RESUMEN
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout syndrome in freshwater salmonid fish worldwide, generating injuries and high mortality rates. Despite several studies on this bacterium, the infection mechanism remains unknown due to limitations in the employed animal models. In this work, we propose using zebrafish (Danio rerio) as a model for studying bacterial pathogenicity. To substantiate this proposal, zebrafish infection by F. psychrophilum strain JIP 02/86 was characterized. Zebrafish larvae were infected using the bath method, and morphological changes and innate immune system activation were monitored using transgenic fish. Salmonid-like infection phenotypes were observed in 4.74% of treated larvae, as manifested by fin, muscle and caudal peduncle damage. Symptomatic and dead larvae accounted for 1.35% of all challenged larvae. Interestingly, infected larvae with no infection phenotypes showed stronger innate immune system activation than specimens with phenotypes. A failure of function assay for myeloid factor pu.1 resulted in more infected larvae (up to 43.5%), suggesting that low infection rates by F. psychrophilum would be due to the protective actions of the innate immune system against this bacterium in zebrafish larvae. Our results support the use of zebrafish as an infection model for studying F. psychrophilum. Furthermore, the percentage of infected fish can be modulated by disturbing, to varying extents, the differentiation of myeloid cells. Using this evidence as a starting point, different aspects of the infection mechanism of F. psychrophilum could be studied in vivo.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/fisiología , Pez Cebra , Animales , Infecciones por Flavobacteriaceae/microbiologíaRESUMEN
Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/ß-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/ß-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous ß-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/ß-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.
Asunto(s)
Trastorno del Espectro Autista/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Trastorno del Espectro Autista/fisiopatología , Células Cultivadas , Embrión de Mamíferos , Femenino , Células HEK293 , Hipocampo/fisiopatología , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-DawleyRESUMEN
Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), which cause significant worldwide losses in aquaculture. Juvenile rainbow trout are particularly susceptible to F. psychrophilum infection, the main external clinical signs of which are extensive necrotic myositis and ulcerative lesions. Despite the economic relevance of this pathogen in aquaculture, little is known about the molecular mechanisms underlying F. psychrophilum infection and pathogenesis. In this study, cultured skeletal muscle cells from rainbow trout (Oncorhynchus mykiss) were co-incubated with the virulent strain of F. psychrophilum JIP02/86 (ATCC 49511). Trypan blue exclusion analysis at 48h post-incubation revealed decreased cellular viability. Direct bacteria-myoblast contact was found a key factor in inducing F. psychrophilum cytotoxicity. Apoptosis was characterized by nuclear DNA fragmentation, decreased plasma membrane integrity, increased caspase activity, and the proteolytic cleavage of poly(ADP-ribose)polymerase-1 (PARP-1). Moreover, bacterial infection induced an early inhibition of NF-κB signaling, as well as a differential expression of the pro- and anti-apoptotic genes, bax and bcl-2. These findings suggest that F. psychrophilum induces rainbow trout muscle apoptosis through the modulation of the NF-κB signaling as a mechanism for nutrient acquisition and survival.
Asunto(s)
Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/fisiología , Oncorhynchus mykiss/microbiología , Animales , Apoptosis , Acuicultura , Supervivencia Celular , Infecciones por Flavobacteriaceae/microbiología , Músculo Esquelético/microbiología , Mioblastos/microbiologíaRESUMEN
Neutrophils play an essential role during an inflammatory response, which is dependent on their rapid recruitment from the bone marrow to the vasculature. However, there is no information about the molecular signals that regulate neutrophil entry to circulation during an inflammatory process in humans. This is mainly due to the lack of a suitable model of study that contains similar set of molecules and that allows in vivo analyses. In this study, we used the zebrafish to assess the role of Cxcl8a, Cxcl8b, and Cxcr2 in neutrophil migration to blood circulation after injury. Using Tg(BACmpx:GFP)i114 transgenic embryos and two damage models (severe and mild), we developed in vivo lack of function assays. We found that the transcription levels of cxcl8a, cxcl8b, and cxcr2 were upregulated in the severe damage model. In contrast, only cxcr2 and cxcl8a mRNA levels were increased during mild damage. After knocking down Cxcl8a, neutrophil quantity decreased at the injury site, while Cxcl8b decreased neutrophils in circulation. When inhibiting Cxcr2, we observed a decrease in neutrophil entry to the bloodstream. In conclusion, we identified different functions for both Cxcl8 paralogues, being the Cxcl8b/Cxcr2 axis that regulates neutrophil entry to the bloodstream, while Cxcl8a/Cxcr2 regulates the migration to the affected area.
