Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2019 Oct 8;213(4):1431–1446. doi: 10.1534/genetics.119.302687

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

Justinn Barr *, Rudolf Gilmutdinov †, Linus Wang *, Yulii Shidlovskii †, Paul Schedl *,†,1
PMCID: PMC6893371  PMID: 31594794

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, Egalitarian, CPEB protein, Cytoplasmic polyadenylation, mRNA cargo complex, mRNA localization, oocyte specification, orb, positive autoregulation

CELL fate specification is central to the differentiation of tissues and cell types that make up multicellular eukaryotes. The critical steps in fate specification can be mediated by extrinsic signals or intrinsic pathways and can be orchestrated by imposing new patterns of chromatin organization, transcription, splicing, or even translation. The initial step often involves a choice between alternative cell fates or developmental pathways. The choice can be stochastic or determinative. A classic example of an extrinsic and stochastic choice would be the anchor cell (AC) or ventral uterine cell (VU) decision in Caenorhabditis elegans (Greenwald 2005; Sternberg 2005). Two initially equivalent cells utilize the Lag-2 (Delta)/Lin-12 (Notch) cell-cell signaling pathway to choose either the AC or VU fate (Kimble and Hirsh 1979; Seydoux and Greenwald 1989). The choice is competitive and depends upon small differences between the signaling and receiving activities of the two cells. An example of a determinative mechanism would be sex identity in Drosophila (Salz 2011). In this pathway, the cell fate decision is intrinsic and depends most critically upon the number of X chromosomes in precellular blastoderm nuclei. Nuclei that are 1X/2A (autosome) choose male identity, while nuclei that are 2X/2A select female. The choice depends upon a twofold difference in the zygotic expression of X-linked transcriptional activators. In 2X nuclei, the activators turn on the Sex-lethal establishment promoter, Sxl-Pe, while this promoter remains off in 1X cells (Cline 1983; Keyes et al. 1992).

While the mechanisms deployed for choice in the AC/VU decision in C. elegans and the sex determination pathway in Drosophila are very different, in both cases the establishment and maintenance of cell identity ultimately depends upon the positive autoregulatory activity of one of the key specification factors. In the AC/VU decision the Lag-2–activated Lin-12 receptor positively autoregulates its own transcription and this drives the process of determination (Seydoux and Greenwald 1989; Wilkinson et al. 1994). In the sex determination pathway, Sxl proteins produced by Sxl-Pe messenger RNAs (mRNAs) activate female-specific splicing of transcripts from the Sxl maintenance promoter, Sxl-Pm (Keyes et al. 1992). The proteins translated from the resulting female spliced Sxl-Pm mRNAs then positively autoregulate their own expression by directing the female splicing of new Sxl-Pm transcripts (Bell et al. 1988, 1991). In this pathway, the positive autoregulatory activity of Sxl not only drives the initial establishment of female identity, but also is required for maintenance of the determined state.

Since positive autoregulatory loops provide robust mechanisms for establishing/maintaining distinct identities, we wondered whether a feed-forward loop might be deployed in the specification of oocyte identity in the Drosophila egg chamber. The egg chamber consists of 16 interconnected cells, of which 15 are nurse cells while the remaining cell is the oocyte. The egg chamber arises from the asymmetric division of a germline stem cell located at the anterior of each ovariole in a structure called the germarium. While one daughter remains a stem cell, the other daughter, the cystoblast, undergoes four mitotic divisions with incomplete cytokinesis to generate a cyst consisting of 16 cells interconnected by ring canals (Spradling et al. 1997; Huynh and St Johnston 2004; Bastock and St Johnston 2008). At each mitotic division, a new ring canal is formed between the daughter cells leading to a network of interconnected cells. Two of the cells (the daughters of the cystoblast) are connected to each other and have four ring canals. The 14 remaining cells have three, two, or one ring canal. The two cells with four ring canals are the pro-oocytes and one of these will become the oocyte, while all of the remaining cells assume a nurse cell identity.

Two models have been proposed for the choice of oocyte identity. In one, the pro-oocytes compete with each other for some limiting factor(s), the oocyte determinant(s) (Carpenter 1994). The cell that wins becomes the oocyte, while the other cell reverts to the default nurse cell identity. Consistent with this model, both pro-oocytes initially form synaptonemal complexes, and accumulate many cytoplasmic markers of oocyte identity. The second model proposes that the oocyte is predetermined (Storto and King 1989; Lin and Spradling 1995; Deng and Lin 1997; de Cuevas and Spradling 1998). In this model a membranous structure called the fusome that runs through the ring canals and interconnects all 16 cells in the cyst is responsible for selecting the oocyte. The fusome is thought to organize a polarized microtubule (MT) network that mediates the targeting of oocyte-specific proteins and mRNAs (Theurkauf et al. 1993; McGrail and Hays 1997; Grieder et al. 2000). The fusome is initially assembled in the newly formed cystoblast from a germline stem cell–derived organelle, the spectrosome. When the cystoblast divides, the old fusome is partitioned to only one of the daughters, while the other daughter assembles a new fusome, which then fuses with the old fusome through the ring canal. This process is repeated at each division. At the 16-cell stage, one of the two cells with four ring canals retains the original fusome and also has the largest amount of fusome material. In this model, the mRNAs or proteins that determine oocyte identity are preferentially targeted to this cell and it becomes the oocyte (Cox and Spradling 2003).

However, in either model, the identity of the oocyte determinant(s) remains elusive. While mutations in fusome protein genes like hu-li-tai-shao (hts) and α-spectrin interfere with oocyte specification (Yue and Spradling 1992; de Cuevas et al. 1996), it is unlikely that the oocyte determinant is a component of the fusome as the fusome begins to degenerate as the oocyte is selected from the two pro-oocytes (Huynh and St Johnston 2004). Other potential candidates include Bicaudal-D (BicD), egalitarian (egl), orb, cup, half-pint, encore (enc), stonewall (stwl), and ovarian tumor (otu) (Suter et al. 1989; Wharton and Struhl 1989; Suter and Steward 1991; Lantz et al. 1992, 1994; Clark and McKearin 1996; Hawkins et al. 1996; Keyes and Spradling 1997; Mach and Lehmann 1997; Van Buskirk and Schupbach 2002; Wong and Schedl 2011). Of these, the most clear-cut in terms their oocyte specification phenotypes are BicD and egl. For both genes, mutant chambers fail to form an oocyte, and instead contain 16 nurse cells (Schupbach and Wieschaus 1991; Ran et al. 1994; Mach and Lehmann 1997). Cytoplasmic markers of oocyte fate, such as orb and osk mRNAs, Orb, BicD, or Egl protein, fail to accumulate in a single cell, the presumptive oocyte. In the case of egl mutants, all cells in the cyst enter meiosis, before reverting to a nurse cell fate, while for BicD mutants none of the cells enter meiosis (Huynh and St Johnston 2000). However, since BicD and Egl are components of a complex that loads mRNAs onto dynein motors, their function in oocyte specification is most likely in the transport of mRNAs encoding the actual oocyte determinants (Bullock and Ish-Horowicz 2001; Dienstbier et al. 2009).

