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Temporal description |
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Expr11524
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By antibody staining, MCM-4 was found to be expressed in dividing cells during all stages of development in wild-type animals. Embryos showed the highest levels of MCM-4 expression, in agreement with the fact that more than half of the somatic cells are formed during embryogenesis. Even dauer larvae that had been arrested in cell division for 2 weeks still contained detectable MCM-4 protein levels. These results suggest that a pool of MCM-4 is retained during prolonged periods of quiescence, so that MCM-4 might function in the re-initiation of DNA synthesis when conditions improve. Immunostaining of wild-type animals for MCM-4 showed strong nuclear staining in the gonad, embryos and postembryonic lineages. MCM-4 was detectable in sperm and accumulated during oocyte maturation in the nucleus but did not show overlap with the condensed chromosomes in diakinesis of meiotic prophase. MCM-4 was not chromatin-associated during MeiosisI of the fertilized oocyte, and the first polar body did not contain MCM-4. This finding is consistent with the absence of S phase between Meiosis I and -II. The second polar body and maternal pronucleus received some MCM-4. Subsequently, embryonic cells in interphase showed strong nuclear staining. In prophase, MCM-4 localization did not overlap with the condensing chromosomes. Upon nuclear envelope degradation, MCM-4 became diffusely localized throughout the cell and clearly did not co-localize with the metaphase-aligned chromosomes. MCM-4 remained cytoplasmic at the onset of anaphase; however, chromatin association became apparent in late anaphase. These data show that chromosome association of MCM-4 is tightly controlled, consistent with origin licensing taking place at the end of mitosis and disappearing during S phase. Similar observations were made during larval divisions. Matching the MCM-4::mCherry reporter, endogenous MCM-4 expression was detectable prior to and during mitosis. Staining of synchronized L1 animals revealed the timing of MCM-4 expression, which in general preceded mitosis by 1-2 h. After 5 h of L1 development at 20 C, MCM-4 immunostaining was predominantly detected in the epithelial seam cells, Q neuroblast daughters and gonad primordium. The somatic gonad precursor cells Z1 and Z4 showed nuclear staining, while the mitotically arrested germline precursor cells Z2 and Z3 showed diffuse cytoplasmic staining. At 6 hours of L1 development, the mesoblast (M) also stained strongly as well as the most anterior ventral cord precursors cells (W, P1 and P2). Subsequently at 7 h, additional P cells showed nuclear MCM-4 expression, which became apparent prior to migration of the nucleus into the ventral nerve cord. At 8 h of L1 development, the intestinal nuclei showed MCM-4 expression, which preceded nuclear division by at least 4 h. At subsequent time points, daughter cells that continued division, such as the Pn.a and M descendants, retained strong nuclear staining. L2 animals stained at 16 h of larval development showed strong MCM-4 expression in the gonad, the H1.a, H2.p, V1-6.p and T.ap seam cells and, weakly, the intestinal nuclei (data not shown). Importantly, MCM-4 staining did not overlap with DNA in prophase and metaphase, while in late anaphase co-localization with the chromosomes was clearly detectable. Similar to our observations with the MCM-4::mCherry reporter, we could not detect any asymmetry in MCM-4 segregation. Thus, even if only one daughter cell continued cell division, both daughters received a similar amount of MCM-4in mitosis. Furthermore, the MCM-4protein became undetectable during quiescence, i.e. the P3.p-P8.p daughter cells that resume DNA replication in the L3 stage did not show detectable expression in the L2 stage. Altogether, our reporter gene and antibody staining analysis show that MCM-4 is dynamically expressed and localized during larval development as well as during different phases of the cell cycle. Expression of MCM-4::mCherry was specifically induced in all postembryonic blast cell lineages well before mitotic entry, at the expected time of S-phase induction. The fusion protein localized to the cell nucleus until degradation of the nuclear envelope in prometaphase, at which point MCM-4 became diffusely localized through the cell. This diffuse localization indicates that MCM-4 is not chromatin-associated in mitosis. MCM-4::mCherry did not disappear upon completion of mitosis but was segregated to both daughter cells. Even cells that permanently withdrew from cell division, such as the motor neurons of the ventral nerve cord, initially retained MCM-4::mCherry expression. However, this expression subsequently disappeared in differentiated cells as well as in cells that temporarily arrested cell division, such as the Pn.p vulval precursor cells in the ventral cord. These experiments indicate that mcm-4 is transcriptionally activated at approximately the time of G1/S transition and that MCM-4 protein is segregated to both daughter cells in mitosis. |
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Expr15538
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Microscopic examination of animals showed clear and reproducible expression of GFP in many tissues, such as the somatic gonad, vulval precursor cells, seam cells, body wall muscle cells, tail and head neurons, as well as, the P lineage |
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Expr13898
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Using small molecule fluorescent in situ hybridization (smFISH), we detected oef-1 transcript throughout wild-type dissected gonads, including in oocytes, as well as in early embryos prior to OEF-1 protein expression. In addition, oef-1 transcript becomes entirely restricted to the P lineage by the formation of Z2 and Z3. |
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Expr10765
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In early wild-type embryos, GPR-1/2 were localized both on the asters and the cell cortex. The cortical localization of GPR- 1/2 changed with the cell cycle. In one-cell embryos, GPR-1/2 were uniformly present at a low level on the cortex from early prophase to metaphase. The level of cortical localization of GPR-1/2 increased and became weakly enriched at the posterior pole of most embryos during anaphase. In some embryos with enriched posterior staining, GPR-1/2 also appeared to be slightly enriched at the anterior pole of the embryo, compared with lateral regions. GPR-1/2 asymmetry became more pronounced during cytokinesis and interphase of the two-cell stage. In the P1 blastomere during interphase, GPR-1/2 were highly enriched around the posterior pole of the cell, and were present at low levels uniformly around the cortex of AB. As the cell cycle progressed, GPR-1/2 asymmetry in P1 disappeared; GPR-1/2 were uniformly localized around the cortex through out prophase, metaphase and early anaphase. During late anaphase and telophase, GPR-1/2 were once again enriched at the posterior part of the P1 cell. These results indicate that GPR- 1/2 is asymmetrically localized in the P lineage. |
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Expr13896
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Examination of the GFP-tagged strain (referred to as F49E8.2:GFP) revealed nuclear expression restricted to the P lineage in embryos beginning in the P4 primordial germ cell. In larval stages, F49E8.2:GFP is present in all germ cells. In adults, F49E8.2:GFP localizes to the nuclei of germ cells in the mitotic zone through entry into meiosis. Strikingly, F49E8.2:GFP expression abruptly disappears in the late pachytene region, and is absent from developing oocytes. OEF-1 expression is detected in the proximal germ cells of L4 animals undergoing spermatogenesis, but not in the proximal germ cells of adult hermaphrodites undergoing oogenesis. |
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Expr13897
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To examine endogenous OEF-1 expression, we generated a polyclonal antibody to OEF-1. The endogenous antibody recapitulates the expression pattern of the OEF- 1:GFP transgene in larval stage 4 (L4) and adult dissected gonads. In embryos, endogenous OEF-1 protein expression is first visible even earlier than OEF-1:GFP, in the P2 blastomere rather than P4. However, OEF-1 protein expression is not detectable in maturing oocytes, implying that little to no OEF-1 is maternally loaded. |
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