Novel Genes Expressed in the Developing Medial Cortex

Yumiko Hatanaka and Edward G. Jones1

Laboratory for Neuronal Recognition Molecules, Brain Research Institute, The Institute of Physical and Chemical Research, RIKEN, 2–1 Hirosawa, Wako, Saitama 351–0198, Japan and , 1 Center for Neuroscience, University of California, Davis, CA 95616, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two cDNAs, M1 and M2, recently isolated by the differential display method from embryonic rat cerebral hemisphere were characterized and their patterns of spatiotemporal expression analysed in developing rat forebrain by in situ hybridization histochemistry and correlative immunocytochemistry. Neither gene bears any sequence homology to other known genes. Both genes are particularly expressed in medial regions of the cerebral hemisphere and M2 in the roof of the adjacent diencephalon. M1 expression is highly localized and confined to the neuroepithelium of the hippocampal rudiment from embryonic day (E) 12 onward. Its location corresponds to the fimbrial anlage, and immunocytochemical localization of M1 protein indicates its expression in radial glial cells. M2 expression at E12 is more extensive in the medial cerebral wall, extending into the preoptic region and beyond the hippocampus into dorsal hemisphere and into the dorsal diencephalon, with caudal extension along the dorsal midline and in the zona limitans intrathalamica. Later, M2 expression is found in association with the corpus callosum, hippocampal commissure, fimbria, optic nerve, stria medullaris, mamillothalamic tract and habenulopeduncular tract. M1 and M2 expression domains corresponding to the locations of fiber tracts are present prior to the arrival of the earliest axons, as identified by neuron specific markers. These findings suggest M1 and/or M2 genes are involved in early regional specification of the hippocampus and related structures in paramedian regions of the forebrain, and that cell populations expressing these genes in advance of developing axonal pathways may be involved in the early specification of tract location.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The anatomical complexity of the cerebral cortex is evident in its regional division into neocortex, palaeocortex and hippocampal formation and in the further division of these regions into areas of distinctive cytoarchitecture. The cytoarchitectonic areas which hold the keys to understanding the functional complexity of the cerebral cortex are conserved among individuals, indicating that their basic construction is genetically determined. One of the major problems in neurobiology is understanding the molecular mechanisms that underlie this regional and areal parcellation of the cerebral cortex.

The cerebral cortex originates from the neuroepithelium of the lateral ventricles, a cytologically homogeneous sheet of epithelial cells. Each distinct cortical area arises from a relatively localized part of the neuroepithelium, although the extent to which the whole cortical map is pre-determined in a neuroepithelial protomap is controversial. Recent studies show that many putative transcription factors are expressed in the telencephalic neuroepithelium in a temporally and spatially specific manner. These factors include: Emx1/2 (Simeone et al., 1992aGo,bGo; Gulisano et al., 1996Go), Otx1/2 (Simeone et al., 1992aGo), BF-1 (Tao and Lai, 1992Go), Tbr-1 (Bulfone et al., 1995Go), Dlx1/2 (Bulfone et al., 1993Go) and Pax6 (Walther and Gruss, 1991Go; Stoykova et al., 1996Go). Disruptions of Emx1 (Qiu et al., 1996Go; Yoshida et al., 1997Go), Emx2 (Pellegrini et al., 1996Go; Yoshida et al., 1997Go), Otx1 (Acampora et al., 1996Go), Otx2 (Acampora et al., 1995Go; Matsuo et al., 1995Go; Ang et al., 1996Go), BF-1 (Xuan et al., 1995Go), and Tbr-1 (Shi et al., 1998Go) lead to malformations of the cerebral cortex. Disruptions of Dlx-1/2 (Anderson et al., 1997Go) and of Otx1 (Weimann et al., 1998Go) will cause defects in cell type specificity of cortical neurons and disruptions of Pax-6 (Gotz et al., 1998Go) to abnormalities of radial glial cells. Genes coding for many diffusible factors are also expressed in restricted regions of the developing telencephalon. These include the Wnt gene family (Parr et al., 1993Go; Grove et al., 1998Go) and BMP family (Furuta et al., 1997Go) in the dorsal portion of the telencephalon, and Shh (Ericson et al., 1995Go; Shimamura et al., 1995Go) in the ventral portion. Their specific expression within the telencephalon and similarly restricted expression in the diencephalon provides a framework of the patterning of the whole forebrain in the chick (Figdor and Stern, 1993Go) and mouse (Bulfone et al., 1993Go; Rubenstein et al., 1994Go), and suggest a neuromeric organization of the forebrain akin to that observed in the hindbrain (Guthrie, 1996Go). However, molecular markers that define the regional subdivisions of the developing cerebral hemisphere and its off-shoots such as the olfactory bulb remain undefined. The extent to which distinct molecular determinants set up boundary constraints within the early cerebral cortex, leading to its subdivision into areas, is also unknown.

