EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells

Abstract

Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. We found that, in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, epidermal growth factor receptor (EGFR) signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, we identified a novel connection between EGFR signaling and the cell-polarity regulator liver kinase B1 (LKB1), which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity. The development of apical-basal polarity is the earliest visible difference between FSCs and their daughters, and our findings demonstrate that the EGFR-mediated regulation of apical-basal polarity is essential for the segregation of stem cell and daughter cell fates.

Keywords: D. melanogaster; EGFR; cell biology; cell polarity; developmental biology; epithelial cells; niche; stem cells.

Figure 1.. The EGFR pathway is upregulated specifically in FSCs.

(A) Diagram of the germarium of the Drosophila ovary. The germarium is divided into four regions as indicated; anterior is to the left. Two follicle stem cells (FSCs, light grey) are maintained in the germarium at the Region 2a/2b border. Escort cells (blue) are anterior to the FSCs and support development of the early germline (orange). As they mature, germline cysts move posteriorly out of Region 2a and into the follicle epithelium. Each FSC divides once per incoming cyst, producing prefollicle cells (dark grey) that encapsulate the germline as it moves into Region 2b. (B) Quantification of pErk staining of FSCs and prefollicle cells just downstream from the niche within a wildtype or Egfr^f24 FSC clone. (C–D) Wildtype (C) or Egfr^f24 (D) FSC clones stained for pErk (red), GFP (green) and DAPI (blue). Boxed regions of C–D are magnified in C′–C′′′ and D′–D′′′. White arrows indicate the FSC, which is the anterior-most GFP⁽⁻⁾ follicle cell in the clone. White dashed line in (C′) indicates prefollicle cells in which pErk is undetectable compared to the FSC. Scale bar represents 5 μm in C–D and 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.003

Figure 1—figure supplement 1.. pErk staining in the ovariole A.

(A) Wildtype ovariole with bright pErk staining (red) detectable in escort cells, early (stage 2 and 3) follicles, and at the position of the FSC niche (white arrows). (B) A wildtype GFP⁽⁻⁾ FSC clone with bright pErk in FSCs (white arrows), and in prefollicle cells that recently divided from the FSC in the clone (blue asterisks in B′–B′′′). All tissues stained with DAPI (blue). Anterior is to the left. Boxed region in B is magnified in B′–B′′′. Scale bars represent 5 μm in A–B and 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.004

Figure 2.. EGFR is required for FSC maintenance in the niche.

(A–B) Germaria with a mature wildtype (A) or Egfr^f24 (B) GFP⁽⁻⁾ FSC clone stained for Dlg (red) and GFP (clone marker, green). (C–E) Graphs indicating the frequencies of the Egfr^f24 or control FSC clones at 2, 4, 7, and 11 dpci (C); all Egfr^f24 or control clones, including polarity-defective Egfr^f24 prefollicle cell (PFC) clones, at 2 dpci (D); and the Egfr^λtop or control FSC clones at 7, 14, and 21 dpci (E). (F) Polarity-defective Egfr^f24 prefollicle cell clone at 2 dpci, stained for Dlg (red) and GFP (green); F′ shows the GFP channel alone; boxed regions are magnified in F′′–F′′′. GFP⁽⁻⁾ clones are indicated by dashed yellow lines, and by white asterisks in F′′–F′′′. White arrows indicate the position of the FSC niche. All tissues stained with DAPI (blue). Anterior is to the left. Scale bar represents 5 μm in A–F and 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.005

Figure 2—figure supplement 1.. Quantification of marked control and Egfr^f24 FSC clone frequencies at 2, 4, 7, and 11 dpci.

Values reflect the percent of germaria that have the indicated the number of GFP⁽⁻⁾ FSCs at the indicated timepoints. The GFP⁽⁻⁾ cells are either wildtype (control column) or mutant for Egfr (Egfr^f24 column). DOI: http://dx.doi.org/10.7554/eLife.04437.006

Figure 2—figure supplement 2.. Quantification of marked control and Egfr^λtop FSC clone frequencies at 7, 14, and 21 dpci.

