The glp-1 gene functions in two inductive cellular interactions and in development of the embryonic hypodermis of C. elegans. We have isolated six mutations as recessive suppressors of temperature-sensitive (ts) mutations of glp-1. By mapping and complementation tests, we found that these suppressors are mutations of known dumpy (dpy) genes; dpy genes are required for development of normal body shape. Based on this result, we asked whether mutations previously isolated in screens for mutants defective in body shape could also suppress glp-1 (ts). From these tests, we learned that unselected mutations of eight genes required for normal C. elegans morphogenesis, including the four already identified, suppress glp-l(ts). All of these suppressors rescue all three mutant phenotypes of glp-l(ts) (defects in embryonic induction of pharyngeal tissue, in embryonic hypodermis development, and in induction of germline proliferation). However, they do not rescue putative glp-1 null mutants and therefore do not bypass the requirement for glp-1 in development. In the light of current ideas about the molecular nature of the glp-1 and suppressor gene products, we propose an interaction between the glp-1 protein and components of the extracellular matrix and speculate that this interaction may impose spatial constraints on the decision between mitosis and meiosis in the germline.

Communication between cells is crucial in the development of multicellular organisms. Inductive interactions, in which cells of one tissue regulate cell fate in a second tissue, are particularly important to the development of whole structures and tissues (e.g. Spemann & Mangold, 1924). However, the molecular mechanism(s) by which inductive interactions are mediated remain unknown. Recently, important clues have been obtained using both biochemical and genetic approaches. In Xenopus laevis, specific growth factors that influence embryonic induction have been identified in in vitro assays (Kimmelman & Kirshner, 1987; Smith, 1987; Slack et al. 1987). And in the nematode Caenorhabditis elegans, a gene required for two inductive events has been identified by mutation (Austin & Kimble, 1987; Priess et al. 1987); this gene is called glp-1 (for germ/ine proliferation defective).

During C. elegans development, glp-1 functions in two distinct inductive interactions. In embryogenesis, descendants of one blastomere (P1) induce descendants of a different blastomere (AB) to generate pharyngeal tissue (Priess & Thompson, 1987) by a mechanism that requires maternal glp-1 gene function (Priess et al. 1987; Austin & Kimble, 1987). Similarly, in postembryonic development, two somatic cells, the distal tip cells, stimulate germ cells to proliferate (Kimble & White, 1981) by a mechanism that requires glp-1 gene function (Austin & Kimble, 1987; Priess et al. 1987). In the absence of either the relevant cell interaction or glp-1 gene function, AB does not produce pharyngeal tissue and the germline is virtually nonexistent. In the interaction between distal tip cell and germline, an analysis of genetic mosaic animals indicates that glp-1 is required in the induced tissue and therefore may mediate the response to the inductive signal (Austin & Kimble, 1987). Besides its role in known inductive events, glp-1 is required during embryogenesis for hypodermal development (Priess et al. 1987). The morphogenesis of an embryo from an aggregate of cells into a worm depends on the hypodermis (Priess & Hirsh, 1986). However, no inductive interactions are known to influence hypodermal development or embryonic morphogenesis (Priess & Thompson, 1987).

To identify other genes necessary for inductive interactions, we generated extragenic suppressors of glp-1 mutations. Surprisingly, several suppressors were defective in larval morphogenesis and had an altered body shape. Mutations in numerous genes affect body shape (Brenner, 1974; Higgins & Hirsh, 1977; Cox et al. 1980; Rose & Baillie, 1980; Hodgkin, 1983; Meneely & Wood, 1984). These suppressors have a ‘dumpy’ mutant phenotype; animals are shorter than normal but have the same diameter. Previous studies of 25 dumpy (dpy) genes indicated that six are involved in the process of dosage compensation (Hodgkin, 1983; Meneely & Wood, 1984; Wood et al. 1985; Meyer & Casson, 1986) but that the remaining nineteen, which include those discussed here, do not affect dosage compensation. These other dpy genes are thought to be required for larval morphogenesis. Other genes that affect body morphology include squat (sqt), small (strut), long (Ion), and roller (rol) genes. Here we show that mutations in eight of these genes (seven dpy and one sqt) suppress both germline and embryonic defects of glp-l(ts) mutations.

Strains and culture methods

Worms were maintained on agar-filled Petri dishes seeded with E. coli as described (Brenner, 1974). The wild-type strain C. elegans var. Bristol, (strain N2) and mutant strains used in this study were obtained either from the Cambridge collection (Brenner, 1974) or from the Caenorhabditis Genetics Center. All mutants are described in Hodgkin et al. (1988) except where indicated.

Our nomenclature follows the guidelines of Horvitz et al. (1979). Mutant phenotypes are designated by a capitalized abbreviation (e.g. Dpy for a dumpy phenotype) and genes are designated by a lower case, italicized abbreviation (e.g. dpy for dumpy genes). Where appropriate, abbreviations are placed after an allele number to indicate that it is temperature sensitive (ts), amber (am), or semidominant (sd). For each gene, one null or putative null allele has been designated as the ‘reference’ allele (see Hodgkin et al. 1988); in general, we used reference alleles in our suppression tests.

