Drosophila photoreceptor neurons (R cells) project their axons to one of two layers in the optic lobe, the lamina or the medulla. The transcription factor Runt (Run) is normally expressed in the two inner R cells (R7 and R8) that project their axons to the medulla. The relationship between Run and the ubiquitously expressed nuclear protein Brakeless (Bks), which has previously been shown to be important for axon termination in the lamina, has been examined. Bks represses Run in two of the outer R cells: R2 and R5. Expression of Run in R2 and R5 causes axonal mistargeting of all six outer R cells (R1-R6) to the inappropriate layer, without altering expression of cell-specific developmental markers (Kaminker, 2002).
As an axon navigates toward a target region during development, it alters its course based on attractive or repulsive molecular signals in its environment. There are at least two phases in the establishment of neuronal connections. First, axons project to and distinguish between regions or layers and second, once within the target layer, axons fine-tune their projections. This second step involves precise interactions between growth cones and target cells and has been well studied, particularly for the participation of cell-surface molecules and their associated signal transduction machinery. This study investigates the role of two transcription factors, Run and Bks, in the first phase of target layer selection for differentiating R cells in the Drosophila optic lobe (Kaminker, 2002).
The expression pattern of several R cell-specific differentiation markers is normal in bks mutants. In striking contrast to other markers, however, Run is ectopically expressed in two extra R cells per cluster in somatic loss-of-function clones of bks mutant tissue. In bks clones, Run expression is expanded from its normal R7/R8 pattern to also include R2 and R5. This suggests that Bks represses Run in R2 and R5 cells. Cells along the edges of bks clones were analyzed for the expression of Run. In 196 ommatidia counted along clone boundaries, R2/R5 expression of Run was never seen in a cell that is wild type for bks. It is concluded that the repression of run by Bks is cell-autonomous (Kaminker, 2002).
R1-R6 photoreceptor axons misproject to the medulla in bks loss-of-function mutants. To determine whether this axonal targeting defect is due to the relief of Run repression in R2 and R5, the GAL4/UAS system was used to express Run in these cells. When Run is expressed in R2, R5 and R8 using the MT14-GAL4 driver, all innervating R-cell axons bypass the lamina and projected through to the medulla. When Run is misexpressed in R2 and R5 alone, the defect is as severe as when Run is misexpressed in all R cells using the GMR-GAL4 driver. Run over-expression in R8 alone, where it is normally expressed, does not affect axonal projections. In addition, misexpression of Run in R1, R6 and R7 using lz-GAL4 or in R3 and R4 using sal-GAL4 does not give rise to a comparable axonal misprojection phenotype. The severe axonal mistargeting phenotype in MT14-GAL4/UAS-run flies is attributable to Run expression in R2 and R5 (Kaminker, 2002).
Unlike the ordered wild-type array, thick bundles of axons are seen entering the medulla when Run is mis-expressed in R2 and R5. The axons do not project into deeper areas of the brain, but stop within the medulla. The phenotype observed in this genetic background is very similar to that for bks. Therefore, in both bks loss-of-function and MT14-GAL4/UAS-run genetic backgrounds, Run expression in R2 and R5 results in the mistargeting of all retinal axon types to the medulla. This also suggests that the targeting of R2 and R5 axons affects axonal pathfinding of other outer R cells. For technical reasons, it was not possible to generate marked, double-lethal clones of run and bks (Kaminker, 2002).
The mistargeting of R-cell axons could, in principle, result from the conversion of all R-cell fates to R7 and R8. Markers for every R-cell type and for cone cells were therefore analyzed, both in mosaic clones of null bks mutant tissue and in the context of MT14-GAL4/UAS-run. In each of these backgrounds, the Run-expressing R2 and R5 cells did not express the R8-specific antigen, Bride of Sevenless (Boss) or the R7 marker, Prospero (Pros). Therefore, the projection phenotype of R cells to the medulla in these backgrounds does not result from the conversion of these R cells to the R7/R8 type during their development. The R1/R6 marker Bar and the R2/R5/R3/R4 marker Rough are also unaffected in these backgrounds. It is concluded that Run reprograms the projection pattern of outer R cells without affecting the expression of developmental markers of cell identity (Kaminker, 2002).
