Cis-inhibition of Notch by Delta controls follicle formation in Drosophila melanogaster

Cis-inhibition of Notch by Delta controls follicle formation in Drosophila melanogaster | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Cis-inhibition of Notch by Delta controls follicle formation in Drosophila melanogaster View ORCID Profile Caroline Vachias , View ORCID Profile Muriel Grammont doi: https://doi.org/10.1101/2025.03.19.644164 Caroline Vachias 1 CNRS 6293, Clermont University , Inserm U1103, UMR GReD, UFR Médecine, Clermont-Ferrand F-63001, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Caroline Vachias Muriel Grammont 2 Univ Lyon, ENS de Lyon, CNRS UMR 5239, INSERM U1210, Laboratory of Biology and Modeling of the Cell , 15, Parvis Rene Descartes, F-69007, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Muriel Grammont For correspondence: muriel.grammont{at}ens-lyon.fr Abstract Full Text Info/History Metrics Preview PDF Abstract Daughters of stem cells often differentiate sequentially in response to inputs from various signalling molecules. We focus on the regulation of Notch signalling in the Drosophila germarium, which contains several somatic stem cells. Stem cell siblings produce polar cell (pc) or main body follicular cells (MBFC) precursors as they surround the germline and form follicles. Notch has been shown to be activated in at least one of the stem cell siblings and in the pc precursors by a Delta signal produced by the germline cells. However, removing N in the soma leads to abnormal follicles while removing Delta from the germline does not, indicating that the tissue-specific requirement needs to be re-examined. Here, we demonstrate that Delta in the soma downregulates Notch activity via a cis-inhibition mechanism. Somatic Delta prevents Notch from being strongly activated by germline Delta, resulting in the maintenance of an undifferentiated state. In addition, we show that somatic Delta is required to activate Notch in the pc precursors and that its activity is more efficient in initiating pc differentiation than germline Delta. Thus, Notch activity in the germarium depends on both germline and somatic Delta, explaining why removing Delta from the germline does not phenocopy Notch phenotype. Finally, our work provides a novel example of the importance of the regulation of Notch activity through a cis-inhibitory mechanism. Significance Statement Stem cells produce daughter cells that progressively adopt different identities through the activities of signalling pathways, such as the Notch pathway. The activity of this pathway depends on the ability of the ligand produced by a cell to activate the Notch receptor expressed by a neighbouring cell. In vertebrates and invertebrates, Notch has been shown to either promote cell fate acquisition or maintenance of an undifferentiated state, depending on its regulation. Here, we investigate the regulation of Notch in stem cell daughter cells in Drosophila . We demonstrate that its activity is downregulated in these cells through an mechanism, called the cis-inhibition mechanism, meaning that Notch is blocked by the ligand produced within the same cell. Introduction Stem cells stand out from other cells by their ability to both self-renew and produce differentiated daughter cells. Differentiation of daughter cells is controlled by intrinsic or extrinsic cues and occurs either rapidly or after several divisions. Ovarian follicle formation in Drosophila is an ideal system to address this process, as it has been shown that differentiation of the daughter cells of the somatic stem cells does not depend of any lineage but rather is established progressively through the activities of various signalling pathways ( Dai et al., 2017 ; Melamed and Kalderon, 2020 ; Reilein et al., 2017a ). A Drosophila ovarian follicle is composed of a monolayer of epithelial cells surrounding a cyst of 16 germline cells (fifteen nurse cells and one oocyte). Follicles are continuously produced within the germarium, a structure where both the germline and the somatic stem cells reside ( Figure 1A ). The germarium is functionally divided into four regions. First, in the anterior part of the germarium (region I), divisions of germline stem cells give rise to 16-cell cysts ( King, 1970 ; Spradling, 1993 ). Second, in region IIa, the cysts interact with the somatic stem cells (ssc). This population is controlled by the opposite activities of the Wnt and the Jak/Stat pathways. The ssc and their daughters may migrate anteriorly, posteriorly or radially and as a function of their position in the germarium, they either remain ssc or differentiate as escort cells (esc), follicular cell (fc) precursors or polar-stalk cell (pc-sc) precursors ( Fadiga and Nystul, 2019 ; Melamed and Kalderon, 2020 ; Nystul and Spradling, 2010 , 2007 ; Reilein et al., 2017b ). Anterior ssc migration leads to esc whereas significant posterior migration leads to fc or pc-sc. The