The maternal effect detected by the genetic interaction of vrille with easter and dpp clearly predicts maternal and early embryonic accumulation of VRI mRNA, but makes no prediction for other developmental functions. A developmental Northern analysis was carried out. Poly A+ RNA from Oregon R embryos, larvae, pupae and adults raised at 25° was hybridized to the 2.8 kb cDNA7. RNA expression is dynamic throughout development. In embryos aged from 0 to 4 hours (stages 1 to 8: germ band elongation completed) two major transcripts of 4.9 and 6.2 kb are present at a low level. In 4 to 8 hour embryos (stages 8 to 11: beginning of germ band retraction) two major types 3.3 and 3.8 kb long were detected along with minor transcripts with higher molecular weights (up to more than 10 kb). The long transcripts do not hybridize with a 15 kb genomic probe mapping upstream cDNA1 and therefore the vri locus extends over at least 20 kb. The large number of bands observed in embryos aged between 0 to 8 hours is also observed when using a genomic probe mapping 5' to cDNA7 with no bZIP domain and no OPA repeats. These bands are, therefore, not due to cross hybridization. In older embryos aged from 8 to 24 hours (stages 11 to 17), the transcripts are more abundant and are represented exclusively by the two 3.3 and 3.8 kb major types. In third instar larvae, the 3.3 and 3.8 kb transcripts are still present, together with an abundant 1.5 kb transcript. In pupae (6 to 7 days of development), and in female adult flies, only the 3.3 and 3.8 kb transcripts are present at a high level. In males, an abundant 1.6 kb transcript is present. The 3.3 and 3.8 kb transcripts probably correspond respectively to the 3340 and 3807 bp putative cDNAs previously described. cDNAs corresponding to longer or shorter RNAs were not recovered and the origin of these RNAs is thus unknown. The expression of RNAs throughout development implicates many other functions besides those giving early embryonic phenotypes (George, 1997).
In situ hybridizations performed in embryos show a uniformly distributed maternal product at preblastoderm stage. From stage 10 (germ band fully elongated), the transcripts begin to localize and can be seen at higher levels in the primordium of the foregut. At stage 13, transcripts are present at high levels in the hypopharyngal lobe at the ventral opening of the stomodeum, the foregut, the proventriculus primordium, the hindgut, anal pads and posterior spiracles. At stage 14, during head involution and dorsal closure, the transcripts are located in stripes along the epidermis in the anterior part of each segment. The stomodeum, hindgut and anal pads are still strongly labeled and a longitudinal stripe is observed dorsally along the epidermis. At stage 15 (end of dorsal closure) thin stripes are seen dorsally across the closing epidermis and the amnioserosa is weakly labeled. The ventral most regions of the epidermis and the central nervous system are not labeled, although 50% of the ventral epidermis shows vri expression. At stage 16, the transcripts are still present in the stomodeum, anal pads and in a network of lateral and dorsal cells probably corresponding to the tracheal track (George, 1997).
vri transcripts are not expressed in ovarian stem cells, oogonia or early cysts and are first detectable at stage 8 in the nucleus and cytoplasm of nurse cells, which is consistent with maternally provided RNAs, and in the columnar follicular epithelial cells. The transcripts are also expressed in gut, brain and imaginal discs of third instar larvae. Similar localizations or sub-patterns were observed with the betagal staining of all the PlacZ mutant alleles, the differences between the five alleles being only quantitative. In embryos, the foregut, posterior spiracles and anal pads express lacZ: in ovaries, the border cells and columnar epithelial cells are stained, but not the nurse cells. This could reflect the fact that the P elements are localized in transcripts provided only zygotically or, on the contrary, that these P elements completely abolish the production of the maternal RNAs. The staining in border cells is not observed with in situ hybridizations and it is not known whether this is an ectopic localization unrelated to the real expression of the transcripts. Staining is observed in larval gut in a pattern very similar to that observed by in situ hybridization and in the central region of imaginal discs. These results are consistent with the hypothesis whereby the P element mutations alter the bZIP function (George, 1997).
