Volume 5, Issue 3, Pages 581-587 (March 2000) The JAK/STAT Signaling Pathway Is Required for the Initial Choice of Sexual Identity in Drosophila melanogaster  Timothy M. Jinks, Alexandros D. Polydorides, Gretchen Calhoun, Paul Schedl  Molecular Cell  Volume 5, Issue 3, Pages 581-587 (March 2000) DOI: 10.1016/S1097-2765(00)80451-7

Figure 1 Sxl-Pe Constructs and Their Patterns of Expression in Early Embryos (Left) structure of the D. melanogaster Sxl-Pe region and the Sxl-Pe transgene constructs. Sxl-Pe extends ∼3 kb upstream of the transcription start site. In the Sxl-Pe constructs, Sxl-Pe3.0kb, Sxl-Pe1.7kb, Sxl-Pe1.4kb, and Sxl-Pe0.4kb sequences extending upstream of +32 are fused to LacZ. In Sxl-PeGOF, four tandem copies of the 72 bp conserved region from −284 to −212 are inserted at the end of the smallest sex-specific promoter, Sxl-Pe0.4kb. Sxl-PeLOF has a deletion of the multimerized sequence. Sequences in D. melanogaster Sxl-Pe conserved in D. subobscura and D. virilis are indicated by white blocks. Triangles show the position of three potential STAT sites. The sequences of these sites closely match the optimal in vitro D-STAT site as well as the D-STAT sites in the eve promoter (Hou et al. 1996; Yan et al. 1996). (Right) Sxl-Pe is expressed specifically in female embryos, while the expression of the Sxl-PeGOF is not sex specific. (A) Expression of Sxl-Pe3.0kb is high throughout the soma of 2X/2A embryos. This promoter is active in only half of the embryos. (B) Although Sxl-Pe0.4kb is active in all female embryos, LacZ expression is less than Sxl-Pe3.0kb. (C) Sxl-PeGOF expression in early female embryos. Note that staining is low at the poles. (D) Sxl-PeGOF expression in early male embryos. Note the stripes. (E) Expression of Sxl-PeGOF in stage 9–10 female embryos is high in most somatic cells. (F) Sxl-PeGOF in stage 9–10 male embryos is lower and is spatially restricted. Embryos in (A)–(F) were generated from fly stocks homozygous for the respective transgene. The embryos shown in the figure are representative of all embryos of the same sex within each population. Molecular Cell 2000 5, 581-587DOI: (10.1016/S1097-2765(00)80451-7)

Figure 2 Sxl-Pe Is Activated by X-Linked Transcription Factors and Is Dependent on Gene Dosage Sxl-Pe transgene constructs are used to monitor the effects of mutations on the regulation of expression of Sxl-Pe. (A) Female embryos heterozygous for sis-a− express the Sxl-Pe3.0kb reporter at levels that are essentially indistinguishable from wild-type female embryos. In this experiment, half of the embryo population is stained, and they are presumed to correspond to 2X/2A embryos. (B) Female embryos trans-heterozygous for sis-a− and the deficiency Df(1)N19 express Sxl-Pe3.0kb at significantly lower levels than their heterozygous siblings and display regional variation of expression. Half the embryos express β-galactosidase, and these fall into two equal classes. One class is weakly stained like the example shown here while the other resembles the sis-a− control. (C) Females heterozygous for sis-b− express the Sxl-Pe3.0kb reporter at levels roughly equivalent to that of wild-type females. (D) Female embryos trans-heterozygous for sis-b− and Df(1)N19 express Sxl-Pe3.0kb at a lower level than heterozygous siblings. As with sis-a−, half the embryos express β-galactosidase, and these fall into two equal classes. One class is weakly stained like the embryo shown here while the other resembles the sis-b− control. (E) Sxl-Pe1.4kb female embryos are not sensitive to a reduction in the dose of genes uncovered by Df(1)N19. (F) The expression of Sxl-PeGOF in males requires a gene(s) deleted in Df(1)N19. (G) The gain-of-function activity of Sxl-PeGOF is abolished in males hemizygous for upd−. The crosses to generate these embryos (shown as female × male) were wild type × sis−;Sxl-Pe3.0kb/Sxl-Pe3.0kb for (A) and (C), Df(1)N19/FM7 × sis−;Sxl-Pe3.0kb/Sxl-Pe3.0kb for (B) and (D), Df(1)N19/FM7 × Sxl-Pe1.4kb/Sxl-Pe1.4kb for (E), Df(1)N19/FM7 × Sxl-PeGOF/Sxl-PeGOF for (F), and upd−/FM7 × Sxl-PeGOF/Sxl-PeGOF for (G). Molecular Cell 2000 5, 581-587DOI: (10.1016/S1097-2765(00)80451-7)

Figure 4 Sxl Expression Is Dependent on D-STAT Activity in the Medial Regions of Female Embryos SXL protein detected by antibody staining. (A and B) SXL protein is expressed uniformly in female soma at the blastoderm stage and at later stages. In female embryos from mrl− germline clones, SXL protein expression is not uniform at the blastoderm stage (C and E), and the autoregulatory feedback loop is not properly initiated (D and F). X-gal staining shows that Sxl-Pe3.0kb is active throughout the soma in female progeny of wild-type mothers (G), but only in the anterior of mrl− embryos (H). Sxl-PeGOF is active at high levels in wild-type 2X (I) and 1X (K) embryos but shows little activity in 2X (J) and 1X (L) mrl− embryos. Molecular Cell 2000 5, 581-587DOI: (10.1016/S1097-2765(00)80451-7)

Figure 3 The Activity of the JAK Pathway Affects the Response of Sxl to the X/A Signal Either wild-type (A, C, and E) or hopTum-1/FM7 (B, D, and F) females were crossed to P[Sxl-Pe]/P[Sxl-Pe] males. The Sxl-Pe reporters were: (A and B) Pe0.4kb, (C and D) Sxl-Pe1.4kb, and (E and F) Sxl-Pe3.0kb. To visualize the increased activity of the different promoters from the hop mutant mothers, embryos carrying the Sxl-Pe1.4kb and Sxl-Pe3.0kb promoters were stained for 1.5 hr rather than 5 hr as in the other experiments. All of the stained embryos from each collection showed essentially the same level of staining. Molecular Cell 2000 5, 581-587DOI: (10.1016/S1097-2765(00)80451-7)