escort cells surround the germline cysts in the region I, the polar cells will be localised at either extremity of S1 follicles whereas the stalk cells will intercalate between adjacent follicles. In region IIb, cysts individually stretch out in between the walls of the germarium and fc surround them and form an epithelium. Posteriorly, in region III, the follicle takes on a rounded shape (at which point it is called a stage 1 follicle) and exits the germarium, extending the chain of follicles. Although, for simplicity, we refer to the cells surrounding the cysts in region IIa/IIb as precursors, lineage analyses have shown that the fc, the pc and the sc fates remain labile up to stage 2. The identities of these cells depend on the balance between the Eyes Absent (Eya) and Castor (Cas) proteins, which are both expressed at low levels in ssc prior to becoming restricted to the fc precursors (in the case of Eya) or to the pc-sc precursors (in the case of Castor) ( Bai and Montell, 2002 ; Chang et al., 2013 ; Dai et al., 2017 ). Eya and Cas expression in the germarium depend on both the Wnt and the Hedgehog signalling pathways( Dai et al., 2017 ; Reilein et al., 2017a ). The fate of the polar and of the stalk cells continue to be progressively established in region III, through the activities of the Notch (N) and Jak/Stat pathways. Polar cells then start expressing markers such as Fasciclin III (Fas III), or the PZ80 (in Fas III ) and A101 (in neuralized ) reporters and the maintenance of this fate depends on the Notch pathway throughout oogenesis ( Assa-Kunik et al., 2007 ; Grammont and Irvine, 2001 ; Lopez-Schier and St Johnston, 2001 ; McGregor et al., 2002 ). Download figure Open in new tab Figure 1. neur is required for follicle formation. In all figures, anterior is to the left, mutant clones are marked by the absence of Myc or GFP and roman numerals indicate the different regions of the germarium. (A) Schematic representation of a germarium and a stage 2 follicle and of the different steps of cell differentiation (A’). The arrangement and identities of the different cell types are shown: somatic stem cells (green), somatic stem cell siblings (blue, pink), MBFC precursors (grey) MBFC (black outline), polar-stalk cell precursors (orange), polar cells (red), stalk cells (yellow) and germline cells (brown outline). (B) WT stage 6 follicle. The oocyte, marked by Orb, is localized at the posterior (arrow). (C-F) Stage 6 neur -mosaic-soma follicles. Each oocyte (Orb, arrows) bear 4 ring canals (marked by Actin). Oocytes are localized either at each extremity (C), or one at the posterior end and one laterally (D) or both at the posterior end (E, F). D’’ and D’’’ are magnified views of the boxed areas in D’. (C, E) Follicles through 2 z-confocal sections (z1 and z2). (G) WT germarium. (H-I) neur -mosaic-soma germaria. Arrows point to cysts in region IIb that do not display a lens shape and that are not separated from the older (H) or younger (I) cyst. Despite these findings, the role of N activity in separating cysts and in pc differentiation remain largely unclear for the following reasons: N reporter activity is detected in the ssc, in the pc-sc precursors and in the pc. Removing N leads to an absence of radial migration of the ssc, to a lack of polar cells and to the formation of swollen germaria with improperly encapsulated cysts ( Grammont and Irvine, 2001 ; Lopez-Schier and St Johnston, 2001 ; Nystul and Spradling, 2010 ; Xu et al., 1992 ). Although both the germline and the soma express Delta, it has been shown that Dl is required only in the germline for all these N activities. One might then expect that removing Dl from germline should phenocopy the N phenotype, but, surprisingly Dl-germline clones do not affect follicle formation ( Lopez-Schier and St Johnston, 2001 ; Nystul and Spradling, 2010 ; Torres et al., 2003 ). Thus, the mechanism of N activation within the ssc and the pc for cyst enclosure remains to be identified. We re-analysed the role of the Dl ligand and of one of its modulators, neuralized (neur) . Neur encodes an E3-ubiquitine ligase required for upregulating Dl signalling activity through endocytosis( Lai et al., 2001 ; Pavlopoulos et al., 2001 ; Yeh et al., 2001 ). Our results indicate that Dl is required in the soma throughout the germarium to down-regulate N activity through a cis-inhibition mechanism and this mechanism is required to prevent premature and ectopic pc differentiation. We show that Dl somatic expression prevents N from responding to Dl from the germline in region IIa, whereas it prevents N from being activated by the Dl signal from the neighbouring MBFC in region IIb/III. This restricts pc fate to the pc-sc precursors and prevents abnormal cyst enclosure. Results neur is required in the somatic cells for follicle formation neur is known to be specifically expressed in the polar cells. In order to understand its function during early oogenesis, we generated