Tbx1 ortholog org-1 is required to establish testis stem cell niche identity in Drosophila

Tbx1 ortholog org-1 is required to establish testis stem cell niche identity in Drosophila | 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 Tbx1 ortholog org-1 is required to establish testis stem cell niche identity in Drosophila View ORCID Profile Patrick Hofe , View ORCID Profile Ariel Harrington , Tynan Gardner , View ORCID Profile Stephen DiNardo , View ORCID Profile Lauren Anllo doi: https://doi.org/10.1101/2025.05.09.653127 Patrick Hofe 1 Department of Biology, East Carolina University , Greenville, NC 27858 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Hofe Ariel Harrington 1 Department of Biology, East Carolina University , Greenville, NC 27858 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ariel Harrington Tynan Gardner 2 Perelman School of Medicine at the University of Pennsylvania , Philadelphia, PA 19104 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen DiNardo 2 Perelman School of Medicine at the University of Pennsylvania , Philadelphia, PA 19104 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stephen DiNardo Lauren Anllo 1 Department of Biology, East Carolina University , Greenville, NC 27858 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lauren Anllo For correspondence: anllol22{at}ecu.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Stem cells require signals from a cellular microenvironment known as the niche that regulates identity, location, and division of stem cells. Niche cell identity must be properly specified during development to form a tissue capable of functioning in the adult. Here, we show that the Tbx1 ortholog org1 is expressed in Drosophila testis niche cells in response to Slit and FGF signals. org1 is expressed during niche development and is required to specify niche cell identity. org1 mutants specified fewer niche cells, and those cells showed disruption of niche-specific markers, including loss of the niche adhesion protein Fas3 and reduced hedgehog expression. We found that org1 expression in somatic gonadal precursors is capable of inducing formation of additional niche cells. Disrupted niche identity in org1 mutants resulted in niche assembly and functionality defects. We find the conserved transcription factor islet is expressed in response to org1 and show that islet functions downstream to mediate niche identity and assembly. This work identifies a novel role for org1 in niche establishment. Summary Statement Specification of niche identity is crucial to establish a functional stem cell microenvironment. Using a translatable model, we show that niche expression of org1 influences islet to specify niche identity. Introduction Stem cells play a vital role in maintaining functional tissues. The preservation of stem cells in many tissues is controlled by the stem cell niche, a local cellular microenvironment that supplies signals to maintain stem cell identity, location, and division rates ( Li & Xie, 2005 ). A key concept in stem cell biology is use of diffusible signals to regulate stem cell behaviors. In models like the mammalian intestinal stem cell (ISC) niche, diffusible signals such as Wnt, Hh, and BMP regulate stem cells ( Walker et al., 2008 ). These signals have varying origins since there are multiple cell types that comprise the intestinal stem cell niche ( Walker et al., 2008 ). Because various cell lineages can comprise the niche, identification of niche cells is often challenging. In some tissues even the identity of the stem cells is unclear ( Sato et al., 2011 ; Kaestner, 2019 ; Li et al., 2023 ). Thus, identifying regulators that specify niche identity during tissue development has remained a challenge in stem cell biology. Because of its readily identifiable, well-defined niche, we leverage the Drosophila testis to study how niche cells acquire their identity and adopt proper function during development ( de Cuevas & Matunis, 2011 ; Le Bras & Van Doren, 2006 ; Anllo et al., 2019 ; Okegbe & DiNardo, 2011 ; Wingert & DiNardo, 2015 ). The Drosophila testis is a paradigmatic niche in which many concepts at the foundation of stem cell biology continue to be elucidated ( Herrera & Bach, 2019 ; Leatherman & DiNardo 2008 and 2010 ; Lenhart & DiNardo 2015 ; Inaba et al., 2015 ; Tulina & Matunis 2001 ; Yamashita et al., 2003 ; Zheng et. al., 2011 ). The singular cell type that comprises the testis niche, somatic “hub” cells, regulates germline and somatic stem cells required to maintain sperm production throughout the adult life of the fly ( Lenhart and DiNardo, 2015 ; Roach and Lenhart, 2024 ). The wealth of powerful genetic tools available to study this niche make it an ideal model to identify regulators of niche cell fate. Prior to niche formation, the embryonic Stage 15 Drosophila testis is a spherical arrangement of somatic gonadal precursors (SGPs) interspersed among and encysting germ cells ( Jenkins et al., 2003 ; Le Bras & Van Doren, 2006 ). SGPs are specified from a subset of mesodermal cells adjacent to muscle precursors in embryonic parasegments 10-13 earlier in embryogenesis ( Boyle et al., 1995 ; Boyle et al., 1997 ; Riechmann et al., 1998 ). Niche, or “hub,” cells derive from SGPs, and are initially specified through Notch signaling ( Kitadate & Kobayashi, 2010 ; Okegbe & DiNardo, 2011 ; DiNardo et al., 2011 ). By stage 11 of embryogenesis, SGPs contacting the primordial midgut receive Delta ligands which initiate Notch signaling and niche specification ( Okegbe & DiNardo, 2011 ). Continued specification, or further induction of niche cell identity after the initial acquisition of niche cell fate, is required to establish the niche. As part of continued specification, SGPs at the gonad anterior receive the Notch ligand Serrate from other gonadal cells, and SGPs at the posterior receive EGFR antagonistic signals that repress niche identity ( Kitadate & Kobayashi, 2010 ). Defects in Notch signaling result in fewer niche cells specified ( Okegbe & DiNardo, 2011 ; DiNardo et al., 2011 ; Kitadate & Kobayashi, 2010 ; Wingert & DiNardo, 2015 ). Specified niche cells downregulate the conserved Maf transcription factor Traffic jam (Tj) in response to Notch ( Wingert & DiNardo, 2015 ). A role for Notch signaling in niche formation has translated to other niches in Drosophila and in mammals ( Zamfirescu et al., 2022 ). Outside of Notch signaling, there is little known about how testis niche cells acquire their identity. After the initiation of specification, embryonic niche cells are compartmentalized to the gonad anterior in a process called niche assembly ( Le Bras & Van Doren, 2006 ; Anllo et al., 2019 ). During assembly, specified prospective (pro) niche SGPs move peripherally onto the gonadal extracellular matrix (ECM), where they then migrate to their anterior assembly position ( Anllo et al., 2019 ). Our previous work revealed that extrinsic Slit and FGF signals from neighboring visceral muscle (Vm) are required for this migration ( Anllo & DiNardo, 2022 ). At the gonad anterior, niche cells first exhibit F-actin polarization to niche-niche interfaces, which later re-polarizes to niche-stem cell interfaces as niche cells compact into a spherical arrangement with smooth contour ( Anllo & DiNardo, 2022 ; Warder et al., 2024 ). Germline stem cells contacting the assembled niche contribute to niche morphogenesis through actomyosin networks that result in tension around the compacting hub periphery ( Warder et al., 2024 ). The assembled and compacted niche expresses niche specific markers such as fasciclin3 and hedgehog and is radially surrounded by germline stem cells (GSCs) and later, cyst stem cells (CySCs) ( Le Bras & Van Doren, 2006 ; Sheng et al., 2009 ; Sinden et al., 2012 ; Wingert & DiNardo, 2015 ). The niche makes