Of the remaining genes implicated in oocyte specification, several factors make orb the most promising candidate. First, orb encodes one of the two fly cytoplasmic polyadenylation element binding (CPEB) proteins. CPEB proteins are translational regulators that can activate or repress translation of target mRNAs depending on the specific biological context (Huang and Richter 2004; Ivshina et al. 2014; Khan et al. 2015). Second, orb mRNA and protein are among the earliest markers of oocyte identity. Third, included in its known or potential regulatory targets are mRNAs encoding several of the proteins implicated in some aspect of oocyte specification, including BicD, Egl, Cup, Enc, and Stwl (Lantz et al. 1992, 1994; Stepien et al. 2016). Four, strong loss-of-function orb alleles fail to specify an oocyte, while orb nulls arrest during the formation of the 16-cell cyst and then undergo cell death (Lantz et al. 1994). Finally, unlike the other genes implicated in oocyte specification, orb has a positive autoregulatory activity. In this autoregulatory loop, Orb binds to sequences in the orb mRNA 3′UTR and activates its own localized expression (Tan et al. 2001; Costa et al. 2005; Wong et al. 2011). During midoogenesis this positive autoregulatory activity is important for localizing orb mRNA within the oocyte and for its proper translation once localized (Tan et al. 2001). Disrupting the autoregulatory activity also compromises the regulation of two orb target mRNAs, gurken and oskar, indicating that it is important for the functioning of the orb gene in the development of the egg chamber.

Were this positive autoregulatory loop operational in newly formed 16-cell cysts it would not only ensure that orb mRNA and protein accumulate specifically in the presumptive oocyte after the 16-cell cyst is formed, but might also provide a mechanism that could help drive the process of oocyte specification. Unfortunately, it has not been possible to test this idea with the previously available genetic tools. Here, we have addressed this question using Crispr/Cas9 to delete the orb 3′UTR. We show that the 3′UTR is required not only for the localized accumulation of orb mRNA and protein in newly formed 16-cell cysts, but also for oocyte specification. Using a 3′UTR replacement strategy, we identified a partially functional sequence from the orb 3′UTR that retains autoregulatory activity, but is not properly localized in newly formed 16-cell cysts. In this 3′UTR replacement, oocyte specification is delayed until stage 2, which is long after the fusome has disappeared. Finally, we show that when orb is knocked down by RNA interference (RNAi) after the oocyte is selected, oocyte identity is not maintained.

Materials and Methods

Drosophila stocks

W1 flies were used as a wild-type control. orbDec and orbF343 were previously described, and are available at the Bloomington Drosophila Stock Center (#145, 10326). Stocks obtained from the Bloomington Drosophila Stock Center include orb RNAi lines (#43143/TRiP.GLO1484 and #64002/TRiP.HMJ30315), otu-Gal4 (#58424), and UAS-dicer (#24646). For stains, ovaries were dissected from adult females fed with yeast paste for 2–3 days. Eggs were collected by placing flies into cups and by providing fresh apple juice and yeast plates.

Generating orb 3′UTR deletion

For orb 3′UTR deletion, 1-kb homology arms for the target sequences within the orb 3′UTR were cloned into pHD-DsRed-attP vector and guide sequences were cloned into pU6-BbsI-chiRNA (Gratz et al. 2013). These vectors were purified and sent to Best Gene and injected into attP40-nos-Cas9 (#TH00788.N; BestGene) embryos (Ren et al. 2013). Transformants were screened for DsRed expression using a NightSea fluorescence adaptor. The DsRed marker was removed using Cre recombinase and the deletion was confirmed by sequencing (see Supplemental Material, Methods S1).

Generating orb overexpression stock

orb complementary DNA (cDNA) was obtained from the Drosophila Genomics Resource Center (FI20148). The orb cDNA sequence was subcloned into pattB-UASp-ΔK10-3′UTR vector (gift of Liz Gavis).

Immunostaining and antibodies

Antibodies were used as follows: mouse anti-Orb (4H8, 6H4) at 1:30 each; mouse anti-Dynein (P1H4, gift of Tom Hayes) at 1:50; mouse anti–alpha-tubulin-FITC (clone DM1A; Sigma, St. Louis, MO) at 1:1000; rabbit anti-Corolla (gift of Scott Hawley) at 1:1000; rabbit anti-Egl (gift of Ruth Lehmann), preincubated at 1:2000; rabbit anti-pTyrosine (Santa Cruz Biotechnologies) at 1:500; and mouse anti-BicD (1B11, 4C2) at 1:20 each. Alexa Fluor 546 phalloidin (Invitrogen, Carlsbad, CA) was used at 1:500. Alexa Fluor wheat germ agglutinin 633 (Invitrogen) was used at 1:500.

Ovaries were dissected in 1 × PBS from young female flies that were fed yeast paste for 2–3 days. Ovaries were fixed for 25 min in 4% paraformaldehyde (Electron Microscopy Services). Ovaries were washed 3 times for 15 min in 1 × PBST (0.1% Triton X-100 in 1 × PBS) and blocked for at least 1 hr at room temperature in 1 × PBST with 1% BSA. Ovaries were incubated overnight with primary antibody, washed 3 times with 1 × PBST for 15 min and incubated with secondary antibody at room temperature for 1 hr, washed 3 times for 15 min, and ovarioles were separated on a slide and most egg chambers older than stage 10 were removed from the slide. Samples were mounted in Aqua-polymount.

Fluorescence in situ hybridization

Oligonucleotide probes for orb and BicD (Methods S2) were ordered from LGC Biosearch Technologies and coupled to Atto NHS Ester 565 or 633 dyes (Sigma) and purified using HPLC. oskar oligoFISH probes were a gift of Shawn Little (Little et al. 2015).

In situs were performed as previously described (Little et al. 2015). Ovaries were dissected in 1 × PBS from young female flies that were fed yeast paste for 2–3 days. Ovaries were fixed for 30 min in 4% paraformaldehyde (Electron Microscopy Services). Ovaries were then rinsed 4 times in 1 × PBSTw (0.1% Tween-20 in PBS) and dehydrated through a methanol series, stored at −20° for 10 min in 100% methanol, and rehydrated into 1 × PBSTw. Ovaries were rinsed 4 times with 1 × PBSTw and incubated in wash buffer (4 × SSC, 35% formamide, 0.1% Tween-20) for 15 min at 37°. Samples were incubated with oligoFISH probes overnight at 37° in hybridization buffer (10% dextran sulfate, 0.01% salmon sperm single-strand DNA, 1% vanadyl ribonucleoside, 0.2% BSA, 4 × SSC, 0.1% Tween-20, and 35% formamide). The following day ovaries were washed twice for 1 hr in wash buffer at 37° and mounted in Aqua-polymount.

Microscopy

A Nikon A1 scanning confocal microscope was used for imaging. A ×60 or ×40/1.3 NA Plan-Apo oil objective was used. Nikon Elements, ImageJ, and Adobe Photoshop software was used.

Quantification and statistical analysis

Orb protein and mRNA enrichment was measured using maximum intensity projections of stage 1 egg chambers using Fiji/ImageJ (Figure 2C and Figure 5, C and F) or average intensity projections of the same stage 1 egg chambers (Figure S1). To calculate enrichment, the ratio of orb mRNA or Orb protein levels in the oocyte compared with the nurse cells was determined by averaging three regions of interest of mean fluorescence intensity of the oocyte, divided by the average of three regions of interest of mean fluorescence intensity of the nurse cells.

Figure 2.

Figure 2

Open in a new tab

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 5.

Figure 5

Open in a new tab

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.

To compare Orb protein levels between control and orbΔ3′UTR (Figure S2), H2Av-GFP and orbΔ3′UTR ovaries were stained for Orb protein in the same tube and ovarioles were mounted and imaged on the same slide. To calculate Orb levels, three regions on interest were taken from both the anterior nurse cell region of the stage 1 egg chamber and the background for each egg chamber. The average background mean fluorescence intensity was subtracted from the average nurse cell mean fluorescence intensity to calculate the level of Orb in the cyst. The results were normalized to the average of the control group.

Two-sided, unpaired Student’s t-test was used to determine statistical significance (Figure 2C, Figure 5, C and F, and Figure 6B legend, 6D legend, Figure S1). For all panels, * P < 0.05, ** P < 0.005, *** P < 0.0005, and ns indicates not significant. To determine how many cells in an egg chamber are positive for markers of oocyte fate, z-stacks were analyzed and levels were compared relative to other germline cells in the cyst. RStudio was used to calculate R2 (Figure 8 legend).

