Guo Sk1995

Date post:03-Apr-2018
Category:
View:218 times
Download:0 times
Share this document with a friend
Transcript:
  • 7/28/2019 Guo Sk1995

    1/10

    Cell, Vol. 61, 61 l-620, May 19, 1995, Copyright 0 199 5 by Cell Press

    par-$ a Gene Required for Establishing Polarityin C. elegans Embryos, Encodes a Putative Ser/ThrKinase That Is Asymmetrically DistributedSu Guo and Kenneth J. KemphuesSection of Genetics and DevelopmentCornel l Universi tyIthaca, New York 14853

    SummaryThe f i rst cleavage of C. elegans is asym metr ic, gener-ating daughter cel ls wi th di fferent sizes, cytoplasmiccompon ents, and fates. Mutations in the par-7 genedisrupt this asym metry. We report here that par-7 en-codes a putative Ser/Thr kinase w ith simi lar i ty to ki-nases from yeasts and mam mals. Two strong al leleshave mutations in the kinase domain, suggesting thatkinase activi ty is essential for par-l fun ction. PAR-1protein is localized to the posterior periphery of thezygo te and is distributed in a polar fashion precedingthe asym metr ic divisions of the germl ine l ineage. Be-cause PA R-1 distr ibution in the germl ine correlateswith the distribution of germl ine-speci fic P granules,i t is possible that PAR-1 functions in germl ine develop-ment as wel l as in establ ishing embryonic polar i ty.IntroductionAsym metr ic cel l division, by which the two daughter cel lsadopt di fferent fates, plays an important role in the genera-t ion of cel lular d iversi ty dur ing developm ent. Daughtercel ls wi th di fferent fates can ar ise via extr insic cel l -cel ls ignaling or by intr insical ly asym metr ic divisions (Horvi tzand Herskow itz, 1992). Intr insical ly determined asym met-r ic divisions occur in a number of invertebrates and appearto play crucial roles in establ ishing embryonic axes orspeci fying part icular di fferentiated cel l fates (Davidson,1986; Slack, 1991). Mos t evidence supports the model ofcytoplasm ic local ization as the basis for such intr insicallypatterned asym metr ic divisions. In cytoplasm ic local iza-t ion, maternal ly provided determinants are unequal lypartitioned to particular cells through ase riesof reproduci-bly or iented cleavage divisions (Davidson, 1986).

    The nematode Caenorhabdit is elegans provides an op-portuni ty to study the mecha nisms responsible for intrinsi-cal ly asym metr ic divisions. C . elegans em bryos have afixed cel l l ineage, and a precise cel l fate map has beenestabl ished (Sulston et al ., 1983). The asymme tr ic divi -sions leading to the formation of the germl ine play an im-portant role in establ ishing embryonic organization. Eachof these divisions is asym metr ic, producing daughter cel lswith di fferences in size, cel l division t iming, cleavage pat-tern, and ult imate fate (see Figure 1A for a summ ary ofthe cell l ineage). In addition, germline -specific P granulesare localized to one pole of the cell prior to each divisionand partitioned into the sma ller da ughter cell (Strome andWood, 1982, 1983).Al though the mecha nisms under lying this ser ies ofasym metr ic divisions are largely unknown, microfi laments

    appear to play a role in at least the firs t division. Briefpulses of the microfi lament-disrupting drug cytochalasinduring a critical period of the first cell cycle preven t theposter ior local ization of the P granules (Strome and Wood,1983; Hi ll and Strome, 1988). Cytochalasin pulses duringthis same per iod som etimes also lead to symm etr ic divi -sions producing daughter cel ls of equal sizes, simi lar cel lcycle rates, and variable spindle orientations (Hill andStrome, 1988, 1990).

    Maternal-effect lethal mutations in the par genes pro-duce phenotypes simi lar to the effects of cytochalasintreatments (Kemphues et al ., 1988). In par mutant e m-bryos, normal ly unequal divisions are equal, cleavagespindlesare misor iented, Pgranules are mislocal ized, andthe blastomere fates are al tered. This impl ies that the pargenes function in both spindle placement and cytoplasmiclocal ization (Kemphues et al ., 1988; Kirby et al ., 1990;Morton et al ., 1992; Cheng et al ., 1995).

    The par-7 gene is required for several aspects of ear lyembryonic polar i ty (Kemphues et al ., 1988). As schema ti-cal ly diagrammed in Figure 1 B, par-7 m utant embryos ex-hibit equal first cleavage and fail to localize P granules.The 2-cel l stage blastomeres divide synchronously, andthe cleavage patterns are aberrant for al l subsequentstages. The par-7 embryos arrest as amorphous mass eswith large numbers of di fferentiated cel ls. Mos t embryoscontain e xcess b ody wal l and pharyngeal muscle ce l ls(normal ly produced by the MS lineage) and are completelylacking intestine. The putative MS cel l fate determinant,SKN-1, normal ly distr ibuted asymm etr ical ly among ear lyblastomeres, is found in equal amoun ts in al l the cel ls atthe 2- and 4-cel l stages in par-7 mutant embryos (see Fig-ure 1 B). The excessive pharyngeal and body wal l musclecel ls observed in par-7 mutant embryos may ar ise as aconsequence of the abnormal distribution of SKN-1 (Bow-erman et al., 1993).We demonstrate here that par-7 encodes a conservedputative Ser/Thr kinase that is asymm etr ical ly local ized tothe posterior periphery of the l-cell emb ryo and differen-t ial ly distr ibuted, preceding al l the asym metr ic divisionsof the germl ine l ineage. Mutations in the kinase domainel iminate PAR-1 function, suggesting PAR-1 kinase activ-i ty is essential for i ts role in establ ishing asym metry.ResultsMolecular Cloning of the par- f GeneIdentificatio n of the par-l RegionAs summ arized in Figure 2A , par-7 m aps to chromosom eV between genetic markers rol-4 and uric-76, an intervalof -3 m.u . Tel fer (1991) had placed the par-7 gene onthe C. elegans physical map within or near the region cov-ered by the yeast ar t i fic ial chrom osome (YAC) Y39Hl. Wefurther mapped par-7 relative to two closely l inked genes,ogr-7 and him-5 The mappingdataputpar-7 -0.075 m.u.(- 60 kb) to the right of him-5 in a region only partiallycovered by cosmid clones (descr ibed in Exper imental Pro-

