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Integrin-dependent anchoring of a stem-cell nicheGuy Tanentzapf1,2,3,4, Danelle Devenport1,3, Dorothea Godt2 and Nicholas H. Brown1

Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem-cell properties1. the adult Drosophila testis contains a morphologically discrete stem-cell niche, the ‘hub’2–4. the small cluster of non-dividing, somatic hub cells at the anterior tip of the fly testis is contacted by the germline stem cells (GsCs)5, which retain their stem-cell character through the direct association with the hub6. Here we show that integrin-mediated adhesion is important for maintaining the correct position of embryonic hub cells during gonad morphogenesis. the misplaced hub in integrin-deficient embryos directs the orientation of cell divisions in the presumptive GsCs, a hallmark of the active germline stem-cell niche. A decrease in integrin-mediated adhesion in adult testes, which resulted in a loss of the hub and the stem-cell population, revealed the importance of hub-cell anchoring. Finally, we show that an extracellular matrix (eCM) is present around the gonad during late embryogenesis and that this eCM is defective in integrin-deficient gonads. On the basis of our data, we propose that integrins are required for the attachment of the hub cells to the eCM, which is essential for maintaining the stem-cell niche.

The hub cells in adult gonads are located at the anterior tip of the testes, where they directly contact about nine GSCs5. Adult testes derive from a pair of embryonic gonads that form during the later stages of embryo-genesis (stages 12–17). After compaction (coalescence), the embryonic gonads are spherical structures comprising primordial germline cells that are intermingled with and ensheathed by mesodermally derived somatic gonadal precursor cells (SGPs)7. Recent studies revealed that hub cells arise during embryogenesis, shortly after gonad compaction, from a subset of anterior SGPs8,9. We noticed that the Drosophila filamin Cheerio (Cher) was highly expressed in a group of somatic cells located at the anterior of the gonad in half of wild-type embryos from late stage 16 onwards. We confirmed that the anti-Cher antibody recognizes embryonic hub cells by

using the colocalization of Cher and β-galactose (β-Gal) staining in hub cells in the LacZ reporter fly line 254 (ref. 10; Supplementary Information, Fig. S1), enabling us to use it to study hub development.

Morphogenesis of most tissues in metazoans requires cell-to-matrix adhesion mediated by the integrin class of adhesion receptors11,12. Integrins are heterodimeric, transmembrane receptors, comprising an α and a β subunit that mediate adhesion by simultaneously binding ECM proteins and the actin cytoskeleton13. In Drosophila, integrins contain-ing the βPS intracellular domain are essential for many morphogenetic processes12. To test the function of integrins in the gonad, we examined myospheroid mutant (mysXG43) embryos, which are hemizygous for a null mutation in the gene encoding the βPS integrin subunit and therefore lack all βPS-containing heterodimers. In mysXG43 embryos, the hub cells were mislocalized (Fig. 1). In wild-type embryos, the hub cells are at the anterior edge of the gonad, forming a cap that contacts the anterior germ-line cells (Fig. 1a, b). In mysXG43 embryos, the hub cells were embedded at the centre of the gonad surrounded by germline cells on all sides (Fig. 1c, d). Although mislocalized, the hub cells formed a tight cluster, suggesting that integrin is not required for adhesion between the hub cells.

In addition to the hub cells, embryonic gonads contain SGPs that are interspersed with the germline cells7. To test whether loss of βPS affects the distribution of all somatic cells within the gonad, embryonic gonads were labelled with an antibody against talin, a protein that displays ele-vated expression in SGPs14. Talin-expressing SGPs were interspersed with germline cells throughout the gonad in both wild-type (Fig. 1bʹ) and mysXG43 (Fig. 1dʹ) embryos. To test whether integrins were required during earlier stages of gonad development, gonads in mysXG43 embryos were labelled with Vasa, to mark germline cells, and the SGP mark-ers Traffic Jam15, Eya and ZFH1 (ref. 7) (Supplementary Information, Fig. S2; data not shown). No differences were detected between wild-type and mysXG43 mutant gonads until late embryonic stages (stage 17), when the hub cells were observed as a cluster of cells in the middle of mysXG43 gonads (Supplementary Information, Fig. S2). Thus, integrins seemed to be specifically required for hub cells to localize at the anterior pole of male embryonic gonads.

