Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila

Abstract

Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. Here, we show that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-βPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation.

Figure 1. Cell differentiation and proliferation defects in ddlc1^ins1 hemizygous mutant testes.

(A) Wild type (a, c, e, g, i, k) and ddlc1^ins1(b, d, f, h, j, l) hemizygous testes stained with antibodies against Vasa (a, b), α-Spectrin (c, d), Traffic Jam (i, j) and Eyes absent (k, l), or, marked by nosGal4; UAS-eGFP (e, f) and sa-CD8GFP (g, h) expression, indicate the state of germline differentiation. (a, b) Arrowheads indicate location of spermatogonia and arrow points to spermatocytes. (c, d) Arrowhead points to a spectrosome and arrows point to branched fusomes. Yellow asterisk indicates a spermatocyte fusome. (e, f) Arrows mark the early spermatogonial cells. (g, h) Arrow marks the onset of Sa-CD8GFP expression in the spermatocytes. (i–j) Arrows point to the Traffic Jam (TJ) positive nuclei of early stage cyst cells. (k, l) Arrow indicates the location of Eya-positive cyst cells. Scale bars (except for c, d): 100 μm. Scale bars (c–d): 25 μm. (B) Four-day-old wild type (a, d, g), ddlc1^ins1(b, e, h), and ddlc1^DIIA82 (c, f, i) testes, labeled with one-hour BrdU pulse, and stained with DAPI (a–c) and anti-BrdU (d–f) are shown. Arrows depict the region intensely stained with DAPI and BrdU. (g–i) Wild type and ddlc1 testes labeled with Vasa (blue), TJ (green), and phospho-Histone H3 (PH3, red) antibodies. (j) Average number of PH3 positive nuclei co-labeled with Vasa or TJ in wild type, ddlc1^ins1, and ddlc1^DIIA82 testes. Error bars indicate +/- SEM. (k–m) Two-day-old wild type (k), and two (l) and three-day old (m) ddlc1^ins1testes were stained with anti-Vasa (green) and anti-BrdU (red) antibodies after a brief (5 minutes) BrdU pulse labeling. (k, l) arrows indicate individual BrdU labeled cysts. (m) Arrows indicate the region containing large cyst-like aggregates with few BrdU labeled nuclei. Scale bars (a–f): 100 μm. Scale bars (g–i and k–m): 50 μm. ddlc1^DIIA82 is a relatively stronger hypomorph than ddlc1^ins1 mutant.

Figure 2. Tissue-specific requirement of ddlc1 during the transit amplification of spermatogonia.

(A) Testes from four-day-old ddlc1^ins1; UAS mycPIN/+ (a), ddlc1^ins1; UAS mycPIN/+; nosGal4/+ (b), ddlc1^ins1; UAS mycPIN/ptcGal4 (c) and ptcGal4/+; UAS-ddlc1^dsRNA/+ (d) adults were pulse-labeled with BrdU for one hour before anti-BrdU staining. Arrows indicate regions containing BrdU positive nuclei. (e) Histograms show average S-phase indices (average number of BrdU positive nuclei per testes) at four days post-eclosion from different genetic backgrounds (mentioned on y-axis). The number of samples used for each genotype is indicated on the bars. Data are shown as mean +/- SEM. (B) UAS-GFP expression due to the tjGal4 (a), updGal4 (b) and esgGal4 (c) variedly marked the hub (arrowhead), somatic stem cells (arrows), and the early (yellow arrow) and late cyst (yellow asterisk) cells. The GFP expression due to the daGal4 (d) labeled the somatic cyst cells until the 4-cell spermatogonia stage (arrowheads), excluded the 8–16 cell cysts (arrow), and resumed in the early spermatocytes (yellow asterisk). Testes from four-day-old tjGal4/+; UAS-ddlc1^dsRNA/+ (e), updGal4/+; UAS-ddlc1^dsRNA/+ (f), esgGal4/+; UAS-ddlc1^dsRNA/+ (g), and daGal4/UAS-ddlc1^dsRNA(h) flies were stained with anti-Vasa (red) and anti-BrdU (green) antibodies after one-hour BrdU pulse-labeling. (i) Average S-phase indices resulting from ddlc1^dsRNAknockdown in different subsets of somatic cells in the testis are plotted. The genotypes used are depicted on the left margins of the plot. The number of samples used for each genotype is indicated on the bars. Data are plotted as mean +/- SEM. Scale bars indicate 50 μm and ** indicates p < 0.001 obtained by the Mann-Whitney test.

