Drosophila abl and genetic redundancy in signal transduction

[~EVIEWS 51 Moran, M.F. et aL (1991) Mol. Cell. Biol. 11, 1804-1812 52 Yu, C.L., Tsai, M.H. and Stacey, D.W. (1990) Mol. Cell. Biol. 10, 6683--6689 53 Serth, J. et al. (1991) EMBOJ. 10, 1325-1330 54 Marshall, C.J. (1991) Trends Genet. 7, 91-95 55 Yatani, A. etal. (1990) Cell61, 769-776 56 Frech, M. et al. (1990) Science 249, 169-171 57 DeClue, J.E. etal. (1991)Mol. Cell. Biol. 11, 3132-3138

41 Huang, Y.K., Kung, H.F. and Kamata, T. (1990) Proc. Natl Acad. Sci. USA 87, 8008-8012 42 Wolfman, A. and Macara, I.G. (1990) Science 248, 67--69

4.3 Downward, J., Riehl, R., Wu, L. and Weinberg, R.A. (1990) Proc. Natl Acad. Sci. USA 87, 5998-6002 44 Jones, s., Vignais, M.L. and Broach, J.R. (1991) Mol. Cell. Biol. 11, 2641-2646 45 Rey, I. et al. (1991) Oncogene6, 347-349 46 Smith, M.R., DeGudicibus, S.J. and Stacey, D.W. (1986) Nature 320, 540-543 47 Kolch, W.. Heidecker, G., Lloyd, P. and Rapp, U.R (1991) Nature 3"49, 426--428 48 Satoh, T. et aL (1990) Proc. Natl Acad. Sci. USA 87, 5993-5997 49 Gibbs, J.B. et aL (!1990)"J. Biol. Chem. 265, 20437-20442 50 Molloy, C0. etal. (1989) Nature342, 711-714

D . R Lowr,, K ZHANG AND J,E. I)ECLUE ARE IN THE LABORATORY OF CELLULAR ONCOLOGY, NATIONAL CANCER INSTITUTE, BETHESDA, M D 20892, USA," R M . WILLUMSEN IS IN THE UNIVERSITY INSTITUTE OF MICROBIOLOGY, D K 1353 COPENHAGEN K, DENMARK.

T h e non-receptor or cytoplasmic tyrosine kinases are encoded by the abl, arg and Jps genes, and by the nine src-related genes src, yes, fyn, fgr, lck, lyn, bck, blk and tkl (Ref. 1). The role of these molecules in normal signal transduction is not well characterized, although recent studies on lck and f y n indicate that their products may be involved in the response of T cells to antigen 2,3. The discovery of non-receptor tyrosine kinase homologs in Drosophila - there are currently four known Drosophila homologs, abl, Dsrc29A, Dsrc64B and fps (Refs 4-6) - permits the application of genetic strategies to the question of what kinds of developmental events are regulated by non-receptor tyrosine kinase-mediated signal transduction. Recently, gene disruption has been used to ask this question for the mouse c-abl and c-src genes 7-0. The surprising result from the studies on Drosophila abl and murine c-src and c-abl is that only subtle phenotypes result from mutations in these genes. To understand this result better, we have exploited Drosophila genetics to screen for second-site modifiers of the abl mutant phenotype. The Drosophila abl gene was isolated by DNA cross-hybridization with the murine v-abl oncogene 4,1°. The deduced amino acid sequence is more similar to the murine and human c-Abl and c-Arg proteins than to any other known tyrosine kinase in vertebrates or Drosophila TM. The Drosophila Abl protein is most similar to mammalian c-AN in the catalytic domain and in the regulatory domains SH2 and SH3 (Fig. 1). The carboxy-terminal domain of the Drosophila

