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Dev Biol
2011 Feb 15;3502:382-92. doi: 10.1016/j.ydbio.2010.12.003.
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A conserved function of the chromatin ATPase Kismet in the regulation of hedgehog expression.
Terriente-Félix A
,
Molnar C
,
Gómez-Skarmeta JL
,
de Celis JF
.
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The development of the Drosophila melanogaster wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior-posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. In this work we identified Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior-posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. We also show that knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression.
Fig. 1. Loss-of-function phenotypes of kis mosaic wings. (A) Wild type wing showing the longitudinal veins (L2–L5). (B–B′) Wings of salEPv-Gal4 / +; kis1 FRT40A / M(2)z FRT40A; UAS-FLP / + (B) and 638-Gal4 / +; kis1 FRT40A / M(2)z FRT40A; UAS-FLP / + (B′) genotype. (C–C′) Wings of salEPv-Gal4 / +; al dp kis172A3FRT40A / M(2)z FRT40A; UAS-FLP / + (C) and 638-Gal4 / +; al dp kis172A3 FRT40A / M(2)z FRT40A; UAS-FLP / + (C′) genotype. (D–D′) Wings of salEPv-Gal4 / +; al dp kis165A1 FRT40A / M(2)z FRT40; UAS-FLP / + (D) and 638-Gal4 / +; al dp kis165A1FRT40A / M(2)z FRT40A; UAS-FLP / + (D′) genotype. (E–E′) Wings of salEPv-Gal4 / +; al dp kis61C FRT40A / M(2)z FRT40A; UAS-FLP / + (E) and 638-Gal4 / +; al dp kis61C FRT40A / M(2)z FRT40A; UAS-FLP / + (E′) genotype. (F–F′) Wings of salEPv-Gal4 / +; al dp kis59C3 FRT40A / M(2)z FRT40A; UAS-FLP / + (F) and 638-Gal4 / +; al dp kis59C3 FRT40A / M(2)z FRT40A; UAS-FLP / + (F′) genotype. In all cases, kis clones were induced in the domain of salEPv-Gal4 expression (B–F) or in the entire wing (B′–F′). The phenotype of mutant kis mosaic wings consists in the differentiation of ectopic veins (B–F′), the loss of L2 stretches (B′, C′, D′, E′ and F′) and the increase in the distance between the L3 and L4 veins (B–F′).
Fig. 2. Phenotype of kis loss-of-function clones in the wing. (A–C) Effects of the expression of interference RNA against kis (ikis). Expression of ikis in the wing blade and hinge (nub-Gal4/UAS-ikis; A) or in the dorsal compartment (ap-Gal4/UAS-ikis; B) cause the formation of ectopic longitudinal veins. (C–C′) Expression of Kis (red) in ap-Gal4 UAS-GFP/UAS-ikis third instar wing disc. Kis (red in C–C′) is not detected in the dorsal compartment (labelled in green in C). (D–E) Example of two kis1 clones (enclosed by black lines) in the dorsal side of the L3 vein, causing non-autonomous formation of L3 vein by wild type cells. (F–F′) Loss of Kis expression (red) in kis1 clones located in the wing blade. Clones are labelled by the expression of GFP (green), and were induced in hsFLP1.22 act-Gal4 UAS-GFP; kis1FRT40A/tubGal80 FRT40A larvae. Kis is not detected in kis1mutant cells. (G) Mosaic wing in which the posterior compartment is formed by kis61C mutant cells generated in kis61C FRT40A/M(2)z FRT40A; hh-Gal4/UAS-FLP flies. The posterior compartment is smaller and differentiates ectopic veins. (H–H′) High magnification of the dorsal (H) and ventral (H′) side of the L3–L4 intervein showing the formation of ectopic veins by kis1 mutant cells (labelled with forked). (I–K) High magnification of the L3–L4 intervein in a wild type wing (I), and in wings carrying kis clones occupying a small part of the intervein (kis29D4; J) and a large fraction of the same intervein (kis1; K). In both cases there is an increase in the distance between the L3 and L4 veins.
