Copyright 0 1991 by the Genetics Society of America

Four Distinct Regulatory Regions of the cut Locus and Their Effect on Cell Type Specification in Drosophila

Su Liu, Elizabeth McLeod, and Joseph Jack

Molecular Biology Program, Sloan-Kettering Institute and Cornell University Graduate School of Medical Sciences, New York, New York 10021

Manuscript received June 18, 1990 Accepted for publication September 24, 1990

ABSTRACT The cut gene in Drosophila is necessary in at least one cell type, the external sensory organs, for

proper cell type specification and morphogenesis. It is also expressed in a variety of other tissues, where its function is less well characterized. Previous work has demonstrated that mutations affecting all the tissues map in the transcribed and translated portion of the gene, while mutations that are tissue specific in their effects map in the 140 kb upstream of the most 5’ exon known. Within that 140 kb, the mutations fall into four subregions, two of which contain mutations affecting unique sets of tissues and the other two of which contain mutations that affect a third set. Our examination of the defects of mutants, their complementation behavior, and their effect on the distribution of the cut protein in embryos, alters the picture in three important ways. First, some mutations convert the cells of the Malpighian tubules into what appear to be gut cells, suggesting that cut is necessary for cell type specification and morphogenesis in a variety of tissues. Second, mutations in each of the four subregions in the 140 kb of upstream DNA cause a different set of phenotypes, suggesting that the regulatory region contains at least four separate units with different tissue specific functions. And third, mutations have now been identified that map in the transcribed and translated portion of the gene but that have tissue specific effects.

T HE cut locus in Drosophila is a complex gene whose function is necessary for the proper cell

type specification of at least one group of cells, the external sense organs. In embryos that lack cut func- tion, 95% of the cells destined in normal embryos to form sense organs with external cuticular structures, instead differentiate into another class of sense organs called chordotonal organs (BODMER et al. 1987). The chordotonal (CH) organs are morphologically distinct from the external sensory (ES) organs. They have attachments on the inside of the epidermis, and ex- press different antigens. Each sense organ of either type is thought to originate from a single ectodermal precursor cell which produces one or sometimes more neurons and usually three support cells (BATE 1978; HARTENSTEIN 1988).

An analysis of the tissue and cellular distribution of the cut gene product gives us some insight into its function. The protein is localized in the nuclei of a number of tissues including the external sensory or- gans, but it is absent from the CH organs (BLOCHLIN- GER et al. 1988). The cut protein is expressed also in the Malpighian tubules, the nonneural cells of the spiracles and in the sense organs of the antenna- maxillary organs. The expression in these tissues is reflected in spiracle and antenna-maxillary organ de- fects that have been observed in mutant embryos lacking cut function (WIESCHAUS, NUSSLEIN-VOLHARD

Genetics 127: 151-159 (January, 1991)

and JURGENS 1984). In addition, we will describe mutant affects on the Malpighian tubules that suggest that cut has an important role in cell type specification and morphogenesis in this tissue. The sequence of the cut locus protein has been deduced from the sequence of cut cDNAs, and that protein is a 2175 amino acid protein, which contains a homeobox (BLOCHLINGER et al. 1988), a motif that has been found to be a DNA binding domain associated with proteins that regulate the transcription of specific genes (DESPLAN, T.HEIS and O’FARRELL 1985; WINSLOW et al. 1989; KRAS- NOW et al. 1989). Since cut antigen is found in the nuclei of ES but not CH organs, the cut protein is apparently expressed in cells determined to form ES organs, perhaps being required in normal morpho- genesis to regulate genes that execute the develop- mental program in those cells. In the absence of cut function, these cells proceed down the developmental pathway that is normally followed by the cells of the CH organs.

Null alleles of cut display all of the embryonic phe- notypes and fail to complement alleles with an array of adult phenotypes as well. However, other cut mu- tations have tissue specific affects, and flies homozy- gous for these mutations are viable and fertile but have visible phenotypes. One group of alleles, called kinked femur (kf ), causes adult flies to have bent legs and crumpled wings. Another group, the cut wing ( c t )

152 S. Liu. E. McLeod and J. Jack

alleles, causes loss of tissue from the wing margin and head capsule of adult flies. Both ct and kf mutations fail to complement a group of mutations designated cut lethal ZZ (JACK 1985), which are separable from ct and kf by recombination (JOHNSON and JUDD 1979). These groups together form a single complex locus. Other alleles of the locus complement kc ct, or both, while failing to complement the lethality of the lethal ZZ alleles (JACK 1985).

