and phenotypic analysis of thirteen essential genes in...

Copyright 0 1994 by the Genetics Society of America

Genetic and Phenotypic Analysis of Thirteen Essential Genes in Cytological Interval 22F1-2; 23B1-2 Reveals Novel Genes Required for

Neural Development in Drosophila

J. Troy Littleton and Hugo J. Bellen Division of Neuroscience, Howard Hughes Medical Znstitute, Department of Molecular and Human Genetics,

Baylor College of Medicine, Houston, Texas 77030 Manuscript received March 1, 1994

Accepted for publication May 17, 1994

ABSTRACT In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new

rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aber- rations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.

T HE cytological region 22F1-2; 23B1-2 has been studied extensively in our laboratory in an effort to generate mutations in synaptotagmin ( s y t ) . syt encodes a synaptic vesicle specific protein, Syt, which contains two repeats within its sequence that are homologous to the C2 domain of protein kinase C. This domain has been implicated in calcium dependent membrane interac- tions (PERIN et al. 1990). In addition, Syt has been reported to interact with several other synaptic proteins (BENNETT et al. 1992; LEVEQUE et al. 1992; PETRENKO et al. 1991), suggesting a role in docking and/or fusion of synaptic vesicles with the presynaptic membrane. A Dro- sophila syt clone was isolated through homology cloning and shown to be homologous to rat Syt, suggesting a conserved role in neural function (PERIN et al. 1991). syt is a pan-neural gene in Drosophila whose protein prod- uct is specifically localized to synapses (LITTLETON et al. 1993a). syt was mapped to cytological bands 23AGB1, and ethyl methanesulfonate (EMS) and P element mutageneses were undertaken to obtain mutations in s ~ t . Mutations were obtained and their phenotypic analyses demonstrate that Syt plays a central role in calcium activation of neurotransmitter release (LI-ITLETON et al. 1993b).

In the process of generating mutations in syt, we have obtained a large collection of deletions and mutations within the 22F1-2; 23B1-2 interval. Using ?rays, EMS, and P element insertion and excision mutagenesis, we have isolated new deletions as well as recessive lethal point mutations. These mutations as well as those generated by others (W. GELBART and J. SEKELSKY, unpub-

Genetics 138: 111-123 (September, 1994)

lished; DIANTONIO et al. 1993) identify 13 complementation groups in this cytological interval. These include decapentaplegic, syt and Curly, as well as 10 other essential complementation groups. The deletions within the region have allowed us to map and order many of the complementation groups within the 22F1-2; 23B1-2 interval. In addition, we provide a preliminary phenotypic characterization of the mutantswithin this interval.

MATERIALS AND METHODS

Genetic strains and chromosomes: All fly strains were raised at 25" on standard cornmeal-molasses medium supple- mented with live yeast. Most rearrangements used in this study are described in LINDSLEY and ZIMM (1992) or SPENCER et al. (1982). A summary of the second chromosome rearrangements used in this study is shown in Table 1. Additional strains used in this study include: (1) y w; P[lacZ, w+]B8-2-30, y w; P[lacZ,w+]El0-2-19, yw; P[w+,kinesin-lacZ] KZ1941/ In(2LR)Gla and yw; KZ1941 excision#2 /In(2LR) Gla gen- erously provided by Y. N. JAN, G. FEGER and E. GRELL; (2) P[lacZ, ry]PZ, 1(2)03728; ry/ry (WEN and SPRADLINC 1992) ; (3) a group of previously uncharacterized EMS-induced mutations that fail to complement Df(2L)DTD2 Dp(2;2) DTD48 which uncovers cytological interval 22F3;23B1-2. These mutants were generated in the laboratory of W. GELBART and provided by J. SEKELSKY. They include: dppd-blk 56.12/Gla, dppd 55.5/Gla, dppd 55.1/Gla, dppd 77.3/CyO, dpphr4 54.6/ Gla, dppd 67.3/Gla, dpphr4 66.4/Gla, ast dppd-h" 5 8 . 2 ed d cl/Gla, dppd-bih 56.1/Gla, ast dPpd-lro 58.3 ed dp cl/Gla, dpp l 55.6/Gla, ast dppd-ho 58.14 ed dp cl/Gla, dpphr4 66.14/Gla, ast dppd-ho 58.15 ed dp cl/Gla and dpphr4 66.5/Gla; (4) published EMS-induced mutations belonging to seven genes mapping to the interval between the breakpoints of Df(2L)Hin34 and Df(2L)DTD2 provided by T. SCHWARZ and A. DIANTONIO and described in DIANTONIO et al. (1993).

112 J. T. Littleton and H. J. Bellen

TABLE 1 TABLE 2

Second chromosome rearrangements used in this study Summary of genetic screens for recessive mutations in region 22F1-2; 23B1-2

Rearrangement Cytology Reference a

Vf(2L)V TD2 22D4-5; 23B1-2 1 Df(2LjC144 D f ( 2 L ) d p p ~ ~ n " 4 Df(2L)dPP 22E4F2; 22F3-23Al 1 Df(2L? 22F1-2; 22F3-4 1

Df ( 2 0 dPP 22A, 23A 2 D f ( 2 L ) T L I 22F1-2; 23B 4 Df(2L)N6 23A6; 23B1 3 D f ( 2 L ) N l 9 23A4; 23B1 3 Df(2L)NlO nvb (23B1) 4 Df(2L)N28 nv (23B1) 4 Df(2LjN13 nv (23B1) 4 Df(2L)D31 nv (23A1; 23A3) 4 Df(2L)D20 nv (22F4; 23A1) 4

23A1-3; 23C 2 22E2-3; 23A34 2

Df(2L?dP!JHin39 22F1-2; 23A1-2 1

Dp(2;2)dpp2' 22A2-3; 22F1-2 into 52F 1 In(2L)54Cy" 23B1-2; 25C 4

a References: 1, SPENCER et al. (1982); 2, J. SEKELsKYand W. GELBART (personal communication); 3 , LITTLETON et al. (19938); 4, this work.

nv = not visible, cytology inferred from complementation analysis.

Mutagenesis: The various types of mutageneses carried out in this and accompanying studies (LITTLETON et al. 199313, 1994) are summarized in Table 2 and the crosses used to generate these mutations are outlined in Figure 2. Mobilization of the P[lacZ,w+]BS line into syt allowed us to isolate the P[lacZ,w+]T77 insertion in syt . Imprecise excisions of the P[lacZ, w+]T77 were generated as described previously (BELLEN et al. 1989). Excision strains were examined by po- lymerase chain reaction (PCR) using primers 5' and 3' of the insertion into syt, as well as by Southern analysis and complementation analyses (LITTLETON et al. 199313).

