Cajal—Retzius cells, Reelin, and the formation of layers

570

Cajal-Retrius

cells, Reelin, and the formation

of layers

Michael Frotscher Early-generated cortex synthesize

Cajal-Retzius and secrete

reelin gene is deleted alterations

in cortical

cells in the marginal the glycoprotein

in reeler mice, which show characteristic lamination.

Recent

some light on the role of Cajal-Retzius formation

zone of the

Reelin. The

studies

have shed

cells and Reelin in the

of cell and fiber layers in the neocortex

Before

Addresses Institute of Anatomy, University of Freiburg, PO Box 1 11, D-79001 Freiburg, Germany; e-mail: [email protected] Current Opinion in Neurobiology

1998,8:570-575

http://biomednet.com/elecref/0959438800800570 Biology

Ltd ISSN

0959-4388

Abbreviations

CR

Cajal-Retzius

EGF GABA

epidermal growth factor y-aminobutyric acid

Introduction Following their generation in the ventricular zone, postmitotic neurons migrate along radial glial fibers to their definitive positions [ 11. Through the sequential generation, migration and deposition of neurons, cortical cell layers are formed. It is an intriguing feature of cortical development that early-generated neurons form deep layers near the ventricular zone, whereas late-generated neurons establish superficial layers. This means that later-generated neurons have to migrate longer distances than their predecessors. We are beginning to understand the molecular mechanisms governing the directed migration of neurons along radial glial fibers [Z-4] and the termination of the migration process [S], but many questions remain concerning the formation

lum [13-151. The animals survive despite developmental malformations, but show deficits in motor coordination.

of the inside-out

pattern

of cortical

lamination.

In addition to being arranged in layers, cortical regions display a lamination of fibers. This is seen most clearly in the hippocampus, where extrinsic and intrinsic fibers show a robust layer-specific termination [6-o]. Little is known about the signals that guide different fiber systems to their appropriate layers and keep them there. In this review, I summarize recent findings important role for Cajal-Retzius (CR) cells are early-generated transient neurons of the (future layer I), in both the formation of the ering of neurons in the neocortex and the fiber projections to the hippocampus.

suggesting an [l&l 11, which marginal zone inside-out laylamination of

reeler mutant mice lack Reelin, a glycoprotein synthesized by Cajal-Retzius cells In the mouse mutant r&er(rl-i-), in 1951 [12], the characteristic

these severe characteristic

and

hippocampus.

0 Current

cortex is perturbed and there are characteristic alterations in the lamination of cells in the hippocampus and cercbel-

which was first described inside-out layering of the

the

factor

missing

in reeler mutants

was discov-

ered, the analysis of the cortical malformation remained at a descriptive level, and further insights into the normal developmental mechanisms leading to the inside-out lamination of cortical neurons were limited. Recently, the gene deleted in 1-eeekr mice was cloned and called reelin [16,17]. In situ hybridization localized reel;, mRNA to CR cells in the marginal zone [16,17]. Similarly, an antibody (CR-SO) raised by immunizing reeler mice with wild-type homogenates immunostained CR cells [18]. Later, it was shown that Reelin is a secreted glycoprotein and that the CR-50 antibody recognizes an epitope near the amino terminus of Reelin [lY*]. Reelin is a large molecule comprising 3461 amino acids and has a relative molecular mass of 388 kDa [16]. ‘lhc reeh gene is conserved in many vertebrate species; analysis of the human reeh gene revealed that mouse and human amino acid and nucleotide sequences are 94.2% and 87.2% identical, respectively [20]. Reelin shares sequence similarities, such as several EGF-like motifs, with other extracellular matrix proteins known to be involved in cell adhesion [16]. Thus, as a component of the extracellular matrix in the marginal zone, Reelin may be involved in neuronal adhesion and migration during critical stages of development (161.