adhesive connection to neighboring stem cells and extracellular matrix to maintain its shape, location, and function ( Lee et al., 2008 ; Vida et al., 2024 ; Tanentzapf et al., 2007 ; Tseng et al., 2022 ; Amoyel et al., 2013 ). After niche establishment, the niche functions throughout the life of the animal. The niche’s location, critical to maintain GSCs and CySCs, is maintained through Integrin-mediated connections to the ECM ( Anllo et al. 2019 ; Lee et al., 2008 ; Tanentzapf et al., 2007 ). In its established position, the niche sends maintenance signals to its surrounding stem cells, including Unpaired (Upd) and Hedgehog (Hh). Upd ligands activate JAK/STAT signaling in GSCs, which regulates their adhesion to the niche and oriented cell divisions ( Chen et. al., 2018 ; Leatherman & DiNardo 2008 and 2010 ; Tulina & Matunis, 2001 ; Wawersik et al., 2005 ; Yamashita et al., 2003 ). Niche-secreted Hedgehog regulates CySC self-renewal (Amoyel et al., 2012). Niche signals are important for establishing the stem cell populations, and for long term stem cell maintenance ( Sheng et al., 2009 ; Sinden et al., 2012 ). Because Notch signaling required to specify niche cells is initiated at stage 11 of embryogenesis ( Okegbe & DiNardo, 2011 ), and niche-specific markers such as Fas3 accumulation are not observed until stage 15 of embryogenesis ( Le Bras & Van Doren, 2006 ; Anllo et al., 2019 ), there are likely other factors influencing the specification of niche cells during this period. Our work here identifies the Tbx1 ortholog org1 as one of those factors. Tbx1 and its orthologs play a conserved role across phyla in specifying mesodermal cell types ( Marcellini et al., 2003 ). Like Tbx1 , org1 encodes a transcription factor with a T-box DNA binding domain and is a known regulator of mesodermal muscle fate during embryonic development ( Schaub et al. 2012 ; Schaub & Frasch, 2013; Boukhatmi et al., 2014 ). Our previous work identified that the conserved transcription factor islet is expressed in niche cells and required for both niche assembly and cytoskeletal polarization downstream of Vm assembly cues ( Anllo & DiNardo, 2022 ). islet expression is known to be regulated by org1 during specification of alary musculature ( Boukhatmi et al., 2014 ). Here, we find that a parallel mechanism regulates continued specification of niche cells and directs their anterior assembly. We show that org1 is expressed in the embryonic niche and reveal its requirement in establishing a full complement of functional, assembled niche cells. We further show that Org1 accumulation is affected by Slit and FGF signals, and that islet acts downstream of org1 . These data reveal org1 as a novel regulator of niche identity and assembly for the Drosophila testis niche. Results org1 is expressed in the niche and is induced by Slit and FGF signals Our previous work has shown that the conserved transcription factor islet is expressed in the developing niche ( Anllo & DiNardo, 2022 ). Because Org1 is a known regulator of islet in other tissues ( Boukhatmi et al., 2014 ), and single nuclear RNA sequence data (snRNAseq) suggested that org1 was enriched in the adult testis niche (Li et al., 2021; Raz et al. 2023), we tested whether org1 was expressed in the developing gonadal niche. Using an Org1::GFP transgenic BAC reporter that rescues org1 lethality ( Kudron et al., 2018 and data not shown), we observed Org1 in prospective niche cells beginning prior to assembly at embryonic stage 15/16 and after niche assembly in stage 17 at the gonad anterior ( Figure 1A - 1C’ ). Org1::GFP detection in some prospective niche cells coincided with the timing of Traffic jam downregulation ( Figure 1A’ , 1B’ ). Additionally, we also saw SGPs without downregulated Traffic jam that exhibited lower levels of Org1::GFP elsewhere in the gonad ( Figure 1B-B” , yellow arrows). We confirmed the stage 17 Org1::GFP expression pattern using an Org1 antibody ( Schaub et al., 2012 ) that revealed a signal in gonadal niche cell nuclei ( Figure 1D, D’ ) which was absent in org1 mutants ( Figure S1 ). Our findings reveal that org1 is expressed in the gonad niche, and its expression begins before niche assembly. Download figure Open in new tab Figure 1. org1 is expressed in the niche in response to Slit and FGF signals. (A-B’) Gonads (dotted line) from Stage 15 animals with the Org1::GFP BAC reporter, immunostained for GFP (green) and Traffic jam (red). Fas3 (white) accumulates at assembling niche cell interfaces. Org1::GFP is brightest when Traffic jam (red) is downregulated. Prospective niche cells with downregulated Traffic jam (white arrows) and with Traffic jam not downregulated (yellow arrows). (C-H) Gonads dissected from Stage 17 animals aged 20-22 hours at 25 degrees C. (C, C’) Org1::GFP reporter (red) showed org1 expression in the assembled niche (dotted white line; Fas3, white). (D, D’) Org1 antibody (magenta) revealed accumulation in the niche (dotted white line; Fas3, white) with spurious non-specific signal at the posterior (see Suppl Fig S1 ). (E-H) Dissected Stage 17 gonads immunostained to reveal Org1 (red) and the Niche (Fas3, white). (E, E’) Control gonads showed Org1 (red) accumulation in the niche (white). (F, F’) slit, fgf mutants had diminished accumulation of Org1. Gonads are outlined with dotted lines. (G-G’) fgf mutants had diminished niche Org1 accumulation. (H, H’) slit mutants had diminished niche Org1 accumulation. Gonads are outlined in yellow; niches are outlined in white. (I) Quantification of Org-1 accumulation in niche cells in controls and Slit and FGF signaling mutants (p<0.0001, Ctrl n=39 niche cells from 13 gonads; slit, fgf n= 30 niche cells, from 10 gonads Mann-Whitney Test). (J) Quantified Org1 accumulation in fgf mutants and heterozygous sibling controls (Het) (p= 0.0136, Het n=45 niche cells from 15 gonads; fgf n=48 niche cells from 16 gonads, Mann-Whitney Test). (K) Quantified Org1 accumulation in slit mutants and heterozygous sibling controls (Het) (p= 0.0090, Het n=57 niche cells from 19 gonads; slit n=81 niche cells from 27 gonads, Mann-Whitney Test). All quantifications are shown for individual niche nuclei. Scale bars, 10um. n≥3 trials. Since islet is regulated by Slit and FGF signals from the visceral muscle (Vm), we tested if org1 is also regulated by these signals ( Anllo and DiNardo, 2022 ). If org1 is induced by these signals, then loss of Slit and FGF Vm signals required for niche assembly would result in loss of Org1 accumulation in niche cells. We assessed Org1 accumulation in double mutant animals with the slit[2] mutation and the small chromosomal deficiency Df(2R)BSC25 that removes both genes encoding FGF Heartless ligands, pyramis and thisbe (see Methods). Indeed, loss of both signals together resulted in diminished Org1 accumulation ( Figure 1E - 1F’ ). Additionally, loss of either Slit or FGF separately resulted in diminished Org1 accumulation ( Figure 1G - 1H’ ) and our fluorescence quantifications supported these findings ( Figure 1I - 1K ). Together, our data confirm that org1 is expressed in the niche and accumulates in response to Slit and FGF. org1 is necessary and sufficient for aspects of niche cell identity Given the known role of org1 in embryonic muscle specification, we asked if there were niche identity defects in org1 mutants ( Schaub & Frasch, 2012 ; Boukhatmi et al., 2014 ). We hypothesized that if org1 had a role in specifying niche fate, we would see disruption of identity markers. We assayed for niche identity markers Fasciclin3 (Fas3) accumulation and hedgehog ( hh ) expression ( Le Bras & Van Doren, 2006 ; Wingert & DiNardo, 2015 ) in an org1 mutant allele lacking the translation initiation codon and T-box domain ( Schaub et al., 2012 ). org1 mutants did not accumulate the niche specific adhesion