A switch in the mode of Wnt signaling orchestrates the formation of germline stem cell differentiation niche in Drosophila

Germline stem cell (GSC) self-renewal and differentiation into gametes is regulated by both intrinsic factors in the germ line as well as extrinsic factors from the surrounding somatic niche. dWnt4, in the escort cells of the adult somatic niche promotes GSC differentiation using the canonical β-catenin-dependent transcriptional pathway to regulate escort cell survival, adhesion to the germ line and downregulation of self-renewal signaling. Here, we show that in addition to the β-catenin-dependent canonical pathway, dWnt4 also uses downstream components of the Wnt non-canonical pathway to promote escort cell function earlier in development. We find that the downstream non-canonical components, RhoA, Rac1 and cdc42, are expressed at high levels and are active in escort cell precursors of the female larval gonad compared to the adult somatic niche. Consistent with this expression pattern, we find that the non-canonical pathway components function in the larval stages but not in adults to regulate GSC differentiation. In the larval gonad, dWnt4, RhoA, Rac1 and cdc42 are required to promote intermingling of escort cell precursors, a function that then promotes proper escort cell function in the adults. We find that dWnt4 acts by modulating the activity of RhoA, Rac1 and cdc42, but not their protein levels. Together, our results indicate that at different points of development, dWnt4 switches from using the non-canonical pathway components to using a β-catenin-dependent canonical pathway in the escort cells to facilitate the proper differentiation of GSCs.

