 and found that only the homozygotes developed diabetes, suggesting that these genotypes represent those of lddm/kdp1. We then tried to maintain KDP rats by mating between the heterozygotes, which resulted in a segregating inbred strain. Within 210 d of age, about 80% of lddm/kdp1 homozygotes developed diabetes with severe insulitis, while neither heterozygotes nor wild-types developed diabetes. The phenotypic characteristics of the homozygotes are the same as those of progeny of diabetic parents in the original KDP rats. The segregating inbred KDP rat strain described here would serve as a useful animal model for autoimmune diseases, including type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Ratos Endogâmicos/genética , Animais , Doenças Autoimunes/genética , Códon sem Sentido , RatosRESUMO
The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap's TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1-mediated transactivation of photoreceptor cell genes during zebrafish retinogenesis.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Células Fotorreceptoras/fisiologia , Proteínas Serina-Treonina Quinases/genética , Retina/fisiologia , Células-Tronco/fisiologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , Morfogênese/fisiologia , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/fisiologia , Fosfoproteínas/genética , Pigmentação/genética , Pigmentação/fisiologia , Rodopsina , Serina-Treonina Quinase 3 , Transdução de Sinais/genética , Fatores de Transcrição/genética , Peixe-Zebra/fisiologiaRESUMO
The liver has an unusual capacity to regenerate after a loss of mass and function caused by surgical resection or toxic liver injury. Over the last 10 years there have been major advances in our understanding of the molecular and cellular mechanisms underlying liver development and regeneration. The numerous factors crucial to these phenomena have been identified mainly by using knockout mice. Forward-genetics studies using zebrafish and medaka have also generated many mutants with liver disorders or defects in liver formation. Our goal is to translate knowledge gained from laboratory work and animal models into novel therapies for human liver diseases. Exciting progress has been achieved using human partial liver transplantation and autologous cell therapy.