Journal: bioRxiv
Article Title: Drosophila oocyte specification is maintained by the dynamic duo of microtubule polymerase Mini spindles/XMAP215 and dynein
doi: 10.1101/2023.03.09.531953
Figure Lengend Snippet: (A-C’) Phalloidin and smiFISH staining of msps mRNA in control (A-A’), Dhc-RNAi (B-B’), and egl-RNAi (C-C’). Cy5-labeled FLAP-X smiFISH probes recognize the 3’UTR of msps mRNA. msps mRNA is concentrated in control oocytes but heavily reduced in Dhc-RNAi and egl-RNAi oocytes. (D) Average fluorescence intensity of msps mRNA (by smiFISH against msps 3’UTR) in control, Dhc-RNAi , and egl-RNAi . The average msps mRNA intensity in control oocytes: stage 5, 3842.6 ± 365.1 A.U. (N=38); stage 6, 5208.2 ± 367.1 A.U. (N=41); stage 7, 3903.1 ± 552.1 A.U. (N=19); stage 8, 2317.8 ± 325.8 A.U. (N=23); stage 9, 1390.5 ± 161.7 A.U. (N=24). The average msps mRNA intensity in Dhc-RNAi oocytes: stage 5, 996.5 ± 113.1 A.U. (N=54); stage 6, 908.6 ± 106.7 A.U. (N=47); stage 7, 896.2 ± 158.3 A.U. (N=38); stage 8, 837.0 ± 122.4 A.U. (N=32); stage 9, 871.2 ± 135.0 A.U. (N=30). The average msps mRNA intensity in egl-RNAi oocytes: stage 5, 500.8 ± 35.0 A.U. (N=71); stage 6, 469.4 ± 34.5 A.U. (N=56); stage 7, 369.8 ± 39.0 A.U. (N=55); stage 8, 308.3 ± 37.8 A.U. (N=32); stage 9, 270.4 ± 43.0 A.U. (N=25). Unpaired t-tests with Welch’s correction were performed between the following groups: control and Dhc-RNAi stage 5-stage 9, all p<0.0001 (****); control and egl-RNAi , stage 5-stage 9, all p<0.0001 (****); Dhc-RNAi and egl-RNAi , stage 5-stage 9, all p<0.0001 (****). (E) Total fluorescence intensity of msps mRNA (by smiFISH against msps 3’UTR) in control, Dhc-RNAi , and egl-RNAi . The total msps mRNA intensity in control oocytes: stage 5, 482091.0 ± 71442.2 A.U. (N=38); stage 6, 1494695.9 ± 211967.4 A.U. (N=41); stage 7, 2282849.2 ± 504102.6 A.U. (N=19); stage 8, 3292804.4 ± 590320.8 A.U. (N=23); stage 9, 8410028.3 ± 1821593.5 A.U. (N=24). The total msps mRNA intensity in Dhc-RNAi oocytes: stage 5, 65031.2 ± 7828.3 A.U. (N=54); stage 6, 65176.3 ± 7707.7 A.U. (N=47); stage 7, 94359.4 ± 21229.0 A.U. (N=38); stage 8, 110487.8 ± 24462.6 A.U. (N=32); stage 9, 146230.2 ± 52093.7 A.U. (N=30). The total msps mRNA intensity in egl-RNAi oocytes: stage 5, 43861.3 ± 3831.3 A.U. (N=71); stage 6, 55754.7 ± 4478.7 A.U. (N=56); stage 7, 78525.1 ± 10578.5 A.U. (N=55); stage 8, 166950.2 ± 87307.5 A.U. (N=32); stage 9, 703163.2 ± 543799.2 A.U. (N=25). Unpaired t-tests with Welch’s correction were performed between the following groups: control and Dhc-RNAi stage 5-stage 9, all p<0.0001 (****); control and egl-RNAi , stage 5-stage 9, all p<0.0001 (****); Dhc-RNAi and egl-RNAi , stage 5, p<0.0001 (****); stage 6, p =0.0369 (*); stage 7, p=0.1825 (n.s); stage 8, p= 0.2122 (n.s); stage 9, p=0.0458 (*). (F-G) The mRNA localizations of sfGFP-msps 3’UTR (F) and CFP-Msps.CDS (G) via the Cy5-labeled FLAP-X smiFISH probes against sfGFP and CFP, respectively. Both mRNAs show clear oocyte enrichments. All samples with one copy of maternal αtub-Gal4 [V37] . Oocytes are indicated by the yellow arrowheads or yellow brackets. A small (5 µm) (A-C’) or a large (>25 µm) (F-G) Max-intensity Z projections were used to show the mRNA localization. Scale bars, 50 µm. Data are represented as mean ± 95% confidence intervals (D, E).
