Proto-oncogenes in pattern formation

TIG-

October 1987, Vol. 3, no. 10

moo

Proto-oncogenes in pattern formation Isabel Guerrero Imperial Cancer Research Fund, Developmental Biology Unit, Department o/Zoology, University of Oxford, South Parks Road, Oxford OXI 3PS, UK.

There is now a considerable body of evidence suggesting that protoonc~genes are cellular genes whose products play a fundamental role in the proliferation and differentiation of cells. Mutations in these genes convert them to active elements in the cooperative process of tumorigenesis. Specific functions have been assigned to certain proto-uncogenes on the basis of their structural similarities, biochemical properties and cellular localization. For the most part they seem to be growth factors, growth factor receptors, G-like proteins or other proteins involved in signal transduction, though some have properties of DNA binding proteins (reviewed in Ref. 1). Some of these genes are generally expressed in all tissues but others exhibit a temporally regulated or spatially restricted expression during development. The int-1 and int-2 proto-oncogenes provide good examples of the latter phenomena. These genes are transcriptionally activated in mouse mammary carcinomas by the insertion of proviral DNA of the mouse mammary tumor virus (MMTV)2'3. It now seems clear that int-1 plays a functional role in this mammary tumorigenesis, since it is able to alter the phenotype and growth properties of some mammary epithelial cell lines that express an exogenous int-I gene in culture4's. In normal development, expression of int-1 is restricted to days 8.5-13.0 of mouse embryogenesis. There is no expression in normal adult tissues, with the exception of the testis of sexually mature mices. Very recently, two independent groups ~'e have presented

evidence suggesting a role for int-1 in the early stages of central nervous system development in the mouse embryo. Wilkinson et al. 7, using computer-aided reconstruction of tissue sections hybridized in situ with an int-1 probe, have &'~lysed the localization of i~t-1 U~NA in the developing mouse embryo. The onset of int-1 transcription is restricted to a subset of neural plate cells in the 8.5-day embryo, implying that regional differentiation of the neuroepithelium occurs before new rai tube closure. After closure, the general pattern of int-1 expression in the brain and spinal cord is also complex, but does not appear to be associated with either neuronal or glial cell formation. In a parallel study, involving isolation of RNA from dissected mid-gestational embryos, Shacldeford and Varmuss have obtained results that are consistent with these findings. In addition, using germ cell fractiunation, spermatogenesis mutants and correlating the onset of int-1 expression with the appearance of cell types during testicular development, Shackleford and Varmus s have also found that int-1 RNA is detectable only in postmek-.'~' gerra cells. These multiple effectz should not be surprising, since it has also been observed that some oncogenes and growth factors are able to promote either neoplastic transformation or cellular differentiation, depending upon the target cell. While the above studies do not in themselves reveal the function of lot], Varmus a.d his colieagues favour the possibility that the int-1 protein is a secreted factor that may serve in intercellular communication. This

hypothesis is based on a ~,etailed analysis of the int-1 product. The gene encodes a protein of 370 amino acids with a high content of cysteine residues, a hydrophobic ieader and a possible amino terminal signal pep~ tide. By use of antibodies raised against different int-1 synthetic peptides it has been shown that the protein is modified by glycosylation and it is found associated with membranes 9. All these properties are expected for proteins that enter the secretory pathway. In fact, an extracelhilar role is more easily reconciled with the apparently diverse and cell-type dependent functions of int-1. Now a quite remarkable discovery from an entirely different source provides additional support for such a function for the int-1 product. In an effort to gain an understanding of the function of the int-1 gene at the organismai level, Rijssewijk et ai. 1°, like several other oncogene research laboratoriesu, turned their attention to Drosophila. The sophistication of the techniques for genetic analysis in this organism are well known; if homolognes of the uncogenes can be found in the fly, the potential for a genetic analysis of their function is enormous. With this in mind, Rijssewijk and co-workers have looked for and found a Drosophila homologue of the mouse oncogene, @t-1. This Dint-1 gene has been cloned and sequenced. The predicted Dint-1 protein, deduced from the nucleotide sequence, is 468 amino acids long and also starts with a hydrophobic leader (Fig. I). The homology with the mouse protein is 54% and the cysteine residues are conserved. A major difference is an extra sequence of 85 amino acids in Drosophila, encoded by an add/t~onal exon. The most significant feature, h~wever, is that, unlike previously cloned Drosopkila uncogene homologues, Dint-1 corresponds to a known geae, called wingless (wg), which has a well charactefized developmental function. This is the first case in which a proto-

I

M

r

46S

I I ~To

Fig. 1. Comparison betweenthepre&¥~edamino acid seq~nces of tks 370 amino acid mouseint-I protein (M) a ~ Db*t-1, its 468 amino mid ~omologue in Drosophila I'D.'. Tke ~,-n bars r@rese~tthepro~n sequee~.es,~med for maximum homolo~ Oosig~ of idoe~ical residues shown by connecting black ~bees)LCysteiue residues are shown a.¢ black dots and putative signcl peplide r@~mnsare marked by stars. Vertical liues with inverted arrow ~e,uls @dicatepossible N-linked glycosylalion sites. ReproducedttomRef. 10, with permissiorL © xss7.Ee~i~ ~ . ~

