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Figures
- Fig. 1.
Major components of SJs co-localize during early and late stages of spermatogenesis. (A) Apical tips of testes showing co-localization of the SJ proteins – Lac-GFP (b), ATPα-GFP (c), NrxIV-GFP (d) and Nrg-GFP (e) – with anti-Dlg1 (red) immunostaining at the interface of the germline and somatic cells. Dlg1-GFP testes (red) were immunostained with anti-Cora (green; a). All specimens were stained with Hoechst dye (blue) marking the nuclei. Scale bar: 50 µm. (B) The SJ proteins also localize caudal to the compact nuclei bundle (NB) of the mature spermatids during the late stages. Hoechst staining, marking all nuclei, is in blue. (g) The X-Z digital section through the top (t) and bottom (b) parts of the specimen shown in panel f. It indicates that the SJ proteins localize all around the NB. Scale bar: 10 µm. (C) Schematic describes the position of the junction between the head and tail cyst cells.
- Fig. 2.
Morphogenesis of the SJ protein Nrg during spermatogenesis. (A–C) Nrg-GFP (green) expressing testes stained with the anti-Vasa antibody (magenta). (A) Low magnification image of Nrg-GFP testis shows the presence of Nrg at different stages. (B) Apical end of the testis shows Nrg-GFP localization around individual spermatogonia (arrowheads) at the initial stage. It is then restricted to the cyst perimeter (arrows) of the primary spermatocyte stages. (C) A post-meiotic cyst shows the presence of Nrg-GFP along the cyst perimeter. Nrg-GFP is excluded from the germ cell perimeter inside the cyst enclosure from the spermatocyte stage onwards. (D–F) Squash preparation: Nrg-GFP (green) testes immunostained with the anti-Spectrin antibody (red, D), Hoechst dye (blue) and Phalloidin (red, F). (D) An early elongating cyst (outlined by white boundary) shows polarization of the spermatid nuclei (blue) and tails (red), and localization of Nrg-GFP (arrows) at the HCC–TCC interface. (E–F) Elongated spermatid cysts from Nrg-GFP testes were isolated and stained for the IC (red) and nucleus (blue), present at the rostral ends of the cyst (arrowheads). The HCC and TCC are highly extended at this stage, and a condensed form of Nrg-GFP (arrows) between these two cells was seen in the middle region. (G) Coiled stage spermatids from Nrg-GFP testes, stained for F-actin (marking the actin cap, red) and nucleus (blue). The arrows indicate localization of Nrg caudal to the NB (arrowheads, blue). Note that the position of the Nrg-GFP has changed post-individualization. (H) Time-lapse images of Nrg-GFP testis show the movement of the Nrg-GFP structure (yellow and white arrowheads) towards the basal end of the testis. The red arrowhead indicates the direction of the SV. Scale bars: 50 µm. (I) Schematic illustrates the morphogenesis of domains marked by SJ proteins in adult testis. The SJ protein is marked in green, spermatid tail in grey, spermatid nuclei in maroon and IC in blue. For simplicity, only one spermatid is shown within a cyst enclosure. The arrows indicate the position of SJs proteins during the spermatogonial stage (1), elongated stage (2) and coiled stage (3). Schematic is not to scale.
- Fig. 3.
Ultrastructural analysis of the germ–soma and soma–soma interfaces in adult testis. (A) Electron micrograph of the spermatogonial stages shows a somatic cyst cell (SCC) and germ cells (GCs). The junction between the SCC and GC is seen (arrowhead, A′). (B) Section through an elongated cyst, as can be identified by the spermatid tails with major and minor mitochondria, along with the associated SCC. Note that the junction between the SCCs does not resemble an SJ (arrowhead, B′). (C) Electron micrograph through the tails of a more mature, pre-individualized cyst shows the presence of a septa-like pattern between the two surrounding cyst cells. C′ shows the magnified image of the boxed region in C. Arrows indicate a ladder-like SJ between the plasma membranes of the two SCCs. (D) Section through the spermatid heads at the coiled stages, with surrounding HCC and TCC. D′ shows a magnified image of the boxed region in D. Ladder-like arrangement of SJs between the two cyst cells can be seen around the sperm head. Note that similar to the results obtained by confocal microscopy, the junction was relocated just caudal to the sperm heads. Scale bars: 1 µM.
- Fig. 4.