Figure 3. Excessive germline proliferation due to cyst cell-specific knockdown of Dhc64C and didum (myosin V).

(A) Four-day old tjGal4/UAS-Dhc64C^dsRNA (a, c) and tjGal4; UAS-didum^dsRNA (b, d) testes were pulse-labeled with BrdU for one hour and stained with anti-BrdU (a, b) and anti-Vasa (c, d) antibodies. Arrows in (a, b) indicate regions containing BrdU positive nuclei, and arrowheads in (c, d) indicate small-sized spermatogonia. (B) Histograms depict average S-phase indices from various genotypes (indicated at the left margin) after a one-hour BrdU-pulse labeling. The number of samples used for each genotype is indicated on the bars. Data are plotted as mean +/- SEM. Scale bars indicate 50 μm in all Figures and ** indicates p < 0.001 obtained by the Mann-Whitney test.

Figure 4. Analysis of Bam expression in the ddlc1^ins1 testes.

(A) Testes from four-day old wild type (a) and ddlc1^ins1; UAS-mycPIN/tjGal4 (d) as well as two (b) and three days old (c) ddlc1^ins1 flies were stained with anti-Bam antibody (arrows). (B) bamP-GFP expression (green) in wild type (a) and ddlc1^ins1 (b-d) testes, aged for one day (b), three days (c), and five days (d) after eclosion. (C) BamP-Bam::GFP expression (green) in wild type (a) and ddlc1^{ins 1}(b–d) testes aged for one day (b), three days (c), and five days (d) after eclosion. (D) GFP expression (green) driven by bam promoter in testes from 4 days old tjGal4/+; bmP⁷⁰²-GFP/+ (a), tjGal4/+; UAS-ddlc1^dsRNA/bmP⁷⁰²-GFP (b), tjGal4/+; UAS-didum^dsRNA/bmP⁷⁰²-GFP (c), and tjGal4/UAS-Dhc64C^dsRNA; bmP⁷⁰²-GFP/+ (d) flies are shown. Nuclei in panels B, C, and D are stained with the Hoescht DNA labeling dye (blue). Scale bars in B, C and D represent 50 μm and scale bars in A represent 25 μm.

Figure 5. Disruption of rab11 in somatic cyst cells using tjGal4.

Germ cell status was checked in testes from 4 day old tjGal4/+; UAS-rab11^dsRNA/+ flies by anti-Vasa (b, green in c) and anti-α-Spectrin (red in c) staining. Testes were also stained with the Hoescht dye (a) and anti-BrdU (d) after one hour BrdU pulse-labeling. Testes from tjGal4/+; UAS-rab11^dsRNA/+ flies were unusually small and stained brightly with the Hoescht dye (yellow circle, a). In addition, the BrdU label was observed throughout the testis (yellow circle, d) which consisted of only small Vasa-positive cells (arrow, b) bearing spectrosomes (arrowhead in c, inset c') and dumbbell-shaped fusomes (arrow in c, inset c”). Scale bars: 50 μm.

Figure 6. Status of cell adhesions in wild type and ddlc1^ins1 testes.

Testes from 4 days old wild type (A, C, E, G, I) and ddlc1^ins1 (B, D, F, H, J) flies were stained with antibodies against Discs large (C, D), Armadillo (E, F), DE-cadherin (G, H) and Integrin-βPS (I, J). Cyst cell cytoplasm was also visualized by ptcGal4, UASeGFP expression in (A) wild type and (B) ddlc1^ins1 testes. Insets show magnified regions of the testes marked with the yellow dashed squares. Scale bars: 25 μm.