Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary

Abstract

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. We show that one daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis.

Figure 1.—

Prefollicle cells associate with cysts in an ordered fashion downstream from the FSCs. (A) A diagram of the Drosophila germarium showing the four subregions: 1, 2a, 2b, and 3. Two GSCs (orange) reside in region 1 and produce cysts (yellow ovals). Two FSCs reside at the border of regions 2a and 2b and produce follicle cells that encapsulate region 2b and region 3 cysts. (B) A diagram of two follicles that have budded from the germarium showing their pairs of anterior and posterior polar cells as well as the interconnecting 4–6 stalk cells. (C) Germaria stained with anti-traffic jam (green) to mark somatic cells, anti-vasa (red) to mark germ cells, and DAPI (blue). The numbers of somatic cells associated with each cyst (indicated) were reconstructed from three-dimensional image stacks. (D–F) Small transient clones stained with anti-LacZ (green, the clonal marker), anti-FasIII (red), and DAPI (blue). Regions 2b and 3 cysts are outlined in white. Pink dots indicate labeled FSC daughters; however, not all labeled cells are marked because some are not visible in the presented plane of focus. (D) A 4-cell clone associated with the first cyst in region 2b. (E) An 8-cell clone associated with the second region 2b cyst. (F) A 15-cell clone associated with the region 3 cyst. (G) Model of follicle layer acquisition. One FSC daughter, the cmc (light green, left) contacts the anterior face of the incoming cyst (2a/b, orange) and founds mostly anterior follicle cells (light green). Another FSC daughter, the pmc (dark green, left) contacts the posterior cyst face and founds mostly posterior follicle cells (dark green). Bar, 10 μm; anterior is to the left.

Figure 2.—

A/P spatial organization and cell type differentiation downstream from the FSCs. (A) An “anterior-biased” clone (green, LacZ), which encompasses ∼50% of total follicle cells (red, FasIII). (B) A “posterior-biased” clone, which encompasses ∼50% of the follicle cells. (C) An FSC clone 8 dphs (green, LacZ) stained with FasIII (red). Neither the FSC (open triangle) nor the daughter cmc (solid triangle) is labeled above background with FasIII. C′ shows the red (anti-FasIII) channel only. (D) An ovariole expressing Imp-GFP stained with anti-GFP (green) and DAPI (blue). Imp-GFP expression is absent in the FSCs (open triangles) and immediate daughters (solid triangle). D′ shows the anti-GFP (green) channel only. (E) A wild-type germarium stained with anti-slit (red) and anti-vasa (green). The FSCs (open triangles) and cmc (solid triangle) have high levels of slit. E′ shows the red (anti-slit) channel only. (F) A clone (green, lacZ) 3 dphs that includes both a polar cell (asterisk) and several follicle cells (curved line) but not stalk cells. (G) A clone (green, lacZ) 3 dphs that contains both stalk cells (solid triangle) and follicle cells (curved white lines) but not polar cells (asterisks). The follicle cells in this clone extend from the posterior of one follicle to the anterior of the adjacent older follicle. Bars, 10 μm; anterior is to the left, and DAPI is pseudocolored in blue.

Figure 3.—

A Notch signaling reporter is activated at multiple steps in the FSC lineage. Gbe-Su(H) LacZ (green) is expressed in (A) a cross-migrating FSC daughter, (B) an FSC (open triangle) and cross-migrating daughter (solid triangle), (C) a cell between the two region 2b cysts (the anterior 2b cyst, indicated by dotted line, is in a different plane of focus), (D) a cell at the posterior of the second region 2b cyst, (E) multiple stage 6 follicle cells (curved line) and polar cells (asterisk), and (F) cap cells (solid triangle). (G) A diagram summarizing sites in regions 2b and 3 where activity was observed. The types of cells and frequency with which they are labeled are indicated (n = 139). FSC, follicle stem cell; cmc, cross-migrating cell; fc, follicle cell. Ovarioles were stained with anti-FasIII (red), anti-β-galactosidase (green), and DAPI (blue). Bar, 10 μm; anterior is to the left.

Figure 4.—

A Notch signal from anterior region 2b germ cells is required for cmc migration. (A) Graph summarizing the percentage of FSC clones of the indicated genotypes that contain a cross-migrating FSC daughter cell. (B–F) Germaria with wild-type or Notch mutant FSC clones, 5 dphs stained with anti-GFP (white, clonal marker). Region 2a cysts (purple dashed lines), negatively marked FSC clones (white dotted lines), FSCs (green asterisks), and cross-migrating FSC daughters (green ovals) are indicated. (B) Wild-type negatively marked clone with a cross-migrating FSC daughter. (C) N^(55e11) negatively marked clone with no cross-migrating FSC daughter. (D) wild-type MARCM clones with a single cross-migrating daughter. (E) N^DN MARCM clones with no cross-migrating FSC daughter. (F) N^act MARCM clone with several cross-migrating daughters. (G) Graph summarizing the frequency of FSC daughter cell cross-migration in wild-type and Delta germline mutant clones 7–9 dphs. (H–J) Germaria with wild-type or Delta mutant germline clones 7–9 dphs stained with anti-GFP (green, clonal marker), traffic jam (red, somatic cell marker) and DAPI (blue). Cross-migrating FSC daughters (solid triangles) and the region 2a cysts (purple dashed lines) are indicated. Cross-migrating FSC daughters are present when a negatively marked wild-type cyst is positioned posterior to the migration path (H) or a negatively marked Delta^−/− cyst is positioned anterior to the migration path (I), but not when a negatively marked Delta^−/− cyst is positioned posterior to the migration path (J). H′, I′, and J′ show the red (somatic cell marker) channel only. Bars, 10 μm; anterior is at the top.

Figure 5.—

Notch signaling is required for FSC replacement. The percentage of ovarioles with one labeled FSC (A and C) or two labeled FSCs (B and D) is shown at 5, 9, and 14 days after clone induction by heat shock. (A and B) Negatively marked wild-type or N^(55e11) clones. (C and D) MARCM wild type and Notch dominant negative (N^dN). Notch mutant FSCs are not replaced by wild-type cells.

Figure 6.—

Polar cells are specified at multiple points in the germarium. (A and B) Anterior (A) and posterior (B) polar cell-only clones coincident with large follicle cell-only clones that cover ∼12.5% (one-eighth) of the follicle. (C and D) Anterior (C) and posterior (D) polar cell-only clones coincident with small follicle cell-only clones that cover ∼3% (1/32nd) of the follicle cell population. Solid white triangles indicate polar cell-only clones. Ovarioles were stained with anti-FasIII (red), anti-β galactosidase (the clonal marker, green), and DAPI (blue). Bar, 50 μm; anterior is to the left. (E) Quantification of polar cell-only transient clones in stage 9–10 follicles at 3–10 dphs. Follicle cell-only clone size was used to estimate the number of cells per follicle at clone induction for each time point. (F) Number of anterior polar cell-only and posterior polar cell-only clones found coincident with follicle-cell only clones. The sizes of the follicle cell-only clones were used to estimate the number of cells per follicle at the time of clone induction. (G) Model showing the timing of Notch/Delta signaling in the germarium. A signal from cysts near the region 2a/2b border (left) induces FSC daughters to cross-migrate. A signal from the posterior region 2b cyst initiates the first round of polar cell specification (middle) and a later signal (right) maintains polar cell fate and initiates additional rounds of polar cell specification.