Fig. 1 is a partial genetic map showing the loci used in this paper. LG/: dpy-5(e61), dpy-14(el88ts), dpy-24(s71). LG//: dpy-2(e8), dpy-10(el28), dpy-25(e817sd), rol-l(e91), rol-6(el87), rol-8(sc!5) (Kusch & Edgar, 1986), sma-6(el482), sqt-l(scl, scl3, sclOO, e!350), sqt-2(sc3), mnDf30, mnCl. LGIII: dpy-l(el), dpy-17(e!64), dpy-18(e364am), dpy-19(el259ts), glp-l(q35, q50, ql58, q224ts, q231ts), lon-l(el85), sma-2(e502), sma-3(e491), sma-4(e729), unc-32(el89), unc-36(e873). LGZV: dpy-4(e!166), dpy-9(e!2),dpy-13(el84sd), dpy-20(e2017am). LGV: dpy-ll(e224), dpy-21(e428), him-5(el490), rol-3(e754), sma-1 (e30), sqt-3(sc63ts). LGK: dpy-3(e27, el82) (originally called dpy-12(el82) but later shown to be allelic to dpy-3; see Meneely & Wood, 1987), dpy-6(e!4), dpy-7(e88), dpy-8(el30), lon-2(e678), sma-5(n678), unc-6(e78), unc-18(e81), vab-3(ef>48).

Fig. 1.

Partial genetic map of C. elegans indicating the relative positions of genes used in this study. The six linkage groups are represented by a heavy horizontal line with well-mapped genes placed on the line and less precisely mapped genes indicated by bars placed above the line. Deficiencies are represented by an open bar placed below the line, glp-1 and genes that interact with glp-1 are indicated in bold-faced type.

Fig. 1.

Partial genetic map of C. elegans indicating the relative positions of genes used in this study. The six linkage groups are represented by a heavy horizontal line with well-mapped genes placed on the line and less precisely mapped genes indicated by bars placed above the line. Deficiencies are represented by an open bar placed below the line, glp-1 and genes that interact with glp-1 are indicated in bold-faced type.

Isolation of extragenic suppressors o/glp-1

L4 hermaphrodites homozygous for glp-l(ts) and a closely linked marker mutation, unc-32(el89), were raised at permissive temperature [12°C for glp-l(q224) and 15°C for glp-l(q231)], mutagenized with ethyl methane sulphonate (Brenner, 1974), and returned to plates at permissive temperature. To isolate recessive suppressors, F1 progeny were picked (3 animals/plate) and grown at permissive temperature. F2 progeny were shifted to restrictive temperature (20 °C) as late embryos or LI larvae. Suppressors were sought by screening plates visually for production of an F3 generation. Among the suppressed glp-1 animals isolated, six contained a mutation with a Dumpy phenotype: two from 2100 glp-l(q224) Fi and four from 1900 glp-1 (q231) F1. Other suppressors will be described elsewhere.

Genetic mapping and complementation tests

Linkage and complementation were determined by standard tests (see Tables 1 and 2 in Results), dpy mutations on LGX were roughly positioned by three-factor mapping: Lon and Une recombinants from lon-2 unc-6/dpy and lon-2 unc-18/ dpy heterozygotes were examined. q288 was positioned more precisely by additional mapping: two-factor mapping established the distance between unc-18 and q288 (Table 1). Three-factor mapping established the position of q288 relative to unc-18: Une and Dpy recombinants were recovered from a strain that was unc-18 q288/vab-3 and examined for a vab-3 phenotype, dpy mutations q291 and q292 were initially linked to unc∼4(el20) and then mapped to the region deleted by mnDf30 (Sigurdson et al. 1984). mnDf30 deletes both dpy-2 and dpy-10’, mnCl, a balancer for half of LG// including the mnDf30 region, carries a dpy-10 mutation. q291 and q292 were placed over mnDf30 by mating q291 /+ or q292/ + males to mnDf30/mnCl hermaphrodites.

Table 1.

Complementation analysis of glp-1 (ts) suppressors with a Dpy phenotype

Complementation analysis of glp-1 (ts) suppressors with a Dpy phenotype
Complementation analysis of glp-1 (ts) suppressors with a Dpy phenotype
Table 2.

(A) Three-factor mapping

(A) Three-factor mapping
(A) Three-factor mapping

Construction of double mutants

Double mutants with glp-1 (ts) and one of several mutations that change body shape were constructed in one of two ways. 1) Males heterozygous or hemizygous for the mutation altering body shape were mated to unc-32 glp-1 (ts) hermaphrodites at permissive temperature and non-Unc F1 crossprogeny picked. Double mutants (e.g. une,dpy) were isolated from the F2. 2) unc-32 glp-l(ts)/ ++ males were mated with hermaphrodites homozygous for the mutation that altered body shape and cross-progeny picked. Again, double mutants were isolated from the F2. This second scheme was used with sqt mutations, as their heterozygous Rol phenotype effectively prohibits males from mating. In some cases, a Dpy phenotype was so extreme that animals could barely move and, therefore, Une Dpy animals could not be distinguished from Dpy animals; in such cases, Une non-Dpy F2 animals were picked to isolate Une Dpy animals from the F3. For two genes, rol-3 and sma-5, we constructed and tested stocks that did not contain unc-32(el89).

