J. Genet., Vol. 75, Number 2, August 1996, pp. 161-172. �9 Indian Academy of Sciences

Genetic interaction of Drosophila homologue of abe&on (abl) proto,oncogene (D-abl) and lethal(2)giant larvae (lgl) tumour suppressor gene during embryonic development

D I B Y E N D U S A H A and P R A D I P S INHA Drosophila Stock Center, School of Life Sciences, Devi Ahilya Vishwavidyalaya, Khandwa Road Campus, Indore 425001, India

MS received 14 May 1996; revised received 23 August 1996

Abstract. The Drosophila homologue (D-abI) of the mammalian abelson proto-oncogene (c-abl) encodes a cytoplasmic protein tyrosine kinase which localizes to axons of the develop- ing embryonic central nervous system (CNS) and has been shown to be required for redundant functions during axonogenesis. These redundant functions become indispensable when other components of the redundant pathway, such as those encoded by disabled (dab), fasciclin I (fas I) or failed axon connection (.fax) are removed from abl mutants. Second-site mutations can thus uncover redundant aspects of abl-mediated axonogenesis. We used this strategy, and present evidence to suggest a redundant function of the cytoskeletal protein encoded by the lethal(2)yiant larvae (lgl) turnout suppressor gone during embryonic axonogenesis. Simulta- neous mutation in I~.ll and abl shifts lethality of the mutations to late embryogenesis while mutation in only one of these genes permits development up. to late larval/pupal or pharate adult stages. The lgl-; abI- embryos show defective development of the CNS, characterized by loss of axonal commissures and longitudinal axonal tracts. Lethality of the double mutation is aggravated or suppressed by disabled (dab) or enabled (ena) mutations, which act, respectively, as dominant enhancers or suppressors ofabl. The redundant fnnction of lgl turnout suppressor gone during axonogenesis therefore appears to involve aspects of D-abl-mediated signalling.

Keywords. abelson proto-oncogene; 1.ql tumour suppressor gone; neurogenesis; Drosophila.

1. Introduction

Cellular signalling is critical for regulation of growth and differentiation during animal development. Cytoplasmic protein tyrosine kinases are impor tant components of cellular signalling (for review, see Hunte r 1987). Several vertebrate proto-oncogenes encode cytoplasmic tyrosine kinases, suggesting that transduction of cellular signals for growth and differentiation are normal developmental functions of these proteins (for review, see Bishop 1987; Hunter 1987). The search for Drosophila homologues of these vertebrate proto-oncogenes was initiated nearly two decades ago (Shilo and Weinberg 1981). The fruit fly model offers the advantage of its rich genetics to permit exploration of the non:nal developmental roles of proto-oncogenes and thereby allows tracing of their signalling pathways. This approach has now met with considerable success in the case of the Drosophila homologue of the h u m a n abelson (c-abl) proto-oncogene, namely D-abl (see Hoffmann 1991). The gone was originally isolated on the basis of its partial sequence similarity with vertebrate src oncogene (Hoffmann etal. 1983). Its subsequent characteriz- ation represents an excellent case of reverse genetics (Henkemeyer etal . 1987, 1990; Gertler et al. 1989, 1990, 1993). Isolat ion of mutan t alleles of this locus using classical mutagenesis techniques revealed its developmental roles (Henkemeyer etal . i987), abl mutan t animals

