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Gene name - abstrakt Synonyms - Cytological map position - 82A1--3 Function - RNA helicase Keywords - Bolwig's organ, CNS, axonogenesis |
Symbol - abs FlyBase ID: FBgn0015331 Genetic map position - Classification - DEAD-box subfamily ATP-dependent helicase protein Cellular location - nucleus and cytoplasm |
abstrakt, a gene originally identified genetically by its effect on axon outgrowth and fasciculation of Bolwig's nerve (Schmucker, 1997), encodes a DEAD-box helicase that is essential for survival at all stages throughout the life cycle of the fly. Mutants show specific defects in many developmental processes, including cell-shape changes, localization of RNA, apoptosis and axon guidance. Abstrakt is not globally required for RNA splicing, transport, subcellular localization or translation. Nevertheless, there is a widespread requirement for Abstrakt during post-transcriptional gene expression. Abstrakt must affect processing of specific subsets of RNAs, suggesting that differential post-translational control is important to the development of the fly (Irion, 2000).
In Drosophila, 12 genes are known that code for DEAD-box proteins, including the Drosophila homologs of P68 (Dm62) and eIF-4a. One of the best studied, vasa, was originally identified as a member of the posterior class of maternal effect genes in Drosophila. It is required for the translation of Oskar and Nanos mRNAs during the assembly of the pole plasm and for the localization and translation of Gurken mRNA during oogenesis. In addition to vasa, mutants exist for belle (bel), hel25E (hel), pitchoune (pit) and eIF-4a. A common feature of the phenotypes of these mutants are larval growth defects, but most mutants also show specific characteristics. For example, strong loss-of-function alleles in eIF-4a affect segmentation of the embryo, a process that is particularly sensitive to changes in protein levels; mutations in hel lead to lethality when homozygous, but when heterozygous act as enhancers of position-effect variegation (Irion, 2000 and references therein).
In Drosophila, both neural and muscle progenitors divide asymmetrically. In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation. Abstrakt (Abs) is a DEAD-box protein that regulates aspects of cell polarity in oocytes and embryos. A conditional allele of abs was used to investigate its role in neural and muscle progenitor cell polarity. In neuroblasts loss of apical Insc crescents, failure in basal protein targeting, and defects in spindle orientation were observed. In the GMC4-2a cell loss of apical Insc crescents, defects in basal protein targeting, and equalization of sibling neuron fates are observed; muscle precursors show a similar equalization of sibling cell fates. These phenotypes resemble those of insc mutants; indeed, abs mutants show a striking loss of Insc protein levels but no change of insc RNA levels. Furthermore, the Abs protein physically interacts with insc RNA. These results demonstrate a novel role for Abs in the posttranscriptional regulation of insc expression, which is essential for proper cell polarity, spindle orientation, and the establishment of distinct sibling cell fates within embryonic neural and muscle progenitors (Irion, 2003).
Mitotic neuroblasts form an apical cortical protein complex containing Bazooka (the Drosophila homolog of nematode and mammalian Par-3), Par-6, atypical Protein Kinase C, Inscuteable (Insc), Partner-of-Inscuteable, and Gαi proteins . These apical proteins have three functions: to promote basal cell fate determinant localization, to orient the mitotic spindle along the apical/basal axis, and to promote the formation of an asymmetric spindle leading to the generation of daughters of unequal size. The basally localized determinants include Miranda (Mir) and Numb (Nb), which were used as markers in this study. Their basal localization ensures their preferential segregation into the basal daughter cell, called ganglion mother cell (GMC), during neuroblast division and ensures proper GMC fate specification (Irion, 2003).
