A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis elegans

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1 Copyright by the Genetics Society of America A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis elegans Elizabeth A. Bucher and Iva Greenwald Department of Molecular Biology, Princeton University, Princeton, New Jersey Manuscript received November 0, 1990 Accepted for publication February 16, C ABSTRACT We have devised asimple genetic mosaic screen, whichcircumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific earlyblastomeres, suggesting thatmany zygotic genes contribute to specificearlyprocesses.we discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome. LASSICALLY, genes that have specific roles in development have been identified through analysis of mutant phenotypes. For example, the analysis of specific alterations in the complex cuticular patterns in mutant Drosophila embryos has successfully identified many zygotic genes (it?., the gene products are produced by the developing embryo itself) that control early development (NUSSLEIN-VOL- HARD and WIESCHAUS 1980) and anatomical and cell lineage analyses have been useful for characterizing mutants in C. elegans postembryonic development (for example SULSTON and HORVITZ 1981; GREENWALD, STERNBERC and HORVITZ 198). However, analysis of mutant anatomy has been of limited utility studying C. elegans early development because of the paucity of easily scored morphological markers and the impracticality of routine cell lineage analysis of embryonic mutants (for reviews see: WILKINS 1986; KEM- PHUES 1987; WOOD 1988). In addition to these essentially technical problems, there may in some cases be an additional problem of recognizing from the terminal phenotype that an essential gene has a defined role in specific developmental processes (for example, see WATERSTON 1989). For these reasons, we needed other criteria to distinguish between genes that are required for general processes such as intermediary metabolism and those required for more specific developmental processes such as determination, differentiation and morphogenesis. In this paper, we report the results of an alternative approach for identifying zygotic genes that may control early development in C. elegans. We developed a strategy to classify essential zygotic Genetics 128: (June, 1991) genes that does not rely upon terminal phenotype. The essence of our strategy is to determine if activity of an essential zygotic gene is required in all cells or only in specific cells for viability. Thus, we used genetic mosaic analysis to define the lethal foci of zygotic lethal mutations. Genetic mosaics contain defined cells that are genotypically mutant and defined cells that are genotypically wild type. The lethal focus is the cell(s) which, when mutant, causes the animal to die. The known, invariant early lineage (SULSTON et ul. 198) and the availability of appropriate, easily scored visible markers enabled us to use mosaic analysis to map the lethal focus to early embryonic blastomeres. We predicted that many genes that have general housekeeping functions would be recognized by the requirement for wild-type gene activity in any or all early blastomeres and/or their descendants for viability. We also predicted that we could define genes with specific developmental functions by the requirement for wild-type gene activity in only one or a subset of early embryonic blastomeres. Such genes would be interesting to us, since they might be genes that encode products unique to differentiated cells,genes that specify cell fates, or genes involved in other developmental processessuchas pattern formation and morphogenesis. In this paper, we discuss the implementation of this strategy, and what we learned from its application to a set of 20 zygotic lethal mutations. MATERIALS AND METHODS Strains and genetic analysis: General methods for the handling, culturing and ethyl methanesulfonate (EMS) mutagenesis of C. elegans are as described by BRENNER (1974).

2 282 E. A. Bucher and I. Greenwald EMS \ Po ncl uncglp; qdp f X glptsd c pick individual WT FI progeny ncl unc glpl+ + glpts; qdp WT ncl unc glp &I+ + glpts+ ; qdp WT ncl unc glp letl+ + glpts+ ; qdp WT ncl unc gp &ti+ + glpts + GlP ++glpts+;qdp WT + + GiP, ncl unc glp let; qdp WT F1 waself progeny of Po no linked lethal linked lethal (no Ncl Unc Glp class) FIGURE 1.-Screen for EMS-induced lethal mutations. glp-l(e2141ts); him-5(e1467) males (glpts) were crossed to mutagenized JK80 hermaphrodites. (The him-5 mutation was used only for male production; HODGKIN, HORVITZ and BRENNER 1979). F1 WT progeny were picked individually to Petri plates. The absence of the Ncl Unc Glp FP progeny indicated the presence of a linked lethal mutation (let).*wt Fv progeny were picked from candidate plates to isolate a ncl-1 unc-6 glp-1 let; gdp homozygote. We grew strains at 20" unless otherwise indicated. The C. elegans variety Bristol strain N2 was the wild-type parent for all strains. Strain JK80 ncl-i(e1865)unc-6(e25i)glp-i(q46); qdp (AUSTIN and KIMBLE 1987) provided the marked chromosome and the free duplication qdp to derivatives carrying EMS induced lethal mutations. Another allele of glp-i used was: glp-i(e214its) (PRIESS, SCHNABEL and SCHNA- BEL 1987); and on LGV the mutation him-5(e1467) (HODG- KIN, HORVITZ and BRENNER 1979) to construct the strain GS16 of the genotypeglp-l(e2141); him-5(e1467). Mutations were generated by the procedure shown in Figure 1. Twenty-six independent lethal alleles were generated: ar4, ar44, ar45, ar46, ar47, ar48, ar49, ar50, ar51, ar5, ar54, ar55, ar56, ar57, ar59, ar60, ar61, ar62, ar64, ar65, ar66, ar67, ar68, ar69, ar70, ar7. The lethal mutations were not assigned gene names. Throughout the selection, worms were maintained at 25" to recover any potential heat sensitive mutations. Mutagenized chromosomes carrying lethal mutations were backcrossed once during the F1 isolation strategy and then a second time to glp-l(e2141ts); him-5(el467) males. The lethal mutations are complemented by qdp (an approximately -5 map unit interval) and have not been mapped more precisely to LGIII. Complementation tests were performed by crossing individual lethal strains to N2 males. The males from this cross having the genotype ncl-1 unc-6 glp-1 Let/++++ were crossed to the hermaphrodites of the control strain JK80, and of all 26 lethal strains. The presence of Ncl Unc Glp cross progeny indicated that the alleles complement. The absence of any Ncl Unc Glp cross progeny indicated