neur somatic clones. When neur clones encompass 25% - 75% of somatic epithelial cells, follicles containing more than 16 germline cells are frequently observed (78%, n=62). Such compound follicles could result either from abnormal division of germline cells, from abnormal enclosure by the follicular cells in the germarium, or from a collapsing of the stalk after follicle formation. To distinguish between these possibilities, we first analysed the expression of the Orb oocyte marker and counted the number of ring canals connecting the oocytes. All compound follicles (n=16) contained two Orb-positive oocytes, connected to 4 nurse cells each ( Figure 1B-F ). Because these oocytes are not always adjacent to each other (they are either one at each end of the follicle (82.7%, Figure 1C ); one posterior and one lateral (11.5%, Figure 1D ); or both at the posterior end (5.8%, Figure 1E-F ), we can rule out that compound follicles are caused by abnormal germline divisions. Compound follicles are thus not due to abnormal division of the germline cells as they always enclose two 16-cell cysts. Next, we investigated whether abnormal cyst enclosure occurs during follicle formation in the germarium or later in follicle development. Germaria with neur somatic clones show two phenotypes: compound follicles in region III or the presence of two round-shaped cysts in region IIb, instead of a single, lens-shaped cyst that spreads throughout the depth of the germaria (Compare Figure 1G with 1H and 1I). Together, these observations show that neur is somatically required in the germarium for cyst enclosure. The germline and soma are redundant sources of Dl for follicle formation As neur is required for the production of active Dl ligands, its requirement for follicle formation suggests that Dl may also be required in the soma for this process. We re-examined the tissue-specific requirement of Dl during follicle formation by counting the number of compound follicles when either all the germline cells or all the somatic cells (henceforth referred to as Dl -germline follicles or Dl -soma follicles, respectively) were mutant ( Figure S1 ). We found that Dl -germline follicles were always WT (n=20) ( Figure 2A , B) whereas Dl- soma follicles were occasionally compound (13%, n=23) ( Figure 2C , S2A-B). We then analysed ovarioles where both the germline and the soma were mutant for Dl . This consistently led to the formation of swollen germaria and huge compound follicles, a phenotype similar to N mutants (100%, n=14) ( Figure 2D ). These data prove that Dl is required for follicle formation, and that this role can be fulfilled by either a somatic or a germline production (that we refer to as DlS and DlG, respectively). Download figure Open in new tab Figure S1. The different types of mutant follicles or germaria. Brown and black outlines correspond to germline and somatic cells, respectively. (A) A Dl -germline follicle is composed of a mutant cyst surrounded by WT somatic cells. A Dl -germline germarium contains at least one Dl cyst. (B) A Dl -soma follicle (or neur -soma follicle) is composed of a WT cyst surrounded by mutant somatic cells. A Dl- or neur -soma germarium contains WT cysts and only mutant somatic cells. (C) A Dl -mosaic-soma follicle (or neur -mosaic-soma follicle) is composed of a WT cyst surrounded by mosaic somatic cells. A Dl- or neur -mosaic-soma germarium contains WT cysts and mosaic somatic cells. (D) A Dl -mosaic-soma-germline follicle is composed of a mutant cyst surrounded by mosaic somatic cells. A Dl -mosaic-soma-germline germarium contains at least one mutant cyst and mosaic somatic cells. Download figure Open in new tab Figure 2. DlG and DlS are redondant for follicle formation Ovarioles with Dl -germline clones (A-B) or Dl -soma clones (C) or with both types of clones (D). The arrow in D points to adjacent cysts in a compound follicle. Cis-interactions between N and DlS downregulate N activity during follicle formation To further analyse the redundancy of Dl produced by the germline and the soma, we analysed follicles with somatic clones for Dl (subsequently referred to as Dl -mosaic-soma follicles; Figure S1 ). These were frequently compound or fused (74%, n=55) ( Figure 3A ), revealing that the juxtaposition of somatic mutant cells with WT germline or WT somatic cells is more deleterious for follicle formation than the complete removal of Dl from the germline or from the soma. We hypothesized that this phenotype could come from inappropriate N activation due to the perturbation of a regulatory mechanism called cis-inhibition, which is observed in cells that express both N and Dl ( Del Álamo et al., 2011 ; Miller et al., 2009 ). In such cells, Dl endogenously blocks N and thus prevents it from being activated by exogenous Dl. In contrast, in cells lacking Dl, N is free to be trans-activated by a Dl signal produced by neighbouring WT cells. Because both Dl and N are expressed in the soma of the ovary, a cis-inhibition mechanism may exist. To test this possibility, we analysed the expression of several reporter for N activity in Dl -mosaic-soma germaria. 