1

F, MICHAELHOFFMANN

Genetic studies on Drosophila Abl and, more recently, on mouse c-Abl and c-Src indicate that the functions of these non-receptor tyrosine kinases may duplicate activities of other molecules within signal transduction pathways. In Drosophila, second-site mutations have been recovered that disrupt the redundant functions so that the Abl tyrosine kinase is essential to the formation of axonal connections in the embryonic central nervous system and for attachment of embryonic muscles to the body wall Molecular isolation and analysis of the genes identified by these second-site mutations should define the molecular basis for the genetic redundancy. protein has weak but statistically significant sequence similarity with the analogous region of tile mouse c-Abl protein. The abl gene is expressed in a broad variety of tissues and times during Drosophila development. Its mRNA is initially supplied to the early embryo maternally. The first zygotic synthesis of abl mRNA and protein is detected midway through cmbuogenesis. The zygotic mRNA and protein are most abundant in the

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Domains of the Drosophila Abl protein and their amino acid sequence similarities and identities to routine c-AN (tspe lb) a> determined by the University of Wisconsin Genetics Computer Group Bestfit program. TIG NOVf:,MBER/1)FCEN[BER1991 VOL. 7 NO. 11/12 g D)Ol Flst'xicl Scicn~v' Puhlisht,rs l i d ~1 K (1168 0179 91 $O2 I)~l

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( FIG[] A Drosopbila embryo midway through embryogenesis stained with antibodies to the Drosophila Abl protein (a). Abl protein is detected in most cells in the embryo at this stage but the signal is highest in the ladder-like array of axon bundles in the CNS, Mutations in abl alone have no detectable effect on the architecture of the wild-type CNS and the abl mutant embryos are indistinguishable from wild-type embryos such as that shown in (b) stained with monoclonal antibody BP102. Homozygous mutations in both abl and dab disrupt the axonal architecture (c) visualized with monoclonal antibody BP102. Note that the rectangle shape outlined by the two commissural axonal bundles in each segment in the embryo in (b) is absent in (c). developing central nervous system (CNS) and somatic musculature, although protein is detected in most cells of the embryo. The Abl protein is asymmetrically localized in these cells, most obviously in the axons of the neurons 13,~4 and at the ends of the somatic muscle fibers attached to the body wall (Fig. 2). After embryogenesis, the larvae have low levels of abl mRNA until pupariation ~5. The pupal expression may be associated with the differentiation of the adult CNS and musculature during this period of development. As in the embryo, Abl protein is detected in most cells of the differentiating imaginal disks but higher levels are detected in cells differentiating as neurons in the eye and wing disks.

Recovery of abl mutant alleles The abl sequence was localized to the 73B region of the Drosophila genome by in situ hybridization to polytene chromosomes. Multigenic deletions of this region were used to screen for new recessive visible

and lethal point mutations in the 73A,B region. The mutagenesis resulted in mutant alleles forming 18 lethal complementation groups ~6. The abl gene was assigned to one of these groups by the molecular mapping of deletion breakpoints to the gene and by complementation with a P element transposon (Tnabl) containing DNA spanning the abl transcriptional unit 17. All five abl alleles permit normal development through metamorphosis. With strong alleles, mutant adult flies fail to escape (eclose) from the pupal case. Flies mutant for mild alleles eclose, but die after a few days ~7. These flies have poor fertility and roughened eyes but no other visible morphological defect. Examination of sections of the roughened eyes by electron microscopy revealed defects in the number and arrangement of retinal cells. The subtlety of the mutant phenotypes was a surprise. We had suspected from the strong evolutionary conservation of abl sequences that the absence of the Abl tyrosine kinase would be lethal to cells or severely disrupt early development. Although early embryonic functions of abl would have been masked by the maternally provided mRNA, the zygotic expression in the later embryo and during pupation was eliminated by the abl mutant alleles. We concluded that the zygotic expression of abl was essential for viability of the organism but did not severely disturb the development of any particular stage or tissue because the functions of the Abl protein were masked by redundant processes.

