Copyright 0 1996 hy the Genetics Society of America
Zygotic Lethal Mutations With Maternal Effect Phenotypes in Drosophila meZamgaster. 11. Loci on the Second and Third Chromosomes Identified by P-Element-Induced Mutations
Norbert Perrimon, Anne Lanjuin, Charles Arnold and Elizabeth Noll
Department of Genetics, Howard Hughes Medical Institute, Hamard Medical School, Boston, Massachusetts 021 15 Manuscript received July 10, 1996 Accepted for publication September 17, 1996
ABSTRACT Screens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the Xchromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal “FLP-DFS” technique to screen a collection of 496 Pelement-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.
HE genetic approach to identify the mechanisms is required for the establishment of the embryonic ter- T underlying embryonic pattern formation in Drc- mini (NUSSLEIN-VOLHARDet al. 1987), would not have sophila mlanogaster has led to a comprehensive view of been isolated from screens for loci associated with fe- the various steps involved in the establishment of the male sterility if its product was also necessary zygotically body plan (see reviews by INGHAM1988; ST. JOHNSTON for production of a viable animal. Similarly, some zy- and NUSSLEIN-VOLHARD1992). These analyses have dem- gotic genefunctions important for embryonic pat- onstrated that the egg contains spatial cues that are de- terning can be missed if the gene is also expressed posited during oogenesis.Following fertilization these maternally because the maternal product can mask the maternal cues regulate and coordinate the expression of zygotic requirement. a small number of genes that are further involved in To determine whethersome genes involved in critical controlling subsequent steps ofbody patterning. The patterning events have not been identified because of identification of the maternal and zygotic gene products their developmental pleiotropy, a screen to analyze the involved in specific patterning events has been the out- maternal effects of X-linked zygotic lethal mutationshas come of large genetic screens. Maternal functions have beenconducted (PERRIMONet al. 1984, 1989). From been identified via screens for female sterility, while zy- thisanalysis, it has been estimated that gene activity gotic genes have been detected via screens for embryonic of 75% of the essential loci is required for either the lethal mutations (GANSet al. 1975; MOHLER 1977; NUS formation of a normal eggor of a wild-type larvae.This LEIN-VOLHARDand WIESCHAUS 1980;JURGENSet al. 1984; represents asignificant fraction of the genomebecause NUSSLEIN-VOLHARDet al. 1984,1987; WIESCHAUSet al. in Drosophila it is estimated that 5000 loci are mutable 1984; PERRIMONet al. 1986; SCHUPBACHand WIESCHAUS to a visible phenotype and that 95% of these are essen- 1986, 1989). These screens have identified -40 maternal tial for viability (see PERRIMONet al. 1989). From the X- and 140 zygotic functions that are instrumental in con- linked studies, a number ofzygotic lethal mutations trolling specific embryonic decisions. This is a small num- have been identified that were associated with specific ber of genes considering that the Drosophila genome maternal effect phenotypes. Analyses of some of them has been estimated to potentiallycode for 10,000-20,000 have revealed that they encode components of the sig- different transcripts (see JOHN and MIKLOS 1988). naling machinery required for interpretationof the ma- The assumption underlying these screens is that the ternal/zygotic cues. The specificity of the embryonic expression of genesthat encode “decision making” phenotypes associated with these mutants reflects the functions is tightly restricted to the corresponding de- selective utilization of the gene products by specific velopmental stage. Indeed, some of the maternal gene pathways. For example, genes involved in the transduc- functions could be missed if the gene products were tion of the signal received by the Torso receptor tyro- sine kinase (0-raJ corkscrm, D-sorl, see review by DUFFY used at multiple times during the development of the and PERRIMON1994) have been identified. In addition, animal. For example, mutationsin the torso gene, which mutations correspondingto molecules involved in wing- less signaling (dishevelled, porcupine and zeste-white 3, see C;o?wsponding author: Norbert Perrimon, Department of Genetics, Howard Hughes Medical Institute, 200 Longwood Ave., Boston, MA review by PERRIMON1994) have been characterized.An- 021 15. E-mail: [email protected] other outcome of these screens has been the identifica-
Genetics 144 1681-1692 (December, 1996) 1682 N. Perrimon et al.
