Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.


INTRODUCTION
Morphogens are long-range signaling molecules that often establish gradients within developing tissues by traveling up to a few dozen cell diameters from the secreting cell source (Christian, 2012). Along the gradient, cells receiving different concentrations of morphogen signal will display different target gene expression profiles and differentiate into distinct cell types. The establishment of such morphogen gradients depends largely on tightly regulated expression of morphogen receptors and negative regulators, though other mechanisms may participate as well. One important family of morphogens is the Wnt proteins, which control various aspects of tissue development and stem cell function in many adult tissues (Clevers, 2006;MacDonald et al., 2009). However, relatively few studies have been conducted to describe the role of Wnt signaling in the development of ovaries, and the mechanisms controlling Wnt territory in the ovaries are unclear.
Wnt signaling is a highly conserved process that includes both canonical and non-canonical pathways (Nusse, 2005;MacDonald et al., 2009). Canonical Wnt signaling is initiated by the binding of Wnt to Frizzled (Fz) receptors and Lipoprotein Receptor Protein (LRP) coreceptors.
Wnt6 (vertebrate Wnt6), Wnt8 (vertebrate Wnt8), and Wnt10 (vertebrate Wnt10) (Janssen et al., 2010). Several of these Wnt ligands have been shown to participate in primordial germ cell (PGC) development in different species. For example, Wnt3 functions in mouse PGC specification (Aramaki et al., 2013), while Wnt4 functions in female sex differentiation (Kim et al., 2006) and Wnt5 controls PGC migration (Cantu et al., 2016). The role of Wnt signaling in the adult germarium has been extensively studied (Luo et al., 2015;Wang et al., 2015;Mottier-Pavie et al., 2016;Waghmare et al., 2020); however, fewer studies have examined the role of Wnt signaling during ovary development. In flies, Wg and Wnt8 respectively control embryonic PGC proliferation and migration (Sato et al., 2008;McElwain et al., 2011), whereas Wnt4 mediates non-canonical Wnt signaling to control the PGC-soma interaction in larval ovaries (Upadhyay et al., 2018) and to stimulate apical cell migration during germarium formation in pupal ovaries (Cohen et al., 2002). Therefore, the role of canonical Wnt signaling in the development of ovaries is not fully understood.
We used the Drosophila ovary as a model to address the potential role and functional effects of canonical Wnt signaling in ovary development because of its simple cell composition and well-characterized cell biology ( Figure 1A) (Xie and Spradling, 2000;Gilboa, 2015;Lai et al., 2017). The gonad of first instar larvae (L1; right after hatching) contains only a few PGCs and somatic gonad precursors (SGPs). Beginning at the early second instar larval stage (L2), a first morphogenesis occurs along the anterior-posterior and medial-lateral axes. A twodimensional array of 16-20 stacks of somatic cells called terminal filaments (TFs) is generated by the end of thirdinstar larval (L3) stage, and the remaining SGPs differentiate into various somatic cell types. Apical cells are positioned above TFs. Intermingled cells (ICs) intermingle with PGCs, and basal cells are located at the bottom of the gonad. During pupal stages, apical cells migrate basally between TFs and through both ICs and basal cells to form 16-20 ovarioles (Cohen et al., 2002). Each ovariole bears six to seven sequentially developing egg chambers and is considered to be a functional unit for producing eggs (Wu et al., 2008). The anterior structure of the ovariole is called the germarium. Within this region, the ICs closest to the basal TFs differentiate into cap cells (Song et al., 2007), whereas more distal ICs become escort cells (ECs). The cap cells, anteriormost ECs, and one TF constitute a GSC niche (Eliazer and Buszczak, 2011), which produces Decapentaplegic (Dpp, a Drosophila BMP), for the maintenance of two or three GSCs (Xie and Spradling, 1998). Each GSC is located directly adjacent to cap cells and contains a membranous organelle, called a fusome, which is found near the GSCcap cell interface (Snapp et al., 2004). Each GSC division gives rise to one daughter GSC and one cystoblast (CB) that subsequently undergoes four rounds of incomplete division to become a 2-, 4-, 8-, and then 16-cell cyst (de Cuevas et al., 1997) as it migrates through regions 1 and 2 of the germarium (Drummond- Barbosa and Spradling, 2001).
During these divisions, the fusome grows to interconnect the germ cells within the cyst, and it takes on a branched morphology in 4-, 8-and 16-cell cysts. Around the CB and cyst, ECs extend long cellular membrane protrusions to wrap and facilitate the differentiation of GSC progeny (Kirilly et al., 2011). At the 2A/2B boundary, follicle cells substitute for ECs to wrap 16-cell cysts, which take on a lens-like shape. After acquiring a monolayer of follicle cells (derived from follicle stem cells at the 2A/2B boundary), the 16-cell cyst becomes a newly formed egg chamber and buds off from the germarium, eventually developing into a mature egg.
In this study, we report that canonical Wnt signaling functions in the formation of ECs and maintains their long cellular protrusions to prevent Dpp and Wg signaling to GSC progeny, which would interfere with their proper differentiation. In germaria bearing ECs with blunted cellular protrusions, Dpp and Wg leak from the soma into the germ cell region. In the germ cells of these germaria, Dpp activates Dpp stemness signaling and Wg activates canonical Wnt signaling to promote transcription of CycB3, a G2/M regulator (Jacobs et al., 1998), and disrupts germ cell differentiation. Our results not only document a role for canonical Wnt signaling in EC formation during ovary development, but the findings also demonstrate a functional requirement for a somatic cell permeability barrier to prevent morphogen signals from reaching germ cells.

