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doi: 10.1242/10.1242/dev.00527

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Splits ends is a tissue/promoter specific regulator of Wingless signaling

¹ Department of Molecular, Cellular and Developmental Biology, University of Michigan, Natural Science Building, Ann Arbor, MI 48109, USA
² Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA

View larger version (152K): [in a new window]   Fig. 1. spen dominantly suppresses a P[sev-wgts] eye bristle phenotype. Micrographs are SEMs of adult fly heads. (A,B) Flies contain the P[sev-wgts] transgene and are heterozygous for either the parental chromosome iso5A1 (A) or spen9C7 (B), both reared at 17.6°C. The spen heterozygotes display significant suppression of the P[sev-wgts] partial loss of interommatidial bristles. A similar suppression was also seen with spen14C2 (data not shown).

View larger version (75K): [in a new window]   Fig. 2. Spen protein is severely reduced in spen14C2 clones. (A-C) Confocal images of a third instar eye imaginal disc containing spen14C2 clones generated by ey-FLP. Clones were marked by the absence of nuclear GFP (A) and stained for Spen (B); the merged image is shown (C). Spen signal is predominantly nuclear (C) and is greatly reduced in spen14C2 clones.

View larger version (80K): [in a new window]   Fig. 3. spen is required for maximal Wg-dependent repression of proneural genes in the eye. (A-D) Confocal images of a P[sev-wg] pupal eye (3-6 hours after pupal formation–APF) containing spen3 clones generated by ey-FLP. Clones of spen3 were marked by the absence of GFP (A; clonal boundaries shown by the white lines in B-D) and were stained for Ac (B) and Wg (C); the merged image is shown (D). P[sev-wg] eyes have high levels of Wg behind the morphogenetic furrow (C) and low levels of Ac (B) outside the spen3 clones. Ac is derepressed in much of the clone (B; note that cells in and ahead of the MF are not competent to express Ac) but significant Ac repression still occurs inside the clone (arrows in B). Transgenic Wg levels are unaffected in spen3 clones (C). Similar Ac derepression and P[sev-wg] expression are also seen in spen14C2 clones (data not shown). Occasional non-autonomous derepression of Ac is observed adjacent to spen clones (B, arrowhead). (E,F) Confocal images of a pupal eye (3-6 hours APF) containing spen14C2 clones generated by ey-FLP. Clones of spen14C2 were marked by the absence of GFP (E) and were stained for Ac (F). Ac expression is elevated in spen14C2 clones (arrows in F; note that the laser intensity is lower than in B).

View larger version (87K): [in a new window]   Fig. 4. spen potentiates Wg-dependent repression of the morphogenetic furrow. (A-C) SEM images of adult eyes of wild-type (A), dppblk (B) and spenk07721, dppblk/spenk13624, dppblk (C) flies. (D-L) Confocal images of eye imaginal discs with the following genotypes: spenk07721-rev/spenk13624-rev (D,G,J), spenk07721-rev, dppblk/spenk13624-rev, dppblk (E,H,K) and spenk07721, dppblk/spenk13624, dppblk (F,I,L). The k07721-rev and k13624-rev alleles are homozygous viable revertants of the spen P-element alleles k07721 and k13624 resulting from precise excisions. Late third instar eyes (D-I) and early third instar eyes (J-L) are stained for Wg (D-F,J-L) and Elav (G-L). Ectopic Wg in dppblk eyes inhibits the furrow, thereby reducing Elav-positive photoreceptors (compare G with H) and resulting in a small eye phenotype (compare A with B). Reduced spen dosage in transheterozygotes of hypomorphic alleles increases the number of photoreceptors (I) and the adult eye size (C); the higher than normal Wg level in dppblk eyes (compare D with E and J with K) is unaffected in the dppblk, spen mutants at early third instar (L) and slightly reduced at late third instar (F). (M,N) Confocal images of third instar eye imaginal discs showing Elav (red) and either lacZ (M, green) or GFP (N, green). Clones of pygo10 (M) and spen14C2 (N) (marked by the absence of lacZ or GFP) were generated by ey-FLP. pygo clones at the edge of the eye block Wg-dependent furrow inhibition and produce ectopic photoreceptors (arrow in M). By contrast, ectopic photoreceptors do not form in spen clones at similar positions (arrow in N; spen14C2: n=16; spen3: n=11).