Advances in the techniques of molecular biology enable the identification and subsequent isolation of differentially expressed transcripts from limited amounts of RNA. Differential display is a PCR-based technique that facilitates direct comparison of genes expressed in multiple samples (Liang and Pardee, 1992Go). We have applied this method to isolate genes expressed in the cerebral hemisphere in a region-specific manner in the embryonic day (E) 13 rat (Hatanaka, 1997aGo, 1997bGo). At this stage, the neuroepithelium is the principal component of the cerebral hemisphere, the cortical plate being non-existent or highly rudimentary and its afferents not yet having arrived. Dorsal and lateral aspects of the later cortical plate have the capacity to produce specific molecular makers, such as lamp (Ferri and Levitt, 1993Go) and latexin (Arimatsu et al., 1992Go). We began searching for differentially expressed genes in the dorso-lateral and dorso-medial aspects of the early hemispheric neuroepithelium, since these are destined to form the neocortex and the hippocampal formation, respectively. We found that two genes, designated M1 and M2, are preferentially expressed in the medial telencephalon. Examination of their precise expression patterns by in-situ hybridization histochemistry indicated that these genes are differentially expressed in the paramedian region of the telencephalic neuroepithelium in a manner that suggests their involvement in specifying the location of future axonal pathways such as the fornix. Immunohistochemistry for M1 gene product suggested its association with an early subpopulation of radial glial cells in the medial cerebral wall. Potential roles of M1 and M2 in the formation of CNS tracts are discussed.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissue Preparation

Wistar rats were used. The day when a vaginal plug was observed in the morning was designated E0. E13 rats were used to obtain RNA and E12, 13, 15, 16, 17 and E19 rats were used to perform in-situ hybridization histochemistry and immunocytochemistry.

Differential Display

Total RNA samples were obtained from dorso-lateral and dorso-medial portions of the cerebral hemisphere of E13 rats (Fig. 1AGo), and digested with DNase I to remove chromosomal DNA. The RNA was reverse transcribed with Superscript II (GIBCO), priming with downstream primers synthesized as GT15MN (M, a mixture of A, G and C; N, a mixture of A, G, C or T). Each sample of dorso-lateral and dorso-medial cDNA was amplified with 80 combinations of arbitrary upstream and downstream primers by using PCR. The PCR products were separated on a 6% denatured polyacrylamide wedge gel (0.3–0.6 mm), following which the gel was dried on filter paper and exposed to X-ray film. Bands of interest were cut out to recover the cDNA from the gel. The cDNA was reamplified and subcloned into Bluescript II vector (Hatanaka, 1997aGo).



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Figure 1.  M1 and M2 genes are expressed in the paramedian region of the developing cerebral hemisphere. A Genes expressed in dorso-lateral (L) and dorso-medial (M) portions of the telencephalon were compared by a modified differential display method. a Coronal slices were obtained from the middle portion of the telencephalon of E13 rats, and L and M indicated by shading are the regions dissected out from the slices. b Result of differential display. Out of more than 4000 cDNA fragments, four fragments termed M1, M1', M2 and M3 were found only after amplification of cDNA derived from the M portion of the telencephalon. For confirmation of reproducibility, each experiment was carried out in duplicate. B Schematic drawings of serial frontal sections from an E13 rat forebrain showing the location of hybridization signal for M1 (a) and M2 (b) mRNAs. a Distribution of M1 mRNA. M1 signal was found in a portion of hippocampal rudiment and in the caudal diencephalon. b Distribution of M2 mRNA. M2 signal, which was much less restricted in distribution than that of M1, was detected in the medial cerebral wall from the most anterior telencephalic level through the adjacent portion of the dorsal diencephalon, in the zona limitans intrathalamica (zl), in the developing eye and in the caudal diencephalon. cp, choroid plexus; HI, hippocampus; HT, hypothalamus; p, preoptic area; TH, thalamus.

