Maternal effects and temperature-sensitive period of mutations affecting embryogenesis in Caenorhabditis elegans

We have used standard tests to investigate the nature of gene expression of a new set of temperature-sensitive mutants defining 30 emb genes (essential for embryogenesis) in the nematode Caenorhabditis elegans. The mode of gene expression as determined by progeny tests for parental effects divides the genes into four classes. For 18 genes maternal gene expression is necessary and sufficient for normal embryogenesis; for 2 genes zygotic expression is necessary and sufficient; for 7 genes either maternal or zygotic expression is sufficient; for 3 genes both maternal and zygotic expression are necessary. One mutant displayed partial paternal sufficiency. The results of temperature-shift experiments define two “execution stages,” corresponding to the limits of the temperature-sensitive period (TSP), and indicate the nature and the time of action or synthesis of the gene products. Most of the maternally expressed genes have very early execution stages indicating translation before fertilization, but some are temperature sensitive late in embryogenesis. Early execution stages for 2 zygotically necessary genes demonstrate that the zygotic genome can be active in the earliest stages of embryogenesis. All taken together, the mode of gene expression, TSP, and arrest stage (terminal phenotype) allow us to classify functionally and begin to order the genes essential for embryogenesis. The results indicate a preeminent role for maternal genes and gene products in embryogenesis, in agreement with the results of others.     

Parental effects and phenotypic characterization of mutations that affect early development in Caenorhabditis elegans

Genetic tests for parental effects were performed on 24 temperature-sensitive embryonic-lethal mutants of the nematode Caenorhabditis elegans. For 21 of these mutants, maternal expression of the wild-type allele is sufficient for embryonic survival, regardless of the embryo's genotype. For 11 of these 21 mutants, maternal expression of the wild-type allele is necessary for embryonic survival (strict maternals). For the remaining 10, either maternal or embryonic expression is sufficient for survival (partial maternals). One mutant shows a paternal effect; that is, a wild-type extragenic sperm function appears to rescue homozygous mutant embryos. Similar parental-effect tests were performed on 11 larval-lethal mutants. In 4 mutants, 1 of which blocks as late as the second larval stage after hatching, maternal contributions still can rescue mutant larvae. The remaining 3 embryonic lethals and 8 larval lethals show no parental effects; that is, zygotic expression of the wild-type allele is necessary and sufficient for embryonic survival. Temperatureshift experiments on embryonic-lethal embryos showed that all but 1 of the strict maternal mutants are temperature sensitive only before gastrulation. One of the partial maternal mutants is temperature sensitive prior to gastrulation, suggesting that some zygotic genes can function early in embryogenesis. At the nonpermissive temperature, 7 of the strict maternal mutants either show cleavage abnormalities in early divisions or stop cleavage at less than 100 cells, or both.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal.     

Morphogenetic networks which determine the spatial expression of zygotic genes in early <Emphasis Type="Italic">Drosophila</Emphasis> embryo

This review deals with the recent studies expanding the idea of positional information in the early embryogenesis of Drosophila melanogaster. Previous studies showed that, in the course of segment determination in Drosophila, information created by gradients of products of maternal coordinate genes is not “read” statically, being interpreted by their zygotic target genes via regulatory interactions. This leads to spatial shifts in the expression of target genes relative to the original positions as well as to dynamic reduction in the zygotic expression variability. However, according to recent data, interpretation of positional information includes the interaction between not only zygotic target genes but also the maternal coordinate genes themselves. Different systems of maternal coordinate genes (maternal systems)—the posterior-anterior, terminal, and dorsoventral—can interact with each other. This is usually expressed in the regulation of zygotic target genes of one maternal system by other maternal systems. The concept of a “morphogenetic network” was introduced to define the interaction of maternal systems during determination of spatial gene expression in the early Drosophila embryo.     

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.     

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

Zygotic transcription and cell proliferation duringDrosophila embryogenesis

The zygotic expression of only a fewDrosophila genes is known to be required for completion of the normal embryonic mitoses. Molecular genetic analyses of these genes reveal that they fall into two classes, those whose mRNA levels are regulated in a stage and/or tissue-specific fashion to control cell cycle events during embryogenesis, and those in which, in the absence of functional zygotic expression, the maternal mRNA contribution does not provide sufficient product to complete the normal embryonic mitoses. Genes that comprise the first class are involved in the developmental control of the cell cycle, while those of the second class identify components of the cell cycle machinery.     