161

162 Dibyendu Saha and Pradip Sinha

normally survive to adulthood and show a rough-eye phenotype, poor viability and fecundity (Henkemeyer etaI. 1987). These phenotypes have been interpreted as pleiotropic effects of abl mutations (Henkemeyer etaI. 1987). During embryonic development Abl protein is localized in the developing central nervous system (CNS). However, surprisingly, abl null embryos develop normally during embryogenesis and show normal neuronal development (Gertler et al. 1989). The significance of the localization of the Abl protein to embryonic CNS can be understood from the interaction of abl with its dominant enhancers (Gertler er al. 1989; Hill et al. 1995) or suppressor (Gertler et at. 1990). disabled (dab), a dominant enhancer ofabl (Gertler et al. 1989), encodes a tyrosine kinase substrate (Gertler et al. 1993). abl homozygotes that are also dab heterozygous die as embryos showing defective CNS development (Gertler et aI. 1989). Thus, while normal axonogenesis can progress in the absence of Abl, the abl mutant embryos with a lowered dose of dab failed to sustain normal neurogenesis. Similarly, mutations in failed axon connection (fax) act as dominant enhancers of abl mutation by shifting the lethal phase of the mutation to the prepupal stage (Hill er al. 1995). Mutations in enabled (ena) on the other hand act as a dominant suppressor of abl and rescue abl mutants (Gertler et aI. 1990). These studies therefore show that redundant aspects of abI-mediated signalling can be uncovered by second-site muta- tions in genes involved in its signalling pathway. Using this strategy it was also shown that flies homozygous for mutations in fascicIin I (fasI), which encodes a neural cell adhesion protein, fail to show normal neurogenenis when they are also mutant for abI (Elkins et aI. 1990).

The genetic identities of all components that directly or indirectly participate in abl-mediated signalling have not been established so far. The Abl protein, for instance, has been predicted to form a macromolecular complex (Henkemeyer et al. 1990). This raises the possibility of requirement of several macromolecules for the transduction of abl-mediated signalling. In this regard cytoskeletal proteins are likely to play important roles in view of their requirement in different aspects of intracellular and intercellular signal transduction (see Peifer et al. 1993; Strand et al. 1994a). The product of the human c-abl proto-oncogene, for instance, has been shown to associate with the cytoskeletal matrix (Graziani et al. 1989). We argued therefore that, in Drosophila, participation of cytoskeletal proteins in abl- mediated signalling can be unravelled by second-site mutations in genes encoding cytoskeletal proteins in abl mutant animals. We used this strategy and report here a requirement of the cytoskeletal protein encoded by the lethal (2) giant larvae (lgl) tumour suppressor gene (Strand et al. 1994a) in aspects of abI-dependent axonogenesis in Drosophila.

2. Materials and methods

2.1 Mutant stocks

cinnabar (cn) brown (bw): This stock represents a wild-type control for embryonic lethality tested in different genetic combinations. It represents an isogenic line with the cn bw marker on the second chromosome and shows less than 0.4 per cent embryonic lethality among its progeny. Before testing the embryonic lethality of any given mutant genotype, fries were crossed to this lethal- free line. This was done to eliminate embryonic lethality caused by homozygosity of the balancer chromosomes (see below). Embryonic lethality was subsequently scored in the progeny of the heterozygous parents.

Genetic interaction of Drosophila abl and Igl 163

lethal (2) giant larvae (lgl): The mutant allele of lgI tumour suppressor gene used in the present study w a s Igl 4. This amorphic allele represents deletion of the chromosomal band 2tA at the tip of the left arm of the second chromosome (Mechler et al. 1985). The chromosome was also marked with the orange (or) marker, based on which lgl 4 homozygous larvae could be identified owing to their colourless Malpighian tubules. The IgP homozygotes, which live up to late third larval instar, show maligant tumours of the imaginal discs and brain ganglia (Gateff 1978). The stock was maintained using the CyO balancer (Lindsley and Zimm 1992). For simplicity, in the subsequent part of the text this allele is referred to as IgF.

abelson (abl): The different mutant alleles of the Drosophila homologue of the mammalian abI proto-oncogene used in the present study were abI 1c2s~c), abl 2 and abl ~ (Henkemeyer et al. 1987). abl ~(254c) and abl ~ are believed to be hypomorphic alleles while abl 2 is presumably an amorphic allele (Henkemeyer et al. 1987). The stocks were maintained over the TM6B balancer. Normally, abI homozygotes live up to pupal/pharate adult stages (Henkemeyer et al. 1987). For simplicity, in the subsequent part of the text all mutant abl alleles are referred to as abl-.