Asunto(s)
Interleucina-8/metabolismo , Neutrófilos/fisiología , Receptores de Interleucina-8B/metabolismo , Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Circulación Sanguínea , Movimiento Celular , Regulación de la Expresión Génica , Humanos , Inflamación/sangre , Inflamación/inmunología , Interleucina-8/deficiencia , Interleucina-8/genética , Infiltración Neutrófila , Neutrófilos/inmunología , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/inmunología , Transducción de Señal , Pez Cebra/embriologíaRESUMEN
Flavobacterium psychrophilum is a Gram-negative bacterium, responsible for the bacterial cold-water disease and the rainbow trout fry syndrome in freshwater salmonid fish. At present, there is only one commercial vaccine in Chile, made with two Chilean F. psychrophilum isolates and another licensed in Europe. The present study analyzed neutrophil migration, as a marker of innate immune activation, in zebrafish (Danio rerio) in response to different F. psychrophilum bath vaccines, which is the first step in evaluating vaccine effectiveness and efficiency in fish. Results indicated that bacterins of the LM-02-Fp isolate were more immunogenic than those from the LM-13-Fp isolate. However, no differences were observed between the same bacteria inactivated by either formaldehyde or heat. Importantly, the same vaccine formulation without an adjuvant only triggered a mild neutrophil migration compared to the complete vaccine. Observations also found that, after a year of storage at 4°C, the activation of the innate immune system by the different vaccines was considerably decreased. Finally, new vaccine formulations prepared with heat and formaldehyde inactivated LM-02-Fp were significantly more efficient than the available commercial vaccine in regard to stimulating the innate immune system.
Asunto(s)
Vacunas Bacterianas/inmunología , Quimiotaxis de Leucocito/inmunología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/inmunología , Inmunidad Innata , Neutrófilos/inmunología , Animales , Pez CebraRESUMEN
G-CSF is an essential cytokine that regulates proliferation and differentiation of granulocytes from hematopoietic stem and progenitor cells. In mammals G-CSF has been identified as a key factor that promotes the release of neutrophils from the bone marrow into the blood circulation. In silico analysis indicates that zebrafish has two gcsf genes, gcsf-chr12 in chromosome 12 and gcsf-chr19 in chromosome 19. Gcsf-Chr12 participates in emergency myelopoiesis, but, in contrast to its mammalian orthologue, is not involved in neutrophil migration toward damaged tissue. In turn, the function of Gcsf-Chr19 has not been examined yet. In this study, we analyzed the role of Gcsf-Chr19 in regulating neutrophil migration toward the wound. Our results indicated that during the first h after caudal fin transection, neutrophils migrate from the hematopoietic tissue toward the injury, using the extracellular matrix as a substrate. Later, between 3 and 4 h postdamage, the recruitment mainly occurs through the bloodstream, and only a few neutrophils still use the extracellular matrix to migrate. During this process, the transcriptional levels of gcsf-chr19 are considerably increased, reaching a peak 1 h postdamage. The knockdown of Gcsf-chr19 indicated that the percentage of neutrophils that reach the wound decreased after the first h postinjury, suggesting that the knockdown specifically affects neutrophils that travel to the wound through blood vessels. Together, our data provide novel information about the regulation of neutrophil migration in zebrafish, positioning Gcsf-Chr19 as a key signal during the course of an inflammatory process triggered by severe damage.