Tests of glp-1 (ts) suppression

In the initial test of glp-1 suppression by mutations that affect body shape, (dpy, sqt, rol, Ion, or sma), >200 embryos homozygous for unc-32 glp-1 (ts) and the mutation to be tested were shifted to 20 °C after the embryonic temperature-sensitive period for glp-1 (4-28 cells). These embryos were allowed to develop and then examined for their ability to produce embryos; any embryos made were in turn examined for hatching, development to adulthood, and ability of these progeny to produce offspring.

Later suppression tests were done using stocks without unc-32. To remove unc-32, glp-l(q231); him-5(el490) males were mated with hermaphrodites homozygous for unc-32 glp-l(q231) and the suppressor mutation. Non-Unc heterozygous cross-progeny were isolated, F2 individuals picked to individual plates, and a stock homozygous for glp-1 and the suppressor obtained.

DAP1 staining

Chromosome morphology was visualized by staining with diamidinophenolindole (DAPI) as described by Austin & Kimble (1987). From a 20°C stock, adults were picked just after moulting and before the onset of embryo production; these adults were maintained for 24,48, 72, 96,120, or 144 h at 20°C before fixation. For each timepoint, 4-10 animals (8-20 ovotestes) were examined. These animals are designated as 1-, 2-, 3-, 4-, 5-, or 6-day adults in Table 5.

Determination of brood sizes and per cent hatching

L4 hermaphrodites homozygous for glp-1 (q231) and a suppressor mutation were placed individually on Petri dishes at 20 °C and transferred to a fresh plate every 24 h. The total number of embryos produced by each hermaphrodite was counted; eggs were scored for hatching —36 h after the hermaphrodite had been transferred. Hatched progeny were observed until at least L3 to assess their development. As a control, brood sizes and per cent hatching of glp-1 (q231 ) were determined in a similar fashion at both 20 °C (to determine brood size) and 17 °C (to determine viability on successive days). At 17°C, we counted the brood sizes of eleven animals: they made an average of 175 embryos; 21 % of those progeny hatched and developed normally. No decrease in per cent hatching was observed as animals aged.

Tests for suppression of other glp-1 alleles

Double mutants possessing one of several glp-1 alleles that are not temperature sensitive and a suppressor of glp-1 (ts) were constructed. The partial loss-of-function glp-1 (q35) and putative null glp-1 (ql58) alleles each were marked with unc-32(el89), the partial loss-of-function allele glp-l(q50) originally was induced on an eTl chromosome and hence was marked with unc-36(e873). The germline phenotype of glp-l(q35) shows some cold sensitivity (J. Austin, personal communication). Therefore, all experiments using glp-1 (q35) were done at an intermediate temperature of 20 °C and Une, non-Dpy siblings were assayed in each experiment as a control for minor fluctuations in incubator temperature. Suppression of glp-l(q35) was retested in the absence of unc-32(el89) to eliminate the possibility of a marker effect. unc-32(el89) was removed by mating glp-l(q35)/eTl-, him-5(el490) males to suppressor hermaphrodites. Fi cross-progeny segregating Glp (and no Une) animals in the F2 were obtained. These were heterozygous both for glp-1 (q35) and the suppressor. F2 animals either with or without the suppressor were picked (as L3 or L4 animals) and scored as producing (1) no eggs, (2) dead eggs only, or (3) viable F3 progeny (and no dead eggs). All animals producing no eggs or only dead eggs were presumed to be glp-1 (q35)/glp-1 (q35).

Tests for maternal effects on the suppression of glp-l(ts)

To examine dpy/+ progeny of dpy homozygous mothers, dpy-10(el28)’, glp-l(q231) hermaphrodites were mated to glp-l(q231f, him-5(el490) males and examined for production of non-dpy \dpy-10(el28) / +; glp-1/glp-1’, him-5(el490)/+] cross-progeny at 20°C. These matings were done en masse in an effort to increase mating efficiency of the glp-1 males. Unfortunately, 5 of the 14 mated hermaphrodites died prematurely, and therefore the average brood size in this experiment is lower than expected; normally, mated hermaphrodites have larger broods than unmated ones, yet the control broods averaged 147 embryos and the mated broods 122 embryos. To look at dpy homozygous progeny of dpy/+ mothers, dpy-10(el28)/ + glp-1 (q231)/glp-l(q231); him-5(el490)/+ hermaphrodites were examined for production of dpy [dpy-10(el28);glp-l(q231)] progeny at 20°C. As a control in this experiment, the survival of dpy progeny from dpy mothers was examined using dpy-10(el28)’, glp-l(q231) at 20 °C.

Isolation and initial characterization o/glp-l(ts) suppressors with a dumpy left roller phenotype

We have isolated recessive suppressors of two temperature-sensitive alleles of glp-1, glp-1 (q224ts) and glp-l(q231ts). Loss of glp-1 gene function causes germ cells that normally would be in mitosis to enter meiosis instead. [Although the C. elegans germline is syncytial (Hirsh et al. 1976), for simplicity we refer to germline nuclei as germ ‘cells’.] As a result of the glp-1 effect on germline mitoses, only 4−7 germ cells are produced in severe glp-1 mutants rather than the 1000−2000 made in wild-type animals. Because hermaphrodites normally produce ∼300 sperm before switching to oogenesis, the few gametes produced by glp-1 mutant hermaphrodites are sperm and the animals are sterile. If glp-l(ts) is shifted to restrictive temperature at any time during postembryonic development, germ cells at the distal end of the gonad that would normally be mitotic enter meiosis; therefore the glp-1 product must function throughout development for continued mitoses. A second glp-1 mutant phenotype is seen in conditional or leaky glp-1 mutants which can make enough germ cells to produce some embryos. Progeny of a homozygous glp-1 mutant mother inevitably die during embryogenesis; a glp-1 (+) allele introduced from the father cannot rescue the lethal phenotype. The temperature-sensitive period for lethality is early in embryogenesis (4-28 cells).