Consistent with the R cell marker expression in larval tissue, plastic sections of adult eyes show that Run misexpression in R2 and R5 during development does not perturb adult R cells or ommatidial structure. The correct complement and arrangement of R cells was found. Strikingly, these seemingly normal adult R cells misproject their axons to the inappropriate optic layer. Axon termination in the lamina region is virtually absent and all R-cell axons project to the medulla. This adult phenotype is also identical to that reported in bks mutant clones in which R cells are unchanged in the expression pattern of Rhodopsins. These data provide strong evidence that Run expression in R2 and R5 causes mistargeting of all outer R-cell axons without changing their individual R-cell fates, as determined by developmental markers and by the adult morphology of rhabdomeres. In spalt (sal) mutants, cell fate is altered without changes in axonal connectivities. Similarly, in sal-GAL4/UAS-run flies, some change in R3/R4 fate to R7 cell type is evident without a significant perturbation of outer R-cell projections. Hence, transcriptional events that control cell identity are separable from those that control axonal targeting (Kaminker, 2002).
R-cell axons provide critical anterograde signals to the lamina target region to induce the proliferation and differentiation of lamina neurons, and to induce the differentiation and migration of glial cells to their correct position adjacent to the lamina plexus. In turn, the glial cells provide positional information that directs R1-R6 axons to terminate in the lamina, and in the absence of glia these axons project into the medulla. The development of the lamina target region was assessed using neuronal and glial cell differentiation markers. Wild-type brains stained with anti-Dachshund (Dac) antibody show a large area of labeled cells corresponding to maturing lamina precursor cells (LPCs) and differentiated lamina neurons. This remains unchanged in GMR-GAL4/UAS-run brains, although the R-cell axons do not target properly. Additionally, the three rows of glial cells that delineate the lamina plexus in wild type also remain unaltered when Run is misexpressed. It is concluded that the abnormal pathfinding of R cells is due to defects intrinsic to R cells and not to a global disruption of lamina target neurons or glia (Kaminker, 2002).
Thus, this study highlights two important characteristics of neuronal pathfinding in the optic lobe. (1) A mechanism involving Bks exists in wild-type flies for repressing Run expression specifically in R2 and R5 cells. The bks loss-of-function causes relief of run repression in these two cells, which entirely abolishes the distinct targeting of R cells to the two optic ganglia. Perhaps additional genes are responsible for repressing run in the other R cells. (2) The mistargeting of R2 and R5 alone is sufficient for all outer R cells to project to the medulla. It appears that R2 and R5 cells, the first two outer R cells to be specified, provide pioneering axons whose tracts the other axons follow. In rough loss-of-function, R2 and R5 are converted to R8 cells. The resulting phenotype, however, is not as severe as that described in this study. Presumably, a residual pioneering function is maintained. The mechanism underlying the interaction between R2/R5 and R3/R4/R1/R6 axons in regulating target layer selection is unclear, although interaction between R1-R6 axons regulates a later step in axonal pathfinding: the fine-tuning of R-cell connections in the lamina (Kaminker, 2002).
See the embryonic expression pattern of sbb at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
The 3-kb EST clone LD13770 (called sbb-cDNA LD13770) was used in Northern blot analysis to probe wild-type (forR) poly(A)+ RNA isolated from larvae, pupae, adult head, and adult body. sbb RNA is expressed at all stages of development. Three transcripts (10.5, 3.6, and 1.6 kb) were detected in larvae, pupae, and adult heads, whereas the 7.8-, 3.6-, and 1.6-kb transcripts were detected in adult body. These different-sized transcripts suggested the involvement of alternative splicing, transcript initiation, or termination. Northern analysis using the same sbb-cDNA LD13770 probe has shown a reduction in the abundance of mRNA in homozygous sbbEP(2)0328 compared to larvae of the control strain. Similar results are found for sbbl(2)03432 and sbbJ2, the early larval lethal excision line, which carries a >10-kb deletion (Yang, 2000).
In situ hybridization to CS embyros with antisense RNA from sbb-cDNA LD13770 reveals that sbb is expressed in the embryonic central nervous system. Wild-type sbb expression is strong in early stage embryos (stage 5) before gastrulation. In larvae, wild-type sbb RNA expression is found in the nervous system including the brain, the optic lobes, the ring gland, and the medial region of ventral ganglion. Expression is also found in the eye-antennal, wing, and leg discs. RNA is expressed at normal levels in heterozygous embryos, but expression is reduced in homozygous mutant embryos of sbbl(2)04440, sbbl(2)03432, and sbbEP(2)0328. sbbl(2)04440 appears to have a more severe reduction in transcript abundance than does sbbl(2)03432 and sbbEP(2)0328. Expression of sbb RNA in sbbEP(2)0328 larvae is reduced in the brain and missing in the medial region of the ventral ganglion (Yang, 2000).