To assay abs function, a temperature-sensitive allele was used in combination with a small deficiency uncovering the abs locus (abs14B/Df(3R)231-5, hereafter referred to as abs14B embryos) in which the maternally contributed Abs protein can be inactivated by a shift to the restrictive temperature. Wild-type embryos at the restrictive temperature and abs14B embryos at the permissive temperature show normal apical (Insc) and basal (Mir) cortical protein crescents in mitotic neuroblasts, as well as normal apicobasal orientation of the mitotic spindle. In contrast, abs14B embryos that are shifted to the restrictive temperature display severe defects in neuroblast polarity: Mir frequently shows uniform cortical distribution or occasionally accumulates as mispositioned lateral crescents. Furthermore, mitotic spindles occasionally fail to orient along the apical-basal axis. The similarity of these phenotypes and those that were previously reported for mutations affecting components of the Insc complex prompted the assaying of Insc protein localization in abs mutants. Interestingly, Insc protein is not detectable above background levels at the restrictive temperature in abs14B mutant neuroblasts, although apical Insc localization is not affected in abs14B embryos at the permissive temperature or in wild-type embryos at the restrictive temperature. It is concluded that loss of abs function leads to the loss of detectable Insc protein in neuroblasts and generates the phenotype previously seen in insc mutants. The simplest interpretation is that Insc expression and/or Insc protein stability is impaired in abs mutants, which leads to the observed defects in neuroblast asymmetric cell division (Irion, 2003).
To determine if abs has a more general role in regulating Insc levels and asymmetric cell division, asymmetric division of the ganglion mother cell GMC4-2a, which produces a pair of identified sibling neurons, RP2 and RP2sib, was examined. During wild-type GMC4-2a divisions, the mitotic spindle is apicobasally oriented; Insc is localized to the apical cortex, whereas Numb is localized as a basal cortical crescent and segregates preferentially into the more basal daughter cell, where it acts to downregulate Notch (N) signaling and induce the RP2 cell fate. The RP2 sibling cell does not inherit Numb, cannot downregulate N signaling, and adopts the secondary RP2sib fate. GMC4-2a and RP2 express the Even-skipped (Eve) transcription factor, but RP2sib does not; thus, there is only one Eve+ cell at the RP2 position in wild-type embryos. However, in abs14B embryos shifted to the restrictive temperature prior to GMC4-2a division, approximately 32% of the hemisegments had a duplicated Eve+ cell at the RP2 position. This phenotype was rarely seen either in control embryos (from a stock homozygous for abs14B along with two copies of a functional abs+ transgene, henceforth referred to as abs24:14B, that rescues the abs lethality subjected to the same temperature-shift regime or in abs14B embryos at the permissive temperature. The duplicated Eve+ cells are likely to be duplicated RP2 neurons because they express two additional markers (22C10 and Zfh1) for mature RP2 neurons (Irion, 2003).
To elucidate the origin of the duplicated RP2 neurons, anti-Eve staining was used to follow the development of the GMC4-2a lineage in wild-type and abs14B embryos. The results indicate that the extra RP2 neuron arises as the result of a transformation of the RP2sib to the RP2 cell fate (Irion, 2003).
Pon directly binds Numb protein and reflects the localization of Numb in all cells analyzed so far. In control abs24:14B and in wild-type embryos shifted to the restrictive temperature (33°C), Pon localizes as a basal crescent in mitotic GMC4-2a. In abs14B embryos subjected to the same temperature shift regime, approximately 50% (18/34) of metaphase GMC4-2a cells show cortical distribution, misplaced crescents, or weak basal crescents of Pon, and approximately 25% of the cells show no obvious Pon crescents. Hence, the symmetric segregation of Numb to both daughter cells in a proportion of the dividing GMC4-2a cells could account for the RP2 duplication phenotype seen in the abs14B embryos (Irion, 2003).
Because the abs phenotype is similar to the insc phenotype in both neuroblasts and GMC4-2a, Insc localization during the GMC4-2a cell division was investigated. In control embryos, Insc always forms an apical crescent in metaphase GMC4-2a cells. In contrast, at the restrictive temperature, the majority of the abs14B mutant GMC4-2a cells showed no clear apical crescents of Insc. Consistent with the finding that Insc localization is affected in abs14B, the duplicated RP2 cells seen at the restrictive temperature exhibit equal nuclear size, as is also seen in insc embryos but not in mutants that disrupt sibling cell fate choice at the postmitotic level (Irion, 2003).
