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First published online 22 March 2006
doi: 10.1242/dev.02338

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Senseless and Daughterless confer neuronal identity to epithelial cells in the Drosophila wing margin

Hamed Jafar-Nejad^1,*, An-Chi Tien², Melih Acar² and Hugo J. Bellen^1,2,3,*,

¹ Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
² Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
³ Division of Neuroscience, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

View larger version (61K): [in a new window]   Fig. 1. A requirement for ac and sc in AWM mechanosensory organ development after pI selection. (A-E) ac, sc and sens regulate the number of AWM stout bristles. Close-up views of the AWM of (A) Canton-S (wild type, wt), (B) sc10-1/Y, (C) sc10-1/Y; sensE2/+ and (D) sc10-1/Y; src/+ are shown. src, sens genomic rescue construct; these flies have three copies of the wild-type sens gene. Arrow and arrowhead in A point to a chemosensory bristle and a stout bristle, respectively. (E) Quantification of the number of stout bristles in A-D; 10-14 wings were quantified for each genotype. One-way ANOVA with Scheffe error protection indicates that the difference between sc10-1/Y and sc10-1/Y; src/+ is not statistically significant. Moreover, a t-test for independent samples shows that sc10-1/Y (*) and sc10-1/Y; sensE2/+ (**) are significantly different from all of the other genotypes (P<0.0001). Error bars indicate s.e.m. (F-F'') Double-staining of the AWM of an 8- to 10-hour APF A101-lacZ pupa for ß-Gal (green) and Sens (red) indicates colocalization of the two proteins in the mechanosensory pIs and the internal cells of the presumptive chemosensory organs (asterisks in F). (G-I) Sens staining of the AWM of (G) A101-lacZ (wild type), (H) sc10-1/Y and (I) sc10-1/Y; sensE2/+ pupae at 12-14 hours APF does not show a significant difference in the number of mechanosensory pIs between these genotypes. Note the absence of chemosensory clusters in H and I.

View larger version (64K): [in a new window]   Fig. 2. Ac and Sc promote the survival of the AWM mechanosensory lineages. (A-E) Loss of ac and sc results in a dramatic decrease in the number of AWM mechanosensory neurons. Double-staining of the AWM of (A) y w (wild type), (B) sc10-1/Y, (C) sc10-1/Y; sensE2/+ and (D) sc10-1/Y; src/+ pupae at 24 hours APF for Su(H) (green) and Elav (red) indicates that the loss of neurons and socket cells in a sc10-1 background is quite sensitive to sens gene dosage. Quantification of the number of mechanosensory neurons in A-D is shown in E. Five wings were analyzed for each genotype. The number of neurons in sc10-1 is less than 10% of wild type, and is significantly different from sc10-1/Y; sensE2/+ (*P<0.0001) and sc10-1/Y; src/+ (**P<0.005). Error bars indicate s.e.m. (F-K') Overexpression of the anti-apoptotic protein P35 in the WM rescues the sc10-1 stout SO phenotype. (F,H) Close-up views of the AWM from a sc10-1/Y (F) and a sc10-1/Y; C96-GAL4 UAS-P35/+ (H) fly; (G,G',I,I',J-K') wings of 36- to 42-hour APF pupae doubly stained with anti-HRP (green) and anti-Elav (red). Comparison of sc10-1/Y (G,G') and sc10-1/Y; C96-GAL4 UAS-P35/+ (I,I') indicates that neurons undergo apoptosis in sc10-1 flies. Note the extra neurons in the PWM (compare with J). Also, unlike the PWM of a y w (wild-type) wing (J,J'), which is devoid of neurons, a C96-GAL4 UAS-P35/+ wing (K,K') is lined with cells that express neuronal markers.

View larger version (82K): [in a new window]   Fig. 3. sens and da are required for AWM mechanosensory and PWM bristle formation. (A) A MARCM clone of Df(1)260-1 in the AWM. The mutant bristles are yellow and singed. (B,B') A MARCM clone of Df(1)260-1 in the wing of an 8- to 10-hour APF pupa stained for Sens (red) and Atonal (green). CD8-GFP (blue) marks the membranes of the mutant cells. Note that the accumulation of Sens in mechanosensory pIs is not affected by the loss of the ASC. However, despite the background staining of Ato along the physical margin due to the high gain used in the scans, no Ato staining is detected in mechanosensory pIs. (C,D) AWM (C) and PWM (D) bristles are lost in sens clones. The mutant tissue in C is marked with multiple wing hairs (mwh). Note that upon loss of stout bristles, a chemosensory bristle (arrow) is misplaced to the stout row, as reported previously (Couso et al., 1994). (E,F) da3 clones lack AWM (E) and PWM (F) bristles. The mutant tissues are marked by crinkled. (G-I') sens expression is activated by Wingless in the WM. All panels show the central part of the wing pouch in third instar wing imaginal discs. Arrows point to the WM; the dashed line depicts the anteroposterior (AP) boundary of the wing disc; anterior to the left. (G) Sens expression in a y w (wild type) wing disc. (H,H') Loss of Wg (green) and Sens (red) expression in a wgCX4 clone. The mutant tissue lacks GFP (blue). Note that even in the heterozygous tissue flanking the mutant clone, Sens expression is decreased, indicating that sens requires very high levels of Wingless signaling for proper expression. (I,I') Overexpression of Wg-GFP (green) along the AP boundary induces Sens (red) expression.

