mCD24 expression in the developing mouse brain and in zones of secondary neurogenesis in the adult

~ Pergamon Neuroscience Vol. 73, No. 2, pp. 581-594, 1996

Copyright © 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain

S0306-4522(96)00042-5 0306-4522/96 $15.00 + 0.00

mCD24 EXPRESSION BRAIN A N D IN ZONES

IN

IN THE DEVELOPING MOUSE OF SECONDARY NEUROGENESIS THE ADULT

V. C A L A O R A , * G. C H A Z A L , * t P. J. NIELSEN,~ G. R O U G O N * and H. M O R E A U *

*Laboratoire de G~nrtique et Physiologie du Drveloppement, CNRS/Universit6 Aix-Marseille II, UMR 9943, Facult6 des Sciences de Luminy, Case 907, Marseille cedex 9, France

~Max Plank Institute ffir Immunbiologie, Stubeweg, 51, D7800 Frieburg, Germany

Abstract--Interactions mediated by cell surface glycoproteins are considered to be crucial during the formation of the nervous system. Using a monoclonal antibody directed to mCD24, a glycosylphos- phatidylinositol-anchored membrane glycoprotein, we have mapped its distribution throughout the mouse cerebral cortex during development and in young adult. Before birth, mCD24 immunoreactivity was observed in the intermediate zone, the cortical plate and the marginal zone, whereas the ventricular zones were immunonegative. After birth, mCD24 expression declined rapidly in the cortex, except in the corpus callosum (and other commissures in the brain) where immunoreactivity was still found until P20. Furthermore, mCD24 expression was maintained in young adults (until P60, at least) in zones of secondary neurogenesis, such as the granule cells of the dentate gyrus, the subventricular zone lining the anterior part of the lateral ventricles and a zone of cells extending between the striatum and the corpus callosum to the centre of the olfactory bulb. In this area mCD24 and polysialic acid neural cell adhesion molecule stainings were superimposed, and this corresponded to the pathway of migration of the olfactory immature neurons (subependymal layer). A layer of ciliated ependymal cells, lining all the ventricular walls, was also immunoreactive for mCD24.

Thus, except for these epithelial-like cells, mCD24 was essentially found associated with differentiating postmitotic neurons. Its spatiotemporal expression, both during development and in the adult, is compatible with a role for this glycoprotein in cell surface recognition and in signalling events occurring during neuronal migration and axonal growth. Copyright © 1996 IBRO. Published by Elsevier Science Ltd.

Key words: brain development, mCD24, secondary neurogenesis.

Informative interactions between cell surfaces are thought to be essential to the processes of cell recognition, sorting and migration during development of the nervous system. A variety of cell surface molecules with restricted spatial or temporal patterns involved in these processes have been described. Among these, an important group is composed of proteins anchored to membranes by a glycosyl- phosphatidylinositol, thought to be involved in axonal growth and guidance. These include proteins of the immunoglobulin super-family such as F3 ~° or T A G - l , 9 but also the recently identified emerging family of Eph receptor tyrosine kinase ligands, termed EFLs. 3'8

In a search for possible novel GPI-anchored molecules involved in the ontogeny of mouse nervous

tTo whom correspondence should be addressed. Abbreviations: BrdU, bromodeoxyuridine; DMEM/FCS,

Dulbecco's modified Eagle's medium/fetal calf serum; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; HSA, heat stable antigen; PBS, phosphate- buffered saline; PSA-NCAM, polysialic acid neural cell adhesion molecule; TRITC, tetramethylrhodamine B isothiocyanate.

system, we selected monoclonal antibodies character- ized by their ability to recognize such lipid-anchored structures. Several of these antibodies detected antigens of 28,000-33,000 and 50,000-55,000 mol.wt in developing mouse brain and thymus, respectively) ° It has been siabsequently shown that the antigen recog- nized in thymus is the heat-stable antigen (HSA) first described by Symington and Hakomori . 36 Its e D N A encoded a small peptide, predicted to contain only 30 amino acid residues in its mature form, 16,4° It is now well established that the antigen expressed in brain, that we initially described as P31, shares the same proteic core as HSA. 27 Hence, differences in molecular weight observed between neural and immune tissues are probably due to differences in the extent of glycosylation of the proteic backbone. In the human, an equivalent has also been identified as CD24, ~5 showing 57% homology in nucleic acid sequence. In order to clarify the nomenclature of this protein, independently discovered by several groups in mouse, and termed P31, 27 nectadrin 14 or HSA, 36 it has been suggested that the mouse antigen be designated mCD24, 42 and this term will be used here.