The role of abs during embryonic muscle progenitor divisions was investigated. The muscle progenitor P15 divides asymmetrically to produce two daughter cells with distinct identities. Numb is asymmetrically localized in the dividing P15 and preferentially segregates to the daughter cell that will become the founder for the single Eve-positive muscle DA1; the sibling cell is Eve-negative. abs14B embryos subjected to a 45 min pulse at the restrictive temperature showed duplications of the Eve-positive DA1 in 34% (23/68) of the hemisegments. In the control abs24:14B embryos, 135/136 of the hemisegments showed a single Eve-positive DA1. Thus, abs is also required for the asymmetric division of some muscle progenitors (Irion, 2003).
The abs and insc mutant phenotypes in asymmetrically dividing cells are very similar, and abs mutants show a loss of Insc protein crescents in neuroblasts, in GMCs, and throughout the embryo. Thus, the abs phenotype can be most simply modeled as a defect in establishing or maintaining normal levels of apical Insc protein in all of these cell types. The loss of Insc crescents could be caused either by an overall decrease in the levels of Insc or by a failure to localize Insc correctly in these cells. In situ hybridization experiments revealed no reduction in insc RNA expression, so abs does not appear to regulate insc at the transcriptional level. Western blots were used to test whether the total amount of Insc protein was affected in abs mutant embryos. The Insc protein migrates as an approximately 100 kDa band. Wild-type and abs14B embryos were shifted to the restrictive temperature and analyzed after 0, 30, and 60 min. The levels of Insc protein decreased progressively in abs14B embryos until they were nearly undetectable at 60 min, whereas they remained constant or even increased (depending on the age distribution of embryos at the beginning of the experiment) in wild-type embryos. Other proteins remain constant, and several proteins can be translated de novo at the restrictive temperature, indicating that abs function is not generally required for protein synthesis. Together, these data indicate that the most upstream defect associated with a reduction in abs function is a reduction in the levels of the Insc protein (Irion, 2003).
If Abs indeed acts on asymmetric cell divisions by maintaining high levels of Insc, it should be possible to circumvent the requirement for Abs at least in part by raising Insc levels experimentally. To test this, the GAL4-UAS system was used to express high levels of insc within neuroblasts in embryos lacking functional Abs protein. This led to a marked rescue of the RP2 phenotype (Irion, 2003).
Because Abs is a DEAD-box protein, it seemed conceivable that it might exert its effect on Insc protein levels by a direct interaction with insc RNA. A yeast-three hybrid assay was used to test this. The assay is based on the interaction of the HIV-1 RNA binding protein Rev with RNA molecules containing a Rev responsive element (RRE). Rev is fused to the GAL4 DNA binding domain, whereas the putative RNA binding protein, in this case Abs, is fused to the activation domain. The two fusion proteins are then bridged by a hybrid RNA consisting of an RRE-containing sequence fused to the RNA to be tested, in this case insc RNA. insc RNA is clearly able to interact with Abs in this system. Both the full-length RNA and a construct lacking the 5' third of the RNA show an interaction. However, no single fragment of the 3' part of the RNA was found to be able to interact with Abs (Irion, 2003).
Thus Abs directly binds Insc mRNA in vitro; loss of Abs leads to lowered Insc protein levels but not lowered mRNA levels, and loss of Abs leads to a failure to properly localized cell fate determinants in at least three asymmetrically dividing cell types: neuroblasts, GMCs and muscle progenitors. It is concluded that Abs has a role in controlling cell polarity and asymmetric cell division in multiple cell types, in part through the posttranscriptional regulation of Insc levels (Irion, 2003).
RT-PCR showed that Abs transcripts are found throughout all developmental stages, but no ABS RNA could be detected by in situ hybridizations or on Northern blots, nor is Abstrakt protein detectable in normal embryos stained with antibodies against Abstrakt. Thus, ABS RNA and protein does not appear to be very abundant. When Abstrakt is expressed at high levels using the GAL4/UAS ectopic expression system, a clear signal is seen by immunofluorescence in the areas in which the UAS-abs transgene is expressed. In older embryos (> stage 12), the signal is restricted to the nuclei. In younger embryos, the subcellular localization of Abstrakt varies between predominantly nuclear localization, homogeneous distribution throughout the cell, and enrichment in the cytoplasm. No correlation between Abstrakt localization and specific stages of the cell cycle has been observed (Irion, 1999).