1The Gurdon Institute and Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 1QN, UK. 2The Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5. 3Present addresses: Department of Cellular and Physiological Sciences, Life Science Center, University of British Columbia, 2350 Health Science Mall, Vancouver, BC, Canada V6T 1Z3 (G.T.); Laboratory of Mammalian Genetics and Development, Rockefeller University, Box 300, 1240 York Avenue, New York, NY 10021, USA (D.D.).4Correspondence should be addressed to G.T. ([email protected])

Received 20 March 2007; accepted 27 September 2007; published online 4 November 2007; DOI: 10.1038/ncb1660

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To understand the function of integrins in hub development we inves-tigated which integrins were expressed in the gonad and how they were distributed. Of the five αPS integrin subunits encoded by the Drosophila genome12, antibodies exist for αPS1, αPS2 and αPS3. The αPS3 subu-nit was expressed in all SGPs (Fig. 2e), whereas neither αPS1 nor αPS2 was detected in embryonic gonads (Fig. 2f; data not shown). Because mutations in the gene encoding αPS3 did not affect hub development

(data not shown) it is likely that αPS4 and/or αPS5 are expressed in the gonad, and act redundantly there with αPS3. There are two β integrin subunits in Drosophila: βPS integrin is expressed in all SGPs (Fig. 2d), whereas the second β subunit, βν, is not detected in SGPs

16. Moreover, the hub cells form normally in embryos lacking βν, and the mutant hub phenotype is not enhanced in embryos lacking both βν and βPS (Fig. 1i, j). It is therefore likely that βPS integrin is the only β integrin subunit that functions in the gonad.

Talin is an integrin-binding cytoskeletal linker that is essential for integrin-mediated adhesion in Drosophila14. In embryos, talin was highly expressed in the gonadal mesoderm and was concentrated at the plasma membrane of all SGPs, including in hub cells (Fig. 2c). Embryos lacking talin had a mislocalization of the hub identical to that seen in embryos

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Figure 1 The hub cells are mispositioned in integrin-mutant embryos. (a, b) In a wild-type (WT) stage 17 embryo, the hub cells (labelled with Cher, red) are positioned at the anterior edge of the male gonad (germline cells labelled with Vasa (green); talin (blue) labels SGPs). (c, d) In a mysXG43 embryo, which lacks the βPS integrin subunit, the hub cells are partitioned to the middle of the testis, nestled between the germline cells. (e–g) Confocal Z-series of 2-µm intervals (labelled with Vasa (green) and Cher (red)) confirmed that, in contrast with the wild type (e), in which the hub cells are at the anterior pole of the gonad, the hub cells are located in the centre of the gonad in embryos lacking integrins (f, mysXG43) or the integrin-associated protein talin (g, rhea79A). (h, hʹ) Normal positioning of the hub cells in embryonic gonads lacking integrin-linked kinase (ilk1) or (i) the second integrin β subunit βν. (j) A gonad lacking both integrin subunits (mysXG43;βν

− double mutant) with an identical phenotype to that seen in a mysXG43 gonad. Scale bars, 20 µm.

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Figure 2 Integrin and talin expression in somatic cells in the gonad. (a–c) Talin protein (aʹ–cʹ; shown in red in a–c, with germline cells (Vasa) in green), encoded by the gene rhea, is expressed in all the SGPs starting at stage 14 (a) and persisting through stage 15 (b) to the end of embryogenesis (c, stage 17). (d, e) Expression of integrins in the gonad: the βPS subunit (d) and the αPS3 subunit (e) are expressed in all SGPs. (f) The αPS2 subunit (fʹ; shown in red in f, with germline cells (Vasa) in green) is expressed in the muscle attachment sites (f, arrows) but is not detected in the gonad. Scale bars, 20 µm.

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lacking integrin (Fig. 1g). By contrast, mutations that eliminate the integrin-associated proteins ILK and PINCH17,18 did not perturb hub-cell positioning (Fig. 1h; data not shown). Analysis of the temporal and spatial aspects of βPS integrin and talin expression showed that they were both expressed in gonads starting at stage 14 and remained uniformly expressed in the SGPs throughout embryonic development (Fig. 2a–c and data not shown).

In wild-type Drosophila embryos, ECM markers were detected around the outer perimeter of the gonad at stage 16. LamininA (LanA) and Nidogen (Ndg), a laminin-associated protein, were abundantly expressed in the gonadal ECM at this stage (Fig. 3a, c). The distribution of these proteins suggests that the ECM is assembled at a time when integrin-mediated adhesion is important for gonad morphogenesis. The ECM is in direct contact with the outermost SGPs, and the hub cells are positioned such that they contact both the ECM and germline cells (Figs 1a, b and 3aʹ, cʹ).

