A human homologue of Drosophila minibrain (MNB) is expressed in the neuronal regions affected in Down syndrome and maps to the critical region
A human homologue of Drosophila minibrain (MNB) is expressed in the neuronal regions affected in Down syndrome and maps to the critical regionJordi Guimerá1, Caty Casas1, Carles Pucharcòs1, Asun Solans1, Anna Domènech1, Anna M. Planas2, Jennifer Ashley3, Michael Lovett3, Xavier Estivill1,* and Melanie A. Pritchard1,*
1Molecular Genetics Department, Cancer Research Institute, Hospital Duran i Reynals, Avia. de Castelldefels Km 2.7, L'Hospitalet de Llobregat, 08907 Barcelona, Catalonia, Spain, 2Pharmacology and Toxicology Department, Centro de Investigación y Desarrolo, CSIC, Jordi Girona 18-26, 08034 Barcelona, Catalonia, Spain and 3Department of Biochemistry and the McDermott Centre, University of Texas Southwestern Medical Centre, 5323 Harry Hines Blvd., Dallas, Texas 75235-8591, USA
Received April 23, 1996;Revised and Accepted June 19, 1996
The minibrain (mnb) gene of Drosophila melanogaster encodes a serine-threonine protein kinase with an essential role in postembryonic neurogenesis. A corresponding human gene with similar function to mnb could provide important insights into both normal brain development and the abnormal brain development and mental retardation observed in many congenital disorders. Trisomy 21 or Down syndrome (DS) is the most frequent human birth defect. It is associated with mental retardation and a broad spectrum of physical abnormalities. A region on human chromosome 21 has been designated the Down syndrome critical region (DSCR) and when present in three copies, this is responsible for many of the characteristic features of DS, including mental retardation. We have isolated a human homologue of mnb from the DSCR. MNB encodes a 6.1 kb transcript which is expressed in foetal brain, lung, kidney and liver. Using a human probe, two major transcripts (6.1 and 3.1 kb) were identified in mouse and expression was detected in situ in several regions of the mouse brain, including the olfactory bulb, the cerebellum, the cerebral cortex, the pyramidal cell layer of the hippocampus and several hypothalamic nuclei. This expression pattern corresponds to the regions of the brain that are abnormal in individuals with DS and suggests that overexpression of MNB could have detrimental consequences in DS patients.
Down syndrome (DS) is one of the most frequent congenital defects. There is a broad spectrum of physical abnormalities associated with the syndrome, including anomalies of the gastrointestinal tract, increased risk of leukemia, defects of the immune and endocrine systems, early onset of Alzheimer's dementia and distinct facial and physical features, but perhaps the most debilitating is a rather severe mental retardation (1 ,2 ). In most cases DS is due to three full copies of human chromosome 21 which arise primarily during maternal non-disjunction, but occasionally DS occurs in people carrying unbalanced translocations, which result in the triplication of only a part of chromosome 21. By correlating phenotype with genotype in patients with partial trisomies a region has been defined, named the DSCR (Down syndrome critical region) which, when present in three copies, is responsible for many of the characteristic features of DS including mental retardation (3 ).
The phenotypic consequences of DS presumably result from the overexpression and subsequent interactions of a subset of chromosome 21 genes and the future challenge is to correlate overexpression of these genes, singly or in combination, with the presence of the DS phenotype. Expression maps of chromosome 21 are currently being developed (4 -8 ) which will help in the identification of all the genes. The first step is to identify the genes in the DSCR and then assess their potential contributions to the pathophysiology of DS. Assigning a function to a gene, particularly in humans, is not simple. Investigators rely on finding clues to function by analysing the expression pattern of a gene, by looking for protein domains or motifs with known functions or by extrapolating from another species in which the function of the homologous gene is known. Any gene with a role in early neurogenesis is potentially important with respect to the abnormal brain development and mental retardation seen in DS. In Drosophila, the mnb gene appears to play an essential role during postembryonic neurogenesis in regulating the numbers of distinct types of neuronal cells (9 ). Mutant mnb flies are characterised by a marked reduction in size of the adult optic lobes and the central brain hemispheres. This is caused by the abnormal spacing of neuroblasts and hence a reduction in the production of neuronal progeny (10 ). We have isolated a human homologue of mnb from the DSCR and we show, by in situ RNA hybridisation studies in mouse brains, that Mnb is normally expressed in regions of the brain which are abnormal in individuals with DS.
