Cloning and developmental expression analysis of the murine homolog of the spinocerebellar ataxia type 1 gene (Sca1)
Cloning and developmental expression analysis of the murine homolog of the spinocerebellar ataxia type 1 gene ( Sca1 )Sandro Banfi1,+,, Antonio Servadio1,, Ming-yi Chung3, Fiorentino Capozzoli1, Lisa A. Duvick3, Robert Elde4, Huda Y. Zoghbi1,2 and Harry T. Orr3,5,*
Departments of 1Pediatrics and 2Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA, 3Department of Laboratory Medicine and Pathology, and Institute of Human Genetics, 4Department of Cell Biology and Neuroanatomy and 5Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA
Received August 25, 1995; Revised and Accepted October 20, 1995
Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat which encodes glutamine in the novel protein ataxin-1. In order to characterize the developmental expression pattern of SCA1 and to identify putative functional domains in ataxin-1, the murine homolog (Sca1) was isolated. Cloning and characterization of the murine Sca1 gene revealed that the gene organization is similar to that of the human gene. The murine and human ataxin-1 are highly homologous but the CAG repeat is virtually absent in the mouse sequence suggesting that the polyglutamine stretch is not essential for the normal function of ataxin-1 in mice. Cellular and developmental expression of the murine homolog was examined using RNA in situ hybridization. During cerebellar development, there is a transient burst of Sca1 expression at postnatal day 14 when the murine cerebellar cortex becomes physiologically functional. There is also marked expression of Sca1 in mesenchymal cells of the intervertebral discs during development of the spinal column. These results suggest that the normal Sca1 gene, has a role at specific stages of both cerebellar and vertebral column development.
Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder characterized by progressive and selective loss of neurons within the cerebellar cortex, brainstem and degeneration of the spinocerebellar tracts. The mutation responsible for SCA1 has been identified as the expansion of an unstable CAG triplet repeat located within the coding region of the SCA1 gene (1 ,2 ). This CAG triplet is highly polymorphic in the population and varies from 6 to 39 repeats on unaffected chromosomes and from 40 to 83 repeats on SCA1 chromosomes (1 ,3 -5 ). The protein encoded by the SCA1 gene, ataxin-1, is predicted to consist of 792-869 amino acids, depending on the number of glutamine residues encoded by the CAG repeat (2 ). mRNA transcribed from an expanded SCA1 allele has been shown to be translated (6 ). To gain insight into the functional significance of the polyglutamine stretch in ataxin-1 and to identify potential functional domains of this molecule, we isolated and characterized the murine Sca1 homolog. The murine Sca1 gene shows very strong conservation with the human gene, according to their general structure, as well as at the nucleotide and amino acid levels, suggesting that the gene organization may be relevant to its regulation. Previous studies using Northern analysis, showed that the SCA1 mRNA is present in a wide variety of human tissues (1 ,2 ). A wide expression pattern has also been observed at the protein level by immunohistochemical analysis (6 ). To further characterize the expression pattern of SCA1 at the cellular level during different stages of development, we analyzed the expression of Sca1 mRNA in adult and embryonic mice using in situ hybridization. During cerebellar development we demonstrate that in Purkinje cells, a primary site of SCA1 neuropathology (7 ), there is a dramatic and transitory burst of Sca1 expression around postnatal day 14. Developmentally, this corresponds to the time when the murine cerebellar cortex becomes physiologically functional. In addition, we show that Sca1 expression is also prominent in the mesenchymal cells of the developing intervertebral discs of the spinal column. These results suggest that the normal product of the Sca1 gene, has a role at specific stages of both cerebellar and vertebral column development.