10 % to 15% of WT germaria expressed the (P( GbeSu(H)m8 )) reporter in region IIa (in ssc and daughter cells) and region IIb/III (in pc-sc precursors). In contrast, 100% of Dl -mosaic-soma germaria displayed N reporter expression when Dl cells were present in region IIa and 65% in region IIb/III, respectively ( Figure 3B , C). This confirms that inappropriate N signalling occurs in Dl -mosaic-soma germaria. In region IIa, N activity was detected in all Dl cells independently of the presence of neighbouring WT somatic cells, whereas in region IIb/III, expression occurred only in Dl cells that were in contact with WT cells ( Figure 3B , C). This was confirmed by examining Dl -soma germaria, which always contained several cells expressing GbeSu(H)m8 in region IIa but none in region IIb/III (Figure D). This implies that N is activated by DlG in region IIa, but by DlS in region IIb/III. To confirm this, we analysed N reporter expression in germaria with somatic Dl clones and Dl germline cysts (that we refer to as Dl -mosaic-soma-germline follicles; Figure S1 ). In region IIa, no expression of the reporter was observed, confirming that inappropriate N activity depends on DlG ( Figure 3E ). In region IIb/III, Dl cells next to WT somatic cells and mutant germline still expressed the N reporter, demonstrating that inappropriate N activity depends on a Dl signal produced by the neighbouring somatic cells (n=12) ( Figure 3E ). Altogether, these data show that all the somatic cells in the germarium express Dl and that this expression is crucial to prevent N from responding to DlG in region IIa, and to DlS in region IIb/III. These data also demonstrate that germline cells are unable to activate N reporter in the MBFC in region III of the germarium. Download figure Open in new tab Figure 3. DlS down-regulates N activity throughout the germarium (A) Dl -mosaic-soma ovariole with compound (arrow) and fused follicles (arrowhead). (B-G) Germaria from females carrying the GbeSu(H)m8 reporter, through three representative z-sections (z1 to z3). A schematic of each germaria is presented on the left. The position of each germline cyst (white dotted line) in regions IIa, IIb and III is shown. Arrows indicates the GbeSu(H)m8- expressing cells in different z sections (white for z1, yellow for z2 and blue for z3). (B) Dl -mosaic-soma germarium with three GbeSu(H)m8 cells (arrows) in region IIa. (C) Dl -mosaic-soma-germline germarium. Three GbeSu(H)m8 MBFC (arrows) are visible in region III. A Dl cyst is present in region IIb. (D) Dl -soma germarium. Four GbeSu(H)m8 cells (arrows) are visible in region IIa. (E) Dl -mosaic-soma-germline germarium. Dl cysts are present throughout the germarium. No Dl cells (arrowheads) in region IIa express GbeSu(H)m8 whereas at least four MBFC expressing GbeSu(H)m8 are present in region III (arrows). (F) neur -mosaic-soma germarium with MBFC expressing GbeSu(H)m8 (arrow) in region III. (G) neur -soma germarium. No cells expressed GbeSu(H)m8 throughout the germarium. We next analysed N reporter expression in neur -mosaic-soma germaria. We did not detect any N reporter expression in regions IIa and IIb of neur -mosaic-soma germaria, in contrast to region III, where expression is detected in neur MBFC that are in contact with WT MBFC ( Figure 3F ). The latter activation depends on the signal produced by neighbouring WT MBFC and not by the germline, as no expression is detected in region III when all somatic cells are mutant for neur ( Figure 3G ). Thus, neur -mosaic-soma germaria induce no phenotype in region IIa, but do induce a phenotype similar to that of Dl -mosaic-soma germaria in region III. From these experiments, we conclude that DlS is cis-inhibiting N in region III in a neur -dependant manner. DlS prevents premature and ectopic polar cell differentiation Since strong N activity correlates with polar cell fate induction, we then asked whether too many cells adopt a polar cell fate in a Dl -mosaic-soma germaria by looking at Castor and PZ80 expression. In such germaria, Cas was strongly expressed in Dl cells in regions IIa and IIb ( Figure 4A , B). In Dl ssc, the fluorescence intensity was about four times more than the WT ssc ( Figure S3A , S3B ) and correlated with high levels of N reporter expression ( Fig 4C ). This strong expression depends on N activity, as it is lost in Dl -germline germarium ( Figure 4D , S3D). In the MBFC and pc-sc precursors mutant for Dl, Cas expression was also high compared to that in WT ( Figure 4A-B , E-F). The fluorescence intensity in pc-sc precursors was about twice that of WT pc ( Figure S3A and S3C ). Download figure Open in new tab Figure S2. DlG and DlS are redundant for follicle formation (A-B) Dl -soma follicles (A) and Dl -soma ovariole (B). The arrows point to compound (A) or fused follicles (B). Download figure Open in new tab Figure S3. DlS controls Castor expression The use of a color gradient allows the visualization of differences in intensities of accumulation from none (blue) to strongest (red) (A’, D’’, E1’’, E2’’). (A) WT germarium and stage 2 follicle showing the different levels of Cas expression in the ssc (weak level, arrows), the MBFC/pc-sc precursors (intermediate level), the pc and the sc (high level). (B, C) Fluorescent intensity of Cas expression: ratio between the ssc and the pc (B) or between the precursors (MBFC or pc-sc) and the pc (C). Error bars indicate s.e.m. *, p<0.05; **, p<0.01 t-test versus control. (D) Dl -mosaic-soma-germline germarium. A Dl cyst is present in region IIb. No Cas-positive cells are detected in regions IIa and IIb. (E) Dl -mosaic-soma-germline germarium through 2 consecutive z-confocal sections (z1 to z2). A Dl cyst is present in region IIb. Cas-positive cells are detected in region III in Dl MBFC close to IIb (arrows). A Dl MBFC (visible in z2) in contact to WT MBFC, expresses high levels of GbeSu(H)m8 (arrowhead), but does not express Cas. Download figure Open in new tab Figure 4. DlS controls polar cell marker expression in the germarium The use of a color gradient allows the visualization of differences in intensities of accumulation from none (blue) to strongest (red) (A’’, B’’’, C’’’, E’’’, F’’’). (A-F) A schematic of each germaria is presented on the left. The position of each germline cyst (white dotted line) in regions IIa, IIb and III is shown. (A) WT germarium showing the different levels of Cas expression in the ssc (weak level, arrows), the MBFC/pc-sc precursors (intermediate level, brackets), the pc and the sc (high level, arrowhead). (B) Dl -mosaic-soma-germline germarium. A Dl cyst is present in region III. The arrow points to a stem cell expressing high levels of Castor. High Cas expression is also seen in MBFC/pc-sc precursors (brackets). (C) Dl -mosaic-soma-germline germarium from a female carrying the GbeSu(H)m8 reporter. The arrow points to cells expressing intermediate levels of Castor and high levels of N activity in region IIa. High Cas expression is also seen in MBFC/pc-sc precursors (brackets). A Dl cyst is present in region IIb. (D) Dl -mosaic-soma-germline germarium through 5 consecutive z-confocal sections (z1 to z5). No cells are expressing Cas before region IIb. All the cysts present in regions IIa and IIb are mutant for Dl . (E) Dl -mosaic-soma germarium. The arrow points to MBFC expressing high levels of Castor in region III. (F) Dl -mosaic-soma-germline germarium. The arrow points to MBFC expressing high levels of Castor in region III. A Dl cyst is present in region III. (G) Dl -mosaic-soma germarium from a female carrying the PZ80 enhancer-trap. The arrows point to MBFC expressing high levels of □-gal in region III. These results indicate that DlS is required to prevent high Cas expression in the ssm, the ssm daughters, the pc-sc and MBFC precursors. In region III of Dl -mosaic-soma germarium, Cas was strongly expressed in mutant MBFC when they were close to region IIb. This was observed independently of the genotype of the neighbouring cells or of the germline ( Figure 4E , F). No correlation was observed in Dl MBFC between high level of Cas expression and of N activity, indicating that Cas up-regulation is not driven by increased N signalling ( Figure S3E ). This is consistent with previous results that showed that Cas expression is not modified in MBFC mutant for N ( Chang et al., 2013 ). Upregulation of Cas expression was less detectable in Dl cells close to the end of germarium, and was completely absent from stage 2 onwards ( Figure S3E ). These results indicate that DlS helps to switch off Castor in the MBFC of region IIb/III. Finally, we analysed PZ80 expression, which is specifically expressed in the polar cells from stage 2 follicles onwards in WT. In Dl -mosaic-soma germarium, no expression was detected in regions IIa and IIb. However, we did observe premature PZ80 expression in Dl cells in region III, but only when they were in contact with WT MBFC, indicating that DlS prevents polar cell differentiation in the MBFC ( Figure 4G ). To conclude, DlS prevents N from being activated by DlG in all region IIa/IIb cells, in order to maintain Cas at low levels. In region III, DlS prevents N from being activated by DlS produced by the neighbouring MBFC, which prevents them from adopting a polar cell fate. Robust polar cell differentiation depends sequentially on DlS and DlG production The literature showed that DlG is required to induce polar cell fate ( Lopez-Schier and St Johnston, 2001 ; Torres et al., 2003 ). However, this conclusion was reached by examining the effect of Dl -germline clones without accounting for the presence or absence of Dl clones in the soma. As our data indicate that polar cell differentiation is impaired in Dl -mosaic-soma germaria, we decided