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First published online 2 November 2005
doi: 10.1242/dev.02093

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Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing

¹ Department of Biology, Box 35 1800, University of Washington, Seattle, WA 98195, USA
² Drosophila Genetics and Epigenetics, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris, France

View larger version (111K): [in a new window]   Fig. 1. Knockdown of EcR leads to precocious BR-Z1 expression and sensory neuron differentiation in contrast to the effects of dominant negative EcR. (A-A'') C96-GAL4 driving UAS-GFP and UAS-IR-EcR in the margin of a wing disc from a late wandering larva. In A GFP identifies areas of C96-GAL4 expression; in A'' low levels of EcR are seen in the region of GAL4 expression. A' is the merged image. (B-B'') en-GAL4 driving UAS-IR-EcR reduces the levels of EcR in the posterior compartment of a wing disc from a wandering larva (B) and leads to precocious up-regulation of BR-Z1 (B''). B' is the merged image. (C-C'') Wing disc 2-3 hours APF with MS1096-GAL4 driving the dominant negative ecdysone receptor EcR-B1W650A. The EcR-B1 antibody also recognizes the dominant negative isoform. At 2-3 hours APF EcR-B1 is normally low, thus the label in C reflects the domain of expression of the dominant negative ecdysone receptor which blocks the up-regulation of BR-Z1 (C''). C' is the merged image. (D,D') Control disc 2 hours APF with C96-GAL4 driving UAS-GFP (D). Sensory neuron differentiation has begun in the margin as seen with 22C10 (D'). (E,E') C96-GAL4 driving UAS-GFP and UAS-IR-EcR. At 2 hours APF 22C10 labeling (E') shows advanced sensory neuron differentiation in the margin (arrows). (F,G) Sensilla on the third vein are also affected by loss of EcR function. 22C10 label shows the first axons (arrow) elongating in a 0 hour APF control (dpp-GAL4 driving UAS-GFP) disc (F). The GFP reporter shows the domain of dpp expression. (G) Axons differentiate prematurely (arrow) in a 0 hour APF disc with dpp-GAL4 driving UAS-IR-EcR. The schematic drawings indicate the morphology of the discs and the expression domain of the drivers used (stippled area). The dorsal/ventral boundary (margin) is indicated in magenta in discs from wandering larvae. In later stages the margin moves to the periphery as the disc elongates. All discs are oriented with anterior to the top and wing anlage to the right.

View larger version (118K): [in a new window]   Fig. 2. Effect of loss-of-function USP on the expression of proneural and neurogenic genes in the wing margin [usp2 clones in a Minute (RpS52) background]. Absence of GFP (green) marks usp2 tissue. (A,A') Wing disc from a late feeding larva. Expression of the ac-lacZ reporter is not affected by loss of USP function. (B,B') The neur reporter A101 in a disc from an early wandering larva is precociously expressed in the absence of USP function. (C,C') Senseless (SENS) is expressed precociously in the mutant clone in a disc from an early wandering larva. Note the accumulation of SENS in the mutant SOPs. In wild-type tissue low levels are detected anteriorly. (D) Diagram gives the orientation of the disc with the area depicted in A-C. Arrowheads indicate estimated intersection of the A/P boundary with the margin. A'-C' show the expression of the different markers.

View larger version (65K): [in a new window]   Fig. 3. sens and br-Z1, but not sc expression can by-pass repression of SOP formation in the wing margin by the unliganded EcR/USP complex. (A,A') Wing discs 0 hours APF with C96-GAL4 driving UAS-sc and UAS-GFP (A). sc misexpression does not by-pass EcR/USP repression. 22C10 expression (A') reveals only a slight advancement in sensory neuron differentiation. (B,B') Wing disc 0 hours APF with C96-GAL4 driving UAS-sens and UAS-GFP (B). 22C10 expression shows precocious differentiation of the sensory neurons (B'). (C,C') Control wing disc 2.5 hours APF with C96-GAL4 driving UAS-GFP. 22C10 is beginning to be expressed in the margin (C'). (D,D') Wing disc 2.5 hours APF with C96-GAL4 driving UAS-br-Z1 and UAS-GFP (D). Labeling with 22C10 shows the precociously differentiating neurons in the margin (D'). Note that their axons are projecting abnormally instead of following along the anterior margin. A differentiating sensory neuron is also seen in the posterior margin (arrow).