The Developing Ovarian Soma Displays Active Canonical Wnt Signaling
A previous study reported that canonical Wnt signaling is undetectable in the late larval ovary, but it is activated in germarial ECs of late pupae (Upadhyay et al., 2018). This finding was made using the fz3RFP reporter, which consists of the promoter region of frizzled3 (fz3) followed by an open reading frame for red fluorescent protein (RFP) (Barolo et al., 2004;Olson et al., 2011), as fz3 is a validated downstream target of canonical Wnt signaling (Sato et al., 1999). Since a complete assessment of canonical Wnt signaling activation in the ovary throughout development was still lacking, we first examined the fz3RFP expression in the ovary at different developmental stages. In the L1 gonad ( Figure 1B and B'), fz3RFP was not detectable, but its expression was clearly observable in ICs of the L2 gonad ( Figure 1C and C'). In the late-L3 gonad ( Figure 1D and D'), fz3RFP was weakly expressed in developing TFs but more highly expressed in a subset of ICs and basal cells. Similar patterns were also observed in late-L3 gonads ( Figure 1E and E') when using another canonical Wnt signaling reporter, 3GRH4TH-GFP, which contains three Grainyhead (GRH) and four classic HMG-Helper (4TH) site pairs followed by green fluorescent protein (GFP) (Zhang et al., 2014). At the early-pupal stage, ICs in the germarium (considered to be ECs) expressed low levels of fz3RFP ( Figure 1F), while fz3RFP expression became strong in ECs at the mid-pupal stage ( Figure 1G) and remained in adult gemaria of newly eclosed flies (1-day-old) ( Figure 1H). Interestingly, in the 1-day-old adult germarium 3GRH4TH-GFP was expressed only in one or two ECs that were in direct contact with GSCs ( Figure 1I), although a previous report showed 3GRH4TH-GFP expression in posterior ECs and follicle stem cells (Kim-Yip and Nystul, 2018). Even so, our results indicate that canonical Wnt signaling is active in the developing ovarian soma and becomes strong in germarial ECs from the midpupal stage.  show only Fax channel. (P-S) tj-GAL4>gfp RNAi (III) (P), tj > dsh RNAi (Q), tj > arm RNAi (V1) (R) and tj > pygo RNAi germaria (S) with LamC (green) and Hts (green). (T-W) tj-GAL4>gfp RNAi (III) (T), tj > dsh RNAi (U), tj > pygo RNAi (N) and tj > arm RNAi (V2) germaria (W) with LamC (green) and Hts (green). Solid lines, GSC-cap cell junction; dashed lines, the 2A/2B boundary (or the junction between escort cells and follicle cells when 2A/2B boundary is missing). Arrowheads and an asterisk in panel 2B respectively mark GSCs and cystoblast. Two asterisks in F, H, K, and Q denote two side-by-side egg chambers in the ovariole. Scale bars are 10 μm; B-D and H-K, E-G, L-O, P-S, and T-W have the same scale bar. In the canonical Wnt signaling pathway, binding of Wnt ligands to Fz receptors leads to recruitment of Dsh and consequent disruption of the Axin destruction complex. This action stabilizes Arm, which subsequently enters the nucleus and interacts with Pygo (co-activator) and TCF to regulate expression of the downstream targets ( Figure 2A) (Staal et al., 2008). It has been previously concluded that canonical Wnt signaling does not play a role during ovary development, as very low fz3RFP expression was detected in the larval ovary (Upadhyay et al., 2018). In addition, arm knockdown throughout development only causes a very mild increase in the number of undifferentiated cells carrying round-shape fusomes (spectrosome-containing cells; SCCs) (Mottier-Pavie et al., 2016). However, our data showed that fz3RFP and 3GRH4TH-GFP expression is detectable in ICs. Furthermore, no canonical Wnt signaling components other than Arm had been tested for functional effects in the developing ovary. We thus individually disrupted dsh, arm and pygo expression in the ovarian soma throughout development and examined 1-day-old germaria. For this purpose, we used UAS-RNAi lines driven by tj-GAL4, which is expressed in ICs of the larval ovary (Supplementary Figure S1) . At the anterior tip of the control 1-dayold germarium ( Figure 2B), two GSCs can be identified by their fusomes, i.e., the membrane-enriched organelle (yellow arrows) adjacent to niche cap cells (cap cell-GSC junction is indicated by a solid line). Additionally, 1 ± 0.7 spectrosome-containing CBs were designated as SCCs (n = 15 germaria) (indicated by the asterisk in Figure 2B). Differentiating germ cells containing branched fusomes were located posterior to the GSCs and wrapped by EC protrusions (marked by fz3RFP; Figure 2B). Between cap cells (indicated by a yellow line) and the 2A/B boundary (indicated by a dashed line, Figure 2B'), we observed 26.2 ± 3.3 ECs (n = 21 germaria) expressing Traffic jam (Tj, a Maf transcription factor (Li et al., 2003)). In contrast, the dsh-and arm-knockdown (KD) germaria (Figures 2C,D), fewer cysts with branched fusomes were found posterior to the GSCs, and this decrease in cysts was accompanied by SCC accumulation [dsh-KD, 4.7 ± 1.9 SCCs (n = 14 germaria), p < 0.001; arm-KD, 6.6 ± 4.6 SCCs (n = 14 germaria), p < 0.001; some SCCs were even observed within egg chambers]. As evidence for the disruption of Wnt signaling activity, fz3RFP expression in ECs was dramatically decreased in dsh-and arm-KD germaria ( Figures  2B-D). dsh-and arm-KD germaria also carried fewer ECs (dsh KD, 9.0 ± 3.8 ECs (n = 21); arm KD, 6.8 ± 4 ECs (n = 15), p < 0.001), resulting in shortened EC regions (space between cap cells and the 2A/B boundary, or space between cap cells and follicle cells if the 2A/ B boundary is lost) ( Figure 2C' and D'). In addition, Failed axon connections (Fax)-labeling was used to mark EC protrusions . The germ cells in the control germarium were completely wrapped by ECs ( Figure 2E and E9), whereas the wrapping was disrupted in dsh-KD germaria ( Figure 2F and F9). Similar results could be obtained by disrupting pygo expression ( Figure 2G and G') or by using another somatic GAL4 driver, c587-GAL4 (Tseng et al., 2018), to drive expression of dsh RNAi or another independent arm RNAi throughout developmental stages ( Figures  2H,I). Moreover, compared to dsh-KD germaria ( Figure 2J), overexpressing a constitutively active form of Arm, arm S10 (Pai et al., 1997), in the dsh-KD soma throughout development expanded the EC region, decreased SCC accumulation [63% of dsh-KD & mCD8gfp germaria (n = 26) carrying more than 4 SCCs vs 0% of dsh-KD & arm S10 germaria (n = 20) carrying more than 4 SCCs, p < 0.001], and increased cyst cells bearing a branched fusome [0% of dsh-KD & mCD8gfp germaria (n = 26) carrying more 4-6 16-cell cysts vs 95% of dsh-KD & arm S10 germaria (n = 20) carrying 4-6 16-cell cysts, p < 0.001] ( Figure 2K). Given that these phenotypes were observed after knockdown of nearly every canonical Wnt signaling component, we concluded that canonical Wnt signaling is required for formation of ECs and their protrusions during ovary development, and that promotes germ cell differentiation.
To understand the temporal requirement of somatic canonical Wnt signaling during ovary development, we knocked down dsh, arm and pygo at different developmental times by modulating GAL4 activity with GAL80 ts ; in these lines, GAL4 activity is suppressed at 18°C but not at 29°C (McGuire et al., 2004). Knockdown of dsh, arm and pygo from late-L3 to adult stage caused similar phenotypes, which were less severe than those seen with continuous knockdown ( Figure 2P-S). Suppressing dsh and pygo in the larval soma before the late-L3 stage did not result in aberrant germaria ( Figure 2T-V), while arm-KD germaria exhibited SCC accumulation ( Figure 2W), likely due to a requirement for the interaction between Arm and E-cadherin to promote PGC-IC intermingling (Supplementary Figure S2) . Taken together, these results show that canonical Wnt signaling acts on ICs, probably as early as late-L3, to maintain the EC population and promote germ cell differentiation.