protein Fasciclin 3 ( Figure 2B - 2B’ ). Additionally, org1 mutants had lower expression of a hh- lacZ reporter in niche cells compared to controls ( Figure 2C - 2E ). Interestingly, hh expression is known to be restricted to the adult niche ( Amoyel et. al., 2013 ). We find that while hh expression is highest in control embryonic niche cells, we detect low levels of hh in some control SGPs outside the niche ( Figure S2 ). Since these niche identity markers were disrupted, we used N-cadherin (N-cad) accumulation to detect niche cells in org1 mutants. N-cad accumulation is enriched at the cortex of properly assembled anterior niche cells, with low accumulation in other SGPs, affording a reasonable measure of identification of niche cells at the anterior ( Le Bras & Van Doren, 2006 ). When visualizing gonads with N-cad immunofluorescence, we detected fewer N-cad accumulating niche cells in org1 mutants compared to controls ( Figure 2F - 2G’ ). The detection of N-cad in some cells indicates that not all aspects of niche identity are disrupted, suggesting a role for org1 in continued induction of niche fate after their initial specification. Download figure Open in new tab Figure 2. org1 is necessary and sufficient for aspects of niche cell identity. (A-D) Dissected gonads immunostained to show germ cells (green), Fas3 (white), hh -lacZ (magenta), DNA (blue). (A, A’, C) Control niche cells accumulated niche specific markers Fas3 and hedgehog reporter expression ( hh -lacZ, separate gonad from A, A’). (B, B’, D) org1 mutants did not accumulate Fas3 and had diminished expression of hh -lacZ (D is a separate gonad from B). (E) Quantified hh -lacZ expression in niche cells accumulating N-cad (p= 0.0294, Ctrl n=24 niche cells from 7 gonads; org1 n=30 niche cells from 10 gonads, Mann-Whitney Test). (F, F’) Control gonads accumulated enriched N-cad at the cortex of niche cells. (G, G’) org1 mutant niches had fewer cells accumulating enriched N-cad. (F-G’) Germ cells (green), N-cad (white), DNA (blue). (H) Quantification of N-cad accumulating niche cells per gonad (p<0.0001, Ctrl n=15 gonads; org1 n=20 gonads, Mann-Whitney Test). (I, I’) Control gonad with six4 moesinGFP marking somatic F-actin. (J, J’) SGP driven org1 overexpression in gonads generated niches that had more cells in comparison to controls. (I-J’) Somatic cells (green), Niche (white), DNA (blue). (K) Quantification of niche cell count (p<0.0001, Ctrl n=26 gonads; org1 n=26 gonads, Mann-Whitney Test). Dotted lines, niche. Scale bars, 10 um. n≥3 trials. To confirm that the fewer N-cad accumulating niche cells in org1 mutants resulted from failure of continued niche cell specification in org1 mutants, and not simply a reduction in the number of SGPs, we quantified the total number of SGPs in org1 mutants. We assayed the SGP-specific transcription factor Traffic jam (Tj) to count all SGPs ( Wingert & DiNardo, 2015 ). While there was a slight decrease in number of SGPs, the fraction of these cells specified into niche cells was significantly lower in org1 mutants compared to control gonads ( Figure S3 ). Together with our data indicating a loss of the niche specific adhesion protein Fas3 and reduction of hh gene expression in the niche, these results support a role for org1 in continued specification of niche identity ( Figure 2 , S3). Interestingly, org1 mutant niche cells still down regulated tj as in normal niches, indicating that some aspects of niche specification occur ( Figure S3 ). Since org1 mutants had fewer niche cells, we tested whether overexpression of org1 in all SGPs would create more niche cells. Indeed, when overexpressing org1 with a somatic lineage-specific driver ( six4- gal4; Anllo et. al., 2019), we generated additional N-cad and Islet accumulating niche cells at the gonad anterior ( Figure 2I-K ; 5E-F ). Together, our data suggest that org1 plays a role in specifying niche identity and is sufficient to induce niche identity in additional SGPs. org1 is required SGP intrinsically for niche identity and assembly The data above were collected from a whole organism org1 mutant line in which all T-box binding domains were excised ( Schaub et al., 2012 ). To test whether org1 is required autonomously in SGPs, we disrupted org1 using two somatic lineage-specific drivers that varied in strength, six4 VP16-gal4 ( Warder et al., 2024 ) and six4 -gal4. Using the weaker driver ( six4- gal4), we found an expected decrease in niche cell number, and Fas3 accumulation appeared reduced ( Figure S4 ). Assuming this driver generates only a partial knockdown, this suggests a stringent requirement of org1 in niche cell specification (See Discussion). Excitingly, using the stronger driver ( six4 VP16-gal4), we saw both fewer niche cells ( Figure 3A - 3C ), and dispersed niche cells accumulating low levels of Fas3 ( Figure 3B’ - 3B’’ , 3D ). It is possible this dispersed phenotype was not something we could observe in the global org1 mutants due to the complete loss of Fas3 ( Figure 2 ). Other niche cell markers, including E-cad and N-cad, accumulate on control non-niche SGPs to a degree, so dispersed niche cells might not have been apparent ( Figure S5 ). Our data suggest that org1 is primarily a specification factor during niche establishment and secondarily affects niche assembly. Download figure Open in new tab Figure 3. org1 is required SGP intrinsically for niche identity and assembly. (A, A’, A’’) Control gonads with N-cad (white) and Fas3 (red) accumulating at the anterior. (B, B’, B’’) org1 knockdown with six4gal4VP16 gonads showed fewer niche cells that accumulate N-cad (white) and Fas3 (red) at the anterior, with displaced niche cells (yellow arrow). Brightness and contrast were adjusted in A” and B” to enable visualization of Fas3. (C) Niche cell count per gonad in org1 knockdown (p<0.0001, Ctrl n=23 gonads; six4VP16-gal4 org1 RNAi n=22 gonads, Mann-Whitney Test). (D) Quantification of niche assembly in six4VP16-gal4 org1 RNAi (p= 0.0006, Ctrl n= 10 gonads; org1 RNAi n=14 gonads, Fisher’s Exact Test) (E) Quantified Fas3 accumulation in org1 rescue niches (p<0.0001, org1 n= 14 gonads; org1; org1 OE n= 16 gonads, Fisher’s Exact Test). (F-H) Yellow arrows indicate niche-GSC F-actin interfaces and blue arrows indicate niche-niche F-actin interfaces. (F, F’) Control gonad with Fas3 (green) accumulating at the anterior. (G, G’) org1 knockout mutant gonad with no Fas3 (green) accumulation. (H, H’) org1 knockout mutant with org1 overexpression in somatic gonadal cells (red) with rescued Fas3 (green) accumulation. (F-H) arrows indicate F-actin enrichment at specific SGP interfaces. (I) Quantified F-actin polarization in control gonads (p<0.0001, n-n interfaces n=23; n-gsc interfaces n=20, from 7 gonads, Mann-Whitney Test). (J) Quantified F-actin polarization in org1 mutants (not significant, n-n interfaces n=49; n-gsc interfaces n=38, from 14 gonads, Mann-Whitney Test). (K) Quantified F-actin polarization in org1 mutant rescued gonads (p<0.0001, n-n interfaces n=47; n-gsc interfaces n=38, from 16 gonads, Mann-Whitney Test). Scale bars, 10 um. n≥3 trials. To further support an SGP intrinsic requirement for org1 during niche establishment, we performed a tissue-specific rescue experiment. We expressed org1 with our somatic lineage specific driver, six4- gal4, in whole organism org1 mutants and observed rescued Fas3 accumulation ( Figure 3E - 3H’ ). Our rescue experiment simultaneously expressed the F-actin label Ftractin-tdTomato in SGPs, allowing analysis of niche cytoskeletal polarity. Our previous work has shown that F-actin polarizes to niche-stem cell interfaces after assembly is complete ( Anllo & DiNardo, 2022 ; Warder et al., 2024 ). While F-actin polarization at niche-stem cell interfaces is disrupted in org1 mutant niche cells, this polarization was