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doi: 10.1242/10.1242/dev.00323

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distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila

B. Starling Emerald^1,*,, Jennifer Curtiss^2,, Marek Mlodzik² and Stephen M. Cohen^1,

¹ Developmental Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
² Department of Molecular Cell and Developmental Biology, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York NY 10029, USA
^* Present address: Institute of Molecular and Cell Biology, 30 Medical Drive, 117609 Singapore

View larger version (58K): [in a new window]   Fig. 1. Expression of Dan and Danr induces leg to antenna transformation. (A) Cuticle preparation showing wild-type tarsal segments and claws. (B) Scanning EM of the distal leg of a DllGal4 EPg3-220 fly. Note the absence of claws and presence of two aristae (arrow). Note that each claw produced an arista when Dan was expressed. In contrast to the leg, the antenna disc is not separated into A and P compartments by a lineage restriction until larval stages. The distalmost elements of A and P origin probably merge in antenna to make a single arista, which they cannot do in leg. (C,D) Distal legs from dppGal4 UAS-dan and dppGal4 UAS-danr animals. One claw was replaced by an arista in each case (arrows). (E) Wild-type antenna.

View larger version (43K): [in a new window]   Fig. 2. EPg J3-220 and EPg 35635 target the dan and danr genes, which encode related pipsqueak-motif-containing proteins. (A) (Top) the genomic region containing the dan and danr genes. The insertion points of EPg J3-220 and EPg 35635 (green triangles) lie 45 kb apart. Predicted genes in the region are shown in blue. (Middle) Red arrowheads indicate the positions of the primer pairs used for genomic PCR to determine the breakpoints of excision mutations; broken red lines between each primer pair indicate the predicted wild-type sizes of the genomic PCR fragments. (Bottom) Boxes show the extent of the genomic regions deleted in the dan danrex56 and danrex35 alleles. (B) Alignment of the Dan and Danr sequences showing regions of similarity. Note that the two proteins are most similar at the N terminus. The amino acid change in danems3 is indicated in red above the alignment. (C) Alignment of pipsqueak motifs from Dan, Danr, Drosophila Pipsqueak, and the transposases Drosophila Pogo and human CENP-B. Asterisks below the alignment indicate identical residues; colons and periods indicate conserved residues (ClustalW). The arrow indicates the residue mutated in danems3.

View larger version (98K): [in a new window]   Fig. 3. Dan and Danr expression. (A) Eye-antenna disc showing Dan protein expression. (B) Dan (green) and Danr (red) expression in the antenna region of an eye-antenna disc. (C) Dan expression in the leg disc. A small group of cells in the location of sense organ precursors are labeled late in development. (D) Dan and Hth (red) expression in the antenna. (E) Dan and Dll (blue) expression in the antenna. (F) Dan and Cut (purple) in the antenna. An optical cross-section across the region indicated by the arrow is shown below. (G) Schematic representation of the expression domains of Hth, Dll, Cut, Dan/Danr and the inferred domain of spineless (SS) expression with reference to antenna segments.

View larger version (103K): [in a new window]   Fig. 4. Regulation of Dan by Hth and Dll. (A) Dan expression in hthc1 mutant clones. (Boxed region) The three channels are shown separately at right. The clone was marked by the absence of GFP (green) and Hth protein (red). (B) Dan expression in DllSA1 mutant clones. The three channels are shown separately on the right of the region indicated by an arrowhead. The clone was marked by the absence of GFP (green) and Dll protein (red). Other Dll mutant clones sorted out towards the edge of the Dan domain (arrows). (C) Ectopic expression of Dan (blue) in the distal leg when Hth was misexpressed (Hth expression was marked by co-expression of GFP. Hth-expressing cells sort out from the distal region by this stage (see Wu and Cohen, 1999). Dan expression shown alone in the inset. (D) Ectopic expression (arrow) of Dan (green) in the proximal leg [overlapping Hth (red)], when Dll was misexpressed (Dll not shown).

