aldolase c/zebrin ii and the regionalization of the cerebellum

Journal of Molecular Neuroscience Copyright © 1996 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/95 / 6:147-158/$8.00

MINIREVIEW

Aldolase C/Zebrin I! and the Regionalization of the Cerebellum

Richard Hawkes *,1 and Karl Herrup 2

1 Department of Anatomy and Neuroscience Research Group, Faculty of Medicine, The University of Calgary, Calgary, Alberta, Canada; 2AIzheimer Research Laboratory,

Case Western Reserve Medical School, Cleveland, OH

Received December 1, 1995; Accepted December 5, 1995

Abstract

The cerebellum is comprised of multiple bands ~ of cells, each with characteristic afferent and efferent projections, and patterns of gene expression. The most studied example of a striped pattern of expression is the antigen recognized by monoclonal antibody antizebrin II. Zebrin II is expressed by subsets of Purkinje cells that form an array of parasagittal bands that extend rostrocaudally throughout the cerebellar cortex, separated by similar bands of Purkinje cells that do not express zebrin II. Recent cloning studies have revealed that the zebrin II antigen is the respiratory isoenzyme aldolase C. This article reviews the cellular and molecular compartmentation of the cerebellum together with the molecular biology of the aldolase C gene, and speculates on possible reasons for a striped pattern of expression.

Index Entries: Purkinje cell; pattern formation.

Introduction

For over a century, anatomists and patholo- gists have developed a picture of the regionalization of the adult central nervous system (CNS). Relying on the appearance of structural features, visible after s taining wi th basophil ic dyes, early workers proposed the existence of

a var ie ty of subdivis ions in d i f fe ren t bra in regions. An example of this type of t axonomy is the regional map of h u m a n cerebral cortex proposed by Brodman (1909), who used differences in cell density a n d / o r thickness of the six cortical layers to propose near ly four dozen different cytoarchitectonic areas. Dur ing the last two decades, increasing use of biochemi-

*Author to whom all correspondence and reprint requests should be addressed.

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148 Hawkes and Herrup

cal and molecular approaches has added con- siderable depth to this picture of regionalization. Antibodies to known or unknown molecules or in situ hybridizat ion probes for mRNAs of known or unknown function have provided a new lens through which we now view the structure of the nervous system. Yet although the newer approaches occasionally reveal group- ings that were missed by previous morphological observation, more often than not the molecularly defined "borders" simply validate regions defined previously based on the morphological picture alone. One area that serves as an exception to this congruence of structure and biochemistry, however, is the cortex of the cerebellum.

The microarchitecture of the cerebellum is very uniform. In par t icular , there are no apparent cytoarchitectonic boundaries compa- rable to those found in the neocortex. Although early tract tracing studies (e.g., Jansen and Brodal, 1940) hinted at a functional pattern of sagittal modules that underlie the seem- ingly monotonous cellular structure of the cortex, it was not until the late 1960s that the first formal proposal of multiple parasagit- tally stacked compartments in the cerebellum was made (Voogd, 1967, 1969). Voogd described alternating bundles of large and small diameter axons in the cerebellar white matter of numerous species, indicative of a longitudinal parcellation of cerebellar afferents. On the basis of the myeloarchitecture, a reproducible family of parasagittal compartments was proposed. Since then, it has become well established that both the climbing fiber projection and several mossy fiber projections terminate in the cerebellum in the form of parasagittal bands (reviewed in Ito, 1984), yet it could be argued that the sagittal orga- nizations of the fiber tracts are not properties directly attributable to the cells of cerebellar cortex themselves. The organization of these inputs and outputs could well reflect patterns that are resident in the afferent populations; the cerebellar cortex might be merely a pas-

sive recipient of these ordered projections. It is here that the advent of molecular markers has proven invaluable in our unders tanding of the regionalization of cerebellar cortex. Through these markers we now know that the cells of the cortex themselves participate in a pattern of sagittally stacked regions, and, by virtue of the early time at which these regional variations can be detected, it seems likely that they are achieved independent ly of the organization of the afferents.

