Curing the shiverer mutant

mately six times more permeable to calcium than to sodium. They point out that calcium ions entering via stretch-activated channels might play a second messenger role in the synthesis and release of substances that regulate vasomotor activity, such as prostacyclin and endothelium-derived relaxing factor. There are further differences in the gating kinetics of bacterial and animal stretch-activated channels. The bacterial channels exhibit slow kinetics, remaining open for seconds at a time, while the channels in animal cells open on a millisecond time scale. A comparison of the kinetic data from skeletal muscle, molluscan cardiac muscle and mammalian endothelial cells suggests some similarities in the gating processes in these animal cells. All three channel types exhibit at least three closed states and one or two open states. The channels open in bursts, separated by long periods of closure. In each case, membrane stretch appears to reduce the period of time between bursts, rather than increasing the burst duration or channel open time. The mechanism of activation has not been elucidated. Guharay and Sachs 4 calculate that a stretch-activated channel in chick skeletal muscle must gather energy from a relatively large area of membrane, greater than 80 nm in diameter. They propose that the energy is delivered to the channel via a network of cytoskeletal fibers. The optimal

channel density is calculated to be Selected references on the order of 1-10 per square 1 Martinac, B., Buechner, M., Delcour, micron 16, which is comparable to A. H., Adler, J. and Kung, C. (1987) Proc. Natl Acad. Sci. USA 84, 2297the densities found in both chick 2301 skeletal muscle and molluscan 2 Lansman, J. B., Hallam, T. J. and Rink, cardiac muscle. It appears unlikely T. J. (1987) Nature 325, 811-813 that calcium or other second 3 Martinac, B., Saimi, Y., Gustin, M. C. messengers are involved in activaand Kung, C. in Ion Channel Modulation of the channels in either tion (Grinnell, A. D., ed.), Plenum Press bacteria or animal cells, since (in press) channel activity is not affected by 4 Brehm, P., Kullberg, R. and Moodyexcision of the patches or by Corbett (1984) J. Physiol. (London) altering calcium concentrations on 350, 631-648 either side of the membrane (with 5 Guharay, F. and Sachs, F. (1984) J. the possible exception of erythroPhysiol. (London) 352, 685-701 cyte channelsn). In some cases, 6 Guharay, F. and Sachs, F. (1985) J. channel activity decreases with Physiol. (London)363, 119-134 membrane hyperpolarizafion 1'6'13. 7 Brezden, B.L., Gardner, D.R. and Although there is much diverMorris, C. E. (1986) J. Exp. Biol. 123, 175-189 sity in the properties of stretch8 Sigurdson, W. J., Morris, C. E., Brezactivated channels, it is conceivden, B. L. and Gardner, D. R. (1987)./. able that they share some comExp. Biol. 127, 191-209 mon features in their structure 9 Sigurdson, W.J., Bedard, E. and and function. An intriguing quesMorris, C. E. (1987) Biophys. J. 51,50a tion concerns the place of 10 Yang, X. C., Guharay, F. and Sachs, F. stretch-activated channels in the (1986) Biophys. J. 49, 373a evolution of ion channels. One of 11 Hamill, O. P. (1983)in Single-Channel the most essential and primitive Recording(Sakmann, B. and Neher, E., cellular functions is regulation of eds), pp. 451-471, Plenum Press cell volume, and it is reasonable 12 Cooper, K. E., Tang, J. M., Rae, J. L. to speculate that ion channels and Eisenberg, R. S. (1986) Biophys. J. involved in osmoregulation may 49, 6a have been among the first to 13 Methfessel, C., Weitzmann, V., evolve. It is not surprising, from Takahashi, T., Mishina, M., Numa, S. that point of view, that stretchand Sakmann, B. (1986) PflOg. Arch. activated channels are present in 407, 577-588 prokaryotic membranes. As we 14 Gustin, M. C., Zhou, X. L., Matinac, B., learn more about the molecular Culbertson, M. R. and Kung, C. (1987) structure of ion channels, it will Biophys. J. 51,251a be of interest to see what evolu- 15 Falke, L., Edwards, K. L., Pickard, B. G. tionary relationships are revealed and Misler, S. (1987) Biophys. J. 51, 251a between the stretch-activated channels of prokaryotes and the 16 Sachs, F. (1986) Membr. Biochem. 6, 173-195 ion channels of eukaryotic cells.

Curingtheshiverer mutant Robert 1. Milner Division of Predinical Neuroscienceand Endocrinology, ResearchInstitute of ScrippsClinic, 10666 North TorreyPines Road, LaJolla, CA 92037, USA.