Figure 6.

Figure 6

Open in a new tab

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 8.

Figure 8

Open in a new tab

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.

Data availability statement

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures and tables. Supplemental material available at figshare: https://doi.org/10.25386/genetics.9946979.

Results

orb 3′UTR is required for orb mRNA and protein localization

The major orb mRNA species in fly ovaries is ∼4900 nt and contains a 1200 nt 3′UTR. In previous studies we showed that an 815 nt sequence defined by HindIII and NdeI restriction sites in the 3′UTR is sufficient to direct the localization of an Escherichia coli lacZ coding sequence in a temporal and spatial pattern that appears to faithfully reproduce that of the endogenous orb mRNA (Lantz and Schedl 1994). Moreover, during midoogenesis, both the localization and translation of the chimeric lacZ-orb 3′UTR mRNA requires orb (Tan et al. 2001). If the orb 3′UTR has a similar function earlier in oogenesis during oocyte specification, then removal of the 3′UTR should hamper if not eliminate oocyte-specific accumulation of orb mRNA. Since activation of the orb autoregulatory loop in the presumptive oocyte also depends upon the 3′UTR, only background (nurse levels) of Orb protein should cell be observed in deletion mutant cysts and egg chambers. To test these predictions, we used Crispr/Cas9 to introduce a 1069 bp deletion within the 3′UTR of the orb gene that spans the genomic sequence encoding the 815 nt HindIII-NdeI 3′UTR sequence (Figure 1).

Figure 1.

Figure 1

Open in a new tab

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)].

In wild type, orb mRNA is first readily detected in 16-cell germline cysts in region 2a of the germarium. At this stage it is typically present at high levels in the two cells that have four ring canals, the pro-oocytes. Subsequently, in late region 2a and early region 2b, orb mRNA largely disappears from one of the two pro-oocytes, and concentrates in the cell destined to become the oocyte. If the 3′UTR is critical for orb mRNA localization in the germarium, this stepwise process should be disrupted in orbΔ3′UTR ovaries. Figure 2A compares the localization of orb mRNA in the germarium and early-stage egg chambers in ovaries from wild-type and orbΔ3′UTR females. Unlike wild type, orbΔ3′UTR mRNAs show no evidence of localized accumulation in the germarium or in early egg chambers. Instead, the mutant mRNAs are distributed uniformly in all 16 germline cells in region 2a and 2b cysts and in stage 1 and older chambers (Figure 2C). These findings show that the 3′UTR is essential for the proper localization of orb mRNA.

The second prediction is that the orb 3′UTR is required for the expression of high levels of Orb protein in the two pro-oocytes in region 2a and in the oocyte in region 2b and in stage 1 and older egg chambers. We found this to be the case. Figure 2B shows that the normal pattern of Orb protein expression is disrupted in orbΔ3′UTR ovaries. Unlike wild type, it does not accumulate at high levels in the two pro-oocytes in region 2a, nor is it enriched in a single cell (the oocyte) in region 2b, or in stage 1 egg chambers (Figure 2C). Instead, the cells in region 2b cysts and in stage 1 chambers have levels of Orb protein similar to that observed in the nurse cells of wild type chambers (Figure S1). Thus, while the basal level of translation of orb mRNA in nurse cells does not seem to be greatly perturbed, the upregulation of Orb protein expression that is normally observed in the two pro-oocytes in region 2a, and in the oocyte in region 2b cysts and in stage 1 chambers, depends upon sequences in the orb 3′UTR. In the vast majority of older egg chambers in orbΔ3′UTR ovaries, Orb protein is also distributed at uniform levels in all cells. However, as illustrated in Figure S2, we infrequently (∼1/75) observe older chambers in which Orb concentrates in a few cells in puncta that resemble P bodies or stress granules or is enriched near ring canals. While this would suggest that there are mechanisms that can promote a local accumulation of Orb that are independent of the 3′UTR, the normal pattern of expression and localization clearly requires the 3′UTR.

orb 3′UTR is required for oocyte specification

If orb functions as a key oocyte determinant, then the failure to properly localize orb mRNA and express high-level Orb protein in orbΔ3′UTR mutant ovaries would be expected to disrupt the process of oocyte specification. Consistent with this prediction, germline cells in orbΔ3′UTR are polyploid (Figure S2) and mutant females are completely sterile and do not lay eggs. While most orbΔ3′UTR chambers have 16 polyploid cells, we infrequently (1/75) observe chambers in which there is a single condensed nucleus (Figure S3B). However, as indicated in Figure S3C, the synaptonemal complex protein Corolla is not maintained in later-stage egg chambers.

These findings would functionally link the 3′UTR-dependent localization of orb mRNA and protein to the specification of oocyte fate. To test this idea, we asked if expressing a wild-type orb cDNA will restore the normal pattern of orb mRNA and protein localization and also rescue the defects in oocyte specification. Figure 2, A and B shows that expressing a full-length orb mRNA using a nos-GAL4 driver rescues the defects in orb mRNA localization and Orb protein expression. Moreover, since orbΔ3′UTR females expressing the orb cDNA not only form egg chambers containing 15 nurse cells and an oocyte but also lay eggs, the full-length cDNA must rescue the early oogenesis defects of the orbΔ3′UTR mutant including the failure to specify an oocyte.

The orb 3′UTR is required at an early step in oocyte specification

The results in the previous section indicate that oocyte specification is disrupted by the orbΔ3′UTR mutation. To better understand the nature of the defect we compared the expression and distribution of several oocyte specific markers in wild-type and orbΔ3′UTR ovarioles.

Oocyte localization of BicD and osk mRNAs

In wild-type ovaries, BicD and osk mRNAs are initially detected in region 2a of the germarium where they are present at highest levels in the two pro-oocytes. These mRNAs then begin to localize to a single cell, and in cysts in region 2b, both mRNAs are concentrated in the oocyte. This progressive pattern of localization is disrupted in the orbΔ3′UTR mutant. Although localized accumulation of BicD and osk is observed in wild type cysts, there is no evidence of localization in regions 2a or 2b in the orbΔ3′UTR mutant (Figure 2, D and E). In ∼60% of the stage 1 orbΔ3′UTR chambers, BicD mRNAs are either unlocalized or localized in two cells (Figure 2F). osk mRNA also fails to concentrate in a single cell in stage 1 chambers, and instead is distributed in two or more cells (Figure 2, E and F).

Localization of Egl and BicD protein

In newly formed 16-cell cysts, Egl and BicD are unlocalized; however, as the cyst progresses through region 2a, both proteins concentrate in the two pro-oocytes. Accompanying the restriction of Egl and BicD to the pro-oocytes, orb mRNAs and proteins also begin to concentrate in these two cells. In region 2b and in stage 1 chambers, both Egl and BicD are localized in the oocyte with highest levels at the posterior in stage 1 chambers (Figure 3A and Figure S3). This progressive enrichment of Egl and BicD is disrupted in orbΔ3′UTR ovaries. In >80% of late region 2b cysts, Egl is localized in two or more cells, while in stage 1 it is either localized in two or three cells or is unlocalized in ∼95% of the chambers (Figure 3B). Similar results are observed for BicD (Figure S5).

Figure 3.

Figure 3

Open in a new tab

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.

Localization of Dynein

BicD and its partner Egl convey mRNAs on Dynein motors. Like these two proteins, the localization of Dynein to the oocyte is also a stepwise process. In region 2a cysts, the two pro-oocytes have the highest levels of Dynein. As the 16-cell cyst matures, Dynein largely disappears from all of the cells except for the two pro-oocytes, while by stage 2b, it is concentrated in the oocyte. This progressive restriction of Dynein localization does not occur in orbΔ3′UTR ovaries (Figure 3C). Levels of Dynein remain high in multiple cells in regions 2b, while in stage 1 chambers the protein is distributed in two or more cells or is unlocalized (Figure 3D).