tion of novel regulatory networks. For example, muta- For P-element insertions on 2L, males were collected from tions in a Drosophila JAK gene has been identified as each of the P-element lines and crossed to Tft FRT2'.=4"A/Cy0 females. From this cross eight Tft FRT2L-40A/mwere collected a component of a system that regulates the expression and crossed to w; CyO/Sco males, in G418 treated vials (0.25 of pair rule genes (BINARIand PERRIMON1994). g geneticin/40 ml dH20). Cy, Tft+ recombinant males that The identification of zygotic lethal mutations with spe- survived the G418 selection were collected and mated in sin- cific maternal effect phenotypes has only been con- gle pairs to w; CyO/Sco females. For P-element lines that were ducted systematically on the X chromosome (PERRIMON proximal to the Tft marker, six to 10 males that have lost the dominant marker were collected. Tft maps at 37A3-6. For P- et al. 1984, 1989), which represents one-fifth of the Drc- element lines that were distal to the dominant marker, one sophila genome. These screens have been possible be- to three males without the dominant marker were collected. cause of the unique properties of the X-linked germline- To establish lines, m FRT2'2~40A/Cy0virgins and males from dependent,dominant female sterile (DFS) mutation each of the potential recombinants were collected and back- ovoD' (BUSSONet al. 1983; PERRIMONand CANS1983; crossed. The presence of the original P-element mutation on the FRT chromosome was determined by checking for the PERRIMON1984). This mutation allows the easy detection absence of homozygous animals in the stock. We also used a of female germline mosaics. Further, the application of Sco FRT2'.-40*chromosome for recombination, but realized the FLP-recombinase technology to promote chromo- that this chromosome carried a lethal mutation outside of somal site-specific exchange (COLIC1991) to this system the Sco region; therefore it was not used further. has led to the development of the "FLP-DFS" technique For Pelement insertions on 2R, males were collected from each of the Pelement lines and crossed to w; L FRT2"'"3/Cy0 (CHOU and PERRIMON 1992; see accompanying paper) females. From this cross eight w; L FRT2"G'3/mfemales were that allows the efficient production of germline mosaics. collected and crossed to w; CyO/Sco males. Putative 7~; m We have conducted a large screen using these chromo- FRTZR-GI~ /Cy0 male recombinants with orange eyes from the somes and have identified 64 new autosomal loci that FRT element that had lost the L dominant marker were se- encode gene functions involved in various steps of oo- lected and crossed as single pairs to w; CyO/Sco females. For P-element lines that were proximal to the dominant marker genesis and/or embryonic development. I, six to 10 putative m FRT2K-"'3/Cy0recombinant lines were established. For P-element lines that were distal to the domi- MATERIALSAND METHODS nant marker, one to three lines were established. Production of germline mosaics using the autosomal FUP- Recombinations of the ?L P-element mutations followed DFS technique: Stocks used togenerate germline clones the same strategy as described for 2K recombinants. The (GLCs) are described in CHOU and PERRIMON(see accompa- stocks used for these experiments were w; D FRT3',~zA/TM?, nying paper). To generate homozygous GLCs of a specific Sb and w; TM?, Sb/I,y. For Pelement lines that were proximal mutation (m),- 15 females of genotype CyO/FRT m or TM?, to thedominant marker D (which is inseparablefrom Sb/FRT m were crossed with five males of genotype FLP"/Y; In(?L)h9D?-El; 7OCl?-D1), 10-20 individual lines were estab- CyO/P[ ouo'"] FRT or FLPz2/Y; TM?, Sb/P[ avou'] FRT. These lished since it was found that the rate of recombination was males were generated by crossing females from the y w FLP"; significantly reduced with this chromosome. Otherwise, one CyO/Sco and y w FUZZ;TM?, Sb/CxD stocks with the appro- to three lines were kept. priate P[ ovd"] FRT males (CHOUand PERRIMON,see accom- Recombinations of the 3R P-element mutations followed panying paper). Females were allowed to lay eggs for 1 day the same strategy as described for 2L recombinants. The in glass vials and their progeny heat shocked twice for 2 hr stocks used for these experiments were Sb FRT3"82A/TM6,Ubx at 37" in a circulatingwater bath over a period of 2 days when and w; TM?, Sb/Ly. For Pelement lines that were proximal they reached late L2 to L3 larval stages. Subsequently, -40 to the dominant markerSb (at 89B),six to 10 males that lost females of genotype w/w FLP; FRT m/P[0~8'1 FRTmated with the dominant markerwere collected. For Pelement lines that either CyO/FRT m or TM3, Sb/FRT m males were analyzed for were distal to the dominant marker, one to three males that the presence of GLCs. All of the P[ovo"'] FRT recombinant lost the dominantmarker were collected. The G418 selection chromosomes are associated with a fully penetrant DFS phe- used was 1.0 g geneticin/40 ml dHzO. notype such that all eggs laid by these females are derived It should be noted that we usually analyzed a single FRT m from germline recombination events. Due to the efficiency of recombinant line for each mutation. Thus, if the original mu- the FLP-DFS technique (CHOUand PERRIMON,accompanying tant chromosome carried an additional mutation, the GLC phe- paper), the analysis of 40 females is usually sufficient to allow notype would represent the