A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage

Abstract

In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. Here we show that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism.

Figure 1. mei-P26 regulates TA cell proliferation in male germ cells

A) Diagram of Drosophila spermatogenesis. Bam protein (blue), somatic cyst cells (red), cyst stem cells (CySC), germline stem cell (GSC), gonialblast (GB), hub (H). B, C) Phase contrast images of apical tips of squashed B) wild type (arrows = spermatocytes) and C) mei-P26^mfs1 mutant testes (arrowheads = refractile dying cysts). D–I) Immunofluorescence images of apical tips of testes. D) Wild type and E) mei-P26^mfs1 mutant testis stained with DAPI (red) and α-Vasa (green). F) Wild type (arrow = 8-cell cyst) and G) mei-P26 ^mfs1 mutant testis (arrow = >16-cell cyst) stained with DAPI (red), and EdU (green) to mark cysts in S-phase. H) Wild type and I) mei-P26^mfs1 mutant testes stained with α-Vasa (red) and α-Fibrillarin (green). J) Quantification of nucleolar size marked by Fibrillarin in wild type (black line) and mei-P26^mfs1 mutant (dotted line) cysts at indicated stages. Error bars = SEM. Scale bars = 50µm. See also Figure S1.

Figure 2. mei-P26 promotes spermatocyte differentiation in the testis

A) Wild type and B) mei-P26^mfs1 mutant testes stained with α-Sa (red above, white below) and α-Fibrillarin (green), (arrow = Sa positive cyst). C) Within the spermatocyte region, percent of cysts that fall into the indicated categories in wild type (black), and mei-P26^mfs1 mutant (gray) testes (dif=differentiation). D–F) Phase contrast images of D) wild type spermatocyte cyst with 16 spermatocytes, E) mei-P26^mfs1 mutant overproliferating undifferentiated cyst, and F) mei-P26^mfs1 mutant spermatid cyst with elongating tails (arrow) and 16 large nuclei indicating failure of meiosis before terminal differentiation (arrowhead). Scale bars = 50µm.

Figure 3. mei-P26 facilitates timely accumulation of Bam protein

A) Wild type and B) mei-P26^mfs1 mutant testes stained for EdU (green) after a short pulse to label cells in S-phase and Bam (red above, white below) (arrow = overproliferating cyst with low levels of Bam expression). C) Quantification of average α-Bam fluorescence per unit of cytoplasm from the indicated cyst stages in wild type (dotted line) and mei-P26^mfs1 mutant (solid line) testes (bf = before, af = after, S = S-phase EdU positive). Error bars = SEM. D-F) Phase contrast images of apical tips of squashed testes. D) mei-P26^mfs1 mutant (white bracket = region with overproliferating cysts of small cells, arrowhead = differentiated spermatocytes). E) mei-P26^mfs1; alpha-Bam/+ (white bracket = spermatocyte cysts, indicating rescue of the mei-P26^mfs1 overproliferation phenotype). F) mei-P26^mfs1; BamΔPEST/+ (white bracket = spermatocyte cysts, arrowheads with “8” = 8-cell spermatocyte cysts). G) Percentage of cysts from the normal spermatocyte region that fall into the indicated stages and phenotypic classes: mei-P26^mfs1 (light gray), mei-P26^mfs1; alpha-Bam (dark gray), mei-P26^mfs1; BamΔPEST (black) (dif = differentiation). H, I) Elongating spermatid cysts from H) wild type (arrowhead = red dash under small nucleus), and I) mei-P26^mfs1 mutant (arrowhead = red dash under large nucleus). Scale bars = 50µm. See also Figure S2.

Figure 4. Reciprocal expression of Mei-P26 and Bam in TA cells

A-A″) Apical tip of wild type whole mount testis and B-B″) wild type flattened testis stained for Mei-P26 (red), Bam (green in the cytoplasm), and the S-phase marker EdU (green in the nucleus in B″) (white arrow = 8-cell cyst positive for Mei-P26, red arrow = 8-cell cyst with lower level of Mei-P26, and dotted outline = 16-cell cyst in S-phase positive for EdU and negative for Mei-P26). C) Quantification of average α-Mei-P26 (red) and α-Bam (green) fluorescence per unit cytoplasm at the indicated cyst stages in wild type testes (16e = early 16-cell cyst and 16l = late 16-cell cyst). Error bars = SEM. D) High magnification view of A′ showing punctate distribution of Mei-P26 protein in early TA cells (arrows). E–G) High magnification of a testis apical tip showing 1 and 2-cell cysts stained for E) GW182 and F) Mei-P26. G) Merge. H–J) Apical tips of testes stained for Mei-P26 (red above, white below) and the S-phase marker EdU (green). H) Wild type showing gap in Mei-P26 staining (arrow) (dotted outlines = two 8-cell S-phase cysts). I) bam^Δ86/bam1 mutant (dotted outlines = 8-cell (upper) and 16-cell (lower) S-phase cysts). J) bgcn^z2-0702/bgcn¹ mutant (dotted outlines = 8-cell (left) and 16-cell (right) S-phase cysts). Scale bars A, B = 50µm, D = 25µm E–G = 10µm and H–J = 50µm. See also Figure S3 and S4.

