Pattern formation and developmental mechanisms.
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Animals↗
Biomedical subjects
J Kimble
Publications and source records attributed to J Kimble.
lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H).
The homologous receptors LIN-12 and GLP-1 mediate diverse cell-signaling events during development of the nematode Caenorhabditis elegans. These two receptors appear to be functionally interchangeable and have sequence similarity to Drosophila Notch. Here we focus on a molecular analysis of the lag-1 gene (lin-12 -and glp-1), which plays a central role in LIN-12 and GLP-1-mediated signal transduction. We find that the predicted LAG-1 protein is homologous to two DNA-binding proteins: human C Promoter Binding Factor (CBF1) and Drosophila Suppressor of Hairless (Su(H)). Furthermore, we show that LAG-1 binds specifically to the DNA sequence RTGGGAA, previously identified as a CBF-1/Su(H)-binding site. Finally, we report that the 5' flanking regions and first introns of the lin-12, glp-1 and lag-1 genes are enriched for potential LAG-1-binding sites. We propose that LAG-1 is a transcriptional regulator that serves as a primary link between the LIN-12 and GLP-1 receptors and downstream target genes in C. elegans. In addition, we propose that LAG-1 may be a key component of a positive feedback loop that amplifies activity of the LIN-12/GLP-1 pathway.
Amino Acid Sequence↗
APX-1 can substitute for its homolog LAG-2 to direct cell interactions throughout Caenorhabditis elegans development.
The homologous LAG-2 and APX-1 membrane proteins are putative signaling ligands in the GLP-1/LIN-12 signal-transduction pathway in Caenorhabditis elegans. Normally, LAG-2 and APX-1 mediate distinct cell interactions. Here, we demonstrate that APX-1, which normally interacts with GLP-1 in the early embryo, can substitute for LAG-2 throughout development. When expressed under control of the lag-2 promoter, an apx-1 cDNA can completely rescue a lag-2 null mutant. To substitute for LAG-2, APX-1 must be able to interact with both GLP-1 and LIN-12 receptors and to mediate a variety of cell interactions during development. Therefore, APX-1 and LAG-2 are essentially equivalent in their ability to influence receptor activity. On the basis of this result, we suggest that the existence of multiple-signaling ligands in the LIN-12/GLP-1 signal transduction pathway does not reflect the evolution of functionally distinct proteins but rather the imposition of distinct controls of gene expression upon functionally similar proteins. Finally, we propose that the specification of distinct cell fates by the LIN-12/GLP-1 signal-transduction pathway relies on activities functioning downstream of the ligand and receptor, rather than on specific ligand-receptor interactions.
Amino Acid Sequence↗
The fog-3 gene and regulation of cell fate in the germ line of Caenorhabditis elegans.
In the nematode Caenorhabditis elegans, germ cells normally adopt one of three fates: mitosis, spermatogenesis or oogenesis. We have identified and characterized the gene fog-3, which is required for germ cells to differentiate as sperm rather than as oocytes. Analysis of double mutants suggests that fog-3 is absolutely required for spermatogenesis and acts at the end of the regulatory hierarchy controlling sex determination for the germ line. By contrast, mutations in fog-3 do not alter the sexual identity of other tissues. We also have characterized the null phenotype of fog-1, another gene required for spermatogenesis; we demonstrate that it too controls the sexual identity of germ cells but not of other tissues. Finally, we have studied the interaction of these two fog genes with gld-1, a gene required for germ cells to undergo oogenesis rather than mitosis. On the basis of these results, we propose that germ-cell fate might be controlled by a set of inhibitory interactions among genes that specify one of three fates: mitosis, spermatogenesis or oogenesis. Such a regulatory network would link the adoption of one germ-cell fate to the suppression of the other two.
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gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans.
We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1 (null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1 (null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells.
Animals↗
A predicted membrane protein, TRA-2A, directs hermaphrodite development in Caenorhabditis elegans.