The coregulation of PER, TIM, and VRI mRNA levels makes it likely that all three genes are expressed in the same cells. In situ hybridizations to adult head sections reveal that vri and tim are expressed in identical regions of the head. vri and tim are both expressed in the photoreceptor cells, which contain functional clocks, and in two clusters of cells in the central brain corresponding in position to the ventral and dorsal lateral neurons (LNs), which are the pacemaker cells responsible for circadian locomotor behavior. Double labeling experiments were performed with third instar larval brains to confirm that vri is expressed in LNs. An antibody was used against crustacean pigment dispersing hormone, which cross-reacts with the highly related Drosophila pigment dispersing factor) (PDF) and labels only the LNs in each brain lobe. Coexpression of LacZ and Tim with PDF was found, although in the case of vri, the pattern is not limited to LNs, consistent with Vrille's role in development (George, 1997). vri expression in larval LNs suggests that vri may play a role in the clock during much of development (Blau, 1999).
Vrille RNA levels oscillate, cycling with a phase and amplitude (10- to 12-fold changes) comparable to tim. Like TIM, VRI mRNA accumulates constitutively and at an intermediate level in per01 mutants. Northern blots of head RNA have resolved two species of vri RNA of approximately 3.4 and 3.8 kb that differ only in their 3' untranslated regions, and which both oscillated in a clock-dependent manner. VRI mRNA also oscillates robustly in wild-type flies maintained in constant darkness following entrainment to light-dark cycles, further confirming vri regulation by the clock. Since oscillations of per promoter activity and Per and Tim gene products can be detected throughout the body of Drosophila, oscillations in RNA isolated from bodies of male flies were sought. Clock-dependent cycling is observed for VRI and TIM, with 4- to 5-fold oscillations detected for both RNAs in LD cycles (Blau, 1999).
Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, the complement of circadian transcripts in adult Drosophila heads was determined. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for ~400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression is associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per0, tim01, and ClkJrk), are consistent with both known and novel regulatory mechanisms controlling circadian transcription (Claridge-Chang, 2001).
A genome-wide expression analysis was performed aimed at identifying all transcripts from the fruit fly head that exhibit circadian oscillations in their expression. By taking time points every 4 hr, a data set was obtained that has a high enough sampling rate to reliably extract 24 hr Fourier components. Time course experiments spanning a day of entrainment followed by a day of free-running were performed to take advantage of both the self-sustaining property of circadian patterns and the improved amplitude and synchrony of circadian patterns found during entrainment. 36 RNA isolates from wild-type adult fruit fly heads, representing three 2 day time courses, were analyzed on high-density oligonucleotide arrays. Each array contained 14,010 probe sets (each composed of 14 pairs of oligonucleotide features) including ~13,600 genes annotated from complete sequence determination of the Drosophila genome. To identify different regulatory patterns underlying circadian transcript oscillations, four-point time course data was colleced from three strains of mutant flies with defects in clock genes (per0, tim01, and ClkJrk) during a single day of entrainment. Because all previously known clock-controlled genes cease to oscillate in these mutants but exhibit changes in their average absolute expression levels, the analysis of the mutant data was focused on changes in absolute expression levels rather than on evaluations of periodicity (Claridge-Chang, 2001).
To organize the 158 statistically significant circadian transcripts in a way that was informed by the data, hierarchical clustering was performed. Both the log ratio wild-type data (normalized per experiment) and the log ratios for each of the three clock mutants (normalized to the entire data set) were included to achieve clusters that have both a more or less uniform phase and a uniform pattern of responses to defects in the circadian clock. One of the most interesting clusters generated by this organization is the per cluster. This cluster contains genes that have an expression peak around ZT16 and a tendency to be reduced in expression in the ClkJrk mutant. Strikingly, all genes previously known to show this pattern of oscillation (per, tim, vri) are found in this cluster. In fact, the tim gene, which has multiple representations on the oligonucleotide arrays, has two independent representations in this cluster. Together with the novel oscillator CG5798, per, tim, and vri form a subcluster (average phase ZT14) that shows upregulation in both the per0 and tim01 mutants. The fact that per, tim, and vri all function in the central circadian clock raises the possibility that several other genes from this cluster, including the ubiquitin thiolesterase gene CG5798 and the gene coding for the channel modulator Slowpoke binding protein (Slob) may function in the circadian clock or directly downstream of it (Claridge-Chang, 2001).
