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First published online 12 January 2005
doi: 10.1242/dev.01610

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Specification of individual Slouch muscle progenitors in Drosophila requires sequential Wingless signaling

Program in Developmental Biology, Weill Graduate School of Medical Sciences at Cornell University and Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA

View larger version (76K): [in a new window]   Fig. 1. Wingless is required for proper development of Slouch-expressing founder cells (FCs). (A) Schematic showing the temporal progression of Slouch-positive FC development from early (left) to late (right) stages of embryogenesis. Brackets indicate position of the Wg-expressing epidermal cells. Slouch is first expressed in a single progenitor cell during early stage 11 (5 hours AEL) of embryonic development; this cell divides to give rise to two FCs (purple), which together form cI located posterior to Slouch-expressing CNS cells (black). During late stage 11 (6 hours AEL), two additional Slouch-positive progenitors appear and divide sequentially to form four FCs that make up cII (green). At stage 12, a single progenitor arises dorsally and divides to give rise to cIII (red). Slouch expression is maintained in a subset of these founder cells that give rise, in the final muscle pattern, to muscle VT1 (25 from cI), VA2 (27 from cII) and DT1 (18 from III). In the figure, all muscles formed from these three clusters, as well as the ventral adult progenitor that comes from cII, are shown in color. Remaining gray muscles arise from non-Slouch-expressing FCs. (B-E) Ventral views of stage 11 embryos are shown; black arrows mark the midline, black arrowheads mark cI, white arrowheads mark cII. Abdominal segments 5-8 are shown; posterior is right. (B) High magnification of a wild-type embryo. cII is in line with the Slouch-expressing CNS cells, while cI is posterior to cII. (C) High magnification of a wgCX4 embryo. All mesodermal Slouch expression is absent, indicating that Wg is necessary for the formation of these clusters. Slouch-expressing cells in the CNS remain. (D) High magnification of a wgCX4, twiGAL4>UASarmS10, wgIG22 embryo shows normal patterning of all Slouch-expressing FCs. The ability of activated Armadillo to support normal patterning of both cI and cII suggests that the Wg pathway is sufficient to pattern these mesodermal clusters. (E) High magnification of a CyO, wglacZ embryo. Wg-expressing cells (brown, white arrowheads) and Slouch-expressing cells (black) are shown. The ectodermal Wg stripe specifically overlaid Slouch cII.

View larger version (60K): [in a new window]   Fig. 2. Slouch-expressing cells in cluster II require more Wingless than Slouch cluster I. (A-D) In this and all subsequent figures ventral views of late stage 11 abdominal segments 5-8 are shown. Insets depict one representative hemisegment at a higher magnification. Black arrowheads mark cI; white arrowheads mark cII. Black arrows denote the midline in panels; black arrows in A'-D' and white arrows in A''-D'' indicate Slouch-expressing CNS cells. (A) Wild-type embryo shows stereotypic repeating pattern of Slouch-positive clusters. (A') Immunocytochemistry of a single hemisegment reveals that cII (white arrowhead) aligns with the CNS, while cI (black arrowhead) localizes just posterior to cII. (A'') Confocal micrograph of a wild-type hemisegment; in cII (white arrowhead) Kr (red/Cy3) and Slouch (green/FITC) co-localize (yellow), while in cI (black arrowhead) Slouch (green/FITC) alone is expressed. Slouch is also expressed in the CNS (white arrow). Kr is also expressed in the CNS. Arrowheads and colors are maintained for all figures. (B) wgPE6 embryo at the nonpermissive temperature (25°C) lost Slouch expression. (B') Immunocytochemistry reveals an example in which both cII and cI are missing. (B'') Confocal micrograph of a single hemisegment shows an example in which Slouch staining is absent from the position where cII is normally found, but is present for cI. Slouch expression does not overlap with Kr. The identity of these Kr-positive cells is unknown. (C) wgPE6 at the permissive temperature (18°C) showed expression of cI and cII in some hemisegments. (C') Immunocytochemistry reveals an example where both cII (white arrowhead) and cI (black arrowhead) are present. (C'') Confocal micrograph shows another example in which Slouch staining is absent from the normal cII position, and Slouch does not co-localize with Kr in cI (black arrowhead). The identity of these Kr-positive cells is unknown. (D) twiGAL4; twiGAL4>UASNTcf embryos in which dominant-negative Tcf is expressed pan-mesodermally; this construct specifically repressed expression of cII and did not affect cI. (D') Immunocytochemistry shows that cII is missing but cI (black arrowhead) is present. (D'') Confocal micrograph of a single hemisegment shows that Slouch staining is absent from its normal position of cII, and Slouch expression does not overlap with Kr in cI (black arrowhead). (E) Quantification of Slouch cluster loss in Wg pathway partial loss-of-function embryos. Graphs show percentage of hemisegments in which cI (blue) or cII (red) are present. n, number of hemisegments counted.

