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doi: 10.1242/10.1242/dev.00202

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Localized JAK/STAT signaling is required for oriented cell rearrangement in a tubular epithelium

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095-1606, USA

View larger version (58K): [in a new window]   Fig. 1. Signaling molecules expressed in the hindgut. The hindgut epithelium (pink) elongates during stages 11 to 16, changing from a short, wide tube to a long, narrow tube (A); the cell rearrangement that is a major contributor to this process is shown in B. The three domains of the hindgut (SI, small intestine; LI, large intestine; RE, rectum), and the genes encoding signaling molecules expressed therein, are indicated in (C); anterior is towards the left, dorsal is upwards and the gray region indicates boundary cells. The points of attachment of the renal tubules are indicated near the anterior of the SI by `V'. Hindgut morphology, outlined by apical staining with anti-Crb (red) and nuclear staining with anti-ß-Gal (green), is shown in stage 16 wild-type (D), drm (E), bowl (F) and (G) lin embryos, all carrying the bynapro enhancer trap that drives expression of lacZ in the nuclei of the hindgut. (H-K) upd expression, detected by in situ hybridization in stage 13 embryos, is seen only in the anterior hindgut (small intestine) of wild-type (H, black arrowhead), is missing from drm (I) and bowl (J) embryos (white arrowheads), and is expanded throughout most of the hindgut in lin embryos (K, black arrowhead).

View larger version (61K): [in a new window]   Fig. 2. upd and JAK/STAT pathway components are expressed at the anterior of the elongating hindgut. The components of the Drosophila JAK/STAT signaling pathway (ligand Upd, receptor Dome, JAK Hopscotch and STAT) are shown in A and discussed in text. Beginning at stage 9, upd expression is detected in a narrow ring of cells at the anterior of the developing hindgut (at junction with posterior midgut) (B), while expression of dome and Stat92E in this domain is weaker and more diffuse (E,H). By stage 11, expression of upd in the prospective small intestine is strong (C), and increased expression of dome and Stat92E is observed in domains overlapping with, but more extensive than that of upd (F,I, respectively). These expression patterns for upd, dome and Stat92E are maintained throughout embryogenesis (D,GJ, respectively; stage 14). Expression in the anterior hindgut, detected by in situ hybridization, is indicated by brackets (B,C,E,F,H,I) and arrowheads (D,G,J).

View larger version (109K): [in a new window]   Fig. 3. Spatially localized JAK/STAT signaling is required for hindgut elongation and cell rearrangement. Compared with wild type (A), the hindgut is shorter and wider in updos1A (B), hopm-z- (C) and Stat92Em-z- (D) embryos. As seen in transverse section, compared with wild-type (E), there are more cells in the hindgut circumference of updos1A (F), hopm-z- (G) and Stat92Em-z- (H) embryos. The hindgut is also shorter and wider when bynGAL4 is used to drive uniform hindgut expression of UAS constructs that activate JAK/STAT signaling, namely UAS-upd (J) and UAS-hopTML (K); the defect is more severe when both UAS-hopTML and UAS-Stat92E are driven together (L). Inhibition of JAK/STAT signaling by bynGAL4 driven expression of the dominant negative UAS-domeCYT3.2 also results in a shorter, wider hindgut (I); transverse sections of embryos of these genotypes similarly reveal a greater than normal number of cells in the hindgut circumference (M-P). All embryos shown are at stage 16; wholemounts are stained with anti-Crb and sections are of the large intestine. Averages of total number of hindgut cells, cells in hindgut circumference, and hindgut lengths observed in the loss-of-function mutants and in bynGAL4 embryos expressing various JAK/STAT components are summarized in(Q.