In preparation for isolating human chromosome 21 expressed sequences we constructed contiguous cosmid sub-librariesfrom YACs from various regions of chromosome 21, including YACs from the DSCR. Pools of these cosmids were used for the isolation of partial cDNAs by direct selection (11 ,12 ) and for exon trapping experiments (13 ,14 ). A total of 576 clones were isolated and arrayed. Of these 576 selected clones, 107 mapped back to human chromosome 21. In total we have 41 non-redundant putative cDNAs, of which 24 are novel, i.e. do not match known genes or ESTs in the databases (manuscript in preparation). We have isolated two known chromosome 21 genes, DSC1 and GIRK2 and another 13 partial cDNAs have significant matches with entries in the databases. Using the blastx program, two non-overlapping partial cDNAs (D7-X4 and D1-34) showed significant similarity to a Drosophila melanogaster serine/threonine kinase (accession no. X70794) [D7-X4 P(N) = 3.0e-18; D1-34 P(N) = 2.5e-36]. This serine/threonine kinase is the product of the Drosophila minibrain(mnb) gene. Using the blastn program, D1-34, D7-X4 and a third partial cDNA (D2-34) had almost complete sequence identity [P(N) = 5.3e-60 for D1-34] with the Dyrk (Dual specificity Yak1-related kinase) mRNA from Rattus norvegicus (accession no. X79769). In addition, D2-34 also matched a previously mapped chromosome 21 EST (L25452) (4 ) and a mouse EST (Z31282). Using the partial cDNAs as probes, we subsequently isolated five overlapping clones from a human foetal brain library (Clontech) The combined sequence of these cDNAs spanned 2568 bp and included a complete coding region of 763 amino acids. We have designated the new gene MNB, a human homologue of mnb. Figure 1 shows an alignment of the amino acid sequences of MNB, mnb and Dyrk.
Southern blot analysis of total human genomic DNA digested with EcoRI and TaqIshowed a single band when hybridised with probe D2-34 (data not shown). Therefore, MNB is a single copy gene. Hybrid cell line DNA, containing human chromosome 21 as its only human component, showed the same bands when probed with D2-34 but with an additional mouse band. The partial cDNAs (D7-X4 from exon trapping, D1-34 and D2-34from cDNA selection) were mapped by hybridisation to the chromosome 21 YAC 336G11 and to cosmids shown in Figure 2 . YAC 336G11 was shown by FISH to be non chimaeric. This places the MNB gene within the proximal half of the DSCR (21q22.2), between D21S17 and D21S55. MNB spans the cosmid containing marker D21S270, is approximately 200 kb from marker D21S55 and is proximal to marker D21S337.
Northern blot analysis using D1-34 as a probe identified one transcript of 6.1 kb in human foetal brain, liver, lung and kidney (Fig. 3 a). There was no evidence of smaller transcripts in the human tissues with a prolonged exposure of the autoradiograph. In mouse there were two major transcripts of 6.1 kb and 3.1 kb (Fig. 3 b), similar to the results of Kentrup et al. in rat (15 ). RNA in situ hybridisation studies of mouse brains were carried out using a 40-mer antisense oligonucleotide derived from cDNA D1-34. Expression of Mnb was evident in the olfactory bulb, the cerebellum, the cerebral cortex, the pyramidal cell layer of the hippocampus and several hypothalamic nuclei (Fig. 4 ).