rescued with org1 overexpression in SGPs ( Fig 3F-K , see arrows). These data suggest that org1 in SGPs is sufficient to initiate downstream cell biological events that polarize F-actin in the assembled niche. org1 is important for niche to stem cell communication Given the defects in niche identity and organization seen when org1 is compromised, we tested if the function was compromised without org1 . We used known steady state niche signaling pathways and centrosome position to assay for niche function deficits ( Leatherman & DiNardo, 2010 ; Kiger et al., 2001 ; Tulina & Matunis, 2001 ; Yamashita et al., 2003 , 2005 ). Stat accumulates in GSCs in response to the niche signal Upd1 ( Chen et al., 2018 ; Kiger et al., 2003). We assessed upd1 expression in niche cells by quantifying levels of upd1 -Gal4 driven RFP fluorescence and found significantly reduced expression in niche cells in whole organism org1 mutants ( Figure 4A-C ). Additionally, we measured reduced Stat accumulation in GSCs ( Figure 4D - 4E’ , 4F ), which was consistent with loss of upd1 expression and impaired niche-GSC communication. Niche cells are also responsible for sending Hh signals to prospective CySCs to enable their self-renewal ( Michel et al., 2012 ; Amoyel et al., 2013 ). We examined localization of Patched, a Hh receptor that accumulates in vesicles in the niche and CySCs in response to signaling ( Amoyel et al., 2013 ; Michel et al., 2012 ; Wingert & DiNardo, 2015 ) ( Fig 4G - 4H’ ). org1 mutants had significantly reduced Patched accumulation in the niche and never accumulated Patched in nearby somatic cells ( Figure 4G - 4H’ , 4I ). This finding aligns with the reduced hh expression we detected in niche cells when org1 was disrupted ( Figure 2C-E ), correlating reduced ligand expression to disruption in niche-CySC communication. Download figure Open in new tab Figure 4. org1 is important for niche to stem cell communication. (A, A’) upd1 -Gal4 drives RFP expression in control niche cells, but (B, B’) not in org1 null animals. (C) Quantification of RFP fluorescence in niche cells (p < 0.0001, Ctrl n = 100 niche cells from 20 gonads; org1 n = 140 niche cells from 28 gonads) (D, D’) STAT (red) accumulated in germline stem cells (GSCs, dotted line) in control gonads, but (E, E’) not in org1 mutant germline stem cells. (F) Quantification of STAT accumulation (p= 0.0041, Ctrl n=44 GSCs from 11 gonads; org1 n=52 GSCs from 16 gonads, Mann-Whitney Test). (G, G’) Hedgehog signaling target Patched (red) accumulated in the niche (white dotted line) and Cyst stem cells (yellow arrow). (H, H’’) Patched did not accumulate in org1 mutant gonads in the niche or cyst stem cells. (I) Quantified Patched accumulation in the niche (p<0.0001, Ctrl n=9 gonads; org1 n= 15 gonads, Mann-Whitney Test). (J, J’) In control gonads with duplicated GSC centrosomes, one centrosome was aligned adjacent to the interface with the niche (yellow bracket). Insets show germline stem cells outlined in white, and the niche boundary (Fas3, white). (K, K’) In org1 mutant gonads, duplicated centrosomes were often not aligned adjacent to the niche interface (yellow bracket). (L) Quantified centrosome alignment (p<0.0001, Ctrl n=64 GSCs from 18 gonads; org1 n= 68 GSCs from 21 gonads, Fisher’s Exact Test). (J’, K’) Insets show centrosome pairs. (M-N) Hedgehog signaling target Patched accumulated in niche cells and CySCs in controls (M-M’) but not in six4VP16-gal4 org1 RNAi gonads (N-N’). (O) Quantified Patched accumulation in the niche (p<0.0001, Ctrl n=34 gonads; org1 RNAi n= 17 gonads, Mann-Whitney Test). (P, P’) STAT (red) accumulated in GSCs (dotted line) in control gonads, but (Q, Q’) not in six4VP16-gal4 org1 RNAi germline stem cells. (R-T) Quantified STAT accumulation (R), STAT+ GSCs (S), and percentage of GSCs that are STAT+ (T) (p<0.0001, Ctrl n=246 GSCs from 28 gonads; org1 RNAi n=146 GSCs from 19 gonads, Mann-Whitney Test). (U) Number of GSCs contacting N-cad positive niche cells in control and six4VP16-gal4 org1 RNAi gonads (p = 0.033, Ctrl n = 29 gonads, six4VP16-gal4 org1 RNAi n = 19 gonads). For all panels: N-cad (white), scale bars, 10 um. Germ cells are indicated with nos -lifeactin::tdTomato (green, panels M-N) or Vasa (green, all other panels). n≥3 trials. We also tested stem cell behavior in the absence of org1 . During normal GSC divisions, one centrosome is anchored close to the niche-germline stem cell interface, while the other centrosome moves to the opposite GSC pole. The alignment of one centrosome at the niche interface enables GSC divisions perpendicular to the niche ( Yamashita et al., 2003 ). Gonads from org1 null animals showed defects in centrosome alignment, with both GSC centrosomes often displaced from the interface with the nearby niche cell. This displacement positioned centrosomes in orientations that would preclude perpendicular GSC divisions from the niche ( Figure 4J - 4K’ , 4L ). Together, our data suggest that org1 is required to generate niche cells capable of communication with stem cells, further supporting a role for org1 in niche cell specification. To ask whether org1 is required cell autonomously in the somatic gonad to enable niche communication with the stem cells, we assessed STAT and Patched accumulation in the six4VP16-gal4 org1 RNAi condition. Consistent with org1 null animals, somatic gonadal knockdown with org1 RNAi resulted in failed accumulation of Patched in the niche and neighboring somatic cells ( Figure 4M-O ), and failed accumulation of STAT in neighboring GSCs ( Figure 4P-R ). These data indicate a cell autonomous requirement for org1 in communication with stem cells. We hypothesized that failed communication to neighboring stem cells would adversely affect stem cell maintenance. Neither org1 null nor six4VP16-gal4 org1 RNAi animals are viable past embryogenesis, precluding analysis of later stem cell maintenance. We thus assessed GSC maintenance by quantifying the number of STAT+ GSCs adherent to the embryonic niche. We determined a threshold level of STAT+ accumulation quantified as one standard deviation below the mean in control GSCs (see methods). Our data revealed significantly fewer STAT+ GSCs ( Figure 4S-T ), and fewer total GSCs adherent to the niche ( Figure 4U ) in the six4VP16-gal4 org1 RNAi somatic cell knockdown, which disrupts org1 during niche specification and prior to and through niche assembly. These data suggest functional defects in GSC maintenance when org1 is autonomously disrupted in the somatic gonad. islet regulates niche cell specification downstream of org1 Our previous work has shown that islet is expressed downstream of Vm signals Slit and FGF and is important for niche assembly ( Anllo & DiNardo, 2022 ). Here, we show org1 is also expressed downstream of Slit and FGF and has a role in niche assembly (see Figure 1 , 3 ). Because org1 is known to induce islet expression in embryonic muscle ( Boukhatmi et al., 2014 ), we next tested if org1 regulated islet in the embryonic gonad. Indeed, we saw that Islet was no longer enriched in org1 mutant niche cells ( Figure 5A - 5C ). We also hypothesized that if org1 is regulating islet , we would see Islet accumulation in the additional niche cells specified with our SGP lineage specific org1 overexpression. Indeed, we saw that additional niche cells at the anterior also accumulated Islet ( Figure 5D - 5F ). These data indicate that Islet accumulates in response to org1 expression. Download figure Open in new tab Figure 5. org1 is important for Islet accumulation. (A, B) Germ cells (Vasa, green), Niche (Ncad, white), DNA (Hoechst, blue). (A, A’) Control gonads showed Islet (magenta) accumulation enriched in the niche. (B, B’) org1 mutant gonads exhibited diminished Islet accumulation. (C) Islet accumulation quantification in niche cells (p<0.0001, Ctrl n=63 from 21 gonads; org1 n=63 from 21 gonads, Mann-Whitney Test). (D, D’, D’’) Control gonads with N-cad (white) and Islet (red) accumulating in niche cells. (E, E’, E’’) org1 overexpressing gonad with additional niche cells (white) that also accumulated Islet (red). (F) Quantification of number of Islet accumulating cells (p= 0.0013, Ctrl n=16 gonads; org1 OE n=18 gonads, Mann-Whitney Test). Scale bars, 10 um. n≥3 trials. The accumulation of Islet in response to org1 parallels the transcriptional cascade that specifies embryonic alary muscle specification ( Boukhatmi et al., 2014 ). We thus hypothesized that islet may also play a role in continued specification of niche identity in addition to the role we previously found during assembly ( Anllo & DiNardo, 2022 ). To test this hypothesis, we quantified the number of Fas3 accumulating niche cells in islet mutants compared to controls. Indeed, we counted fewer Fas3 accumulating cells in islet mutants, supporting our hypothesis ( Figure 6A - 6C ). This reduction in Fas3 accumulating cells is less striking than the complete loss of Fas3 accumulation in the majority of org1 mutant gonads ( Figure 2A-B ; 3E ), suggesting a more prominent role for org1 in continued niche specification. Download figure Open in new tab Figure 6. islet regulates niche cell specification downstream of org1 . Germ cells (Vasa, green), DNA (Hoechst, blue), Niche cells (white). Scale bars, 10 um. (A, A’) Control gonads with niche (Fas3, white) at the anterior. (B, B’) islet mutant gonad with disrupted niche (Fas3, white). (C) Quantification of niche cells per gonad in islet mutants (p= 0.0019, Ctrl n=23 gonads; islet n=19 gonads, Mann-Whitney Test). (D, D’) Control gonads with N-cad (white) and Islet (red) accumulating in niche cells. (E, E’) org1 mutant gonad with fewer niche cells accumulating N-cad at the anterior. (F, F’) An org1 mutant with islet overexpression in somatic gonadal cells had more niche cells than org1 mutants, and a displaced niche cell (yellow arrow). (G) Number of niche cells per gonad for org1 rescue with islet overexpression experiments ( org1 mut vs org1 sib, p<0.0001, org1 mut n=32 gonads; org1 sib n=21 gonads. org1 mut vs org1 mut islet OE, p=0.0005, org1 mut n=32 gonads; org1 mut islet OE n=40 gonads. org1 sib vs org1 mut islet OE, p=0.0338, org1 sib n=21 gonads; org1 mut islet OE n=40 gonads. org1 sib vs org1 sib islet OE, not significant, org1 sib n=21 gonads; org1 sib islet OE n=22 gonads) Mann-Whitney Tests. Scale bars, 10 um. n≥3 trials. To further support a role for islet in niche specification downstream of org1, we asked whether we could rescue org1 mutant niche cell number with islet overexpression. We overexpressed islet with our SGP lineage-specific driver six4- gal4. In gonads with both the org1 mutation and SGP-specific restoration of islet , we saw increased numbers of niche cells compared to org1 mutants alone ( Figure 6G - 6J’ ), supporting an ability to rescue niche cell identity. While niche cell number increased with islet overexpression, this number was still not fully rescued to control levels, and islet overexpression in control genetic backgrounds does not significantly increase niche cell number ( Figure 6G ). These results suggest that while islet clearly contributes to niche identity, its expression alone is not fully sufficient to induce niche cell fate. The partial rescue also suggests that islet is not the only relevant org1 target for continued specification. Taken together, our results support a role for islet in niche cell specification and suggest that it acts downstream of org1 in this role. Discussion An impressive body of work has explored stem cell and niche functions ( Anllo & DiNardo, 2022 ; Kaestner et. al., 2019; Losick et al., 2011 ; Morrison & Spradling, 2008 ; Sato et al., 2011 ; Vida et al., 2024 ; Warder et. al., 2024; Wei et al., 2023; Yamashita et al., 2005 ), yet how niche cells acquire their identity and are positioned to properly regulate resident stem cells is largely unknown. Prior to our work, Notch signaling and a downstream transcriptional regulator, traffic jam, were identified as inputs to Drosophila testis niche identity ( Kitadate & Kobayashi, 2010 ; Okegbe & DiNardo, 2011 ; Wingert & DiNardo, 2015 ). These inputs are first induced by early contact with the developing gut ( Okegbe & DiNardo, 2011 ). Our previous work studying extrinsic signals important for niche establishment has linked niche assembly with niche function in this system ( Anllo & DiNardo, 2022 ), and prior work has linked niche cell identity and assembly ( Wingert & DiNardo, 2015 ). Here, we have identified a novel regulator that further supports a connection between the processes of niche identity and assembly, the Tbx1 ortholog org1 . We show that org1 is expressed in response to Slit and FGF signals, which we previously identified as niche assembly cues from neighboring visceral muscle ( Anllo & DiNardo, 2022 ; this work). In addition, we have defined islet as a regulator of niche identity operating downstream of org1 ( Fig 7 , model). The combination of these factors suggests that niche assembly and identity are controlled through overlapping processes. Our work addresses a knowledge gap in mechanisms establishing a functional stem cell niche. Download figure Open in new tab Figure 7. Predicted model of establishment of niche identity and assembly. Diffusible signals Slit and FGF regulate org1 . The Visceral muscle (Vm) is a neighboring tissue source of both signals. org1 functions to regulate islet for niche identity and assembly. Testis niche identity and assembly are linked Our previous work showed that the Visceral muscle signals Slit and FGF are required for niche assembly. When niche assembly was compromised, niche function was also disrupted even though these manipulations did not directly affect niche identity ( Anllo & DiNardo, 2022 ). Previous work suggested a connection between niche identity and assembly ( Wingert & DiNardo, 2015 ). In response to the niche specification signal Notch, the Maf transcription factor traffic jam ( tj ) is downregulated. traffic jam mutants specify additional niche cells that do not express all niche identity markers, and as a result do not assemble at the anterior ( Wingert & DiNardo, 2015 ). Interestingly, activating other requirements of niche identity such as Bowl places the additional tj mutant niche cells at the gonad anterior ( Wingert & DiNardo, 2015 ; DiNardo et al., 2011 ). This suggests that reduction of traffic jam alone is not sufficient for niche assembly at the anterior, supporting a role for additional regulators specifying niche cells capable of assembly. Perhaps the signals necessary for anterior assembly (Slit and FGF) are unable to be received by ectopic niche cells in tj mutants. We have shown that niche assembly signals Slit and FGF are important for regulating the novel niche identity regulator org1 . Gonads with compromised org1 show disruptions to markers of niche identity, including complete loss of Fas3, reduction of hh , loss of upd1 expression, and fewer cells accumulating N-cadherin. The few niche cells that do accumulate N-cad exhibit assembly defects in which a portion of these cells do not arrive at the anterior (this work). The loss of continued niche specification after earlier Notch signaling might account for incomplete assembly when org1 is disrupted. traffic jam mutants show additional cells expressing some niche identity markers, and a portion of these cells are also located in positions away from the gonad anterior ( Wingert & DiNardo, 2015 ). Together with work presented here, these data link proper specification of niche identity with anterior assembly. While these data support that niche identity is important for aspects of niche