Feature Article: What is a Cajal–Retzius cell? A Reassessment of a Classical Cell Type Based on Recent Observations in the Developing Neocortex

Gundela Meyer, André M. Goffinet1 and Alfonso Fairén2

Departamento de Anatomía, Universidad de La Laguna, 38071 Tenerife, Spain, , 1 Neurobiology Unit, FUNDP Medical School, B5000 Namur, Belgium and , 2 Instituto de Neurociencias, CSIC, Universidad Miguel Hernández, 03550 San Juan de Alicante, Spain

Address correspondence to Dr Gundela Meyer, Departamento de Anatomía, Facultad de Medicina, Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain. Email: gmeyer{at}ull.es.


    The Need for a Revised Definition of the ‘Cajal–Retzius cell'
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The term ‘Cajal–Retzius cell' is applied to neurons of the human embryonic marginal zone which display, as a salient feature, radial ascending processes that contact the pial surface, and a horizontal axon plexus located in the deep marginal zone. These cells were first described by Retzius (Retzius, 1893Go, 1894Go) (see the cell labelled ‘Retzius, 1893' in Fig. 1Go). There is consensus that homologous elements are present in the non-primate neocortex, where their morphology is much simpler, as initially described in 1891 by Cajal (Fig. 2Go) (Cajal, 1891Go). In spite of several reviews (Duckett and Pease, 1968Go; König, 1978Go; Huntley and Jones, 1990Go; Marín-Padilla, 1978Go, 1990Go; Supèr et al., 1998Go), the definition of these cells has remained somewhat confusing, in part because Cajal and Retzius studied different species and different developmental stages, and also because their original publications have not been generally available. In fact, the cells drawn by Cajal (Cajal, 1899aGo,bGo, 1911Go) from his human material (see the cell labelled A, ‘Cajal, 1899' in Fig. 1Go) appear morphologically so different from those described by Retzius (Retzius, 1893Go, 1894Go) that they must belong to different, although possibly related, cell classes. The discrepancies in the observations by Retzius and Cajal could reflect vagaries of the Golgi method, or different magnifications or drawing techniques, rather than actual morphological differences in the cell types represented. However, recent data have confirmed the accuracy of the original observations. The morphological definition of Cajal–Retzius cells is thus based on observations of rather disparate cells that may not be homologous in all mammalian species.



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Figure 1.  Selected drawings by Retzius, Cajal, and Meyer and González-Hernández (Retzius, 1983; Cajal, 1899; Meyer and González-Hernández, 1993). Drawings by Cajal and Retzius show that both authors referred to different cell types present at different developmental moments and in different species. Retzius depicted the exuberant morphologies of the neurons present in the marginal zone of human fetuses around mid-gestation (Retzius, 1893Go); we consider these neurons the ‘Retzius cells'. Cajal drew the neurons in layer I of the human fetus at term and newborn (Cajal, 1899). These cells are smaller, and have a more superficial location than Retzius cells; we refer to them as ‘Cajal cells'. The drawings have been scaled with reference to the drawings by Meyer and González-Hernández of DiI-stained Retzius (cells a–c) and Cajal (cell d) cells (Meyer and González-Hernández, 1993). The drawing by Cajal has been altered with Photoshop software to eliminate cell processes coming from other cellular elements represented in the original drawing. Scale bar: 50 µm.

 


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Figure 2.  Cajal described in 1891 slender horizontal bipolar cells in the developing marginal zone of lagomorphs. These cells were considered by Retzius as homologues to the cells he found in humans and in other mammals (Retzius, 1893Go, 1894Go). Similar cells are also present in the rodent marginal zone, and are usually termed ‘Cajal–Retzius cells'.

 
In particular, DiI tracing visualizes cellular details in fixed human specimens in a Golgi-like fashion. This technique has given an exhaustive catalogue of Cajal–Retzius cell morphologies present in the human fetus, and their changes during different phases of gestation (Meyer and Gonzalez-Hernández, 1993) (Fig. 1Go, cells labelled ‘Meyer, 1993'). Similarly, immunohistochemical work in human fetuses (Verney and Derer, 1995Go; Belichenko, 1995; Uylings and Delalle, 1997Go) as well as in non-human primates (Huntley and Jones, 1990Go; Hendrickson et al., 1991Go; Yan et al., 1995aGo,bGo) confirmed the descriptions by Cajal and Retzius (see below).

