RESUMEN
Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Empleo , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Neuronas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Coordination of neurite morphogenesis with surrounding tissues is crucial to the establishment of neural circuits, but the underlying cellular and molecular mechanisms remain poorly understood. We show that neurons in a C. elegans sensory organ, called the amphid, undergo a collective dendrite extension to form the sensory nerve. The amphid neurons first assemble into a multicellular rosette. The vertex of the rosette, which becomes the dendrite tips, is attached to the anteriorly migrating epidermis and carried to the sensory depression, extruding the dendrites away from the neuronal cell bodies. Multiple adhesion molecules including DYF-7, SAX-7, HMR-1 and DLG-1 function redundantly in rosette-to-epidermis attachment. PAR-6 is localized to the rosette vertex and dendrite tips, and promotes DYF-7 localization and dendrite extension. Our results suggest a collective mechanism of neurite extension that is distinct from the classical pioneer-follower model and highlight the role of mechanical cues from surrounding tissues in shaping neurites.
Asunto(s)
Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Epidermis/metabolismoRESUMEN
C. elegans cell divisions that produce an apoptotic daughter cell exhibit Daughter Cell Size Asymmetry (DCSA), producing a larger surviving daughter cell and a smaller daughter cell fated to die. Genetic screens for mutants with defects in apoptosis identified several genes that are also required for the ability of these divisions to produce daughter cells that differ in size. One of these genes, ham-1, encodes a putative transcription factor that regulates a subset of the asymmetric cell divisions that produce an apoptotic daughter cell. In a survey of C. elegans divisions, we found that ham-1 mutations affect primarily anterior/posterior divisions that produce a small anterior daughter cell. The affected divisions include those that generate an apoptotic cell as well as those that generate two surviving cells. Our findings suggest that HAM-1 primarily promotes DCSA in a certain class of asymmetric divisions.
Asunto(s)
División Celular Asimétrica/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , División Celular/genética , Proteínas del Tejido Nervioso/genética , Animales , Apoptosis/genética , Linaje de la Célula/genética , Tamaño de la Célula , Supervivencia Celular/genética , Modelos Biológicos , Mutación , Neuronas/metabolismo , FenotipoRESUMEN
Formation and resolution of multicellular rosettes can drive convergent extension (CE) type cell rearrangements during tissue morphogenesis. Rosette dynamics are regulated by both planar cell polarity (PCP)-dependent and -independent pathways. Here we show that CE is involved in ventral nerve cord (VNC) assembly in Caenorhabditis elegans. We show that a VANG-1/Van Gogh and PRKL-1/Prickle containing PCP pathway and a Slit-independent SAX-3/Robo pathway cooperate to regulate, via rosette intermediaries, the intercalation of post-mitotic neuronal cell bodies during VNC formation. We show that VANG-1 and SAX-3 are localized to contracting edges and rosette foci and act to specify edge contraction during rosette formation and to mediate timely rosette resolution. Simultaneous loss of both pathways severely curtails CE resulting in a shortened, anteriorly displaced distribution of VNC neurons at hatching. Our results establish rosette-based CE as an evolutionarily conserved mechanism of nerve cord morphogenesis and reveal a role for SAX-3/Robo in this process.
Asunto(s)
Polaridad Celular/fisiología , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Caenorhabditis elegans , Movimiento Celular/fisiología , Proteínas RoundaboutRESUMEN
Developmental systems biology is poised to exploit large-scale data from two approaches: genomics and live imaging. The combination of the two offers the opportunity to map gene functions and gene networks in vivo at single-cell resolution using cell tracking and quantification of cellular phenotypes. Here we present Digital Development (http://www.digital-development.org), a database of cell lineage differentiation with curated phenotypes, cell-specific gene functions and a multiscale model. The database stores data from recent systematic studies of cell lineage differentiation in the C. elegans embryo containing â¼ 200 conserved genes, 1400 perturbed cell lineages and 600,000 digitized single cells. Users can conveniently browse, search and download four categories of phenotypic and functional information from an intuitive web interface. This information includes lineage differentiation phenotypes, cell-specific gene functions, differentiation landscapes and fate choices, and a multiscale model of lineage differentiation. Digital Development provides a comprehensive, curated, multidimensional database for developmental biology. The scale, resolution and richness of biological information presented here facilitate exploration of gene-specific and systems-level mechanisms of lineage differentiation in Metazoans.
Asunto(s)
Caenorhabditis elegans/embriología , Diferenciación Celular , Linaje de la Célula , Bases de Datos Factuales , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Modelos Biológicos , FenotipoRESUMEN
The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis.