Mutations were obtained by feeding isogenized 3-5daydd adult cn bw sp/m bw sp males with EMS as previously described (hws and BACHER 1968). Males were crossed to CyO/Gla virgins and flies transferred once after 4 days. Males were allowed to mate an additional 4 days and then discarded. Single male progeny carrying the mutagenized chromosome over Gla were then crossed to Df(ZL)D?2)2 Bl SpDp(2;Z) dP/Glafemales at 28". Gla is an inversion that balances most of the left arm of the second chromosome. It was used instead of other second chromosome balancers since Cy fails to complement Df(ZL)D72)2. Progeny were scored for the absence of cn bw sp*/D7D2 flies as outlined in Figure 2. Stocks were balanced and maintained at 25". Flies were retested over Df(2LJDTD2 at 18" to identify any temperature sensitive mutations. A total of 5400 chromosomes were tested and 58 homozygous lethal mutations were recovered that fail to complement Df(2L)D7D2. No temperature-sensitive mutations were recovered.

Given the apparent cytological localization of Cy (SPENCER et al. 1982) and syt (LITTLETON et al. 199310) we were interested to establish whether syt and Cy were the same gene. A Cy chromosome free of cytological rearrangements had previously been isolated by TINDERHOLT (LINDSLEY and ZIMM 1992) and was used in this study to revert the Curly phenotype. A 'y-ray mutagenesis to obtain revertants of the dominant Curly phenotype was carried out as outlined in Figure 2C and following a protocol described by ASHBURNER et al. (1983). The mutagenized males were crossed to Cy ed/Gla virgins. The progeny was screened for non-Cy flies and revertants were crossed back to Cy ed/Gla to establish a stock of Cy'" ed/Gla. Of 39,850 chromosomes scored, 6 revertants were obtained, correspond- ing to a reversion rate of 0.015%.

Screen Chromosomes Mutations

scored recovered

EMS for recessive lethals in Vf(2LIDTD2 5,400 58

Local P element hop for insertion into syt 3,200 1

P element excision of P[lacz ,wi] T77 300" 5

CY reversion with X-irradiation 39.850 6 a -

711 revertants.

Complementation analysis: Flies carrying EMSinduced mutations were crossed to flies carrying deficiencies, allowing their assignment to specific subregions of the interval. Muta- tions were then assigned to specific complementation groups by crossing them to each other. All complementation tests were originally performed at 28" until the lack of temperature sensitivity was established. Crosses were then performed at 25". All mutations were balanced over the Cy0 or Gla chromosomes.

Lethal phase determination: The lethal phase was determined by outcrossing mutant X and a deficiency uncovering locus X to Canton S flies. Outcrossed flies were mated (mut/CS X Df/CS) and allowed to lay eggs for 2-hr intervals. From 200 to 500 eggs were then aligned on grape juice agar plates, and hatching frequency was determined 48 hr after egg-laying. To determine larval and pupal lethality, mutations were rebalanced over a chromosome carrying a translocation between the second chromosome balancer SM5 and the third chromosome balancer TMGB-Tb. Mutant strains were then crossed to a strain carrying the deficiency uncovering the locus. The presence of non-Tubby larvae and pupae was analyzed at various time points.

Cytology: Flies carrying deficiency chromosomes of the genotype Df(2L)X/Gla Bc were outcrossed to Canton S flies at 18" and the polytene chromosomes of third instar larvae lacking the Bc marker present on the GZn balancer chromosome were prepared for cytological analysis as described by ASHBURNER (1989). In situ hybridization to polytene chromosomes was carried out using digoxigenin labeled probes.

Molecular biology: Southern analysis and chromosomal walking was performed as described by ( SAMBROOK et al. 1989). Genomic phages were isolated and mapped with a combination of three restriction enzymes: HznIII, EcoRI and XbaI. Iso- lated deficiencies and P element insertions were mapped with Southern analysis.

Immunocytochemical staining Staining of whole mount embryos with monoclonal antibody (mAb) 22C10 was performed as described by LITTLETON et al. (1993a).

RESULTS

Deficiencies uncovering cytological bands within the 22F1-2; 23B1-2 interval. The chromosomal area defined by cytological interval 22F1-2; 23B1-2 contains two well characterized genes to date: decapen tap leg ic (dpp) (SPENCER et al. 1982; ST. JOHNSTON et al. 1990) and syn- a p t o t a g m i n ( s y t ) (LITTLETON et al. 1993a,b). The d p p gene maps to polytene bands 22F1-2 (SPENCER et al. 1982), has been studied extensively at the genetic and

Genetics of Interval 22Fl-23 23B1-2 113

Df(2L)C144 Canton S

I I Df(2L)TLl

Frcw l.-Cytological localization of syt in relationship to deficiencies in the 22F1-2; 23B1-2 interval. Polytene chromosomes were hybridized with a digoxigenin labeled syt cDNA probe containing the entire ORF of the gene. Flies carrying chromosomes that uncover deficiencies were outcrossed to wild-type Canton S flies and heterozygous larvae carrying a wild-type second chromosome and a deficiency were selected and analyzed. The deficiency stained is indicated below each frame. The arrows highlight the syt staining. syt maps to polytene chromosomal bands 23A6; 23B1. syt is uncovered by Df(ZL)DTDZ, Df(ZL)C144 and Df(ZL)TLl, but not by Df(ZL)Hin59 and Df(ZL)Hin34. The extent of these deficiencies are listed in Table 1.

molecular level (ST. JOHNSTON et al. 1990), and has been shown to play a key role in morphogenesis (FERGUSON and ANDERSON 1992). Numerous rearrangements with breakpoints around 22F1-2 have been isolated previously (IRISH and GELBART 1987) and were used extensively in this study. The boundaries of the region examined in this study are defined by the centromere proximal breakpoint of Df(2L)DTD2 at 23B1-2 and the cytological interval covered by duplication Dp(2:2)dppZ1 which includes 22F1-2. Hence, the cytological region covered in this study encompasses 13 cytological bands in the 22F1-2; 23B1-2 interval. Many of the previously isolated deficiencies and their breakpoints, as well as newly obtained deficiencies described in this study, are shown in Table 1.

As our study of this cytological interval was prompted by our interest in syt, we first mapped syt cytologically. As shown in Figure 1, syt is uncovered by Df(2L)DTD2, Df(2L)C144, Df(2L)TLl, but is not uncovered by Df(2L)Hin59and Df(2L)Hin34. Hence, syt maps to the 23A3-4; 23B1-2 interval. A P[lacZ,wi]T77 insertion into syt (abbreviated T77) was obtained by mobi- lizing the P[lacz, w+]B8 insertion at 23A46 as shown in

Figure 2B (LITJUTON et al. 1993b). This P element insertion into syt maps to 23A6B1, in agreement with the cytological mapping data obtained with the deficiencies. To generate smaller deficiencies in the region proximal and distal to syt, excision mutagenesis of P[lacZ, #+IT77 was undertaken. Approximately 300 revertants were identified and partially characterized using PCR to determine whether the excisions were precise or not. Imprecise excisions were further characterized by Southern analysis. Five excision events proved quite useful to define the null phenotype of syt and to order the genes which map in proximity of syt. Two imprecise excisions of T77, Df(2L)N6and Df(2L)Nl9, uncover syt and extend distally of the syt locus (Figure 3); one deficiency, Df(2L)Nl3, extends distally of T77 and only affects syt; two deficiencies, Df(2L)NlO and Df(2L)N28, extend proximal of syt and T77, and also affect syt only.