P[lgI + ry+]-13 transposon: This transposon carries the complete genomic sequence of IerhaI(2)giant larvae (Igl) inserted at chromosomal position 22F-23A on the left arm of the second chromosome which also carried the IgP v275 mutation (Opper et aI. 1987). The latter allele represents an insertion of B104 or 'roo' transposon (Lutzelschwab et al. 1986) in the lgl locus and normally induces tumorous growth of the imaginal discs in homozygous larvae. However, the presence of the homozygous viable P [lgl § ry + 7-13 insertion rescues the IgP v275 animals to normal adulthood and fertility (Opper et al. 1987).

Df(3L)std11: This deficiency spans the cytogenetic interval 73B5-7 in the polytene chromosome map on the left arm of the third chromosome (Lindsley and Zimm 1992) and uncovers two tightly linked loci, namely abl and disabled (dab) (Henkemeyer et al. 1987; Gertler et al. 1989). The stock was maintained using the TM6B balancer (Lindsley and Zimm 1992). For simplicity, this stock is referred to as abl- dab- in the subsequent part of the text.

enabled (ena): The lethal allele of ena used in the present study was ena zT~, which represents a strong allele of this locus isolated during a chemical mutagenesis screen (Gertler et al. 1990) for dominant suppressors of abl. ena z J~ heterozygosity rescues flies from the pupal lethality due to homozygosity for different abl alleles (Gertler et al. 1990). This stock was maintained using the CyO balancer. For simplicity, this stock is referred to as ena- in the subsequent part of the text.

2.2 Genetic crosses

Generation of lgl-; abl- embryos: To generate lgl-; abl- progeny, lgl- /CyO;abl- / TM6B flies were first outcrossed to the lethal-free stock marked by cn bw markers. Mating of Ig l - /enbw;abl - /+ heterozygous flies was expected to generate lgl-;abl-embryos in Mendelian ratio (see table 1A). As controls, phenotypes of putative Igl- or abl- mutant embryos were examined from the eggs collected, respectively, from Igl-/cn bw and abI - /+ heterozygous parental flies. To score embry- ordc lethality, eggs were collected for one hour from the desired parental flies and all the

164 Dibyendu Saha and Pradip Sinha

embryos that failed to hatch within 36 h of oviposition were counted and processed for clearing of the first-instar larval cuticle (see below).

The specificity of lgI-;abl- interaction was further tested by examining the fre- quency of embryonic lethality in lgl-; abI- double mutants carrying a single dose of P [lgl + ry+]-13 transposon. Crossing Igl D~w P(Igl § ry + ) cn bw/cn bw; abl"/+ flies with Igl4/cn bw; abt2/+ flies generated these progeny (see table 1B).

Influence of enhancers and suppressors of abl signalling on the genetic interaction of I9l and abh dab (Gertler et al. 1989) and ena (Gertler et aI. 1990) are haplo-dominant enhancer and suppressor of abl mutation respectively. To test their influence on interaction of Igt and abI, haploid doses of dab or ena mutations were introduced in lgl-;abl- mutant or other genetic backgrounds (see table 1C). The 191-ena-/lgl- ena+;abI - progeny were generated by mating lgPena'-'l~ males to lgl4/cn bw; abI"/ + females, lgl4/cn bw males were crossed to lgI 4 enaZl~ bw females to generate Igl- ena- /Igl- ena + progeny. Mating of Igl4/cn bw; Df (3L)stdl 1/+ flies with Igl4/cn bw; abl3/ + generated lgl-; abI- dab-/abl- dab + progeny. To generate lgl-; abI- dab-/abl + dab + progeny, IgI4/cn bw males were mated to Igl4/cn bw; Df (3L)stdI I/ + females. Mating of lgI 4 ena'-J~ bw; abl3/ + males with lgl4/cn bw; Df(3L)std11/ + females generated progeny of genotype lgl- ena-/lgl- ena+; abI- dab-/abl- dab +.

