The Journal of Neuroscience, January 4, 2006, ():
Dystrophin Is Required for Appropriate Retrograde Control of Neurotransmitter Release at the Drosophila Neuromuscular Junction
J. Neurosci. van der Plas et al.
26: 333
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1
dystrophin isoform mRNA expression patterns in wild type and dystrophin mutants. Wild type (A-L), dysE6 (M, N) and dysGE20705 (O, P) embryos (A-F, M-P) or third instar larvae (G-L) were used for RNA in situ analyses using isoform specific DLP1, DLP2 and Dp186 probes (indicated in the panels). DLP1 is present in the embryonic visceral mesoderm (A), but absent from the embryonic (D) or larval (G) body walls or the larval neuropile or brain (J). DLP2 is present in the embryonic visceral mesoderm (B) and the embryonic (E) and larval (H) musculature and eye-discs (K), but absent in the larval neuropile and brain (K). Dp186 is expressed in the embryonic CNS (C) and larval brain and neuropile (L), but absent from embryonic muscle (F) and at a low level at the larval musculature (I). DLP2 expression is absent in dysE6 (M) and reduced in dysGE20705 (O), while Dp186 is at a normal level in dysE6 (N) and reduced in dysGE20705 (P). Arrows indicate the ventral nerve cord or neuropile, open arrows indicate eye-discs and arrowheads indicate brain. There is some nonspecific trapping of probe in the tracheal system (panels J and K), which is also observed in the sense probe control RNA in situ hybridizations (data not shown).
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Supplemental Figure 2
Semi-quantitative RT-PCR analysis of DLP1, DLP2, Dp186 and CG6255 expression in dystrophin mutants. Total mRNA was prepared from dissected 3rd instar larval body walls (A), whole larvae (B) or dissected brain-imaginal disc complexes (C), reversed transcribed and serial dilutions of first-strand cDNAs (2-fold in A and 3-fold in B and C) were PCR amplified as described in the Materials and Methods with DLP1, DLP2, Dp186 or RP49 primer sets. All samples in a given set were treated identically and electrophoresed and visualized at the same time in the same gel. The sizes of the products (indicated by labeled arrows) were as predicted from the cDNA sequences. Similar RP49 PCR product band intensities observed in titrations of input first strand cDNA across each set (A-C, bottom rows) indicate that equivalent amounts of total first-strand cDNA were present in each sample. The DLP2-specific RT-PCRs (A, top row) indicate that the dysE31 precise excision control expresses DLP2 at wildtype levels, however dysE6 does not detectably express DLP2. dysGE20705 exhibits an ~ 4-fold reduction in DLP2 expression; pan muscle expression of UAS-RNAi-dysNH2, > 8-fold and the dysE6/TM6 heterozygotes, between 2- and 4-fold. The DLP1-specific RT-PCRs (B, top row) indicate that dysE6 exhibits no significant reduction from dysE31/wildtype levels of DLP1, whereas dysGE20705 DLP1 levels are ~ 3-fold reduced and the expression of UAS-RNAi-dysNH2 throughout the muscle does not significantly reduce DLP1 expression as was expected since DLP1 is not detectably expressed in the muscle. CG6255 (B, middle row) is not detectably expressed in dysE6 but is at wildtype levels in the other genotypes, including dysGE20705. Dp186 expression (C, top row) is reduced ~ 27-fold in dysGE20705, but it is expressed at wildtype levels in the other genotypes, including those expressing UAS-RNAi-dysNH2 either throughout the muscle (24B-Gal4) or pan-neuronally (Elav-Gal4).
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Supplemental Figure 3
Muscle and synapse terminal size and morphology are not substantially affected in dystrophin Mutants. Wild type, dysE31 control and the dystrophin mutants dysE6 and dysGE20705 3rd instar larval body walls were stained with anti-HRP and subsequently muscle area (A), number of boutons (B), terminal length (C) and number of branches (D) were determined. The mean values ± SEM were calculated from thirty muscles 6/7, segment A3, for fifteen larvae of each genotype. The mean muscle area of dysE31 is slightly larger compared to the other groups. There are somewhat less branches in dysE31 and dysE6 compared to the w1118 and dysGE20705 terminals, however, the differences are small.
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Supplemental Figure 4
Morphometric analysis of bouton morphology and shape of dystrophin mutants. Morphometric analyses of bouton midline area (A), ratio SSR/bouton midline area (B), area of bouton occupied by vesicles (C) and bouton shape (D) are indicated for Type Ib boutons at muscle 6 of segment A3 to A5 of third instar larvae for the genotypes w1118, dysE31, dysE6 and dysGE20705. The mean values were calculated from fifteen boutons from 3 different larvae. There are two significant differences between the dysGE20705 mutant and control boutons indicated by an asterix: 1) the bouton area filled with vesicles is enlarged in dysGE20705 animals and 2) the ratio of the long versus the short diameter of the mutant dysGE20705 boutons is altered, reflecting the long extended shape of the boutons occasionally observed on the EM micrographs.
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Supplemental Figure 5
PMad expression is not altered when postsynaptic Dystrophin or CaMKII levels are either reduced or increased. Stage 17 embryos of the following genotypes were stained with anti-PMad: w1118 (A), witB11 (B), dysE6 (C), GS12472/24B-Gal4 (D), UAS-Ala /+; 24B-Gal4/+ (E) and UAS-CaMKIIT287D/+; 24B-Gal4/+ (F). PMad staining is undetectable in the absence of wit; however, PMad protein levels are unaltered in the other genotypes.