~

0lea- 9s~,nrse,oo

rn on itor ~-"

TIG - - October 1987, VoL 3, no. 10

oncogene isolated in Drosophila has been found to be associated with a known mutation. The segmental pattern of the Drosophila body is determined in the first 3-4 h of development. Many of the genes that control the establishment of this pattern have been identified. The segment polarity genes constitute a class that is required for the specification of the pattern within each segmental unit~z. This means that mutations in these loci cause pattern alteration~ that are repeated in each segment. Wingless is one such gene and mutations in this locus can affect both the adult and the embryo.tic pattern. Lethal alleles that completely inactivate the wg gene function~z'~s produce a bizarre cuticle pattern in the embryos, which die before hatching. In normal

embryos, each segment is marked by l a belt of cuticle processes called

denticles, which have a defined distribution and polarity (Fig. 2a). In the absence of tog+ this pattern is disrupted and the embryo is covered with a continuous lawn of denticles (Fig. 2b). The first tog allele described x4 only partially inactivates the gene. Mutant individuals survive to adulthood but frequently lack wings; in their place they develop extra thorzcic s ~ c t u r e s , usually in mirror-image symmetry. Early analysis of this weak allele, by production of mutant cell clones in wild-type fliesIs, suggested that the effect of wg mutant cells is nonautonomous when they grow in small patches in a wild-type wing. However, tog- clones that populate the Fig. 2. Dark-field photograph of (a) wild.type first instar Drosophila larva and (b) wg- larva. (c) Bright-field photograph of in situ hybridization of wg probe to wild-13/pe 6-hour embryo section. (Photographskindlyprovided by Phil Ingham.)

~I~

~. "

i "

t'

r

entire thoracic segment do show the mutant phenotypeTM. This apparent contradiction can be resolved if it is supposed that t.h.e-~jild-typeproduct has a role in cooperative decisions made by groups of cells during development. Small groups of togcells could be rescued by their tog+ neighbours, whereas an entire segment or wing disc lacking tog÷ cells would be incapable of normal development. Thus the togproduct may affect a process involving communication between cells both in the larval segments and in imaginal discs. The tog region has recently been cloned by Baker x6, employing the transposon tagging technique. He found that several tog mutants map within a single transcription unit, and determined the spatial distribution of this transcript in embryos. Cabrera etaL 17have now shown that injection of antisense copies of this 3 kbp RNA into wild-type eggs can produce tog phenocopies. Baker's analysis shows tog to be expressed in a narrow strip of cells close to the posterior border of each parasegmentxe (Fig. 2c). An identical pattern of expression was obtaino~ ~ t h the Dint-1 probe I°, providing the first indication that Dint-1 and tog are identical. Final proof came from evidence that tog (Ref. 17) and Dint-1 (Ref. 10) are at the same chromosomal location and have identical nucleotide sequences. More significantly, the homology between int-1 and tog may go further than just sequence homology. In the formation of the cuticle pattern of Drosophila, there is evidence that the function of tug extends beyond the domain in which the gene is transcribedxe. In situ hybridization studies have shown that the wingless product regulates the expression of other segment polarity genes that are not expressed in the same cells. (A. Martinez-Arias, N. E. Baker and P. Ingham, pets. commun.). These results are consistent with the tog product being a diffusible factor, as suggested for the mouse int-I protein9. Finally, it is not difficult to imagine that genes that interact with tog and act in a cooperative way in the differentiation of cells within each sem~ent might also have homologues in the mouse; such genes could have a specific role in the development of the neural tube, spermatogenesis and mammary tumorigenesis. It is now clear that

monito

TIG ~ October 1987. Vol. 3, no. I0

for his helpful criticism and stimution of these genes by tissue culture lating discussions. of differentDrosophila mutants. This approach will enable genetic studies with the whole organism to be complemented by a cellular and References I Bishop, J. M. (1987)Sc/emce 235, 305--311 biochemical analysis of the system. A number of developmental genes 2 Nusse, R. and Varnms, H. E. (1982) Cell 31, 99-109 in Drosophila have now been shown 3 Peters, G., Brookes, S., Smith, R. and to be related to growth factors or Dickson, C. (1983) Cell 33, 369-377 growth factor receptors zs-~°. These 4 Brown, A. M. C., Wildm, R. S., Prendergast, T.J. and, Varmus, H. E. findings, together with rids con(1986) Cell 46, 1001-1009 clusive new evidence for a develop- 5 RUsewijk, F. et al. (1987)EMBO ]. 6, mental role of a proto-oncogene, are 127-~31 lending to a new experimental 6Jakobovits, A., Shaddefotd, G. M., Yarmus, IL E. and Martin, G. R. (1986) approach to the genetic analysis of itwill be important to study the func-

cancer.