The two initial vri mutations, l(2)jf23Sz7 and l(2)jf23Sz36, were recovered in a screen as lethal EMS induced mutations on the 2nd chromosome (Szidonya, 1988). They have been renamed vri1 and vri2, respectively. Five other vri alleles were recovered by testing the P induced lethal stocks. Df(2L)tkvSz2 totally deletes vri and represents a null allele for vri and the nearby tkv, l(2)03771 and probably l(2)jf24 genes. Df(2L)tkvSz2 homozygous embryos show the typical tkv null phenotype with no dorsal closure and head involution resulting in the absence of dorsal epidermis. In developing embryos, no defects are observed before dorsal closure. Principally, at the blastoderm stage the expression of the Zen protein is normal and no obvious gastrulation defects are observed. vri lethal mutants die as embryos and, except with vri8, no dominant maternal lethality is detectable when vri females are crossed to wild type males. vri1, vri2 as well as vri1/vri2 homozygous embryos have very similar phenotypes. The embryos are shortened and the dorsal epidermis often appears wrinkled and reduced, and tracheae are interrupted. This dorsal shortening leads to a slight 'tail up' phenotype. Filzkorper are often internal. Less frequently the head skeleton is abnormal and ventral denticles are fused or missing. The vriP alleles result in similar phenotypes but these latter are stronger than those described above. The head skeleton is almost always defective. In some embryos the germ band remains extended, suggesting defects in germ band retraction. Some embryos are convoluted, with ventral denticles extended laterally and Filzkorper internal and presenting an abnormal morphology. This phenotype is similar to those observed in weakly ventralized embryos. These latter phenotypes have a low penetrance, however, and for this reason it could not be determined (using the expression of zen) whether they result from defects occurring in the early stages of establishment of dorsoventral polarity or later on. The vriP alleles are probably not null alleles since the mutants show apparent defects in dorsoventral polarity, not observed with the tkvSz2 null allele.
The zygotic phenotype of the total absence of the vri gene is difficult to detect, due to the presence of at least three other embryonic lethal genes within Df(2L)tkvSz2, the smallest available deficiency. Df(2L)tkvSz2 deleting mainly the thick veins gene itself presents strong head and dorsal cuticular defects. l(2)03771, represented by a single P induced mutation, shows weakly ventralizing embryonic phenotypes and is clearly not a null allele. In order to obtain a better view of the null vri phenotype, the Df(2L)tkvSz2 homozygous phenotype was observed in the presence of a tkv cDNA transgene, P[Ubi-tkv-2], which is capable of the total rescue of tkv null mutants. Such tkv rescued deficiency embryos die and possess a phenotype very similar to the homozygous phenotypes of vri1 or vri2, which can therefore be considered null alleles. However, vri/Df(2L)tkvSz2 hemizygous progeny hatch and die as larvae. Thus, the phenotype appears weaker when hemizygous than when homozygous. Although this delayed lethality could be due to a background effect, it would suggest that the alleles are not null or even hypomorphic, but rather neomorphic or antimorphic. Alternatively, it could be that one gene within the deficiency acts as a dominant suppressor of the embryonic lethality. The same results are observed with the P induced alleles. One hypothesis to explain why the hemizygous progeny die as larvae whereas homozygotes do not hatch is that the product of these alleles, including vri1 and vri2, is able to antagonize itself or the wild type maternal product and therefore has a stronger effect than the total absence of Vri (George, 1997).