Introduction Stem cell self-renewal and differentiation are critical for maintaining the organ systems of multicellular organisms. Loss of stem cell self-renewal leads to aging, due to an inability to replenish these organs; while loss of differentiation leads to tumors, which can progress toward diseases such as cancer [1][2][3]. Thus, identifying triggers of stem cell differentiation is pivotal for understanding the etiology of degenerative diseases and cancer. Germline stem cells (GSCs) self-renew and differentiate to produce gametes [4][5][6][7]. The balance between selfrenewal and differentiation is critical for a steady supply of gametes for increased reproductive success. Dysregulation of GSC self-renewal and differentiation manifests itself as changes in fecundity without markedly altering the organism's growth or survival. Additionally, processes regulating GSC differentiation are conserved in other stem cell systems [8][9][10]. Therefore, GSCs make an excellent system to identify triggers of stem cell differentiation.
Drosophila GSCs are well characterized and genetically tractable [5]. GSC development starts during the larval stages. The female larval gonad is made up of the somatic niche and primordial germ cells (PGCs) [11,12]. The late larval somatic niche is comprised of intermingled cells (ICs), the terminal filament and the cap cells [11][12][13][14]. The PGCs that give rise to the GSCs in the adults are interspersed with ICs, precursors of the adult escort cell [11,15,16] (Fig 1A). Most of the PGCs remain undifferentiated during the larval stage [15,17,18]. As development progresses, the larval gonad transforms first into a pupal gonad and then into an adult ovary. During this transition, the PGCs closest to the cap cells acquire a stem cell fate and become GSCs while the rest directly differentiate [15,18]. Each adult ovary consists of 16-18 individual units called ovarioles. GSCs are located in the germarium, present at the anterior end of each ovariole [19]. GSCs divide to give rise to a self-renewed GSC and a stem cell daughter, the cystoblast (CB) (Fig 1B). The CB expresses a differentiating factor called Bag of marbles (Bam), undergoes differentiation and four incomplete divisions to form a sixteen-cell cyst. Of the sixteen cells, fifteen become nurse cells and one is specified as an oocyte [20][21][22][23][24] (Fig 1B). Loss of GSC differentiation results in loss of or delayed progression towards becoming an oocyte.
Soma-germ line interaction is critical for proper GSC self-renewal and differentiation [8,25,26]. Both PGCs in the larval gonad and GSCs in the adults are surrounded by somatic cells that constitute the somatic niche. Close contact and coordinated signaling between the surrounding somatic niche and the germ line is pivotal for self-renewal and differentiation [26,27]. In the larval stages, ICs regulate PGC proliferation [11]. Additionally, proper intermingling of ICs in the larval gonad promotes proper escort cell function in the adults [28,29]. In the adults, the somatic niche can be broadly divided into two regions; the self-renewal niche and the differentiation niche [26,30]. The terminal filament and cap cells comprise the selfrenewal niche that is required for regulating GSC self-renewal (Fig 1B) [24,26,[31][32][33][34][35][36]. Loss of differentiation (Fig 1B) [39][40][41]. Thus, soma-germline contact throughout development is a critical extrinsic cue that controls GSC differentiation.
Signaling from the somatic niche coordinates the balance of self-renewal and differentiation. In the larval gonad, EGF/Spitz signaling from the PGCs is required for IC survival and intermingling [11]. In the adult germaria, decapentaplegic (dpp) signaling in the self-renewal niche regulates GSC self-renewal by phosphorylating the transcriptional regulator, mothers against dpp (pMAD), to repress expression of the differentiation factor, Bam [20,24,31]. Ecdysone and dWnt4, among others, have been shown to function in the differentiation niche to promote the differentiation of the CB. Ecdysone signaling in the escort cells regulates the formation of escort cell protrusions through an unknown mechanism [42,43]. dWnt4 autocrine signaling in the escort cells is required for escort cell survival, repression of dpp expression and for formation of escort cell protrusions to encapsulate the CB [44][45][46].
Canonical Wnt signaling plays a critical role in the adult differentiation niche. Wnts can signal through either a canonical or a non-canonical pathway to affect downstream changes [47]. In the escort cells, dWnt4 is known to regulate differentiation through the canonical pathway [44][45][46]. dWnt4 binds to the receptor, Frizzled2 and co-receptor, Arrow (Arr), and relays a signal to the multidomain protein, Dishevelled (Dsh), leading to stabilization of β-catenin [47][48][49][50]. β-catenin, a cytoskeletal protein that is also a transcription factor, translocates into the nucleus and initiates transcription of downstream targets including Frizzled3 (Fz3) [51][52][53][54][55][56][57]. Depletion of any of the canonical pathway factors, such as β-catenin or co-receptor arr, specifically in the escort cells leads to loss of CB differentiation by regulating escort cell number [44][45][46]. Additionally, loss of dWnt4 leads to downregulation of a Wnt canonical reporter, Fz3 promoter fused to RFP (Fz3RFP), and adhesion molecules, β-catenin, Innexin2 and DE-Cadherin [46]. These adhesion molecules promote escort cell encapsulation of the CB that is required for its differentiation [46]. Thus, dWnt4 uses components of the canonical pathway to control both the number and function of escort cells.
Wnt ligands, such as dWnt4, can modulate the Planar Cell Polarity (PCP) of cells [58]. Polarity within a plane of individual cells or of tissues is known as PCP. PCP can specify the proximal and distal end of a cell, as in the Drosophila wing, or the dorsal-ventral and anteriorposterior side of a cell, as in the Drosophila eye [58][59][60][61][62][63][64]. Two systems are known to independently regulate PCP-the core components and the Daschous (Ds)/Fat (Ft) system [65,66]. The core components consist of six proteins-Frizzled (Fz), Dishevelled (Dsh), Diego (Dgo), Van Gogh (Vang)/Strabismus (Stbm), Prickle (Pk), and Flamingo (Fmi). These proteins form two complexes that are asymmetrically arranged on the opposite ends of a cell-the Fz, Dsh and Dgo, and the Vang/Stbm, Pk complex. Fmi is enriched on both sides of the cell and stabilizes the complexes [59][60][61]67]. Small Rho GTPases such as Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and Cell division control protein 42 (cdc42) can function as the downstream effectors of the core complex [60,[68][69][70]. The Ds/ Ft system consists of two atypical transmembrane cadherin proteins-Ds and Ft. Ds and Ft are present on opposite ends of each cell and bind to each other at cell-cell junctions. A golgi associated kinase, Four Jointed (Fj) phosphorylates the extracellular domains of Ds and Ft and thereby promotes their binding [66,71]. The role of the core components and the Ds/Ft system has been well established in specifying PCP in the wing and the eye.
Here, we find that RhoA, Rac genes and cdc42, the downstream Wnt non-canonical components, but not members of the core components that define PCP, such as vang, ds, fz and ft, regulate CB differentiation. We find that dWnt4 genetically interacts with the downstream non-canonical components: dsh 1 , DAAM1, RhoA, Rac1 and cdc42. RhoA, Rac1 and cdc42 are expressed at higher levels and are more active in the ICs of the late larval gonad than in the adult escort cells. Conversely, the Wnt canonical reporter is not expressed in the late larval stages but is expressed in the adults. Consistent with this observation, we find that these downstream non-canonical pathway components are required in the late larval stages for regulating intermingling of ICs with the germ cells but their function is not essential in the adults to regulate differentiation. Additionally, we find that dWnt4 regulates the activity, but not the protein levels, of RhoA, Rac1 and cdc42 in the larval gonad. Thus, dWnt4 regulates assembly of the somatic differentiation niche of the GSCs by switching the mode of signaling during development.