to re-examine polar cell differentiation in Dl -germline or in Dl -soma follicles. The vast majority of WT stage 2 follicles (90-100 %) display polar cell marker expression at the anterior and posterior ends. We observe that both mutant conditions lead to delays in the appearance of polar cell markers in the anterior and posterior ( Figure 5A ), but that polar cell markers can be detected in the absence of DlG or of DlS ( Figures 5B and 5C , S4A). Up to stage 4, the phenotype is more severe when Dl is removed from the soma than when Dl is removed from the germline. Notably, polar cell differentiation rarely occurs at the posterior of Dl -soma follicles prior to stage 4. However, DlG is required and sufficient to maintain this fate after stage 4. In its absence, as it has been previously described, follicles fuse with anterior ones and the expression of some polar cell-specific markers, such as PZ80, become undetectable ( Figure S4B ). We also analysed the expression of the N activity reporter in young Dl -germline and Dl -soma follicles, which marks with the selection of the second polar cell. In Dl -soma clones, most of the follicles did not show expression before stage 4 (compare Figures 5D and 5E ), confirming that DlS production is required to turn on N reporter expression before this stage and that DlG becomes efficient only at this stage. In Dl -germline clones, follicles often showed expression at both poles at stages 3 or 4, but the expression was weaker than in WT ( Figure 5F ). This indicates that DlG contributes to the activation of the N reporter before stage 4. Altogether, these data show that Dl must be produced in both tissues to ensure robust polar cell formation, and that DlS plays a preponderant role from stages 1 to 3 whereas DlG is essential to maintain this fate from stage 4. Download figure Open in new tab Figure S4. Robust polar cell differentiation requires DlG and DlS (A-B) Dl -germline follicles from females carrying the PZ80 enhancer trap. (A) Stage 3 follicle at 3 consecutive z-confocal sections (z1 to z3). Aa’ - Aa’’’ and Ap’ - Ap’’’ are magnified views of the anterior (a) and posterior (p) boxed areas in A. The arrows point to polar cells expressing PZ80. (B) Fused follicles between an anterior follicle with a WT cyst and a posterior follicle with a Dl cyst. B’-B’’’ are magnified views of the posterior (p) boxed area in B. No posterior cells express PZ80. Download figure Open in new tab Figure 5. Robust polar cell differentiation requires DlG and DlS (A) Percentages of follicles containing one or more cells expressing at least two of the following polar cell markers (Fas III, PZ80, Cas, GbeSu(H)m8 ) in Dl -germline follicles (blue) or in Dl -soma follicles (red) from stages 2 to 5. Over 14 clusters were counted per stage for each type of mutant follicles. (B) Stage 3 Dl -germline follicle from a female carrying the PZ80 enhancer-trap. One anterior and two posterior cells express PZ80 (arrow). Ba’ - Ba’’’ and Bp - Bp’’’ are magnified views of the anterior (Ba) and posterior (Bp) boxed areas in B. (C) Stage 2 and 3 Dl -soma follicles from a female carrying the PZ80 enhancer-trap, through 2 consecutive z-confocal sections (z1 and z2). One posterior cell of the stage 2 follicle and one anterior cell (arrows) of the stage 3 follicle display PZ80 expression. C1’-C1’’ and C2’-C2’’ are magnified views of the boxed areas in C1 and C2, respectively. (D) WT ovariole from a female carrying the GbeSu(H)m8 reporter through 3 consecutive z-confocal sections (z1 to z3). The arrows point to polar cells expressing GbeSu(H)m8 . (E) Dl -soma ovariole from a female carrying the GbeSu(H)m8 reporter through 2 consecutive z-confocal sections (z1 to z2). No expression is detected up to the anterior of stage 4 follicle (arrow). (F) Stage 3 Dl -germline follicle from a female carrying the GbeSu(H)m8 reporter. N activity is detected at the anterior and posterior polar cells (arrows). Fa’, Fa’’, Fp’, Fp’’ are magnified views of the anterior (a) and posterior (p) boxed areas in F. Discussion Previous analyses have shown that N is required for the migration of sss daughters in region IIa of the germarium and for the differentiation of the pc starting in region IIb ( Grammont and Irvine, 2001 ; Lopez-Schier and St Johnston, 2001 ; Nystul and Spradling, 2010 ). Though both processes are known to depend on DlG production, the discrepancies in N -soma and Dl -germline phenotypes suggest that the current understanding of the activation of this pathway is incorrect ( Lopez-Schier and St Johnston, 2001 ; Nystul and Spradling, 2010 ; Torres et al., 2003 ). First, our study demonstrates that DlS is required to downregulate N in all the cells located in region IIa of the germarium and in the MBFC through a cis-inhibition mechanism. In region IIa, DlS prevents N from responding to DlG, whereas in region IIb/III, DlS prevents MBFC from responding to DlS produced by the neighbouring MBFC ( Figure 6 ). This