As a first step toward understanding the relation- ship between these complex genetics on the one hand, and the function and regulation of the gene on the other, many of the cut mutations have been physically mapped in cut DNA. A striking pattern emerges, each of the phenotypic groups described above occupying a unique region of the gene (see Figure 1 ; JOHNSON and JUDD 1979; JACK 1985). Null alleles map in the transcribed portion of the gene (BLOCHLINGER et a l . 1988). In this paper we analyze a new group of alleles that has not been thoroughly studied before. In ad- dition, we describe the embryonic phenotypes and expression of cut protein in alleles of each of the other complementation groups. These analyses demonstrate that the 140 kb of DNA upstream of the cut open reading frame consists of at least four discrete blocks, each of which directs expression in a different set of tissues. Furthermore, we provide evidence that cut mutations also transform the cells of the Malpighian tubules into cells resembling gut, indicating that the gene is involved in specifying cell type in a number of tissues that are quite different from each other.

MATERIALS AND METHODS

Genetic mapping and complementation tests: The lethal ZV mutations were mapped with respect to ctK b mating, for example, c ~ " ~ ' / F M ~ X cm ctK sn3/Y and then ctX2'/cm ctK sn3 X FM7/Y and scoring the male progeny of the second cross for ct+ recombinants among their brothers with the parental ctK alelle. Males carrying any of the four lethal ZV alleles except for ctll8' do not survive to adulthood. The

from another X-linked lethal mutation. The direction of the mutations from ctK was determined by noting which flanking marker was associated with the ct+ recombinant chromo- somes. The distance from ctK was determined by calculating the percentage of ct+ recombinants among all the males. Because ctK is less viable than wild type, the adjustment in the recombination frequency described below was made to account for the c tK/Y zygotes that die and are not observed amon the adult males. Males from the cross cm ctK sn'/ c t+R(KEZ'~n3 X FM7/Y were scored to determine the relative frequency that the two X chromosomes from the mothers were observed in their sons since these are the two chro- mosomes whose frequency is compared to calculate the recombination frequency. If the viability of the two chro- mosomes is the same, the number of males with each would be expected to be approximately equal. However, the num- ber of ct+R(K""2) sn3/Y males recovered was 2.33 times the number of cm ctK sn3/Y males out of a total of 385 males counted. So, the recombination frequency is calculated as (No.ct+)/(No.ctK)(2.33).

ctllX" allele proved to be hemizygous viable when separated

Complementation tests between cut lethal alleles were done by mating cta/FM7 or FM6 X ctb/ct+Y. The number of offspring was compared to the number of FM6 or FM7 offspring to determine whether or not the ct' and ctb alleles complement. If hemizygous males of one of the alleles were viable, that allele was used as the male parent. Table 1 shows (number of ct"/ctb offspring)/(number of ctb/FM6 offspring) outside parentheses. The number of control ctb/FM6 off- spring is shown in parentheses.

Antibody staining: For antibody staining of lethal mu- tants, flies of the cross ct"/FM6 or FM7 X FM6 or FM7/Y were put in bottles with standard cornmeal agar medium with yeast paste on the side for 12-24 hr and were then placed on apple juice plates with yeast paste to lay overnight. The procedure for staining whole mount embryos is a modification of a procedure described by MITCHISON and SEDAT (1983). Embryos were dechorionated, fixed, re- moved from their vitelline membranes, and stained with primary and secondary antibodies as described in THOMAS, CREWS and GOODMAN (1988). The embryos were then treated with Vectastain Rabbit ABC Kit (Vector Labs) and developed in 0.5 mg/ml diaminobenzidine (Polysciences) and 0.03% H202 in 0.12 M Tris (pH 7.6) for 2-5 min. The embryos were then dehydrated in an ethanol series, cleared in xylene, and mounted in Permount (Sigma).

For analyzing the sense organ transformation phenotypes of cut mutants, whole mount embryos were stained sepa- rately with the monoclonal antibody 21A6 (gift of SEYMOUR BENZER) or anti-horseradish peroxidase (Cappel). The cut product was detected in embryos with a polyclonal rabbit antiserum (gift of K. BLOCHLINCER of the JAN laboratory) directed against amino acids 567-581 of the predicted cut sequence (BLOCHLINGER et a l . 1988) diluted 1:2000.