Ovary-autonomous maternal inheritance at a developmental locus in Drosophila

The sex-linked temperature-sensitive mutation shibire^ts of Drosophila melanogaster shows a maternal effect causing embryonic lethality at 29°C. The maternal influence is due to gene action autonomous to the ovary. Embryos carrying the paternally derived wild-type gene can survive at 29°C but only if heat pulses are begun at least 9 hr after oviposition. The paternal rescue is presumably due to zygotic gene action at this locus beginning part way through embryogenesis. A maternal wild-type genome, however, can produce shi embryos that have sufficient shi⁺ product to support embryogenesis up to the hatching stage even at 29°C.     

Type IV Collagen Is Detectable in Most, but Not All, Basement Membranes of Caenorhabditis elegans and Assembles on Tissues That Do Not Express It

Type IV collagen in Caenorhabditis elegans is produced by two essential genes, emb-9 and let-2, which encode α1- and α2-like chains, respectively. The distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is detected in all basement membranes except those on the pseudocoelomic face of body wall muscle and on the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were shown to be expressed in the same cells, primarily body wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with basement membranes that contain type IV collagen, these tissues do not express either type IV collagen gene. Using an epitope-tagged emb-9 construct, we show that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes. Additionally, we show that expression of functional type IV collagen only in body wall muscle cells is sufficient for C. elegans to complete development and be partially fertile. Since type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface–associated molecule(s) is required to facilitate type IV collagen assembly.     

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.     

DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence

Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.     

Functional Analysis of the Cathepsin-Like Cysteine Protease Genes in Adult Brugia malayi Using RNA Interference

Background

Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1) and cathepsin Z (Ce-cpz-1) has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting.

Methods and Findings

RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages.

Conclusions

Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.     

Caenorhabditis elegans polo-like kinase PLK-1 is required for merging parental genomes into a single nucleus

Before the first zygotic division, the nuclear envelopes of the maternal and paternal pronuclei disassemble, allowing both sets of chromosomes to be incorporated into a single nucleus in daughter cells after mitosis. We found that in Caenorhabditis elegans, partial inactivation of the polo-like kinase PLK-1 causes the formation of two nuclei, containing either the maternal or paternal chromosomes, in each daughter cell. These two nuclei gave rise to paired nuclei in all subsequent cell divisions. The paired-nuclei phenotype was caused by a defect in forming a gap in the nuclear envelopes at the interface between the two pronuclei during the first mitotic division. This was accompanied by defects in chromosome congression and alignment of the maternal and paternal metaphase plates relative to each other. Perturbing chromosome congression by other means also resulted in failure to disassemble the nuclear envelope between the two pronuclei. Our data further show that PLK-1 is needed for nuclear envelope breakdown during early embryogenesis. We propose that during the first zygotic division, PLK-1–dependent chromosome congression and metaphase plate alignment are necessary for the disassembly of the nuclear envelope between the two pronuclei, ultimately allowing intermingling of the maternal and paternal chromosomes.     

Cytochrome P450-dependent metabolism of PCB52 in the nematode Caenorhabditis elegans

There are 75 full length cytochrome P450 (CYP) genes known in the genome of the nematode Caenorhabditis elegans. The individual biological functions of the vast majority are mostly as yet unknown. Here the impact of cytochrome P450 isoforms on the metabolism of PCB52, an ortho-substituted, non-coplanar 2,2′,5,5′-tetrachlorbiphenyl, as a model PCB of these worldwide distributed pollutants is investigated. Organic extracts, isolated from treated worms and analyzed by GC/MS, contained two obvious PCB52-derived products which have been identified as C3-, C4- and/or C6-hydroxy-PCB52. Moreover, these hydroxylase reactions strictly required the functional expression of the NADPH-dependent cytochrome P450 reductase (CPR) encoding emb-8 gene, which was recently shown to be essential also for several other cytochrome P450-dependent enzymatic reactions. Multiple and subsequent single RNAi-gene silencing experiments, as well as the use of cyp-mutant strains, identified members of the CYP-14A subfamily and CYP-34A6 as the major isoforms contributing to PCB52 metabolism in C. elegans. In the gene-silenced worms and mutants, the reduction in formation of hydroxylated products ranged from 55% to 78%. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent PCB metabolites and may thus facilitate future studies on biotransformation.