The scheme used to isolate suppressors of glp-1 (ts) is outlined in Materials and methods. In brief, after EMS mutagenesis of glp-1 (ts), F2 animals were tested for production of live progeny that developed into fertile adults at the restrictive temperature of 20°C. At 20°C, glp-1 (ts) animals are sterile, but make more germ cells than at the more stringent restrictive temperature of 25°C. This intermediate temperature was used with the idea that some functional glp-1 product might be present and that a bypass of glp-1 function would not be required to achieve suppression. Among the suppressors isolated were six with a dumpy left roller phenotype, termed simply dumpy (Dpy). The six suppressors include q287 and q291, suppressors of glp-1 (q224), and q288, q289, q290, and q292, suppressors of glp-1 (q231). Other recessive suppressors isolated will be described elsewhere.

To confirm that the dpy mutations (rather than a second mutation with a more subtle phenotype) were indeed suppressing glp-1, each dpy was separated from glp-1 (ts) by multiple crosses with wild-type (N2) animals. Then, glp-1 (ts)’, dpy double mutants were reconstructed. To retest each double mutant, we shifted embryos with more than 28 cells to 20 °C and looked both for development into fertile adults and for continued production of fertile animals over several generations. In every case, the germline and embryonic defects associated with glp-1 were suppressed (see below). The dpy mutations were isolated as recessive suppressors of glp-1 and remained recessive upon retesting: dpy/+’, glp-1/glp-1 animals were not suppressed at 20°C (see below and Table 6). Moreover, no suppression of the dumpy left roller phenotype by glp-l(ts) was observed. Each dpy suppressor was further tested at the more stringent restrictive temperature of 25°C. At this temperature, suppression of glp-1 embryonic lethality was not observed. However, the germline phenotype was partially suppressed: some glp-l(q231)’, dpy animals produced many more germ cells than the 6-25 typical of glp-1 (q231) alone (data not shown).

To test for allele specificity, each suppressor of glp-l(q231) was tested for suppression of glp-1 (q224) and vice versa. In every case, the suppressor rescued both glp-1 alleles. (However, they do not suppress all glp-1 mutations, see below and Table 4). Because suppression is not specific for either glp-1 (ts) allele, many of the tests described below were done using only glp-l(q231).

glp-1 suppressors are alleles of known dpy genes Numerous dpy genes have been identified (Brenner, 1974; Cox et al. 1980; Rose & Baillie, 1980; Hodgkin, 1983; Meneely & Wood, 1984). The glp-1 suppressors might identify a novel class of dpy genes or they might be new alleles of known dpy genes. We have done both complementation tests (Table 1) and mapping experiments (Table 2) to distinguish between these two possibilities.

(C) Deficiency mapping

Four suppressors, q287, q288, q289, and q290, map to EGA. Three dpy genes on EGA, dpy-3, dpy-7, and dpy-8, have numerous alleles, many of which are left rollers as well as dumpy. A fourth dpy gene on LG A”, dpy-6, is represented by only two, non-roller alleles. Complementation tests and mapping of q287 and q289 suggest that both are alleles of dpy-8. The q287/q289 heterozygote is slightly Dpy, and both mutations fail to complement dpy-8(el30) and complement other dpy genes on EGA (Table 1). Although both map between lon-2 and unc-6 (Table 2A), q287 maps precisely to dpy-8 whereas q289 maps ∼0·5 map units to the left of dpy-8. The discrepancy in q289 map position may result from statistical error or may indicate an association with a small DNA rearrangement that changes the apparent map position of q289 by suppressing recombination. The other two JV-linked suppressors, q288 and q290, fail to complement dpy-7(e88) and do complement other X-linked dpy genes (Table 1). Although q288 and q290 complement each other (Table 1), both suppressors map to dpy-7 between unc-6 and unc-18 (Table 2A and B). These two mutations are therefore assigned to dpy-7.

The remaining two suppressors, q291 and q292, map to LGZZ. Similarly, dpy-2 and dpy-10 map to LGZZ and have some left roller alleles. q291 fails to complement dpy-10(el28), whereas q292 fails to complement dpy-2(e8) (Table 1). Furthermore, dpy-2, dpy-10, q291, and q292 all map to a deficiency on LG/Z, mnDf30 (Sigurdson et al. 1984; this paper: Table 2C). On the basis of this complementation and map data, we assign q291 to dpy-10 and q292 to dpy-2.

