RESUMEN
Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.
Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Células Epiteliales/citología , Fagocitos/citología , Fagocitosis , Pez Cebra/embriología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Apoptosis , Movimiento Celular , Forma de la Célula , Extensiones de la Superficie Celular , Inmunidad Innata , Ratones , Fosfatidilserinas/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. METHODS: We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. RESULTS: Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. CONCLUSIONS: Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.
Asunto(s)
Cardiomiopatía Hipertrófica , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Edición GénicaRESUMEN
Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.
Asunto(s)
Proteínas , Segmento Externo de las Células Fotorreceptoras Retinianas , Factores de Empalme Serina-Arginina , Visión Ocular , Animales , Exones , Eliminación de Gen , Humanos , Proteínas/genética , Proteínas/fisiología , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/fisiología , Transcriptoma , Visión Ocular/genética , Visión Ocular/fisiología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days postamputation) time point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.
Asunto(s)
Matriz Extracelular/fisiología , Corazón/fisiología , Miocardio/citología , Regeneración/fisiología , Proteínas de Pez Cebra/análisis , Animales , Fenómenos Biomecánicos , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/metabolismo , Microscopía de Fuerza Atómica , Proteómica/métodos , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Novel organismal structures in metazoans are often undergirded by complex gene regulatory networks; as such, understanding the emergence of new structures through evolution requires reconstructing the series of evolutionary steps leading to these underlying networks. Here, we reconstruct the step-by-step assembly of the vertebrate splicing network regulated by Nova, a splicing factor that modulates alternative splicing in the vertebrate central nervous system by binding to clusters of YCAY motifs on pre-RNA transcripts. Transfection of human HEK293T cells with Nova orthologs indicated vertebrate-like splicing regulatory activity in bilaterian invertebrates, thus Nova acquired the ability to bind YCAY clusters and perform vertebrate-like splicing modulation at least before the last common ancestor of bilaterians. In situ hybridization studies in several species showed that Nova expression became restricted to CNS later on, during chordate evolution. Finally, comparative genomics studies revealed a diverse history for Nova-regulated exons, with target exons arising through both de novo exon creation and acquisition of YCAY motifs by preexisting exons throughout chordate and vertebrate history. In addition, we find that tissue-specific Nova expression patterns emerged independently in other lineages, suggesting independent assembly of tissue-specific regulatory networks.
Asunto(s)
Empalme Alternativo , Antígenos de Neoplasias/metabolismo , Encéfalo/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Vertebrados/genética , Animales , Antígenos de Neoplasias/genética , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Antígeno Ventral Neuro-Oncológico , Proteínas de Unión al ARN/genéticaRESUMEN
Regulation of microtubule (MT) dynamics is key for mitotic spindle assembly and faithful chromosome segregation. Here we show that polyglutamylation, a still understudied post-translational modification of spindle MTs, is essential to define their dynamics within the range required for error-free chromosome segregation. We identify TTLL11 as an enzyme driving MT polyglutamylation in mitosis and show that reducing TTLL11 levels in human cells or zebrafish embryos compromises chromosome segregation fidelity and impairs early embryonic development. Our data reveal a mechanism to ensure genome stability in normal cells that is compromised in cancer cells that systematically downregulate TTLL11. Our data suggest a direct link between MT dynamics regulation, MT polyglutamylation and two salient features of tumour cells, aneuploidy and chromosome instability (CIN).
Asunto(s)
Segregación Cromosómica , Neoplasias , Animales , Humanos , Cinetocoros , Huso Acromático/genética , Pez Cebra/genética , Microtúbulos/genética , Neoplasias/genéticaRESUMEN
The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment.