581

582 V. Calaora et al.

In a previous study aimed at detect ing m C D 2 4 expression by i m m u n o b l o t of total bra in extracts, we showed that expression was at its m ax i m um between embryonic day 17 and postnata l day 5, a per iod tha t cor responds to the format ion of neurona l networks. 27 Fur the rmore , light and electron microscopic obser- vat ions in rat and mouse cerebel lum showed tha t m C D 2 4 is part icular ly a b u n d a n t on postmitot ic precursors of granule cells, as well as on newly formed parallel fibres and unmyel ina ted axons of the white mat te r dur ing a period extending from day 5 to day 15 of pos tna ta l life. l?

In this report , we studied, by immunoh i s tochem- istry, the spat io temporal expression of m C D 2 4 in developing and adult mouse cerebral cortex. To investigate whether m C D 2 4 expression might be linked to neurogenesis occurr ing in the adult , we searched for its expression in h ippocampal format ion and olfactory system of young adults, and compared its d is t r ibut ion to tha t of polysialic acid neural cell adhes ion molecule (PSA-NCAM) . The specificity of

the staining observed was assessed by the absence of labelling in a mCD24-nega t ive mouse obta ined by genetic recombinat ion. ~t,4~

EXPERIMENTAL PROCEDURES

Tissue preparation

Embryos from Swiss mice (IFFA CREDO, France) were obtained at 8.5, 10.5, 12.5 and 16.5 days after fertilization. Timed pregnancies were obtained by mating a single male with two females overnight. The presence of a vaginal plug the following morning was considered evidence of mating and was counted as day 0.5 (E0.5) of gestation. For studies on early stages (between E8.5 and El0.5), the uterus was removed, cut at the beginning and the end of each embryo, and immersed for 48 h in 4% paraformaldehyde phosphate- buffered saline (PBS) pH 7.2. For ages between E12.5 and E16.5, pregnant females were killed (lethal injection of xylazin/ketamin) and embryos quickly removed from uterus. Total embryos or heads were immersed 48 h in PBS-4% paraformaldehyde. Postnatal mice of different ages (P2, P6, P12, P20, P30 and P60) were deeply anaes- thetized with xylazin/ketamin and perfused intracardiacally with a solution of 4% paraformaldehyde in PBS. The brain

A E 1 0 - E 1 2 E 1 2 - E 1 7

Mz f

qD

Iz f ~ ~ Mz / ~

Vz Vz

LV Lv

Fig. 1. Immunocytochemical (FITC staining) localization of mCD24 in sagittal frozen sections (about 15-/~m thick) of the anterior part of the cerebral cortex, during prenatal development. (A) Schematic drawing of the development of the mouse cortical layers. In B (El0.5), C (E14.5) and D (E16.5), the labelling appeared in the molecular zone (Mz) and the cortical plate (Cp). Note that the ventricular zone (Vz) is immunonegative. Iz, intermediate zone; Lv, lateral ventricle; Ov, olfactory ventricle; Ob, olfactory bulb; arrows, limit of the ventricle; S, limit of the skull. Scale bars: B - 100/~m; C and D ~ 250 pm.

mCD24 expression in mouse brain 583

Fig. 2. Postnatal mCD24 expression (peroxidase staining) in Vibratome sagittal brain sections (about 50-/~m thick). At P2 (A) and P6 (B), the cortex and zones in the hippocampal formation (H) are immunoreact ive for mCD24. At P12 (C) and P20 (D), mCD24 labelling decreases in the cortex. Note an intense labelling in the migratory pathway between the anterior part of the lateral ventricle and the olfactory bulb (arrow). The dentate gyrus (D) is also immunoreactive. Cx, cerebral cortex; T, thalamus; S, striatum; Sv, subventricular zone; O, olfactory bulb; *, lateral ventricle. Scale bars: A = 1.5 mm; B and

