RESUMEN
Temporal regulation of gene expression is a crucial aspect of metazoan development. In the roundworm Caenorhabditis elegans, the heterochronic pathway controls multiple developmental events in a time-specific manner. The most downstream effector of this pathway, the zinc-finger transcription factor LIN-29, acts in the last larval stage (L4) to regulate elements of the larval-to-adult switch. Here, we explore new LIN-29 targets and their implications for this developmental transition. We used RNA-sequencing to identify genes differentially expressed between animals misexpressing LIN-29 at an early time point and control animals. Among 230 LIN-29-activated genes, we found that genes encoding cuticle collagens were overrepresented. Interestingly, expression of lin-29 and some of these collagens was increased in adults with cuticle damage, suggesting a previously unknown function for LIN-29 in adult cuticle maintenance. On the other hand, genes involved in fat metabolism were enriched among 350 LIN-29-downregulated targets. We used mass spectrometry to assay lipid content in animals overexpressing LIN-29 and observed reduced fatty acid levels. Many LIN-29-repressed genes are normally expressed in the intestine, suggesting cell-nonautonomous regulation. We identified several LIN-29 upregulated genes encoding signaling molecules that may act as mediators in the regulation of intestinally expressed genes encoding fat metabolic enzymes and vitellogenins. Overall, our results support the model of LIN-29 as a major regulator of adult cuticle synthesis and integrity, and as the trigger for metabolic changes that take place at the important transition from rapid growth during larval life to slower growth and offspring production during adulthood.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Unión al ADN/genética , Metabolismo de los Lípidos/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Larva/genética , Larva/crecimiento & desarrollo , RNA-Seq , Vitelogeninas/genética , Dedos de Zinc/genéticaRESUMEN
The cuticle, the outer covering of the nematode C. elegans, is synthesized five times during the worm's life by the underlying hypodermis. Cuticle collagens, the major cuticle component, are encoded by a large family of col genes and, interestingly, many of these genes express predominantly at a single developmental stage. This temporal preference motivated us to investigate the mechanisms underlying col gene expression and here we focus on a subset of col genes expressed in the L4 stage. We identified minimal promoter regions of <300 bp for col-38, col-49, and col-63. In these regions, we predicted cis-regulatory sequences and evaluated their function in vivo via mutagenesis of a col-38p::yfp reporter. We used RNAi to study the requirement for candidate transcription regulators ELT-1 and ELT-3, LIN-29, and the LIN-29 co-factor MAB-10, and found LIN-29 to be necessary for the expression of four L4-specific genes (col-38, col-49, col-63, and col-138). Temporal misexpression of LIN-29 was also sufficient to activate these genes at a different developmental stage. The LIN-29 DNA-binding domain bound the col-38, col-49, and col-63 minimal promoters in vitro. For col-38 we showed that the LIN-29 sites necessary for reporter expression in vivo are also bound in vitro: this is the first identification of specific binding sites for LIN-29 necessary for in vivo target gene expression.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , ADN de Helmintos/metabolismo , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
The development of the single cell layer skin or hypodermis of Caenorhabditis elegans is an excellent model for understanding cell fate specification and differentiation. Early in C. elegans embryogenesis, six rows of hypodermal cells adopt dorsal, lateral or ventral fates that go on to display distinct behaviors during larval life. Several transcription factors are known that function in specifying these major hypodermal cell fates, but our knowledge of the specification of these cell types is sparse, particularly in the case of the ventral hypodermal cells, which become Vulval Precursor Cells and form the vulval opening in response to extracellular signals. Previously, the gene pvl-4 was identified in a screen for mutants with defects in vulval development. We found by whole genome sequencing that pvl-4 is the Paired-box gene pax-3, which encodes the sole PAX-3 transcription factor homolog in C. elegans. pax-3 mutants show embryonic and larval lethality, and body morphology abnormalities indicative of hypodermal cell defects. We report that pax-3 is expressed in ventral P cells and their descendants during embryogenesis and early larval stages, and that in pax-3 reduction-of-function animals the ventral P cells undergo a cell fate transformation and express several markers of the lateral seam cell fate. Furthermore, forced expression of pax-3 in the lateral hypodermal cells causes them to lose expression of seam cell markers. We propose that pax-3 functions in the ventral hypodermal cells to prevent these cells from adopting the lateral seam cell fate. pax-3 represents the first gene required for specification solely of the ventral hypodermal fate in C. elegans providing insights into cell type diversification.