To explore the molecular basis for the hub-cell mislocalization in integrin mutants, we examined the organization of ECM components in mysXG43 gonads. The amount of LanA and Ndg around the gonad was greatly reduced in mysXG43 mutants (Fig. 3b, d). Integrins therefore have an active role in ECM accumulation around the gonad. This find-ing raises the possibility that the main role of integrins is to assemble an ECM around the gonad, and a different ECM receptor anchors the hub cells to the ECM. It is difficult to rule this out completely, but we did not detect dystroglycan, the other known ECM receptor present in Drosophila19 in embryonic hub cells (data not shown).

Cells with different adhesive properties can undergo cell sorting, in which cells that adhere most strongly to one another become separated from — and enveloped by — less adhesive cells20. Two of our observations suggested that differential adhesion-mediated cell sorting takes place dur-ing hub development. First, the hub cells form a tight cluster, separate from other SGPs. Second, in integrin mutants the hub becomes envel-oped by other SGPs and germline cells. Cell sorting can be achieved by modulating cadherin-mediated adhesion21,22. Both DE-cadherin and DN-cadherin are expressed at higher levels in the hub than in other gonadal cells (Supplementary Information, Fig. S3; ref. 8). To test whether hub cells segregated from other SGPs and the germline cells because of differ-ential cadherin expression, we modulated DE-cadherin or DN-cadherin

levels in integrin-mutant embryos. We found that reducing or increas-ing cadherin levels (Supplementary Information, Figs S3 and S4) did not affect the position or segregation of the hub cells. These results suggest that the molecule that mediates adhesion between hub cells and their mispositioning in integrin mutants is not a classical cadherin, but do not exclude the possibility of a contribution by these cadherins. Moreover, the homophilic cell-adhesion molecule Fas3 is expressed on the surface of embryonic hub cells8, which is consistent with the idea that other adhe-sion molecules contribute to adhesion between hub cells.

By morphological and molecular criteria the hub cells in embryonic gonads seem similar to adult hub cells (Supplementary Information, Fig. S1; ref. 8). In adult testes, cell divisions of GSCs are oriented perpen-dicular to the hub, and this helps to regulate the balance between GSC self-renewal and differentiation6. GSCs in early interphase have a single centrosome that is always positioned near the cell cortex adjacent to the site of contact with the hub. After centrosome duplication, one daugh-ter centrosome migrates to the opposite side of the nucleus so that the spindle is oriented perpendicular to the hub6. To test whether germline cell divisions in the embryonic gonad are also oriented towards the hub, centrosomes were revealed with a centrosomin (CNN)-specific antibody, and dividing cells were labelled with phospho-histone H3. Serial confo-cal sections were used to reconstruct the orientation of dividing germline cells. In wild-type gonads, in every germline cell that directly contacted the hub, one centrosome was positioned on the side that contacted the hub (n > 100) (Fig. 4a). During mitosis, the centrosomes were oriented perpendicular to the hub (n = 23), resulting in a regular pattern of cell divisions along the anterior–posterior axis (Fig. 4a). These results sug-gest that hub cells already control the spindle orientation of dividing GSCs in the embryo.

We examined whether the misplaced hub in integrin mutants could orient the mitotic spindle during GSC division. In interphase germline cells that contacted the hub, the centrosome was always positioned towards the hub, even though the hub was in the centre of the gonad (n > 100). In addition, mitotic germline cells adjacent to the hub cells divided perpendicular to the hub in all cases (n = 21). However, because the hub was in the centre of the gonad, the regular anterior–posterior orientation of dividing GSCs was not maintained (Fig. 4b), resulting in an abnormal cell division pattern compared with that in wild-type gonads.

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Figure 3 Integrins assemble an ECM around the embryonic gonad. (a, c) ECM proteins Ndg (a, aʹ, red) and LanA (c, cʹ, red) are localized around the embryonic gonad of a wild-type (WT) embryo. The hub cells (labelled

with Cher, green in aʹ and cʹ) are in contact with the ECM. In integrin-mutant (mysXG43) embryos, staining of Ndg (b, bʹ, red) and LanA (d, dʹ, red) around the gonad is substantially reduced. Scale bars, 20 µm.



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These results indicate that hub cells in embryonic testes direct spindle orientation even when mislocalized, implying that integrin-dependent hub positioning is critical for the normal anterior–posterior orientation of germline cell divisions.

