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

Abstract

In 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 alpha-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.

A Spindle Checkpoint Functions during Mitosis in the EarlyCaenorhabditis elegansEmbryo

“…A single allele of dnc-1 has a lesion in the predicted gene ZK593.5, as shown below exon 12. We identified dnc-1(or404ts) in a temperature-sensitive screen for embryonic lethal mutants described in Encalada et al (2000). stage zyg-9(RNAi) mutant embryos, as expected, astral microtubules were abnormally short and the first mitotic spindle was mispositioned (our unpublished data).…”

“…apo-5(or358ts) and dnc-1(or404ts) were isolated in a screen for temperaturesensitive (ts) maternal-effect embryonic-lethal mutations, using methods described previously (Encalada et al, 2000). Both or358 and or404 were backcrossed five times by using either him-8 or N2 males.…”

“…For live imaging of wild-type, apo-5(or358) and dnc-1(or404) embryos by using differential interference contrast (DIC) microscopy, gravid hermaphrodites were dissected and embryos were mounted in M9 buffer (Brenner, 1974) on 3% agarose pads, covered with a coverslip, and filmed as described previously (Encalada et al, 2000). To measure the duration of mitotic stages, embryos from hermaphrodites carrying GFP strains were mounted on 3% agarose pads, covered with a coverslip, and DIC and fluorescence time-lapse movies of some GFP strains were made using a spinning disk confocal microscope (PerkinElmer Life and Analytical Sciences, Boston, MA) as described in Hamill et al (2002).…”

“…GFP expressed in ␤-tubulin labels microtubules and centrosomes (Praitis et al, 2001); pJH4.52: his-11::GFP. GFP expressed in H2B histones throughout the cell cycle (Strome et al, 2001).apo-5(or358ts) and dnc-1(or404ts) were isolated in a screen for temperaturesensitive (ts) maternal-effect embryonic-lethal mutations, using methods described previously (Encalada et al, 2000). Both or358 and or404 were backcrossed five times by using either him-8 or N2 males.…”

Death and more: DNA damage response pathways in the nematode C. elegans

“…42 In the germ line, however, DNA damage-induced signaling induce two clear responses -cell cycle arrest and apoptosis -that are spatially separated (Figure 1). Exposure of worms to ionizing radiation causes a transient halt in cell cycle progression in the proliferating zone, resulting in a decreased number of mitotic germ cells.…”