phenotypes of both mutants. We unambiguous determination of the GLC phenotype of the believe that this is not agreat concern as many of the Pelements mutation tested. that we analyzed have been shown to revert to wild type (Berke- Recombination of theP-element mutations on the FRT ley Drosophila Genome Project, personal communication). chromosomes: The collection of P element-induced muta- Determination of the GLC phenotype of the zygotic lethal tions characterized by SPRADLINGet al. (1995) as part of the mutations: P element-induced zygotic lethal mutations were Berkeley Drosophila Genome Project was obtained from the classified into three groups based upon their GLC phenotypes. Bloomington Stock Center. These mutations are kept with Group 1 corresponds to those that do not lay eggs. Group 2 either the Cy0 or TM?, Sb balancerchromosomes. The P corresponds to those where eggs are laid and most hatch, and elements carry either the rosy or white genes as markers. P- Group 3 corresponds to those that do not lay eggs but possess element insertions were recombined onto the FRT chromc- developed ovaries, those that lay abnormal eggs, or those where somes. To facilitate the recombination events, we constructed at least 20% of the eggs fail to hatch. Mutations that belong to different stocks that contain the FKT elements and dominant Group 3 were set up a second time for GLC analyses and the visible mutations. These are as follows: Tft FRT'2'.-4M/Cy0,Sco embryonic phenotypes further characterized. FRTzI.-4&!/CyO, FRTZR""' L/T0, G1 FRT"."/TM?, Sb, D Females carrying homozygous mutant GLCs are crossed FRT31.-2.4 /TM?, Sb and FRT3K-8A Sb/TMG, Ubx. The dominant with either CyO/FRT m or TM3, Sb/FRT m fathers. TWOclasses markers we use are described in LINDSLEYand ZIMM(1992). of embryos are produced: embryos that lack both maternal M aternal EffectsMaternal 1683 Mutations of Lethal and zygotic copies of the wild-type gene, and embryos that genes whose expression during oogenesis is not critical lack only the maternal copy. To determine the extentto which to embryonic development. One hundred ninety-nine the introduction of a wild-type copy of the gene from the father influences the maternal effect phenotype, theembry- of the mutations (40%) examined fell into this class. onic phenotypes of 200 eggs derived from females carrying Group 3 corresponds to those mutations that do not homozygous GLCs crossedwith either m/+ or +/+ males lay eggs but possess developed ovaries as well as those were examined. In addition,to distinguish between an embry- where a substantial fraction of the eggs were either onic zygotic lethal mutation with no maternal effect and a abnormal or failed to hatch. We placed 143 mutations fully paternally rescuable maternal effect, the stagesof lethal- ity of the zygotic lethal mutations were determined. In the (29%) in this group. A number of additional tests were case of an embryonic lethal mutation that does not exhibita done with these mutants to further characterize the maternal effect phenotype, 50% embryoniclethality is ob phenotype of the clones. These include the determina- served when females with m/m GLCs are crossed with hetero- tion of the stages at which the mutations cause zygotic zygous +/m fathers and 0% when crossed with wild-type +/ lethality. This testis especially important because it + fathers. Lethal phase determination: To determine the stagesof allows us to distinguish between embryonic lethal muta- lethality (ie., lethal phases) of the mutations associated with tions with no maternal effect and zygotic lethal mutants zygotic lethality, a minimum of 200 eggs from parentsof geno- associated with a fully paternally, rescuable maternal type +/mare lined up on an agarplate and allowed to develop effect phenotype (see MATERIALS AND METHODS). In ad- at 25". Since embryos that are homozygous for the Cy0 and dition, we carefully determined the extentof the pater- TM3, Sb balancer chromosomes are associated with embry- onic lethality, we first outcrossed each individual stock with nal contributions to the maternal effect phenotypes. a wild-typestrain to eliminatethe balancer chromosome. This was done by examining eggs derived from females After a period of 24 hr, the number of unhatched eggs are that carry homozygous GLCs crossed to eitherwild-type counted. The numberof unhatched and unfertilizedeggs was males or males heterozygous for the mutation tested. determined after dechorionationwith a 50% bleach solution The 143 mutations inthis group were set up for GLC (PERRIMONet al. 1989). If 25% of the developed eggs fail to hatch, it indicates that the mutationis associated with embry- analysis at least one additional time and the specificity onic lethality. Thezygotic phenotype of the mutation was and penetrance of the phenotypes examined carefully. examined by cuticle preparation of the eggs. The develop- From the original collection of 143, 78 were associated ment of hatched larvae is followed at 25" and examined every with distinctive phenotypes (Table 2, Figure 1) and 2 days. During this time the numberof dead larvae are scored were thus retained. Results from the screen are shown as well as the number of pupae that form. After 11 days of development, emerging adults are counted and the number in detail in Tables 1, 2 and Figure 1. Among the 143 of dead pupae scored. Group 3 mutants,65 had very variable phenotypes and Examination of embryos: Larval