2.3 Developmental studies

First-instar larval cuticular phenotype: All unhatched embryos generated by different combinations of crosses were collected 36 h after oviposition, dechorionated, fixed in 1:4 glycerol : acetic acid overnight according to standard protocol (van der Meer 1977), and mounted in Hoyer's mountant and allowed to clear at 40~ for 3-5 days.

lmmunostaining of embryos: For immunostaining, egg collections were made for short durations and all embryos failing to emerge as first-instar larvae 24 h after oviposition were immunostained with MAb22C10 as described earlier (Gertler et al.. 1989). The primary antibody used in the present study was MAb22C10 (Fujita et al. 1982) at a dilution of I: 50 in phosphate-buffered saline containing 0.1% Triton X- 100 and 0.1% BSA (PBTX), while the secondary antibody (biotinylated horse anti-mouse antibody) was used at a dilution of 1:400 in PBTX. The embryos were examined and photo- graphed under Nomarski optics.

3. Results

3.1 Embryonic lethality of Igl; abI double-mutant animals

Individual mutations in lgl (Gateff 1978) and abI (Henkemeyer et al. 1987) do not disturb normal embryogenesis and the mutants grow till the third larval instar and pharate adult stages respectively. Mating of Igl-/+; a b I - / - heterozygous flies is expected to generate Igl-; abl- double mutants. Embryos from these crosses showed excessive lethality, suggesting a shift in the lethal period of these double mutants (table 1A). In contrast, progeny of parents heterozygous for lgl 4 or any of the three abl alleles, namely ctbt I(254cJ, abl 2 and abl 3, were free from embryonic lethality (table 1A).

Genetic interaction of Drosophila abl and Igl 165

Table 1. Genetic interaction of abl and lgl mutations.

Cross number

Genotype of the parental files a

Doses of enhancer Total no. (dab) or suppressor of embryos

(ena) introduced scored

No. of Percentage Lethality as embryonic of embryonic percentage of lethals in lethals in the expected the total the total Mendelian

population population ratio b

1,

2,

3.

4.

5.

6.

7.

8.

9.

cn bw lal4/ +

abll~Z~4c)/ +

abl2/ +

abl~/ + Igl4/ + ; abl1~2s"c)/ +

laP/+; abl2/ +

lgl4/ + ; abI~/ +

lal~ + ry + )

cnbw/ + ;abl"/ + •

lg14/ + ; abl2/ +

A. Lethality of Igl- ; abl- double-mutant embryos

Wild 1258 4 0.32 1.27

Wild 1081 7 0-65 2"59

Wild 618 2 0.32 1.29

Wild 811 3 0"37 1.47

Wild 779 8 1"02 4'10

Wild 918 14 1"56 24'40 c

Wild 864 18 2-08 33"3Y

Wild 927 32 3-45 55"23 c

B. Reduction of lethality by P(lgl +)

Wild 643 4 0"62 NA d

C. Effect of dab and ena on the genetic interaction of tgl and abl

10. lgl%naZ1~ ena haplo 2054 11 0.53 2"14 ~ •

tgl4/+

11. lgl4enaZt~ abl3/+ ena haplo 1732 12 0.69 2-21 .r •

lgl4/ + ; abP/ +

12. Df(3L)std11/+ dab mutant 1533 377 24'60 98'36 ~ •

Df(3L)std11/ +

13. abl2/+ dab haplo 1410 33 2'34 9"36 h X

Df(3L)stdIl/ +

14, lgl4/+;abI3/+ dab haplo 1317 123 9,34 149'1 e •

Igi4/+;Df(3L)stdl 1/+

15. lgl4/+; + / + abI haplo 1427 40 2.80 22"42 j x dab haplo

IoI4/ + ;Df(3L) std11/ +

16. Igl4enaZln/+; abl3/+ ena and 1170 53 4.52 10'35 k x dab haplo

IO14/ + ;Df(3L)std11/ +

(a) Heterozygous flies were derived by crossing the desired mutant balanced flies with isogcnized cn bw flies which display a very low level of embryonic lethality. These flies are designated as Igl/+, abl/+ and so on since the cn bw markers are of no consequence in this cross.

