hbs expression was analyzed by in situ hybridization in embryos and in imaginal discs. hbs transcripts were first detected at stage 8 in precursors of the amnioserosa and in the mesectoderm, where they are maintained in progeny of these midline cells throughout embryogenesis. By stage 10, hbs was expressed in the visceral mesoderm in a pattern reminiscent of bagpipe. These cells maintained hbs expression while forming the visceral mesoderm surrounding the gut. By late stage 11, hbs is expressed in somatic mesoderm where its expression peaks between stage 12 and early stage 13, and subsequently declines, disappearing by stage 14. By stage 12 there is expression in the precursors of the heart, where hbs is maintained throughout the remainder of embryogenesis. Late hbs expression is detected in the epidermis at putative muscle attachment sites, hindgut and pharyngeal muscles (Artero, 2001).
Immunohistochemical staining reiterated the same embryonic expression pattern of the Hbs protein. Notably, Hbs localization at the cell membrane is consistent with the protein structure predicted by the sequence data. Hbs is detected weakly in the somatic mesoderm by late stage 11. This expression increases around stage 12 and 13, yet declines thereafter, and mature muscles are completely devoid of Hbs. Hbs is expressed at specific points in the myoblast membrane, as described for its paralog, Sns (Bour, 2000). This localized expression is detected also in the hindgut, at putative epidermal attachment sites and in the midline (Artero, 2001).
Transgenic flies carrying a 4 kb fragment of hbs genomic sequence fused to a lacZ reporter reproduce aspects of hbs expression and provide an additional tool with which to study hbs. Embryos containing this reporter inserted in the hbs locus (P[w+]36.1) show expression in visceral and somatic mesoderm, mesectoderm, pharyngeal muscles and hindgut in a pattern similar to that found with the anti-Hbs antibody. To reinforce further that P[w+]36.1 recapitulates hbs expression in the mesoderm, ß-galactosidase expression and hbs transcription in P[w+]36.1 embryos were simultaneously detected, confirming there is overlap between both signals. Because of the high degree of conservation between Hbs and the kidney protein Nephrin, hbs expression was analyzed in Malpighian tubules in the reporter P[w+]36.1. Interestingly, weak expression was detected at stage 12/13 in a subset of tubule cells that, based on their morphology, appear to be stellate cells (Artero, 2001).
Since these results suggested that hbs was excluded from founder cells and was present in fusion-competent cells, several additional experiments were performed to confirm this possibility. Co-localization experiments using either P[w+]36.1 that reiterates Hbs expression in the mesoderm or Hbs antibody and various founder cell markers reveal that Hbs is absent from founder cells. These experiments also suggest that Hbs is not expressed in all fusion-competent cells during myoblast fusion. It was therefore asked whether Hbs and Sns, a protein known to be expressed exclusively in fusion-competent cells (Bour, 2000), co-localize in fusion-competent cells. Wild-type embryos double labeled with antisera against Sns and Hbs show several examples of co-localization of these proteins. During stage 11, when muscle progenitors are being specified and the fusion process has not begun, Hbs expression is initially more widespread in somatic and visceral mesoderm than that of Sns. Subsequently, as the process of muscle fusion begins, Hbs expression is less widespread than Sns. It is noted that at this stage, although overlap was detected in Hbs and Sns expression, cases of Sns and Hbs-specific expression are found. At later stages of the fusion process, Hbs protein is no longer detected in the remaining fusion-competent cells, in contrast to the continued presence of Sns. Co-localization of both Hbs and Sns in the visceral mesoderm is also found. It is concluded that Hbs expression initially precedes Sns, but then largely overlaps with Sns, suggesting that Hbs could modulate the fusion-promoting properties of Sns in fusion-competent cells during the course of myoblast fusion (Artero, 2001).
hbs is expressed in a highly dynamic manner across tissue types and life stages. At stage 5, along the dorsal surface of the cellularized embryo, a strong narrow band of hbs expression is present that extends along approximately two-thirds the length of the embryo. This band of expression broadens laterally, decreases in length, and becomes confined to the dorsal furrows. By stage 8, dorsal expression is still present at the furrows, and hbs expression also begins ventrally, where it is associated with the mesectodermal cells. Expression strengthens in the mesectodermal cells as they move into close juxtaposition with one another at the ventral midline, forming neighboring columns (across stages 9 and 10). Expression continues during stage 11 as the mesectodermal cells intermingle, divide and move internally. By stage 12, as the midline axonal scaffold is forming, a subset of midline cells posterior to the developing posterior commissure continue to express hbs. The number of hbs-expressing cells at the midline decreases so that by late stage 14 there are two to three hbs-expressing cells below the posterior commissure. Expression in these cells is absent by stage 15. Double-labeling of glial cells with anti-Repo mAb reveals that a subset of the exit glia at the edge of the CNS are hbs positive from stages 12 to 15 (Dworak, 2001).