failure to complement. Reciprocal complementation tests were performed to confirm these results. The stage at which ncl-i unc-6 glp-i let animals arrest or die was determined in synchronized populations of embryos. Ten gravid hermaphrodites were placed on solid media for 12 hr and then removed. The progeny were scored approximately 15, 24, 6 and 48 hr later (all embryos should have hatched by 14 hr after being laid) for the presence of dead eggs and/or larvae. The stages were confirmed under Nomarski optics. Mosaic analysis: Living mosaic animals identified under the dissecting microscope were observed under Nomarski differential interference contrast microscopy as described bysulston and HORVITZ (1977). We specifically scored: body wall muscle nuclei derived from ABpr, MS, C and D; pharyngeal muscle nuclei derived from ABa and MS;MS derived coelomocytes; the excretory cell nucleus derived from ABpl; neuronal nuclei in the head and some hypodermal nuclei (SULSTON et al. 198). Two double mosaics (two observable independent duplication losses within the same animal) and 9 consecutive mosaics (loss of the duplication in two consecutive divisions) were observed. Recombinant animals of the following genotypes were occasionally found while screening for mosaic animals: (1) ncl-i unc-6 glp-1, (2) ncl-i unc-6, () unc-6 glp-1, (4) unc-6, (5) ncl-i glp-i. glp-i recombinants probably also occurred but we could not confirm their genotype (see RESULTS section) and, in fact, increased recombination at the ends of qdp or terminal deletions of qdp (HERMAN 1984) may account for the high number of potential P4 lossesas compared to duplication losses in the other divisions, or relatively few isolates of the other recombinant classes (see above). Mosaic analysis has not been completed on: ar46ts, ar50, ar60, ar61 and ar68. Mosaic analysis cannot be done on ar70, a mutation that fails to complement ar51. Although ar70 is slow growing, at high frequency viable Ncl Unc Glp animals are seen, suggesting that ar70 is a hypomorphic allele of ar51. ar46ts is a heat-sensitive allele: culturing at 20" results in adult Ncl Unc Glp progeny whereas culturing at 25" results in arrested L1 larvae. We expect that some of our mutations may have maternal effects (see RESULTS and DISCUSSION). In the absence of conditional mutations, one way to test for maternal contributions is to generate viable mosaics unable to provide maternal products to the embryo. This approach is not possible in existing strains, since the glp-i mutation results in the absence of a germ line. In addition, the strains do not

3 Zygote ' r, unc-6 As E I, I focus, intestine Screen Genetic Mosaic 28 germ line g'p" zygotic focus FIGURE 2,"Early embryonic lineage (modified from SULSTON et al. 198). The numbers and types of cells derived from each of these founder cells at hatching are listed below each founder cell. The foci of unc-6 (ABp; KENYON 1986) and glp-1 (P4; AUSTIN and KIMBLE 1987) are indicated. For other information, see text. contain a marker for EMS, which gives rise to the intestine and somatic gonad, likely sources of maternal products in addition to P4 (e.g., yolk is synthesized in the intestine; KIMBLE and SHARROCK 198). Even if the strains did carry appropriate markers, most of the lethal mutations have a lethal focus in EMS.Since we are interested in essential zygotic genes independent of whether they are also required maternally, this is not relevant to our screen. RESULTS Early embryonic cell divisions: The C. elegans embryonic lineage (pattern of cell divisions and cell fates) is essentially invariant (SULSTON et al. 198). Figure 2 shows a simplified early lineage. Asymmetric and asynchronous divisions of the zygote, PI, PB, Ps and EMS give rise to the 6 founder cells AB, MS, E, C, D and P4. ABa and ABp are the anterior and posterior daughters of AB. In this paper, we use the term "early blastomeres" to include ABa and ABp as well as the six founder cells. The invariant lineage enables precise knowledge of the numbers and types of cells derived from each early blastomere at any point in development. Zygotic lethal mutations complemented by qdp: We screened for mutations in essential zygotic genes in a portion of linkage group 111 (LGZZZ) that is complemented by the free duplication qdp (AUSTIN and KIMBLE 1987). We chose this region for two reasons. First, qdp, like other free duplications, is a chromosomal fragment that is generally maintained during mitotic division; however, occasional mitotic loss results in genetic mosaicism (see below). Second, qdp complements the major gene "cluster" of LGIZZ (MA- TERIALS AND METHODS), including recessive cell autonomous mutations in the ncl-i, unc-6 and glp-1 genes. unc-6 and glp-1 mutations cause defects visible under the dissecting microscope. The unc-6 mutation results in an uncoordinated (Unc) phenotype (BRENNER 1974), and theglp-1 mutation results in a sterile, germ line proliferation defective (Glp) phenotype (AUSTIN and KIMBLE 1987; PRIESS, SCHNABEL and SCHNABEL 1987). The ncl-i mutation results in enlarged nucleoli (Ncl phenotype) and is visible under Nomarski differential interference contrast optics (E. HEDCECOCK, personal communication; HERMAN 1989). These mutations were useful for the isolation of lethal mutations and for subsequent mosaic analysis. Animals of the genotype ncl-1 unc-6 glp-1; qdp (strain JK80) are phenotypically wild type since qdp complements the recessive markers. Figure shows the expected self progeny of a hermaphrodite of this genotype: approximately 70% of the self-progeny have qdp and are phenotypically wild type, and 0% lack qdp and have enlarged nucleoli, are uncoordinated, and sterile (Ncl Unc Glp). Figure also shows the expected self progeny of a hermaphrodite of genotype ncl-1 unc-6 glp-i let; qdp, containing a recessive zygotic lethal mutation (let) complemented by qdp. The presence of a let mutation on the marked chromosome can be easily recognized by the absence of Ncl Unc Glp self progeny (Figure ). These lethal mutations are easily maintained since they are complemented by qdp. We isolated 26 recessive zygotic lethal mutations by the screen described in MATERIALS AND METHODS and Figure 1. Complementation tests (MATERIALS AND METHODS) showed that the 26 lethal mutations fall into 22 complementation groups. The fact that most complementation groups are identified by single alleles suggests that we are far from saturation of this region of the genome for zygotic lethal mutations. Nevertheless, these mutations provide a