 
RNA Probe Preparation and In Situ Hybridization

Since the cDNA fragments obtained from differential display were relatively short, longer corresponding cDNAs were isolated by the 5' RACE method or by screening of a cDNA library (Hatanaka and Jones, 1998Go). Both antisense and sense RNAs were synthesized by in vitro transcription using digoxigenin (DIG)-11-UDP with T7 or T3 RNA polymerase, using the longer cDNAs as templates. The RNAs were fragmented into an average size of 150 nucleotides by alkali treatment.

Whole embryos at E12 and E13 or whole brains at E15, E16, E17 and E19 were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffered saline at 4°C for 24 h, then cryoprotected in 30% w/v sucrose in the same buffer. The tissues were embedded in OCT compound (Miles, Elkhart, IN, USA), sectioned at 10 µm in a cryostat and collected on silane-coated glass slides. Non-radioactive in situ hybridization was perform as described previously (Hatanaka and Jones, 1998Go).

Immunocytochemistry

Rabbit polyclonal antiserum to M1 protein was raised against a synthetic C-terminal peptide of M1 (Hatanaka, unpublished data) and affinity purified on a peptide conjugated column. M1 protein was detected with anti-M1 (0.2 µg/ml), then with Cy3-conjugated anti-rabbit antibody (Jackson, 1:300). Neurons were identified by using rabbit anti-neuronspecific ß-tubulin antiserum (1:1000, Hatanaka and Jones, 1998) with the ABC ‘Elite’ kit (Vector). For double immunostaining of M1 protein and either type III ß-tubulin or nestin, a monoclonal antibody to type III ß-tubulin (Sigma, 1:200) or the monoclonal antibody, Rat-401 (Developmental Studies Hybridoma Bank, 1:5), were used. Bound antibodies were detected with biotinylated anti-mouse secondary IgG (Vector, 1:200) followed by Cy2-conjugated streptavidin (Jackson, 1:300). Fluorescent images were collected by laser confocal microscopy.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Isolation of Region-specific Genes from Developing Cerebral Hemisphere by Differential Display

The dorsal portion of the E13 rat cerebral hemisphere contains the anlagen of both isocortex and allocortex whose delineation is not yet clear at this stage (Bayer and Altman, 1991Go). Using differential display, we compared cDNA derived from the dorso-lateral aspect, containing presumptive neocortex and lateral limbic cortex, and cDNA derived from the dorso-medial aspect, containing medial limbic cortex and the archicortex of the hippocampal formation (Bayer and Altman, 1991Go) (Fig. 1AaGo).

Using a combination of 80 primer sets, we observed four cDNA bands, named M1, M1', M2 and M3, preferentially amplified from cDNA derived from the dorso-medial region of the hemisphere (Fig. 1AbGo). Since sequence analysis showed that these included alternatively spliced forms and PCR dependent derivatives (Liang and Pardee, 1992Go; Hu, 1993Go), we concluded that two types of cDNAs, M1 and M2, had been isolated (Hatanaka, 1997aGo; Hatanaka and Jones, 1998Go). The lengths of these cDNA fragments were 117 bp and 121 bp, respectively. In-situ hybridization analysis showed that their expression was concentrated in the paramedian region of the cerebral wall, although their individual expression profiles differed (see below).

Since both cDNAs were relatively short and the information obtained from them was limited, we cloned near-full length cDNAs by screening an E15 rat brain cDNA library with a combination of the 5' RACE method. A M1 cDNA of 3.1 kb and a M2 cDNA of 1.1 kb were isolated and proved to be previously unknown. No known genes were related to them except for certain EST sequences (Y. Hatanaka, unpublished data). M1 encodes 250 amino acids with a high content of cystein residues (Hatanaka, 1998Go), and M2 is expressed as a circular RNA (Hatanaka, 1997bGo). Their function remains to be determined.