Asunto(s)
Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Biología Computacional/métodos , Sistema Nervioso/citología , Sistema Nervioso/embriología , Neuronas/fisiología , Programas Informáticos , Animales , Rastreo Celular/métodos , Curaduría de DatosRESUMEN
BACKGROUND: Imaging and image analysis advances are yielding increasingly complete and complicated records of cellular events in tissues and whole embryos. The ability to follow hundreds to thousands of cells at the individual level demands a spatio-temporal data infrastructure: tools to assemble and collate knowledge about development spatially in a manner analogous to geographic information systems (GIS). Just as GIS indexes items or events based on their spatio-temporal or 4D location on the Earth these tools would organize knowledge based on location within the tissues or embryos. Developmental processes are highly context-specific, but the complexity of the 4D environment in which they unfold is a barrier to assembling an understanding of any particular process from diverse sources of information. In the same way that GIS aids the understanding and use of geo-located large data sets, software can, with a proper frame of reference, allow large biological data sets to be understood spatially. Intuitive tools are needed to navigate the spatial structure of complex tissue, collate large data sets and existing knowledge with this spatial structure and help users derive hypotheses about developmental mechanisms. RESULTS: Toward this goal we have developed WormGUIDES, a mobile application that presents a 4D developmental atlas for Caenorhabditis elegans. The WormGUIDES mobile app enables users to navigate a 3D model depicting the nuclear positions of all cells in the developing embryo. The identity of each cell can be queried with a tap, and community databases searched for available information about that cell. Information about ancestry, fate and gene expression can be used to label cells and craft customized visualizations that highlight cells as potential players in an event of interest. Scenes are easily saved, shared and published to other WormGUIDES users. The mobile app is available for Android and iOS platforms. CONCLUSION: WormGUIDES provides an important tool for examining developmental processes and developing mechanistic hypotheses about their control. Critically, it provides the typical end user with an intuitive interface for developing and sharing custom visualizations of developmental processes. Equally important, because users can select cells based on their position and search for information about them, the app also serves as a spatially organized index into the large body of knowledge available to the C. elegans community online. Moreover, the app can be used to create and publish the result of exploration: interactive content that brings other researchers and students directly to the spatio-temporal point of insight. Ultimately the app will incorporate a detailed time lapse record of cell shape, beginning with neurons. This will add the key ability to navigate and understand the developmental events that result in the coordinated and precise emergence of anatomy, particularly the wiring of the nervous system.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Sistema Nervioso/citología , Análisis de la Célula Individual/métodos , Programas Informáticos , Interfaz Usuario-Computador , Animales , Bases de Datos FactualesRESUMEN
The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Proteínas Contráctiles/genética , Proteínas de Microfilamentos/genética , Fosfoinositido Fosfolipasa C/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Señalización del Calcio/fisiología , Proteínas Contráctiles/metabolismo , Femenino , Filaminas , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Proteínas de Microfilamentos/metabolismo , Oocitos/metabolismo , Oocitos/fisiología , Fosfoinositido Fosfolipasa C/metabolismo , Espermatozoides/metabolismo , Espermatozoides/fisiologíaRESUMEN
Mechanical properties of the microenvironment are fundamental in orchestrating normal tissue function, disease progression, and organismal development. Studies of mechanotransduction in cultured cells on artificial substrates have revealed underlying principles, but the in vivo roles of mechanotransduction remain unclear. We recently reported that the Caenorhabditis elegans spermatheca-a myoepithelial tube composed of a cell monolayer-may be mechanosensitive. Live imaging with the genetically encoded calcium indicator GCaMP revealed that oocyte-induced stretching of the spermatheca resulted in calcium oscillations and constriction of the tube. FLN-1/filamin, a mechanosensitive cytoskeletal scaffolding protein, is required to correctly trigger the calcium transients. PLC-1/phospholipase C-epsilon and ITR-1/IP3 receptor are required to produce the calcium transients, and may function downstream of filamin. In addition to providing important insights into the biology of C. elegans, our studies offer a novel and genetically tractable model for studying mechanotransduction in a myoepithelial tissue.