Wnt4 and Wnt6 in the Developing Soma Stimulate Canonical Wnt Signaling in Escort Cells
Five Wnts are known to be expressed in adult germarial somatic cells. Wg and Wnt6 are highly expressed in cap cells, while Wnt2 and Wnt4 are expressed in both cap cells and ECs (Luo et al., 2015;Wang et al., 2015). Wnt5 is also expressed at low levels, according to RNA-seq analysis of isolated ECs . To determine which Wnt activates the somatic canonical Wnt signaling required for EC formation and germ cell differentiation, we individually knocked down Wnt ligands throughout development using tj-GAL4 and examined germarial phenotypes and fz3RFP expression in 1-day-old ovaries. However, we did not observe overt phenotypes similar to those seen in dsh-or arm-KD ( Figures 3A-H). However, we did observe side-by-side cysts or egg chambers (indicated by  asterisks in Figure 3D and D') after knockdown of wnt4, and we found slightly increased SCC numbers upon knockdown of wnt4, wnt5 and wnt6 ( Figure 3K). Except wnt2 (only one RNAi line was available), similar results were obtained similar results from an independent RNAi lines (Supplementary Figure S3). Somatic knockdown of wg, wnt2, wnt5, wnt8 or wnt10 increased fz3RFP, somatic knockdown of wnt4 or wnt6 decreased fz3RFP expression in ECs of 1-day-old germaria ( Figure 3L). Co-knockdown of wnt4 and wnt6 in the developing soma caused germaria to exhibit nearly absent fz3RFP expression, a shortened EC region, SCC accumulation (wnt4 & 6 coKD germaria: 4.2 ± 3.7 SCCs, n = 22 germaria; gfp-KD germaria: 1.6 ± 1.0 SCCs, n = 20 germaria; p < 0.005), and side-by-side cysts or eggs ( Figure 3I-L), reminiscent of somatic dsh-KD germaria. We did not know why developmental knockdown of wg, wnt2, wnt5, wnt8, and wnt10 would increase canonical Wnt signaling in ECs, perhaps other Wnt ligands are increased for compensation when those Wnts are decreased. Nevertheless, our data suggested that Wnt4 and Wnt6 appear to be positive regulators of canonical Wnt signaling in ECs.

