The Drosophila CPEB Protein Orb Specifies Oocyte Fate by a 3'UTR-Dependent Autoregulatory Loop

Abstract

orb encodes one of the two fly CPEB proteins. These widely conserved proteins bind to the 3'UTRs of target messenger RNAs (mRNAs) and activate or repress their translation. We show here that a positive autoregulatory loop driven by the orb gene propels the specification of oocyte identity in Drosophila egg chambers. Oocyte fate specification is mediated by a 3'UTR-dependent mechanism that concentrates orb mRNAs and proteins in one of the two pro-oocytes in the 16-cell germline cyst. When the orb 3'UTR is deleted, orb mRNA and protein fail to localize and all 16 cells become nurse cells. In wild type, the oocyte is specified when orb and other gene products concentrate in a single cell in region 2b of the germarium. A partially functional orb 3'UTR replacement delays oocyte specification until the egg chambers reach stage 2 of oogenesis. Before this point, orb mRNA and protein are unlocalized, as are other markers of oocyte identity, and the oocyte is not specified. After stage 2, ∼50% of the chambers successfully localize orb in a single cell, and this cell assumes oocyte identity. In the remaining chambers, the orb autoregulatory loop is not activated and no oocyte is formed. Finally, maintenance of oocyte identity requires continuous orb activity.

Keywords: 3’UTR; Bicaudal D; CPEB protein; Cytoplasmic polyadenylation; Egalitarian; mRNA cargo complex; mRNA localization; oocyte specification; orb; positive autoregulation.

Figure 1

orb 3′UTR deletion design and replacement strategy. (A) Diagram of the endogenous orb gene and sites targeted by Cas9 within the 3′UTR. Homology-directed repair was used to engineer an attP site within the orb 3′UTR. (B) Fragments of the orb 3′UTR and summary of the localization activity of these fragments when fused to a lacZ transgene [from Lantz et al. (1994)].

Figure 2

Enrichment of orb mRNA and protein requires the orb 3′UTR. (A) Maximum intensity projections of orb mRNA in region 2b, stage 1 and stage 2 of oogenesis for WT, orbΔ3′UTR, and orbΔ3′UTR overexpressing (o/e) a full-length orb cDNA driven by nanos-Gal4::VP16. (B) Maximum intensity projections of Orb protein in region 2b, stage 1 and stage 2 of oogenesis for WT, orbΔ3′UTR, and orbΔ3′UTR overexpressing (o/e) a full-length orb cDNA driven by nanos-Gal4::VP16. (C) Top: plot of the ratio of orb mRNA enrichment in the oocyte compared with nurse cells at stage 1 for WT (n = 14), orbΔ3′UTR (n = 16), and orbΔ3′UTR overexpressing (o/e) full-length orb mRNA by nanos-Gal4::VP16 (n = 14). Bottom: box plot of the ratio of Orb protein enrichment in the oocyte compared to the nurse cells at stage 1 for WT (n = 21), orbΔ3′UTR (n = 18), and orbΔ3′UTR overexpressing (o/e) full-length orb mRNA by nanos-Gal4::VP16 (n = 16). For all panels, *** P < 0.0005, ns = not significant. (D) Maximum intensity projections of BicD mRNA in WT and orbΔ3′UTR germariums and stage 1. Arrowheads indicate localized BicD mRNA WT cysts. (E) Maximum intensity projections of oskar mRNA in WT and orbΔ3′UTR germariums and stage 1. Arrowheads indicate oskar mRNA localized within WT 16-cell cysts. In orbΔ3′UTR, arrowheads indicated oskar mRNA localized to multiple cells at stage 1. (F) Localization of BicD mRNA (n = 11) and orbΔ3′UTR (n = 13) and oskar mRNA at stage 1 in WT (n = 32) and orbΔ3′UTR (n = 23). Bar for all panels, 10 µm. WT, wild type.

Figure 3

The orb 3′UTR is required for oocyte specification. (A) Egl localization in WT and orbΔ3′UTR region 2b cysts and stage 1 egg chambers. (B) The number of cells in which Egl is enriched is quantified for WT (2b n = 13; stage 1 n = 13) and orbΔ3′UTR (2b n = 47; stage 1 n = 50). (C) Dynein localization in WT and orbΔ3′UTR germariums and stage 1 egg chambers. (D) The number of cells in which Dynein is enriched is quantified for WT (2b n = 35; stage 1 n = 35) and orbΔ3′UTR (2b n = 35; stage 1 n = 36). WT, wild type.

