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Genomic Sequences of Aldolase C (Zebrin II) Direct Lacz Expression Exclusively in Non-Neuronal Cells of Transgenic Mice

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Genomic Sequences of Aldolase C (Zebrin II) Direct Lacz Expression Exclusively in Non-Neuronal Cells of Transgenic Mice Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2615–2620, March 1998 Neurobiology Genomic sequences of aldolase C (Zebrin II) direct lacZ expression exclusively in non-neuronal cells of transgenic mice ERNST U. WALTHER*†,MARTIN DICHGANS*†,STEPHEN M. MARICICH†‡,RITA R. ROMITO‡,FEI YANG‡, i SUZAN DZIENNIS‡§,SAUL ZACKSON¶,RICHARD HAWKES¶, AND KARL HERRUP‡ *Neurologische Klinik, Marchioninstrasse 15, 81377 Munich, Germany; ‡Alzheimer Research Laboratory, Case Western Reserve Medical School, 10900 Euclid Avenue, Cleveland, OH 44106; and ¶Department of Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada Communicated by Richard L. Sidman, Harvard Medical School, Southborough, MA, December 23, 1997 (received for review May 9, 1997) ABSTRACT Aldolase C is regarded as the brain-specific Aldolase B, found on human chromosome 9 (9), generally is form of fructose-1,6-bisphosphate aldolase whereas aldolase considered to be a liver enzyme, although it also is found in A is regarded as muscle-specific. In situ hybridization of mouse kidney proximal tubule cells and enterocytes. This form of central nervous system using isozyme-specific probes revealed aldolase favors gluconeogenesis, befitting its higher concen- that aldolase A and C are expressed in complementary cell tration in the liver. During hepatic development, aldolase A types. With the exception of cerebellar Purkinje cells, aldolase and C are expressed earlier, but aldolase B soon takes over. A mRNA is found in neurons; aldolase C message is detected Unlike the other isozymes, aldolase B is transcriptionally in astrocytes, some cells of the pia mater, and Purkinje cells. activated by signals from hormones and dietary factors (e.g., We isolated aldolase C genomic clones that span the entire ref. 10). A proximal promoter region, approximately 200 bp in protein coding region from 1.5 kb 5* to the transcription start length, has been identified that contains many of the elements site to 0.5 kb 3* to the end of the last exon. The bacterial gene, (including overlapping hepatic nuclear factor binding sites) lacZ, was inserted in two different locations and the constructs necessary for properly regulated expression if it is paired with tested in transgenic mice. When the protein coding sequences sequences in the first intron (11–13). were replaced with lacZ, three of five transgenic lines ex- Aldolase C, found on human chromosome 17 (1), has kinetic pressed b-galactosidase only in cells of the pia mater; one line properties that are intermediate between those of the A and also expressed in astrocyte-like cells. When lacZ was inserted B isoforms. In the adult, aldolase C is considered to be the into the final exon (and all structural gene sequences were brain-specific isozyme, with low but detectable activity in fetal retained) transgene expression was observed in astrocytes in tissues and hepatocarcinomas (8, 14–17). The exact cellular all regions of the central nervous system as well as in pial cells. location of aldolase C is controversial. Studies of mRNA by Thus, with the exception of Purkinje cell expression, the Northern blot analysis, in situ hybridization, or transgene behavior of the full-length transgene mimics the endogenous expression studies indicate that the gene is transcribed mostly aldolase C gene. The results with the shorter transgene in neurons (8, 14, 18). On the other hand, earlier studies using suggest that additional enhancer elements exist within the isozyme-specific antibodies report its location in gray matter intragenic sequences. The absence of Purkinje cell staining astrocytes and cells of the pia mater (16, 19). All reports agree suggests that the cis elements required for this expression that Purkinje cells of the cerebellum contain by far the highest must be located outside of the sequences used in this study. levels of the enzyme. By sequence analysis, the aldolase C promoter region is typical of a ‘‘housekeeping’’ type of pro- The temporal and spatial regulation of the expression of moter. A normal TATA box is missing; instead a GC-rich aldolase (fructose-1,6-bisphosphate aldolase) represents a region can be found just upstream of the transcription start site classic problem in developmental genetics. Three isozymes of (20). A CAAT box is present, but it is located far upstream this phylogenetically ancient enzyme exist: aldolases A, B, and from its expected position (21). Northern analysis using a C, each of which is encoded by a separate gene. The kinetics probe specific for aldolase C mRNA demonstrates a 1.6-kb of aldolase A favor the cleavage of fructose-1,6-bisphosphate band; there is no evidence of alternative splicing. Two DNase to glyceraldehyde-3-phosphate and dihydroxyacetone phos- I-sensitive sites flank the transcription unit: one immediately phate, and its gene is found on human chromosome 16 (1). 