MT organization

A key step in oocyte specification is the assembly of a MT organizing center in the presumptive oocyte. This process begins in cysts in region 2a with the organization of a polarized MT network in association with the fusome. As the network grows, an MT organizing center starts to assemble initially in the two cells that have four ring canals, and subsequently in a single cell, the oocyte. MTs emanating from the oocyte pass through the oocyte ring canals into the neighboring nurse cells and are thought to play a critical role in the transport of key oocyte factors. As shown in Figure 4, α-tubulin is not properly organized in the presumptive oocyte in stage 1 orbΔ3′UTR egg chambers. Instead, in most orbΔ3′UTR stage 1 chambers, MTs are distributed more or less evenly between the two cells (pro-oocytes) with four ring canals, and this network appears to assemble in association with the ring canal connecting these two cells (Figure 4). In other orbΔ3′UTR chambers the MT network is distributed between more cells than just the pro-oocytes.

Figure 4.

Figure 4

Open in a new tab

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.

Restricting the localization of the synaptonemal complex

Newly formed 16-cell cysts in region 2a of wild-type ovaries typically assemble a synaptonemal complex in four cells, the two cells with four ring canals (pro-oocytes) and the two cells that have three ring canals. The synaptonemal complex–like structures in the two cells with three ring canals disappear shortly thereafter, while the two cells with four ring canals retain the synaptonemal complex until region 2b of the germarium when it is disassembled from the pro-oocyte that differentiates as a nurse cell. As shown in Figure 4 for the synaptonemal complex marker, Corolla, only the oocyte has a synaptonemal complex in wild-type stage 1 chambers. By contrast, the synaptonemal complex is not properly restricted to one of the cells with four ring canals in orbΔ3′UTR stage 1 chambers. Instead, most of orbΔ3′UTR chambers have Corolla in two or more cells (Figure 4), while in older chambers Corolla disappears.

orb mRNA and protein localization to the presumptive oocyte in a 3′UTR replacement

The findings in the previous sections suggest that the 3′UTR-dependent localization of orb mRNA and protein functions to drive oocyte specification. To test this model we used the attP site in the orbΔ3′UTR deletion mutant to introduce two different sequences, CE and XN, derived from the orb 3′UTR, that were used previously in transgene reporter experiments (Lantz and Schedl 1994) (Figure 1). In these experiments, the CE sequence directed the localization of a heterologous lacZ mRNA in a pattern that recapitulated the endogenous orb mRNA: localization to the presumptive oocyte in the germarium, localization to the posterior of the oocyte in stage 1–7 chambers, and localization to the anterior margin of the oocyte in older chambers. Unexpectedly, the CE replacement failed to recapitulate the patterns of mRNA and protein localization expected from our studies on the lacZ reporter. Instead, we found that the orb-CE 3′UTR encoded mRNAs and proteins are unlocalized and all cells in the mutant chambers assume a nurse cell fate (data not shown). We suspect that the discrepancy between the CE reporter and the orb-CE 3′UTR replacement is that the reporter was tested in a wild-type orb background, while the orb-CE 3′UTR replacement is the only source of orb gene products. This supposition is supported by analysis of the XN replacement.

In the transgene assay, the XN sequence was partially functional. Like the full-length 3′UTR, it directed the localization of lacZ mRNA to the presumptive oocyte in the germarium, while in stage 1–7 chambers it localized lacZ mRNA to the posterior of the oocyte. However, it failed to localize lacZ transcripts to the anterior margin of the oocyte after the repolarization of the MT network in older egg chambers. This is also true for the orb-XN 3′UTR replacement mRNAs; the mRNAs are unlocalized after stage 7 (data not shown). On the other hand, the XN replacement differs from CE in that it is partially functional at earlier stages, and exhibits a temporal pattern of mRNA and protein accumulation that is highly unusual.

In wild type, orb mRNAs and proteins undergo a progressive restriction in their pattern of localization in the germarium. As described above, the localization of orb gene products commences in cysts in region 2a, initially the orb gene products are present at highest levels in the two pro-oocytes; however, by the time the cysts reach region 2b, orb mRNAs and protein are concentrated in the cell destined to become the oocyte. orb mRNA localization in region 2a and 2b of the germarium is not observed in orb-XN 3′UTR ovaries. As shown in the examples in Figure 5, there is little evidence of localized orb-XN 3′UTR mRNA or protein in cysts in region 2a, while in cysts in region 2b, mRNA and protein is weakly enriched in multiple cells. Since lacZ-XN mRNAs are properly localized in the germarium of wild-type flies, this finding implies that the early localization activity of the XN sequence depends upon a wild-type orb gene. Only in stage 1 chambers is there any hint of localized orb-XN 3′UTR transcripts and protein. However, as indicated in Figure 5, the extent of enrichment in stage 1 chambers is much less than in wild type. Subsequently, at stage 2 or 3, a subset of the mutant chambers successfully accumulate near levels of orb mRNA and protein in a single cell, while in other chambers, orb mRNA and protein are either found in two or more cells or are unlocalized. By stages 4–7 about half of the chambers contain an oocyte and 15 nurse cells, while the remaining chambers have 16 nurse cells. In those that contain an oocyte, this cell is not always the most posterior cell in the chamber, and instead can be displaced to the side or internally (Figure S5).

Oocyte specification is delayed in orb-XN 3′UTR mutant ovaries

If orb plays a pivotal role in the specification of oocyte fate, this decision in orb-XN 3′UTR ovaries should be delayed until orb mRNAs and proteins begin to localize in a single cell. Consistent with this prediction, we find that other markers of oocyte identity also show a delayed pattern of localized accumulation. While osk mRNA is localized in a single cell (the oocyte) in wild type in nearly all region 2b cysts, orb-XN 3′UTR resembles orbΔ3′UTR at this stage in that osk is localized in two or more cells in region 2b cysts as well as in stage 1 chambers (Figure 6). Only in stage 2 do we observe a significant number of chambers in which osk mRNA is found in only a single cell (Figure 6). By way of contrast, in orbΔ3′UTR mutant ovaries rescued by expressing the full-length orb cDNA, osk mRNA is localized in a temporal pattern that resembles wild type (Figure 6).

A delay is also observed for a nuclear marker of oocyte identity, the synaptonemal complex protein Corolla (Figure 6). In wild type, the restriction of the synaptonemal complex to a single cell (the oocyte) lags behind cytoplasmic markers like osk and orb mRNA. In about half of the cysts in region 2b Corolla is found not only in the oocyte nucleus, but also in the other cell with four ring canals. It can also be detected in two cells in a subset of the stage 1 chambers, and in those chambers strong and weak levels of staining are correlated with identity. As was observed for osk mRNA, the distribution of Corolla in orb-XN 3′UTR cysts in region 2b is similar to that of the orbΔ3′UTR mutant: in most of the cysts Corolla is found in three or four cells. At stage 1, Corolla is found in two cells in most of the orb-XN 3′UTR chambers, while even in stage 2 Corolla is localized in two cells in a significant fraction of the chambers. Again this differs from the Corolla distribution in orbΔ3′UTR mutant egg chambers rescued by expressing the full-length orb cDNA. In the rescued ovaries, Corolla localizes to a single cell in all stage 2 chambers (Figure 6).