(Continued)

166 Dibyendu Saha and Pradip Sinha

(b) Expected numbers of mutant embryos were a quarter of the total embryos in a monohybrid cross and one-sixteenth of the total embryos in a dihybrid cross. The proportion of embryonic lethality was calculated from these Mendelian ratios as

Number of lethal embryos x 100 %

Expected number

(c) Note the higher incidence of embryonic lethality in crosses expected to generate lgl-; abl- progeny. (d) Note the decline in embryonic lethality in the presumptive lgl- and abl- embryos in the pre-

sence of a single copy of the Igl + gone. For comparison see embryonic lethality in the progeny of cross no. 7.

(e) A haple dose of ena does not affect the embryonic viability of lgP mutant embryos, (f) Progeny of the genotype lgl-; ena-/lgt- ena+; abl-/ctbI- are expected to be one-sixteenth of the offspring

from the cross. A haplo dose of ena enhances embryonic viability of the otherwise lgl-; ab1- mutant animals. For comparison, see the frequency of embryonic lethality in cross no. 8.

(g) One quarter of the total population scored are expected to be abl- dab- from this cross. (h) A haplo dose of dab in abt- embryos causes excessive embryonic lethality. This is a reconfirmation of the

earlier work of Henkemeyer et al. (1987). abI- embryos display only about 2% embryonic lethality, see crosses 3, 4 and 5.

(i) The mutant progeny class of lgl-;abt-dab-/abl-dab + are expected to be one-sixteenth or 6% of the total population scored. Excessive lethality resulting from this cross is thus contributed by the genotypes abl- dab-/abl- dab + (Gertler etal. 1989) and lgl-;abl- dab-/abl + dab + (this work; see cross no. 15).

(j) The progeny with lgl-;abI- dab-/abl + dab + genotype are expected to be one-eighth of the offspring from the cross. The observed lethality implies that all lgl mutant animals carrying haplo doses of both abl and dab die during embryogenesis.

(k) Note the overall reduction in embryonic lethality in the progeny of this cross compared to the lethality for cross no. 14. Heterozygosity for ena therefore has a positive influence on survival of several classes of progeny in this cross, including Igl-; abl- dab-/~bl- dab +.

NA, Not applicable in the present case.

Moreover , the cross expected to generate tgl-; abl- double mutan ts with a copy of a P Elgl + ry +]-13 t r ansposon gave rise to p rogeny with a marked decline in embryon ic lethality (table 1B). This suggested that the presence of a copy of the wild-type lgl + gene alleviates embryonic lethality in the presumpt ive lgI- ;abl- progeny.

3.2 Influence o f modifiers of abl on the interaction o f lgl and abl

abl- mutan ts show embryonic lethality in dab heterozygous (dab/+ ) background . Muta t ions in dab thus act as hap lo -dominan t enhancers of abl muta t ions (Gertler etal. 1989). Given that muta t ion in dab interferes with aspects of cellular signalling med ia t ed by abl (Henkemeyer etal. 1987; Gert ler et a/.1989), we argued that the lethali ty in lgl-; abl- embryos would be aggravated by heterozygosi ty for dab mutat ion. True to this presumption, lethality in p rogeny of the cross expected to generate Igl-; abl- dab- /abI - dab + was exaggerated (table 1C) compared to the cross expected to generate a b l - d a b - ; a b l - d a b + progeny. This exaggerated embryonic lethal i ty could not, however, be entirely accounted for by the lethality in the p resumpt ive Igl-;abt- dab- /ab l - dab + progeny in a cross of tgl4/+ ;Df (3L)s td11/+ flies wi th Igl4/+;ablJ/+ flies (see table 1C), which is also expected to generate p rogeny wi th other genotypes tha t are likely to suffer f rom embryonic lethality. One cand ida te genotype generated in the above cross, namely Ig I - ;abI -dab- /ab l+dab + (i.e.