388

he generation of transgenic animals by introduction of DNA T sequences into the germ line has rapidly become a standard tool of molecular biology. Yet the application of this procedure to neurobiological problems is still relatively novel. Recentlyl'z, Carol Readhead and her colleagues in Lee Hood's laboratory have introduced the gene for myelin basic protein (MBP) into the mutant mouse shiverer. This mutant carries a deletion in the MBP gene and displays severe defects in CNS myelination. Shiverer animals that were homozygous for the MBP transgene formed compact myelin and showed none of the gross pheno-

typic features of the mutant. Apart from providing formal proof that the shiverer mutant is due to a defect in the MBP gene, these studies also indicate an interesting relationship between the level of expression of the MBP gene and the degree of myelination. Shiverer (gene symbol: shz) is an autosomal recessive mutation first observed in 1973 by Biddle et aL 3 Affected animals display a generalized tremor, beginning at about 12 days after birth, progressing to convulsions after 30 days, and death at 90-150 days4. There is a striking deficiency of myelin in the CNS, with most axons unmyelinated or with only a few turns of

© 1987, ElsevierPublications, Cambridge 0378-5912/87/$O2,00

uncompacted myelin5. The myelin that is present has several abnormal features, particularly an absence of the major dense line, formed by apposition of the cytoplasmic faces of the myelin membrane and believed to be mediated by MBP. Not surprisingly, most myelin components are reduced in amount, but MBP in particular is essentially absent from both central and peripheral myelin6. It is interesting to note that the absence of MBP has little effect on PNS myelin, which is virtually normal in both structure and function6. The cloning of MBP mRNA7'8 spurred studies of MBP gene expression in mutant animals. MBP mRNA could not be detected in shiverer mice 7 and the MBP gene TINS, Vol. 10, No. 10, 1987

in these animals was shown to contain a massive deletion, beginning within the second intron and extending beyond the last exon7'9-11. Five of the seven exons of the MBP gene are absent, and the shiverer mouse is therefore incapable of synthesizing any of the five protein forms of MBP, which are generated in mice by alternative splicing of exons two, five and six12-14. The presence of similar repeating nucleotide sequences flanking the site of the deletion suggested that the deletion might have been produced by recombination within the MBP gene n. Both the MBP gene 1° and the shiverer gene is have been mapped to chromosome 18 of the mouse. In order to provide formal proof that the shiverer phenotype was indeed due to a defect in the MBP gene and to examine the details of MBP gene expression, Redhead and her colleagues chose to express a normal MBP gene in shiverer mice 1. [The procedure used was essentially similar to that described for the transfer of the gonadotrophic releasing hormone gene into hypogonadal (hpg) mice, recently reviewed in TINS16.] Several hundred mutant and normal mouse eggs were microinjected with approximately 200 copies each of a cosmid clone that included all of the MBP gene, together with flanking sequences. The injected eggs were then implanted in foster mothers and allowed to develop. Viable offspring were tested for the transferred MBP gene by analysis of DNA taken from their tails. Two transgenic mice were produced but only one, a normal C57BL/6J female, expressed the MBP transgene, which was given the gene symbol MBP 1. To examine the effects of the MBP transgene on the shiverer phenotype, the single successful transgenic animal was mated with shiverer mice. Through a series of breedings, shiverer mice carrying one (shi/shi MBP1/-) or two copies (shi/shi MBPI /MBP I) of the transgene were generated. Some effects of a single MBP transgene were observed: the shi/shi MBP1/mice did live slightly longer but still suffered convulsions beginning at about 2-3 months (Table I). However, two copies of the transgene appeared to correct most of the gross features of the shiverer phenotype: shi/shi MBp1/MBP 1 TIN& Vol. 10, No. 10, 1987

TABLE I. Phenotypes of shiverer, myelin deficient and MBP transgenic mice MBP mRNA Genotype + I + (wild type) shi/shi shi/mld rnld/mld shi/shiMBP 11shi/mldMBP1/shi/shiMBW/MBP 1

level(%)

Degree of myelination

Convulsion onset (months)

(months)

Lifespan

100 0 I 2 12.5 13.5 25

normal none slight slight low low intermediate

never 2-3 2-3 2-3 2-3 >4 a >7 a

24-36 2-5 >4 d 3-6 6 >4 d >7 ~

The data is combined from tables in Refs 1 and 2. The degree of myelination in the different genotypes, determined by ultrastructural analysis, was classified as follows: none: complete absence of compact myelin; slight: some myelin lamellae but no compact myelin; low: myelination of larger diameter axons only, limited lamellae but with visible major dense lines; intermediate: most axons have compact myelin with prominent major dense lines but with fewer turns than wild type animals. aThese animals were still alive or had exhibited no convulsions at the time of publication.