A human SCA1 cDNA clone, designated 31-5 (2 ), spanning the entire coding region was used as a probe to screen three mouse cDNA libraries derived from thymus, brain and retina (see Materials and Methods). Five clones were identified and isolated: two from the thymus cDNA library, two from the brain cDNA library and one from the retina library. Northern analysis using independent murine cDNA clones identified a transcript similar in size to the human transcript (~11 kb) with a wide pattern of expression (data not shown). Sequence analysis of the cDNA clones allowed the assembly of a contig encompassing 3616 bp of the Sca1 transcript. This sequence has been deposited in the EMBL database, accession no. X83542. Analysis of the sequence revealed a coding region of 2376 bp predicted to encode a 792 amino acid protein. The coding sequence revealed 85.8% identity with the human DNA sequence and 88.9% identity at the amino acid level (Fig. 1 A). The most striking difference between the murine and human homologs pertains to the CAG repeat which is limited to two repeat units in the mouse. This difference was confirmed by sequencing of the region from three different strains of mice: B6/CBAF1, BALB/c and 129/Sv. Another notable difference between the mouse and the human sequences involves the presence of three proline residues next to the two glutamines in the murine protein. Overall, the conservation between human and murine ataxin-1 is quite uniform along the entire predicted protein sequences. However, a region of highest homology is detected between amino acids 544 and 685 (99%) of the mouse protein. In this stretch of 141 residues there are only two amino acid differences.
By Northern analysis, SCA1 mRNA is expressed in all tissues examined to date (2 ). To determine if the regional and/or cellular expression pattern of SCA1 mRNA within the CNS might provide a basis for the cellular specificity of Sca1 neuropathology, the distribution of Sca1 expression was assessed in adult mouse brain by in situ hybridization using SCA1-specific riboprobes and oligonucleotide (Fig. 2 ).
Development of the cerebellar cortex is well characterized for several species including the mouse (9 ). To determine if the pattern and level of Sca1 mRNA expression varied during cerebellar cortical development, sections of murine cerebellum were examined at various times of development using the Sca1 riboprobe. Sca1 mRNA expression was detected in developing cerebellar cortex beginning at the earliest time examined, embryonic day 14.5, E14.5, (data not shown). At E18.5, Fig. 4 B, Sca1 mRNA expression was detected in cells of the external granule layer (EGL) and in cells having a location consistent with developing Purkinje cells. These latter Sca1 expressing cells are positioned just below the EGL and express message from the Purkinje cell-specific gene Pcp-2 (10 ; data not shown). No Sca1 mRNA was detected in the deep cerebellar nuclei. The pattern and level of Sca1 mRNA expression remained constant through the first week of postnatal cerebellar development (Fig. 5 B). At around postnatal day 14, P14, there was a dramatic increase in the expression of Sca1 by the Purkinje cells (Fig. 5 D). Expression of Sca1 within Purkinje cells, as detected by in situ hybridization, was the highest at this time of development. However, by P21 (Fig. 5 G), the level of Sca1 mRNA in Purkinje neurons was the same as detected in adult mouse cerebellar cortex (Fig. 5 I). Thus, there is a transient surge in Sca1 mRNA in Purkinje neurons during cerebellar development ~P14.
Sca1 mRNA expression was detectable in several somatic tissues at E14.5, for example the developing gut, liver, kidney and ventricular zone of the cerebral cortex (Fig. 6 A). A particularly noticeable pattern of Sca1 mRNA expression in the developing spinal column was seen using in situ hybridization. At the earliest stage examined, E14.5, prominent expression of Sca1 mRNA was detected along the vertebral column using the Sca1 riboprobe (Fig. 6 A). At E16.5 and E18.5, expression of Sca1 mRNA in the vertebral column continued to be high and was particularly prominent using the oligonucleotide probes to Sca1 (Fig. 6 B-D). Sca1 mRNA was also detected in a region of the sternal column analogous to the intervertebral discs of the vertebral column (Fig. 6 C).
Examination of emulsion dipped sections of E18.5 embryos revealed that within the developing vertebral column, Sca1 expression was restricted to the inner cell zone, the mesenchymal cells that surround the remnant of the notochord, the nucleus pulposus (Fig. 7 ). During development of the intervertebral disc, there is a progressive transition to a fibrocartilaginous state (11 ). The inner cell zone around the notochord remains more cellular for a longer portion of vertebral disc development than other regions of the disc. Expression of Sca1 mRNA in the intervertebral discs continued to be detected into the first postnatal week and then declined as development of the collagen fibers in the intervertebral disc proceeded (data not shown).
