Biomedical subjects
Publications and source records attributed to M Labouesse.
How do animal tissues resist the shearing forces to which they are exposed during locomotion or harsh encounters with the environment? Genetic analysis in Caenorhabditis elegans is furthering our understanding of the nature and function of the attachments that preserve tissue integrity.
Epithelial cells are essential and abundant in all multicellular animals where their dynamic cell shape changes orchestrate morphogenesis of the embryo and individual organs. Genetic analysis in the simple nematode Caenorhabditis elegans provides some clues to the mechanisms that are involved in specifying epithelial cell fates and in controlling specific epithelial processes such as junction assembly, trafficking or cell fusion and cell adhesion. Here we review recent findings concerning C. elegans epithelial cells, focusing in particular on epithelial polarity, and transcriptional control.
How epithelial cell fates become specified is poorly understood. We have previously shown that the putative C2H2 zinc-finger transcription factor LIN-26 is required for the differentiation of ectodermal and mesodermal epithelial cells in Caenorhabditis elegans. Here, we report that ectopic LIN-26 expression during early gastrulation transforms most blastomeres into epithelial-like cells. Specifically, LIN-26 induced the expression of three epithelial markers: the adherens junction protein JAM-1; DLG-1, which is essential for the assembly of JAM-1 at junctions; and CHE-14, which is involved in apical trafficking. Furthermore, ultrastructural studies revealed that ectopic LIN-26 expression induced the formation of adherens-like junctions. However, ectopic lin-26 expression did not confer any tissue-specific cell fate, such as the epidermal cell fate, as evidenced from the observation that several epidermal-specific genes were not induced. Conversely, we show that epidermal cells displayed some polarity defects in lin-26 mutants. We conclude that lin-26 can induce epithelial differentiation and that epitheliogenesis is not a default pathway in C. elegans.
The aim of our study was to investigate whether a human neural cell line could be used as a reliable screening tool to examine the functional conservation, in humans, of transcription factors involved in neuronal or glial specification in other species. Gain-of-function experiments were performed on DEV cells, a cell line derived from a human medulloblastoma. Genes encoding nine different transcription factors were tested for their influence on the process of specification of human DEV cells towards a neuronal or glial fate. In a first series of experiments, DEV cells were transfected with murine genes encoding transcription factors known to be involved in the neuronal differentiation cascade. Neurogenins-1, -2, and -3; Mash-1; and NeuroD increased the differentiation of DEV cells towards a neuronal phenotype by a factor of 2-3.5. In a second series of experiments, we tested transcription factors involved in invertebrate glial specification. In the embryonic Drosophila CNS, the development of most glial cells depends on the master regulatory gene glial cell missing (gcm). Expression of gcm in DEV cells induced a twofold increase of astrocytic and a sixfold increase of oligodendroglial cell types. Interestingly, expression of tramtrack69, which is required in all Drosophila glial cells, resulted in a fourfold increase of only the oligodendrocyte phenotype. Expression of the related tramtrack88 protein, which is not expressed in the fly glia, or the C. elegans lin26 protein showed no effect. These results show that the Drosophila transcription factor genes tested can conserve their function upon transfection into the human DEV cells, qualifying this cell line as a screening tool to analyze the mechanisms of neuronal and glial specification.
Specialised subapical junctions play a critical role in maintaining epithelial cell polarity and tissue integrity, and provide a platform for intracellular signalling. Here we analyse the roles of C. elegans genes let-413 and dlg-1, a homologue of Drosophila lethal discs large, in the assembly of the C. elegans apical junction (CeAJ), and provide the first characterisation of this structure. We have identified dlg-1 as an essential gene in an RNA interference screen against C. elegans homologues of genes encoding proteins involved in tight or septate junction formation. We show that DLG-1 colocalises with the junctional protein JAM-1 at CeAJs in a unit distinct from HMP-1/alpha-catenin, and apical to the laterally localised LET-413. Loss of dlg-1 activity leads to JAM-1 mislocalisation and the disappearance of the electron-dense component of the CeAJs, but only mild adhesion and polarity defects. In contrast, loss of let-413 activity leads to the formation of basally extended discontinuous CeAJs and strong adhesion and polarity defects. Interestingly, in LET-413-deficient embryos, CeAJ markers are localised along the lateral membrane in a manner resembling that observed in wild-type embryos at the onset of epithelial differentiation. We conclude that the primary function of LET-413 is to correctly position CeAJ components at a discrete subapical position. Furthermore, we propose that DLG-1 is required to aggregate JAM-1 and other proteins forming the electron-dense CeAJ structure. Our data suggest that epithelial adhesion is maintained by several redundant systems in C. elegans.