View larger version (88K): [in a new window]   Fig. 4. BR function is required for sens expression in the wing margin. (A,A') A large npr3 clone in a Minute (Rp5S) background. SENS protein (A') is not detected in the npr3 clone (GFP negative area, A) in the wing margin, but is expressed normally in the region along the third vein [see Murray et al. (Murray et al., 1984) for nomenclature of these sensilla]. Image is a composite of projections of the dorsal epithelium only. (B-F) Misexpression of br isoforms and their effect on SENS expression in the wing margin. (B) Normal pattern of SENS expression in control disc. (C) dpp-GAL4 driving UAS-br-Z1 leads to ectopic SENS expression and early SOP differentiation (arrow). (D) ddp-GAL4 driving UAS-br-Z2 leads to ectopic SENS expression (arrow). However SENS has not accumulated in the SOPs in the area of ectopic SENS expression. (E) Ectopic SENS is expressed in a wing disc where dpp-GAL4 drives UAS-br-Z3 but does not lead to precocious accumulation of SENS in the SOPs. (F) Misexpressing br-Z4 using the dpp-GAL4 driver does not induce ectopic SENS expression. Asterisks in all panels indicate the approximate axis of dpp-GAL4 expression.

View larger version (121K): [in a new window]   Fig. 5. In vitro culture in the absence of ecdysone reveals the step at which neurogenesis is blocked. (A) Disc from an early wandering A101 larva cultured in vitro for 24 hours in the presence of 20E shows the differentiation of the normal complement of neurons and outgrowth of their axons (22C10). (B) Disc from an early wandering larva cultured in vitro for 24 hours without ecdysone. Only the early born SOPs are visible with the A101 reporter line. Insets in A and B show higher magnification of the L3-2 region. (C-C'') Disc from an early wandering A101 larva cultured in vitro without ecdysone for 24 hours. The SOPs are recognizable with A101-ß-gal (C) but SENS fails to accumulate in them (C''). (C') merged image of A101 and SENS expression. (D-E) Changing pattern of SENS expression in the margin from two broad bands in the early wandering stage (D) to the accumulation in the SOPs by the mid-late wandering stage (E). (F) Block of SENS accumulation requires a functional ecdysone receptor: wing disc from an early wandering larva expressing UAS-IR-EcR under the control of dpp-GAL4 cultured in vitro in the absence of ecdysone for 24 hours. SENS only accumulates in the margin (arrow) where it is intersected by the dpp-domain (compare with control in C''). (C-F) Area shown is the same as in Fig. 2D. In all panels, anterior is to the top.

View larger version (19K): [in a new window]   Fig. 6. Frequency of wings cultured in vitro without 20E that have 1, 2, 3, 4 or more A101-positive nuclei in two early born sensilla (GSR and ACV). Grey bars show the data from discs at the onset of culture, black bars show the data from discs cultured for 22-24 hours in vitro without 20E. A and C show the results from discs of early wandering larvae; B and D show discs from mid-wandering larvae. Frequencies are based on analysis of 19-24 discs.

View larger version (25K): [in a new window]   Fig. 7. Repression by the unliganded ecdysone receptor is lifted at different steps in neurogenesis for different sets of SOPs. For the chemosensory neurons of the margin we speculate that BR-Z2 activates SENS whereas BR-Z1 is required for the accumulation of SENS in the mature SOP. BR-Z1 is repressed by the unliganded EcR/USP complex. The control of br-Z2 expression has not been established. The early born SOPs accumulate SENS prior to the rise of 20E and are independent of BR function. EcR/USP repression inhibits the division of the SOP. The rise in 20E relieves repression and allows the SOP to divide. Grey represents low levels of SENS protein [adapted from Jafar-Nejad et al. (Jafar-Nejad et al., 2003)]; black shows the mature SOPs that have accumulated SENS. Open arrow marks the rise in 20E.