As of 2001, a total of 89 ran expressed sequence tags (ESTs) have been reported in the Berkeley Drosophila Genome Project database while only a single ran-like EST has been identified. The ran like EST (GH25818) was isolated from an adult head cDNA library. In comparison, ran ESTs have been isolated from both embryo and adult tissues (61, embryo; 18, adult head; and 1 cell culture). The differences in the number of reported ran and ran-like ESTs suggest that the two genes may have different spatial/temporal expression dynamics during development. To test this hypothesis, their embryonic expression patterns were tested on 0- to 18-h embryo collections via whole-mount in situ hybridizations (Koizumi, 2001).
Localization of ran mRNA during embryonic development revealed two temporal phases of expression. Initially, ran expression is found in the pre-cellular blastoderm, suggesting that it is supplied maternally. Localization of ran transcripts in progressively older embryos revealed that shortly after the onset of gastrulation, there is a significant and rapid loss of ran mRNA throughout the embryo. By embryonic stage 7, no expression was observed. The second temporal phase of ran expression occurs in CNS neuroblasts but only during their late sublineage-forming mitotic divisions. No expression was observed in NBs or any other cell during stages 8-11 indicating that ran gene expression is restricted from NBs during the formation of their early sublineages. During germ-band contraction (stage 12), ran transcripts were detected in large-diameter cells that line the cephalic lobes and the ventral/ventral-lateral surface of the developing ventral cord ganglia. The size and location of ran-expressing cells suggest that they are predominantly NBs. By stage 16, ran transcripts were detected in only a few putative neural precursor cells (NBs and/or ganglion mother cells). The fact that ran CNS expression is progressively reduced, starting at early stage 14, further suggests that its expression is predominately restricted to NBs and ganglion mother cells and not to post-mitotic neurons or glia. In situ hybridization of third instar larval tissues with ran -specific probes reveals that ran is also expressed ubiquitously, albeit at low levels, in both larval tissues and imaginal discs (Koizumi, 2001).
ran-like in situ hybridization analysis of embryos, obtained from overnight collections, also identified a maternal and zygotic pattern of gene expression. Similar to ran, ran-like transcripts were detected uniformly distributed in syncytial blastoderm embryos, and starting just after cellularization there was a rapid decline in the ran-like in situ signal. By stage 5, little or no ran-like in situ staining was observed. However, differing from the neuronal precursor gene expression pattern of ran, zygotic ran-like expression was detected in salivary gland cells and in the epithelial cells lining the tracheal tubular network (Koizumi, 2001).
The GTPase Ran regulates multiple cellular functions throughout the cell cycle, including nucleocytoplasmic transport, nuclear membrane assembly, and spindle assembly. Ran mediates spindle assembly by affecting multiple spindle assembly pathways: microtubule dynamics, microtubule motor activity, and spindle pole assembly. Ran is predicted to facilitate spindle assembly by remaining in the GTP-bound state around the chromatin in mitosis. This study directly tests the central tenet of this hypothesis in vivo by determining the cellular localization of Ran pathway components in Drosophila embryos. During mitosis, RCC1, the nucleotide exchange factor for Ran, is associated with chromatin, while Ran and RanL43E, an allele locked in the GTP-bound state, localizes around the spindle. In contrast, nuclear proteins redistribute throughout the embryo upon nuclear envelope breakdown (NEB). Thus, in vivo RanGTP has the correct spatial localization within the cell to modulate spindle assembly (Trieselmann, 2002).
To investigate the significance of ran-restricted CNS expression, its misexpression was targeted to different temporal windows of CNS development. In addition, a dominant- negative mutant form of ran was targeted to the developing CNS and to the larval eye/antenna imaginal disc to assess the role of ran-dependent functions. Embryonic CNS misexpression of the mutant, but not wild-type, ran results in larval death. Neither wild-type nor mutant ran misexpression had any detectable effect on embryonic CNS lineage specification, nuclear transport of a number of CNS-specific transcription factors or axonal guidance. However, expression of the dominant-negative mutant ran in the developing eye/antenna disc did result in a severe adult eye phenotype marked by apoptosis of photoreceptor, cone and pigment cells (Koizumi, 2001).
