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All of the reporter constructs produced the same cell-specific expression pattern as transgenes. |
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Expr1438
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The reporter transgenes express ubiquitously in the early embryo starting at about the 100 cell stage during gastrulation. In late embryogenesis and posthatching, expression is more limited. Strongest expression is observed in migrating cells and growing neurons as these cells undergo movements on the epidermis. At hatching, the reporters express in many neurons throughout the animal, in several cells of the pharynx including some pharyngeal neurons, in the elongated processes of the excretory cells, in the amphid and phasmid sheath and socket cells, in the tail hypodermis, and at later stages in intestine, muscles, vulva, and somatic gonad including the gonad sheath and hermaphrodite distal tip cells. The neurons expressing unc-73 include the PLM, ALM, PDE, HSN, CAN, PHC, and PVN neurons and the ventral cord motorneurons. Expression in the HSNs is absent in early larval stages, but begins late in the second larval stage (L2), precisely when axon outgrowth is initiated from the HSN cell bodies. The Q neuroblasts, Pn neuroectoblasts, sex myoblasts (SMs), and canal associated neurons (CANs) express unc-73 reporters. The left and right Q cells begin to express the GFP reporter as they initiate their migrations along the longitudinal axis of the epidermis during the early first larval (L1) stage, and expression in these cells continues beyond the completion of their first division. The unc-73 reporters express in the Pn cells just before this second phase of movemen. The distal tip cells also express the unc-73/reporters during their migration. |
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A similar ceh-34 expression pattern has been independently observed by T. Hirose and H. R. Horvitz (personal communication). Picture: Fig. 2. |
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Expr8715
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Both constructs showed identical patterns of GFP expression and nuclear localization. ceh-34::gfp expression begins late during embryogenesis and persists in a few anterior body wall muscle cells and other unidentified cells in the head throughout postembryonic development. ceh-34::gfp is transiently expressed in the M lineage, specifically in M.dlpa and M.drpa, before they terminally differentiate into coelomocytes. ceh-34::gfp expression is no longer detectable in these two cells after they differentiate into coelomocytes. |
nuclear localization |
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Expr3994
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All reporters show qualitatively similar expression patterns. In transgenic embryos, gfp expression is detected in the developing gut by the comma stage and up to the 2-fold stage. By the late 3-fold stage of embryogenesis, dig-1 reporter constructs become strongly expressed in mesodermal cells. In larvae of all stages and adults, dig-1 reporter genes remain strongly expressed in mesodermal cells, including body wall, head, vulval, uterine, pharyngeal muscles, and in enteric and anal depressor muscles. Additional mesodermal cells expressing the dig-1 reporter construct include support cells in the head (GLR cells), the head mesodermal cell, coelomocytes and sex myoblasts. Expression could also be observed in the hypodermis. No expression could be observed in the nervous system. |
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Expr2347
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CYE-1 is present in adult animals and is restricted to the germline, which is the only proliferative tissue in adults. CYE-1 levels vary in the germline. Mitotic germ cells in the distal region of the gonad have easily detectable levels of nuclear CYE-1. Germ cells in the initial stages of meiosis (proximal to the mitotic germ cells) have lower CYE-1 levels. Finally, as oocytes cellularize in the loop region of the gonad, CYE-1 levels increase with mature oocytes having the highest levels of nuclear CYE-1. These results demonstrate that a significant portion of maternal cye-1 contribution to the embryo is CYE-1 protein. CYE-1 level was assayed postembryonically to determine whether CYE-1 could be detected and if levels of CYE-1 correlated with mitotic proliferation. CYE-1 protein is detectable in larval blast cells that give rise to all tissue types, including, germline, intestine, hypodermis, neurons, and muscle. During larval stages, the level of CYE-1 protein is much lower than that found in germ cells or in the early embryo. CYE-1 antibody staining is restricted to the developmental time when the blast cells are undergoing active proliferation. For example, in the L1 stage, proliferating P blast cells that produce ventral nerve cells have relatively high levels of nuclear CYE-1. In contrast, during the L2 larval stage, the nonproliferating neuronal descendents of the P blast cells have CYE-1 levels that are only barely detectable above background. Further, while a subset of the P cell descendents, the vulva precursor cells (VPCs), will proliferate in the L3 larval stage to produce the vulva, these cells do not have appreciable CYE-1 levels while they are quiescent in the L2 larval stage. Nuclear CYE-1 becomes detectable in the VPCs during the L3 larval stage when they begin proliferation. CYE-1 becomes undetectable in the VPC descendents after completion of cell divisions in L4 larval stage animals. Monoclonal anti-CYE-1 antibody was used to assay CYE-1 levels from fertilization to the end of embryogenesis. In the zygote, CYE-1 is observed in the maternal and paternal pronuclei as soon as they form. The specificity of antibody staining was confirmed by cye-1 RNAi treatment of adult hermaphrodites that abolishes both oocyte nuclei and embryonic anti-CYE-1 protein staining. In early embryos, CYE-1 is enriched in nuclei, and levels appear constant with no evidence of cell cycle fluctuations other than during mitosis. During mitosis, CYE-1 antibody staining appears diffuse once nuclear envelope breakdown occurs, but resumes nuclear localization upon reformation of the nuclear envelope in telophase. CYE-1 is present equally in all cells of the early embryo. The level of CYE-1 declines during embryogenesis and disappears from most cells in comma-stage embryos coincident with the completion of the majority of embryonic cell divisions. |
nuclear |
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A similar pattern of EGL-15 expression has been observed for many other arrays containing transgenic egl-15 constructs. The expression in the hypodermis is very similar to that observed in Pe15*2::GFP animals, in which expression was observed in the major hypodermis but was excluded from the seam cells. A similar expression pattern can be observed in rescued egl-15(null) animals expressing an egl-15 transgene driven by a hypodermal promoter (Prol-6 and Pdpy-7). Hypodermal expression of EGL-15 is also reported in animals expressing Pegl-15::lacZ, consistent with the antibody staining in this paper. Endogenous EGL-15 was not detected in wild-type animals, probably due to the low-level expression of the endogenous receptor. Therefore, a strain overexpressing a chromosomally integrated egl-15(+) array (ayIs29) was generated , and immunofluorescence staining was performed on this strain using affinity-purified anti-EGL-15 antibodies. |
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Expr2943
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EGL-15 expression was observed in hypodermal cells as well as the sex myoblasts, the type I vulval muscles and some unidentified neurons in the head . The staining is specific to EGL-15, since no signal is observed with secondary antibody alone. Hypodermal expression is obvious throughout all four larval stages, with stronger expression in the early larval stages. |
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