View larger version (65K): [in a new window]   Fig. 3. Gain of function in the Wingless pathway specifically increases the size of Slouch cluster II. Ventral views of late stage 11 embryos were stained with Slouch antibody; ventral views are shown and anterior is left. cI (black arrowhead) and cII (white arrowhead) are indicated in both panel and insets. Black arrows denote the midline in panels and Slouch-expressing CNS cells in the insets. Insets show one hemisegment for each condition. The effect of different levels of Wg signaling were assayed in embryos of the following genotypes: (A) wild-type, (B) twiGAL4>UASwgE, (C) twiGAL4>UASarms10, (D) dAPC2d40, (E) twiGAL4;twiGAL4>UASwgE and (F) twiGAL4;Dmef2GAL4>UASwgE. As shown in B-F, increased Wg signaling led to an increase in cII size (white arrowhead) to 5-15 cells, mode=12. cI size (black arrowhead) was unaffected. Four cells constitute cII in wild-type; two cells make up cI. Higher Slouch expression was detected in the visceral mesoderm in F. We detect displacement of cI towards the midline in these experiments. This effect may be due to the GAL4 drivers used to manipulate Wg signaling. (G) Quantification of cluster expansion in Wg pathway gain-of-function experiments. Graphs show percentage of hemisegments that show cluster expansion, cII (red) and cI (blue). **, conditions where cI was expanded from two cells; n, number of hemisegments counted. The mode number of cells increased in cII under gain-of-function conditions was 12 for each condition.

View larger version (66K): [in a new window]   Fig. 4. Wingless acts sequentially to specify Slouch clusters I and II. (A-F) Ventral views of late Stage 11 embryos stained with Slouch; anterior is left. cI (black arrowhead) and cII (white arrowhead) are indicated in both panel and insets. Black arrows denote the midline in panels and Slouch-expressing CNS cells in insets. (A) Loss of hh function causes a loss of cII in most hemisegments but does not effect cI expression as strongly. (B) hh embryos in which wg is now expressed in the mesoderm reveals that Hh does not have a specific input to cII. cII and cI are specified normally. (C) Embryos carrying the hypomorphic allele wgIL114 raised at 18°C for 15 hours show Slouch expression in both cI and cII. (D) Embryos carrying the hypomorphic allele wgIL114 raised at 25°C for 8 hours show complete loss of mesodermal Slouch expression, although Slouch continues to be expressed in the CNS as in wgCX4 embryos. (E) Embryos carrying the hypomorphic allele wgIL114 raised at 18°C for 13 hours and shifted to 25°C for 2 hours show partial loss of Slouch expression in cII. (F) Embryos carrying the hypomorphic allele wgIL114 raised at 18°C for 12 hours and 25°C for 3 hours show complete loss of Slouch cII and no loss of cI. (G) Quantification of Slouch cluster loss when Wg levels are manipulated by hh alleles and temperature shifts. Graphs show percentage of hemisegments in which cI (blue) or cII (red) are present. **, conditions in which cI and cII appeared in the same hemisegment; n, number of hemisegments counted.

View larger version (60K): [in a new window]   Fig. 5. Wingless sets up a region competent to express Slouch and is required later to specify the fate of cluster II. (A) Late stage 11 wgCX4 mutant embryos fail to maintain Twist at high levels. (B) Ectopic Twist expression in wgCX4 mutant embryos maintains Twist expression through stage 11. (C) Late stage 11 wgCX4 mutant embryo showed loss of all mesodermal Slouch, although some expression remained in the CNS. cI (black arrowhead) and cII (white arrowhead) are indicated in both the panel and the inset. Black arrows denote the midline in the panel; black arrows in C',D' and white arrows in C'',D'' show Slouch-expressing CNS cells in the insets. (C) Immunocytochemical staining of a single hemisegment shows that both cII and cI are missing. (C'') Confocal micrographs of embryos stained with antibodies to Slouch (green/FITC) and Kr (red/Cy3). No co-localization of Kr and Slouch is detected in the mesoderm. White arrow indicates Slouch CNS expression. (D) wgCX4,twiGAL4>UAStwi embryos showed rescue of mesodermal Slouch expression in positions corresponding to cI. (D') Immunocytochemical staining shows that cII is absent but cI is present (black arrowhead). (D'') Confocal micrograph of a single hemisegment shows that Slouch staining is absent from the normal position of cII, and Slouch (green/FITC) does not co-localize with Kr (red/Cy3), supporting the identity of this cluster as cI (black arrowhead). We note that the amount of Twist maintained in these cells after specification is detrimental. While Twist is necessary for the specification of the Slouch clusters, maintained elevated expression can lead to repression of these clusters (V.T.C. and M.K.B., unpublished). (B) Quantification of Slouch cluster rescue in wg mutant embryos that overexpress twist. Graphs show percentage of hemisegments in which cI (blue) or cII (red) are present under conditions listed at the bottom of the graph. n, number of hemisegments counted.

View larger version (19K): [in a new window]   Fig. 6. A model for Wingless specification of Slouch clusters I and II. An illustration of the two-step process by which Wg specifies Slouch cI and cII in the developing mesoderm. Wg provides two contributions for Slouch cII: one through Twist and a second that is Twist independent. The Twist-independent Slouch activation is likely to involve transcriptional regulation by the Wg transcriptional effector Pangolin/Tcf. cI needs Wg only to generate a myogenic competency domain through Twist.