Copyright 0 1995 by the Genetics Society of America
Mutations that Alter the Timing and Pattern of cubitus interruptus Gene Expression in Drosophila melanogush Diane C. Sl~sarski,”~ Cynthia Kelsey Motzny‘ and Robert Holmgren Department of Biochemisty, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500 Manuscript received June 8, 1994 Accepted for publication September 15, 1994
ABSTRACT The cubitus intenuptus ( c i ) gene is a member of the Drosophila segment polarity gene family and encodes a protein with a zinc finger domain homologous to the vertebrateGli genes and the nematode tra-l gene. Threeclasses of existing mutations in the d locus alter the regulation ofci expression and can be used to examineci function during development. The first class of cimutationscauses interruptions in wing veins four andfive due to inappropriate expressionof the ci product in the posterior compartment of imaginal discs. The second classof mutations eliminatesci protein early in embryogenesis and causes the deletion of structures that are derived from the region including and adjacent to the engrailed expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks theformation of theventralnakedcuticle.Thelossof d expressionatthese two different stages in embryonic development correlates with the subsequent elimination of wingless expression. Adults heterozygous for the unique mutation have deletions between wing veins three and four. A similar wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative serine/threonine kinase. In cia/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both the ci mRNA and protein appears to be critical for normal Drosophila development.
ci“
D
URING Drosophila embryogenesis the antero-pos-
terior body axis is divided into a series ofsegments that are the basic units for establishing the pattern of structures within the developing embryo. The segment polarity genes play a critical role in specifymg pattern along the antero-posterior axis ofeach segment (NUSSLEIN-VOLHARD and WIESCHAUS 1980). The initial expression ofseveral segment polarity genes is controlled by pair rule genes, but continued expression depends on interactions among the segment polarity genes and their protein products (reviewed in INCHAM 1991; HOOPER and Scorn 1992). Central to the pattern formationprocess is the maintenance of wingless (wg) gene expression in the cells (MARTIjust anterior to the parasegmental boundary NEZ-ARIAZand LAWRENCE 1985; BAKER1987, 1988;VAN DEN HEUVEL et al. 1989). wgencodes a Drosophila member of the Wnt family of secreted growth factors and may act as a morphogen to establish pattern within segments [VAN DEN HEUVEL et al. 1989; BEJSOVEC and MARTINEZ-ARIAS1991; GONZALEZ et al.
1991; BEJSOVEC
and WIESCHAUS 1993; SAMPEDRO et al. 1993 (who sugCorresponding author: Robert Holmgren, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208-3500, E-mail:
[email protected] Present address:Department of Biology, Johns HopkinsUniversity, Baltimore, MD 21218-2685. Co-first authors.
’
Genetics 139: 229-240 (Januav, 1995)
gest that wg is not a morphogen)]. In animals lacking the wg protein, most of the pattern elements within a segment are eliminated and replaced with a lawnof denticles. In animals deficient for the ci region of the fourth chromosome, wg expression in the epidermis is and eliminated during germ bandextension (HIDALGO INCHAM1990; C. MOTZNYand R. HOLMGREN, u n p u b lished results) and the ventral cuticle is covered with denticles (NUSSLEIN-VOLHARD and WIESCHAUS1980;
ORENIC et al. 1987). The cubitus i n t m p u s (ci) region of the fourth chromosome contains three genetically distinct classes of mutations with a complex pattern of complementation (Table 1) (HOCHMAN 1973; ORENIC et al. 1987). In this work it is shown that all of these mutations alter the expression of a single transcription unit, which is referred to as ci. Conceptual translation of the ci transcript et al. predicts a protein with five zinc fingers (ORENIC 1990). The zinc finger domain has a high degree of amino acid sequence identity with the vertebrate Gli genes (84-93%) (KINZLER et al. 1987; RUPPERT et al. 1990) and a lower degree of identity with the tra-1 gene of Caenorhabditis elegans (63%) (ZARKOWERand HODG KIN 1992). Because ci was the first mutation to be isolated in this locus, we have given all of the mutations ci allele designations. class of mutations causes breaks in the fourth The and fifth wing veinsand includes therecessive mutation the semidominant mutation c i w and the dominant
ci’
n‘’,
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C.