Cajal-Retzius cells, Reelin, and the inside-out layering of the neocortex In addition to having perturbed cortical layers, rpe/(J/. mutant mice display a loss of the characteristic vertical orientation of the main apical dendrites of pyramidal cells. How could the lack of Reelin cause these severe alterations? It has recently been hypothesized that Reelin acts as a stop signal for migrating neurons [Zl]. If one accepts this hypothesis, then it is possible to develop a model for how the inside-out layering of the neocortex develops. CR cells, being among the earliest neurons formed [22,23”], do not have to migrate far from the ventricular zone towards the marginal zone of the still thin dorsal telencephalic wall. They are probably maintained beneath the pia by a trophic factor supplied by meningeal cells, as destruction of meningeal cells causes the rapid degeneration of CR cells and severe migration defects [24’]. Another class of early-generated neurons, the subplate cells, are stopped early in their migration by Reelin secreted from CR cells [lo”]. Being repelled by Reelin, subplate neurons start to differcntiate, while the marginal zone containing CR cells moves

Cajal-Retzius

away:

the

telencephalic

wall

increases

in

thickness

(Figure la,b) as the cortical plate develops between cells and subplate neurons [23”,25] (Figure lb-d).

CR

A crucial point of this hypothesis [Zl] is that the early neurons of the cortical plate are allowed to migrate along radial glial fibers for a short distance only, because they soon arrive at the marginal zone where they are stopped in their migration by Reelin (Figure lb). As the marginal zone containing CR cells and Reelin moves further outward with increasing cortical thickness, late-generated neurons of the cortical plate can migrate for longer distances before they meet the Reelin-containing extracellular matrix of the marginal zone. This way, cortical layers will form in an inside-out manner (Figure lc,d).

In the

cells,

Reelin,

absence

and the formation

of Reelin,

of layers

subplate

Frotscher

neurons

571

are not pre-

vented from reaching the marginal zone (Figure le). Consequently, the cortical plate does not develop between the two types of early-generated neurons (i.e. subplate neurons and CR cells) [26]. Rather, because the first neurons of the cortical plate cannot penetrate the dense accumulation of early-generated neurons, they assemble underneath them in the order in which they arrive (Figure If-h). Reelin’s role as a stop signal may also explain the vertical organization of the apical dendrites of pyramidal cells. Early-generated cortical plate neurons layer V pyramidal cells) migrate towards the where they are stopped by Reelin; they then entiate processes that anchor these neurons zone (Figure lb). With increasing cortical

(presumptive marginal zone, start to differin the marginal thickness, the

Figure 1

(d)

(a)

(h) (9)

o/b

..” PI

rl+

p2

Vv‘SP

Current ODinion in Neurobioloav

Schematic diagram illustrating a hypothesized role of CR cells and Reelin in the formation of the inside-out pattern of cortical cell layers in wild-type

mice (r/+‘+) and cortical

p3

malformation

in reeler mutants

(r/-I-).

(a) Reelin (gray-shaded

area), which is synthesized by CR cells, acts as a stop signal for migrating neurons, thereby preventing subplate cells (SP) from reaching the marginal zone. P, pial surface; V, ventricular surface. (b) With increasing cortical thickness, the marginal zone (containing CR cells and Reelin) moves further outward; in between CR cells and subplate neurons, the cortical plate starts to develop. Early-formed cortical plate neurons (PI) migrate along radial glial fibers (RF) until they reach the marginal zone, where they are stopped by Reelin. The postmigratory cell differentiates by forming dendritic processes, anchoring the cell to the marginal zone. (c,d) With further increases in cortical thickness, a stem dendrite is extended, forming the vertically oriented main apical dendrite. Later-generated

cortical

plate neurons

(P2 and P3) are allowed

to migrate

for increasing

distances along radial glial fibers before they meet Reelin. This way, early-generated cortical plate neurons give rise to deep layers, and late-generated

cortical

plate neurons

give rise to superficial

layers of

the cerebral cortex. (e-h) In reeler mutant mice, CR cells are present but do not secrete Reelin. Subplate cells are not stopped in their migration, but accumulate in the marginal zone. Cells of the cortical plate (PI-P,) cannot traverse this dense layer of early-generated CR cells and subplate neurons, so they accumulate underneath in the sequence of their generation. The inside-out lamination is reversed. Also, cortical plate cells not getting in contact with the marginal zone at the end of the migration process are not anchored there by their initial processes. As a result, the vertical extension of apical dendrites does not take place. Modified from [21].

572

Neuronal and glial cell biology

marginal zone moves away from the location of the postmigratory cell, resulting in the extension of an apical dendrite that has retained its contact with the marginal zone (Figure lc,d) [‘23”]. In reder mice (which lack Reelin), postmigratory neurons are positioned in the order in which they are generated, their dendrites are not initially anchored in the marginal zone, so the characteristic vertical orientation of the main apical pyramidal cell dendrites is altered (Figure If-h). A role for Reelin as a stop signal for migrating neurons, as hypothesized here, is compatible with its sequence similarities to other extracellular matrix proteins involved in cell adhesion and with observations made in the cortex of reel’er

mice, but has not yet been demonstrated experimentally. As the reel& gene has been cloned [16,17], it will now be possible to design in vitro experiments aimed at testing this directly.