In order to identify putative functional domains in the SCA1 gene product, ataxin-1 and to investigate the significance of the long 5' and 3' UTRs of the gene, we isolated and characterized its murine homolog. The murine Sca1 gene is very similar to the human homolog, both at the nucleotide and at the amino acid levels. The two homologs share a high degree of identity over the entire amino acid sequence, failing to provide clues for the identification of putative functional domains. The only significant difference in the murine homolog is the drastic reduction of the polyglutamine tract. The presence of only two glutamines in the murine ataxin-1 argues that the glutamine repeat does not serve a key function in the wild-type protein, at least in the mouse. Notable reductions in the size of the polyglutamine tracts or unstable trinucleotide repeats in general and significant conservation at the amino acid level, have been previously observed in mouse homologs of other genes involved in human disorders caused by trinucleotide repeat expansions like the Huntington disease gene (IT15) (12 ), the FMR-1 gene (13 ), the androgen receptor gene involved in spinobulbar muscular atrophy (14 and pers. comm.) and the dentatorubropallidoluysian atrophy gene (15 ). In the murine homolog (hd) of the IT15, the amino acid sequence is highly conserved and is nearly identical to its rat homolog (rhd), including the reduced polyglutamine stretch (16 ).
It is interesting to note that the CAG repeat which is highly polymorphic in the human SCA1 gene, is not polymorphic and is limited to two repeats in all the three different mouse strains analyzed. It is questionable whether or not the lack of polymorphic and unstable repeats in mice is due to the very low number of CAG triplets within the Sca1 gene: such a low number of repeats might not be susceptible to the mutational mechanism that operates on longer repeats. To date, there are no known natural animal models of the human dynamic mutations of trinucleotide repeats in general (17 ). Therefore, the stability of CAG repeats in non-humans may suggest that the mechanism causing triplet repeat expansions is restricted to the human genome.
Overall, the murine Sca1 gene organization shows the same features of the human homolog (2 ). We have analyzed over 1200 of the 8600 DNA sequences of the 3' and 5' UTRs of Sca1 and a high level of conservation was found in both UTRs of this gene. While the portion of the Sca1 3' UTR yet to be sequenced may differ from the sequence of the human gene, the high homology found in the regions of the UTR that have been sequenced is not a common finding, corroborating the hypothesis that the sequences of these regions might play an important role in transcriptional or/and translational control of the murine and human SCA1 homologs (2 ).
The isolation of the murine homolog allowed subsequent examination of the cellular and developmental expression patterns of Sca1 gene in the mouse. In adult mouse brain, in situ hybridization demonstrated that Sca1 mRNA is expressed in neurons throughout the brain. While the cerebellar cortex, a primary site of neurodegeneration in SCA1, is one region of the brain expressing higher levels of Sca1 mRNA, other regions of the brain not affected by SCA1 also express high levels of Sca1 mRNA. For example, the hippocampal formation expresses high levels of Sca1 mRNA. Like the cerebellar cortex, the hippocampal formation is a region of high neuronal density. Thus, neuronal cell density seems to be one factor that correlates with the intensity of the Sca1 in situ hybridization signal. Throughout the brain, Sca1 expression was detected in what appears to be select populations of neurons. This was best exemplified by the pattern of Sca1 mRNA levels seen in cells of the neocortex. In the neocortex, Sca1 hybridization was clustered over large neurons distributed throughout this region (Fig. 3 D).
These in situ hybridization data demonstrate clearly that the regional specificity of neuropathology in SCA1, most prominent in the cerebellar cortex and brainstem (7 ), cannot be accounted for by the regional or cellular pattern of SCA1 mRNA expression. An important caveat is that there may be regional specificity of ataxin-1 protein expression which could explain the cellular pattern of neurodegeneration in SCA1.
It is interesting to note that the regional pattern of Sca1 message expression is very similar to that found for the expression of the Huntington disease (HD) gene (18 ,19 ). Both Sca1 and HD are caused by the expansion of an unstable CAG triplet repeat within the coding region of genes that are expressed widely throughout the brain. While the normal functions of the proteins encoded by these two genes are still unknown, the high expression of Sca1 and HD mRNA in neurons throughout the brain suggests that both could be important for the normal function of neurons. The fact that the regional expression patterns of both Sca1 and HD do not correlate with the patterns of neuropathology seen in the two diseases suggests the possibility that pathology in these two diseases is not simply due to an alteration of the normal function of either protein. Although a gain in function mutation for SCA1 is supported by these data, several points remain unclear which could alter this interpretation. First, the regional expression pattern of this gene has been determined for only the mouse. Thus, if there are differences in the expression patterns between mouse and human, these might reconcile discordances between expression pattern and pathology. It is also possible that functions redundant to those of ataxin-1 may be active in some cells and not in other cells of the brain. If true, this could also explain why not all neurons expressing ataxin-1 are subjected to neurodegeneration in the disease.
