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The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling

Molecular Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

View larger version (57K): [in a new window]   Fig. 1. Genetic map and molecular characterization of hbs. (A) Co-ordinate ‘0’ corresponds to the beginning of cDNA D1, the ORF of which is denoted with black boxes. The differential display cDNA fragment (P1/T9(1)) used to clone hbs is also indicated. P[w+]36.1 denotes a reporter lacZ construct inserted 680 nucleotides upstream of the gene. The extent of deficiencies is shown as lines, with broken ends indicating breakpoint uncertainty. (B) hbs transcription detected by Northern blot on mutant embryos Toll10b and twist-GAL4 driving expression of activated Ras (ras*) or activated Notch (N*). (C) Developmental Northern blot of wild-type embryos at indicated ages (in hours). Using a hbs probe, we detected a 6.2 kb transcript (arrowhead) with peak expression around mid-embryogenesis. The absence of expression in 0-1 hour embryos suggested that hbs was not maternally loaded. (D) Conceptual translation of cDNA D1 (Accession Number, AF210316) revealed an ORF 3708 nucleotides long with the first ATG at position 373. The encoded protein was 1235 amino acids in length and had a predicted molecular weight of 134.4 kDa. Nine Ig-C2 repeats are underlined, Fibronectin type III repeat is shown in bold and transmembrane region is in lowercase. Predicted signal peptide cleavage site is indicated by a triangle. (E) ClustalW alignment between Hbs and Sns cytoplasmic domains. Predicted transmembrane domain is indicated in lowercase, a cAMP- and cGMP-dependent protein kinase phosphorylation site is underlined, protein kinase C phosphorylation sites are indicated in bold lowercase and casein kinase II phosphorylation sites are indicated in bold uppercase. Two direct repeats in Hbs are underlined twice. Conserved motifs are boxed and, when conserved, phosphorylation sites in Sns are also indicated as in the Hbs sequence. Asterisks denote amino acid identity, colons indicate conserved amino acid changes and periods indicate related amino acids.

View larger version (103K): [in a new window]   Fig. 2. Embryonic expression of hbs transcript and protein. In all panels anterior is towards the left and dorsal upwards. Lateral views (A-E,G,H,M,N), dorsal views (F,I,J) and ventral view (K). (A) hbs was first detected by stage 8 in the amnioserosa anlagen and in the mesectoderm (arrow). Inset shows a ventral view of the same embryo. Expression in the mesectoderm or its progeny remained throughout embryogenesis (see also B-D). (B,C) By early stage 10, transcription started in patches in precursors of the visceral musculature (B, arrowhead) where it continued to be expressed as these cells internalize (arrowhead, C). (D,E) Stage 11 embryo shows hbs expression in the somatic mesoderm (D, arrow). This expression increased as founder and fusion-competent cells fuse during germ band retraction (arrowhead, E). (F) The late expression pattern (stage 15) consisted of epidermal stripes at putative muscle attachment sites (straight arrow), heart precursors (black arrowhead), hindgut (white arrowhead) and pharyngeal muscles (bent arrow). (G-N) An anti-Hbs antibody reproduced the same pattern of expression and revealed finer details. (G) At stage 11 Hbs protein was detected in the dorsalmost part of the somatic mesoderm (arrow). (H,I) This expression was found throughout the somatic musculature by germ band retraction (arrowhead, H) and stage 13 (bent arrow, I). By this stage, Hbs was still detected in the visceral musculature (arrowhead, I). (J) By stage 16, Hbs was found in pharyngeal muscles (bent arrow), dorsal vessel (arrowhead) and epidermal attachment sites (straight arrow). (K-N) High magnification micrographs of several tissues show signal at cell border (arrowhead, K) and polarized expression of the protein. Hbs was restricted to the membranes in contact between mesectoderm cells (asterisk in germ band extended embryo K), to the lumenal side of the hindgut (inset, J), to the contact side between cells at the epidermal attachments (arrowhead, L) and to discrete points at the myoblast membranes while fusion is in progress (arrowheads, M,N). I,N,M are confocal micrographs.

View larger version (98K): [in a new window]   Fig. 3. hbs is a Notch and Ras signaling pathway target in vivo. hbs transcription detected in situ in wild-type embryos (A,D), zygotically null N55e11 (C), twist-GAL4 driving dominant negative Ras (UAS-ras1N17; B), activated Ras (UAS-ras1V12; E) or activated Notch (UAS-Nintra; F). All pictures are lateral views at mid stage 12 showing segments T2-A2. Embryos A-C and D-F were hybridized in parallel experiments. Ras signaling represses (vertical arrowheads point to somatic mesoderm; compare A,B and D,E) while Notch signaling activates hbs transcription (compare A,C and D,F). Note CNS hyperplasia (horizontal arrowhead indicates lateral edge of the CNS) and lack of staining in the somatic mesoderm (vertical arrowhead) in C.

