miR-965 controls cell proliferation and migration during tissue morphogenesis in the Drosophila abdomen
- Institute of Molecular and Cell Biology, Singapore
- University of Copenhagen, Denmark
Figures
Figure 1 with 3 supplements
The miR-965 mutant.
(A) miR-965 is located within the first intron of the kismet gene. The targeting strategies used to produce two independent miR-965 deletion mutants by ends-out homologous recombination are shown …
Figure 1—figure supplement 1
Evidence that kismet and miR-965 arise from a common transcription unit.
Above: diagram of the kismet locus showing miR-965 in the first intron. kismet and miR-965 are transcribed in the same direction. Below: quantitative miRNA PCR showing the level of miR-965 miRNA. miR…
Figure 1—figure supplement 2
Phenotype classification.
Images showing the three classes of defect: gaps, or lack of tissue; fusion, and polarity reversal. Right panels show higher magnification views of the bristle pattern to illustrate the polarity …
Figure 1—figure supplement 3
Penetrance of defects in miR-965 mutants, shown as % of affected individuals.
Large gaps associated with fusion of adjacent segments occurred in ∼10% of cases. Gap penetrance per segment is shown at right. n = 200 flies. Refers to Figure 1E.
Figure 2
miR-965 expression in histoblasts.
Top: design of the control and miR-965 sensor transgenes. EGFP was under control of the tubulin promoter. For the miR-965 sensor, 1 copy of a perfect miR-965 target sequence was placed into the SV40 …
Figure 3 with 3 supplements
Abnormal histoblast proliferation and migration in the miR-965 mutant.
(A) Still images taken from time-lapse videos of control, miR-965 mutant (KO1/KO2) and rescued mutant showing the reduction divisions of the early histoblast proliferation phase. M1, M2 and M3 …
Figure 3—figure supplement 1
Rate of histoblast nest expansion measured from time-lapse videos.
Each data point corresponds to one histoblast nest. The leading edge of each histoblast nest was tracked using imageJ. Speed was calculated measuring total distance covered (micrometer/hour). …
Figure 3—figure supplement 2
Large polyploid cells in miR-965 mutant histoblast nests.
Histoblast nests were labeled with esg-Gal4-directed expression of UAS-GFP at 24 hr APF. Note the presence of large polyploid cells in the histoblast nest in the miR-965 mutant (arrows). At the …
Figure 3—figure supplement 3
Pupal survival assays.
Pupal survival was assayed for flies of the indicated genotypes. 6 batches of pupae were sampled/genotype. The data present the total number of surviving adults (live) and the total number of dead …
Figure 4 with 1 supplement
miR-965 regulates string and wingless.
(A, B) string (stg) and wingless (wg) transcript levels measured by quantitative real time RT-PCR in RNA isolated from w1118 control, KO1/KO2 and 965-rescue pupae at 21 hr APF. Data represent the …
Figure 4—figure supplement 1
(A) Predicted miR-965 sites in the string 3′UTR.
Based on the potential for strong 3′ pairing in the Seed 1 mutant (shown in Figure 4D), as well as the presence of a second nearby non-canonical seed match (seed 2), a more extensively mutated UTR …
Figure 5 with 5 supplements
Overexpression of string and wg contributes to the miR-965 mutant phenotype.
(A) Dorsal views of abdomens from adult female esg-Gal4 UAS-string flies illustrating the segment gap, segment fusion and polarity reversal phenotypes. (B) Penetrance of abdominal defects of all …
Figure 5—figure supplement 1
The proportion of flies with defects caused by string overexpression.
Penetrance of the three types of abdominal defect in flies overexpressing UAS-String under esg-Gal4 control. n = 102 flies. Refers to Figure 5A.
Figure 5—figure supplement 2
Still images from a time-lapse video of esg-Gal4>UAS-string histoblasts.
Left: rapid proliferation phase. Note the presence of cells of different sizes, indicative of asynchronous division. Images represent 0 hr and mitosis M1, M2 and M3. Genotype: esg-Gal4, UAS-string, …
Figure 5—figure supplement 3
Speed of histoblast nest migration.
n = 15 for control, n = 18 for KO1/KO2 and n = 11 for esg-GAL4>UAS-stg. p < 0.001 comparing control and esg-GAL4>UAS-stg using one-way ANOVA. esg-GAL4>UAS-stg is not significantly different from the …
Figure 5—figure supplement 4
Rescue of the migration defect of miR-965 mutants with reduced levels of string.
Left: still images taken from a time-lapse video showing histoblast divisions in the miR-965 mutant (KO1/KO2) with reduced levels of string transcript using the stgEY12388 allele. M1 and M2 indicate …
Figure 5—figure supplement 5
Speed of histoblast migration restored by reduced string activity.
