A Deficiency Screen for Zygotic Loci Required for Establishment and Patterning of the Epidermis in Caenorhabditis Elegans

To identify genomic regions required for establishment and patterning of the epidermis, we screened 58 deficiencies that collectively delete at least ~67% of the Caenorhabditis elegans genome. The epidermal pattern of deficiency homozygous embryos was analyzed by examining expression of a marker specific for one of the three major epidermal cell types, the seam cells. The organization of the epidermis and internal organs was also analyzed using a monoclonal antibody specific for epithelial adherens junctions. While seven deficiencies had no apparent effect on seam cell production, 21 were found to result in subnormal, and five in excess numbers of these cells. An additional 23 deficiencies blocked expression of the seam cell marker, in some cases without preventing cell proliferation. Two deficiencies result in multinucleate seam cells. Deficiencies were also identified that result in subnormal numbers of epidermal cells, hyperfusion of epidermal cells into a large syncytium, or aberrant epidermal differentiation. Finally, analysis of internal epithelia revealed deficiencies that cause defects in formation of internal organs, including circularization of the intestine and bifurcation of the pharynx lumen. This study reveals that many regions of the C. elegans genome are required zygotically for patterning of the epidermis and other epithelia.     

Essential embryonic roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and morphogenesis in C elegans

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.     

Par-4, a Gene Required for Cytoplasmic Localization and Determination of Specific Cell Types in Caenorhabditis Elegans Embryogenesis

Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4; par-2 double mutant suggests that par-4 and par-2 gene products interact in this system.     

Combined activities of hedgehog signaling inhibitors regulate pancreas development

Hedgehog signaling is known to regulate tissue morphogenesis and cell differentiation in a dose-dependent manner. Loss of Indian hedgehog (Ihh) results in reduction in pancreas size, indicating a requirement for hedgehog signaling during pancreas development. By contrast, ectopic expression of sonic hedgehog (Shh) inhibits pancreatic marker expression and results in transformation of pancreatic mesenchyme into duodenal mesoderm. These observations suggest that hedgehog signaling activity has to be regulated tightly to ensure proper pancreas development. We have analyzed the function of two hedgehog inhibitors, Hhip and patched 1 (Ptch), during pancreas formation. Our results indicated that loss of Hhip results in increased hedgehog signaling within the pancreas anlage. Pancreas morphogenesis, islet formation and endocrine cell proliferation is impaired in Hhip mutant embryos. Additional loss of one Ptch allele in Hhip-/-Ptch+/- embryos further impairs pancreatic growth and endodermal cell differentiation. These results demonstrate combined requirements for Hhip and Ptch during pancreas development and point to a dose-dependent response to hedgehog signaling within pancreatic tissue. Reduction of Fgf10 expression in Hhip homozygous mutants suggests that at least some of the observed phenotypes result from hedgehog-mediated inhibition of Fgf signaling at early stages.     

Systematic Screening of Drosophila Deficiency Mutations for Embryonic Phenotypes and Orphan Receptor Ligands

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification.     

Identification of genes required for cytoplasmic localization in early C. elegans embryos

We have isolated and analyzed eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to absence of functional germ cells. We propose that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos.     

A Screen for Genetic Loci Required for Body-Wall Muscle Development during Embryogenesis in Caenorhabditis Elegans

We have used available chromosomal deficiencies to screen for genetic loci whose zygotic expression is required for formation of body-wall muscle cells during embryogenesis in Caenorhabditis elegans. To test for muscle cell differentiation we have assayed for both contractile function and the expression of muscle-specific structural proteins. Monoclonal antibodies directed against two myosin heavy chain isoforms, the products of the unc-54 and myo-3 genes, were used to detect body-wall muscle differentiation. We have screened 77 deficiencies, covering approximately 72% of the genome. Deficiency homozygotes in most cases stain with antibodies to the body-wall muscle myosins and in many cases muscle contractile function is observed. We have identified two regions showing distinct defects in myosin heavy chain gene expression. Embryos homozygous for deficiencies removing the left tip of chromosome V fail to accumulate the myo-3 and unc-54 products, but express antigens characteristic of hypodermal, pharyngeal and neural development. Embryos lacking a large region on chromosome III accumulate the unc-54 product but not the myo-3 product. We conclude that there exist only a small number of loci whose zygotic expression is uniquely required for adoption of a muscle cell fate.     