To determine the expression pattern and the subcellular localization of Sbb/Brakeless, antibodies to it were generated. A glutathione S-transferase fusion protein containing a fragment of the Bks protein (amino acids 623-818) was used as an immunogen in rabbits. Western blot analysis of extracts made from wild-type third instar eye-brain complexes using the affinity-purified antibody detected a single band at ~80 kDa, which is close to the predicted size of the Bks protein. That this band corresponds to Bks is supported by its loss in bks mutant extracts. The distribution of the Bks protein in the eye imaginal disk was determined in whole-mount preparations. Neuronal differentiation occurs posterior to a dorsoventral groove called the morphogenetic furrow. Bks was detected in the nuclei of developing R-cells and undifferentiated precursor cells lying in the basal region of the disk. Weak nuclear staining was also seen in cells located anterior to the morphogenetic furrow. This nuclear staining was missing in bks mutants and was greatly enhanced in larvae overexpressing bks, confirming the specificity of the antibody. In addition to the staining in the developing eye disk, Brakeless protein was also detected in the nuclei of glial cells in the optic stalk. High background staining precluded an assessment of Bks protein in the central nervous system. Because bks mutations are lethal, however, bks must be required for functioning outside the eye. Transgene rescue experiments indicate that bks is not required in these glial cells (Rao, 2000).
To examine the cellular localization of Bks proteins, antisera were generated recognizing a region of high sequence complexity at the C terminus of BksA, corresponding to the central region of BksB. These sera detect a widely expressed nuclear antigen in eye-antennal imaginal discs. This antigen is not restricted to R1-R6 cells, or even to photoreceptors, but appears to be present in all cells of the imaginal disc. As the Bks antisera do not distinguish the two Bks isoforms, transgenic animals expressing c-myc epitope-tagged versions of either BksA or BksB were generated under the control of the eye-specific promoter GMR. Using anti-c-myc antibodies to stain the eye discs of animals carrying either of these GMR-myc bksA and GMR-myc bksB trangenes, it was confirmed that indeed both Bks isoforms are predominantly nuclear proteins (Senti, 2000).
Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).
An unanticipated intricacy of the independent, versus Dpp-mediated, Hh patterning activity in the wing arose from comparison of the gradient of Dpp activity with dpp expression. It revealed that Dpp signaling is downregulated in the AP organizer and upregulated in both A and P flanking cells. This downregulation of Dpp activity was shown to result from the localized transcriptional repression of tkv, which is itself due to activation of the transcriptional repressor Master of thick vein (Mtv: Scribbler) in response to Hh signaling. Col activity is required for the downregulation of Dpp signaling in the AP organizer and this regulation is linked to Col requirement for positioning L3 vein and formation of L4 vein. The relative functions of these two genes remain to be examined in detail, although the observation that mtv transcription is downregulated in col mutant discs and tkv is upregulated in clones of col mutant cells suggests that col may act upstream of mtv in the regulatory cascade, thereby attenuating Dpp signaling in the AP organizer (Crozatier, 2002).
It has been been proposed that Hh does directly control the position of L3 vein, although the molecular mechanisms of this control have not been firmly established. In both col and mtv mutant clones, the position of L3 vein is shifted posteriorwards. That both col and mtv control the position of L3 vein suggests that this position is defined by Hh signaling through the modulation of Dpp signaling. iro is required for rho activation in the L3 primordium and formation of L3 vein. iro is activated by both Dpp and Hh signaling and its anterior border of expression is under control of sal/salr, a target of Dpp. The patterns of col, iro and rho expression are intimately connected. Both an increased number of cells expressing rho and a posterior shift of the anterior border of iro expression are observed in col1 mutant discs. This posterior shift is interpreted as reflecting a modified range of Dpp signaling relayed, at least in part, by sal/salr activity. The increased number of rho-expressing cells, for its part, indicates that Col is able to antagonize rho activation by iro in cells, which express both iro and Col. This correlates well with the wing phenotype – anteriorwards shift of the L3 vein, together with gaps in its distal region – which results from anterior extension of Col expression, in UAS-Col/dpp-Gal4 wing discs. The distal gaps could reflect the complete absence of rho expression close to the DV border, because of the complete overlap between col and iro expression where iro expression is narrower. From col loss- and gain-of function experiments, it is therefore concluded that the primordium of L3 vein corresponds to cells that express iro but not col. Col thus appears to play a dual role in defining the position and width of L3 vein: activating Blistered and repressing EGFR in the wing AP organizer cells, endows these cells with an intervein fate, while attenuating Dpp signaling indirectly positions the anterior limit of iro expression domain, and L3 vein competence anterior to the AP organizer (Crozatier, 2002).
