Copyright 0 1995 by the Genetics Society of America Phenotypic and Molecular Analysis of mes-3, a Maternal-Effect Gene Required for Proliferation and Viability of the Germ Line in C. eleguns Janet E. Paulsen,' Elizabeth E. Capowski2 and Susan Strome Department of Biology, Indiana University, Bloomington, Indiana 47405 Manuscript received July 24, 1995 Accepted for publication September 14, 1995 ABSTRACT mes-3 is one of four maternaleffect sterile genes that encode maternal components required for normal postembryonic development of the germ line in Caenorhabditis elegans. mes-3 mutant mothers produce sterile progeny, which contain few germ cells andno gametes. This terminal phenotype reflects two problems: reduced proliferation of the germ line and germ cell death. Both the appearanceof the dying germ cells and the results of genetic tests indicate that germ cells in mes-3 animals undergo a necrotic-like death, not programmedcell death. The few germ cells that appear healthyin mes-3 worms do not differentiateinto gametes, even after eliminationof the signaling pathway that normally maintains the undifferentiated population of germ cells. Thus, mes-3 encodes a maternally supplied product that is required both for proliferation of the germ line and for maintenance of viable germ cells that are competent to differentiate into gametes. Cloning and molecular characterizationof mes-3 revealed that it is the upstream gene in an operon. The genes in the operon display parallel expression patterns; transcripts are present throughout development and are not restricted to germ-line tissue. Both mes-3 and the downstream gene in the operon encode novel proteins. HE germ line enables metazoan organisms to pro- germ cell, P4, at the 16-24cell stage. During larval T duce offspring and thus is responsible for the per- development, the two daughters ofP4 (22 and 23) petuation of species. In most animal species, the germ undergo extensive proliferation within a syncytium in- line is set apart from the soma relatively early in em- side the somatic gonad. The proliferating gonadal cells bryogenesis. Primordial germ cells display a prolifera- form a cylindrical tube that extends bothanteriorly and tive stage and then undergo meiosis and subsequent posteriorly to form the two arms of the hermaphrodite gametogenesis. Two special features of germ cells are gonad (the endsof the tube are definedas distal). Start- their totipotency and potential immortality; egg cells ing in the L3 stage, the proximal-most germ nuclei have the potential to generate all of the different cell cease their mitotic divisions and entermeiosis. The first types of the organism, including more germ cells to germ cells to enter meiosis differentiate into sperm dur- carry on the cycle and give rise to future generations ing the L4 stage. In adult hermaphrodites, the germ (for a review, see MARSH and GOODE1994). Recent line converts to oogenesis. insights into germ-line specification and development Laser ablation studies and genetic screens identified have come from combinedmolecular and genetic anal- some of the cells and factors that control germ-line ysis of germ-line-required genes in Drosophila and Carno- development in C. elegans larvae and adults. The gZp-4 rhabditis elegans (reviewed in LEHMANN1992; MARSH and gene is required for normal proliferation of the germ GOODE 1994). We present molecular genetic analysis line. glp-4 mutant adults contain very few germ cells, of a gene required to make a functional germ line in which appear to be arrested in prophase ofmitosis C. elegans. (BEANANand STROME1992). A signal from the somatic The germline of C. elegans is a clonally derived tissue distal tip cell (DTC), located at the distal end of each gonad arm, controls themitosis/meiosis decision: germ that is generated by a series of unequal stem-cell-like cells located near the DTC remain mitotic, whereas divisions of the zygote (for a review, see WOOD 1988). germ cells that have proliferated and moved away from Each unequal division produceslargea somatic the influence of the DTC enter meiosis and differenti- founder cell and a smaller germ-line progenitor cell (P ate intogametes (KIMBLE and WHITE1981). If the DTC cell), which again divides unequally. The fourth and is eliminated by laser ablation, the germ cells in that last stem-cell-like division producesthe primordial gonad arm precociously enter meiosis and differentiate into gametes (KIMBLE and WHITE1981). Thus, theDTC Cwespondingauthm Susan Strome, Departmentof Biology, Indiana University, Bloomington, IN 47405. E-mail: [email protected] helps maintain thedistal-to-proximal progression of mi- ' Present address: Genetics Institute, Cambridge, MA 02140. totic, meiotic, and differentiated gametes in each gonad Present address: Fred Hutchinson Cancer Research Center, Seattle, arm. Theglpl gene encodes the likely germ-line recep- WA 98104. tor for the DTC signal (AUSTINand KIMBLE 1987,1989; Genetics 141: 1383-1398 (December, 1995) 1384 