59 and the other about 1.5 kb 39 to the nine exons of the gene. Generally considered to be a muscle enzyme, the transcrip- Several tissue-specific methylation sites have been identified in tional regulation of this gene is unusually complex with three the immediate vicinity of the transcription unit (22). In alternative promoters (2–5) that result in three different transgenic mice (18, 22) large rat genomic fragments (or mRNA species. Curiously, the three transcripts differ only in fragments with short marker tags inserted) have been shown their 59-untranslated regions; each mRNA encodes the same to direct central nervous system (CNS)-specific expression. structural enzyme. Of the three promoters, the 59-most and Expression outside of the CNS is suppressed. Even a short, 39-most are expressed broadly in fetal tissues as well as in adult 115-bp promoter element contains sufficient cis-active se- muscle and red blood cells. The central promoter (M-type or quences to drive marker gene expression solely in the CNS H-type) is highly active in skeletal muscle, particularly in (23). fast-twitch fibers (6, 7). The 59-most promoter is active, albeit The interest of our laboratories in the aldolase isozymes at lower levels, in a range of other tissues, including brain (4). grew out of the discovery that aldolase C is the antigen In the nervous system, the exact cellular location of the identified by the Zebrin II mAb (24). Zebrin II has long been aldolase A isozyme is uncertain; Northern analysis of cultured cells suggests that it is present in both neurons and glia (8). Abbreviations: CNS, central nervous system; GFAP, glial fibrillary acidic protein; X-Gal, 5-bromo-4-chloro-3-indolyl-b-D-galactoside. † The publication costs of this article were defrayed in part by page charge E.U.W., M.D., and S.M.M. contributed equally to the work. Their names are listed in the order in which they began work on the project. payment. This article must therefore be hereby marked ‘‘advertisement’’ in §Present address: Beth Israel Hospital, 99 Brookline Avenue, Boston, accordance with 18 U.S.C. §1734 solely to indicate this fact. MA 02115. i © 1998 by The National Academy of Sciences 0027-8424y98y952615-6$2.00y0 To whom reprint requests should be addressed. e-mail: kxh26@po. PNAS is available online at http:yywww.pnas.org. cwru.edu. 2615 Downloaded by guest on September 25, 2021 2616 Neurobiology: Walther et al. Proc. Natl. Acad. Sci. USA 95 (1998) recognized as a Purkinje cell biochemical marker of the AgTTCT-39; and down, 59-CACTACgCgTACTgTgAgCCA- sagittal compartments of the adult cerebellar cortex (see for gAg-39. review refs. 25–29). As part of our effort to define the cis-acting Lines were established from DNA-positive founders and elements that regulate compartment-limited cerebellar gene maintained by continued backcrossing to the C57BLy6J strain. expression, we wished to learn more about the aldolase C The copy number of the transgene was determined by South- promoter and its cell type-specific features. We report here a ern blot using a radiolabeled probe against lacZ. Each line detailed in situ hybridization study of the endogenous pattern harbors 10 or fewer copies of the transgene. of aldolase A and C expression, and the behavior of two Reverse Transcription–PCR. To analyze transgene expres- transgene constructs in which sequences from mouse aldolase sion, RNA was isolated from adult brain, liver, or muscle using C are joined with the histochemical marker gene, b-galacto- the guanidinium thiocyanate method. After DNase I digestion sidase, in various configurations. The expression pattern of to remove contaminating genomic DNA, reverse transcription these constructs in multiple independent transgenic lines was performed under standard conditions, and PCR was done reveals significant information on the spatiotemporal regula- by using the specific lacZ primers listed previously. tion of this intriguing gene. Histology. Postnatal transgenic mice were anesthetized and perfused transcardially with ice-cold 3% paraformaldehyde in MATERIALS AND METHODS 0.1 M Pipes (pH 6.9). Embryonic mice were decapitated and immersion-fixed in the same solution. Tissues were cryo- In Situ Hybridization. Animals were perfused with 4% sectioned at 10 mm and stained at 37°C overnight for lacZ paraformaldehyde, and their brains were postfixed, equili- activity by using 1 mgyml X-Gal substrate (5-bromo-4-chloro- b brated with 18% sucrose in PBS, embedded in OCT com- 3-indolyl- -D-galactoside) in 35 mM K3Fe(CN)6,35mM m z pound, and cryo-sectioned at 10 m. Cerebellar RNA was K4Fe(CN)6 3H2O, 1.0 ml 1 M MgCl2, 0.01% desoxycholic acid, reverse-transcribed, then amplified by 35 cycles of PCR using 0.02% Nonidet P-40. For immunocytochemistry, mice were these primers containing T3 or T7 polymerase sites (under- perfused and brains postfixed in 4% paraformaldehydey0.125 lined) at their 59 ends: M phosphate buffer. Polyclonal rabbit anti-b-galactosidase Aldolase C (258 bp)—(T3): 59-AATTAACCCTCACTAA- (59339, 1:400 dilution) and monoclonal mouse anti-glial fibril- AgggggCAgAgATgAACgggCT-39; and (T7): 59-TgTAATA- lary acidic protein (GFAP; Chemicon, 1:400 dilution) were CgACTCACTATAgggCgAATCCTgTggTCTCgggTA-39.
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