dWnt4 acts through the downstream non-canonical pathway components to regulate differentiation
To determine if the Wnt non-canonical pathway components regulate differentiation, we depleted these components in the escort cells. We made use of RNA interference (RNAi) in conjunction with c587-GAL4, which is expressed in the escort cells, to deplete dsh, DAAM1, RhoA, Rac1 and cdc42 [43,83,84]. Both control and mutant germaria were stained with 1B1 and Vasa. 1B1 marks the endoplasmic reticulum rich organelle, the spectrosome, in undifferentiated GSCs and CBs as well as in differentiating CBs; and it marks the branched structures, the fusomes, in differentiated cysts [85]. 1B1 also marks somatic cell membranes [86]. Vasa, an RNA helicase, marks the germ line [87]. We assayed for differentiation defects, defined as an accumulation of greater than 3 undifferentiated cells marked by spectrosomes. Depletion of dsh, DAAM1, RhoA, Rac1 and cdc42 resulted in germline differentiation defects compared to control (Fig 1D-1J) (S1A Fig) ( Table 1). We also depleted the mediators that define the PCP of a cell utilizing previously validated RNAi lines in conjunction with c587-GAL4. We found that for vang, ds, ft and fz, we did not observe any significant differentiation defect [42,[88][89][90] (S1B-S1I Fig) (Table 1). These results are consistent with Cohen et al's observation that mutations in genes that regulate PCP of a cell, such as fz, stbm, fmi, dgo and pk, do not lead to defects in oogenesis [82]. Thus, we can conclude that downstream components of the Wnt noncanonical pathway such as dsh, DAAM1, RhoA, Rac1 and cdc42 are required in the escort cells for promoting GSC differentiation. Additionally, these results also suggest that the core components that mediate PCP such as vang/stbm, ds, fz and ft do not play a significant role in the escort cells to regulate GSC differentiation. DAAM1, RhoA, Rac1 and cdc42 are critical proteins required for various basic cellular processes, such as progression of the cell cycle and cell movement [76][77][78][79][80]91]. It is not surprising that they are required in the escort cells for proper function. We asked if these proteins act downstream of dWnt4 to promote differentiation. To answer this, we generated trans-heterozygous flies that contained one genetically reduced copy of dWnt4 and one reduced copy of dsh, DAAM, RhoA, Rac1 or cdc42 [92][93][94][95][96]. Dsh is a multi-domain protein that uses distinct domains to interact with either the Wnt canonical or the Wnt non-canonical proteins [97].

Downstream Wnt non-canonical components regulate CB encapsulation to promote differentiation
If dWnt4 acts through the downstream Wnt non-canonical components to regulate GSC differentiation, then dWnt4 mutants and RhoA, Rac1 and cdc42 depleted escort cell mutants should phenocopy each other. Loss of dWnt4 leads to an accumulation of pre-CBs that do not express Bam due to loss of escort cell encapsulation and a reduction of escort cell number [44][45][46]. To test if depletion of the downstream non-canonical components in the escort cells also results in an accumulation of pre-CBs, we stained control and mutants for pMAD, which marks the GSCs, and used GFP under the control of bam promoter to analyze bam transcription and hence mark the differentiating progeny [99]. In addition, we also stained control and mutants for pMAD and BamC that marks proper translation of bam in the differentiating progeny. Similar to dWnt4 mutants, RhoA, Rac1 and cdc42 depleted escort cell mutants showed an accumulation of pMAD negative, and BamGFP and BamC negative cells, suggesting that these mutants accumulate pre-CBs (S3A- S3E Fig) (S4A-S4D2 Fig). Interestingly, in addition to early pre-CBs, cdc42 mutants also showed an accumulation of BamGFP and BamC positive CBs, suggesting that cdc42 may have an additional role in promoting CB differentiation.
Pre-CBs that accumulate in dWnt4 mutants are capable of differentiation upon ectopic expression of bam. To test if the undifferentiated germ cells that accumulate due to depletion of downstream components of PCP in the escort cells are also capable of differentiating, we ectopically expressed bam by using a transgene that expresses bam under the control of heatshock promoter (hs-bam) [21]. We found, post heat-shock, these mutants showed loss of undifferentiated cells and an accumulation of cysts, as marked by the presence of fusomes comparable to the control (90% for c587-GAL4; 86% for c587-GAL4>RhoA RNAi, P-value = 0.5353; 76% for c587-GAL4>Rac1 RNAi, P-value = 0.0629 and 92% for c587-GAL4>cdc42 RNAi, P-value = 0.7263 {for all n = 50}) (S5A- S5E Fig). We also analyzed the expression of Bruno in RhoA, Rac1 and cdc42 depleted escort cell mutants that carry the hs-bam transgene, without heat-shock and post heat-shock. Bruno, a translational repressor, is expressed at very low levels in the undifferentiated cells but is expressed at high levels post-differentiation from 16-cell cysts onwards [100,101]. We found, post heat-shock, cysts in Rac1 and cdc42 mutants that carry the hs-bam transgene expressed Bruno, while cysts in RhoA mutants carrying the hsbam transgene only weakly expressed Bruno (S5F- S5M1 Fig). Altogether, these results suggest that the downstream Wnt non-canonical pathway components are required extrinsically in escort cells, to promote differentiation in the germ line.
Loss of escort cell number and their encapsulation results in loss of pre-CB differentiation [39][40][41]102]. To determine if loss of the downstream Wnt non-canonical pathway components leads to loss of encapsulation, we visualized the cytoplasmic protrusions using FaxGFP, a somatic cell membrane marker [39]. It has been previously demonstrated that overexpression in the escort cells of dominant-negative Rho (Rho DN ), that disrupts Rho function, leads to loss of escort cell encapsulation [40]. We found that, similar to dWnt4 mutants and germaria where Rho DN was overexpressed in the escort cells, depletion of RhoA, Rac1 and cdc42 in the escort cells also resulted in loss of encapsulation (Fig 2A-2D1). To determine if this was due to decreased number of escort cells, we counted the number of Tj positive escort cells in these mutants. We found that these mutants exhibited significantly lower number of escort cells (16.5 ± 2.1 for c587-GAL4; 9.9 ± 3.7 for c587-GAL4>RhoA RNAi, P-value = 0.00013; 11 ± 1.5 for c587-GAL4>Rac1 RNAi, P-value = 4.1403E-06 and 9.3 ± 2.2 for c587-GAL4>cdc42 RNAi, P-value = 9.5478E-07 {for all n = 10}). However, the escort cells that were present in the germaria failed to extend cytoplasmic protrusions. These results demonstrate that like dWnt4, downstream Wnt non-canonical pathway components in the escort cells regulate both encapsulation and their numbers, and thus CB differentiation [44][45][46]. These results taken together suggest that dWn4 acts through Dsh and the downstream Wnt non-canonical pathway components to regulate differentiation.