During the course of this work Kentrup and co-workers published the identification and functional studies of Dyrk. They speculated that Dyrk is involved in cell cycle control and is the rat homologue of mnb (15 ). Futhermore, based on the high similarity of Dyrk with the human EST (L25452), Kentrup et al suggested that the human homologue of Dyrk maps to 21q22.2. This human homologue of Dyrk is MNB. Human MNB is expressed as a 6.1 kb transcript and there is no evidence of a smaller transcript analogous to the 3.1 kb transcript found in rat and mouse. The Drosophilamnb gene encodes three alternatively spliced transcripts (5.5, 4.4 and 4.2 kb) (9 ). It is unknown whether the two transcripts observed in rodents are alternative splicing products. The deduced amino acid sequence of our partial MNB cDNA exhibits structural features which are shared with Dyrk. The core domains which contain amino acids found in the catalytic sites of protein kinases (16 ) are identical with the exception of two residues and both proteins have a potential nuclear translocation signal (Fig. 1 ).
Genes which show temporal or high levels of expression during the development of the central nervous system may be of special importance in DS, especially in the pathogenesis of mental retardation. In Drosophila, the mnb gene appears to play an essential role during postembryonic neurogenesis in regulating the numbers of distinct types of neuronal cells (9 ). Mutant mnb flies are characterized by a marked size reduction of the adult optic lobes and the central brain hemispheres. This is caused by the abnormal spacing of neuroblasts and hence a reduction in the production of neuronal progeny (10 ). The mnb gene encodes a serine-threonine protein kinase which is expressed in distinct neuroblast proliferation centres during Drosophila postembryonic neurogenesis. The minibrain kinases (mnb, MNB, and Dyrk) share sequence similarity with the cyclin-dependent kinases, which are known to regulate cellular proliferation, suggesting a role for mnb in the correct mitosis of neuroblast progeny (9 ). Although the overall scheme of neuronal development is quite different in invertebrates and vertebrates, molecular studies on vertebrate neurogenesis have revealed a remarkable evolutionary conservation of the cellular mechanisms of neuronal development (17 ). Moreover, cyclin-dependent kinases are known to regulate cellular proliferation in various species, suggesting a more universal regulatory mechanism (18 ). It is conceivable that MNB has a role in the processes which generate neuronal cells in the brain during postembryonic development.
The detection of MNB in the DSCR on chromosome 21 suggests that it may be involved in the altered neuronal development observed in DS. Although in Drosophila the mnb phenotype was due to a reduction in the level of expression of the mnb gene, we expect that in DS, MNB is overexpressed. At a gross morphological level, DS brains are smaller than normal (19 ) and there is a decrease in the number of neurons. Neuronal number is reduced in distinct regions, including the cochlear nuclei, cerebellum, hippocampus, the cholinergic neurons of the basal forebrain, the granular layers of the cerebral cortex, and in areas of the brain stem (20 ). These abnormalities occur in regions where the Mnb gene is normally expressed and are consistent with the view that altered expression is in some way detrimental. During the search for the gene which is responsible for the weaver phenotype in mice (Girk2), Patil et al. assessed a partial sequence, which they called Mmb (mouse minibrain), as a possible candidate (21 ). Mnb and Mmb are probably the same gene.
Although a critical region on chromosome 21 has been defined which, when present in three copies, is responsible for the main features of DS, including mental retardation (3 ), others have challenged this concept (22 ). In spite of this controversy, the major efforts to identify genes are being centred on this region (5 ,6 ). Several new genes with potential relevance in brain development and/or function have been isolated from the DSCR (GIRK2, SIM2 and MNB) and from the region just proximal (DSC1) (21 ,23 -25 ). The generation of transgenic mice that overexpress each of these genes, either singly or in combination, should permit an evaluation of their involvement in the pathophysiology of DS. In a recent report, transgenic mice were generated that overexpress Ets2, a transcription factor and proto-oncogene which is located on chromosome 21 within the distal boundary of the DSCR. These mice develop skeletal anomalies reminiscent of those observed in DS (26 ).
