RESUMEN
The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.
Asunto(s)
Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Mesodermo , Factores de Transcripción , Animales , Ratones , Diferenciación Celular , Cromatina/metabolismo , Expresión Génica , Mesodermo/metabolismo , Somitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Esbozos de los Miembros , Animales , Ratones , Adenosina Trifosfato/metabolismo , Glucosa/metabolismo , Glucólisis/genética , Esbozos de los Miembros/metabolismo , Ratones NoqueadosRESUMEN
Endolichenic fungi are expecting for new bioresources of pharmacological compounds. However, the number of investigations targeting antioxidant compounds produced by endolichenic fungi remains limited. To discover new antioxidant compounds, we analyzed the antioxidant activity of the methanol extracts derived from isolated lichen mycobionts or endolichenic fungi induced from Pyxine subcinerea. We performed this analysis using the oxygen radical absorbance capacity (ORAC) method. As a result, we isolated from an endolichenic fungus identified as Penicillium sp.-stain 1322P in Pyxine subcinerea. This fungus produced a red pigment, and its chemical structure was determined to be sclerotioramine based on the analytical data obtained from NMR, LC-MS/MS, and HPLC-PDA. Sclerotioramine exhibited high antioxidant activity, and the ORAC values (mean ± SD) of sclerotioramine and sclerotiorin were 11.4 ± 0.36 and 4.86 ± 0.70 mmol TE per gram of the respective pure compound. Thus, the antioxidant activity of sclerotioramine was greater than twice that of sclerotiorin. This work represents the first report that the antioxidant activity of sclerotioramine is higher than that of the sclerotiorin.
Asunto(s)
Ascomicetos , Penicillium , Antioxidantes/farmacología , Cromatografía Liquida , Espectrometría de Masas en Tándem , Ascomicetos/química , Penicillium/químicaRESUMEN
The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common preference for the AATA tetranucleotide shared by ZFC4 of SALL1, SALL3, and SALL4. Furthermore, our cell biology experiments demonstrate that the DNA-binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family proteins in controlling cell fate via preferential targeting to AT-rich sites within genomic DNA during cell differentiation.
Asunto(s)
Proteínas de Unión al ADN , Factores de Transcripción , Animales , Ratones , ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc , Nucleótidos/químicaRESUMEN
While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.
Asunto(s)
gamma-Glutamilciclotransferasa , Animales , Humanos , Ratones , gamma-Glutamilciclotransferasa/química , Células MCF-7 , Colorantes Fluorescentes/químicaRESUMEN
Zebrafish have a remarkable ability to regenerate the myocardium after injury by proliferation of pre-existing cardiomyocytes. Fibroblast growth factor (FGF) signaling is known to play a critical role in zebrafish heart regeneration through promotion of neovascularization of the regenerating myocardium. Here, we define an additional function of FGF signaling in the zebrafish myocardium after injury. We find that FGF signaling is active in a small fraction of cardiomyocytes before injury, and that the number of FGF signaling-positive cardiomyocytes increases after amputation-induced injury. We show that ERK phosphorylation is prominent in endothelial cells, but not in cardiomyocytes. In contrast, basal levels of phospho-AKT positive cardiomyocytes are detected before injury, and the ratio of phosphorylated AKT-positive cardiomyocytes increases after injury, indicating a role of AKT signaling in cardiomyocytes following injury. Inhibition of FGF signaling reduced the number of phosphorylated AKT-positive cardiomyocytes and increased cardiomyocyte death without injury. Heart injury did not induce cardiomyocyte death; however, heart injury in combination with inhibition of FGF signaling caused significant increase in cardiomyocyte death. Pharmacological inhibition of AKT signaling after heart injury also caused increased cardiomyocyte death. Our data support the idea that FGF-AKT signaling-dependent cardiomyocyte survival is necessary for subsequent heart regeneration.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regeneración/genética , Transducción de Señal/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cromonas/farmacología , Factores de Crecimiento de Fibroblastos/genética , Lesiones Cardíacas/metabolismo , Morfolinas/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Regeneración/efectos de los fármacosRESUMEN
Bi-potential neuromesodermal progenitors (NMPs) produce both neural and paraxial mesodermal progenitors in the trunk and tail during vertebrate body elongation. We show that Sall4, a pluripotency-related transcription factor gene, has multiple roles in regulating NMPs and their descendants in post-gastrulation mouse embryos. Sall4 deletion using TCre caused body/tail truncation, reminiscent of early depletion of NMPs, suggesting a role of Sall4 in NMP maintenance. This phenotype became significant at the time of the trunk-to-tail transition, suggesting that Sall4 maintenance of NMPs enables tail formation. Sall4 mutants exhibit expanded neural and reduced mesodermal tissues, indicating a role of Sall4 in NMP differentiation balance. Mechanistically, we show that Sall4 promotion of WNT/ß-catenin signaling contributes to NMP maintenance and differentiation balance. RNA-Seq and SALL4 ChIP-Seq analyses support the notion that Sall4 regulates both mesodermal and neural development. Furthermore, in the mesodermal compartment, genes regulating presomitic mesoderm differentiation are downregulated in Sall4 mutants. In the neural compartment, we show that differentiation of NMPs towards post-mitotic neuron is accelerated in Sall4 mutants. Our results collectively provide evidence supporting the role of Sall4 in regulating NMPs and their descendants.