Asunto(s)
Movimiento Celular/inmunología , Factor Estimulante de Colonias de Granulocitos/inmunología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/inmunología , Animales , Movimiento Celular/genética , Técnicas de Silenciamiento del Gen , Factor Estimulante de Colonias de Granulocitos/genética , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/inmunología , Infiltración Neutrófila/genética , Neutrófilos/patología , Transducción de Señal/genética , Transducción de Señal/inmunología , Factores de Tiempo , Transcripción Genética/genética , Transcripción Genética/inmunología , Heridas y Lesiones/genética , Heridas y Lesiones/inmunología , Heridas y Lesiones/patología , Pez Cebra/genética , Proteínas de Pez Cebra/aislamiento & purificaciónRESUMEN
Genome-wide association studies (GWAS) have successfully identified several risk loci for Alzheimer's disease (AD). Nonetheless, these loci do not explain the entire susceptibility of the disease, suggesting that other genetic contributions remain to be identified. Here, we performed a meta-analysis combining data of 4,569 individuals (2,540 cases and 2,029 healthy controls) derived from three publicly available GWAS in AD and replicated a broad genomic region (>248,000 bp) associated with the disease near the APOE/TOMM40 locus in chromosome 19. To detect minor effect size contributions that could help to explain the remaining genetic risk, we conducted network-based pathway analyses either by extracting gene-wise p-values (GW), defined as the single strongest association signal within a gene, or calculated a more stringent gene-based association p-value using the extended Simes (GATES) procedure. Comparison of these strategies revealed that ontological sub-networks (SNs) involved in glutamate signaling were significantly overrepresented in AD (p<2.7×10(-11), p<1.9×10(-11); GW and GATES, respectively). Notably, glutamate signaling SNs were also found to be significantly overrepresented (p<5.1×10(-8)) in the Alzheimer's disease Neuroimaging Initiative (ADNI) study, which was used as a targeted replication sample. Interestingly, components of the glutamate signaling SNs are coordinately expressed in disease-related tissues, which are tightly related to known pathological hallmarks of AD. Our findings suggest that genetic variation within glutamate signaling contributes to the remaining genetic risk of AD and support the notion that functional biological networks should be targeted in future therapies aimed to prevent or treat this devastating neurological disorder.
Asunto(s)
Enfermedad de Alzheimer/genética , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Glutamatos/metabolismo , Transducción de Señal/genética , Encéfalo/metabolismo , Encéfalo/patología , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Neuroimagen , Reproducibilidad de los Resultados , Sinapsis/metabolismoRESUMEN
The necessary replacement of fish meal with other protein source in diets of commercially important fish has prompted the study of the effect of the inclusion of different vegetable proteins sources on growth performance and on the gastro-intestinal tract. Currently, soybean meal is the primary protein source as a fish meal replacement because of its low price and high availability. Likewise, it is been documented that the ingestion of soybean meal by several fish species, such as salmonids and carp, triggers a type of intestinal inflammation called enteritis. In this paper, we analyzed the effects of the ingestion of soybean meal and two of its components, soy protein and soy saponin, on zebrafish to establish the basis for using zebrafish larvae as a model for fish nutrition. We took advantage of the existence of different transgenic lines, which allowed us to perform in vivo analysis. Our results indicated that larvae that were feed with soybean meal developed a clear intestinal inflammation as early as two day after beginning the diet. Moreover, we determined that is not the soy protein present in the diet but the soy saponin that is primarily responsible for triggering the immune response. These findings support the use of zebrafish screening assays to identify novel ingredients that would to improved current fish diets or would formulate new ones.
Asunto(s)
Enteritis/veterinaria , Enfermedades de los Peces/inmunología , Glycine max/efectos adversos , Pez Cebra/inmunología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Movimiento Celular , Dieta , Modelos Animales de Enfermedad , Enteritis/etiología , Enteritis/inmunología , Enteritis/patología , Enfermedades de los Peces/etiología , Enfermedades de los Peces/patología , Intestinos/inmunología , Intestinos/patología , Larva , Neutrófilos/fisiología , Saponinas/administración & dosificación , Saponinas/efectos adversos , Proteínas de Soja/administración & dosificación , Proteínas de Soja/efectos adversos , Proteínas de Soja/inmunología , Glycine max/inmunologíaRESUMEN
One of the most important mechanisms that promotes metastasis is the stabilization of Hif-1 (hypoxia-inducible transcription factor 1). We decided to test whether Hif-1α also was required for early embryonic development. We focused our attention on the development of the neural crest, a highly migratory embryonic cell population whose behavior has been likened to cancer metastasis. Inhibition of Hif-1α by antisense morpholinos in Xenopus laevis or zebrafish embryos led to complete inhibition of neural crest migration. We show that Hif-1α controls the expression of Twist, which in turn represses E-cadherin during epithelial to mesenchymal transition (EMT) of neural crest cells. Thus, Hif-1α allows cells to initiate migration by promoting the release of cell-cell adhesions. Additionally, Hif-1α controls chemotaxis toward the chemokine SDF-1 by regulating expression of its receptor Cxcr4. Our results point to Hif-1α as a novel and key regulator that integrates EMT and chemotaxis during migration of neural crest cells.