Identification of second-site modifiers Candidate genes for functions redundant to the Abl tyrosine kinase were identified in our search for second-site modifiers of the abl mutant phenotype. We noticed in our initial characterization of the lethal phase caused by abl mutant alleles that deletions encompassing abl and the polytene region 73B5-7 caused a late embryonic/early larval lethality when heterozygous with abl point mutations or abl deletions. When abl function was restored by introducing the Tnabl transposon, the late embryonic/early larval lethality was rescued, indicating that the lethality required both the absence of abl function and the heterozygous deletion of a gene or genes in the 73B5-7 region. We called this synthetic embryonic lethality the HDA effect (for haploinsufficient dependent on abl mutations) 13. Analysis of the dying embryos revealed gaps in the CNS axon bundles, a phenotype that correlated well with the abundant levels of Abl protein in the embryonic CNS axons. A screen was carried out for dominant mutations that would cause lethality before the pupal stages of development in the absence of abl function. The goals of this screen were to identify the specific gene in 73B5-7 that was haploinsufficient in the absence of abl function, and to determine how many other genes on chromosome 3 (40% of the genome) exhibited similar effects. In a screen of approximately 4000 mutagenized chromosomes, we recovered four such mutations, two of which mapped to the 73B5-7 HDA region 13. Because of the detrimental effect of mutations in the 73B5-7 gene on abl mutant embryos, we called this gene disabled (dab). The other two HDA mutations

TIG NOVeMBER/DECEMBER1991 VOL. 7 NO. 11/12

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]REVIEWS were mapped to the regions 72EF and ['or 86E1-2 and named failed axon connections (fax) and prospero (pros), respectively. The pros gene was discovered independently in ~ w ~ lanai. an enhancer-trap screen for genes expressed Defectl~ Active during embryonic neural development 18. The molecular analysis of all three genes is ab/Pupal lethal/rough-eyed Yes Yes in progress. Recent mutagenesis efforts adults using a similar strategy to screen both Partial Yes chromosomes 2 and 3 (80% of the genome) abl- dab recovered five ~ew mutations, two of which + mapped to the 72EF HDA region containing abl- dabNo Partial ,fax and one that failed to complement dab and muscle attachments alleles. The fact' that 'the mutations were recovered at moderate frequency and that abl-faxPartial Yes multiple alleles were recovered indicated + that mutations in only a few genes could No Yes fulfill the requirements of the screen, abl- dab- + Embryonic lethality: Mutagenesis screens to recover mu+ ~ - - defects in cuticle, CNS tations that alleviate rather than exacerbate the abl mutant phenotype were also successful. Three mutant alleles of a gene named enabled the axon bundles and in the pathfinding of specific (ena) were recovered that permitted homozygous neurons were observed only when both copies of abl mutant abl animals to survive as fertile adult flies and both copies o f f a s / w e r e mutant. indistinguishable from wild-type flies in appearance 19. The ena alleles act as dominant suppressors of the abl Characterization of a kinase-defective Abl protein Although we were surprised by the mild phenolethality but are themselves recessive embryonic lethals. type of the abl mutations, we were stunned to find that defects specifically affecting the tyrosine kinase Revealing a role for abl in embryonic development domain of Abl caused no phenotype at all under The availability of point mutations in dab permitted laboratory conditions. We had assumed that the abl the analysis of mutant phenotypes in embryos homo- mutant phenotypes were caused by the absence of the zygous for both an abl mutation and the dab mutation, Abl tyrosine kinase activity. To test this assumption, and in embryos homozygous for the dab mutation we used site-directed mutagenesis to introduce two alone 13. In the double homozygous mutant embryos, all kinds of mutations into the region of abl encoding the axon connections in the embryonic CNS were severely catalytic domain 2°. One of these changed the invariant disrupted (Fig. 2c). The double mutant embryos also lysine, involved in ATP binding, to an arginine, exhibited defects in the pattern of the somatic muscu- (Tnabl K-N) and the other duplicated changes in a temlature late in embryogenesis. The muscle fibers had perature-sensitive Src protein in order to make the activity of Abl temperature sensitive pulled away from the attachments at the body wall. In kinase contrast, these animals had no detectable defects in (TnablRV-Hn). These mutant forms of Abl were introthe structures or patterns of structures formed by the duced on P element transposons and tested for the epidermis during embryogenesis. Embryos mutant for absence of kinase activity. When crossed genetically dab, but with abl function, exhibited a much milder into abl mutant backgrounds, they provided full rescue phenotype than the double mutant. The dab mutant of the pupal lethality/rough-eyed adult abl mutant phenotypes at all temperatures! embryos had gaps in some of the CNS axon bundles. Severe defects in embryogenesis were also obThe availability of the second-site modifier HDA served when a combination of two heterozygous HDA mutations was critical to establishing that tyrosine mutations, for example dab and f a x or dab and pros, kinase activity was an important function of the Abl were present in an abl mutant background. The protein. The Tnabl K-N or the Tnabl RYnH constructs embryos exhibited a range of defects in gastrulation, provided only partial rescue of the late embryonic/ the CNS, and the structures present on the cuticle. early larval lethality of animals mutant for abl and These defects were all dependent on the absence of hemizygous for dab (Table 1). In contrast, full rescue abl, indicating that the synergistic interaction between was obtained by a non-mutant abl transposon or by the two heterozygous HDA mutations could be com- Tnabl RY-~tn at the permissive temperatures (18°C and 22°C). Tnabl K-N did not provide any phenotypic rescue pensated for by the Abl tyrosine kinase. Further support for a role for abl in the develop- of the disrupted axonal architecture caused by homoment of the embryonic CNS was obtained by examin- zygous mutations in both abl and dab. Abl tyrosine ing the phenotype of embryos doubly mutant for abl kinase activity was also required to compensate for the and a gene encoding a cell adhesion molecule associ- severe defects caused by two heferozygous HDA muated with axon fasciculation 14. The fasciclin I protein tations. Indeed, in these severe genetic backgrounds mediates homophilic cell-cell adhesion in cell culture abl itself was a dosage-sensitive gene, indicating that and is expressed, like abl, during the formation of higher levels of Abl tyrosine kinase activity were reaxon connections in the embryonic CNS. Defects in quired to compensate for the effects of the two HDA TIG NOVEMBER/DECEMBER1991 VOL.7 NO. 11/12