Next we examined the importance of integrin-dependent anchoring of the hub for spermatogenesis. In adult testes, hub cells occupy the distal tip of the gonad and contact somatic and GSCs on one side and ECM on the other5. The ECM overlaying the adult hub is thicker than around the rest of the testis and has a convoluted appearance, sug-gesting that it makes extensive contacts with the hub cells5. It has been proposed that hub-cell–ECM contacts in the adult maintain the hub at the anterior tip of the gonad5, which is consistent with the function of integrins in embryonic testes. We could not study integrin func-tion in adults by using integrin-null mutants because these die during embryogenesis. Moreover, hub cells in adult testes do not divide5,23, which prevents the generation of integrin-deficient hub-cell clones. We therefore used double-stranded RNA interference (RNAi) to disrupt integrin-mediated adhesion.

talin dsRNA was expressed in somatic cells of the testes under the control of a tj-GAL4 driver (P{GawB}NP1624-5-1; ref. 24) to disrupt integrin-mediated adhesion. The activity pattern of the tj-GAL4 driver reflects that of tj (see Supplementary Information, Fig. S5; ref. 15). In tj-GAL4/+;UAS-talin RNAi/+ (talin RNAi) embryos we observed no decrease in talin, and the hub cells localized normally (Fig. 5a). However, when talin RNAi males were allowed to develop to adult-hood they developed progressive defects in spermatogenesis. In adult control flies (tj-GAL4/tj-GAL4), aged 1, 3 and 14 days, the testes con-tained a hub, GSCs, and differentiating germline cells at all stages of spermatogenesis (Fig. 5b, bʹ, dʹʹ, f). In contrast with control testes, in which talin staining was detected in all somatic cells including the hub (Fig. 5dʹ), talin staining was not detected in somatic cells in talin RNAi testes (Fig. 5eʹ). Most one-day-old talin RNAi adult testes had normal spermatogenesis but a small number (9%; n = 100) seemed to lack a

hub. In three-day-old talin RNAi flies, the number of testes that did not have a hub had substantially increased (33%; n = 100). In testes lacking a hub, no GSCs or germline cells at early stages of differentiation were observed; instead, the distal region contained germline cells at mid-to-late stages of spermatogenesis (Fig. 5g). Moreover, even when the hub was present it was sometimes mislocalized (Fig. 5h). After two weeks, more than half the testes from talin RNAi adults (56%; n = 100) lacked a hub, GSCs and differentiating germline cells and instead contained only differentiated sperm, which filled the entire gonad (Fig. 5e). Inspection of the distal region showed that expressing talin RNAi caused a com-plete loss of GSCs (compare Fig. 5e” with Fig. 5d”). Although tj-GAL4 is expressed in both cyst cells and hub cells, our data suggested that cyst cells are not affected by talin RNAi expression because the germline cells continue to differentiate into sperm. It is unclear why the hub disappearance is gradual, but because hub cells are non-dividing it may take a long time to obtain a significant decrease in talin protein level if its turnover is low. Alternatively, there may be another mechanism to retain the hub in place that is only partly effective and fails to retain the hub over time. Thus, on the basis of our data we propose that the deple-tion of talin leads to a progressive loss of the hub in adult testes, which results in the gradual differentiation of all GSCs into sperm.

In summary, our results show that integrins have an essential function in maintaining the position of the germline stem-cell niche within fly testes. Multiple lines of evidence presented here suggest that the germ-line stem-cell niche in Drosophila testes is anchored to the anterior of the gonad by adhering to the ECM. First, an ECM assembles around the gonad late in embryonic development at a time when the hub cells become morphologically distinct. Second, the hub cells line up along the anterior periphery of the gonad and contact this ECM directly. Third, integrins and their associated proteins, such as talin, are expressed in SGPs, including hub cells. Fourth, integrins are necessary for the accu-mulation of ECM components around the gonad. Fifth, integrin and talin are required for maintaining the hub cells in an anterior position. Last, loss of integrin-mediated adhesion by depleting talin in adult testes results in loss of the hub.

As a model, we propose that the hub cells, derived from cells that form at the anterior of the embryonic gonad8 and are restricted to it9, sort out from other somatic and germline cells, form a cohesive unit, and attach to the ECM adjacent to the anterior of the gonad. The func-tion of the ECM attachment is to anchor the hub cells and prevent their sorting to the middle of the gonad. The mispositioning of the hub in integrin-mutant embryos and the loss of the hub and the GSCs in talin-RNAi-treated adult testes illustrate the severe consequences of a failure to anchor the stem cell niche to the ECM.