Three otherrearrangements, Df(2L) TLl, Df(2L)D20 and Df(2L)D31, were obtained through EMS mutagenesis experiments (Table 1 and Figure 3): Df(2L)TLl uncovers cytological bands 22F2-4, 23B2-6 whereas Df(2L)D20 and Df(2L)D31 are not cytologically visible.


A. Ts EMS 6. Pelement Insertion ~ ~ ~~~~

C. Cy Reverslor1

28 0 cn bw SP* x pIDz Sp yw; p r W + m . K ; , A 2 - 3 p x y w cp Gla Gla + + +lethal

I CY

4 Dn + discard

Gla

cn bw sp* Gla

stock

18 + retestat

+ * new eye color

y w ; p l w + l , L x y w ; Cved + ’

+ 4 Y W Plw+f stock for

Gla

PCR analysis

- G‘a +lethal Gla

LL + c y Gla score for

Gla + non-Curly

cn bw sp* score for

DTDZ Sp these progeny “-c lackof

FIGURE 2.-Cenetic screens. A summary of the crosses used to generate mutations within the 22F-23B interval is shown. (A) An EMS mutagenesis to detect temperature sensitive or nonsensitive lethals was initiated by mutagenizing an isogenized cn. bw sp/cn bw sp strain. Mutations obtained were then tested for complementation with Df(2L)DTD2 at 28”. Flies were scored for the lack of non- GZa progeny. (B) The crosses used to obtain a new P element insertion into syt are indicated. Balanced lines containing new insertions were pooled into groups of 10-50 flies and mass screened with PCR using primers from syt and the P element ( L I ~ L E T O N et al. 1993b). (C) Reversion of the dominant Curly phenotype was achieved by irradiating Cy ed/Gla males with 4 kR X-rays and selecting non-Cy flies from the F, progeny.

These mutations may correspond to other types of subtle rearrangements ( e . g . , inversions), but are inferred to be small deficiencies on the basis of complementation tests which indicate that they affect adjacent complementation groups at 22F423A1 and 23A1-3, respectively (see below). Figure 3 shows the extent of many deficiencies isolated in this and previous studies. This map is tentative as it is mostly based on our complementation data, previously published breakpoints, and some molecular data derived from the walk (see below). The precise cytological breakpoints shown in Figure 3 were inferred by combining cytological, genetic, and in situ hybridization data.

EMSiduced mutations in 22F1-2; 23B1-2: To obtain mutations that map to the 22F1-2; 23B1-2 region, 5400 EMStreated chromosomes were tested over Df(2L)DTD2 S p Bl Dp(2;2)dppd21/Gla. Fiftyeight homozygous lethal mutations were recovered in this screen. An additional 15 previously uncharacterized mutations isolated in W. GELBART’S laboratory that map to this cytological interval were also included in this study. Finally, 26 homozygous lethal EMSinduced mutations that map to cytological interval 23A2-4;23Bl-2 were isolated by DIANTONIO et al. (1993), and one representative allele of each of the seven complementation groups identified in their study was also included in this study. Hence, a total of approximately 100 EMSinduced homozygous lethal mutations are now available for the 23F1-2; 23B1-2 cytological area. 23F1-2; 23B1-2 contains 13 essential genes: To assign

the lethal mutations to complementation groups,

chromosomes carrying mutations uncovered by Df(2L)DTDZ were tested by complementation with a series of chromosomes that carry deficiencies (Table 1 and Figure 3). Complementation tests were then performed among all the mutant chromosomes carrying mutations mapping to the same interval (data not shown). This strategy was also used for mutations isolated in W. GELBART’S laboratory and those isolated by DIANTONIO et al. (1993). The data from these complementation tests are summarized in Figure 3. Most genes, with the exception of dpp, syt and Cy, were named according to standard procedures described by LINDSLEY and ZIMM (1992). The 100 mutations represented by this study identify 13 essential complementation groups in a cytogenetic interval that contains 13 cytological bands. This corresponds to an average of 7.7 alleles per gene, indicating it is likely that all essential genes within 23F1-2; 23B1-2 have now been isolated.

Based on the complementation data and the cytogenetic data many genes were ordered with respect to each other, the deficiency breakpoints, and a walk carried out in this interval (Figure 3). Here we briefly discuss some of the evidence underlying the mapping assignment of the specific complementation groups. A more complete description of the phenotypic analysis of these complementation groups is provided below (see “Phenotypic characterization of the lethal complementation groups in 22F1-2; 23B1-2”). Two genes map centromere distal of the breakpoints of Df(2L) Cl44:dpp (22Fa) and 22Fb (= l (2 ) NDl ) (LINDSLEY and ZIMM 1992). Eleven new

Genetics of Interval 22Fl-2; 23B1-2 115

22F 23A 238 23C 1 2 3 4 1 2 3 4 56 7 1 2 3 4 5 6 7 8 1 2

II I I II I1 I I II I I II V @P SYt CY

Df(2L)C144 4 i D f ( 2 L ) d ~ p ~ ~ ” ~ ~ 4 1 Df(2L)dppHinS9

Df(2L)dppt4 I

[-wf(ZL)NC I D f ( 2 L ) N I S

Df(2L)dppTg

4”+ I L W ~ L ) rL I D P ( ~ ; ~ ) ~ P P ~ ’

H Df(2L)DZO H Df(2L)D31

- 0 f l P L ) d ~ p ~ ~ Legend dpp - decapentaplegic syt - synaptotagmin m Genomic Walk E l cy - cur&

22 Fb Fc Fd 23 Pb k W Pe Af pS Ba Bb Fa pa

SYt CY

A A A A A A A A I\ A A A A 22Fa 22Fb 22Fc 22Fd 23Aa 23Ab 23Ac 23Ad 23Ae 23Af 23Ag 230a 23Bb

T I 0 T3 H30 T I 54.6 58.2 AD11 AD20 T9 T60 AD25 T7 66.5 T14 T28 T9 020 58.3 T21 55.1 TI1 66.14 T24 T12 T26 031 58.14 T29 55.5 T41 67.3 T33 T57 020 046 55.6 027 A016 66.4 T16 T39 T58 T7 1 AD5 02 T18 T4 0 03 1 03 T25 T50 037 T31 T65 039 T32 T66 045 T37 01 1 T38 036 T4 3 042 T5 1

