TIC, - - October 1988, Vol. 4, no. 10

No Mickey Mouse genetics lan J. Jackson Developmental Genetics Group, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

When it comes to genetics, and in particular developmental genetics, the mouse just isn't in the same league as the fruit fly or the nema- tode. It probably never will be, but this is not for want of endeavour, as it is the major mammalian model, organ- ism. Progress can be measured at two-yeariy intervals at the Inter- national Workshop on Molecular Genetics of the Mouse. The 5th, held in Wurth, Switzerland, was reviewed in TIG 1. The 6th was at Girton College, Cambridge, 3-8J~uly, 1988, and the abstracts will be p~tblished in Mouse News Letter.

The most informative experi- ments are those in which a molecular study of a gene can be coupled with an examination of the effects of mu- tations in that gene, preferably in whole animals. These studies are being done in the mouse in several ways. Firstly, mutations known from 'classical' genetics are being cloned, by testing candidate genes or by moving in from linked DNA markers. Secondly, mutations are being made by random insertion of exogenous DNA. Some of these turn out to be alleles of known mutations, but most are novel mutations. A new mutation means more work in characterizing the biological nature of the defect, but, as there are few, if any, tlue 'developmental' mutations in the mouse, these might prove the most fruitful in the lohg term.

The third approach is to direct mutations to the genes correspond- ing to an interesting piece of Cloned DN,~ The easiest to make are dominant mutations, where in- creased expression, ectopic expres- sion (neomorphs) or expression of a product that will interfere with the wild-type product (an antimorph) can be tailored into transgenic mice. Somewhat more difficult to achieve is the knocking out of a gene, to pro- duce a loss-of-function mutation, ex- pressed either dominantly (a haplo- insufficiency) or recessively (an amorph). It seems tha~ this should now be possible using embryonal stem cells (ES cells).

Old mutations One of the best known classical

mutations is albino. Several groups have now cloned the tyrosinase gene, the product of the wild type allele at the locus 2'3 (Kelsey and Schiitz, Heidelberg). It is a large (85 kb) gene, with a high frequency (>10%) of aberrant splicing. The interest in the locus comes not or.ly from the several alleles that affect pigmentation, but also from the large number of deletions that have homo- zygous effects on development. Small deletions on the proximal side of albino result in failure to turn on several liver enzymes around birth (Kelsey). The larger deletions have effects much earlier in development, and a map of genes around albino has been derived 4's. Efforts are being made to clone these genes, by jump- ing or walking around the region (Kelsey; Rinchik, Oak Ridge). In addition to probes for tyrosinase, other, random, DNA probes have been isolated, and mapped within the deletions (Johnson, Oak Ridge). Ethyl nitrosourea (ENU) is also being used to mutagenize the region encompassed by one of the largest deletions (Rinchik). These point mutations are an additional com- ponent of the genetic analysis of the region, which will eventually be saturated with mutations and with cloned DNA, and the genetic and physical maps should be super- imposable.

Another region where detailed physical and genetic maps are being developed together is the t-complex. This is a region which, by virtue of unusual genetic properties (suppres- sion of recombination with the wild- type chromosome, transmission ratio distortion in favour of the mutant-hearing sperm and sterility of homozygouG malese), has accumu- lated nattwally a number of recessive embryonic lethals. It is also being subjected to ENU mutagenesis (Ref. 7; King and Dove, Wisconsin) to add m the rich genetic resource of feces- sires provided by nature. It is a 30 megabase segment of chromosome 17, and detailed physical mapping (Lehrach, London) has so far shown that the recombination suppression is apparently caused by s two large chromosomal inversions.

© 1988, Elsevier Publkalions, C,mlbddge 0168 - 957.5/~$02.00

monito Genes from the region are being

cloned and identified. The preferen- tial transmission of t-bearing sperm is mediated by a number of transmis- sion ratio distorter (Tcd) genes act- ing on a transmission ratio responder (Tcr). Silver (Princeton) has cloned a gene family encompassing a few hun- dred kilobases of DNA which must contain Tcr, and which encode male germ-cell specific products. These are strong candidates for the product of Tcr, and it will be interesting to see if and how the response to Tedis mediated through them.

