Co-expressed Cyclin D variants cooperate to regulate proliferation of germline nuclei in a syncytium

Abstract

The role of the G1-phase Cyclin D-CDK 4/6 regulatory module in linking germline stem cell (GSC) proliferation to nutrition is evolutionarily variable. In invertebrate Drosophila and C. elegans GSC models, G1 is nearly absent and Cyclin E is expressed throughout the cell cycle, whereas vertebrate spermatogonial stem cells have a distinct G1 and Cyclin D1 plays an important role in GSC renewal. In the invertebrate, chordate, Oikopleura, where germline nuclei proliferate asynchronously in a syncytium, we show a distinct G1-phase in which 2 Cyclin D variants are co-expressed. Cyclin Dd, present in both somatic endocycling cells and the germline, localized to germline nuclei during G1 before declining at G1/S. Cyclin Db, restricted to the germline, remained cytoplasmic, co-localizing in foci with the Cyclin-dependent Kinase Inhibitor, CKIa. These foci showed a preferential spatial distribution adjacent to syncytial germline nuclei at G1/S. During nutrient-restricted growth arrest, upregulated CKIa accumulated in arrested somatic endoreduplicative nuclei but did not do so in germline nuclei. In the latter context, Cyclin Dd levels gradually decreased. In contrast, the Cyclin Dbβ splice variant, lacking the Rb-interaction domain and phosphodegron, was specifically upregulated and the number of cytoplasmic foci containing this variant increased. This upregulation was dependent on stress response MAPK p38 signaling. We conclude that under favorable conditions, Cyclin Dbβ-CDK6 sequesters CKIa in the cytoplasm to cooperate with Cyclin Dd-CDK6 in promoting germline nuclear proliferation. Under nutrient-restriction, this sequestration function is enhanced to permit continued, though reduced, cycling of the germline during somatic growth arrest.

Keywords: CAK, CDK Activating Kinase; CDK, Cyclin-Dependent Kinase; CKI, CDK inhibitor; CREB, CRE Binding protein; CRM, Chromosome Region Maintenance; ERK, Extracellular signal-regulated kinases; G-phase, Gap phase; GA, Growth Arrest; GFP, Green Fluorescent Protein; GSC, Germline Stem Cell; IdU, 5-Iodo-2′-deoxyuridine.; M-phase, Mitotic phase; MAPK p38; MAPK, Mitogen Activated Protein Kinase; MSK, Mitogen and Stress activating Kinase; NLS, Nuclear Localization Sequence; PCNA, Proliferating cell nuclear antigen; Rb, Retinoblastoma protein; S-phase, DNA Synthesis phase; SCF complex, Skp, Cullin, F-box containing complex; TOR signaling; TOR:Target Of Rapamycin; cyclin D splice variants; cyclin-dependent kinase inhibitor; cytoplasmic sequestration; growth arrest; niche; stem cell; syncytium; urochordate.

Figure 1.

Functional domains in human and Oikopleura dioica Cyclin D splice variants. (A) Human Cyclin D1 and mouse Cyclin D2 and their splice variants, D1b and D2SV, respectively. (B) O. dioica Cyclin Db splice variants cover all presence/absence combinations (+/+, −/+, +/−, −/−) of the Retinoblastoma(Rb)-binding and phosphodegron domains, respectively. (C) O. dioica Cyclin Dd. NLS, Nuclear Localization Sequence; CRM, (Chromosomal maintenance 1 or Exportin 1) interaction motif; LxCxE Rb-binding motif. All motifs were predicted by SMART and ELM. Cyclin-dependent Kinase 6 (CDK6) and CDK inhibitor (CKI) binding regions on O. dioica Cyclin D splice variants were based on Zwicker et al. Unique C-terminal sequences arising in splice variants are indicated by different stippling patterns. BLAST and secondary structure assessments (PsiPred) of the C-termini of O. dioica Cyclin D variants show residual signatures of a second Cyclin box with low confidence scores. Hs, Human; Mm, Mouse; Od, O. dioica.

Figure 2.

Asynchronous proliferation of germ nuclei with a distinct G1-phase. (A) Immunostaining of asynchronous mitotic germ nuclei show different levels of Cyclin Dd expression (n = 10 animals). Actin network within the single-cell coenocyst is stained in green. Cyclin Dd expression peaks in G1 (arrows), persisted at reduced levels in early S (arrowheads; Campsteijn et al.²⁴) and was absent thereafter. Cyclin Dd expression was not seen in nuclei expressing the mitotic marker H3pS28. (B) Larger overview of germline nuclei indicating proportions in G- (no staining), S- (IdU incorporation) and M-phase (H3pS28 staining) prior to meiotic entry. (C) Proportion of germ nuclei in each of the proliferative cell cycle phases was assessed (n = 10 animals) by immunostaining in combination for markers: Cyclin Dd (G1), short IdU pulse (S), and H3pS28 (M). Nuclei that were negative for all of these markers were in G2. Error bar indicates standard error. Scale bars = 10 µm.

Figure 3.

