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.     

Molecular Genetics of the Caenorhabditis Elegans Heterochronic Gene Lin-14

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.     

Identification of heterochronic mutants in Caenorhabditis elegans. Temporal misexpression of a collagen::green fluorescent protein fusion gene.

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 specify the timing of lateral hypodermal seam cell terminal differentiation in Caenorhabditis elegans. We devised a screen to identify additional genes involved in this developmental timing mechanism based on identification of mutants that exhibit temporal misexpression from the col-19 promoter, a downstream target of the heterochronic gene pathway. We fused the col-19 promoter to the green fluorescent protein gene (gfp) and demonstrated that hypodermal expression of the fusion gene is adult-specific in wild-type animals and temporally regulated by the heterochronic gene pathway. We generated a transgenic strain in which the col-19::gfp fusion construct is not expressed because of mutation of lin-4, which prevents seam cell terminal differentiation. We have identified and characterized 26 mutations that restore col-19::gfp expression in the lin-4 mutant background. Most of the mutations also restore other aspects of the seam cell terminal differentiation program that are defective in lin-4 mutant animals. Twelve mutations are alleles of three previously identified genes known to be required for proper timing of hypodermal terminal differentiation. Among these are four new alleles of lin-42, a heterochronic gene for which a single allele had been described previously. Two mutations define a new gene, lin-58. When separated from lin-4, the lin-58 mutations cause precocious seam cell terminal differentiation and thus define a new member of the heterochronic gene pathway.     

A Screen for Genetic Loci Required for Hypodermal Cell and Glial-like Cell Development during Caenorhabditis Elegans Embryogenesis

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.     

Physical mapping of the rice genome with BACs

The development of genetics in this century has been catapulted forward by several milestones: rediscovery of Mendel's laws, determination of DNA as the genetic material, discovery of the double-helix structure of DNA and its implications for genetic behavior, and most recently, analysis of restriction fragment length polymorphisms (RFLPs). Each of these milestones has generated a huge wave of progress in genetics. Consequently, our understanding of organismal genetics now extends from phenotypes to their molecular genetic basis. It is now clear that the next wave of progress in genetics will hinge on genome molecular physical mapping, since a genome physical map will provide an invaluable, readily accessible system for many detailed genetic studies and isolation of many genes of economic or biological importance. Recent development of large-DNA fragment (>100 kb) manipulation and cloning technologies, such as pulsed-field gel electrophoresis (PFGE), and yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning, has provided the powerful tools needed to generate molecular physical maps for higher-organism genomes. This chapter will discuss (1) an ideal physical map of plant genome and its applications in plant genetic and biological studies, (2) reviews on physical mapping of the genomes of Caenorhabditis elegans, Arabidopsis thaliana, and man, (3) large-insert DNA libraries: cosmid, YAC and BAC, and genome physical mapping, (4) physical mapping of the rice genome with BACs, and (5) perspectives on the physical mapping of the rice genome with BACs.     

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.     

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.     

Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines.

Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular rearrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene.     

Genetic Analysis of Adult-Specific Surface Antigenic Differences between Varieties of the Nematode Caenorhabditis elegans

We have studied developmental stage-specificity and genetic specification of surface antigens in the nematode Caenorhabditis elegans. Rabbit antisera directed against the adult C. elegans cuticle were used in conjunction with antiserum adsorption experiments to obtain antibody reagents with specificity for the adult surface. Adult-specific antibodies were used to identify several varietal strains of C. elegans that display antigen-negative phenotypes as adults. Genetic mapping results using the surface antigen phenotype as a marker indicated that a single gene (designated srf-1) or cluster of genes on linkage group II determines the adult surface antigen phenotype.     

Correlation of the physical and genetic maps in the lin-12 region of Caenorhabditis elegans.

We describe the assembly of a set of overlapping clones from the lin-12 III chromosomal region that spans approximately 600 kb, and the identification of two restriction fragment length polymorphisms, eP6 and eP7, that flank the lin-12 locus. A comparison of the physical map and the genetic map yields approximate measurements of 930 kb/map unit for the eP6--lin-12 interval and 830 kb/map unit for the lin-12--eP7 interval. We interpret these values as supporting the proposal that the apparent clustering of genes observed for C. elegans autosomes results from decreased recombination frequency in clusters and not from nonrandom distribution of genes on the physical map.     