The currently prevailing view on Cajal–Retzius cells is based on the hypothesis of the dual origin of the cerebral cortex, initially proposed by Marín-Padilla (Marín-Padilla, 1971Go, 1972Go, 1978Go). It holds that the cerebral cortex derives from an early neural network, originally named the ‘primordial plexiform layer' (Marín-Padilla, 1971Go, 1972Go, 1978Go) and subsequently renamed the ‘preplate' (Rickmann and Wolff, 1981Go). The preplate contains mutually interconnected early generated neurons and is split apart by the cortical plate (CP) that will contribute layers II–VI, with the result that preplate derivatives settle superficially, in the marginal zone (MZ), or future layer I, as well as deeply, in the subplate, at the interface with the future white matter. It is generally inferred that the derivatives of the preplate that settle in the MZ are the Cajal–Retzius cells. According to Marín-Padilla, early Cajal–Retzius cells send axonal projections into the subplate. The descending axons, together with ascending fibers from Martinotti-type cells, are essential components of his concept of a primordial cortical neuropil (Marín-Padilla, 1971Go, 1978Go, 1998Go).

The hypothesis of an early birth of neurons in the MZ and subplate, originating from the preplate, followed by the division of the preplate when the CP appears, has been amply confirmed (König et al., 1977Go; Rickmann et al., 1977; Raedler and Raedler, 1978Go; Luskin and Shatz, 1985Go; Chun and Shatz, 1989Go; Wood et al., 1992Go). These studies provided unequivocal evidence that the oldest neurons of the cortex do in fact settle above the cortical plate, in layer I, as well as below, in the superficial white matter. However, while simple and attractive, the inference that the preplate elements in the MZ correspond to the classical Cajal– Retzius cells is far from obvious. In the present paper, we wish to propose that this view is too restricted to account for some observations in the human and rodent developing cortex (Meyer and Gonzalez-Hernandez, 1993Go; Meyer and Goffinet, 1998Go; Meyer et al., 1998Go, 1999Go; Soria et al., 1998Go; Soria and Fairén, 1999Go) and needs to be significantly modified.

Recently, so-called Cajal–Retzius cells were shown to secrete Reelin (Reln), the product of the reeler gene (Ogawa et al. 1995Go; D'Arcangelo et al., 1995Go, 1997Go; Ikeda and Terashima, 1997Go; Schiffmann et al., 1997Go; Alcántara et al., 1998Go; Meyer and Goffinet, 1998Go; Meyer et al., 1998Go). The availability of new markers provides an opportunity to study in more detail the typology of developing neurons in the neocortical marginal zone. We studied brains from various species, namely human fetuses (Meyer and Goffinet, 1998Go), pre- and postnatal mice and rats (Meyer and Fairén, 1996; Meyer et al., 1998Go; Soria et al., 1998Go) and late fetal and postnatal kittens (unpublished). We used anti-Reelin antibodies (de Bergeyck et al., 1998Go), as well as other immunohistochemical markers such as calretinin, calbindin, GABA and GAD antibodies, and bromodeoxyuridine (BrdU) birthdating. Our data show that Reln is expressed by different neuronal populations in the developing marginal zone MZ, some of which appear quite different from the cells described in the classical studies of Cajal and Retzius. Although some preplate derivatives do express Reln, others do not. Furthermore, at variance with the dual-origin hypothesis of Marín-Padilla, we have observed that a substantial contingent of Reln-positive cells in the MZ seem to derive from a discrete sector of the neuroepithelium in the basal forebrain and to invade the MZ by tangential subpial migration. In addition, some early-born neurons in the marginal zone, which do not express Reln, give rise to the first pioneer axon projections of the embryonic cortex reaching as far as the lateral ganglionic eminence before subplate neurons are generated; we named these cells ‘pioneer neurons' of the MZ (Meyer et al., 1998Go). Together with subplate cells, these pioneers contribute an early efferent projection from the preplate.