Embryos homozygous for the non-conditional abstrakt alleles or the deficiency Df(3R)231-5 develop without apparent gross defects, but fail to hatch. To assay functions at other stages, the temperature-sensitive allele abs14B was used. abs14B animals were transferred from room temperature to 32°C at various stages of development. Shifts to restrictive temperature lead to lethality at all stages of the life cycle. Although embryos homozygous for the non-conditional alleles develop until hatching, abs14B embryos cease to develop and show gross morphological defects at restrictive temperature. Thus, abs is essential for embryogenesis. The embryos homozygous for the non-conditional alleles must therefore use the wild-type gene product provided by their heterozygous mothers to complete embryogenesis in the absence of functional zygotic Abstrakt. A wild-type paternal copy of the gene is sufficient to rescue abs14B embryos if they are shifted to restrictive temperature after gastrulation. The temperature-sensitive phenotype is identical in progeny from mothers homozygous for abs14B allele and mothers heterozygous for abs14B and a deletion of the gene. No dominant effects of the abs14B allele were seen in animals heterozygous for the abs14B and one or more wild-type copies of the gene (Irion, 1999).
It seemed possible that Abstrakt, as a DEAD-box protein, might be involved in general RNA processing or metabolism, and that cells in the mutant animals eventually run out of essential components and commit apoptosis. When TUNEL assays were performed on abs14B mutants, however, no increased rate of apoptosis is seen. Instead, apoptosis is almost completely suppressed, and even the wild-type pattern of cells undergoing programmed cell death is not seen in mutant embryos (Irion, 1999).
To test at which level the apoptotic pathway is interrupted in abs mutants, the expressions of reaper (rpr), a nuclear regulator of apoptosis in Drosophila, and dredd, a target gene of reaper encoding a caspase were examined. Both reaper and dredd are expressed properly in abs14B mutant embryos. This shows that the apoptotic regulators are activated in the mutant and components of the apoptotic pathway are expressed, but the program is not completed (Irion, 1999).
Ectopic cell death induced by overexpression of reaper is also greatly suppressed in abs mutants. The amnioserosa is the only tissue in abs mutants in which extensive apoptosis was found after overexpressing reaper. Ectopic apoptosis induced by overexpression of a second regulator, hid, is not completely suppressed in abs mutants, consistent with previous findings that hid is a more potent inducer. Rather than being blocked, apoptosis in abs14B mutant ovaries occurs prematurely in follicle cells and nurse cells. This points to different modes of regulation of apoptosis during embryogenesis and oogenesis, in line with recent findings that reaper, hid and grim are not essential for programmed cell death of the nurse cells (Irion, 1999).
As noted above, Abstrakt is essential for survival at all stages throughout the life cycle of the fly: mutational defects in many embryonic developmental processes include cell-shape changes, localization of RNA and apoptosis. To understand which cellular and developmental processes are affected by loss of abs function, defects were analysed in more detail at various stages of embryogenesis and during oogenesis. When embryos mutant for temperature sensitive Abstrakt are shifted to the restrictive temperature at any point from egg laying up to approximately 3 hours, they continued to develop normally up to gastrulation, producing a fully cellularized blastoderm without any demonstrable defects. Abstrakt, therefore, is not essential for nuclear cleavages, migration of the nuclei to the periphery of the egg, or cellularization of the pole cells. Significantly, it is also not needed for the first process directed by the zygotic genome, the cellularization of the syncytial blastoderm. This shows that the genes required for cellularization must be transcribed, and their RNAs exported and translated normally in the absence of functional Abs. Moreover, normal expression patterns of the early zygotic pattern formation genes (for example, twist, snail, hairy, eve) are established by the cellular blastoderm stage, demonstrating that abs is not required for any of the steps in the production of functional proteins from these genes or their upstream regulators. Because the mRNAs of several of these genes need to be spliced, this result excludes a general role for Abstrakt in splicing. Nevertheless, although a normal cellular blastoderm is produced, development at restrictive temperature is significantly slower than in wild-type embryos (Irion, 1999).