The R1-R6 subclass of photoreceptor neurons (R cells) in the Drosophila compound eye form specific connections with targets in the optic ganglia. brakeless (bks: accepted FlyBase name scribbler) is essential for R1-R6 growth cone targeting. In brakeless mutants, R1-R6 growth cones frequently fail to terminate migration in their normal target, the lamina, and instead project through it and terminate in the second optic ganglion, the medulla. Genetic mosaic analysis and transgene rescue experiments indicate that bks functions in R cells and not within the lamina target region. It is proposed that it participates in a gene expression pathway regulating one or more growth cone components controlling R1-R6 targeting (Rao, 2000).
A collection of lethal P element insertions on the second chromosome was screened for mutations that disrupt the R-cell projection pattern. One insertion line, l (2)k06903, causes defects in both R-cell projections and R-cell patterning in the eye, as assessed in preparations of third instar eye-brain complexes stained with an antibody recognizing R-cell bodies and their axons. l (2)k06903 carries two P-element insertions at polytene chromosome bands 47D1-D2 and 55C1-C5. Genetic experiments have demonstrated that the connectivity and patterning defects are genetically separable and that the connectivity defects result from the insertion at 55C1-C5. Initially, the projection, but not the eye, phenotype is uncovered by a deficiency [Df(2R)PC4] that selectively removes 55C1-C5. Next, the two P-element insertions are separated by meiotic recombination; larvae homozygous for the 55C1-C5 insertion exhibit the projection, but not the patterning, defect. Finally, excision of the P element at 55C1-C5 restores R-cell projection pattern in 88 of 90 excision lines analyzed. Because many R1-R6 growth cones fail to stop in the lamina, the mutation was named brakeless (bks) and the initial P allele bksP1. Two other lethal P-element insertions, l (2)k0702 and l (2)04440, have been shown to be allelic to bksP1 and are designated bksP2 and bksp3, respectively. Two imprecise excision mutant alleles are designated bks4 and bks5 (Rao, 2000).
The R-cell axon projection patterns in wild-type and mutant third-instar larval eye-brain complexes were visualized by using mAb24B10. In the wild type, bundles of R-cell axons converge at the posterior end of the eye disk, where they enter the optic stalk. On exiting the stalk, axon bundles elaborate a topographic map in the developing lamina and medulla. The R1-6 growth cones terminate in the lamina with their expanded growth cones, forming a dense continuous layer of immunoreactivity in a region referred to as the lamina plexus. R7 and R8 growth cones project through the lamina and terminate in an array in the medulla neuropil. In bks mutant larvae, R-cell axons project through the optic stalk and into the optic lobe, largely as they do in the wild type. Projection defects in both the lamina and medulla are observed. In contrast to the wild type, the lamina plexus is discontinuous, with regions of dense staining separated by weakly stained regions or gaps. Thick bundles of axons are frequently observed between the lamina and medulla, in striking contrast to the lightly stained thin bundles in the wild type. In the most severe cases, the lamina plexus is largely missing and the medulla neuropil is hyperinnervated. In addition, disruptions in the normally smooth topographic map in the medulla are observed. However, R-cell projections do not extend through the medulla into deeper layers of the optic lobe, suggesting that R8 neurons terminate, as in the wild type, within the medulla neuropil. As R7 axons project into the optic lobe only later, and no early R7-specific axon markers are available, the initial projections of these neurons to their medulla targets could not be assessed. These data are consistent with a strong mistargeting defect, where R1-R6 neurons fail to terminate in the lamina and, instead, project into the medulla neuropil. These phenotypes are completely penetrant in bks4, bksP1, bksP2, and bks5 and partially penetrant in bksP3. Individuals homozygous for bks4 and bksP1 show the most severe phenotype. These phenotypes are not enhanced in trans to a deficiency for the region, Df(2R)PC4, suggesting that bks4 and bksP1 are strong loss-of-function, if not null, mutations. The relative strength of the connectivity defects is bks4>bksP1>bks5=bksP2>bksP3. Stronger alleles not only increase penetrance, they also increase the severity of the mistargeting phenotype. Increasing strength of the alleles also correlates with progressive decrease in larval motility (Rao, 2000).