Dna2 requirement for normal reproduction of Caenorhabditis elegans is temperature-dependent

To determine the function of Dna2 in a multicellular organism, the Caenorhabditis elegans Dna2 expression was probed and deletion mutant phenotypes were analyzed. Dna2 was localized to the nuclei of C. elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging. A homozygous dna2 deletion mutant showed a reduced brood size and embryonic lethality, and the phenotypes greatly depended on growth temperature and aggravated in the succeeding generation. The mutant embryos also showed delayed cell divisions, which together with temperature-dependence of the mutant phenotypes supported the well-conserved role of Dna2 in DNA replication.     

Loss of the mouse ortholog of the shwachman-diamond syndrome gene (Sbds) results in early embryonic lethality

Mutations in SBDS are responsible for Shwachman-Diamond syndrome (SDS), a disorder with clinical features of exocrine pancreatic insufficiency, bone marrow failure, and skeletal abnormalities. SBDS is a highly conserved protein whose function remains largely unknown. We identified and investigated the expression pattern of the murine ortholog. Variation in levels was observed, but Sbds was found to be expressed in all embryonic stages and most adult tissues. Higher expression levels were associated with rapid proliferation. A targeted disruption of Sbds was generated in order to understand the consequences of its loss in an in vivo model. Consistent with recessive disease inheritance for SDS, Sbds(+/-) mice have normal phenotypes, indistinguishable from those of their wild-type littermates. However, the development of Sbds(-/-) embryos arrests prior to embryonic day 6.5, with muted epiblast formation leading to early lethality. This finding is consistent with the absence of patients who are homozygous for early truncating mutations. Sbds is an essential gene for early mammalian development, with an expression pattern consistent with a critical role in cell proliferation.     

Site of action of imprinted genes revealed by phenotypic analysis of parthenogenetic embryos

The phenotypes of early postimplantation parthenogenetic embryos were examined. The spectrum of phenotypes suggested that three stages are adversely affected by imprinting—implantation, pregastrulation, and postgastrulation. Survival of parthenogenetic embryos past these developmental blocks can be improved but not completely overcome by experimental asynchrony. These results suggest that imprinting may be “leaky” at early stages. © 1993Wiley-Liss, Inc.     

CDC-25.1 regulates germline proliferation in Caenorhabditis elegans

The cell cycles in C. elegans are tightly controlled but appear to use the same regulators found in other organisms. Four homologues of the dual-specificity phosphatase Cdc25 are present in the C. elegans genome. In our study, we have characterized a deletion mutant for one of these orthologues. We show that embryonic defects are absent in cdc-25.1 homozygous mutants, presumably because of maternally contributed CDC-25.1 product. These embryos hatch and develop into sterile adults. The adults do not appear to have any somatic defects. The sterility results from inadequate germline proliferation. Germline precursors divide slowly and produce abnormally sized daughter cells. Only three to four rounds of germ-cell division occur before they die during the L3 and L4 larval stages.     

Mapping the distribution of differentiation potential for intestine, muscle, and hypodermis during early development in Caenorhabditis elegans

We have analyzed the differentiation potential of cells in early embryos of Caenorhabditis elegans by assessing the production of markers for intestinal, muscle, and hypodermal cell differentiation in cleavage-arrested blastomeres. Our results show that differentiation potential does not always segregate during cleavage in a linear fashion, i.e., a blastomere can express a differentiation potential that is absent in its parent blastomere and vice versa. Furthermore, the expression of a particular differentiation program by certain cleavage-arrested blastomeres is an exclusive event in that each cell will express only one program of differentiation, even though it may have the potential to express several.     

Requirement for tumor suppressor Apc in the morphogenesis of anterior and ventral mouse embryo

Tumor suppressor Apc (adenomatous polyposis coli) is implicated in the Wnt signaling pathway that is involved in the early embryonic development and tumorigenesis in vertebrates. While the heterozygous null mutant mice develop intestinal polyps, the homozygous embryos die before gastrulation. To investigate the role of Apc in later embryonic development, we constructed a novel hypomorphic Apc allele whose expression was attenuated by approximately 80%. In the hypomorphic Apc homozygous ES cells, reduction in Apc expression caused beta-catenin accumulation and Wnt signaling activation. The homozygous mutant mouse embryos survived 3 days longer than the null mutant embryos. Interestingly, they showed anterior truncation, partial axis duplication, and defective ventral morphogenesis. To determine the tissues where Apc functions for anterior and ventral morphogenesis, we constructed chimeric embryos whose epiblast was derived predominantly from the Apc hypomorphic homozygous cells but the visceral endoderm was from the wild type. Although these chimeric embryos still showed some anterior defects, their ventral morphogenesis was rescued. In addition, marker studies indicated that the axial mesendoderm was also defective in the homozygous embryos. Our results provide genetic evidence that expression of Apc at the normal level is essential for both anterior and ventral development, in the epiblast derivatives and visceral endoderm.     