Blocking Ran function(s) in mammalian cultured cells has been achieved via the misexpression of different dominant-negative mutant forms of Ran. The mutant Ran proteins have single or double amino acid substitutions (G19V and/or Q69L) that alter the Ran protein in such a way that it can bind but not hydrolyze GTP (Coutavas, 1993; Ren, 1994; Lounsbury, 1996). These dominant-negative mutant alleles have been shown to have profound effects on multiple nuclear events, including progression of the cell cycle, RNA transport, and DNA replication (Ren, 1994; Melchior, 1998).
The restricted expression of ran to CNS NBs undergoing late lineage-generating divisions and its absence from post-mitotic neurons or glia suggests that ran may be required for a temporally defined event in neural development. Alternatively, ran may be required in all neural cells, but due to the high rate of NB mitotic divisions, the maternal supply is insufficient for CNS stem cells to complete their lineage development (Koizumi, 2001).
As an initial step toward determining the significance of the temporally restricted CNS expression, the effects of ran misexpression outside the temporal boundaries of its wild-type expression window was studied. The effects of the dominant-negative ran mutant, ranG19V;Q69L, on different aspects of embryonic development and on adult eye development was studied. The targeted ectopic expression was accomplished by using the yeast Gal4/UAS system. Both wild-type and mutant ran ORFs were inserted into the P-element based UAS vector to create the following constructs: P[UAS.ranwild-type] and P[UAS.ranG19V;Q69L], respectively. Multiple independent transformant lines were generated with the above P-element constructs with integration sites on either the second or third chromosomes. For misexpression in the developing nervous system, Gal4 drivers were used that can activate the expression of the UAS transgenes in either NBs or post-mitotic neurons. For NB expression, pros.Gal4 and pdm.Gal4 drivers were used; for post-mitotic neurons an elav.Gal4 driver was used. Pan-neural embryonic transgene expression was also achieved using scabrous. Gal4. The effects of UAS.ranwild-type and UAS.ranG19V;Q69L misexpression on early segmentation was also studied using a hairy.Gal4 driver. The effects of wild-type and mutant ran misexpression were assessed by three criteria: viability, changes in the sub-cellular localization of nuclear factors, and defects/changes in nervous system structure as assessed by axonal patterning. To confirm that these Gal4 drivers activated the different UAS.ran transgenes, misexpression was monitored via in situ hybridization (Koizumi, 2001).
The misexpression of wild-type ran with the Gal4 drivers used in this study did not result in any detectable abnormal phenotype in either the CNS or in other aspects of Drosophila biology. However, expression of UAS.ranG19V;Q69L under the control of each of the above Gal4 drivers, with the exception of the scabrous driver, resulted in either late first, early second instar larval or pupal mortality. No abnormal behavioral phenotypes were detected in the first instar larva harboring any of the different promoter-driven mutant ran transgene combinations. After hatching from the egg case, larva exhibited wildtype motility and eating behavior. The prosperoGal4/UAS.ranG19V;Q69L transformant adults failed to exit the pupa case and inspection of the pharate adults failed to identify any morphological defects. The scabrous.Gal4/UAS.ranG19V;Q69L transgene combination proved to be viable (Koizumi, 2001).
To determine if ectopic expression of the wild-type or mutant ran transgenes leads to defects in the subcellular distribution of developmentally regulated nuclear proteins, embryos were stained with antibodies to the Castor, Pdm1, Hb, Prospero and Even-skipped transcription factors. Immunostains of all Gal4/UAS combinations (both wild-type and mutant Ran) failed to reveal any defects in the subcellular distribution of these proteins. To assess if other aspects of neural development were affected, such as axonal outgrowth, axon markers (monoclonal antibodies BP102 and 22C10) were employed to immunostain embryos containing the different Gal4/UAS combinations. Again, within the resolution of this analysis, no defects in axonal outgrowth or axon fascicle development during embryonic development were detected in any of the Gal4/UAS combinations tested. However, the possibility can not be ruled out that misexpression of mutant ran triggers an embryonic defect, albeit subtle, which ultimately results in larval lethality (Koizumi, 2001).