Components of Wnt non-canonical pathway are expressed at high levels and are active in the larval gonad
Wnt proteins are known to regulate signaling through either the canonical or the non-canonical pathway [47]. Surprisingly, our data suggests that dWnt4 also acts through the non-canonical pathway components in the escort cells. There is little precedent for canonical and noncanonical Wnt pathways acting in the same cell at the same time. We therefore hypothesized that either dWnt4 signals via the canonical or non-canonical arm in distinct subsets of escort cells or at different stages of escort cell development. If there are subsets of cells that respond to canonical signaling but not to non-canonical signaling, then we could observe the canonical reporter to be present only in some escort cells. If the regulation is temporal, we hypothesized that we could see differences in either levels or activity of canonical and non-canonical reporters as a function of development.
To test these hypotheses, we analyzed the expression patterns of downstream Wnt noncanonical pathway components and a Wnt canonical reporter during late larval stages, late pupal gonads and in the adult ovaries. Transgenic lines with GFP tagged RhoA, Rac1 or cdc42 that report endogenous expression were used for the Wnt non-canonical components [103]. These reporters were stained for their respective fluorescent proteins and Tj, which marks ICs in the larval gonad and all somatic cells, except for the terminal filament, in the adult gonad [29]. Vasa was used to mark the germ line. We found that RhoA, Rac1 and cdc42 showed high expression in the larval gonad RhoA, Rac1 and cdc42 are members of the Rho family of small GTPases. These proteins are inactive when bound to GDP and active when bound to GTP [104,105]. The active forms of these proteins regulate various downstream signaling cascades [76,77,106,107]. We observed that downstream components of the Wnt non-canonical pathway, RhoA, Rac1 and cdc42 are expressed at high levels in the larval gonad. We asked if these proteins are present in their activated form in the larval gonad. To test this, we performed live-imaging of larval gonads of transgenic lines that report the active form of RhoA/Rac and cdc42 [108]. In these biosensors, GFP is fused to the Rho family GTPase Binding Domain (RBD) of downstream effector proteins. Expression of GFP suggests the presence of active forms of these proteins. For RhoA/Rac activity, the downstream effector protein, Protein Kinase N (Pkn) was fused to GFP and placed under squash (sqh) promoter [108]. For cdc42 activity, the cdc42 binding domain of Wiskott-Aldrich Syndrome protein (WASp) was fused with GFP and this was placed under the control of sqh promoter [108]. In order to observe the germ cells in these biosensors, we used kusabiraorange (KO) fused germline marker, Vasa-KO [109,110]. Similar to the expression pattern of the GFP tagged RhoA, Rac1 and cdc42 we observed the presence of active form of RhoA, Rac1 and cdc42 in the ICs of the larval gonad (Fig 3E-3F2). We also asked if these proteins are present in their active forms in the escort cells of the adult germaria. We found that RhoA/Rac activity was attenuated and cdc42 was present in its activated state in the escort cells, albeit at lower levels compared to the larval gonad (S7A- S7C Fig). Together, these results demonstrate that the Wnt non-canonical components are not only present, but also active in the ICs. To determine the activity of the canonical pathway in the larval gonad, we used Fz3RFP [57]. We found that while Fz3RFP showed no expression or was expressed at background levels in the ICs of the larval gonad, it was expressed at higher levels in the escort cells of pupal and adult stages [46,111,112] (Fig 3G-3I) (S7D-S7D1 Fig). Additionally, all the escort cells in the adult germarium expressed the canonical reporter (n = 10, 99.5% Tj positive cells expressed Fz3RFP). Thus, we concluded that Wnt canonical and Wnt non-canonical pathway components, and their activity, are temporally regulated in escort cells and their precursors during oogenesis.