assembly, some niche cells still assemble anteriorly in these instances when continued specification is disrupted. Perhaps differential adhesive sorting enables partially specified pro-niche cells at the gonad anterior to assemble. Interestingly, the loss of slit and fgf regulators do not completely mimic phenotypes seen in org1 mutants. For instance, slit and fgf signaling mutants are capable of accumulating Fas3, whereas org1 mutants do not. We detect residual Org1 accumulation in niche cells from slit and fgf mutants ( Figure 1 ) suggesting that additional regulators are impacting org1 expression. We suspect that while Slit and FGF both regulate org1 , Slit and FGF signaling have a primary role in facilitating niche cell anterior placement, while org1 has a primary function in continued specification of niche cell identity. islet has dual roles in niche identity and assembly downstream of org1 To further link niche identity and assembly, we present an exciting new role for the transcription factor islet in niche specification in addition to the role we previously revealed for islet in niche assembly. Together with our previous study, this work reveals that Slit and FGF signals induce both org1 and islet expression in niche cells ( Anllo & DiNardo, 2022 ; this work). The visceral muscle (Vm) is an exciting candidate source of the Slit and FGF ligands inducing org1 , as previous work revealed Vm as an important tissue supplying these signals for niche assembly ( Anllo & DiNardo, 2022 ). In addition, we show that org1 regulates islet in the niche, as org1 mutant niches lack Islet, and niche cell numbers are increased by overexpressing islet in SGPs in an org1 mutant background. These data support the model of a transcriptional cascade regulating islet first through Slit and FGF signals inducing org1, which in turn induces islet expression ( Figure 7 ). org1 has a more prominent role in continued niche specification than islet , as org1 mutants often lack Fas3 accumulation ( Figure 2A-B ; 3E ), whereas islet mutants have fewer Fas3 positive niche cells ( Figure 6B ). This result, along with the finding that islet overexpression only partially rescues niche cell number ( Figure 6E ), suggests that other genes act downstream of org1 in addition to islet . Interesting questions that remain include identifying these additional org1 targets that contribute to continued specification, and revealing how islet influences the number of niche cells that accumulate Fas3 and N-cad. The detection of dispersed, unassembled niche cells in our six4 VP16-gal4 driven org1 RNAi was unexpected. It is reasonable to suggest earlier and stronger knockdown driving org1 RNAi with the enhanced six4 VP16-Gal4 driver compared to that observed with six4 -Gal4. Since we only observed assembly defects under the presumed stronger conditions, we infer that org1 has a primary role in niche cell identity and a secondary role in niche assembly. org1 regulates testis niche identity In alignment with Tbx1 orthologs and their conserved role in specifying mesodermal cell types, we found it fitting to test if org1 had a role in specifying the mesodermally-derived niche cells. Using the niche identity markers Fasciclin3 (Fas3), unpaired1 ( upd1), and hedgehog ( hh ), we saw that org1 mutants were unable to properly accumulate these markers ( Le Bras & Van Doren, 2006 ; Okegbe & DiNardo, 2011 ). hh and its expression have been linked to niche assembly downstream of Notch ( Wingert & DiNardo, 2015 ). Using the hh -lacZ reporter, we detected high hh expression in the niche, and low expression in SGPs outside the niche ( Figure S2 ). While we find that hh expression is not solely restricted to the niche in the embryonic gonad as it is in the adult ( Amoyel et. al., 2013 ), our ability to detect higher hh expression in niche cells enables us to use its upregulated expression as a reliable marker for niche identity. There are residual niche cells in org1 mutants, which suggests that other inputs of niche identity are partly active. It has been previously described that Notch signaling is necessary for specifying niche cells ( Kitadate & Kobayashi, 2010 ; Okegbe & DiNardo, 2011 ). The average number of niche cells in Notch mutants is far fewer than we see in org1 mutants, which suggested to us that org1 is either one of multiple effectors downstream of Notch or involved in a parallel niche specification mechanism. One of the effects of Notch signaling in specifying niche cells is to downregulate Traffic jam (Tj) accumulation in these cells. Previously, it was not known when downregulation of traffic jam began in prospective niche cells ( Wingert & DiNardo, 2015 ). We detected downregulation of tj coinciding with the beginning of org1 expression in prospective niche cells after gonad coalescence. Since org1 -expressing cells had lowered levels of Tj, we considered whether org1 might repress Tj. However, we have shown that Tj is not regulated by org1 ( Figure S2 ). Alternatively, Tj might block org1 expression. This relationship could explain the higher accumulation or Org1::GFP we detect in SGPs with reduced Tj ( Figure 1B-B’’ , yellow arrows). Future work should assess whether Tj regulates org1 downstream of Notch. Our work has established org1 as a novel regulator of identity and assembly during testis niche establishment. Multiple factors identified in the Drosophila testis stem cell niche have translated into other systems, including a role for Notch in niche specification ( Zamfirescu et al., 2022 ). Because of the numerous cell biological concepts elucidated in this niche that have translated to other systems, our work lays the foundation for future studies to investigate a conserved capacity of Tbx1 to specify gonadal mesoderm or niche cell fate. Study limitations While we reveal a role for org1 in continued niche specification during development, our genetic manipulations do not survive past embryogenesis. This precludes our ability to assess later effects of losing org1 during development. This work importantly reveals a role for org1 during initial niche formation, and future work will be required to study a role for org1 in adult niche maintenance. Finally, while assessing double mutant animals would be one way to support evidence of islet downstream of org1 , genetic odds of conducting this experiment are not feasible. Methods Embryonic gonad dissection and immunostaining Embryos were collected for 2 hours and then aged for 20-22 hours in a humidified chamber at 25 C to stage 17. Embryos were then sorted for mutant and control genotypes using fluorescently marked balancer chromosomes (FM7DfdYFP or TM6DfdGFP). Sorted embryos were dissected and immunostained as previously described (Anllo et. al. 2019). Dissected tissue was incubated at 4C overnight with primary antibodies. Secondary antibodies were used at 3.75 ug/mL (Alexa488, Cy3, or Alexa647; Molecular Probes; Jackson Immunoresearch) for 1.5-2 hours at room temperature. DNA was stained with Hoechst33342 (Sigma) at 0.2-0.3 ug/mL for 6 minutes. We used goat anti Vasa 1:800 (Santa Cruz Biotech -Discont.); rabbit anti Vasa 1:5000 (BosterBio DZ41154); mouse anti Fasciclin III 1:50 (DSHB AB_528238); rat anti N-cadherin 1:20 (DSHB AB_528121); rat anti E-cad 1:20 (DSHB AB_528120); rabbit anti Stat92E 1:200 ( Flaherty et al., 2010 ); rabbit anti RFP 1:500 (Abcam ab62341); mouse anti Islet 1:200 (DSHB AB_528313); guinea pig anti Traffic jam 1:10,000 (Gift from D. Godt); rat anti Org-1 1:200 (gift from C. Schaub); mouse anti Gamma Tubulin 1:200 (Sigma AB_477584); chicken anti Beta-gal 1:1000 (Abcam ab9361-250); mouse anti Patched 1:50 (DSHB AB_528441); and chicken anti GFP 1:1000 (Aves labs AB_2307313). Tissue was imaging using a Zeiss AxioImager.Z1 microscope equipped with and ApoTome.2, X-cite Xylis fluorescent