MetHOdsFly stocks and genetics. The following mutants and transgenic lines were used: rhea79a (ref. 14), mysXG43, cadN-cadN2∆14 (this deficiency removes both cadN genes25), shg317, twist-GAL4, nanos-GAL4-VP16, UAS-DE-cadherin (a gift from Ulrich Tepass (CSB Department, University of Toronto, Canada), UAS-DN-cad-herin (a gift from Tadashi Uemura (Kyoto University, Japan) and 254-LacZ10. UAS-talin RNAi lines were obtained from the Vienna Drosophila RNAi Center26 (Transformant IDs 40399 and 40400, Construct ID 12050). tj-GAL4 (P{GawB} NP1624-5-1) was obtained from the Drosophila Genomic Resource Centre, Kyoto Institute of Technology, Japan (ref 24).

Female germline clones lacking talin were generated with the FRT/FLP OvoD method27. Female flies of the genotype hs-FLP/+;FRT2A OvoD/FRT2A rhea79a were heat shocked and their female progeny crossed to rhea79a/TM3, GFP males. To

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Figure 4 Spindle orientation in wild-type and integrin-mutant embryonic gonads. (a) In mitotic GSCs of a wild-type (WT) gonad (labelled with phospho-histone H3 (blue)) the centrosomes (labelled with CNN (green)) are oriented so that the cell division is perpendicular to the anterior hub (labelled with Cher (red)) and are aligned along the anterior–posterior axis of the embryo. (b) In an integrin-mutant (mysXG43) gonad, the centrosomes align perpendicular to the mispositioned hub. However, because the hub is in the centre of the gonad the divisions are no longer strictly oriented along the anterior–posterior axis. Scale bars, 10 µm.



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drive expression in the gonadal mesoderm, twist-GAL4 (on the second chromo-some) was used and nanos-GAL4-VP16 (on the third chromosome) was used for germ-cell overexpression. talin RNAi testes were obtained by crossing tj-GAL4 (P{GawB}NP1624-5-1 DGRC; ref. 24) to UAS-talin RNAi lines. Adult testes were dissected 1, 3, 5 and 14 days after emergence of the adult fly.

Immunohistochemistry and microscopy. The following antibodies were used: anti-βPS (CF6G11 (ref. 28), mouse monoclonal antibody (mAb); 1:10 dilution), anti-αPS2 (7A10 (ref. 29), rat mAb; 1:10 dilution), anti-αPS3 (gift from R. Davis, Baylor College of Medicine, Houston, TX), rabbit polyclonal antibody (pAb); 1:300 dilution), anti-talin (E16B14, mouse mAb; 1:10 dilution), anti-Ndg (gift from S. Baumgartner, Lund University, Sweden, rabbit pAB; 1:200 dilution), anti-LanA (gift from S. Baumgartner, rabbit pAB; 1:500 dilution), anti-Cher (gift from Lynn Cooley (Yale University, New Haven, CT), rat pAB N-fil and C-fil; 1:1000 dilution), anti-DN-cadherin (Developmental Studies Hybridoma Bank, University of Iowa, IA), DN-ex#8, rat mAb; 1:50 dilution), anti-DE-cadherin (Developmental Studies Hybridoma Bank, DCAD2, rat mAb; 1:50 dilution), anti-Vasa (gift from R. Lehmann, Skirball Institute, New York City, NY, rab-bit pAb; 1:5000 dilution), anti-β-Gal (Cappel, Westchester, PA, mouse mAb; 1:1000 dilution), anti-CNN (gift from Jordan Raff and Renata Basto, Gurdon Institute, Cambridge, UK, rabbit mAb; 1:4000 dilution), anti-phospho-histone H3 (Upstate Biotechnology, Lake Placid, NY, mouse mAb; 1:2000 dilution) and anti-Tj (G5 (ref. 15), guinea-pig pAb; 1:2500 dilution). Fluorescently conjugated Alexa 488 fluorophore, and Cy3 and Cy5 secondary antibodies were used at 1:400 dilution (Molecular Probes, Eugene, OR).

Antibody staining of adult testes and embryos was performed in accord-ance with standard procedures. For stage 17 embryos, at which point the cuticle interferes with standard fixation, we employed a heat-fixation protocol. In brief, embryos were dechorionated in 50% bleach for 1 min, rinsed in water, immersed in boiling 1 × E-wash buffer (100 mM NaCl, 0.1% Tween 20) for a few seconds, then immediately cooled by adding 3 volumes of ice-cold E-wash and placed on ice. The vitelline membrane was removed in methanol/heptane. For anti-βPS staining, embryos were fixed in standard 4% formaldehyde/PBS solution and dechorionated in ethanol. For DE-cadherin staining, phosphate buffer was used instead of PBS. For every gonad imaged, a confocal Z-series extending through the entire gonad was collected. Confocal images were collected with a LSM510 (Carl Zeiss MicroImaging, Bernreid, Germany) confocal microscope. The lenses used were Neofluar 16×/0.5 numerical aperture (NA), 40×/1.3 NA and Apochromat 63×/1.4 NA. Images were processed with Adobe Photoshop 8.0.