Given the lack of detectable abnormal phenotype accompanying the wild-type or mutant ran transgene expression, the possibility exists that the levels of transgene expression were not sufficient to trigger cellular or developmental embryonic defects. To address this possibility, transformants were generated that contained multiple wild-type or mutant Ran Gal4/UAS transgene combinations. Analysis of these lines revealed no detectable differences between embryos and larva harboring single or multiple copies of the Gal4/UAS combinations. Nevertheless, embryonic expression of Ran G19V;Q69L does produce larval and pupal lethality, confirming that the Gal4/UAS.ranG19V;Q69L transgene combinations did in fact work. The data suggest that larval cells and/or postmitotic neurons may have a greater requirement for Ran dependent processes. Currently it is not known why the effects of the ectopic expression are delayed. Perhaps the high levels of endogenous Ran expression are sufficient to overcome the immediate effects of the different Gal4 driven UAS.ranG19V;Q69L combinations. It is also possible that other ran -independent nucleo-cytoplasmic transport mechanisms may compensate for a deficiency in ran function during embryonic development. Ran-independent nuclear transport has been observed in other systems (Nakielny, 1997; Fagotto, 1998; Sachdev, 2000; Koizumi, 2001 and references therein).
During embryonic development, perdurance of Ran function from the maternal expression of both ran genes may be sufficient to over-ride the misexpression of the dominant-negative mutant Ran. To avoid this possibility, we assessed the effects of ran misexpression during larval development in imaginal cells that should have little or no maternal contribution. To accomplish this, the misexpression of wild-type and mutant ran was targeted to different stages of adult eye development. The targeted misexpression of UAS.ran transgenes to the eye-imaginal disc takes advantage of the fact that the eye is not essential for viability and that disruption of the ommatidial organization is a sensitive indicator of phenotypic effects. In addition, different Gal4 drivers are available to target misexpression within different temporal windows of eye development. Three different spatial/temporal eye disc Gal4 drivers were employed: (1) dppdisc-.Gal4 to target misexpression to undifferentiated cells within the morphogenetic furrow, (2) pGMR.Gal4 to target all cells behind the morphogenetic furrow, and (3) sevenless.Gal4 to drive misexpression in a subset of differentiating photoreceptor cells and non-neuronal cone and pigment cells. As in the embryo, the ectopic misexpression of wildtype ran during eye development resulted in no detectable mutant phenotype. In all cases the ommatidial organization of the various Gal4/UAS.ran combinations was indistinguishable from wild-type eyes. In addition, the misexpression of mutant ran in retinal precursors within the morphogenetic furrow, P[dppdisc.Gal4]/P[UAS.ranG19V;Q69L], did not induce detectable changes in eye development. Expression of Ran G19V;Q69L in developing photoreceptors using a sevenless promoter resulted in an eye phenotype: close examination of eye cross-sections showed a disrupted ommatidial structure with absence of photoreceptor cells including the R7 cell. Expression of mutant ran behind the morphogenetic furrow via the P[GMR.Gal4] transgene resulted in a severe eye phenotype characterized by a complete loss of photoreceptor, cone and pigment cells. The severe phenotype was partially reverted in the presence of a constitutively active p35 genetic background. p35 has been shown to prevent programmed cell death in Drosophila, and acts by binding to and inhibiting the proteolytic activity of caspases. The partial rescue suggests that the loss of ommatidial structures is caused by programmed cell death. However, misexpression of mutant ran via the GMR promoter did not appear to affect the development of interommatidial bristles (Koizumi, 2001).
Interommatidial bristles are mechanosensory organs composed of four cells that are derived from a single sensory organ precursor (SOP). Unlike the photoreceptor and cone cells, the appearance of SOPs is not related to the distance from the morphogenetic furrow. The final cell divisions generating the interommatidial bristles take place between 6 h and 15 h after puparium formation. Photoreceptor cell determination occurs behind the furrow, during third instar development. Because all eye lineages come from equivalent precursors, cell death induction due to Ran G19V; Q69L expression occurs after the transit of the morphogenetic furrow and subsequent to or parallel to the induction of interommatidial bristles. It is not clear from these results whether the bristles cells are more resistant to RanG19V;Q69L or whether Ran is specifically required for a transport step in photoreceptor cells that does not take place in bristle cells (Koizumi, 2001).
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date revised: 25 July 2007
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