Fig. S1. Temperature shifts reveal that wg is required in the hindgut only prior to stage 10. At stage 9, Wg expression, detected with a-Wg antibody, is observed throughout much of the hindgut primordium (A, arrowhead). By stage 11, Wg is expressed only at the extreme anterior of the hindgut, in the region of the everting renal tubules (B, black arrowhead), and is no longer detected in the central portion of the hindgut (B, white arrowhead). In a wg null mutant (wg^7L74), a-Crb staining reveals a smaller, shorter hindgut (C); consistent with a previous report (Skaer and Martinez Arias, 1992), the wg null hindgut consists of only 151±13 (n=5) epithelial cells (data not shown). To ask whether wg might be required for the cell rearrangement that drives hindgut elongation, we carried out temperature shift experiments: a temperature-sensitive wg mutant shifted to the restrictive temperature during stage 10 (before hindgut elongation occurs) has an almost completely elongated hindgut (D). Hindgut lengths after shift-up and shift-down are shown in E.

The temperature shift experiments were carried out as follows. wg^IL114, a temperature sensitive allele with permissive and restrictive temperatures of 17ºC and 25ºC, respectively, was used. For upshifts, embryos were collected for 2.6 hours on a yeasted Nitex mesh screen on an apple juice agar plate at 17ºC, aged at 17ºC, then shifted by transferring the mesh to a 25ºC plate; development was allowed to continue at 25ºC to stages 16/17, after which embryos were dechorionated, fixed, and stained with a-Crb. Downshift experiments were similar, withembryo collection at 25ºC for 1 hour, followed by transfer to and further development at 17ºC. As embryogenesis was determined to occur 2.6 times more rapidly at 25ºC than at 17ºC, this factor was used to normalize developmental times at 17ºC to those at 25ºC. Hindgut lengths in wg mutant embryos (solid squares and circles, identified by characteristic segmentation and head defects) were measured using Axiovision software. Open squares and circles represent populations containing both wild-type and mutant embryos. Numbers measured (n) are above data points.

Fig. S2. JAK/STAT signaling in the hindgut visceral mesoderm is not required for elongation of the hindgut epithelium. A section through the small intestine at stage 16 shows that the a-Stat92E antibody labels cells of both the hindgut epithelium (HE) and the hindgut visceral mesoderm (HVM) (A; HE and HVM separated by a dotted line). The hindgut epithelium in both wild-type and upd^os1A embryos is surrounded by visceral mesoderm, as shown by staining with a-Con (B,C). The twi hindgut lacks visceral mesoderm, as shown by staining with a-Con (D); nevertheless, when compared with wild type (E), the hindgut in twi embryos elongates almost normally, as shown by staining with a-Crb (F).

Skaer, H. and Martinez Arias, A. (1992). The wingless product is required for cell proliferation in the Malpighian tubule anlage of

Drosophila melanogaster. Development 116, 745-754.