RESUMEN
In contrast to other mammals, the spiny mouse (Acomys) regenerates skin and ear tissue, which includes hair follicles, glands, and cartilage, in a scar-free manner. Ear punch regeneration is asymmetric with only the proximal wound side participating in regeneration. Here, we show that cues originating from the proximal side are required for normal regeneration and use spatially resolved transcriptomics (tomo-seq) to understand the molecular and cellular events underlying this process. Analyzing gene expression across the ear and comparing expression modules between proximal and distal wound sides, we identify asymmetric gene expression patterns and pinpoint regenerative processes in space and time. Moreover, using a comparative approach with nonregenerative rodents (Mus, Meriones), we strengthen a hypothesis in which particularities in the injury-induced immune response may be one of the crucial determinants for why spiny mice regenerate whereas their relatives do not. Our data are available in SpinyMine, an easy-to-use and expandable web-based tool for exploring Acomys regeneration-associated gene expression.
Asunto(s)
Murinae , Cicatrización de Heridas , Animales , Cicatrización de Heridas/genética , Murinae/genética , Transcriptoma , Regeneración/genética , Piel , Mamíferos/genéticaRESUMEN
In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression. Many studies have described the conserved functions of CBP/p300 in stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model for the in vivo study of the molecular mechanisms underlying stem cell differentiation during regeneration. However, how this process is regulated genetically and epigenetically is not well-understood yet. We identified 5 distinct Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses revealed that Smed-cbp-2 appears to be essential for stem cell maintenance. On the other hand, the silencing of Smed-cbp-3 resulted in the growth of blastemas that were apparently normal, but remained largely unpigmented and undifferentiated. Smed-cbp-3 silencing also affected the differentiation of several cell lineages including neural, epidermal, digestive, and excretory cell types. Finally, we analysed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their functions in planarian stem cell biology. Our results indicate that planarian cbp genes play key roles in stem cell maintenance and differentiation.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Diferenciación Celular/genética , Planarias/genética , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Cromatina/metabolismo , Histonas/metabolismo , Planarias/metabolismo , Regeneración/genética , Células Madre/citología , Células Madre/metabolismo , Células Madre/fisiología , Factores de Transcripción/metabolismoRESUMEN
Flatworms (Platyhelminthes) are a basally branching phylum that harbours a wealth of fascinating biology, including planarians with their astonishing regenerative abilities and the parasitic tape worms and blood flukes that exert a massive impact on human health. PlanMine (http://planmine.mpi-cbg.de/) has the mission objective of providing both a mineable sequence repository for planarians and also a resource for the comparative analysis of flatworm biology. While the original PlanMine release was entirely based on transcriptomes, the current release transitions to a more genomic perspective. Building on the recent availability of a high quality genome assembly of the planarian model species Schmidtea mediterranea, we provide a gene prediction set that now assign existing transcripts to defined genomic coordinates. The addition of recent single cell and bulk RNA-seq datasets greatly expands the available gene expression information. Further, we add transcriptomes from a broad range of other flatworms and provide a phylogeny-aware interface that makes evolutionary species comparisons accessible to non-experts. At its core, PlanMine continues to utilize the powerful InterMine framework and consistent data annotations to enable meaningful inter-species comparisons. Overall, PlanMine 3.0 thus provides a host of new features that makes the fascinating biology of flatworms accessible to the wider research community.
Asunto(s)
Biodiversidad , Bases de Datos Genéticas , Platelmintos/genética , Transcriptoma/genética , Animales , Perfilación de la Expresión Génica , Genoma/genética , Genómica/tendencias , Humanos , Internet , FilogeniaRESUMEN
In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3'-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3'-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.
Asunto(s)
Células Madre Adultas/fisiología , Planarias/genética , Planarias/fisiología , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Regeneración/genética , Regeneración/fisiología , Células Madre Adultas/citología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Autorrenovación de las Células/genética , Autorrenovación de las Células/fisiología , Modelos Biológicos , Conformación de Ácido Nucleico , Planarias/citología , Interferencia de ARN , Procesamiento Postranscripcional del ARN , Empalme del ARN , ARN de Helminto/química , Homología de Secuencia de Ácido NucleicoRESUMEN
Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating ß1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Integrina beta1/fisiología , Neurogénesis/fisiología , Planarias/fisiología , Regeneración/fisiología , Animales , Tipificación del Cuerpo , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Hibridación in Situ , Neuronas/citología , Filogenia , Interferencia de ARN , Transducción de Señal , Células Madre/citologíaRESUMEN
A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.