animals showing no convulsions have survived to at least seven months of age in perfect health. A subtle tremor could still be detected in these animals, particularly on initiation of movement. The MBP transgene displayed the expression patterns of a normal MBP gene. Transgenic shiverer animals expressed MBP mRNA of normal size in brain but not in several non-neural tissues; this mRNA peaked in abundance in brain at 18 days after birth, identical to the pattern in wild-type animals. Furthermore, three of the four forms of MBP protein could be detected in transgenic mice, suggesting that MBP gene transcripts are spliced normally. (The rarer 21.5 kDa MBP was also assumed to be present, although it could not be detected in either normal or transgenic animals.) This suggests that the transferred MBP gene carries all of the necessary signals regulating correct tissue and developmental expression, as well as alternative RNA splicing, in the environment of the myelinating oligodendrocyte or Schwann cell. The expression of the MBP transgene, however, was considerably reduced compared to the normal gene: mice carrying one and two copies of the gene expressed approximately 12.5% and 25%, respectively, of the normal amounts of MBP mRNA. A lower level of gene expression is not unusual for transgenes and probably reflects the environment of the chromosome where the transgene is integrated. The levels of MBP mRNA correlated fairly well with the levels of MBP protein detected in transgenic animals and with the degree of myelination observed ultrastructurally. Shiverer mice carrying a single MBP transgene

showed limited myelin wrappings around large axons in the optic nerve. However, with two copies of the transgene, there was considerable myelination: most axons were clearly surrounded by wellcompacted myelin sheaths with prominent major dense lines. There thus appeared to be a good correlation between the expression level of the MBP gene and the degree of myelination. This relationship was explored further in studies of the related mutant myelin deficient (told) by Popko et al. 2 The told mutation is allelic to shiverer, is also recessive, and displays a very similar phenotype, except that told homozygotes live slightly longer17. The told mice, however, do express low (1-2%) but detectable amounts of both MBP protein TM and mRNA2. Popko and his colleagues have demonstrated, by genomic blotting and cloning studies, that mld mice conrain several, probably adjacent, copies of the MBP gene 2. At least one of these appears to be normal but two others contain internal deletions and rearrangements. At present, it is not clear what the molecular defect in told is or why an apparently normal MBP gene should be expressed at such low levels. Introduction of the MBP transgene into mid~mid homozygotes and shi/mld heterozygotes, by appropriate crosses, produced mice with additional, intermediate levels of MBP gene expression2. Analysed together with the shiverer transgenic animals (Table I), these mice demonstrated that increasing levels of MBP mRNA resulted in increasing levels of myelination, with successive elimination of the phenotypic traits of the told and shi mutations. With increasing levels of 389

MBP mRNA, more axons become myelinated, the number of turns increases and the myelin appears compacted, with prominent major dense lines. Axons with larger diameters appear to be more readily myelinated than smaller axons and to contain more myelin turns. Even small differences in MBP gene expression resulted in distinct phenotypes: for example, shi/mld MBP1/- mice shiver less than shi/shi MBP1/- animals, do not have convulsions, and live longer, although the abundances of MBP mRNA in these animals are estimated to be 13.5% and 12.5% of normal, respectively, and there are no apparent differences in the myelin ultrastructure. These elegant studies provide formal genetic proof that the phenotype of the shiverer mouse is the consequence of a deletion in the gene for MBP. In addition, this work indicates that myelination is not an all-or-none event but that the degree of myelination is dependent on the levels of expression of at least one of its protein components. The production of lines of mice with varying degrees of myelination will be an important resource for the study of myelination and the physiological consequences of dysmyelination. One also assumes that similar studies will be carried out t !H , i

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ma

for other dysmyelinating mutants as their molecular defects are defined. The mutant mouse jimpy, for example, displays severe CNS hypomyelination as a result of aberrant splicing of proteolipid protein (PLP) gene transcripts 19'2°. KlausArmin Nave in this correspondent's laboratory has recently demonstrated that the defect results from a single base change in a consensus splice site of the PLP gene 21. We eagerly await a similar molecular genetic analysis of other dysmyelinating mutants to increase our understanding of the vital process of myelination.

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Selected references

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1 Redhead, C., Popko, B., Takahashi, N., Shine, H. D., Saavedra, R. A., Sidman, R. L. and Hood, L. (1987) Ce1148,703712 2 Popko, B., Puckett, C., Lai, E., Shine, H.D., Redhead, C., Takahashi, N., Hunt, S. W., Sidman, R. L. and Hood, L. (1987) Cell 48, 713-721 3 Biddle, F., March, E. and Miller, J. R. (1973) Mouse News Lett. 48, 24 4 Chernoff, G. F. (1981) J. Hered. 72, 128 5 Bird, T. D., Farrell, D. F. and Sumo, S.M. (1978) J. Neurochem. 31, 387-391 6 Kirschner, D.A. and Ganser, A.L. (1980) Nature 283,207-210 7 Roach, A., Boylan, K., Horvath, S., Prusiner, S. B. and Hood, L. E. (1983) Cell 34, 799-806 8 Zeller, N. K., Hunkeler, M. J., CampagJ