In the periphery during stage 10, laterally located clusters of cells begin expressing hbs and are distinct patches at stage 11. These cells are visceral mesoderm, as their nuclei co-label with the anti-myocyte enhancer factor 2 polyclonal antibody (anti-MEF2). By late stage 11, the somatic and visceral mesoderm expresses hbs. During stage 12, hbs is clearly present in the fusion competent myoblasts. This expression is truly restricted to fusion competent myoblasts. In Notch mutant embryos, all myoblasts adopt a founder cell fate and in NXK11 mutant embryos, there are no hbs-positive myoblasts. Similarly, co-labeling with anti-Kruppel antibody reveal that the fusion-competent myoblasts, but not the founder cells, are hbs positive. By stage 14, expression in the somatic mesoderm has ceased. As mesodermal expression decreases, epidermal expression begins. At stage 12, this expression is several cells broad and occurs at the segment boundary and in lateral patches. It becomes confined to the muscle attachment sites by stage 14. Along the dorsal edges of the embryo lie some hbs-positive cells. These cells are MEF2 negative, identifying them as the pericardial cells (Dworak, 2001).
In third instar larvae, in the eye/antennal disc, hbs expression is strong behind the morphogenetic furrow, and also as clusters within the furrow. In the larval brain there is hbs expression in the optic lobes. Expression in the larval wing disc consists of a striking cruciform pattern, corresponding to the regions that abut the presumptive wing margin, and those areas destined to be wing veins L3 and L4. More proximally expression is found in the region destined to be wing veins L0 and L1. There is also light expression in the presumptive notum region. In leg discs expression is seen in concentric circles (Dworak, 2001).
hbs is expressed in imaginal tissues, most notably in the eye-antennal, wing, leg and haltere discs (Artero, 2001).
To assay the effects of loss of hbs function, both new deficiencies were generated around region 51D7 and EMS-induced mutations were generated. For two of the EMS-induced mutations recovered, 2593 and 459 (hereafter hbs2593 and hbs459), nucleotide changes were identified in the hbs transcription unit, therefore establishing that they were hbs alleles. hbs2593 mutation is a T to A transversion in the second nucleotide of the intron between exons 9 and 10. The mutation leads to an aberrantly spliced transcript that retains the whole intron and results in a prematurely truncated protein. hbs459 is an A to T transversion in exon 7 that generates a stop codon (Artero, 2001).
Immunohistochemistry provided further confirmation that these mutations led to loss of hbs function. In these experiments, hbs2593 embryos showed a dramatic decrease in Hbs protein, while hbs459 homozygous embryos show no detectable protein. Allele hbs361 shows protein expression at levels indistinguishable from wild type but behaves at least as strongly as hbs459 in phenotypic assays (Artero, 2001).
In order to determine the function of Hbs during embryonic development, a series of deletions were generated by irradiation of nearby P-elements. For EP(2)2590, over 50 w- flies were isolated from the 71,700 progeny screened and 13 stable lines were successfully established, while for l(2)k04218, 55,600 progeny were screened and 29 lines established. Deletion 12 removes hbs expression as assayed by in situ hybridization with cDNA4, and is lethal over l(2)k06403 but not l(2)k04218. As the smallest deletion removing hbs, this line has a phenotype where the ventral muscle pattern is abnormal in two to three hemisegments per mutant embryo. The abnormality consists of a loss of some muscle fibers from the ventral muscle group. In hemisegments where the muscle patterning is normal, motor innervation is also normal. Deletion 11 does not remove or disrupt hbs expression, yet also shows the ventral muscle defect that was seen in Deletion 12 (as does Deletion 6). Furthermore, in deletions 11 and 12 transheterozygotes, the muscle phenotype is present. As such, the ventral muscle phenotype maps to a gene(s) in the region other than hbs. It appears that hbs mutant embryos do not have an overt muscle phenotype because: (1) the muscle phenotype in deletion 12 is no different from that in the transheterozygote deletions 11 and 12 embryos; (2) muscle number, insertion sites and innervation are normal in the unaffected hemisegments; and (3) unfused myoblasts are barely evident in late stage 16. The nervous system was also examined across stages 12-17 with mAbs BP102, 1D4, 22C10, anti-Wrapper and anti-Repo, and no defects were found. As such, Hbs appears functionally redundant in the development of the embryonic somatic mesoderm and central nervous system. Deletion 12 in trans with sns, irreC-rst or the duf/irreC-rst deletion does not produce phenotypes in the embryonic muscles or the adult (Dworak, 2001).