D.
Slusarski, C.
TABLE 1
Classes of ci mutations Class
ci'
Phenotype Disruptions in wing veins 4 and 5
and R. Holmgren
Motzny Kelsey
Complementation properties
The distribution of the ci protein in ci""/+ mutant imaginal discs is similar to that of the wild-type ci protein in imaginal discs mutant for the putative serine/ threonine kinase gene fused (PREATet al. 1990).
Complements the segmentation defects ~ of the ~ 2 " ~ "and d1(4)17 classes Complements the segmentation defects of the ciL(4j17class and the wing vein defects of the ci' class Complements the segmentation defects 'j of the ~ i ' ( ~ jclass and the wing vein defects of the ci' class
vu)
MATERIALS AND METHODS
Stocks: The ci" and alleles were obtained from B. HOCHMAN, the ciu, Mhz< ci' and ciw alleles were provided by #) 13 the Bowling Green Drosophila stock center, the tip'"" allele Deletion of ventral was provided by T. KORNBERGand the su(Hd) and su(Hw") naked cuticle in alleles were from V. CORCES. The gamma-ray revertant gRS0 embryos is a partial revertant of c i D (ORENIC et al. 1987).fu" is a strong EMS mutation of f u generated by R.HOLMGREN. Generating c i P h revertants: y q tip'"' females were crossed to P[q+A2-3] Sb/TM3; spaFi males. y q P[q+A2-3] S b / + ; &4j 1 7 Deletion of the first row ciphc/spupoL males were crossedto yw, spaPo'females. Each indiof ventral denticles vidual excision event was crossed to yw; s p P l flies and the y q and variable deletions sPaP"'+/sPuP0' sibling progeny crossed to identify both embryof ventral naked onic and adult mutant phenotypes. Each lethal line was charcuticle acterized by examining cuticle pheno pes and by testing complementation with ci", ci1(4'17 and ci . Mapping ci mutants: Mutations were mapped by performing a series of restriction digests with DNA isolated from appropriate mutants, blotting the gel fractionated fragments onto nitrocellulose and probing withvarious cloned fragmutation ci". is an unusual allele because it is also ments that spanned the ci gene region. Fragments from ci a member of the c2zf41z3class of mutations. This class cDNA clones were particularly useful because they did not includes the and l(4)13 ( ci1f4113) mutations and causes contain repetitive sequences that are present in flanking gerecessive segmentation defects in which the ventral nanomic regions. ked cuticle of each segment is deleted and replaced Cuticlepreparations: Theprocedure of VAN DER MEER (1977) was used. The cuticles were mounted in a 1:l solution with a mirror image duplication of the denticle belt. The ci"4'17cla~~contains the Cz (ci") and Z ( 4 ) 1 7 ( ~ i ' ( ~ ) . ' ~ ) of Hoyers:lactic acid and photographed with phase-contrast microscopy. mutations. These mutantsalso have recessivesegmentaWingbladepreparations: Wingswere removed, dehytion defects. In ~i''~).'' mutants, the first row of denticles drated with propanol and mounted in euparal. Antibody stainings: Antibody staining of embryos was peris removed from the abdominal segments, and there formed according to PATELet al. (1989) using the anti-ci rat are variable deletions of naked cuticle. The ci" mutants monoclonal antibody 2A1 (C. MOTZNYand R. HOLMGREN, are more extreme, and thenaked cuticle is removed in unpublished data), the anti-engrailed (en) mouse monclonal every segment. The ci" mutation also causes variable antibody 4D9 (gift