regulation prevents premature and ectopic induction of polar cells. Second, we show that DlS plays a more important role than DlG in pc differentiation in the germarium. Download figure Open in new tab Figure 6. A model for N activity during follicle formation Schematic representation of a germarium and a stage 2 follicle with the different steps of cell differentiation in WT. The arrangement and identities of the different cell types are as in Figure 1A . (B) In region IIa of WT follicles, DlS cis-inhibits N in all the cells to prevent N to be highly activated by DlG (arrow). In region IIb/III, DlS cis-inhibits N to prevent it to be activated from the neighboring somatic cells (light grey arrow). DlG is unable to promote activation on its own. Only the pc-sc precursors activate N, possibly due to the activity of Fringe in these cells which renders the cells more efficient to respond to Dl. In young follicles, DlS and DlG promote N activation on the polar cells (black arrows) DlS still inhibits N in the MBFC (ref). (C) In region IIa of Dl -germline follicles, N is off in all the cells. In region IIb/III, N is activated only the pc-sc precursors. In young follicles, DlS promote N activation on the polar cells until stage 4, but is unable to maintain this fate on its own after this stage. (D) In region IIa of Dl -soma follicles, N is on in many cells. In region IIb/III, N is off in the pc-sc precursors. DlG promotes strong N activation in the polar cells from stage 4 onwards. Evidence for a cis-inhibitory mechanism during Drosophila oogenesis has already been shown in MBFC between stages 2 and 6 ( Palmer et al., 2014 ; Poulton et al., 2011 ). Here DlS blocks N to prevent its ligand-independent activation and alter the cell cycle ( Deng et al., 2001 ). In the germarium, our data show that the N reporter is not active in the absence of DlG and DlS, implying that ligand-independent N activation does not occur during follicle formation. Thus, downregulation of N is permanent from region IIa of the germarium to stage 6 follicle, but the role of this regulation changes from maintenance of an undifferentiated state to control of the cell cycle. In addition, our data show that neur is also required for the cis-inhibtion of N in the MBFC, indicating that the mechanism used in these cells works differently from that described by Miller et al. ( Miller et al., 2009 ). Several analyses have allowed the establishment of a temporal and spatial sequence of events leading to follicle formation. The process begins with the migration of some of the ssc daughters around the germline cysts, followed by the selection of MBFC and pc-sc precursors ( Fadiga and Nystul, 2019 ; Melamed and Kalderon, 2020 ; Nystul and Spradling, 2010 , 2007 ; Reilein et al., 2017b ). Our results reveal a redundant N function during follicle formation ( Figure 6 ). Indeed, in absence of Dl s, N activity is detected only in the cells of region IIa, which corresponds to ssc and their immediate siblings and this activity is sufficient to assure follicle formation. In the absence of DlG, the only cells that display N activity are the pc-sc precursors, implying that these cells are sufficient to achieve follicle formation. Thus, we conclude that the first two steps of N activation are redundant for follicle formation, but are both necessary for the timely differentiation of polar cells. Altogether, our data support a functional link between the ssc siblings and pc-sc precursors for follicle formation. The literature suggests that N is also required later for follicular cell fate specification, and that the role of the Hedgehog pathway on Cas and Eya expression is responsible for initiating that process ( Bai and Montell, 2002 ; Chang et al., 2013 ). One important aspect of their work was to show the repression exerted by Eya on Cas, which leads to mutually exclusive expression patterns. However, it is unclear why both proteins can be detected at low levels in the cells that reside in region IIa. Based on our results showing that N activity is required in region IIa for Cas expression, we propose that low levels of N activity counter-balances Eya in these cells. Our results also show that DlS is required for downregulating Cas in these cells, which implies that both Notch and Hedgehog signalling work together to prevent early follicle cell fate specification, and to allow the maintenance of an undifferentiated state. With respect to region IIb/III, our data show that DlS works in concert with Eya to down-regulate Cas in the MBFC. Finally, our data show that DlG alone is unable to activate N in any cells of region IIb/III and that DlS is more efficient to initiate polar cell differentiation during these stages. One might consider that the somatic cells in the region 2b are kept undifferentiated until few of them start differentiate as pc-sc precursors. One possibility to separate the pc-sc precursors from the MBFC precursors is to render the