The developmental pattern of Sca1 expression in the cerebellar cortex is intriguing when considered in relation to the development of the cerebellar cortex and, in particular, in relation to the developmental time course of Purkinje cells. Development of Purkinje cells in the mouse and other vertebrates can be classified into five main phases (9 ,20 ). The first phase is embryonic when Purkinje cells originate from the germinal zone of the roof of the fourth ventricle and migrate to form the first neurons of the cerebellar plate. The remaining four phases of Purkinje cell development are postnatal. Phase two, during the first postnatal week in the mouse, is the period when Purkinje cells initially form a multicell layer which progressively develops into a single cell layer. Phase three, beginning around the postnatal day 8 is when the apical dendrite of the Purkinje cell becomes apparent. The next phase, phase four beginning around postnatal day 11, is marked by rapid growth and development of the Purkinje cell dendritic tree which results in a dramatic increase in the width of the molecular layer. This is also the period of migration of the granule cells from the external granule cell layer, past the Purkinje cells, to the inner granule cell layer. There is considerable evidence that as the granule cells migrate past the Purkinje cells there is an interaction between these two cell types which provides an important stimulation for the growth and development of the Purkinje cell dendritic tree. For example, Purkinje cells dendrites do not develop normally and remain stunted after external granule cells are destroyed by X-rays (21 ) and in cerebellum of weaver mutant mice in which the postmitotic granule cells fail to migrate from the external granule layer (22 ). This period of pronounced development of the Purkinje cells dendritic tree is completed by the end of the second postnatal week in the mouse. The final stage of Purkinje cell development, day P15 and beyond in the mouse, is characterized by the Purkinje cells having reached their mature structure and attaining a fully developed dendritic tree. At about day P14-P15 the cerebellar cortex of a mouse becomes physiologically functional. Prior to the second postnatal week, there is little difference neurologically between a mouse with a cerebellum versus a mouse lacking a cerebellum (23 ). Around P14, one is first able to determine if a mouse has a normal functioning cerebellum.
Interestingly, the transient burst of SCA1 expression detected around postnatal day 14 in the mouse corresponds to a time in Purkinje cell development when the growth of the dendritic tree is being completed and synaptic connections are being completed and becoming functional (9 ,20 ). While the expression of other Purkinje cell markers usually increases during the second week of postnatal development (e.g. Pcp-2 and calbindin), expression of these genes remains high throughout adulthood. In contrast to other Purkinje cell-specific genes, the developmental surge of Sca1 expression during the second postnatal week is transitory. Thus, the enhanced expression of Sca1 mRNA in murine Purkinje cells which occurs in passing at this developmental timepoint suggests that wild type ataxin-1 protein has a function that might be particularly important during the final stages of Purkinje cell development, as the cerebellar cortex begins to function physiologically. Whether such a proposed developmental function for ataxin-1 in Purkinje cells relates in some way to the specific loss of Purkinje cells in individuals affected with SCA1 remains to be determined.
The prominent expression of Sca1 mRNA in mesenchymal cells of the developing intervertebral discs suggests that ataxin-1 may also function during the development of the spinal column. In the developing intervertebral discs, Sca1 mRNA expression is restricted to the mesenchymal cells of the inner cell zone immediately surrounding the nucleus pulposus, the notochord remnant. The inner zone cells are believed to interact with cells of the notochord and play a critical role in intervertebral disc growth and development (24 ). The vertebral column is formed by an aggregation of mesenchyme around the notochord (11 ). During early vertebral development, the mesenchyme differentiates into embryonic hyaline cartilage which subsequently develops into a fibrous cartilage. The outer zone of the developing disc constitutes the annulus fibrosus which early in development becomes almost entirely collagen fibers with a loss of cellularity. In contrast, the inner cell zone around the notochord remains more cellular for a longer period during development. The inner cell zone does not show a lamellar arrangement as found in the annulus fibrosus and expresses an embryonic or hyaline cartilage. This zone becomes fibrocartilaginous late in fetal development and may contribute to the growth of both the outer annulus fibrosus and the inner nucleus pulposus.