View larger version (144K): [in a new window]   Fig. 5. spen is required for Wg signaling in the wing imaginal disc. (A-C) Confocal images of third instar wing imaginal discs containing randomly generated spenXIE1796 clones (marked by the absence of GFP, boundary shown by white lines). Samples were stained for Sens (A) and Wg (B); the merged image is shown (C). Reduction or total lack of Sens (A) was observed in some spen clones (spen9C7: 23% and 26%, respectively, n=31; spen14C2: 50% and 6.7%, n=30; spen3: 27% and 0%, n=30; spenXIE1796: 30% and 8.6%, n=70), and Wg level is unaffected or slightly derepressed (B), consistent with attenuated Wg signaling in cells that lack spen. (D-G) Confocal images of third instar wing imaginal discs containing the transgenes P[en-Gal4] and P[UAS-spenDN]II stained for Sens (D,F), Wg (E) and Fz2 (DFz2 in figure) (G). Flies were reared at 18°C. Expression from P[UAS-spenDN] in the posterior compartment of the wing reduces (D) or eliminates (F) Sens expression (the posterior compartment is towards the right of the arrows in F and G; 44% are similar to D, 27% are more severe but some Sens remains and 29% are as in F, n=68) and derepresses Wg (E, 97%, n=31). By contrast, expression of Fz2 is either unaffected (compare arrowheads; G, 73%, n=45) or reduced throughout the posteior compartment (data not shown, 27%).

View larger version (127K): [in a new window]   Fig. 6. spen is required for Wg signaling in the leg imaginal disc. (A-I) Confocal images of third instar leg imaginal discs of wild type (A-C) and animals containing transgenes P[ptc-Gal4], P[dpp-lacZ] and P[UAS-spenDN]II (D-I) reared at 21°C. Samples were stained for Wg (A,D,G) and lacZ (B,E,H); merged images are shown (C,F,I). All panels are shown with the same magnification. Ventral (v) expression of Wg (A) restricts lacZ expression to the dorsal (d) half (B) in the wild type. Expression from P[UAS-spenDN] along a stripe overlapping both the wg and dpp domains either has no effect on the restriction of lacZ expression (D-F) or leads to derepression of lacZ in the ventral region (G-I; note the overlap of Wg and lacZ in I; 9% are similar to I, 45% have significant but incomplete derepression of lacZ, and 46% are similar to F, n=35). This derepression is consistent with a block in Wg signaling.

View larger version (154K): [in a new window]   Fig. 7. spenDN acts downstream of Arm stabilization. Micrographs are SEMs of adult fly heads from flies containing P[GMR-Gal4], P[GMR-arm*]F36 and either P[UAS-lacZ] (A) or P[UAS-spenDN]II (B) reared at 25°C. Expression of an activated form of Arm activates Wg signaling and produces an eye that is severely reduced in size and is suppressed by P[UAS-spenDN].

View larger version (99K): [in a new window]   Fig. 8. spen is not required for Wg signaling in the embryo. (A,D) Micrographs of cuticles of wild type (A) and embryos containing transgenes P[da-Gal4] and P[UAS-spenDN]III (D) reared at 25°C. Expression from P[UAS-spenDN] has variable effects on cuticle patterning, ranging from wild type (P[UAS-spenDN]II at 25°C, data not shown), moderate reduction of denticles (D) to complete disruption of cuticle formation (P[da-Gal4], P[UAS-spenDN]III containing embryos reared at 29°C, data not shown). (B,C,E,F) Confocal images of stage 11 embryos containing P[prd-Gal4] and either P[UAS-TCFDN] (25°C; B,C) or P[UAS-spenDN]III (29°C; E,F). Samples were stained for En (B,E) or Slp1 (C,F). En and Slp1 stripes remain wild type in spenDN-expressing embryos. (G-I) Confocal images of stage 13 wild-type embryos (G) and embryos containing transgenes P[twi-Gal4] and P[UAS-spenDN]II (H,I) reared at 25°C. Expression from P[UAS-spenDN] has variable effects on the Eve pericardial expression, ranging from wild type (H) to an increase in Eve-positive cells (I). Some embryos containing transgenes P[twi-Gal4], P[24B-Gal4] and P[UAS-spenDN]II reared at 25°C or 29°C also display disorganization of Eve-expressing cells or occasional gaps missing Eve expression, in addition to an overall increase in Eve-positive cells (data not shown). These effects are qualitatively different from a blockage of Wg signaling [ectopic denticles, loss of En (B) and Slp-1 (C) stripes and loss of Eve in pericardial cells].

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