RESUMO
The architecture of gene regulatory networks determines the specificity and fidelity of developmental outcomes. We report that the core regulatory circuitry for endoderm development in Caenorhabditis elegans operates through a transcriptional cascade consisting of six sequentially expressed GATA-type factors that act in a recursive series of interlocked feedforward modules. This structure results in sequential redundancy, in which removal of a single factor or multiple alternate factors in the cascade leads to a mild or no effect on gut development, whereas elimination of any two sequential factors invariably causes a strong phenotype. The phenotypic strength is successfully predicted with a computational model based on the timing and levels of transcriptional states. We found that one factor in the middle of the cascade, END-1, which straddles the distinct events of specification and differentiation, functions in both processes. Finally, we reveal roles for key GATA factors in establishing spatial regulatory state domains by repressing other fates, thereby defining boundaries in the digestive tract. Our findings provide a paradigm that could account for the genetic redundancy observed in many developmental regulatory systems.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Endoderma/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Lógica , Fatores de Transcrição/metabolismoRESUMO
Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN in Caenorhabditis elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.
Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Redes Reguladoras de Genes , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In addition to performing digestion and nutrient absorption, the intestine serves as one of the first barriers to the external environment, crucial for protecting the host from environmental toxins, pathogenic invaders, and other stress inducers. The gene regulatory network (GRN) governing embryonic development of the endoderm and subsequent differentiation and maintenance of the intestine has been well-documented in C. elegans. A key regulatory input that initiates activation of the embryonic GRN for endoderm and mesoderm in this animal is the maternally provided SKN-1 transcription factor, an ortholog of the vertebrate Nrf1 and 2, which, like C. elegans SKN-1, perform conserved regulatory roles in mediating a variety of stress responses across metazoan phylogeny. Other key regulatory factors in early gut development also participate in stress response as well as in innate immunity and aging and longevity. In this review, we discuss the intersection between genetic nodes that mediate endoderm/intestine differentiation and regulation of stress and homeostasis. We also consider how direct signaling from the intestine to the germline, in some cases involving SKN-1, facilitates heritable epigenetic changes, allowing transmission of adaptive stress responses across multiple generations. These connections between regulation of endoderm/intestine development and stress response mechanisms suggest that varying selective pressure exerted on the stress response pathways may influence the architecture of the endoderm GRN, thereby leading to genetic and epigenetic variation in early embryonic GRN regulatory events.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/embriologia , Endoderma/embriologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Longevidade , Estresse Fisiológico , AnimaisRESUMO
Our ability to explore the cosmos by direct contact has been limited to a small number of lunar and interplanetary missions. However, the NASA Starlight program points a path forward to send small, relativistic spacecraft far outside our solar system via standoff directed-energy propulsion. These miniaturized spacecraft are capable of robotic exploration but can also transport seeds and organisms, marking a profound change in our ability to both characterize and expand the reach of known life. Here we explore the biological and technological challenges of interstellar space biology, focusing on radiation-tolerant microorganisms capable of cryptobiosis. Additionally, we discuss planetary protection concerns and other ethical considerations of sending life to the stars.
RESUMO
We report that Notch signaling is essential for the switch from developmental plasticity to commitment during Caenorhabditis elegans embryogenesis. The GLP-1 and LIN-12 Notch receptors act to set a memory state that affects commitment of cells arising from the major ectodermal progenitor (AB blastomere) several cell divisions later, thereby preventing their forced reprogramming by an endoderm-determining transcription factor. In contrast to Notch-dependent cell fate induction, this activity is autonomous to the AB lineage, is independent of the known cell fate-inducing Notch ligands, and requires a putative secreted Notch ligand, Delta Serrate Lag-3 (DSL-3). Thus, Notch signaling promotes developmental commitment by a mechanism that is distinct from that involved in specifying cell fates.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Diferenciação Celular , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Reprogramação Celular , Desenvolvimento Embrionário , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
