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First published online 5 November 2003
doi: 10.1242/dev.00843

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Notch and Ras signaling pathway effector genes expressed in fusion competent and founder cells during Drosophila myogenesis

Ruben Artero^1,*,, Eileen E. Furlong^2,,, Karen Beckett¹, Matthew P. Scott² and Mary Baylies^1,

¹ Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
² Departments of Developmental Biology and Genetics, Howard Hughes Medical Institute, Beckman Center, B300, 279 Campus Drive, Stanford University School of Medicine, Stanford, California 94305-5329, USA

View larger version (71K): [in a new window]   Fig. 2. Toll10b mutant embryos specify the FC and FCM fate, and respond to alterations in the Ras and Notch signaling pathways. (A) Representation of the steps involved in FC specification (1, 2, 3, 4) and some aspects of terminal muscle differentiation (5). Dark blue indicates clusters of equipotent myoblasts in which the Ras signaling pathway is active, whereas an increase in the thickness of the outline of the cell represents activation of the Notch signaling pathway. Cells (in 1) are depicted in a transitional state when the coordinated activities of the Notch and Ras pathways (and Argos activity, not represented) single out the muscle progenitor cell (green in 2). In these clusters (1), all cells show some Notch activity, but a particular cell achieves a stronger Delta signaling ability, which leads to a strong activation of the Notch pathway in surrounding cells (dark blue, 2). Surrounding cells are therefore prevented from becoming muscle progenitors. Concomitantly with this process, a burst of Ras signaling activity in the progenitor cell (green in 2) leads to activation of progenitor cell markers, such as Eve, and feedback loops, such as Argos. Muscle progenitor cells (P1 and P2 in 3) undergo asymmetric division giving rise to two FCs represented as green and orange cells in 4. Finally, FCs fuse to surrounding FCMs (represented as blue cells with processes in 5) to form syncytial muscles, growth cones extend to innervate the muscles, and muscle precursors extend towards their normal epidermal attachment sites. (B-J) All panels show 5- to 9-hour AEL embryos with anterior to the left. Anti-Slouch (B-D) and anti-Vg (E-G) antibody staining of Toll10b mutant embryos (B,E), and similar embryos expressing an activated form of Notch (UAS-Nintra; C,F) or Ras (UAS-rasV12; D,G) under the control of the twist-Gal4 driver at 29°C. Activation of the Notch pathway throughout the embryo led to a complete inhibition of the FC fate (note lack of staining in C,F), whereas activation of the Ras pathway enhanced FC fate as shown by an increased staining (D,G; arrowheads). Conversely, the FCM marker Sns was readily expressed in Toll10b embryos (arrowheads, H), and showed more (bent arrow, I), or less (J, bent arrow indicates residual staining), expression upon Notch or Ras activation, respectively. Cartoons beneath the panels represent schematically the results of experimental manipulation. Color scheme is the same as in A.