These data suggest that it would be inappropriate and confusing to label all neurons in the MZ as ‘Cajal–Retzius cells'. We would rather define ‘Cajal–Retzius cells' loosely, as the family of Reln-immunoreactive (ir) neurons in the marginal zone, and reserve the term of pioneer neurons for the early, Reln-negative preplate derivatives that settle in the MZ and project to sub-cortical levels. This terminology avoids confusion and reflects the complexity of early corticogenesis.

The early descriptions by Cajal and Retzius referred to the neocortex. Similarly, the present account deals with the neo-cortical MZ, even though the term ‘Cajal–Retzius cell' has been extended to neurons in the marginal zone of the hippocampus (Soriano et al., 1994Go; Drakew et al., 1998Go; Supèr et al., 1998Go; Alcántara et al., 1998Go). Although these hippocampal neurons do express Reln (D'Arcangelo et al., 1995Go; Ikeda and Terashima, 1997Go; Drakew et al., 1998Go), studies of the fetal human hippocampus suggest that their development is largely independent from that of the neocortical Cajal–Retzius cell family (Meyer, 1998Go).


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Initially, Retzius described neurons present in the human MZ around mid-gestation, characterized by large horizontal, sometimes vertically oriented somata located at some distance from the pia (Retzius, 1893Go). These ‘Retzius cells' give rise to a rich axonal arborization oriented horizontally, situated below the perikaryon, and their horizontal dendrites extend characteristic comb-like ascending processes that contact the pial surface. Although this may seem a convenient summary of the morphology of these cells, in practice it is not, since they show an extraordinary variety of neuronal shape and orientation (Fig. 1Go, cell labelled ‘Retzius, 1983'). Meyer and Gonzalez-Hernandez named the transient cell population present around midgestation ‘polymorphic Cajal–Retzius cells' (Fig. 1Go, DiI-stained cells labelled ‘Meyer, 1993, a–c') (Meyer and Gonzalez-Hernandez, 1993Go). Our more recent study (Meyer and Goffinet, 1998Go) showed that these cells are Reln-positive (Fig. 3Go). We propose here the term ‘Retzius cells' because it is simple and more in accord with the images drawn by Retzius.



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Figure 3.  Reelin-immunoreactive (ir) neurons in preplate (PP) and marginal zone (MZ) of the human cortex. (A) Preplate stage, 6 gestational weeks (GW). The arrows point to reelin-ir neurons. (B) Reelin-ir neurons in the MZ of a fetus at 8.5 GW. They display at this moment a simple monopolar morphology; they are located immediately below the pia. The cortical plate (CP) is devoid of reelin-immunoreactivity. (C,D) 18 GW. Reelin-ir Retzius cells separating from the subpial granular layer (SGL). In (D), Retzius cells appear in clusters. (E) Retzius cells of a human fetus at 23 GW. Retzius cells have a protean shape and may be horizontally or vertically oriented. The soma of the vertical neuron shows numerous vacuoles (arrows) suggestive of incipient degeneration. (FH) Cajal cells of the human cortex at 29 GW. Cajal cells are smaller and more superficially located than Retzius cells, but they also express reelin. H represents a subpial pyriform neuron. Arrows in (G) and (H) point to the thick descending process, frequent in these cells. Bar in (A), for (A) and (B) 30 µm; in (C,D) 25 µm; in (E), for (F) and (H) 15 µm.

 
Shortly after mid-gestation, Retzius cells display signs of degeneration (Meyer and Gonzalez Hernandez, 1993Go; Meyer and Goffinet, 1998Go). In these studies, signs of degeneration were evident with Nissl and DiI staining, AChE histochemistry and immunohistochemistry for calretinin or Reln, and consisted of shrunken, vacuolated dendrites and cell bodies (Fig. 3Go) and the appearance of extremely extravagant forms [other authors have given similar interpretations (Wahle and Meyer, 1987Go; Valverde and Facal-Valverde, 1988Go)]. In our material, Retzius cells disappear from the MZ within a short period of 2 or 3 weeks, presumably by cell death. This time lapse is hardly compatible with a dilution by tangential cortical growth (Marín-Padilla, 1990Go, 1998Go; Spreafico et al., 1999Go), the more so since the maximal surface expansion of the cortex occurs during cortical folding, i.e. the formation of secondary sulci and gyri, that takes place much later, during the last two months of gestation, after Retzius cells have disappeared. Downregulation of Reln expression cannot account for the apparent decrease in the number of Reln-ir cells, as Retzius cells also decrease drastically in Nissl-stained sections after the 25th gestational week (GW), suggesting that they die.