At later stages of embryogenesis, embryos mutant for temperature sensitive abs14B show defects within 30 minutes of a shift to 32°C. Serious morphological defects are seen during gastrulation. The ventral cells of the blastoderm, which normally undergo a series of cell-shape changes, are unable to do this properly. Even where weak apical cell constrictions result in the formation of an indentation, one of the earliest processes during ventral cell shape changes, the movement of nuclei from the apical towards the basal side of the cell, does not occur in the mutants. Interestingly, this phenotype does not resemble that of mutants in either twist or snail, genes encoding the two essential transcription factors required for this process. This shows that Abstrakt either affects only a subset of the gene products controlled by Twist and Snail, or the genes that act in parallel to Twist and Snail (Irion, 1999).
The behaviour of the neighboring ectodermal cells demonstrates that a loss of Abstrakt function does not simply result in a cessation of all cellular activity, or constitute a block to cell-shape changes in general. In wild-type embryos, neuroblasts delaminate from the neuro-epithelial layer, while the epithelial cells become cuboidal. In the mutant, no delamination of neuroblasts occurs, and the epithelial cells become extremely tall and thin. Gene expression patterns, again, appear normal as far as they have been tested (Irion, 1999).
When embryos are shifted after the neuroblasts have segregated, the neuroblasts continue to develop, to activate expression of the neural differentiation marker 22C10 and to extend neurites. The first cells to express 22C10, the ventral midline precursors vMP2 and dMP2 were examined in greater detail. These cells normally extend their neurites toward the posterior and the interior of the embryo. In mutants, neurite outgrowth appears to be randomized, with a preference for growth towards the exterior of the embryo (Irion, 1999).
The pole cells in wild-type embryos are internalized during gastrulation, then pass through the posterior midgut epithelium, contact the mesoderm and migrate anteriorly toward the somatic gonadal precursor cells. They are guided to their correct targets by a combination of repellants and attractants, controlled by the genes wunen and columbus (see HMG Coenzyme A reductase). In abs14B mutants that were shifted to restrictive temperature after gastrulation, the pole cells that have passed through the midgut epithelium disperse widely within the embryo. In mutants shifted before gastrulation, the pole cells are not internalized and they distribute over the surface of the embryo. This phenotype is stronger than those observed for any of the genes known to be required for pole cell migration. Abstrakt might be involved in the production of one of the guiding signals by interfering with the function of Wunen or Columbus or other, similarly acting proteins (Irion, 1999).
Both of the above cases show that lack of abs function does not compromise the ability of cells to undergo their differentiation program or cellular behaviour typical of their differentiated state. Thus, neurite outgrowth still occurs, and pole cells are able to migrate over large distances, but both types of cells appear to fail to recognize or react to the cues that direct their morphogenetic behavior in the spatially appropriate manner (Irion, 1999).
One reason for this defect might be a loss of subcellular order or polarity. Various aspects of cell polarity were indeed affected in abs mutants. The first abnormality detected was in the localization of mRNAs in the blastoderm. In the wild-type blastoderm, the mRNAs of several genes are distributed unevenly in the cell. For example, the transcripts of short gastrulation (sog) and crumbs (crb) are tightly apposed to the apical cell surface. In abs14B mutants, CRB mRNA is not seen apically but predominantly at the level of the nuclei, while SOG mRNA forms a gradient from the apical towards the basal side of the cell. In the case of crb, the mislocalization of RNA is also seen at later stages and in other tissues. For example, the tight apical localization in the hindgut epithelium is disturbed in abs mutants. Remarkably, the Crumbs protein is synthesized properly and targeted to the apical cell surface, showing that apical-basal cell polarity in the epithelial cells is correctly established and maintained in abs mutants (Irion, 1999).
Other unevenly distributed mRNAs are those of the pair-rule genes fushi tarazu, even-skipped and hairy, which are localized in the area between the nucleus and the apical cell surface. The localization of these transcripts is not affected in abs mutants. This shows that lack of Abstrakt does not lead to a breakdown of subcellular RNA sorting in general, but that only a subset of transcripts require Abstrakt for their correct targeting (Irion, 1999).
A further manifestation of cell polarity is the regular orientation of mitotic spindles in the post-blastoderm mitotic domains. These are groups of synchronously dividing cells, and in most of them the spindles are oriented parallel to the surface of the embryo. In abs mutants, both the synchrony of division and the regularity of spindle orientation are disrupted. Although the patterning of the mitotic domains, controlled through the transcriptional regulation of String (Cdc25), occurs properly, not all cells in a domain divide at the same time and the mitotic spindles are positioned randomly. In the mispositioned spindles, the centrosomes are mispositioned, together with the spindles (Irion, 1999).