Although bks mutations lead to severe defects in neuronal connectivity, several markers indicated that development of cells in the target region occurs normally. As in the wild type, lamina neuronal precursor cells are driven through their final division, as assessed by BrdUrd incorporation, and differentiate as assessed by the expression of the lamina neuronal markers, Dachshund and Elav. Lamina glia, the intermediate targets for R-cell growth cones, migrate into the target region and differentiate largely as in the wild type (Rao, 2000).
R-cell projection defects in bks mutants may reflect a role for bks in R cells, in their targets, or both. To distinguish between these possibilities, genetic mosaic analysis was carried out. Mutant eye tissue, homozygous for bksP1, was generated in otherwise heterozygous eyes by x-ray-induced mitotic recombination. R1-R6 targeting from mutant tissue into the lamina was assessed using an R1-R6-specific axonal marker, Rh1-LacZ. Cryostat sections of adult heads prepared from genetic mosaics and wild-type controls were stained with a rabbit anti-beta-galactosidase antibody. In the wild type, all R1-R6 axons terminate in the lamina; no staining is observed in the medulla. In contrast, in all mosaic individuals examined, many R1-R6 axons fail to terminate in the lamina and, instead, project through the lamina and terminate in the medulla. In support of these findings, the bks mutant phenotype is largely rescued by expressing a bks cDNA under the control of an eye-specific promoter, GMR. These data indicate that expression of bks in the eye is not only necessary for normal R1-R6 connectivity, it is also sufficient (Rao, 2000).
In the Senti (2000) study, brakeless/sbb was identified in a large scale genetic screen to isolate mutations that autonomously disrupt photoreceptor axon targeting. Two EMS-induced alleles of bks, bks1 and bks2 were isolated. In this screen, photoreceptor projections were analyzed in genetic mosaics in which the developing eye, but not the optic lobe, was genetically homozygous for a newly induced mutation. Eye-specific mosaics were generated by using the FLP/FRT system for site-specific recombination together with an eyFLP transgene to provide FLP recombinase activity exclusively in proliferating eye imaginal disc cells. In animals of the genotype eyFLP;FRTbks/FRTcl2R11, for example, FLP recombinase activity in a dividing cell in the developing eye induces recombination at the FRT sites to create two daughter cells, one homozygous for the bks mutation and the other homozygous for both bks+ and the cl2R11 mutation. The latter is a recessive cell lethal mutation, introduced to eliminate these homozygous bks+ cells. Thus, a heterozygous bks cell is effectively replaced by its homozygous bks daughter. The continuous high levels of FLP recombinase activity in the developing eye ensure that this occurs with almost 100% efficiency. As a result, almost all heterozygous cells in the developing eye are replaced by homozygous bks cells during the proliferative phase of eye development, which precedes photoreceptor cell fate specification and axon targeting. In the visual system of such animals, axonal connections are therefore established between homozygous bks mutant photoreceptors in the eye and wild-type (bks/+) target cells in the brain. These animals are referred to as bks mosaics (Senti, 2000).
bks mosaics show a striking defect in their photoreceptor axon projections, as visualized in third instar larvae using MAb24B10 to label all photoreceptors and their axons. In wild type, photoreceptor axon fascicles project through the optic stalk to their appropriate topographic locations in the optic lobe. R1-R6 axons terminate in the lamina, where their growth cones expand to form a dense layer of staining, the lamina plexus, that lies between two glial cell layers. R7 and R8 axons continue beyond this layer to terminate in the medulla. In bks mosaics, photoreceptor axons project normally through the optic stalk to the brain, but fail to segregate into their distinct target layers. Only a vestigial lamina plexus is formed, while thick bundles of photoreceptor axons project through to the medulla. These features suggest that the primary defect in these mutants is the failure of ingrowing R1-R6 axons to stop in the lamina, which inspired the name brakeless. The projection defects observed in bks1 and bks2 mosaics, as well as bks1/bks2 transheterozygotes, are indistinguishable (Senti, 2000).
To examine the projection errors of bks mutant photoreceptors in further detail, a series of markers to label the axons of specific photoreceptor subclasses were introduced into bks mosaics. It was first confirmed that R1-R6 axons extend through the lamina to terminate in the medulla in bks mosaics. To label specifically these axons, a rough-taulacZ marker was used to visualize R2-R5 axons in larvae and an Rh1-taulacZ marker to label all R1-R6 axons in adults. In wild-type animals, all photoreceptor axons expressing these markers terminate in the lamina, whereas in bks mosaics most of these axons project through the lamina and terminate instead in the medulla. This observation confirms that many R1-R6 axons are indeed mistargeted to the medulla in bks mosaic larvae, and further demonstrates that these inappropriate projections are maintained in the adult. Within the medulla, mistargeted R1-R6 axons all appear to make R7-like projections (Senti, 2000).