Asunto(s)
Forma de la Célula , Mecanotransducción Celular , Membrana Nuclear/fisiología , Fosfolipasas A2 Citosólicas/metabolismo , Actomiosina/metabolismo , Animales , Movimiento Celular , Lipasa/metabolismo , Miosina Tipo II/metabolismo , Pez CebraRESUMEN
Tyrosine kinase (TK) proteins play a central role in cellular behavior and development of animals. The expansion of this superfamily is regarded as a key event in the evolution of the complex signaling pathways and gene networks of metazoans and is a prominent example of how shuffling of protein modules may generate molecular novelties. Using the intron/exon structure within the TK domain (TK intron code) as a complementary tool for the assignment of orthology and paralogy, we identified and studied the 118 TK proteins of the amphioxus Branchiostoma floridae genome to elucidate TK gene family evolution in metazoans and chordates in particular. Unlike all characterized metazoans to date, amphioxus has members of all known widespread TK families, with not a single loss. Putting amphioxus TKs in an evolutionary context, including new data from the cnidarian Nematostella vectensis, the echinoderm Strongylocentrotus purpuratus, and the ascidian Ciona intestinalis, we suggest new evolutionary histories for different TK families and draw a new global picture of gene loss/gain in the different phyla. Surprisingly, our survey also detected an unprecedented expansion of a group of closely related TK families, including TIE, FGFR, PDGFR, and RET, due most probably to massive gene duplication and exon shuffling. Based on their highly similar intron/exon structure at the TK domain, we suggest that this group of TK families constitute a superfamily of TK proteins, which we termed EXpanding TK, after their seemingly unique propensity to gene duplication and exon shuffling, not only in amphioxus but also across all metazoan groups. Due to this extreme tendency to both retention and expansion of TK genes, amphioxus harbors the richest and most diverse TK repertoire among all metazoans studied so far, retaining most of the gene complement of its ancestors, but having evolved its own repertoire of genetic novelties.
Asunto(s)
Cordados no Vertebrados/enzimología , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Cordados no Vertebrados/clasificación , Cordados no Vertebrados/genética , Evolución Molecular , Exones , Duplicación de Gen , Código Genético , Variación Genética , Genoma , Humanos , Intrones , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Oncogénicas/genética , Filogenia , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas , Seudogenes , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Familia-src Quinasas/genética , Tirosina Quinasa del Receptor AxlRESUMEN
The discovery that most regulatory genes were conserved among animals from distant phyla challenged the ideas that gene duplication and divergence of homologous coding sequences were the basis for major morphological changes in metazoan evolution. In recent years, however, the interest for the roles, conservation and changes of non-coding sequences grew-up in parallel with genome sequencing projects. Presently, many independent studies are highlighting the importance that subtle changes in cis-regulatory regions had in the evolution of morphology trough the Animal Kingdom. Here we will show and discuss some of these studies, and underscore the future of cis-Evo-Devo research. Nevertheless, we would also explore how gene duplication, which includes duplication of regulatory regions, may have been critical for spatial or temporal co-option of new regulatory networks, causing the deployment of new transcriptome scenarios, and how these induced morphological changes were critical for the evolution of new forms. Forty years after Susumu Ohno famous sentence 'natural selection merely modifies, while redundancy creates', we suggest the alternative: 'natural selection modifies, while redundancy of cis-regulatory elements innovates', and propose the Duplication-Degeneration-Innovation model to explain the increased evolvability of duplicated cis-regulatory regions. Paradoxically, making regulation simpler by subfunctionalization paved the path for future complexity or, in other words, 'to make it simple to make it complex'.
Asunto(s)
Evolución Biológica , Duplicación de Gen , Modelos Genéticos , Secuencias Reguladoras de Ácidos Nucleicos/genética , AnimalesRESUMEN
Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases.
Asunto(s)
Sistemas CRISPR-Cas , Neuronas Dopaminérgicas/metabolismo , Edición Génica , Células Madre Pluripotentes Inducidas/metabolismo , Imagen Molecular , Tirosina 3-Monooxigenasa/genética , Calcio/metabolismo , Diferenciación Celular , Rastreo Celular , Células Cultivadas , Neuronas Dopaminérgicas/citología , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Mesencéfalo/citología , Mesencéfalo/fisiologíaRESUMEN
Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(-/-) cells, descendants from the tumor originating cell. These NF1(-/-)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(-/-) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.
Asunto(s)
Carcinogénesis/genética , Reprogramación Celular/genética , Predisposición Genética a la Enfermedad/genética , Neurofibroma Plexiforme/genética , Neurofibromatosis 1/genética , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Proliferación Celular/genética , Niño , Femenino , Genes Supresores de Tumor/fisiología , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Cresta Neural/fisiología , Neurofibroma Plexiforme/sangre , Neurofibromatosis 1/sangre , Células de Schwann/fisiología , Adulto JovenRESUMEN
In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term "CardioSlice." PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients.
Asunto(s)
Miocardio , Ingeniería de Tejidos , Andamios del Tejido , Técnicas de Cultivo Celular por Lotes , Fenómenos Biomecánicos , Reactores Biológicos , Diferenciación Celular , Células Cultivadas , Fenómenos Electrofisiológicos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocardio/citología , Miocardio/metabolismoRESUMEN
In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Hematopoyesis , Mesodermo/metabolismo , Diferenciación Celular , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica , Hemangioblastos/citología , Hemangioblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mesodermo/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Unión Proteica , Análisis de la Célula IndividualRESUMEN
We report the generation-characterization of a fetal liver (FL) B-cell progenitor (BCP)-derived human induced pluripotent stem cell (hiPSC) line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the "safe locus" AAVS1 (iCas9-FL-BCP-hiPSC). Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure. T7-endonuclease assay demonstrated that iCas9 induces robust gene-edition when gRNAs against hematopoietic transcription factors were tested. This iCas9-FL-BCP-hiPSC will facilitate gene-editing approaches for studies on developmental biology, drug screening and disease modeling.