T6 1 T62 012 Dl 3 02 1 025

&p H39 Hl2 HZ2 77.3 56.1 T63 028 56-72 T6 035 ~ y t CY

FIGURE 3.4ytological map of deficiencies and complementation map of recessive lethals in 22F-23B. The locations of 13 identified lethal complementation groups are shown with respect to previously described and newly identified deficiencies. EMS alleles obtained or identified within each group are shown. Only EMS alleles used in this study are indicated. The approximate position of the genomic walk is also shown. I t should be noted that complementation groups 23Acand 23Ad have not been ordered with respect to each other. In addition, the ordering of the 23Ae, 23Afand 23Ag complementation groups is also unknown with respect to each other. Gray bars indicate uncertain cytology.

putative alleles of dpp and five new alleles that fail to complement a 22F6 mutation (allele l(2)NDlH39) were isolated. The 22Fb gene is known to be centromere proximal to dpp (SPENCER et al. 1982). Mutations in two other loci, 22Fc (= 1(2)ND3) and 22Fd (= 1(2)ND2) (LINDSLEY and ZIMM 1992) fail to complement Df(2L)C144 and Df(2L)Hin59, but complement Df(2L)dppd”9 The 22Fcgene is likely to be proximal and adjacent to 22Fb because one of the mutations isolated

in the EMS mutagenesis, 0 2 0 , fails to complement all 22Fd (= 1(2)ND3) alleles and one allele of the more proximal 23Aa complementation group, suggesting that the order is 22Fb (1(2)NDl) , 22Fc (1(2)ND3) and 22Fd (1(2)ND2). However, the 0 2 0 mutation could be a rearrangement other than a deficiency and this order is therefore tentative. Four new alleles were isolated that fail to complement the 22Fd mutation ( l(2)ND2H22). The 23Aa gene has been mapped between the proximal


2 3 B + X 4 T77 + 2 3 A RRR R R R R R R R R R R R R R R R H R X H H H H XH H H X X H X X HH H H X X H

I I unl R7 I I I I I I I II I I I I I II II I II nI I I I1 I I I l l Ill I x x

L d " - kkR ' R R R R R R R R R RIR R R R R R R R R R R ), ), RR R7

I I I I I I I II I I I I I II II I II nI I I I1 I I I l l Ill I

+ 2 3 A

I I unl H X X H

- 4 kb

R €COR1 H: Hindlll

x xbd

5' 3 '

D f ( 2 L ) N G 4 , D f ( Z L ) N 1 9 4 ,?

FIGURE 4 . 4 e n o m i c walk surrounding the syt locus. A contiguous walk spanning approximately 100 kb and encompassing 11 A DASH phages is shown. A restriction map of the region is indicated below the phages. The location of the exon-intron boundaries of the syt gene is shown below for reference (LITTLETON et al. 199313). The location of three P elements described in this study are also shown. The breakpoint of a Cy revertant that was mapped to the walk is also shown, as well as the extent of two deficiencies mapped within the walk.

breakpoint of Df(2L)Hin59 and the distal breakpoint of Df(2L)Nl9 four alleles have been assigned to this complementation group. The 23Abgene maps between the proximal breakpoints of Df(2L)Hin34 and the distal breakpoint of Df(2L)N19. Five alleles from W. GELBART'S collection and two new alleles identify this complementation group. Two additional loci, 23Ac (= 23AB3, DIANTONIO et al. 1993) and 23Ad (= 23AB5, DIANTONIO et ul. 1993), map proximal to the breakpoints of Df(2L)Hin34 and fail to complement Df(2L)Nl9, but not Df(2L)N6. We have been unable to order these loci. Si alleles have been identified in each of the complementation groups 23Ac and 23Ad five previously isolated alleles (DIANTONIO et al. 1993) and one newly is@ lated allele. The next four complementation groups, 23Ae (= 1(2)23AB2), 23Af (= Z(2)23AB4), 23Ag (= Z(2J23-6) and syt(23Bu) fail to complement Df(2L)N6, which is esti- mated to be approximately 30 kb (Figure 3). Four new alleles of 23Ae, four new alleles of 23Aj and one new allele of 23Agwere isolated. These three genes were not ordered but most likely map to a genomic fragment that is only 15 kb (see below). syt(23Bu) also fails to complement Df(2L)N6, but is centromeric to 23Ae, 23Af and 23Ag as the proximal breakpoint of Df(2L)N6 is near the putative syt transcription start site (LITKETON et al. 199310). We have isolated eight EMS induced alleles of syt (LITTLETON et aZ. 1993b, 1994). Finally, the Cy(23Bb) locus is uncovered by Df(2L)DZD2 (SPENCER et al. 1982) but not by Df(2L)Nl9. Eighteen new EMS alleles of Cy were isolated in this study. An additional complementation group, Z(2)23AB7, was

_ _

previously reported to map between the breakpoints of Df2L)Hin34 and Df(2L)DZD2 (DIANTONIO et al. 1993). Our complementation data show that this mutation is viable over Of(2L)D71>2 but fails to complement Df(2L) C144, thus placing the gene centromeric to Cy and not within the 22F1-2; 23B1-2 interval.

To determine where Cy maps with respect to syt, and to correlate the cytological location of Cy with respect to the walk shown in Figure 4, a yray mutagenesis was performed to isolate revertants of the dominant Curly wing phenotype. For this analysis we used the Cy ed chromosome which carries no visible rearrangements (LINDSLEY and ZIMM 1992). Cy ed/GZa flies were mutagenized as outlined in Figure 2C. A total of 39,850 chromosomes were scored, and six Cy revertants were obtained and examined for cytological rearrangements. Four chromosomes ( 11 Cy"', 2 7Cy"', 6 7Cy" and KCydrvl) display no obvious rearrangements. One chromosome, 54Cy"', contains an inversion with breakpoints at 23B1-2 and 25 C/D. A second line, 34Cy", carries a more complex rearrangement with a breakpoint at 23B1-2. These data suggest that at least a portion of the Cy locus indeed resides in 23B1-2. This is in agreement with the complementation data. One of the breakpoints associated with the Cy revertant chromosome, 54Cy", has been mapped to the genomic walk shown in Figure 4, indicating that Cy is proximal of syt.