Programmed cell death occurs widely during development, particularly in the subplate (Wahle and Meyer, 1987Go; Valverde and Facal-Valverde 1988Go; Kostovic and Rakic, 1990Go; Spreafico et al., 1995Go; Price et al., 1997Go) and in the cortical plate (Meyer and Wahle, 1988Go; Wahle and Meyer, 1987Go, 1989Go; Blaschke et al., 1996Go), and Retzius cells in the MZ may provide a further example of this phenomenon (Ranke, 1910Go; Sas and Sanides, 1970Go; Bradford et al., 1977Go; Derer and Derer, 1990Go; Meyer and Gonzalez-Hernandez, 1993Go; Del Rio et al., 1995Go). However, a recent report by Spreafico et al. using the terminal dUTP nick-end labelling (TUNEL) method to assess apoptosis in the fetal human MZ failed to identify dying Cajal–Retzius cells after mid-gestation (Spreafico et al., 1999Go). Two explanations may be offered for this discrepancy with our own results: Retzius cells may die through a mechanism not visualized by the TUNEL method; or, alternatively, the apoptotic neurons identified by these authors as subpial granule cells would actually correspond to dying Retzius cells, which shortly before their death display shrunken and thus smaller nuclei (Meyer and Gonzalez-Hernandez, 1993Go).

The cells described by Cajal in humans were Golgi-impregnated in the cortex of newborn infants (Cajal, 1899aGo,bGo, 1911Go); similar neurons were also described by Retzius in a paper that included material from a 9-month-old fetus (Retzius, 1894Go). These ‘Cajal cells' differ from Retzius cells in that they lie closer to the pia and display smaller, often triangular or pyriform somata, and less complex processes that lack the ascending branchlets (see Fig. 1Go, cell labelled ‘Cajal, 1889', A). Small horizontal bipolar forms are also common. The pyriform Cajal cells show, as their most peculiar morphological trait, a thick descending process from which thin horizontal processes with short collaterals originate. The neuron in Figure 1Go, cell d, ‘Meyer, 1993', is a DiI-labelled Cajal-type neuron, and shows surprising similarities with the neurons in Cajal's drawings.

In a previous study (Meyer and Gonzalez-Hernandez, 1993Go), this cell population was termed ‘persisting Cajal–Retzius cells', to set it apart from the transient polymorphic cells. Our recent studies (Meyer and Goffinet, 1998Go) show that Reln-ir neurons, morphologically similar to those drawn by Cajal (Fig. 3Go), are found in the MZ only from the last trimester of gestation onwards. When Cajal cells appear, Retzius cells have already largely disappeared from the MZ; while Cajal cells are occasionally found in adult brains, Retzius cells are not. A theoretical possibility is that transformation of Retzius cells into Cajal cells accounts for the changes of neuronal populations in the MZ (Cajal, 1911Go; Marín-Padilla, 1998Go). However, we based our observations and conclusions on a large gapless ontogenetic series of fetal human brains labelled by DiI or stained for acetylcholinesterase and Reln (Meyer and Gonzalez-Hernandez, 1993Go; Meyer and Goffinet, 1998Go), which visualize the entire populations of Retzius and Cajal cells, in contrast to the Golgi method which stains at random only a few neurons in each preparation. Therefore, we favour the idea that Retzius and Cajal cells develop sequentially in the MZ.

In accordance with our proposed timetable, immunohistochemical studies performed at advanced developmental stages of human and macaque cortex, or in adult humans, visualize mostly triangular or small bipolar MZ neurons, our Cajal cells (Belinchenko et al., 1995; Uylings and Delalle, 1997Go; Huntley and Jones, 1990Go). In contrast, studies of the human mid-gestation period clearly describe the Retzius type (Verney and Derer, 1995Go).

How do the cells depicted by Marín-Padilla (Marín-Padilla, 1978Go, 1998Go) fit in our scheme? First of all, the Golgi-stained human material of this author, similarly to Cajal's sections, covers predominantly the late gestation or early postnatal period. It is not surprising, therefore, that most of his Cajal–Retzius cells look like the Cajal type rather than the Retzius type.