Components of the Wnt non-canonical pathway play a critical role in the larval gonad
To determine if the different expression patterns of the downstream Wnt non-canonical pathway components mirrored their role in the two developmental stages, we used the UAS-GAL4 dWnt4 switches signaling modes system to deplete genes using RNAi in the escort cells at specific developmental time points. The UAS-GAL4 system is temperature dependent and therefore, different temperatures can be used to attenuate genes at various developmental time points. Maximal RNAi activity is attained at 29 o C whereas minimal RNAi activity is attained at 18 o C [113]. c587 is expressed in the larval gonad, including the ICs, but is only expressed in the escort cells of the adult inner germarium (S8A-S8C1 Fig). To elucidate if the downstream Wnt non-canonical pathway components are required in the adults to regulate CB differentiation, the flies were kept at 18 o C until they eclosed. Once eclosed, the flies were shifted to 29 o C and kept at this temperature for 7 days [114] (Fig 4A). The germaria were then stained for 1B1 and Vasa. This strategy has been previously used to show that the dWnt4 canonical pathway is required in the adults to regulate CB differentiation [45]. We depleted dWnt4, RhoA, Rac1, cdc42 and also expressed cdc42 DN in the escort cells to assay for differentiation defects. We found that compared to control, 18 o C-29 o C temperature shift dWnt4 mutants exhibited a differentiation defect (Fig 4B  and 4C and Fig 4G). In contrast, compared to control, 18 o C-29 o C temperature shift RhoA, Rac1, cdc42 and cdc42 DN mutants did not exhibit a significant accumulation of undifferentiated cells, suggesting that the downstream Wnt non-canonical pathway components do not play a critical role in the adult to regulate CB differentiation (3 ± 1 for c587-GAL4; 4 ± 1 for c587-GAL4>dWnt4 RNAi, P-value = 0.002958; 3 ± 2 for c587-GAL4>RhoA RNAi, P-value = 0.431434; 3 ± 1 for c587-GAL4>Rac1 RNAi, P-value = 0.91346, 3 ± 1 for c587-GAL4>cdc42 RNAi, P-value = 0.54991 and 3 ± 1 for c587-GAL4>cdc42 DN , P-value = 0.242545 {for all n = 50}) (Fig 4B and Fig 4D-4G) (S9A and S9B Fig, S9E-S9G Fig). To elucidate if the downstream Wnt non-canonical pathway components are required in the larval gonad to regulate CB differentiation, the flies were kept at 29 o C until they eclosed. Once eclosed, the flies were shifted to 18 o C and kept at this temperature for 7 days [114] (Fig 4H). Compared to control, 29 o C-18 o C temperature shift dWnt4, RhoA, Rac1, cdc42 and cdc42 DN mutants resulted in an accumulation of undifferentiated cells (2.5 ± 1 for c587-GAL4; 5.6 ± 2 for c587-GAL4>dWnt4 RNAi, P-value = 1.04811E-17; 5 ± 1.5 for c587-GAL4>RhoA RNAi, P-value = 1.43965E-15; 5 ± 1.5 for c587-GAL4>Rac1 RNAi, P-value = 1.53586E-18; 5 ± 2.5 for c587-GAL4>cdc42 RNAi, P-value = 2.34120E-18 and 5 ± 1 for c587-GAL4>cdc42 DN , P-value = 1.49752E-17{for all n = 50}) (Fig 4I-4N) (S9C-S9F and S9H Fig). Taken together, these results demonstrate that the Wnt non-canonical pathway components act primarily prior to the adult stage to regulate CB differentiation.
As RhoA, Rac1 and cdc42 are highly expressed and active in the larval gonad and were not required in the adult germaria for CB differentiation, we asked if these proteins are required earlier in development. To test this we used an IC driver traffic jam-GAL4 (tj-GAL4). Tj is expressed only in ICs in the late larval gonad, therefore tj-GAL4 is a more restricted driver for ICs [11,29]. We depleted dsh, DAAM1, RhoA, Rac1, cdc42 and expressed RhoA DN and cdc42 DN versions in the ICs, in the late larval stages using tj-GAL4. Because we posited that RhoA, Rac1 and cdc42 act downstream of dWnt4, we also monitored the late larval gonads of dWnt4 mutants for defects, by staining for Vasa, Tj and 1B1. We found that compared to dWnt4 heterozygotes, dWnt4 mutants and dsh, DAAM1, RhoA, RhoA DN , Rac1, cdc42 and cdc42 DN mutants showed loss of intermingling of ICs with PGCs (n = 25) (Fig 5A-5H) (S10A-S10C Fig). Although loss of Rac1 in ICs led to loss of intermingling, the phenotype was weaker than dWnt4 mutants, RhoA and cdc42 depleted ICs. Additionally, depletion of Rac1 in the ICs resulted in a decrease in the size of the larval gonad. It has been shown Rac1, Ras-related C3 botulinum toxin substrate 2 (Rac2) and Mig-2-like (Mtl) act redundantly to regulate PCP [115]. For this reason, we also depleted Mtl and Rac2 specifically in the ICs and found that Mtl depleted ICs resulted in a strong loss of ICs intermingling but Rac2 depleted ICs did not exhibit any intermingling defect (Fig 5I and 5J). Although we find that depletion of Rac2 does not lead to a differentiation defect, we do not know if this is due to a feeble RNAi mediated depletion, or in fact Rac2 does not play a role. These results suggest that dsh, DAAM1, RhoA, Rac1, Mtl and cdc42 regulate intermingling of ICs in the larval gonad.
Together, these results suggest that RhoA, Rac genes (Rac1 and Mtl) and cdc42 regulate intermingling of ICs.
dWnt4 mutants and RhoA, Rac1 and cdc42 depleted escort cell germaria exhibit reduced number of escort cells and loss of CB encapsulation [46]. Additionally, depletion of RhoA, Rac1 and cdc42 in the ICs resulted in reduced number of ICs. We asked if reduction in the escort cell number and loss of encapsulation of CB in the adults is a consequence of reduced number of ICs in the larval gonad. To answer this question, we counted the number of Tj positive escort cells in the adult flies, of 29 o C-18 o C temperature shift escort cell depleted RhoA, Rac1 and cdc42 mutants. We found that these mutants exhibited significantly lower number of escort cells (15.4 ± 2.6 for c587-GAL4; 10.4 ± 2.6 for c587-GAL4>RhoA RNAi, P-value = 0.00048; 10.6 ± 2 for c587-GAL4>Rac1 RNAi, P-value = 0.00068 and 6.9 ± 1.6 for c587-GAL4> cdc42 RNAi, P-value = 8.1045E-08 {for all n = 10}). These results suggest that RhoA, Rac1 and cdc42 act in the ICs to regulate the escort cell number and therefore, proper GSC differentiation in the adults.