To extend this analysis we examined the localization of Dynein in orb-XN 3′UTR mutant ovaries. Figure 7, C and D shows the germarium and stage 1 and 2 chambers from two different orb-XN 3′UTR ovarioles. In both orb-XN 3′UTR ovarioles, the Dynein localization pattern in the germarium and stage 1 chambers resembles that observed in orbΔ3′UTR: instead of localizing to the presumptive oocyte, Dynein remains distributed in multiple cells in region 2b cysts (Figure 7). This is also true for nearly all orb-XN 3′UTR stage 1 chambers. The pattern of Dynein localization changes in stage 2. As was seen for orb mRNA and protein, Dynein becomes localized to a single cell in a subset of the orb-XN 3′UTR stage 2–3 chambers (Figure 7C), while in the remaining stage 2–3 chambers it is distributed between two or more cells (Figure 7D). Similar results are obtained for BicD (Figure S5) and Egl (Figure S6).

Figure 7.

Figure 7

Open in a new tab

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.

While these findings indicate that the specification of oocyte fate is delayed in orb-XN 3′UTR, the choice is still between the two cells, the pro-oocytes, which have four ring canals (as visualized by actin staining). In those orb-XN 3′UTR chambers in which orb mRNA and protein successfully accumulates at high levels in a single cell (which assumes oocyte identity), this cell always has four ring canals. Similarly, in chambers in which orb mRNA and protein are localized in two cells (either at unequal or equal levels), both of these cells also have four ring canals. Additionally, we found that the delay in oocyte specification can also disrupt the differentiation of the pro-oocyte that develops as a nurse cell (Figure S7). This cell often fails to undergo the normal rounds of endoreplication, and can also retain markers of oocyte identity.

Oocyte specification is correlated with orb mRNA and protein localization

The delay in the localized accumulation of orb gene products in orb-XN 3′UTR ovaries is accompanied by a delay in oocyte specification. Moreover, only about half of the chambers manage to specify an oocyte. If localized orb mRNA/protein is needed to specify oocyte identity, then the orb-XN 3′UTR egg chambers that contain an oocyte and 15 nurse cells should correspond to chambers in which orb mRNA and protein have successfully accumulated at high levels in a single cell. Conversely, those that fail to localize orb mRNA and express only basal levels of Orb protein should not contain an oocyte. To test this prediction we compared orb and osk mRNA localization and Orb and Egl protein localization in stage 3–7 egg chambers (Figure 8).

The two most frequent classes for orb mRNA are (i) chambers in which orb mRNA is localized to a single cell (40%) and (ii) chambers in which orb mRNA is unlocalized (36%) (Figure 8C). In the former case, osk mRNA is also localized to the same cell and this cell corresponds to the oocyte. In the latter case, osk mRNA is either unlocalized, or (with one exception) enriched in multiple cells (Figure 8, B and C). These chambers have 16 nurse cells and no oocyte. In the remaining chambers orb mRNA is either localized weakly in a single cell or is localized either equally or unequally in two cells. In most of the chambers in which orb mRNA is weakly localized in a single cell, this cell also has a low level of osk mRNA but is a nurse cell, not an oocyte. For those chambers in which orb is found in two cells, osk is also found in two cells and its pattern of accumulation in these two cells is the same as that of orb mRNA. Thus, in chambers in which there is an orb “winner”, the winner also has the highest levels of osk and appears to be developing as an oocyte (Figure 8C).

Similar results were obtained for Orb and Egl proteins. The two most frequent classes were (i) chambers in which Orb and Egl proteins accumulate at high levels in a single cell (32%) and (ii) chambers in which neither Orb nor Egl were localized (54%) (Figure 8F). Chambers in the former class had 15 nurse cells and an oocyte containing high levels of Orb and Egl. Chambers in the latter class had 16 nurse cells.

Taken together these findings argue that successful activation of the orb autoregulatory loop in stage 2 orb-XN 3′UTR chambers (as evidenced by the localized accumulation of orb mRNA and protein) is able to induce oocyte specification. In contrast, in chambers in which the autoregulatory loop is not successfully activated in a single cell, orb mRNA and protein remain unlocalized and oocyte identity is not established.

Oocyte specification by orb-XN 3′UTR is dose-dependent

A threshold model could potentially explain why orb-XN 3′UTR is able to induce oocyte specification in only a subset of the stage 2 chambers. In chambers that fail to form an oocyte, the level of orb mRNA and/or protein in the pro-oocytes would be below the threshold needed to initiate and/or sustain the orb autoregulatory loop. In chambers that differentiate properly, the level of orb mRNA and protein would exceed this threshold value in one of the pro-oocytes and this would trigger the autoregulatory loop. Once successfully initiated, the feed-forward loop would ensure further accumulation of orb mRNA and protein and in turn recruit other factors needed to properly specify and sustain oocyte fate.

If this threshold model is correct, then oocyte specification in orb-XN 3′UTR ovaries should depend on gene dose. When the orb-XN 3′UTR gene dose is reduced in half, the frequency of chambers containing an oocyte should be diminished. This prediction is met. Whereas orb-XN 3′UTR females lay eggs, females trans-heterozygous for orb-XN 3′UTR and one of the two orb null alleles, orb343 or orbDec, do not, and this phenotype is fully penetrant (Figure 9). Moreover, unlike orb-XN 3′UTR, the egg chambers in orb-XN 3′UTR/orb343 and orb-XN 3′UTR/orbDec ovaries have 16 nurse cells and no oocyte (Figure 9).

Figure 9.

Figure 9

Open in a new tab

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/orbDec egg chambers. Maximum intensity projection of Orb protein shown below. Bar, 10 µm.

Orb is required for the maintenance of oocyte fate

The experiments above link oocyte specification to the 3′UTR-dependent orb autoregulatory loop. If the orb positive feedback loop plays an important role in oocyte fate determination, it might be expected to function not only in the initial specification of oocyte identity, but also in maintenance. If this expectation is correct, disruption of the orb autoregulatory loop after a cell has assumed oocyte identity should lead to a failure in maintaining oocyte identity. To test this prediction we used otu-GAL4 to drive expression of two different orb RNAi lines, 43143 and 64002. The otu-GAL4 driver first comes on in region 2b cysts after the initial steps in oocyte specification have occurred (Figure 10C). In the orb RNAi 43143 line, near wild-type levels of Orb protein (Figure 10A) are localized in the oocyte in cysts in region 2b, while levels are reduced compared to wild type in stage 1 chambers. In the orb RNAi 64002 line, accumulation of Orb protein in the oocyte is already somewhat reduced in region 2b cysts (Figure 10A). In both RNAi lines, osk mRNA is localized in a single cell in ∼40% of the region 2b cysts, while it is present in two cells in nearly 60% of the cysts (Figure 10, B and D). For the RNAi 64002 line, the effects of the knockdown are evident at stage 1, where osk mRNA is dispersed in three cells or shows little evidence of localization in about one third of the chambers (Figure 10E). By stage 4, osk mRNA is unlocalized in all chambers (Figure 10H). For the RNAi 43143 line, the number of chambers with osk mRNA in a single cell actually increases to nearly 60% in stage 1 chambers; however, by stage 2, osk mRNA is localized to a single cell in <20% of the chambers (Figure 10, E and F). Localization is progressively lost thereafter and by stage 4, osk mRNA is unlocalized in nearly 60% of the chambers (Figure 10, G and H). These findings indicate oocyte fate in stage 1–4 chambers is not maintained in the absence of continued orb activity.

Figure 10.

Figure 10

Open in a new tab

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.