cuticles were prepared in were discarded, 18 were associated with oogenesis de- Hoyers' mountant as described by VAN DER MEER(1977). cuti- fects (AO), nine were embryonic lethals with no mater- cles were examined using dark-field and phase illumination. nal effects (NME) and 51 were associated with maternal effects (ME, PMER, FMER,or WE).The GLC pheno- RESULTS types associated with several of the zygotic lethal muta- tions were not fully penetrant (Table 2). Nevertheless, Analysis of P-element mutations: To identify autoso- we have included them in this analysis because >30% mal zygotic lethal mutations (m) associated with novel of unhatched embryos with specific mutant phenotypes maternal effect lethalphenotypes, we analyzed the are obtained from females with GLCs. The variability collection of P-element mutations, established by of the maternaleffects observed may reflect the residual SPRADLINGet al. (1995) as part of the Berkeley Drosoph- activity associated with the P-element mutations ana- ila Genome Project. We recombined each P-element lyzed or the simultaneous occurence of both GLCs and mutation with the FRT element located on the same presence of follicle cell clones (see DISCUSSION). Re- chromosomal armby meiotic recombination (see MATE- gardless, it suggests that the gene function is involved RIALS AND METHODS). Then, we prepared GLCs for each in a specific developmental processs. of the m FRT chromosomes (see MATERIALS AND METH- Of the 18 loci in which GLCanalysis revealed an ODS for protocols)and analyzed the phenotypes of eggs abnormal oogenesis (AO) phenotype, we identified two derived from the clones. The 496 mutations examined previously knowngenes based on informations available were classified into three groups (Table 1) based on in Flybase. One of these is squid, which encodes a puta- their GLCs phenotypes. tive RNA binding protein involved in the establishment Group 1 corresponds to those mutations that do notlay of the dorsoventral axis of the egg chamber (KELLEY eggs and do notpossess obviousdeveloped egg chambers. 1993). The other is an allele of the Protein Kinase A This group corresponds to gene functions that are re- catalytic subunit (Pka),which plays a role in the reorga- quired for germ cell viabilityor early oogenesis.We found nization of the microtubulenetwork during mid-oogen- that 154 mutations (31%) fell into this class. esis (LANE and KALDERON 1994; RONGOand LEHMANN Group 2 corresponds to those mutations thatlay eggs 1996). The functions of the remaining 16 genes have where most of the eggs hatch. This group corresponds not been characterized. to genes that are either not expressed maternally or For the A0 phenotype, females with GLCs develop I(i83 X. Pt*rrimon rt (11.
Fl(;[.l
group are embryonic lethals. While two of these pro- Mutations in 42 loci were identified that laid normal duce embryos that do notexhibit obvious cuticular phe- eggs but produce embryos with specific cuticular pat- notypes (1(2)01152 and 1(3)06737), forthe third, terning defect.. . Various patterning processes such as 1(2)06825, animals derived from heterozygous mothers segmentation, dorsal closureor head formation arerep die as embryos and exhibit a poorly organized cuticle resented in this group of mutants. Among these muta- (data not shown). The embryonic phenotypeof unres- tions, we identified nine loci that are associated with cued embryos derived from 1(2)06825 GLCs, however, embryonic lethality but do notexhibit a maternal effect is much more severe (Figure 1). phenotype. Mutations in these loci (wingkss, goosebpny, 1686 N. Perrimon et al.
TABU 1 the tail region (1(2)09049),and dorsal defects inthe amni- Results from the screen oserosa (1(3)7Q303).
No. in No. in No. in DISCUSSION Total group 1 group 2 group 3 ZL 90 22 34 34 In this paper we report the results of a screen to 2R 122 31 42 49 identify the maternal effect of autosomal zygotic lethal 3L 116 44 43 29 mutations. We have analyzeda collection of 496 autoso- 3R 168 34 73 61 mal P element-induced mutations generated by the Total 496 154 199 143 Berkeley Drosophila Genome Project and identified a The GLC phenotypes of the mutations were classified into number of mutants associated with novel maternal ef- three groups. group 1: No eggswere laid and no obvious fect phenotypes. Seventy-eight of the mutations were developed eggs could be found following brief inspection selected for detailed analysis of their GLC phenotypes of the adult abdomen. group 2: Most eggs hatch. group 3: because they were found to generate reproducible and Oogenesis defects in which no eggs are laid but vitellogenic penetrant phenotypes. Fourteen of them disrupted the oocytes are present, as well as those where a substantial frac- tion of the eggs were either abnormal or failed to hatch. gene activity of previously known genes while others appearto identify loci not previously characterized based on the information available from the Berkeley hairy, string, schnum; spitz, trachealess, zipper and serpent) Drosophila Genome Project (see Table 2). have been previously characterized in screens for em- The collection of P element-induced mutations was bryonic lethal mutants ( JURGENS et al. 1984; NUSSLEIN- chosen for analysis because it provides a number of VOLHARDet al. 1984). advantages over a collection of randomly induced muta- Among the mutations that exhibit maternal effect tions (SPRADLINGet al. 1995). First, the P