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mutations. The second-site modifiers therefore not only revealed a function for the Abl protein in the embryonic CNS, they also e x p o s e d a specific requirement for the tyrosine kinase activity o f Abl.

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W h e n the genes identified by second-site modifying mutations were in their wild-type diploid state, Abl tyrosine kinase activity was completely redundant. Yet abl mutant animals died as adults in the pupal case or soon after eclosion. A clue to the region of Abl required for viability came from experiments in which portions of the abl gene in a P element transposon were substituted with the analogous portions of mammalian abl sequences e°. These experiments revealed a function for the Abl protein i n d e p e n d e n t of its kinase activity that was required for the viability of the adult fly. Both the carboxy-terminal domain of Abl and p r o p e r subcellular localization of the Abl protein were required for this function. The carboxy-terminal domain of the product of the bcr-abl o n c o g e n e has also b e e n implicated in the subcellular localization to the cytoskeleton, which correlates with the transforming activity of the gene 21.

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FIG~ Hypothetical model for the involvement of Drosophila Abl tyrosine kinase in the interactions between cell adhesion molecules and the cytoskeleton. The subcellular localization of Abl in axons and at the attachment sites of muscles, as well as the mutant phenotypes affecting axonal architecture and musculature, provide circumstantial evidence that Abl may be involved in regulating changes in the cytoskeleton at sites of contact between cells. Immunochemical methods have been used to localize abl and dab gene products in the cytoplasm and fasciclin I at the cell surface. The subcellular localizations of the products of the pros, fax and ena genes are not currently known.

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Redundancy in signal transduction Two molecular mechanisms that could account for the redundancy of abl function are (1) a s e c o n d tyrosine kinase with overlapping substrate specificity

(a)

