The Arp2/3 complex and the formin, Diaphanous, are both required to regulate the size of germline ring canals in the developing egg chamber

Abstract

Intercellular bridges are an essential structural feature found in both germline and somatic cells throughout the animal kingdom. Because of their large size, the germline intercellular bridges, or ring canals, in the developing fruit fly egg chamber are an excellent model to study the formation, stabilization, and growth of these structures. Within the egg chamber, the germline ring canals connect 15 supporting nurse cells to the developing oocyte, facilitating the transfer of materials required for successful oogenesis. The ring canals are derived from a stalled actomyosin contractile ring; once formed, additional actin and actin-binding proteins are recruited to the ring to support the 20-fold growth that accompanies oogenesis. These behaviors provide a unique model system to study the actin regulators that control incomplete cytokinesis, intercellular bridge formation, and growth. By temporally controlling their expression in the germline, we have demonstrated that the Arp2/3 complex and the formin, Diaphanous (Dia), coordinately regulate ring canal size and growth throughout oogenesis. Dia is required for successful incomplete cytokinesis and the initial stabilization of the germline ring canals. Once ring canals have formed, the Arp2/3 complex and Dia cooperate to determine ring canal size and maintain stability. Our data suggest that nurse cells must maintain a precise balance between the activity of these two nucleators during oogenesis.

Keywords: Arp2/3 complex; Diaphanous (Dia); Drosophila; Egg chamber; Oogenesis; Ring canal.

Fig. 1:. The Arp2/3 complex is required to regulate ring canal size throughout oogenesis.

(A) Maximum intensity projections of control and arpC2-RNAi egg chambers (stages 4/5, 6, and 7 are shown). Arrowheads indicate examples of small ring canals in the arpC2-RNAi egg chambers. (B) Magnified view of the ring canals indicated by the boxed regions in the stage 7 egg chambers in (A). Arrowheads indicate the three ring canals in each region; yellow arrowhead indicates a lumenless ring canal. (C) Box and whiskers plot showing the 10-90^th percent values for the diameter of ring canals connecting nurse cells. Individual points represent values outside of that range; lumenless ring canals were excluded from this analysis. n = 81-149 ring canals/stage for each condition. Asterisks indicate significant difference compared to control (p<0.05, K-S test). (D) Average number of normal and lumenless ring canals in the arpC2-RNAi egg chambers at each stage. In controls, all egg chambers contain 15 ring canals, all with a clear lumen. n=8-12 egg chambers per stage. Error bars represent SEM. (E) Box and whiskers plot showing the 10-90^th percent values for the volumes of mature eggs. n=44 eggs for control and n=38 eggs for arpC2-RNAi. Asterisk indicates significant difference compared to control (p<0.0001, 2-tailed t-test). Examples of mature eggs for each condition are shown. Scale bar is 100 μm. (F,G) Average fluorescence intensity of phalloidin and Hts-RC staining in ring canals of (F) stage 5 and (G) stage 10 control and arpC2-RNAi egg chambers (n=32 for control, n=39 for arpC2-RNAi in F; n=16 for control, n=17 for arpC2-RNAi in G). Error bars are SEM. Average full width at half maximum +/− SEM is shown for each stain in each condition. Examples of a single plane image of a ring canal for control (top) and arpC2-RNAi (bottom) at each stage are shown. Scale bars are 2 μm in F and 5 μm in G. For all experiments, ArpC2 was depleted throughout oogenesis using the maternal triple driver (MTD-GAL4).

Fig. 2:. Depletion or mutation of the formin, Diaphanous, leads to reduced nurse cell number, multinucleation, and increased ring canal diameter.

(A) Maximum intensity projections of stage 10a control and dia-RNAi egg chambers. (B) Magnified view of the ring canals indicated by the boxed regions in A. (C) Box and whiskers plot showing the 10-90^th percent values for the diameter of ring canals connecting nurse cells in control, dia-RNAi, and dia⁵/dia⁵ mutant germlines. Individual points represent values outside of that range. The DFS technique was used to generate dia⁵/dia⁵ mutant germlines. This technique does not utilize a visible marker to identify mutant or wild type cells; instead, egg chambers containing nurse cells that have not undergone mitotic recombination will be heterozygous for the ovo^D1 mutation, which will cause them to degenerate around stage 5 of oogenesis. Therefore, only stage 6-10b egg chambers could be analyzed. n = 11-152 ring canals/stage for each condition. Asterisks indicate significant difference compared to control (p<0.05, K-S test); + indicates significant difference compared to dia-RNAi (p<0.05, K-S test), and double asterisk indicates significant difference compared to both control and dia-RNAi (p<0.05, K-S test). (D,E) Average fluorescence intensity of phalloidin and Hts-RC staining in ring canals of (D) stage 5 and (E) stage 10 control and dia-RNAi egg chambers (n=49 for control, n=51 for dia-RNAi in D; n=12 for control, n=19 for dia-RNAi in E). Error bars are SEM. Average full width at half maximum +/− SEM is shown for each stain in each condition. Examples of a single plane image of a ring canal for control (top) and dia-RNAi (bottom) at each stage are shown. Scale bars are 2 μm in D and 5 μm in E. (F) Average number of ring canals connecting nurse cells (blue) or nurse cells and the oocyte (orange) at each stage in dia-RNAi egg chambers. There should be a total of 15 ring canals per egg chamber at each stage (11 ring canals connecting nurse cells and 4 ring canals connecting nurse cells to the oocyte; dotted blue line indicates the number of nurse cell-nurse cell ring canals that each egg chamber should have at each stage). n=6-12 egg chambers/stage. Error bars are SEM. (G) Box and whiskers plot showing the 10-90^th percent values for the volumes of mature eggs. n=41 eggs for control and n=34 eggs for dia-RNAi. Asterisk indicates significant difference compared to control (p<0.0001, 2-tailed t-test). Examples of mature eggs for each condition. Scale bar is 100 μm. For all experiments, Dia was depleted using the nanos-GAL4 driver under the control of the temperature sensitive repressor (GAL80^ts); nanos-GAL4 shows a peak of expression within the germarium and again during mid-oogenesis.