Cyclin Dbβ/δ cytoplasmic foci and the G1/S transition in germline nuclei. (A) Immunostaining of mitotic germline nuclei with antibodies specific to C-terminal regions of Cyclin Db splice variants β/δ. Cyclin Dbβ/δ were present as foci in the syncytial germline cytoplasm adjacent to nuclei. These foci were not observed adjacent to nuclei in M-phase (H3pS28 staining) and were also not preferentially associated with nuclei staining most intensely for Cyclin Dd (n = 10 animals). Inset depicts magnification of a cytoplasmic Cyclin Dbβ/δ focus adjacent to a nucleus. (B) Cytoplasmic foci of Cyclin Dbβ/δ were adjacent to nuclei with low amounts of IdU incorporation (arrows) but were not adjacent to nuclei with no IdU incorporation (G-phase; empty arrowheads) or nuclei with large amounts of IdU incorporation (filled arrowheads). (C) Cyclin Dbβ/δ foci were selected (n = 10 animals; 1000 foci) and the cell cycle phase of the closest nucleus was assessed based on extent of IdU incorporation and H3pS28 staining. (C') Reciprocally, nuclei in G1 (no incorporation of IdU, Cyclin Dd staining), G1/S (weak incorporation of IdU, weak Cyclin Dd staining) S or G2 (increased IdU staining, no Cyclin Dd staining) or M (H3pS28 staining) were selected (n = 10 animals; 900 nuclei) and assessed for the presence or absence of adjacent Cyclin Dbβ/δ foci. Error bars indicate standard errors. Scale bars = 10 µm.

Figure 4.

Cyclin Db splice variants interact with CDK6 and CKIa. Anti-CDK6 antibody co-immunoprecipitated Cyclin Dd, Cyclin Db splice variants and CKIa from whole cell lysates of Oikopleura dioica cultured at standard densities.

Figure 5.

Cyclin Dd levels in the germline are gradually reduced when growth arrest occurs in response to nutrient limitation. Oikopleura dioica were pulsed with the S-phase marker IdU for 1 h at day 3 when cultured under standard conditions or for 1 h at days 3, 6 and 12 when cultured under dense conditions. IdU incorporation and Cyclin Dd immunostaining were then assessed. Levels of Cyclin Dd during the early period of growth arrest remained similar to that observed under standard conditions but then gradually decreased as growth arrest persisted (n = 8 animals). Scale bars = 5 μm.

Figure 6

(See previous page). Increased cyclin Dbβ levels during TOR inhibition require MAPK-p38 signaling. (A) Cyclin Db expression increased (*P < 0.05) and persisted at higher levels in O. dioica cultured under dense conditions where growth-arrest occurs. Under these same conditions, no significant change in expression of cyclin Da, cyclin Dc, cyclin Dd was observed. (B) The observed increase in cyclin Db expression during growth arrest was restricted to the β splice variant, as levels of the α, γ and δ splice variants were unaffected. (C) When animals were cultured under standard conditions but in the presence of the TOR inhibitor CCI-779 for 24 h, cyclin Db transcripts were upregulated (*P < 0.05) whereas cyclin Da, Dc, and Dd transcripts were not. (D) Similar to the results in (B) under growth arrest, at dense culture conditions, inhibition of TOR signaling under standard culture conditions resulted in upregulation of the Cyclin Dbβ splice variant (*P < 0.05), whereas levels of the α, γ and δ splice variants were unaffected. (E) When animals were cultured under standard conditions, but in the presence of the MAPK p38 inhibitor, SB203580, for 24 h at day 3, cyclin Db transcript levels were reduced (*P < 0.05), whereas cyclin Da, cyclin Dc, cyclin Dd transcript levels were not. Expression levels are relative to those observed in day 3 animals cultured under standard conditions (with addition of DMSO in controls when chemical inhibitors were used). Error bars indicate standard errors. (F) Western blots showed that Cyclin Dbα/γ levels were similar under standard culture or growth arrested (GA) conditions, whereas Cyclin Dbβ/δ levels increased under the latter conditions. Levels of all Cyclin Db splice variants decreased in the presence of MAPK p38 inhibitor SB203580.

Figure 7.

Cyclin Dbβ/δ foci colocalize with CKIa in the syncytial germline cytoplasm and the number of these foci increase during growth arrest. (A) Antibody staining revealed that Cyclin Dbβ/δ foci co-localized with CKIa (n = 8 animals). Upper panels show an overview of colocalisation in the gonad; lower panels show colocalization at higher magnification. (B) Cytoplasmic foci were also observed when capped mRNAs generated for GFP-fused Cyclin Dbβ or CKIa constructs were microinjected into the gonads of day 3 animals (n = 8 animals). (C) GFP-fused CKIa colocalized with endogenous Cyclin Dbβ foci in germline cytoplasm (n = 8 animals). (D) The number of colocalized Cyclin Db-CKIa foci relative to the number of nuclei increased (*P < 0.05) in the cytoplasm of mitotic germ line during growth arrest (GA). Error bars indicate standard errors. Scale bars = 10 µm.