Asunto(s)
Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Proteínas de Unión al ADN/fisiología , Mesodermo/citología , Mesodermo/embriología , Células-Madre Neurales/citología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular/genética , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Mesodermo/metabolismo , Ratones , Células-Madre Neurales/fisiología , Embarazo , Vía de Señalización Wnt/fisiologíaRESUMEN
'Gain' of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent 'gene desert' of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Amplificación de Genes/genética , Dosificación de Gen/genética , Genes myc/genética , Proteína Oncogénica p55(v-myc)/genética , ARN Largo no Codificante/genética , Animales , Transformación Celular Neoplásica , Cromosomas Humanos Par 8/genética , Modelos Animales de Enfermedad , Células HCT116 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína Oncogénica p55(v-myc)/metabolismo , FenotipoRESUMEN
Isl1 is required for two processes during hindlimb development: initiation of the processes directing hindlimb development in the lateral plate mesoderm and configuring posterior hindlimb field in the nascent hindlimb buds. During these processes, Isl1 expression is restricted to the posterior mesenchyme of hindlimb buds. How this dynamic change in Isl1 expression is regulated remains unknown. We found that two evolutionarily conserved sequences, located 3' to the Isl1 gene, regulate LacZ transgene expression in the hindlimb-forming region in mouse embryos. Both sequences contain GATA binding motifs, and expression pattern analysis identified that Gata6 is expressed in the flank and the anterior portion of nascent hindlimb buds. Recent studies have shown that conditional inactivation of Gata6 in mice causes hindlimb-specific pre-axial polydactyly, indicating a role of Gata6 in anterior-posterior patterning of hindlimbs. We studied whether Gata6 restricts Isl1 in the nascent hindlimb bud through the cis-regulatory modules. In vitro experiments demonstrate that GATA6 binds to the conserved GATA motifs in the cis-regulatory modules. GATA6 repressed expression of a luciferase reporter that contains the cis-regulatory modules by synergizing with Zfpm2. Analyses of Gata6 mutant embryos showed that ISL1 levels are higher in the anterior of nascent hindlimb buds than in wild type. Moreover, we detected a greater number of Isl1-transcribing cells in the anterior of nascent hindlimb buds in Gata6 mutants. Our results support a model in which Gata6 contributes to repression of Isl1 expression in the anterior of nascent hindlimb buds.
Asunto(s)
Embrión de Mamíferos/embriología , Factor de Transcripción GATA6/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Miembro Posterior/embriología , Proteínas con Homeodominio LIM/biosíntesis , Modelos Biológicos , Motivos de Nucleótidos , Factores de Transcripción/biosíntesis , Animales , Embrión de Mamíferos/citología , Factor de Transcripción GATA6/genética , Miembro Posterior/citología , Proteínas con Homeodominio LIM/genética , Ratones , Ratones Transgénicos , Factores de Transcripción/genéticaRESUMEN
Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone. Abbreviations: AAPH: 2,2'-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography.
Asunto(s)
Antioxidantes/farmacología , Ascomicetos/metabolismo , Líquenes/microbiología , Xantonas/metabolismo , Xantonas/farmacología , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Capa Delgada/métodos , Análisis Espectral/métodos , Xantonas/aislamiento & purificaciónRESUMEN
Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod.