The Discovery of the Biochemical Markers

The first molecular marker of compartmentation was the glial ectoenzyme 5'-nucle- otidase (5'N: Scott, 1963, 1964). When sections of mouse cerebe l lum are h i s tochemica l ly stained for 5'N, an array of parasagittal bands of high and low enzyme activity is revealed (Marani, 1982). Following this initial discovery, however, it was nearly two decades before addi t ional molecules were ident i f ied that shared the property of a parasagittal distri- but ion ( reviewed in Hawkes et al., 1992). Some of these display a pattern similar to 5'N (e.g., zebrin I, Hawkes et al., 1985; Hawkes and Leclerc, 1987; Eisenman and Hawkes , 1990; zebrin II, Brochu et al., 1990; Eisenman and Hawkes, 1993), some display the inverse pattern (i.e., high expression where 5'N is low, e.g., P-path, Leclerc et al., 1992; Map-la, Touri et al., 1995), and some reveal a different, more subtle cerebellar parcellation (e.g., HNK-1, Eisenman and Hawkes, 1993; nicoti- namide adenosine dinucleotide diaphorase: Yan et al., 1993; Hawkes and Turner, 1994). In many cases a topographical relationship has been identif ied be tween afferent terminal fields and Purkinje cell bands where in the fiber projections tend to respect the boundaries defined by the molecular markers (Gravel et al., 1987; Gravel and Hawkes, 1990; Wassef et al., 1992; Ji and Hawkes, 1994).

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Aldolase and Cerebellar Compartments 149

Development of Cerebellar Regionalization

The distinctions among cerebellar regions appears very early in cerebellar embryogenesis (reviewed in Wassef et al., 1993; Hawkes and Mascher, 1994). Purkinje cell progenitors arise from both mesencephalon and metencephalon (Martinez and Alvarado-Mallart, 1989; Hal- lonet et al., 1990), but whether this distinction has an impact on adult Purkinje cell phenotype (e.g., zebrin- cells from the mesencephalon, zebrin ÷ from the metencephalon) is unknown. Purkinje cells in rats are born between embryo age (E)13-E16 with a peak of production at E15 (Altman and Bayer, 1978). Almost from the first, clusters of Purkinje cells can be distin- guished by their patterns of gene expression (Wassef and Sotelo, 1984; Wassef et aL, 1985, 1992a). During the prenatal period the Purkinje cell clusters seem to organize both the climbing fiber (Sotelo et al., 1984; Sotelo, 1987; Wassef et al., 1992) and the mossy fiber (Arsenio- Nunes et al., 1985; Ji and Hawkes, 1995) afferents. At birth, the embryonic Purkinje cell clusters can still be identified, although the dif- ferential gene expression is all but gone, and the afferent topography resembles that of the adult.

At this stage, another set of compartmentation markers is expressed, including rhombotin (Greenberg et al., 1990) and L7 (Oberdick et al., 1990, 1993; Smeyne et al., 1991; Varidoele et al., 1991). L7 is a gene isolated from a cDNA library constructed from P13 mouse cerebellum. The L7 gene is expressed in the Purkinje cells, with mRNA first appearing between P4 and P8 and continuing into adulthood (Oberdick et al., 1988). Three transgenic lines carrying an L7-~-galactosidase fusion gene exhibit ~-galactosidase expression in all adult Purkinje cells (Oberdick et al., 1990). However, the development of the L7 transgene reveals a novel spatiotemporal pattern. At E17, the earliest time at which the transgene can be detected, four parasagittal bands of Purkinje cells are identified, two on each side of the midline. The number of Purkinje

cells expressing the gene increases in a nonuni- form manner until by P9 all the Purkinje cells express both L7 and the transgene (Smeyne et al., 1991). Promoter mutation experiments have identified a complex combination of positive and negative control elements that contribute to the pattern of L7 expression (Oberdick et al., 1993).

As the cerebellum expands the Purkinje cell clusters undergo an elaborate morphological transformation, spreading to form a monolayer of long parasagittal bands. During this period novel compartmentat ion markers are first expressed that are retained throughout life, including zebrin I and zebrin II (reviewed in Hawkes and Gravel, 1991; Hawkes et al., 1992).