View larger version (102K): [in a new window]   Fig. 5. Regulation of Dan by ss and Cut. (A) Dll and Dan expression in a spinelessaristapedia mutant antenna. Note the partial loss of Dan expression. (B) Loss of Dan expression in a spinelessaristapedia mutant antenna correlates with ectopic Antp expression (red). (C) Regions of a wing and leg disc. Ectopic expression of ss in clones of Gal4-expressing cells (labeled by co-expression of GFP, shown in red) induced ectopic Dan expression (green). (D) Ectopic ss expression in the antenna under ptcGAL4 control repressed Cut (purple) and caused ectopic expression of Dan (green). Arrow and arrowhead show reduced Cut expression where ss was expressed. (E) Dan expression in cut145 mutant clones (marked by the absence of ß-gal, red). Boxed area: Dan and Dan + Dll expression shown at higher magnification at right. Arrow indicates mutant clone in which Dan was not ectopically expressed proximally.

View larger version (61K): [in a new window]   Fig. 6. Dan acts downstream of ss. (A,B) Cuticle preparation of the antenna from a ss mutant. The arista is transformed to tarsus. (B is a higher magnification of A.) (C) Cuticle preparation of the antenna from a ss mutant expressing Dan under ptcGal4 control. Much of the distal arista is normal in morphology (compare with Fig. 1F). (D) ss mutant eye-antenna disc expressing Dan under ptcGal4 control. Dan (green) was repressed distally where Antp (red) was ectopically expressed (compare with Fig. 3A,B; Fig. 5F). Note that ectopic Dan (in the ptcGal4 expressing cells, blue) did not repress Antp expression.

View larger version (111K): [in a new window]   Fig. 7. dan danr mutant phenotypes. (A) Wild-type antenna. (B,C) Antennae from danrex35 and danr danex56 homozygous mutants. Arrowheads indicate reduced third antennal segments with ectopic bristles. Arrow indicates ectopic bristles on the enlarged basal capsule of the arista. (D) Wild-type arista: basal capsule. (E) Basal capsule of the arista from an eyFlp; FRT82B danrex35/FRT82B M+ armlacZ animal. Note the two-segmented appearance and the bracted bristles (arrow). (F) Basal capsule from an eyFlp; FRT82B dan danrex56/FRT82B M armlacZ animal. Note the clear segmentation of the basal capsule and the bracted bristles (arrow). (G,H) Antenna region of eye-antenna discs carrying danrex35 or dan danrex56 homozygous mutant clones (arrows). Clones were labeled by the absence of ß-gal protein (blue). Danr protein is shown in red; Dan protein in green. (G) Note upregulation of Dan in the Danr mutant clone. (H) Dan and Danr were both absent.

View larger version (50K): [in a new window]   Fig. 8. dan mutant phenotype. (A) Antenna from a danrems3 homozygous mutant. Note the single ectopic bristle in the third segment (arrow). (B) Antenna from a fly expressing dan double stranded RNA under DllGal4 control. Arrows indicate ectopic bristles. (C) Eye-antenna disc from a larva expressing dan double stranded RNA under dppGal4 control (green). Dan protein (red) was reduced. Danr protein (blue) was unaffected. Dan and Danr expression in the boxed area is shown separately on the right.

View larger version (81K): [in a new window]   Fig. 9. Genetic interaction between ss, Dll, dan and danr. (A) Basal capsule of spineless114.4 heterozygous arista shows a mild defect (compare with Fig. 7D). (B) spineless114.4/danrex35 double heterozygote. (C) spineless114.4/dan danrex56 double heterozygote. Note the two-segmented appearance and bracted bristles. (D) DllSA1/+ heterozygote. The basal capsule appears nearly normal. (E) DllSA1/+ danrex35/+ double heterozygote. (F) DllSA1/+ dan danrex56/+ double heterozygote. Stout ectopic bristles were formed on the basal capsule. (G) spineless114.4 dan danrex56/dan danrex56 antenna. Note the more extensive transformation of arista to tarsus.

View larger version (12K): [in a new window]   Fig. 10. Genetic regulatory hierarchy in the antenna. Summary of the regulatory relationships controlling antenna identity. spineless plays a central role in specification of distal antenna by repression of Antp and induction of Dan and Danr expression. Dan and Danr can direct distal antenna development when misexpressed in distal leg, and can override the effects of ectopic Antp in the antenna.