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myoblasts incompetent encodes a zinc finger transcription factor required to specify fusion-competent myoblasts in Drosophila

¹ Centro de Biologia Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain
² Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

View larger version (79K): [in a new window]   Fig. 1. Differentiation of mesodermal derivatives in wild-type and minc mutant embryos. (A,B) Anti-Krüppel staining confirms normal distribution of Kr-expressing founder cells in minc mutant embryos. Stage 14 embryos: wild type (A) and minc[A388] (B). (C) Diagram to show normal pattern of Kr-expressing muscles (black) in an embryonic hemisegment. (D-F) Anti-myosin staining in wild-type (D) and mutant (E,F) embryos to show normal muscle pattern (D), complete lack of myoblast fusion and differentiating mononucleated muscles (E,F) and normal heart (F). Inset in D shows enlarged view of pharynx and inset in F shows enlarged view of unfused pharyngeal muscles in this minc[A388] embryo. Note absence of myosin expression in unfused fusion-competent cells in E and F. (G-I) Expression of Dmef2 in wild-type (G), minc[A388] (H) and mbc1 (I) embryos. Note absence of Dmef2 expression in fusion-competent myoblasts in H; compare with expression of Dmef2 in these cells in I. Insets show enlarged views of regions indicated by asterisks in F-I.

View larger version (95K): [in a new window]   Fig. 2. Abnormal patterns of gene expression in putative fusion-competent cells in minc mutant embryos. (A,B) Anti-Twist staining in wild-type (A) and minc[A388] (B) embryos at stage 14. Note maintained expression of twist in fusion-competent population in B. (C,D) Anti-Hairy in wild-type (C) and minc[A388] (D) embryos. Hairy is present in fusion-competent cells in C but absent from these cells in the mutant (D). Note hairy expression is unaffected in other tissues in these embryos. (E-H) RNA in situ hybridisations for sns and anti-ß-galactosidase staining in rP298;minc[A388]/TM3ftzlac (E,G) or rP298;minc[A388]/minc[A388] (F,H) embryos. Note loss of sns expression from somatic mesoderm in mutant embryos (F) but maintained presence in visceral mesoderm in the mutants (H). Arrows in G and H indicate midgut visceral mesoderm.

View larger version (49K): [in a new window]   Fig. 3. (A) Predicted amino-acid sequence of Minc protein showing four C2H2 zinc finger motifs (boxes). The shaded, underlined box indicates the position of the amino acid change associated with the minc[A388] mutation. In addition a conservative change of F to I at amino acid 140 was also present in the mutant. (B) Northern blot prepared from samples of embryonic (E), larval (L) and adult (A) poly(A)+ RNA hybridised to DNA probe LD 47926. A single transcript 3 kb long is revealed uniquely in RNA from embryos.

View larger version (84K): [in a new window]   Fig. 4. Embryonic pattern of minc expression. (A,B,E,F) RNA in situ hybridisations with the LD47926 probe. (C,D) as other panels but with anti-ß-galactosidase (brown) staining to reveal rP298 expression. (A) Embryo at late stage 10 showing minc expression in repeated clusters of cells corresponding to primordia of trunk visceral mesoderm. There is faint expression in mesodermal cross bridges below the clusters (arrowheads). (B) Stage 11 embryo showing minc expression in both founders (arrow) and fusion-competent cells (arrowhead) of visceral mesoderm. (C) Superficial (left) and deeper (right) planes of focus in stage 11 embryo to show transition from expression in visceral to expression in somatic fusion-competent cells. Expression in visceral mesoderm (arrows in right panel) is reduced as strong expression begins in somatic mesoderm (arrows in left panel). The location of the visceral mesoderm is indicated by the ribbon of rP298-expressing visceral founder cells. (D) Late stage 12 embryo: founder myoblasts express rP298 (arrows) but little if any minc; non rP298-expressing fusion-competent cells express minc at high levels (arrowheads). (E) Stage 13 embryo showing continued expression of minc in the somatic mesoderm. (F) By stage 15, minc expression is lost from the somatic mesoderm but now appears in the mesodermal cells of the gonad (arrow).

View larger version (91K): [in a new window]   Fig. 5. Regulation of minc expression. RNA in situ hybridisation using LD 47926 and anti-ß-galactosidase staining. (A) Control (twistID96/TM3lac) and (B) twistID96 embryos, showing minc expression is absent from twist mutant embryos. (C) Control (N55e11/FM7lac) and (D) N55e11 embryos to show marked reduction of minc expression in the N mutant. (E) Control (rP298; DlFX3/TM3lac) and rP298;DlFX3 to show residual minc expression as in D and enlarged population rP298-expressing (brown) founders, which do not express minc.

View larger version (70K): [in a new window]   Fig. 6. Rescue of minc and ectopic minc expression. (A-F) Twi-Gal4 used to drive UAS-minc in minc[A388] mutant background. A and D are controls for C and F, in which UAS-minc rescues minc mutant phenotype. B and E are minc mutant embryos for comparison. (A-C) Anti-myosin staining, (D-F) anti-Twist staining to illustrate (C,F) partial rescue of minc mutant phenotype shown by both recovery of fusion and myosin expression (C, compare B) and loss of twist expression from fusion-competent cells (F, compare E). (G,H) En-Gal4/UAS-minc drives ectopic Dmef2 expression (arrows, G) in the CNS and sns expression in the ectoderm (arrows, H). Sca-Gal4/UAS minc drives CNS expression of sns (arrows, I).

View larger version (25K): [in a new window]   Fig. 7. Diagram illustrating the mode of action of minc in the myogenic pathway and patterns of gene expression underlying myoblast diversification within the somatic mesoderm. Fusion-competent myoblasts (red; fcm) and founder myoblasts (green; fm) segregate from unspecified somatic myoblasts (blue) through the activation of the N signalling pathway in the fusion-competent myoblasts. This leads to minc being expressed selectively in these cells, thus initiating patterns of gene expression that are characteristic of either fusion-competent cells (red) or founder myoblasts (green). minc expression in fusion-competent cells is required both for specific patterns of gene expression (h, sns) and for the implementation of myogenic differentiation in these cells (myosin).