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Characterization of Drosophila hibris, a gene related to human nephrin

Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University Medical School, 540 First Avenue, New York, NY 10016, USA

View larger version (117K): [in a new window]   Fig. 1. Proteins of the Nephrin subfamily from Drosophila (Hibris and Sticks-and-Stones (Sns)), and worm, human, mouse and rat aligned with Clustal V. For Hbs, single lines lie over regions between conserved cysteines of Ig domains, the broken line is above the FN type-III like domain, the double line is over the putative transmembrane region and asterisks are over potential asparagine-linked glycosylation sites. The black indicates amino acid identity with respect to Hbs, while the gray indicates identity with respect to human Nephrin.

View larger version (45K): [in a new window]   Fig. 2. (A) Kyte-Doolittle hydrophobicity plot for Hbs shows strongly hydrophobic regions for the putative signal sequence and transmembrane regions (asterisks). The schematic beneath shows the corresponding organization of domains. (B) The genomic region containing the hbs in cDNA4 is spanned by P1s DS00087 and DS04940. Location of the 12 exons contained in cDNA4 is shown (thick black lines) relative to the genomic area. (C) S2 cells fail to aggregate when Hbs-transfected and RmHa3-transfected cells are mixed, form aggregates containing only red cells when Duf-transfected (red) and RmHa3-transfected (green) cells are mixed, and form aggregates containing both red and green cells when Duf-transfected (red) and RmHa3-transfected (green) cells are mixed. (D) Matrix of S2 cell aggregation responses: –, no aggregation; R, aggregates with only cells of the one color; RG, aggregates with cells of both colors.

View larger version (136K): [in a new window]   Fig. 3. Expression of hbs in embryonic tissues and larval imaginal discs. (A) A late stage 10 embryo has the mesectodermal cells aligned at the midline (arrowhead) and is hbs positive. Lateral patches of mesodermal cells (arrow) also express hbs. (B) This pattern is revealed at the protein level with the Hbs antibody. Arrow and arrowhead indicate equivalent tissues from A. (C) hbs-positive cells lie posterior and internal to the posterior commissure (pc) as it and the anterior (ac) are developing during stage 12. (D) By stage 14, only a subset of cells below the pc are still Hbs positive (arrowheads). (E) Stage 13 embryo. hbs is expressed by fusion competent myoblasts. (F) hbs is not expressed in muscle founder cells as shown in the age matched Notch mutant embryo. (G) At stage 11 hbs is expressed in fusion competent myoblasts (arrowheads) but not the Kruppel-positive founder cell (arrow). (H) Expression of hbs in the muscle attachment sites of a stage 14 embryo. (I) hbs is expressed in cells within and behind the morphogenetic furrow of the larval eye/antennal disc. (J) In the larval brain, hbs is expressed in the optic lobes in the locations where the photoreceptors terminate (arrows). In the wing disc (K), hbs expression is present in stripes that bound the presumptive wing margin (arrowhead), the stripes corresponding to wing veins L3 and L4 (downward arrows) and L0 and L1 (horizontal arrow). (L) In the leg disc, there are broad concentric circles of hbs.

View larger version (44K): [in a new window]   Fig. 4. (A) Map showing location of genes and P-elements in the region of hbs, and the regions removed by x-ray-induced deletion of EP(2)2590. Deletion 12 embryos show perturbed ventral musculature in a subset of hemisegments (B, arrow), and this phenotype is also observed in Deletion 11/Deletion 12 embryos (C, arrow). (D) Innervation of muscles is normal (arrows show innervation at muscle 12) in hemisegments without defects, and also in the muscles present in the disrupted regions.

View larger version (111K): [in a new window]   Fig. 5. Misexpression of hbs in the embryonic mesoderm disrupts myoblast fusion. (A) Wild type pattern of the ventral musculature. (B) UAS-hbs;twi-GAL4 embryo showing partial failure of myoblast fusion. Arrowheads indicate unfused myoblasts. (C) UAS-hbs/+;da-GAL4/+showing both partial failure of myoblast fusion (arrowheads), and absence of a muscle fiber in the ventral muscle group (arrow). Preparations are stained with mAb FMM5.

View larger version (157K): [in a new window]   Fig. 6. Misexpression of hbs in the embryonic epidermis disrupts muscle attachment. (A) Wild-type embryonic somatic musculature in three hemisegments. Arrows indicate a segmentally reiterated group of three muscle fibers in the lateral musculature, while arrowheads indicate the external ventral muscles. (B) UAS-hbs/en-GAL4 embryo show occasional failure of the three muscle fibers (shorter arrows) to insert at their dorsal attachment sites. More ventral muscles fibers are also occasionally defective (arrowhead). (C) The ability of the three lateral muscle fibers to insert at their dorsal attachment sites is comprehensively overturned in the UAS-hbs/sca-GAL4 embryo (shorter arrows). Ventral muscles fibers sometimes no longer span the hemisegment correctly (arrowheads). (D) Overexpression of hbs in the dorsal epidermis with the pnr-GAL4 driver results in dorsal muscle fibers failing to extend across the segment (arrows), or sending a thinned process (arrowhead). Many fibers remain aligned with the segment boundary. Preparations are stained with mAb FMM5.

View larger version (130K): [in a new window]   Fig. 7. Misexpression of hbs in imaginal discs leads to adult phenotypes. Distal wing margin in (A) wild type, (B) omb-GAL4/+;UAS-hbs/+ and (C) UAS-hbs/+;da-GAL4/+. The misexpression conditions lead to abnormal development of the wing margin (B) and notching (C). (D,E) The microchaetes on the scutum of wild type flies are linear (D, arrows) but disorganized on UAS-hbs/+;da-GAL4/+ flies (E, arrows). Wild-type flies show an orderly arrangement of eye structures (F,J,N), while in UAS-hbs/GMR-GAL4 (G,K,O), UAS-hbs/sca-GAL4 (H,L,P) and (I,M,Q) UAS-hbs/+;da-GAL4/+ flies, patterning is disrupted. Gain-of-function flies show rough eye phenotypes (G-I) and disorganization of ommatidia with anti-Elav staining (K-M). Fusion of ommatidia is evident as absence of pigment cells under darkfield microscopy (O-Q). Arrows in O-Q indicate examples of fused ommatidia.

View larger version (136K): [in a new window]   Fig. 8. hbs expression depends on sim, Notch and bap, but not mef2. (A) Wild-type stage 11 embryo showing characteristic line (arrowhead) of hbs expression in the mesectoderm. (B,C) This expression is absent in the sim mutant embryo (B, arrowhead), and expanded in UAS-sim/sca-GAL4 embryos (C, arrowhead). (D) In a stage 12 Notch embryo, hbs expression is absent at the CNS midline (arrowhead; compare with Fig. 3F). (E,F) Stage 11 mef2 embryos show normal expression of hbs in visceral mesoderm cells (E, arrowhead), but this is greatly reduced in bap mutant embryos (F, arrowhead).

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