Canonical Wnt Signaling Is Activated in the Germline When Thickveins Is Suppressed in the Soma During Development
We have previously shown that during ovary development, germ cell differentiation requires somatic Tkv, a Dpp receptor (Xie and Spradling, 1998), which maintains EC protrusions via a Smadindependent pathway (Tseng et al., 2018). In this line when tkv is disrupted in the developing ovarian soma, EC protrusions are disrupted, and SCC accumulation occurs, but the EC region is not shortened ( Figures 4A,B, and see Supplementary Figure  S8A,B), as compared to the germaria developed from the gonad with disrupted Canonical Wnt signaling. Intriguingly, analysis from our previous RNA-seq result (Tseng et al., 2018) showed an increased fz3 mRNA transcript level in 1-day-old c587>tkv RNAi ovaries ( Figure 4C), whereas, fz3RFP expression was not increased in ECs of 1-day-old tkv-KD germaria, as compared to controls (see inset images in Figures 4A,B). Coknockdown of dsh and tkv in the developing ovarian soma did not prevent SCC accumulation but did cause a shortened EC region ( Figures 4D,E). These results indicate that canonical Wnt signaling in the soma is not involved in the impairment of germ cell differentiation in somatic tkv-KD germaria; instead, canonical Wnt signaling may be elevated within the germ cells of somatic tkv-KD germaria. We were not able to perform knockdown of Wnt signaling specifically in the germline of somatic tkv-KD germaria by the current genetic tools because the targeted signaling component would be knocked down both in the germline (by nos-GAL4) and in the soma (by tj-GAL4, which was used to knockdown tkv). Therefore, we knocked down Wnts in the developing soma of somatic tkv-KD ovaries. Somatic knockdown of tkv with wnt2, wnt4, wnt5 or wnt6 did not suppress SCC accumulation (Supplementary Figure S4). Strikingly, simultaneous knockdown of wg and tkv in the developing soma partially rescued the germ cell differentiation defect, as evidenced by reduced SCC numbers, and increased 16-cell cyst numbers ( Figures 4F-H), indicating that Wg may activate canonical Wnt signaling in the germline of somatic tkv-KD germaria.
We did not detect fz3RFP expression in germ cells, perhaps because fz3RFP only effectively reports Wnt signaling in somatic cells. We thus examined fz3 mRNA expression in somatic tkv-KD germaria by in situ hybridization. In the control germariA ( Figure 4I), fz3 transcripts were detected in the cytoplasm of anterior germ cells and ECs, while fz3 transcript levels were dramatically increased in the germ cells of somatic tkv-KD germaria ( Figure 4J,O). Of note, no fz3 mutants or UASp-RNAi lines were available to test the specificities of fz3 antisense probes we used. Instead, we overexpressed arm or knocked down axin in the germline to force canonical Wnt signaling activation, and then we examined fz3 expression. We found that fz3 transcripts were significantly increased in the germline with arm overexpression ( Figure 4K,L,O), and in axin-KD germ cells (Supplementary Figure S5). As expected, increased fz3 transcripts in the germline of somatic tkv-KD germaria could be suppressed by knockdown of wg, using two independent RNAi lines ( Figure 4M-O). These results suggested that Wg from the soma contributes to germ cell differentiation defects in somatic tkv-KD germaria. It is likely that when tkv is disrupted in the soma, the receipt of Wg by germ cells activates canonical Wnt signaling, which is deleterious for germ cell differentiation.

Decreasing CycB3 Expression in the Germline of Somatic Tkv-KD Germaria Partially Rescues Germ Cell Differentiation
From the previously obtained RNA-seq data (Tseng et al., 2018), we noticed that transcripts of some Cyclins (e.g., CycB, CycB3, and CycE) and Cyclin-dependent protein serine/threonine kinase regulators (e.g., CycT) were significantly increased in 1-day-old somatic tkv-KD germaria ( Figure 5A). Among the increased transcripts, CycB, CycB3 and CycE are known to be required for GSC maintenance (Ables and Drummond-Barbosa, 2013;Chen et al., 2018;Wang and Lin, 2005), and increased CycB3 expression delays CB differentiation (Chen et al., 2018). Given the continuous proliferation of SCCs in somatic tkv-KD germaria (Supplementary Figure S6), and since an antagonism between cell cycle regulators and differentiation genes has been proposed (Ruijtenberg and van den Heuvel, 2016), we next asked if reducing Cyclins could suppress germline differentiation defects in somatic tkv-KD germaria. We individually knocked down cycB, cycB3 and cycE in the germline using nos-GAL4, along with tkv knockdown in the soma using tj-GAL4 throughout developmental stages (Figures 5B-E). Of note, the tkv RNAi(V) line used in this study was not effectively expressed in the germline due to its UASt promoter (see Figures 4H, Figure 5F), and therefore the role of Tkv in the germline is uncertain with regard to GSC maintenance. Knockdown of cyclins in the developing soma did not cause obvious defects in germaria, but knockdown of cycB and cycE in the germline throughout development respectively caused GSC loss and SCC (I) Box plot shows expression of CycB3-GFP in GSCs or SCCs in the indicated genotypes. (J) Schematic shows how β-catenin (β-Cat) interacts with TCF, which binds to HMG and Helper sites of the cycB3 promoter through its HMG and C domains, respectively. ChIP analysis of TCF binding in 1-day-old ovaries; the chromatin from nos > gfp and nos > arm-mgfp6 cells was precipitated with GFP-Trap beads. Co-precipitated DNA was analyzed by qPCR using two sets of primers (P1 and P2) against the region between Helper and HMG sites. The amplicons of two different coding regions were used as negative controls. (K and L) One-day-old bab1>mcherry RNAi (K) and bab1>wg RNAi (V) (L) with cycB3P-CycB3-GFP (gray) and CellMask (Magenta, cell membrane). (M) Average of CycB3-GFP expression in GSCs of indicated genotypes. Number of GSCs analyzed is shown above each bar. Differences in F and F′ were analyzed by one-way ANOVA; data in I and M were analyzed by Student's t test, and in J were analyzed by two-way ANOVA. Solid line in the box of I and M is median; cross in M is Mean. Error bars represent SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001. RNAi was expressed throughout development until dissection. Scale bar is 10 μm. Figure S7). Furthermore, knockdown of cycB in the germline of somatic tkv-KD germaria did not rescue the germ cell differentiation defect ( Figures 5B-F and F'), and germ cells were completely lost from tj&nos > tkv RNAi &cycE RNAi germaria, which displayed a thin tubular-like shape ( Figure 5D). Strikingly, knockdown of cycB3 in the germline of somatic tkv-KD ovaries decreased SCCs and increased 16-cell cysts ( Figures 5E,F and F'), and it partially rescued EC protrusions (Supplementary Figure S8). These effects suggested that the increase of CycB3 in the germline suppressed germ cell differentiation in the somatic tkv-KD germaria.