light source, and Axiocam 705 mono camera, using 40x 1.2 N.A. plan apo lens. Org1::GFP reporter images in Stage 15 embryos were obtained with a Crest X-Light V3 spinning disk confocal on an Olympus IX83 platform with a 60x 1.3 N.A. plan apo silicone oil immersion objective. Visualization of Stage 15 embryonic gonads Embryos were collected on grape juice agar plates supplemented with a dab of fresh yeast paste. After allowing adult flies to lay fertilized eggs for 16 h, the embryos were transferred to a nylon mesh strainer by rinsing with de-ionized water and using a paintbrush. Embryos were de-chorionated with 50% bleach for no longer than 5 min. Embryos were fixed in a glass vial at 23 degrees C in a 1:1 mixture of 4% paraformadelhyde (PFA) in PBS:heptane for 18 min while on a nutator ( Mitchison and Sedat, 1983 ). The PFA was removed and replaced with an equal volume of 100% methanol. The vial was vigorously shaken for 30-60 seconds to devitellinize the embryos. Heptane was then removed, and devitellinized embryos were washed with 100% methanol three times. Embryos were washed once with 50% methanol in phosphate buffered saline (PBS), then again with 100% PBS with 0.1% Triton for gentle rehydration. Embryos were blocked for at least 1 hour in 4% Normal Donkey Serum, prior to immunostaining and imaging as described above. Stage 15 embryos selected for imaging based on Campos-Ortega and Hartenstein (1985) . Drosophila strains All Drosophila lines used are listed in the Key Resources Table. org1[OJ487] is a null allele lacking the translation initiation codon and T-box domain ( Schaub et al., 2012 ). Org1::GFP is a bacterial artificial chromosome (BAC) including the org1 coding sequence and nearby genetic regulatory elements upstream of an in-frame GFP sequence ( Kudron et al., 2018 ). We have confirmed that this BAC rescues org1 lethality (data not shown). slit[2] is a null allele with no detectable protein product ( Battye et. al., 2001 ; Nusslein-Volhard et. al., 1984 ). To remove pyr and ths FGF ligands together, we used a small chromosomal deficiency, Df(2R)BSC25, which completely deletes the genes encoding both ligands that bind the Heartless FGF receptor ( Stathopoulos et al., 2004 ). When generating org1[OJ487] , upd1 -Gal4 recombinant chromosomes, we screened for homozygous lethality and the ability to drive upd1 -Gal4, UAS-redStinger expression in larval tissues. Identification of male gonads, and the niche We identified male gonads by the presence of male specific SGPs at the posterior ( De Falco et. al., 2003 ) which were visualized with Vasa (BosterBio) or six4moeGFP (Sano et al., 2012). Additionally, male gonads can be identified by the fact that they have more proliferative germ cells and therefore have more germ cells than female gonads (Anllo et. al., 2019; Wawersik et. al., 2005). Niches in stage 17 embryos were identified via immunofluorescence by niche cell specific accumulation of Fasciclin 3 (DSHB AB_528238), or enrichment of N-cadherin (DSHB AB_528121) or E-cadherin (DSHB AB_528120). Characterization of niche phenotypes When assessing morphology and proper assembly of the niche, we characterized a niche as either “assembled” or “unassembled”. “Assembled” niches show all niche cells contacting one another at the gonad anterior and have clearly smoothened cell boundaries around the niche periphery. “Unassembled” niches have niche cells anterior and at the gonad periphery, but with irregular boundaries, and/or have dispersed niche cells that are not contacting the anterior cluster of niche cells. Rigor and Reproducibility All experiments were conducted at least in triplicate (n≥3 trials) with sufficiently large sample sizes that previously enabled detection of significant differences (significance level of less than or equal to 0.05%). Genetic controls were always used, comparing perturbed conditions to the most genetically similar control background feasible. Quantification of niche cell count When quantifying the number of cells that express N-cad, Fas3, or any other niche marker, we use the ImageJ Cell Counter Plugin to record cells that we counted manually. A niche cell will accumulate Fas3 when in contact with other niche cells and will accumulate N-cad on all interfaces. Niche cells that are counted with either Fas3 or N-cad are co-labeled with a nuclear marker such as Traffic jam or Hoechst, to enable clear identification of distinct cells. Dispersed niche cells that accumulate Fas3 do not show polarized enrichment to specific cell interfaces. Quantification of normalized niche cell expression/accumulation When quantifying accumulation of proteins and reporters of gene expression, we used ImageJ software to measure mean gray value fluorescence intensity within regions of interest (ROIs). We selected ROIs including a circular region within somatic cell boundaries, using either Fas3 or N-cad immunofluorescence to delineate niche cell boundaries and Hoechst dye to delineate nuclei. ROIs were in a single Z plane in which the relevant cell was in focus. For all experiments except upd measurements, 3 niche cell ROIs were measured from each gonad. For upd measurements, 5 niche cell ROIs were measured per gonad. To quantify background fluorescence, an ROI was selected to encompass the unlabeled region of a single germ cell within each gonad, and a separate background ROI drawn from a region where no tissue was present. Background-subtracted fluorescence values for each niche cell were normalized, dividing by the background-subtracted value from the neighboring unlabeled germ cell for the respective gonad. Relative fluorescence values for each niche cell were plotted. Mann-Whitney tests were used to evaluate comparisons. Quantification of fluorescence at cell junctions To quantify fluorescence intensity of F-actin between cell interfaces, we used ImageJ software to measure mean gray value fluorescence intensity within regions of interested (ROIs). ROIs were either niche-niche interfaces or niche-GSC interfaces. We selected 4-pixel-wide segmented line ROIs between niche cells and between niche cells and GSCs. 2-4 niche-niche and niche-GSC interfaces were measured in every gonad using N-cad to visualize each interface. We measured the mean fluorescence intensity of our Ftractin-tdTomato blindly with N-cad as a boundary. To estimate background fluorescence, a separate ROI was drawn from a region where no tissue was present. Background-subtracted fluorescence values for each niche cell were normalized, divided by the background-subtracted average value of all cell interfaces. Relative fluorescence values for each interface were plotted. Mann-Whitney tests were used to evaluate comparisons. Quantification of Stat accumulation To quantify Stat accumulation, we stained gonads with a Stat antibody (E. Bach, 1:200) and used ImageJ to measure the mean gray value fluorescence intensity within regions of interest (ROIs). During dissections and staining, we used the phosphatase inhibitor PhosStop (Sigma – Cat#4906845001). We selected ROIs including a circular region to sample germ cells, using Vasa immunofluorescence as a marker to delineate cell boundaries. Whole germ cells were measured in a single Z plane in which the relevant cell was in focus. We selected ROIs by only using the germ cell marker to mark sample regions to avoid biased observations towards brighter or dimmer fluorescence of Stat. For each gonad, we sampled all GSCs we could clearly identify and 3-7 neighboring germ cells in the second tier, further from the germline stem cells. After background subtraction, we measured the ratio of Stat accumulation within each GSC relative to the neighboring germ cell average for that gonad. Relative Stat enrichment values were plotted for each GSC. We obtained measurements on mutants for org1 and their