A 10:17 translocation into the t- region causes a mutation at the Steel (Sl) locus on chromosome 10. This interesting locus affects the dewlop- meut of three cell types: melano- cytes, e .rythropoietic stem cells and germ cells. One t-complex clone is within a few megabases of the trans- location, and working on the assump- tion that the breakpoint is in the Sl gene, Stubbs (London) is u~ing chromosomal jumping technology 9 to move towards the breakpoint and therefore clone the gene.

Insertional mutat ions Insertion of a defined piece of DNA

into a gene to cause a mutation is a rather easier way of getting a probe for cloning it. Retroviral insertions occur naturally, and such an event produced the dilute coat-colour mutation. This locus, and associated lethal deletions, has been the subject of a great deal of study by Nancy Jenkins (Frederick) TM. Many other alleles of the vitally induced dilute have been identified, all of which (unlike the original insertioaal mutant) have severe neuromuscular defects in addition to the pigmenta- tion effect. Multiple transcripts from the gene have now been identified, both in melanocytes and in the ner- vous system. The effects of different mutations on the different tran- scripts should enab!e them to be discriminated functionally.

There is only a fairly small number of ecotropic retroviral insertions, such as the one that caused dilute, in the mouse genome, and so there are few chances of a mutation being caused by one. On the other hand, over a hundred different non- ecotropic retroviral integrants have been identified. By use of specific oligonucleotide probes, a number of families of viruses can be distin- guished, any one mouse strain having 10-15 proviruses from each family

o n i t o r (Stoye, Boston). The majority of these proviruses have been mapped by use of recombinant inbred strains; a number of them map very close to (if they are not allelic with) interest- ing mutations, and provide access to those regions. One of them has caused the mutation hairless 1~. This is proved by a reversion of hairless to wild-type that is accompanied by loss of the provirus by recombination be- tween the LTRs, exactly as is seen in dilute revertants. The gene, which has been cloned, is interesting, for not only do homozygous mutants lose their hair at a few weeks of age, but they also have abnormal T-cell differentiation, and are predisposed both to thymic leukaemia and to skin cancers.

The production of transgenic mice has a useful spin-off in the mutations caused by the transgenes. A mu- tation affecting development which turned out to be an insertional allele of lfmb-deformily has been picked up in this way TM. Another transgene was reported at the Cambridge meeting to have produced a new allele o~ downless (Overbeek, Houston). Homozygotes for this allele lack cer- tain hair types in their coat, and the wild-type gene seems to be involved in morphogenesis of the dermal layer. The existence of the old feces- sire mutation, as well as a dominant, Sleek, at the same locus, will facili- tate determination of the function of the gene.

The chances of an insertion bitting a known gene are fairly slim, but the probability of hitting any gene is quite high (5--15% by most estimates). One previously undescribed gene found by Costantini (New York) in a transgenic line seems to be essential for the developing embryo proper (but not extra-embryonic tissues) soon after gastrulation; mutants arrest at this stage (about day 7 after fertilization). A flanking probe detects mRNAs which are candi- dates for transcripts from the ~evelopmental gene.

Jaenisch (Cambridge, Mass.) has for several years used retroviru~es as mutagens in the search for novel developmental genes 13. He now has two with mutatioas early in embryo- genesis, one of which has been cloned and sequenced. Another is a homozygous lethal in young adult mice, which die of kidney failure.

This slow accumulation of new mu~tions steadily turns up interest-

2 ~ i n g genes. The use of ES cells should

accelerate the rate of finding new mutations 14 (Evans, Cambridge, UK; Lovell-Badge, London). ES cells with multiple retroviral inser- tions can be generated and intro- duced into mice, enabling the screen- ing of tens of insertions per animal and considerably increasing the chances of a 'hit'. A screen set up specifically to find mutations in the testis-determining gene on the Y chromosome (Tdy) was successful (Lovell-Badge), but unfortunately the gene does not seem to be tagged by a retrovirus. Nevertheless, this is the first time an XY female has been seen in the mouse, and the fine should be invaluable for working out the testis-determination pathway.