To obtain embryos without either maternal Abstrakt protein or RNA, X-rays were used to induce homozygous mutant germline clones. No mutant clones were obtained, suggesting that abs function is necessary for oogenesis. To test this, ovaries of females homozygous for abs14B that were shifted to restrictive temperature immediately after eclosion were analysed (pupae kept at restrictive temperature do not eclose). Even after 2 days, the ovaries of these females contained no mature eggs. In most cases, the egg chambers had not developed beyond stage 9. Frequently, the nucleus in abs14B oocytes does not reach its anterior dorsal position within the oocyte and oskar (osk) mRNA is mislocalized to the center of the oocyte. Less often, a mislocalization of bicoid (bcd) mRNA to the anterior and posterior pole of the oocyte is found. The oldest egg chambers appear to be degenerating, and on analysis by TUNEL staining, their follicle cells are found to be undergoing apoptosis (Irion, 1999).
As a member of the DEAD-box protein family, Abstrakt probably participates in some aspect of RNA processing. The gene was initially identified by a very specific phenotype, the failure of Bolwig's nerve to fasciculate and project normally. By contrast, the variety of phenotypes and tissues affected in the temperature-sensitive mutants suggest, at a first glance, a widespread function for abs. These results do not, however, support a general, ubiquitous function in cellular RNA metabolism for Abstrakt. A general function for Abstrakt in the production of protein from all mRNAs is excluded by several findings, the most important being that morphogenesis and pattern formation up to gastrulation are largely unaffected in abs14B embryos. This means that the proteins required for these processes are translated properly and, in the case of genes transcribed from the zygotic genome, their mRNAs are synthesized, processed and exported from the nucleus properly. The proteins made in the absence of Abstrakt include at least the maternal determinant Bicoid, the cyclins CycA and CycB, Twist, Snail, Decapentaplegic (Dpp), the segmentation gene products, and the cellularization proteins Nullo, Serendipity-alpha and Bottleneck. At later stages of development, inactivation of Abstrakt also leads to serious defects, but does not interfere with the production of functional mRNA and protein of a number of genes, for example, 22C10, Engrailed, Even-skipped and Twist. Finally, in cases in which experiments have found a more direct effect of abs on RNA, namely, on the subcellular localization of specific mRNAs, only a subset of RNAs responded to the loss of abs function. In summary, these findings indicate that the role of abs is likely to be restricted to a subset of RNAs. The pool of mRNAs expressed in each cell may be less homogeneous, and different transcripts might have more options for processing than might have been assumed. The variety of phenotypes described here should provide assays for the further investigation of the cell biological function of Abstrakt. Conversely, the biochemical and cell biological investigation of Abstrakt, including the identification of its protein and RNA partners, should reveal new levels of control of gene activity during development (Irion, 1999).
The larval visual system consists of a pair of photoreceptive organs: Bolwig's organs (BO) and their optic nerves, Bolwig's nerves (BN). Each BO consists of only 12 photoreceptors, unipolar neurons, whose axons fasciculate to form the BN. The BN projects ipsilaterally along the surface of the optic lobe anlagen (OLA), the primordia of the adult optic lobes, from where it leaves the surface and grows toward a small target area in the central brain. The growth of the BN proceeds in three phases, during which the nerve changes direction at two intermediate targets, P1 and P2, which are located on the surface of the OLA (Schmucker, 1997).