Expression of SCA1 mRNA by cells thought to be important in the development of the vertebral column is interesting in light of the fact that there are no spinal column abnormalities in SCA1 patients. While an understanding of how Sca1 expression contributes to the development of the intervertebral discs should provide insight into its normal function, the absence of vertebral column abnormalities in SCA1 suggests that the normal function of the SCA1 encoded protein, ataxin-1, is not affected substantially by the expansion of the CAG triplet repeat which causes SCA1. This further indicates that the normal functions of ataxin-1 may be quite distinct from the function it acquires after expansion of the CAG triplet into the disease size range.
Three cDNA libraries were screened: a BALB/c mouse neonatal brain library and a B6/CBAF1 adult thymus library from Stratagene; and a BALB/c mouse adult retina library constructed in [lambda]-ZapII (kindly provided by Wolfgang Baehr, Baylor College of Medicine, TX). Library platings and screenings were carried out using previously published protocols (2 ). The probes used to screen these libraries included 31-5, a cDNA insert spanning the coding region of the human SCA1 gene and M6, a murine cDNA clone isolated from the thymus library. Sequencing of the various cDNA clones was done either manually using a Sequenase sequencing kit (US Biochemicals) or an Applied Biosystems, ABI 370A, automated fluorescent sequencer. Data base searches were carried out using the GCG software package and the BLAST network service from the National Center for Biotechnology Information. Northern blot analysis on total and poly(A)+ mouse brain RNA was carried out by standard procedures (25 ) using clone M6 as probe.
A riboprobe, designated M1, was generated using a 311 bp MboI fragment from the mouse Sca1 gene subcloned into pBluescriptII KS+ (Stratagene). This portion of Sca1 includes nucleotides 1018-1329 of the murine cDNA located 3' to the CAG triplet repeat (EMBL accession no. X83542). In vitro transcription was carried out to generate sense and anti-sense probes of this segment of the murine Sca1 cDNA using [alpha][35S]UTP (Amersham) according to the method described (26 ). After removal of the DNA template, probes were resolved on a 4% polyacrylamide/8 M urea gel. Probe bands were excised, eluted in 300 [mu]l of 0.5 M NaOAC, 1 mM EDTA and 0.1% SDS for 16 h at 4oC, precipitated by 1.5 vol of absolute ethanol and resuspended in 10 mM DTT. For each slide, 2 * 106 c.p.m. of probe was used in 100 [mu]l of hybridization solution. In addition to the riboprobe, in situ hybridizations were performed using two complementary oligonucleotides synthesized to two segments from the region 5' to the CAG triplet repeat of the murine Sca1 cDNA (Fig. 2 ). These oligonucleotides, 112 and 111, consisted of 38 and 48 bp and begin at positions 500 and 790 respectively in the mouse Sca1 cDNA (EMBL accession no. X83542). For use as in situ hybridization probes, oligos 112 and 111 were end labeled by using terminal deoxynucleotidyl transferase (TdT) and [alpha][35S]dATP (1000-1500 Ci/nmol).
Mouse embryos, strain FVB/N (27 ), at various stages of development and adult brains were collected and quick frozen. A series of 14 [mu]m sections were cut and stored at -20oC until analyzed by in situ hybridization. All steps of pre-hybridization were carried out at room temperature. Tissues were first fixed using 4% paraformaldehyde, 0.1 M sodium phosphate buffer pH 7.4 for 10 min and washed three times in the same phosphate buffer, followed by proteinase K digestion (20 [mu]g/ml) for 8 min at room temperature. The tissues were washed again before a second fixation in 4% paraformaldehyde for 5 min, followed by a wash before acetylation in 0.1 M triethanolamine and 0.25% acetic anhydride for 10 min. Slides were dipped in DEPC-treated water prior to dehydration steps in 50, 70, 95 and 100% ethanol for 1, 2, 2 and 2 min respectively. Dehydration was followed by a 5 min incubation in chloroform and a 1 min incubation in 95% ethanol. Slides were air dried for at least 1 h before the addition of hybridization solution.