Genomic walk at 23AB: A genomic walk was initiated to determine the structure and size of syt and to define

Genetics of Interval 22F1-2: 23B1-2 117

its precise location with respect to the other loci that map within this cytogenetic interval. As shown in Figure 4,11 overlapping phages covering approximately 100 kb were isolated and mapped with three restriction enzymes. Four of the 11 phages covering the syt locus were previously published (LIITLETON et al. 199313). Here we show the extended walk surrounding syt. This walk encompasses the breakpoint of the 54Cy" inversion, suggesting that at least a portion of Cy is included in the walk. We were also able to cytogenetically and molecu- larly map three Pelement enhancer detector insertions (BELLEN et al. 1989; BIER et al. 1989; O'KANE and GEHRINC 1987) to 23A46 and one to 23B1-2. The extent of the walk, as well as a restriction map of the region is shown in Figure 4. The approximate location of the genomic walk with respect to the complementation map is shown in Fig- ure 3. The proximal and distal phages of this walk hybridize to 23B1-2 and 23A3-5. Two other walks in cytogenetic interval 22F1-2; 23B1-2 have been carried out previously. A genomic walk at dpP (22F1-2) (ST. JOHNSTON et al. 1990) extends over approximately 200 kb (W. GELBART and R. BLACKMAN, personal communication) and probably covers most of 22F1-4. This walk is known to include dpp and 22Fb (= l(2)NDl). Another walk (approximately 60 kb) was initiated by M. HOFFMAN and probably covers the 23A3; 23A7 bands (M. HOW, personal communication). Hence, these three walks cover a large portion of this cytological interval and should provide an entry point to clone any of the remaining loci which were not cloned in this or other studies.

Df(2L)N6 fails to complement mutations in four essential genes, syt (= 23Ba), 23Ae, 23Af and 23Ag. Df(2L)N6 uncovers approximately 30 kb within the genomic walk, indicating that these four genes (or portions thereof) reside within our walk. As shown in Fig- ures 3 and 4, we have mapped the syt gene to a 15-kb interval at 23A6; 23B1. It is thus likely that the 23Ae, 23Af and 23Ag genes (or portions thereof) map to a 15-kb region, distal of syt. However, we cannot exclude the possibility that one or more of these genes map within the syt gene.

An additional 40 kb of contiguous sequence was obtained 5' of the syt locus. Several deletions map to this region, including Df(2L)NlO and Df(2L)N28. How- ever, only the syt complementation group is affected by these deficiencies, indicating that the region immedi- ately 5' of the syt promoter region does not contain an essential gene. The only additional chromosomal aber- ration mapped to this region is the 54Cy". Shortly be- yond this breakpoint is a stretch of repetitive DNA, com- plicating a molecular analysis. In situ hybridization to whole mount embryos with fragments derived from this portion of the walk failed to reveal an expression pattern. It is therefore unclear whether this breakpoint resides directly in the transcribed region or in regulatory

sequences of Cy. The remaining five Cy revertants have no visible rearrangements within the walk. P element enhancer detector insertions at 23AB:

Four P element enhancer detector strains have been mapped to the genomic walk shown in Figure 4. These include P[lacZ, w' /B8, PllacZ, wi ]El 0, P[lacZ,ly' IPZ 03728 and an insertion in syt, P[lacZ, wi lT77. P element insertions B8 and El 0 map within a few hundred base pairs of each other, while PZ03728 maps a few kilobases away from these two insertions. All three enhancer detector strains exhibit a very similar Pgalactosidase expression pattern indicating that they report the expression pattern of the same gene. The GacZgene is expressed in the developing central nervous system (CNS) at stage 11, just following maximum germ band extension. At later developmental stages, the lac2 gene is expressed abundantly in the CNS (Figure 5A), and to a lesser degree in the peripheral nervous system (PNS). It is therefore likely that a gene specifically expressed in the nervous system lies within this region. This gene corresponds to 23Ac or 23Ad based on the mapping data shown in Figure 3 and the molecular data shown in Figure 4.

Another insertion, P[w', kinain lacZ]El941, which causes a dominant Cy phenotype at 28" but not 18", was obtained from ED GEE. We have mapped this insertion at 23C, indicating that the Curly phenotype may be associated with a P element insertion in Cy. Given that cyte logical rearrangements affecting the Cy phenotype map to 23B1-2 (SPENCER et al. 1982; this study), and 23B3-8 (ASHBURNER et al. 1983), it is likely that the Cy gene encompasses most of 23B and some bands of 23C. The E1941 enhancer detector confers Pgalactosidase expres sion to fat body cells, suggesting that the Cy gene may be expressed in fat bodies (Figure 5B).

Phenotypic characterization of the lethal complementation groups in 22F1-2; 23B1-2: Preliminary analysis of the mutant phenotype associated with some of the isolated deficiencies and mutations, is provided below. An initial analysis of two deficiencies covering most of the 22F1-2;23B1-2 region, Df(2L)Hin34 and Df(2L)N19, was performed to identify obvious phenotypes caused by lack of the genes within these two deficiencies. Absence of visible phenotype in either of those two deficiencies would indicate that they do not uncover genes required for proper development. These deficiencies cover 12 of the 13 complementation groups as Cy is not uncovered. Both Df(2L)Hin34 and Df(2L)N19 cause homozygous embryonic lethality. However, embryos homozygous for Df(2L)Hin34 show obvious morphological defects including defects in gastrulation, head involution, and nervous system development, whereas embryos homozygous for Df(ZL)N19show no obvious morphological defects and can elicit reduced but coordinated muscle propagation waves within the egg case (LITTLETON et al. 1993b). Hence, Df(BL)Nl9 is unlikely to uncover genes


8

FIGURE 5.Whole mount embryos stained for Pgalactosidase activity or immunocytochemically stained with mAb 22C10. Em- bryos are shown dorsal up, anterior to the left. (A) The expression pattern in embryos carxying the P[ZacZ, w+]S8 insertion at 23A44. Abundant expression of the lacZ gene is seen in the C N S of this stage 16 embryo. Faint staining in the PNS can also be observed (not shown). (B) walactosidase expression pattern in embryos containing the P[w+, kinesin lacZ]KZl941 insertion at 23C. This enhancer detector insertion is probably in the Cy gene. Expression is restricted to fat bodies in stage 16 embryos. (C) Canton4 embryo immunocytochemically stained with mAb 22C10. Focus is on the neurons of the PNS. D. T28/Df(2L)Hin34 embryo (22Fb). The arrow indicates a growth cone guidance defect as axonal tracts descend from a dorsal PNS cluster into the lateral cluster of a neighboring segment. The arrowhead points to a dorsal cluster of PNS neurons which lacks many neurons. (E) A 77.3/Df(2L) C144 embryo of the 23Aa complementation group (neural disrupted) stained with mAb 22C10. This embryo shows a dramatic decrease in the number of C N S and PNS neurons, and a disorganization of the PNS. Gaps within the CNS are also evident. (F) 58.2/Df(2L) C144 (23Ab) embryo stained with mAb 22C10. This embryo shows an approximate twofold reduction of the number of PNS neurons. Gaps in the CNS of these embryos are also evident.

required for proper development of the embryo. As we are particularly interested in neural development of the embryonic PNS, embryos homozygous for these deficiencies were stained with monoclonal antibody mAb 22C10. This antibody labels dendrites, cell body and ax- ons of neurons of the PNS and a subset of cells of the CNS (GOODMAN et al. 1984; ZIPURSKY et al. 1984). Ho- mozygous Df(2L)NI 9 embryos showed no obvious defects in number, pattern, or axonal projections of neurons. However, homozygous Df(2L)Hin34 embryos showed numerous defects in neuronal patterning and

connections, in addition to germ band retraction and head involution defects. A brief description of mutations in each of the thirteen complementation groups is provided below, and a summary of the data is shown in Table 3. All alleles described below are homozygous lethal except for syt"' (-TON et al. 1993b).

dpp(22Fa): Twelve homozygous lethal mutations were obtained that failed to complement Df(2L)DTD2 and Df(2L)Hin34 and complement Df(2L)C144. These mutations all complement mutations in 22Fb (= Z(2) NDl ) . Hence, we infer that they probably cor-

Genetics of Interval 22F1-2; 23B1-2 119

TABLE 3 .