good sample for applying the genetic mosaic screen. The approximate stage of arrest or death was de- termined by synchronizing populations of embryos and observing the terminal stage of progeny having the genotype ncl-i unc-6glp-1 let under the dissecting microscope and Nomarski optics (MATERIALS AND METHODS). We observed three general terminal stages. Most mutants die as a mixed population of embryos and larvae; four mutations (ar46, ar50, ar58 and ar60) are strict embryonic lethals, and three mutations (ar44, ar64 and ar67) cause death as L1 larvae. The terminal stages of the mutations complemented by qdp result from the zygotic gene requirement. It is possible that qdp supplies an essential maternal contribution (ie., the gene products provided by the mother to the embryo) and removal of the maternal dose may result in an earlier time of lethality. In fact, we expect that maternal expression is required for many of our essential genes since previous studies of temperature sensitive maternal effect mutations revealed that in addition to early embryonic lethality, most maternal effect mutations have late embryonic or postembryonic zygotic requirements (HIRSH and

4 284 and E. A. Bucher Starting strain JK80: ncl-i uric-6 g41-i; qdp 1. Greenwald Derivatives carrying linked, EMS-induced lethal mutations i -70% ncl-1 unej6glpl; qdp -0% ncl-i uncj6glp-1-70% ncl-1 unc-6glpl let; qdp -0% ncl-1 uncl-6glpl kt phenotypically WT phenotypically mutant phenotypically WT DEAD lothll balanced by qdp la. no Nd Une Clp segregants FIGURE.--Segregation of qdp to self-progeny in a strain having the genotype ncl-i unc-6 glp-i; qdp (strain JK80). ncl = ncl-i(e1865); unc = unc-6(e251); glp = glp-i(q46). Approximately 70% of the self-progeny contain qdp, which complements the recessive mutations, and are phenotypically wild type (WT). 0% of the progeny lack qdp and are phenotypically mutant (Ncl Unc Glp). Lethal mutations (let) that are linked to the recessive markers and complemented by qdp (genotype ncl-i unc-6 glp-i let; qdp) can be identified by the failure to segregate Ncl Unc Glp progeny. VANDERSLICE 1976; VANDERSLICE and HIRSH 1976; MIWA et al. 1980; WOOD et al. 1980; CASSADA et al. 1981; DENICH et al. 1984). We were not able to assess if the lethal mutations we generated have a maternal effect (see MATERIALS AND METHODS), but it is irrelevant for the screen described below. Strategy for using mosaic analysis to classify essential zygotic genes: We wanted to analyze all of these lethal mutations, including those that result in postembryonic death, since disruption of an early developmental event may not be manifested until later developmental stages. To analyze further these mutations, we developed a genetic mosaic screen to distinguish among essential zygotic genes independent of terminal phenotype. As described below, the screen relies on mosaic analysis to define the lethal focus of a let mutation, i.e., the cell(s) which, when mutant, results in death. Mosaics were generated by the spontaneous rare mitotic loss of qdp during embryonic cell divisions (HERMAN 1984, 1989). The result of this rare somatic loss is a mosaic individual in which defined cells are genotypically let and defined cells are genotypically let(+). We first examined the parental strain JK80 (ncl-1 unc-6 glp-1; qdp) for rare mosaicsby screening under the dissecting microscope using the linked visible unc-6 and glp-1 markers. An essential aspect of the mosaic screen is that the foci of unc-6 and glp-1 are in different, nonoverlapping parts of the lineage (see Figure 2). The unc-6 focus is in the early blastomere ABp, so if qdp is absent from AB or ABp, the animal is Unc, but if qdp is absent from PI or its descendants, the animal is non-unc (KENYON 1986). The glp-1 focus is in P4, so if qdp is absent from AB, the animal is fertile, but if qdp is lost in PI, Pp, Ps or P4, the animal is Glp (sterile) (AUSTIN and KIMBLE 1987). Thus, random loss of qdp during early embryonic cell division in the parental strain results in Unc non-glp mosaic animals (if qdp loss occurred in AB or ABp) or Glp non-unc mosaic animals (if qdp loss occurred in PI, Pp, PS or P4). The ncl-1 marker enables the division at which the duplication was lost to be precisely defined: the nucleoli of cells retaining qdp are wild type, whereas nucleoli of cells lacking qdp are enlarged. Figure 5 and Table 1 show that we indeed recovered all possible identifiable mosaics resulting from losses at each early cell division in the parental strain. The presence of a let mutation in the strains may affect the mosaic types arising in strains of the genotype ncl-1 unc-6 glp-1 let; qdp. We expected that most strains containing let mutations would not yield all the mosaic types observed in the parental strain, since viable animals must require let(+) activity in all or part of the lineage. The pattern of duplication loss for each let mutation would, therefore, indicate the lethal focus, ie., the cell(s) which, when mutant, cause the animal to die. We expect that many of these lethal mutations reduce or eliminate gene activity, so that the lethal focus may also be considered to be the cell(s) that require gene activity for the animal to survive. The genetic mosaic screen classifies mutations based on their lethal foci. We reasoned that gene activities required in all lineages might be involved in general processes, whereas gene activities required inonly defined parts of the lineage might be involved in specific developmental processes. Figure 4 illustrates the three classes of let mutations we predicted that we