C = 2 m m ; D = 2 . 5 m m .

was dissected out and immersed for 48 h in the same fixative solution at 4°C. For frozen sections, prenatal samples were then incubated 48 h in PBS-2% paraformaldehyde with 15% (w/v) sucrose at 4°C. Brains and whole embryos were immersed with the appropriate orientation in O.C.T. (Bayer SA), and frozen in isopentane at - 4 0 ° C . Coronal and sagittal sections were cut at various thicknesses (5-20 #m) with a cryostat, and collected on gelatin-coated slides. Postnatal brains were coronally or sagitally sectioned (50- /~m thick) using a Vibratome. For each age, at least three animals were studied.

Antibodies

An ascitic fluid of the rat monoclonal IgG antibody against mCD24 (clone 194-4-3) diluted 1/100 in Dulbecco modified Eagle's medium supplemented with 10% fetal calf serum (DMEM/FCS; Gibco) was used. The specificity of this antibody has been described elsewhere) 7,3° PSA-NCAM was revealed with a mouse monoclonal lgM antibody raised against the capsular polysaccharides of meningococcus group B (anti-Men B) that share ~-2,8-PSA residues with PSA-NCAM; 3' an ascitic fluid was used at a 1/500 dilution. Bromodeoxyuridine (BrdU) incorporation was revealed by a commercial monoclonal mouse IgG antibody (Dako) specific to the BrdU epitope, and used at a dilution o f 1/250. Fluorescein isothiocyanate (FITC) or horseradish peroxidase (HRP)-conjugated antibodies against rat lgG or mouse IgM were purchased from Immunotech S.A. (Marseille, France) and were diluted 1/200 in D M E M / F C S .

Immunocytochemistry

Sections were first incubated for 1 h at room temperature with D M E M / 1 0 % FCS to reduce non-specific labelling. They were then incubated overnight at 4°C with the first antibody. Sections were rinsed three times in D M E M / F C S and incubated for 1 h at room temperature with the second fluorescent or HRP-conjugated antibody. After three rinses in DMEM/FCS , twice more in PBS, slides were mounted with mount ing medium (Sigma). HRP-conjugated antibodies were revealed with the diaminobenzidine method, using a colouration kit (Biosys). Sections were examined with a Zeiss microscope (Axioskop), with epifluorescence for FITC-labelled sections. Controls were performed either by omitt ing the first antibody, or by replacing the first specific antibody with a non- immune rat serum. In addition, sections corresponding to equivalent area of the brain of mice bearing a specific deletion for mCD24 gene H.4~ were examined as control. In this case, both first and second antibodies were added in the reaction.

For double-labelling experiments, Vibratome sections (50-/~m thick) were first incubated overnight at 4°C with both ant i -mCD24 (rat IgG) and ant i -PSA-NCAM (mouse IgM) or ant i-BrdU (mouse IgG) antibodies. Secondary antibodies [anti-rat IgG fluorescein isothiocyanate with either ant i-mouse IgM-tetramethylrhodamine B isothiocyanate (TRITC) or ant i-mouse IgG-TRITC] were added together for 2 h at room temperature. For BrdU staining, sections were treated as described below. Saturation, washes and mount ing steps were similar to those described for simple labelling experiments.


A " r ' r - V l

, ~ . ~ . t ~ "41

L ~e~ ~ " ' l v

Fig. 3. mCD24 expression in postnatal cerebral cortex (peroxidase staining). (A) Schematic representation of the zone studied. At P2 (B), the labelling is found in the different layers (I V1) of the cortex.The higher magnification (C) shows that the labelling appeared located at the periphery of the cells. Between P6 (D) and P20 (F), the intensity of the labelling decreased in the cortical layers and became restricted to the more ventral part of the Cc (see higher magnifications in E and G). Note in (E) immunonegat ive cell bodies (arrows) oriented perpendicularly to the fibres of the corpus callosum (Cc). Sz, subependymal zone; Ez,

ependymal zone; Lv, lateral ventricle. Scale bars: B, D and F = 250/~m; C, E and G = 50/ tm.