Genetic interaction of Drosophila abl and Igl 167

Igl4; Df(3L)stdl 1 /+ ), is likely to contribute to the excessive embryonic lethality in the cross since these embryos now have haploid doses of both abl and dab products. Simultaneous reduction in dosage of both these genes is likely to aggravate the genetic interaction between lgl and abI since abl and dab are known to act in the same redundant genetic pathway (Henkemeyer et al. 1987, 1990; Gertler et al. 1989, 1993). To test this possibility, suitable crosses were made to generate progeny that were Igl-; abl- dab-/abl + dab + (table 1C). True to the expectation, the progeny showed excessive embryonic lethality compared to Igl mutant embryos (table 1C).

abl mutants, which otherwise die as pupae or pharate adults, when heterozygous for ena mutation show a suppression of the mutant phenotype and eclose as fertile adults (Gertler et al. 1990). Also, flies heterozygous for ena and homozygous and hetero- zygous, respectively, for abl and dab (i.e. ena-/ena +;abl- dab-/abl- dab +) show rescue from embryonic lethality and survive as fertile adults (Gertler et al. 1990), whereas, normally, mutants homozygous for abl and heterozygous for dab die as embryos (Gertler et al. 1989; also see table 1C). ena, however, does not interact with Igl and does not affect the survival or phenotype of Igl mutants (table 1C). When influence of the ena mutation on survival of IgI homozygotes alone and in combinations with abl, or dab, or both mutations was tested, a marked reduction in embryonic lethality was observed (table 1C). Thus the cross expected to generate Igl- ena-/lgl-ena+; abl-/abI- progeny resulted in a striking reduction in embryonic lethality (table 1C) compared to those that generated Igl -; abl- progeny. In agreement with these results the presumptive Igl and abl homozygotes and dab heterozygotes also showed reduced embryonic lethality (table 1C) when heterozygous for ena. Taken together, these results suggest that lgl interacts with abl and that this genetic interaction is sensitive to gene dosage of both ena and dab.

3.3 Aberrant C N S development in Igl; abl double-mutanr embryos

First-instar larval cuticle of the Igl-; abl- mutants showed normal segmentation with defective cephalopharyngeal skeleton (figure 1). These cuticular defects, however, do not provide any clue to the cellular and other defects that the presumptive Igl-; abl- mutants suffer from. Localization of the Abl protein in axons of the CNS (Gertler et al.

Figure 1. Cuticular phenotype of (A) wild-type embryo and (B) a presumptive Igl4;abt 2 double-mutant embryo derived by crossing lgI4/cn bw; abl2/+ parents. Note the deformed cephalopharyngeal skeleton (arrow) in the double-mutant embryo.

168 Dibyendu Saha and Pradip Sinha

1989; Henkemeyer et al. 1990) and expression of Lgl protein in neuroectoderm during early embryonic development (Strand er al. 1994a) raised the possibility of origin of the embryonic lethality in lgl-;abl- embryos in defective development of their nervous system. Moreover~ since ablis involved in embryonic neurogenesis, the consequences of perturbation of abI signalling by Igl mutation are likely to be seen primarily in CNS. Examination of the CNS by MAb22C10 immunostaining confirmed this suspicion. In wild-type embryos each neuromere consists of two commissures spanning the ventral midline joined together by the longitudinal connectives giving it a ladder-like appear- ance (figure2A). In contrast, lgl-;abl- double-mutant embryos derived from lgl-/+ ; abI-/+ parents showed high incidence of defective CNS development charac- terized by absence or reduction of the longitudinal axonal connections and defective commissures (figure 2,C&D). In these mutant embryos axons are formed but the scaffold of the developing CNS is disorganized. The segmented arrangement of the neuromeres was apparent even in the absence of the connecting axons. The peripheral nervous system of these mutants appeared unaffected since the clasters of cell bodies along the lateral wall and axonal processes in the intersegmental nerves were clearly observed (see figure 2C). The defects in the embryonic CNS were consistently seen in all the embryos that failed to produce viable first-instar larvae. The defects were also distinct from those seen rarely in some of the lethal embryos of the control samples expected to generate cn bw, IgI or abl homozygous embryos. These latter category of defects were characterized only by clumping of neural material (not shown here).