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The subpial granular layer (SGL) is a transient cell layer, visible from the 14th GW onwards and located immediately below the pial surface, thus far considered specific to the human cortex. First described by Ranke (Ranke, 1910Go), it originates from a discrete sector of the neuroepithelium, the retrobulbar area in the human basal forebrain (Fig. 4A,BGo), although contributions from other sources are possible. The SGL spreads over the entire neocortex through tangential, subpial migration (Brun, 1965Go; Gadisseux et al., 1992Go). While the neuronal nature of the granule cells in the SGL has been questioned (Marin-Padilla, 1995Go), histochemical and immunohistochemical studies showed that most, if not all, are neurons (Gadisseux et al., 1992Go; Meyer and Gonzalez-Hernandez, 1993Go; Spreafico et al., 1999Go; Meyer and Wahle, 1999Go). Our observations suggest that the SGL stands as a precursor pool for Reln-producing neurons that complement earlier-generated cells during the critical period of cortical migration, thus keeping pace with the surface expansion of the growing cortex (Meyer and Goffinet, 1998Go). As radial migration is arrested at the interface of the cortical plate and marginal zone (Anton et al., 1996Go), addition of neurons to the MZ after the appearance of the cortical plate most probably occurs by tangential migration. Thus, through the SGL, granule cells are continuously added to the MZ from the 14th GW; these granule cells, which are initially Reln-negative — but express the calcium binding protein calretinin — then gradually differentiate into Reln-producing neurons. Interestingly, from the 14th to the 24th GW, SGL-derivatives assume the phenotype described by Retzius (Fig. 1Go, cells labelled ‘Meyer, 1993', a–c; Fig. 3Go), while the neurons differentiating after this period resemble the cells described by Cajal (Fig. 1Go, cell labelled ‘Meyer, 1993', cell d; Fig. 3Go). The SGL would thus provide a continuous supply of precursors of Reln-ir neurons: Retzius-type cells during the period of maximal neuronal migration into the cortical plate, and Cajal-type cells during the time of maximal surface expansion of the cortex, when the secondary sulci and gyri begin to form. However, both Retzius and Cajal cells express Reln, and could in this sense be considered members of a loosely defined Reln-producing Cajal–Retzius family.



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Figure 4.  The retrobulbar area. (A,B) The retrobulbar area of a human fetus at 8.5 GW, after the appearance of the cortical plate. The retrobulbar ventricular zone gives rise to reelin-ir neurons which migrate into the cortical marginal zone, following medial and lateral routes, indicated by arrows. The olfactory bulb (ob) lacks the superficial layer of Reln-ir neurons characteristic of the cortex. (C) Coronal section of an E13 mouse brain, showing calretinin-ir neurons in the retrobulbar area in continuity with the preplate (white arrows). Calbindin-ir cells are visible in the medial aspect of the ventricle (asterisk). (D) Front view of an E12 rat brain wholemount immunostained for calbindin, showing the olfactory bulb (ob) and retrobulbar area. Fibers of the vomeronasal (vn) and terminal (nt) nerves mark the initial migration route of subpial granular cells; ge, ganglionic eminence. Bars: (A) 325 µm; (B) 50 µm; (C,D) 120 µm in (C).

 
We have recently observed Reln-ir cells in the human preplate, at 6 GW, i.e. before the emergence of the cortical plate, which do not display the complex morphology of Retzius cells (G. Meyer and A.M. Goffinet, unpublished) (Fig. 3AGo). Instead, these early Reln-expressing cells distribute diffusely within the preplate; they have the simple horizontal monopolar morphology of tangentially migrating neurons (O'Rourke et al., 1992Go, 1995Go; Tamamaki et al., 1997Go). Although most of these early neurons may derive from the local neuroepithelium, some of them may derive from distant non-cortical sources as seems to be the case in rodents (Lavdas et al., 1999Go). The early Reln-ir neurons may differentiate into the Retzius-cells of later stages, but as long as direct evidence is lacking, we would prefer the term ‘Reln-ir preplate neurons'.