View larger version (108K): [in a new window]   Fig. 1. Toll10b mutant embryos differentiate largely as somatic mesoderm. Anterior is to the left and dorsal up unless otherwise noted. All stages are according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985). All views are lateral except for those shown in G,H,U,V,AA,BB, which are ventral. Wild-type and Toll10b mutant embryos were stained with the indicated antibodies, subjected to in situ hybridization (E,F) or processed to reveal the larval cuticle (U,V), as described in Materials and methods. (A-L) The somatic mesoderm is the chief mesodermal tissue present in Toll10b mutant embryos. All embryos were 5- to 9-hour AEL except for those shown in A,B and K,L, which were stage 5 and stage 16, respectively. (A,B) Arrowhead denotes lack of dorsal Twist staining in a wild-type embryo (A) and ubiquitous expression of Twist in Toll10b mutant embryos (B). (C,D) Tinman was expressed in heart precursors (straight arrow, C), dorsal somatic muscle and foregut (bent arrow, C). Only some putative foregut expression remained in Toll10b mutant embryos (bent arrow, D). (E,F) bagpipe transcripts were expressed in circular visceral mesoderm precursors (straight arrow, E), foregut and hindgut precursors (bent arrow, E). Only the expression in putative hindgut precursors was maintained in Toll10b mutant embryos (bent arrow, F). Straight arrow in F denotes lack of the circular visceral mesoderm marker bagpipe expression in the trunk. (G,H) Fasciclin III, a marker for differentiated visceral mesoderm, labeled the muscle sheet surrounding midgut (arrow, G) and pharyngeal muscles (arrowhead, G). Toll10b embryos showed expression in the putative remains of the pharyngeal musculature (arrowhead, H). Arrow in H denotes the absence of Fasciclin III expression in the trunk where the visceral mesoderm would have developed. (I,J) A serpent-lacZ reporter revealed that pro-hematocytes migrating from the head mesoderm (arrowhead, I) were also present in Toll10b mutant embryos (arrowhead, J). (K,L) Myosin heavy chain staining of wild-type (K) and Toll10b mutant (L) embryos. The final muscle pattern was disrupted in Toll10b mutant embryos but there was an abundance of Myosin-positive myoblasts and muscle fibers (arrow, L). (M-V) Ectodermally derived tissues were missing from Toll10b mutant embryos. All embryos were 5- to 9-hour AEL, except for those shown in Q,R, which were 7.5- to 10.5-hour AEL, and those shown in U,V, which were first instar larvae. (M,N) Single-minded expression, an early mesectoderm marker, was maintained in the Toll10b mutant background but showed an aberrant pattern. However, further mesectoderm differentiation did not occur, as antibodies to neuronal markers such as HRP failed to detect differentiated neurons (not shown). (O,P) Crumbs, a marker for apical-basal polarization of epidermal cells, was expressed in ectodermal stripes (arrow) and in foregut ectoderm in wild-type embryos (O). All the ectodermal stripe expression was absent in Toll10b mutant embryos (arrow) but foregut ectoderm expression remained (arrow, P). (Q,R) The anti-22C10 antibody labeled all neurons in the peripheral nervous system in wildtype embryos (Q) but was almost completely absent in Toll10b mutant embryos (R). (S,T) Anti-Trachealess, a marker for tracheal cell fate, revealed invaginating ectodermal cells (arrowhead in S) in wild-type embryos but showed only marginal posterior expression in Toll10b mutant embryos (arrowhead, T). (U,V) A terminal ectodermal specialization, the larval cuticle, was readily detected in wild-type larvae (U) but was completely absent in Toll10b mutant larvae. Image shows vitelline membrane and absence of cuticle (V). (W,X) An endoderm-specific enhancer-trap line crossed into the Toll10b mutant background revealed that the endodermal cell fate was readily specified in these mutant embryos. Panels show 5- to 9-hour AEL embryos. (Y-BB) Several paracrine signaling pathways remain active in Toll10b mutant embryos. All embryos were 5- to 9-hour AEL. (Y-Z) Htl expression in wild-type stage 12 embryos was restricted to clusters of myoblasts (arrow, Y). Toll10b mutant embryos similarly aged also showed expression in clusters (arrow, Z). (AA,BB) Wg expression in Toll10b mutant embryos was found in stripes reminiscent of wild-type expression.

View larger version (98K): [in a new window]   Fig. 3. Northern analysis confirmed the differential regulation of genes identified by microarray. Blots containing 1 µg of embryonic polyA+-selected RNA from the cross twist-Gal4; Toll10b X UAS-Nintra (N); twist-Gal4; Toll10b X UAS-rasV12 (R) and twist-Gal4; Toll10b X yw (Toll10b) at 29°C were hybridized to the indicated genes, using the probes described in Material and methods. Equal loading was assessed using -tubulin. The inclusion of the Toll10b lane allows an assessment of why a gene is enriched in a particular condition. For genes enriched in the microarray under activated Notch signaling conditions, the major contribution to the differential expression of the genes tested came from Ras signaling acting as an inhibitory signal. By contrast, the behavior of genes enriched under activated Ras conditions revealed greater complexity. Whereas phyl and trn displayed strong repression by Notch signaling and little activation by Ras signaling, CG17492 showed two transcripts, one that did not change, and a smaller transcript that showed a strong regulation by Ras and Notch signaling. Finally, CG6024 and nidogen exhibited repression by both signaling pathways; however, the repression by Notch was stronger than that by Ras, which led to the observed differential expression. Approximate sizes are: parcas, 2.8 kb; CG4136, 5 kb; gol, 3.5 kb; dei, 2.6 and 2.8 kb; CG8503, 1.9 kb; CG6024, 5.5 kb; phyl, 3.7 and 4.2 kb; CG17492, 3.8 and 4.2 kb; trn, 3.7 kb; nidogen, 5.3 kb; and -tubulin 2 kb.