Zebrin Development

Zebrin I and II are first expressed during the perinatal stage, and follow an identical developmental timetable (Leclerc et al., 1988; Dor6 et al., 1990; Lannoo et al., 1991a). Four stages in the expression of the zebrins have been identified in rodents:

1. An initial phase in which no Purkinje cells express detectable levels of zebrin;

2. First expression of zebrin by patches of Purkinje cells in the posterior lobe of the vermis;

3. An intermediate stage in which all Purkinje cells express zebrin; and

4. The final, adult stage of selective expression in parasagittal bands.

Zebrin induction begins in clusters of immunoreactive Purkinje cells in the median posterior lobe of the vermis at postnatal day (P)5. Expression next extends both anteriorly and laterally i n t w o stages, first to the rest of the vermis, then throughout the hemispheres, so that by P12 all Purkinje cells are zebrin +. The spread of zebrin expression is not smooth, but rather involves several clearly defined stages, with sharply defined mediolateral and rostro- caudal boundaries: For example, the first signs of expression during development, in the most



caudal lobules of the vermis, stop abrupt ly m i d w a y along lobule VIII (consistent with the arrest of d e v e l o p m e n t in the Lc/+ mutant : Tano et al., 1992). Between P6 and P12, a transient pat tern of zebrin compartmentat ion can be seen (Leclerc et al., 1988) but this has disap- peared by around P12. At this stage all Purkinje cells are zebrin ÷. The adult array of zebrin ÷/- compartments appears from about P15 onward through the suppression of zebrin expression in those Purkinje cells dest ined to be zebrin- in the adult. The pattern of alternating bands is mature by around P24. Therefore, zebrin is first expressed in the cerebellum too late for the protein itself to be responsible for regionalization.

Adult Zebrin Distribution

Of the parasagi t ta l markers ident i f ied to date, the best s tudied is zebrin II, recognized by a monoclonal ant ibody raised against the cerebellum of the weakly electric fish Apteronotus (Brochu et al., 1990). Both zebrin II and the earlier zebrin I share a common adult distribution (Brochu et al., 1990). Immunocy tochemis t ry reveals the presence of significant levels of zebrin antigens exclusively in Purkinje cells. No other neurons are immunoreactive, although wi th zebrin II there is a variable glial background staining that may either be weak expression or nonspecific background. In the rest of the brain there is only weak, diffuse deposition of reaction product . Within the Purkinje cell the antigen is found throughout the cytoplasm, including the dendrites, somata, and axons.

The immunoreact ive Purkinje cells are spatially organized in an elaborate array of bands (Fig. 1). In lobule VI of the vermis, all of the Purkinje cells are zebrin ÷. Both rostrally and caudal ly , parasagit tal bands of zebrin + Pur- kinje cells (PI÷-P4 +) a l ternate w i th s imilar zebrin- bands (P1--P4-). In addi t ion to this mediolateral variation, there are also anterior- posterior differences in staining. Although not as d ichotomous as the sagittal bands, there is

IV-V

IX

IV-V

Fig. 1.

nonetheless a distinct gradient from the posterior lobe, where zebrin ÷ cells predominate , to the anterior lobe, where most Purkinje cells are zebrin-. Because all Purkinje cells in lobule VI are immunoreactive, it is not clear how (or even if) the individual bands in the posterior lobe are continuous with those in the anterior lobe. In the hemispheres, alternating zebrin +/- bands are also found (P4 +/- to P7+/-). In the more complex topology of the lateral cerebellum, the clusters of zebrin ÷ and zebrin- Purkinje cells



are not strictly parasagittal. Instead, they tend to run perpendicular to the transverse fissures.

The pattern of bands is reproducible between individuals, and highly conserved across species (e.g., rat, Brochu et al., 1990; mouse, Eisen- man and Hawkes, 1993; opossum, Dor6 et al., 1990). The dis t inct ion be tween zebrin II+/- Purkinje cells is conserved in all main vertebrate taxa (fish, Lannoo et al., 1991a,b; Meek et al., 1992; reptiles, Hawkes , in preparat ion; birds, Holst and Hawkes, unpubl ished obser- vations); to date, no zebrin immunoreactivi ty has been demonstrated in amphibia, including Xenopus and Rana).

Zebrin Expression Is Cell Autonomous

Several lines of evidence point to the Pur- kinje cell zebrin phenotype being a cell-autonomous p rope r ty that is es tabl ished early in cerebellogenesis. First, lesions of cerebellar afferent pathways, either in the newborn or the adult, do not affect the pattern of zebrin expression (Leclerc et al., 1988). Second, when the cer-