Unselected alleles of dpy-2, dpy-7, dpy-8, and dpy-10 also suppress glp-l(ts)

All six glp-1 suppressors are mutations of previously identified dpy genes. It is possible that only specific rare alterations in these genes lead to glp-1 suppression. We tested alleles obtained in a screen for visible mutants to ask whether these unselected alleles would also suppress glp-l(ts). Double mutants containing a reference allele (see Materials and methods) of dpy-2, dpy-7, dpy-8, or dpy-10 plus glp-l(ts) were shifted to 20°C as embryos of more than 28 cells and examined for continued production of fertile animals over several generations (Table 3). Unselected alleles of all four dpy genes suppressed both germline and embryonic abnormalities of both glp-1 (q224) and glp-1 (q231) at20°C. As found with the selected suppressors, these reference alleles partially suppressed the germline defect, but did not suppress the embryonic defect, at 25°C. We conclude that the selected suppressors are not unusual mutations of their respective genes.

Table 3.

Tests for suppression of glp-1 (q231) by unselected alleles of dpy, sqt, sma, rol and Ion mutations

Tests for suppression of glp-1 (q231) by unselected alleles of dpy, sqt, sma, rol and Ion mutations
Tests for suppression of glp-1 (q231) by unselected alleles of dpy, sqt, sma, rol and Ion mutations
Table 4.

Tests for suppression ofnon-ts glp-1 alleles glp-l(ql58)* glp-l(q50)l glp-l(q35)t

Tests for suppression ofnon-ts glp-1 alleles glp-l(ql58)* glp-l(q50)l glp-l(q35)t
Tests for suppression ofnon-ts glp-1 alleles glp-l(ql58)* glp-l(q50)l glp-l(q35)t
Table 5.

Suppression of the germline and embryonic phenotypes of glp-1 at 20°C

Suppression of the germline and embryonic phenotypes of glp-1 at 20°C
Suppression of the germline and embryonic phenotypes of glp-1 at 20°C
Table 6.

Suppression of glp-1 embryonic lethality by dpy-10 in mother and/or zygote (20°C)

Suppression of glp-1 embryonic lethality by dpy-10 in mother and/or zygote (20°C)
Suppression of glp-1 embryonic lethality by dpy-10 in mother and/or zygote (20°C)

Tests for glp-1 suppression by other mutations that alter body shape

Given the result that unselected mutations in four dpy genes suppress glp-l(ts), we next tested mutations in other genes required for normal body morphogenesis to learn whether these too might affect the phenotype of glp-1 (ts). We tested mutations in essentially all dumpy (dpy), roller (rol), squat (sqt), small (sma), and long (Ion) genes. Only one of seven dpy genes implicated in control of dosage compensation, dpy-21, was tested, rol mutants roll helically as they move forward or backward (Brenner, 1974; Higgins & Hirsh, 1977; Cox et al. 1980). Most sqt mutants roll as heterozygotes and are dumpy as homozygotes (Cox et al. 1980); they were therefore tested for suppression as heterozygotes as well as homozygotes. Several phenotypically different alleles of sqt-1 were tested (see below), sma mutants are proportionally smaller (i.e. both shorter and thinner) and Ion mutants are longer than wild type (Brenner, 1974). When available, an amber mutation was used because such alleles are probably null.

Mutants homozygous for unc-32 glp-1 (ts) plus a mutation in one of the genes described above were assayed for suppression of glp-1 (ts) at restrictive temperature (Table 3). We found that mutations in three additional dpy genes, dpy-1, dpy-3 and dpy-9, and one sqt gene, sqt-1, were recessive suppressors of glp-l (q231) at 20 °C. At 25 °C, these suppressors prolonged germline proliferation but did not prevent embryonic lethality. Mutations in other genes neither suppressed nor enhanced the mutant phenotypes of glp-1 (ts) (enhancement data not shown).

There is no correlation between severity of dumpy phenotype (i.e. length of the animal) and degree of suppression. Both dpy-1 and dpy-5 are extremely dumpy, for example, but dpy-1 suppresses glp-1 (ts) and dpy-5 does not. Conversely, both dpy-3 and dpy-18 have a less severe dumpy phenotype, and dpy-3 suppresses and dpy-18 does not. Suppression by sqt-1 is unique in that only one sqt-1 allele suppresses glp-1 (ts). Although sqt-l(scl) and sqt-1 (el350) have similar phenotypes (dominant Rol and recessive Dpy), only the latter suppresses glp-1 (q231). Further, neither the recessive Rol allele sqt-1 (scl5) nor the phenotypically wild type, putative null allele sqt-1 (sclOO) is a suppressor.

Tests for suppression of other glp-1 alleles

The greater suppression of glp-1 (ts) at 20°C than at 25 °C suggested that the dpy mutations might suppress partial loss-of-function but not null alleles of glp-1. We therefore tested each suppressor with a putative null allele, glp-l(ql58), and two partial loss-of-function alleles,glp-l(q35) and glp-l(q50). Whereasglp-1 (ql58) mutants produce only 4-7 germ cells, glp-1 (q35) mutants produce a variable number of germ cells (47-198) and glp-1 (q50) mutants produce either 5-8 or about 800 germ cells (Austin & Kimble, 1987). Double mutants homozgyous for one glp-1 allele, glp-1 (x) and a suppressor were compared to single mutants of glp-l(x) (Table 4). For glp-1 (ql58), the germline defect was not suppressed and the embryonic defect could not be tested because too few germ cells are generated to produce embryos. For glp-1 (q50) neither the germline nor embryonic defect was suppressed. In contrast, though the embryonic defect of glp-1 (q35) was not suppressed, the germline defect was partially suppressed. Three suppressors, dpy-7(q288), dpy-8(q287), and dpy-10(q291), were retested with glp-1 (q35) in the absence of unc-32(e!89) to eliminate the possibility of a marker effect. Similar germline suppression was observed in all three cases (data not shown).

