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First published online 11 April 2007
doi: 10.1242/dev.003830

Development 134, 1845-1852 (2007)
Published by The Company of Biologists 2007
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Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development

Javier Terriente Félix1, Marta Magariños2 and Fernando J. Díaz-Benjumea1,*

1 Centro de Biología Molecular-C.S.I.C., Facultad de Biología, Universidad Autónoma-Cantoblanco, 28049 Madrid, Spain.
2 Dpto. Fisiología Animal, Facultad de Biología, Universidad Autónoma-Cantoblanco, 28049 Madrid, Spain.


Figure 1
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  		Fig. 1. Proximodistal domains of the Drosophila wing imaginal disc. (A,A') vg (green) and wg (red) expression patterns in wing discs of early (A) and late (A') third instar larvae. In the early third instar the inner ring (IR) of wg expression abuts on vg expression in the wing pouch, but in the late third instar the two domains are several cells apart. (B,C) Wing disc (B) and adult wing (C) showing the expression domains of genes involved in proximodistal patterning relevant to this work. vg (green), rn (blue) and nub (yellow) are expressed in concentric domains, and wg (red) is expressed in a stripe that defines the wing margin (not shown) and in two rings - the IR and the outer ring (OR). Bars in B represent the extension of the domains on the proximodistal axis. The color scheme in C shows the corresponding expression domains in the adult wing. (D) nubGal4/+; EP#13/+ wing. Note that the wing hinge (arrow) is deleted. (E) Molecular organization of nab. The domains NCD1 and NCD2 and the sites of insertion of the alleles used in this work are shown. (F-F'') Wing imaginal discs of second instar (F) and early (F') and late (F'') third instar larvae labeled with Nab (green, here shown as white in combination with blue), Wg (red) and Rn (ß-galactosidase, blue). The right-hand panel shows separately the different channels of a view on the z-axis. Note how from the initial situation in the early third instar (F'), nab expression abuts on wg expression in the IR. Scale bars show the relative sizes of the discs (white bars) and wings (black bars). Hereafter, imaginal discs are always shown in the same orientation: dorsal up and anterior to the left.

 

Figure 2
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  		Fig. 2. The Drosophila gene vestigial is necessary and sufficient to activate nab expression. (A) nab expression in a vg283b27r wing disc. Expression in the wing pouch is lost; only basal expression remains in the notum. (B,B') Clones of vg-expressing cells (green) showing expression of wg (red) and nab (blue): y w hsFLP122; Act5C>y+>Gal4 UASGFP/UASvg. Insets at the bottom show the individual channels of the selected areas in B and B'. (B) vg misexpression activates nab nonautonomously in a broader domain.

 

Figure 3
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  		Fig. 3. nab limits distal expression of wg in the inner ring. (A) nabSH143 FRT80 clones in the Drosophila wing pouch labeled by the loss of GFP (green) and stained with Wg antibody (red). wg is misexpressed in these clones (arrows). (B,C) nubGal4/UASGFP (B) and nubGal4/UASGFP; UASnab/+ (C) wing discs stained with Wg antibody (red). nab misexpression represses activation of wg expression by the IR enhancer. Arrows, IR; arrowheads, OR. (D,D') Clones of nab misexpression (green): y w hsFLP122; Act5C>y+>Gal4 UASGFP/+; UASnab/+. wg expression (red) in the IR is cell-autonomously repressed. D' shows the individual channels for the selected areas in D.

 