RESUMEN
The Caenorhabditis elegans distal tip cells (DTCs) are an in vivo model for the study of developmentally regulated cell migration. In this study, we characterize a novel role for CCDC-55, a conserved coiled-coil domain containing protein, in DTC migration and larval development in C. elegans. Although animals homozygous for a probable null allele, ccdc-55(ok2851), display an early larval arrest, RNAi depletion experiments allow the analysis of later phenotypes and suggest that CCDC-55 is needed within the DTC for migration to cease at the end of larval morphogenesis. The ccdc-55 gene is found in an operon with rnf-121 and rnf-5, E3 ubiquitin ligases that target cell migration genes such as the ß-integrin PAT-3. Genetic interaction studies using RNAi depletion and the deletion alleles rnf-121(ok848) and rnf-5(tm794) indicate that CCDC-55 and the RNF genes act at least partially in parallel to promote termination of cell migration in the adult DTC.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/citología , Movimiento Celular , Alelos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Larva/citología , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Morfogénesis , Estructura Terciaria de Proteína , Interferencia de ARN , Homología de Secuencia de Aminoácido , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Filamins are long, flexible, multi-domain proteins composed of an N-terminal actin-binding domain (ABD) followed by multiple immunoglobulin-like repeats (IgFLN). They function to organize and maintain the actin cytoskeleton, to provide scaffolds for signaling components, and to act as mechanical force sensors. In this study, we used transcript sequencing and homology modeling to characterize the gene and protein structures of the C. elegans filamin orthologs fln-1 and fln-2. Our results reveal that C. elegans FLN-1 is well conserved at the sequence level to vertebrate filamins, particularly in the ABD and several key IgFLN repeats. Both FLN-1 and the more divergent FLN-2 colocalize with actin in vivo. FLN-2 is poorly conserved, with at least 23 IgFLN repeats interrupted by large regions that appear to be nematode-specific. Our results indicate that many of the key features of vertebrate filamins are preserved in C. elegans FLN-1 and FLN-2, and suggest the nematode may be a very useful model system for further study of filamin function.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas Contráctiles/química , Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/genética , Secuencia Conservada , Proteínas Contráctiles/genética , Filaminas , Regulación de la Expresión Génica , Genómica , Humanos , Inmunoglobulinas/química , Proteínas de Microfilamentos/genética , Modelos Moleculares , Estructura Terciaria de Proteína , Transporte de Proteínas , Homología Estructural de ProteínaRESUMEN
Filamin, known primarily for its actin cross-linking function, is a stretch-sensitive structural and signaling scaffold that binds transmembrane receptors and a wide variety of intracellular signaling proteins. The Caenorhabditis elegans filamin ortholog, FLN-1, has a well conserved overall structure, including an N-terminal actin-binding domain, and a series of 20 immunoglobulin (Ig)-like repeats. FLN-1 partially colocalizes with actin filaments in spermathecal and uterine cells. Analysis of phenotypes resulting from a deletion allele and RNAi depletion indicates FLN-1 is required to maintain the actin cytoskeleton in the spermatheca and uterus, and to allow the exit of embryos from the spermatheca. FLN-1 deficient animals accumulate embryos in the spermatheca, lay damaged and unfertilized eggs, and consequently exhibit dramatically reduced brood sizes. The phospholipase PLC-1 is also required for the exit of embryos from the spermatheca, and analysis of doubly mutant animals suggests that PLC-1 and FLN-1 act in the same pathway to promote proper transit of embryos from the spermatheca to the uterus. Given the modular protein structure, subcellular localization, genetic interaction with PLC-1, and known mechanosensory functions of filamin, we postulate that FLN-1 may be required to convert mechanical information about the presence of the oocyte into a biochemical signal, thereby allowing timely exit of the embryo from the spermatheca.
Asunto(s)
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Proteínas Contráctiles/fisiología , Filaminas/fisiología , Proteínas de Microfilamentos/fisiología , Oocitos/fisiología , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Contráctiles/antagonistas & inhibidores , Proteínas Contráctiles/química , Proteínas Contráctiles/genética , Femenino , Fertilidad/genética , Fertilidad/fisiología , Filaminas/antagonistas & inhibidores , Filaminas/química , Filaminas/genética , Genitales/crecimiento & desarrollo , Genitales/fisiología , Masculino , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Interferencia de ARN , Transducción de Señal/fisiologíaRESUMEN
The two specialized C. elegans distal tip cells (DTCs) provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC migration. RNAi depletion experiments and analysis of the hypomorphic allele cacn-1(tm3126) indicate that CACN-1 is required during DTC migration for proper pathfinding and for cessation of DTC migration at the end of larval morphogenesis. Strong expression of CACN-1 in the DTCs, and data from cell-specific RNAi depletion experiments, suggest that CACN-1 is required cell-autonomously to control DTC migration. Importantly, genetic interaction data with Rac GTPase activators and effectors suggest that CACN-1 acts specifically to inhibit the mig-2/Rac pathway, and in parallel to ced-10/Rac, to control DTC pathfinding.