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Epidermis/metabolismo , Factores de Transcripción Paired Box/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linaje de la Célula/genética , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Células Epidérmicas , Epidermis/embriología , Femenino , Larva/citología , Larva/genética , Larva/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Factores de Transcripción Paired Box/genética , Interferencia de ARN , Vulva/citología , Vulva/embriología , Vulva/metabolismoRESUMEN
In metazoans, stem cells in developing and adult tissues can divide asymmetrically to give rise to a daughter that differentiates and a daughter that retains the progenitor fate. Although the short-lived nematode C. elegans does not possess adult somatic stem cells, the lateral hypodermal seam cells behave in a similar manner: they divide once per larval stage to generate an anterior daughter that adopts a non-dividing differentiated fate and a posterior daughter that retains the seam fate and the ability to divide further. Wnt signaling pathway is known to regulate the asymmetry of these divisions and maintain the progenitor cell fate in one daughter, but how activation of the Wnt pathway accomplished this was unknown. We describe here our recent work that identified the GATA transcription factor EGL-18 as a downstream target of Wnt signaling necessary for maintenance of a progenitor population of larval seam cells. EGL-18 was previously shown to act in the initial specification of the seam cells in the embryo. Thus the acquisition of a Wnt-responsive cis-regulatory module allows an embryonic fate specification factor to be reutilized later in life downstream of a different regulator (Wnt signaling) to maintain a progenitor cell population. These results support the use of seam cell development in C. elegans as a simple model system for studying stem and progenitor cell biology.
RESUMEN
The evolutionarily conserved Wnt/ß-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector ß-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/ß-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismo , Alelos , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Diferenciación Celular , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Genes Reporteros , Larva/citología , Larva/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Temperatura , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vulva/crecimiento & desarrollo , Vulva/metabolismo , Proteínas Wnt/genética , beta Catenina/genética , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
BACKGROUND: Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo. RESULTS: We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. CONCLUSIONS: These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción GATA/metabolismo , Proteínas de Homeodominio/genética , Tejido Subcutáneo/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ADN de Helmintos/genética , ADN de Helmintos/metabolismo , Proteínas de Unión al ADN/genética , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Femenino , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Larva/genética , Larva/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tejido Subcutáneo/embriología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Vulva/citología , Vulva/embriología , Vulva/metabolismoRESUMEN
The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Colágeno/genética , beta Catenina/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Larva/metabolismo , Fenotipo , ARN/aislamiento & purificación , ARN/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
The C. elegans seam cells are lateral epithelial cells arrayed in a single line from anterior to posterior that divide in an asymmetric, stem cell-like manner during larval development. These asymmetric divisions are regulated by Wnt signaling; in most divisions, the posterior daughter in which the Wnt pathway is activated maintains the progenitor seam fate, while the anterior daughter in which the Wnt pathway is not activated adopts a differentiated hypodermal fate. Using mRNA tagging and microarray analysis, we identified the functionally redundant GATA factor genes egl-18 and elt-6 as Wnt pathway targets in the larval seam cells. EGL-18 and ELT-6 have previously been shown to be required for initial seam cell specification in the embryo. We show that in larval seam cell asymmetric divisions, EGL-18 is expressed strongly in the posterior seam-fated daughter. egl-18 and elt-6 are necessary for larval seam cell specification, and for hypodermal to seam cell fate transformations induced by ectopic Wnt pathway overactivation. The TCF homolog POP-1 binds a site in the egl-18 promoter in vitro, and this site is necessary for robust seam cell expression in vivo. Finally, larval overexpression of EGL-18 is sufficient to drive expression of a seam marker in other hypodermal cells in wild-type animals, and in anterior hypodermal-fated daughters in a Wnt pathway-sensitized background. These data suggest that two GATA factors that are required for seam cell specification in the embryo independently of Wnt signaling are reused downstream of Wnt signaling to maintain the progenitor fate during stem cell-like divisions in larval development.