A Survey of New Temperature-Sensitive, Embryonic-Lethal Mutations in C. elegans: 24 Alleles of Thirteen Genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci

A Spindle Checkpoint Functions during Mitosis in the Early Caenorhabditis elegans Embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis

Conditional Dominant Mutations in the Caenorhabditis elegans Gene act-2 Identify Cytoplasmic and Muscle Roles for a Redundant Actin Isoform

Animal genomes each encode multiple highly conserved actin isoforms that polymerize to form the microfilament cytoskeleton. Previous studies of vertebrates and invertebrates have shown that many actin isoforms are restricted to either nonmuscle (cytoplasmic) functions, or to myofibril force generation in muscle cells. We have identified two temperature-sensitive and semidominant embryonic-lethal Caenorhabditis elegans mutants, each with a single mis-sense mutation in act-2, one of five C. elegans genes that encode actin isoforms. These mutations alter conserved and adjacent amino acids predicted to form part of the ATP binding pocket of actin. At the restrictive temperature, both mutations resulted in aberrant distributions of cortical microfilaments associated with abnormal and striking membrane ingressions and protrusions. In contrast to the defects caused by these dominant mis-sense mutations, an act-2 deletion did not result in early embryonic cell division defects, suggesting that additional and redundant actin isoforms are involved. Accordingly, we found that two additional actin isoforms, act-1 and act-3, were required redundantly with act-2 for cytoplasmic function in early embryonic cells. The act-1 and -3 genes also have been implicated previously in muscle function. We found that an ACT-2::GFP reporter was expressed cytoplasmically in embryonic cells and also was incorporated into contractile filaments in adult muscle cells. Furthermore, one of the dominant act-2 mutations resulted in uncoordinated adult movement. We conclude that redundant C. elegans actin isoforms function in both muscle and nonmuscle contractile processes

DNA Replication Defects Delay Cell Division and Disrupt Cell Polarity in Early Caenorhabditis elegans Embryos

AbstractIn early Caenorhabditis elegans embryos, asymmetric cell divisions produce descendants with asynchronous cell cycle times. To investigate the relationship between cell cycle regulation and pattern formation, we have identified a collection of embryonic-lethal mutants in which cell divisions are delayed and cell fate patterns are abnormal. In div (for division delayed) mutant embryos, embryonic cell divisions are delayed but remain asynchronous. Some div mutants produce well-differentiated cell types, but they frequently lack the endodermal and mesodermal cell fates normally specified by a transcriptional activator called SKN-1. We show that mislocalization of PIE-1, a negative regulator of SKN-1, prevents the specification of endoderm and mesoderm in div-1 mutant embryos. In addition to defects in the normally asymmetric distribution of PIE-1, div mutants also exhibit other losses of asymmetry during early embryonic cleavages. The daughters of normally asymmetric divisions are nearly equal in size, and cytoplasmic P-granules are not properly localized to germline precursors in div mutant embryos. Thus the proper timing of cell division appears to be important for multiple aspects of asymmetric cell division. One div gene, div-1, encodes the B subunit of the DNA polymerase α-primase complex. Reducing the function of other DNA replication genes also results in a delayed division phenotype and embryonic lethality. Thus the other div genes we have identified are likely to encode additional components of the DNA replication machinery in C. elegans

The phenotypes of the TS mutants when grown at the restrictive temperature from the L1 larval stage.

1<p>We determined the L1 upshift phenotype by plating hypochlorite-synchronized L1 larvae and incubating them at 26°C until they reached adulthood. Abbreviations: Egl: egg laying-defective, Emb: embryonic lethal, Pvl: protruding vulva, Sb: small broods, Ste: sterile.</p

Summary of the TS mutant loci and comparison of previously available alleles.

1<p>Information obtained from: <a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>.</p

<i>par-2</i> mutants.

<p>A. DIC time-lapse images of wild-type <i>par-2(or373 </i>ts<i>)</i>, <i>par-2(or539 </i>ts<i>)</i>, and <i>par-2(or640 </i>ts<i>)</i> embryos. The blastomeres in the <i>par-2</i> mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The <i>par-2(or373 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The <i>par-2(or539 </i>ts<i>)</i> and par-<i>2(or540 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 30 minutes prior to imaging. Arrows indicate mitotic spindles at the two cell stage. Times in min:sec are given relative to AB NEBD. Scale bar, 10 µm. B. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; Normal one cell embryo; 2; assymetric two cell embyro, 3; asynchronous two cell divisions. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites transferred to the restrictive temperature for 30 minutes. C. Amino acid alteration in the <i>par-2(or373 </i>ts<i>)</i> mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

Determination if the TS mutations are potentially fast-acting.

1<p>We determined if an allele was potentially fast-acting in the following manner: We mounted embryos produced at 15°C on microscope slides and immediately made time-lapse videomicrographs at a room maintained at 24°C. If defects similar to those observed after long temperature shifts were found in at least 20% of the embryos and if there was little embryonic lethality at 15°C, we conclude that the allele may be fast-acting. We have labeled these cases as “Yes”. However, if there was significant embryonic lethality at 15°C, we cannot conclude that the presence of cellular defects after short upshifts is due to the upshift or to defects that occur even at 15°C. We have labeled these cases as “Unclear”.</p>2<p>For <i>mei-1, par-2,</i> and <i>zyg-1</i>, we incubated mutant worms at 26°C for 30 minutes prior to imaging (instead of the usual∼1 min. upshift) because the gene products appeared to be required prior to when we started our imaging (pronuclear migration).</p>3<p>High lethality at the permissive temperature precludes making a determination.</p>4<p>The low penetrance of severe defects precludes making a determination.</p

A <i>plk-1</i> mutant.

<p>A. DIC time-lapse images of wild-type and <i>plk-1(or683 </i>ts<i>)</i> embryos. In the <i>plk-1</i> mutant the nuclear centrosomal complex (NCC) failed to rotate, a transverse P₀ spindle assembled, and the daughter blastomeres were binucleate. The <i>plk-1(or683 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 6 hours prior to imaging. Black dots represent centrosomes/spindle poles and asterisks denote multiple nuclei per cell at the two cell stage. Times in min:sec are given relative to NEBD. Scale bar, 10 µm. B. Amino acid alteration in the mutant. Asterisk indicates changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein. C. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; nuclear centrosomal complex rotation, 2; spindle alignment, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.</p