Discussion

While many genes are known that function at one step or another in the process of oocyte determination in the fly germarium, the identity of the factors that serve as oocyte determinants has not been established. Most of the genes that affect oocyte specification appear to have supportive or ancillary roles in that they contribute to or are needed for oocyte fate, but do not actually specify oocyte identity. For example, the fusome has been implicated in the choice of which of the two pro-oocytes assumes oocyte identity; however, the available evidence suggests that it does not function directly in oocyte specification, but instead helps ensure that the oocyte determinant accumulates preferentially in one of the two pro-oocytes. Unlike the fusome, the BicD:Egl mRNA cargo complex is required for oocyte fate specification. BicD functions as a Dynein motor adaptor protein and its association with the Dynein motor is dependent upon Egl binding to target mRNAs (Vazquez-Pianzola et al. 2017). A clear inference from the biological functions of the Egl:BicD complex is that the key determinants of oocyte identity are localized mRNAs. While BicD and egl mRNAs accumulate in the oocyte, this is likely a consequence of oocyte specification rather than a cause. A far better candidate for a gene that could act as an oocyte specification factor is orb. orb is required for oocyte fate and it encodes a translation factor responsible for activating or repressing the onsite translation of many different localized mRNAs. The known orb regulatory targets include BicD (and likely egl), as well as the polarity axis determinants gurken and osk (Chang et al. 1999, 2001; Castagnetti and Ephrussi 2003). In addition, orb has a positive autoregulatory activity that directs the localized accumulation of its own mRNA and protein (Tan et al. 2001) and, as is the case in other biological contexts, this feed-forward loop could help drive fate specification.

The requirement for the BicD:Egl mRNA cargo complex in oocyte specification predicts that mRNAs encoding key oocyte determinants must be localized to establish oocyte fate. We have tested this prediction by deleting the orb 3′UTR. Consistent with previous transgene studies, the localization of the endogenous orb mRNA requires the 3′UTR and orbΔ3′UTR mRNAs are unlocalized. As the orb autoregulatory loop also depends on the 3′UTR, Orb proteins are expected to be expressed at only basal levels from the orbΔ3′UTR mutant mRNAs and to be unlocalized as well. This expectation is also correct; Orb protein is distributed throughout the orbΔ3′UTR cysts egg chambers at levels similar to that in the nurse cells of wild-type chambers. The failure in the localized accumulation of orb mRNA and protein also has the predicted developmental consequence: oocyte fate specification is disrupted in the orbΔ3′UTR mutant. While these findings implicate the orb 3′UTR and orb autoregulation in oocyte specification, it is worth noting that orb null alleles arrest oogenesis at an earlier stage, before oocyte specification. This means that orb must have other functions during early oogenesis (Lantz et al. 1994; Rojas-Ríos et al. 2015).

Taken together, our findings suggest a model in which the BicD:Egl mRNA cargo complex localizes orb mRNA to the two pro-oocytes in region 2a cysts. Accompanying localization, the orb autoregulatory loop is activated, and Orb protein begins to accumulate. As the newly formed 16-cell cysts mature, the feed-forward loop drives the accumulation of higher levels of Orb protein in one of the pro-oocytes. The initially small differences in Orb protein levels could then be amplified by the orb-dependent translation of factors such as BicD, Egl, Lis-1 (Lissencephaly-1), and Dhc64C (Dynein heavy chain 64C) (Stepien et al. 2016) that could serve to further augment the localization and subsequent translation of orb mRNA. Supplementing the feed-forward loop, there could be mechanisms that suppress orb mRNA and protein accumulation in the second pro-oocyte and in other cells in the cyst (Wong and Schedl 2011). As Orb binds to and potentially regulates a gamut of different mRNA species (Stepien et al. 2016), it could potentially activate the onsite translation of mRNAs encoding other factors critical for oocyte differentiation. By region 2b, orb mRNA and protein, as well as other markers of oocyte fate, are restricted to a single cell, and that cell has assumed an oocyte identity.

The idea that the localized activation of the orb autoregulatory loop might drive the specification of oocyte fate is supported by the XN 3′UTR replacement. In this mutant, the initial steps in oocyte specification that normally take place as the cyst transitions through region 2a and 2b of the germarium fail. Instead of concentrating in a single cell, orb mRNA and protein are distributed more or less evenly in all cells in the cyst or are present at only marginally higher levels in several of the cells. This distribution pattern persists until around stage 2, when a subset of chambers begins to accumulate high levels of orb mRNA and protein in a single cell. The delay in orb localization is accompanied by a delay in localization of other key cytoplasmic markers of oocyte identity, like BicD and osk mRNAs and Egl, Dynein, and BicD proteins. Only when orb mRNAs and proteins begin to accumulate to high levels in a single cell do these other oocyte markers follow suit and oocyte fate is established. In those XN 3′UTR chambers in which orb mRNA and protein fail to concentrate in a single cell, the oocyte is not formed and all of the cells assume a nurse cell fate.

In transgene assays, the XN sequence localized lacZ mRNA in the germarium and in stage 1–7 chambers in a pattern that mimicked the endogenous orb mRNA (Lantz and Schedl 1994) (Figure 1). Since the orb-XN 3′UTR mRNAs are mislocalized in the germarium and stage 1 chambers, it would appear that the proper functioning of the XN UTR sequence at these early stages requires a wild-type orb gene. This requirement would suggest a mechanism for activating the orb autoregulatory loop in a subset of stage 2 chambers even though the normal fusome-dependent pathway for oocyte fate determination in region 2a of the germarium is disrupted. In these stage 2 chambers, Orb protein (and mRNA) in one of the cells with four ring canals would reach a critical threshold required to engage the impaired autoregulatory functions of the XN sequence. Once engaged, the feed-forward loop, together with the orb-dependent translational activation of mRNAs encoding other important factors, would propel oocyte specification. A prediction of this model is that activation of the autoregulatory loop would be dose-dependent. We found this to be the case. When the dose of the orb-XN 3′UTR gene is reduced in half, the autoregulatory loop is not activated and oocyte fate is not established.

In orb-XN 3′UTR egg chambers, oocyte fate specification is delayed until long after the fusome degenerates in late region 2a of the germarium. This would imply that the fusome does not play a direct role in fate specification in this mutant. While it is apparently possible to bypass the fusome, it is important to note that the choice is still between one of the two cells with four ring canals and in this sense is predetermined by a mechanism that is independent of orb mRNA localization. At least two factors could contribute to this restriction. First the polarized cytoskeleton that is assembled before the disappearance of the fusome likely direct orb and other oocyte localized mRNAs toward the two pro-oocytes. Second, the distribution and arrangement of ring canals may help generate an intrinsic polarity. Consistent with a preexisting intrinsic polarity, BicD and osk mRNAs and BicD, Egl and Dynein proteins are not uniformly distributed in most XN 3′UTR cysts. Instead they tend to concentrate in a subset of cells including the two that contain four ring canals. Since these factors also tend to concentrate in a subset of cells in the orbΔ3′UTR mutant, it would appear that this preexisting “polarity” is independent of whatever polarity is generated by the orb 3′UTR-dependent localization of orb gene products. This, of course, does not exclude the possibility that sensing this preexisting polarity is contingent upon a functional Orb protein. We also expect that many of the factors that promote the establishment and/or maintenance of the orb autoregulatory loop in region 2a of the germarium will also contribute to oocyte specification when the decision is made in XN 3′UTR chambers. It will be of interest to determine if their functions in stage 2 are also dose sensitive like the XN 3′UTR replacement. The delay in oocyte specification seen in XN 3′UTR ovaries is not unprecedented (Gonzalez-Reyes et al. 1997; Huynh and St Johnston 2000). For example, in several double-mutant combinations of several of the spindle genes, stage 4 chambers with two oocytes are observed.

