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First published online March 6, 2009
doi: 10.1242/10.1242/dev.032383

Development 136, 1137-1145 (2009)
Published by The Company of Biologists 2009
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The co-regulator dNAB interacts with Brinker to eliminate cells with reduced Dpp signaling

Oren Ziv1,*, Yaron Suissa1,*, Hadar Neuman1, Tama Dinur1, Peter Geuking2, Christa Rhiner3, Marta Portela3, Fidel Lolo3, Eduardo Moreno3,{dagger} and Offer Gerlitz1,{dagger}

1 Developmental Biology and Cancer Research, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
2 Institut fur Molekularbiologie, Universitat Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
3 Spanish National Cancer Institute (CNIO), Melchor Fernandez Almagro 3, E-28029 Madrid, Spain.


Figure 1
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  		Fig. 1. dnab is a Dpp target gene expressed in the Drosophila wing pouch. (A) Wing imaginal disc (anterior to the left, dorsal up, in all figures) showing the activity of the S149 Gal4 enhancer-trap insertion as revealed by UAS-GFP (green). (B) In situ hybridization with an RNA probe derived from the dnab ORF. (C,D) dNAB and Brk expression pattern in second and third instar larvae. Double staining of wing discs with antibodies directed against Brk (red) and dNAB (green) shows no overlap in second instar (C) and a slight overlap in third instar (D) larvae. (E-J) Dpp signaling positively regulates dnab expression. (E,F) dnab-lacZ (red) is ectopically expressed in clones overexpressing TkvQ235D (green, arrowheads); in all figures, overexpression clones (marked with GFP, green) are derived from activation of a Flp-able actin promoter (unless stated otherwise). Dad (G,H) and Brk (I,J) overexpression (using the hh-Gal4 driver, green) downregulates the expression of the dnab-lacZ reporter (red).

 

Figure 2
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  		Fig. 2. dNAB overexpression represses Dpp target genes, but is not required for Dpp-dependent growth and patterning in the Drosophila wing disc. (A-C) dNAB-overexpressing clone (A, green) represses Sal expression as revealed by immunostaining (B, red). (C) Merge of A and B. (D-F) hh-Gal4 driver was used to overexpress dnab in the posterior compartment (D, green). vg expression, as visualized by means of the vg-lacZ reporter, is abolished by dnab overexpression (E, red). (F) Merge of D and E. (G-O) In dNAB loss-of-function clones (G,J,M), marked by the loss of GFP (arrowheads), the expression of the Dpp target genes omb (H, red), sal (K, red) and vg (N, red) is unaffected. (I,L,O) Merges.

 

Figure 3
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  		Fig. 3. dNAB overexpression induces apoptosis through the JNK signaling pathway. (A-D) dNAB-overexpressing clones are apoptotically eliminated from the Drosophila wing disc in a lateral-to-medial pattern. Clones overexpressing dNAB (green) 24 (A) and 48 (C) hours after induction. (B,D) Immunodetection of activated Caspase 3 (red) as a marker of apoptosis. (E-H) dNAB-dependent cell elimination is associated with, and caused by, activation of the JNK pathway. Clones overexpressing dNAB (E, green, arrowheads) upregulate puc-lacZ reporter expression (F, blue, arrowheads). Non-autonomous JNK and Caspase 3 activation is occasionally seen around the border of dNAB-overexpressing clones (B,D,F). Co-expression of Puc with dNAB (G, green) abolishes Caspase 3 activation (H, red).

 

Figure 4
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  		Fig. 4. The cell death-promoting activity of dNAB is Brk-dependent and positioned downstream of brk transcriptional regulation. (A-C) dNAB requires Brk to induce cell death. Overexpression of dNAB in the posterior compartment using the hh-Gal4 driver (marked by GFP, green, A) leads to apoptosis as visualized by activated Caspase 3 (red, B). Caspase 3 activation induced by dNAB is abolished in the large brk loss-of-function clone marked by the lack of arm-lacZ expression (blue, C, outlined by the dashed line). (D-I) dNAB acts downstream of the Dpp receptor complex. Drosophila wing imaginal discs overexpressing dnab (D,G), tkvQ235D (E,H) or both (F,I) using the sd-Gal4 wing pouch driver (marked by GFP, green, D-F). Apoptosis is detected by staining for active Caspase 3 (red, G-I). (J-L) dNAB activates Caspase 3 without inducing brk expression. A dNAB-overexpressing clone (J, green) showing Caspase 3 activation (J,K, red). brk expression is visualized by brk-lacZ staining (J-L, blue).