5 ~ Genetic Mosaic Screen 285 Lethal No. alleles screened ABP AB (A) Class I mutations (unrestricted foci) Control 175,000 6 ar45 195,000 ar47 215,000 ar54 218,000 Aar65 188,000 Aar69 250,000 Array64 215,000 AAar67 195,000 (B) Class 111 mutations (restricted foci) Control 175,000 6 or56 225,000 a ,000 ar62 224,000 ar57 187,000 ar4 215,000 ar48 212,000 ar5 22,000 ar59 24,000 ar55 285,000 ar66 210,000 ar7? 28,000 1 ar51 160,000 5 (C) One complementation group with class I and class 111 alleles Control 175,000 6 AAar44 150,000 AAar64 215,000 Array67 195,000 TABLE 1 Mosaics obtained from JK80 and derivatives carrying let alleles Unc ABpr ABpl P, pz PS PI " 2 LVNT IV 1" 22v 6 5 f Y T IV 2 21V (M Sx?) 1 1 (MSa) 1 '" 1 VNT The lethal allele and the number of adult hermaphrodites screened is indicated in the left column. Unc or Glp indicates the visible phenotype by which each mosaic was initially detected. The first blastomere found to be missing qdp, based on the Ncl phenotype of representative descendants (MATERIAIS AND METHODS) is indicated just below the Unc and Glp designations. The numbers listed below the founder cells (and ABpl and ABpr) indicate the number of mosaics of that class identified. For example AB indicates that qdp was lost in the first division such that AB and its descendants were missing qdp? but that P, retained qdp?, as ascertained by the Ncl phenotype. We organized the alleles by placing mutations having duplication losses in similar lineages in close proximity. The number of Glp animals that lacked qdp in PI was disproportionately high in the sample screened for JK80 compared to a previous study (AUSTIN and KIMBLE 1987), so we believe this high number is a statistical fluctuation. Since we could not distinguish recombinants from true P4 losses (see Figure 5), all candidates are included in the data in the table. The number of potential Pq losses is high, probably because it includes recombinants and terminal deletions (HERMAN, 1984) as well as true mosaics. The results of the complementation tests are shown: alleles that fail to complement are indicated by similar symbols, A, AA, AAA. * indicates consecutive mosaics in which the duplication was lost in consecutive divisions (HERMAN 1984, 1989), and thus lack the duplication in C and P4 but not D. The number of * indicates the number of consecutive mosaics. We found several "semi-unc" mosaics in let containing strains resulting from losses of qdp in ABpr or ABpl cells. Potential semi-unc mosaics were probably missed in the control strain since semi-uncs are harder to identify when Ncl Unc Glp animals are present (Figure ). Some viable mosaics had morphological abnormalities, indicated as superscripts, in the vulva (V), nose (N) and/or tail (T). The strain containing ar4 segregated one Glp non-unc that lacked qdp in MSap as well as in Pq suggesting that the lethal focus must be elsewhere in EMS. ar7 segregated a mosaic which was uncoordinated and unusually small and in which qdf was lost within the MS lineage. We could not determine the precise point of loss within MS, and specific abnormalities including gaps at the cuticle were evident. The strain containing ar51 gave rise to viable mosaics lacking qdp in AB resulting in abnormal nucleolar morphology (see text). We fortuitously found an MS-derived mosaic for ar51, which exhibited similar abnormalities. would find. A class I mutation would not have a class I11 mutation would have a restricted focus: lerestricted focus: no early blastomeres may be mutant, thality results only if a specific early blastomere is so viable mosaics would not be isolated since loss of mutant so only asubset of mosaictypeswouldbe gene activity in any part of the lineage will be lethal. obtained. The pattern of duplication losses, and es- A class I1 mutation also would not have a restricted pecially the failure to find viable animals having dufocus, but in this case, any early blastomere may be plication losses within a particular lineage, should mutant, so all mosaic types would be isolated since identify the lethal focus. Although we note that some loss of gene activity in any part of the lineage would classi or class I1 mutations may define genes with be compensated for by presence in another part. A developmental roles, and that some class I11 mutations 10 * * 1 * 2 1 I* * GlP 6* 5* 6** *

6 286 E. A. Bucher and I. Greenwald Control: J KBM Class 111: ars6, ar49 (B) Class1 Po pa AB No mosaics (C) classn class Ill: 065 P AB P All mosnic types Aid ABm Class In: ur7 P Class Iff: ur51 I class III: ad4 (D) Class 111 (example) PO AB D P4 Subset of mosnic types FIGURE 4.-Predicted patterns of duplication loss yielding viable mosaics in strains of the genotypes ncl-i unc-6 glp-i; qdp and ncl- I unc-6 glp-i let; qdp. Solid lines indicate early divisions at which duplication loss is lethal to an animal. Slashed lines indicate early divisions at which duplication loss is not lethal. A, JK80 has the genotype ncl-i unc-6 glp-1; qdp. Viable mosaics can be recovered after duplication loss in any early blastomere. B, Class I mutations cause lethality if let(+) activity is missing in any early blastomere and do not have restricted foci. No viable mosaics are expected (except mosaics lacking qdp in P4; see RESULTS). C, Class I1 mutations cause lethality only if let(+) activity is missinginall early blastomeres and do not have restricted foci. Viable mosaics can be recovered if the duplication is lost anywhere in the lineage, as is observed in JK80. D, Class 111 mutations cause lethality only if let(+) activity is missing in a specific part of the lineage and have restricted foci. A specific pattern of the inability to lose the dupliration defines the lethal focus. In the example shown, the lethal focus is in P,, that is the duplication may be lost in AB but not in PI. (A class Ill mutation may have a non-essential gene function in addition to the essential function, which may be revealed by additional non-lethal phenotypes in the mosaic animals.) may define genes with metabolic functions, we believe this classification provides a valid criterion to identify genes involved in cell-type and/or lineage specific processes for further study. Classification of 20 lethal mutations by mosaic analysis We used mosaic analysis to classify 20 mutations as either having a restricted focus (class 111) or not having a restricted focus (classes I and 11). In addition, we further distinguished among class I11 mutations by different patterns of duplication loss, defining different lethal foci. The data for the twenty mutations are FIGURE 5.