Neurons in the early human preplate showing similar distribution and morphology have been shown to express GABA (Zecevic and Milosevic, 1997Go), and the possibility that they coexpress Reln needs to be explored further. This is suggested by the fact that Reln is co-expressed with GABA in an early preplate cell population of the rat cerebral cortex (Meyer et al., 1998Go).

Slightly later in development, from the 8th to the 13th GW, Reln-ir cells in prospective neocortical territories are closely apposed to the pial surface and display a very simple mono- or bipolar horizontal morphology (Fig. 3BGo). Their location and shape, together with the presence of a latero-medial gradient in their packing density apparently originating at the retrobulbar neuroepithelium, strongly suggest tangential subpial migration (Meyer and Wahle, 1999Go). If this hypothesis is correct, both radial and tangential migration would contribute Reln-positive neurons to the embryonic and early fetal MZ. This point, however, remains to be demonstrated further.


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The hypothesis outlined above proposes that, in addition to Reln-ir preplate derivatives, different Reln-ir neurons migrate tangentially in the MZ and differentiate sequentially. This hypothesis is best demonstrated in the human neocortex (Meyer and Goffinet, 1998Go; Meyer and Wahle, 1999Go), but we have suggested that it also applies to non-primate species (Meyer et al., 1998Go).

Birthdating studies in rat (Raedler and Raedler, 1978Go; Bayer and Altman, 1990Go; Rickman et al., 1997), mouse (Fairén et al., 1986Go) and ferret (Peduzzi, 1988Go) show that neurons are added to the MZ during the entire period of corticogenesis. The simplest way for these neurons to reach layer I would be by tangential migration. Furthermore, the SGL and neuronal elements with features of early subpial Reln-ir neurons, Retzius cells and Cajal cells can be defined in all species examined.

Contrary to the assumption that the SGL is specific to the human or primate cortex, our observations (Meyer et al., 1998Go) have confirmed the existence of a rodent SGL as a distinct cellular component of the MZ. Granule cells in the rodent SGL most likely arise from a specific sector of the neuroepithelium behind the olfactory bulb, the retrobulbar area (Fig. 4C,DGo), from where they appear to spread through tangential subpial migration into the MZ of the neocortex.

The limited spatiotemporal resolution in these species compared to human fetuses makes it difficult to distinguish the early preplate neurons from later SGL neurons. Several subpopulations can be defined in the preplate and MZ, however, on neurochemical grounds. In the rat, GABA is expressed by early neuron cohorts of the preplate (Van Eden et al., 1989Go; Cobas et al., 1991Go) [reviewed by Fairén et al. (Fairén et al.,1998Go)], and these early GABA-ir cells co-express Reln (Meyer et al., 1998Go). In addition, SGL cells express calbindin and calretinin. The rat SGL neurons start expressing Reln before they differentiate into Retzius or Cajal cells (Meyer et al., 1998Go), but we have chosen to designate as Retzius or Cajal cells only MZ Reln-ir cells in postnatal animals. In this aspect we differ from most current studies on rodent cortical development.

The rationale of our choice is based on the initial description by Cajal (Cajal, 1891Go) of these cells in postnatal lagomorphs (see Fig. 2Go), and the different time-course of corticogenesis in primates compared to rodents. Although the ascending processes are less visible than in humans, Retzius-type cells are characterized by vertical processes that ascend to the pia. By contrast, SGL cells look like the undifferentiated, possibly migratory cells in the human SGL. The transformation of SGL cells into Retzius cells concurs with their descent in the MZ (Meyer et al., 1998Go), a process that does not take place in reeler mice (Derer, 1985Go). Retzius cells attain their highest differentiation in the first postnatal week and die during the second week. Contrary to the case in the mouse (Weisenhorn et al., 1994Go; del Rio et al., 1995Go), calretinin is not a permanent marker of Retzius cells in rats, where an important cohort of calretinin-expressing cells does not express Reln and dies during the early postnatal period. Other Retzius cells, co-expressing Reln and calretinin, survive for longer periods in layer I. During the early postnatal period, GABA downregulates in layer I or, alternatively, the cells expressing it die, so that Retzius cells do not express GABA (Meyer et al., 1998Go).