It is presumed that the structural alterations observed in the brain, together with the accompanying functional changes may account for the subsequent physiological and cognitive abnormalities associated with DS. Therefore, it is likely that genes involved in neurogenesis and which have altered expression in DS might account, at least partly, for the alterations that lead to mental retardation. The location of MNB in the DSCR together with its probable function in neurogenesis, supports MNB as a strong candidate gene to produce some of the neurological abnormalities present in DS patients. With the help of neuro- pathological, neurochemical and behavioural studies in transgenic animals, we may be able to dissect the components contributing to the mental retardation and to the complexity of the DS phenotype.
Selected human chromosome 21 YACs were tested for chimaerism by FISH. YACs were grown on AHC selective medium and were encapsulated in agarose beads using a modification of the method described in ref. 27 .Total yeast DNA containing YACs was partially digested with MboI and ligated into the BamHI site of SuperCos (Stratagene). Cosmids were packaged using Stratagene Gigapack II Plus packaging extracts. Clones containing human inserts were identified by screening with a radiolabelled total human DNA probe. Cosmid contigs were generated using riboprobe and linear PCR strategies.
Probes were labelled with [[alpha]-32P]dCTP by random priming. Colony and plaque hybridizations were in 7% SDS/0.5 M sodium phosphatebuffer (pH 7.0). cDNA probes were hybridized to filters containing the hybrid cell line WAV17, containing human chromosome 21 as its only human component (28 ).
A pool of 502 cosmid DNAs from the libraries made from seven human chromosome 21 YACs (manuscript in preparation) was used for cDNA selection, essentially as described (11 ,12 ). The cDNA source was human foetal brain mRNA which had been reverse transcribed using random hexamers and oligo dT. Linkers were ligated and these cDNAs were then hybridized with the pool of biotinylated cosmids. The selected cDNAs were amplified using the linker primer (5'-CTCGAGAATTCTGGATCCTC-3') with a (CUA)4 tail and subcloned in pAMP10 (GIBCO-BRL). Exon trapping was as described (13 ) using a pool of 12 overlapping, non-redundant cosmids from YAC 336G11 subcloned in pSPL3 (14 ). Amplified exons were directionally subcloned in pAMP1 (GIBCO-BRL). Using a Biomek 1000 station (Beckman), colonies were gridded at a high density onto nylon filters. Clones were sequenced with the M13R and M13D primers using fluorescent DyeDeoxy Terminators on an ABI373A automatic DNA sequencer (Applied Biosystems).
Northern blots (Clontech) containing poly(A+) mRNA from human foetal tissues or adult mouse tissues were hybridized with D1-34 according to the manufacturer's protocol. RNA in situ hybridization studies on sections of mouse brain were carried out with a 40-mer antisense oligonucleotide (5'-GGAATACCCAGAACTTCCACTATTTTATTCATCTGATCTA-3'), essentially as described previously (25 ), except that following hybridization, sections were washed twice in 1 * SSC (150 mM sodium chloride and 15 mM sodium citrate, pH 7.0) at 52oC for 1 h each. A 40-mer sense oligonucleotide was used as a control under the same conditions, and gave no signals in the hybridization experiments performed.
We thank M. Dierssen for useful contributions to the manuscript, M. Lynch for useful comments, M. Nadal for FISH and M. L. Yaspo for advice on the application of exon trapping. This work was supported by the European Union (Grants CEC/BIOMED GENE-CT93-0015, GENE-CT93-0037 and GENE-PL95-0554); Fundació Catalana Síndrome de Down / Marató de TV3 - 1993; the Fondo de Investigaciones Sanitarias de la Seguridad Social (94/1905E) (Spanish Government); National Center for Human Genome Research HG00368 to ML. JG was granted a HUGO Travel Award.