-Pattern of duplication loss in strains carrying let mutations. The patterns of duplication loss are schematically represented as described in Figure 4 for the control strain JK8O (ncl- I unc-6 glp-i; qdp) and for individual let mutations for strains of the genotype ncl-i unc-6 glp-i let; qdp. The slashed lines represent positions where qdp may be lost to yield cells of genotype ncl- I unc-6 glp-i let (ncl-i unc-6 glp-1 for JK80) in viable mosaic animals. Solid lines indicate the lethal focus, i.e., positions at which qdp cannot be lost [cells must be let(+)] without resulting in lethality. Solid lines marked with a * indicate a semilethal focus, Le., a rare duplication loss can occur (see RESULTS and Table IB): the frequency is significantly lower than the control loss frequency, suggesting an essential function in that lineage. For this reason ar7 may be a class I mutation. (Note that in this paper a statement that the duplication can be lost in a founder cell with no deleterious effect will also imply that it can also be lost in the descendant of that founder cell.) The data are shown in Table 1. We expected that all strains would give rise to viable mosaics that had duplication loss in P4, since P4 gives rise solely to the germ line, and laser ablation experiments have demonstrated that P4 is not necessary for viability, but only for fertility (SULSTON et al. 198). This mosaic type is of no consequence to the mosaic screen. Furthermore, we cannot determine the Ncl phenotype in P4 (AUSTIN and KIMBLE 1987) and these Glp non-unc animals could be either mosaics or recombinants (Glp animals cannot be progeny tested since they are sterile). Since we could not distinguish recombinants from true Pq losses, all candidates are included in the data in Table 1. We expected that a low frequency of viable mosaics lacking the duplication in P would also be observed, since occasional animals hatch after laser ablation of P1. Pa is thought to be important for viability not because of the 20 muscle cells that it generates (SULSTON et al. 198), but because of its influence on spatial cell patterning necessary for proper embryogenesis (E. SCHIERENBERG, personal communication). shown in Table 1 and schematically represented in Figure 5. Class I mutations (no restricted foci): The pattern for 7 mutations (ar45, ar47, ar54, ar64, ar65, ar67, ar69) is strikingly different from the parental pattern (Table 1 and Figure 5). We never isolated Unc non-

7 Screen Mosaic Genetic 287 Glp mosaics. The Glp non-unc animals isolated never had mutant Ncl nuclei in MS, C, or D derived cells, suggesting that loss of qdp? occurred in the P4 founder cell or that these animals were recombinants or terminal deletions of qdp? (HERMAN 1984). Since no viable mosaics were identified we conclude that these are class I mutations. Statistical analysis supports the assignment of these mutations to class I: chi square analysis for small numbers (STANSFIELD 198) was performed for each mutation, using as the null hypothesis that no difference exists between the frequency of qdp? loss in the control and lethal-bearing strains for a particular early blastomere (data not shown). The failure to isolate AB or ABp losses when 175,000 animals are scored is significant (P < 0.025) and the failure to isolate P2 or Ps mosaics is also significant (P < 0.05). These values are in fact underestimates of the significance of failure to find mosaics since we screened more animals in most of the lethal strains than in the parental strain. Complementation tests show that these seven mutations represent five genes (MATERIALS AND METHODS and Table 1). Class I1 mutations (no restricted foci): We did not identify any class I1 mutations but other investigators have (B. WIGHTMAN, D. GREENSTEIN and G. RUVKUN, personal communication). Class I11 mutations (restricted foci): We identified 1 class I11 mutations (ad?, ar44, ar48, ar49, ar51, ar5?, ar55, ar56, ar57, ar59, ar62, ar66 and ar7). These 1 mutations complement one another and exhibit 9 distinct mosaic patterns (Table 1 and Figure 5), which represent at least four distinct foci (see DISCUSSION). For example, strains containing ar56 or ar49 must retain qdp? in AB, but not in PI for viability, so the lethal focus is inab. Three other mutations, ar57, ar48 and ar4, are lethal if the duplication is lost in AB or EMS, but not in P2, so the lethal focus is inab and EMS. The failure to get duplication losses in specific lineages is significant by chi square analysis (for low frequency mosaics see below). These results demonstrate that not only can we distinguish class I genes from class 111 genes, but we can also distinguish among class 111 genes based upon differential requirements (distinct restricted foci) within the embryonic lineage. The class 111 mutation ar44 fails to complement the class I mutations ar64 and ar67 (Table 1C). The different foci of ar44 and ar64 or ar67 may illustrate a potential problem of the genetic mosaic screen: alleles that do not greatly reduce or eliminate gene activity may appear to be class I11 mutations if different blastomeres require different levels of gene activity. Thus, ar44 may partially reduce the activity of a gene that is required in all early blastomeres, while ar64 and ar67 may be null alleles of that gene; if ABp requires less gene activity, then residual activity of ar44 may allow animals that have lost the duplication from ABp to survive. Alternatively, ar44 may specifically affect gene activity in PI and not in ABp, for example by affecting one domain of the protein. Mosaics that arise at low frequency: A surprising number of strains containing class 111 mutations segregated some viable mosaics lacking let(+) in ABp at a lower frequency than the control strain. The low frequency of these mosaics is a common feature of all of the strains that segregate mosaics lacking let(+) in ABp with the exceptions of strains containing ar51 and ar44. The low frequency suggests that these genes have essential activities within AB and Poisson analysis suggests that this low frequency recovery of mosaics is meaningful. In addition, only some strains containing class 111 mutations that segregate mosaics lacking let(+) in ABp mosaics do so at low frequency, and the strains that do, segregate mosaics lacking let(+) in P2 at control frequencies. We therefore think that this result suggests that these mutations do not have a fully penetrant lethal focus in ABp: some animals lacking the duplication in ABp die, while others live. We term this behavior a semilethal focus, which suggests that gene activity is at least partially required in an early blastomere. We consider a semilethal focus as indicating an essential function for a gene in that early blastomere. In these cases, there is a semilethal focus in ABp and lethal foci in ABa and EMS, implying that let(+) functions in ABp, ABa and EMS.Also, ar59 might actually be a class I mutation, since it has a lethal focus in P1 and in ABa, and a semilethal focus in ABp. Alternatively, a semilethal focus may reflect an underlying variable penetrance or expressivity of the lethal mutations, which may be true even if the mutation is null. At present it is difficult to interpret the meaning of a semilethal focus. Additional phenotypes observed in viable mosaics: Viable mosaics for several class I11 mutations had additional phenotypes. This observation emphasizes how mosaic analysis may be able to distinguish different function in different places (and in principle, at different times) during development. In particular, the strain containing ar51 segregated viable mosaics displaying a specific and reproducible phenotypic abnormality. Although the ar51 lethal focus is in PI, it has a nonessential function in AB (Table 1 and Figure 5). This non-essential function is revealed since all viable mosaics that lack qdp? in AB, ABp, ABpl or ABpr display a pitted appearance instead of the characteristic Ncl phenotype in let(ar51) nuclei (data not shown). (Nuclei that could be scored as Ncl subsequently exhibit this pitted appearance.) We do not know whether the nonessential function in AB is similar to the essential function in P1. In this regard, a single mosaic lacking qdp in MSa was fortuitously found. This mosaic did not have a gonad and exhib-