Summary of 22F1-2; 23B1-2 complementation groups

No. of Lethal Locus Synonym allelesn phase Comment

22Fa dpp 11 E 22Fb l(2)NDl 6 E 22Fc 1(2)ND3

CNS and PNS disorganization, neuronal fasciculation defects 2 P

22Fd 1(2)ND2 19 L 23Aa nrd 4 E 23A b 7 L Structural defects in CNS, loss of neurons in PNS

Lack parts of CNS and PNS, defects in germ band retraction and head involution

23Ac 6 P 22Ac or 22Ad is likely a pan-neuronal gene 23Ad 6 L 23Ae 10 P Within genomic walk 23A f 8 L/P Within genomic walk 23Ag 2 L/P Within genomic walk 23Ba synaptotagmin 19 E 23Bb Curly 24 E Multimer, antimorphic allele, expressed in embryonic fat bodies

Defects in calcium activation of neurotransmitter release

‘ E = embryonic, L = larval, P = pupal. Lethality of most severe allele is shown. Alleles found in previous studies are also included in this column (except for dpp).

respond to new dpp alleles. However, complementation analysis between these alleles and several previously identified dpp mutations indicate that some can easily be classified as dpp alleles, whereas others show a complex complementation behavior. Allele T24 fails to complement dpp’63 Cy (ST. JOHNSTON et al. 1990) but complements dppshu4 (SEGAL and GELBART 1985) mutations, suggesting that T24 is a dppdisk mutation. T24 fails to complement the 10 other mutations isolated here, except 0 4 2 , indicating that these 10 mutations are indeed dpp alleles. However, allele 0 4 2 fails to complement Df(2L)dppdv14 and Df(2L)dPpt6’, and the remaining 10 mutations complement dppt6’ and dppshu4, indicating a highly complex complementation pattern. Three of these 10 alleles, TI 0, TI 4 and T24 were chosen for further study to verify whether they cause a known dpp phenotype. Alleles T I 0 and T14 in combination with Df(2L)Hin34 show 15% embryonic lethality. Few pupae of these genotypes can be recovered, but they die and contain few adult imaginal structures, suggesting that they are indeed hypomorphic dpp alleles (SPENCER et al. 1982). Allele T24 is embryonic lethal and shows numerous morphological defects. The remaining eight alleles were not examined further.

22Fb (l(2)NDl): Alleles of this complementation group fail to complement Df(2L)Hin34, but are viable in combination with Df(2L)Cl44 and Df(2L)TLl, placing the locus centromeric to dpp. Five new alleles which fail to complement l(2)NDlH39 were obtained. Three alleles, T3, TI2 and T28, were chosen for further study. All three alleles cause homozygous embryonic lethality. Embryos carrying alleles T 3 and T28 were stained with mAb 22C10 to identify neuronal defects. T28/ Df(2L)Hin34 embryos exhibit growth cone guidance defects in some segments, primarily in tracts descending from the dorsal to lateral PNS clusters (Figure 5D). In addition, some segments exhibit dorsally located lateral chordotonal neurons, and some dorsal and lateral clus-

ters of the PNS have a reduced number of neurons (Fig- ure 5D). T3/T3 mutant embryos also exhibit aberrant neuronal connection defects. In addition, these embryos exhibit germ band retraction defects and a severe disorganization of the CNS and PNS (data not shown), It is possible that these defects are due to other mutations on the chromosome that carries the T3mutation as we did not stain T3/Df embryos. How- ever, given the similarities in the observed defects it is possible that the 22Fb gene is required for proper neural development.

22Fc (1(2)ND3): No new alleles of this complementation group were obtained in our screens. Two published alleles of this complementation group are available (H12 and H30) (SPENCER et al. 1982). H30/ Df(2L)Hin34 third instar larvae are viable and early pupae could be recovered of this genotype, suggesting that allele H30 is an early pupal lethal.

22Fd (1(2)ND2): Four alleles that fail to complement 1(2)N02H22 were obtained. Alleles T 9 and T26 in combination with Df(2L) C144 exhibit higher than normal levels of embryonic lethality (approximately 15%). No pupae were observed with any of the alleles over a deficiency. Indeed, most alleles cause death at the bound- ary of the first/second instar molt. Second instars that are recovered are extremely sluggish. Immunocyto- chemical staining of embryos mutant for T I , T 9 and T26with mAb 22C10 failed to reveal any defects in neuronal development in the embryos, and no other obvious defects were observed.

23Aa: This complementation group maps between the proximal breakpoint of Df(2L)Hin59and the distal breakpoint of Df(2L)N19. Two previously isolated mutations, 77.3 and 54.6 , along with three newly isolated alleles, 0 2 0 , 0 3 1 and 0 4 6 , map to this complementation group. Allele 0 3 1 fails to complement allele 77.3, although rare adult survivors are sometimes recovered. Allele 0 3 1 does complement the two other alleles, 0 4 6


and 54.6, indicating that D31 is a partial loss of function allele of 23Aa. The D31 mutation also fails to complement all mutations in 23Ab, suggesting that it is a small rearrangement affecting both loci. Allele 0 2 0 fails to complement allele 77.3, but complements the remaining alleles of 23Aa. 0 2 0 fails to complement all alleles of 22Fd. The partial lack of complementation with 77.3 suggests that allele 0 2 0 is a partial loss of function allele of the 23Aa gene and a severe loss of function allele of 22Fd. The most naive interpretation of these data is shown in Figure 3: 0 2 0 and D31 are probably small deficiencies which extend in opposite orientations. Both affect a severe loss of function allele, 77.3 of 23Aa, but not the other alleles of 23Aa. In addition, as both 0 2 0 and 0 3 1 complement each other, they are unlikely to overlap. These data are supported by the observation that D31 fails to complement Df(2L)dppHinj4 but complements Df(2L)dppZ4 and Df(2L)dppj3, whereas 0 2 0 fails to complement the two latter deficiencies and Df(2L)dppH"59. The possibility that 0 2 0 and 0 3 1 contain two lethal hits cannot be ruled out.