 

Figure 5
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  		Fig. 5. dNAB binds Brk via its NCD2 domain. Brk binds to dNAB derivatives that contain the NCD2 domain. (A,B) dNAB binds Brk via its NCD2 domain in a GST pull-down assay. (A) GST-Brk full-length (GST-Brk FL) fusion protein was incubated with either Gro or with dNAB derivative proteins (35S-labeled, in vitro translated). Following washing, retained proteins were separated by SDS-PAGE and exposed for autoradiography. (B) GST-dNAB fusion proteins were incubated with 35S-labeled full-length Brk and processed as above. (C) Schematic representation of the various dNAB peptides fused to GST.

 

Figure 6
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  		Fig. 6. dNAB interacts with Brk to eliminate cells with reduced Dpp signaling and is required for dmyc-induced cell competition. (A-E) dNAB loss-of-function decreases the elimination of Dad-overexpressing clones. (A-D) dNAB loss-of-function clones overexpressing Dad (A, green) visualized 72 hours after induction are significantly more abundant in the pouch region compared with control clones (C, green). Activated Caspase 3 levels in control clones (D, red) are higher than in dNAB loss-of-function clones (B, red). (E) Bar chart showing a 2-fold increase (P<0.005) in the average number of dNAB loss-of-function clones overexpressing Dad (left column, blue, n=51) that survived in the wing pouch region compared with wild-type control clones (right column, red, n=54). (F-H) dNAB enhances the killing activity of Brk in the wing pouch region (outlined by dashed line). The C765-Gal4 driver was used to drive ubiquitous (mild) expression of brk and dnab in the wing disc. (F) Wing disc overexpressing Brk shows mild Caspase 3 activation (red) in the wing pouch region. (G) Wing disc overexpressing dNAB shows strong Caspase 3 activation (red) at the periphery around the wing pouch. (H) Wing disc overexpressing both Brk and dNAB shows dramatic enhancement of Caspase 3 activation (red) in the pouch region. The delineation of the pouch is based on the natural folds of the disc seen in bright-field microscopy. All discs were photographed under the same magnification (x200). Discs that co-express dnab and brk (H) are much smaller than those that express either gene alone (F,G). (I-L) Stability of dNAB protein in brk-overexpressing clones (generated by Flp-able abx-ubx promoter). (I,J) dNAB protein levels in the wing pouch, as revealed by immunostaining (red), are not affected in GFP-marked brk-overexpressing clones (arrowhead, green) 30 hours after induction. (K,L) Sal protein levels, as revealed by immunostaining (red), are diminished in brk-overexpressing clones (arrowhead, green) 30 hours after induction. (M-P) Brk and dNAB are required for dMyc-induced cell competition. (M-O) GFP-marked clones of wild-type cells (green) generated in a tub>dmyc genetic background and monitored for survival 72 hours after induction. (M) Most clones have disappeared from the wing pouch, and only a few persist in the periphery (n=50). (N,O) When expressing a UAS-RNAi construct that knocks down the expression of either brk (N) or dnab (O), many clones persist in the wing pouch (n=50). (P) Bar chart showing the percentage of the wing pouch area occupied by GFP-marked wild-type cells recovered in M-O. To measure the percentage of wild-type cells that survived, each wing pouch was subdivided into four quadrants and the surviving GFP-marked clones were fitted into the quadrants. Discs were analyzed either by computer imaging or by hand and were assigned to one of four groups according to the percentage of quadrants occupied: 0-10, 25, 50 or 75%.

 

Figure 7
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  		Fig. 7. Differential effects of the Brk co-repressors dNAB and Gro on JNK activation and cell survival. (A-C) Clones overexpressing Gro (marked with GFP, green, A) are not eliminated from the Drosophila wing disc, and do not show Caspase 3 activation (red, B). (C) Merge of A and B. (D-I) hh-Gal4 driver was used to overexpress dnab (D-F) or gro (G-I) in the posterior wing compartment (marked by GFP, green, D,G). Overexpression of dNAB activates both Caspase 3 (red, E) and the JNK pathway (puc-lacZ, blue, F). Gro overexpression activates neither Caspase 3 (red, H) nor the JNK pathway (puc-lacZ, blue, I).

 

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© The Company of Biologists Ltd 2009