Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche

Abstract

In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. We have found that the phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period.

Keywords: Drosophila; EGFR; Epithelial stem cell; Groucho; Ovary; Six4.

Fig. 1.

RNA-seq of follicle cells expressing constitutively active EGFR implicates the transcription factor Six4 in follicle cell differentiation. (A) Map of cell lineages in the follicle epithelium, including some of the known signaling inputs. Numbers at the bottom of the diagram indicate the approximate generation in the FSC lineage of each transition: the FSC division (generation 1) produces a pFC; pFCs divide 1–3 more times (generations 2–4) before committing to the polar cell fate; and differentiation towards the stalk and main body fates occurs over subsequent generations. EGFR promotes FSC self-renewal, Notch promotes the polar fate and Upd promotes the stalk fate. Expression of Cas or Eya is indicated on each cell type. (B) Schematic presentation of a germarium and the most anterior budded follicles, color coded to match the lineages of A. The regions of the germarium and stages of follicle development are indicated below. (C) Morphology of a wild-type germarium. Fas3 (red) staining outlines cell membranes in early follicle cells. Vasa (green) staining marks the germline cysts of the developing follicles. (D) Differentiation status of wild-type follicle cells, as monitored by staining for Cas (green) and Eya (red). Undifferentiated prefollicle cells express both Cas and Eya (solid line), whereas main body cells express only Eya and polar/stalk cells (arrowheads) express only Cas. (E) Follicle cells expressing EGFR^λtop show an expansion of follicle cell staining for both Cas and Eya (solid line and arrowheads). D′,E′ and D″,E″ show the Cas and Eya channels, respectively. (F,F′) Six4 staining is uniform in the follicle cells of regions 2b and 3 of the germarium (solid lines), nuclear in the main body follicle cells of budded follicles (dotted lines) and absent from stalk cells (arrowheads). Scale bars: 10 μm. DAPI is in blue.

Fig. 2.

Six4 is required for polar and stalk cell formation. (A) Knockdown of Six4 prevents stalk formation between adjacent follicles (arrowheads). (B) Overexpression of Six4 causes the accumulation of extra cells between adjacent follicles (arrowheads). (C-C″,E-E″) Knockdown of Six4 (C-C″) or Notch (E-E″) by RNAi causes some follicle cells (arrowheads) to remain Cas⁺ Eya⁺ in follicles that have budded from the germarium. (D-D″,F-F″) Overexpression of Six4 (D-D″) or N^intra (F-F″) leads to ectopic Cas⁺ Eya^– cells (arrowheads) between adjacent follicles. (G,H) Using the polar cell driver upd-Gal4, neither knockdown of Six4 nor overexpression of EGFR^λtop cause follicle cell phenotypes, indicating that polar cell maintenance is unaffected by these inputs. Insets in C-F″ are enlarged to the right of each panel. Scale bars: 10 μm. DAPI is in blue.

Fig. 3.

Six4 promotes the polar cell lineage via Notch signaling. (A,A′) Expression of neur-lacZ (red, arrowheads) in wild type. Expression is low in two to four pFCs on the anterior face of a region 3 follicle and high in pairs of polar cells at subsequent stages. (B,B′) Knockdown of Six4 by RNAi eliminates neur-lacZ reporter expression in cells positioned to become polar cells (arrowheads). (C,C′) Overexpression of Six4 causes ectopic activation of neur-lacZ (red) in main body (arrowheads) and stalk cells (arrow) near the polar cells, but not in all follicle cells. (D,D′) Expression of NRE-GFP (green) in wild type. GFP is detected in four to six cells at the anterior face of a follicle budding from the germarium, but is restricted to pairs of polar cells in later stages (arrowheads). (E,E′) Knockdown of Six4 by RNAi causes loss of NRE-GFP activity in cells positioned to become polar cells (arrowheads). Occasional follicle cells can still activate NRE-GFP (arrow), but fail to induce stalk formation. (F,F′) Overexpression of Six4 causes ectopic activation of NRE-GFP (green) in many cells near the border between adjacent follicles (e.g. yellow dashed lines) but not in all follicle cells. (G-G″) Expression of N^intra is epistatic to Six4 RNAi, indicating that Six4 is likely upstream of Notch cleavage. Many ectopic Cas⁺ Eya^– cells are observed (arrowheads). Scale bars: 10 μm. DAPI is in blue.