Asunto(s)
Dineínas Axonemales/metabolismo , Cilios/patología , Síndrome de Kartagener/genética , Proteínas Asociadas a Microtúbulos/fisiología , Mutación/genética , Animales , Dineínas Axonemales/genética , Axonema/genética , Células Cultivadas , Cilios/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Exoma/genética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Hibridación in Situ , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Linaje , Fenotipo , Técnicas del Sistema de Dos Híbridos , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun-NH2-kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.
Asunto(s)
Apoptosis/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Planarias/genética , Células Madre Pluripotentes/fisiología , Cicatrización de Heridas/fisiología , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular , Proliferación Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mitosis , Planarias/citología , Interferencia de ARN , ARN Interferente Pequeño , Regeneración , Ubiquitina-Proteína Ligasas/biosíntesis , Cicatrización de Heridas/genéticaRESUMEN
In contrast to adult vertebrates, which have limited capacities for neurogenesis, adult planarians undergo constitutive cellular turnover during homeostasis and are even able to regenerate a whole brain after decapitation. This enormous plasticity derives from pluripotent stem cells residing in the planarian body in large numbers. It is still obscure how these stem cells are programmed for differentiation into specific cell lineages and how lineage identity is maintained. Here we identify a Pitx transcription factor of crucial importance for planarian regeneration. In addition to patterning defects that are co-dependent on the LIM homeobox transcription factor gene islet1, which is expressed with pitx at anterior and posterior regeneration poles, RNAi against pitx results in islet1-independent specific loss of serotonergic (SN) neurons during regeneration. Besides its expression in terminally differentiated SN neurons we found pitx in stem cell progeny committed to the SN fate. Also, intact pitx RNAi animals gradually lose SN markers, a phenotype that depends neither on increased apoptosis nor on stem cell-based turnover or transdifferentiation into other neurons. We propose that pitx is a terminal selector gene for SN neurons in planarians that controls not only their maturation but also their identity by regulating the expression of the Serotonin production and transport machinery. Finally, we made use of this function of pitx and compared the transcriptomes of regenerating planarians with and without functional SN neurons, identifying at least three new neuronal targets of Pitx.
Asunto(s)
Linaje de la Célula , Factores de Transcripción Paired Box/metabolismo , Planarias/citología , Planarias/metabolismo , Neuronas Serotoninérgicas/citología , Neuronas Serotoninérgicas/metabolismo , Animales , Apoptosis/genética , Biomarcadores/metabolismo , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Transdiferenciación Celular/genética , Regulación de la Expresión Génica , Proteínas con Homeodominio LIM/metabolismo , Datos de Secuencia Molecular , Planarias/genética , Interferencia de ARN , Regeneración/genética , Serotonina/biosíntesis , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.
Asunto(s)
Tipificación del Cuerpo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Cabeza/fisiología , Planarias/fisiología , Regeneración/fisiología , Células Madre/fisiología , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in Situ , Hibridación Fluorescente in Situ , Microscopía Confocal , Modelos Biológicos , Datos de Secuencia Molecular , Interferencia de ARN , Análisis de Secuencia de ADN , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo , Dedos de Zinc/genética , Dedos de Zinc/fisiologíaRESUMEN
Understanding the cellular and molecular determinants of mammalian tissue regeneration and repair is crucial for developing effective therapies that restore tissue architecture and function. In this review, we focus on the cell types involved in scarless wound response and regeneration of spiny mice (Acomys). Comparative -omics approaches with scar-prone mammals have revealed species-specific peculiarities in cellular behavior during the divergent healing trajectories. We discuss the developing views on which cell types engage in restoring the architecture of spiny mouse tissues through a co-ordinated spatiotemporal response to injury. While yet at the beginning of understanding how cells interact in these fascinating animals to regenerate tissues, spiny mice hold great promise for scar prevention and anti-fibrotic treatments.
Asunto(s)
Regeneración , Cicatrización de Heridas , Animales , Regeneración/fisiología , Cicatrización de Heridas/genética , Cicatriz/patología , Murinae , Mamíferos , RatonesRESUMEN
Since the establishment of planarian species as laboratory models, investigation of molecular pathways has relied heavily on visualization of transcripts using in situ hybridization (ISH). ISH has revealed various aspects ranging from anatomical details of different organs to distribution of planarian stem cell populations and signaling pathways involved in their unique regenerative response. High-throughput sequencing techniques including single-cell approaches have allowed us to investigate gene expression and cell lineages in more detail. One application that could provide important new insights into more subtle intercellular transcriptional differences and intracellular mRNA localization is single-molecule fluorescent in situ hybridization (smFISH). In addition to obtaining an overview of the expression pattern, this technique allows for single-molecule resolution and hence quantification of a transcript population. This is achieved by hybridization of individual oligonucleotides antisense to a transcript of interest, all carrying a single fluorescent label. This way, a signal is produced only when the combination of labelled oligonucleotides, targeting the same transcript, are hybridized, minimizing background and off-target effects. Moreover, it requires only a few steps compared to the conventional ISH protocol and thus saves time. Here we describe a protocol for the tissue preparation, probe synthesis, and smFISH, combined with immunohistochemistry, for whole-mount Schmidtea mediterranea samples.
