Organismos rectores de la calidad a nivel nacional

To elucidate the mechanisms regulating Dscam splicing in adaptation to pathogen exposure or during neuronal development, it was thought to test mutants in candidate genes for splicing regulators. Since Dscam splicing in mushroom bodies and dendritic arborization neurons changes such that individual cells acquire a unique set of Dscam isoforms, it is possible that Dscam splicing is generally variable. To exclude that such variability is a key feature of Dscam, the pattern of Dscam splicing at various developmental stages and in individual flies was analysed. Consequently, Dscam exon 4 and 9 splicing pattern was analyzed between nine independent pools of ten Canton S embryos, ten individual Canton S males and eight individual yw females (Figures 25 and 26). These results of this analysis revealed that the choice of exon variants in Dscam exon 4 and 9 splicing followed a different trend between different developmental stages, wild type strains and sexes. For Dscam exon 4 splicing, exon 4.9 accounted for 10.3% of all spliced exons in Canton S embryos, where as in Canton S males and yw females it accounted for only 3.9%. In Canton S males, exon 4.5 represented 12.7% of all splicing events, which was only 6.9% in yw females. Similarly, exon 4.8 accounted for 12.5% of all spliced variable exons in yw females, which was only 6.9% in Canton S males (Figures 25A-G).

To attribute variation observed between Canton S males and yw females to strain or sex differences, and also to get a better understanding about tissue specific splicing, neuron rich head-thoraces were compared with neuron poor abdomens between Canton S and yw females. The results revealed that differences in proportions of exon variants included in Canton S and yw flies were due to strain differences as

Figure 25 (A,B): Variation in Dscam exon 4 splicing pattern between different developmental stages, strains, sexes and tissues. (A) Analysis of Dscam exon 4 splicing pattern using RNA extracted from independent pools of ten 14-18 h old Canton S embryos (Lanes 1-9). (B) Heat map representation of Dscam splicing changes observed in A. p values for each exon variant are mentioned alongside.

Dscam exon variants were separated as explained in figure legend 21. Samples were run on an 8%

denaturing polyacrylamide gel. M=50bp ladder (NEB).

Figure 25 (C,D): Variation in Dscam exon 4 splicing pattern between different developmental stages, strains, sexes and tissues. (C) Analysis of Dscam exon 4 splicing pattern using RNA extracted from single Canton S males (Lanes 1-10). (D) Heat map representation of Dscam splicing changes observed in C. p values for each exon variant are mentioned alongside. Dscam exon variants were separated as explained in figure legend 21. Samples were run on an 8% denaturing polyacrylamide gel. M=phiX174 DNA/HinfI marker (Biotools).

C D

Figure 25 (E,F,G): Variation in Dscam exon 4 splicing pattern between different developmental stages, strains, sexes and tissues. (E)Analysis of Dscam exon 4 splicing pattern using RNA extracted from single yw females (Lanes 1-8). (F) Heat map representation of Dscam splicing changes observed in E. p values for each exon variant are mentioned alongside. Dscam exon variants were separated as explained in figure legend 21. Samples were run on an 8% denaturing polyacrylamide gel. (G) Graphical representation of percentage inclusion levels of each exon 4 variant observed in A, C and E.

M=phiX174 DNA/HinfI marker (Biotools).

E F

G

Figure 25 (H,I,J): Variation in Dscam exon 4 splicing pattern between different developmental stages, strains, sexes and tissues. (H) Analysis of Dscam exon 4 splicing pattern using RNA extracted from head-thorax from single Canton S female (Lane 1) and yw female (Lane 2); abdomen from single Canton S female (Lane 3) and yw female (Lane 4). (I and J) Graphical representation of percentage inclusion levels of each exon 4 variant observed in H. Dscam exon variants were separated as explained in figure legend 21. Samples were run on an 8% denaturing polyacrylamide gel. M=phiX174 DNA/HinfI marker (Biotools).

H

I

J

observed in exons 4.5, 4.8 and 4.12. The pattern between head-thoraces and abdomens was however, very similar within the same strain (Figures 25H-J). Similar levels of exon variants such as 4.2, 4.9 and 4.10 were also observed between Canton S males and yw females. Only exons 4.6 and 4.11 showed comparable levels of inclusion between all the three sample types (Figure 25G). For Dscam exon 9 splicing, exons 9.7 and 9.8 both accounted for 15.5% of all exon 4 variants in Canton S embryos, which were expressed at levels less than 8.6% and 4.1% in Canton S males and yw females, respectively. Exon 9.24 was maximally included in yw females representing 33.4% of all exons included within the cluster, where as in Canton S embryos and flies it was only 9.2% and 19.32% respectively. Overall, exon 9 cluster showed less variation between the three sample sources as compared to the exon 4 cluster (Figures 26A-G).

The splicing pattern between individual pools of Canton S embryos, Canton S males and yw females was highly reproducible. To investigate reproducibility of Dscam splicing pattern between independent experiments a 2-tailed t-test was performed for each of the resolved exon 4 and 9 variants by comparing their inclusion levels across all experiments. The data set was randomly divided into 3 different pairs of arrays and their mean p value was calculated. The obtained p values for all exon 4 and 9 variants were >0.05, which suggested that the results obtained between independent experiments are very similar. Hence, Dscam splicing pattern is highly reproducible between individual samples of the same source such as embryo pools and individual flies. The p values are shown adjacent to each exon 4 and 9 variants in figures 25 and 26B, D and F, respectively. Between individual Canton S males, exons 4.4, 9.2,

Figure 26 (A,B): Variation in Dscam exon 9 splicing pattern between different developmental stages, strains, sexes. (A) Analysis of Dscam exon 9 splicing pattern using RNA extracted from independent pools of ten 14-18 h old Canton S embryos (Lanes 1-5). (B) Heat map representation of Dscam splicing changes observed in A. p values for each exon variant are mentioned alongside. Dscam exon variants were separated as explained in figure legend 22. Unspecific bands are indicated by asterisks (*). Samples were run on an 8% denaturing polyacrylamide gel. M=phiX174 DNA/HinfI marker (Biotools).

Figure 26 (C,D): Variation in Dscam exon 9 splicing pattern between different developmental stages, strains, sexes. (C) Analysis of Dscam exon 9 splicing pattern using RNA extracted from single Canton S males (Lnes 1-5). (D) Heat map representation of Dscam splicing changes observed in C. p values for each exon variant are mentioned alongside. Dscam exon variants were separated as explained in figure legend 22. Unspecific bands are indicated by asterisks (*). Samples were run on an 8%

denaturing polyacrylamide gel. M=50bp ladder (NEB).

C D

Figure 26 (E,F,G): Variation in Dscam exon 9 splicing pattern between different developmental stages, strains, sexes. (E) Analysis of Dscam exon 9 splicing pattern using RNA extracted from single yw females (Lanes 1-5). (F) Heat map representation of Dscam splicing changes observed in E. p values for each exon variant are mentioned alongside. (G) Graphical representation of percentage inclusion levels of each exon 9 variant observed in A, C and E. Dscam exon variants were separated as explained in figure legend 22. Unspecific bands are indicated by asterisks (*). Samples were run on

G

E F

9.5 and 9.33 showed the most variation. Females from the yw strain showed complete absence of exon 9.33.

3.5. Analysis of Dscam splicing in mutants of genes