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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. |