While our studies are consistent with a model in which oocyte fate is driven at least in part by an orb dependent feed-forward loop that is analogous to those deployed in the specification of AC identity in the worm and female identity in the fly, there are many unanswered questions. One is to what extent is the decision-making process in oocyte specification stochastic or determinative? For not only wild type, but also XN 3′UTR, there must be “determinative” mechanisms in place as the only cells in the cyst that can assume oocyte identity are the two cells with four ring canals. On the other hand, the fact that we occasionally observe XN 3′UTR chambers that contain two “oocyte-like” cells (Figure S8) would argue that the choice between these two cells in this genetic background is likely stochastic. Since this decision in XN 3′UTR is also temporally divorced from the fusome, the possibility remains open that in wild-type ovaries the choice between these two cells is determinative and not stochastic. Another important question is what drives the activation of the orb autoregulatory loop in one (but typically not both) of the cells containing four ring canals? Is this an entirely autonomous process, or are there communication mechanisms in place that serve to enhance the response in the “winning” cell and/or dampen the response in the “losing” cell? Potentially affecting this process is the fact that the translational regulatory functions of CPEB proteins are controlled by phosphorylation (Wong et al. 2011). Thus, activation of the orb feed-forward loop in one cell but not the other might depend on restricting the activity of some unknown kinase(s). This could be a fusome-dependent process. Finally, are there any other factors besides orb that can promote oocyte specification by a mechanism requiring BicD/Egl-mediated mRNA localization?

Acknowledgments

The authors thank Girish Deshpande, Tsutomu Aoki, Jasmin Imran Alsous, and Julie Merkle for helpful discussions; Gary Laevsky and the Confocal Imaging Facility; Anna Schmedel for administrative support; and Gordon Grey for fly media. We would also like to thank Tom Hayes, Scott Hawley, and Ruth Lehmann for key antibodies used in these studies. This study was supported by the Russian Science Foundation (project no. 18-74-10051 to Y.S.) and the National Institutes of Health (NIH; grant R35GM126975 to P.S.), and an NIH Training Grant supported J.B. (T32GM007388). A clone from the Drosophila Genomics Resource Center (NIH grant 2P40OD010949) was used in this study. Stocks obtained from the Bloomington Drosophila Stock Center (NIH grant P40OD018537) were used in this study.

Footnotes

Supplemental material available at figshare: https://doi.org/10.25386/genetics.9946979.