J. E. Paulsen, E. E. Capowski and S. Strome YOCHEMand GREENWALD1989; CRITTENDENet al. 1994), were performed as described in BRENNER(1974). Strains were and lag-2 was recently identified as encoding the likely cultivated at thepermissive temperature of 16”.The restrictive temperature used in this study was 25”. ligand for GLP-1 in the germ line (HENDERSONet al. C. eleguns N2 variety Bristol was the wild-type parent of all 1994; TAXet al. 1994). Interestingly, GLP-1 is homolo- mutant strains. The mes-? mutant alleles bn2lts, bn35, and gous to Drosophila Notch, and LAG2 is homologous to bn53 were isolated by EMS mutagenesis as previously reported Drosqbhila Delta and Serrate. Notch, Delta, and Serrate in CAPOWSKI et al. (1991). The mes-? alleles bn86 and bn88 are membrane-spanning proteins required for numer- were isolated by gamma radiation mutagenesis (Is7Cs at 10 ous cell-cell interactions during Drosophila develop- krad) using the screendetailed in CAPOWSW et al. (1991). Genetic mapping and complementationanalysis of bn86 and ment (reviewed by ARTAVANIS-TSAKONASet al. 1995). bn88 were performed as described in CAPOWSW et ul. (1991). The gld-1 gene is required for oogenesis and progres- The following genes and mutations were used: sion through meiotic prophase. Germ cells in gld-1 null LGI: mes-3, unc-11 (e47), dpy-5(e61), bLi-4(e937), dpy-l4(el88), mutanthermaphrodites undergo the normal switch unc-73(rh40),unc-l3(e51), glp4(bn2ts) from mitosis to meiosis but thenexit meiosis and return LGII: rol-b(n1178),ced-3(n 717), unc-26(e205) LGIII: unc-32(eI89),glpl(q175), fm-2(b245ts), unc-79(e1068), to the mitotic cell cycle, resulting in “tumorous” germ ced-4(nll62) lines (FRANCISet al. 1995). Tumorsuppression by GLD- LGN fem-?(q20ts) 1 may involve RNA binding (JONES and SCHEDL1995). The following chromosomal rearrangements were used: We sought to identify genes required for the early hDp20 (I;V,f ), nDp4 (I,f ), sDp2 (I,f ), eT1 (IZI, V). Some strains stages of germ-line development. Because we expected were provided by the Caenorhabditis Genetics Center. hDp20 and sDp2 were provided by KIM McKIM and ANN ROSE. rol- that at least some of these genes would be maternally 6(n1178)was provided by JIM KRAMER. expressed, we screened for maternal-effect sterile (Mes) Microscopy and photography: A ZeissICM405 inverted or grandchildless mutants (CAPOWSKIet al. 1991). We microscope with a Leitz micromanipulator was used for mi- identified 31 mutations in five complementation groups croinjections (Carl Zeiss Inc., Batavia, IL). Specimens were that result in a Mes phenotype (CAPOWSKIet al. 1991; examined ona Zeiss Axioscope equipped with Nomarski dif- ferential interference contrast and epifluorescence optics. C. GARVIN,I. KORF, P. MARTIN, E. WOWSKI,and J. Specimens stainedwith the DNA dyes diamidinophenylindole PAULSEN,unpublished data). Thematernal-effect steril- (DAE’I) or Hoechst were viewed with 365-nm epiillumination. ity associated with one of these genes, mes-1, is a result Photographs were taken on T-max film and developed with of embryonic defects that transform the primordial Diafine Twc-Bath developer (Acufine Inc., Chicago, IL). germ cell, P4, into a muscle precursor (STROMEet al. Analysis of germ-he proliferation: The developmental stages of larvae were determined by examining the ventral 1995). In contrast to mes-1, embryonic germ-line devel- hypodermal cells and the vulval cells with Nomarski DIC op opment is apparently normal in progeny from mothers tics (SUI.STONand HORVITZ1977). The numberof germ nu- containing mutations in any of the other four mes loci. clei present in larvae and adults was determined by counting The maternal-effect sterility observed inthese individu- DAF’I-stained germnuclei as describedin CAPOWSKIet al. als appears to result from defective postembryonic (1991). Laserablation of DTCs: The nuclei of DTCs were de- germ-line development. stroyed by the laser microbeam method of SULSTONand We present a detailed analysis of mes-3, one of the WHITE (1980)using a VSL-337 laser (Laser Science, Inc.) and four “postembryonic” mes genes. The progeny of mes-3 coumarin dye (AVERYand HORVITZ1987). The DTC in one mutant mothers containfew germ cells and nogametes. gonad arm of L3 stage mes-? mutant worms was ablated. Dur- This terminal phenotype reflects two perhaps related ing each session of ablations, wild-type L3 worms were also operated on toverify the effectiveness of cell killing. Forty to problems: reduced proliferation of the germ line (CA- 48 hr (at22“) after the ablations, operated worms were placed POWSKI et UL. 1991) and germ cell death. Both the ap- in a 5-pl drop of water on a slide. The slide was slowly heated pearance of the dying germ cells and the results of until the water evaporated. Subsequently,the heat-fixed speci- genetic tests indicate that germ cells in mes-3 animals mens were stained with Hoechst solution [40 mM NaCI, 10 are undergoing a necrotic-like death, not programmed mM Tris, pH 8.0,2.5 mM EDTA, 10 pg/ml Hoechst (33342)l.