Mad acts as a dominant enhancer of vri phenotypes in wing. About 10% of Mad6+/+ vri2 flies show a wing phenotype. The L5 vein is shortened and sometimes the posterior cross vein is also shortened and extra vein material is observed along the L2 vein. The same phenotype is observed with vri1 whereas with the other alleles the effect is weaker. Since this phenotype in not observed in the Mad/+ and vri/+ controls, it is concluded that it is due to the association of both genes. In order to investigate a possible interaction between vri and dpp in wing, a dominant effect of vri was sought in a dpp- context. The dpphr4/dppd6 phenotype consists of a reduction of wing to about one half of the wild type size and no defects in eyes or legs. When one dose of vri is associated with this genotype in dpphr4 vri2/dppd6 + flies, a further reduction in wing size is observed with reduction of veins. Furthermore, eyes are smaller with a rough aspect and legs are truncated. The enhancement of dpp phenotypes by vri2 is always observed, although the strength of the enhancement is variable. The same phenotypes are observed with vri1 (George, 1997).
Vri is closely related to bZIP transcription factors involved in growth or cell death. vri clonal and overexpression analyses reveal defects at the cellular level. vri clones in the adult cuticle contain smaller cells with atrophic bristles. The phenotypes are strictly cell autonomous. Clones induced in the eye precursor cells lead to individuals with smaller eyes and reduced number of ommatidia with an abnormal morphology and shorter photoreceptor cell stalks. Overexpression of vri is anti-proliferative in embryonic dorsal epidermis and in imaginal discs, and induces apoptosis. On the wing surface, larger cells with multiple trichomes are observed, suggesting cytoskeletal defects. In salivary glands, vri overexpression leads to smaller cells and organs. vri has been shown to be involved in locomotion and flight and interacts genetically with genes encoding actin-binding proteins. The phenotypes observed are consistent with the hypothesis that vri is required for normal cell growth and proliferation via the regulation of the actin cytoskeleton (Szuplewski, 2003).
The functional analysis of vri performed by mutant clone induction observed in adults indicates that vri is cell autonomous and involved in hair and cell growth. The fact that vri acts in a strict cell-autonomous manner suggests that it does not regulate the expression of a diffusive molecule such as a growth factor or a hormone. Smaller cells are recovered on the whole cuticle with shorter thinner or atrophic bristles. In the wing, clones have an abnormal shape and degenerative tissues are observed. These defects could be due to cytoskeleton defects. Clones induced in the eye precursor cells result in smaller eyes with a significantly reduced number of ommatidia with an atrophic morphology and a reduced size with the stronger allele. The photoreceptor cell stalks are shorter and atrophic. These results suggest that vri cells grow more slowly and are less viable than vri+ cells, even when they are not surrounded by wild-type cells (Szuplewski, 2003).
vri overexpression phenotypes suggest a role in cell cycle and proliferation. However, these phenotypes are not rescued by simultaneous overexpression of the genes encoding activators of proliferation, Drosophila E2F, cyclin E or string. Therefore, it is unlikely that Vri is either a direct repressor of genes that activate proliferation or an activator of those acting as inhibitors of proliferation like rbf or dacapo. It could act upstream in the Ras/MAPK or PI3K pathways regulating growth and involved in the regulation of the mammalian homolog of Vri (NFIL3A) acting mostly as a repressor. Genetic interactions have been tested in double-heterozygotes with available members of these pathways and vri, but neither reduction in viability nor any strong phenotypes were recovered. This could result from genes with non-limiting products and/or be due to the functional redundancy of vri. Alternatively, vri may control cell size independently of growth signals (Szuplewski, 2003).
vri loss-of-function and overexpression phenotypes, more probably, could result from primary defects in cytoskeletal actin network. Although cytoskeletal integrity and adhesion are altered in mutants of regulators of cell growth and proliferation, these effects are indirect. New vri phenotypes affect wing shape flight and locomotion. Locomotory defects could result from neurological or muscular alteration. Interaction was found with the alpha actn and bent genes involved in muscle actin function, which suggests that the effect is rather at the muscular level, although no gross defect was observed in indirect flight muscle. However these defects appear degenerative and must be studied in more detail. Hair atrophic phenotypes are observed in interaction with these two genes, suggesting an effect in other cell types. Although the locomotory and hair defects are not necessarily related, it is notable that the genes interacting with vri affect different types of actin, muscle and non-muscle actin. It will be interesting to search for the direct targets of Vri to understand its implication in locomotion and cytoskeletal integrity (Szuplewski, 2003).
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date revised: 30 November 2007
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