  • 7/28/2019 Guo Sk1995

    2/10

    Cell61 2

    0 0 zygote

    AB($h & heI1+AB o

    EM SFigure 1. Cell Lineages and Early Div is ions in C. elegans(A) Diagram showing a s implif ied cell l ineag e and majo r cell typesproduced by the founder cells (Sulston et al., 1963). PO is the zygote.Posterior is to the right. The blastomeres of the P lineage , namely, POto 22 and 23, are outl ined . Those P lineag e blastomeres that undergoasymmetric divisions are marked with asterisks. Note that asymmetricdiv is ion of EMS segregates the abil ity to produce intestine to the Edaughter and the abil ity to produce pharyngeal and body wall musclesto the M S daughter.(B) Schematic drawing of I-, 2-, and 4-cell wild-type ( left) and par-lmutant (r ight) embryos, showing the div is ions and the distr ibution ofP granules (represented by dots in the cytoplasm) and transcriptionfactor S KN-1 (represented by wavy l ine s in the nucleus; the extentof shading represents the relative concentration of SKN-1 protein).Posterior is to the r ight. In wild-type embryos, f irst and second div is ionsproduce daughter cells differing in s ize, div is ion t iming, c leavage pat-tern, and cell fates. P granules are localized to the posterior end beforethe first div is ion; they are segregated into the Pl blastomere by f irstasymmetric c leavage and further loca lized to the P2 blastomere(Strome and Wood, 1963). SKN-1 protein is highly concentrated in thePl nucleus a nd remains concentrated in the s ister blastomeres EMSand P2 derived from Pi (Bowerman et al., 1993). In par-l mutantembryos (from homozy gous par-l mothers), asymmetry in size, divi-s ion t iming, a nd c leavage pattern is disrupted, and cell fates are dra-matically altered. P granules fail to localize. Since the P granules areundetectable in the late l-cell par-7 embryos and in the 2-cell par-lembryos, they are only shown in the 4-cell embryo. SKN-1 protein isfound in equa l concentration in all blastomeres at 2-, 4-, and &cellstages.

    cedures). We were able partially to rescue par-7 mutantsby transformation with the YAC, Y51 F3 (see Exper imentalProcedures). Rescued par-7 homozygous animals pro-duced about f ive hatching progeny per worm, but all ofthese progeny were agametic. This agametic phenotypeis a maternal effect, s ince par-7 homozygous hermaphro-dites derived from par-l/+ mothers produced gametes.We interpret this result to mean that the expression of

    ALGVgeneticp:,- - I, /,fl, ofFI im-5 py-I\ ,,2!lc -camids Md YACS:

    E2sEu \D.1t

    0 \00 Y-0 khl m \ \, YyF3 (-2Sa kb)YFA1.4 Ad YFAlsa-

    cDNA clone: ZC22(4 W

    I3 100 kb probeN2 bn2c ZC22 probe

    Bcl IAlw39 N2~.,

    Figure 2. Identif ication of par-7(A) Placemen t of par-7 on the physical map. The diagram shows thegenetic map and physical map in the par-l region. Genetic ma ppingputs par-7 -0.075 mu. (estimated - 60 kb) to the r ight of him-5 (seeExperimenta l Procedures). The YAC Y5lF3 rescued par-7 mutant em-bryos in a germline transformation assay. Y51F3 was cut with therestr ic tion enzyme Ascl, and two fragments, YFAlOO and YFA15 0,were generated.(B) Northern blot analysis of maternally enriched transcripts presentin the 100 kb fragment of Y5lF3. Poly(A)t RNA was isolated fromwild-type (N2) and the temperature-sensitive germline-defic ient straing/p-4(bn2), resolved by electrophoresis, blotted, and probed with the100 kb fragments. A s ingle maternally enriched transcript is detectedand indicated with an arrowhead. It is -4.4 kb in length.(C) Genomic Southern blot analysis of par-l(lw39) and wild type (N2).Genomic DNA was isolated from wild-type (N2) and par-l( lw39) hetero-zygous animals, digested with Bell, resolved by electrophoresis, blot-ted onto n ylon fi l ter, and probed with the cDNA clone ZC22. Threebands, 1.6 kb, 7.5 kb, and 10 kb, were detected in N2; in par-l(l

Embed Size (px)
Recommended