The results of our genetic crosses showed that heterozygosity for mutation in ena substantially reduced embryonic lethality in/g/-; abI- mutants (table 1C). To test if this reduction involved alleviation of the defects in CNS, we examined the embryos of the cross expected to generate Igl- ena-/Igl- ena +; abl-/abl- progeny. Consistent with their improved embryonic survival (table 1), these embryos showed partial restoration (figure2, E&F) from the CNS defects generally seen in lgl-;abl- double-mutant embryos (figure 2, C & D).

4. Discussion

The results presented in this report reveal that simultaneous loss of lgl and abl causes late-embryonic lethality. The lethality during late embryonic development, however, is not seen in the entire population of double mutants (see table 1), suggesting incomplete penetrance of the phenotype. This is likely to be caused by an abundance of maternally derived transcripts of IgI, abl or both in the developing embryos. Lgl protein is localized in the nurse cells and in the growing oocyte (Strand et al. 19940, suggesting maternal contribution of lgl transcript. Recently, further evidence in favour of maternal contri- bution oflgl during embryonic development was shown with the l'/elp of a temperature- sensitive allele of Igl (Manfruelli et al. 1996). Eggs derived from mothers homozygous for the temperature-sensitive allele lgl '~3 and grown at restrictive temperature failed to complete normal embryonic development when these were also zygotically deficient for lgl and incubated at restrictive temperature. This result, as well as normal embryonic development of lgl mutant embryos, appeared to suggest that zygotic lgl function is dispensable for normal embryonic development. In contrast, our results reveal a re- quirement of zygotic function of lgl, albeit redundant (see below), during embryonic axonogenesis.

Genetic interaction of Drosophila abl and Igl 169

A

Figure 2. Defective development of the central nervous system (CNS) of putative Igl-; abl- double-mutant embryos revealed by immunostaining with MAb22C10. In all panels, anterior is to the left. (A) A wild-type embryonic CNS and (B) enlarged view to reveal a highly ordered ladder-like patterning of the axonal commissures, namely anterior (ac) and posterior commis- sure (pc), the longitudinal tract (1), and the intersegmental nerve (is). (C) A presumptive Igl-; abl- embryo showingdefective CNS and (D) enlarged view to display lack of connections and fasciculations of the axons constituting the commissures. Note the absence of the longitudinal tract (dark arrow) and eommissures (open arrow). (E) A presumptive Igl- ena-/Igl- ena +;abI- mutant embryo and (F) enlarged view revealing partial restoration from the mutant phenotype normally seen in Igl-; abl- double mutants as seen in (C) and (D).

4.1 Synergistic interaction between Igl and abl

The phenotypes exhibited by the presumptive lgl-; abI- embryos are different from those of Igl or abl mutant homozygotes. The axonal defects observed in the/g/-; abl- double mutants appeared more acute than those seen earlier in a small proportion of abI mutant animals (see Elkins et al. 1990). Moreover, only the lethal embryos derived

170 Dibyendu Saha and Pradip Sinha

from lgI and abl heterozygous parents displayed the neuronal defects, suggesting that the lethality indeed originated from the homozygosity for both lgI and abI mutations. These observations strongly argue in favour of a synergistic interaction of lgl and abl during embryogenesis (for a detailed discussion on genetic interactions, see Martinez- Arias 1994).