Values reflect the percent of germaria that have the indicated number of GFP⁽⁻⁾ FSCs at the indicated timepoints. The GFP⁽⁻⁾ cells are either wildtype (control column) or express Egfr^λtop (Egfr^λtop column). P-values were determined using a two-tailed t-test. DOI: http://dx.doi.org/10.7554/eLife.04437.007

Figure 3.. Egfr^f24 FSC clones have epithelial polarity defects.

(A–J) Wildtype (A–E) and Egfr^f24 (F–J) FSC clones stained for polarity markers (red) Dlg (A and F), aPKC (B and G), Baz (C and H), DE-cad (D and I) and β-integrin (β-int) (E and J); GFP (green); and DAPI (blue). Panels A′–J′ show the red channel only. All polarity markers are able to localize properly in wildtype GFP⁽⁻⁾ FSC clones and in the GFP⁽⁺⁾ follicle cells within germaria that contain either wildtype or Egfr^f24 FSC clones; all polarity markers are undetectable in GFP⁽⁻⁾ Egfr^f24 FSC clones. GFP⁽⁻⁾ clones are indicated by dashed yellow lines. Images are oriented with the apical surface of the follicle cells on the bottom. Scale bar represents 1 μm. DOI: http://dx.doi.org/10.7554/eLife.04437.008

Figure 3—figure supplement 1.. Positively marked Egfr^f2 FSC clones have epithelial polarity defects.

(A–D) Wildtype (A, C) and Egfr^f2 (B, D) prefollicle cell clones (A–B) and FSC clones (C–D) marked by the presence of GFP and stained for polarity marker Dlg (red); GFP (green); and DAPI (blue). Dlg is able to localize properly in wildtype GFP⁽⁺⁾ FSC clones, in the GFP⁽⁻⁾ follicle cells within germaria that contain either wildtype or Egfr^f2 FSC clones, and in Egfr^f2 GFP⁽⁺⁾ prefollicle cell clones; Dlg is undetectable in Egfr^f2 GFP⁽⁺⁾ FSC clones. (E–F) Germaria with wildtype (E) or Egfr^f2 (F) GFP⁽⁺⁾ FSC clones stained for Dlg (red). GFP⁽⁺⁾ clones are indicated by dashed yellow lines. Images in (A–D) are oriented with the apical surface of the follicle cells on the bottom; in (E–F) anterior is to the left. Scale bar represents 1 μm in (A–D), and 5 μm in (E–F). Panels A′–F′ show the red channel only. DOI: http://dx.doi.org/10.7554/eLife.04437.009

Figure 3—figure supplement 2.. Quantification of the frequency of polarity phenotypes in positively marked control FSC clones, Egfrf2 FSC clones, and Egfrf2 prefollicle cell clones.

Values reflect both the percent and fraction of each clone type in which polarity is disrupted or notdisrupted as indicated. DOI: http://dx.doi.org/10.7554/eLife.04437.023

Figure 3—figure supplement 3.. Polarity defects in Egfr^DN follicle cells.

(A) Germarium in which GFP (green) is expressed using 109-30-Gal4 to indicate the expression pattern of the 109-30-Gal4 driver in follicle cells. (B–C) Germaria containing UAS-Egfr^DN/UAS-Egfr^DN but no Gal4 driver (B), or 109-30-Gal4 and UAS-Egfr^DN/UAS-Egfr^DN (C), stained for Dlg (red) and Vasa (green). Dlg localization is disrupted in the germarium overexpressing Egfr^DN (C), which phenocopies the polarity defects seen in Egfr^f24 FSC clones. All tissues stained with DAPI (blue). White arrows indicate the position of the FSC niche. Anterior is to the left. Boxed regions of B′–C′ are magnified in B′′–C′′. Scale bar represents 5 μm in (A–C), and 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.010

Figure 4.. Loss of EGFR does not cause cell death or loss of follicle cell identity.