Figure 4

Microtubule organization and restriction of Corolla to one cell requires 3′UTR directed enrichment of orb. Top: in WT stage 1 egg chambers, α-tubulin is enriched in one cell (the oocyte), and Corolla is localized in nucleus of that cell. In orbΔ3′UTR and orb-XN 3′UTR, α-tubulin is enriched between two cells, and Corolla is localized to both cells. Bottom: quantification of defects in α-tubulin localization for WT (n = 12), orbΔ3′UTR (n = 15), and orb-XN 3′UTR (n = 13). See Figure 6 for quantification of defects in Corolla restriction. Bar for all panels, 10 µm. WT, wild type.

Figure 5

Localization of orb mRNA and protein in the orb-XN 3′UTR replacement. (A and B) Maximum intensity projections of orb mRNA in WT and orb-XN 3′UTR, showing young egg chambers within an ovariole (A) or focused on localization within the germarium and stages 1 and 2 (B). (C) Plot of the ratio of orb mRNA enrichment at stage 1 in the oocyte compared to the nurse cells in WT (n = 14), orbΔ3′UTR (n = 16), orb-XN 3′UTR (n = 16), and orbΔ3′UTR overexpressing (o/e) full-length orb mRNA by nanos-Gal4::VP16 (n = 14). (D and E) Maximum intensity projections of Orb protein in WT and orb-XN 3′UTR, showing young egg chambers within an ovariole (D) or focused on localization within the germarium and stages 1 and 2 (E). (F) Plot of the ratio of Orb protein enrichment at stage 1 in the oocyte compared to the nurse cells in WT (n = 21), orbΔ3′UTR (n = 18), orb-XN 3′UTR (n = 20), and orbΔ3′UTR overexpressing (o/e) full-length orb mRNA by nanos-Gal4::VP16 (n = 16). For all panels, *** P < 0.0005, ns = not significant. Bar for all panels, 10 µm. WT, wild type.

Figure 6

Oocyte specification is delayed in XN 3′UTR. (A) Maximum intensity projections of oskar mRNA in WT, orb-XN 3′UTR, and orbΔ3′UTR overexpressing (o/e) full-length orb mRNA by nanos-Gal4::VP16. Arrowheads point to osk mRNA localization in multiple cells in orb-XN 3′UTR. Arrow points to a stage 2 orb-XN 3′UTR chamber in which osk mRNA is localized to a single cell. Bars, 10 µm. (B) Plots show the localization pattern of osk mRNA during region 2b, stage 1 and stage 2. Top, region 2b: WT (n = 30), orbΔ3′UTR (n = 24, P < 0.0001), orb-XN 3′UTR (n = 34, P < 0.0001), and orbΔ3′UTR overexpressing (o/e) full-length orb (n = 14, P-value is not significant). Middle, stage 1: WT (n = 32), orbΔ3′UTR (n = 23, P < 0.0001), orb-XN 3′UTR (n = 36, P < 0.0001), and orbΔ3′UTR overexpressing (o/e) full-length orb (n = 14, P-value is not significant). Bottom, stage 2: WT (n = 32), orbΔ3′UTR (n = 15, P < 0.0001), orb-XN 3′UTR (n = 30, P < 0.0001), and orbΔ3′UTR overexpressing (o/e) full-length orb (n = 12, P-value is not significant). P-values are for each group compared with wild type. (C) Maximum intensity projections of Corolla in WT, orb-XN 3′UTR, and orbΔ3′UTR overexpressing (o/e) full-length orb mRNA by nanos-Gal4::VP16. Arrows in WT indicate Corolla localization to a single cell. Arrowheads in orb-XN 3′UTR indicate Corolla enrichment in multiple cells in a cyst. Bars, 10 µm. (D) Plots show localization pattern of Corolla in region 2b, stage 1 and stage 2 WT, orbΔ3′UTR, orb-XN 3′UT, and orbΔ3′UTR (o/e) overexpressing full-length orb. Top, region 2b: WT (n = 37), orbΔ3′UTR (n = 30, P < 0.0001), orb-XN 3′UTR (n = 31, P < 0.0001), and orbΔ3′UTR overexpressing (o/e) full-length orb (n = 17, P-value is not significant). Middle, stage 1: WT (n = 38), orbΔ3′UTR (n = 36, P < 0.0001), orb-XN 3′UTR (n = 34, P < 0.0001), and orbΔ3′UTR overexpressing (o/e) full-length orb (n = 15, P < 0.001). Bottom, stage 2: WT (n = 32), orbΔ3′UTR (n = 36, P < 0.05), orb-XN 3′UTR (n = 34, P-value is not significant), and orbΔ3′UTR overexpressing (o/e) full-length orb (n = 15, P-value is not significant). P-values are for each group compared with wild type. WT, wild type.