An Rh4-taulacZ marker was used to examine R7 projections and it was found that these axons are correctly targeted to the medulla in bks mosaics. It was also noticed that both mistargeted R1-R6 axons and correctly targeted R7 axons appear to project in topographically correct fashion along the anteroposterior axis. To also examine retinotopic mapping along the dorsoventral axis, an omb-taulacZ reporter expressed in the dorsalmost and ventralmost photoreceptors in the eye imaginal disc was used. These photoreceptors project their axons retinotopically to the dorsal and ventral extremes of the optic lobe, respectively, in both wild-type and in bks mosaic animals. It is therefore concluded that bks is specifically required for the targeting of R1-R6 axons to the lamina; it is not required for targeting of R7 axons to the medulla, nor for the retinotopic mapping of photoreceptor axons along the anteroposterior and dorsoventral axes (Senti, 2000).
The specific failure of R1-R6 axons to terminate in the lamina in bks mosaics might be due to either (a) a transformation of R1-R6 cells towards an R7 cell fate; (b) a failure of lamina cells to issue a 'stop' signal to R1-R6 growth cones, or (c) a failure of R1-R6 growth cones to respond to this stop signal. To distinguish between these possibilities, cell fate specification was examined in both the retina and lamina in bks mosaics. The specification of retinal cell fates was assessed by examining plastic sections of bks mosaic eyes. In both wild-type and bks mutant ommatidia, R1-R6 cells form large-diameter rhabdomeres that are arranged in trapezoidal fashion around the smaller R7 and R8 rhabdomeres. 96% of bks mutant ommatidia examined show the normal complement and arrangement of photoreceptors, consistent with the observation that the R1-R6-specific markers rough-taulacZ and Rh1-taulacZ are still expressed in bks mosaics. Thus, by both morphological and molecular criteria, R1-R6 cells are not transformed towards an R7 cell fate in bks mosaics. Indeed, the most common abnormality in bks mutant ommatidia is a cell fate transformation in the opposite direction: in 40 of the 63 abnormal bks ommatidia observed, the R7 cell appears to be transformed to an R1-R6 cell, as judged by the size and position of its rhabdomere (Senti, 2000).
Lamina precursor cells require retinal innervation for their final differentiation, including, most likely, their ability to instruct R1-R6 growth cones to seek targets within the lamina. Lamina cell fates were examined in bks mosaics using the neuronal marker Elav and the glial marker Repo. Lamina neurons were found to differentiate normally in bks mosaics. More importantly, the glia that are thought to provide targeting signals for R1-R6 growth cones migrate into the developing lamina and express Repo in bks mosaics just as they do in wild type. However, the ordered arrangement of these glia into rows is slightly disrupted in bks mosaics, but it is suspected that this minor irregulatity is a consequence rather than cause of the continued growth of R1-R6 axons through this layer. Thus, the lamina glial cells are in place and appear to be differentiating normally in bks mosaics. Since bks function is not disrupted in these cells, they presumably still issue their normal targeting instructions to incoming retinal axons (Senti, 2000).
It is therefore concluded that the R1-R6 targeting errors in bks mosaics are not due to the transformation of these cells towards an R7 fate, nor the failure of lamina cells to provide targeting instructions to R1-R6 growth cones. Rather, the data strongly suggest that, in bks mosaics, R1-R6 growth cones are unable to sense or respond to a specific stop signal produced in the lamina (Senti, 2000).
