Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila

Abstract

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a 'molecular corset' mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ.

Keywords: Drosophila; Egg chamber; Laminin; Morphogenesis; Muscular dystrophy; Vitellogenesis.

Fig. 1.

Introduction to Drosophila ovary structure and Laminin isoforms. (A) A pair of ovaries with two ovarioles highlighted. Modified from Miller (1950), used with permission from Cold Spring Harbor Laboratory Press. (B) An ovariole showing egg chamber morphology at several stages. Ovarian muscles are not shown. (C) Micrographs showing how the muscle sheath envelops the egg chambers. All cells are stained with Phalloidin. The muscle sheath has been false colored red. Ovariole (top panel), transverse (bottom left) and surface views of the same egg chamber (bottom right). (D) Schematic of Drosophila's two Laminin isoforms, LamininA and LamininW.

Fig. 2.

Wb is required in the muscle sheath to shape the egg. (A) Schematic showing how egg aspect ratio was measured. (B) tj-Gal4 drives UAS-mCD8-eGFP in the follicle cells and muscle sheath (arrowheads). (C) tj-Gal4 driving wb-RNAi decreases the egg's aspect ratio. (D) Mef2-Gal4 drives UAS-mCD8-eGFP in the muscle sheath (arrowheads), not the follicle cells. (E) Mef2-Gal4 driving wb-RNAi also decreases the egg's aspect ratio. (F) tj-Gal4, Mef2-Gal80 drives UAS-mCD8-eGFP in the follicle cells, but not the muscle sheath (arrowheads). (G) tj-Gal4, Mef2-Gal80 driving LanA-RNAi decreases the egg's aspect ratio. Driving wb-RNAi has no effect. Scale bars: 20 µm. Data represent mean±s.d., Student's t-test; N.S., not significant; ***P<0.001; n=9-10 eggs per condition. Newly eclosed females were cultured for 6 days at 29°C.

Fig. 3.

Wb depletion largely eliminates muscle sheath contractions. (A) The sliding filament model for muscle contraction. (B,C) Effects of Mef2>wb-RNAi(5d) on sarcomere length. (B) Representative images of sarcomere structure in the muscle sheath overlying stage 10 egg chambers. A-bands (green) are marked with myosin heavy chain GFP (MHC-GFP) and Z-discs (red) are marked with an anti-integrin-βPS. (C) Average Z-disc distance is increased under Mef2>wb-RNAi(5d), whereas A-band length is unchanged. n=100 sarcomeres per condition. (D-F) Mef2>wb-RNAi(6d) dampens ovarian contractions. (D) Movie still of an ovary in culture. The purple line corresponds to the control kymograph in E. (E) Representative kymographs showing ovarian contraction frequency. Contractions are largely eliminated under Mef2>wb-RNAi(6d). (F) Quantification of the data in E. Data represent mean±s.d., Student's t-test; N.S., not significant; ***P<0.001. Scale bar: 5 µm in B, 100 µm in D.

Fig. 4.

Wb depletion reduces egg length and oocyte volume. (A) Representative images showing that egg length is reduced under Mef2>wb-RNAi(6d) (arrow), whereas the width is unchanged. Orange and yellow reference lines are the same length on both images. (B,C) Quantification of the data shown in A. (D) Representative images showing that oocyte size is reduced at stage 10b (arrow) under Mef2>wb-RNAi(6d). Green and pink lines are the same distance apart on both images. (E,F) Measurements of germ cell volume at stage 10b. Under Mef2>wb-RNAi(6d), nurse cell volume is normal (E), but oocyte volume is reduced (F). Data represent mean±s.d., Student's t-test; N.S., not significant; ***P<0.001; n=9-10 eggs/egg chambers per condition. Scale bars: 50 µm.

Fig. 5.

Wb depletion reveals a role for the muscle sheath in vitellogenesis. (A) Representative images showing that the density and mean autofluorescence of yolk granules is reduced under Mef2>wb-RNAi(6d) at stage10b. The yellow boxes on the images on the left indicate regions shown in higher magnification on the right. Scale bars: 20 µm. (B) Graphs quantifying the effect shown in A. n=7 egg chambers per condition. (C) Western blot showing that yolk protein levels are increased in the hemolymph under Mef2>wb-RNAi(6d). Two independent experiments are shown. Please see Materials and Methods for a discussion of loading controls. (D-G) Measurements performed on egg chambers from females where yolk protein levels have been reduced genetically (Yp1^ts/Df(1)C52). This condition has a similar effect to Mef2>wb-RNAi(6d), as egg length (D,E) and stage 10b oocyte volume (F,G) are both selectively reduced. Data represent mean±s.d., Student's t-test; N.S., not significant; ***P<0.001; n=9-10 eggs/egg chambers per condition in D-G.