The most likely homologues of the Cajal-type cells appear as small subpial pyriform elements subjacent to the pial surface; they differentiate from the SGL by the end of the first week; some of them persist and continue to express Reln into adult life. Subpial pyriform cells express Reln and calretinin (Meyer et al., 1998Go) and also GABA (Imamoto et al., 1994Go).

It may seem ironic that the first description of Retzius cells (as defined here) was indeed due to Cajal (Cajal, 1891Go) (Fig. 2Go). Therefore, we suggest maintaining the name Cajal–Retzius to designate the Reln-expressing cells of the early postnatal period of the rodent (Meyer et al., 1998Go).

Derer and Derer stained Cajal–Retzius cells in late prenatal and postnatal mice using HRP in vitro labelling and in tangential sections (Derer and Derer, 1995). These authors confirmed the existence of the ascending appendages reaching the pia in this species, and described very long axons of Cajal–Retzius cells, in the range of millimetres. The axons, at variance with primates, extend within the same depth as the cell bodies and dendrites. These morphological features imply advanced differentiation, which is not present in SGL cells.

Reln-ir Retzius cells are also present in the perinatal cat cortex (G. Meyer, unpublished results) (Fig. 6AGo). Although there are considerable morphological differences between the human Retzius cells and the much simpler cat homologues, the defining features such as the comb-like ascending appendages and the large soma are evident (Meyer and Ferres-Torres, 1984Go). At post- natal day 20, most Retzius cells have disappeared, and instead the MZ is now populated by Cajal cells (Fig. 6B,CGo), along with many small non-pyramidal cells, all of which secrete Reln. As in the human cortex, the cat Cajal cells have a subpial position, triangular or pyriform somata, and a single descending process (Fig. 5B,CGo, arrows).



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Figure 6.  (A) Panoramic view of a coronal section of the cortical vesicle of a rat embryo at E13, immunostained for calbindin. Composite photomicrograph modified from Figure 5Go in Meyer et al. (Meyer et al., 1998Go). It has been altered with Photoshop software to enhance contrast of the descending axonal projection of pioneer neurons. This projection descends into the incipient internal capsule (ic) in the lateral ganglionic eminence. (B) Enlarged portion of (A) Arrows in (A) and (B) point at identical pioneer neuorns, which are located immediately below the preplate surface and form a single row of cells. (C) The cortical primordium of a rat fetus at E16. Below the subpial granular layer (SGL), pioneer neurons are seen in the upper tier of the cortical plate, and maintain a descending axon (arrow). Subplate cells are also visible. Bars: (A) 150 µm; (B) 50 µm; (C) 25 µm.

 


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Figure 5.  Retzius cells and Cajal cells in the cat. (A) Retzius cells are present prenatally (cat fetus, E55) as well as during the first postnatal days. They are strongly reelin- immunoreactive. (B,C) Cajal cells in a kitten at P20, when Retzius cells are no longer present. Cajal cells are characterized by their smaller size, superficial location and reelin expression. Arrows point to the descending processes. Bar: 50 µm.

 

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 The Reelin-negative Pioneer...
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As outlined above, so-called Cajal–Retzius cells are generally considered preplate derivatives and the oldest neurons of the cerebral cortex, and are sometimes referred to as ‘pioneer Cajal–Retzius neurons' (Supèr et al., 1998Go). However, the data summarized above suggest that this simple view that Cajal– Retzius cells and pioneer preplate derivatives in the MZ are one and the same should be modified. The term ‘pioneer neuron' was originally introduced to define neurons that send pioneer axons (Harrison, 1910Go; Bate, 1976Go), i.e. axons that guide the growth of follower axons. In this sense, as the axon of Cajal–Retzius cells does not leave the MZ, the term of pioneer neurons might not be adequate. In addition, from a historical standpoint it is worth noting that neither Retzius nor Cajal looked at early fetal stages (the youngest fetus examined by Retzius was 4 months old, while the earliest fetus in Cajal's material was 8 months old). They could not know whether they described the earliest neurons of the developing cortex and never claimed so. As mentioned above, we found Reln-positive cells in the human preplate from the 6th GW, the first age examined, while typical Retzius cells develop their typical shape around the 14th GW, and then increase in number over the following weeks. This is in keeping with the original description by Retzius, but contradicts the assertion that their number is fixed at the preplate stage, before the formation of the cortical plate (Marin-Padilla, 1998Go), which in man takes place around the 8th GW. Although the preplate contains Reln-ir neurons, it cannot give rise to all Reln-positive cells in the MZ. Conversely, some early preplate derivatives in the MZ are Reln-negative. In the rat, early-born Reln-negative neurons begin to develop in the MZ prior to the appearance of Reln-positive cells. While the axons of Cajal–Retzius cells do not leave the marginal zone (Derer and Derer, 1990Go; Meyer and Gonzalez-Hernandez, 1993Go; Verney and Derer, 1995Go), these early Reln-negative cells emit the first efferent axonal projections of the preplate, and in that sense would correspond to genuine pioneer cells (Fig. 6Go).