Figure 4
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  		Fig. 4. Nab represses the transcriptional activity of Rn. (A-E) Leg, wing and a high magnification of the wing hinge area of wild-type (A), rn5/rn20 (B), rnGal4/EP#13 (C), UASrn/+; rnGal4/EP#13 (D) and DllGal4/+; EP#13/+ (E) Drosophila. T1-5, tarsal segments. Arrows point to the sex combs at the distal end of the first tarsal segment (T1), and arrowheads point to the claw organs at the distal end of the leg. Note that the rn lack-of-function phenotypes (B) are identical to the nab misexpression phenotypes (C): tarsal segments (T2-T4) are lost or much reduced and the wing hinge is deleted. In D, the leg and wing phenotypes shown in C are partially rescued by overexpression of UASrn. Note that although nab is expressed in a broader domain in E than in C (from mid-tibia to the distal end of the leg) the phenotype is the same. (F,G) Wg antibody staining (red) in rnGal4/EP#13 (F) and UASrn/+; rnGal4 UASGFP/EP#13 (G) wing discs. Nab antibody staining is shown in blue and rn expression in green (GFP). Arrows, IR; arrowheads, OR. Note that in F, wg expression in the IR is lost. The simultaneous misexpression of Rn and Nab (G) partially rescues wg expression in the IR. In the right-hand panel, individual channels of a view in the z-axis are shown to indicate that in flies with this genotype, nab is co-expressed with wg in the IR.

 

Figure 5
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  		Fig. 5. Nab is a co-activator of Sqz in embryonic CNS development. (A,B) Ventral (A) and lateral (B) views of stage-17 Drosophila embryos showing nab (red) and sqz (green) expression patterns. (A',B') Magnified views of the segmental pattern showed in A,B. (C,C') Stage-17 embryo stained with antibodies to Ap (red) and Nab (green). Three cells in the ap cluster express nab (C', a magnified view of the T1 segments), one of them showing stronger expression. (D,D') First instar larvae stained for Ap (red), Nab (green) and FMRFa (blue). Only the Tv neuron, detected by anti-FMRFa staining, expresses nab in the ap cluster (D', a magnified view of the T1 segments). (E,E') nab first instar larvae showing expression of ap (red), dimm (green) and FMRFa (blue). More cells occupy the ap cluster (T3 on the right is out of focus), FMRFa expression is mostly lost and is present only in T3, and additional cells express dimm (E', a magnified view of the T1 segments). (F) The larval CNS. The green square indicates the area shown in D and E, the red square the area shown in G and G'. (G,G') Wild-type (G) and nab mutant (G') second instar CNS showing FMRFa expression. In all panels, arrows indicate the first (T1) thoracic segment and arrowheads the second (T2) and third (T3) thoracic segments. (H,I) Averages (x) and s.d. (d) of the number of cells that express ap and dimm (H) and FMRFa (I) in the T1 segments of wild-type, sqzlacZ and nabSH143 embryos; n, number of larvae scored; *, data from Allan et al. (Allan et al., 2005Go).

 

Figure 6
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  		Fig. 6. Drosophila Nab physically interacts with Rn and Sqz via their conserved C-terminal domains. (A) Results of the GST-Nab pull-down assays. GST-Nab pulls down intact [35S]Rn and [35S]Sqz proteins, indicating direct binding. Binding was greatly reduced when the Rn protein lacked its C-terminal domain ([35S]Rn{Delta}854). Each experiment was repeated three times with identical results. (B) Comparison of Rn and Sqz proteins showing the two conserved domains: the zinc-finger domain (black box) with 90% homology, and the C-terminal domain (gray box). (C) Amino acid alignment of the conserved C-terminal domains of Rn and Sqz. *, identical; (:) conserved substitution; (.) semi-conserved substitution. (D) Clones of rn{Delta}854 misexpression (green, Act5C>Gal4>UASGFP UASrn{Delta}854). wg expression (red) is activated throughout in the wing pouch (arrows). (E) Clones of rn misexpression (green, Act5C>Gal4>UASGFP UASrn) only activate wg expression (red) in the wing hinge (arrows) where Nab is not expressed, but not in the wing pouch (arrowheads).

 

Figure 7
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  		Fig. 7. Sqz represses Rn function in Drosophila wing and leg. (A) sqz expression in rn20 wing disc as monitored by in situ hybridization. The expression is strong in the center and fades off laterally. The proximal limit coincides with the distal-most fold that is the limit of vg expression. (B-D) Wing (B), leg (C) and wing disc (D) showing wg expression (red, D) in rnGal4/UASsqz UASGFP. The wing hinge is reduced and tarsal segments are deleted (B,C, black arrows). wg expression in the IR is much reduced (D, arrow) but expression in the OR is not affected (D, arrowhead; here partially out of focus).

 

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© The Company of Biologists Ltd 2007