Molecular Cloning and Duplication of the Nematode Sex-Determining Gene Tra-1

The autosomal sex-determining gene tra-1 plays a major role in controlling sexual phenotype in the nematode Caenorhabditis elegans. This gene is the terminal global regulator in a well-characterized cascade of sex-determining genes. It governs all aspects of somatic sexual differentiation, and it also has important functions in governing germ-line differentiation. Previous genetic analyses have led to the characterization of many loss-of-function (masculinizing) and gain-of-function (dominant feminizing) alleles, and to models for the functions and regulation of tra-1. The gene was cloned by identifying linked transposon insertions, about 200 kb away from tra-1. From this starting point a series of YAC, cosmid and phage clones were assembled into a genomic walk covering over 400 kb. Much of this region was found to be unrepresented in the cosmid database that covers most of the C. elegans genome. This deficit is largely or wholly due to the presence of sequences that cannot be cloned in rec(+)bacterial hosts. The ratio of physical map distances to recombinational map distances in the tra-1 region of the genome appears to be unusually low, indicating considerable local map expansion. The location of tra-1 within the cloned region was determined using a variety of tra-1 mutations that are associated with physical rearrangements of the gene. One of these is a 14-kb deletion, which behaves as a null allele. Another rearrangement, eDp24, is a tandem duplication of 22 kb. Genetic analysis demonstrates that eDp24 carries two incomplete copies of tra-1, and that these copies appear to interact, suggesting some form of negative autoregulation at this locus. Three variant forms of the tra-1 locus have been identified in different natural isolates of C. elegans.     

Rice-barley synteny and its application to saturation mapping of the barley Rpg1 region.

In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric region of barley chromosome 1P and rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large insert genomic YAC and cosmid clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared with the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped in rice or isolated from YAC or cosmid clones from this region, were mapped in barley. All, except one, were in synteny with the rice gene order. The exception, probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1P. The barley probes from this region could not be mapped to rice, but two of them were inferred to be in a syntenic location based on their position on a rice YAC. This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives.     

Use of repetitive DNA probes as physical mapping strategy in Caenorhabditis elegans.

A method for linking genomic sequences cloned in yeast artificial chromosomes (YACs) has been tested using Caenorhabditis elegans as a model system. Yeast clones carrying YACs with repeated sequences were selected from a C. elegans genomic library, total DNA was digested with restriction enzymes, transferred to nylon membranes and probed with a variety of repetitive DNA probes. YAC clones that overlap share common bands with one or more repetitive DNA probes. In 159 YAC clones tested with one restriction enzyme and six probes 28 overlapping clones were detected. The advantages and limitations of this method for construction of YAC physical maps is discussed.     

Cloning by synteny: identifying C. briggsae homologues of C. elegans genes.

Phylogenetic comparisons of gene and protein sequences between related species are often used to identify evolutionarily conserved elements that are important for gene expression, function, or regulation. However, homologoues may sometimes be difficult to identify by conventional low stringency hybridisation techniques, if they have undergone substantial sequence divergence. A new approach, cloning by synteny, is described that was used to identify the C. briggsae homologue of the C. elegans sex-determining gene tra-2. We show that four genes tra-2, ppp-1, art-1, and sod-1 are organised in a syntenic cluster and suggest that extensive conservation of gene linkage may exist between C. briggsae and C. elegans. We have also constructed a C. briggsae cDNA library to facilitate characterisation of these genes. Given the rapid progress in the physical mapping and sequencing of the C. elegans genome, cloning by synteny may provide the fastest method for identifying C. briggsae gene homologues, especially for genes encoding novel proteins.     

A potential role for fatty acid biosynthesis genes during molting and cuticle formation in Caenorhabditis elegans

Caenorhabditis elegans undergoes a developmental molting process that involves a coordinated interplay among diverse intracellular pathways. Here, we investigated the functions of two fatty acid biosynthesis genes; pod-2, encoding acetyl-CoA carboxylase, and fasn-1, encoding fatty acid synthase, in the C. elegans molting process. Although both the pod-2 and fasn-1 genes were expressed at constant levels throughout C. elegans development, knockdown of the proteins encoded by these genes using RNA interference produced severe defects in triglyceride production, molting, and reproduction that were coupled to suppression of NAS-37, a metalloprotease. An assessment of the structure and integrity of the cuticle using a COL-19::GFP marker and Hoechst 33258 staining showed that downregulation of either pod-2 or fasn-1 impaired cuticle formation and disrupted the integrity of the cuticle and the hypodermal membrane.