1 Hassold, T. and Jacobs, P. Trisomy in man. (1984) Annu. Rev. Genet., 18, 69-97.MEDLINE Abstract
2 Epstein, C.J.(1986) The consequences of chromosome imbalance: principles, mechanisms and models. Cambridge University Press, New York.
3 Delabar, J.M., Théophile, D., Rahmani, Z., Chettouh, Z., Blouin, J.L., Prieur, M., Nöel, B. and Sinet, P.M. (1993) Molecular mapping of twenty-four features of Down syndrome on chromosome 21. Eur. J. Hum. Genet., 1, 114-124.MEDLINE Abstract
4 Cheng, J-F., Boyartchuk, V. and Zhu, Y. (1994) Isolation and mapping of human chromosome 21 cDNA: progress in constructing a chromosome 21 expression map. Genomics, 23, 75-84.MEDLINE Abstract
5 Peterson, A., Patil, N., Robbins, C., Wang, L., Cox, D.R. and Myers, R.M. (1994) A transcript map of the Down syndrome critical region on chromosome 21. Hum. Mol. Genet.,10, 1735-1742.
6 Lucente, D., Chen, HM., Shea, D., Samec, SN., Rutter, M., Chrast, R., Rossier, C., Buckler, A., Antonarakis, SE. and McCormick, MK. Localization of 102 exons to a 2.5 Mb region involved in Down syndrome. (1995) Hum. Mol. Genet., 4, 1305-1311.MEDLINE Abstract
7 Yaspo, M-L., Gellen, L., Mott, R., Korn, B., Nitzetic, D., Poutska, A. and Lehrach, H. (1995) Model for a transcript map of human chromosome 21: isolation of new coding sequences from exon and enriched cDNA libraries. Hum. Mol. Genet., 4, 1291-1304.MEDLINE Abstract
8 Kao, F.T., Yu, J., Tong, S., Qi, J., Patanjali, S.R., Weissman, S.M. and Patterson, D. (1994) Isolation and refined regional mapping of expressed sequences from human chromosome 21. Genomics, 23, 700-703.MEDLINE Abstract
9 Tejedor, F., Zhu, X.R., Kaltenbach, E., Ackermann, A., Baumann, A., Canal, I., Heisenberg, M., Fischbach, K.F. and Pongs, O. (1995) Minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila. Neuron, 14,287-301.MEDLINE Abstract
10 Fischbach, K.F. and Heisenberg, M. (1984) Neurogenetics and behaviour in insects. J. Exp. Biol., 112, 65-93.
11 Lovett, M., Kere, J. and Hinton, L.M. (1991) Direct selection: A method for the selection of cDNAs encoded by large genomic regions. Proc. Natl Acad. Sci. USA, 88, 9628-9632.MEDLINE Abstract
12 Morgan, J.G., Dolganov, G.M., Robbins, S.E., Hinton, L.M. and Lovett, M. (1992) The selective isolation of novel cDNAs encoded by the regions surrounding the human interleukin 4 and 5 genes. Nucleic Acids Res.,20, 5173-5179.MEDLINE Abstract
13 Buckler, A.J., Chang, D.D., Graw, S.L., Brook, J.D., Haber, D.A., Sharp, P.A. and Housman D.E. (1991) Exon amplification: A strategy to isolate mammalian genes based on RNA splicing. Proc. Natl Acad. Sci. USA, 88, 4005-4009.MEDLINE Abstract
14 Church, D.M., Stotler, C.J., Rutter, J.L., Murrell, J.R., Trofatter, J.A. and Buckler, A.J. (1994) Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nature Genet., 6, 98-105.MEDLINE Abstract
15 Kentrup, H., Becker, W., Heukelbach, J., Wilmes, A., Schuermann, A., Huppertz, C., Kainulainen, H. and Joost, H.G. (1996) Dyrk: A dual-specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII. J. Biol. Chem., 271, 3488-3495.MEDLINE Abstract
16 Hanks, S.K., Quinn, A.M. and Hutter, T. (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science, 241, 42-52.MEDLINE Abstract
17 Purves, D. and Lichtman, J.W. (1992) Early events in neural development. In Principles of neuronal development. (ed Sinauer) pp. 3-72.