Distribution of M1 and M2 mRNA in the Cerebral Hemisphere and Other Portions of the Forebrain During Prenatal Ages

M1 mRNA is expressed uniformly in the medial telencephalon of the E13 rat, spanning the full thickness of the neuroepithelium and its areal extent having distinct expression boundaries (Fig. 1BaGo) (Hatanaka, 1997aGo; Hatanaka and Jones, 1998Go). Its anterior boundary is located just anterior to the developing choroid plexus of the lateral and third ventricles, and its posterior boundary is confined within the medial telencephalon. Its medio-lateral expression also showed distinct dorsal and lateral boundaries and appeared to be restricted to the anlage of the hippocampal formation, not extending into the medial diencephalon, although there was an isolated patch of weak M1 expression in the caudal diencephalon, apparently corresponding to the mamillary neuroepithelium (Hatanaka and Jones, 1998Go). At later ages, the dorsal boundary of M1 expression in the telencephalon corresponded to a point just beneath the dentate notch, an evaginated neuroepithelial patch in the developing hippocampus (Altman and Bayer, 1990Go). After appearance of the fimbria at E16, M1 expression is restricted to the thin layer of the neuroepithelium outlined by fimbrial fibers. M2 mRNA expression is also observed in the medial telencephalon at E13, extending across the full thickness of the neuroepithelium like that of M1, but its overall expression profile is different from that of M1 (Fig. 1BbGo) (Hatanaka, 1997aGo; Hatanaka and Jones, 1998Go). The M2 expression area is more extensive than the M1 expression area, and its expression pattern showed less sharp boundaries. Anteriorly, the zone of M2 expression extends beyond the level of the developing choroid plexus, and includes the commissural plate, where the hippocampal commissure and corpus callosum cross the midline at later developmental stages (Valentino and Jones, 1982Go). Posteriorly, expression of M2 is concentrated within the medial cerebral wall but the dorsal and ventral expression boundaries are not clear-cut, simply fading out over a relatively extended zone. The M2-positive area extends somewhat beyond the M1-positive zone dorsally. In the medial hemisphere, M2 expression extends into the presumptive preoptic region and into the dorsal diencephalon adjacent to the M2-positive zone in the medial telencephalon, with caudal extension along the dorsal midline, and into the zona limitans intrathalamica and presumptive mamillary region. M2 expression in the paramedian region included the hippocampal rudiment as well as the diencephalic roof of the paraphysis and related choroid plexus and was at its highest at the level of the eminentia thalami. From there, it decreased anteriorly and posteriorly. At later ages, M2 expression was found in association with the developing corpus callosum, hippocampal commissure, fimbria, optic nerve, stria medullaris, tract of the zona limitans, and habenulopeduncular tract (Hatanaka and Jones, 1998Go) (see below).

Although the expression profiles of the two genes differed in detail, the results show that the paramedian region of the cerebral hemisphere is delineated as a molecularly distinct region in early development. The two zones of expression possibly implicate at least two functional components in the early differentiation of this part of the cerebral wall and adjacent part of the diencephalon.

Expression of M2 in the Developing Eye

Outside the brain, M2 hybridization signal was detected in the developing eye. Expression was weak at E11 but became more prominent at E12 when expression was high in the optic stalk and moderate in adjacent portions of the optic vesicle and in the preoptic area of the brain. In the retina, the distribution of M2 transcript expression, initially homogeneous, became more heterogeneous with stronger expression in the ventral–anterior region and weaker expression in the dorsal–posterior region. By E16, M2 signal in the retina had declined considerably and was only intense in the portion of retina most removed from the optic stalk (Hatanaka and Jones, 1998Go).

M1 and M2 Expressing Cells in Relation to Neurons and Axons

M1 expression associated with the fimbria, and M2 expression associated with the fimbria and other axonal tracts, were usually detectable before the arrival of the earliest axons in these tracts. The association of the M1 expression domain with the most ventral portion of the hippocampal rudiment and thus with the original location of the fimbria, and the coincidence of the M2 expression domain with the locations of other major axonal tracts in the early stages of development raised the possibility that M1 and/or M2 expressing cells were of neuroglial lineage and associated with the specification and/or early development of these tracts prior to the appearance of axons in them. To examine this possibility, we identified neurons and axons by immunohistochemical staining of sections adjacent to those hybridized for M1 or M2 mRNA, using antibodies specific for class III ß-tubulin, an early marker for differentiating neurons (Burgoyne et al., 1988Go; Lee et al., 1990Go), for TAG-1, a cell adhesion molecule expressed on a subpopulation of neuronal processes in the optic nerve, corpus callosum, hippocampal commissure, stria medullaris thalami, mamillothalamic tract and habenulopeduncular tract (Yamamoto et al., 1986Go; Wolfer et al., 1994Go), or for GAP-43 which is a major protein of axonal growth cones.