from N. PATEL)and an anti-wg rabbit antidominant adult defects, including partial fusions beWith ci" it is possible to unambigubody (gift from R. NUSSE). ously identify mutant embryos because the ci" allele is viable tween wing veins three and four and the deletion of and fertile over ciplnc. Progeny from c i D / c i p l " " parents were the ocelli. double labeled with anti-ciantibodies and anti-@galactosidase The mutations were placed into these three groups antibodies; embryos that fail to stain with the anti-p-galactosion the basis of their complementation properties. dase antibody are homozygous for the c 'imutation. The other mutations fail to complement the ci" recessive segmenci embryonic lethal mutations are notfertile over tip"'. Mutant embryos wereidentified by the distinct staining patterns prestation defect but complement the ci.' wing vein defect. ent in approximately one quarter of the embryos. For the Mutations in the ciLf4"jr class and the ~i"".'~ class compleanti-en stainings, the ci" mutant embryos were identified by ment each other's segmentation defect and are viable double labeling with anti-ci antibodies. Embryos were staged in certain combinations. Deletions of the locus have a according to CAMPOSORTECA and HARTENSTEIN (1985). phenotype similar to that of cia and fail to complement Imaginal discs werestained in a similar manner. Wandering third instar larvae were collected, rinsed in BSS (CHm and both the and ci1'4'17 classes. GEHNNG 1971), cut in half and turned inside out. Animals Here it is shown that mutations in the ci locus cause were fixedfor 20 min in phosphate-buffered saline containing a variety of alterations in the expression of the ci pro2% paraformaldehyde. Stained discs were removed from the tein. The two classes of embryonic mutations eliminate larval carcass and dehydrated for mounting. In situ hybridizations: In situ hybridization to ci mRNA was expression of the ci protein at different developmental performed using DIGlabeled RNA probes (TAUTZand stages. The adult wing veindefects of the class mutaPFEIFLE 1989). tions are caused by misexpression of the ci protein in Protein gels:Wing imaginal discs were dissectedfrom wanthe posterior compartment of imaginal discs. The tic' dering third instar larvae and dissolved in sodium dodecyl mutation produces a truncated ci protein with an alsulfate (SDS) gel loading buffer. fu" mutants were marked with y and recognized by their yellow mouth hooks. Samples tered distribution pattern in the anterior compartment.
c 'i
c 'i
~i"~'.'~
ci'
2
23 1
Regulation of ci Expression were fractionated ona 7.5% acrylamide gel and blotted onto nitrocellulose. Protein levels were examined by staining the nitrocellulose with PonceauS solution (Serva).ci protein was visualized using the 2A1 monoclonal antibody, an HRPcoupledgoatanti-ratIgGsecondary(Sigma)andtheelectrochemiluminescence (ECL) detection system from Amersham. RNA analysis: Late third instar larvae were homogenized in an SDSurea solution, phenol extracted and ethanol precip itated (MCKENZIEet al. 1975). PolyA+RNAwas selected using oligo dT cellulose. Equal amounts ofRNA were loaded onto a formaldehyde gel. After blotting, the RNAswere probed with the 5' end of a n' cDNA. The hybridization intensities an rp49 standard usinga Molecular were quantified relative to Dynamics phosphorimager.