Figure 3:. Both Diaphanous and the Arp2/3 complex are required to modulate ring canal size after formation of the germline cyst.

(A) Maximum intensity projections of stage 10b control, arpC2-RNAi, and dia-RNAi egg chambers. Arrowheads indicate small, lumenless ring canals. (B) Box and whiskers plot showing the 10-90^th percent values for the diameter of ring canals connecting nurse cells in control, arpC2-RNAi, and dia-RNAi egg chambers. Individual points represent values outside of that range. n = 33-165 ring canals/stage for each condition. Asterisks indicate significant difference compared to control (p<0.05, K-S test). (C) Box and whiskers plot showing the 10-90^th percent values for the volumes of mature eggs. n=30 eggs for control, n=35 eggs for arpC2-RNAi, and n=39 eggs for dia-RNAi. Asterisk indicates significant difference compared to control (p<0.0001, one-way ANOVA with Tukey’s post hoc). Examples of mature eggs for each condition. Scale bar is 100 μm. ArpC2 or Dia was depleted beginning at stage 2 of oogenesis using matαTub-GAL4.

Figure 4:. Expression of a constitutively active form of Diaphanous leads to ring canal collapse and nurse cell fusion.

(A) Maximum intensity projections of stage 10a control, Dia^ΔDAD, and Dia^FH3FH1FH2-expressing egg chambers. White arrowheads indicate small, lumenless ring canals; yellow arrowheads indicate abnormal actin structures. (B) Average number of nurse cell nuclei in Dia^ΔDAD- or Dia^FH3FH1FH2 - expressing egg chambers. Control egg chambers have 15 nurse cell nuclei at each stage. (C) Average number of normal and collapsed/lumenless ring canals in egg chambers expressing Dia^ΔDAD or Dia^FH3FH1FH2 in the germline. Control egg chambers contain a total of 15 ring canals per egg chamber at each stage (11 ring canals connecting nurse cells and 4 ring canals connecting nurse cells to the oocyte; dotted blue lines indicate the number of nurse cell-nurse cell ring canals that each egg chamber should have at each stage.). Error bars are SEM. n = 4-20 egg chambers/stage/condition. Dia^ΔDAD and Dia^FH3FH1FH2 were expressed beginning at stage 2 of oogenesis using matαTub-GAL4.

Figure 5:. Reducing Dia levels provides a modest rescue of the arpC2-RNAi phenotype.

(A) Maximum intensity projections of stage 9 control, dia⁵/+, arpC2-RNAi, and dia⁵/+; arpC2-RNAi egg chambers. Arrowheads indicate small, lumenless ring canals. (B) Box and whiskers plot showing the 10-90^th percent values for the diameter of ring canals connecting nurse cells in control, dia⁵/+, arpC2-RNAi, and dia⁵/+; arpC2-RNAi. Individual points represent values outside of that range. n = 60-264 ring canals/stage for each condition. Asterisks indicate significant difference compared to control; double asterisk indicates significant difference compared to arpC2-RNAi (p<0.05, K-S test). (C) Average number of normal and lumenless ring canals in arpC2-RNAi and dia⁵/+; arpC2-RNAi egg chambers in stages 9, 10a, and 10b. Each egg chamber should contain 15 ring canals at each stage. Error bars are SEM. Asterisk indicates significant difference in the number of lumenless ring canals when compared to arpC2-RNAi alone (p<0.05, 2-tailed t-test). (D) Box and whiskers plot showing the 10-90^th percent values for the volumes of mature eggs. n = 58 eggs for control, n=58 eggs for dia⁵/+, n = 69 eggs for arpC2-RNAi,and n = 63 eggs for dia⁵/+; arpC2-RNAi. Asterisk indicates significant difference compared to control (p<0.0001, one-way ANOVA with Tukey’s multiple comparison post hoc). Examples of mature eggs for each condition are shown. Scale bar is 100 μm. All crosses were done using MTD-GAL4. (E) Schematic summarizing the proposed role for the Arp2/3 complex and Dia in regulation of incomplete cytokinesis and ring canal growth.