Article Snippet: The following transgenic fly stocks were generated in this study using PhiC31-mediated integration (BestGene Inc.): UASp-sfGFP-msps 3’UTR and UASp-sfGFP-K10CT 3’UTR (inserted at 89E11, III); UASp-MoxMaple3-K10subregionA 3’UTR and UASp-Msps (RNA-resistant)-MoxMaple3-K10subregionA 3’UTR (inserted at 75A10, III); UASp-Msps (RNA-resistant)-YFP-BLID-K10subregionA 3’UTR (inserted at 55C4, II).
Techniques: Staining, Control, Labeling, Fluorescence
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![(A-C’) Phalloidin and smiFISH staining of msps mRNA in control (A-A’), Dhc-RNAi (B-B’), and egl-RNAi (C-C’). Cy5-labeled FLAP-X smiFISH probes recognize the <t>3’UTR</t> of msps mRNA. msps mRNA is concentrated in control oocytes but heavily reduced in Dhc-RNAi and egl-RNAi oocytes. (D) Average fluorescence intensity of msps mRNA (by smiFISH against msps 3’UTR) in control, Dhc-RNAi , and egl-RNAi . The average msps mRNA intensity in control oocytes: stage 5, 3842.6 ± 365.1 A.U. (N=38); stage 6, 5208.2 ± 367.1 A.U. (N=41); stage 7, 3903.1 ± 552.1 A.U. (N=19); stage 8, 2317.8 ± 325.8 A.U. (N=23); stage 9, 1390.5 ± 161.7 A.U. (N=24). The average msps mRNA intensity in Dhc-RNAi oocytes: stage 5, 996.5 ± 113.1 A.U. (N=54); stage 6, 908.6 ± 106.7 A.U. (N=47); stage 7, 896.2 ± 158.3 A.U. (N=38); stage 8, 837.0 ± 122.4 A.U. (N=32); stage 9, 871.2 ± 135.0 A.U. (N=30). The average msps mRNA intensity in egl-RNAi oocytes: stage 5, 500.8 ± 35.0 A.U. (N=71); stage 6, 469.4 ± 34.5 A.U. (N=56); stage 7, 369.8 ± 39.0 A.U. (N=55); stage 8, 308.3 ± 37.8 A.U. (N=32); stage 9, 270.4 ± 43.0 A.U. (N=25). Unpaired t-tests with Welch’s correction were performed between the following groups: control and Dhc-RNAi stage 5-stage 9, all p<0.0001 (****); control and egl-RNAi , stage 5-stage 9, all p<0.0001 (****); Dhc-RNAi and egl-RNAi , stage 5-stage 9, all p<0.0001 (****). (E) Total fluorescence intensity of msps mRNA (by smiFISH against msps 3’UTR) in control, Dhc-RNAi , and egl-RNAi . The total msps mRNA intensity in control oocytes: stage 5, 482091.0 ± 71442.2 A.U. (N=38); stage 6, 1494695.9 ± 211967.4 A.U. (N=41); stage 7, 2282849.2 ± 504102.6 A.U. (N=19); stage 8, 3292804.4 ± 590320.8 A.U. (N=23); stage 9, 8410028.3 ± 1821593.5 A.U. (N=24). The total msps mRNA intensity in Dhc-RNAi oocytes: stage 5, 65031.2 ± 7828.3 A.U. (N=54); stage 6, 65176.3 ± 7707.7 A.U. (N=47); stage 7, 94359.4 ± 21229.0 A.U. (N=38); stage 8, 110487.8 ± 24462.6 A.U. (N=32); stage 9, 146230.2 ± 52093.7 A.U. (N=30). The total msps mRNA intensity in egl-RNAi oocytes: stage 5, 43861.3 ± 3831.3 A.U. (N=71); stage 6, 55754.7 ± 4478.7 A.U. (N=56); stage 7, 78525.1 ± 10578.5 A.U. (N=55); stage 8, 166950.2 ± 87307.5 A.U. (N=32); stage 9, 703163.2 ± 543799.2 A.U. (N=25). Unpaired t-tests with Welch’s correction were performed between the following groups: control and Dhc-RNAi stage 5-stage 9, all p<0.0001 (****); control and egl-RNAi , stage 5-stage 9, all p<0.0001 (****); Dhc-RNAi and egl-RNAi , stage 5, p<0.0001 (****); stage 6, p =0.0369 (*); stage 7, p=0.1825 (n.s); stage 8, p= 0.2122 (n.s); stage 9, p=0.0458 (*). (F-G) The mRNA localizations of sfGFP-msps 3’UTR (F) and CFP-Msps.CDS (G) via the Cy5-labeled FLAP-X smiFISH probes against sfGFP and CFP, respectively. Both mRNAs show clear oocyte enrichments. All samples with one copy of maternal αtub-Gal4 [V37] . Oocytes are indicated by the yellow arrowheads or yellow brackets. A small (5 µm) (A-C’) or a large (>25 µm) (F-G) Max-intensity Z projections were used to show the mRNA localization. Scale bars, 50 µm. Data are represented as mean ± 95% confidence intervals (D, E).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8982/pmc10028982/pmc10028982__nihpp-2023.03.09.531953v1-f0003.jpg)