Asunto(s)
Linfocitos B/metabolismo , Proteínas Bacterianas/biosíntesis , Dependovirus , Endonucleasas/biosíntesis , Expresión Génica , Sitios Genéticos , Células Madre Pluripotentes Inducidas/metabolismo , Linfocitos B/citología , Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.
Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Mucopolisacaridosis III/patología , Red Nerviosa/patología , Neuronas/patología , Técnicas de Cultivo de Célula , Células Cultivadas , Humanos , NeurogénesisRESUMEN
Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.
Asunto(s)
Dopamina/metabolismo , Neuronas/patología , Neuronas/fisiología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Células Madre Pluripotentes/patología , Células Madre Pluripotentes/fisiología , Autofagia , Células Cultivadas , Humanos , Receptores Dopaminérgicos/metabolismo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Pallid anchovy fillet, friendly filtering, peacefully laying and little lancelet are some of the nicknames and adjectives the cephalochordate amphioxus has received throughout the last two centuries. Traditionally regarded as the living representative of the last ancestor of vertebrates, amphioxus has recently been promoted to the privileged position of being the most ancient chordate. The preliminary analysis of its prototypical genome is nearly completed, and its hidden secrets towards the understanding of the primitive chordate and deuterostome genomes will soon see the light. Amphioxus embryonic development and body plan have remained in evolutionary stasis since the cephalochordate lineage split from the chordate ancestor about 500 million years ago. In contrast, amphioxus research is far from being at a standstill; in Europe, thanks to the international cooperation and the Banyuls Oceanographic Station, amphioxus embryos are obtained on demand during the spawning season. We summarise here our progress towards the dream of the experimental manipulation of the amphioxus embryo, to enter the era of Experimental Evo-Devo.
Asunto(s)
Cordados no Vertebrados/embriología , Cordados no Vertebrados/genética , Evolución Molecular , Américas , Animales , Tipificación del Cuerpo/genética , Biología Evolutiva/métodos , Biología Evolutiva/tendencias , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Europa (Continente) , Genoma/genética , Genómica/métodos , Genómica/tendencias , Proteínas de Homeodominio/genética , Modelos Biológicos , Familia de Multigenes , Investigación/tendencias , Proyectos de InvestigaciónRESUMEN
The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies. It has been shown that an increase of 3-4 degrees C in water temperature triggers spawning of the European amphioxus (Branchiostoma lanceolatum) in captivity, however, very little is known about the natural spawning behavior of this species in the field. In this work, we have followed the spawning behavior of the European amphioxus during two spawning seasons (2004 and 2005), both in the field and in captivity. We show that animals in the field spawn approximately from mid-May through early July, but depending on the year, they show different patterns of spawning. Thus, even if temperature has a critical role in the induction of the spawning in captivity, it is not the major factor in the field. Moreover, we report some improvements on the methodology for inducing spawning in captivity (e.g. in maintenance, light cycle control and induction of spawning in a laboratory without running sea water system). These studies have important implications for amphioxus animal husbandry and for improving laboratory techniques to develop amphioxus as an experimental animal model.
Asunto(s)
Crianza de Animales Domésticos , Cordados no Vertebrados/fisiología , Animales , ReproducciónRESUMEN
Amphioxus Hairy genes have gone through a number of lineage-specific duplications, resulting in eight members, some of which are differentially expressed in the embryo. In order to gain insights into the evolution and function of this gene family we have compared their genomic structure and searched for conserved non-coding sequence elements. We have found that introns have been lost independently from these genes at least twice and after the duplication events. By carrying out phylogenetic footprinting between paralogues expressed in the embryo, we have found a differential distribution of conserved elements that could explain the limited overlap in expression patterns of Hairy genes in the amphioxus embryo. Furthermore, clustering of RBP-Jk binding sites in these conserved elements suggests that amphioxus Hairy genes are downstream targets of the Notch signaling pathway, as occurs in vertebrates. All of this evidence suggests that amphioxus Hairy genes have gone through a process of subfunctionalization shortly after their duplication, representing an extreme and rapid case of the duplication-degeneration-complementation model.