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Factores de Transcripción GATA/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Wnt/metabolismo , Alelos , Animales , Diferenciación Celular , Linaje de la Célula , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Wnt signaling is an evolutionarily ancient pathway used to regulate many events during metazoan development. Genetic results from Caenorhabditis elegans more than a dozen years ago suggested that Wnt signaling in this nematode worm might be different than in vertebrates and Drosophila: the worm had a small number of Wnts, too many ß-catenins, and some Wnt pathway components functioned in an opposite manner than in other species. Work over the ensuing years has clarified that C. elegans does possess a canonical Wnt/ß-catenin signaling pathway similar to that in other metazoans, but that the majority of Wnt signaling in this species may proceed via a variant Wnt/ß-catenin signaling pathway that uses some new components (mitogen-activated protein kinase signaling enzymes), and in which some conserved pathway components (ß-catenin, T-cell factor [TCF]) are used in new and interesting ways. This review summarizes our current understanding of the canonical and novel TCF/ß-catenin-dependent signaling pathways in C. elegans.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Proteínas del Citoesqueleto/metabolismo , Inducción Embrionaria/fisiología , Endodermo/embriología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Movimiento Celular/fisiología , Modelos Biológicos , Tejido Nervioso/citología , Tejido Nervioso/embriologíaRESUMEN
Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the ß-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas del Citoesqueleto/fisiología , Proteínas de Unión al ADN/fisiología , Células Epiteliales/citología , Proteínas del Grupo de Alta Movilidad/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/fisiología , Proteínas Wnt/fisiología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Recuento de Células , Diferenciación Celular , División Celular , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Genes Reporteros , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Proteínas del Grupo de Alta Movilidad/deficiencia , Proteínas del Grupo de Alta Movilidad/genética , Larva , Interferencia de ARN , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Proteínas Represoras/fisiología , Transducción de Señal/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
Although the epidermal growth factor receptor (EGFR) signaling pathway is overactive in more than half of human cancers and mediates resistance to cytotoxic therapy, the molecular mechanisms of EGFR pathway-mediated resistance have remained elusive in cancer research. This difficulty partly stems from the lack of tissue models enabling clear separation of the many forms of cell death that the downstream signaling pathways of EGFR affect. We have created a model in Caenorhabditis elegans of radiation-induced reproductive cell death ("Radelegans") in isolation of all other forms of cell death. We have employed Radelegans to genetically define the role of the EGFR signaling pathway in protection from reproductive cell death, the primary form of tumor stem or clonogen cell death postirradiation. We have found that the RAS/mitogen-activated protein kinase (MAPK) downstream signal transduction pathway of EGFR is critical for protection from reproductive cell death in Radelegans. In addition, we have shown that RAS/MAPK pathway signaling is genetically linear with the DNA damage response pathway and acts downstream of the DNA damage checkpoint in the radioresponse, implicating this pathway in DNA repair post-cytotoxic therapy. These findings support the hypothesis that enhanced repair is a mechanism of RAS/MAPK pathway-mediated resistance to cytotoxic therapy through its interaction with the DNA damage response pathway postirradiation. We postulate that these findings also help explain why current treatment strategies, based on the presumption that tumors have ineffective repair compared with normal tissues, are ineffective in EGFR/RAS/MAPK pathway-mediated tumors. Radelegans is a platform to further define the genetic basis of the radiation response in tissues.