Fig. 6.

Eggs that develop outside the muscle sheath have reduced length. (A) Schematic for germarium transplantation. Modified from Miller (1950), used with permission from Cold Spring Harbor Laboratory Press. (B,C) Eggs that develop outside the muscle sheath have reduced length (B) and normal width (C); n=20-24 eggs per condition. (D,E) At stage 10b, nurse cell volume is normal (D) and oocyte volume is reduced (E); n=5 egg chambers per condition. Data represent mean±s.d., Student's t-test; N.S., not significant; **P<0.01, ***P<0.001.

Fig. 7.

Weak defects in nurse cell dumping reduce egg length. (A) Representative images showing that egg length is reduced under two conditions that cause weak nurse cell dumping defects (arrows), whereas the width is unchanged. (B,C) Quantification of the data shown in A. Data represent mean±s.d., Student's t-test; N.S., not significant; **P<0.01, ***P<0.001; n=14-16 eggs per condition. Scale bars=50 µm.

Fig. 8.

Wb depletion can also cause muscle sheath hyper-contraction. (A,B) Depletion of either Wb or Dg from the muscle sheath can both increase and decrease the aspect ratio of the egg. Wb (A) and Dg (B) were depleted from the muscle sheath and egg aspect ratio was measured on a daily basis for one to seven days. Aspect ratio is initially increased, but decreases over time until it is below that of controls. n=9-10 eggs per genotype per day. (C,D) Effects of Mef2>wb-RNAi(1d) on sarcomere length in the muscle sheath overlying stage 10 egg chambers. (C) Representative images where A-bands (green) are marked with myosin heavy chain GFP (MHC-GFP) and Z-discs (red) with anti-integrin-βPS. (D) The average Z-disc distance is decreased under Mef2>wb-RNAi(1d), whereas A-band length is unchanged. n=100 sarcomeres per condition. (E-G) Effects of Mef2>wb-RNAi(1d) on ovarian contractions. (E) Movie still of an ovary in culture. The purple line corresponds to the control kymograph in F. (F) Representative kymographs showing ovarian contraction frequency is increased under Mef2>wb-RNAi(1d). (G) Quantification of the data in F. Data represent mean±s.d., Student's t-test; N.S., not significant; **P<0.01, ***P<0.001. Scale bar: 5 µm in C, 100 µm in E.

Fig. 9.

A hyper-contractile muscle sheath enhances egg chamber elongation. (A) Representative images showing that egg width is reduced under Mef2>wb-RNAi(1d) (arrow), whereas length is unchanged. Orange and yellow reference lines are the same length on both images. Scale bar: 50 µm. (B,C) Quantification of egg length and width data in A. (D,E) Measurements of germ cell volume at stage 10b. Under Mef2>wb-RNAi(1d), nurse cell volume is normal (D), but oocyte volume is reduced (E). (F-H) Wb depletion affects the way the muscle sheath envelops the egg chambers. (F) Muscle sheath morphology can be binned into three categories; tight, normal and loose. Arrowheads mark gaps between the muscle sheath and the egg chambers. (G,H) Quantification of muscle sheath morphology. (G) In controls, the normal morphology predominates. Under Mef2>wb-RNAi(6d), the loose morphology predominates. (H) Under Mef2>wb-RNAi(1d), the tight morphology predominates. (I,J) Aspect ratio measurements across stages 4-14. (I) The aspect ratio for Mef2>wb-RNAi(6d) egg chambers does not fall below controls until stage 9, after vitellogenesis begins. (J) By contrast, the aspect ratio for Mef2>wb-RNAi(1d) egg chambers is higher than controls from stage 4. Data represent mean±s.d., Student's t-test in B-E,I,J, Chi squared test in G,H; N.S., not significant; **P<0.01, ***P<0.001; n=9-10 egg chambers per condition except where noted in G,H.