element-in- phenotypes, we identified two previously known genes, duced mutations have been mapped cytologically facili- thickveins and punt, where embryos derived from GLCs tating further genetic characterization of the loci. exhibit embryonic defects along the dorsoventral axis. Second, zygotic lethality has been shown in most cases These genes have been shown to be involved in signal- to be associated with the P-element insertion (Berkeley ing by Decapentaplegic (NELLENet al. 1994; LETSOUet Drosophila Genome Project, personal communica- al. 1995; RUBERTEet al. 1995). In addition, we character- tion), thus we expect the mutant phenotype identified ized an allele of Downstream of receptor tyrosinekinase (Drk, in GLCs to map at the site of the P-element insertion. OLMERet al. 1993; SIMONet al. 1993) that has been Third, the availability of P insertions will greatly facili- shown to be involved in patterning of the embryonic tate future molecular characterization of the genes. termini (HOUet al. 1995). It is of interest to compare theresults from this screen Four new loci with segment polarity GLC phenotypes with results obtained from the previous analysis of X- ( 1(2)00681, 1(3)03844, 1(3)08310, 1(3)02619)were recov- chromosome loci (PERRIMONet al. 1989). These num- ered. These genesmay be implicated in aspectsof Wingless bers compare as follows: cell lethal mutations Xchro- or Hedgehog signaling (see PERRIMON 1994 for review). mosome screen (X: 40%), autosome screen (A31%), Fifteen loci were associated withdeletions of segments. mutations with no maternal effect (X: 31%, A 40%), Three mutations exhibited a pair rule-like phenotype abnormal oogenesis (X: 8.5%, A 3.6%), mutations with (1(3)03649,1(2)00632,1(3)04556)while others were associ- maternal effects (X: 9.5%, A 10.2%), or with variable ated withvariable deletions of segments (1(2)00255, maternal effects (X: 11%,A 15.2%). Numbers regard- 1(2)06214, 1(3)04837, 1(3)02414, 1(3)03463, 1(3)03719, ing the Xchromosome study are obtained from Table 1(3)03550, 1(3)01618, 1(3)07013, 1(3)06346, 1(3)j5A6and 4 of PERRIMONet al. (1989) that was derived from the 1(3)01164). Within this group, the only locus that has analysis of211 random X-linked lethal mutations. Num- been previously characterized is 1(3)06346, which we have bers regarding the autosome screen are derived from shown encodes a Drosophila STAT protein, Marelle, the data presented in Tables 1 and 2. The numbers which operates in the Hopscotch/JAK signaling pathway obtained from the different screens are quite similar (Hou et al. 1996). Characterization of the functions of and differ only for the class of mutations that result in the other loci may identlfy maternally stored proteins A0 phenotypes. This class is underrepresented in the that act in concert with gap or pair rule zygotic genes to autosomal screen because we did not systematically ex- speclfy proper segmentation. amine females that lay no eggs for oogenesis defects. Various phenotypes are exhibited by the remainer of Further, the frequency of mutant loci associated with the loci. Theseinclude head and dorsal open phenotypes cell lethality vs. no maternal effect is higher in the EMS (1(2)05836,1(3)00281),head defects (1(3)~2172,1(3)05113), collection. This may reflect the nature of the mutagen subtle head and tail defects (1(2)01482),twisted or tail on used in inducing the mutations. The Xchromosome dorsal side phenotypes (1(2)06214, 1(3)03349, 1(3)05430),study screen utilized EMSinduced mutations, while we ventral hypoderm absent (1(2)04291),necrotic patch in used Pelement-induced mutations in the present analy- Maternalof Effects Lethal Mutations 1687
TABLE 2 Description of the mutants
G ene nameGene Mutation tested Stock number (Bloomington number) References Location Lethal phase phenotype Germline clone Classification I. Abnormal eggs 1(2)06955 P2330 21F1-2 L2 Misshapened eggs A0 (100) Pka* 1(2)01272 P1030 3 3OC1-2 E-L1 Eggs collapsed A0 (100) 1(2)04?29 P1371 49D1-3 L2-3 tiny Few collapsed eggs A0 (100) 1(2)rG270 P2060 58F1-2 L1-2 Few collapsed eggs A0 (100) fi(?)07084 P1713 64E08-12 L1-2 No eggs laid, few vitellogenic oocytes A0 (100) l(?)j9B4 P2082 68AO4-05 L3-P No eggs laid, vitellogenic oocytes A0 (100) 1(?)0?802 P1607 72D01-02 Pol Tiny eggs, few eggs laid A0 (100) 1(?)09904 P1740 82F8-9 E (no defects) Collapsed eggs A0 (100) squid* l(?)j6E? P2133 10 87F2-3Small L3-P dorsalized eggs A0 (100) l(?)j4B4 P2134 87F3-4 L2-P Arrest during vitellogenesis A0 (100) l(?)jlE7 P2135 88A4 - 5 Pol Tiny collapsed eggs A0 (100) 1(?)02102 P1555 91F10-11 L1-2 Few eggs laid, defective appendages A0 (100) 1(?)07086 P1714 93B8- 11 L1-2 Few abnormal eggs laid A0 (100) l(?)jWl P2 148 9361-2 L1-2 Collapsed eggs A0 (100) 1(3)05241 P1654 93D4-7 L3-P Few eggs laid, vitellogenic oocytes A0 (100) 1(?)03852 P1612 93El-2 Pol with Eggs fused filaments A0 (100) 1(?)0?921 P1614 94E3-7 E (head defects) Few eggs laid, vitellogenic oocytes A0 (100) 1(?0)07207 P1718 95Fll-12 E (ventral hypoderm absent) Small collapsed eggs A0 (100)
11. Normal eggs with no cuticle development 1(2)01?51 P1045 30A3- 5 L1 No cuticle ME (100/100) 1(2)04431 P1375 32El-2 P-A No cuticle ME (100) 1(2)0937? P2361 60B10-11 E-L1-2 84% with no cuticle, others have variable defects ME (86/76) 1(?)01658 P1542 74B01-02 L3 No cuticle ME (100/100) 1(?)04629 P1634 86E16-19 L1-2 68% with no cuticle, others have variable defects ME (82/100) 1(?)05203 P1652 1688 N. Perrimon et al.