formers more sensitive to DlS from the latters. This could be achieved by controlling Fringe expression, which is known to facilitate N activation when Dl is expressed by the neighbouring cells ( Grammont and Irvine, 2001 ; Moloney et al., 2000 ). Our results also demonstrate that the juxtaposition of somatic cells with different levels of Dl is more deleterious to follicle formation than the complete absence of Dl would be. This juxtaposition could delay pc differentiation, as this process is likely to be influenced by Dl levels in the pc-sc precursors, but also lead to the formation of ectopic polar cells in the MBFC as it is observed in Dl or neur compound follicles. Thus, compound and fused follicles may derive not only from a delay in polar cell differentiation but also from the formation of ectopic polar cells. Our results highlight the importance of protecting cells from inappropriate N activation by signals that may originate in surrounding cells. Although only a few cases of developmental processes using cis-regulation of N by endogeneous Dl have been uncovered so far in both vertebrates and invertebrates ( Becam et al., 2010 ; Del Álamo et al., 2011 ; Miller et al., 2009 ), this work points to the conclusion that this mechanism is likely to be more common than expected. Materials and methods Drosophila Stocks and Crosses CantonS is used as WT. neur if65 and Dl rev10 are null alleles ( de Celis et al., 1993 ; de-la-Concha et al., 1988 ; Heitzler and Simpson, 1991 ; Sun and Artavanis-Tsakonas, 1996 ). The Notch activity reporter line used is GbeSu(H)m8-lacZ ( Furriols and Bray, 2001 ). Fly stocks were cultured at 25°C on standard food. Clones were generated by Flipase-mediated mitotic recombination on FRT82D chromosomes carrying either GFP or Myc as markers ( Golic and Lindquist, 1989 ; Xu and Rubin, 1993 ). Flipase expression was induced by heat shocking two-day old females at 38°C for 1 hour, then the females were placed at 25°C and were dissected 9 to 12 days after the heat shock. Myc expression was induced by heat shocking females at 37C for 1 h, four hours before dissection. Follicle Staining Pc identity was assayed by the expression of the FasIII protein and of the PZ80 enhancer trap, inserted in the fasIII gene, ( Karpen and Spradling, 1992 ), which are specifically expressed in the pc from stage 1 onwards. The A101 marker (an enhancer-trap within neur ) was not used in order to prevent feedback from affecting marker expression when the N pathway was manipulated. The following primary antibodies were used: goat anti-ß-galactosidase (1:1000, Biogenesis), rabbit anti-ß-galactosidase (1:2000, Cappel), rabbit anti-Myc (1:100, Santa Cruz), mouse anti-GFP (1:500, Sigma-Aldrich), goat anti-GFP (1:1000, AbCam), mouse anti-FasIII 7G10 (1:200, DSHB), Rabbit anti-Castor (1:5000; a gift from Dr Ward D. Odenwald) and mouse anti-orb 6H4 and 4H8 (1:100, DSHB). Actin staining is realized by using Rhodamin-Phalloidin (Molecular Probes). Secondary antibodies coupled with Cy5, Cy3 (Jackson Immunoresearch) or Alexa Fluor 488 (Molecular Probes) are used at 1/1000. Ovaries from females were dissected directly into fixative 3 to 4 days after Flipase induction and stained following the protocol described in ( Grammont and Irvine, 2001 ). To avoid fluctuations of the depth of the follicles that are squeezed by the coverslip, each slide contains 15 ovaries, from which S11 to S14 are removed. After dissection of the follicles, most of the PBS is removed and 20µl of the Imaging medium (PBS/Glycerol (25/75) (v/v)) is added before being covered by a 22/32 mm coverslip. Confocal Microscopy Preparations were examined using confocal microscope (LSM 710 and LSM 700; Carl Zeiss MicroImagin, Inc.) with 40x/NA 1.3 plan-Neofluar and 63x/NA 1.4 plan-Apochromat. Imaging was performed at RT. Images were examined using ImageJ ( http://imagej.nih.gov/ij/ ). Statistical analysis All data graphs are reported as mean ± standard deviation. Statistical analysis was performed using R software (RStudio, Inc), with alpha levels for all statistical tests set to 5%. Acknowledgments We thank DSHB, Bloomington Stock Center; Lyon Bio Image and Arthrotools of the SFR Bioscience (UMS3444/US8) for flies and reagents, Dr W. Odenwald for reagents, P. Das for critical comments on the manuscript. References ↵ Assa-Kunik , E. , Torres , I.L. , Schejter , E.D. , Johnston , D.S. , Shilo , B.-Z ., 2007 . 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Share Cis-inhibition of Notch by Delta controls follicle formation in Drosophila melanogaster Caroline Vachias , Muriel Grammont bioRxiv 2025.03.19.644164; doi: https://doi.org/10.1101/2025.03.19.644164 Share This Article: Copy Citation Tools Cis-inhibition of Notch by Delta controls follicle formation in Drosophila melanogaster Caroline Vachias , Muriel Grammont bioRxiv 2025.03.19.644164; doi: https://doi.org/10.1101/2025.03.19.644164 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)