Asunto(s)
Quimiotaxis/genética , Transición Epitelial-Mesenquimal/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Cresta Neural/citología , Animales , Hipoxia de la Célula , Embrión no Mamífero/citología , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Cresta Neural/metabolismo , Receptores CXCR4/genética , Proteína 1 Relacionada con Twist/genética , Xenopus laevis , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
We previously found that single nucleotide polymorphisms in the low-density lipoprotein receptor-related protein 6 (LRP6) gene are associated with Alzheimer's disease (AD). Here, we studied the posttranscriptional metabolism of the LRP6 message scanning sequentially the 23 LRP6 exons in human tissues and found a novel LRP6 isoform that completely skips exon 3 (LRP6Δ3) in all tissues examined and was also conserved in mice. Expression levels of the LRP6 isoforms were determined in 47 cortical brain messenger (m)RNA samples including 22 AD cases, 11 control subjects, and 14 individuals with other neurological disorders. LRP6Δ3 mRNA levels were significantly augmented in AD brains compared with controls (1.6-fold; p = 0.037) or other pathological samples (2-fold; p = 0.007). Functional analysis in Wnt/ß-catenin signaling assays revealed that skipping of exon 3 reduced significantly the signaling activity of the LRP6 coreceptor. We conclude that the LRP6Δ3 isoform is a novel splice variant, which shows diminished Wnt/ß-catenin signaling activity and might have a functional role in individuals with AD.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Estudios de Asociación Genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Isoformas de Proteínas/genética , Vía de Señalización Wnt/genética , Anciano , Anciano de 80 o más Años , Empalme Alternativo/genética , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.
Asunto(s)
Muerte Celular/efectos de los fármacos , Sulfato de Cobre/toxicidad , Estrés Fisiológico/efectos de los fármacos , Pez Cebra , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunohistoquímica , Larva/efectos de los fármacos , Dosificación Letal Mediana , Factores de Tiempo , Pruebas de Toxicidad Aguda , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/embriologíaRESUMEN
Insulin-like growth factor-1 and insulin-like growth factor-1 receptor (IGF-1 and IGF-1R) play main roles in vertebrate growth and development. In fish, besides contributing to somatic growth, both molecules exhibit pleiotropic functions. We isolated complete cDNAs sequences encoding for both IGF-1 and IGF-1R in the Chilean flounder by using RT-PCR and rapid amplification of cDNAs ends (RACE) techniques. We analyzed gene expression in pre-metamorphic larvae and different organs of juvenile fish through whole mount in situ hybridization and RT-PCR, respectively. In addition, we studied the presence of calcified skeletal structures in pre-metamorphic larvae through the fluorescent chromophore calcein. The IGF-1 cDNA sequence displays an open reading frame of 558 nucleotides, encoding a 185 amino acid preproIGF-1. Moreover, IGF-1R contains an open reading frame spanning 4239 nucleotides, rendering a 702 amino acid subunit alpha and a 676 amino acid subunit beta. The deduced mature IGF-1 and IGF-1R exhibited high sequence identities with their corresponding orthologs in fishes, especially those domains involved in biological activity. RT-PCR showed expression of IGF-1 and IGF-1R transcripts in all studied tissues, consistent with their pleiotropic functions. Furthermore, we observed IGF-1 expression in notochord and IGF-1R expression in notochord, somites and head in larvae of 8 and 9 days post fertilization. Complementarily, we detected in larvae of 8 days post fertilization the presence of calcified skeletal structures in notochord and head. Interestingly, both mRNAs and calcified structures were found in territories such as notochord, an embryonic midline structure essential for the pattern of surrounding tissues as nervous system and mesoderm. Our results suggest that IGF-1 and its receptor play an important role in the development of the nervous system, muscle and bone-related structures during larval stages.
Asunto(s)
Lenguado/genética , Lenguado/metabolismo , Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina , Receptor IGF Tipo 1 , Animales , Chile , Clonación Molecular , Perfilación de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.