Left: speed of histoblast nest migration in the third abdominal segment. p < 0.05 comparing KO1/KO2 with KO1/KO2; stgEY12388. Control (n = 15), KO1/KO2 (n = 18) and KO1/KO2; stgEY12388 (n = 14). The …
Figure 6 with 2 supplements
Regulation of miR-965 by ecdysone at the beginning of pupariation.
(A) Quantitative RT-PCR showing levels of miR-965 primary transcript, EcR, and string mRNAs in RNA isolated from pupae expressing esg-GAL4 (control) and esg-GAL4 driving UAS-EcR-RNAi to deplete EcR …
Figure 6—figure supplement 1
Mature miR-965 miRNA regulation by EcR.
miRNA quantitative RT-PCR showing the levels of mature miR-965 in RNA extracted from 0 hr pupae. esg-GAL4 was used to direct UAS-EcRRNAi expression in histoblasts. Data were normalized to U27 and …
Figure 6—figure supplement 2
EcR 3′ UTR reporter expression in the miR-965 mutant.
A reporter transgene containing the n EcR 3′ UTR linked to GFP was introduced into the miR-965 KO1/KO2 mutant background. GFP expression (green) did not increase in the histoblast nests in the miRNA …
Author response image 1
Author response image 2
Videos
Video 1
Control, division phase.
Early synchronous divisions of histoblast nests in Esg-GAL4>UAS-nuclear GFP controls. GFP was used to track anterior and posterior dorsal histoblast nests during the early division phase. Animals …
Video 2
miR-965 mutant division phase.
Early asynchronous divisions in the miR-965 mutant (KO2, esg-GAL4>UAS-nuclear GFP/KO1). Esg-GAL4>UAS-nuclear GFP was recombined onto miR-965 RMCE mutant (KO2) chromosome. ADHN and PDHN indicate …
Video 3
miR-965 mutant apoptosis.
Apoptotic cells are seen during the early histoblast division phase in the miR-965 mutant. Esg-GAL4>UAS-nuclear GFP was recombined onto miR-965 RMCE mutant (KO2) chromosome. ADHN and PDHN indicate …
Video 4
miR-965-Rescue division phase.
Early synchronous divisions of histoblasts in miR-965 RMCE rescue. Esg-GAL4>UAS-nuclear GFP was recombined onto miR-965 RMCE rescue chromosome. ADHN and PDHN indicate anterior and posterior dorsal …
Video 5
Control, growth phase.
Migration of histoblast nests during the growth phase in an Esg-GAL4>UAS-cytoplasmic GFP control pupa. GFP (green) is used to monitor growth and migration of histoblast nests. H2-RFP (red) marks the …
Video 6
miR-965 mutant growth phase.
Delayed migration of histoblast nests during the growth phase in the miR-965 mutant. Esg-GAL4>UAS-cytoplasmic GFP was recombined onto both KO1 and KO2 mutant chromosomes. This video shows a KO2, …
Video 7
miR-965 Rescue growth phase.
Migration of histoblast nests during the growth phase in the miR-965 RMCE rescue genotype. Esg-GAL4>UAS-cytoplasmic GFP was recombined onto the miR-965 RMCE rescue chromosome. H2-RFP (red) marks the …
Video 8
Esg-Gal4 UAS-Stg division phase.
Early divisions in a pupa overexpressing String under esg-GAL4 control (genotype: esg-GAL4, UAS-nuclear GFP/UAS-stg). ADHN and PDHN indicate anterior and posterior dorsal histoblast nests. Animals …
Video 9
Esg-Gal4 UAS-Stg growth phase.
Growth and migration phase in a pupa overexpressing String under esg-GAL4 control, as in Video 8. ADHN and PDHN indicate anterior and posterior dorsal histoblast nests. Scale bar: 100 µM. Refers to F…
Video 10
miR-965 mutant with reduced string levels division phase.
The string mutant allele, stgEY12388 was used to reduce string levels in the miR-965 mutant (KO1/KO2) background. Atpα-GFP (green) was used to mark cell membranes. H2-RFP (red) marks the nuclei. …
Video 11
miR-965 mutant with reduced string levels with reduced string levels growth phase.
Normal growth and migration of histoblast nests in the miR-965 mutant with reduced string levels. The string mutant allele, stgEY12388 was used to reduce string levels in the miR-965 mutant …
Video 12
miR-965 overexpression.
Early division arrest in a pupa overexpressing miR-965 under esg-GAL4 control (genotype: esg-GAL4, UAS-nuclear GFP/UAS-miR-965). Animals were collected for imaging at 0 hr APF. Scale bar: 50 µM. …