Wnt canonical pathway and PCP regulators do not regulate the intermingling of ICs in the larval gonad
To determine if PCP core components regulate intermingling in the larval gonad we depleted vang/stbm, ds, fz and ft in ICs and found that depletion of these genes did not exhibit any intermingling defects (S12A-S12E Fig). To ascertain that the canonical pathway is not required in the larval gonad, we depleted the canonical specific co-receptor, arr in ICs and found that these mutants also did not exhibit any intermingling defect (S12A and S12F Fig). We used arr as β-catenin has roles outside of Wnt signaling and loss of arr in the adult escort cells results in CB differentiation defects [45,116]. These results suggest that the Wnt canonical pathway and vang/stbm, ds, fz and ft, the core components and Ds/Ft systems that define PCP, do not play a critical role in the ICs.

dWnt4 regulates RhoA, Rac and cdc42 activity in the larval gonad
Similar to dWnt4 mutants, RhoA, Rac1 and cdc42 depletion in the ICs showed a defect in intermingling of ICs. To determine if RhoA, Rac1 and cdc42 act downstream of dWnt4, we analyzed the expression and activity of RhoA, Rac1 and cdc42 with the help of the GFP tagged lines, in larval gonads of both control and dWnt4 mutants. We found that compared to the control, expression of RhoA, Rac1 and cdc42 was not altered in the ICs of dWnt4 mutants, suggesting that dWnt4 does not regulate the protein levels of RhoA, Rac1 and cdc42 (P-value = 0.64182 for RhoAGFP, P-value = 0.79908 for Rac1GFP, and P-value = 0.63675 for cdc42GFP in dWnt4 mutants {for all n = 3}) (S13A-S13G Fig). The activity level of RhoA/Rac and cdc42 was determined using biosensors in dWnt4 mutants. To ensure that there is no background, we also imaged larval gonads, without the biosensors (S13H-S13I1 Fig). We found that RhoA/Rac and cdc42 activity was significantly downregulated in ICs (P-value = 0.02067, n = 25 for RhoA/Rac activity and P-value = 0.01369, n = 25 for cdc42 activity) (Fig 6A-6F). We also observed cdc42 activity in the terminal filaments and found that it was not altered in dWnt4 mutants (P-value = 0.35901, n = 3) (S13J Fig). These results suggest that dWnt4 regulates the activity of RhoA, Rac and cdc42 in the ICs to regulate intermingling of ICs with PGCs.