Note: Supplementary Information is available on the Nature Cell Biology website.

AcknowledGemenTsWe thank S. Baumgartner, L. Cooley, R. Lehmann, J. Raff, R. Basto and the Developmental Studies Hybridoma Bank (University of Iowa, IA) for antibodies; B. Dickson and F. Schnorrer for RNAi fly stocks, the Drosophila Genomics Resource Center (Kyoto Institute of Technology) for the tj-GAL4 line; T. Clandinin (Stanford University, Palo Alto, CA) and U. Tepass for cadherin fly stocks; and S. Choksi, L. Jones (UCSD, San Diego, CA) and U. Tepass for critical reading of the manuscript. This work was supported by a Natural Sciences and Engineering Research Council of Canada grant to D.G., Wellcome Trust grants to N.H.B. (69943) and D.D. (72817), a Human Frontiers Science Program Long Term Fellowship and a Development Travelling Fellowship to G.T.

compeTinG finAnciAl inTeresTsThe authors declare no competing financial interests.

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Figure 5 Phenotypes of talin RNAi in adult testes. UAS-talin RNAi was expressed in embryonic and adult testes by using tj-GAL4. (a) In a tj-GAL4/+;UAS-talin RNAi/+ (talin RNAi) embryo the hub cells are at the anterior of the gonad (Vasa, green; Cher, red). (b) In a two-week-old adult tj-GAL4/tj-GAL4 testis, the distal region of the testis holds the hub (bʹ; Cher, red), the GSCs, dividing gonial cells and differentiating spermatocytes (bʹ; Vasa, green), and the rest of the testis is filled with differentiated sperm bundles (the arrow shows the distal tip of the testis). (c) A two-week-old adult talin RNAi testis contains only differentiated sperm; there is no hub at the distal testis tip (arrow) (cʹ; Vasa, green; Cher, red). (d, e) A close-up view of the distal tip region of two-week-old testes. (d) In a tj-GAL4/tj-GAL4 testis, Vasa (green) stains the GSCs, gonial cells and primary spermatocytes; no differentiated sperm bundles are observed. (e) In a talin RNAi testis the distal region contains only differentiated sperm bundles (arrow in e) that extend to the tip of the testis (tip marked with an asterisk in d and e). (dʹ, eʹ) Somatic cells express talin in a tj-GAL4 testis (dʹ) but talin staining is absent in a talin RNAi testis (eʹ), although the gonadal sheath is stained. (dʹʹ, eʹʹ) In a tj-GAL4/tj-GAL4 testis (dʹʹ), the hub is located at the tip but in a talin RNAi testis (eʹʹ) the hub is missing (Vasa, green; Cher, red; talin, blue). (f, g) In a three-day-old tj-GAL4;tj-GAL4 testis (f), the tip of the testis contains GSCs and germline cells at early stages of differentiation (arrow) (g) but these are absent in a three-day-old talin RNAi testis. (h) A three-day-old talin RNAi testis, in which the hub is mispositioned (asterisk). In f–h, Vasa is in green and Cher is in red. Scale bars, 100 µm (b, c), 20 µm (a, d–h).


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13. Hynes, R. O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).

14. Brown, N. H. et al. Talin is essential for integrin function in Drosophila. Dev. Cell 3, 569–579 (2002).

15. Li, M. A., Alls, J. D., Avancini, R. M., Koo, K. & Godt, D. The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila. Nature Cell Biol. 5, 994–1000 (2003).

16. Devenport, D. & Brown, N. H. Morphogenesis in the absence of integrins: mutation of both Drosophila beta subunits prevents midgut migration. Development 131, 5405–5415 (2004).

17. Clark, K. A., McGrail, M. & Beckerle, M. C. Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes. Development 130, 2611–2621 (2003).

18. Zervas, C. G., Gregory, S. L. & Brown, N. H. Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane. J. Cell Biol. 152, 1007–1018 (2001).

19. Deng, W. M. et al. Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila. Development 130, 173–184 (2003).

20. Steinberg, M. S. Reconstruction of tissues by dissociated cells. Some morphogenetic tissue movements and the sorting out of embryonic cells may have a common explana-tion. Science 141, 401–408 (1963).