Asunto(s)
Extremidades/crecimiento & desarrollo , Factor de Transcripción GATA6/genética , Regulación del Desarrollo de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas del Tejido Nervioso/genética , Organogénesis/genética , Células Madre/metabolismo , Animales , Tipificación del Cuerpo/genética , Línea Celular , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Esbozos de los Miembros/crecimiento & desarrollo , Esbozos de los Miembros/metabolismo , Ratones , Células 3T3 NIH , Polidactilia/genética , Transducción de Señal/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Proteína Gli3 con Dedos de ZincRESUMEN
BACKGROUND: Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. RESULTS: By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. CONCLUSIONS: The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/genética , Elementos Reguladores de la Transcripción/genética , Animales , Encéfalo/metabolismo , Células Cultivadas , Embrión de Pollo , Secuencia Conservada/genética , Electroporación/métodos , Embrión no Mamífero , Esbozos de los Miembros/metabolismo , Mesodermo/citología , Regiones Promotoras GenéticasRESUMEN
Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.
Asunto(s)
Huesos/embriología , Proteínas de Unión al ADN/metabolismo , Miembro Anterior/embriología , Miembro Posterior/embriología , Factores de Transcripción de Tipo Kruppel/metabolismo , Esbozos de los Miembros/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo , Huesos/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/genética , Epistasis Genética , Miembro Anterior/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Miembro Posterior/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Proteína Gli3 con Dedos de ZincRESUMEN
A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAß1-3Xylα1]n-3GlcAß1-4Xyl-Rbo5P-1Rbo5P-3GalNAcß1-3GlcNAcß1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylß1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (ß1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAß1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate ß1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan.
Asunto(s)
Distroglicanos/metabolismo , Proteínas de la Membrana/metabolismo , Distrofias Musculares/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Línea Celular , Distroglicanos/química , Distroglicanos/genética , Glicosilación , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Distrofias Musculares/genética , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Pentosiltransferasa , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1âß-cateninâFgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.
Asunto(s)
Miembro Posterior/embriología , Anomalías Maxilomandibulares/embriología , Proteínas con Homeodominio LIM/metabolismo , Osteogénesis/genética , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Animales , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Región Branquial/embriología , Linaje de la Célula/genética , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Fosfatasa 6 de Especificidad Dual/biosíntesis , Embrión de Mamíferos/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Factor 8 de Crecimiento de Fibroblastos/biosíntesis , Factor 8 de Crecimiento de Fibroblastos/deficiencia , Factor 8 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Miembro Posterior/anomalías , Proteínas de Homeodominio/biosíntesis , Anomalías Maxilomandibulares/genética , Factores de Transcripción de Tipo Kruppel/biosíntesis , Proteínas con Homeodominio LIM/genética , Mandíbula/embriología , Mesodermo/embriología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Transducción de Señal/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Regulación hacia Arriba , Proteína Gli3 con Dedos de Zinc , beta Catenina/genéticaRESUMEN
Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.