Wg Signaling Promotes CycB3 Expression in Spectrosome-Containing Cells Upon Somatic Knockdown of Tkv
We next examined CycB3 expression in live germaria, using a cycB3 promoter (P)-CycB3-GFP transgene (Chen et al., 2018). We used this approach due to a lack of anti-CycB3 antibody and high background from anti-GFP staining. In the control germarium ( Figures 5G,I), CycB3-GFP was expressed in GSCs, germ cells posterior to GSCs, and some follicle cells, but it was absent in late-differentiating cysts (marked by asterisks). In somatic tkv-KD germaria, CycB3-GFP expression was further enhanced in GSCs and prospective SCCs ( Figures  5H,I), which were identified by the nucleus size being comparable to control GSCs. These results raise the possibility that canonical Wnt signaling may promote CycB3 expression at the transcriptional level.
Activation of Wnt signaling induces β-catenin nuclear translocation and interaction with TCF, turning on target gene transcription (MacDonald et al., 2009). We found that the cycB3 promoter includes a putative Helper-HMG pair element (Helper: -1529-1529bp; HMG: -1178-1173bp) ( Figure 5J); Helper and HMG sequences are respectively recognized by the C-clamp and HMG domain of TCF (Ravindranath and Cadigan, 2016). We used chromatin immunoprecipitation (ChIP) to examine whether the Arm (tagged with GFP)-TCF complex binds to the Helper-HMG pair element of the cycB3 promoter in 1-day-old ovaries carrying nos > arm-gfp. To determine the Arm-TCF complex occupancy on the Helper-HMG pair element, we used qPCR to amplify two fragments (P1 and P2) located in the promoter region between the Helper and HMG sites ( Figure 5J). The amounts of amplified P1 and P2 fragments from nos > arm-gfp ovaries were 6-to 7-fold higher than those from nos > gfp ovaries, while the amounts of PCR product amplified from the coding region of cycB3 in nos > gfp ovaries showed no difference ( Figure 5J). Furthermore, knockdown of wg using another somatic driver (bab1-GAL4, which is expressed in ICs and enriched in cap cells where Wg is generated after pupal stages (Tseng et al., 2018), significantly reduced cycB3-GFP expression ( Figure 5K-M). These results indicate that cycB3 is a novel target of canonical Wnt signaling. Taken together, the data suggests that when tkv expression is disrupted in the developing ovarian soma, germline canonical Wnt signaling is upregulated and transcriptionally promotes expression of CycB3, which in turns suppresses germ cell differentiation.

Wnt-cycB3 Regulation in the Germline Promotes Germ Cell Differentiation in Normal Germaria
To determine if enhanced Wnt signaling promotes SCC accumulation via loss of somatic Tkv function in the soma, we directly overexpressed Arm or suppressed Axin (a negative regulator of Wnt signaling (Kishida et al., 1998)) in the germline throughout development and examined 1-day-old germarial phenotypes. These germaria did not exhibit SCC accumulation, but we observed increased cycB3-GFP expression and higher numbers of 16-cell cysts located in the anterior germarium compared with the numbers of 16-cell cysts present in region 2 of the control germaria (Supplementary Figure S9A-I); these findings were in agreement with a previous study (König and Shcherbata, 2015). In addition, disruption of canonical Wnt signaling in the germline was previously shown to slightly increase SCC numbers, suggesting a delay of CB differentiation (König and Shcherbata, 2015). Furthermore, knockdown of cycB3 in the germline of nos > axin RNAi germaria decreased 16-cell cysts (Supplementary Figure 9J-L). These results further confirm the existence of a Wnt signaling-CycB3 regulatory axis that is important for germline homeostasis. However, somatic cells appear to play a direct or indirect role in promoting or suppressing germ cell differentiation by Wnt signaling-CycB3 regulation, at least in part through the action of Tkv.

