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. |
Lineage expression: P lineage. Lineage expression: T lineage. Lineage expression: V lineage. |
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Expr1894
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The pattern of gfp expression in hermaphrodites carrying the gfp reporter was dynamic but at all stages restricted to hypodermal cells. Early in the L1 stage, expression was seen in H0, H1, and H2, in the anterior V cells, and in the T cells. Weak expression was also seen in hypodermal nuclei in the head and the tail, including those in hyp5, hyp6, hyp8, hyp9, and hyp10. Expression was not seen at this stage, however, in the P cells or in nuclei in the hyp7 syncytium. After division of H1, both H1.a, a seam cell, and H1.p, which joins hyp7, expressed gfp. Likewise, after division of V1V4 both the anterior daughters (which join hyp7) and the posterior daughters (which remain seam cells) expressed gfp. This pattern was repeated at each of the larval molts with the result that in adult worms, all descendents of H1, H2, and V1V4 expressed gfp. At the end of the L1 stage, V5.p could be seen to express gfp but not V5.a, which is a neuroblast. In the P cell lineages, expression was first seen toward the end of the L1 stage in P1.p, P2.p, P9.p, P10.p, and P11.p nuclei. These cells fuse with hyp7 during the L1 stage. Expression of gfp was not seen, on the other hand, in the daughters of P3 to P8 or P12 (all of which remain separate from hyp7). By the end of the L1 stage most nuclei in hyp7 expressed gfp, as did those in hyp5, hyp6, hyp8, hyp9, hyp10, and hyp11. After division of P3.p, P4.p, and P8.p during the L3 stage, all six daughters started to express gfp concomitant with their fusion with hyp7. The daughters of P5.p, P6.p, and P7.p, the progenitors of the vulva, remained negative for gfp expression as did all the cells they subsequently gave rise to. In adult worms, no hypodermal nuclei that failed to express gfp could be identified. Conversely, no nonhypodermal nuclei were observed at any stage that expressed the gfp marker. |
nuclei |
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Expr13569
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Transcripts for elt-1 were detected in WT animals in both the anterior and posterior V1-V4 daughters following the L2 symmetric division and mostly in the posterior daughters of H2. |
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Expr13571
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In WT animals, egl-18 is enriched in the posterior daughter cell following the L2 asymmetric division of H2, and also enriched in the posterior daughter cells following the subsequent V1-V4 asymmetric division. |
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