4.2 abl-mediated axonogenesis and redundant function of LgI protein

The observations presented here reveal a genetic interaction of a Drosophila proto- oncogene, abl, which encodes a cytoplasmic tyrosine kinase (Henkemeyer et al. 1987), and a tumour suppressor gene, lgI, which encodes a cytoskeletal protein (Strand et aI. 1994a). These results are not surprising in the context of earlier biochemical studies that revealed association of mammalian c-AN protein with proteins of membrane cyto- skeleton (Graziani et al. 1989). The Drosophila Abl protein too, on account of its localization at specialized regions of cell-cell interaction (Bennett and Hoffmann 1992), has been thought to participate in a presumptive macromolecular complex that mediates signal transduction (Henkemeyer er al. 1990). Lgl protein on the other hand has been shown to tightly associate with the cytoskeletal network of the cell and also the inner face of the plasma membrane at septate junctions (Strand et al. 1994a). Moreover, Lgl binds to a serine kinase which in turn aids its binding to nonmuscle myosin II heavy chain and plasma membrane (Kalmes et al. 1996). These features of the Lgl protein are strongly suggestive of its role in signal transduction, both intracellular and intercellular (Strand et aI. 1994a). That lgl indeed plays a role in intercellular signalling is revealed by the phenotype of adult cuticle of flies mosaic for Igl. For instance, aberrant growth and development are seen in wild-type cuticle neighbouring Igl mutant cells (Agrawal et aI. 1995). The results presented in this study suggest, in agreement with its proposed role in transduction of cell-cell signalling, that Lgl is a likely candidate protein involved in transduction of abl-mediated signalling. The following features of the observed interaction of lgl and abI further suggest that Lgl protein is likely to be involved in the transduction of abl-mediated signals. First, the defect in Igl-; abl- embryos were seen in embryonic CNS, where abl is known to be required for aspects of axonogenesis (Gertler et aI. 1989). Secondly, heterozygosity for mutation in dab, which is directly involved in abI-mediated axonogenesis and acts as a dominant enhancer of abl (Gertler et at. 1989), aggravates the lethality in IgI-; abl- embryos.

The results presented here suggest that the cytoskeletal Lgl protein, namely p127 (Strand et al. 1994a, b), performs redundant functions during embryonic development. It is redundant because the defective axonogenesis is seen in embryos that are mutant for both lgl and abl and not when abl is functional (also see Gertler et aI. 1989). The redundant aspect of lol function during embryonic axonogenesis is further revealed by the fact that heterozygosity for ena mutation, which alleviates abl-induced defects (Gertler et al. i990), almost completely suppresses the lethality in Igl-;abl- embryos. Finally, a large number of IgI- homozygotes showed a shift in lethal period to late embryogenesis when heterozygous for both abl and dab mutations. Haploid doses of both abl and dab are likely to make the heterozygotes further sensitive to loss of other redundant components of ab[ signalling. In the present case this is revealed by loss of the Lgl protein.

Genetic interaction of Drosophila abl and lol 171

We also note that the observed interaction of lgI and abl is reminiscent of that of abl and fasI genes (Elkins et al. 1990), which apparently takes place without direct binding of the two interacting proteins. The phenotype of the 191-;abl- double mutants, however, appears to be distinct from that of abl- fasI- embryos (Elkins et al. 1990). Instead, the embryonic CNS phenotype of the Igl-; abl- embryos is similar to that of abl- dab-~ abl- dab + embryos (Gertler et al. 1989), with a characteristic thinning of the axonal bundles and gaps in the commissures and longitudinal axon bundles (figure 2). These features of neuronal defects in Igl-; abl- embryos further suggest their origin from aberrations in aspects of axonogenesis mediated by abl (Gertler et al. 1989, 1990, 1993; Henkemeyer et al. 1990).

Acknowledgements

This work was supported by financial assistance from the Department of Biotechnology, New Delhi, for the Drosophila Stock Center and a European Commission, Brussels, grant (No. CII* CT92 0109) to P.S. The authors are grateful to Michael Hoffmann for kindly providing the various abl mutant stocks, L.S. Shashidhara for the MAb22C10 and help in immunostaining, and Sherly Varghese for help in preparation of the manuscript.

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