View larger version (60K): [in a new window]   Fig. 4. Da and Sens synergize in vitro and in vivo, and bind to each other. (A-D) Co-expression of Da and Sens in the wing results in a synergistic increase in the number of ectopic SOs generated. The third wing vein region of (A) sca109-68-GAL4/UAS-da52, (B) sca109-68-GAL4 UAS-sensC12 and (C) sca109-68-GAL4 UAS-sensC12/UAS-da52 flies are shown. In D, the number of extra SOs are quantified for each genotype (n17). Arrows in insets indicate the extra campaniform (dome-shaped) sensilla. (E) Sens strongly increases the level of ac-luciferase transcription induced by Da. (F) In vitro-translated Sens can be pulled-down using GST-Da, but not with GST alone. Western blotting with an anti-Sens antibody was used for detection.

View larger version (122K): [in a new window]   Fig. 5. Overexpression of Sens and Da in the thorax can generate bristles in ASC clones. (A) Loss of Ac and Sc leads to a loss of all bristles in the thorax. (B,C) Overexpression of (B) Da or (C) Sens using Eq-GAL4 results in the formation of numerous bristles in a sc10-1 background. Note that overexpression of Sens in the midline region causes some thorax closure defects and abnormal midline morphology. (D) Overexpression of Sens using a moderate UAS-sens transgene is able to induce a few SOs in a sc10-1 background. (E) Co-expression of Sens and Da results in the generation of a large number of SOs in sc10-1 flies. Note that because flies used in D and E were taken out of the pupal case as pharate adults, their cuticle is somewhat wrinkled. (F-H) MARCM clones of Df(1)260-1. Mutant bristles are yellow and singed. In clones of the ASC in the thorax, which are normally devoid of bristles (F), overexpression of (G) Da or (H) Sens induces SO formation.

View larger version (92K): [in a new window]   Fig. 6. Da is able to induce pI formation in the absence of Sens function. (A) A MARCM da3 clone that overexpresses sens using tub-GAL4. Note the absence of microchaetae in the mutant clone (the closed line in the close-up view), which is marked by crinkled. (B,C) Low (B) and high (C) magnification views of two MARCM clones of sensE2 that overexpress da using tub-GAL4. No microchaetae are formed in the clone, which is marked by mwh. (D-D'') Overexpression of da in a MARCM sens clone in the thorax using tub-GAL4 results in the generation of numerous additional Elav+ cells (red), often clustered. Note that the neurons are usually well spaced in the wild-type tissue. The image is from a 36-hour APF pupa. GFP (blue) marks the nuclei of the sens mutant cells.

View larger version (100K): [in a new window]   Fig. 7. sens regulates cell-fate specification in the microchaetae pI progeny. (A-A'') Sc expression is restricted to single cells in a sens clone. Shown is a sensE2 clone in a 12-hour APF pupal thorax stained for Sc (red). GFP (blue) marks the wild-type tissue. (B,B') A MARCM clone of sens in the thorax 24-30 hours APF stained for Elav (red) and Prospero (green). Note the multiple neurons and an occasional sheath cell in mutant clusters. (C,C') A MARCM clone of sens in the thorax around 28-30 hours APF, stained for Elav (red) and HRP (green). Because of the presence of several neurons in each cluster, some axonal tracts in the mutant tissue are thicker than their wild-type counterparts. (D-D'') Microchaetae pI progeny undergo cell fate transformation in sens clones. A wild-type (left) and a sensE2 (right) sensory cluster is shown at 24-26 hours APF, stained for Elav (red) and Su(H) (green). Note that the mutant cluster contains multiple neurons but no socket cells. (E) Simplified model of the microchaetae lineages in wild-type and sens- animals. Note that in addition to the pIIa-to-pIIb transformation, many mutant clusters also exhibit a sheath-to-neuron transformation in the pIIb progeny. so, socket; sf, shaft; st, sheath; n, neuron.

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