Bks proteins are necessary but not sufficient for lamina targeting. The nuclear localization of Bks proteins excludes them from playing a direct role in axon targeting, but raises the interesting possibility that they might control the choice of target layer by regulating the expression of specific guidance receptors. Target layer specificity might even be determined by the specific Bks isoform(s) expressed in each of the different photoreceptor classes. To test such a model, it was asked whether the expression of either BksA or BksB alone would restore lamina targeting of R1-R6 axons in bks mosaics, and possibly even retarget R7 or R8 axons to the lamina. The GMR-mycbksA and GMR-mycbksB transgenes were introduced into bks mosaics and R1-R6 projections were assayed using the Rh1-taulacZ marker. GMR-mycbksA transgene partially rescues the R1-R6 targeting defects in bks mosaics. Most R1-R6 axons now correctly target the lamina. However, a small number of R1- R6 axons in each hemisphere, predominantly those in the posterior regions of the eye, still project through the lamina to terminate in the medulla. Complete rescue of the bks targeting defect was only obtained with the GMR-mycbksB transgene. Identical results were obtained for both the protein null bks1 allele and the protein-positive bks2 allele. Thus, R1-R6 cells expressing only one of the two Bks isoforms still target their axons to the lamina, arguing against models in which the different isoforms specify different target layers. Evidently, the unique regions of BksB, including the putative zinc finger domain, are also largely, though not entirely, dispensible for Bks function in photoreceptor axon targeting (Senti, 2000).
Finally, R8 projections were examined (using MAb24B10 to stain larval eye-brain complexes) and R7 projections were examined (using the adult marker Rh4-taulacZ) in both wild-type and bks mosaic animals carrying either the GMR-mycbksA or GMR-mycbksB transgene. Despite the high levels of expression provided by the GMR promoter, neither BksA nor BksB is sufficient to retarget R7 or R8 axons to the lamina. Thus, Bks proteins are necessary, but not sufficient, for lamina targeting (Senti, 2000).
The Drosophila larva is extensively used for studies of neural development and function, yet the mechanisms underlying the appropriate development of its stereotypic motor behaviors remain largely unknown. Mutations in scribbler (sbb), a gene encoding two transcripts widely expressed in the nervous system, cause abnormally frequent episodes of turning in the third instar larva. Hypomorphic sbb mutant larvae display aberrant turning from the second instar stage onwards. Focus was placed on the smaller of the two sbb transcripts; its pan-neural expression during early larval life, but not in later larval life, restores wild type turning behavior. To identify the classes of neurons in which this sbb transcript is involved, transgenic rescue experiments were carried out. Targeted expression of the small sbb transcript using the cha-GAL4 driver is sufficient to restore wild type turning behavior. In contrast, expression of this sbb transcript in motoneurons, sensory neurons or large numbers of unidentified interneurons is not sufficient. These data suggest that the expression of the smaller sbb transcript may be needed in a subset of neurons for the maintenance of normal turning behavior in Drosophila larvae (Suster, 2004).
In eukaryotes, the ability of DNA-binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified, including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Strong genetic interactions are seen between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. Mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests that they do not function together, but instead that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity (Wehn, 2006; full text of article).
Previous studies demonstrated that Atro acts as a corepressor in Drosophila, the most convincing of these being the demonstration that fusion of Atro to a heterologous DNA-binding domain confers repressive activity to the chimera. Atro has been shown to interact directly with two transcription factors, Even-Skipped (Eve) and Huckebein, and the repressive ability of Eve is compromised in Atro mutants, probably accounting for the loss of en expression in even-numbered parasegments in Atro mutant embryos (Wehn, 2006).
These studies here are consistent with Atro acting as a corepressor since it was shown that several genes, including run, tkv, al, and B, are completely or partially derepressed in Atro mutant clones in imaginal discs, suggesting that transcriptional repressors required to silence these genes recruit Atro. Atro-dependent repression of Bar (B) in the center of the leg disc is very likely due to interaction with the transcription factor C15, which is expressed in the center of the leg and is required for repression of B. Similarly, Atro-dependent repression of al in the posterior of the wing is very likely due to interaction with En, which is expressed in the posterior and required to exclude al from this compartment. At present it is unclear which transcription factors recruit Atro to repress run in the eye or tkv in the wing, although a strong candidate for run would be the Rough homeodomain protein, which is expressed in the same cells, R2 and R5, that exhibit ectopic run expression in Atro mutant clones. Whether Atro can, in fact, bind directly to C15, En, and possibly Rough, needs to be tested biochemically, since previous studies with Eve and Hkb did not identify a possible interaction motif for Atro nor do sequence comparisons among C15, En, Eve, and Hkb suggest a common motif (Wehn, 2006).
The sbb gene encodes a nuclear protein with unknown function. sbb mutations have many different phenotypes affecting multiple tissues. sbb and Atro interact very strongly genetically and that many of the phenotypes of sbb mutants are very similar to those of Atro mutants, including derepession of run, tkv, al, and B in imaginal discs. Thus, Atro is unable to silence these genes in the absence of Sbb, suggesting that it is required for Atro activity either to recruit Atro to transcription factors or possibly to assist binding of these factors to DNA. Since these transcription factors appear to function normally in some respects in the absence of Sbb (or Atro), it appears more likely that Sbb and Atro function together in a corepressor complex (Wehn, 2006).