TABLE 2 Continued
Ge ne nameGene Mutation tested Stock number (Bloomington number) References Lethal phase Germline clone GermlineLocation phase Lethal phenotype Classification 11. Normal eggs with no cuticle development 89B12-13 L1-2 No cuticle ME (100/100) l(3j2B10 P2 142 90F6 - 7 No cuticle ME (100/100) 1(3)01207 PI524 96B10- 11 E (abnormal denticle bands) No cuticle ME (99/95)
111. Poor cuticle development 1(2)06825 P2324 29F1-2 E (poor cuticle development) Very poor cuticle development FMER (50/5) 1(2)02516 P1198 48C1-2 L1-2 Poor cuticle development, U shaped ME (100/100) 1(2)07214 P2339 51B7-10Unrescued P poor cuticle development,rescued head PMER (l00/80) and tail defects 1(2)03050 PI261 57B13-14 P Poor cuticle development, paternally rescued embryos WME (81/63) show variable cuticle defects 1(3)00274 PI491 73B01-02 L1-3 Poor cuticle development PMER (88/87) 1(3)04226 PI627 89B6- 7 Pol Poor cuticle development ME ( loo/ 100) 1(3)05089 PI 646 91A1-2 L2 Poor cuticle development ME ( 100/ 100) 1(3)07551 PI721 91B5-6 L2 Poor cuticle development ME (100/100) 1(3)01152 P1522 95B5-6 E (no cuticle defects) Poor cuticle development PMER (96/36) 1(3)06737 P1697 95El-2 E (no cuticle defects) Poorly differentiated cuticle MER (59,’ 15)
IV. Patterning defects 1(2)00632 P944 23C1-2 L1-2 20% of embryos with variable pair rule phenotype WME (83/65) thick vein? 1(2)04415 P1373 1 25D1-2 L2 Ventralized embryos PMER wingless* 1(2)02657 P1205 2 27F1-2 Segment E polarity mirrormutant, image duplication of NME denticle bands 1(2)05836 PI451 28E1-2 L-P and Head dorsal open FMER (56/3) 1(2)01482 P1062 29C3-5 Subtle E-LI and head tail defects PMER (91/44) 1(2)00255 P936 33E7-8 L1-2 Variable segmentation defects PMER (100/83) spitz* 1(2)s3547 P2049 2 37F1-2 Deletion E of medianparts all of denticle belts NME schnum* 1(2)04738 P1386 2, 4, 5 47El-2 E absentDorsal hypoderm NME drk* 1(2)10626 P2378 6, 7, 8 50A12-14 L Defectsandhead in both tail regions FMER (45/5) 112)00681 P949 Maternal Effects of Lethal Mutations 1689
TABLE 2 Continued
G ene nameGene Mutation tested Stock number (Bloomington number) References L ethal phase Germline clone GermlineLocation phase Lethal phenotype Classification N. Patterning defects 51B1-5 L-P Segment polarity mutant, unrescued: mirror image FMER (57/5) duplication of denticle bands, rescued: wild type 1(2)06214 P1469 53C1-2 P Tail on the dorsal side WME (66/38) L(2)05428 P1407 53C9-10 L1 Variable segmentation defects, head defects WME (67/42) 1(2)04291 P1369 53F1-2 L1-2 Ventral hypoderm absent PMER ( loo/ 100) 1(2)09049 P2360 59F1- 2 L2 Small necrotic patch in the tail region FMER (47/0) gooseberry* 1(2)01155 P999 2 60F1- 3 E Segment polarity mutant, mirror image duplication of NME denticle bands zipper” 1(2)02957 P1215 2 60F1- 3 E-L Dorsal closure defective NME trachaeles? 1(3)10512 P1747 9 61CO1-02 E No trachea NME 1(3)04556 P1633 64CO1-02 Pol Variable pair rule segmentation defects WE(75/50) l(3Oj03844 P1610 65C1-2 L3-P Segment polarity mutant, unrescued: mirror image FMER (58/1) duplication of denticle bands, rescued: wild type 1(3)08310 P1731 65D4-5 L1-2 Segment polarity mutant, unrescued: mirror image PMER (85/30) duplication of denticle bands, rescued: partial fusion of denticle bands or wild type haily* 1(3)08247 P1730 9 66D10-12 E Pair rule segmentation defects NME 1(3)03349 P1587 66EO6-07 P Embryos twisted, variable cuticle differentiation WME (36/13) 1(3)s2172 P2089 71B04-05 L1-2 Head open WE(38/25) 1(3)02619 P1572 74CO1-02 L2-3 Segment polarity mutant, unrescued: mirror image PMER (81/25) duplication of denticle bands, rescued: partial fusion of denticle bands or wildtype 1(3)03649 P1598 75DO4-05 L2-3 Pair rule segmentation defects ME (100/100) 1(3)00281 P1492 85B8-9 L3-P Head defects, variable dorsal open WE(30/16) 1(3)04837 P1639 85D5-6 L1-2 Variable segmentation defects WME (44/38) 1(3)054?0 P1659 85D8-9 Pol Embryos twisted, variable cuticle differentiation WME (56/43) 1(3)02414 P1568 85F12-13 Pol No abdominal segments, poor layer ME ( 100/ 100) 1(3)03463 P1590 87D7-9 Pol Ventral holes, segment fusion ME ( loo/ 100) punP 1(3)10460 P1745 9, 11, 12 88C9-10 E (dorsal open) Ventralized embryos PMER ( 100,’ 100) 1(3)03719 P1605 1690 N. Perrimon et al.