Alleles 77.3 and 0 4 6 both cause embryonic lethality in trans to Df(2L) C144. 77,3/Df(2L) C144 embryos, when freed from the egg case, showed some movements of the mouth hooks and contractions of individual muscle fibers. However, no coordinated muscle propagation waves were observed. When examined with mAb 22C10, 77.3/Df(2L)C144 embryos lack significant portions of the CNS and PNS (Figure 5E). Gaps in the CNS were more apparent in the posterior portion of the embryo. In the PNS we observe numerous fasciculation defects. In addition, defects in head involution and/or germ band retraction are apparent. D46/D46 mutant embryos show subtle germ band retraction defects, but CNS and PNS development appear normal. Mutant D2O/Df(2L) C144 embryos show subtle fasciculation defects in the embryonic PNS (not shown), but 54.6/ Df(2L) C144 embryos exhibit no morphological defects, and rare adult escapees are found. We conclude that the 23Aa locus is essential for neuronal development and hatching. Based on the mapping position and the phenotype of allele 77.3, this complementation group probably corresponds to a previously described gene named neural disrupted (A. MAHOWALD and D. WRIGHT, personal communication; LINDSLEY and ZIMM 1992).

23Ab: The 23Ab complementation group consists of five previously isolated mutations 56.1, 58.2, 58.3, 55.6 and 58.14, and two newly isolated alleles, T71 and D31. These mutations map between the distal breakpoints of Df(2LjN19 and the proximal breakpoint of Df(2L)Hin34. Approximately 10% of the T71/Df2L)N19 embryos die, but most T71/Df(2L)N19 embryos survive to third instar larvae. Alleles 58.2 and 58.3 also cause larval lethality. Al- leles T71, 55.6, 56.1, 58.3and 58.2in trans to Df(2L)C144 were examined for defects in neural development using

mAb 22C10 as a marker. As shown in Figure 5F, 58.2/ Df2L)C144 embryos lack approximately half the neurons in the PNS and exhibit gaps in the CNS. In the PNS some thoracic chordotonal neurons are absent, and many lateral chordotonal neurons are missing or associated with the dorsal cluster. Both the dorsal and lateral PNS clusters exhibit a severe decrease in the number of neurons. 55.6/ Df(2L)Cl44 and T71/Df(2L)C144 embryos show less severe defects in the CNS and PNS. We observed a decreased number of neurons in the dorsal PNS clusters, as well as a reduction in the number of chordotonal neurons. Abnor- mal positioning of the dorsal and lateral chordotonal neurons is also evident. Alleles 58.3 and 56.1 do not cause any obvious developmental defects when tested in trans to Df(2L)Cl44, suggesting that they are hypomorphic alleles. Finally, Df(2L)Nl9is also a weak allele of 23Abas we observe adult escapees in trans to several 23Ab alleles, In addition, we observe no obvious morphological defects in homozygous Df(2L)Nl9embryos. We conclude that the 23Abgene plays a key role in CNS and PNS development and based on our genetic and phenotypic analyses we propose the following allelic series with decreasing strength 58.2 > 55.6 > T71 > 58.3 = 56.1 = Df(2L)Nl9.

23Ac and 23Ad: These complementation groups complement Df(2L)Hin34 and Df(2L)N6, but fail to complement Df(2L)Nl9 and could not be ordered with respect to each other. Mutations in neither gene cause obvious embryonic visible phenotypes. It is likely that one or both of these genes is expressed in the CNS and PNS, since three P element enhancer detector insertions which express &alactosidase in CNS and PNS map to this region (Figure 5A). The 23Acgroup consists of five previously isolated alleles ( l (2) 23AB3; DIANTONIO et al. 1993) and one new allele, T63. T63/Df(2L)Nl9 animals die as pupae. The 23Ad complementation group consists of five alleles from the DIANTONIO collection (1(2)23AB5) and one new allele, 028. D28/ Df(2LjNl9 animals die as larvae.

23Ae, 23Af and 23Ag: Mutations in these genes fail to complement Df(2L)N6 and are telomeric to syt. As homozygous Df(2L)N6 embryos display no obvious morphological defects when stained with mAb 22C10, these three genes do not seem to play a role in embryonic morphogenesis. The 23Ae complementation group consists of six 1(2)23AB2 alleles (DIANTONIO et al. 1993), one allele from W. GELBART'S collection 56.12, and three newly isolated alleles, T21, T29 and 027. Alleles T21, T29,56.12 and 0 2 7 cause pupal lethality. Complemen- tation group 23Af consists of six previously isolated alleles, four 1(2)23AB4 alleles, 55.1 and 55.5, and two new alleles, T6 and T60. Alleles 55.1,55.5, T6 and T60 in trans to Df(2L)N6 cause late larval or pupal lethality. Complementation group 23Ag consists of one allele from the DIANTONIO collection (1(2)AB6) and one new allele, 035. Allele D35 in combination with Df(2L)N6 causes larval or pupal lethality.

synaptotagmin (23Ba): Eight EMS alleles of syt were isolated: T7, TI 1, T41 (LITTLETON et al. 1993b); and 0 2 , 03, 0 3 7 , 0 3 9 and 0 4 5 (LITTLETON et al. 1994). One allele was obtained from W. GELBART, 66.4, and four were obtained from A. DIANTONIO and T. SCHWARZ, 1(2)23ABI. Additional alleles of syt were obtained by excision mutagenesis of the T77 P element insertion. Two excisions, NIO and N28, remove parts of the P element and a small portion of genomic DNA 5‘ of the P element. Both imprecise excisions alter syt transcription. Excision N13 deletes the 3’ region of the T77 insertion and a small portion of the genomic DNA en- coding the 5’ portion of the syt transcript. Homozygous N23 embryos do not produce detectable levels of Syt. Most syt null alleles are embryonic lethal, but many hypomorphic mutations survive as first instars. None of the syt alleles cause obvious morphological defects. Electro- physiological analysis of syt mutants demonstrates that Syt functions as a calcium sensitive activator of neurotransmitter release and an inhibitor of spontaneous synaptic vesicle fusion (LITTLETON et al. 1993b, 1994).

Cy (23Bb): Seventeen EMS alleles fail to complement the Cy mutation and complement Df(2L)N6. Six Cy revertants were obtained after y-irradiation. Two of these revertant chromosomes show rearrangements in 23B1-2, in agreement with the mapping position determined through deficiency mapping. Additionally, a line containing a P element insertion causing a conditional dominant Cy wing phenotype was obtained from E. GRELL (University of California, San Francisco). The insertion strain and some excision strains are homozygous viable and exhibit a temperature sensitive Curly phenotype. A complementation analysis of most alleles is shown in Table 4. Note that a significant number of alleles partially complement each other. However, most surviving adult flies show a severe reduction in body size compared to wild-type flies, and exhibit a slight curva-

ture of the wing blade. All of these adult flies show be- havioral defects and are unable to fly.