Discussion
Here, we find that dWnt4 regulates CB differentiation through the downstream non-canonical pathway components. We show that RhoA, Rac1 and cdc42 are expressed at high levels in the ICs of the larval gonad and are active while the expression of Fz3RFP, a β-catenin-dependent transcriptional reporter, is not detectable. Conversely, we find low levels of RhoA, Rac1 and cdc42 in the adults but the β-catenin-dependent canonical reporter is expressed at high levels. Consistent with this, we find a role for RhoA, Rac1 and cdc42 but not the β-catenin-dependent canonical pathway in the larval gonad for promoting intermingling of ICs (Fig 7). Our results, in conjunction with Mottier-Pavie et al. and Wang et al., that show the canonical pathway is required in the adult, point towards a switch from utilizing Wnt non-canonical components to utilizing a canonical pathway to regulate the formation of the differentiation niche [44,45]. Our findings that the depletion of vang/stbm, ds, fz and ft in either ICs or escort cells does not lead to differentiation defects, suggests that PCP does not play a critical role in regulating CB differentiation. These results are consistent with previous literature, where Cohen et al. showed that mutations in, fz, fmi, dgo, vang/stbm and pk, genes required for PCP of a cell, do not lead to significant disruption of ovarian morphology [82]. We show that the other PCP system components, ds and ft, are also not required to regulate differentiation. Together, Cohen et al. and our results suggest that it is not the PCP system, but the downstream Wnt non-canonical components that play a critical role in the differentiation niche to regulate differentiation. We propose that dWnt4 uses the downstream Wnt/Fz non-canonical pathway to regulate formation of the differentiation niche.
We find that RhoA, Rac1 and cdc42 regulate intermingling of ICs in the larval gonad. In addition, we find that loss of RhoA leads to death of ICs and loss of Rac1 leads to decrease in the larval gonad size. dWnt4 mutants do not show either of these outcomes. Together, these results suggest that RhoA and Rac1 have roles in the larval gonad independent of Wnt signaling. It has been extensively shown that Rac1, Rac2 and Mtl have redundant roles in axon growth, guidance and PCP in the eye and the wing [93,95,115]. Indeed, we found that Rac1 depleted ICs resulted in loss of intermingling but this phenotype was not as strong as dWnt4, RhoA and cdc42 depleted ICs. Rac2 depleted ICs resulted in no intermingling defect. However, depletion of Mtl in the ICs resulted in a strong loss of intermingling phenotype. This suggests that small GTPases play a critical role in ICs function, both independently and in coordination with Wnt signaling. In the adult, escort cells extend dynamic cytoplasmic protrusions to encapsulate the GSCs, the differentiating daughter and differentiating cysts, a function necessary for proper differentiation [28,[39][40][41]102,117]. It was recently shown that in adult female germaria, stat regulates the formation of these protrusions through cdc42. Loss of stat or woc, a component of the stat pathway, leads to formation of smaller protrusions instead of longer stable protrusions. Loss of woc also leads to an accumulation of undifferentiated cells. Overexpression of cdc42 in woc mutants rescued the small protrusion defect and partially rescued the phenotype. However, Banisch et al report that expression of dominant active or dominant negative cdc42 in the escort cells only mildly affects protrusions and does not lead to an accumulation of undifferentiated cells. Our temperature shift experiments also suggest that expression of dominant negative cdc42 exclusively in the adults does not result in an accumulation of undifferentiated cells, while its expression prior to eclosion does (S9C and S9D and S9E and S9F Fig). Moreover, Banisch et al. also show that active cdc42 is expressed in the escort cells and active RhoA could not be detected in most of the escort cells using Dia-RBD:GFP but could be detected mainly in the escort cell body and weakly in the escort cell protrusion using Capu-RBD:GFP [117]. We think that cdc42 may have a role in the adults by controlling protrusive activity of escort cells, but it does not affect CB differentiation in a biologically meaningful way. This suggests that cdc42 and Rho activity in the adult is attenuated and their activity is independent of dWnt4.
Cell movement requires dynamic actin-myosin polymerization at the leading edge, driven by Rac1 and cdc42, and actin-myosin contraction at the lagging end, driven by RhoA [118,119]. In our study, we find that both RhoA/Rac and cdc42 are present and active in the ICs. Loss of RhoA, Rac1 and cdc42 leads to loss of intermingling of ICs, suggesting that these proteins drive ICs cell movement and intermingling in the larval gonad. In contrast, only cdc42, but not RhoA, is active in the adult escort cells, consistent with the fact that these cells are stationary [41,117]. Thus the switch in the module used by Wnt signaling to regulate the activity of RhoA/Rac and cdc42 parallels the developmental demands of these cells to first move between germ cells in the larval gonad, then to stop and create long, stable protrusions that promote differentiation in the pupal and adult gonad.
Cancer metastasis requires cell migration. The invasion of metastatic cancer cells requires cells to lose epithelial properties and gain mesenchymal properties. During this process, known as Epithelial-mesenchymal transition (EMT), the epithelial cells, which are otherwise polarized, non-motile and have strong cell-cell interaction, subsequently lose polarity, cell-cell adhesion and become motile [120,121]. RhoA, Rac1 and cdc42 are conserved from lower eukaryotes to mammals and are key players that affect cell division, cytoskeletal rearrangement, cell polarity, and cell motility [76,77,79,80,91,106,118]. It has been previously discovered that during cancer, these proteins are upregulated and therefore help initiate metastasis [122]. We have discovered that RhoA, Rac1 and cdc42 are turned on in the ICs of the larval gonad and switched off in the adult escort cells. This is fascinating because the ICs and the escort cells are essentially the same cells at different developmental time points. A better understanding of how this switch is mediated may give us an insight into cancer and aid in developing mechanisms to block metastasis.