21. Gumbiner, B. M. Regulation of cadherin-mediated adhesion in morphogenesis. Nature Rev. Mol. Cell Biol. 6, 622–634 (2005).

22. Godt, D. & Tepass, U. Drosophila oocyte localization is mediated by differential cad-herin-based adhesion. Nature 395, 387–391 (1998).

23. Gonczy, P. & DiNardo, S. The germ line regulates somatic cyst cell proliferation and fate during Drosophila spermatogenesis. Development 122, 2437–2447 (1996).

24. Hayashi, S. et al. GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4 enhancer traps. Genesis 34, 58–61 (2002).

25. Prakash, S., Caldwell, J. C., Eberl, D. F. & Clandinin, T. R. Drosophila N-cadherin medi-ates an attractive interaction between photoreceptor axons and their targets. Nature Neurosci. 8, 443–450 (2005).

26. Dietzl, G. et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156 (2007).

27. Chou, T. B. & Perrimon, N. The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144, 1673–1679 (1996).

28. Brower, D. L., Wilcox, M., Piovant, M., Smith, R. J. & Reger, L. A. Related cell-surface antigens expressed with positional specificity in Drosophila imaginal discs. Proc. Natl Acad. Sci. USA 81, 7485–7489 (1984).

29. Bogaert, T., Brown, N. & Wilcox, M. The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell 51, 929–940 (1987).


S U P P L E M E N TA RY I N F O R M AT I O N

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Figure S1. Cheerio highlights hub cells throughout development. (a) The Drosophila filamin Cheerio (Cher: a’, green in a’’) is highly expressed in stage 17 embryos in cells at the anterior tip of male gonads (germline cells stained with Vasa in a, red in a’’). (b-d) The enhancer trap line 254-LacZ contains an insertion in the gene esg and marks hub cells in embryonic, larval, and adult gonads1. Testis from embryo (b), larva (c), and adult (d) of the 254-LacZ line were co-stained with β-gal (b-d, red in b’’-d’’) and Cher (b’-d’, green in b’’-d’’) antibodies. (b) In embryos strong Cher and 254-lacZ expression overlap, marking the same

anterior cells, but the distribution of Cher was more restricted, labelling only the anterior most cells that were β-Gal positive. In 3rd instar larva (c) and adult testis (d) hub cells identified by 254-LacZ show strong Cher staining though Cher was also weakly expressed in the cyst cells. (e, f) Schematic of the embryonic and adult gonads. In both embryonic and adult testis the hub cells are restricted to the anterior tip where they contact the GSCs. (Legend: SGP- somatic gonadal precursor, PGC- primordial germline cell, GSC- germline stem cell, GB – gonialblast, cyst cells in blue). Scale bars are 20 μm.



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Figure S2. Gonad development in wildtype and integrin mutants. Somatic gonadal precursors cells (SGPs) and Germline Stem Cells (GSCs) were marked in Wildtype (a-c) and mysXG43 (d-f) embryos during stages 14 (a, d), 15 (b, e), and 17 (c, f). In both wildtype and mysXG43 gonads the germline cells (marked using Vasa, red) are surrounded by SGPs (marked using the

nuclear SGP marker Traffic Jam in green) though occasionally single SGPs intermingle with the germline cells. However, in mysXG43 gonads during stage 17 the hub cells can be observed as a cluster of cells in the middle of the gonads (arrow). Images shown are individual sections from the middle of the gonad. Scale bars are 20 μm.




Figure S3. DN and DE cadherin expression in hub cells in wildtype and integrin mutants. (a) DN Cadherin (red, a’) is strongly expressed in cells at the anterior of the gonad (mesoderm highlighted by talin, green). (a’’) DN-Cadherin staining overlaps with β-gal expressing hub cells in 254-LacZ embryos. (b) DE-Cadherin (b’, red in b and b’’) is strongly expressed in the hub cells and is also expressed in the germline cells (b’’, labelled with Vasa, blue). (c) In mys mutants the mispositioned hub cells express normal levels of DN-Cadherin (c’, red in c). Double mutants for mys and cadN

(d) or mys and shg (which encodes DE-Cadherin (e) display the same hub cell defect observed in single mys mutants (d, e, Vasa in blue, and her in green; d’, e’ Cher in white; d’’, e’’ Vasa in white). However, it is not possible to completely remove maternally contributed DE-cadherin2, and enough DE-cadherin is made to potentially support hub cells sorting in integrin mutants. The compaction defects in the mys, shg double mutants in (e) are consistent with the previously reported defects of shg mutant gonads3. Scale bars are 20 μm.