No part of the M1 expression area in the hippocampal rudiment at E12 was class III ß-tubulin immunopositive (Fig. 2Go), although a few immunopositive cells were present in neighboring dorsal regions. In the hippocampal rudiment, M1 was exclusively expressed in cells of the neuroepithelium. This was also true of M1 expression in the caudal diencephalon. At E12, GAP-43 and TAG-1 immunoreactivity was not detectable in the cerebral wall. By E15, the segregated expression of M1 and class III ß-tubulin in the ventral portion of the hippocampal anlage and the adjacent choroidal epithelium became more apparent than at E12, and this continued throughout development. GAP-43 immunoreactivity first appeared at E15 in the dorsal margin of the M1 expressing region. From E16 onward, class III ß-tubulin and GAP-43 immunopositive fibers started to occupy the fimbria, located in the ventral region of the hippocampal anlage, and M1 signal was observed in the part of the neuroepithelium bordered by the tract but independent of the fibers in the tract.



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Figure 2.  Summary of the spatiotemporal appearance of M1 and M2 expression and of axonal tracts, as defined by immunocytochemical staining for neuron-specific ß-tubulin, GAP-43 and/or TAG-1. Regions of expression include the optic stalk, tract of the zona limitans, habenulopeduncular tract, fimbria, hippocampal commissure and corpus callosum. Light bars, M1 expression; medium bars, M2 expression; dark bars, presence of axons defined by neuron-specific ß-tubulin, GAP-43 and/or TAG-1 immunoreactivity. Broken bars indicate weak expression. Note that M1 and M2 expression appears earlier than or at least contemporaneously with the appearance of immunopositive nerve fibers in the tracts.

 
The majority of the M2-expressing areas also lacked ß-tubulin immunoreactivity at early developmental stages. At E12, these included the preoptic area, the medial portion of the telencephalic–diencephalic fissure, where the highest expression of M2 was observed, and the region along the zona limitans intrathalamica. GAP-43 and TAG-1 immunoreactivity was also lacking from these regions at this age. TAG-1 immunopositive fibers were first detected along the zona limitans intrathalamica at E13, 1 day after the appearance of M2 signal in the corresponding region. Association of M2 hybridization signal with TAG-1 immunopositive fibers was evident in the region between the dorsal and ventral thalami until E15, but both M2 and TAG-1 immunoreactivity was not clearly recognizable in this region after E16. TAG-1 positive fibers, were found in the habenulopeduncular tract at E13 but only weak M2 expression was detectable in the same location. By E15, an association of M2 signal and TAG-1 immunoreactivity in the habenulopeduncular tract became very evident and continued to E19. In the medial cerebral region where M2 expression was observed from E12 onward, axons of the fimbria were recognizable by anti-GAP-43 immunostaining only from E16 onward, as described above. Those in the hippocampal commissure and corpus callosum were recognizable by anti-TAG-1 immunostaining from E17 and E19, respectively. These ages at which axons could be labeled by immunocytochemistry in the commissures are the same as those at which axons can first be detected by electron microscopy (Valentino and Jones, 1982Go). Although there was a clear separation of M2 expression and the arrival of axons in the tracts, as development proceeded, ß-tubulin staining of the axonal tracts became superimposed upon the M2 expression areas.

In the developing eye where retinal axons appear from E15, M2 signal was observed from E12, but neuron specific class III ß-tubulin, GAP-43 and TAG-1 immunoreactivity was not detectable. Immunoreactivity for these three became detectable in the optic stalk and retina at E15. At E16, TAG-1 immunoreactivity was observed in retinal ganglion cell axons distributed through most of the central retina, but not in the peripheral portion, in which the most intense M2 hybridization signal was observed at this stage.

Localization of M1 Protein in the Hippocampal Rudiment

M1 gene was found to encode a predicted protein of 250 amino acids but the coding region for M2 gene is not yet clear (Hatanaka, 1998Go). We developed a polyclonal antiserum against the C-terminal peptide of M1 to examine the precise localization of M1 protein in the hippocampus and for the characterization of M1 expressing cells.

