MSP and GLP-1/Notch signaling coordinately regulate actomyosin-dependent cytoplasmic streaming and oocyte growth in C. elegans

Abstract

Fertility depends on germline stem cell proliferation, meiosis and gametogenesis, yet how these key transitions are coordinated is unclear. In C. elegans, we show that GLP-1/Notch signaling functions in the germline to modulate oocyte growth when sperm are available for fertilization and the major sperm protein (MSP) hormone is present. Reduction-of-function mutations in glp-1 cause oocytes to grow abnormally large when MSP is present and Galpha(s)-adenylate cyclase signaling in the gonadal sheath cells is active. By contrast, gain-of-function glp-1 mutations lead to the production of small oocytes. Surprisingly, proper oocyte growth depends on distal tip cell signaling involving the redundant function of GLP-1 ligands LAG-2 and APX-1. GLP-1 signaling also affects two cellular oocyte growth processes, actomyosin-dependent cytoplasmic streaming and oocyte cellularization. glp-1 reduction-of-function mutants exhibit elevated rates of cytoplasmic streaming and delayed cellularization. GLP-1 signaling in oocyte growth depends in part on the downstream function of the FBF-1/2 PUF RNA-binding proteins. Furthermore, abnormal oocyte growth in glp-1 mutants, but not the inappropriate differentiation of germline stem cells, requires the function of the cell death pathway. The data support a model in which GLP-1 function in MSP-dependent oocyte growth is separable from its role in the proliferation versus meiotic entry decision. Thus, two major germline signaling centers, distal GLP-1 activation and proximal MSP signaling, coordinate several spatially and temporally distinct processes by which germline stem cells differentiate into functional oocytes.

Fig. 1.

GLP-1 regulates MSP-dependent oocyte growth. (A) Penetrance of the Looc phenotype in glp-1, fbf and lag-1 mutants, measured 16 hours post-L4 at 25°C in hermaphrodite or female backgrounds. In this and subsequent figures, gonad arms were scored as having a Looc phenotype if oocyte volumes were at least 20% larger than that of the -1 oocyte in the wild type. (B) DIC images showing oocyte size phenotypes in glp-1 mutants, and after DTC ablations or distal oil injection (see Table 1). Inset shows the distal region in which oil was injected. Scale bar: 10 μm.

Fig. 2.

Time-course analysis of sperm-dependence and glp-1 contributions to oocyte growth. To examine the sperm response, glp-1(tn777ts); fog-2(oz40) L4-stage larvae were transferred to 25°C for 24 hours and then mated with CB4855 wild-type males. The Looc penetrance increases rapidly after mating (blue line). To examine the glp-1 requirement, glp-1(tn777ts); fog-2(oz40) day-1 adult females were mated at 15°C and then transferred to 25°C. The Looc penetrance was measured at different times after mating with CB4855 males (red line).

Fig. 3.

An apx-1 reporter is expressed in the DTC after the L2 stage, and APX-1 functions redundantly with LAG-2 to promote germline proliferation. (A-C) apx-1 expression was examined using an apx-1 promoter::lacZ fusion (Chen and Greenwald, 2004) in the early L1 stage (A, 1 hour), in the early L3 stage (B, 21 hours) and in the L4 stage (C, 31 hours). All times are post-hatch at 25°C; white arrow indicates the DTC. White bar underscores the gonad in A. (D) Time-course analysis of apx-1::lacZ expression in the DTC. The somatic primordium of the gonad (SPG) forms at the beginning of the L3 stage. (E) Penetrance of Glp-1-like phenotype (all germ cells in a gonad arm displaying nuclear morphology consistent with meiotic prophase or gametogenesis). Synchronous L1 larvae were transferred to RNAi plates, and fixed and stained with DAPI ∼50 hours later at 20°C. Control RNAi is L4440 vector. In addition to scoring the Glp-1-like sterile phenotype, less severe phenotypes were also observed (for a classification of all phenotypes and a complete description of the RNAi conditions, see Table S2 in the supplementary material).

Fig. 4.

GLP-1 signaling regulates MSP-dependent cytoplasmic streaming. (A) Measurements of cytoplasmic flow rates (DIC particle speed) at 16-20 hours post-L4 in glp-1 mutant backgrounds. glp-1(rf) mutations increase flow; glp-1(gf) mutations decrease flow. Flow rates were also analyzed at the same timepoints in the other genetic backgrounds and experimental treatments, except oil injections were also analyzed 12 hours post-injection. (B) MSP is sufficient to cause elevated flow rates in glp-1 mutant females. Hermaphrodites or unmated fog-2 females were analyzed 1 hour after MSP injection or mating. Flow rates were compared with the wild type: ^*P<0.001; ^#P<0.01; ^‡P<0.05.

Fig. 5.

glp-1 affects oocyte cellularization. Fluorescence images showing cortical actin staining (red) to visualize the connection of proximal oocytes to the cytoplasmic core. DNA is blue. Animals of the indicated genotypes were examined 16 hours post-L4. Arrow indicates the most proximal oocyte connected to the core. Scale bar: 10 μm.

Fig. 6.

glp-1 control of MSP-dependent oocyte growth and cytoplasmic streaming involves gap junction proteins and germ cell apoptosis. (A-D) Genetic epistasis analysis of glp-1(ts) oocyte growth defects. Looc penetrance (A and D), cytoplasmic flow rates (B) and DIC phenotypes (C) were analyzed. Because mpk-1(ga111ts) causes severe gonad disorganization after transfer to 25°C for prolonged periods, day-1 adults were shifted and analyzed within 4 hours. ^*P<0.0005 in comparison to glp-1(tn777ts). Scale bar: 10 μm.

Fig. 7.

MSP signaling triggers rMLC phosphorylation throughout the gonad arm. (A-D) Detection of cortical actin (red) and P-rMLC using PSer19-MLC antibodies (green) in dissected gonads from hermaphrodites (A), unmated fog-2(oz40) females (B), MSP-injected fog-2(oz40) females 15 minutes post-injection (C) and glp-1(tn777ts) hermaphrodites (D). Arrowheads indicate P-rMLC staining in ring channels of the proximal oocytes. DNA is blue. Scale bar: 10 μm. (E) A model for the control of germline proliferation, oocyte growth and meiotic maturation by GLP-1 and MSP signaling (see text for details). P-rMLC is shown in brackets to indicate that MSP signaling is sufficient to promote rMLC phosphorylation in the germline, whereas the glp-1 pathway appears dispensable.