View larger version (79K): [in a new window]   Fig. 4. upd has only minimal effect on hindgut patterning. Spatially localized patterns of gene expression in distinct domains of the stage 14 hindgut and posterior midgut are summarized in A; points of renal tubule attachment are indicated by `V'. Gene expression patterns in A are shown in wholemounts of wild-type (wt), upd loss-of-function (updos1A) and upd gain-of-function (byn-GAL4; UAS-upd) embryos (B). In the absence of upd activity (updos1A), expression of Wg at the most anterior of the small intestine is reduced (white arrowhead), while expression patterns of hh and Ser in small intestine, En in large intestine, dri in boundary cells and hh, Ser and Wg in rectum are not affected. When upd is expressed uniformly throughout the hindgut (byn-GAL4; UAS-upd), expression of Wg in the anterior small intestine is slightly upregulated (black arrowhead), while all other spatially localized gene expression patterns examined appear normal. Expression of hh, Ser and dri was detected by in situ hybridization, that of Wg and En by antibody staining.

View larger version (83K): [in a new window]   Fig. 5. Anteriorly localized upd expression is required for hindgut elongation. When upd is expressed throughout the hindgut (using bynGAL4) in an updos1A mutant background, the hindgut is shorter and more deformed (A) and has as many or more cells in transverse section (B) than that seen in updos1A embryos (compare with Fig. 3B,F). When upd is expressed only in the small intestine (using drmGAL4) in an updos1A mutant background, the length of the hindgut and number of cells in transverse section is almost indistinguishable from that seen in wild-type (C,D; compare with Fig. 3A,E). The drm3/drm6 hindgut is short and wide (E) and has an excessive number of cells in its circumference (F). When upd is expressed only in the small intestine (using drmGAL4) in a drm3/drm6 mutant background, the hindgut is more elongated (G), and has fewer cells in its circumference (H) than seen in drm3/drm6. This phenotype is similar to the rescue of the drm3/drm6 hindgut that is seen when drmGAL4 is used to drive UAS-drm (R. B. Green, PhD thesis, UCLA, 2002). Hindgut apical surfaces are outlined by anti-Crb staining; embryos are stage 16; GAL4-driven expression patterns are shown on the right of each pair of panels. Length measurements and circumference counts are summarized in I.

View larger version (120K): [in a new window]   Fig. 6. upd activates the JAK/STAT pathway in cells posterior to the small intestine. Sagittal sections through the hindgut at stages 11 (A) and 12 (B) show outlines of the everting renal tubules (black dashes), small intestine primordium (red dots), large intestine primordium (yellow dots) and rectum primordium (orange dots). Estimates of the lengths of the small intestine (SI) and the large intestine (LI) are shown based on analysis of the sets of serial sections to which A and B belong. Scale bar: 50 µm. Levels of Stat92E protein and mRNA are barely detectable in updos1A embryos (C and E, respectively), but dramatically upregulated in bynGAL4; UAS-upd embryos (D and F, respectively); Stat92E protein is also upregulated in bynGAL4, UAS-hopTML embryos (L). Stat92E protein is detected along a significant region of the length of the anterior hindgut, as observed in both whole-mount (G,I) and sagittally sectioned embryos (H,J) at stages 11 (G,H) and 12 (I,J). The hindgut is outlined by black dots (H,J), and red dots indicate the observed anteroposterior gradient of Stat92E protein (H,J). Consistent with this, double staining for both upd mRNA (in situ hybridization) and Stat92E protein (antibody staining) shows expression of Stat92E in an anterior-to-posterior gradient (brown, posterior extent marked with open arrowheads) that extends posterior to the domain of upd expression (blue, posterior extent marked with black arrowheads) (K). Expression of UAS-upd with drmGAL4 in an upd mutant background rescues the upd hindgut elongation defect (M), while expression of UAS-hopTML with drmGAL4 in an upd mutant background fails to rescue (N); Crb is used to outline hindgut morphology. In C-G,I, the lumen of the hindgut is indicated with dots; the anterior limit of the small intestine is indicated with a larger dot.

View larger version (8K): [in a new window]   Fig. 7. Model for hindgut elongation by Upd/JAK/STAT signaling. Expression of upd in the small intestine (SI) under the control of Drm and Lin results in the formation of a gradient of Upd protein in cells posterior to the small intestine. In response to signaling by Upd, a gradient of Stat92E is established; this leads to the orientation of cell rearrangement in the hindgut.