The nematode C. elegans naturally develops as either an XO male or XX hermaphrodite. The sex-determining gene, tra-2, promotes hermaphrodite development in XX animals. This gene encodes a predicted membrane protein, named TRA-2A, which has been proposed to provide the primary feminising activity of the tra-2 locus. Here, we show that transgenic TRA-2A driven from a heat shock promoter can fully feminise the somatic tissues of XX tra-2 loss-of-function mutants, which would otherwise develop as male. TRA-2A is thus likely to provide a component of the tra-2 locus that is both necessary and sufficient to promote female somatic development. Transgenic TRA-2A driven by the heat shock promoter can also transform XO animals from male to self-fertile hermaphrodite. This result establishes the role of tra-2 as a developmental switch that controls somatic sexual cell fate. We show that a carboxy-terminal region of TRA-2A, predicted to be intra-cellular, can partially feminise XX tra-2 loss-of-function mutants and XO tra-2(+) males. We suggest that this intra-cellular domain of TRA-2A promotes hermaphrodite development by negatively regulating the FEM proteins.
Animals↗
An ancient molecular mechanism for establishing embryonic polarity?
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Animals↗
Translational control of maternal glp-1 mRNA establishes an asymmetry in the C. elegans embryo.
In C. elegans, the glp-1 gene encodes a membrane receptor that is required for anterior cell fates in the early embryo. We report that GLP-1 protein is localized to anterior blastomeres in 2- to 28-cell embryos. By contrast, glp-1 mRNA is present in all blastomeres until the 8-cell stage. Furthermore, the glp-1 3' untranslated region can restrict translation of a reporter mRNA to anterior blastomeres. Therefore, the translation of maternal glp-1 mRNA is temporally and spatially regulated in the C. elegans embryo. The regulation of maternal glp-1 mRNA has striking parallels to the regulation of maternal hunchback mRNA in the Drosophila embryo. Thus, the establishment of embryonic asymmetry in diverse organisms may involve conserved mechanisms of maternal mRNA regulation.
Animals↗
Control of germ cell differentiation in Caenorhabditis elegans.
The germline of Caenorhabditis elegans is organized in a linear fashion--the most distal germ cells remain in mitosis, those in the middle enter meiosis and proximal cells differentiate as sperm or oocytes. Two signal transduction pathways control germ cell fates. The glp-1 gene mediates a signal that promotes mitosis and the genes of the sex determination pathway mediate a signal that determines if germ cells will develop as sperm or oocytes. Information from these pathways acts through terminal regulators to specify cell fate. For example, fog-1 and fog-3 are required to initiate spermatogenesis and gld-1 appears to be required to initiate oogenesis. Study of these terminal regulators suggests that the decisions about sexual identity and mitosis are linked in germ cells. We propose a tripartite interaction that forces germ cells to adopt one of only three fates--mitosis, spermatogenesis or oogenesis.
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GLP-1 is localized to the mitotic region of the C. elegans germ line.
In C. elegans, germline mitosis depends on induction by the somatic distal tip cell (DTC) and on activity of the glp-1 gene. Using antibodies to GLP-1 protein, we have examined GLP-1 on western blots and by immunocytochemistry. GLP-1 is tightly associated with membranes of mitotic germline cells, supporting its identification as an integral membrane protein. Furthermore, GLP-1 is localized within the germ line to the mitotic region, consistent with the model that GLP-1 acts as a membrane receptor for the distal tip cell signal. Unexpectedly, GLP-1 and the zone of mitosis extend further than the DTC processes. We present three models by which the DTC may influence GLP-1 activity and thereby determine the zone of mitosis. The spatial restriction of GLP-1 appears to be controlled at the translational level in hermaphrodites. We suggest that down-regulation of GLP-1 may be required to effect the transition from mitosis into meiosis.
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lag-2 may encode a signaling ligand for the GLP-1 and LIN-12 receptors of C. elegans.
The C. elegans lag-2 gene is required for several cell-cell interactions that rely on the receptors GLP-1 and LIN-12. In this paper, we report that lag-2 encodes a putative membrane protein with sequence similarity to Drosophila Delta, a proposed ligand for the Notch receptor. Furthermore, we show that the lag-2 promoter drives expression of a reporter protein in the signaling distal tip cell (DTC) of the DTC/germline interaction. By in situ hybridization, we have found that endogenous lag-2 mRNA is present in the DTC but not the germ line. One fusion protein, called LAG-2::beta-gal(intra), rescues a lag-2 null mutant and can be detected in both DTC and germ line. Taking these results together, we propose that lag-2 may encode a signaling ligand for GLP-1/LIN-12 and that the entire LAG-2 protein may be taken up into the receiving cell during induction by GLP-1 and lateral signaling by LIN-12.
Amino Acid Sequence↗
The pha-4 gene is required to generate the pharyngeal primordium of Caenorhabditis elegans.
In the 4-cell Caenorhabditis elegans embryo, two blastomeres are destined to generate pharyngeal cells, each by a distinct developmental strategy: one pathway is inductive, while the other is autonomous. Here, we identify the pha-4 locus. In animals lacking pha-4 activity, an early step in pharyngeal organogenesis is blocked: no pharyngeal primordium is formed and differentiated pharyngeal cells are absent. Most other tissues are generated normally in pha-4 mutants, including cells related to pharyngeal cells by cell lineage and position. Thus, pha-4 activity is required to form the pharyngeal primordium. We propose that pha-4 marks a convergence of the inductive and autonomous pathways of pharyngeal development and suggest that establishment of pharyngeal organ identity is a crucial step for pharyngeal organogenesis.
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Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C. elegans.
C. elegans hermaphrodites make sperm and then oocytes in an otherwise female animal. Gain-of-function mutations in the sex-determining gene tra-2 (tra-2(gf)) transform hermaphrodites into females (spermless hermaphrodites). The tra-2(gf) mutations map to a perfect direct repeat in the 3' untranslated region; each repeat is called a direct repeat element (DRE). Three experiments demonstrate that DREs repress tra-2 at the translational level. First, tra-2(gf) mRNAs are associated with larger polysomes than are their wild-type counterparts. Second, translation of a reporter RNA is inhibited by DREs. Third, disruption of DREs does not increase tra-2 mRNA levels. An RNA binding activity specifically associates with the DREs. We propose that tra-2 translation is inhibited by association of an RNA binding-factor with the DREs and that this translational control is essential for development of C. elegans as a hermaphrodite/male species.
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Control of cell fate in C. elegans by a GLP-1 peptide consisting primarily of ankyrin repeats.
The homologous proteins GLP-1 and LIN-12 are required for cell interactions during nematode development. glp-1 and lin-12 are members of a gene family that includes Drosophila Notch and several vertebrate homologues. The members of this family have a single transmembrane domain and a similar arrangement of repeated amino-acid motifs (see Fig. 1). The mechanism by which proteins in this family function is not understood. One hypothesis is that these proteins are receptors, each with an extracellular domain that binds a ligand and an intracellular domain that influences the activity of downstream cell fate regulators. Here we report that a region of the GLP-1 intracellular domain, consisting primarily of six ankyrin repeats, is sufficient to direct cell fate. The cell fate transformations seen are similar to transformations caused by gain-of-function mutations in either glp-1 or lin-12 and do not rely on endogenous lin-12 or glp-1 activity. We propose that the ankyrin repeat region of GLP-1 is responsible for controlling downstream regulators of cell fate.
Amino Acid Sequence↗
More mog genes that influence the switch from spermatogenesis to oogenesis in the hermaphrodite germ line of Caenorhabditis elegans.
The Caenorhabditis elegans XX animal possesses a hermaphrodite germ line, producing first sperm, then oocytes. In this paper, we report the genetic identification of five genes, mog-2, mog-3, mog-4, mog-5, and mog-6, that influence the hermaphrodite switch from spermatogenesis to oogenesis. In mog-2-mog-6 mutants, spermatogenesis continues past the time at which hermaphrodites normally switch into oogenesis and no oocytes are observed. Therefore, in these mutants, germ cells are transformed from a female fate (oocyte) to a male fate (sperm). The fem-3 gene is one of five genes that acts at the end of the germline sex determination pathway to direct spermatogenesis. Analyses of mog;fem-3 double mutants suggest that the mog-2-mog-6 genes act before fem-3; thus these genes may be in a position to negatively regulate fem-3 or one of the other terminal regulators of germline sex determination. Double mutants of fem-3 and any one of the mog mutations make oocytes. Using these double mutants, we show that oocytes from any mog;fem-3 double mutant are defective in their ability to support embryogenesis. This maternal effect lethality indicates that each of the mog genes is required for embryogenesis. The two defects in mog-2-mog-6 mutants are similar to those of mog-1: all six mog genes eliminate the sperm/oocyte switch in hermaphrodites and cause maternal effect lethality. We propose that the mog-2-mog-6 mutations identify genes that act with mog-1 to effect the sperm/oocyte switch. We further speculate that the mog-1-mog-6 mutations all interfere with translational controls of fem-3 and other maternal mRNAs.
Alleles↗
The mog-1 gene is required for the switch from spermatogenesis to oogenesis in Caenorhabditis elegans.
Caenorhabditis elegans hermaphrodites make first sperm, then oocytes. By contrast, animals homozygous for any of six loss-of-function mutations in the gene mog-1 (for masculinization of the germ line) make sperm continuously and do not switch into oogenesis. Therefore, in mog-1 mutants, germ cells that normally would become oocytes are transformed into sperm. By contrast, somatic sexual fates are normal, suggesting that mog-1 plays a germ line-specific role in sex determination. Analyses of double mutants suggest that mog-1 negatively regulates the fem genes and/or fog-1: mog-1; fem and mog-1; fog-1 double mutants all make oocytes rather than sperm. Therefore, we propose that wild-type mog-1 is required in the hermaphrodite germ line for regulation of the switch from spermatogenesis to oogenesis rather than for specification of oogenesis per se. In addition to its role in germline sex determination, maternal mog-1 is required for embryogenesis: most progeny of a mog-1; fem or mog-1; fog-1 mother die as embryos. How might the roles of mog-1 in the sperm/oocyte switch and embryogenesis be linked? Previous work showed that fem-3 is regulated post-transcriptionally to achieve the sperm/oocyte switch. We speculate that mog-1 may function in the post-transcriptional regulation of numerous germ-line RNAs, including fem-3. A loss of mog-1 might inappropriately activate fem-3 and thereby abolish the sperm/oocyte switch; its loss might also lead to misregulation of maternal RNAs and thus embryonic death.
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Suppressors of glp-1, a gene required for cell communication during development in Caenorhabditis elegans, define a set of interacting genes.
The glp-1 gene is essential for two cell interactions that control cell fate in Caenorhabditis elegans: induction of anterior pharynx in the embryo and induction of mitotic proliferation in the germ line. To identify other genes involved in these cell interactions, we have isolated suppressors of two temperature sensitive alleles of glp-1. Each of 14 recessive suppressors rescues both embryonic and germline glp-1(ts) defects. These suppressors are extragenic and define a set of six genes designated sog, for suppressor of glp-1. Suppression of glp-1 is the only obvious phenotype associated with sog mutations. Mutations in different sog genes show allele-specific intergenic noncomplementation, suggesting that the sog gene products may interact. In addition, we have analyzed a semidominant mutation that suppresses only the glp-1 germline phenotype and has a conditional feminized phenotype of its own. None of the suppressors rescues a glp-1 null mutation and therefore they do not bypass a requirement for glp-1. Distal tip cell function remains necessary for germline proliferation in suppressed animals. These suppressor mutations identify genes that may encode other components of the glp-1 mediated cell-signaling pathway or regulate glp-1 expression.
Alleles↗
The Caenorhabditis elegans sex determining gene fem-3 is regulated post-transcriptionally.
The fem-3 gene of Caenorhabditis elegans is required for male development. Both maternal and zygotic fem-3 activities are required for spermatogenesis in the XX hermaphrodite germline and for male development in somatic and germline tissues XO (male) animals. Here we show that fem-3 RNA is contributed to embryos as a maternal product and that this RNA is degraded early in embryonic development. The poly(A) tail of embryonic fem-3 RNA is substantially longer than that of adult hermaphrodites which indicates that poly(A) tail lengthening probably occurs at or soon after fertilization. During subsequent development, fem-3 poly(A) tails shorten. The amount of fem-3 RNA in XX and XO embryos is equivalent, suggesting sex-specific regulation of maternal fem-3 activity occurs post-transcriptionally. The sequence of fem-3 predicts an open reading frame that could encode a soluble protein; putative fem-3 null mutants truncate this open reading frame. We discuss the implications of these results for the regulation and function of fem-3.
Amino Acid Sequence↗