8 288 E. A. Bucher and I. Greenwald ited large gaps, especially at pharyngeal and body wall muscle positions, that were reminiscent of the phenotype ofviablemosaics lacking qdp inab.we conclude that ar51 is a class 111 mutation having an essential function in PI and a nonessential function in AB. Many strains containing class 111 mutations with semilethal foci in ABp (ar5, ar59, ar66 and ar7) and ar44 segregated some viable mosaics that lacked let(+) in AB or its descendants that exhibited variable vulval, nose and tail abnormalities (Table 1, B and C). Since these phenotypes occur in mosaics of five different complementing mutations and are variable, these mutations maymay be an indirect effect on vulval, nose or tail development. These major morphological structures in the wormmightbe more sensitive to perturbations in a variety of processes by different types of mutations or perhaps are more readily apparent upon inspection than more subtle anatomical defects (see also DISCUSSION). More interestingly, it is possible that some of these abnormalities directly reflect the requirement of let(+) function for vulval development. For example, ar66 mosaic individuals lacked a fully formed vulva. An important class of vulval gene has essential functions but was initially defined by non-null alleles; this class includes let-60, which is now known to encode a ras homolog (HAN and STERNBERC 1990; HAN, AROIAN and STERNBERC 1990; BEITEL, CLARK and HORVITZ, 1990). The null phenotypes of several genes affecting vulval development that map in the region complemented by qdp are not known (FERGUSON and HORV- ITZ 1985, 1989). Some of the mosaics exhibiting vulval defects also had nose and tail morphological abnormalities, characteristics not previously reported for genes having specific roles in vulval development; however, since mosaic analysis lethal of alleles of genes such as let-60 has not been done we cannot comment on the relevance of these additional phenotypes. DISCUSSION We developed the genetic mosaic strategy to circumvent the difficulties of phenotypic analysis of zygotic lethal mutations. We reasoned that class 111 mutations, which have restricted foci (the animaldies only if a defined subset of early blastomeres is mutant) might define genes involved in cell type- and/or lineage-specific developmental processessuchas determination, differentiation or morphogenesis. For example, we would expect that mutations in essential genes involved in muscle development, including genes encoding potential transcription factors (KRAUS et al. 1990) or muscle components suchasmyosin heavy chain A (WATERSTON 1989), would have a lethal focus in PI, the progenitor of 80 of the 8 body 1 wall muscle cells, and would not have a lethal focus in AB, the progenitor of only 1 body wall muscle cell. Conversely, we would expect that mutations in some genes that encode proteins involved in general processes would have a lethal focus in any early blastomere, i.e., be class I mutations without a restricted focus (but see also below). Most zygotic mutations in our set have restricted lethal foci: Based on the fact that most essential genes function in both embryogenesis and later stages (HIRSH and VANDERSLICE 1976; VANDERSLICE and HIRSH 19 76; SCHIERENBERC, MIWA and VON EHREN- STEIN 1980; MIWA et al. 1980; WOOD et al. 1980; CASSADA et al. 1981) and the known complexity of cellular metabolism, it was proposed that most essential zygotic genes would be involved general in housekeeping processes. The recent finding that many genes are transcribed throughout embryogenesis has also been interpreted as suggesting that most zygotic genes are involved in housekeeping (SCHAUER and WOOD 1990). Our results suggest an alternative view. We found that 1 of20 lethal mutations analyzed have restricted lethal foci. Curiously, this proportion is comparable to findings in Drosophila, where 60% of the lines containing reporter gene insertions asso- ciated with recessive zygotic lethal mutations have cell type specific expression (BIER et al. 1989). If weassume that most of the mutations in our set are null or near null and account for the results of the complementation tests, we conclude that we have identified 5 genes required in all early blastomeres and 12 genes required in only specific blastomeres. Thus, the genetic mosaic screen suggests that many essential zygotic genes have specific functions and are not necessarily involved in general housekeeping. At present, there is no way to resolve the two different interpretations of the contribution of zygotic gene activity to early C. elegans development. In some cases, it may be that housekeeping genes have spe- cific foci if they have functionally redundant counterparts expressed or active in different cell types. However, insomecases, a developmental gene may be required more than one time during development; if it functions at different times and/or in different cells during embryogenesis, then the sum of its activities may appear general. Maternal us. zygotic gene requirements: It has beendifficult to distinguishbetweengeneshaving specific developmental roles from housekeeping genes. KEMPHUES et al. (1988) reasoned that strict maternal-effect or strict zygotic genes are good can- didates for genes having specific developmental roles. The majority ofknowngenes,which are required both maternally and zygotically may encode housekeeping functions (HIRSH and VANDERSLICE 1976; VANDERSLICE and HIRSH 1976; MIWA et al. 1980; WOOD et al. 1980; CASSADA et al. 1981). Analysis of

