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A distinct set of founders and fusion-competent myoblasts make visceral muscles in the Drosophila embryo

Beatriz San Martin^*, Mar Ruiz-Gómez^*,, Matthias Landgraf and Michael Bate

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
^* Both authors contributed equally to the work
Present address: Centro de Biología Molecular ‘Severo Ochoa’, UAM-CSIC, Madrid, Spain

View larger version (60K): [in a new window]   Fig. 1. Single cell dye fills (red) of midgut circular and longitudinal muscles. (A) Projection of a confocal Z-series showing four midgut circular muscles (red) in an early stage 14 embryo. The lower two muscles are adjacent – see inset. The embryo carries the RP298 enhancer trap (Nose et al., 1998) which marks founder cell nuclei (green) but is otherwise wild type. All four midgut circular muscles are binucleate and contain one RP298-positive (arrowheads in A) and one RP298-negative nucleus (arrows in A and B), thus demonstrating that these muscles are the product of a fusion between a founder cell and a fusion-competent non-founder myoblast. (B) A montage of single confocal sections (2 µm thickness) of the same preparation as shown in A. The nuclear label TOTO-3 shows nuclei of the midgut circular muscles. Arrows indicate RP298-negative nuclei of fibres seen in A. (C) Part of a wild-type midgut longitudinal muscle filled at late stage 16. Anti-MEF2 staining (green) reveals three nuclei in the syncytium (arrowheads). (D) Midgut muscle fills of a late stage 16 mbc mutant embryo (no myoblast fusion) carrying the RP298 enhancer trap. Midgut longitudinal (arrowheads) and circular (arrows) muscles form in non-fusion mutants, but are always mononucleate and positive for the founder cell marker RP298 (green). n10. Scale bar, A,B, 20 µm; C,D, 50 µm; inset in A, 10 µm.

View larger version (95K): [in a new window]   Fig. 2. Cell populations in the midgut visceral mesoderm.A,B embryos at late stage 12 (see inset B) stained to reveal migratory longitudinal muscle precursors. RNA in situ hybridizations show bHLH54F expression (A) and duf expression (B) on the midgut primordium. Note distribution of cells along palisade formed by immature circular muscles. (C,D) Dorsolateral views of part of stage 11 embryos to show differentiating midgut visceral mesoderm. RNA in situ hybridizations show duf expression in columnar marginal cells (C) and sns expression (D) in distinct but adjacent population of cells. Arrows indicate marginal columnar cells in these figures. (E) Early stage 11 embryo; DNA in situ hybridization to show bap expression in the invaginated primordia of the midgut visceral mesoderm. (F) Two different focal planes of these bap-expressing cells at later stage 11 showing the orderly arrangement of columnar cells with low expression (arrow, left) and relatively higher expression in adjoining cells (arrowhead, right).

View larger version (89K): [in a new window]   Fig. 3. Fusion-competent visceral myoblasts contribute to circular and longitudinal muscles. (A,B) Embryos (stage 11 A; early stage 12 B) double stained for antibodies against Fas III (brown) and Hairy (gray). (A) Hairy expression is not present in the marginal columnar cells but is present in adjoining aggregates of Fas III-expressing cells. (B) The marginal cells have divided to produce two rows of adjacent, columnar cells. Expression of Hairy remains confined to adjacent cells. (C) Embryo from strain HairyL43 stained with anti-ß-gal antibody. A posterior region of the forming midgut is shown with persistent ß-gal expression in circular (arrow) and longitudinal muscle (arrowhead) precursors.

View larger version (56K): [in a new window]   Fig. 4. Localised patterns of gene expression in visceral muscle founders. (A-C) Embryos stained with antibodies against Connectin. (A) Dorsolateral view of stage 11 embryo showing consecutive sets of Connectin-expressing visceral founders in adjacent segments. (B) Stage 13 embryo showing the dorsoventrally expanded domain of Connectin expression. (C) sns mutant embryo at stage 13 showing restriction of Connectin expression to the circular founders (arrow). Note the adjacent population of fusion-competent myoblasts (arrowhead) that are not recruited to expression in the absence of fusion. (D-F) Stage 11 embryos stained to reveal expression of (D) wg (anti-Wg), (E) dpp (in situ hybridisation), (F) abd-A (anti-Abd-A) in subsets of circular visceral muscle founder cells.

View larger version (112K): [in a new window]   Fig. 5. Midgut phenotypes in wild-type and non fusion mutant embryos revealed by Fas III expression. (A-F) Midgut region of stage 13 embryos stained with antibody to Fas III to show, (A) palisade of forming circular visceral muscles in wild type; (B) elongated founders (arrow) and closely adherent but unfused myoblasts (asterisk) in an mbc mutant embryo; (C-F) Low and higher power views of (C,D) Df(1)w67k30 and (E,F) snsZF1.4 embryos showing palisade of circular muscle founders (arrow) distinctly separated from adjacent population of fusion-competent cells (asterisk). Note similarity of the phenotypes in C,D and E,F.

View larger version (14K): [in a new window]   Fig. 6. Diagram to illustrate the formation of longitudinal and circular visceral muscles from founders and fusion-competent cells. (A) Visceral mesoderm of late stage 11 embryos with fusion-competent cells (orange) and associated circular founders (blue and red nuclei) together with inwardly migrating longitudinal founders (green nuclei). Local patterns of gene expression in circular founders indicated by red and blue nuclei. (B) Stage 12 embryo showing forming palisade of circular muscle precursors (red and blue nuclei) and associated longitudinal precursors (green nuclei) together with fusion-competent myoblasts (orange) which contribute to both kinds of muscles. (C) The completed lattice of circular and longitudinal visceral muscles, with localised patterns of gene expression as indicated.

View larger version (58K): [in a new window]   Fig. 7. Longitudinal visceral founders require visceral myoblasts to complete muscle formation. (A,B) DNA in situ hybridisation with probe for bHLH54F in stage 13/14 embryos showing longitudinal muscle precursors in (A) wild-type and (B) eveR13 mutant embryos. Note that the precursors are syncytial in A but that fusion fails to occur in the absence of trunk visceral mesoderm in the eve mutant embryo (B).