Asymmetric division of cyst stem cells in Drosophila testis is ensured by anaphase spindle repositioning - PubMed (original) (raw)
Asymmetric division of cyst stem cells in Drosophila testis is ensured by anaphase spindle repositioning
Jun Cheng et al. Development. 2011 Mar.
Abstract
Many stem cells divide asymmetrically to balance self-renewal and differentiation. In Drosophila testes, two stem cell populations, germline stem cells (GSCs) and somatic cyst stem cells (CySCs), cohere and regulate one another. Here, we report that CySCs divide asymmetrically through repositioning the mitotic spindle around anaphase. CySC spindle repositioning requires functional centrosomes, Dynein and the actin-membrane linker Moesin. Anaphase spindle repositioning is required to achieve high-fidelity asymmetric divisions in CySCs, thus maintaining both GSC and CySC numbers. We propose that dynamic spindle repositioning allows CySCs to divide asymmetrically while accommodating the structure of the GSCs they encapsulate.
Figures
Fig. 1.
The CySC mitotic spindle is not oriented with respect to the hub. (A) Schematic of Drosophila spermatogenesis. A germline stem cell (GSC) is encapsulated by a pair of cyst stem cells (CySCs), both of which contact the hub. The GSC daughter, known as gonialblast (GB), begins to differentiate. A pair of CySC daughters, known as cyst cells (CCs), encapsulates a developing GB and spermatogonia. (B,C) The GSC spindle is oriented nearly perpendicular to the hub (B), while the CySC spindle is not consistently oriented and is often far away from the hub (C). Spindle orientations are indicated by double-headed arrows. Asterisks indicate the hub. (B′,C′) Diagrams of images shown in B and C, respectively, showing the hub and dividing GSC or CySC. Red, Fasciclin III [hub (*)] and γ-tubulin (centrosome); blue, Thr 3-phosphorylated histone H3 (Phospho-H3) (mitotic chromatin); green, GFP-α-tubulin. Ubi-GFP-α-tubulin flies were used. Scale bar: 10 μm. (D) CySC spindle angle (ϕ, defined in the top panel) is random with respect to the hub. This is in striking contrast to the consistently oriented GSC spindles.
Fig. 2.
The CySC spindle is associated with the hub-CySC interface during anaphase. (A,B) Two examples of CySC anaphase spindles (double-headed arrows) with one pole closely associated with the hub (arrowhead), but at a random angle (in A, the spindle is relatively perpendicularly oriented, while in B the spindle is almost parallel to the hub). (A′,B′) Diagrams of images shown in A and B, respectively, illustrating the hub and CySC spindles. Spindle orientations are indicated by double-headed arrows; arrowheads show proximal spindle poles associated with the hub (asterisk). Red, Fasciclin III [hub (*)] and γ-tubulin (centrosome); Blue, Thr 3-phosphorylated histone H3 (Phospho-H3) (mitotic chromatin); green, GFP-α-tubulin. Ubi-GFP-α-tubulin flies were used. Scale bar: 10 μm. (C) Quantification of GSC and CySC spindle association with the hub interface throughout mitosis. Pro, prophase and prometaphase; Meta, metaphase; Ana, anaphase; Telo, telophase. Error bars are s.d. (D) The CySC spindles do not have a consistent angle with respect to the hub, even during anaphase, when one spindle pole is closely associated with the hub.
Fig. 3.
Dynamic movement and repositioning of the CySC spindle revealed by live imaging. (A) Selected frames of live-imaging of CySC mitosis (see Movie 1 in the supplementary material). CySC shape is indicated with broken lines; arrowheads indicate spindle poles; hub is indicated by an asterisk. Times are in minutes. Ubi-GFP-α-tubulin flies were used. Scale bar: 10 μm. (B) The onset of anaphase (marked by a yellow line) coincides with the association of one spindle pole with the hub. The blue line shows proximal spindle pole position and the green line spindle length, representing the data in A. (C) Schematic of CySC mitosis. A CySC becomes rounder in mitosis and repositions the spindle pole (red) close to the hub during anaphase.