Further characterization of glp-1 suppression

Our initial test of suppression relied on continued production of progeny after shifts of animals homozygous for unc-32(el89) glp-1 (ts) and a suppressor to 20°C. For subsequent experiments, we removed unc-32 to eliminate possible marker effects.

Suppression of the glp-l(q231) germline proliferation defect (Table 5, Fig. 2 )

In wild-type adults, meiotic nuclei are not observed in the most distal portion of the gonad (Klass et al. 1976; Kimble & White, 1981) and mitotic figures are still visible six days after the moult to adulthood (Table 5). In contrast, in glp-l(q231) animals raised at 20°C, all germline nuclei have entered meiosis two days after this final moult (Table 5, Fig. 2A). To examine the suppression of the glp-1 (ts) germline phenotype, we compared the state of distal germline nuclei in double mutants homozygous for glp-1 (ts) plus a suppressor to single mutants carrying the suppressor alone. Germline chromosomes were examined at 24 hour intervals starting with newly moulted adults. In contrast to glp-l(q231) animals, mitotic nuclei were still observed in the distal gonad after two days of adulthood in the double mutant (Table 5, Fig. 2B). However, by four or five days after the last moult, all germline nuclei in the distal gonad of the double mutant were in pachytene (Table 5). Therefore, the suppressors delay the abnormal entry into meiosis caused by glp-1 (q231 ) but do not prevent it. In control animals, mitotic nuclei were present in the distal gonad of single mutants homozygous for the suppressor alone even after six days of adulthood (Table 5). Hence, the suppressor mutations themselves do not block germline mitoses. Consistent with this finding that a suppressed glp-1 (ts) germline continues mitoses longer than does a glp-1 (ts) germline, the number of progeny produced by the suppressed glp-1 (is) hermaphrodite is greater than that of the glp-1 (ts) hermaphrodite (Table 5).

Fig. 2.

Suppression of glp-1 germline phenotype (20°C). The distal region of a single ovotestis is shown in each picture with the distal tip to the left; virtually only germline nuclei are shown. Animals were stained with DAPI after 2 days of adulthood (see Materials and methods). (A) glp-l(q231): all germline nuclei are in pachytene; (B) glp-I(q231);dpy(q288): mitotic germline nuclei are present. A metaphase plate (right) and a pair of late anaphase nuclei (left) are indicated by arrows.

Fig. 2.

Suppression of glp-1 germline phenotype (20°C). The distal region of a single ovotestis is shown in each picture with the distal tip to the left; virtually only germline nuclei are shown. Animals were stained with DAPI after 2 days of adulthood (see Materials and methods). (A) glp-l(q231): all germline nuclei are in pachytene; (B) glp-I(q231);dpy(q288): mitotic germline nuclei are present. A metaphase plate (right) and a pair of late anaphase nuclei (left) are indicated by arrows.

Suppression of glp-1 (q231) embryonic lethality (Table 5, Fig. 2 )

Almost all wild-type embryos hatch (>99 %) (Hodgkin et al. 1979), but only an occasional glp-l(q231) embryo produced at 20°C hatches and none develops to adult-hood (Table 5). When the percentages of embryos that hatch and grow to adulthood were compared among double mutants homozygous for glp-1 (q231 ) and one of the suppressors, we found that the suppressors vary in strength (2% to 38%; Table 5). We also observed in this experiment that the percentage of embryos that hatch and develop to adulthood decreases as mothers age (Fig. 3). This decrease might have been associated with the phenotype of either single mutant, i.e. glp-l(q231), or the suppressor, rather than reflecting a property of suppression. When we examined glp-l(q231) at 17°C, where approximately 21% of the embryos hatch and develop to adulthood, we found no decrease in per cent hatching with maternal age (Fig. 3). Similarly, when we counted the per cent of embryos that hatch on successive days from mothers homozygous for each suppressor, we found viability to be high (Table 5) with no decrease over maternal age (data not shown). The drastic drop in per cent hatching observed in suppressed glp-l(ts) embryos must therefore be associated with suppression.

Fig. 3.

Suppression of glp-1 embryonic phenotype decreases with maternal age. Histograms indicate the percentage of embryos that hatch; the number below each bar represents the maternal age in days after the moult to adulthood. A glp-1 (q231) control shows essentially no change in per cent hatching with maternal age. In contrast, seven of the eight double mutants with glp-1 plus a suppressor show a drastic decrease in progeny viability with maternal age. Note that the glp-l(q231) control was raised at 17°C, an intermediate temperature at which some hatching could be observed, while the double mutants were raised at the restrictive temperature of 20 °C.

Fig. 3.