RESULTS

A number of cut locus mutations have been classi- fied into complementation groups, and their positions in the gene have been determined (JOHNSON and JuDD 1979; JACK 1985). A diagram of the locations of the different types of mutations is shown in Figure 1. Each group of mutations displays a different set of effects. The kf mutations have bent legs and folded wings and fully complement ct mutations, which cause tissue to be missing from the wing margin. The lethal ZZ mutations are essentially null. They are mutant for all cut locus phenotypes including the transformation of ES to CH organs, and they map within a known transcribed region (JOHNSON and JUDD 1979; JACK 1985; BLOCHLINGER et a l . 1989). A second lethal group, the lethal Z group, behaves the same as the lethal ZZ mutations except that they complement kf (JACK 1985; BODMER et a l . 1987). Two other muta- tions behave like lethal Z mutations in complementa- tion tests but map 30 kb away, and as described below, are different from the lethal Z mutations in their effects on cell type transformation. These mutations will be designated lethal ZZZ mutations. In addition, we have analyzed another group of lethal alleles that complement both the kf and ct phenotypes and have designated them lethal ZV alleles.

Complementation behavior of the lethal IV group of mutations: Four mutations fail to complement the

cut Locus Regulatory Mutations

TABLE 1

Complementation of cut locus mutations

153

Lethal I11 Lethal 1V Lethal I Lethal I 1

kf ~t L-39 L-59 IS2 1188 122 1 1242 L- 1 L-5 C145 1s kf kf + + + + + + + + + kf kf Ct Ct Ct Ct + + + + L-59 IS2 1188 122 1 1242 L- 1 L-5 C145 1s (kf) kinked femur phenotype, (ct) cut wing phenotype, (ND) not done. The numbers outside parentheses indicate the fraction (number

heterozygous mutants surviving)/(number of wild type controls). This is the fraction of what is expected if the mutations fully complement. T h e number in parenthesis is the number of control flies.

Ct Ct ct Ct L-39 - 0 (300) 0.23 (210) 0.95 (395) 0.23 (244) 1.3 (443) 0.01 (148) 0.08 (155) ND ND

- 0.05 (311) 0.12 (427) 0.35 (171) 0.61 (298) 0 (153) 0 (290) 0 (156) 0 (134) - 0.78 (198) O.OZ(183) 0.46 (245) 0 (63) 0 (75) 0 (177) 0.006(156)

- 0.18 (219) 0.92 (339) 0.99 (279) 0.17 (327) 0.02 (213) 0 (236) - 0.55 (406) 0 (115) 0.13 (146) 0 (189) 0 (199)

- 0.52 (265) 0.09 (348) 0 (405) 0.003 (343) - ND ND 0 (320)

- 0 (50) 0 (334) - 0 (35)

-

1242 c flS2

0.09 ":;-I Recombination k f c f 6 C f

in centimorgans map

" l e tha l 11-

~ 0 . 0 4 ~ 0 . 1 6 - - - - " 0 . 0 . 0 5 ~ cM 0.1

Lethal

kinked femur cut wing 111 I I 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - 6 0 - 4 0 - 2 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 160 kilobases

Centromere + FIGURE 1 .-Map of mutations and transcripts in cut. Both a recombination map in centimorgans and a physical map in kilobases are shown.

Each block of mutations-kinked femur, cut, lethals 111, I , and 11-is composed of mutations with a different set of phenotypes as described in the text. Mapping data are taken from JOHNSON andJuDD (1979), JACK (1985), BLOCHLINGER et al. (1988), and this report.

lethality of cut lethal ZZ alleles but complement both cut wing and kinkedfemur alleles. One, ctlS2, is a spon- taneous mutation (A. SCHALET, personal communica- tion), and the others, ct1lS8, ~ t ' ~ ~ ' , c t " ~ ~ , were recovered in a screen for hybrid dysgenesis induced, X-linked lethal mutations (F.-M. SHEEN and M. SIMMONS, per- sonal communication). All but ct"" are lethal in hem- izygous males. We have tested the complementation of these four mutations among themselves and with a variety of other cut alleles, and the results of the complementations tests are shown in Table 1. All four mutations are completely lethal in combination with

the lethal ZZ alleles ~ t ~ ' ~ ~ and ctiS, but they are comple- mented to varying degrees by alleles from other com- plementation groups. In particular, the four muta- tions show a fair amount of viability as heterozygotes with each other in most combinations. This partial complementation among the four mutations makes it ambiguous as to whether they are members of the same or different complementation groups. Because d S 2 and ctLZ2' complement each other very little and show very similar complementation behavior with the lethal I and IZI alleles, these two might be considered to be in the same complementation group while ct""

154 S. Liu, E. McLeod and J. Jack

TABLE 2

Recombination of lethaZ N mutations with c l

Mutation from c f Recombinants Total males Frequency

ctlS2 Centromere 11 1984 0.0024 Centromere 5 2831 0.0008

ct 1221

a The recombination frequency is adjusted as described in MA- TERIALS AND METHODS to account for the reduced viability of the c t K chromosome relative to the ct+ recombinant chromosomes.