View larger version (91K): [in a new window]   Fig. 4. Hbs co-localizes with Sns at distinct points in the cell membrane of fusion-competent and visceral mesoderm myoblasts. Expression of Hbs (A,D,G,J in red) and Sns (B,E,H,K in green) was analyzed by immunofluorescence with confocal microscopy (A-L). Co-localization was assessed in images combining both channels (C,F,I,L) by presence of the yellow color. All panels are lateral views showing expression in somatic (A-I) or visceral mesoderm (J-L). Late stage 11 embryos (A,C) showed stronger and more widespread Hbs expression in somatic (arrowhead) and visceral mesoderm (arrow) than Sns. Both signals overlapped at the myoblast cell membrane (yellow in C). Late stage 12 embryos (D-F) showed characteristically punctate expression for both proteins, with yellow spots (arrowhead, F) indicating co-localization. Note that some foci of expression appeared to be Hbs (straight arrow) or Sns (bent arrow) specific. Stage 15 embryos (G-I) no longer express Hbs (G, arrowhead) but do express Sns (arrowheads, H,I) in putative unfused myoblasts. Stage 12 embryos (J-L) showed co-expression of Hbs and Sns in precursors of visceral mesoderm. (M-O) P[w+]36.1 embryos analyzed for co-expression of the hbs enhancer trap (in red) and Krüppel founder cell marker (in green). Ventrolateral views of mid-stage 12 embryos showed ß-galactosidase expression (bent arrow, O) adjacent to a Krüppel-positive cell (arrowhead, O). No other mesodermal ß-galactosidase-expressing nuclei could be seen above or below the indicated cluster, suggesting that this reporter was expressed in a subset of fusion-competent cells.

View larger version (63K): [in a new window]   Fig. 5. Mutations mapped to the hbs transcription unit. (A) The donor/acceptor splice site nucleotide sequence (intron boxed) from the mutagenized stock is shown at the center. Wild-type splice event is shown above with its conceptual translation on top of the nucleotide sequence. cDNA clones from hbs2593 mutant flies showed a T to A transversion (large letters) in the donor splice site. The hbs2593 cDNA retained the intron between exon 9 and 10, apparently owing to mutation in the donor splice site. This, in turn, leads to a prematurely truncated protein as indicated by an asterisk. We detected a dramatic decrease in hbs immunoreactivity when comparing wild type (C) with hbs2593 homozygous mutant embryos (E). Note remaining expression in somatic mesoderm and midline (arrowheads) in C,E. (B) hbs459 was an A to T transversion at nucleotide 2029 in the cDNA sequence that leads to a non-sense codon and truncates the protein in position 553. (D) Antibody staining in wild-type embryos detected Hbs in visceral muscle precursors (arrowhead) and midline (bent arrow), while hbs459 homozygous mutant embryos showed a complete lack of Hbs (F).

View larger version (112K): [in a new window]   Fig. 6. hbs loss of function leads to a partial fusion block in somatic muscles and a visceral mesoderm phenotype. Wild-type (A,C,E,G,I) and hbs459/Df(2R)X28 embryos (B,D,F,H,J) probed with anti-Myosin (A-H) or anti-Fasciclin III antibody (I,J). Anti-Myosin staining revealed that the overall pattern of somatic muscles was normal in hbs-null embryos (compare A with B). We detected a partial fusion block in embryos lacking hbs function as demonstrated by the presence of free myoblasts around the CNS (arrowheads, D) and around the heart (bent arrow, F). Note that unfused myoblasts can extend filopodia (arrowheads, D) suggesting that these free myoblasts can scan for founder cells but have failed to fuse. Asterisks indicate the CNS in C,D. hbs mutant embryos also showed a gut phenotype consisting of an enlarged first gut chamber (arrowheads, G,H), which pushes the dorsal muscles and heart up (black arrowheads in E,F). Asterisk in F denotes the gut. The gut phenotype may stem from an earlier reduction in the number of visceral mesoderm precursors, as suggested by the presence of gaps in embryos homozygous mutant for hbs (bent arrows, J)