Directed mutations Manipulating the expression of

cloned genes is an essential tool for investigating gene function in disease and development. Introducing an or(I) collagen gene with a specific glycine--~cysteine change into mouse embryos causes a dominant pheno- type which kills the animals at birth 15 (Jaeuisch). The mice have very little collagen in their bones, because the mutant peptide chains interfere with normal collagen assembly. This is known as 'protein suicide', is a model for certain types of osteogenesis imperfecta in man, and is a classic example of an antimorph mutation.

The homeobox genes are receiv- ing close and exciting attention. Two cases of dominant mutations in trans- genics were reported. Gruss (G6t- tingen) reported that some mice carrying the Hox-l.1 gene driven by the 13-actin promoter died soon after birth (although others with ectopic expression in the brain had no pheno- type). Wolgemuth (New York) has put the Hox-l.4 gene into trans- genlcs, driven by its own promoter but removed from the context of the Hox-1 cluster. In a number of cases she sees expression of the gene in the colon, where it is not normally seen, and where it apparently gives rise to a megacolon condition. It is hard to interpret this type of experi- ment - bear in mind that ectopic or over-expression of oncogenes can give rise to tumours which are not in any obvious way related to the nor- real function of the gene. On the other hand, the classical Antenna- pedia phenotype of Drosophila can be simply caused by ubiquitous expres- sion of the Antp gene.

The megacolon mice will be useful,

TIC, - - October 1988, Vol. 4, no. 10

but the best analysis of Hox gene function should come from the loss- of-function mutations. These can be made by introducing a defect into a particular gene by homologous re- combination in ES cells, which are then introduced into the germ line. Gruss described the identification of such a mutation in the Hox-l.1 gene in ES cells. It should now be possible to derive mice carrying the mutation, and to test for its effect on develop- ment. The technique is universally applicableXe, and the impetus the H ox genes have given to the area will quickly spill over into other fields.

This report can only be a superfi- cial look at the current state of mouse molecular genetics. It is intended to show the advance~ that are being made, and I hope communicates the excitement. The 7th Workshop in Prague in 1990 is eagerly awaited: Czech it out!

Acknowledgement The author is a Fellow of the Lister

Institute for Preventive Medicine.

References 1 Hastie, N. (1986) Trends Genet. 2,

249-250 2 Kwon, B. S. el al. (1988) Biochem.

B i~kys. R es. C ommun. 153, 1301-1309

3 Mtiller, G., Ruppert, S., Schmid, E. and Schlitz, G. (1988)EMBO J. ?, 2723-2730

4 Russell, L. B., Montgomery, C. S. and Rayner, G. D. (I~82) Genetics 100, 427--453

5 Niswander, L. et aL (1988) Develop- ment 102, 45-53

6 Willison, K. (1986) Trends Genet. 2, 305--306

7 Shedlovsky, A., Guenet, J-L., Johnson, L.L. and Dove, W.F. (1986) Genet. Res. 47, 135-142

8 Rogers, J. H. (1986) Trends Genet. 2, 145

9 Poustka, A. and Lehrach, H. (1986) Trends Genet. 2, 174-179

10 Rinchik, E. M., Russell, L.B., Copeland, N. G. and Jenkins, N. A. (1986) Genetics 112, 321-342

II Stoye, J. P. et al. (1988) Cell 54, 383-391

12 Woychik, R. P. et al. (1985) Nature 318, 36--40

13 Gridley, T., Soriano, P. a~d Jael~isc'n, R. (1987) Trends Genet. 3, 162-166

14 Robertson, E. S., Bradley, A., Kuehn, M. and Evans, M. J. (1986) Nature 323, 445--448

15 Stacey, A. et al. (1988) Nature 332, 131-13~

16 Jackson, L J. 0987) Trends Genet. 3, 119-120