Asunto(s)
Quimiocina CXCL12/biosíntesis , Corazón/fisiología , Miocitos Cardíacos/fisiología , Receptores CXCR4/biosíntesis , Regeneración , Proteínas de Pez Cebra/biosíntesis , Pez Cebra , Animales , Animales Modificados Genéticamente , Movimiento Celular , Proliferación Celular , Quimiocina CXCL12/genética , Lesiones Cardíacas/fisiopatología , Ventrículos Cardíacos , Miocardio/metabolismo , Receptores CXCR4/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
How divergent genetic systems regulate a common pathway during the development of two serial structures, forelimbs and hindlimbs, is not well understood. Specifically, HAND2 has been shown to regulate Shh directly to initiate its expression in the posterior margin of the limb mesenchyme. Although the Hand2-Shh morphoregulatory system operates in both the forelimb and hindlimb bud, a recent analysis suggested that its upstream regulation is different in the forelimb and hindlimb bud. A combination of all four Hox9 genes is required for Hand2 expression in the forelimb-forming region; however, it remains elusive what genetic system regulates the Hand2-Shh pathway in the hindlimb-forming region. By conditional inactivation of Islet1 in the hindlimb-forming region using the Hoxb6Cre transgene, we show that Islet1 is required for establishing the posterior hindlimb field, but not the forelimb field, upstream of the Hand2-Shh pathway. Inactivation of Islet1 caused the loss of posterior structures in the distal and proximal regions, specifically in the hindlimb. We found that Hand2 expression was downregulated in the hindlimb field and that Shh expression was severely impaired in the hindlimb bud. In the Hoxb6Cre; Islet1 mutant pelvis, the proximal element that is formed in a Shh-independent manner, displayed complementary defects in comparison with Pitx1(-/-) hindlimbs. This suggests that Islet1 and Pitx1 function in parallel during girdle development in hindlimbs, which is in contrast with the known requirement for Tbx5 in girdle development in forelimbs. Our studies have identified a role for Islet1 in hindlimb-specific development and have revealed Islet1 functions in two distinct processes: regulation upstream of the Hand2-Shh pathway and contributions to girdle development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Proteínas Hedgehog/metabolismo , Miembro Posterior/embriología , Proteínas con Homeodominio LIM/metabolismo , Pelvis/embriología , Factores de Transcripción/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Hibridación in Situ , Proteínas con Homeodominio LIM/genética , Ratones , Ratones Noqueados , Microscopía Confocal , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The zebrafish heart regenerates after various severe injuries. Common processes of heart regeneration are cardiomyocyte proliferation, activation of epicardial tissue, and neovascularization. In order to further characterize heart regeneration processes, we introduced milder injuries and compared responses to those induced by ventricular apex resection, a widely used injury method. We used scratching of the ventricular surface and puncturing of the ventricle with a fine tungsten needle as injury-inducing techniques. RESULTS: Scratching the ventricular surface induced subtle cardiomyocyte proliferation and responses of the epicardium. Endothelial cell accumulation was limited to the surface of the heart. Ventricular puncture induced cardiomyocyte proliferation, endocardial and epicardial activation, and neo-vascularization, similar to the resection method. However, the degree of the responses was milder, correlating with milder injury. Sham operation induced epicardial aldh1a2 expression but not tbx18 and WT1. CONCLUSIONS: Puncturing the ventricle induces responses equivalent to resection at milder degrees in a shorter time frame and can be used as a simple injury model. Scratching the ventricle did not induce heart regeneration and can be used for studying wound responses to epicardium.
Asunto(s)
Proliferación Celular/fisiología , Lesiones Cardíacas/fisiopatología , Ventrículos Cardíacos/fisiopatología , Miocitos Cardíacos/fisiología , Regeneración/fisiología , Pez Cebra/fisiología , Animales , Células Endoteliales/metabolismo , Ventrículos Cardíacos/cirugía , Técnicas Histológicas , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Retinal-Deshidrogenasa/metabolismo , Proteínas de Dominio T Box/metabolismo , Proteínas WT1/metabolismo , Cicatrización de Heridas/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
The transcriptional basis of vertebrate limb initiation, which is a well-studied system for the initiation of organogenesis, remains elusive. Specifically, involvement of the ß-catenin pathway in limb initiation, as well as its role in hindlimb-specific transcriptional regulation, are under debate. Here, we show that the ß-catenin pathway is active in the limb-forming area in mouse embryos. Furthermore, conditional inactivation of ß-catenin as well as Islet1, a hindlimb-specific factor, in the lateral plate mesoderm results in a failure to induce hindlimb outgrowth. We further show that Islet1 is required for the nuclear accumulation of ß-catenin and hence for activation of the ß-catenin pathway, and that the ß-catenin pathway maintains Islet1 expression. These two factors influence each other and function upstream of active proliferation of hindlimb progenitors in the lateral plate mesoderm and the expression of a common factor, Fgf10. Our data demonstrate that Islet1 and ß-catenin regulate outgrowth and Fgf10-Fgf8 feedback loop formation during vertebrate hindlimb initiation. Our study identifies Islet1 as a hindlimb-specific transcriptional regulator of initiation, and clarifies the controversy regarding the requirement of ß-catenin for limb initiation.
Asunto(s)
Miembro Posterior/embriología , Miembro Posterior/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Animales , Retroalimentación Fisiológica , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Proteínas con Homeodominio LIM/genética , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Mutantes , Ratones Transgénicos , Transducción de Señal , Factores de Transcripción/genética , beta Catenina/genéticaRESUMEN
Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.