View larger version (86K): [in a new window]   Fig. 7. Functional mapping of domains important for hbs function. Wild type (A,E,I,M) or twist-GAL4; Dmef2-GAL4-driven overexpression of UAS-hbsFL (B,F,J,N), UAS-hbsECD (C,G,K,O) and UAS-hbsICD (D,H,L,P) in embryos stained for Myosin (A-H), Krüppel (I-L) and Fasciclin III (M-P). All panels are lateral views of stage 16 (A-H), stage 14 (I-L) and stage 12 (M-P) embryos. Overexpression of UAS-hbsFL or UAS-hbsECD constructs throughout the mesoderm resulted in partial fusion block (arrowheads, E-G) and muscle losses (white asterisk, B; note also losses in C). Bent arrow in B indicates dorsal muscles that are abnormally fused together. Specification of the founder cell marker Krüppel was normal initially yet later showed an abnormal pattern (I-K). Bent arrows in J,K designate LT and DA1/DO1 muscle precursors showing an abnormal morphology, respectively. By contrast, UAS-hbsICD overexpression in forming somatic muscles did not appreciably interfere with myogenesis (D,H,L). Overexpression of either UAS-hbsFL, UAS-hbsECD or UAS-hbsICD constructs in the visceral mesoderm resulted in defects in Fasciclin III expression (arrowheads), suggesting abnormal segregation of visceral mesoderm precursors (M-P). (Q) Schematic representation of the constructs used in this study. UAS-hbsFL is a full-length hbs protein, UAS-hbsICD is a 1093 amino acid long version that lacks the cytoplasmic region, and UAS-hbsECD is a 277 amino acid long construct deleting the extracellular domain of the protein. Light gray boxes represent Ig-C2 domains, dark gray boxes represent Fibronectin type III domains and black boxes represent the transmembrane region.

View larger version (111K): [in a new window]   Fig. 8. hbs antagonizes sns function during myoblast fusion and gut morphogenesis. Wild type (A-C) or embryos with genotypes hbs2593/Df(2R)X28 (D-F), hbs459/Df(2R)X28 (G-I) and snsXB3 hbs2593/hbs459 (J-L) are shown. Heart (black arrowheads, A,D,G,J) and gut (B,E,H,K) views of stage 16 embryos stained for Myosin, and lateral views of stage 12 embryos stained for Fasciclin III (C,F,I,L). hbs homozygous mutant embryos showed not only a partial fusion block, particularly conspicuous around the heart (compare A with D,G white arrowheads), but also an aberrant morphology of the gut chambers (asterisks) in late embryos (compare B with E,H), and gaps in the visceral mesoderm (black arrowheads; compare C with F,I). Both aspects were suppressed by halving the sns genetic dose (compare D-F with J-L).

View larger version (65K): [in a new window]   Fig. 9. hbs overexpression phenotype is sensitive to the sns genetic dose. Ventrolateral views of stage 16 embryos stained with anti-Myosin (A,C,E,G) and lateral views of stage 12 embryos stained with anti-Fasciclin III (B,D,F,H) antibody. Wild-type embryos (A,B) and embryos overexpressing UAS-hbsFL under the control of twist-GAL4;Dmef2-GAL4 in a wild-type background (C,D), or snsXB3 heterozygous background (E,F). snsXB3 homozygous embryos (G,H) are shown as control. Hbs overexpression throughout the mesoderm leads to an increase in the number of free myoblasts (arrowheads, C) and abnormalities in early stages of visceral mesoderm development (arrowheads, D). This phenotype was dominantly enhanced by sns. Note increased numbers of free myoblasts (arrowheads, E; compare with C) and presence of more defects in the visceral mesoderm (arrowheads, F; compare with D). sns mutant embryos showed complete myoblast fusion block (G) and a weak visceral mesoderm phenotype (arrowhead, H). (I) Representation of mean number of Fasciclin III expression gaps per embryo at mid-stage 12 in the indicated genotypes. Bars designate standard error. Fasciclin III expression gap phenotype caused by overexpression of hbs was dominantly enhanced by lowering sns dose (compare UAS-hbsFL with snsXB3/+; UAS-hbsFL values). This increase was statistically significant at P<0.0001 (Student’s t-test). A deficiency that removed kirre and roughest, Df(1)w67k30, did not appreciably modify the hbs overexpression phenotype in the visceral mesoderm. Note also presence of Fasciclin III expression gaps in embryos homozygous for snsXB3 (0.53 per embryo) and hbs (1.04 per embryo).