1kb
E EX
5'
-
Molecular analysis of the ci', ciw, ciD, dLC and c i C e mutations: Genetic analysis of the ci', c i D and cite mutations did not distinguish whether they represented distinct genes or partially complementing alleles of a single locus. Previous work showed that each of these mutations is associated with alterations in a 6-kb BglII fragment that contains the 5' end of the ci transcript (ORENIC et al. 1990) (Figure 1).These alterations could be due to polymorphisms, but the lack of recombination on the fourth chromosome makes that possibility unlikely (BERRYet al. 1991).Therefore, we mapped these mutations in more detailand assessed changes in the expression pattern of the ci protein. Sequences 5' to the ci transcript were conveniently divided into four regions using the restriction enzymes Nsd and BglII (Figure 1).The ci' mutation is caused by the insertion of a gypsy element into region 3. The ci" mutation is associated with the insertion of foreign DNA into region 2. The insertion appears to be an I element (FINNEGAN 1989), because an I element probe hybridizes in situ to theci region (101F) in ci" mutant polytene chromosomes and region 2 DNA hybridizes to a band that comigrates with an I element containing fragment from tic" mutants (data not shown). The c i D mutation results from a small inversion (ORENIC et al. 1990). We have mapped the breakpoints to two regions: region 1 and 10 kb 3' to the ci transcription unit (not shown in Figure 1). The breakpoint is represented by a dashed line because the exact position has not been determined. The spontaneous mutation ciw also has alterations in these same two regions, but the nature of the alterations has not been characterized. The ciphc line containsaninsertion of a whitei (w') enhancer-trap (EATON and KORNBERC1990) into region 3. Analysis of ci protein expression in ci mutantembryos: Expression of the cigenewas assessed in mutants using an anti-ci ratmonoclonal antibody, 2A1 (C. MOTZNYand R. HOLMGREN, unpublished results). The antibody is specific to the ci protein product because there is no labeling of A f Z f mutant embryos in which the ci region is deleted (data not shown).
v
X El
94
183
217 228
RESULTS
E
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FIGURE 1.-Mapof the 5' end of the ci gene. The long horizontal line representsthe DNA from the 5' region of the ci gene. The 6kb BgnI (B) fragment has been divided into foursegmentsusing the restrictionenzyme NstI (N). The locations of insertions (V) and DNA alterations (- - -) associated with each mutation are indicated. The positions of these aberrations have been determined by Southern blot analysis and are mapped to the resolution of the restriction map (with the exception of the insertion, which has been precisely mapped).The exon patternof the ci transcript is shown below the map. The 5' end corresponds to the end of the longest ci cDNA clone. The extent of deletion mutations generated by imprecise excisionof the ciPlac element is also shown below the map of the region. The endpointsof the excision events were determined by probing Southern blotsof DNA from the appropriate mutants and are mapped relative to the indicated restriction sites. The distal break in mutation 183 is in fragment 4, whereas the distal breaks for deletions 94, 217 and 228are all within fragment 3. Deletions 183, 217and 228end within the element, whereas deletion94 eliminates the entire P element andhas a proximal breakpointin fragment 1. The extent of the deletions within the P element have not been indicated on this map. The NsiI sites are only shown within the 6kb BgZII fragment. B, BgZII; E, EcoRI; N, NsiI; X, XhoI. ciPlnC
In wild-type animals, expression of the ci protein initiates during stage 5 and rapidly becomes expressed throughout the embryo. During stage 10 ci mRNAis eliminated from the posterior compartment of each segment (EATONand KORNBERG1990; ORENICet al. 1990) and thereis a correspondingdecay of the protein (Figure 2B). During stage 11 the wide stripe of ci protein expression begins to split, with higher levelsof antibody staining on themargins of the stripe and lower level staining in the middle (Figure 2C). In ci" mutants, lowlevel uniform expression ofci protein is observed at stage 5 and this expression peaks at or shortly after gastrulation (Figure 2D). With the initiation of germ band extension, the ci protein level begins to decrease relative to wild type (Figure 2E; compare the mutantembryo in the centerwith the adjacent wildtype siblings) and is barely visible by the end of stage 11 (Figure 2F). rice and cil(4)17 mutant embryos fail to express the ci protein until stage 7, but as development proceeds,
232
D. C. Slusarski, C.Motzny Kelsey
and R. Holmgren C
FIGURE 2.-Pattern of ci protein expression in embryonic development. Anterior is to the left and dorsal is up. Embryos were stained with the 2A1 rat antici monoclonal. (A-C) Stages 8, 10 and 11 wild-type embryos. Initial expression ofci protein is uniform throughout the segment; during stage 10 ci expression is eliminated in the en expressing cells andduring stage 11 the cells bracketing the m expressing cells show a higher level of antici antibodv labeling. (D-F) Stages 7, 8 and early stage 11 n'" mutant embryos. The stage 7 embryo shows uniform expression of ci protein though the levels are somewhat lower than wild type. With the initiation of germ band extension, protein levels begin to decrease, and bv stage 11 staining for the ci protein has decreased to nearly background levels. (The embryo collections were specifically overstained to allow visualization of the low ci protein levels in the mutants.) ( G I ) Stages 8, 10 and early stage 11 c;''~''' mutant embryos. At stage 7 expression of the ci protein is not detected, but during stage 8 cells begin expressing the ci protein, and by stage 11 the pattern of expression, though not wild type, is quite robust.