A Cx

mCD24 expression in mouse brain

C Cx

Fig. 4. Labelling (peroxidase staining) of the cell migratory pathway between the anterior part of the lateral ventricle and the olfactory bulb on Vibratome sagittal sections (about 50-/~ m thick) of young adults (P30). (A, B) mCD24 labelling; (C, D) BrdU labelling (after intraperitoneal injections described in methods); (E, F) PSA-NCAM labelling. Note that the labelling of mCD24 (B) and PSA-NCAM (F) is located at the periphery of the cells, whereas it appears intracellular for BrdU (D). Cx, cerebral cortex; Ob, olfactory bulb; Lv, lateral ventricle. A, B, C, D and E, F are adjacent sections. Scale bars: A, C and

E = 500/~m; B, D and F = 50/~m.


Bromodeoxyuridine injections and staining

To label dividing cells of the postnatal brain, a sterile solution of BrdU (Sigma) at 10 mg/ml in PBS was injected intraperitoneally in mice at P30 (2 g/kg of body weight). Six injections (0.1ml each) were given in 72 h, and mice were killed 6 h after the last injection. Brains were fixed as described above. Because the antibodies did not recognize the BrdU incorporated in double strand DNA, it was first necessary to denaturate the DNA under single strand. Sections were treated for 30 min at 37C with HCI 2 N in PBS containing 0.5% Triton X-100. Then, sections were rinsed three times in sodium tetraborate buffer (0.1 M, pH 8.5). Immunolabelling steps were as described above.

RESULTS

mCD24 immunoreactivi ty was widely and transiently distributed in different areas of the developing brain according to their schedule of morphogenesis. Mesencephalon and rhombencephalon appeared labelled at E14-E16, and became negative at E l8 El9. The intense immunolabell ing found in cerebral cortex, striatum and colliculus of embryos decreased rapidly after birth (P5-P10) and was not detectable in the adult, except in discrete areas. In contrast, in cerebellum, immunoreactivity was low before birth, increased between P0 and P6 and declined progressively thereafter. We will focus, in this paper, on the distribution of mCD24 in the cerebral cortex during development, and on its restricted expression in several discrete brain areas after birth and in the young adult.

Cerebral cortex and ventricular zones

In embryos, mCD24 expression followed the development of the cerebral cortical layers (Fig. 1A). Immunolabell ing appeared immediately after the formation of brain vesicles at embryonic day 10.5 (Fig. I B). At this stage of development, mCD24 was distributed as a thin layer on the external part of the neuroepithelium (Fig. 1B), corresponding to the marginal zone (Mz); weak or no immunoreactivi ty was seen in the ventricular zone (Vz). At older ages (between E14.5 and E16.5) the intermediate zone, the cortical plate and the marginal zone were immuno- positive (Fig. lC and D), whereas the ventricular zone was devoid of staining.

After birth, the six different layers of the cerebral cortex develop (Fig. 3A), and at P2, mCD24 was still widely expressed in all layers of the cerebral cortex (Figs 2A and 3B). Immunostaining appeared located

at the periphery of the cells (Fig. 3C, see also Figs 6 and 7). At P6 and P12, the expression of mCD24 decreased progressively in these different layers, although a weak expression of mCD24 could still be detected (Figs 2B, C and 3D-G), and at P20, immunoreactivity disappeared in all of them (Fig. 2D).

Underneath the cortical layers and lining the dorsal part of the lateral ventricle, a zone corresponding to the corpus callosum (Cc) was strongly immunoposi- tive at P2 and P6 (Figs 2A, B, 3D). After P6, mCD24 expression in Cc decreased, and appeared, at P20, essentially restricted to the more ventral part of the Cc (Figs 2C, D, 3F, G). At P6, we observed in this structure, perpendicularly to the labelled fibres, un- labelled cells (Fig. 3E). After P20, mCD24 immunoreactivity in Cc became very weak and negative at P30 and later (Figs 5H and 6).