Suppression of glp-1 embryonic phenotype decreases with maternal age. Histograms indicate the percentage of embryos that hatch; the number below each bar represents the maternal age in days after the moult to adulthood. A glp-1 (q231) control shows essentially no change in per cent hatching with maternal age. In contrast, seven of the eight double mutants with glp-1 plus a suppressor show a drastic decrease in progeny viability with maternal age. Note that the glp-l(q231) control was raised at 17°C, an intermediate temperature at which some hatching could be observed, while the double mutants were raised at the restrictive temperature of 20 °C.

The maternal dpy genotype is crucial for suppression o/glp-l(ts) embryonic lethality

The embryonic lethality of glp-1 is dependent on the maternal genotype: progeny of homozygous glp-1 mothers die whether heterozygous or homozygous for glp-1 (Austin & Kimble, 1987; Priess et al. 1987). We asked if suppression of the glp-1 embryonic lethality is also dependent on the maternal genotype. We chose dpy-10(e!28) for this study because it is most likely to be a null mutation based on dosage studies (Sigurdson et al. 1984). Progeny of mothers homozygous for both glp-l(q231) and dpy-10(el28) are rescued. This rescue is independent of whether the progeny themselves are heterozygous or homozygous for the suppressor (Table 6). Conversely, progeny of mothers homozygous for glp-l(q231) but heterozygous for dpy-10(el28) are never rescued. This lack of rescue is again independent of whether the progeny themselves are heterozygous or homozygous for the suppressor (Table 6). These results indicate that suppression of glp-1 (ts) depends on the genotype of the mother and not that of the progeny.

The glp-1 gene functions in two inductive interactions and in development of the embryonic hypodermis of C. elegans (Austin & Kimble, 1987; Priess et al. 1987). In this paper, we show that the mutant phenotypes of two temperature-sensitive (ts) alleles of glp-1 are suppressed by mutations in eight genes required for normal morphogenesis during larval and adult stages. Further, one of the mutant phenotypes of a third, partial loss-of-function allele is suppressed. These suppressor genes include seven dpy (dpy-1, dpy-2, dpy-3, dpy-7, dpy-8, dpy-9, and dpy-10) and one sqt (sqt-1) gene. The dpy mutants are shorter than normal and many, but not all, roll helically as they move (Brenner, 1974); the sqt mutant is shorter than normal as a homozygote and rolls as a heterozygote (Cox et al. 1980). However, neither a shorter body nor the ability to roll per se is sufficient to suppress glp-1. Mutations in most dpy, sma, rol, and sqt genes do not suppress glp-1. In addition, there is no correlation between body length and ability to suppress. Certain severe dpy mutants do not rescue glp-l(ts), and Ion mutants, which have an increased body length, neither enhance nor suppress glp-1.

From our genetic characterization of the interactions between glp-1 mutations and the suppressor mutations that alter larval body shape, we draw three main conclusions. First, the suppressors must affect a process that is common to all three functions of glp-1 (induction of the pharynx, induction of the germline, and development of the embryonic hypodermis), because all three glp-1 defects are suppressed. Second, the suppressors do not bypass the requirement for glp-1 since they do not rescue a putative null mutant of glp-1, and since they are better suppressors at 20°C, a moderate restrictive temperature, than at 25°C, a stringent restrictive temperature. Instead, the suppressors probably allow disabled glp-1 product to function more efficiently. Third, the interaction of these genes with glp-1 suggests that the suppressor genes constitute a functional subclass distinct from other genes affecting body morphology.

In addition, our results show that these suppressors are not unusual mutations of their respective loci. Suppression of glp-1 (ts) is obtained not only by mutations isolated as suppressors but also by mutations isolated in visual screens for phenotypes altering body shape. However, we cannot conclude that these mutations are due to complete loss of the suppressor gene product. The null phenotype of sqt-1 is wild-type (Kusch & Edgar, 1986; Kramer et al. 1988). In contrast, the suppressor sqt-1 mutation has a visible phenotype indicating that its product is probably present but defective. Although the null phenotypes of the dpy suppressor genes are not known, two lines of evidence support the idea that the dpy suppressors are at least partial loss-of-function mutations: they are fully recessive and they arise at a frequency similar to loss-of-function alleles in genes understood at the molecular level.

Suppression of glp-1 by mutations in seven dpy genes and sqt-1 indicates that these genes are not only required for body morphogenesis, but that they are also involved at some level in the decision between mitosis and meiosis. Because the glp-1 mutations do not suppress (or enhance) the Dpy or Sqt phenotypes, we do not propose a role of glp-1 in larval morphogenesis. However, an interaction between genes required for larval morphogenesis and glp-1 is not inconsistent with known glp-1 functions: the hypodermis of glp-1 mutant embryos develops incorrectly resulting in defective embryonic morphogenesis (Priess et al. 1987).

How might the glp-1 and suppressor gene products interact? An understanding of the molecular nature of these gene products provides insight into this problem. The glp-1 gene has now been isolated and its DNA sequence obtained (Austin & Kimble, unpublished; J. Yochem & I. Greenwald, personal communication). The deduced amino acid sequence of glp-1 is extremely similar to that of lin-12 (J. Yochem & I. Greenwald, personal communication); the sequence of lin-12 predicts a membrane protein with an extracellular portion composed primarily of EGF-like repeats (Yochem et al. 1988). Analysis of genetic mosaic animals indicates that glp-1 functions in the germline and may be part of the receiving mechanism in the regulatory interaction that controls its growth (Austin & Kimble, 1987). These data together place glp-1 in the germline as a membrane protein that mediates induction of germline growth by the somatic distal tip cell.