BACKGROUND: Polarised trafficking of proteins is critical for normal expression of the epithelial phenotype, but its genetic control is not understood. The regulatory gene lin-26 is essential for normal epithelial differentiation in the nematode Caenorhabditis elegans. To identify potential effectors of lin-26, we characterised mutations that result in lin-26-like phenotypes. Here, we report the phenotypic and molecular analysis of one such mutant line, che-14. RESULTS: Mutations in che-14 resulted in several partially penetrant phenotypes affecting the function of most epithelial or epithelial-like cells of the ectoderm, including the hypodermis, excretory canal, vulva, rectum and several support cells. The defects were generally linked to the accumulation of vesicles or amorphous material near the apical surface, suggesting that secretion was defective. The CHE-14 protein showed similarity to proteins containing sterol-sensing domains, including Dispatched, Patched and NPC1. A fusion protein between full-length CHE-14 and the green fluorescent protein became localised to the apical surface of epithelial cells that require che-14 function. Deletions that removed the predicted transmembrane domains or extracellular loops of CHE-14 abolished apical localisation and function of the protein. CONCLUSIONS: We propose that CHE-14 is involved in a novel secretory pathway dedicated to the exocytosis of lipid-modified proteins at the apical surface of certain epithelial cells. Our data raise the possibility that the primordial function of proteins containing a sterol-sensing domain is to control vesicle trafficking: CHE-14 and Dispatched in exocytosis, Patched and NPC1 in endocytosis.
Recently, a novel family of TATA binding protein (TBP)-like factors (TLFs) have been described in metazoan organisms; however, their function has not yet been elucidated. Using Caenorhabditis elegans (Ce) as a model, we demonstrate that CeTLF is required in vivo for zygotic transcription during embryogenesis. Elimination of CeTLF expression by RNA interference caused embryonic lethality either due to the lack of expression of early patterning genes or to their ectopic expression. Moreover, the absence of CeTLF in vivo prevented the correct soma-specific phosphorylation of RNA polymerase II (Pol II). Thus, CeTLF may positively or negatively regulate Pol II transcription, depending on the developmental stage of the embryo.
In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. The apparently widespread nature of RNAi in eukaryotes, ranging from trypanosome to mouse, has sparked great interest from both applied and fundamental standpoints. Here we review the technical improvements being made to increase the experimental potential of this technique. We also discuss recent advances in uncovering the proteins that act during the RNAi process, discoveries that have revealed enticing links between transposition, transgene silencing and RNAi.
Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.
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The Caenorhabditis elegans embryo undergoes a series of stereotyped cell cleavages that generates the organs and tissues necessary for an animal to survive. Here we review two models of embryonic patterning, one that is lineage-based, and one that focuses on domains of organ and tissue precursors. Our evolving view of C. elegans embryogenesis suggests that this animal develops by mechanisms that are qualitatively similar to those used by other animals.
lin-26, which encodes a unique Zn-finger protein, is required for differentiation of nonneuronal ectodermal cells in Caenorhabditis elegans. Here, we show that the two genes located immediately upstream of lin-26 encode LIN-26-like Zn-finger proteins; hence their names are lir-1 and lir-2 (lin-26 related). lir-2, lir-1, and lin-26 generate several isoforms by alternative splicing and/or trans-splicing at different positions. On the basis of their trans-splicing pattern, their intergenic distances, and their expression, we suggest that lir-2, lir-1, and lin-26 form two overlapping transcriptional operons. The first operon, which is expressed in virtually all cells, includes lir-2 and long lir-1 isoforms. The second operon, which is expressed in the nonneuronal ectoderm, includes short lir-1 isoforms, starting at exon 2 and lin-26. This unusual genomic organization has been conserved in C. briggsae, as shown by cloning the C. briggsae lir-2, lir-1, and lin-26 homologs. Particularly striking is the sequence conservation throughout the first lir-1 intron, which is very long in both species. Structural conservation is functionally meaningful as C. briggsae lin-26 is also expressed in the nonneuronal ectoderm and can complement a C. elegans lin-26 null mutation.
In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles.