Overexpression of hbs in the mesoderm in homozygous UAS-hbs;twi-GAL4 embryos partially disrupts myoblast fusion, but not muscle fiber number or sites of muscle attachment. This phenotype is evident in all hemisegments of all embryos. When hbs is misexpressed using the da-GAL4 driver, unfused myoblasts are again present, and in all hemisegments, some muscle fibers are inserted at inappropriate attachment locations. twi-GAL4 expression is restricted to mesoderm, while da-GAL4 is expressed in all tissues, suggesting that aberrant muscle fiber attachments may be due to hbs misexpression in the epidermis. To further test this idea, hbs was misexpressed in the epidermis with additional GAL4 drivers. Driving in the Engrailed pattern in the epidermis with en-GAL4 disrupts attachments made by several lateral muscle fibers in most hemisegments of all embryos. This is strongly exacerbated, occurring in all hemisegments of all embryos, by overexpressing more broadly and strongly in the lateral epidermis with sca-GAL4, a GAL4 line that drives in the sca pattern in the epidermis and CNS. Driving with pnr-GAL4 in the dorsal ectoderm, dorsal muscle attachments sites are radically disrupted, with the muscle fibers often failing to cross the segment and instead aligning with the segment boundary. Unfused myoblasts are also seen in the epidermal gain-of-function embryos (Dworak, 2001).
The effects of increased hbs expression in imaginal discs were assessed using drivers giving general or specific domains of expression. The distal wing margin was abnormal in omb-GAL4/+;UAS-hbs/+ and UAS-hbs/+;da-GAL4/+ flies. There was a blistered appearance at the distal wing edge in all omb-GAL4/+;UAS-hbs/+ flies, and blistered or notched distal wing margins in the UAS-hbs/+;da-GAL4/+ flies. On the dorsal thorax (notum) the large anterior section (scutum) has two mechanosensory bristle populations: macrochaetes (large bristles) and microchaetes (small bristles). Microchaetes on the central scutum are in 10 longitudinal rows and fairly constant numbers. Misexpression of hbs in the wing discs with pnr-GAL4, sca-GAL4 or da-GAL4 perturbs the linear arrangement of the microchaetes, but their number is unaltered (Dworak, 2001).
Driving UAS-hbs with GMR-GAL4 or sca-GAL4 in the eye-antennal disc gave a rough eye phenotype with disorganization of the ommatidia and bristles. Driving with the da-GAL4 driver gave the strongest rough eye phenotype with occasional fusion of ommatidia. Examination with anti-Elav antibody shows that the photoreceptor clusters are irregularly placed, and pigment cells are absent at sites of ommatidia fusions. Larval photoreceptor pathfinding and targeting appears normal when examined with mAb 24B10 (Dworak, 2001).
Overexpressing a secreted form of Hbs with all of the aforementioned GAL4 drivers did not generate gain-of-function phenotypes. In addition, the adult hbs gain-of-function phenotypes were not suppressed as transheterozygotes with sns, irreC-rst or the duf/irreC-rst deletion (Dworak, 2001).
Thus misexpression of hbs in the wing disc yields abnormal distal wing margins and disorganized microchaetes on the notum. At first glance, these phenotypes look like mild loss-of-function Notch defect; however, microchaete numbers do not deviate from normal. Hence, the microchaete phenotype appears to be due to displacement of cells, rather than changes in cell number caused by disturbance of lateral inhibition. This could arise if: (1) the cell-cell associations within proneural clusters are slightly perturbed, resulting in subtle changes in the location of the founder cells; or (2) founder cells are normally specified but subsequent alterations in cell-cell associations lead to their being slightly displaced (Dworak, 2001).
Misexpression of hbs in the eye disc results in a rough eye phenotype, which is reminiscent of that seen when irreC-rst is absent or misexpressed in the eye disc. Yet neither gain-of-function eye phenotype is suppressed by a 50% decrease in the expression of the other gene, and no rough eye phenotype, is observed in the hbs and irreC-rst transheterozygotes. Since Hbs and IrreC-Rst did not give a positive result in the S2 cell aggregation assay, and the overexpression of irreC-rst in the wing imaginal disc causes a notal microchaete phenotype that differs from that for hbs, there is still no direct support that Hbs and IrreC-Rst interact directly with one another in a simple one-to-one trans binding relationship. More suitable interaction analyses await elucidation of whether Kirre and Sns have roles in eye (Dworak, 2001).
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date revised: 15 December 2005
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