Direction

ct 1242

0 95 1

and d Z 4 ' could each be considered a member of a second and third group. Nevertheless, since all four are similar in being lethal mutations but wild type for kj and ct, we are designating all four as lethal ZV mutations. The partial complementation of the lethal ZV alleles with each other cannot easily be explained by supposing that the alleles are simply leaky because they are almost completely lethal as homozygotes or as heterozygotes with lethal ZZ mutations. Rather, each lethal ZV mutation may interrupt one of a group of units in the gene that partially overlap in function. This would leave the other units functioning and able to complement, at least partially, any of the other lethal ZV or lethal ZZZ alleles. These units might be functional domains of the protein, regulatory se- quences in the gene, or both.

Recombination mapping of lethal N alleles: In order to find out how the lethal ZV mutations alter cut expression, we wanted to know the location of the mutations in the gene, particularly with respect to the transcription coding sequences. However, Southern blots yielded no evidence of any rearrangements of the 225 kb of cloned cut locus genomic DNA in any of the four mutations. Nevertheless, recombination mapping can locate the mutations roughly with re- spect to the transcribed region. We began by mapping the mutations with respect to ctK, which is caused by an insertion of the transposable element gypsy at about +72, among the other lethal Z mutations (JACK 1985) about 6 kb upstream of the most 5' exon identified for the cut locus transcript (Figure 1). Re- combinants were identified as ct+ male progeny among the offspring of ct'r'ha' '"lcm ctK sn3 females. The results are summarized in Table 2 and shown with the recombination mapping data OfJoHNSON and JUDD (1979) in Figure 1 . Both ctlS2 and ctlZ4' map downstream of ctK, with cttZ4' about 0.09 cM away and ctlS2about 0.24 cM from ctK. The recombination fre- quency for ct"" is about the same as JOHNSON and JUDD (1979) reported for the distance between ctK and some lethal ZZ alleles, placing it roughly in the transcribed portion of the gene. However, the ctK-ctlS2 distance is very large, equal to the distance they re- ported for the entire gene and about 3 to 5 times farther to the right of ctK than the distance they reported for the lethal ZZ mutations (JOHNSON and

JUDD 1979). These results suggest that ctlS2 might be located well past the most 3' end identified for the transcription unit. However, other evidence, dis- cussed below, indicates that ct"' is within a translated portion of the gene. We found no recombinants be- tween ct"" and ctK out of 95 l chromosomes suggest- ing that ctl"' is located nearer the lethal Z group of mutations than are the other lethal ZV alleles. Recom- bination mapping of was not completed due to complications described in MATERIALS AND METHODS. Nevertheless, the mapping data indicate that at least two of the lethal ZV alleles, which are capable of complementing a number of cut locus phenotypes, are located within the transcribed region of the gene.

Sense organ phenotype of the mutants: The lethal ZV, kf, and ct alleles each fail to complement other cut alleles for only one phenotype. Since kf and ct appear to be tissue specific in their effects, the lethal ZV alleles could also affect only specific tissues. None of the four lethal ZV mutations transform the ES organs into CH organs in homozygous embryos. This observation was originally made by R. BODMER, L. Y. JAN, and Y. N. JAN (personal communication) for embryos hemizy- gous for the mutations, and we have confirmed this result with all four lethal ZV mutations. In order to test embryos with the strongest possible phenotypes, we have examined heterozygotes for the deletion Df(1)ctJ'over ct"" and ctlZ4', the strongest of the lethal ZV alleles in complementation tests for lethality. Nei- ther of the two heterozygotes, Df( l )~ tJ~ /c t ' " ' or D f ( I ) ~ t J ~ / c t " ~ ' , show any tranformation of the ES organs. Thus, the lethal N alleles affect some function of the cut locus necessary for viability but not for specifying the cell types of the embryonic sense or- gans.

The lethal ZZZ mutations are similar to the lethal Z mutations in their complementation behavior with the hf, ct, and lethal ZZ alleles. The only marked difference in complementation behavior is that the lethal ZZZ mutations complement the lethal ZV mutations to a higher degree than do the lethal Z mutations (Table 1). These differences in complementation for lethality could be due to differences in expressivity of the alleles. However, we find an additional qualitative difference with regard to the ES organ phenotype. While lethal Z mutations transform ES to CH organs (BODMER et al. 1987), the lethal ZZZ mutations have no effect on this phenotype. Thus, lethal Z and ZZZ muta- tions differ in three respects: their location, their degree of complementation with lethal ZV alleles, and their effect on sense organ transformation.