Fig. 3. Expression of Hh-target genes in kis mutant clones. (A–B′) Expression of En (red) in a third instar wild type wing disc (A) and in discs bearing kis1 clones (B–B′; labelled in green). The white line in (A) labels the extent of En expression in the anterior compartment. (C–D′) Expression of Ptc (red) in a third instar wild type wing disc (C) and in discs bearing kis1 clones (D–D′; labelled in green). (E–F′) Expression of Iro in a third instar wild type wing disc (E) and in discs bearing kis1 clones (F–F′; labelled in green). En, Ptc and Iro are ectopically expressed in kis mutant cells in a cell autonomous manner (arrowheads in B′, D′ and F′). Iro is also expressed in the surrounding wild type cells (arrowhead in F′). (G–H′) Expression of Smo in a third instar wild type wing disc (G) and in discs bearing kis1 clones (H–H′; labelled in green). Smo is not ectopically expressed in anterior kis mutant cells.
Fig. 4. Expression of hh in kis mutant cells. (A) Expression of βGal (red) in the posterior compartment of hh-lacZP30 / + third instar wing disc. (B–B′) Ectopic expression of hh-lacZP30 (red) in kis1 clones located close to the antero-posterior compartment boundary (arrowheads in B′). hh-lacZP30 is not expressed in kis mutant cells located in more anterior regions of the anterior compartment (arrow in B). (C) Expression of βGal (red) in the posterior compartment of hh-lacZ / + third instar wing disc. (D) Expression of βGal (red) in the anterior compartment of kis61C / +; hh-lacZ / + third instar wing disc. hh-lacZ is now also detected at low levels in an anterior domain close to the A/P boundary (bracketed). (E–E′) Expression of βGal (red) at higher than background levels in the anterior compartment of hh-lacZ / + third instar wing discs bearing kis1 clones (labelled by the absence of green). (F–F″) Cell autonomous de-repression of hh-lacZ in small kis mutant clones induced in hsFLP1.22 / +; kis61C FRT40A / tubGFP FRT40A; hh-lacZ / + larvae. In E–F′ LacZ is detected at higher levels in all kis mutant cells located close to the A/P compartment boundary. (G–I) Expression of hh mRNA in the posterior compartment of wild type discs (G) and de-repression of hh in anterior cells of kis1 homozygous (H) and kis1 / Df(2L)ED21 (I) mutant discs. The position of the A/P boundary is indicated by a black arrow in G–I.
Fig. 5. Interactions between Kis and Ci in the regulation of Hh-target gene expression. (A–L) Expression of En, Ptc and Iro proteins in kis clones induced 48–72 AEL in larvae of the following genotypes:
–
hsFLP1.22 actGal4 UAS-GFP; FRT40A / tubGal80 FRT40A; UAS-iCi / + (iCi: A, E and I).
–
hsFLP1.22 actGal4UAS-GFP; kis1FRT40A / tubGal80 FRT40A;UAS-iCi / + (iCi kis1: B, F and J).
–
hsFLP1.22 actGal4 UAS-GFP; FRT40A / tubGal80 FRT40A; UAS-ismo / + (ismo: C, G and K).
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hsFLP1.22 actGal4UAS-GFP; kis1FRT40A / tubGal80FRT40A;UAS-ismo / + (ismo kis1: D, H and L). In all cases, mutant clones were labelled by the expression of GFP (green). The expression of En (A–D and A′–D′), Ptc (E–H and E′–H′) and Iro (I–L and I′–L′) is shown in red. The red channels of A–L are shown in A′–L′, respectively. The reduction of Ci (iCi) causes loss of En (A–A′) and Ptc (E–E′) and cell-autonomous ectopic expression of Iro (I–I′). When kis is also mutated, the main difference is the strong non-autonomous expression of Ptc (F–F′) and Iro-C (J–J′). The reduction in Smo causes loss of En (C–C′), Ptc (G–G′) and Iro (K–K′). The same changes are observed in ismo cells mutant for kis1 (D–D′, H–H′ and L–L′).