Figure 5. Mei-P26 is under translational control via its 3′UTR in TA cells

Immunoflorescence images of apical tips of whole mount wild type testes expressing A-A″) control eYFP reporter or B- B″) reporter with the mei-P26 3′UTR, stained for A, B) Bam and A′, B′) GFP. A″, B″) Merge (dotted outline = region of Bam expression). C) 3′UTR reporters showing repression (red) or not showing repression (white) (asterisks = mutations). D) Percent of Bam positive cysts with eYFP expression (numbers in parentheses = total number of Bam positive cysts scored per construct, n = number of insertion lines scored per construct). E) RNAhybrid binding prediction for let-7 miRNA (green) and mei-P26 3′UTR (red) site 2 (gray box = nucleotide residues mutated within the mei-P26 3′UTR in reporter constructs). F–G′) Immunofluorescence images of apical tips of squashed testes stained on the same slide for Bam and Mei-P26 in F, F′) wild type and G, G′) let-7-C^GK1/let-7C^K01; let-7-C^Δlet-7-C mutant testes. H) Quantification of Mei-P26 protein levels from the indicated cyst stages in wild type (boxes) and let-7-C^GK1/let-7C^K01; let-7-C^Δlet-7-C mutant (asterisks) testes. Scale bars= 50µm. Error bars= SEM. See also Figure S5 and S6.

Figure 6. Bam protein specifically binds the mei-P26 3′UTR

A-A″) Immunofluoresence images of whole mount testes expressing Bam-HA (red) and Bgcn-GFP (green). A) α-HA, A′) α-GFP, A″) merge. Scale bar = 50um. B) Immunoprecipitation with α-GFP from extract of Bam-HA; Bgcn-GFP testes. Western blot probed with α-HA and α-GFP (arrow = Bam-HA co-immunoprecipitated with Bgcn-GFP). C) Diagram of RNA pull-down assay with biotinylated mei-P26 3′UTR probe. D) RNA pull-down competition experiment. Lysates from Drosophila S2 cells transfected with both Bam-HA and Bgcn-Myc were incubated with: no probe (lane 2), biotinylated mei-P26 3′UTR with no competitor (lane 3), or biotinylated mei-P26 3′UTR plus varying ratios of unlabeled mei-P26 3′UTR (1-, 10-, 90-, 100- fold excess) to unlabeled don juan 3′UTR (unrelated competitor) (lanes 4–7). The total amount of competitor RNA (400pmols) remained constant. Blots probed with α-HA (top) or α-Myc (bottom). E, F) Quantification of bands for E) Bam-HA and F) Bgcn-Myc blots. G) RNA pull-down competition experiment. Lysates from Drosophila S2 cells transfected with Bam-HA alone were incubated with: no probe (lane 2), biotinylated mei-P26 3′UTR with no competitor (lane 3), or biotinylated mei-P26 3′UTR plus varying ratios of unlabeled mei-P26 3′UTR (0-, 1-, 10-, 100- fold excess) to unlabeled don juan 3′UTR (lanes 4–7). Blot probed with α-HA. H) Quantification of bands normalized to unlabeled don juan 3′UTR competitor alone (DJ only, lane 4). I) RNA pull-down competition experiment. Lysates from Drosophila S2 cells transfected with Bam-HA alone were incubated with: no probe (lane 2), biotinylated mei-P26 3′UTR with no competitor (lane 3), or biotinylated mei-P26 3′UTR plus varying ratios of unlabeled mei-P26 3′UTR (0-, 0.5-, 5-, 50-, 100- fold excess) to unlabeled mutated mei-P26 3′UTR (lanes 4–8). Blot probed with α-HA. J) Quantification of bands, for either 50× total competitor (solid line) or 100× total competitor (dotted line), normalized to no competitor (lane 3) (brackets = statistically analyzed points). n= 3 independent experiments for all pull-down assays. Error bars= SEM. See also Figure S7.

Figure 7. Model

In early TA cells Mei-P26 facilitates accumulation of Bam protein. As Bam protein accumulates, it acts with Bgcn to repress translation of Mei-P26 so that Mei-P26 levels fall to below the level of detection by immunofluorescence in late 8-cell/ early 16-cell cysts. Mei-P26 (red) and Bam (green) protein levels.