Cajal-Retzius cells and the layer-specific termination of hippocampal afferents As part of the cortex, the hippocampus has a marginal zone containing CR cells. The marginal zone of the hippocampus proper corresponds to the future stratum lacunosum-moleculare and that of the fascia dentata to the dentate outer molecular layer - well-known termination zones of afferents from the entorhinal cortex to the hippocampus [9,27]. Could it be that the laminar specificity of CR cells and entorhinal fibers is causally related? Interestingly, entorhinal fibers arrive in their termination zones very early (around embryonic day 17) [28], long before the majority of their target neurons in the dentate gyrus, the granule cells, are born or have grown dendrites as far as to the outer molecular layer [29]. Could it be that early-generated CR cells serve as transient targets for the entorhinal fibers and keep them in place? By using slice co-cultures of entorhinal cortex and hippocampus, it has indeed been shown that entorhinal fibers find their appropriate layers in hippocampal target cultures [6-81 and establish transient synapses with CR cells [30”]. As in the neocortex [31], the majority of CR cells in the hippocampus undergo degeneration later in postnatal development [32]. The entorhinal afferents to the dentate establish their definitive contacts with distal granule cell dendrites, which by then have arrived in the outer molecular layer. A role for CR cells as transient targets of entorhinal afferents has been confirmed by another series of co-culture experiments in which CR cells in hippocampal cultures were selectively eliminated by excitotoxic lesions [30”]. LJnder these conditions, fibers from the entorhinal co-culture no longer found their target layers, whereas commissural fibers from a co-cultured hippocampal slice terminated as normal in the dentate inner molecular layer [30”]. It is not clear what are the molecular interactions between entorhinal fibers and CR cells during the development of

the entorhino-hippocampal projection. Could it be that Reelin secreted by CR cells is involved? When Reelin is blocked by the CR-SO antibody [18,19”,33”], the fibers from an entorhinal co-culture still found their way to the outer molecular layer [30”]; however, individual fibers followed a straight course and gave rise to very few collaterals compared to control co-cultures [30”]. Similar observations were made when the entorhinal projection was traced in reeler mice [30”]. These findings suggest that Reelin acts as a branching signal for entorhinal fibers but that it is not involved in pathfinding. As elimination of CR cells prevented the entorhinal axons but not commissural fibers from reaching their appropriate target layers, CR cells appear to exert a specific, Reelinindependent effect on the pathfinding of entorhinal afferents. LJsing infrared videomicroscopy in combination with the patch-clamp technique, we recently recorded from CR cells in the outer molecular layer, filled them with biocytin, and found that they gave rise to an early projection to the entorhinal cortex, both in acute slices from young postnatal animals and in co-cultures of entorhinal cortex and hippocampus (K Ceranik ef al., unpublished data). This projection was confirmed by retrograde tracing experiments using DiI and by double-labeling experiments in which outgrowing entorhinal fibers were seen to grow along the axons of CR cells (K Ceranik eta/., unpublished data). We hypothesize from these data that CR cells provide an early projection to the entorhinal cortex that is used by the entorhinal fibers as a guide to find their way to the hippocampus. Entorhinal cells do, in fact, appear to have a specific affinity to CR cells and their axons. When entorhinal cells are dissociated, labeled with a fluorescent marker, and allowed to settle on freshly prepared slices of neonatal hippocampus, they show a specific adhesion to the outer molecular layer (E FGrster, C Kaltschmidt, J Deng, H Cremer, M Frotscher, Sor Neurosri Abstr 1997. 23:1698). This adhesion assay may be used in future experiments to determine the molecular interactions between entorhinal

axons and CR cells.