View larger version (89K): [in a new window]   Fig. 4. FCM and FC specific expression of genes from array validates genetic and microarray strategy. (A-F) Simultaneous fluorescent detection of dei and gol, two genes predicted to be enriched in FCMs. dei (A,C) and gol (D,F) transcripts detected by in situ hybridization and anti-ß-Gal staining (B,E) in stage 12 rP298 embryos. The enhancer trap insertion rP298 marks FCs in somatic (Ruiz-Gómez et al., 2000) and visceral mesoderm (Klapper et al., 2002; SanMartin et al., 2001). All panels are confocal images with C and F showing the red and green channels merged. dei transcripts in somatic (arrowhead, A,C) and visceral (arrow, C) mesoderm cells were detected in non ß-Gal-expressing FCs (red nuclei; B,C), which were therefore putative FCMs. gol transcripts in somatic (bent arrow, D,F) and visceral (arrowhead, D,F) mesoderm did not overlap with ß-Gal-expressing FCs (E,F). A similar situation was detected in visceral mesoderm: ß-Gal-expressing nuclei (arrow, E) were not observed in gol-expressing cells (arrowhead, F). (G-I) Confocal images of wild-type embryos at mid-stage 12 immunostained for Kr (G) and Ase (H); the corresponding merged image is shown in I. Ase was detected transiently in Kr-positive FCs in close proximity to Kr, suggesting that Ase was expressed in FCs immediately after the progenitor cell had divided (arrows). (J-O) Confocal images of rP298 embryos at late stage 12, showing immunoreactivity to Ubx (J), Trn (M) and ß-Gal (K,N). The corresponding merged images are shown in L,O. The yellow color (bent arrow, L) indicates expression of Ubx in FC. Trn was expressed in a punctate pattern in close proximity to nuclei expressing the rP298 reporter (arrowheads, O), suggesting that FCs express Trn. (P,R) nidogen transcripts detected by fluorescent in situ hybridization in a late stage 11 embryo (green, arrows in P) tightly surrounded ß-Gal expressing FCs (arrows, Q), as shown in the merged image (R).

View larger version (67K): [in a new window]   Fig. 5. Embryonic phenotype of a subset of genes from the screen reveal specific morphological defects. Lateral views (A-C) of late stage 16 embryos of the genotypes wild-type (A), ast1/Df(2R)ast4 (B) and trn25.4/In(3LR)C190 (C) stained with an anti-Mhc antibody (see Materials and methods for details). The same symbols are used in both A and B to designate the same muscles. A diagramatic representation of visible muscles is included within each panel, with missing muscles colored red and muscles showing morphological abnormalities colored blue. Loss of ast (B) function leads to muscle-specific losses, as exemplified by loss of muscle LL1 and DO4. Mutations in trn (C) specifically blocked normal muscle morphogenesis, without interfering with the initial specification. This is illustrated by two complete sets of lateral transversal muscles showing aberrant morphologies (1 to 4 indicate LT1-4, and 5 indicates DT1).

View larger version (88K): [in a new window]   Fig. 6. phyl is expressed in FCs and both loss-of-function and overexpression cause specific muscle defects. (A-F) Confocal images of rP298 embryos in which phyl expression has been detected by fluorescent in situ hybridization (A,D; green), and FCs have been detected using the ß-Gal reporter (B,E; red). Both channels are shown merged in C and F. phyl expression in the somatic (arrowheads; C,F) and visceral (bent arrow; F) mesoderm closely followed the ß-Gal expression (C,F), indicating that both signals originated from FCs. Based on this level of analysis, phyl is expressed only in a subset of FCs. Phenotypic analysis of phyl mutant embryos and embryos overexpressing phyl revealed specific morphological defects in the muscle pattern. Lateral views of anti-Mhc-stained late stage 16 embryos (G,I,K) and ventrolateral views of anti-Krstained late stage 12 embryos (H,J,L) of the genotypes wildtype (G,H), phyl2/Df(2R)Trix (I,J) and twi-Gal4; Dmef2-Gal4 driving UAS-phyl (K,L). The same symbols are used to designate the same muscles in G-L. A diagrammatic representation is included with muscle loss indicated in red and morphological abnormalites indicated in blue. Null mutations in phyl reiterated some defects found in ast mutant embryos, such as losses of the LL1 (bracket, I) and DO4 (black arrow, I) muscles. In addition to muscle losses, the final muscle morphology in these embryos was compromised. Overexpression of phyl throughout the mesoderm lead to specific morphological problems (K). The LL1 and DO4 muscles were usually present in these embryos, but LT4 (arrowheads, K) frequently showed an abnormal shape. These muscles contained more than one nucleus, indicating that a fusion block was not responsible for the observed defect. Similar problems were found in the DT1 (bent arrow, K) and SBM (not shown) muscles. Loss of phyl function interfered with Kr expression in the LL1 muscle FC. The LL1 FC was absent in some hemisegments (black bent arrow, J) or showed reduced Kr expression in others (asterisk, J), whereas other FCs, such as LT2-4 and VA1, were present. These data suggest that the muscle losses detected stem from defects in initiation or maintenance of muscle FC determinants such as Kr. Conversely, phyl overexpression in the mesoderm (L) did not reproducibly affect Kr expression in LT4 FCs and, therefore, the morphological defects found in the final LT4 muscle must stem from a later interference of Phyl during the morphogenetic process.