l(1)pole hole is required maternally for pattern formation in the terminal regions of the embryo

Maternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.     

Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression

Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.     

Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.     

Mutants Affecting Processing of DNA in Macronuclear Development in Paramecium

In Paramecium tetraurelia, stock 51, the A surface protein is coded by the wild type A51 gene, present in micronuclei in two copies and in macronuclei in about 1500 copies. DNA processing, comprised of DNA cleavage, copy number amplification and telomere addition occurs at autogamy and conjugation when old macronuclei degrade and new macronuclei are formed from micronuclei. In this paper we characterize mutants with macronuclear A gene deletions. These mutants are notable in three respects. First, the mutants do not appear to be simple micronuclear deletions. Although genetic analysis shows that the d12 mutant d12(-1300) is homozygous for the allele A-1300 and the mutant d12(+1) for A+1, analysis by the polymerase chain reaction indicates that the micronuclei in these two mutants contain intact, but presumably altered, micronuclear A genes. They undergo deletion during DNA processing when new macronuclei are formed. Second, the position of the deletions in these alleles has been shown to change. The deficiency present in the d12 allele A-1300 was originally determined to extend from position -1300 (relative to the start of translation of the A gene) to the end of the chromosome. Later, a derivative of this strain, homozygous for the d12 allele A+1 was isolated in which the start site of the deletion was found to have moved from -1300 to +1. Third, a surprising interaction occurs in crosses between a line homozygous for the d12 allele and one homozygous for the wild-type A51 allele. Previous work on the non-Mendelian d48 mutant (which has intact A51 genes in its micronucleus, but has truncated A51 genes in its macronucleus) has shown that intact A51 alleles must be present in the old macronucleus in order for A51 alleles to undergo proper processing. We find that d12 alleles act on A51 alleles in heterozygotes such that intact macronuclear A genes are no longer required for proper processing of A51. Thus, in crosses of 51 x d12 (either +1 or -1300) d12 exconjugants, as well as 51 exconjugants, give rise to clones carrying both intact A51 and truncated d12 alleles. Remarkably the d12 alleles, which are themselves deleted during processing, are capable in the heterozygote of fostering normal processing of the A51 allele.     

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that “denticleless” is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.     

Inactivation of Cdc7 kinase in mouse ES cells results in S-phase arrest and p53-dependent cell death

Cdc7-related kinases play essential roles in the initiation of yeast DNA replication. We show that mice lacking murine homologs of Cdc7 (muCdc7) genes die between E3.5 and E6.5. We have established a mutant embryonic stem (ES) cell line lacking the muCdc7 genes in the presence of a loxP-flanked transgene expressing muCdc7 cDNA. Upon removal of the transgene by Cre recombinase, mutant ES cells cease DNA synthesis, arresting growth with S-phase DNA content, and generate nuclear Rad51 foci, followed by cell death with concomitant increase in p53 protein levels. Inhibition of p53 leads to partial rescue of muCdc7(-/-) ES cells from cell death. muCdc7(-/-)p53(-/-) embryos survive up to E8.5, and their blastocysts generate inner cell mass of a significant size in vitro, whereas those of the muCdc7(-/-)p53(+/-) embryos undergoes complete degeneration. These results demonstrate that, in contrast to cell cycle arrest at the G(1)/S boundary observed in yeasts, loss of Cdc7 in ES cells results in rapid cessation of DNA synthesis within S phase, triggering checkpoint responses leading to recombinational repair and p53-dependent cell death.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.     

hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo

The locus hunchback (hb) is a member of the gap class of segmentation genes of Drosophila. A number of X-ray-induced deletions locate the hb locus at the chromosomal site 85A3-B1, to the right of the pink locus, which maps in the same interval. A total of 14 EMS and 3 X-ray-induced hb alleles have been studied. Homozygous mutant embryos show deletions of segments in two separate regions. In the six strong alleles, the labium and all three thoracic segments are deleted anteriorly while posteriorly the 8th abdominal segment and adjacent parts of the 7th abdominal segment are lacking. The eight weak alleles show smaller deletions both in the thoracic and posterior abdominal region. In the weakest allele only part of the mesothorax is deleted. Three hb alleles produce a homoeotic transformation: superimposed on a strong or weak deletion phenotype, head or thoracic segments are transformed into abdominal segments, respectively. This suggests that hb might also be involved in the regulation of genes in the Bithorax complex (BX-C). Fate mapping of the normal-appearing segments in strong mutant embryos using the UV-laser beam ablation technique (Lohs-Schardin et al., 1979) shows that these segments arise from the normal blastoderm regions. The mutant phenotype can be recognized soon after the onset of gastrulation in a failure to fully extend the germ band. In 6-hr-old mutant embryos, two clusters of dead cells are observed in the thoracic and posterior abdominal region. These observations indicate region specific requirement of hb gene function. The analysis of germ line chimeras by transplantation of homozygous mutant pole cells shows that hb is already expressed during oogenesis. Homozygous mutant embryos derived from a homozygous mutant germ line have a novel phenotype. The anterior affected region is enlarged, including all three gnathal segments and the anterior three abdominal segments. In addition three abdominal segments with reversed polarity are formed between the remaining head structures and the posterior abdomen. Heterozygous mutant embryos derived from a homozygous mutant germ line develop normally, indicating that maternal gene expression is not required for normal development.     

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication     

TopBP1 deficiency causes an early embryonic lethality and induces cellular senescence in primary cells

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.