Different target cells determine fiber segregation in the hippocampus As mentioned above, the layer-specific termination of entorhinal fibers is retained in reeler mutant mice and earlygenerated CR cells are present in the outer molecular layer to guide the entorhinal fibers and serve as transient targets (Figure 2). In the same animals, however, a dramatically altered, broad projection of commissural axons to the molecular layer and hilus is observed (T Deller, A Drakew, B Heimrich, M Frotscher, Sor Neurosci A&r 1997, 232103). In normal animals, commissural fibers give rise to a compact, sharply delineated projection to the inner molecular layer [9]. Commissural fibers arrive late in development, largely postnatally in rodents [28], when the majority of the granule cells are born and have sent their dendrites into the molecular layer. Thus, in contrast to the early-arriving entorhinal axons, the commissural fibers to the fascia

Cajal-Retzius

Figure

cells,

Reelin,

and the formation

of layers

Frotscher

573

2

(a) r/+1+

OML

+ IML 0

GC

(b)

O

rl-/-

0

Current Op~mon I” Neurobiology

Role of CR cells in the layer-specific

to

the fascia dentata.

(r/-I-)

mice, early-arriving

transient synaptic proper termination

termination

In both (a) wild-type entorhinal

fibers

of entorhinal

afferents

(r/+1+) and (b) reeler mutant (black lines) establish

zone of the fascia dentata). At the time of entorhinal fiber ingrowth, the majority of the granule cells (GC) have not yet been formed and have into the molecular

are loosely

layer. (b; left) In the mutant,

afferents.

distributed,

postsynaptic

contacts with CR cells and are thus kept in their zone, the outer molecular layer (OML; marginal

not sent their dendrites

the entorhinal

partners.

their postsynaptic

cells resulting

layer (IML). Because

of their late arrival, many postsynaptic

partner

like

dentata do not need a transient target but are likely to establish synapses directly with their definitive partners, the proximal dendrites of granule cells (Figure 2). This assumption is supported by the abnormal broad commissural projection in reeler mice: as a result of a migration defect in the mutant, the granule cells do not form a dense-

ly packed cell layer, but are loosely distributed the hilar region in a manner similar to their partners, the commissural fibers (Figure 2).

throughout presynaptic

hypothesized commissural

fibers

of their primary when granule

and now establish

synapses

with distal

contacts

of a compact entorhinal termination zone, fibers follow the loose distribution of the granule

from an altered

migration

in the absence

of Reelin. It is

that the segregated termination of entorhinal and afferents to the fascia dentata is determined by initial

with different

target

cells at the time of fiber ingrowth.

afferents. As both the entorhinal and the commissural projections to the hippocampus and fascia dentata are heterogeneous [34,35], it is reasonable to assume that additional types of primary target cells are involved in the establishment of these connections. In fact, Sup&r et a/. [36”] have recently shown that GABAergic neurons play a role as early target neurons for commissural fibers to the hippocampus.

Conclusions It appears that the distribution of primary target cells -CR cells for the early-arriving entorhinal fibers and granule cells for the late-arriving commissural axons - at the time of fiber ingrowth is mimicked by the two projections. Thus, the different types of target cells are an important factor for the segregated, layer-specific termination of hippocampal

(a; right) Later on in development,

partner

fibers (CF; red) arrive and establish synapses directly with the yet short, proximal dendrites of the granule cells (thick black lines) in the fibers do not need a transient

commissural

granule cell dendrites. (b; right) The regular presence of CR cells in the development of the entorhino-hippocampal projection in the mutant leads to the formation whereas commissural

commissural

In the mutant,

the broad distribution

cell dendrites have grown into the outer molecular layer and the majority of CR cells have disappeared, the entorhinal fibers change

granule cells do not form a compact layer, but are loosely distributed as a result of a migration defect. (a; middle) After birth, commissural

inner molecular

(b; middle) reflecting

and outlook

CR cells and Reelin are essential components of the marginal zone in the developing cerebral cortex. By virtue of its sequence similarities with other extracellular matrix proteins involved in cell adhesion, Reelin may act as a stop signal for migrating neurons, thereby exerting an important function in the formation of the inside-out pattern of cortical cell layers.

574

Neuronal and glial cell biology

In the hippocampus, Reelin has been found to affect the branching pattern of entorhinal afferents. By projecting early into the entorhinal cortex, CR cells in the outer molecular layer and stratum lacunosum-moleculare (marginal zones of the fascia dentata and hippocampus proper, respectively) serve as guideposts and transient targets of the early-arriving entorhinal fibers. A similar guidance function has been suggested for the subplate cells in the development of thalamocortical projections [37,X3] and for hippocamposeptal cells in the formation of the septohippocampal pathway [39,40].