Fig. 4.
CySC spindle repositioning requires centrosome, Dynein, Myosin and Moesin. (A-G) Examples of defective anaphase repositioning in CySCs from _cnn_HK21/_cnn_mfs3 mutant flies (A), c587-Gal4>Gl-RNAi flies (C), _lis1_k11702/ _lis1_k13209 mutant flies (D), c587-Gal4>Moe-RNAi flies (E), c587-Gal4>Moe-TA flies (F) and c587-Gal4>Moe-TD flies (G). Example of normal anaphase repositioning in CySCs from _apc2_d40/_apc2_Δs mutant flies (B). (A′-G′) Diagrams of images shown in A-G, highlighting anaphase spindle and cell shape of CySCs. Hub is indicated by an asterisk; arrowheads indicate centrosomes; dotted lines outline CySC cell shape. Red in A-E indicates Adducin (cell membrane of cyst lineage and spectrosome of germline lineage); red in F,G indicates Fasciclin III [hub (*)] and γ-tubulin (centrosome); green in A-E indicates Thr 3-phosphorylated histone H3 (Phospho-H3) (mitotic chromatin); (F) green indicates Moe-TA-GFP; (G) green indicates Moe-TD-GFP; blue indicates Vasa (germ cells); white in F,G indicates Thr 3-phosphorylated histone H3 (Phospho-H3) (mitotic chromatin). Spindle orientations are indicated by double-headed arrows. Scale bar: 10 μm. (See also Fig. S2 in the supplementary material for defective spindle repositioning with their spindle poles marked.)
Fig. 5.
Failure in anaphase spindle repositioning leads to symmetric CySC division and increases CySC and GSC numbers. (A) Experimental scheme for detecting outcome of CySC division. Expression of GFP and Pav-GFP was activated by a heatshock (hs-FLP; Actin>FRT-stop-FRT>Gal4) to induce CySC clone. Pav-GFP, which localizes to the contractile ring/midbody, allows identification of twin daughters of a single CySC division. The division outcome was analyzed 24 hours post-heatshock (when many CySCs have divided once but not more than once). (B,C) Examples of asymmetric CySC division in control flies (B) and symmetric CySC division in flies expressing Moe-TA (C). (B′,C′) Diagrams of images shown in B and C, respectively. Arrows indicate the attachment of CySCs to the hub; arrowheads indicate the contractile ring/midbody between twin daughters, demonstrating that they are indeed derived from a single CySC division; broken lines outline the cell shapes of the twin daughters. Red indicates Fasciclin III [hub (*)] and Adducin (cell membrane of cyst lineage and spectrosome of germline lineage); blue indicates Vasa (germ cells); green indicates GFP and Pav-GFP in B, and GFP, Pav-GFP and Moe-TA-GFP in C. Flies of hs-FLP; Actin>FRT-stop-FRT>Gal4, UAS-GFP/UAS-MoeTA-GFP; UAS-Pav-GFP were used, and control flies were hs-FLP; Actin>FRT-stop-FRT>Gal4, UAS-GFP; UAS-Pav-GFP. Scale bar: 10 μm. (D) Frequency of CySC asymmetric and symmetric divisions. CySC symmetric division increases upon Moe-TA overexpression. Error bars are s.d. _P_-values are calculated using Student's _t_-test when compared with the control. (E,F) CySC number (E) and GSC number (F) increase upon expression of Moe-TA, Moe-RNAi or Gl-RNAi in CySCs using c587-Gal4. c587-Gal4 without UAS-transgenes flies were used as control. _P_-values are calculated using Student's _t_-test when compared with the corresponding control.
References
- Bienz M. (2001). Spindles cotton on to junctions, APC and EB1. Nat. Cell Biol. 3, E67-E68 - PubMed
- Decotto E., Spradling A. C. (2005). The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals. Dev. Cell 9, 501-510 - PubMed
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