Among the glp-1 suppressors, only sqt-1 has been molecularly isolated; the deduced amino acid sequence of sqt-1 shows that it is a collagen gene (Kramer et al. 1988). The molecular identity of sqt-1, its mutant phenotypes, and its genetic interactions with glp-1 together suggest that this gene encodes a collagen in the extracellular matrix of C. elegans. This suggestion does not preclude the possibility that the sqt-1 gene product is also found in the cuticle, which is essentially a specialized form of the extracellular matrix. Indirect evidence suggests that the dpy suppressor genes may also act in formation of the extracellular matrix (Cox et al. 1980, 1984; Kramer et al. 1982; Kusch & Edgar, 1986). The distal arm of the gonad in C. elegans is composed primarily of germ cells plus the somatic distal tip cell (Klass et al. 1976; Kimble & Hirsh, 1979). A basement membrane encases both the germline and somatic distal cells; no extracellular matrix is visible between the distal tip cell and the germ cells (Kimble & Ward, 1988). We suggest that the dpy and sqt-1 gene products participate in formation of the gonadal basement membrane.

The genetic interactions observed between glp-1 and the sqt-1 and dpy mutations may reflect molecular interactions between the glp-1 protein and elements of the extracellular matrix. An influence of the extracellular matrix on inductive interactions has also been proposed from a variety of studies that correlate changes in the composition of the extracellular matrix with changes in induction of developmental fate (reviewed in McClay & Ettensohn, 1987; Ekblom & Thesleff, 1985; Ekblom et al. 1986; Hay, 1983, 1984).

Furthermore, certain growth factors bind components of the extracellular matrix (Gordon et al. 1987; Schweigerer et al. 1987); the EGF-like domains found in the putative extracellular domain of glp-1 (J. Yochem & I. Greenwald, personal communication) may bind components of the extracellular matrix in an analogous fashion.

Fig. 4 shows one mechanism by which interactions between glp-1 and the extracellular matrix may influence the germline decision between mitosis and meiosis. In this model, the glp-1 protein stimulates mitosis directly and is present in both proximal and distal regions of the germline plasma membrane. Contacts between the extracellular domain of glp-1 and the extracellular matrix would block or inactivate glp-1 function so that these proximal germ cells enter meiosis. Distally, where no (visible) extracellular matrix is found between the distal tip cell and the germline plasma membrane, glp-1 would not be inhibited and therefore would be free to stimulate mitosis. Disruption or alteration of the extracellular matrix in suppressor mutants may release glp-1 protein located proximally and rescue the glp-1 phenotype by allowing this additional glp-1 protein to function. Alternative models, of course, remain possible. For instance, glp-1 may not act directly to stimulate mitosis. One mechanism by which it might indirectly stimulate mitosis is to anchor a positive regulator of meiosis to the extracellular matrix and hence to restrict that regulator from the distal region. By this second model, germline mitoses might continue in the distal region simply because the distal tip cell prevents contact between the extracellular matrix and the germline plasma membrane.

Fig. 4.

Model for interaction between glp-1 protein and the extracellular matrix. The distal gonad is schematized with glp-1 protein as an integral membrane protein in the germline plasma membrane. The space between the basement membrane and the germline plasma membrane is greatly exaggerated; in actuality, the plasma membranes of the distal tip cell and the germline are closely apposed. D = distal tip cell. See text for further explanation.

Fig. 4.

Model for interaction between glp-1 protein and the extracellular matrix. The distal gonad is schematized with glp-1 protein as an integral membrane protein in the germline plasma membrane. The space between the basement membrane and the germline plasma membrane is greatly exaggerated; in actuality, the plasma membranes of the distal tip cell and the germline are closely apposed. D = distal tip cell. See text for further explanation.

In summary, suppression of the glp-1 mutant phenotypes by dpy and sqt-1 mutations has led us to propose an interaction between the extracellular domain of glp-1 and components of the extracellular matrix. In the germline, we speculate that this interaction imposes spatial contraints on the decision between mitosis and meiosis. Although a similar process may also occur during embryogenesis, it is complicated by the fact that the embryonic function and suppression of glp-1 depends on maternal products. We therefore focus on the control of germline proliferation by the distal tip cell as an experimental model system for elucidating the function of glp-1 in mediating inductive cellular interactions.

We thank Jim Kramer for providing sqt-1 alleles. We are grateful to many of our colleagues, especially Judith Austin, M. Kathryn Barton, Tim Schedl, and Phil Anderson, for discussions during the course of this work and for critical reading of the manuscript. Leanne Olds did much of the technical illustration. We thank John Yochem and Iva Greenwald for communication of results prior to publication.

This research was supported by a US Public Health Service Grant GM31816 and Research Career Development Award HD00630 to J.K. E.M.M. was supported by Public Health Service Grant GM11569. Many nematode strains used in this study were provided by the Caenorhabditis elegans Genetics Center, which is supported by contract NO1-AG-9-2113 between the National Institutes of Health and the Curator of the University of Missouri.

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