Asunto(s)
Planarias , Animales , Hibridación Fluorescente in Situ , Planarias/genética , Hibridación in Situ , Colorantes/química , InmunohistoquímicaRESUMEN
Although most mammals heal injured tissues and organs with scarring, spiny mice (Acomys) naturally regenerate skin and complex musculoskeletal tissues. Now, the core signaling pathways driving mammalian tissue regeneration are poorly characterized. Here, we show that, while immediate extracellular signal-regulated kinase (ERK) activation is a shared feature of scarring (Mus) and regenerating (Acomys) injuries, ERK activity is only sustained at high levels during complex tissue regeneration. Following ERK inhibition, ear punch regeneration in Acomys shifted toward fibrotic repair. Using single-cell RNA sequencing, we identified ERK-responsive cell types. Loss- and gain-of-function experiments prompted us to uncover fibroblast growth factor and ErbB signaling as upstream ERK regulators of regeneration. The ectopic activation of ERK in scar-prone injuries induced a pro-regenerative response, including cell proliferation, extracellular matrix remodeling, and hair follicle neogenesis. Our data detail an important distinction in ERK activity between regenerating and poorly regenerating adult mammals and open avenues to redirect fibrotic repair toward regenerative healing.
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Cicatriz , Murinae , Animales , Cicatriz/patología , Quinasas MAP Reguladas por Señal Extracelular , Fibrosis , MamíferosRESUMEN
Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.
Asunto(s)
Calcio , Corazón , Miocitos Cardíacos , Regeneración , Sarcómeros , Pez Cebra , Animales , Calcio/fisiología , Proliferación Celular , Corazón/fisiología , Miocitos Cardíacos/fisiología , Sarcómeros/fisiología , Pez Cebra/fisiologíaRESUMEN
Ischemic heart disease and by extension myocardial infarction is the primary cause of death worldwide, warranting regenerative therapies to restore heart function. Current models of natural heart regeneration are restricted in that they are not of adult mammalian origin, precluding the study of class-specific traits that have emerged throughout evolution, and reducing translatability of research findings to humans. Here, we present the spiny mouse (Acomys spp.), a murid rodent that exhibits bona fide regeneration of the back skin and ear pinna, as a model to study heart repair. By comparing them to ordinary mice (Mus musculus), we show that the acute injury response in spiny mice is similar, but with an associated tolerance to infarction through superior survivability, improved ventricular conduction, and near-absence of pathological remodeling. Critically, spiny mice display increased vascularization, altered scar organization, and a more immature phenotype of cardiomyocytes, with a corresponding improvement in heart function. These findings present new avenues for mammalian heart research by leveraging unique tissue properties of the spiny mouse.
RESUMEN
BACKGROUND: Freshwater planarians are an attractive model for regeneration and stem cell research and have become a promising tool in the field of regenerative medicine. With the availability of a sequenced planarian genome, the recent application of modern genetic and high-throughput tools has resulted in revitalized interest in these animals, long known for their amazing regenerative capabilities, which enable them to regrow even a new head after decapitation. However, a detailed description of the planarian transcriptome is essential for future investigation into regenerative processes using planarians as a model system. RESULTS: In order to complement and improve existing gene annotations, we used a 454 pyrosequencing approach to analyze the transcriptome of the planarian species Schmidtea mediterranea Altogether, 598,435 454-sequencing reads, with an average length of 327 bp, were assembled together with the ~10,000 sequences of the S. mediterranea UniGene set using different similarity cutoffs. The assembly was then mapped onto the current genome data. Remarkably, our Smed454 dataset contains more than 3 million novel transcribed nucleotides sequenced for the first time. A descriptive analysis of planarian splice sites was conducted on those Smed454 contigs that mapped univocally to the current genome assembly. Sequence analysis allowed us to identify genes encoding putative proteins with defined structural properties, such as transmembrane domains. Moreover, we annotated the Smed454 dataset using Gene Ontology, and identified putative homologues of several gene families that may play a key role during regeneration, such as neurotransmitter and hormone receptors, homeobox-containing genes, and genes related to eye function. CONCLUSIONS: We report the first planarian transcript dataset, Smed454, as an open resource tool that can be accessed via a web interface. Smed454 contains significant novel sequence information about most expressed genes of S. mediterranea. Analysis of the annotated data promises to contribute to identification of gene families poorly characterized at a functional level. The Smed454 transcriptome data will assist in the molecular characterization of S. mediterranea as a model organism, which will be useful to a broad scientific community.