In a genetic screen for recessive mutations on the third chromosome that disrupt the Bolwig's nerve (BN) projection, mutations in 13 genes were isolated that affect the projection in distinct phases of its development, suggesting that different genes and mechanisms are involved at different steps of the projection (Schmucker, 1997). One of the mutations isolated in this screen was abstrakt (abs). In abs mutant embryos, defects in BN development become evident shortly after the onset of axon outgrowth. Whereas in wild-type embryos the outgrowing BN axons fasciculate rapidly and project as a tightly fasciculated nerve, abs mutant embryos show 2±3 axon bundles, which explore a broad area, show abrupt directional changes and often fail to reach the second intermediate target. Furthermore, the distal-most portions of the BN branches are significantly thicker than in wild-type, containing fine filopodia-like axonal extensions that extend over several cell diameters (Schmucker, 1997). In abstrakt mutants, the formation of the BO is normal with respect to both cell number and differentiation, and the OLA and the second intermediate target P2 also appear morphologically intact, thus excluding the possibility that the axonal projection defects are merely a secondary consequence of abnormalities in cell number, cell fate, or tissue organization (Schmucker, 1997). abs appears to be essential for the directed and fasciculated early outgrowth of the BN, as well as for its navigation at later stages (Schmucker, 1997).
Given its role in the establishment of the larval visual system in the embryo, attempts were made to determine whether abs is also required for the development of the adult visual system. This analysis of the postembryonic requirement for abs function was made possible by the isolation of the temperature sensitive allele absE2. absE2/absLR7 transheterozygotes, which survive to the third instar larval stage at 25°C and thus represent a partial loss-of-function situation, were examined for axonal projection defects in their developing adult visual system using 24B10 antibodies, which specifically label photoreceptor axons. In the wild-type, the photoreceptor axons leave the optic stalk and fan out according to their retinotopic position. The axons of the outer photoreceptors R1-R6 terminate in the first optic ganglion, the lamina, while the axons of the inner photoreceptors R7 and R8 grow deeper into the brain and terminate in the second optic ganglion, the medulla. The overall organization of the retinal projections is disrupted in absE2/absLR7 animals. The fan of retinal projections into the optic lobe is irregular in shape, axons terminate prematurely and often cluster aberrantly. As in the larval visual system, there are no apparent defects in photoreceptor differentiation in the developing adult system under these weak hypomorphic abs conditions, since both Elav and Chaoptin are expressed, and the number of ommatidia appears to be normal as well. However, some irregularities in the photoreceptor cell layer are observed, including misrotation of ommatidia, additional photo- receptors, and sometimes fewer photoreceptors, suggesting that abs function may be pleiotropic. This notion is confirmed by the fact that absE2/absLR7 larvae, apart from their visual system-specific defects, often show melanotic tumors at various positions in their bodies, reflecting a broader requirement for abs function. abs mutant embryos also show abnormal axonal projections in the CNS. These defects may be responsible for the embryonic lethality of abs mutants (Schmucker, 2000).
Thus, during postembryonic stages, abs function is not only required in the developing visual system of the adult, but, given the occurrence of melanotic tumors in the mutant, may be required also for the maintenance of normal cellular behavior in a broad range of tissues. For the larval visual system, there is clear evidence that the effects of (zygotic) loss of abs function are likely to be due to a direct involvement of the abs gene product in axon guidance and not a secondary consequence of abnormalities in cell number, cell fate, or tissue organization (Schmucker, 1997). In the developing adult visual system, it is not clear whether abs has multiple parallel functions or whether some of the effects observed are secondary; the molecular role of Abs as a putative RNA helicase does not help to differentiate between these two possibilities. In any case, the broader requirement for abs function is consistent with the ubiquitous expression of the abs transcript throughout development. Importantly, the abs transcript is already present in early embryos prior to the onset of zygotic expression, which is likely to be due to maternally provided RNA. This maternal contribution could potentially mask a requirement for abs function both during early and later stages of development. It is therefore possible that embryos lacking both maternal and zygotic abs function may show more severe developmental abnormalities. Another question in need of further investigation is the tissue specificity of abs requirement (Schmucker, 2000).
RT-PCR showed that Abs transcripts are found throughout all developmental stages, but no ABS RNA could be detected by in situ hybridizations or on Northern blots, nor is Abstrakt protein detectable in normal embryos stained with antibodies against Abstrakt. Thus, ABS RNA and protein does not appear to be very abundant. When Abstrakt is expressed at high levels using the GAL4/UAS ectopic expression system, a clear signal is seen by immunofluorescence in the areas in which the UAS-abs transgene is expressed. In older embryos (> stage 12), the signal is restricted to the nuclei. In younger embryos, the subcellular localization of Abstrakt varies between predominantly nuclear localization, homogeneous distribution throughout the cell, and enrichment in the cytoplasm. No correlation between Abstrakt localization and specific stages of the cell cycle has been observed (Irion, 1999).