Three other areas of the brain maintained the expression of mCD24 in postnatal ages: the cerebellum, a thin path of cells extending from the lateral ventricle to the olfactory bulb and the dentate gyrus of the hippocampus. In the cerebellum (not shown), we confirmed the previous observations of Kuchler et al.~7 describing mCD24 expression restricted to the internal part of the external granular layer and on the white matter. This expression disappeared at around P10, and corresponded to postmitotic and pre- migratory granule cells.

Migratory pathway from the lateral ventricle to the olfactory bulb

On sagittal sections of P30 mouse brain, mCD24 immunoreactivity appeared to form a pathway that connects the anterior part of the Vz of the lateral ventricle with the ipsilateral olfactory bulb (Fig. 4A and B). This immunoreactivity persisted at least until P60. The anatomical organization of this path was reminiscent of the migratory stream of immature neurons 2~'23 described to be immunoreactive for P S A - N C A M . 32 Immunolabell ing of P S A - N C A M on sagittal sections adjacent to those labelled with anti- mCD24 antibodies showed that its expression pattern was very similar to that of mCD24 (Fig. 4E and F). In addition, after the BrdU injections, it clearly appeared that BrdU-positive cells displayed a distribution pattern similar to that of mCD24 and PSA- NCAM-posi t ive cells (Fig. 4C and D). mCD24 and P S A - N C A M immunoreactivity appeared located at the periphery of the cells (Fig. 4B and F), whereas

Fig. 5. mCD24 and PSA-NCAM staining (peroxidase staining) on Vibratome coronal sections (50-pm thick) between the anterior part of the lateral ventricle and the olfactory bulb of young adults (P30). mCD24 (A, B) and PSA-NCAM (C) labellings appear in the same subventricular zone (Sz) of the olfactory bulb (position 1 in Fig. 4). B and C are adjacent sections, mCD24 (D, E) and PSA-NCAM (F) labellings appear in the same zone (Sz) in the migratory pathway (position 2 in Fig. 4). E and F are adjacent sections. mCD24 (G, H) labelling appears more extended than PSA-NCAM (1) in the anterior part of the lateral ventricle (Lv) (position 3 in Fig. 4). H and I are adjacent sections. Sz, subventricular zone; Gr, granule cell layer; l, internal plexiform layer; M, mitral layer; E, external plexiform layer; GI, glomerular layer; Cx, cerebral cortex; Cc, corpus callosum. Arrows indicate the midline of the brain. Scale bars: A and

D = 500#m; B-I - 250 #m; G = 1000pm.


Fig. 5.


BrdU displayed a dense labelling similar to the intracellular (nuclear) labelling previously described 3v (Fig. 4D).

Adjacent coronal sections of the olfactory bulb (Fig. 5A, position 1 of the migratory pathway in Fig. 4), showed that mCD24 (Fig. 5B) and PSA- NCAM (Fig. 5C) stainings appeared located in the same cell layers: the subependymal zone was strongly immunoreactive, and a dispersed labelling was seen radially from this zone towards the more external structures. In addition, the glomerular layer containing plexus of fibres comprising axons of olfactory receptor neurons was immunoreactive. Other layers of the olfactory bulb such as the granular, mitral and external or internal plexiform granular layers pre- sented weak or no immunostaining. In the median part of the migratory pathway (Fig. 5D, position 2 of the migratory pathway), only a small cluster of cells appeared immunoreactive for both mCD24 (Fig. 5E) and PSA-NCAM (Fig. 5F).

In the anterior part of the lateral ventricle (Fig. 5G, position 3 of the migratory pathway in Fig. 4), an mCD24 immunoreactivity (Fig. 5H) appeared on the cells lining the lateral ventricle, whereas PSA-NCAM labelling appeared restricted to the dorsal edge of the anterior part of the ventricle (Fig. 5I). Higher magnifications of this zone (Fig. 6) showed that two morphologically distinct populations of cells expressed mCD24. One cell type was distributed on the total surface lining the lumen of the lateral ventricles. It appeared to be ciliated epithelium-like cells (Fig. 6A and B, Ez). These correspond to the classical ependymal cells described previously. 2 The second cell type expressing mCD24 was located at the dorsal surface of the anterior part of the lateral ventricle, in the subependymal layer (Fig. 6A and B, Sz). These cells had the same shape as those of the migratory pathway towards the olfactory bulb. Only these cells were positive for PSA-NCAM, whereas the ciliated ependymal cells were negative (Fig. 6C and D). In contrast, in the ependymal layer where most of the cells were positive for mCD24 and negative for PSA-NCAM, only a few cells were BrdU positive (Fig. 6E and F).