9 Screen Genetic Mosaic 289 strict maternal-effect mutations did in fact reveal some mutants that had informative phenotypes consistent withspecific functions in earlycleavages and cytoplasmic partitioning (KEMPHUES et al. 1988; SCHNABEL and SCHNABEL 1990a). We note that in these and in certain other studies terminal phenotypes may be informative (e.g., PRIESS, SCHNABEL and SCHNABEL, 1987; SCHNABEL and SCHNABEL 1990b). However, it remained possible that some genes required both maternally and zygotically do play specific roles, but cannot be recognized in the absence of good phenotypic criteria. The genetic mosaic screen offers an approach to identifying such genes, since it enables the zygotic contribution to be analyzed independently of any maternal contribution and, as described above, does not rely on phenotypic criteria. Thus, the focus of any gene that acts zygotically, either strictly or in combination with a maternal component, may be defined and used to classify that gene as potentially involved in a specific process or in a general one. Limitations of the mosaic screen: We encountered one example of a limitation of this genetic mosaic screen: mosaic analysis of non-null alleles may be misleading if different early blastomere lineages have quantitively different requirements for a gene activity, either because a given lineage may require less activity or a possible maternal contribution is sufficient to bypass the zygotic requirement (see also RESULTS). We expect that many of the lethal mutations greatly reduce or eliminate gene activity, and that the classifi- cation of these mutations reflects the type of gene activity. However, while this expectation may be true for the collection, it may not be true for any given mutation in our collection. Thus, some of the mutations interpreted as class 111may be non-null alleles of class I genes. It is also possible that some mutations we have classified as class I may actually be alleles of class 111 genes. One reason that we might not have been able to recognize these mutations is that they may be inviable in large clones but are viable in small clones, ashasbeenobserved for somelethal mutations in Drosophila (RIPOLL 1977). At present, we do not have convenient visible markers complemented by qdp which would enable us to recognize smaller clones. Another possible reason is that the invariance of the lineage means that certain combinations of mutant blastomeres cannot be obtained by single duplication loss events. Furthermore, mosaic analysis using different lineage markers with different foci may allow us to assess if only a subset of cells within the early blastomeres require the gene activity. The unc-?6 and glp-1 mark- ers have foci in the early blastomeres which enables us potentially to score early requirements, however it does not allow us to assess more limited requirements within the lineages. Currently, we are limited by the availability of lineage markers. Analysis of LGZZZ and the rest of the genome: We specifically designed our strategy for the LGZZZ region of the genome complemented by qdp. The foci for the useful lineage specific markers unc-6, ncl-1 and glp-1 had been defined (KENYON 1986; AUSTIN and KIMBLE 1987; E. HEDCECOCKpersonalcommunication), the ordering ofthis region of LGZZZ into a physical map (COULSON et al. 1986, 1988) would allow rapid cloning of genetically defined genes, and about 80% of the mapped genes on LGZZZ are complemented by qdp? (EDGLEY and RIDDLE 1989). In addition, qdp had been successfully used for mosaic analysis with a measured loss rate of about 1/000 cell divisions (AUSTIN and KIMBLE 1987; HERMAN 1989). Unfortunately, we found that the rate of qdp loss in our strains is an order of magnitude lower than previously published rates (see Table l), so applying this screen until saturation of zygotic lethal mutations is achieved is not practical. We note that the apparent stabilization of qdp that we observed has also been observed for another free duplication, sdp (B. WIGHTMAN, D. GREENSTEIN and G. RUVKUN, personal communication; E. BUCHER, unpublished observations). In addition, the rate of qdp loss appears to be different in different lineages (e.g., SEYDOUX and GREENWALD 1989). Other duplicationscoveringall or part ofthis region mayallow saturation to be achieved. This mosaic strategy can also be applied by other investigators to classify additional zygotic mutations throughout the entire C. elegans genome. Free duplications are available or can be generated that complement regions of the genome having appropriate mark- ers for mosaic analysis (also see: HERMAN 1984, 1989; MCKIM and ROSE 1990; EDCLEY and RIDDLE 1989). Alternatively, techniques are now available to generate free duplications that have desired markers (R. HERMAN, personal communication). For example, the cloning of the ncl-1 gene should allow attachment of this marker to free duplications complementing any part of the genome. Strategies that do not rely upon terminal phenotype to identify genes involved in specific develop- mentalprocesses: BRYANT and ZORNETZER (197) used mosaic analysis to analyze zygotic lethal mutations in Drosophila for a purpose similar to ours. They found that 40% of zygotic lethal mutations did not generate viable gynandromoph (equivalent to our class I) and defined a subset of alleles that generate viable gynandromorphs that alsohavespecificfoci (equivalent to our class111). The gynandromorph approach, however, has not been useful as a routine screen for characterization of zygotic lethals in Drosophila since the gynandromorph mosaic technique

10 290 E. and A. Bucher takes much more time than inspecting terminal phenotypes, especially since many gynandromophs have to be generated and carefully mapped to determine specific foci. In contrast, in C. elegans, the duplication loss mosaic technique is easy to apply (especially if the duplication loss frequency is high), and the invariant lineage means that the duplication loss patterns automatically provide unambiguous assignment of the lethal focus with resolution depending on the markers used. Strategies that rely on timing and/or pattern of gene expression also do not rely on terminal phenotype. For example, in Drosophila, the enhancer trap strategy relies upon random insertion of an enhancerless P element reporter gene construct in the genome, which is not necessarily associated with mutation (O KANE and GEHRING 1987; BELLEN et al. 1989; BIER et al. 1989; WILSON et al. 1989; MLODZIK et al. 1990). Expression of the reporter gene construct is influenced by nearby enhancers and thus may identify developmentally regulated genes. Many tissues and stages can be scored for the