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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00595

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blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development

Sung Bae Lee^*, Kyoung Sang Cho^*, Euysoo Kim and Jongkyeong Chung

National Creative Research Initiatives Center for Cell Growth Regulation and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea

View larger version (34K): [in a new window]   Fig. 3. Characterization of the by mutant phenotype. (A) A schematic drawing of the by genomic locus and the genomic structures of the by mutant alleles. ex10, byex10; ex49, byex49. (B) Comparison of the severity of the blistered wing phenotype between different by mutants. (C) Determination of expression levels of by transcript in various by mutant alleles using RT-PCR method. `a' represents 5' region of the by gene (shown in A). Ribosomal protein 49 (Rp49) was used as an internal control. RT products were amplified 28 times to detect `a' region, and 25 times to detect Rp49 levels in PCR reactions. (D) The by loss-of-function mutants exhibited decreased hatching rates from eggs. Black circles and triangles represent wild type and byex10, respectively. White squares and triangles represent by2 and byex49, respectively. E, egg; L1, first instar larva; L2, second instar larva; L3, third instar larva; P, pupa; A, adult.

View larger version (41K): [in a new window]   Fig. 6. The N-terminal region and the SH2 domain of tensin are required for normal wing development. (A) Various regions of by subcloned into pUAST vector were overexpressed in Drosophila as described in the Materials and Methods. (B) Determination of expression levels of by fragments in overexpression lines using RTPCR method. `a' and `b' represent 5' and 3' regions of the by gene, respectively (shown in A). Ribosomal protein 49 (Rp49) was used as an internal control. FL, overexpression of full-length by. All RT products were amplified 25 cycles in PCR reactions. (C) MS1096/X; UAS-PTB/+; by2/by2. (D) MS1096/X; by2,UAS-N/by2. (E) MS1096/X; UAS-C/+; by2/by2.

View larger version (71K): [in a new window]   Fig. 1. Characterization of by. (A) Genomic structure of by and the corresponding protein domain structure with the amino acid number presented underneath. The P-element insertion site of the by2 mutant allele is noted. (B) Drosophila tensin (Dtensin) exhibits highly homologous C-terminal SH2 and PTB domain structures to its human orthologs. (C) by mRNA was expressed throughout all the developmental stages, and its level was highest in the adult male (upper panel). 18S rRNA was used as a loading control (lower panel). E, Embryo; L1, first instar larva; L2, second instar larva; L3, third instar larva; P, pupa; F, female; M, male. (D) In RNA in situ hybridization analyses, by was abundantly expressed in the wing pouch of wild-type flies (left), while significantly reduced signal was detected in the wing imaginal disc of by2 mutant (right).

View larger version (61K): [in a new window]   Fig. 2. The blistered wing phenotype of various by mutants. (A) by2/by2. (B) MS1096/X; by2, UAS-by/by2. (C) by2/byex10. (D) by2/byrv8. (E) by1/by1. (F) by1/by2.

View larger version (76K): [in a new window]   Fig. 4. Histochemical analyses of wing imaginal discs of by2 flies. (A-D) Prepupal wings 4-6 hours after puparium formation. Wild-type (A) and the by2 mutant (B) wings were stained with phalloidin-TRITC as described in the Materials and Methods. The same samples were optically cross-sectioned (C,D) in the regions indicated by the green horizontal lines (C,D). (E-L) Pupal wings at 30-36 hours after puparium formation. Wild-type (E,I) and the by2 mutant (F,J) wings were stained for actin (E,F) and PS integrin (I,J) as described in the Materials and Methods. The same samples were optically sectioned. The corresponding optical sections for E,F,I,J are presented as G,H,K,L, respectively.

View larger version (83K): [in a new window]   Fig. 5. Time-lapse pictures of the wing blister formation in the by2 mutants. Comparison of wild type (A-E) and the by2 mutants (F-J) at the same time scale. Pictures were taken immediately after eclosion (-) and at the indicated times after the initiation of wing unfolding.

View larger version (69K): [in a new window]   Fig. 7. Genetic interactions between Drosophila integrin and tensin. (A) if3/Y. (B) by2/+. (C) if3/Y; by2/+. (D) if3/Y; by2/by2. (E) MS1096/Y; UAS-rlSem /+. (F) MS1096/Y; UAS-rlSem /by2.

View larger version (58K): [in a new window]   Fig. 8. Functional interactions between tensin and the JNK pathway. (A) bsk1/+. (B) bsk1/+; by2/by2. (C) hep1/hep1; by2/by2 (pharate pupa). (D) hep1/Basc; +/+. (E,F) hep1/Basc; by2/by2. (G) MS1096/Y; UAS-by/+. (H) MS1096/Y; UAS-by/UAS-by. (I) MS1096/Y; UAS-rlSem/+. (J) MS1096/Y; UAS-rlSem/UAS-by. (K) MS1096/Y; UAS-bsk/+. (L) MS1096/Y; UAS-bsk/+; UAS-by/+. (M) MS1096/X; UAS-hep/+. (N) MS1096/X; UAS-hep/+; UAS-by/+.

View larger version (43K): [in a new window]   Fig. 9. Induction of apoptosis and JNK activity by by. (A-C) In immunohistochemical analyses using anti-phosphospecific JNK antibody, JNK phosphorylation was dramatically increased in the wing imaginal discs expressing by (C), and decreased in the wing imaginal discs of homozygous by2 mutant (B) compared with the wild-type control (A). (D) The MS1096-GAL4 fly displayed a normal wing phenotype. (E) Overexpression of by using MS1096-GAL4 turned the wing into a convex shape. (F,G) In Acridine Orange staining experiments, overexpression of by (G) dramatically induced apoptotic cell death in wing imaginal discs, compared with the wild-type control (F).