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rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila

Martin Strünkelnberg^1,*, Bernhard Bonengel^1,*, Livia M. Moda², Alexander Hertenstein¹, H. Gert de Couet^1,3, Ricardo G. P. Ramos² and Karl-Friedrich Fischbach^1,

¹ Institut für Biologie III, Schänzlestr.1, Albert-Ludwigs-Universität, D-79104 Freiburg im Breisgau, Germany
² Departmento de Biologia Celular, Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14.049-900 Ribeirão Preto-SP, Brazil
³ Department of Zoology, University of Hawaii at Manoa, 2538 McCarthy Mall, Honolulu, HI 96822, USA
^* These two authors contributed equally to this work

View larger version (171K): [in a new window]   Fig. 1. Rst overexpression and mutant phenotypes in the stage 16 abdominal muscle pattern. Three dimensional reconstructions of confocal stacks of antibody staining against ß-3-tubulin. Anterior is to the left and dorsal is upwards. (A) Heatshocked yw embryo. Arrowheads indicate ventral acute (va) and ventral oblique (vo) muscles. (B) Heatshocked pCa18Z3.1 embryo. Muscles are thin and some are missing (e.g. ventral acute muscles). (C) Heatshocked yw embryo. Single confocal plane interior to the muscles. (D) Heatshocked pCa18Z3.1 1 embryo. Single confocal plane as in C, showing a large amount of unfused fusion-competent myoblasts. (E) da-Gal4/+;UAS-rst/+ embryo showing unfused fusion-competent myoblasts (asterisk), thin muscles and some muscles with ectopic projections (arrow). Several muscles are missing. lt, lateral transversal muscle. (F) rstirreC1 mutant embryo showing missing and thin muscles (arrowheads). (G) rst6 mutant embryo with misled (arrow) and thin muscles (arrowhead). Asterisks mark unfused fusion-competent myoblasts. Scale bars: 20 µm.

View larger version (69K): [in a new window]   Fig. 2. The kirre and rst genes and proteins. (A) Physical map of the kirre locus. The first exon of kirre resides about 30 kb distal and is not depicted. Unbroken lines symbolize genomic DNA present in the depicted deficiency chromosomes and the cosP479BE transgene. Hatched bars refer to the closest possible breakpoint predictions. Arrowheads symbolize kirre-specific primers used in single fly PCR. Open reading frames are depicted as black bars and orientation of transcription by arrows. Restriction sites: B, BglII; E, EcoRI; H, HindIII; S, SacI; Xb, XbaI; Xh, XhoI. (B) Schematic comparison of the Rst and Kirre proteins. Numbers refer to sizes of Immunoglobulin (Ig) domains and to percentages of sequence identities of paralogous Ig domains, respectively. Arrows indicate the serine- and glycine-rich repeats of Kirre and Rst, respectively. An asterisk marks the sequence stretch separating the autophosphorylation domain and the PDZ-binding motif in Kirre. (C) Alignment of Rst, Kirre, Sns and Hibris. Residues identical in Rst and Kirre are on green background, residues identical within all four sequences are boxed in red. Borders of Ig domains (Ramos et al., 1993) are marked by a vertical bar and an inverted triangle. Arrows indicate cysteines involved in forming a disulphide bond. Serine- and glycine-rich repeats of Kirre and Rst, respectively, are underlined. Putative phosphorylation sites conserved within Kirre and Rst are marked by P in an inverted triangle. Unconserved sites are boxed. APD, autophosphorylation domain with consensus sequence below; IC, intracellular domain; IG, Immunoglobulin domain; PADVI, conserved motif; opa, opa-like repeat; PDZ, PDZ-binding motif; SP, signal peptide; TM, transmembrane domain. Boxed sequence stretches contain the corresponding patterns.

View larger version (106K): [in a new window]   Fig. 3. Comparative analysis of rst and kirre expression patterns. Anterior is towards the left. Dorsal is upwards for A,I,K (lateral view) and F,H,L (ventrolateral view). (B-E,G) Dorsal view. (A-F) rst in situ hybridization. (A) Expression of rst starts during stage 4 in seven stripes enveloping the embryo. Soon thereafter this pattern fades and rst is expressed dorsally in procephalic regions and in the future amnioserosa, while ventrally the striped pattern remains present during invagination of the ventral furrow (data not shown). (B) At stage 8, rst labels a segmental pattern in mesectodermal cells. (C) During stage 9, rst is expressed in the midline (arrow) and mesodermal cell clusters (arrowhead). (D) At stages 10 to 11, the staining in the midline increases and rst starts to label progenitors of the visceral muscles (arrowhead). (E) At stage 12, the midline staining fades and rst is expressed strongly in the majority of mesodermal cells. (F) At stages 13-14, rst labels segmental stripes of mesodermal cells close to the epidermis. (F'out) Focal plane close to the epidermis: rst expression includes regions where founder cells reside. dl, dorsolateral; l, lateral; v, ventral clusters (d, dorsal cluster out of focus in F’out). (F’in) Focal plane somewhat further interior within the mesoderm: rst is expressed where fusion-competent myoblasts reside. (G-L) kirre in situ hybridization. (G) Expression of kirre starts during stage 11 in the progenitors of the visceral muscles (arrowhead). (H) kirre expression can be detected up to stage 16, when kirre labels a ventral u-shaped structure at the posterior end of the head region (arrowhead) and two regions more anterior (arrow). (I) During stages 12 to 13, kirre is expressed in segmental clusters close to the epidermis at positions where founder cells reside. (I’) Higher magnification of the boxed region in I, showing kirre expression in the dorsal group of muscle founder cells (d). (J) Scheme of the larval muscle pattern. Depicted in red are muscle groups that can be identified in K',L'. (K,K',L,L') Expression of kirre in outgrowing muscle founder cells/precursors in stage 13-14. lt, lateral transversal muscle precursor; vt, ventral transversal precursor. (M-O) Rst antibody staining of abdominal hemisegments of embryos of the rP298lacZ enhancer trap line. Green labels ß-galactosidase-expressing nuclei and red highlights Rst signal. (M) Single confocal plane within somatic mesoderm of a stage 13-14 rP298lacZ embryo. Arrowheads indicate cells labelled for Rst and ß-galactosidase. (M') Reconstruction of one hemisegment viewed from anterior. Position and morphology of Rst-labelled cells that do not express ß-galactosidase strongly suggests that these cells are fusion-competent myoblasts. vm, visceral muscles. (N,N') Staining of stage 13 to 14 rP298lacZ embryos in a mbcC1 background are roughly comparable with M,M'. (O) In older embryos of the same genotype, Rst expression can be detected in fibrous cells expressing ß-galactosidase (founder cells), while it is very weak in fusion-competent myoblasts. v, ventral founder cells; sb, founder cell of segment border muscle. Scale bars: 20 µm.