Fig. 4.

Gro is required for the specification of the polar and stalk cell lineages. (A,A′) Gro is expressed most strongly in follicle cells, but also in IGS and germ cells. (B,D-D″) Knockdown of gro causes severe multilayering, fusion of adjacent follicles and ectopic Cas⁺ Eya⁺ cells (arrowheads) beyond the germarium, indicative of excess undifferentiated follicle cells and a lack of stalk cells. (C-E″) Overexpression of gro^AA causes elongated, multilayered stalks and elicits the accumulation of excess Cas⁺ Eya^– cells between adjacent follicles (arrowheads). (F-F″) Stalk-like Cas⁺ Eya⁻ cells (arrowheads) are present between follicles in ovarioles expressing gro^AA and EGFR^λtop. (G) Percentage of ovarioles with Cas⁺ cells between follicles overexpressing gro^AA or EGFR^λtop, or both.***P<0.001 using a two-tailed t-test. Scale bars: 10 μm. DAPI is in blue.

Fig. 5.

Phosphorylation of Gro prevents Notch signaling and polar cell specification in early pFCs. (A,A′) Expression of gro RNAi causes loss of NRE-GFP activity in early stage follicles (arrowheads). A separate wave of Notch activation occurs surrounding stage 6 follicles (solid line), which is beyond the range of 109-30 expression. (B,B′) Expression of gro^AA, which is refractory to ERK-mediated phosphorylation, causes ectopic Notch activity throughout the early follicle cell lineage, including FSCs (arrowhead). (C,C′) Expression of gro^AA causes ectopic activation of neur-lacZ in stalk cells (arrowheads). (D-E″) Phosphorylated Gro (p-Gro) is observed only in FSCs and pFCs within three cell diameters of the niche (arrowheads). (F-F″) Co-staining for Gro (green) and p-Gro (red) indicates that FSCs and early pFCs are p-Gro⁺, Gro⁻ (arrowheads), whereas later pFCs are p-Gro⁻, Gro⁺ (arrows). Scale bars: 10 μm. DAPI is in blue.

Fig. 6.

Six4 and Gro are required to maintain neutral FSC competition. (A) FSC competition assay using MARCM clones. The proportion of germaria that were double labeled, single labeled or unlabeled are indicated by the black, hatched or white bars, respectively. Timepoints observed were 7, 14 and 21 days after clone induction. At least 250 ovarioles scored for each genotype and time point. (B) Rates of FSC clonal extinction or expansion. Error bars indicate the 95% confidence intervals. (C) Competitive bias for the indicated genotypes. FRT 2A and FRT 40A controls exhibit neutral competition. Six4 loss, either by Six4 RNAi or homozygosity for the Six4¹⁰⁸ allele, causes hypercompetition. Six4 overexpression is neutral. Expression of either gro RNAi or gro^AA causes strong FSC hypocompetiton. Error bars indicate the 95% confidence intervals. *P<0.05 for the null hypothesis that b=0. (D) Interactions of Six4 and Gro in the FSC niche. Within the niche, EGFR maintains Gro phosphorylation to inhibit Notch signaling, and thus differentiation. Cells that have recently left the niche temporarily maintain p-Gro, thus resisting differentiation to allow for replacement events and transit amplifying divisions. As the prevalence of p-Gro decreases, non-phosphorylated Gro becomes available to engage in Notch signaling. Six4 and Gro both promote Notch signaling to initiate differentiation of pFCs specifically towards the polar cell fate.