Asunto(s)
Apoptosis/efectos de la radiación , Caenorhabditis elegans/efectos de la radiación , Daño del ADN , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas ras/fisiología , Animales , Receptores ErbB/fisiología , Tolerancia a RadiaciónRESUMEN
In Caenorhabditis elegans, vulval precursor cell (VPC) fate is specified by the action of RTK/Ras, Notch and Wnt signaling pathways. While the identity of signals for the Ras and Notch pathways is known, the source and identity of the Wnt ligand acting on the VPCs are unknown. Single mutations in any of the five Wnt genes (lin-44, cwn-1, cwn-2, egl-20 and mom-2) do not cause strong defects in VPC fate specification, suggesting that functionally redundant Wnts are required. Surprisingly, we found that all five Wnts influence VPC fate. The strongest defects we observed were in the lin-44; cwn-1; egl-20 triple mutant. Anterior VPCs were more strongly affected by loss of Wnt function than posterior VPCs, and expression from WntColon, two colonsGFP transcriptional reporters showed that the Wnts most strongly affecting VPC fate were expressed predominantly in the posterior, suggesting that some of the redundant Wnt ligands act over a distance to affect the VPCs. In addition to ligand redundancy, we found that at least three Wnt receptors, lin-17, mom-5 and mig-1, function in the VPCs. We also examined ligand and receptor function in another Wnt-mediated vulval process, the orientation of the P7.p lineage. Here, too, we found that four of five Wnt receptors can influence P7.p orientation, suggesting that a surprising amount of functional redundancy exists in Wnt signaling during C. elegans vulval induction.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/embriología , Proteínas Wnt/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciación Celular , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Glucógeno Sintasa Quinasa 3/fisiología , Modelos Biológicos , Proteínas Quinasas/fisiología , Transducción de Señal , Células Madre , Vulva/embriología , Vulva/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Caenorhabditis elegans , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Datos de Secuencia Molecular , Neuronas/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de Ácido NucleicoRESUMEN
Extracellular signaling pathways and transcriptional regulatory networks function during development to specify metazoan cell fates. During Caenorhabditis elegans vulval development, the specification of three vulval precursor cells (VPCs) requires the activity of Wnt, Notch, and Ras signaling pathways, and function of the Hox gene lin-39. LIN-39 protein levels are regulated in the VPCs by both Wnt and Ras signaling. In particular, activation of Ras signaling leads to an increase in LIN-39 protein in P6.p at the time of VPC fate specification. We wish to understand the regulation of lin-39 by these pathways. We first show that LIN-39 is a target for MAP kinase in vitro, suggesting that the Ras-dependent LIN-39 upregulation could be mediated post-translationally. To test this idea, we created transcriptional and translational lin-39::GFP fusions that include the entire lin-39 genomic region, allowing observation of lin-39 expression in live animals. The reporters express GFP in most, if not all, sites of expression previously observed by LIN-39 antibody staining. We used these constructs to show that at the time of vulval induction both lin-39::GFP reporters are upregulated in P6.p, indicating that the accumulation of high levels of LIN-39 protein detected previously corresponds to transcriptional upregulation of lin-39 expression. This transcriptional upregulation of lin-39 is dependent on Ras signaling. We tested the requirement for several transcription factors acting downstream of Ras signaling in the VPCs, and found that P6.p upregulation requires the transcription factors LIN-1 and LIN-25, but appears to be independent of LIN-31, SEM-4, EOR-1 and EOR-2.Finally, we found that when the Wnt pathway is over activated, expression from the transcriptional lin-39::GFP increases, suggesting that the Wnt pathway also regulates lin-39 at the transcriptional level.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Vulva/crecimiento & desarrollo , Proteínas ras/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciación Celular/genética , Femenino , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Células Madre/metabolismo , Células Madre/fisiología , Transcripción Genética , Regulación hacia Arriba , Vulva/citología , Proteínas Wnt/metabolismo , Proteínas ras/genéticaRESUMEN