The two GATA transcription factors ELT-2 and ELT-7 function in the differentiation of the C. elegans intestine. ELT-2 loss causes lethality. ELT-7 loss causes no obvious phenotype but enhances the elt-2(-) intestinal phenotype. Thus, ELT-2 and ELT-7 appear partially redundant, with ELT-2 being more influential. To investigate the different regulatory roles of ELT-2 and ELT-7, we compared the transcriptional profiles of pure populations of wild-type, elt-2(-), elt-7(-), and elt-7(-); elt-2(-) double mutant L1-stage larvae. Consistent with the mutant phenotypes, loss of ELT-2 had a>25 fold greater influence on the number of significantly altered transcripts compared to the loss of ELT-7; nonetheless, the levels of numerous transcripts changed upon loss of ELT-7 in the elt-2(-) background. The quantitative responses of individual genes revealed a more complicated behaviour than simple redundancy/partial redundancy. In particular, genes expressed only in the intestine showed three distinguishable classes of response in the different mutant backgrounds. One class of genes responded as if ELT-2 is the major transcriptional activator and ELT-7 provides variable compensatory input. For a second class, transcript levels increased upon loss of ELT-2 but decreased upon further loss of ELT-7, suggesting that ELT-7 actually overcompensates for the loss of ELT-2. For a third class, transcript levels also increased upon loss of ELT-2 but remained elevated upon further loss of ELT-7, suggesting overcompensation by some other intestinal transcription factor(s). In spite of its minor loss-of-function phenotype and its limited sequence similarity to ELT-2, ELT-7 expressed under control of the elt-2 promoter is able to rescue elt-2(-) lethality. Indeed, appropriately expressed ELT-7, like appropriately expressed ELT-2, is able to replace all other core GATA factors in the C. elegans endodermal pathway. Overall, this study focuses attention on the quantitative intricacies behind apparent redundancy or partial redundancy of two related transcription factors.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Endoderma/metabolismo , Fatores de Transcrição GATA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Fatores de Transcrição GATA/deficiência , Fatores de Transcrição GATA/genética , Genes de Helmintos , Genes Reporter , Estudos de Associação Genética , Intestinos/citologia , Larva , Regiões Promotoras Genéticas , Transcrição Gênica , TranscriptomaRESUMO
The differentiated cell identities and structure of fully formed organs are generally stable after their development. In contrast, we report here that development of the C. elegans proximal somatic gonad (hermaphrodite uterus and spermathecae, and male vas deferens) can be redirected into intestine-like organs by brief expression of the ELT-7 GATA transcription factor. This process converts one developing organ into another and can hence be considered "transorganogenesis." We show that, following pulsed ELT-7 expression, cells of the uterus activate and maintain intestine-specific gene expression and are transformed at the ultrastructural level to form an epithelial tube resembling the normal intestine formed during embryogenesis. Ubiquitous ELT-7 expression activates intestinal markers in many different cell types but only cells in the somatic gonad and pharynx appear to become fully reprogrammed. We found that ectopic expression of other endoderm-promoting transcription factors, but not muscle- or ectoderm- promoting transcription factors, redirects the fate of these organs, suggesting that pharyngeal and somatic gonad cells are specifically competent to adopt intestine identity. Although the intestine, pharynx, and somatic gonad are derived from distant cell lineages, they all express the PHA-4/FoxA transcription factor. While we found that post-embryonic PHA-4 is not necessary for pharynx or uterus reprogramming and PHA-4 is not sufficient in combination with ELT-7 to induce reprogramming in other cells types, knock down of PHA-4 during embryogenesis, which abolishes normal pharynx differentiation, prevents pharyngeal precursors from being reprogrammed into intestine. These results suggest that differentiated cell identity determines susceptibility to transdifferentiation and highlight the importance of cellular context in controlling competency for reprogramming.
Assuntos
Caenorhabditis elegans/citologia , Transdiferenciação Celular , Organogênese , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proliferação de Células , Reprogramação Celular , Embrião não Mamífero/citologia , Endoderma/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Imageamento Tridimensional , Intestinos/citologia , Masculino , Músculos/citologia , Faringe/citologia , Fatores de TempoRESUMO
Terminally differentiated post-mitotic cells are generally considered irreversibly developmentally locked, i.e. incapable of being reprogrammed in vivo into entirely different cell types. We found that brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, highly specialized non-endodermal cells of the pharynx into fully differentiated intestinal cells in intact larvae and adult Caenorhabditis elegans. Stable expression of intestine-specific molecular markers parallels loss of markers for the original differentiated pharynx state; hence, there is no apparent requirement for a dedifferentiated intermediate during the transdifferentiation process. Based on high-resolution morphological characteristics, the transdifferentiated cells become remodeled to resemble typical intestinal cells at the level of both the cell surface and internal organelles. Thus, post-mitotic cells, though terminally differentiated, remain plastic to transdifferentiation across germ layer lineage boundaries and can be remodeled to adopt the characteristics of a new cell identity without removal of inhibitory factors. Our findings establish a simple model to investigate how cell context influences forced transdifferentiation of mature cells.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Transdiferenciação Celular , Fatores de Transcrição GATA/metabolismo , Intestinos/citologia , Faringe/citologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Linhagem da Célula , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Faringe/embriologia , Faringe/metabolismoRESUMO