Fig. 1. (previous page) Drawings of three surface views of the adult mouse cerebellum--anterior (top), dorsal (center), and posterior (bottom)--showing the locations of the zebrin + bands (P+) of Purkinje cells. The Purkinje cell bands PI+-P7 ÷ are labeled in the dorsal view (for clarity, only the numerals have been used). Note that in the vermis of the posterior lobe the immunoreactive Purkinje cells form 5-7 bands (posterior and dorsal views) whereas in Iobules VII and Vl all vermal Purkinje cells are immunoreactive (posterior and dorsal views). This pattern gradually changes in the anterior lobe to result in 3-5 very nar- row immunoreactive bands (anterior view). In the hemispheres there are three major immunoreactive bands of Purkinje cells on either side (P5b +, P6 ÷, P7 +) plus two sub-bands in the paravermal area of the paramedian and ansiform Iobules (P4b +, P5a+). Note too that the Purkinje cells are all zebrin ÷ in the nodulus (Iobule X, illustrated as indicated by arrows as reflected out from the ventral surface of the cerebellum), the paraflocculus, and the flocculus. Modi- fied from Eisenman and Hawkes (1993).

ebellar anlagen is r emoved shortly after the Purkinje cells are born, and transplanted to an ectopic location, both zebrin phenotypes still develop (Wassef et al., 1990). Third, both zebrin phenotypes are expressed in long-term explant cultures from newborn rats, and expression is apparently unaffected by the e l iminat ion of most glial cells or by the inclusion of tetrodo- toxin in the culture med ium (Seil et al., 1995). Fourth, genetic mutat ions that cause a cell- au tonomous block in Purkinje cell develop- men t (e.g., staggerer and lurcher) arrest the acquisition of the zebrin phenotype, whereas mutations that affect Purkinje cell development indirectly (e.g., reeler and weaver) allow the full development of normal zebrin levels (e.g., Sotelo and Wassef, 1991; Tano et al., 1992). Finally, pre- liminary studies of murine chimeras suggest that Purkinje cells within zerbrin compartments are lineage related (Herrup et al., 1990).

The Identification of the Zebrin II Antigen: Aldolase C

Western blots of cerebellar proteins stained by using antizebrin II show a single polypeptide antigen, apparent mol wt 36 kDa (Brochu et at., 1990; Hawkes, 1992; Ahn et al., 1994). Monoclonal antizebrin II was used to screen a mouse cerebellar cDNA express ion l ibrary (Ahn et al., 1994). Sequence analysis revealed that the inferred amino acid sequence was 98% identical to rat a ldolase C. Two addi t iona l experiments were performed to confirm that zebrin II was in fact aldolase C (aldC). First, a polyclonal anti-aldC ant ibody was shown to stain bands of Purkinje cells identical to those expressing zebrin II. Second, in situ hybridization with an antisense aldC riboprobe revealed bands of aldC mRNA similar to those seen with antizebrin II immunocytochemist ry (Ahn et al., 1994). Previous published reports with anti-aldC antibodies (Royds et al., 1987) and probes for the distribution of aldC mRNA (e.g., Popovici et al., 1990; Mukai et al., 1991) also


152 Ha wkes and Herrup

describe high levels of Purkinje cell expression, al though these earlier authors did not describe a regional variation in enzyme levels. In sum- mary, it appears that aldC is a highly conserved compartmentation marker in the cerebellum of vertebrates from fish to human.

Metabolic Function of Aldolases

Aldolase is a glycolytic enzyme that cata- lyzes the a ldol hydro lys i s of f ructose- l ,6- biphosphate into dihydroxyacetone phosphate and g lycerol -3-phosphate . There are three aldolase i soenzymes in mammals , aldolase A (aldA), found th roughout most tissues and especially rich in skeletal muscle, aldolase B (aldB), the l iver-specif ic i soform, and the brain-specific isoform aldC. Each isoform is c o d e d by a separate gene and, at least in humans , each is located on a different chro- mosome (Rocchi et al., 1989).

Aldolase exists in a soluble as well as in a s t ructure-bound form. The soluble fraction of aldolase is distributed isotropically throughout the cy toplasm, exc lud ing the nuc leus and vesicles. One striking feature of the aldC/zebrin II d is t r ibut ion in the cerebel lum is that it is expressed in those Purkinje cells that do not express a particular Map-la epitope (Touri et al., 1995). This may be a reflection of interactions of zebrin II wi th the cytoskeleton since there is evidence for aldolase b ind ing to both the microtubules (e.g., Carr and Knull, 1993) and microfilaments (e.g., O'Reilly and Clarke, 1993).