Figure 7

Enrichment of Dynein to one cell is delayed in orb-XN 3′UTR. (A) Dynein is enriched in one cell in 16-cell cysts in region 2b and stages 1 and 2 in WT. (B) Quantification of the number of cells enriched for Dynein in WT (2b n = 35; stage 1 n = 35), orbΔ3′UTR (2b n = 35; stage 1 n = 36), and orb-XN 3′UTR (2b n = 40; stage 1 n = 40). (C and D) Dynein localization in orb-XN 3′UTR. (C) An example of an orb-XN 3′UTR ovariole in which Dynein is restricted to one cell at stage 2. At stage 1, two optical slices are shown (slice #1 and slice #2) in which Dynein is enriched in multiple cells compared to the anterior nurse cells. In region 2b, Dynein is enriched at the anterior of two cells. (D) An example of an orb-XN 3′UTR ovariole in which Dynein is not restricted to one cell in any of the cysts in region 2b, stage 1 or stage 2. In region 2b, two optical slices are shown which demonstrate Dynein enrichment in two cells in the region 2b cyst. At stage 1, Dynein is clearly enriched in two cells within the cyst, and at stage 2 Dynein is enriched in multiple cells of the cyst. Bars, 10 µm. WT, wild type.

Figure 8

Oocyte specification is correlated with orb mRNA and protein localization in orb-XN 3′UTR. (A) orb mRNA and osk mRNA colocalized in most germline cysts. Arrowhead points to a cyst in which orb and osk mRNAs are weakly enriched in multiple cells at the posterior. (B) In most stage 3–7 chambers orb and osk mRNA localize in a similar pattern; however, the arrowhead points to a cyst in which orb mRNA shows little evidence of localization, while osk mRNA is enriched at the anterior of two cells. (C) Plot of the localization patterns of orb and oskar mRNA in the same chamber from stages 3–7 (n = 72, R²=0.88). (D) In most stage 3–7 chambers, Orb and Egl proteins show similar patterns. Arrowhead points to a chamber in which both proteins are unlocalized. (E) Two orb-XN 3′UTR egg chambers. In one, Orb and Egl localize to the same cell (the oocyte), while in the other both proteins are unlocalized. (F) Plot of the lot of the localization patterns of Orb and Egl in the same chamber from stages 3–7 (n = 54, R²=0.94). Bars, 10 µm.

Figure 9

Oocyte specification in orb-XN 3′UTR is dose-dependent. (A) Egg laying of orbΔ3′UTR, orb-XN 3′UTR females, and females trans-heterozygous for orb-XN 3′UTR and either wild type or the orb null alleles, F343 and Dec. Multiple plates were collected over several days to count eggs laid from cups containing ∼40 females. orb-XN 3′UTR/+ females laid 846 eggs; orb-XN 3′UTR females laid 676 eggs, while orbΔ3′UTR, orb-XN 3′UTR/Dec, and orb-XN 3′UTR/F343 laid no eggs. (B) Staining for Orb protein and Corolla protein in orb-XN 3′UTR/orb^Dec egg chambers. Maximum intensity projection of Orb protein shown below. Bar, 10 µm.

Figure 10

orb is required for maintenance of oocyte fate. (A) Top: Orb protein in WT, and in ovaries expressing orb RNAi 43143 or 64002 driven by otu-Gal4, also expressing dicer. Bottom: maximum intensity projections of Orb. (B) Maximum intensity projections of orb and oskar mRNA in ovaries expressing orb RNAi 43143 (top) or 64002 (bottom). Arrowheads point to localization of oocyte markers. (C) GFP protein expressed from UASp promoter driven by otu-Gal4, also expressing dicer. Arrowhead points to a region 2b cyst weakly expressing GFP. (D–H) Quantification of the number of cells within the cyst enriched for oskar mRNA in region 2b through stage 4 when orb is knocked down either by 43143 or 64002 (all n ≥ 20). WT, wild type. Bar for all panels, 10 um.