Blunted Escort Cell Protrusions Allow Wg and Dpp to Signal in the Germline
We next asked how Wg-mediated canonical signaling becomes activated in the germline of somatic tkv-KD germaria. Previous reports showed that Wg is produced from cap cells (Forbes et al., 1996;Luo et al., 2015;Song and Xie, 2003;Wang and Page-McCaw, 2014) and received by follicle stem cells to promote their maintenance and proliferation (Song and Xie, 2003;Wang and Page-McCaw, 2014). EC protrusions wrap germ cells (Sahai-Hernandez and Nystul, 2013) and are disrupted in somatic tkv-KD germaria (see Supplementary Figure S8) (Tseng et al., 2018), raising the possibility that Wg might be normally restricted to somatic cells due to EC protrusions. Therefore, Wg may be able to access germ cells that are not wrapped by EC protrusions. To test this hypothesis, we used GFP-Wg in which GFP is inserted into the wg locus (Port et al., 2014) to examine the distribution of Wg in live 1-day-old control and somatic tkv-KD germaria labeled with CellMask, a cell membrane dye. GFP-Wg granule numbers in TF and cap cells (19.6 ± 3, n = 12), where Wg is produced, were decreased when wg (9.1 ± 3, n = 17, p < 0.001) or gfp (7.8 ± 4.3, n = 14, p < 0.001) were knocked down in the developing soma using c587-GAL4 ( Figures 6A,B, and Supplementary Figure S10), demonstrating that GFP-Wg expression can fairly represents Wg expression. GFP-Wg expression was not altered in somatic tkv-KD ovaries as compared to control ( Figure 6C), while GFP-Wg distribution was altered ( Figures 6D-F). In the control germarium ( Figure 6E,E@ and Supplementary Figure S11), GFP-Wg was mainly present in cap cells (arrows), and it wasv observed in ECs (indicated by yellow asterisks) as well as in follicle cells and germ cells posterior to the 2A/2B boundary; very few GFP-Wg signals were observed in the germ cell zone before the 2A/2B boundary (2.3 ± 1.2 granules, n = 11 germaria). In contrast, in addition to above-mentioned somatic cells and germ cells after follicle cell layer (2A/B boundary was missing), GFP-Wg signals were increased in the germ cell zone before the Representative immunoblot shows that Wg-GFP expression (anti-GFP antibody) is similar in 1-day (D)-old control (ctrl) and c587>tkv RNAi (V) ovaries. Histone (H3) was used as a loading control. Molecular weight markers are indicated to the right of the blots. (D) Schematic of a germarium with a three-axis (X, Y, and Z) coordinate system. The directions of each axis are shown. The Y-axis is defined as anterior to posterior. An XY section is shown as the light green shaded area, and an XZ section is shown as a light pink shaded area. Terminal filament cells, gray; cap cells, dark green; escort cells, red; germ cell, light yellow; follicle cells, light blue. (E-I) Live images of 1-day-old GAL4 control (E), c587>tkv RNAi(v) (F), and bam 1 /bam △86 mutant (G and I), sibling control germaria (H), bearing GFP-wg (green in E-G), Dpp-mcherry (green in H and I) and labelled with CellMask (red, cell membrane). (E"-I") are optical sections in the XZ plane; the corresponding schematic with the cell types is shown in D and corresponds to (E'-I'); green color shows the distribution of Wg (E"-G") or Dpp (H"-I"). Scale bar, 10 μm; A and B, E and F, and H and I share the same scale bar. Germaria were examined for wg-GFP distribution in control (n = 12), c587>tkv RNAi(v) (n = 25), and bam 1 /bam △86 mutant (n = 15). Germaria were examined for Dppmechrry distribution in the control (n = 8), and bam 1 /bam △86 mutant (n = 12). Control genotypes in A, C and E are c587>gfp RNAi(III) , GFP-wg/+; in H is (+/Dpp-mcherry; bam 1 or bam △86 /+). Arrows point to the cap cell region. Dashed lines in E and H mark the 2A/2B boundary, and F, G and H mark in the junction between escort cells and follicle cells (the 2A/2B boundary is lost). Asterisks mark GFP signals present in ECs.
Frontiers in Cell and Developmental Biology | www.frontiersin.org April 2022 | Volume 10 | Article 877047 follicle cell layer of the somatic tkv-KD germarium (10.9 ± 2.3 granules, n = 12 germaria; p < 0.001) ( Figure 6F,F" and Supplementary Figure S11). We further confirmed this explanation by examining the Wg distribution in the bag-ofmarbles (bam) mutant germarium, which display blunted EC protrusions due to defective germ cell differentiation (Kirilly et al., 2011). The GFP-Wg distribution in the germline zone before follicle cell layer (2A/B boundary was missing) of the bam mutant was similar to the distribution in the somatic tkv-KD germaria ( Figure 6G,G" and Supplementary Figure S11), although GFP-Wg expression seemed to be increased. Consistent with this observation, CycB3-GFP expression was also increased in SCCs of bam mutant germaria (Supplementary Figure S12). ECs did not produce wg transcripts (Supplementary Figure S13), indicating that niche-produced GFP-Wg distributed in the germline of somatic tkv-KD germaria is due to the lack of EC protrusions. We next wanted to know if EC protrusions also limit the distribution of Dpp (mammalian BMP; stemness factor that maintains GSC fate), which is produced from cap cells. To answer this question, we used mCherry-tagged Dpp (Dpp-mCherry), which was expressed in cap cells and ECs in the control germarium ( Figure 6H,H"). Remarkably, Dpp-mCherry signal was spread throughout the germ cell zone of the bam mutant germarium (bam mutant; 28.9 ± 7 granules, n = 10 germaria vs sibling control: 2.7 ± 2 granules, n = 18 germaria; p < 0.001) ( Figure 6I,I" and Supplementary Figure S14). These results suggest that EC protrusions wrap germ cells and prevent them from receiving cap cellproduced signals. Furthermore, multiple secretory factors leak to germ cells when EC protrusions are disrupted may explain the differential effect of canonical Wnt signaling-CycB3 regulation on germ cell differentiation in the normal and somatic tkv-KD germaria.

Escort Cell Protrusions Act as a Physical Barrier to Compartmentalize Germ Cells
We next directly tested if the germline is isolated from the external environment by EC protrusions using a previously developed permeability assay (Fairchild et al., 2015). In this assay, ovaries were dissected and incubated in medium containing a fluorescently labeled 10-kDa dextran dye. The dye accessibility to germ cells was assessed. In the control germarium (n = 10 germaria) ( Figure 7A and A"), the fluorescence signal (black) overlapped with EC protrusions (red, marked by CellMask) but was excluded from germ cells. In contrast, in somatic tkv-KD germarium with blunted EC protrusions (n = 10 germaria) ( Figure 7B,B"), the fluorescent dye was observed between all germ cells. This result suggests that EC protrusions isolate germ cells from the external environment.

DISCUSSION
Wnts are critical and conserved morphogens that control development of various organs. However, the nuanced roles of Wnt signaling in ovary development are still undefined, and how Wnt signaling territory is set within the tissue remains largely unclear. Here, we report that canonical Wnt signaling is activated in the ovarian ICs to promote EC formation and maintain EC protrusions. When canonical Wnt signaling is disrupted in ICs, EC number is decreased and EC cellular protrusions are disrupted. In the adult wild-type germarium ( Figure 7C), cap cells express Dpp and Wg signals, which respectively lead to Mad phosphorylation and transcriptional activation of CyCB3 in GSCs to maintain GSC fate. In addition, Dpp and Wg can signal to ECs as well (see Figure 6) (Luo et al., 2015). When EC protrusions are present, GSC progeny are compartmentalized, preventing aberrant activation of Dpp/Wnt signaling in GSC progeny to allow their proper differentiation. By contrast, without EC protrusions ( Figure 7D), Wg and Dpp are no longer restrained in the soma and diffuse to GSC progeny, where they suppress germ cell differentiation. Taken together, our data suggest that canonical Wnt signaling in the developing ovarian soma promotes the development of ECs, and the EC protrusions act as a physical permeability barrier to establish the territory of morphogens produced by the GSC niche. This barrier is necessary for proper differentiation of GSC progeny. Similar physical cell barriers to prevent the reception of morphogen signals by germ cells might exist in other organs and organisms, where they may act as determinants of tissue patterning.