Anatomical left-right (L/R) asymmetry in C. elegans is established in the four-cell embryo as a result of anteroposterior skewing of transverse mitotic spindles with a defined handedness. This event creates a chiral embryo and ultimately an adult body plan with fixed L/R positioning of internal organs and components of the nervous system. While this "dextral" configuration is invariant in hermaphrodites, it can be reversed by physical manipulation of the early embryo or by mutations that interfere with mitotic spindle orientation, which leads to viable, mirror-reversed (sinistral) animals. During normal development of the C. elegans male, the gonad develops on the right of the midline, with the gut bilaterally apposed on the left. However, we found that in males of the laboratory N2 strain and Hawaiian ("Hw") wild isolate, the gut/gonad asymmetry is frequently reversed in a temperature-dependent manner, independent of normal embryonic chirality. We also observed sporadic errors in gonad migration occurring naturally during early larval stages of these and other wild strains; however, the incidence of such errors does not correlate with the frequency of L/R gut/gonad reversals in these strains. Analysis of N2/Hw hybrids and recombinant inbred advanced intercross lines (RIAILs) indicate that the L/R organ reversals are likely to result from recessively acting variations in multiple genes. Thus, unlike the highly reproducible L/R asymmetries of most structures in hermaphrodites, the L/R asymmetry of the male C. elegans body plan is less rigidly determined and subject to natural variation that is influenced by a multiplicity of genes.
Assuntos
Padronização Corporal/fisiologia , Caenorhabditis elegans/embriologia , Gônadas/embriologia , Organogênese/fisiologia , Animais , Desenvolvimento Embrionário/fisiologia , MasculinoRESUMO
The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes.1-3 To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on transcriptional profiling4, 5 and RNA interference (RNAi)6 to define molecular targets. Such studies require efficient, accurate, and robust RNA extraction methods; however, obtaining high-quality quantitative levels of RNA from H. exemplaris has been challenging6, 7. Possessing a layer of firm chitinous cuticle, tardigrade tissues are difficult to disrupt by chemical or mechanical means8. Here we present an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but dramatically reduces the time required for extraction. We show that this RNA extraction method yields robust quantities of cDNA and can be used to amplify multiple transcripts by qRT-PCR. To validate the method, we use it to compare dynamic changes in expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 ß2 (HSP70ß2) and Heat-Shock Protein 90α (HSP90α) by quantifying their expression levels in heat-exposed and cold-exposed individuals using qRT-PCR across long-term and short-term heat stressors. Our method effectively complements existing bulk RNA extraction methods7, permitting rapid examination of individual tardigrade transcriptional data and quantification of phenotypic variations in expression profiles amongst individuals.
RESUMO
The Wnt signaling pathway must be properly modulated to ensure an appropriate output: pathological conditions result from either insufficient or excessive levels of Wnt signal. For example, hyperactivation of the Wnt pathway is associated with various cancers and subnormal Wnt signaling can lead to increased invasiveness of tumor cells. We found that the Caenorhabditis elegans ortholog of the Fer nonreceptor tyrosine kinase, FRK-1, limits Wnt signaling by preventing the adhesion complex-associated ß-catenin, HMP-2, from participating in Wnt-dependent specification of the endoderm during embryogenesis. Removal of FRK-1 function results in relocalization of HMP-2 to the nucleus of epidermal cells, and allows it to substitute for WRM-1, the nuclear ß-catenin that normally transduces the Wnt signal during endoderm development. APR-1, the C. elegans APC ortholog, is similarly required to prevent HMP-2 relocalization and keeps it from participating in Wnt signal transduction; this finding partially explains the paradoxical observation that APR-1 acts either negatively or positively in Wnt signaling, depending on context. The apparent hyperactivation of the Wnt response in the absence of FRK-1 leads to hyperproliferation in the endoderm, as is also seen when WRM-1 is overexpressed in wild-type embryos. The specification and proliferation activities of Wnt signaling are separable: although the Tcf/Lef factor POP-1 acts in Wnt-dependent endoderm specification, it is not apparently required for hyperproliferation resulting from excessive Wnt signaling. These findings highlight a role for a Fer-type kinase in setting the proper levels of Wnt signaling and demonstrate the importance of this modulation in ensuring appropriate cell division.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Tirosina Quinases/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The odd-2 gene in C. elegans is an orthologue of the odd-skipped gene in Drosophila and the odd-skipped related genes in mammals. The mammalian genes have been shown to be expressed in a variety of tissues and cancers. It was previously reported that ODD-2 is expressed in the intestine and shows some expression outside of the intestine in the tail region. Using a partial ODD-2 ::GFP fusion, we hypothesize that the expression outside of the intestine may be in rectal gland cells, and we also report that ODD-2 may be expressed in the germline sheath cells.