spatial and temporal distribution of reporter gene activity, but in contrast to mosaic analysis, the enhancer trap method does not directly assay where the gene activity is essential. The mosaic screen is based on gene function, but does not define the lethal focus beyond the early blastomere cell lineages, because of the foci of the markers that enable a rapid visual screen. Ideally, the enhancer trap and mosaic strategies would be combined in C. elegans. If mutations were generated using a transposable reporter gene, the expression pattern could be analyzed using the reporter activity and the focus of gene activity could be determined by mosaic analysis. Interpretation of class I11 mosaic patterns: The numbers and types of mosaics identified at least 4 different foci for the class 111 mutations. The duplication loss patterns show that three mutations have lethal foci in AB (ar49, ar56, ar62), seven mutations have lethal foci in AB and EMS (ar4, ar48, ar5, ar55, ar57, ar66, ar7), the ar51 lethal focus is in P1 and the ar44 lethal focus is in ABa and EMS. These patterns show that most (12/1) mutations have a lethal focus within AB, which may reflect the fact that the AB blastomere gives rise to 89 out of 58 cells, or approximately 72% of the embryo (SULSTON et al. 198). In addition, many of these (8) also have a focus in EMS. We examined the lethal foci in terms of the cell types that the early blastomeres generate (see Figure 2) to see if we could predict possible functions for the genes. The gene activity may be autonomous (the genotypically mutant cells exhibit a mutant phenotype) or nonautonomous (the presence of genotypically mutant cells causes other genotypically wild-type cells to exhibit a mutant phenotype). For this discus- I. Greenwald sion we will assume that most gene activities will be autonomous. For example, mostclass I11 mutations have foci in AB and EMS. This observation may be a secondary consequence of the fact that the C and D blastomeres give rise to fewer descendants and hence may need less gene activity (see above). However, it might also indicate that these mutations define genes that function in cell types descended in common from those blastomeres, such as neurons. In this context, it is worth noting that many of the Drosophila enhancer trap strains with specific expression patterns were expressed in neurons (O KANE and GEHRINC 1987; BELLEN et al. 1989; BIER et al. 1989). However, the simple interpretation that mutations with foci in AB and EMS define genes required for neural development or function is complicated by the hypodermal defects often observed in mosaics for these mutations (see RESULTS). Since common cell types originate from AB, MS and C, mutations with a lethal focus only in AB (ar56, ar49, ar62) may define genes required in neuronal and hypodermal cell types descended only from AB. This prediction is plausible because only a few neuronal and hypodermal cell types are descended from PI, but many different types are descended from AB. Furthermore, even when similar cell types arise from both AB and P1, they may be produced by redundant processes: different genes may be regulated in nonlineally related cells to give rise to these similar cell types (SULSTON et al. 198). Thus, blastomere specific genes may regulate the expression of common or related developmental programs and might be detected by class 111 mutations. The results thus far do not enable us to distinguish among these and other possibilities. Mutations of particular interest: We find four class III mutations particularly interesting for further study. (1) ar48 gene activity is required in AB and EMS. Although this focus is common, ar48 is unusual in that mutant embryos die in early embryogenesis, at the comma to two fold stage (data not shown). The mosaic pattern, in combination with the stage of death suggests that ar48 may have a specific function early in development. (2) ar62 gene activity is required in the AB lineage (lethal focus in AB including a semilethal focus in ABp). ar62 mutants die as either embryo or L1 larva. The ar62 larvae sometimes have misplaced hypodermal nuclei in the head region and abnormal ventral hypodermal cells divisions suggesting a specific role in these cells. () ar49 is required in the AB lineage, but ar49 does not have a semilethal focus as defined for ar62. Thus, ar49 has an interesting mosaic pattern but also avoids possible complications of interpreting a semilethal focus. (4) ar51 is the only mutation that has a lethal focus in PI. In addition, the gene it defines has an additional, nonessential

11 Genetic Mosaic Screen 29 1 function inab (preventing pitted nuclei; see RE- SULTS). Analysis of individual genes will be important for understanding the types of genes identified in the genetic mosaic screen, as well as for evaluating the utility of the screen for future studies. The four mutations we have chosen for further study represent genes required in different parts of the lineage: ar48 in AB and EMS; ar62 in ABa and a semilethal focus in ABp; ar49 in AB; and ar51 in P1. We will further characterize these genes by isolating null alleles for mosaic analysis to confirm the lineage specificity of the lethal foci. In addition, antibodies to cell type specific proteins (MILLER et al. 198; PRIESS and HIRSH 1986) and lacz reporter constructs (e.g., FIRE, HARRISON and DIXON 1990; KRAUS et al. 1990) will provide us with ways to characterize the phenotype of lethal mutants, which will be useful in conjunction with the mosaic analysis. Ultimately, the molecular cloning of genes that remain good candidates for genes with specific developmental functions after additional genetic and phenotypic analysis will be straightforward by transformation rescue (FIRE 1986) using cosmid clones available from the nearly complete physical map collection (COULSON et al. 1986, 1988). We thankjudith AUSTIN and JUDITH KIMBLE for providing the strain JK80 and JIM PRIES for the glp-l(e2141) allele. We are particularly grateful to BRUCE WIGHTMAN, DAVID GREENSTEIN and GARY RUVKUN for sharing information on their similar mosaic screen and for fruitful discussions. We thank MARK PEIFER, EIN- HARD SCHIERENBERG, TRUDI SCHUPBACH, ERIC WIESCHAUS, members of our laboratory and the anonymous reviewers for discussion and/or comments on the manuscript. We thank GERALDINE SEY- DOUX for even more discussion and her advice on scoring mosaic animals. E.A.B. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. 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