(A) Egfr^f24 FSC clone with normal FasIII (red) and Traffic jam (Tj) (cyan) in the clone, indicated by white asterisks in the magnified regions in (A′–A′′′). (B) Egfr^f24 FSC clone with a Cas3-positive cell (red, yellow arrowhead) in the polar region of a newly budded follicle, but not in the clone. Panel B′ shows the red channel only. (C) Graph indicating the frequency of Cas3-positive follicle cells in Egfr^f24/+ control germaria or in Egfr^f24 FSC clones. GFP⁽⁻⁾ clones are indicated by dashed yellow lines. All tissues stained with DAPI (blue). Anterior is to the left. Scale bar represents 5 μm in (A–B), and 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.012

Figure 5.. Egfr^f24 prefollicle cell clones do not have epithelial polarity defects.

(A–J) Wildtype (A–E) and Egfr^f24 (F–J) prefollicle cell clones stained for polarity markers (red) Dlg (A and F), aPKC (B and G), Baz (C and H), DE-cad (D and I) and β-int (E and J); GFP (green); and DAPI (blue). Panels A′–J′ show the red channel only. All polarity markers are properly localized in both wildtype and Egfr^f24 GFP⁽⁻⁾ prefollicle cell clones. GFP⁽⁻⁾ clones are indicated by dashed yellow lines. Images are oriented with the apical surface of the follicle cells on the bottom. Scale bar represents 1 μm. DOI: http://dx.doi.org/10.7554/eLife.04437.013

Figure 5—figure supplement 1.. pErk is absent from Egfr^f24 prefollicle cell clones.

A. Egfr^f24 prefollicle cell clone (indicated by dashed yellow line) that lacks pErk signal (red). Clone is GFP⁽⁻⁾ and DAPI is in blue. The GFP channel alone is shown in A′ and the pERK channel alone is shown in A″. Images are oriented with the apical surface of the follicle cells on the bottom. Scale bar represents 1 μm. DOI: http://dx.doi.org/10.7554/eLife.04437.014

Figure 6.. Constitutive activation of EGFR disrupts prefollicle cell apical polarity.

(A–D) Control germaria containing UAS-Egfr^λtop but no Gal4 driver (A and C) and experimental germaria in which Egfr^λtop is expressed in follicle cells under the control of 109-30-Gal4 (B and D) and stained for Dlg (red), DAPI (blue), and either aPKC (green, A–B) or Baz (green, C–D). Follicle cells along the Region 3 cyst (R3, yellow dashed line) of control germaria have a cuboidal shape with a clear apical surface (A′′, orange arrowheads); aPKC localizes to the apical surface (A′′), Baz localizes to apical–lateral junctions (C′′), and Dlg localizes to lateral surfaces (A′ and C′). In germaria expressing Egfr^λtop in which the R3 cyst is present, cells have a pointed shape and form narrow contacts with the germline (B′, orange arrowheads). In addition, aPKC is delocalized from the cell surface of follicle cells (B′′), but Dlg is detectable on the cell membrane (B′ and D′) and Baz localizes to apical–lateral junctions (D′′). (E–F) Graphs indicating the frequencies of control or experimental germaria with no R3 cyst, or with localized or delocalized aPKC (E) or Baz (F) in follicle cells along the R3 cyst. Boxed regions of (A–D) are magnified in A′–D′′. Anterior is to the left. Scale bar represents 5 μm in A–D an 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.015

Figure 6—figure supplement 1.. Expression of Egfr^λtop in follicle cells.

(A) Germarium in which Egfr^λtop is expressed in follicle cells using 109-30-Gal4 with bright pErk (red) throughout the follicle epithelium. (B) Germarium stained for aPKC (red) in which GFP (green) is expressed using 109-30-Gal4 to indicate the expression pattern of the 109-30-Gal4 driver in follicle cells. (C–D) Germaria in which Egfr^λtop is expressed in follicle cells using 109-30-Gal4 and stained for Dlg (red) and either Baz (green, C) or aPKC (green, D). Germarium in panel C has no observable R3 cyst. Boxed regions of B and D are magnified in insets. Anterior is to the left. All tissues stained with DAPI (blue). Scale bar represents 5 μm and 1 μm in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.016

Figure 7.. EGFR functions upstream of Ras and LKB1 to establish epithelial polarity.