Aldolase C

The rat aldC gene was isolated from a rat genomic DNA library and completely sequenced (Paolella et al., 1986; Mukai et al., 1991). Simi- lar sequences have been reported for mouse (Ahn et al., 1994) and human (Rottman et al., 1987; Buono et al., 1988) aldC. Southern blot

ATG

V 1 2 3 4 5 6 7 8 9

5' -- _- ~ . -" 3'

0 2 3 4

Krox 24120

-200 -180 -160 -140 -120

bases

Fig. 2. The structure of the rat aldolase C gene. ( T o p ) The nine exons are represented by closed boxed (1-9) covering 3.59 kb. The ATG start site of transcription in exon 2 is indicated by an arrow. (Bot- tom) A proposed regulatory sequence immediately upstream of the ATG site, with three putative binding sites for SP1 (lightly shaded) and two for Krox 24/20 (darker shading) transcriptional complexes (the base pair count upstream from the ATG site in the second exon). Modified from Mukai et al. (1991) and Tho- mas et al. (1993).

analysis showed that a single copy of the gene occurs per haploid genome. The rat gene is comprised of nine exons and spans 3590 base pairs (Fig. 2), with the coding exons showing a 70% sequence identity to aldA but little simi- larity between their noncoding and 5' flanking sequences. Transcription is ini t iated at two different sites.

Despite its high brain specificity, the aldC promoter has many features of a typical housekeeping gene, including multiple start sites of transcription, the absence of TATA and CAAT boxes, and the presence of putative GC boxes (Thomas et al., 1993). The aldC gene is regulated at the transcriptional level, based on run- on assays and in situ hybridization (Vibert et al., 1989; Ahn et al., 1994). The promoter has been studied in transfection assays in cultured pheochromocytoma cells (Thomas et al., 1993): It was shown that a 115-bp fragment from the aldC 5' flanking sequence (between-199 and -84



with respect to the ATG initiation site for trans- lation in the second exon) is sufficient to confer neural cell specificity on a chloramphenicol acetyltransferase (CAT) reporter gene (Fig. 2). Similar studies investigated aldC cis-acting sequences of the human aldC gene by transient transfections into human neuroblastoma cells (Buono et al., 1993). It was found that 420 bp of the 5'-flanking DNA directed the transcription of the CAT reporter gene at high efficiency. A deletion between -420 and -164 bp causes a 60% decrease of CAT activity.

The putative aldC promoter has also been studied in transgenic mice. At one extreme, brain specificity was seen in several lines of transgenic mice where the 115-bp fragment was used to regulate expression of a CAT transgene. The level of expression is very low, but is greatly increased when the transgene consists of a CAT hybrid gene directed by 5.5 kb of aldC 5'-flanking sequences (Makeh et al., 1994). Since CAT activity was used, it is not clear whether the 5.5-kb sequence confers complete cell-type (i.e., Purkinje cell) specificity on the transgene. At the other extreme, a fusion gene comprising a 13-kb sequence of rat aldC gene, including both 5' and 3' regulatory regions, was found to be expressed in a brain-specific manner, with position-independent and high- level expression and correct regional distribution in the brain (although banding was not reported; Arai et al., 1994).

Detailed sequence analysis of the 5' region of the rat aldC gene reveals the presence of both Spl and Krox 24/20 binding sites (Tho- mas et al., 1993: Fig. 2). Any deletion or mutation of these sites that impairs recognition by transacting factors also suppresses promoter activity. A similar arrangement is found in the human aldC promoter (Buono et al., 1993). Interestingly, this structure is reminiscent of the SNN element in several neuron-specific promoters (68-kDa neurofi lament subunit, Lewis and Cowan, 1986; Sehgal et al., 1988; synapsin, Sauerwald et al., 1990), and also in the Hox 1.4 gene (Chavrier et al., 1990).

Evolution of the Aldolase Gene Family

Kukita et al. (1988) have analyzed the evolu- tionary relationships of the vertebrate aldolases based on eight DNA sequences. The ancestral aldA-like gene can be traced back to Drosophila aldolase, which has a 33% homology to all mammalian isoforms. A split occurred in vertebrates between the aldA/C and aldB genes, with aldC diverging from aldA still later. Thus, there is an 81% sequence ident i ty be tween rat aldA and aldC, but only 70% between aldA and aldC with aldB. An early origin for the aldolase lineages is consistent with the antizebrin II immunocytochemistry, which shows Purkinje cell staining in multiple vertebrate taxa (fish, e.g., Lannoo et al., 1991a,b; Meek et al., 1992; reptiles, Hawkes, in preparation; mammals, e.g., Brochu et al., 1990; Dor6 et al., 1990; Eisenman and Hawkes, 1993).