The embryonic lethality of various Cy trans- heterozygotes is shown in Figure 6. Interestingly one of the alleles examined, 67.3, behaves as an antimorphic allele in combination with several alleles such as 66.14 and 66.5. Allele 67.3 causes embryonic lethality over Df(2L) C144, while alleles 66.5 and 66.14 cause lethality at later developmental phases. However, when alleles 66.5 and 66.14 are paired with 67.3, the combination results in embryonic lethality, suggesting that allele 67.3 has a more negative contribution than a deficiency. This is typical of antimorphic alleles (MULLER 1932). mAb 22C10 staining of embryos homozygous for alleles 67.3, T18 and T62 reveal no obvious neuronal or morphological defects in embryos.

DISCUSSION

In this work we have summarized an extensive body of data collected in our laboratory on the 22F1-2; 23B1-2 chromosomal region. The information presented here shows that this cytological interval is now one of the best characterized regions of the Drosophila genome at the genetic level. This domain contains 13 cytological bands and 13 essential genes, represented with an average of 7 alleles per gene. Hence, it is very likely that all essential genes within this interval are described in this work.

Several genes like dpp and syt were previously characterized in detail. Here, we present genetic and molecular data that allowed us to map many of the remaining genes, including Cy and neural disrupted (23Aa). The Cy mutation is the most commonly used dominant marker for second chromosome balancers and was originally identified by WARD (1923). However, little is known about the Cy gene. Here, we describe 18 homozygous recessive lethal Cy alleles. Many of these alleles exhibit


.- V c P a W E

FIGURE 6.-Embryonic lethality of several Cy alleles. The lethality associated with Cy ed as well as a selected set of loss of function mutations in Cy is shown. The number of embryos scored is shown below the genotype. One of these alleles, 67.3, behaves as a dominant antimorphic allele as it causes embryonic lethality in combination with two additional alleles that are not homozygous embryonic lethal. However, 67.3 in combination with the dominant Cy allele, is not em- brvonic lethal.

CY 67.3 66.14 66.5 66.14 66.5 67.3 66.14 66.5 i

"

C144 C144 C144 C144 67.3 67.3 Cy Cy cy (n-370) (549) (608) (255) (466) (605) (164) (170) (215)

-------

Genotype

partial intragenic complementation (Table 4), and the viable trans-heterozygote adults show a severe reduction in body size and a slight curvature of the wing blade. In addition, at least one allele, 67.3, behaves as an antimorphic mutation (Figure 6 ) , suggesting that the Cy protein may function as a multimer. Characterization of a P element enhancer detector insertion which maps to 23C and causes a subtle Curly phenotype, suggests that the Cy gene may encompass most of the 23B cytological band and a portion of 23C. In addition, Pgalactosidase expression in embryonic fat bodies (Figure 5B) suggests that Cy may be expressed in fat bodies. We observe no Pgalactosidase expression in imaginal disks of flies carrying the enhancer detector insertion (data not shown).

In addition to Cy, syt and dpp, one other gene m a p ping to this interval, neural disrupted (nrd, 23Aa) has previously been described in some detail (LINDSLEY and ZIMM 1992; D. WRIGHT and T. "IOWALD, personal communication). The nrd gene was isolated in a differential hybridization screen and is specifically expressed in neurons. The defects that we observe in one of the nrd (23Aa) alleles, 77.3, corresponds to the described phenotype in LINDSLEY and ZIMM (1992). However, other alleles that affect this complementation group exhibit much milder phenotypes, and probably are weak hypomorphic alleles.

In addition to the genes discussed above, nine other additional essential loci are contained within the studied interval. Most of these loci were mapped in relation to the previously identified genes. Interestingly, at least three genes are either required for neuronal develop ment (22F6, 23Aa and 23Ab) or expressed in the nervous system (23Ac or 23Ad). Some alleles of 22Fb alleles cause axonal fasciculation defects, a loss of neurons in the CNS and PNS, and misplacement of the lateral chordotonal neurons. Alleles of the 23Ab complementation group cause an approximate halving of the number of

PNS neurons, as well as gaps in the CNS. In addition, three enhancer detectors with neuronal specific expression of the lacZ gene were also mapped to the interval between Df(2L)N6and Df(2L)Hin34. This interval contains two complementation groups: 23Ac and 23Ad. However, mutations in neither of these genes cause neuronal patterning defects, suggesting a functional rather than developmental role. Hence, the cytological interval studied in this work contains at least 5 genes out of 13 that are either required for neural development (22Fb, nrd, 23Ab) or function (syt , 23Ac and/or 23Ad).

The primary reason for initiating this analysis was the identification of mutations within the synaptic vesicle protein synaptotagmin. Eight new syt mutations not previously reported have now been identified. These include five EMS mutants and three small deletions. Four EMS mutations have been previously characterized (LIITLETON et al. 1993b). Deletions which only affect syt are embryonic lethal and severely disrupt coordinated muscle activity in mutant embryos. These results support our previous observations that Syt functions as a calcium sensitive activator of neurotransmitter release (-TON et al. 1993b). A detailed electrophysiological characterization of these mutations will be published elsewhere (LITLETON et aL 1994).

In summary, we have saturated the 22F1-2; 23B1-2 interval for recessive lethal mutations and have identified 13 complementation groups. Most of these loci have been deficiency mapped and ordered on the cytological map. At least seven genes within this interval have been previously cloned or are shown to reside in the walk presented in this work. This study should greatly facili- tate further genetic and molecular investigation into the loci mapping in this interval.

We would like to thank DANIEL LI-ITLETON, KAREN SCHULZE and DIANA D'EVELW for contributions at various stages of the work. We would like

Genetics of Interval 22F1-2; 23B1-2 123

to thank JEFF SEKELSKY, WILLIAM GELBART, AARON DIANTONIO, TOM SCHWARZ, GEORG FEGER, YUH-NUNC JAN, ALLAN SPRADLING and the Bloom- ington and Bowling Green Drosophila Stock Centers for fly strains. We also would like to thank DAVID WRIGHT and ANTHONY MAHOWALLI for unpublished data. We thank MANZOOR BHAT and ADI SLUBERG for com- ments on the manuscript and J ~ D I COLEMAN for secretarial assistance. J.T.L. was supported by a National Institute of Mental Health fellow- ship and a National Institute of Health (NIH) grant to MARK PERIN, whose support is gratefully acknowledged. This work was supported by an NIH grant to MARK PERIN and H.J.B. H.J.B. is an Assistant Investigator of the Howard Hughes Medical Institute.

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