Symmetry Breaking in an Edgeless Epithelium by Fat2-Regulated Microtubule Polarity

Abstract

Planar cell polarity (PCP) information is a critical determinant of organ morphogenesis. While PCP in bounded epithelial sheets is increasingly well understood, how PCP is organized in tubular and acinar tissues is not. Drosophila egg chambers (follicles) are an acinus-like "edgeless epithelium" and exhibit a continuous, circumferential PCP that does not depend on pathways active in bounded epithelia; this follicle PCP directs formation of an ellipsoid rather than a spherical egg. Here, we apply an imaging algorithm to "unroll" the entire 3D tissue surface and comprehensively analyze PCP onset. This approach traces chiral symmetry breaking to plus-end polarity of microtubules in the germarium, well before follicles form and rotate. PCP germarial microtubules provide chiral information that predicts the direction of whole-tissue rotation as soon as independent follicles form. Concordant microtubule polarity, but not microtubule alignment, requires the atypical cadherin Fat2, which acts at an early stage to translate plus-end bias into coordinated actin-mediated collective cell migration. Because microtubules are not required for PCP or migration after follicle rotation initiates, while dynamic actin and extracellular matrix are, polarized microtubules lie at the beginning of a handoff mechanism that passes early chiral PCP of the cytoskeleton to a supracellular planar polarized extracellular matrix and elongates the organ.

Figure 1. PCP rotation initiates during follicle budding

(A) Stills (opacity projections) from live imaging of germarium through stage 4 follicles, with angular velocity (ω) of each given above. His2AV-mRFP (red) and Vkg-GFP (green) show nuclei and basement membrane respectively; colored dots mark tracked nuclei. The st. 1 follicle initially does not rotate, but between 72 and 132 minutes transitions to st. 2 and begins rotation (B, C) Maximum linear and angular velocity of rotating follicles (n>5 for each stage, bars represent SEM). Color-coded bar indicates relative time length of stages of oogenesis. (D) BM closure during st. 1-2 transition, shown in single confocal section from live imaging. In the non-rotating st. 1 follicle, BM at the anterior is non-continuous (arrowheads). Rotation initiates after BM becomes continuous around the entire follicle. (E, F) Quantitation of relationship between BM completion and follicle rotation (n=11, bars represent SEM).

Figure 2. Germarial PCP revealed by ‘unrolling’ algorithm

(A) Phalloidin-stained st. 6 follicle, shown in (A) single confocal section at basal surface, (A’) maximum intensity projection of confocal stack, and (A”) 3D opacity projections. (B) Diagram of ImSAnE unrolling of the basal surface of the follicle. (C) ImSAnE unrolling of stage 6 follicle stained with phalloidin and (C’) anti-acTub. Nematic order parameters (quantitated in upper left) demonstrate PCP organization. (D) Confocal cross-section of germarium stained with phalloidin (red) and anti-acTub (green). (E) ImSAnE unrolling of region 2b through stage 3 basal surface of ovariole in D. (F) Nematic order quantitation of MTs demonstrate dynamics of PCP organization in the germarium and early follicle. Data for Actin and acetylated Tubulin is shown to the immediate left and right (respectively) of each stage. Scale bars: 10μm.

Figure 3. PCP MTs in the germarium direct rotation

(A) ImSAnE unrolling of basal surface of CLASP-depleted ovariole, showing regions 2b through st. 3 stained with phalloidin and anti-acTub. (B) Nematic order shows intact actin alignment when MTs are disrupted. (n≥5 for each stages, **p<0.01) (C, D) Control stage 9 follicle and mature eggs are elongated, while CLASP-RNAi stage 9 follicles and mature eggs are round. (E) Rotation speeds of st. 6-7 follicles, depleted at specified times using tj> CLASP RNAi tubGAL80ts, or treated with the MT polymerization inhibitor colchicine or the Arp2/3 inhibitor CK-666. (F) Quantitation of tj> CLASP RNAi tubGAL80ts egg shape.

Figure 4. Fat2 regulates rotation initiation and MT chirality

(A) ImSAnE unrolling of basal surface of fat2-depleted ovariole, showing regions 2b through st. 3 stained with phalloidin and anti-acTub. (B) Nematic order shows MT and actin alignment resemble WT until st. 2, but become disrupted following st. 3. (C) Rotation speeds at st. 7-8 of follicles depleted of fat2 by RNAi at specified stages. Follicles depleted of fat2 either prior to or following st. 3 fail to rotate. (D) Aspect ratios of eggs from conditional depletion of fat2, showing strong early and late requirements for egg shape. (E) Egg shapes from fat2-RNAi-mild genetic interaction tests. Heterozygosity for either fat2 or CLASP enhances the round egg phenotype. (F) Still frame from live imaging of MT +end growth in WT st. 1 follicle. (G) Quantification of EB1 growth bias in WT and fat2-depleted st. 1 follicle (0=unbiased direction of growth, 1=fully concordant direction of growth), shows that significant MT growth bias in WT is lost when fat2 is depleted. (H) Population-level MT growth biases at st. 1 and follicle rotation directions at st. 6 show similar proportions of chiralities. (I) Live imaging of EB1-GFP expressing follicles during st.1 to st. 2 transition reveals rotation with a chirality opposite to that of MT growth bias.