To build complex organs, embryos have evolved mechanisms that integrate the development of cells unrelated to one another by cell type or ancestry. Here we show that the pha-4 locus establishes organ identity for the Caenorhabditis elegans pharynx. In pha-4 mutants, pharyngeal cells are transformed into ectoderm. Conversely, ectopic pha-4 expression produces excess pharyngeal cells. pha-4 encodes an HNF-3 homolog selectively expressed in the nascent digestive tract, including all pharynx precursors at the time they are restricted to a pharyngeal fate. We suggest that pha-4 is a key component of a transcription-based mechanism to endow cells with pharyngeal organ identity.
The Caenorhabditis elegans LIN-26 protein is required to specify and/or maintain the fates of all non-neuronal ectodermal cells. Here we show that lin-26 is expressed until the somatic gonad primordium stage in all cells of the somatic gonad, except in distal tip cells, and later in all uterine cells. To determine if lin-26 functions in the somatic gonad, we have generated gonad-specific lin-26 alleles obtained by integration of lin-26 promoter deletion derivatives into a lin-26 null mutant background. In this way, we rescued the lethal phenotype imparted by lin-26 null mutations and uncovered a highly penetrant sterile phenotype. Specifically, the strongest of these new alleles was characterized by the absence of lin-26 expression in the somatic gonad, the presence of endomitotic oocytes, decreased germline proliferation, a protruding vulva and a less penetrant absence of gonad arms. Lineage analysis of mutant somatic gonads and examination of several markers expressed in the spermatheca, sheath cells, distal tip cells and the uterus, suggest that LIN-26 is required in sheath, spermatheca and uterine precursors, and in uterine cells. We conclude that lin-26 performs a similar function in the non-neuronal ectoderm and the somatic gonad, a mesoderm derivative, and we speculate that lin-26 is required to express epithelial characteristics.
The monoclonal antibody MH27 recognizes an adherens junction protein present around hypodermal cells in the pharynx and the intestine. By using this antibody and an antiserum against the LIN-26 protein, which is present in hypodermal and glial-like cells, I have examined the morphogenesis of the embryo in embryos homozygous for 91 chromosomal deficiencies that cover approximately 74% of the Caenorhabditis elegans genome. Most deficiencies were found to affect both the morphogenesis of the embryo and the organogenesis of the pharynx. By contrast, the intestine was generally normal. I have classified deficiencies according to their hypodermal staining abnormalities. I identified a few deficiencies that appeared to affect more specifically anterior-directed migration of hypodermal cells or extension of the margins of ventral hypodermal cells, integrity of hypodermal membranes, elongation of the embryo, and hypodermal cell fusions. This work opens the way for a genetic analysis of morphogenesis in C. elegans.
The Caenorhabditis elegans lin-26 gene is expressed in all nonneuronal ectodermal cells. To identify genes required to specify the fates of ectodermal cells, we have conducted screens designed to identify loci whose zygotic function would be required for normal lin-26 expression. First, we examined 90 deficiencies covering 75% of the genome; second, we examined the progeny of 3600 genomes after EMS mutagenesis. We identified six loci that appear to be required for normal lin-26 expression. We argue that the deficiency eDf19 deletes a gene involved in specifying hypodermal cell fates. The genes emb-29 (previously known) and ale-1 (newly found) could be involved in a cell cycle function and/or in specifying the fates of some precursors within different lineages that generate hypodermal cells and nonectodermal cells. We argue that the overlapping deficiencies qDf7, qDf8 and qDf9 delete a gene required to limit the number of nonneuronal ectodermal cells. We suggest that the deficiencies ozDf2, itDf2 and nDf42 delete genes required, directly or indirectly, to repress lin-26 expression in cells that normally do not express lin-26. We discuss the implications of these findings concerning the generation of the ectoderm.
The C. elegans gene lin-26, which encodes a presumptive zinc-finger transcription factor, is required for hypodermal cells to acquire their proper fates. Here we show that lin-26 is expressed not only in all hypodermal cells but also in all glial-like cells. During asymmetric cell divisions that generate a neuronal cell and a non-neuronal cell, LIN-26 protein is symmetrically segregated and then lost from the neuronal cell. Expression in glial-like cells (socket and sheath cells) is biologically important, as some of these neuronal support cells die or seem sometimes to be transformed to neuron-like cells in embryos homozygous for strong loss-of-function mutations. In addition, most of these glial-like cells are structurally and functionally defective in animals carrying the weak loss-of-function mutation lin-26(n156). lin-26 mutant phenotypes and expression patterns together suggest that lin-26 is required to specify and/or maintain the fates not only of hypodermal cells but also of all other non-neuronal ectodermal cells in C. elegans. We speculate that lin-26 acts by repressing the expression of neuronal-specific genes in non-neuronal cells.