As shown in Figure 3Go, the antiserum stained the neuroepithelium of the hippocampal rudiment of the E12 rat. The location of M1 immunoreactivity was essentially coextensive with that of M1 mRNA, indicating that the predicted M1 protein was translated (Fig. 3AGo). The immunoreactive staining was punctate and pericellular, located on the cell surface rather than in the cytoplasm. Staining was strong in the basal aspect of the neuroepithelium, and expanded beyond the M1 mRNA expression area. As described above, immunoreactivity for neuron specific markers suggested that M1 expressing cells in the hippocampus are not neurons. However, strong immunoreactivity for M1 was located in the basal neuroepithelium among the early hippocampal neurons that appear there.



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Figure 3.  Expression of M1 protein in the hippocampus. A Distribution of M1 mRNA and protein in a sagittal section from an E12 rat. a M1 mRNA, b M1 immunoreactivity, c higher magnification of the hippocampal region in b showing strong M1 immunoreactivity. d Illustration of the whole sagittal section. Localization of M1 protein in the hippocampal rudiment was coextensive with that of M1 mRNA. Note the punctate appearance of the immunoreactivity and its density in the in the basal region, revealing polarity of M1 protein distribution in the hippocampus. Scale bars, a and b 200 µm; c, 100 µm. B Comparison of spatial distribution of M1 mRNA and protein with that of ß-tubulin immunoreactivity in the hippocampus in frontal sections from an E13 rat. a M1 mRNA, b M1 protein, c neuron specific ß-tubulin immunoreactivity. Because distribution of M1 mRNA did not overlap with that of ß-tubulin immunoreactivity, M1 expressing cells are likely to be non-neuronal. However, distribution of M1 protein overlapped with location of ß-tubulin immunopositive cells. Scale bar, 250 µm.

 
The spatial relationship between the distribution of M1 mRNA and immunoreactivity and that of ß-tubulin immunoreactive cells was analysed further in sections of E13 rat. Segregated expression of the M1 mRNA and ß-tubulin immunoreactivity was still observed in the hippocampal neuroepithelium at this stage (Fig. 3Ba, cGo). Superimposed images of the localization of M1 immunoreactivity and that of ß-tubulin immunoreactive cells were obtained by double labeling of E13 horizontal sections with anti-M1 antibody and with anti-class III ß-tubulin antibody. A row of ß-tubulin positive cells appeared at the marginal zone of the hippocampus but not around the choroid plexus primodium. M1 immunoreactivity was observed in neuroepithelium around the choroid plexus and along processes running vertically towards the pia mater then turning and running parallel to the surface and ending in end feet at the surface. The M1 stained processes were intermingled with those immunoreactive for ß-tubulin in the hippocampus but no double labeling was observed (Fig. 4Go).



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Figure 4.  Double labeling of horizontal sections from an E13 rat with anti-M1 antiserum and anti-class III ß-tubulin or Rat-401monoclonal antibody. a ß-Tubulin, b M1 protein, c superimposed images of a and b. df Higher magnification of ac. Section was taken from a middle level along the dorso-ventral axis. Pial endfeet exhibited strong immunoreactivity for M1 protein and are intermingled with ß-tubulin positive cells. Possible role of M1 protein is interaction with early-generated neurons in the hippocampus. g Rat-401, h M1 protein, i superimposed images of g and h. Many Rat-401 positive cells are M1 expressing cells, suggesting that M1-positive cells are a subpopulation of radial glial cells in the cerebral hemisphere. Scale bars, 100 µm.