(A–D) GFP⁽⁻⁾ Ras85D⁻ (A) or lkb1⁻ (C) FSC clones and Ras85D⁻ (B) or lkb1⁻ (D) prefollicle cell clones stained for Dlg (red) and GFP (green). (E) Graph indicating the frequencies of polarity phenotypes in wildtype, Ras85D⁻, and lkb1⁻ FSC clones and in Ras85D⁻ and lkb1⁻ prefollicle cell clones. (F–H) Germaria containing UAS-Egfr^DN/UAS-Egfr^DN but no Gal4 driver (F), 109-30-Gal4 and UAS-Egfr^DN/UAS-Egfr^DN (G), or 109-30-Gal4, UAS-Egfr^DN/UAS-Egfr^DN and UAS-GFP-lkb1^S535E (H) stained for Dlg (red), and either Vasa (green, F–G) or GFP (green, H). Dlg localization is disrupted in the germaria overexpressing Egfr^DN only (G), but it is restored in germaria overexpressing both Egfr^DN and lkb1 ^S535E (H) (I) Graph indicating the frequencies of polarity phenotypes in control, Egfr^DN only, and Egfr^DN and lkb1 ^S535E co-expressing germaria. (J–K) Germaria containing UAS-Egfr^DN/UAS-Egfr^DN but no Gal4 driver (J) or 109-30-Gal4 and UAS-Egfr^DN/UAS-Egfr^DN (K) stained for Dlg (red) and pAMPK (green), which is detectable in prefollicle cells of the control (yellow arrowhead, J) but not in germaria overexpressing Egfr^DN (K). Images in (A–D) are oriented with the apical surface of the follicle cells on the bottom, and GFP⁽⁻⁾ clones are indicated by dashed yellow lines. Panels A′–D′ show the red channel only. Boxed regions of F–H are magnified in F′–H′, and white arrows indicate the position of the FSC niche. All tissues stained with DAPI (blue). Anterior is to the left in F–K. Scale bar represents 5 μm in F–K and 1 μm in A–D and in magnified insets. DOI: http://dx.doi.org/10.7554/eLife.04437.017

Figure 7—figure supplement 1.. Polarity phenotypes of Ras85D− and lkb1− FSC clones.

(A–B) Germaria with Ras85D− FSC clones stained for Dlg (red) and GFP (green). Ras85D− FSC clone in panel A has disrupted Dlg localization whereas in the Ras85D− FSC clone in panel B Dlg localization is not disrupted. (C) lkb1− FSC clone stained for Dlg (red) and GFP (green) with disrupted Dlg localization. (D) lkb1− FSC clone stained for pAMPK (red) and GFP (green) with no detectable pAMPK in the clone. Panels A′–D′ show the red channel only. (E) Graph indicating the frequencies of detectable pAMPK in lkb1− FSC clones and in wildtype prefollicle cells of the same tissue (lkb1−/+). All tissues stained with DAPI (blue). GFP(−) clones are indicated by dashed yellow lines. Anterior is to the left. Scale bar represents 5 μm. DOI: http://dx.doi.org/10.7554/eLife.04437.018

Figure 8.. A model for the role of EGFR in the establishment of epithelial polarity.

High levels of EGFR signaling in the FSC promote maintenance in the niche and the formation of basal and lateral domains while suppressing the formation of an apical domain. EGFR activates both the canonical Ras-mediated pathway leading to the phosphorylation of Erk, and the LKB1–AMPK pathway. Both Erk and AMPK are kinases that can regulate gene activity by activating transcription factors and phosphorylating proteins in the cytoplasm. AMPK directly promotes the lateral identity in polarized cells by activating lateral proteins. PKA is an upstream activator of LKB1 in follicle cells, and PKA can be activated by EGFR signaling, suggesting that EGFR signaling may activate LKB1 via PKA. EGFR signaling may suppress apical polarity either directly by regulating the transcription or activity of apical proteins, or indirectly by enhancing the activity of lateral proteins that suppress the localization of apical proteins. Low levels of EGFR signaling in prefollicle cells relieves this suppression, allowing apical domains to form and permitting differentiation away from the stem cell fate. DOI: http://dx.doi.org/10.7554/eLife.04437.020