View larger version (63K): [in a new window]   Fig. 4. Rst is expressed in the apodemes. Double staining of abdominal hemisegments of the Wß1HI-lacZ apodeme specific enhancer trap line shows overlap during stage 14: (A) Rst channel. (B) Overlay of Rst (red) and ß-galactosidase (green). a, anterior; iapo, intersegmental apodemes; p, posterior. Scale bar: 20 µm.

View larger version (44K): [in a new window]   Fig. 5. kirre is not essential for muscle development. (A) Schematic depiction of the white-Notch region and deficiencies used in this study (see text). Df(1)w67c23 is a viable deletion removing 3C2-5 (Lefevre and Green, 1972) (see discussion). (B) Single fly PCR proves that kirre is not present in the genome of Df(1)N54l9/Y;cosP479BE/+ flies. Lanes 1,3,5: kirre-specific primers. Lanes 2,4,6: rst-specific primers. Lanes 1,2: Df(1)N54l9/Y;cosP479BE/+ as template. Lanes 3,4: wild-type Berlin males as template. Lanes 5,6: no template. (C) Abdominal muscle pattern of Df(1)N54l9/Y;cosP479BE/+ embryo stained for muscle Myosin is indistinguishable from wild type. Anterior is towards the left, dorsal is upwards. Scale bar: 20 µm.

View larger version (126K): [in a new window]   Fig. 6. Df(1)w67k30 is a rst, kirre double mutant and shows severe muscle defects. (A) Wild-type abdominal muscle pattern as revealed by staining against ß-3-tubulin. vl, ventral longitudinal muscles; sb, segment border muscle. (B) Df(1)w67k30 embryos display a drastic myoblast fusion phenotype with thin outgrowing muscle founder cells and many unfused myoblasts (asterisks). (C) Detailed view of two abdominal hemisegments of the same embryo. Scale bars: 20 µm.

View larger version (51K): [in a new window]   Fig. 7. Reintroduction of Rst into the mesoderm rescues the phenotype of Df(1)w67k30, Nfa-g embryos (see Materials and Methods). (A) Df(1)w67k30, Nfa-g/Y embryo displaying a strong myoblast fusion phenotype. (B) Df(1)w67k30, Nfa-g/Y, twi-Gal4 x UAS-rst embryo displaying a nearly wild-type abdominal muscle pattern with minor defects: some muscles are thin (arrow) and some terminate in ectopic positions (asterisk). (C) Hetero- or homozygously balanced embryo out of the same cross (indistinguishable from wild-type). vl, ventral longitudinal muscles. Scale bar: 20 µm.

View larger version (133K): [in a new window]   Fig. 8. Ectopic expression of Rst attracts myoblasts to ectopic sites. Embryos are stained in green for muscle-specific markers ((A-C,J-L) ß-3-tubulin (D-I) muscle myosin) and for Rst in red. (C,F,I,L) Merged images. (A) Df(1)w67k30, Nfa-g embryo: only very few myoblasts can be found close to the epidermis. (B,C) Df(1)w67k30, Nfa-g; dll-Gal4/+, UAS-rst/+ embryo: attraction of myoblasts towards the epidermis around legdiscs (arrowheads). (D-I) Embryos from the same cross, which are either heterozygous or homozygous for the balancer. (D,G) Embryo not misexpressing Rst. (E,F,H,I) dll-Gal4/+, UAS-rst/+ embryos: ectopic myoblasts cluster in ventral regions of the thoracal segments up to stage 16 (E,F, arrowheads) and in the head region (H,I, arrowheads). (J) wg-Gal4/wg-Gal4 control embryo. (K,L) wg-Gal4/UAS-rst embryo: attraction of myoblasts to epidermal sites distal to the ventral acute muscles. (M,N) Two single focal planes through the epidermis: (M) apical position; (N) 2,5 µm below. Rst labels basal membranes of epidermal cells only at sites of ectopic expression (ectopic) and only very weakly in apodemes (apo). Scale bars: 20 µm.