clusters of cells initiate ci protein expression. During stage 10 (Figure 2H), the clustersbegin to coalesce into stripes, and by stage 11 the pattern of expression approximates that of wild type, but at lower protein levels (Figure 21). A distinction between the pattern of ci expression in cif'and embryos is observed in stage 11 embryos. Inci'(4)f7embryosthe wide stripe splits as it does inwild type. In ci" mutants the stripe remains uniform (data not shown). In ci"/cirMembryos thepattern of ci protein expression is a combination of the two mutant patterns. Athough the level of protein expression is lower than that of wild-type, ci protein is expressed throughout embryonic development and the pattern resembles that of wild-type embryos. In n" and c i " ' mutants, which generally have normal embryonic development, the ci protein continues to be expressed throughout the segment and fails to be repressed in the posterior compartment (Figure 3, A and R). Expression of wg and en in ci mutant embryos: T h e consequences of eliminating ci protein expression at different times in development were assayed by following the patterns of wg and en protein expression. In
~i"~"'
ci" mutants, expression of the wg protein decays during stage 12 (Figure a), whereas expression ofthe enprotein appears normal (data not shown). By stage 10, wg protein expression is eliminated in regions of the ventral epidermis of ciN4)" mutant embryos (Figure 4B). In these mutants,ci protein levels rise during stages 10 and 11 and epidermal expressionof the wg protein returns in late stage 12 (Figure 4C). In c i N 4 ) ' i mutants, en protein expression is normal through stage 10 but during stages 11 and 12 breaks in the stripes of en expression become evident (Figure 4D). Generation of deletion mutations withii the ci promoterregion: To define the promoter elements required for proper n'expression,we generated deletions of a marked P element insertion. This approach seemed particularly appropriate given the complex nature of the existing ci alleles. T h e n'/''"' line carries a zu" enhancer trap and has a normal pattern of ci protein expression during embryogenesis. Excision events were induced by crossing this line to P[ly'A2-3](99R), a P element transposase expressing strain (ROBERTSONd al. 1988). Approximately 800 excision events were generated and tested for embryonic segmentation defects.
of
Regulation 233
FIGLIRE S.-ci expression in n" and ci'"mr1tants.Anterior is to the left and dorsal is up. (A) Stage 10 n" mutant embryo. (€3) Stage 1 1 ci"' mutant embryo. In both mutants ci protein
expression persists
in the posterior
ci Expression
patterns of these mutations placed them in the ci""" group (Figure 5). On the ventral surface the first row of denticles is missing from most o f the abdominal segments, and there arevariable deletions of naked cuticle including the Keilin's organs of the thoracic segments (Figure 5, C and D). Dorsally the isolated row of large triangularhairs (visible in abdominalsegments 2-8, see arrowheadin Figure 5E) and flanking naked cuticle are eliminated from most segments, and there is a corresponding expansion of the region of socketed hairs andfinehairs(Figure 5, G and H ) . The molecular nature of the excision eventswas examined by Southern blot analysis and the approximate extent of the deletions mapped (Figure 1). In all cases sequences around the distal inverted-repeat of the transposable element were altered. ci protein expression in imaginal discs: Two types of wing vein defects are observed in a' mutants. Mutations of the n" class all cause disruptions in veins 4 and 5. In n'"/+, n", cri"'and ci'"''"/+mutants (h""5''is a revertant of b"), we find defects ranging from minor interruptions of vein 5 to fusions of veins 4 and 5 (Figure 6, RE). ci';./+ mutants have deletionsbetween veins 3 and 4 (Figure 6F). The ci protein distrihutionsin these mu-
compartment.