The use of Wnt ligands for signaling between cells is a conserved feature of metazoan development. Activation of Wnt signal transduction pathways upon ligand binding can regulate diverse processes including cell proliferation, migration, polarity, differentiation and axon outgrowth. A 'canonical' Wnt signaling pathway has been elucidated in vertebrate and invertebrate model systems. In the canonical pathway, Wnt binding leads to the stabilization of the transcription factor beta-catenin, which enters the nucleus to regulate Wnt pathway target genes. However, Wnt binding also acts through beta-catenin-independent, noncanonical pathways, such as the planar cell polarity (PCP) pathway and a pathway involving Ca2+ signaling. This chapter examines our current understanding of Wnt signaling and Wnt-mediated processes in the nematode C. elegans. Like other species, the C. elegans genome encodes multiple genes for Wnt ligands (five) and Wnt receptors (four frizzleds, one Ryk/Derailed). Unlike vertebrates or Drosophila, the C. elegans genome encodes three beta-catenin genes, which appear to have distinct functions in Wnt signaling and cell adhesion. Canonical Wnt signaling clearly exists in C. elegans, utilizing the beta-catenin BAR-1. However, a noncanonical pathway utilizing the beta-catenin WRM-1 also exists, and to date a similar pathway has not been described in other species. Evidence for beta-catenin independent noncanonical Wnt signaling is currently limited. The role of Wnt signaling in over a dozen C. elegans developmental processes, including the regulation of cell fate, polarity and migration, is described.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismoRESUMEN
Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. Here we describe pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Unión al Calcio/genética , Caspasas/genética , Cisteína Endopeptidasas/genética , Genes de Helminto , Animales , Apoptosis , Muerte CelularRESUMEN
Development of the vulva in C. elegans is mediated by the combinatorial action of several convergent regulatory inputs, three of which, the Ras, Wnt and Rb-related pathways, act by regulating expression of the lin-39 Hox gene. LIN-39 specifies cell fates and regulates cell fusion in the mid-body region, leading to formation of the vulva. In the lateral seam epidermis, differentiation and cell fusion have been shown to be regulated by two GATA-type transcription factors, ELT-5 and -6. We report that ELT-5 is encoded by the egl-18 gene, which was previously shown to promote formation of a functional vulva. Furthermore, we find that EGL-18 (ELT-5), and its paralogue ELT-6, are redundantly required to regulate cell fates and fusion in the vulval primordium and are essential to form a vulva. Elimination of egl-18 and elt-6 activity results in arrest by the first larval stage; however, in animals rescued for this larval lethality by expression of ELT-6 in non-vulval cells, the post-embryonic cells (P3.p-P8.p) that normally become vulval precursor cells often fuse with the surrounding epidermal syncytium or undergo fewer than normal cell divisions, reminiscent of lin-39 mutants. Moreover, egl-18/elt-6 reporter gene expression in the developing vulva is attenuated in lin-39(rf) mutants, and overexpression of egl-18 can partially rescue the vulval defects caused by reduced lin-39 activity. LIN-39/CEH-20 heterodimers bind two consensus HOX/PBC sites in a vulval enhancer region of egl-18/elt-6, one of which is essential for vulval expression of egl-18/elt-6 reporter constructs. These findings demonstrate that the EGL-18 and ELT-6 GATA factors are essential, genetically redundant regulators of cell fates and fusion in the developing vulva and are apparent direct transcriptional targets of the LIN-39 Hox protein.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Vulva/crecimiento & desarrollo , Animales , Sitios de Unión , Proteínas de Caenorhabditis elegans/genética , Diferenciación Celular/fisiología , Fusión Celular , Proteínas de Unión al ADN/genética , Dimerización , Elementos de Facilitación Genéticos , Femenino , Factores de Transcripción GATA , Proteínas Fluorescentes Verdes , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas de Homeodominio/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Neuronas/citología , Neuronas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Vulva/anomalías , Vulva/metabolismoRESUMEN
During Caenorhabditis elegans vulval development, activation of receptor tyrosine kinase/Ras and Notch signaling pathways causes three vulval precursor cells (VPCs) to adopt induced cell fates. A Wnt signaling pathway also acts in cell fate specification by the VPCs, via regulation of the Hox gene lin-39. We show here that either mutation of pry-1 or expression of an activated BAR-1 beta-catenin protein causes an Overinduced phenotype, in which greater than three VPCs adopt induced cell fates. This indicates that pry-1, which encodes a C. elegans axin homolog, acts as a negative regulator of Wnt signaling in the VPCs. Loss of activity of the APC homolog apr-1 increases the penetrance of this Overinduced phenotype, suggesting that APR-1 may play a negative role in Wnt signaling in this process in C. elegans similar to APC proteins in other systems. The Overinduced phenotype is suppressed by reduction of function of the genes pop-1 TCF and lin-39 Hox. Surprisingly, the Overinduced phenotype caused by hyperactivated Wnt signaling is not dependent on signaling through the Ras pathway. These data suggest that hyperactivation of Wnt signaling is sufficient to cause VPCs to adopt induced fates and that a canonical Wnt pathway may play an important role during C. elegans vulval induction.