Knockdown of Dlg1 and NrxIV during spermatogonial stages leads to a defect in proliferation and differentiation. (A–B′) The control eya>dsGFP (eya-Gal4/+; UAS-dsGFP/+) testis stained with the Hoechst dye (white), anti-Dlg1 (green) and anti-Vasa (red) antibodies. (A) Hoechst staining shows tightly packed, condensed nuclei at the apical tip (arrow). (A′) Vasa pattern in control testis. (B) High magnification image of the apical tip shown in A. Arrow marks the condensed nuclear staining of mitotically active cells, while arrowhead marks the transition to meiotic stages, as indicated by comparatively less intense nuclear staining. (B′) Dlg 1 (green) localizes around the germ cells (arrowhead) as indicated by Vasa (red) initially, and then on membranes of the SSCs (arrows). (n=13). (C) DIC image indicates the presence of elongated/individualized cysts (arrow), as well as coiled cysts (arrowhead). (C′) Control testis shows the distribution of Eya (magenta) positive somatic cyst cells. (D–E′) eya>dsDlg1 (eya-Gal4/UAS-dsDlg1) testis stained with the Hoechst dye (white), anti-Dlg1 (green) and anti-Vasa antibodies (red). (D) Brightly stained spermatogonial nuclei are extended to the middle region of the testis. (D′) Pockets of Vasa staining, usually restricted more apically, extend until the middle region of the testes (arrow). (E) High magnification image of the apical tip of the testis shown in D. Note that the apical tip is shrunken as compared to control testes in B. (E′) Arrowheads indicate anti-Dlg1 (green) immunostaining around the germ cells. There is no somatic Dlg1 immunostaining. (n=13). (F) DIC image indicates a lack of elongated/individualized cysts and a decrease in the density of coiled cysts (arrowhead). (F′) Distribution of Eya (magenta) positive somatic cyst cells in eya>dsDlg1 testis. Distribution is similar to control in C′. (G–H) Control (CantonS) testis stained with anti-Vasa (red) and anti-Tj (green) antibodies. Note that the Tj-expressing somatic cells are restricted near the apical tip of the testis (H). (n=10). (I–J) eya>dsNrxIV (eya-Gal4/UAS-dsNrxIV) testis stained with anti-Vasa (red) and anti-Tj (green) antibodies. Patchy expression of Vasa indicates defects in germline differentiation. Tj expression is expanded. Also note that the testis appeared shrunken, similar to what was seen upon the knockdown of Dlg1. (n=10). Scale bars: 50 µm, unless specified otherwise on the image.
- Fig. 5.
Knockdown of SJ components in the cyst cells during late stages affects NB integrity and spermatid coiling. (A) Control PpY>dsGFP (PpY-Gal4/UAS-dsGFP) testes stained with Hoechst (white) to mark the nuclei. Arrows mark the NBs in the TE region (red dashed outline). The inset shows a high magnification image of the testis base. (B,C) PpY>dsDlg1 (UAS-dsDlg1/+; PpY-Gal4/+) and PpY>dsNrxIV (UAS-dsNrxIV/+; PpY-Gal4/+) testes stained with the Hoechst dye. Comparatively fewer intact NBs (arrows) can be seen in the TE zone (red dashed outline), whereas an unusually large number of individual spermatid heads were found inside these testes (insets). (D–F) Bright-field images show the basal end of control (D), PpY>dsDlg1 (E) and PpY>dsNrxIV (F) testes. The arrows indicate intact coiled tail bundles in D, while arrowheads point towards the disrupted tail bundles in E and F. (G–I) Histograms indicate the average intact NBs outside TE (G), the number of early ICs (H) and the number of NBs inside the TE (I) in control (n=23), PpY>dsDlg1 (n=15) and PpY>dsNrxIV (n=27) testes. P-value (**<0.01) was calculated using the Mann–Whitney U-test. ns, not significant; scale bars: 50 µm.
- Fig. 6.
Dlg1 loss from the HCC–TCC interface at the coiling stage causes premature sperm release inside the testis. (A,B) NB orientations in the control PpY>dsGFP (n=13) (A) and the PpY>dsDlg1 (n=18) (B) testes. Testes were stained with the Hoechst dye (red) and Phalloidin (green), and the position of the actin cap was used as an indicator of whether the NBs were facing towards (white arrowhead) or away (asterisk) from the SV (direction of SV marked by blue arrowhead). Note the decrease in bundles facing away from SV in 100–200 µm region in B. (C) Zone-wise distribution and the orientation of NBs in the TE region. Distances from the SV were measured from the proximal end of the testicular duct. ‘A’ denotes the NB orientation away from SV, and ‘T’ denotes orientations towards the SV. P-value (**<0.01) was calculated using the Mann–Whitney U-test. Apart from these two classes, a fraction of NBs were found with intermediate orientations that are not represented on the graph. Spermatids exit testis in the ‘A’ orientation, as shown by Dubey et al. (2016). (D,E) Time series from live imaging of the testes. Protamine A (green) marks spermatid head, while mCD8-RFP (red) marks cyst cell (dashed outline). The white arrowhead in each panel indicates the direction of SV and the curved arrow marks the NB retraction trajectory. In control (ProtA-GFP/UAS-Dicer; PpY-Gal4>UAS-mCD8-RFP/+) testis (D), the spermatid heads retracted (white arrow at time 0′) in the direction of the SV. In the dlg1 RNAi background (ProtA-GFP/UAS-dsDlg1; PpY-Gal4>UAS-mCD8-RFP;+) (E), spermatid heads retracted away from the SV (white arrow at time 0′), even though the cyst has not turned to face away from the SV. (F) Box plots depict the number of pupae produced by individual PpY>dsGFP (control) and PpY>dsDlg1 males in 24 h. P-value (**<0.01) was calculated using the Mann–Whitney U-test.
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