Double-labelling experiments with anti-mCD24 and anti-PSA-NCAM antibodies (Fig. 7) confirmed the results shown with adjacent sections (Fig. 6). Both markers were expressed on the migratory pathway, whereas only mCD24 was present on the zone lining the lateral ventricle (Fig. 7A and B). The different distribution of mCD24 and PSA-NCAM on this zone clearly indicated that no cross-reaction occurred between these two antibodies. This labelling probably corresponded to the ciliated cells described above. Furthermore, in mCD24 gene-deleted mouse, mCD24 immunoreactivity was negative (Fig. 7C). These double-staining experiments showed that mCD24 and PSA-NCAM were co-expressed on the periphery of the cells (Fig. 7 D F ) . Scarce cells seemed to express only mCD24, this could be due to differen-

tial endogenous level of expression of the two markers, or alternatively, to a difference of the state of maturation of these cells. The co-localization of mCD24 and BrdU also appeared closely related, but could not be determined unambiguously, because the cell membranes were damaged after the acidic treat- ment performed on the sections for the BrdU staining (Fig. 7G and H).

Hippocampal formation

Expression of mCD24 was also maintained in the hippocampal formation of the young adult, at least until P60. At P6 (Fig. 8A), mCD24 was mainly expressed in the granule cells of the dentate gyrus, but not in the CA zones of the hippocampal formation. mCD24 labelling was seen throughout the whole thickness of the granular layer, although the intensity of the reaction was stronger on its internal side. At this age, mossy fibres, the granule cell axons growing towards pyramidal cells, were strongly immunoreactive for mCD24. At P12 and P30 (Fig. 8B and C), mCD24 expression dramatically decreased in the granular layer, and the immunoreactivity in the mossy fibre layer was very low or negative. At P30 (Fig. 8C), only the more internal side of the granular layer of the dentate gyrus expressed mCD24. At P30 (Fig. 8D), BrdU immunolabelling showed that mi- totic cells were located at the internal part of the dentate gyrus, as a thin cell layer, which could be superimposed on the mCD24 positive layer, mCD24- positive cells had a rounded shape cell body, with short processes oriented towards the external side of the granular layer (Fig. 8E). A PSA-NCAM immunostaining of this area at P30 showed a pattern identical to that observed for mCD24 (Fig. 8F).

DISCUSSION

In the present study, the expression pattern of the GPI-anchored glycoprotein mCD24 has been investi- gated in developing mouse brain using a monoclonal antibody. This antibody has previously been charac- terized by biochemical 27 and immunohistochemical ~7 studies. The specificity of the staining described was assessed by several controls including the use of mCD24 knock-out mouse brain tissues. During development, mCD24 is widely and transiently expressed on the cells identified as developing postmitotic neurons and on growing axons. In the adult, it is restricted to areas exhibiting neurogenesis and to ciliated ependymal cells. In all the tissue structures observed in this study, mCD24 labelling appeared located at the periphery of the cells, as expected for a GPI-anchored molecule. 22 This is also in agreement with the cell membrane localization of this glycoprotein already described at the electron microscopic level, in the developing cerebellum. ~v

The major indication for a specific expression of mCD24 by developing neurons comes from the corre- lation of its pattern of expression with what is known


on the appearance and d is t r ibut ion of neurons in the ventr icular zone, an area where precursors cerebral cortex dur ing development . Mos t of the undergo intense division (see for example Refs 24 and neurons in the cerebral cortex arise prenata l ly f rom 25). The ventr icular zone, however, does not express

Fig. 6. Labelling (peroxidase staining) of layers lining the lateral ventricle (Lv) in young adults (P30). mCD24 is expressed both in subependymal (Sz) and ependymal (Ez) zones (A and B), whereas PSA-NCAM expression is restricted to the subependymal zone (Sz) (C and D). Most of the dividing BrdU-labelled cells are located in the subependymal layer (E and F). Cc, corpus callosum. BrdU intraperitoneal injections were made as described in Experimental Procedures. Scale bars: A, C and

E = 1 0 0 # m ; B, D and F = 5 0 # m .