Communicating editor: M. Wolfner

Literature Cited

  1. Bastock R., and St Johnston D., 2008.  Drosophila oogenesis. Curr. Biol. 18: R1082–R1087. 10.1016/j.cub.2008.09.011 [DOI] [PubMed] [Google Scholar]
  2. Bell L. R., Maine E. M., Schedl P., and Cline T. W., 1988.  Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell 55: 1037–1046. 10.1016/0092-8674(88)90248-6 [DOI] [PubMed] [Google Scholar]
  3. Bell L. R., Horabin J. I., Schedl P., and Cline T. W., 1991.  Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65: 229–239. 10.1016/0092-8674(91)90157-T [DOI] [PubMed] [Google Scholar]
  4. Bullock S. L., and Ish-Horowicz D., 2001.  Conserved signals and machinery for RNA transport in Drosophila oogenesis and embryogenesis. Nature 414: 611–616. 10.1038/414611a [DOI] [PubMed] [Google Scholar]
  5. Carpenter A. T., 1994.  Egalitarian and the choice of cell fates in Drosophila melanogaster oogenesis. Ciba Found. Symp. 182: 223–246, discussion 246–254. [PubMed] [Google Scholar]
  6. Castagnetti S., and Ephrussi A., 2003.  Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte. Development 130: 835–843. 10.1242/dev.00309 [DOI] [PubMed] [Google Scholar]
  7. Chang J. S., Tan L., and Schedl P., 1999.  The Drosophila CPEB homolog, orb, is required for oskar protein expression in oocytes. Dev. Biol. 215: 91–106. 10.1006/dbio.1999.9444 [DOI] [PubMed] [Google Scholar]
  8. Chang J. S., Tan L., Wolf M. R., and Schedl P., 2001.  Functioning of the Drosophila orb gene in gurken mRNA localization and translation. Development 128: 3169–3177. [DOI] [PubMed] [Google Scholar]
  9. Clark K. A., and McKearin D. M., 1996.  The Drosophila stonewall gene encodes a putative transcription factor essential for germ cell development. Development 122: 937–950. [DOI] [PubMed] [Google Scholar]
  10. Cline T. W., 1983.  The interaction between daughterless and sex-lethal in triploids: a lethal sex-transforming maternal effect linking sex determination and dosage compensation in Drosophila melanogaster. Dev. Biol. 95: 260–274. 10.1016/0012-1606(83)90027-1 [DOI] [PubMed] [Google Scholar]
  11. Costa A., Wang Y., Dockendorff T. C., Erdjument-Bromage H., Tempst P. et al. , 2005.  The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway. Dev. Cell 8: 331–342. 10.1016/j.devcel.2005.01.011 [DOI] [PubMed] [Google Scholar]
  12. Cox R. T., and Spradling A. C., 2003.  A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130: 1579–1590. 10.1242/dev.00365 [DOI] [PubMed] [Google Scholar]
  13. de Cuevas M., and Spradling A. C., 1998.  Morphogenesis of the Drosophila fusome and its implications for oocyte specification. Development 125: 2781–2789. [DOI] [PubMed] [Google Scholar]
  14. de Cuevas M., Lee J. K., and Spradling A. C., 1996.  alpha-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 122: 3959–3968. [DOI] [PubMed] [Google Scholar]
  15. Deng W., and Lin H., 1997.  Spectrosomes and fusomes anchor mitotic spindles during asymmetric germ cell divisions and facilitate the formation of a polarized microtubule array for oocyte specification in Drosophila. Dev. Biol. 189: 79–94. 10.1006/dbio.1997.8669 [DOI] [PubMed] [Google Scholar]
  16. Dienstbier M., Boehl F., Li X., and Bullock S. L., 2009.  Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor. Genes Dev. 23: 1546–1558. 10.1101/gad.531009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gonzalez-Reyes A., Elliott H., and St Johnston D., 1997.  Oocyte determination and the origin of polarity in Drosophila: the role of the spindle genes. Development 124: 4927–4937. [DOI] [PubMed] [Google Scholar]
  18. Gratz S. J., Cummings A. M., Nguyen J. N., Hamm D. C., Donohue L. K. et al. , 2013.  Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194: 1029–1035. 10.1534/genetics.113.152710 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Greenwald I., 2005.  LIN-12/Notch signaling in C. elegans, (August 4, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.10.1, http://www.wormbook.org. 10.1895/wormbook.1.10.1 [DOI] [Google Scholar]
  20. Grieder N. C., de Cuevas M., and Spradling A. C., 2000.  The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127: 4253–4264. [DOI] [PubMed] [Google Scholar]
  21. Hawkins N. C., Thorpe J., and Schupbach T., 1996.  Encore, a gene required for the regulation of germ line mitosis and oocyte differentiation during Drosophila oogenesis. Development 122: 281–290. [DOI] [PubMed] [Google Scholar]
  22. Huang Y. S., and Richter J. D., 2004.  Regulation of local mRNA translation. Curr. Opin. Cell Biol. 16: 308–313. 10.1016/j.ceb.2004.03.002 [DOI] [PubMed] [Google Scholar]
  23. Huynh J. R., and St Johnston D., 2000.  The role of BicD, Egl, Orb and the microtubules in the restriction of meiosis to the Drosophila oocyte. Development 127: 2785–2794. [DOI] [PubMed] [Google Scholar]
  24. Huynh J. R., and St Johnston D., 2004.  The origin of asymmetry: early polarisation of the Drosophila germline cyst and oocyte. Curr. Biol. 14: R438–R449. 10.1016/j.cub.2004.05.040 [DOI] [PubMed] [Google Scholar]
  25. Ivshina M., Lasko P., and Richter J. D., 2014.  Cytoplasmic polyadenylation element binding proteins in development, health, and disease. Annu. Rev. Cell Dev. Biol. 30: 393–415. 10.1146/annurev-cellbio-101011-155831 [DOI] [PubMed] [Google Scholar]
  26. Keyes L. N., and Spradling A. C., 1997.  The Drosophila gene fs(2)cup interacts with otu to define a cytoplasmic pathway required for the structure and function of germ-line chromosomes. Development 124: 1419–1431. [DOI] [PubMed] [Google Scholar]
  27. Keyes L. N., Cline T. W., and Schedl P., 1992.  The primary sex determination signal of Drosophila acts at the level of transcription. Cell 68: 933–943. 10.1016/0092-8674(92)90036-C [DOI] [PubMed] [Google Scholar]
  28. Khan M. R., Li L., Perez-Sanchez C., Saraf A., Florens L. et al. , 2015.  Amyloidogenic oligomerization transforms Drosophila Orb2 from a translation repressor to an activator. Cell 163: 1468–1483. 10.1016/j.cell.2015.11.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kimble J., and Hirsh D., 1979.  The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev. Biol. 70: 396–417. 10.1016/0012-1606(79)90035-6 [DOI] [PubMed] [Google Scholar]
  30. Lantz V., and Schedl P., 1994.  Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis. Mol. Cell. Biol. 14: 2235–2242. 10.1128/MCB.14.4.2235 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lantz V., Ambrosio L., and Schedl P., 1992.  The Drosophila orb gene is predicted to encode sex-specific germline RNA-binding proteins and has localized transcripts in ovaries and early embryos. Development 115: 75–88. [DOI] [PubMed] [Google Scholar]
  32. Lantz V., Chang J. S., Horabin J. I., Bopp D., and Schedl P., 1994.  The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity. Genes Dev. 8: 598–613. 10.1101/gad.8.5.598 [DOI] [PubMed] [Google Scholar]
  33. Lin H., and Spradling A. C., 1995.  Fusome asymmetry and oocyte determination in Drosophila. Dev. Genet. 16: 6–12. 10.1002/dvg.1020160104 [DOI] [PubMed] [Google Scholar]
  34. Little S. C., Sinsimer K. S., Lee J. J., Wieschaus E. F., and Gavis E. R., 2015.  Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nat. Cell Biol. 17: 558–568 [corrigenda: Nat. Cell Biol. 10.1038/ncb3143 10.1038/ncb3143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mach J. M., and Lehmann R., 1997.  An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila. Genes Dev. 11: 423–435. 10.1101/gad.11.4.423 [DOI] [PubMed] [Google Scholar]
  36. McGrail M., and Hays T. S., 1997.  The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila. Development 124: 2409–2419. [DOI] [PubMed] [Google Scholar]
  37. Ran B., Bopp R., and Suter B., 1994.  Null alleles reveal novel requirements for Bic-D during Drosophila oogenesis and zygotic development. Development 120: 1233–1242. [DOI] [PubMed] [Google Scholar]
  38. Ren X., Sun J., Housden B. E., Hu Y., Roesel C. et al. , 2013.  Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc. Natl. Acad. Sci. USA 110: 19012–19017. 10.1073/pnas.1318481110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rojas-Ríos P., Chartier A., Pierson S., Severac D., Dantec C. et al. , 2015.  Translational control of autophagy by orb in the Drosophila germline. Dev. Cell 35: 622–631. 10.1016/j.devcel.2015.11.003 [DOI] [PubMed] [Google Scholar]
  40. Salz H. K., 2011.  Sex determination in insects: a binary decision based on alternative splicing. Curr. Opin. Genet. Dev. 21: 395–400. 10.1016/j.gde.2011.03.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schupbach T., and Wieschaus E., 1991.  Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129: 1119–1136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Seydoux G., and Greenwald I., 1989.  Cell autonomy of lin-12 function in a cell fate decision in C. elegans. Cell 57: 1237–1245. 10.1016/0092-8674(89)90060-3 [DOI] [PubMed] [Google Scholar]
  43. Spradling A. C., de Cuevas M., Drummond-Barbosa D., Keyes L., Lilly M. et al. , 1997.  The Drosophila germarium: stem cells, germ line cysts, and oocytes. Cold Spring Harb. Symp. Quant. Biol. 62: 25–34. 10.1101/SQB.1997.062.01.006 [DOI] [PubMed] [Google Scholar]
  44. Stepien B. K., Oppitz C., Gerlach D., Dag U., Novatchkova M. et al. , 2016.  RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2. Proc. Natl. Acad. Sci. USA 113: E7030–E7038. 10.1073/pnas.1603715113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sternberg P. W., 2005.  Vulval development (June, 25 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.6.1, http://www.wormbook.org. 10.1895/wormbook.1.6.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Storto P. D., and King R. C., 1989.  The role of polyfusomes in generating branched chains of cystocytes during Drosophila oogenesis. Dev. Genet. 10: 70–86. 10.1002/dvg.1020100203 [DOI] [PubMed] [Google Scholar]
  47. Suter B., and Steward R., 1991.  Requirement for phosphorylation and localization of the Bicaudal-D protein in Drosophila oocyte differentiation. Cell 67: 917–926. 10.1016/0092-8674(91)90365-6 [DOI] [PubMed] [Google Scholar]
  48. Suter B., Romberg L. M., and Steward R., 1989.  Bicaudal-D, a Drosophila gene involved in developmental asymmetry: localized transcript accumulation in ovaries and sequence similarity to myosin heavy chain tail domains. Genes Dev. 3: 1957–1968. 10.1101/gad.3.12a.1957 [DOI] [PubMed] [Google Scholar]
  49. Tan L., Chang J. S., Costa A., and Schedl P., 2001.  An autoregulatory feedback loop directs the localized expression of the Drosophila CPEB protein Orb in the developing oocyte. Development 128: 1159–1169. [DOI] [PubMed] [Google Scholar]
  50. Theurkauf W. E., Alberts B. M., Jan Y. N., and Jongens T. A., 1993.  A central role for microtubules in the differentiation of Drosophila oocytes. Development 118: 1169–1180. [DOI] [PubMed] [Google Scholar]
  51. Van Buskirk C., and Schupbach T., 2002.  Half pint regulates alternative splice site selection in Drosophila. Dev. Cell 2: 343–353. 10.1016/S1534-5807(02)00128-4 [DOI] [PubMed] [Google Scholar]
  52. Vazquez-Pianzola P., Schaller B., Colombo M., Beuchle D., Neuenschwander S. et al. , 2017.  The mRNA transportome of the BicD/Egl transport machinery. RNA Biol. 14: 73–89. 10.1080/15476286.2016.1251542 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wharton R. P., and Struhl G., 1989.  Structure of the Drosophila BicaudalD protein and its role in localizing the the posterior determinant nanos. Cell 59: 881–892. 10.1016/0092-8674(89)90611-9 [DOI] [PubMed] [Google Scholar]
  54. Wilkinson H. A., Fitzgerald K., and Greenwald I., 1994.  Reciprocal changes in expression of the receptor lin-12 and its ligand lag-2 prior to commitment in a C. elegans cell fate decision. Cell 79: 1187–1198. 10.1016/0092-8674(94)90010-8 [DOI] [PubMed] [Google Scholar]
  55. Wong L. C., and Schedl P., 2011.  Cup blocks the precocious activation of the orb autoregulatory loop. PLoS One 6: e28261 10.1371/journal.pone.0028261 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wong L. C., Costa A., McLeod I., Sarkeshik A., Yates J. 111, et al. , 2011.  The functioning of the Drosophila CPEB protein Orb is regulated by phosphorylation and requires casein kinase 2 activity. PLoS One 6: e24355 10.1371/journal.pone.0024355 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yue L., and Spradling A. C., 1992.  hu-li tai shao, a gene required for ring canal formation during Drosophila oogenesis, encodes a homolog of adducin. Genes Dev. 6: 2443–2454. 10.1101/gad.6.12b.2443 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures and tables. Supplemental material available at figshare: https://doi.org/10.25386/genetics.9946979.

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES