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doi: 10.1242/dev.00972

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Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways(mili^Alk) as receptor for Jeb signalling

Christiana Stute¹, Kristina Schimmelpfeng^2,*, Renate Renkawitz-Pohl¹, Ruth H. Palmer³ and Anne Holz^4,

¹ Philipps-Universität Marburg, Fachbereich Biologie, Zoologie/Entwicklungsbiologie, Karl-von-Frisch-Strasse, 35039 Marburg, Germany
² Institut für Neuro- und Verhaltensbiologie, Westfälische Wilhelms-Universität Münster, Badestrasse 9, 48149 Münster, Germany
³ Umeå Center for Molecular Pathogenesis, Building 6L, Umeå University, S-90187, Sweden
⁴ Institut für Allgemeine und Spezielle Zoologie, Allgemeine Zoologie und Entwicklungsbiologie, Justus-Liebig-Universität Gießen, Stephanstraße 24, 35390 Gießen, Germany

View larger version (99K): [in a new window]   Fig. 1. Notch signalling is involved in founder cell determination in the visceral mesoderm. The visceral mesoderm is visualised by Fas3 (red in A,B,C,M), ß-galactosidase expression from bap-lacZ (red in H,J) and founder cells are marked by rP298-lacZ expression (green in A,C,M brown in K,L). (A) Visceral mesoderm (vm) of a stage 11 wild-type embryo. (B) In Fas3 staining of Notch55e11 embryos, it seems as if most cells of the vm are converted into founder cells as indicated by the stronger Fas3 expression and the more rectangular shape. (C) In stage 11 DeltaB2 mutants, the number of founder cells also seems to be increased in the visceral mesoderm and fewer fcms are visible (compare C with A). The clusters of rP298-lacZ-positive cells ventral to the visceral mesoderm belong to the somatic mesoderm (sm). (D-F) sns in situ hybridisation comparing the wild type (D) where sns is expressed in two bands with the sns expression in N55e11 (E) and DlB2 (F) embryos where it is reduced. This reduction is more severe in the ventral band of the fcms of the somatic mesoderm but also visible in the dorsal band of the fcms of the visceral mesoderm. (G,H) Notch expression in the visceral mesoderm in stage 11 embryos. (G) Notch is expressed ubiquitously at the membrane of all cells of the visceral mesoderm. (H) Some cells seem to have a lower expression level of Notch (arrowhead in G). These cells also express bap-lacZ, which at this stage is mainly restricted to the fcms (H). (I,J) In contrast to the N expression Delta is expressed in the cells adjacent to the bap-lacZ positive cell clusters of the visceral mesoderm. (K) rP298-lacZ expression in the gut of a stage 14 wild-type embryo. (L) In gut preparations of stage 14 embryos with ectopic expression of UAS-Nintra in the visceral mesoderm, the number of rP298-lacZ positive founder cells of the circular visceral musculature is decreased. Founder cells of the longitudinal visceral muscles are not affected and migrate normally in anterior direction (arrow in L). (M) In stage 11 embryos overexpressing a dominant-negative form of Notch (UAS-dnN) with a bap-GAL4 driverline, the number of rP298-lacZ positive founder cells is increased compared with the wild type (A) and also some cells which are not marked by lacZ expression exhibit a stronger Fas3 expression, which is characteristic for the founder cells in the visceral mesoderm.

View larger version (16K): [in a new window]   Fig. 2. Ectopic expression of Dl, Nintra and N+Dl in the visceral mesoderm results in increased lethality. Lethality and survival rate of the progeny of the UAS-GAL4 crosses (n1000). All crosses were carried out with a bap-GAL4 line (asterisk indicates crossed to bap-GAL4) carrying rP298-lacZ as a founder cell marker.

View larger version (102K): [in a new window]   Fig. 3. An EMS screening approach identifies novel mutations, which display severe defects during visceral mesoderm development. The development of the visceral mesoderm is visualised by Fas3 expression. (A-C) Wild-type development at stages 11 (A), 12 (B) and stage 15 (C, arrowhead shows the midgut encircled by the visceral mesoderm). (D-F) Examples for the different subgroups of mutations are shown. (D) In embryos of the first subgroup (G1), no visceral mesoderm can be detected at stage 11 (arrowhead). (E) In the second group (G2), embryos develop the initial patches of the circular visceral muscles, but these patches subsequently fail to form a continuous band (arrowhead). (F) In the third group (G3), the continuous band is formed but the cells of the visceral mesoderm do not migrate dorsally and ventrally to encircle the entire midgut (arrowhead in F compare with arrowhead in C).

View larger version (100K): [in a new window]   Fig. 4. In jebweli and miliAlk mutants the founder cells of the circular visceral musculature are not determined. The development of the visceral mesoderm in wild-type (WT), jebweli and miliAlk mutants as visualised by Fas3 expression (red in A-I). (A-F) Stage 11, (G-I) stage 15-16 and (J-L) stage 14. The nuclei of the founder cells are marked by rP298-lacZ expression (green in D-F, brown in J-L). In jebweli and miliAlk mutants at stage 11, the more columnar-shaped founder cells are missing. Only cells that display the more globular shape of the fusion-competent myoblasts (fcm) are present (B,C,E,F). Additionally, the founder cell marker rP298-lacZ is absent in the visceral mesoderm in these mutants (E,F). The rP298-lacZ-positive cells, which can be observed over the fcms of the visceral mesoderm in D-F belong to the somatic mesoderm. At later stages, there are no signs of visceral musculature in either mutant (stage 16, arrowheads in H-I). (J-L) The founder cells of the somatic mesoderm are not affected in these mutants as visualised by rP298-lacZ expression (stage 14, K,L compare with J).

View larger version (61K): [in a new window]   Fig. 6. The cells of the visceral mesoderm of jebweli and miliAlk mutants become incorporated in the somatic musculature. Ventrolateral (A,B,C) muscles of stage 16 embryos as visualised by ß3tubulin expression (green in A-F). Cells of the visceral mesoderm are marked by bap-lacZ expression (red in D-F). Compared with the wild type (A) the somatic muscles in both jebweli and miliAlk mutants are thinner and show long thin projections (B,C; arrow in B). Several unfused myoblasts are visible (arrow in C). (D-F) In wild-type embryos, no bap-lacZ expression is found in the somatic mesoderm during all stages of development (stage 14, D). By contrast, jebweli and miliAlk mutant embryos exhibit bap-lacZ expression in the lateral muscles of the somatic mesoderm (stage 14, E,F).

View larger version (83K): [in a new window]   Fig. 5. The fusion-competent myoblasts of the somatic mesoderm do not differentiate in jebweli and miliAlk mutants. (A-C) sns in situ hybridisation of stage 11 embryos marks fcms in the visceral and somatic mesoderm. In wild-type embryos, sns is expressed in two bands along the entire length of the embryo (A). These bands are connected in a ladder-like pattern, where the ventral band represents the fcms of the somatic mesoderm (arrow in A), and the dorsal band consists of the fcms of the visceral mesoderm (arrowhead in A). In jebweli (B) and miliAlk (C) mutant embryos, the fcms of the visceral mesoderm and some cells that connect the two bands are present (arrowheads); however, the ventral band of fcms of the somatic mesoderm shows no sns expression. (D-F) Alk (green) and Fas3 expression (red) in stage 11 wild-type embryos. In addition to expression in the visceral mesoderm, Alk is also transiently expressed in some cells in the neuroectoderm and in patches in the somatic mesoderm (arrows in D,F). (G-I) Lmd expression of stage 12 embryos. In the wild type, Lmd is expressed in two bands in the fcms of the somatic and visceral mesoderm (G). In jebweli (H) and miliAlk (I) mutants, expression in both mesodermal cell types can be observed.