Hybridization was carried out at 60oC in a moisture chamber for 16 h. Post-hybridization steps were as follows: 2 washes in 4 * SSC, 10 mM sodium thiosulfate for 30 min at 37oC; 1 round of RNaseA digestion (50 [mu]g/ml) for 30 min at 45oC, 2 * SSC, 10 mM sodium thiosulfate for 20 min at 37oC; 2 washes in 0.5 * SSC, 10 mM sodium thiosulfate for 20 min at 37oC and a final wash in 0.1 * SSC for 30 min at 37oC. For film autoradiography, slides were dipped in sterile distilled water, followed by dipping in 95% ethanol and air dried for at least 1 h before being exposed to Bio-Max film (Kodak) for 2 days. For microscopic autoradiography, slides were dipped in Kodak NTB-2 nuclear track emulsion (Eastman Kodak Co.). After air drying for 16-24 h, the slides were exposed for 6-12 days at 4oC, developed in Kodak D-19 and counterstained with bis-benzamide. Silver grains were visualized with dark field microscopy.
This work was supported by grants from the National Institutes of Health, National Institute of Neurological Disease and Stroke (NS27699 to H.Y.Z and NS33718 to H.T.O.) and by a fellowship from Italian Telethon to A.S.
1 Orr, H. T., Chung, M., Banfi, S., Kwiatkowski, T. J. Jr, Servadio, A., Beaudet, A. L., McCall, Duvick, L. A., Ranum, L. P. W. and Zoghbi, H. Y. (1993) Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia 1. Nature Genet., 4, 221-226.MEDLINE Abstract
2 Banfi, S., Servadio, A., Chung, M. -y., Kwistkowski, T. J. Jr., McCall, A. E., Duvick, L. A., Roth, E. J., Orr, H. T. and Zoghbi, H. Y. (1994) Identification and characterization of the gene causing type 1 spinocerebellar ataxia. Nature Genet., 7, 513-519.MEDLINE Abstract
3 Ranum, L. P. W., Chung, M. -y., Banfi, S., Bryer, A., Schut, L. J., Ramesar, R., Duvick, L. A., McCall, A., Subramony, S. H., Goldfrb, L., Gomez, C., Sanduijl, L. A., Orr, H. T. and Zoghbi, H. Y. (1994) Molecular and clinical correlations in spinocerebellar ataxia type 1: evidence for familial effects on the age at onset. Am. J. Hum. Genet., 55, 244-252.MEDLINE Abstract
4 Matilla, T., Volpini, V., Genis, D., Rosell, J., Corral, J., Dávalos, A., Molina, A. and Estivill, X. (1993) Presymptomatic analysis of spinocerebellar ataxia type 1 (SCA1) via the expansion of the SCA1 CAG-repeat in a large pedigree displaying anticipation and parental male bias. Hum. Mol. Genet., 2, 2123-2128.MEDLINE Abstract
5 Jodice, C., Malaspina, P., Persichetti, F., Novelletto, A., Spadaro, M., Giunti, P., Morocutti, C., Terrenato, L., Harding, A.E. and Frontali, M. (1994) Effect of trinucleotide repeat length and parental sex on phenotypic variation in spinocerebellar ataxia I. Am. J. Hum. Genet., 52, 959-965.
6 Servadio, A., Koshy, B., Armstrong, D., Anatalffy, B., Orr, H.T. and Zoghbi, H.Y. (1995) Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals. Nature Genet., 10, 94-98.MEDLINE Abstract
7 Schut, J. W. and Haymaker, W. (1951) Hereditary ataxia: a pathologic study of five cases of common ancestry. J. Neuropathol. Clin. Neurol., 1, 183-213.
8 Frohman, M.A. (1990) Race: rapid amplification of cDNA ends. In Innis, M.A., Gelfand, D.H., Sninsky, J.J. and Whit, T.J. (eds) PCR protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA.
