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First published online 11 January 2006
doi: 10.1242/dev.02232

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Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling

Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montréal, Québec H3A 1B1, Canada.

View larger version (67K): [in a new window]   Fig. 1. aak-2 larvae are defective in establishing germline quiescence under compromised insulin-like signalling. (A) Open arrowheads delineate the distal extremities of the gonad of daf-2(e1370) dauer larvae. (B) An integrated lag-2p::GFP transgene (qIs56) is strongly expressed in the DTCs throughout the dauer stage. A lag-2p::CFP transgene (arEx645) containing the full 6.2 kb promoter (Chen and Greenwald, 2004) was also strongly expressed in the DTCs of dauer larvae (data not shown). We similarly found strong GFP expression in the DTCs of qEx308 dauer larvae (data not shown), which contain a functional lag-2p::LAG-2::GFP lag-2(q411) rescuing construct (S. Crittenden and J. Kimble, personal communication). (C) Extruded daf-2(e1370) dauer gonad stained with DAPI (blue) and anti-GLP-1 antibodies (red). (D) Enlarged gonad of daf-2(e1370); aak-2(rr48) dauer larvae. (E) aak-2 is required to progressively suppress germline proliferation during L2d/dauer formation. Grey zone indicates the period during which >90% of the larvae undergo the dauer moult. The dauer moult was delayed by 1 and 3 hours in aak-2(ok524) and aak-2(rr48) mutants, respectively. Error bars represent standard deviation, and the sample size was 25 for each point. (F) In both daf-2(e1370) and daf-7(e1372) larvae, germ cells arrest in mitotic interphase during dauer (arrowhead), as shown in a DAPI-stained, extruded daf-7(e1372) dauer gonad. (G) In daf-2(e1370) glp-1(e2141) dauer larvae, germ cell nuclei arrest in late stage meiotic prophase I, the nuclear size and chromosome morphology being consistent with the pachytene stage (inset). (H) Germ cells often (26/30 animals) progress further through meiosis in daf-2(e1370) glp-1(e2141); aak-2(rr48) dauer larvae, and their chromosomal morphology closely resembles that of spermatids (arrow). Similar defects were observed in daf-2(e1370) glp-1(e2141); aak-2(ok524) (19/40 animals) and in daf-2(e1370) glp-1(e2141); par-4(it57) (25/30 animals) dauer larvae. (I-L) Extruded daf-2(e1370) glp-1(e2141); aak-2(rr48) dauer gonad. Asterisks indicate germ cells undergoing spermatogenesis. Cells were stained with (I) DAPI (blue); (J) anti-P-granule (green), a germ cell marker that is lost during spermatogenesis (Gruidl et al., 1996); (K) anti-membranous organelle (1CB4; red), a sperm marker (Arduengo et al., 1998). A merged image is shown in L. Images in B,C,F-H are single focal planes; condensed z-stacks are shown in I-L. Scale bars: 50 µm in A,B,D; 10 µm in C,F-H,I-L.

View larger version (25K): [in a new window]   Fig. 2. aak-2 positively regulates lifespan and dauer maintenance. (A) daf-2(e1370) (n=100), daf-2(e1370); aak-2(rr48) (n=98) and daf-2(e1370); aak-2(ok524) (n=60) individual larvae were assayed for dauer lifespan (days). (B) Whereas most daf-7(e1372) (n=500) dauer larvae remain at this stage at 25°C, the majority of daf-7(e1372); aak-2(rr48) (n=500), or daf-7(e1372); aak-2(ok524) (n=400) dauer larvae recover prematurely within 5 days. (C) aak-2 is required for the full adult lifespan extension induced by compromised insulin-like signalling. The graph marks the time (day) of the temperature up-shift (corresponding to the L4 larval stage). Each line represents the average of three independent replicate experiments (each with n=50), not performed in parallel, except for daf-2(e1370); aak-2(ok524), for which we did one single experiment (n=50). Error bars represent standard deviation between cohorts of (A) 10 or (B) 100 individuals.

View larger version (23K): [in a new window]   Fig. 3. The dauer lethality in aak-2(rr48) mutants is not due to a germ line defect. Precursors of the germ line (Z2, Z3), and/or of the somatic gonad (Z1, Z4), were ablated using laser microsurgery. Larvae were subsequently plated at 25°C and assayed for dauer lifespan. The success of the ablation was monitored 24 hours after surgery, by examining lag-2::GFP expression.

View larger version (18K): [in a new window]   Fig. 4. The inhibition of germline proliferation in daf-2 mutants largely requires daf-16 activity. L1 animals were grown at 25°C for the specified times, and the total number of germ cells was determined as described in the Materials and methods. Germline proliferation was also examined in daf-16(mgDf50); daf-2(e1370) animals (data not shown), and the results obtained were similar to daf-16(mgDf47); daf-2(e1370). Error bars indicate s.d. Sample size was >10 for each point.

View larger version (8K): [in a new window]   Fig. 5. Regulation of germ cell cycle during dauer development. Under reduced daf-2/insulin-like receptor and/or daf-7/TGF-ß activity, daf-18/PTEN, par-4/LKB1 and aak-1,aak-2/AMPK mediate the downregulation of germline proliferation during dauer development. Although the role of par-4 in aak-1 activity is obvious, the relationship between daf-18, par-4 and aak-2 remains unclear. Arrows and bars represent positive and negative genetic interactions, respectively.

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