Embryos homozygous for the non-conditional abstrakt alleles or the deficiency Df(3R)231-5 develop without apparent gross defects, but fail to hatch. To assay functions at other stages, the temperature-sensitive allele abs14B was used. abs14B animals were transferred from room temperature to 32°C at various stages of development. Shifts to restrictive temperature lead to lethality at all stages of the life cycle. Although embryos homozygous for the non-conditional alleles develop until hatching, abs14B embryos cease to develop and show gross morphological defects at restrictive temperature. Thus, abs is essential for embryogenesis. The embryos homozygous for the non-conditional alleles must therefore use the wild-type gene product provided by their heterozygous mothers to complete embryogenesis in the absence of functional zygotic Abstrakt. A wild-type paternal copy of the gene is sufficient to rescue abs14B embryos if they are shifted to restrictive temperature after gastrulation. The temperature-sensitive phenotype is identical in progeny from mothers homozygous for abs14B allele and mothers heterozygous for abs14B and a deletion of the gene. No dominant effects of the abs14B allele were seen in animals heterozygous for the abs14B and one or more wild-type copies of the gene (Irion, 1999).
It seemed possible that Abstrakt, as a DEAD-box protein, might be involved in general RNA processing or metabolism, and that cells in the mutant animals eventually run out of essential components and commit apoptosis. When TUNEL assays were performed on abs14B mutants, however, no increased rate of apoptosis is seen. Instead, apoptosis is almost completely suppressed, and even the wild-type pattern of cells undergoing programmed cell death is not seen in mutant embryos (Irion, 1999).
To test at which level the apoptotic pathway is interrupted in abs mutants, the expressions of reaper (rpr), a nuclear regulator of apoptosis in Drosophila, and dredd, a target gene of reaper encoding a caspase were examined. Both reaper and dredd are expressed properly in abs14B mutant embryos. This shows that the apoptotic regulators are activated in the mutant and components of the apoptotic pathway are expressed, but the program is not completed (Irion, 1999).
Ectopic cell death induced by overexpression of reaper is also greatly suppressed in abs mutants. The amnioserosa is the only tissue in abs mutants in which extensive apoptosis was found after overexpressing reaper. Ectopic apoptosis induced by overexpression of a second regulator, hid, is not completely suppressed in abs mutants, consistent with previous findings that hid is a more potent inducer. Rather than being blocked, apoptosis in abs14B mutant ovaries occurs prematurely in follicle cells and nurse cells. This points to different modes of regulation of apoptosis during embryogenesis and oogenesis, in line with recent findings that reaper, hid and grim are not essential for programmed cell death of the nurse cells (Irion, 1999).
Search PubMed for articles about Drosophila Abstrakt
Benz, J., Trachsel, H. and Baumann, U. (1999). Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae - the prototype of the DEAD box protein family. Structure 7: 671-679. PubMed Citation: 10404596
Irion, U. and Leptin, M. (1999). Developmental and cell biological functions of the Drosophila DEAD-box protein Abstrakt. Curr. Biol. (23): 1373-81. PubMed Citation: 10607561
Irion, U., et al. (2004). Abstrakt, a DEAD box protein, regulates Insc levels and asymmetric division of neural and mesodermal progenitors. Curr. Biol. 14: 138-144. 14738736
Schmucker, D., Jackle, H. and Gaul, U. (1997). Genetic analysis of the larval optic nerve projection in Drosophila. Development 124: 937-48. PubMed Citation: 9056770
Schmucker, D., Vorbruggen, G., Yeghiayan, P., Fan, H. Q., Jackle, H. and Gaul, U. (2000). The Drosophila gene abstrakt, required for visual system development, encodes a putative RNA helicase of the DEAD box protein family. Mech Dev. 91: 189-96. PubMed Citation: 10704843
Subramanya, H. S., Bird, L. E., Brannigan, J. A. and Wigley, D. B. (1996). Crystal structure of a DExx box DNA helicase. Nature 384: 379-383. PubMed Citation: 8934527
date revised: 6 June 2000
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