Both Canonical and Non-canonical Wnt Signaling in the Soma of Larval Gonads Are Required for Germ Cell Differentiation
A switch between Wnt4-Dsh-mediated non-canonical and canonical Wnt signaling in larval gonads and adult germaria has been proposed (Upadhyay et al., 2018). In their study, Upadhyay and colleagues reported that two non-canonical Wnt signaling reporters were expressed in ICs of late larval gonads, but the reporter expression levels were decreased in adult ECs. Meanwhile, expression of the canonical Wnt signaling reporter, fz3RFP, was observed in a reverse pattern; it was not expressed in ICs, but it became strongly expressed in ECs. Knockdown of non-canonical Wnt signaling components decreased IC number and disrupted IC-PGC intermingling, resulting in a milder germ cell differentiation defects in adult germaria. In contrast, knockdown of non-canonical Wnt signaling components in adult ECs did not cause obvious defects.
In our study, we did not observe obvious defects in newly eclosed flies when canonical Wnt signaling was knocked down in the ovarian soma from embryonic to late-L3 stages, suggesting a dispensable role of canonical Wnt signaling in the soma before the late-L3 stage. However, we could detect the expression of two different canonical Wnt signaling reporters in ICs of L2 and late-L3 gonads. In addition, although Dsh is involved in both canonical and non-canonical Wnt signaling, we did not find IC-PGC intermingling defects when dsh was knocked down in the soma (see Supplementary Figure S2C). Nevertheless, overexpressing a constitutively active form of Arm could not rescue the side-by-side cyst or egg chamber phenotype in somatic dsh-KD germaria (see Figure 2F); this phenotype is also observed in tj > wnt4 RNAi germaria (see Figure 3D). Thus, both canonical and non-canonical Wnt signaling function in the larval soma, and the putative switch between non-canonical and canonical Wnt signaling in the ovarian soma might occur as early as the late-L3 stage.

Escort Cell Protrusions Compartmentalize Germ Cells to Block Cap Cell-Produced Maintenance Cues
The boundaries of Wg signaling contribute to the patterning of various cell types in tissues throughout the organism. Several determinants of Wg territories have been reported. For example, glypicans (cell surface heparan sulfate proteoglycans) were shown to affect cell surface localization of morphogens (Hufnagel et al., 2006), including Wg, Hedgehog (Hh) and Dpp. The fly has two glypicans, Dally and Dally-like protein (Dlp) (Filmus et al., 2008). Dally is expressed in cap cells to facilitate short-range Dpp trans signaling in GSCs (Guo and Wang, 2009;Hayashi et al., 2009), while Dlp is expressed in ECs for Wg long-range travel from cap cells to follicle stem cells (Wang and Page-McCaw, 2014). It has been proposed that Dally acts a classic co-receptor, while Dlp is like a gatekeeper, helping to transfer Wg from the source cells to distal cells (Franch-Marro et al., 2005). Interestingly, overexpression of Dlp in ECs attenuates Wnt signaling and results in the absence of fz3RFP expression, fewer ECs, blunted EC protrusions, and defective germ cell differentiation (Waghmare et al., 2020). On the other hand, knockdown of Dlp in ECs phenocopies Wnt overexpression, resulting in increased EC number and GSC loss without affecting germ cell differentiation (Waghmare et al., 2020). These results suggest that Dlp and Fzs trap Wg, at least partially on the EC surface. However, this explanation cannot fully account for the inactivation of canonical Wnt signaling in germ cells, since germ cells are closely associated with ECs and express low levels of Fzs, according to single cell-sequencing results from the larval gonad (Slaidina et al., 2020) and adult ovaries (Rust et al., 2020). In addition, somatic tkv-KD germaria do not show decreased fz3RFP expression, obvious changes in EC number, or GSC loss (Tseng et al., 2018), suggesting that Wg-trapping molecules are still expressed on the EC surface. In this study, we showed that GFP-Wg is distributed in the germ cell zone of somatic tkv-KD or bam mutant germaria, indicating that the expansion of GFP-Wg territory does not rely on ECs themselves. Therefore, the EC protrusions may generate a compartment to keep germ cells shielded from Wg. In such case, the interfaces between ECs and germ cells would not encounter Wg, while the outer surfaces of ECs (facing sheath cells) would have the opportunity to trap cap cell-secreted Wg. A similar mechanism seems to restrict Dpp in GSCs. In addition to Wg, cap cells also produce Hh, Wnt2, Wnt4 and Wnt6 (Luo et al., 2015;Lai et al., 2017). Unfortunately, we do not have tools available to investigate whether the distributions of these molecules are altered when EC protrusions are blunted. In addition, we do not know how germ cells remain unresponsive to Wnt2 and Wnt4, which are produced by ECs (Luo et al., 2015;Wang et al., 2015). Perhaps ECs display a cell polarity that causes Wnt2 and Wnt4 to be secreted only from the outer surface. Despite these remaining uncertainties, our results show that depletion of Wg alone can partially rescue germ cell differentiation defects in somatic tkv-KD germaria. Furthermore, the results suggest that EC protrusions physically wrap GSC progeny to block receipt of cap cell-derived maintenance cues, allowing germ cells to undergo proper differentiation, in line with the hypothesis made by Banisch and colleagues (Banisch et al., 2017).

