lin-35/Rb cooperates with the SWI/SNF complex to control Caenorhabditis elegans larval development

Null mutations in lin-35, the Caenorhabditis elegans ortholog of the mammalian Rb protein, cause no obvious morphological defects. Using a genetic approach to identify genes that may function redundantly with lin-35, we have isolated a mutation in the C. elegans psa-1 gene. lin-35; psa-1 double mutants display severe developmental defects leading to early larval arrest and adult sterility. The psa-1 gene has previously been shown to encode a C. elegans homolog of yeast SWI3, a critical component of the SWI/SNF complex, and has been shown to regulate asymmetric cell divisions during C. elegans development. We observed strong genetic interactions between psa-1 and lin-35 as well as a subset of the class B synMuv genes that include lin-37 and lin-9. Loss-of-function mutations in lin-35, lin-37, and lin-9 strongly enhanced the defects of asymmetric T cell division associated with a psa-1 mutation. Our results suggest that LIN-35/Rb and a certain class B synMuv proteins collaborate with the SWI/SNF protein complex to regulate the T cell division as well as other events essential for larval growth.     

lin-17 mutations of Caenorhabditis elegans disrupt certain asymmetric cell divisions

The identification of a gene necessary for the asymmetry of cell division would be an important first step toward understanding how sister cells come to differ in their developmental fates. The lin-17 gene of the nematode Caenorhabditis elegans is an excellent candidate for being such a gene. lin-17 mutations cause several blast cells that normally generate sister cells of two distinct types to generate instead sister cells of the same type. Moreover, lin-17 mutations cause sister cells to be equal in size as well as equivalent in developmental fate, suggesting that lin-17 acts at or prior to the asymmetric cell division. The lin-17 gene product is involved in asymmetric cell divisions in a variety of tissues, indicating that lin-17 functions in a general mechanism for the establishment of cellular asymmetry in parent cells.     

Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C. elegans

Secreted proteins of the Wnt family affect axon guidance, asymmetric cell division, and cell fate. We show here that C. elegans Wnts acting through Frizzled receptors can shape axon and dendrite trajectories by reversing the anterior-posterior polarity of neurons. In lin-44/Wnt and lin-17/Frizzled mutants, the polarity of the PLM mechanosensory neuron is reversed along the body axis: the long PLM process, PLM growth cone, and synapses are posterior to its cell body instead of anterior. Similarly, the polarity of the ALM mechanosensory neuron is reversed in cwn-1 egl-20 Wnt double mutants, suggesting that different Wnt signals regulate neuronal polarity at different anterior-posterior positions. LIN-17 protein is asymmetrically localized to the posterior process of PLM in a lin-44-dependent manner, indicating that Wnt signaling redistributes LIN-17 in PLM. In this context, Wnts appear to function not as instructive growth cone attractants or repellents, but as organizers of neuronal polarity.     

The bromodomain protein LIN-49 and trithorax-related protein LIN-59 affect development and gene expression in Caenorhabditis elegans

We have molecularly characterized the lin-49 and lin-59 genes in C. elegans, and found their products are related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, we propose lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes.     

Cell cycle-dependent sequencing of cell fate decisions in Caenorhabditis elegans vulva precursor cells

In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1 degrees ) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1 degrees cell each express secondary (2 degrees ) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non-vulval tertiary (3 degrees ) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two developmental decisions separable by cell cycle phase: lin-12 must act prior to the end of VPC S phase to influence a 1 degrees versus 2 degrees cell fate choice, but must act after VPC S phase to influence a 3 degrees versus 2 degrees cell fate choice. Coupling developmental decisions to cell cycle transitions may provide a mechanism for prioritizing or ordering choices of cell fates for multipotential cells.     

lin-35 Rb and cki-1 Cip/Kip cooperate in developmental regulation of G1 progression in C. elegans.

We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in cyd-1 cyclin D and cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduced the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, as the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that lin-35 Rb and cki-1 Cip/Kip contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals.     

Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the previously demonstrated role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, we have undertaken a functional analysis of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggested that germline stem cells proliferate more rapidly, in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, we propose that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression.     