Collection and Fixation of tissues
3-4 day old fly ovaries were dissected in PBS, fixed for 30 min in PBS plus 5% formaldehyde, incubated for 1 h in PBST (0.2% Tween 20 (Sigma) in PBS) supplemented with 1% Triton X-100 (Sigma), followed by incubation for 2 h in BBT (PBST supplemented with 1% (w/v) bovine serum albumin (BSA; Sigma)). Primary antibodies were added in BBT and incubation was carried out overnight at 4 o C. The following day, ovaries were washed four times for 10 min, 20 min, 30 min in BBT and for 30 min in BBT supplemented with 2% (w/v) donkey serum (Sigma). Secondary antibodies were added in BBT supplemented with 4% (w/v) donkey serum (Sigma) and incubated for 2 h followed by five washes, 10 min each in PBST. VECTASHIELD (Vector Laboratories) with DAPI was added prior to mounting. Fixation and staining of larval gonads was carried out as previously described [123].

Temperature shift experiment
In order to express a specific gene only in the adults, the flies were kept at 18 o C until they eclosed. Once eclosed, the young flies were shifted to 29 o C and kept at this temperature for 7 days. Staining was followed to determine any differentiation defects.
In order to express a specific transgene only in the larval stage, the flies were kept at 29 o C until they eclosed. Once eclosed, the young flies were shifted to 18 o C and kept at this temperature for 7 days. These flies were then dissected and stained for observation.
Unless specified, all experiments that utilized the UAS-GAL4 system were performed constitutively at 29 o C.

Antibodies
Immunostaining of the ovaries and larval gonad was carried out with the following Primary Alexa 488 (Molecular Probes), Cy3 and Cy5 (Jackson Labs) conjugated secondary antibodies were used at a concentration of 1:500.

Fluorescence imaging
The tissues were visualized under 10X, 20X, 40X and 63x objective lenses. The images were acquired using a Zeiss LSM-710 confocal microscope under 20x, 40X and 63x objective.

Live imaging
The tissues were dissected in Schneider's media and mounted. These were then visualized under 20X objective lens. The images were acquired using a Zeiss LSM-710 confocal microscope under 20x objective.

Quantification analysis
A.U of protein levels. In order to calculate intensities for Fz3RFP and GFP in ICs or escort cells, images for both, control and mutant larval gonad and adult germaria were taken using the same confocal settings. Z stack planes were obtained for all images. Specific planes showing Tj positive ICs or escort cells were chosen, the area of Tj positive cell was outlined and analyzed using the 'analyze' tool in ImageJ. The mean and area of the specified region was obtained. An average of all the ratios (Mean/Area), for Fz3RFP, GFP and Tj, per image was calculated for both, control and mutants. The average ratio for Fz3RFP and GFP was normalized with average ratio for Tj. These are the arbitrary units (A.U) for Tj, GFP and RFP for this region. A minimum of 7 germaria and 7 larval gonads (minimum 5 Tj positive cells per image) were considered for the each quantification.
A.U of activity reporters. In order to calculate intensities of activity reporters, images with Z stacks were obtained for both, control and mutant larval gonads using the same confocal settings. For ICs, an area right besides a PGC was outlined and for terminal filament an area in the terminal filament was outlined. All these areas were analyzed using the 'analyze' tool in ImageJ. The mean and area of the specified region was obtained (25 cells were analyzed per image). An average of all the ratios (Mean/Area), per image was calculated for control and mutants. The average ratio for activity was subtracted with average ratio for background. The results are the arbitrary units (A.U) for activity reporters for this region. 25 cells of 2 larval gonads each were quantitated for RhoA/Rac and cdc42.
In order to calculate intensities of activity reporters between larval gonads and adult germaria, A.U of GFP was normalized to Vasa-KO. 5 cells of 3 larval gonads and adult germaria each were quantitated for RhoA/Rac and cdc42.
The number of Tj and Fz3RFP positive cells were calculated manually in all stacks for 10 germaria in ImageJ.

Statistical analysis
P-values were determined by two-tailed equal variance t test in mutants vs. wild type strains. Z-score was determined by a two-tailed test for data represented in percentage.

Materials and reagents
Fly food was created using the procedures from the Ruth Lehmann lab at NYU (summer/winter mix), and used to fill narrow vials to approximately 12mL.  (Fig 6A-6B1) showing no GFP. GFP channel is shown in H1. (I-I1) Larval gonad of dWnt4 heterozygous lacking cdc42 GTPase reporter, imaged under the same confocal settings (Fig 6C-6D1) showing no GFP. GFP channel is shown in I1 (J) Quantification (n = 3) of GFP in the terminal filament showing that cdc42 activity is not altered in dWnt4 mutants. Scale bar for all images is 20μm. (TIF) dWnt4 switches signaling modes