TABLE 2 Continued
Gen e nameGene Mutation tested Stock number (Bloomington number) References Lethal phase Germline clone GermlineLocation phase Lethal phenotype Classification
IV. Patterning defects 88D1-2 P Varible segmentation defects ME (99/100) 1(?)0?550 P1594 88E8-9 L3-P Anterior head defects, variable abdominal WE(38/49) segmentation defects 1(?)01618 P1539 89A8 - 9 L1-2 Variable segmentation defects MER (53/8) serpenP 1(?)01549 P1538 9 89B1-3 E Tail region remains on dorsal side NME 1(3)07013 P1711 91F1-5 L2 Head defects and variable segmentation defects FMER (49/7) 1(?)/5A6 P2144 91F10-11 L3/P Variable deletions of denticle belts, head defects ME (61/83) 1(?)0511? P1647 92A13-14 E-L1 (no cuticle defects) Head defects MER (54/ 17) marelle l(?)06?46 P1681 20 92E2-4 L Defects in T2, A5 and A8 mainly PMER (100/100) 1(3)01164 P1523 93B1-2 E (no cuticle defects) Variable segmentation defects FMER (58/9) l(?)rQ?O? P2154 95D1-2 L1-2 Amnioserosa defects MER (51/13) string* 1(3)012?5 P1525 9 99A5-6 E Some denticle rows are missing NME Since it is likely that some of these genes are currently under investigation in other laboratories, we have decided not to give descriptive names to the loci at the present time. We feel it is more appropriate that these genes, following further characterization, be renamed in subsequent publications. The numbers in parentheses indicate the specificity of the mutant phenotypes. A0 (100) indicates that the abnormal oogenesis phenotype is 100% penetrant. The ratio (A/B) of embryonic lethality observed from females with GLCs crossed with either heterozygous (A) or wild-type (B) males is shown. This ratio allows us to distinguish between the various classes of maternal effects (ME, PMER, FMER, WME or NME). Classification of the GLC phenotypes is as follows: Abnormal Oogenesis (AO), Maternal Effect (ME), Partially (P) orFully (F) Paternally Rescuable Maternal Effect (MER), Weak Maternal Effect (WME), No Maternal Effect (NME). Lethal phases: Embryonic (E), Larval (LI, L2 and L3) and Pupal (P) stages, Polyphasic (Pol). * Previously known genes. References: Indicated is the original reference that describes the gene identified by P-element insertion as well as references on the GLC phenotypes, when available: 1, NELLENet al. (1994); 2, NUSSLEINVOLHARD et al. (1984); 3, LANE and KALDERON (1994); 4, GRIEDERet al. (1995); 5, ARORA et al. (1995); 6, OLMERet al. (1993); 7, SIMONet al. (1993); 8, HOU et al. (1995); 9, JURCENS et al. (1984); 10, KELLEY (1993); 11, RUBERTEet al. (1995); 12, LETSOUet al. (1995). sis. It is unlikely that these discrepencies reflect the variability in penetrance of the mutant phenotypemay number of mutations present on the chromosome as correspond to the simultaneous occurence of follicle both the EMS- and P element-induced collections of cell clones. A number of signaling pathways involved in mutants contain few chromosomes with multiple hits embryonic patterning operatebetween the follicle cells (N. PERRIMON, unpublished data;Berkeley Drosophila and the oocyte (review by RONGOand LEHMANN1996), Genome Project, personal communication). Because P- thus, one expects to identify a number of mutants that element insertions appear to preferentially insert near in folliclecell clones would disrupt embryonic pat- the 5’ end of transcription units (SPRADLINGet al. 1995), terning. Although it is clear that mutantswith fullypen- one might anticipate that the ratio of genetic null us. etrant GLC phenotypes affect germline specific func- hypomorphic mutations to be different. tions, it remains to be determined whethersome of the We have included in Table 2 a number of Pelement- mutants with variable phenotypes may actually corre- induced mutations that exhibit weak maternal effect spond to the simultaneous occurence of clones of mu- phenotypes. Thenature of the variability associated tant follicle cells. Follicle cell clones can be induced with these mutations is unclear. Perhaps these P ele- following FLPmediated mitotic recombination (HAR- ment-induced mutations have residual activity raising RISON and PERRIMON1993; Xu and RUBIN1993; MAR the possibility that a strongerallele may exhibit a more GOLIS and SPRADLINC1995). However, it remains to be severe or penetrant GLC phenotype. Alternatively, the determined how often follicle cell clones are induced M aternal EffectsMaternal Mutationsof Lethal 1691 under the heatshock conditions we used (see also HAR- references Table l), cytoskeletal proteins [Armadillo RISON and PERRIMON 1993 for DISCUSSION). One solu- (PEIFERand WIESCHAUS1990)], RNA binding protein tion to avoid the occurence of follicle cell clones simul- (Squid, see reference in Table l),transcription factors taneously with the GLCs would be to use a FM- [Ulraspiracle (ORO et al. 1990), Marelle (HOU et al. recombinase that only expresses in the germline. 