Localization of cut protein in mutant embryos: Since each group of cut mutations displays a different subset of mutant phenotypes, we determined the ef- fect of each group on the tissue specific expression and localization of cut protein. The mutations used

cut Locus Regulatory Mutations 155

TABLE 3

Classification of mutant alleles tested

Allele tvPe Reference

Kinked femur v WHITNEY and LUCCHFSI (1972) C t / C 2 0 G . LEFEVRE (unpublished)

ct MORGAN, BRIDGES and STURTEVANT (1 925) Ct/':20 G . LEFEVRE (unpublished)

ct L-39 J. K. LIM, J. W. JACK, and B. H. JUDD (unpublished) ct L-19 J. K. LIM, J. W. JACK, and B. H. JUDD (unpublished)

ct L- l LIM et al. (1983) ct LJ LIM et al. ( 1 983)

ct KRIVSHENKO (1 956)

Cut wing

Cut lethal 111

Cut lethal I

L-27 J. K. LIM, J. W. JACK, and B. H. JUDD (unpublished)

ct YE1 I 8 WIESCHAUS, NUSSLEIN-VOHARD and JURGENS

(1 984a) Cut lethal I1

C t C / 4 1 JOHNSON and JUDD (1979) ct IS A. SCHALET (unpublished); JACK (1 985) ct BLOCHLINGER et al. (1988) Of( I ) d 4 LEFEVRE and JOHNSON (1 973)

Cut lethal IV ct IS2 A. SCHALET (unpublished)

ct I221 F.-M. SHEEN and M. SIMMONS (unpublished) Ctl188 F.-M. SHEEN and M. SIMMONS (unpublished)

ct 1242 F.-M. SHEEN and M. SIMMONS (unpublished)

and their type are summarized in Table 3. In partic- ular, since lethal ZZZ and N mutations do not affect ES organs, it is possible that they alter expression of the gene in other embryonic tissues. Recall that in wild- type embryos, cut antibodies stain the Malpighian tubules, central nervous system, and spiracles in ad- dition to the ES organs. We have examined the local- ization of the cut protein in embryos hemizygous for all six lethal ZZZ and N mutations and in selected kf, ct, and lethal Z alleles by histochemical staining with a cut antipeptide antibody (BLOCHLINGER et al. 1988). The ctlcZo mutation, which lacks all of the kf region and part of the ct region and is extreme for both pheno- types, does not alter expression in embryos. This comes as no particular surprise since the only known phenotypes of this allele are adult phenotypes. Like- wise, the lethal ZZZ mutations have no effect on embry- onic expression. This fact is consistent with the obser- vation of normal embryonic nervous systems in these mutants. The lack of a change in the overall embry- onic expression suggests that the lethal ZZZ region, like the ct and kfregions, controls expression of cut after embryogenesis.

In contrast, the lethal Z mutations do affect expres- sion of the protein in embryos. We examined ctL",

these lethal Z alleles causes the Malpighian tubules to be missing in mutant embryos and also causes a re- duction of cut protein levels in all of the embryonic

CtL-55, C P Z 7 , and ctYE"' from the lethal Z group. Each of

tissues in which it is expressed. However, the degree of the reduction and the tissues that are most affected vary with the particular mutation. For instance, in ctL"/Y embryos no antibody staining is evident in the PNS, and only a slight staining remains in the anterior spiracle (Figure 2b). In c ~ ~ - ' ~ / Y embryos (not shown), staining is clearly reduced but still evident in the CNS, PNS, and spiracles. For ~ t ~ - ~ , the staining in the CNS, PNS, and anterior spiracles was barely detectable while the staining in the posterior spiracles was still quite strong (data not shown).

A change in staining pattern qualitatively different from the other three lethal Z mutations is observed in

is reduced in ctYE"' embryos, but contrary to the observations with other lethal Z alleles, the remaining protein is not localized to the nucleus but instead is distributed throughout the cell. This is similar to the ctDB7 mutation, which is shown in Figure 4. The change in cellular localization of the cut protein is unexpected since other lethal Z mutations map pre- dominantly to +70 on the map, which is a position upstream of the known translated portion of the gene (BLOCHLINGER et al . 1988). ctYE"' is grouped with the lethal Z mutations because like the other alleles it complements kf but is mutant for the other cut phe- notypes. However, it is unusual among lethal Z muta- tions in that it was induced by EMS, while other lethal Z mutations are caused by gypsy transposable element insertions. Since ctYE"' has not been mapped, it may well be located centromere proximal to the other lethal Z mutations, in the coding region. The other lethal Z insertion mutations are likely to affect cut transcription since the mutant phenotypes of these can be suppressed by mutations of the gene su(Hw) (JACK 1985).