Mutations in the Caenorhabditis elegans Gene vab-3 Reveal Distinct Roles in Fate Specification and Unequal Cytokinesis in an Asymmetric Cell Division

Asymmetric cell divisions in which a precursor cell distributes fate potential unequally between the two daughter cells represent one of the major mechanisms for fate specification during development. Such mechanisms suggest at least two distinct cellular activities: factors that act to establish asymmetry in the precursor cell and factors that are distributed or activated unequally and function to make the daughter cells different from each other. In Caenorhabditis elegans , cytokinesis of the first division of the male-specific postembryonic blast cell B is unequal, and the two daughters adopt different fates. Others have observed that the genes lin-17 and lin-44 are required, respectively, to establish and to orient this asymmetric division. Mutations in lin-17 and lin-44 coordinately disrupt cytokinesis and fate specification. We describe the function of the gene vab-3 in the B cell lineage. Mutations in vab-3 disrupt the fate of the anterior daughter of B, B.a. However, unlike lin-17 and lin-44 , mutations in vab-3 can disrupt fate without the corresponding disruption of unequal cytokinesis. Analysis of lin-17; vab-3 double mutants suggests that vab-3 acts after lin-17 for B.a fate specification. Double mutant analysis has also identified additional functions of lin-17 in the B lineage subsequent to this first division.     

The let-7 microRNA target gene,Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure

In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) controls the timing of key developmental events and terminal differentiation in part by directly regulating lin-41. C. elegans lin-41 mutants display precocious cell cycle exit and terminal differentiation of epidermal skin cells. lin-41 orthologues are found in more complex organisms including both mice and humans, but their roles are not known. We generated Mlin41 mouse mutants to ascertain a functional role for Mlin41. Strong loss of function Mlin41 gene-trap mutants demonstrated a striking neural tube closure defect during development, and embryonic lethality. Like C. elegans lin-41, Mlin41 also appears to be regulated by the let-7 and mir-125 miRNAs. Since Mlin41 is required for neural tube closure and survival it points to human lin-41 (HLIN41/TRIM71) as a potential human development and disease gene.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

The shut-down gene of Drosophila melanogaster encodes a novel FK506-binding protein essential for the formation of germline cysts during oogenesis

In Drosophila melanogaster, the process of oogenesis is initiated with the asymmetric division of a germline stem cell. This division results in the self-renewal of the stem cell and the generation of a daughter cell that undergoes four successive mitotic divisions to produce a germline cyst of 16 cells. Here, we show that shut-down is essential for the normal function of the germline stem cells. Analysis of weak loss-of-function alleles confirms that shut-down is also required at later stages of oogenesis. Clonal analysis indicates that shut-down functions autonomously in the germline. Using a positional cloning approach, we have isolated the shut-down gene. Consistent with its function, the RNA and protein are strongly expressed in the germline stem cells and in 16-cell cysts. The RNA is also present in the germ cells throughout embryogenesis. shut-down encodes a novel Drosophila protein similar to the heat-shock protein-binding immunophilins. Like immunophilins, Shut-down contains an FK506-binding protein domain and a tetratricopeptide repeat. In plants, high-molecular-weight immunophilins have been shown to regulate cell divisions in the root meristem in response to extracellular signals. Our results suggest that shut-down may regulate germ cell divisions in the germarium.     

LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.     

Developmental timing in C. elegans is regulated by kin-20 and tim-1, homologs of core circadian clock genes

In Caenorhabditis elegans, heterochronic genes constitute a developmental timer that specifies temporal cell fate selection. The heterochronic gene lin-42 is the C. elegans homolog of Drosophila and mammalian period, key regulators of circadian rhythms, which specify changes in behavior and physiology over a 24 hr day/night cycle. We show a role for two other circadian gene homologs, tim-1 and kin-20, in the developmental timer. Along with lin-42, tim-1 and kin-20, the C. elegans homologs of the Drosophila circadian clock genes timeless and doubletime, respectively, are required to maintain late-larval identity and prevent premature expression of adult cell fates. The molecular parallels between circadian and developmental timing pathways suggest the existence of a conserved molecular mechanism that may be used for different types of biological timing.     

The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. We identified a new heterochronic gene, lin-46, from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. Our analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. We also show that lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. Our findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development.     