1996)] and novel proteins [Dishevelled (UINGENSMITH In this study we analyzed 496 independent zygotic le- et al. 1994), Porcupine (KADOWAKI and PERRIMON,un- thal mutations identified by single Pelement mutations. published data), Brainiac,a.k.a. 6P6 (GOODEet al. It is of interest to estimate the scope of this analysis with 1996a), Egghead, a.k.a. Zw4 (GOODE et al. 1996b)l. The regard to the level of saturation achieved. For the entire specificity of the GLC mutant phenotypes reflect the Drosophila genome, BRIDGES(1938) drew 5059 bands. specific utilization of these maternal gene products by The counts for each chromosome are as follows: Xchro- signaling pathways activatedby a small number of zygotic mosome, 1012 bands; 2L chromosomal arm, 804 bands; genes. The mutant phenotypes of the loci we have identi- ZR chromosomal arm, 1136 bands; 3L chromosomal fied mimic the phenotypes of the zygotic genes because arm, 1178 bands; 3R chromosomal arm, 884 bands; and at the time we examine the embryos, the gene products 45 bands are on the fourth chromosome. The number have not been utilized by other pathways. Alternatively, of autosomal loci that mutate to zygoticlethality can the specificity of the embryonic phenotypes may reflect be easily estimated from studies on the X chromosome. the strength of the allele, as different signaling pathways Previously, we (PERRIMONet al. 1989) and others (LEFE- may be more or less sensitive to a certain amount of VRE and WATIUNS1986) estimated that there are -540 gene product.Interestingly, many of the maternal effects vital loci among the 1012 bands on the Xchromosome. we recovered are fully or partially paternally rescuable. Extrapolation to the autosomes estimate that 1048 vital The extent of the paternalrescue reflects a combination loci are linked to the second chromosome and 1114 are of when during embryogenesis the gene product is uti- linked to the third. In the present study we analyzed 496 lized and the onset of zygotic transcription. independent Pelement mutations thus representing a We are indebted to TZEBIN CHOUfor developing the “autosomal 23% level of saturation. FLP-DFS technique” that made this work possible. We thank K. MAT- This analysis, in combination with previous studies of THEWS for sending us the collection of Pelement lethal mutations, X-linked mutations that represented an86% level ofsatu- LIZ PERKINSand SCOTT GOODEfor comments on the manuscript. ration (PERRIMONet al. 1989), indicates that 36% of loci This work was supported by a grant from the National Science Foun- that mutate to zygotic lethality have been analyzed in dation and theHoward Hughes Medical Institute. N.P. is an Associate GLC analysis. The identification of additional loci will Investigator of the Howard Hughes Medical Institute. require additional screens. Our laboratory is currently completing a large EMS screen to achieve this goal. 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Genes Dev. 8: 300-312. cation of five X-linked segment polarity genes (armadillo, BOUROUIS,M., P. MOORE, L. RUEL,Y. GRAU,P. HEITZIXRet al., 1990 3, An early embryonic product of the geneshaggy encodes a serine/ zeste-white dishevelled, porcupine and fwe4. Because the threonine protein kinase related to the CDC28/cdc2+ subfam- autosomal screen is not to saturation, we expect that at ily. EMBO J. 9 2877-2884. least 15 additional loci withsegment polarity phenotypes BRIDGES,C. B., 1938 A revised map of the salivary gland X-chromo- could be identified using the GLC approach. some of Drosophila mlanogaster. J. Hered. 21: 81-86. BUSSON,D., M. GANS,K. KOMITOPOULOUand M. MASSON, 1983 Ge- The GLC approach has led to the identification of netic analysis of three dominantfemale sterile mutations located many components of signaling pathways that are acti- on the X chromosome of Drosophila melanogaster. Genetics 105 309-325. vated during embryogenesis. 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