The Challenge of Aldolase C Expression and Its Relationship to Cerebellar Compartmentation

The distribution of zebrin II/aldolase C in the cerebellum and the known functions of this glycolytic enzyme present two key challenges to the student of cerebellar development. The first is to learn the molecular details of how aldC expression is regulated; the second is to understand the functional consequences of the regionalization of this apparently unremark- able "housekeeping" enzyme.

The first problem crystallizes many of the general issues of developmental gene regulation. The gene for aldC must be sensitive to cell type (only expressed in Purkinje cells), as well as to the anatomy of the brain (only in a specific and highly conserved pattern of bands). These problems are usually dealt with sepa- rately in vertebrate studies. Thus, for example, transgenic mouse studies tend to focus either


154 Ha wkes and Herrup

on cell-type specificity, as in the regulation of the opsin (e.g., Lem et al., 1991), GFAP (e.g., Brenner et al., 1994), or tyrosine hydroxylase (e.g., Banerjee et al., 1992) genes, or on regional variation, as in the regulation of the HOM-C family of transcription factors (for recent reviews, see Rubenstein and Puelles, 1995; Wilkinson, 1993). The cerebellar cortex actually offers one of the best mode l systems in which to com- bine these two issues and study their interplay. The elegant studies of Oberdick et al. (1993) are an example of such an approach, but the regulatory mechanisms required for the expression of aldolase C are different from those for L7; the stripe pattern of L7 is transient whereas the pattern of aldolase C expression, once established, is maintained for the life of the animal.

The second problem presented by the zebrin/ aldC expression pattern, the functional consequences of the banding pattern, may be even more challenging than the first. There is noth- ing obvious in the biochemistry or kinetics of the aldC enzyme that might explain its remark- able distribution in the cerebellum. For example, there are no known functional specializations of the different Purkinje cell subsets that would require a particular set of aldolase properties, and similarly, aldC enzymology is not greatly different from that of the other brain isoform, aldA. An appealing possibility is that whereas a l dC /z e b r in II has aldolase activity in vitro, its actual function in vivo may be quite different. For example, it may function to cleave a substrate other than fructose-l,6-biphosphate. An analogous situation has been reported for a ldehyde dehydrogenase (McCaffery et al., 1991). An alcohol dehydrogenase isoform is expressed in a gradient in the developing retina. A p laus ib le exp lana t ion for this puzz l i ng observation emerged when it was shown that a preferred substrate for the enzyme was retinal- dehyde , which it converted to retinoic acid. Retinoic acid is a morphogen in many systems, inc lud ing l imb b u d deve lopmen t , where a putative gradient in its concentration has been postulated to establish the anteroposterior axis

(Eichele, 1989). A similar role in the retina is entirely plausible. Aldolase C may also have preferred substrate in vivo that plays a crucial role either in Purkinje cell function or in the maintenance of compartmentation. In this con- text it is in t r igu ing to note that s tudies of Xenopus aldC expression have suggested that the enzyme plays an important role in neuru- lation (Atsuchi et al., 1994). To date, however, no alternative aldC substrate has been reported.

Conclusion

Increasingly, the cerebellar cortex is being recognized as a power fu l m o d e l sys tem in which to study the regionalization of the nervous system. A l though init ial ly it appears m o n o t o n o u s because of its near ly un i fo rm cytoarchitecture, many functional, anatomical, and molecular studies have revealed a deeply set pat tern of parasagittal bands. The functional studies by themselves leave open the quest ion of where the information is stored tha~ establishes the pattern (is it imposed by extrinsic forces or is it intrinsic to the constitu- ent cells), but the use of molecular markers has made it clear that the cellular pattern is intrinsic to the cortex. The genes for some of the compartmental ized proteins, such as L7 and aldolase C, are n o w isolated. A s ignif icant future goal is to use these genes as tools to identify and isolate the factors that regulate gene expression both temporally and spatially. Such strategies should lead to a more general understanding of the differentiation of the cells of the brain and offer insight into the causes (and possibly cures) of the large numbers of congenital malformations of the CNS.

Acknowledgments

We gratefully acknowledge the advice of Saul Zackson and the financial support of the Medical Research Council of Canada (R. H.) and the National Institutes of Health (NS18381 to K. H.)



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aldolase c/zebrin ii and the regionalization of the cerebellum

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