Cell Barriers in Setting Morphogen Territories May Be Evolutionary Conserved
Soma-germline interactions are critical for germ cell differentiation, and in the fly ovary, EC-germline interactions are particularly important for germ cell differentiation. The long cellular protrusions of ECs wrap germ cells (Banisch et al., 2017;Kirilly et al., 2011), and disruption of these protrusions causes germ cell differentiation defects. Conversely, blocking germ cell differentiation also impairs EC protrusions (see Figures 6G,I) (Kirilly et al., 2011). In this study, we show that protrusions from ECs act as a somatic-germline barrier that compartmentalizes germ cells to prevent undue influence from GSC niche signals. A similar hypothesis has also been made with regard to fly testes, wherein somatic cyst cells (the counterparts of ECs) wrap GSCs and their progeny to facilitate proper differentiation (Decotto and Spradling, 2005;Fairchild et al., 2015). In mammalian testes, the early phase of spermatogenesis (GSCs and progenitor spermatogonia) occurs at the basal compartment of the seminiferous epithelium. This early phase is physically separated from the later phase of spermatogenesis, which occurs in the apical compartment, by an epithelial layer of somatic Sertoli cells called the blood-testicular barrier (the Sertoli cell barrier) (Cheng and Mruk, 2012;Piprek et al., 2020). Because blood vessels, lymphatic vessels and nerves do not enter into the seminiferous epithelium, the blood-testicular barrier regulates the entry of molecules, such as nutrients and hormones, into the apical compartment in which germ cells enter meiosis (Cheng and Mruk, 2012). Disruption of the bloodtesticular barrier leads to a failure of spermatogenesis (Lui et al., 2003;Cheng and Mruk, 2012).
A similar physical barrier of cells is found in the C. elegans gonad, but this barrier is formed by the germ cells themselves (Cinquin et al., 2015). The germ cells in C. elegans form a syncytium in which the nuclei are enclosed by a partial plasma membrane, which has large openings on a central cytoplasmic core called "rachis"; some germ cells with partial membranes span the rachis and form cell bridges to pattern stemness Notch signaling in the gonad. Overall, these studies and ours strongly suggest that at least in the gonads, physical permeability barriers formed by cells can help to establish morphogen territories for proper cell patterning.

Developmental Stage of Larvae and Pupae
The developmental stages of Drosophila were morphologically defined as previously described (Ashburner, 2005). Flies were transferred to a new vial at 25°C to lay eggs for 3-6 h and then were removed. The vial was kept at 25°C. Newly hatched flies (First instar larvae, L1) were collected for dissection or further culturing. At approximately 50 h after egg laying (AEL), larvae were second-instar larvae (L2). Larvae climbing up and down from the food were considered mid-third instar larvae (ML3, 96 AEF), and larvae that had left the food and began wandering were late-third instar larvae (LL3,~120 AEL). Mid-and late-pupae were collected at around 170 and 194 AEL, respectively. Newly eclosed flies collected within 24 h were referred to as 1-dayold flies.

Cloning and Probe Synthesis for in situ Hybridization
Total RNA was extracted from 20 pairs of 1-day-old ovaries by using the GENEzol ™ TriRNA Pure Kit (Geneaid) according to the manufacturer's manual. Total RNA (1 µg) was reversed transcribed with the Transcriptor First Strand cDNA Synthesis kit (Roche). Fragments of fz3 and wg were amplified and used for the templates for synthesizing antisense probes; primers used are listed in the Supplementary Table S2 mRNA probes labeled with digoxigenin-UTP (Roche) were synthesized from 1 μg of the above PCR product using the ampliCap ™ SP6 high-yield message marker kit (Cell Script).

Imaging Quantification
GSCs were defined by their location directly adjacent to niche cap cells, and their fusome that is juxtaposed to the GSC-cap cell junction. SCCs were identified as cells with round-shaped fusomes (CBs in controls) that are not GSCs. To measure fz3-RFP expression, five confocal z-sections of each germarium carrying a clear EC region (5 sections) were merged and analyzed with ImageJ. The EC region was selected and the mean intensity (arbitrary units) was measured. To assess CycB3-GFP expression, Zen 3.1 (blue edition, ZEISS) was used to measure the mean fluorescence intensity of the confocal z-section with the largest nuclear diameter from each GFPpositive germ cell. To measure fz3 transcript signals in the germ cell, four to five confocal z-sections covering the largest area of the germarium were assessed. The numbers of signals in germ cells marked by Vasa-GFP were counted using ImageJ. To measure GFP-Wg signals, confocal z-sections containing images of TF and cap cells or the germ cell region were merged, and numbers of GFP-Wg granules were counted using ImageJ.
Each experiment was performed with at least two biological replicates. For fixed samples, 10 newly eclosed females with the indicated genotypes were randomly picked from a standard cross; ovaries were dissected and subjected to immunostaining. Ovaries from at least 10 pairs of ovaries were separated, mixed, and mounted for observation; 5-15 images were collected of representative phenotypes for each replicate. Statistical analysis was performed as described in the figure legend.

Live Imaging of Adult Germaria
Live images were captured of germaria carrying Wg-GFP, Dppmcherry or cycB3p-CycB3-GFP, as fixation caused high background that interfered with analysis of signals. To obtain images of live adult germaria, ovaries of 1-day-old flies were dissected in GIM, and the anterior portions of the ovarioles were gently teased apart to separate germaria. Ovaries were then stained with or without CellMask ™ Deep Red Plasma Membrane Stain (1:2000 diluted in GIM, Invitrogen, C10046) for 1 min at RT. A short incubation time was used to prevent/ reduce the staining of germ cell membranes. Note that somatic tkv-KD and bam mutant germaria have blunted EC protrusions, and the CellMask signal within those germaria likely corresponds to germ cell membranes. Ovaries were transferred on to a glass slide, and 15 µl fresh GIM was added; sheath cells were removed from each ovariole using a tungsten filament needle. For the permeability assay, after removing the sheath, 0.3 µl of 5 μg/μl 10-kDa dextran conjugated with Alexa Fluor ™ 488 (Invitrogen, D22910, a gift provided by Dr. Y. Henry Sun, Institute of Molecular Biology, Academia Sinica, Taiwan) was directly added to the 15 µl GIM on the slide (final concentration, 0.2 μg/μl). Finally, the ovarioles were covered with a coverslip and imaged using a Zeiss LSM 900 confocal microscope.

RNA-Seq Data Analysis
The RNA-seq data shown in the manuscript were published before by Tseng et al., 2018 and can be found in the NCBI GEO database (GSE117251).

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.