 
The end feet of M1 expressing cells resemble those of radial glial cells in the medial cerebral wall. To confirm this, sections were double stained with anti-M1 antibody and with the Rat 401 monoclonal antibody that recognizes the intermediate filament, nestin, expressed in radial glial cells (Hockfield and McKay, 1985Go; Lendahl et al., 1990Go). M1 immunoreactive cells are Rat-401 positive and Rat-401 stained fibrous structures perpendicular to the lumen of the cerebral vesicle and parallel to the basal surface from E13. The basal border of M1 expression in the neuroepithelium was coincident with that defined by Rat-401 immunoreactivity.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To isolate molecules whose expression defines discrete regions of the cerebral hemisphere, we applied a modified differential display method (Hatanaka, 1997aGo). This approach led to the isolation of two gene fragments, designated M1 and M2, expressed in the developing hemisphere in a region-specific manner. Both were expressed in the medial cerebral wall, indicating early specialization of the paramedian region of the cortex but their exact expression profiles differed. M1 gene was expressed in a highly restricted portion of the ventral hippocampal rudiment, while M2 gene, although concentrated medially, was expressed weakly over much of the convexity of the hemisphere. Analysis of their expression during prenatal development in relation to markers specific for neurons and developing axonal tracts suggested that these genes were expressed in cells of neuroglial lineage located in the anlagen of future axonal tracts. Distribution of M1 protein as determined immunocytochemically, supported the likelihood of its expression in a subpopulation of neuroglial cells in the developing medial cerebral wall.

Search for Genes Expressed in the Cerebral Cortex in a Region-specific Manner

Identification, cloning, and characterization of differentially expressed genes in the dorsal telencephalon should provide important insights into the molecular process that underlie differentiation of the cerebral cortex. Although there are at present very few data that speak to the early delineation of the subdivisions of the cerebral cortex, it has been speculated that the dorso-lateral and dorso-medial aspects of the early cerebral hemisphere should be fundamentally different because they contain the neocortical and the hippocampal anlagen, respectively. Recent studies using an in vitro culture system (Tole and Grove, 1998Go) showed that in the E12.5 rat, the medial cerebral wall possesses the potential to express a set of distinct molecular markers that identify the hippocampal regions. These findings indicate the early specialization of the medial aspect of the hemisphere. The potential possessed by the lateral and dorsal cerebral wall at E12 for producing region-specific molecular markers has been demonstrated (Arimatsu et al., 1992Go; Ferri and Levitt, 1993Go; Arimatsu and Ishida, 1998Go). The present study provides further evidence for the specification of the medial region at early stages.

The differential display method is one of several molecular biological approaches that permit the isolation and cloning of differentially expressed genes from limited amounts of RNA. In this study, we screened ~4000 species of cDNA fragments and found two kinds of genes expressed in the paramedian region of the developing cerebral hemisphere. The same method failed to reveal genes preferentially expressed in the dorso-lateral region of the cerebral wall. This may result from the extent of the screening being too small to cover all the potentially expressed genes or from the homogeneity of gene expression in all except the molecularly specified medial region of the hemisphere at E13. Recent analyses of the expression of Msx (Mackenzie et al., 1991Go; Wang et al., 1996Go), Wnt (Parr et al., 1993Go; Grove et al., 1998Go) and the BMP family (Furuta et al., 1997Go) showed that these genes are also expressed in the developing medial telencephalon (see below), but they were not isolated in our screening. Improvements in technique, including improved differential display (Matz and Lukyanov, 1998Go), combinations of single-cell cDNA libraries and differential screening (Matsunami and Buck, 1997Go), restriction landmark cDNA scanning (Suzuki et al., 1996Go), or microarray analysis (Schena et al., 1995Go) will provide the basis of higher resolution isolation of region-specific genes in the near future.

M1 and M2 Expressing Cells in Relation to Developing Axonal Tracts

In early developmental stages, M1 was expressed in the ventral portion of the hippocampal rudiment, while M2 was extensively expressed in the dorsal midline of the forebrain, including the commissural plate, hippocampal rudiment, telencephalic– diencephalic junction, zona limitans intrathalamica and extending to the developing eye. All of these regions contain the rudiments of major axonal tracts of the forebrain. Comparison of the timing of appearance of early growing axons and that of M1 and M2 gene expression showed that expression of the two genes preceded the appearance of the first pioneer axons in the tracts examined, namely the optic tract, corpus callosum, fimbria–fornix, tract of the zona limitans and the habenulopeduncular tract. Since the onset of gene expression was not correlated with the appearance of the earliest axons, we believe that the genes are likely to be associated with the specification of tract location rather than directly with guidance of pioneer axons. Lack of immunoreactivity for neuron-specific ß-tubulin in the M1 expression area, and in the greater part of the M2 expressing area at early developmental stages suggests that M1 and M2 may be specifically expressed in cells of neuroglial lineage and this was confirmed for M1 by imunocytochemistry.