Asunto(s)
Animales , Muerte Celular/efectos de los fármacos , Sulfato de Cobre/toxicidad , Estrés Fisiológico/efectos de los fármacos , Pez Cebra , Animales Modificados Genéticamente , Embrión no Mamífero/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , /metabolismo , Inmunohistoquímica , Larva/efectos de los fármacos , Factores de Tiempo , Pruebas de Toxicidad Aguda , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/embriologíaRESUMEN
Growth hormone receptor (GHR) is a critical regulator of growth and metabolism. Although two GHRs have been characterized in many fish species, their functional characteristics, mechanisms of regulation and roles in embryonic development remain unclear. The zebrafish (Danio rerio) is an excellent model organism to study both developmental and physiological processes. In the present work, we characterized the complete cDNA sequences of zebrafish GHRs, ghra and ghrb, and their gene structures. We studied the expression of both receptors in adult tissues, and during embryonic development and larval stages by means of RT-PCR and whole-mount in situ hybridization. We determined that both transcripts are maternal ones, with specific expression patterns during development. Both GHR transcripts are mainly expressed in the notochord, myotomes, anterior structures and in the yolk cell. Interestingly, their expression became undetectable at 96h post-fertilization. Unlike other reports in fish, ghrs expression could not be detected in brain when adult tissues were used, and we detected ghrb but not ghra transcripts in muscle. In addition, we determined alternative transcript sequences for ghra with specific domain deletions, and alternative transcripts for ghrb that generate a premature stop codon and codify for truncated isoforms. These isoforms lack intracellular regions necessary for the activation of signal transducers and activators of transcription (STAT) family transcription factors 5.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Receptores de Somatotropina/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Datos de Secuencia Molecular , Receptores de Somatotropina/genética , Homología de Secuencia de Aminoácido , Pez Cebra/embriologíaRESUMEN
BACKGROUND: The mineralized skeleton is a major evolutionary novelty that has contributed to the impressive morphological diversifications of the vertebrates. Essential to bone biology is the solidified extracellular matrix secreted by highly specialized cells, the osteoblasts. We now have a rather complete view of the events underlying osteogenesis, from a cellular, molecular, genetic, and epigenetic perspective. Because this knowledge is still largely restricted to mammals, it is difficult, if not impossible, to deduce the evolutionary history of the regulatory network involved in osteoblasts specification and differentiation. In this study, we focused on the transcriptional regulators Runx2 and VDR (the Vitamin D Receptor) that, in mammals, directly interact together and stabilize complexes of co-activators and chromatin remodellers, thereby allowing the transcriptional activation of target genes involved in extracellular matrix mineralization. Using a combination of functional, biochemical, and histological approaches, we have asked if the interaction observed between Runx2 and VDR represents a recent mammalian innovation, or if it results from more ancient changes that have occurred deep in the vertebrate lineage. RESULTS: Using immunohistochemistry and in situ hybridization in developing embryos of chick, frog and teleost fishes, we have revealed that the co-expression of Runx2 and VDR in skeletal elements has been particularly strengthened in the lineage leading to amniotes. We show that the teleost Runx2 orthologue as well as the three mammalian Runx1, Runx2 and Runx3 paralogues are able to co-immunoprecipitate with the VDR protein present in nuclear extracts of rat osteoblasts stimulated with 1alpha,25-dihydroxyvitamin D3. In addition, the teleost Runx2 can activate the transcription of the mammalian osteocalcin promoter in transfection experiments, and this response can be further enhanced by 1alpha,25-dihydroxyvitamin D3. Finally, using pull-down experiments between recombinant proteins, we show that the VDR homologue from teleosts, but not from ascidians, is able to directly interact with the mammalian Runx2 homologue. CONCLUSIONS: We propose an evolutionary scenario for the assembly of the molecular machinery involving Runx2 and VDR in vertebrates. In the last common ancestor of actinopterygians and sacropterygians, the three Runx paralogues possessed the potential to physically and functionally interact with the VDR protein. Therefore, 1alpha,25-dihydroxyvitamin D3 might have been able to modulate the transcriptional activity of Runx1, Runx2 or Runx3 in the tissues expressing VDR. After the split from amphibians, in the lineage leading to amniotes, Runx2 and VDR became robustly co-expressed in developing skeletal elements, and their regulatory interaction was incorporated in the genetic program involved in the specification and differentiation of osteoblasts.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Evolución Molecular , Osteogénesis , Receptores de Calcitriol/genética , Vertebrados/genética , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Osteoblastos/citología , Receptores de Calcitriol/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vertebrados/embriología , Vertebrados/metabolismoRESUMEN
The full length cDNA sequence of the myostatin gene was cloned from a teleostean fish, the Chilean flounder (Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5'- and 3'-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including the structural domains responsible for its biological activity. Amino acid sequence comparison revealed high sequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Gene expression analysis showed that cfMSTN mRNA is present in a wide variety of tissues in juvenile fish. In addition, we assessed the spatial expression pattern of the MSTN mRNA during embryos and larval stages through whole mount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days post fertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye, were positive for MSTN mRNA. Our results contribute to the knowledge of the MSTN system in larval and juvenile stages; in particular the strong expression observed in the notochord suggests that MSTN, in synchronization with positive growth signals, may play an important role in the control of the development of larvae somites.