sibling controls, and org1 RNAi knockdowns with their sibling controls. Mann-Whitney tests were used to evaluate comparisons. Quantification of STAT+ GSCs To determine whether a germ cell adherent to the niche was STAT+, we first counted the number of germline stem cells (GSCs) directly adjacent to N-cadherin labeled niche cells. We then quantified the mean fluorescence intensity from each of these adherent germ cells as described above, measuring mean STAT enrichment for each adherent germ cell in org1 RNAi knockdowns and sibling control animals. We established a threshold STAT+ fluorescent value, measured as one standard deviation below the relative mean fluorescence intensity of controls. For controls, the relative mean fluorescence intensity was 2.76, with a standard deviation of 0.97, therefore any adherent germ cells with a mean fluorescence intensity above 1.79 were counted as STAT+ in both control and org1 RNAi knockdown gonads. Data were imported into Prism v10.0 to generate scatterplots where each dot represents a gonad. We plotted the number of STAT+ GSCs per gonad, and separately the percentage of STAT+ GSCs per gonad for org1 RNAi and sibling control conditions. A Mann–Whitney test was used to evaluate the comparison. Centrosome anchoring quantification Centrosome position was visualized with immunofluorescence against Gamma tubulin to label pericentriolar material. GSCs were scored for centrosome position if they had already undergone centrosome duplication, indicating that they had advanced at least to the G2 stage of the cell cycle ( Chen et. al., 2018 ). We quantified how often one of the two centrosomes was positioned closer to the adjacent niche, marked by N-cad, than to other neighboring cells. GSCs with a centrosome located near the niche-GSC interface were scored as appropriately aligned. GSCs with both centrosomes positioned away from the niche were scored as misaligned. Significance was assessed with Fisher’s exact test. Key Resources Table View this table: View inline View popup Supplemental Figures Download figure Open in new tab Figure S1. Org1 antibody validation in org1 mutants. (A, A’) Control gonads with Org1 accumulation (red) in the niche (Fas3, white) at the anterior, along with variable punctate signal in other places in the gonad (yellow arrow). (B, B’) org1 mutant gonads with no Org1 accumulation (red) in the niche (white) at the anterior. Note that the variable punctate signal still persists (yellow arrow) in the mutant and is thus spurious background and cannot be due to Org1. Scale bars, 10 um. Download figure Open in new tab Figure S2. hh is expressed outside of the niche. Somatic cells (green), Niche (white), hh -lacZ (red), DNA (blue). (A-A”) hh- lacZ reporter can be seen at high levels in niche cells, with lower levels of expression outside of the niche (yellow arrows) in control gonads. (B-B”) org1 mutant gonads with low hh -lacZ expression in the niche (quantification in Fig 2 ), and outside of the niche (yellow arrows). Download figure Open in new tab Figure S3. org1 mutants exhibit a lower ratio of niche cells relative to number of SGPs. (A) Control and (B) org1 mutant gonads with Traffic jam accumulation. Niches are outlined with dotted lines. (C) Quantification of number of Traffic jam accumulating SGPs indicated fewer SGPs in org1 mutants versus controls (p = 0.0399, Ctrl n= 14 gonads; org1 n=16 gonads, Mann-Whitney test). (D) Quantification of SGP to Niche cell ratio showed a lower ratio of niche cells relative to SGPs in org1 mutants (p = 0.0010, Ctrl n=13 gonads; org1 n=16 gonads, Mann-Whitney test). Scale bars, 10 um. Download figure Open in new tab Figure S4. org1 RNAi with a Gal4 driver shows identity defects but not assembly defects. Germ cells (green), N-cad (white), Fas3 (red), DNA (blue). (A-A’) Control gonads with assembled niches and clear Fas3 accumulation. (B,B’) six4 gal4 > org1 RNAi gonads with fewer niche cells and reduced Fas3 accumulation. (C) Quantification of niche cell count in six4-gal4 org1 RNAi gonads (p= 0.0022, Ctrl n=13 gonads; six4 -gal4 org1 RNAi n=21 gonads, Mann-Whitney Test). (D) Quantification of assembly defects in six4-gal4 org1 RNAi gonads (p> 0.99, Ctrl n=13 gonads; six4 -gal4 org1 RNAi n=20, Mann-Whitney Test). Scale bars, 10 um. Download figure Open in new tab Figure S5. N-cad and E-cad can be found spuriously in SGPs outside of the niche. (A-B”) Control gonads immunostained to show germ cells (green) and DNA (blue). (A) N-cad (white) and (B) E-cad can be seen in an SGP (yellow arrow) outside of the niche but is not as enriched at the cell cortex as in the anterior niche. (C-D’) Germ cells (red), E-cad (white), DNA (blue). (C, C’) Control gonad with E-cad enriched in the cortex of niche cells. (D, D’) org1 mutant gonad with E-cad enriched to similar levels in a presumptive lost niche cell outside of the anterior niche (yellow arrow). Scale bars, 10 um. View this table: View inline View popup Download powerpoint Table S1. Supplementary Genotype Table. Funding Work was supported by NIH grants R15 GM154246 to L.A. and R35 GM136270 to S.D. Authors have no competing interests to declare. Author Contributions Conceptualization, P.H. and L.A.; methodology, L.A.; validation, P.H. & L.A.; formal analysis, P.H., A.H., T.G., and L.A.; investigation P.H., T.G., and L.A.; resources S.D. and L.A.; data curation P.H. and L.A.; writing – original draft P.H.; writing – review & editing P.H., A.H., S.D., and L.A.; visualization P.H., A.H., and L.A.; supervision S.D. and L.A.; project administration S.D. and L.A.; funding acquisition S.D. and L.A. Acknowledgements We acknowledge Kirklan Naumuk for technical assistance. We thank Drs. Elizabeth Ables and Beth Thompson, and the Lenhart lab for valuable feedback on the manuscript. We thank the Bloomington Drosophila Stock Center (NIH P40OD018537) for stocks, and D. Godt, E. Bach and C. Schaub for antibodies. Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , R15 GM154246 , R35 GM136270 Footnotes This version of the manuscript has been revised to update with additional functional data in Figure 4, textual changes for clarity, and a supplementary genotype table. These changes were made in response to reviewer suggestions pre-publication. We also added an author during these revisions. References 1. ↵ Amoyel , M. , Sanny , J. , Burel , M. and Bach , E. A . ( 2013 ). Hedgehog is required for CySC self-renewal but does not contribute to the GSC niche in the Drosophila testis . Development (Cambridge) 140 , 56 – 65 . OpenUrl Abstract / FREE Full Text 2. ↵ Anllo , L. and DiNardo , S . ( 2022 ). Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche . Dev Cell 57 , 1009 – 1023 .e5. OpenUrl CrossRef PubMed 3. ↵ Anllo , L. , Plasschaert , L. W. , Sui , J. and DiNardo , S . ( 2019 ). 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Magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis . Dev Biol 357 , 202 – 210 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 27, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Tbx1 ortholog org-1 is required to establish testis stem cell niche identity in Drosophila Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Tbx1 ortholog org-1 is required to establish testis stem cell niche identity in Drosophila Patrick Hofe , Ariel Harrington , Tynan Gardner , Stephen DiNardo , Lauren Anllo bioRxiv 2025.05.09.653127; doi: https://doi.org/10.1101/2025.05.09.653127 Share This Article: Copy Citation Tools Tbx1 ortholog org-1 is required to establish testis stem cell niche identity in Drosophila Patrick Hofe , Ariel Harrington , Tynan Gardner , Stephen DiNardo , Lauren Anllo bioRxiv 2025.05.09.653127; doi: https://doi.org/10.1101/2025.05.09.653127 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 (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)