It is known that rhombomere boundaries in the developing brainstem contain cells defined by temporally and spatially distinct patterns of gene expression, and that pioneer axons grow along the boundaries (Heyman et al., 1995Go). This correlation of fiber pathways with rhombomere boundaries has now been extended to the diencephalon, where the trajectories of newly developing axonal pathways follow interneuromeric subdivisions (Figdor and Stern, 1993Go). The tract of the zona limitans follows the boundary between the developing dorsal and ventral thalamus. The axons of the habenulopeduncular tract as they grow posteriorly from the habenula form fascicles along the boundary between the developing dorsal thalamus and pretectum. The locations of these axonal tracts are coincident with M2 positive zones. Interkinetic nuclear migration of neuroepithelial cells is normally significantly reduced in the neuroepithelium forming the boundary regions between rhombomeres (Guthrie et al., 1991Go). The M1 and/or M2 positive regions that include the ventral hippocampal rudiment, the epithalamic region and the vicinity of the optic tract are also regions in which the neuroepithelium lacks typical interkinetic nuclear migration (Altman and Bayer, 1990Go). These observations support the idea that M1 and/or M2 expressing cells possess properties akin to those of rhombomere boundary cells.

M1 Expression and M1 Expressing Cells in the Hippocampal Rudiment

Analysis of the localization of M1 mRNA and protein showed that M1 positive cells are non-neuronal and have many of the characteristics of radial glial cells, including the possession of apical processes that are attached to the pial surface by end feet. Immunostaining with Rat-401 also implied that M1 positive cells are radial glial cells. Removal of the ventral hippocampus, containing M1-positive cells, at E15 followed by culture in vitro for several days, resulted in the appearance of GFAPpositive cells located between neurons and choroid plexus cells (Y. Hatanaka, unpublished observations). This result suggests that glial lineage cells are already present in the peripheral regions of the choroid plexus, although we have not yet confirmed that the GFAP-positive cells are derived from M1-positive cells. The roof plate of the developing spinal cord contains primitive glial cells stainable with the monoclonal antibodies RC1 (Edwardset al., 1990Go) and Rat-401 (Hockfield and McKay, 1985Go), and it is known that these primitive glial cells have processes extending to the pia mater and forming end feet there (Snow et al., 1990Go). The region of the medial cerebral wall in which M1 expressing cells are found is supposed to be the roof plate of telencephalon, and the M1 expressing cells extending end feet to the glia limitans thus resemble those of the spinal roofplate.

In addition to M1, the ventral hippocampal region is also distinguished by differential expression of genes of the Emx family, Emx2 being expressed but not Emx1 (Yoshida et al., 1997Go). Emx2 mutant mice lack a dentate gyrus and show severe defects in the remainder of the hippocampal formation and choroid plexus. In the embryos of these mice the ventral hippocampal formation was greatly reduced, indicating involvement of Emx1 in formation of the dentate gyrus and choroid plexus, but not in formation of the CA fields of the hippocampus which arise more dorsally. This is indicative of the likely involvement of multiple genes in the areal and regional specification of the cerebral hemisphere. The non-neuronal M1 expressing cells in the medial cerebral wall may affect the development of the hippocampus via diffusible or contact mediated cues that influence the growth and differentiation of early-generated hippocampal neurons.

At the time just before the onset of major neurogenesis in the cerebral hemisphere, leading to the development of the cerebral cortex, at least two novel genes, M1 and M2, are expressed in the paramedian region of the cerebral cortex and may be associated with the molecular specification of the derivatives of this region which include the choroid plexus, fimbria, dentate gyrus and other parts of the hippocampal region. Expression profiles of M1 and M2 and their likely specific expression in cells of neuroglial lineage in the anlagen of axonal tracts in advance of the appearance of the earliest axons, suggest that they have an important influence in the early specification of tract location.


    Notes
 
We thank Drs Y. Arimatsu, K. Mori and T. Hashikawa for helpful discussion during the course of this study. This work was supported by the Human Frontier Science Program, by the Special Postdoctoral Researchers' Program from RIKEN and by a Grant-in-Aid for Encouragement of Young Scientists (Y.H.). Y.H. is now at the National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444-8585, Japan.

Address correspondence to Edward G. Jones, Center for Neuroscience, University of California Davis, 1544 Newton Court, Davis, CA 95616, USA. Email: ejones{at}ucdavis.edu.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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