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21 tt ii

Whathappenswhengrowthconesmeetneurites: attractionor repulsion? Paul C. Letourneau

he routes taken by elongating axons to their synaptic targets Departmentof Cell are controlled by the growth cone, Biologyand a sensory-effector system that Neuroanatomy, senses local cues and responds with Universityof five activities that determine neurMinnesota, onal form: elongation, branching, Minneapolis, MN, USA. turning, retraction and synaptogenesis. Recent in-vitro studies show that movements of vertebrate growth cones are both promoted and, surprisingly, inhibited by contacts with axons. Thus, the classical terms, contact guidance and contact inhibition1, have renewed significance in understanding the navigation of growth cones.

T

Axons are pathways for growth cone migration Growth cone navigation involves sensory functions provided by surface receptors for adhesive ligands, 390

growth factors, neurotransmitters, and ions, while the effector system includes transmembrane signals and second messengers that control secretion and extension, adhesion and contraction of motile processes 2. The paths taken by different growth cones result from differences in surface sensitivities that modulate effector functions. The importance of axon bundles as pathways for neurite elongation is clearly illustrated in simple embryos like Daphnia and grasshoppers. Corey Goodman and colleagues at Stanford University have shown that growth cones can recognize a single axon or small group of axons3'4. Do vertebrate growth cones also follow labelled axonal pathways? If so, how many labels exist, and bow do growth cones respond to axonal labels?

© 1987.ElsevierPublications.Cambridge 0378- 5912187150200

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noni, A. T., Sprague, J. and Lazzarini, R. A. (1984) Proc. Natl Acad. Sci. USA 81, 18-22 Kimura, M., Inoko, H., Katsuki, M., Ando, A., Sato, T., Hirose, T., Takashima, H., Inayama, S., Okano, H., Takamutsu, K., Mikoshiba, F., Tsukada, Y. and Watanabe, I. (1985) J. Neurochem. 42, 692-696 Roach, A., Takahashi, N., Pravtcheva, D., Ruddle, F. and Hood, L. (1985) Cell 42, 149-155 Molineaux, S. M., Engh, H., de Ferra, F., Hudson, L. and Lazzarini, R.A. (1986) Proc. Natl Acad. Sci. USA 83, 7542-7546 de Ferra, F., Engh, H., Hudson, L., Kamholz, J., Puckett, C., Molineaux, S. and Lazzarini, R.A. (1985) Cell 43, 721-727 Takahashi, N., Roach, A., Teplow, D.B., Prusiner, S.B. and Hood, L. (1985) Cell 42, 139-148 Newman, S., Kitamura, K. and Campagnoni, A. T. (1987) Proc. NatlAcad. Sci. USA 84, 886-890 Sidman, R. L., Conover, C. S. and Carson, J.H (1985) Cytogenet. Cell Genet. 39, 241-245 Charlton, H.M. (1987) Trends Neurosci. 10, 229-231 Doolittle, D. P. and Schweikart, K. M. (1977) J. Hered. 68, 331-332 Matthieu, J-M. Ginalski, H., Friede, R. L., Cohen, S. R. and Doolittle, D. P. (1980) Brain Res. 191,278-283 Nave, K-A., Lai, C., Bloom, F. E. and Milner, R.J. (1986) Proc. Natl Acad. Sci. USA 83, 9264-9268 Hudson, L. D., Berndt, J. R., Puckett, C., Kosak, C.A. and Lazzarini, R.A. (1987) Proc. Natl Acad. Sci. USA 84, 1454-1458 Nave, K. A., Bloom, F. E. and Milner, R. J. J. Neurochem. (in press) m,,,,i

F r i e d r i c h B o n h o e f f e r and his colleagues at the M a x - P l a n c k d n s t i tfit ~ E n t w i c k l u n g s b i o l o g i e in T f i b i n g e n have d e v e l o p e d elegant

tissue culture paradigms for analysing growth cone navigation5'6. Jonathan Raper, who studied labelled pathways with Goodman, has joined Bonhoeffer and co-authored some interesting papers on interactions of growth cones with neurites 7-1°. In the February, 1987 issue of the Journal of Cell Biology, Chang, Rathjen and Raper described an ingenious method for assessing neurite elongation on axon bundles. Reproducible arrays of fascicles extending from chick sympathetic ganglia were prepared and seeded with fluorescently labelled sympathetic neurons, which rapidly attached to the cables and extended neurites (Figs 1, 2). After one day, the cultures were fixed and labelled neurites were measured. Raper and his colleagues used this system to test the effects of antibodies against three axonal surface glycoTINS, VOI. 10, No. 10, 1987