O ~

Fig. 7. Fluorescent photomicrographs showing double labelling with mCD24 and PSA-NCAM or BrdU on sagittal brain sections of young adults (P30). (A-C) Low magnification of the migratory pathway, near the anterior part of the lateral ventricle, mCD24 (A) and PSA-NCAM (B) double-labelling occurred in the same area for the normal mouse. Note that the zone lining the lateral ventricle [probably corresponding to the ciliated cells (C)] is only mCD24 positive. No mCD24 immunoreactivity is observed on a section from a mCD24 gene-deleted mouse (C). Zone of the migratory pathway near the olfactory bulb labelled with ant i -mCD24 (D). High magnifications of this area after double labelling, mCD24 (E) and PSA-NCAM (F). The two markers appeared localized at the periphery of the same cells. The same double-labelling experiment performed with ant i -mCD24 (G) and anti-BrdU (H) antibodies showed that these two markers are closely

related. Lv, lateral ventricle. Scale bars: A, B, C and D = 200#m; E, F, G and H - 50/~m.


Fig. 8. Expression of mCD24 and PSA-NCAM (peroxidase staining) in the hippocampal formation during postnatal development, mCD24 is strongly expressed in the dentate gyrus (Dg) at P6 (A) and P12 (B), and is dramatically reduced at P30 (C, D). BrdU-labelled cells are located as a thin layer of cells at the internal part of the dentate gyrus (D). At higher magnification, the mCD24 (E) and PSA-NCAM (F) pattern of labelling appeared very similar. E and F are adjacent sections (50-/~m thick). BrdU intraperitoneal injections were made as described in Experimental Procedures. Mf, mossy fibres. Scale

bars: A, B and C = 250/lm; D = 100 ~tm; E and F = 50 l~m.


mCD24, indicating that the dividing neuronal precursors are not expressing this molecule. When the divisions stop, postmitotic immature neurons leaving the ventricular zone migrate along radial glial cells, their cell bodies constituting the future cortical plate. 25 At this stage, mCD24 is heavily expressed on the cells constituting the cortical plate. The expression persists during the formation of the cortex and disappeared after birth. This disappearance probably corresponds with the end of the migration period and axonal growth. It is likely that once synaptogenesis has occurred mCD24 expression is lost, although more detailed studies are required to decide whether there is a causal link between these two events.

Besides being expressed on migrating neuronal cell bodies, mCD24 is also strongly expressed on brain commissures around birth and later, during the period of intense axonal growth. This axonal localization of mCD24 is in agreement with what is known for GPI-anchored molecules. 9'1~ While the precise function(s) of the GPI-linkage is at present unknown, it may confer specific properties suited to the molecules presumed function in cellular interactions. 22 In neurons, it may serve to target these molecules to the axon. 7 In the commissures mCD24 expression decreased at around 20 days after birth. This kinetic of mCD24 extinction correlates with myelinization of these axons. 6'~3 These two events might be coinciden- tal since myelinization is known to occur on axons after completion of growth. Moreover, mCD24 expression is obviously not restricted to axons that will become myelinated.

It is difficult to decide whether all neurons of the CNS transiently express mCD24. Nevertheless, our observations, recorded at different times and in different zones of the developing CNS, strongly favour such a possibility. This is also supported by the observation that zones of secondary neurogenesis expressed mCD24 (see discussion below). Moreover, data obtained on primary neuronal cultures of cerebral cortex, cerebellum or striatum indicated that all neurons present in the culture expressed mCD24 (Ref. 30 and unpublished observations).