Three of the four lethal ZV mutations, ct1118, ctLZz1, and ctlz4', have no clear effect on staining of mutant embryos with anti-cut antibodies, although the level of protein could be reduced somewhat without being detectable in these experiments. In ct"' heterozygotes and homozygotes, the protein is still present in the same tissues as the wild type, however, it is distributed throughout the cells rather than being localized in the nucleus (BLOCHLINGER et al . 1990 and Figure 3). This is similar to the effect of ctYE"'. However, in contrast to ctYEI1', where the Malpighian tubules are com- pletely missing, ct"' apparently does not alter the morphology of the Malpighian tubules.

As we noted above, ct"' maps by recombination far proximal to ctK, at a distance that would seem to be well downstream of the known translated portion of the gene. However, since the mutation affects the cellular localization of the protein, we assume that ctlS2 is in a translated portion of the gene and that the high rate of recombination does not accurately reflect

C t Y E l 1 8 mutant embryos. The general level of staining

156

p A '

S. Liu, E. McLeod a n d J . Jack

c AMC ." 1

..

D

CNS

r-

the distance of ct"' from c t K . On the other hand, an alternatively used exon with translated sequence could be located to the right of where the 3' end of the protein coding sequence has been identified in exist- ing cDNA clones (RI.OCHI.INGER et al. 1988). How- ever, the sequences of four additional cDNAs each

/ HG

F ~ G ~ ~ R K 4.-JlaIpighi~~n tul~ulc pl~c~~otvpc. \Vild-typc ( '4) ; I I I ~

ct""' ( R ) c m I ) ~ . v o z arc st;lirlctI w i t h anti-rut antiImcIy. J1aIpigIlian tul)ules, hindgut. ;Ind posterior spir;des arc shown I;cbclcd i n wild type (A ) . I n rt"'" the antigen is still prcsc-nt i n the tnodifietl Jlalpi- gllian tubule tissue (1nJf-1.). b u t i t is localized i n the cytopl;~sm instead of t h r I I I I C ~ ~ I I S ;IS i t is in wild-type embryos.

contains the same 3' end of the open reading frame (S. LIU, unpublished results). Even though ct'"' must alter the structure of the protein product, it comple- ments both the kf and ct phenotypes and has no embryonic phenotype. This residual activity could be due to leakiness of the mutation. Another possibility is that cut encodes rnore than one protein, and the

cut Locus Regulatory Mutations 157

ctLS2 mutation affects a protein whose function is not necessary far the normal embryonic, ct, and k. phe- notypes. If this is the case, the antibody that we used must recognize a peptide present in both proteins.

We have also analyzed expression in two of the lethal ZZ mutations. The distribution of cut antigen in ctiS and ctDE7 (Figure 4) mutants is similar to ctYE"'. That is, the staining is not nuclear but is distributed throughout the cells, and the Malpighian tubules are missing. Other lethal ZI mutations have been analyzed, and cytoplasmic localization of the antigen proves to be the exception (BLOCHLINCER et al. 1990). Most LethaE ZZ alleles lack detectable cut expression.

Transformation of the Malpighian tubules by Ze- thal Z and ZZ mutations: All of the lethal Z and ZZ mutants that we have analyzed and Df(1)ctJ' and Df(I)ct4" lack Malpighian tubules in homozygous mu- tant embryos. In addition, the wall of the gut is thickened three to four fold near of the junction of the posterior midgut and the anterior hindgut, where the Malpighian tubules would normally attach to the gut.

In wild-type flies the Malpighian tubules form as four outgrowths of the primordium of the anterior hindgut or posterior midgut. We begin to see cut protein expression at that site just before the Malpi- ghian tubules begin to bud, and expression continues in the Malpighian tubules through the time when secretion of cuticle prevents antibody staining of whole mount embryos. In ctLS, ctDE7, and ctYE118, mu- tations that lack Malpighian tubules but retain cyto- plasmic cut staining, the thickened part of the gut stains (Figure 4). So, the thickened portion of the gut in mutant embryos is apparently comprised of cells that in wild-type embryos would form Malpighian tubules. Thus, embryos mutant for lethal I or ZZ ap- parently have a transformation of cell type of the cells normally destined to become Malpighian tubules. These cells are present, but they never bud out to form tubules and instead resemble the gut.