RESUMO
The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). 'Purifying selection' mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1-kb mtDNA deletion mutation, uaDf5, which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNAuaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNAuaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Apoptose/genética , Caspases/genética , Caspases/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Envelhecimento/genéticaRESUMO
Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates movement. Additionally, gravity sensation, which occurs in the mammalian inner ear, is closely related to hearing - thus, understanding this process has implications for auditory and vestibular research. Gravitaxis assays exist for some model organisms, including Drosophila. Single worms have previously been assayed for their orientation preference as they settle in solution. However, a reliable and robust assay for Caenorhabditis gravitaxis has not been described. The present protocol outlines a procedure for performing gravitaxis assays that can be used to test hundreds of Caenorhabditis dauers at a time. This large-scale, long-distance assay allows for detailed data collection, revealing phenotypes that may be missed on a standard plate-based assay. Dauer movement along the vertical axis is compared with horizontal controls to ensure that directional bias is due to gravity. Gravitactic preference can then be compared between strains or experimental conditions. This method can determine molecular, cellular, and environmental requirements for gravitaxis in worms.
Assuntos
Caenorhabditis , Resposta Táctica , Animais , Gravitação , Sensação Gravitacional , Larva , MamíferosRESUMO
The transition from specification of cell identity to the differentiation of cells into an appropriate and enduring state is critical to the development of embryos. Transcriptional profiling in Caenorhabditis elegans has revealed a large number of genes that are expressed in the fully differentiated intestine; however, no regulatory factor has been found to be essential to initiate their expression once the endoderm has been specified. These gut-expressed genes possess a preponderance of GATA factor binding sites and one GATA factor, ELT-2, fulfills the expected characteristics of a key regulator of these genes based on its persistent expression exclusively in the developing and differentiated intestine and its ability to bind these regulatory sites. However, a striking characteristic of elt-2(0) knockout mutants is that while they die shortly after hatching owing to an obstructed gut passage, they nevertheless contain a gut that has undergone complete morphological differentiation. We have discovered a second gut-specific GATA factor, ELT-7, that profoundly synergizes with ELT-2 to create a transcriptional switch essential for gut cell differentiation. ELT-7 is first expressed in the early endoderm lineage and, when expressed ectopically, is sufficient to activate gut differentiation in nonendodermal progenitors. elt-7 is transcriptionally activated by the redundant endoderm-specifying factors END-1 and -3, and its product in turn activates both its own expression and that of elt-2, constituting an apparent positive feedback system. While elt-7 loss-of-function mutants lack a discernible phenotype, simultaneous loss of both elt-7 and elt-2 results in a striking all-or-none block to morphological differentiation of groups of gut cells with a region-specific bias, as well as reduced or abolished gut-specific expression of a number of terminal differentiation genes. ELT-2 and -7 synergize not only in activation of gene expression but also in repression of a gene that is normally expressed in the valve cells, which immediately flank the termini of the gut tube. Our results point to a developmental strategy whereby positive feedback and cross-regulatory interactions between two synergistically acting regulatory factors promote a decisive and persistent transition of specified endoderm progenitors into the program of intestinal differentiation.