One problem with the proposal that Atro activity is dependent upon Sbb is that the phenotypes of Atro and sbb mutants are not identical. For example, embryos lacking both maternal and zygotic Atro have a very severe, almost uncharacterizable phenotype, while embryos lacking both maternal and zygotic Sbb have a much less severe phenotype, characterized by a reduced number of abdominal segments, that is similar to that of embryos lacking only maternal Atro. This could be explained if Atro is partially active in the absence of Sbb, or if it is dependent upon Sbb for repression of some genes but not others. Alternatively, the difference between Atro and sbb mutant phenotypes could be related to Atro having functions other than that of a corepressor. It is has been implicated in positive regulation of Hox gene expression, and it also functions in the cytoplasm to control planar cell polarity. This analysis of sbb mutants does not reveal any potential involvement of Hox gene expression or planar cell polarity and, consequently, if Sbb is required only for Atro to act as a corepressor, then it is not surprising that Atro and sbb mutant phenotypes are not identical. Further experiments are required to determine the nature of the Atro dependence on Sbb for transcriptional repression and how direct any interactions might be (Wehn, 2006).
Mutations in sbb and Atro were originally uncovered in a genetic screen for enhancers of al. It is likely that they act as enhancers because they are utilized by the C15 transcription factor to repress genes such as Bar; C15 is expressed in the same cells as Al and it is thought that they bind together to regulate gene expression. Strong genetic interactions were uncovered among sbb, Atro, and en mutations, that could be explained if En also recruits Atro/Sbb (Wehn, 2006).
Curiously, genetic studies also revealed strong interactions among gro, sbb, and Atro. This could be explained if Gro was also required for Atro activity; i.e., all three proteins may form a corepressor complex. However, this appears to be unlikely because, in contrast to the similar phenotypes of sbb and Atro mutants, there are several distinct differences among the phenotypes of gro mutants and those of sbb and Atro mutants. For example, repression of tkv in the anterior of the wing is dependent on both Sbb and Atro but not on Gro, while repression of run in the antennal disc is dependent upon Gro but not upon Atro or Sbb. This suggests that a specific transcription factor recruits Atro/Sbb to repress tkv in the wing and another transcription factor recruits Gro to repress run in the antenna. The identity of these transcription factors remains to be uncovered (Wehn, 2006).
In some cases gro mutants do have a similar phenotype to those of Atro and sbb; this includes partial derepression of al expression in the posterior of the wing and Bar in the center of the leg. This can be explained if C15 (expressed in the center of the leg) and En (expressed in the posterior of the wing) recruit both Gro and Atro/Sbb and if each imparts some but not all the repressive activity to these transcription factors. Consistent with this, both C15 and En possess eh1-type Gro-interaction motifs and previous studies have revealed that En can repress in the absence of Gro. Further biochemical studies are required to determine if C15 and En can indeed recruit Atro (Wehn, 2006).
At present it is unclear whether Atro and Gro provide all the repressive activity to C15 and En; this will await the generation of Atro gro double-mutant clones. sbb gro double-mutant clones have been analyzed and these reveal that some targets of C15 and En are still at least partially repressed, although En activity appears to be somewhat compromised following the simultaneous loss of Sbb and Gro, in comparison to loss of one of these alone. Either Atro has some activity in the absence of Sbb or C15 and En can use mechanisms other than recruitment of Gro and Atro to repress transcription. Many transcription factors have been shown to have the ability to repress by several mechanisms; for example, although Brk recruits both CtBP and Gro, it can repress some genes in the absence of both, using additional repression domains (Wehn, 2006).
Why do C15 and En need to recruit both Gro and Atro? En can repress some genes completely in the absence of either Gro or Atro, for example, ci and dpp in the wing. However, for repression of al, the activity of En is clearly reduced in the absence of either, indicating that it needs to recruit both to completely repress this gene. This would suggest a quantitative explanation; i.e., En recruits both Gro and Atro to increase its activity, rather than to allow it to repress specific genes repressed more efficiently by one or the other. This is consistent with the suggestion that both corepressors function via a similar mechanism: both Gro and a vertebrate homolog of Atro have been shown to recruit a histone deacetylase. The recruitment of both may decrease histone acetylation to a level that cannot be achieved with either alone (Wehn, 2006).
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date revised: 20 February 2007
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