DISCUSSION

The product of the cut gene is required in a number of different tissues in embryos and adults in order for those tissues to develop properly. In embryos, anti- bodies against the cut protein stain ES organs, Malpi- ghian tubules, spiracles, and the antenna-maxillary complex (BLOCHLINGER et al. 1988), and all of these structures are malformed or absent in mutant em- bryos (WIESCHAUS, NUSSLEIN-VOLHARD and JURGENS 1984; BODMER et al. 1987). In adults cut mutations can either cause the loss of a large part of the wing margin, prevent the newly emerged adults from ex- panding their wings, or both.

The role of cut is best defined in the peripheral nervous system. It is likely to be a transcription factor

that controls the activity of genes that distinguish ES organs from CH organs. Mutations that lack cut activ- ity convert the ES organs to CH organs, and cut protein is localized to the nucleus and contains a homeobox domain characteristic of a group of tran- scriptional regulators (QESPLAN, THEIS and O'FARREL 1985; WINSLOW et al. 1989; KRASNOW et ul. 1989). Since cut antigen is expressed in the ES organs but not the CH organs, the gene is probably turned on in response to signals that determine the precursors of the ES organs. Then, it regulates genes that execute the ES developmental pathway.

Since cut appears to be involved in specifying cell type in the ES organs, it may have a similar role in the other cells in which it is expressed. For instance, the cells of the Malpighian tubules are transformed by lethal Z and ZZ mutations into a thick ring of cells associated with the gut (Figure 4). The Malpighian tubules first appear as four outgrowths of the midgut or hindgut primordium. They grow into long winding tubes as their cells divide ( JANNINC, LUTZ and WISSEN 1986), and they continue to be connected to the gut and empty their excretions into it. In cut Eethal Z and II alleles, the Malpighian tubules are missing and are replaced by a thickening in the wall of the gut. The mutant structure can be identified as transformed Malpighian tubules because it continues to stain with anti-cut antiserum in embryos homozygous for lethal Z and ZZ alleles that do not abolish cut antibody stain- ing. This staining of the thickened portion of the gut is likely to represent the proper determination and subsequent tissue specific expression of cut in the Malpighian tubules because the other cells that con- tain cut antigen in these mutations are the same ones that do in normal embryos. However, the function of the cut product is obviously impaired in these muta- tions since its cellular localization is abnormal, being cytoplasmic rather than nuclear. The mutant product must fail to correctly activate the differentiation path- way in the Malpighian tubules, so the cells instead form an enlargement of the gut. Whether these mu- tant Malpighian tubule cells have been transformed into bona$de midgut or hindgut cells or simply undif- ferentiated cells that remain associated with the gut is a question that we have not yet answered. In either case, these mutations cause a transformation of Mal- pighian tubule cells indicating that cut is involved in the control of morphogenesis of the Malpighian tu- bules as well as the ES organs. Moreover, since cut is involved in cell type specification of both ES organs and Malpighian tubules, it is likely that it has a similar function in some or all of the other cells that express it.

Another issue addressed by these studies is the mechanism by which cut is expressed in a specific set of tissues. Because many of the cut mutations are

158 S. Liu, E. McLeod and J. Jack

TABLE 4

Functional defects of cut complementation groups

Complementation group defects Kinked femur Cut wing Lethal 111 Lethal I Lethal I1 Lethal IV

Folded wings mut + + + mut + Bent legs

Scalloped wings + mut mut mut mut + Post embryonic lethality + + mut mut mut mut

Sense organ transformation + + + mut mut + Malpighian tubule defects Embryonic lethality Antigen expression reduced

(+) indicates that the mutants in the group are wild type for the phenotype and (mut) indicates that they are mutant. For mutations that would cause lethality before a Darticular Dhenotvae could be observed, the determination of the phenotype is made by complementation with other alleles.

I .

located upstream of the translated portion of the gene and affect only a subset of the different tissues in which the gene is normally expressed, they probably block the function of sequences necessary for correct regulation. In this paper, we have described the com- plementation patterns, the embryonic phenotypes, and the location in the gene of a number of different cut alleles. Based on this description and previous work (JOHNSON and JuDD 1979; JACK 1985; BODMER et al . 1988), we are able to classify the different types of alleles into groups, and we have determined the effect on embryonic cut expression of representatives from each of these groups. Table 4 summarizes the phe- notypes and changes in antigen expression of each group. From the previous work, a picture had emerged of an extraordinarily large gene made up of blocks with each block being involved in expression in a different subset of tissues (Figure 1). Mutations in the centromere proximal segment appeared to pre- vent expression in all of the tissues in which cut is active because the mutations displayed all the mutant phenotypes. That segment turns out to be the protein coding region of the gene (BLOCHLINGER et al . 1988). Each block of mutations in the 140 kb upstream of the translated portion of the gene retains activity in at least one tissue. Because these mutations are 5’ of the only known translated sequence in the gene, they probably affect regulation.