(b)

abl Hypothetical molecular model in which Abl _PO4 kinase tyrosine kinase activity modulates the activity of activity some as yet unidentified process, pathway or structure. This model is based on our observations of the genetic interactions between Active process Active process abl, dab, fax and pros. The rectangles, circles and diamonds represent three different gene products involved in an essential biological process. The two units of each shape represent abl _po 4 the level of gene product produced by the two kinase [---] ( ~ < ~ wild-type alleles present in the diploid cells. In activity P°4 a wild-type genetic background (a, b), Abl kinase function is redundant to the function of Active process this multicomponent regulatory pathway or Partially active process macromolecular complex. Reduction in the level of a gene product such as those encoded by dab, pros or fax results in an inefficient (e) abl or defective process (c); however, regulation kinase by the abl tyrosine kinase can fully compensate for these defects, presumably by phosphorylation of one of the components (d). A Poorly active or Inactive Active process 50% reduction in the expression of two of these genes causes more severe defects (e); the synergistic nature of the dosage-sensitive interabl ~ po4 actions between these two genes is consistent kinase with their participation in a common function. activity (~)<~P04 Abl tyrosine kinase activity can compensate for these defects, although the Abl kinase itself Partially active process becomes dosage sensitive in these genetic Poorly active or inactive backgrounds (f). Differences in the degree of rescue are observed when one, two or four copies of abl are introduced via P element transposons. Even the complete absence of one of these gene products (g) can be largely rescued by the Abl tyrosine kinase activity, analogous to the phenotypic rescue of homozygous dab mutations by abl, but the rescue is not sufficient for viability (h).

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T1G NOVEMBER/DECEMBER1991 VOL. 7 NO. 11/12

[]~EVIEWS

expressed in the same tissue-specific and temporal patterns as abl or (2) the processes that abl regulates could also be regulated by other, non-kinase signal transduction molecules. For example, if abl really is involved in the interactions between cell adhesion molecules and the cytoskeleton (Fig. 3), these interactions would probably be subject to many regulatory inputs and feedback loops. Eliminating any one of the regulators would have little effect on the overall process but additional genetic insults, perhaps as subtle as reducing the level of a key protein to half its normal level, would destabilize the process such that the regulatory input is critical. Although it is' currently popular to propose the existence of a second kinase to explain redundancy, we believe the second mechanism described above is more likely to be the basis for the genetic redundancy of Drosophila abl. None of the other non-receptor tyrosine kinase genes identified in Drosophila shows a pattern of temporal and spatial expression identical to that of ahl, although there are overlaps in expression patterns 6,22,23. In addition, none of the HDA mutations maps to a region in which the known tyrosine kinase genes are located. The observation that a 50% reduction in HDA gene dosage is sufficient to reveal a requirement for Abl tyrosine kinase activity is consistent with the destabilization of some multicomponent complex or process (Fig. 4). The fact that different HDA mutations show synergistic effects as heterozygotes in abl mutant backgrounds is also consistent with the idea that the products of the HDA genes participate in a common process (Fig. 4e, f). Such heterozygous interactions have been observed previously for Drosophila genes involved in dorsoventral embryonic pattern formation, neurogenesis and eye development 2~-e8. Initial characterization of the phenotypes of three different HDA genes indicates that all the genes are required for normal formation of axon connections in the embryonic CNS. The phenotypes of some of the HDA genes can be suppressed by mutations in ena, while the phenotypes of other HDA genes are not affected by such mutations. As ena mutations can compensate for the tyrosine kinase-dependent functions of Abl, the product of ena is likely to be a key factor in the process. Additional mutagenesis efforts are underway to try to identify allele-specific suppressors of the kinase-dependent functions of Abl; these may identify genes whose products directly interact with the tyrosine kinase. Functional redundancy in regulatory pathways is pervasive in higher organisms. For example, redundant functions have been found in the pathways regulating the cell cycle z930, and the unexpectedly subtle phenotypes caused by gene knockouts of c-src, c-abl, Wnt-1 or c-myb in the mouse indicate that these highly conserved regulatory molecules are in part redundant to other aspects of signal transduction networks 7-9,3132. It will be interesting to see whether the genes identified in Drosophila that eliminate the genetic redundancies of specific kinases like Abl will be useful in identifying homologous mouse genes. Reduction or elimination of the redundant functions in the mouse might then become feasible and reveal a role for specific tyrosine kinases in mammalian development.