tants were examined in the developing imaginal discs. In normal imaginal development, ci transcript and protein are restricted to the anterior compartment (Figure Six independent embryonic lethal mutations were iso7, A and B). T h e ci mRNA is uniformly expressed in the lated. The nature of thesegmentationdefects,the patanterior compartment (Figure 7A), whereasantibody terns of ci protein expression and the complementation labeling of the ci protein is elevated along the compart-
FICCRE4.-Expression of en and wg proteins in ci mutants. Anterioris to the left. In a stage 12 Yj" mutant embryo, expression of wg is fading (A). In n"'.""animals, wg expression begins to be eliminated in the epidermis (*) bv stage I O (B), but expression in the epidermis returns (*) during late stage 12 ( C ) .Breaks in the stripes of en expression are visible in stage 1 1 n"'"'"embryos and are marked with arrows (D) .
234
D. C . Slusarski, C. Kelsey M o t m y and R. Holmgren
F I ( ; ~ . K.7).-Cuticle E patterns of d d ' " cxrision mutmls. Anterior is u p . (A) \'rntral surfarc. of a wild-typr lawa. The external cuticle has three thoracic segments that contain fine hair denticle belts and eight abtlominal segments with thick haired denticle is located after thc first row of denticles. (B-D) Ventral cuticle of belts. In the ahdominal segments the segmental border homozygous ci'""', ci" and ci"' mutants. The first row of denticles is eliminated from each abdominal segment, and there are variable tleletions of the naked cuticle. The inset in C shows a detail from an additional mutant animal. In this segment nearly all the first row hairs have been eliminated with the exception of a small cluster identified by the arrow. (E)Dorsal surface of a series of wild-tvpe abdominal segments. The segmental boundary is in the region of naked cuticle j u s t posterior of the single row large triangular hairs (marked with an arrowhead). The naked cuticleis followed sequentially bv regions of socketed hairs and fine hairs. In the homozygous ci""'i, c i w and mutants, the triangular hairs and naked cuticle are deleted in most segments. and the regions contilining socketed hairs and fine hairs are exp;lntled (F-H).
n'"'
Regulation of n' Expression . ..
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F1c.t IU;. (i.--M'ing defects in ci mrmnts. Anterior is up. (A) A wild-type wing. From antcrior t o posterior t h r veins are namhcwd 1 through 3, o f ~ v h i c h3 t o 5. arc indic;ltctl. The I X ) U I I ~ ; I I ~ separating t h c anterior ;mcl posterior coI111);1rtl?lrntsruns.just anterior t o \ving vein 4. 111 r i " / + , ci', c.i"md d ' " 5 " /+ mutants there is a range o f vein t I c f c ~ t sin the posterior conl MI tmcnt. I n c.i"/+ mutants. the f o u r t h and filih veins do not reach t h c margin o f t h c wing black (B). n" at 18' ( C ) anti r i h ' ( h ) have :I 111019' estrclne phrnotye i n which w i n s 4 and 5. fuse t o make a singlr w i n t h a t angles toward t h c posterior margin o f ' the wing. vi""5"/ + (E) is a g ; ~ m n ~ : ~rcvcrtant -~xy o f ri" ;and h a s t h c we;tkcst m u t a n t phenotype in which thcrc is a slight tliswption in vein 3.