Fig. 6. Suppression of kis effects by CiCell expression. Expression of En, Ptc and Iro proteins in clones induced 48–72 AEL in larvae of the following genotypes:
–
hsFLP1.22 actGal4 UAS-GFP; FRT40A / tubGal80 FRT40A;UAS-CiCell / + (< CiCell>: A, C and E).
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hsFLP1.22 actGal4 UAS-GFP; kis1FRT40A / tubGal80 FRT40A;UAS-CiCell / + (< CiCell> / kis1: B, D and F). In all cases, mutant clones were labelled by the expression of GFP (green). The expression of En (A and B), Ptc (C and D) and Iro (E–F) is shown in red. The red channels of A–F are shown in A′–F′, respectively. Ectopic expression of CiCell causes loss of En (A–A′), Ptc (C–C′) and Iro (E–E′). These phenotypes are not modified when kis is mutant in CiCell-expressing cells (B–B′ for En, D–D′ for Ptc and F–F′ for Iro). (G) Schematic representation of the effects on Hh target gene expression of Ci-Kis combinations as shown in Fig. 5, Fig. 6A–F. Inner circles represents the mutant clones, outer circles the cells surrounding the mutant clone, green implies gene expression and red gene repression.
Fig. 7. Expression Pax2 and Shh in stage 35 Xenopus tropicalis embryos injected with MOCHD8. (A) RT-PCR with primers from exons 10 and 14 reveals an extra band of 1127 bp (red arrow) in the embryos injected with 7.5 ng of MOCHD8 that is not observed in the control embryos (compare second and third lanes). (B–D) Pax2 expression in MOCHD8 morphant embryos. (B) Lateral view of the control un-injected side. (C) Lateral view of the MOCHD8-injected side. (D) Frontal view of morphant embryos with the injected side to the right. Note the dorsal expansion of the expression of Pax2 in the eye (red arrows), and the reduction of its expression in the pronephros and hindbrain (green and red arrowheads, respectively). The expression in the midbrain-hindbrain boundary (blue arrowhead) is unaffected. (E) Transverse section of the spinal cord showing Shh expression in embryos injected with 7.5 ng of MOCHD8. The injected side is to the right, and the control, uninjected side, to the left. Note the dorsal expansion of Shh expression in the injected side (right arrow).
Supplementary Figure 1. Expression levels of Ci, Smo and CiCell in mosaic wing discs. (A–A′) Expression of Smo (red) in ismo clones (green) induced in hsFLP1.22 actGal4 UAS-GFP;FRT40A / tubGal80 FRT40A; UAS-ismo / + larvae. (B–B′) Expression of Ci (red) in iCi clones (green) induced in hsFLP1.22 actGal4 UAS-GFP;FRT40A / tubGal80 FRT40A; UAS-iCi / + larvae. (C–C′) Expression of HA-CiCell (red) in UAS-CiCell clones (green) induced in hsFLP1.22 actGal4 UAS-GFP;FRT40A / tubGal80 FRT40A; UAS-CiCell / + larvae.
Supplementary Figure 2. Effects of kis over-expression in the wing. (A) Representation of the kis gene (taken from flybase) showing the intron-exon structure of the three mRNA (kis-RA, kis-RB and kis-RC) and the positions of the P-UAS elements (red triangles) used to drive kis expression in the wing disc (EP474, EP2473, EP2288 and EP2240). (B) Adult wing of 638-Gal4 / EP474 genotype. The wing is normal in size and pattern, and similar results were obtained in combinations of sal-Gal4 and the EP474, EP2473, EP2288 and EP2240 insertions. (C) In situ hybridization with a kis-RA / kis-RC specific probe in a third instar wing disc of sal-Gal4 / EP474 genotype. The expression of kis is detected at higher levels in the sal domain (enclosed by white lines). (D) Expression of kis-RA / kis-RC in a wild type third instar disc. (E) Expression of kis-RA / kis-RB / kis-RC in a third instar wing disc of sal-Gal4 / EP2288 genotype. Similar results were obtained in the sal-Gal4 / EP2473 and sal-Gal4 / EP2240 combinations.