Acknowledgements The work on Drosophila abl in my laboratory has progressed through the efforts of Frank Gertler, Mark Henkemeyer, Randy Bennett, Kevin Hill, Mike Clark and Missy Visalli. The research has been supported by the ACS and the NIH. References 1 Cooper, J.A. (1990) in Peptides andProtein Phospho~lation (Kemp, B.E., ed.), pp. 85-113, CRC Press 2 Glaichenhaus, N., Shastri, N., Littman, D.R. and Turner, J.M. (1991) Cell 64, 511-520 3 Cooke, M.P,, Abraham, K.M., Forbush, K.A. and Perlmutter, R.M. (1991) Cell 65, 281-291 4 Hoffman-Falk, H., Einat, P., Shilo, B-Z. and Hoffmann, F.M. (1983) Cell 32, 589-598 5 Simon, M.A., Komberg, T.B. and Bishop, J.M. (1983) Nature 302, 837-839 6 Katzen, A.L. etal. (1991) Mol. Cell. Biol. 11,226-239 7 Soriano, P., Montgomery, C., Geske, R. and Bradley A. (1991) Cell 64, 693-702 8 Schwartzberg, P.L. etal. (1991) Cell65, 1165-1175 9 Tybulewicz, V.L.J. et al. (1991) Cell 65, 1153-1163 10 Hoffmann, F.M., Fresco, L.D., Hoffman-Falk, H. and Shilo, B-Z. (1983) Cell 35, 393-401 11 Henkemeyer, M.J., Bennett, R.L,, Gertler, F.B. and Hoffmann, F.M. (1988) Mol. Cell. Biol. 8, 843-853 12 Kruh, G.D., Perego, R., Miki, T. and Aaronson, S.A. (1990) Proc. Natl Acad. Sci. USA 87, 5802-5806 13 Gertler, F.B., Bennett, R.L., Clark, M.J. and Hoffmann, F.M. (1989) Cel158, 103-113 14 Elkins, T. et al. (1990) Cell 60, 565-575 15 Telford, J., Burckhardt, J., Butler, B. and Pirrotta, V. (1985) EMBOJ. 4, 2609-2615 16 Belote, J.M. et al. (1990) Genetics 125, 783-793 17 Henkemeyer, M.J., Gertler, F.B., Goodman, W. and Hoffmann, FM. (1987) Cell 51,821-828 18 Doe, C.Q., Chu-LaGraff, Q., Wright, D.M. and Scott, M.P. (1991) Cell 65,451-464 19 Gertler, F.B., Doctor, J.S. and Hoffmann, F.M. (1990) Science 248, 857-860 2 0 Henkemeyer, M., West, S.R., Gertler, F.B. and Hoffmann, FM. (1990) Cell 63, 949-960 21 McWhirter, J.R. and Wang, J.Y.J, (1991) Mol. Cell. Biol. 11, 1553-1565 2 2 Wadsworth, S.C., Muckenthaler, F.A. and Vincent, W.S., III (1990) Dev. Biol. 138, 296-312 23 Simon, M.A., Drees, B., Kornberg, T. and Bishop, J.M. (1985) Cell 42, 831-840 24 Simpson, P. (1983) Genetics 105, 615-632 25 Vassin, H., Vielmetter, J. and Campos-Ortega, J.A. (1985) .L Neurogenet. 2, 291-308 2 6 Kennison, J.A. and Tamkun, J.W. (1988) Proc. NatlAcad. Sci. USA 85, 8136--8140 27 Rabinow, L. and Birchler, J.A. (1990) Genetics 125, 41-50 28 Heberlein, U. and Rubin, G.M. (1991) Dev. Biol. 144, 353-361 2 9 Lundgren, K. etal. (1991) Ce1164, 1111-1122 30 Nasmyth, K.A. (1990) Cell63, 1117-1120 31 McMahon, A.P. and Bradley, A. (1990) Ce1162, 1073-1085 32 Mucenski, M.L. et al. (1991) Cell 65, 677-689

F.M. HOFFMANN IS IN THE McARDLE LAI~RATORY FOR CANCER i! RESEARCH, UNIVERSITY OF WISCONSIN, MADISON, W I

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USA.

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