9 Jacobson, M. (1991) Developmental Neurobiology. 3rd edn, Plenum Press, New York.
10 Nordquist, D. T., Kozak, C. A. and Orr, H. T. (1988) cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons. J. Neurosci., 8, 4780-4789.MEDLINE Abstract
11 Christ, B. and Wilting, J. (1992) From somites to vertebral column. Ann. Anat., 174, 23-32.
12 Lin, B., Nasir, J., MacDonald, H., Hutchinson, G., Graham, R.K., Rommens, J.M. and Hayden, M.R. (1994) Sequence of the murine Huntington disease gene: evidence for conservation and polymorphism in a triplet (CCG) repeat alternate splicing. Hum. Mol. Genet., 3, 85-92.MEDLINE Abstract
13 Ashley, C.T., Sutcliffe, J.S., Kunst, C.B., Leiner, H.A., Eicher, E.E., Nelson, D.L. and Warren, S.T. (1993) Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat. Nature Genet., 4, 244-251.MEDLINE Abstract
14 He, W.W., Fischer, L.M., Sun, S., Bilhartz, D.L., Zhu, X.P., Young, C.Y., Kelley, D.B. and Tindall, D.J. (1990) Molecular cloning of androgen receptors from divergent species with a polymerase chain reaction technique: complete cDNA sequence of the mouse androgen receptor and isolation of androgen receptor cDNA probes from dog, guinea pig and clawed frog. Biochem. Biophys. Res. Commun., 171, 697-704.MEDLINE Abstract
15 Onodera, O., Oyake, M., Takano, H., Ikeuchi, T., Igarashi, S. and Tsuji, S. (1995) Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) and regional expressions of the expanded alleles in the central nervous system. Am. J. Hum. Genet., in press.
16 Schmitt, I., Bachner, D., Megow, D., Henklein, P., Hameister, H., Epplen, J. T. and Riess, O. (1995) Expression of the Huntington disease gene in rodents: cloning the rat homologue and evidence for downregulation in non-neuronal tissues during development. Hum. Mol. Genet., 4, 1173-1182.MEDLINE Abstract
17 Sutherland G. R. and Richards, R. I. (1995) Simple tandem DNA repeats and human genetic disease. Proc. Natl. Acad. Sci. USA, 92, 3636-3641.
18 Strong, T. V., Tagle, D. A., Valdes, J. M., Elmer, L. W., Boehm, K., Swaroop, M., Kaatz, K. W., Collins, F. S. and Albin, R. L. (1993) Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nature Genet., 5, 259-265.MEDLINE Abstract
19 Li, S.-H., Schilling, G., Young, W. S., Li, X.-J., Margolis, R. L., Stine, O. C., Wagster, M. V., Abbott, M. H., Franz, M. L., Ranen, N. G., Folstein, S. E., Hedreen, J. C. and Ross, C. A.(1993) Huntington's disease gene (IT15) is widely expressed in human and rat tissues. Neuron, 11, 985-993.MEDLINE Abstract
20 Hendelman, W. J. and Aggerwal, A. S. (1980) The Purkinje neuron: I. a golgi study of its development in the mouse and in culture. J. Comp. Neurol., 193, 1063-1079.MEDLINE Abstract
21 Purpura, D. P., Shofer, R. J., Housepain, E. M. and Noback, C. (1964) Comparative ontogenesis of structure-function relations in cerebral and cerebellar cortex. Prog. Brain Res., 4, 187-221.
22 Rakic, P. and Sidman, R. L. (1973) Sequence and developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice. J. Comp. Neurol., 152, 103-132.MEDLINE Abstract
23 Feddersen, R. M., Ehlenfeldt, R., Yunis, W. S., Clark, H. B. and Orr, H. T. (1992) Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice. Neuron, 9, 955-966.MEDLINE Abstract
24 Taylor, J. R. and Twomey, L. T. (1988) The development of the human intervertebral disc. In P. G. Ghosh (ed.), The Biology of the Intervertebral Disc. vol 1, CRC Press, Boca Raton, FL, pp. 39-82.
25 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. 2nd edn, Cold Spring Harbor Laboratory Press, Cvold Spring Harbor, New York.
26 Ausubel, F. M., Brent, R., Kinston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1992) Short Protocols in Molecular Biology. 2nd edn, John Wiley & Sons, Inc., New York.
27 Taketo, M., Schroeder, A. C., Mobraaten, L. E., Gunning, K. B., Hanten, G., Fox, R. R., Roderick, T. H., Stewart, C. L., Lilly, F., Hansen, C. T. and Overbeek, P. A. (1991) FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc. Natl. Acad. Sci. USA, 88, 2065-2069.MEDLINE Abstract
*To whom correspondence should be addressed+Present address: Telethon Institute for Genetics and Medicine (TIGEM), 20132 Milano, ItalyThese authors contributed equally to the work
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