Asunto(s)
Bases de Datos Genéticas , Perfilación de la Expresión Génica , Planarias/genética , Análisis de Secuencia de ADN/métodos , Animales , Composición de Base/genética , Secuencia de Bases , Ciclo Celular/genética , División Celular/genética , Secuencia de Consenso/genética , Daño del ADN/genética , Reparación del ADN/genética , Ojo/metabolismo , Genoma/genética , Proteínas de Homeodominio/genética , Internet , Proteínas de la Membrana/genética , Anotación de Secuencia Molecular , Neurotransmisores/genética , Planarias/citología , Sitios de Empalme de ARN/genética , Receptores de Superficie Celular/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In metazoans, many developmental and disease-related processes are mediated by Wnt proteins, which are secreted by specific cells to regulate cellular programmes in the surrounding tissue. Although the Wnt-induced signal-transduction cascades are well studied, little is known about how Wnts are secreted. The discovery of Porcupine, an endoplasmic-reticulum-resident acyltransferase, led to closer inspection of the secretory routes of Wnts, and the analysis of Wnt secretion has become an exciting new area of research. Wnt post-translational modifications, interaction partners and subcellular localizations now indicate that Wnt release is tightly regulated. In this review, we summarize recent advances in the field of Wnt secretion and discuss the possibility that separate pathways might regulate the release of lipid-linked morphogens for short-range and long-range signalling.
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Proteínas Wnt/metabolismo , Acilación/fisiología , Animales , Transporte Biológico/fisiología , Humanos , Morfogénesis/fisiología , Transducción de Señal/fisiologíaRESUMEN
Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
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Nucleótidos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas del Citoesqueleto/deficiencia , Situs Inversus/genética , Adolescente , Adulto , Animales , Astenozoospermia/patología , Axonema/ultraestructura , Sistemas CRISPR-Cas/genética , Cilios/metabolismo , Cilios/ultraestructura , Proteínas del Citoesqueleto/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Epidídimo/patología , Femenino , Flagelos/metabolismo , Flagelos/ultraestructura , Humanos , Mutación con Pérdida de Función , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Planarias/citología , Planarias/genética , Planarias/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/patología , Situs Inversus/diagnóstico por imagen , Situs Inversus/patología , Motilidad Espermática/genética , Tomografía Computarizada por Rayos X , Secuenciación del ExomaRESUMEN
SILAC (stable isotope labeling by amino acids in cell culture) proteomics enables the relative quantification of proteins in one or more biological samples by mass spectrometry. This technology is based on the metabolic incorporation of heavy isotope-labeled essential amino acids into nascent proteins in vivo. Here, we describe the preparation of SILAC protein samples from planarians, flatworms with high regenerative potential and tissue plasticity. Applications for SILAC proteomics of planarians include the analysis of protein abundances, protein-protein interactions and turnover rates during stem cell-based regeneration and tissue homeostasis.
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Planarias/citología , Proteínas/metabolismo , Extractos de Tejidos/metabolismo , Aminoácidos/metabolismo , Animales , Técnicas de Cultivo de Célula , Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Mapas de Interacción de Proteínas/fisiología , Proteómica/métodosRESUMEN
Despite the identification of numerous regulators of regeneration in different animal models, a fundamental question remains: why do some wounds trigger the full regeneration of lost body parts, whereas others resolve by mere healing? By selectively inhibiting regeneration initiation, but not the formation of a wound epidermis, here we create headless planarians and finless zebrafish. Strikingly, in both missing-tissue contexts, injuries that normally do not trigger regeneration activate complete restoration of heads and fin rays. Our results demonstrate that generic wound signals have regeneration-inducing power. However, they are interpreted as regeneration triggers only in a permissive tissue context: when body parts are missing, or when tissue-resident polarity signals, such as Wnt activity in planarians, are modified. Hence, the ability to decode generic wound-induced signals as regeneration-initiating cues may be the crucial difference that distinguishes animals that regenerate from those that cannot.
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Sistema de Señalización de MAP Quinasas/genética , Planarias/genética , Regeneración/genética , Vía de Señalización Wnt/genética , Cicatrización de Heridas/genética , Pez Cebra/genética , Aletas de Animales/fisiología , Animales , Cabeza/fisiología , Planarias/fisiología , Regeneración/fisiología , Transducción de Señal , Cicatrización de Heridas/fisiología , Heridas y Lesiones , Pez Cebra/fisiologíaRESUMEN
Over the last years microarray technology has become one of the principal platform technologies for the high-throughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.