18 Nigg, E.A. (1995) Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays,17, 471-480.MEDLINE Abstract
19 Kemper, T.L. Neuropathology of Down Syndrome. (1989) In The Psychobiology of Down Syndrome (ed Nadel, L.) The MIT Press pp.269-289.
20 Becker, L., Mito, T., Takashima, S. and Onodera, K. (1991) Growth and development of the brain in Down Syndrome. In The Morphogenesis of Down Syndrome The Wiley-Liss NY. Prog. Clin. Biol. Res., 373, 133-153.MEDLINE Abstract
21 Patil, N., Cox, D.R., Bhat, D., Faham, M., Myers, R.M. and Peterson, A.S. (1995) A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. Nature Genet.,11, 126-129.MEDLINE Abstract
22 Korenberg, J.R., Chen, X.N., Schipper, R., Sun, Z., Gonsky, R., Gerwehr, S., Carpenter, N., Daumer, C., Dignan, P., Disteche, C., Graham, J.M., Hugdins, L., McGillivray, B., Miyazaki, D., Ogasawara, N., Park, J.P., Pagon, R., Pueschel, S., Sack, G., Say, B., Suchuffenhauer, S., Soukup, S. and Ymanaka, T. (1994) Down syndrome phenotypes: The consequences of chromosomal inbalance. Proc. Natl Acad. Sci. USA, 91, 4997-5001.MEDLINE Abstract
23 Chen, H., Chrast, R., Rossier, C., Gos, A., Antonarakis, S.E., Kudoh, J., Yamaki, A., Shindoh, N., Maeda, H., Minoshima, S. and Shimizu, N. (1995) Single-minded and Down syndrome? Nature Genet., 10, 9-10.MEDLINE Abstract
24 Dahmane, N., Charron, G., Lopes, C., Yaspo, M-L., Maunoury, C., Decorte, L., Sinet, P.M., Bloch, B. and Delabar, J-M. (1995) Down syndrome-critical region contains a gene homologous to Drosophila sim expressed during rat and human central nervous system development. Proc. Natl Acad. Sci. USA, 92, 9191-9195.MEDLINE Abstract
25 Fuentes J-J., Pritchard M.A., Planas A.M., Bosch A., Ferrer I. and Estivil, X. (1995) A new human gene from the Down syndrome critical region encodes a proline-rich protein highly expressed in fetal brain and heart. Hum. Mol. Genet., 4, 1935-1944.MEDLINE Abstract
26 Sumarsono, S.H., Wilson, T.J., Tymms, M.J., Venter, D.J., Corrick, C.M., Kola, R., Lahoud, M.H., Papas, T.S., Seth, A. and Kola, I. (1996) Down's syndrome-like skeletal abnormalities in Ets2 transgenic mice. Nature, 379, 534-537.MEDLINE Abstract
27 Overhauser, J. and Radic, M.Z. (1987) Encapsulation of cells in agarose beads for use with pulsed-field gel electrophoresis. Focus,9, 8-9.
28 Raziuddin, A., Sarkar, F.H., Dutkowski, R., Shulman, L., Ruddle, F.H. and Gupta, S.L. (1984) Receptors for human alpha and beta interferon but not gamma interferon are specified on human chromosome 21. Proc. Natl Acad. Sci. USA, 81, 5504-5508.MEDLINE Abstract
*To whom correspondence should be addressed
This page is maintained by OUP admin. Last updated Thu Oct 31 15:27:05 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