mCD24 is certainly not a neuronal marker s t r i c t u

s enso . Indeed, it is known to be expressed on other cell types such as cells of the haematopoetic lineage 3~ or some populations of epithelial cells, 35 including ciliated ependymal cells lining the ventricular walls (this study). Therefore, it is more likely to be a marker for given stages of differentiation of cells from different lineages. Our study, however, suggests that cells of glial lineage do not express it during either their migration or differentiation. This is supported by several observations. Firstly, radial glial cells that are the first glial cells to appear in development have their cell bodies located in the ventricular zone l~'19 which is consistently mCD24 negative. Secondly, in the cerebral cortex, astrocyte proliferation takes place around birth and later, a time at which mCD24

immunorcactivity decreased dramatically in the cortical layers. Thirdly, mCD24 disappears as soon as myelinization begins, as a further indication that oligodendrocytes and their precursors are also not expressing it. This absence of expression by glial cells was confirmed by the electron microscopic observations made in cerebellum by Kuchler e t al. ~7

In postnatal rodents, the subventricular zone, known as a zone of cell proliferation for glial cells 4'18 also contains in its anterior part (SVZa) proliferating neuronal progenitors able to migrate towards the olfactory b u l b . 21'23'24"26 We have shown that these migrating immature neurons expressed mCD24. The expression pattern was very similar to that of the polysialylated isoform of NCAM, another molecule whose expression is maintained in zones of secondary neurogenesis.

Similarities of expression between PSA-NCAM and mCD24 in the adult brain also extend to the hippocampal formation. In this area, new granule cells are produced after birth and during the adult period. 33 The distribution of mCD24 after birth cor- related with an expression by these newly formed neurons, which eventually migrate into deepest por- tion of the dentate granular layer, where they probably differentiate into granule cells. This very discrete expression in the adult CNS places mCD24 molecule as a new marker for zones of cell neurogenesis in the adult, as recently reported for PSA-NCAM. 32 The similarity of expression between these two molecules incidentally extends further, since during development PSA-NCAM is also heavily expressed on developing neurons and disappears on most of them when synaptogenesis has occurred. 1"29'34 The expression of these molecules, both on primary neurons and on neurons born in the adult, points to a conservation of molecular mechanisms, which might be central to neurogenesis, whatever the place and time at which it occurs. It is difficult to decide at this stage whether these two markers are expressed only on postmitotic neurons, as observed during development, or also on still dividing immature neurons. If this was the case, it would point to a difference in the timing of expression of these molecules. A possible hypothesis would be that mCD24 is necessary for migration regardless of the dividing status of the neuroblasts. Further experiments are necessary to explore this possibility. It should be stressed, however, that, un- like PSA-NCAM, 38 mCD24 is not expressed in the adult CNS in areas exhibiting morphofunctional plasticity such as the hypothalamus. This places mCD24 as a purer marker than PSA-NCAM for secondary neurogenesis in the CNS.

The spatiotemporal expression of mCD24 inbrain development supports a role in neuronal migrations and axonal growth. Its wide distribution, however, is not compatible with that of an informative molecule involved in guidance, but rather with that of a permissive element allowing movement. This is a further similarity with PSA-NCAM which is thought


to play a role in plasticity. 29 Moreover , PSA direct

involvement in cell migra t ion has recently been established by the study of a N C A M knock-ou t mouse.5,28, 39

m C D 2 4 widespread and t rans ient expression on developing neurons is also reminiscent of its expression and role in lymphocytes 2° in which it has been shown to be a cos t imula tory signal. For example, a recent report , H using cel l lines derived from a m C D 2 4 gene targeted d is rupt ion mouse, indicates tha t m C D 2 4 alters an integrin (VLA-4)- media ted adhesion. Moreover , const i tut ive expression of m C D 2 4 in lymphocytes of a t ransgenic mouse induced a defective deve lopment of thymo-

cytes, lz It is possible, however, t ha t m C D 2 4 interacts with different l igands in the immune and nervous systems, since biochemical studies have indicated tha t m C D 2 4 glycosylation differs between the two systems.

Acknowledgements--The authors wish to thank Dr J. Cohen and Dr J. P. Herman for their advice and for stimulating discussions. We also thank G. Monti and A. Ribas for their excellent technical assistance. This work was partly supported by Association Franqaise contre la Myopathie (AFM), EEC Biotech Concerted Action 930326 to G.R., and Ligue D6partementale des Bouches du Rh6ne Contre le Cancer to H.M.

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(Accepted 8 January 1996)

mCD24 expression in the developing mouse brain and in zones of secondary neurogenesis in the adult

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