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Figure S4. Hub cell morphogenesis is not affected by changes in the relative levels of Cadherin. (a, b) Hub cell anchoring is not disrupted by reducing cadherin function. (a) Hub cells are located at the anterior gonad in DE-cadherin mutant embryos (shg317), despite their defects in gonad compaction. (b) Hub cell positioning is normal in DN-cadherin mutants (cadN-cadN2Δ14). (c-f) Hub cell anchoring is not disrupted by ectopic cadherin overexpression. (c) Hub cells remain anchored to the anterior gonad when DE-cadherin is ectopically expressed in the mesoderm using the twist-GAL44 driver. (a, b, c, Cher labels the hub cells; a’, b’, c’ Vasa labels the germline cells; talin is in green an cher is in red in a’’, b’’; talin is in red and

cher is in green in c’’). (d) Hub cell position is normal when DN-cadherin is ectopically expressed either in the mesoderm with twist-GAL4 or (e) in the germline cells with nanos-GAL43. Overexpressed DN-cadherin concentrates at the hub cell-germline cell interface (d, e, DN-cadherin; d’, e’, talin; d’’, e’’, talin is in green, DN-cadherin is in red). (f) The anterior position of the hub cells is not affected when DE-cadherin is ectopically expressed in the germline cells with nanos-GAL4. However, the shape of the gonad is disrupted due to extensive cell-cell contacts between hub cells and germline cells (f, DE-cadherin; f’, talin; f’’, DN-cadherin is in red, talin is in green). Scale bars are 20 μm.




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References

1. Gonczy, P., Viswanathan, S. & DiNardo, S. Probing spermatogenesis in Drosophila with P-element enhancer detectors. Development 114, 89-98 (1992).

2. Godt, D. & Tepass, U. Drosophila oocyte localization is mediated by differential cadherin-based adhesion. Nature 395, 387-91 (1998).3. Jenkins, A. B., McCaffery, J. M. & Van Doren, M. Drosophila E-cadherin is essential for proper germ cell-soma interaction during gonad

morphogenesis. Development 130, 4417-26 (2003).4. Baylies, M. K. & Bate, M. twist: a myogenic switch in Drosophila. Science 272, 1481-4 (1996).

Figure S5. The activity pattern of the tj-Gal4 driver reflects that of tj. tj-GAL4 is expressed in somatic gonadal cells that contact the germline cells, including the hub. (a-c) Adult testes from tj-GAL4/+; UAS-GFP/+ GFP flies:

Cher staining (red in a, white in b) marks the hub, GFP staining (green in a, white in C) reveals tj-GAL4 activity. Scale bars are 20 μm.


Integrin-dependent anchoring of a stem-cell nicheFigure 1 The hub cells are mispositioned in integrin-mutant embryos. (a, b) In a wild-type (WT) stage 17 embryo, the hub cells (labelled with Cher, red) are positioned at the anterior edge of the male gonad (germline cells labelled with Vasa (green); talin (blue) labels SGPs). (c, d) In a mysXG43 embryo, which lacks the βPS(j) A gonad lacking both integrin subunits (mysXG43;βν− double mutant) with an identical phenotype to that seen in a mysXG43 gonad. Scale bars, 20 µm.Figure 2 Integrin and talin expression in somatic cells in the gonad. (a–c) Talin protein (aʹ–cʹ; shown in red in a–c, with germline cells (Vasa) in green), encoded by the gene rhea, is expressed in all the SGPs starting at stage 14 (a) and persisting throFigure 3 Integrins assemble an ECM around the embryonic gonad. (a, c) ECM proteins Ndg (a, aʹ, red) and LanA (c, cʹ, red) are localized around the embryonic gonad of a wild-type (WT) embryo. The hub cells (labelled with Cher, green in aʹ and cʹ) are in contact with the ECM. In integrin-mutant (mysXG43) embryos, stainFigure 4 Spindle orientation in wild-type and integrin-mutant embryonic gonads. (a) In mitotic GSCs of a wild-type (WT) gonad (labelled with phospho-histone H3 (blue)) the centrosomes (labelled with CNN (green)) are oriented so that the cell division is peMethodsAcknowledgementsCompeting financial interestsReferences

integrin-dependent anchoring of a stem-cell nichelabs.bio.unc.edu/peifer/pdfs bio 625 08/tanentzapf...

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