Asunto(s)
Lenguado/crecimiento & desarrollo , Lenguado/genética , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chile , Clonación Molecular , ADN Complementario/genética , Lenguado/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Larva/metabolismo , Datos de Secuencia Molecular , Miostatina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The entire cDNA sequence of the growth hormone receptor (GHR) of the Chilean flounder (Paralichthys. adspersus) was cloned by RT-PCR and RNA ligase rapid amplification of 5' and 3'ends. The deduced amino acid sequence contains 641 residues and codes for the GHR1 form. The receptor includes all the structural domains and motifs responsible for its interaction with the growth hormone and growth signal transduction. Sequence comparison revealed 95 and 88% identity with other flat fish such as the Japanese flounder and Atlantic halibut respectively, but decreased to 41% with the GHR of other teleosts such as salmon. In addition we performed a phylogenetic analysis of this receptor in teleosts. RT-PCR experiments were performed to study the expression of GHR1 mRNA in different tissues of juvenile fish, detecting the transcripts in all tissues investigated with higher expressions in the liver, brain and gonads. Additionally, using whole-mount in situ hybridization in larvae stages, we observed an on and off GHR1 mRNA expression pattern. This novel finding evidences that during early development of a teleost, GHR1 is transiently expressed in somites, a source of muscle, bone and spinal chord precursors cells, suggesting a relevant role of GH in fish development. GHR1 was also temporally detected in the notochord, intestines, brain and retinal layers, before its ubiquitous establishment.
Asunto(s)
Lenguado/embriología , Lenguado/genética , Regulación del Desarrollo de la Expresión Génica , Receptores de Somatotropina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chile , ADN Complementario/genética , Perfilación de la Expresión Génica , Hibridación in Situ , Larva/genética , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Somatotropina/química , Receptores de Somatotropina/metabolismo , Alineación de SecuenciaRESUMEN
Protein kinase CK1 is a ser/thr protein kinase family which has been identified in the cytosol cell fraction, associated with membranes as well as in the nucleus. Several isoforms of this gene family have been described in various organisms: CK1alpha, CK1beta, CK1delta, CK1epsilon and CK1gamma. Over the last decade, several members of this family have been involved in development processes related to wnt and sonic hedgehog signalling pathways. However, there is no detailed temporal information on the CK1 family in embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe for the first time the cloning and detailed expression pattern of five CK1 zebrafish genes. Sequence analysis revealed that zebrafish CK1 proteins are highly homologous to other vertebrate orthologues. Zebrafish CK1 genes are expressed throughout development in common and different territories. All the genes studied in development show maternal and zygotic expression with the exception of CK1epsilon. This last gene presents only a zygotic component of expression. In early stages of development CK1 genes are ubiquitously expressed with the exception of CK1epsilon. In later stages the five CK1 genes are expressed in the brain but not in the same way. This observation probably implicates the CK1 family genes in different and also in redundant functions. This is the first time that a detailed comparison of the expression of CK1 family genes is directly assessed in a vertebrate system throughout development.
Asunto(s)
Quinasa de la Caseína I/genética , Regulación del Desarrollo de la Expresión Génica , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Quinasa de la Caseína I/metabolismo , Clonación Molecular , Cartilla de ADN , Perfilación de la Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Filogenia , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Pez Cebra/genéticaRESUMEN
The Snail genes are implicated in processes that involve cell movement, both during embryonic development and tumour progression. In teleosts, the vertebrate Snail1 gene is represented by two distinct genes, snail1a and snail1b (previously snail1 and snail2). These genes are expressed in complementary mesodermal domains and their combined expression matches that of their mammalian counterpart. By analysing their loss and gain of function, we found that the most-anterior axial mesendodermal cells, the precursors of the polster, move in a cohesive manner directed by the activity of snail1a- and snail1b-expressing cells surrounding these precursors. The cell-autonomous function of Snail1 proteins regulates cell motility and influences the behaviour of Snail-negative neighbouring cells. Snail1a is required by the prechordal plate for it to reach its normal position, whereas Snail1b controls the acquisition of its normal shape. These non-redundant functions of Snail1a and Snail1b in controlling axial mesendoderm migration comply with the duplication-degeneration-complementation model, and indicate that Snail genes not only act as inducers of epithelial-to-mesenchymal transition, but also as more general regulators of cell adhesion and movement.