The distal regulatory region appears to be made up of blocks which are each used in a different set of tissues. Each block of mutations upstream of the start of translation-that is to the left of +78 in Figure 1- is made up of mutations with the same set of pheno- types, and the set phenotypes of each block of muta- tions is different from that of the other blocks. When the physical mapping of the gene was described, only the kinked femur, cut wing, and lethal phenotypes had been described; and on the basis of those pheno- types, the mutations at +40 on the map and those at +70-72 were characterized as having the same set of

phenotypes-that is cut wing and lethality but wild type for kinked femur. However, in this report we have analyzed the sense organ phenotype of the two mutations at +40 and find that the ES organs are not transformed in the mutations as they have been shown to be in the mutations at +70-72 (BODMER et al. 1987). Furthermore, the mutations at +40 have no discernible effect on the embryonic expression of the cut protein, while all the mutations at +70-72 reduce expression. So, on closer inspection, these two widely separated blocks of mutations do not have the same set of phenotypes. We have, therefore, designated the mutations located at +40 lethal ZZZ and those at +72 lethal I . Mutations, then, define four functional units upstream of the start of translation and the most 5’ exon identified, and each unit apparently promotes cut expression in a different subset of tissues. This is reminiscent of the situation at the Ultrabithorax gene, where the regulatory regions that control transcrip- tion of the gene are located 30-50 kb away from the start of transcription (DUNCAN 1987).

This work also suggests that the translated region may be somewhat more complex than previously re- ported (BLOCHLINGER et al. 1988). Most of the muta- tions in that part of the gene are lethal ZZ alleles, that is they are mutant for all known cut phenotypes. We have previously considered this complete lack of func- tion to be associated with all mutations in the coding region. However, a group of four mutations, desig- nated lethal ZV mutations, fail to complement the lethality of the lethal ZZ mutations but are wild type for the kinked femur and cut wing phenotypes and have no obvious effect on either the phenotype or the expression of cut antigen in embryos. At least two of the four mutations are apparently located in the cod- ing region because of their map position as deter- mined by recombination. One of these, ctLSZ, causes the protein to be distributed throughout the cell rather than to be localized to the nucleus as in wild type, suggesting that the protein itself has been altered

cut Locus Regulatory Mutations 159

by the mutation. Furthermore, another allele ctYE1I8, which is wild type for the kinked femur phenotype, also interferes with the nuclear localization of the protein suggesting that the mutation is in the protein coding region. In fact, JOHNSON and JUDD (1979) mapped another mutation wild type for kinked femur ct"' to the region to the right of the lethal I group. So, a number of mutations located in the coding region of the gene are phenotypically wild type for one or more cut functions. The coding region muta- tions that are lethal but retain some wild type function are all apparent point mutations. So, their exact loca- tions within that region have not been determined, and we cannot draw any conclusion as to why they complement certain phenotypes. The simplest expla- nation is that the mutations are just leaky and have lost enough function to express some mutant func- tions but not others. However, this is inadequate to explain all the properties of the mutations. For in- stance, ctlS2 and ctlZ4', which both map to the coding region, complement the lethality of a variety of other mutations and the adult phenotypes of the kfand ct alleles. The complementation by ctLS2 and ctlZ4' of the lethality of other cut alleles cannot be explained by leakiness because both ct"' and ~ t ' ' ~ ~ are tight lethal alleles as homozygotes or heterozygotes. So, the mu- tations must leave some activities at least partially intact in order to complement other cut alleles. One possibility is that the gene codes for more than one protein product, and ctlS' and ~t~~~~ alter some prod- ucts leaving others unaffected. Knowing the position of the coding region mutations more accurately would help to determine whether there are particular exons that might not be used in all tissues or whether there are exons or other functional units to the right of the lethal I group of mutations that we have not yet discovered.

We thank ARE SCHALET for providing us with the ckS2 mutant stock and MICHAEL SIMMONS and FANG-MIIN SHEEN for the ctfJ8', ctfZ2' and stocks. We thank DENNIS BALLINGER for a critical reading of the manuscript and many useful comments, and DALE DORSETT for helpful discussions. We also thank YVONNE DELOTTO for assistance with the photography. This work was supported by National Science Foundation grant DMB 881 1519.

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Communicating editor: W. M. GELBART