Location of specific messenger RNAs in Caenorhabditis elegans by cytological hybridization

We have developed an autoradiographic technique for detecting specific Caenorhabditis elegans messenger RNA molecules in situ by hybridization of labeled, cloned DNA probes to fixed tissue sections and squashes of embryos and adults. We report analyses with probes of actin and collagen gene sequences from a C. elegans genomic clone library. Hybridization is RNase sensitive and tissue specific. In adults the actin probe, which recognizes cytoplasmic as well as muscle actin mRNA, hybridizes strongly to muscle and distal gonad (ovary), somewhat less strongly to maturing oocytes, and weakly to intestine. The collagen probe hybridizes weakly to distal gonad and intestine and very strongly to subcuticular tissues, in particular to the hypodermal cells and syncytial cytoplasm of the lateral hypodermal ridges, which are the sites of cuticle synthesis. In embryos, hybridization to squashes indicates that actin message is present at fertilization, decreases during early cleavage, and then increases again during morphogenesis. By contrast, collagen message is absent until the 100-cell stage and then increases rapidly during morphogenesis. The number of cells labeled is consistent with the view that the collagen probe hybridizes to hypodermal precursor cells. We estimate that our present methods can detect messages representing about 0.2% or more of the total mRNA population, and increases in this sensitivity should be possible. Therefore, the cytological hybridization technique should be useful for determining temporal and spatial patterns of specific mRNA distributions during development, at least for abundant and moderately abundant messages.     

A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis Elegans

We have devised a simple genetic mosaic screen, which circumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific early blastomeres, suggesting that many zygotic genes contribute to specific early processes. We discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome.     

Enhancers of Glp-1, a Gene Required for Cell-Signaling in Caenorhabditis Elegans, Define a Set of Genes Required for Germline Development

The distal tip cell (DTC) regulates the proliferation or differentiation choice in the Caenorhabditis elegans germline by an inductive mechanism. Cell signaling requires a putative receptor in the germline, encoded by the glp-1 gene, and a putative signal from the DTC, encoded by the lag-2 gene. Both glp-1 and lag-2 belong to multigene gene families whose members are essential for cell signaling during development of various tissues in insects and vertebrates as well as C. elegans. Relatively little is known about how these pathways regulate cell fate choice. To identify additional genes involved in the glp-1 signaling pathway, we carried out screens for genetic enhancers of glp-1. We recovered mutations in five new genes, named ego (enhancer of glp-1), and two previously identified genes, lag-1 and glp-4, that strongly enhance a weak glp-1 loss-of-function phenotype in the germline. Ego mutations cause multiple phenotypes consistent with the idea that gene activity is required for more than one aspect of germline and, in some cases, somatic development. Based on genetic experiments, glp-1 appears to act upstream of ego-1 and ego-3. We discuss the possible functional relationships among these genes in light of their phenotypes and interactions with glp-1.     

Developmental potential of fused Caenorhabditis elegans oocytes: generation of giant and twin embryos

With their first cleavage blastomeres in Caenorhabditis elegans are fixed to very different developmental programs going along with differential segregation of maternal gene products. To investigate whether indications for a prelocalization of cytoplasmic components can already be found in unfertilized egg cells, we fused mature C. elegans oocytes with the help of a laser microbeam. Fertilization of two fused oocytes resulting in triploid zygotes showed an essentially normal early cleavage pattern with the establishment of five somatic cell lineages and a germline and also a normal spatial arrangement of blastomeres. A considerable fraction of such embryos hatched and developed into fertile giant nematodes. The numbers of cell nuclei in freshly hatched and adult giant animals were found to be essentially the same as in untreated controls. When three fused oocytes were fertilized, two alternative patterns of early embryogenesis were observed. Half of the embryos followed the normal cleavage mode. The other half, however, developed in a twin-like fashion with all cells present in two copies, apparently due to fertilization by two sperm. In such embryos, two areas of gastrulation were established, resulting in the generation of two separate gut primordia. In summary, our results suggest that (1) in contrast to the uncleaved zygote in the mature oocyte of C. elegans no cytoplasmic regionalization exists, (2) the invariable cell numbers typical for the C. elegans embryo are not controlled via cell size, and (3) the entry of a second sperm can induce a cascade of events in the egg leading to the formation of two complete embryo anlagen.