Assuntos
Padronização Corporal , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Diferenciação Celular , Endoderma/embriologia , Fatores de Transcrição GATA/metabolismo , Animais , Biomarcadores/metabolismo , Caenorhabditis elegans/metabolismo , Linhagem da Célula , Sistema Digestório/citologia , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Junções Intercelulares/metabolismo , Especificidade de Órgãos/genéticaRESUMO
BACKGROUND: Cell-size-control systems, coupled with apoptotic- and cell-proliferation-regulatory mechanisms, determine the overall dimensions of organs and organisms, and their dysregulation can lead to tumor formation. The interrelationship between cell-growth-regulatory mechanisms and apoptosis during normal development and cancer is not understood. The TRK-fused gene (TFG) promotes tumorigenesis when present in chromosomal rearrangements from various human-cancer types by unknown mechanisms. Apaf1/CED-4 is essential for apoptosis but has not been shown to function in cell-growth control. RESULTS: We found that loss of TFG-1, the TFG ortholog in Caenorhabditis elegans, results in supernumerary apoptotic corpses, whereas its overexpression is sufficient to inhibit developmentally programmed cell death. TFG-1 is also required for cells and nuclei to grow to normal size. Furthermore, we found that CED-4 is required for cell-growth inhibition in animals lacking TFG-1. However, caspases, the downstream effectors of CED-4-mediated apoptosis, are not required in TFG-1- or CED-4-regulated cell-size control. CED-4 acts to inhibit cell growth by antagonizing the effects of other conserved cell-size-regulating proteins, including cAMP response element binding (CREB) protein, translation-initiation factor eIF2B, and the nucleolar p53-interacting protein nucleostemin. CONCLUSIONS: These findings show that TFG-1 suppresses apoptosis and is essential for normal cell-size control, suggesting that abnormalities in the cell-growth-promoting and apoptosis-inhibiting functions of TFG might be responsible for its action in tumorigenesis. Also, they reveal that CED-4 plays a pivotal role in activating apoptosis and restricting cell and nuclear size, thereby determining the appropriate overall size of an animal. Thus, these findings reveal links between the control mechanisms for apoptosis and cell growth.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Ligação ao Cálcio/genética , Animais , Apoptose/genética , Apoptose/fisiologia , Tamanho Corporal/genética , Tamanho Corporal/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/fisiologia , Tamanho Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/fisiologia , Genes de Helmintos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proto-Oncogene Mas , Proto-Oncogenes , Interferência de RNARESUMO
We review the application of Caenorhabditis elegans as a model system to understand key aspects of stem cell biology. The only bona fide stem cells in C. elegans are those of the germline, which serves as a valuable paradigm for understanding how stem-cell niches influence maintenance and differentiation of stem cells and how somatic differentiation is repressed during germline development. Somatic cells that share stem cell-like characteristics also provide insights into principles in stem-cell biology. The epidermal seam cell lineages lend clues to conserved mechanisms of self-renewal and expansion divisions. Principles of developmental plasticity and reprogramming relevant to stem-cell biology arise from studies of natural transdifferentiation and from analysis of early embryonic progenitors, which undergo a dramatic transition from a pluripotent, reprogrammable condition to a state of committed differentiation. The relevance of these developmental processes to our understanding of stem-cell biology in other organisms is discussed.
Assuntos
Células-Tronco/citologia , Animais , Caenorhabditis elegans , Linhagem da Célula , Células Germinativas/citologiaRESUMO
Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.
Assuntos
Caenorhabditis elegans , Medições Luminescentes , Animais , Caenorhabditis elegans/genética , Diagnóstico por Imagem , Luciferinas , NeurôniosRESUMO
The endoderm of C. elegans arises entirely from a single progenitor cell, the E blastomere, whose identity is specified by GATA type transcription factors, including END-1. In response to an inductive interaction mediated through Wnt/MAP kinase signaling pathways, POP-1, a Lef/Tcf-type transcription factor, restricts end-1 transcription to the posterior daughter of the mesendoderm progenitor (EMS cell), resulting in activation of endoderm differentiation by the SKN-1 and MED-1/2 transcription factors. We purified a factor from semi-synchronized early embryos that binds to an end-1 cis regulatory region critical for its endoderm-specific expression. Mass spectrometry identified this protein, PLP-1, as a C. elegans orthologue of the vertebrate pur alpha transcription factor. Expression of end-1 is attenuated in embryos depleted for PLP-1. While removal of plp-1 activity alone does not prevent endoderm development, it strongly enhances the loss of endoderm in mutants defective for the Wnt pathway. In contrast, loss of PLP-1 function does not synergize with mutants in the endoderm-inducing MAPK pathway. Moreover, nuclear localization of PLP-1 during interphase requires components of the MAPK pathway, suggesting that PLP-1 is influenced by MAPK signaling. PLP-1 is transiently asymmetrically distributed during cell divisions, with higher levels in the chromatin of the future posterior daughter of EMS and other dividing cells shortly after mitosis compared to that in their sisters. These findings imply that PLP-1 acts as a transcriptional activator of end-1 expression that may be modulated by MAPK signaling to promote endoderm development.
Assuntos
Proteínas de Caenorhabditis elegans , Endoderma/citologia , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição , Proteínas Wnt/metabolismo , Animais , Células/citologia , Indução Embrionária , Endoderma/crescimento & desenvolvimento , Fatores de Transcrição GATA , Regulação da Expressão GênicaRESUMO
The programmed death of particular cells in Caenorhabditis elegans and Drosophila has been shown to occur by non-apoptotic programs regulated by developmental timing. Such alternative programs may be used as a general mechanism to eliminate differentiated, functional cells.