The presumed molecular interactions between entorhinal axons and CR cells may be analyzed by using the adhesion assay described above. In addition to entorhinal cells found to adhere to their termination zones in the hippocampus, other identified cells may be tested for their adhesiveness, as well as entorhinal cells from mutants lacking defined adhesion molecules. The presumed effects of Reelin on neuronal migration and branching can be studied directly experiments

in appropriate in Z.&O assays with tissue from reeler mutants.

and

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14.

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15.

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17.

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18.

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in rescue

Finally, the Reelin receptor needs to be identified. Recent studies on mutants with a reeler-like phenotype, such as .s~~nb/e~ [41’,42], have suggested a mutation downstream to the Reelin receptor in other CNS regions which were not dealt ther as well.

7.

[43”,44”]. The functions of Reelin [45%47,48’] and other tissues [49’], with here, need to be explored fur-

of

D’Arcangelo G, Nakajima K, Miyata T, Ogawa M, Mlkoshiba K, Curran T: Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody. J Neurosci 1997, 17:23-31. This paper is the first to demonstrate that Reelin is a secreted glycoprotein. The authors show that a highly charged carboxy-terminal region is essential for secretion. The data presented in this paper suggest that Reelin is secreted by Cajal-Retzius cells and that it is a component of the extracellular matrix in the marginal zone, where Cajal-Retzius cells are located. Moreover, this paper proves that the CR-50 monoclonal antibody recognizes Reelin, in particular an epitope near the amino terminus. 19. ..

Acknowledgements ‘I’hc author thanks hl \I?ntcr for her help with the drawing5 and A IIrakc\\. ‘1‘ Drllcr, E Fiirbter, R Heimrich. (; Raivnan, and E Soriano for man) stimulating discussion\. This work was supported 1~~LI rcsrnrch gmnt from the 1)eutschc P(rrschungsgcmrins~h~ft (SFB .%.~I.

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36. ..

Sup&r H, Martinez A, Del Rio JA, Soriano E: Involvement of distinct pioneer neurons in the formation of layer-specific connections in the hippocampus. J Neuroso 1998, 18:4616-4626. This paper shows that different types of primary target neurons are involved in the formation of layer-specific connections rn the hippocampus. The paper confirms an important role for Cajal-Retzius cells in the layer-specific termination of entorhinal afferents. In addition, GABAergic cells in the termination

41.

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.

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43. ..

Howell SW, Hawkes R, Soriano P, Cooper JA: Neuronal position in the developing brain is regulated by mouse disabled-l. Nature 1997, 388:733-737. This and the paper by Sheldon et a/. [44”1 show that mutations in mdab-I, a mouse gene related to the Drosophila gene disabled (dab), result in a reelerlike phenotype. mDab-1 appears to function as an intracellular adaptor protein. 44. l*

Sheldon M, Rice DS, D’Arcangelo G, Yoneshima H, Nakajima K, Mikoshiba K, Howell SW, Cooper JA, Goldowitz D, Curran T: Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nafure 1997, 389:730-733. The authors report that the autosomal-recessive mouse mutations, scrambler and yorari, arise from mutations rn mdab-1 (see [43”]). 45.

Terashima T: Distribution of mesencephalic trigeminal nucleus neurons in the reeler mutant mouse. Anat Ret 1996, 244:563-571,

46.

Mryata T, Nakajima K, Mikoshiba K, Ogawa M: Regulation of Purkinje cell alignment by Reelin as revealed with CR-50 antibody. J Neurosci 1997, 17:3599-3609.

47.

Schiffmann SN, Bernier B, Goffinet AM: Reelin mRNA expression during mouse brain development Eur J Neurosci 1997, 9:1055-l 071,

48. .

Soriano E, Alvarado-Mallart RM, Dumesnil N, Del Rio JA, Sotelo C: Caial-Retzius cells reaulate the radial alia _ .ohenotvoe _. in the adult and developing cerebellum and alter granule cell migration. Neuron 1997, 18:563-577. This study shows that transplantation of embryonic Cajal-Retzius cells Into the adult cerebellum induces a rejuvenation of host Bergmann glial cells into a radial glia phenotype. Evrdence is provided that these effects are mediated by diffusible molecules. 49. .

lkeda Y, Terashima T: Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse. Dev Dyn 1997, 210:157-l 72. The authors analyzed reelin expressron in various organs during development and adulthood. In adult animals, reel/n IS expressed rn brain, spinal cord, liver, kidney, testis, and ovary, suggesting yet unknown functions of reelin in the mature organism.