View larger version (115K): [in a new window]   Fig. 7. Alk protein is mislocalised in miliAlk mutant embryos. Stage 11 embryos stained with Fas3 (B,E,H) to mark all cells of the visceral mesoderm or Alk antibodies (green in A,D,G). The stainings are merged in C,F,I. In wild-type embryos (A-C), Alk is expressed in both the founder cells (f) and fusion-competent myoblasts (fcms) of the visceral mesoderm and localises at the cell membrane. (A-C). In jeb mutant embryos (D-F), Alk is also expressed at the membranes of all visceral cells. In miliAlk mutants, the majority of the protein does not localise at the cell membranes but instead can be found in the cytoplasm (G-I). (J-M) In stage 11 wild-type embryos (J,K) Jeb (green in J-M) is taken up by the Alk-expressing cells of the visceral mesoderm (red in K,M), whereas in miliAlk mutants (L,M) where the Alk protein is present but non-functional there is no co-localisation of the two proteins.

View larger version (40K): [in a new window]   Fig. 9. The mode of action of Mili/Alk-mediated Jeb signalling during the determination and differentiation within the visceral and somatic mesoderm. At stage 10 (S 10) ventromedial somatic precursor cells start to express jelly belly (jeb, red cells) and secrete Jeb protein (small red dots) (Weiss et al., 2001). Some of these cells (red with green crescents) also express miliAlk (green) and all visceral precursor cells express miliAlk and bagpipe (bap) at this time. We conclude that Jeb signalling, especially the RTK pathway activation in visceral founder cells, depends on the level of Jeb protein reaching these cells. This activation leads in stage 11 (S 11) in the visceral mesoderm (vm) to the expression of dumbfounded/kin of irre (duf/kirre, blue) in the visceral founder cells (f), whereas the fusion-competent myoblasts (fcms) without an active pathway are characterised by sticks and stones (sns) expression. We further suggest that initial jeb expression in the somatic mesoderm (sm) is maintained mainly in lame duck/myoblast incompetent/gleeful (lmd/minc/glee)-expressing fcms responding on RTK pathway activation with differentiation into sns-expressing fcms. Nothing is known so far about the role of Jeb and Mili/Alk signalling in the ectoderm (ecto) and fat body (fb).

View larger version (112K): [in a new window]   Fig. 8. Ectopic expression of UAS-Alk and UAS-jeb in the mesoderm gives rise to different phenotypes. Wild-type embryos are shown in A,D,G and L. (B,E,H) UAS-jeb is ectopically expressed with a twi-GAL4 driverline. (C,F,I,J,K,M) UAS-Alk is ectopically expressed with a twi-GAL4 (C,F,I,M) or bap-GAL4 driver line (J,K). Founder cells (f) are marked by rP298-lacZ expression (green in A,B,J,K). (D-F) Fusion-competent myoblasts (fcms) are visualised by sns in situ hybridisation. (B) Ectopic expression of UAS-jeb converts all cells of the visceral mesoderm to founder cells. (E) sns expression is absent in these cells of the visceral mesoderm. (H) The somatic mesoderm shows no defects, as indicated by ß3tubulin antibody staining. (C) No visceral founder cells can be detected by Fas3 staining of stage 11 embryos in which UAS-Alk is ectopically expressed in the entire mesoderm. (F) sns in situ hybridisation indicates that through the overexpression of UAS-Alk the fcms of the somatic mesoderm are missing in stage 11 and only the band of the fcms of the visceral mesoderm remains. (I) The dorsal and ventral somatic muscles in stage 16 show a fusion defect phenotype with thin projections (arrow) and unfused myoblasts (arrowhead) as indicated by ß3tubulin staining. When UAS-Alk is expressed only in the visceral mesoderm with a bap-GAL4 driverline, which also carries rP298-lacZ as founder cell marker, the founder cells of the visceral mesoderm are still present and seem to be doubled in number (J,K). In contrast to a single row in the wild type (A), a second row of founder cells is present in these stage 11 embryos. The bands of both halves of the embryo are shown (J,K). In the wild type, jeb is expressed in a continuous band in the mesoderm (L), whereas upon overexpression of UAS-Alk in the entire mesoderm the signal is reduced (M).