Acknowledgement I am ~,ery grateful to Phil Ingham

Proc. NatiAca& Sd. USA 83, 7806-7810 7 Wilkinson, D. G., Bailes, J. A. and MclVl:,-hon,A. P. (1987) Cell 50, 79-88 8 Shaddeford, (3. M. and Varmus, H. E. (1987) Cell 50, 89-95

More V gamma genes! B. Flanagan Irf~erial Cancer Re~earchFu~l Laboratodes, St Bart~lomew's Hospital, Dominion House, Bartholomew Close, London ECIA 7BE, UK.

T lymphocytes have a central role in determining both the nature and magnitude of an immune response. Many questions remain to be answered about the cell surface antigen receptor found on these cells. The T-cell receptor (TCR) was initially identified as a disulphide-linked heterodimer composed of variant o~ and [3 polypeptides always found on the cell surface in association with a second invariant antigen complex, CD3. Both ¢ and [3 polypeptide chains are encoded by gene segments that join through rearrangement events during T-cell ontogeny, m a manner analogous to that of the immunogiobulm genes. These rearrangements form the basis of receptor diversity and the control of receptor expression. During attempts to clone the TCR o~and [5 genes a third gene, y, which rearranges in T cells was also inadvertently cloned]'2. This gene also undergoes rearrangement of V (vat.hie region), J (joining region) and C (constant regign) genes in somatic T cells. Initially the y protein product remained elusive but it has now been identified and is found on a subpopulation of T cells that constitute around 0.1% of adult thymocytes aad 5% of penpher~ blood lymphocytes. These cells express the CD3 antigen but not CD4 or CD8

or the TCR ¢d~ hete;~
differences between the Ig X lightchain gene loci. In man, so far, ithas been shown that two Cy genes 16 kbp apart, ~ach with associated J segments, share a single set of Vy segments, while in the mouse four C., genes have been descn~oed, each having its own assodated Vy and Jy segments (see Fig. I). Recently, new C~genesegments in both mouse and man nave been described, indicating that t~e potential ~or diversity at the ~t loci is not as limited as prevlous]y suggested. The isolation of a further mouse Vy gene, V~4.4, brings the total number of potentially functional V T genes in

© 198"/,E[se~ [~ka[ioo.~C~vdge 0168- 9~M//$02.00

9 Brown, A. M. C. etal. Mol. Cell. Biol. (in press) 10 Pdjsewijk, F. etaL (1987)CeU50, 649--657 11 Shiln, B-Z. (1987) Tremis Genet. 3, 69-72 12 N~sslein-Volhard, C. and Wieschaus, E. (1980) Nature 287, 795-801 13 Balm, P. (1977) MoL Gen. Genet. 151, 289-294 14 Sharma, R, P. (1973) Dvosoph. Inf.. Serv. 50, 134 15 Morata, G. and Lawrence, P. A. (1977) Dev. Biol. 56, 277-/.A0 16 Baker, N. E. 0987)EMBO ]. 6, 17651773 17 Cabrera, C. V. etal. (1987) CEU50, 659663 18 Wharton, IL A., Johansen, IL M., Xu, 1". and Artavanis-Tsakonan, S. (1985) Cell 43, 567--581 19 Padgett, IL W., SL Johnston, IL D. and Gelbart, W. M. (1987) Nature 325, 81--84 20 llaIen, E., Basler, K., F~lstroem, J. E. and ~ubin, G.M. (1987) S ~ 236, 55-63

the mouse to seven6. The new gene was isolated from a panel of thymic hybridomas generated by fusing a thymoma ceil line (BW 5147) and day-15 fetal th~ymocytes.In Southern blot analysis, a C-region probe detected four hybridizing bands in the original BW 5147 cells. In several of the hybridomas, only one of these bands was retained, suggesting that it was derived from one BW 5147 chromosome 13 whose homologue, from which the other three bands originated, had been lost. The remaining rearranged fragment was isolated from the hybridoma and proved to contain a previously unidentified V~ gene. Analysis of germ-line clones demor,~trated the new Vv gene to be located within the Vv4 cluster (see Fig. la). Notably, although the new gene has low nucleotide (32-38%) and amino acid (20-21%) sequence homology to other murine Vv gene segments, it has Fdghhomology (6068% and 42-48% respectively for nuclentides and amino acids) with all eight previously defined human Vy genes of subgroup I. In the human Y loci two new V~ genes, each from a disthlct new subfamily, have been described7. This brings the total number of V~ genes to eight (excluding pseudogenes) and the number of subgroups to four. The first new Vv gene, designated V~10, was isolated from a T-lineage genomic DNA libra:y by screening wi~ a Jy probe. The resulting clone vms examined by sequencing; although it clearly identified a Vy gene on the basis d higifly conserved