Caenorhabditis elegans genes required for the engulfment of apoptotic corpses function in the cytotoxic cell deaths induced by mutations in lin-24 and lin-33

Two types of cell death have been studied extensively in Caenorhabditis elegans, programmed cell death and necrosis. We describe a novel type of cell death that occurs in animals containing mutations in either of two genes, lin-24 and lin-33. Gain-of-function mutations in lin-24 and lin-33 cause the inappropriate deaths of many of the Pn.p hypodermal blast cells and prevent the surviving Pn.p cells from expressing their normal developmental fates. The abnormal Pn.p cells in lin-24 and lin-33 mutant animals are morphologically distinct from the dying cells characteristic of C. elegans programmed cell deaths and necrotic cell deaths. lin-24 encodes a protein with homology to bacterial toxins. lin-33 encodes a novel protein. The cytotoxicity caused by mutation of either gene requires the function of the other. An evolutionarily conserved set of genes required for the efficient engulfment and removal of both apoptotic and necrotic cell corpses is required for the full cell-killing effect of mutant lin-24 and lin-33 genes, suggesting that engulfment promotes these cytotoxic cell deaths.     

The Drosophila tumor suppressors Expanded and Merlin differentially regulate cell cycle exit, apoptosis, and Wingless signaling

Mutations that inactivate either merlin (mer) or expanded (ex) result in increased cell growth and proliferation in Drosophila. Both Mer and Ex are members of the Band 4.1 protein superfamily, and, based on analyses of mer ex double mutants, they are proposed to function together in at least a partially redundant manner upstream of the Hippo (Hpo) and Warts (Wts) proteins to regulate cell growth and division. By individually analyzing ex and mer mutant phenotypes, we have found important qualitative and quantitative differences in the ways Mer and Ex function to regulate cell proliferation and cell survival. Though both mer and ex restrict cell and tissue growth, ex clones exhibit delayed cell cycle exit in the developing eye, while mer clones do not. Conversely, loss of mer substantially compromises normal developmental apoptosis in the pupal retina, while loss of ex has only mild effects. Finally, ex has a role in regulating Wingless protein levels in the eye that is not obviously shared by either mer or hpo. Taken together, our data suggest that Mer and Ex differentially regulate multiple downstream pathways.     

A method for assaying the sensitivity of Drosophila replication checkpoint mutants to anti-cancer and DNA-damaging drugs.

In multi-cellular organisms, failure to properly regulate cell-cycle progression can result in inappropriate cell death or uncontrolled cell division leading to tumor formation. To guard against such events, conserved regulatory mechanisms called "checkpoints" block progression into mitosis in response to DNA damage and incomplete replication, as well as in response to other signals. Checkpoint mutants in organisms as diverse as yeast and humans are sensitive to various chemical agents that inhibit DNA replication or cause DNA damage. This phenomenon is the primary rationale for chemotherapy, which uses drugs that preferentially target tumor cells with compromised checkpoints. In this study, we demonstrate the use of Drosophila checkpoint mutants as a system for assaying the effects of various DNA-damaging and anti-cancer agents in a developing multicellular organism. Dwee1, grp and mei-41 are genes that encode kinases that function in the DNA replication checkpoint. We tested zygotic mutants of each gene for sensitivity to the DNA replication inhibitor hydroxyurea (HU), methyl methanosulfonate (MMS), ara-C, cisplatin, and the oxygen radical generating compound paraquat. The mutants show distinct differences in their sensitivity to each of the drugs tested, suggesting an underlying complexity in the responses of individual checkpoint genes to genotoxic stress.     

lin-35/Rb and ubc-18, an E2 ubiquitin-conjugating enzyme,function redundantly to control pharyngeal morphogenesis in C. elegans

The retinoblastoma gene product has been implicated in the regulation of multiple cellular and developmental processes, including a well-defined role in the control of cell cycle progression. The Caenorhabditis elegans retinoblastoma protein homolog, LIN-35, is also a key regulator of cell cycle entry and, as shown by studies of synthetic multivulval genes, plays an important role in the determination of vulval cell fates. We demonstrate an additional and unexpected function for lin-35 in organ morphogenesis. Using a genetic approach to isolate lin-35 synthetic-lethal mutations, we have identified redundant roles for lin-35 and ubc-18, a gene that encodes an E2 ubiquitin-conjugating enzyme closely related to human UBCH7. lin-35 and ubc-18 cooperate to control one or more steps during pharyngeal morphogenesis. Based on genetic and phenotypic analyses, this role for lin-35 in pharyngeal morphogenesis appears to be distinct from its cell cycle-related functions. lin-35 and ubc-18 may act in concert to regulate the levels of one or more critical targets during C. elegans development.