Pioneer neurons are generated before subplate neurons and their axonal projection is seen when the preplate is formed by a monolayer of cells (Fig. 6A,BGo, arrows). The axons fasciculate as they pass through the intermediate zone of the cortical primordium (Fig. 7Go) to the incipient internal capsule in the lateral ganglionic eminence; axon collaterals also reach the ventricular zone (Meyer et al., 1998Go). In keeping with their pioneer character (Bovolenta and Mason, 1987Go; Kim et al., 1991Go), their axons are cupped with large, elaborate growth-cones. This pioneer axon projection appears before the pioneer descending projection from the subplate (de Carlos and O'Leary, 1992Go; Métin and Godement, 1996Go; Richards et al., 1997Go; Molnár et al., 1998Go). Pioneer cells are clearly different from subplate cells. In tangential views of the cortical surface they appear as a huge neuronal population that is maintained after the appearance of the cortical plate. At this developmental period (Figs 6CGo, 7CGo), MZ cell axons can be followed through the cortical plate to the subplate (Meyer et al., 1998Go) but the projection into the incipient internal capsule seems to be lost, being replaced by the subcortical projection from the subplate.



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Figure 7.  (A,B) Presumptive pioneer neurons in the incipient cortical plate (CP) of a human fetus at 8.5 GW. Calretinin-ir pioneer (p) neurons are located at the upper border of the CP, and emit descending fibers (in B, indicated by an arrow). Subplate (SP) neurons are also calretinin-ir. (C) The early cortical plate in an E16 rat fetus, stained for calbindin, located in the upper border of the CP. In the developing rat, pioneer neurons may be calbindin-or calretinin-ir. Bar in (A) (30 µm) applies to all photomicrographs.

 
In comparison to the first Reln-positive cells, the Reln-negative pioneer neurons are larger and located progressively deeper in the MZ (Figs 6CGo, 7CGo). They disappear before birth, earlier than the Reln-positive cells, and thus represent another transient cell population restricted to a specific period of cortical development. During their short lifespan, pioneer neurons may play a role, together with subplate neurons (McConnell et al., 1989Go; de Carlos and O'Leary, 1992Go), in establishing the first projections of the cortex. Their distinctive tangential distributions might be involved in an early territorialization of the cortical vesicle that clearly precedes the invasion of the subplate by thalamo-cortical axons (Soria et al., 1998Go; Soria and Fairén, 1999Go). On the other hand, the fact that pioneer neurons do not secrete Reln (Meyer et al., 1998Go) sets them apart from the Cajal–Retzius cell group. They are, moreover, early functioning neurons that express functional GABAA and NMDA receptors (Soria et al., 1999Go). Neurons with a similar morphology and axonal projection pattern were described with the Golgi method in the earliest stage of the embryonic cat cortex, but they were thought to represent the early form of Cajal–Retzius cells, which later would retract the descending axon and undergo a ‘horizontalization process' (Marín-Padilla, 1971Go, 1998Go).

An important question concerns the presence of pioneer neurons in the human cortex, in which they have not yet been described. Our preliminary data suggest that some calretinin-ir neurons may be the homologues of the rodent pioneer neurons (Fig. 7A,BGo).They are placed in the lower part of the MZ, have a descending axon and invade the upper tier of the cortical plate. Clearly, the identification of the early preplate derivative of the human or primate MZ requires further investigation.

In sum, the theory that all Cajal–Retzius cells are derivatives of the preplate and remain unchanged from the first moments of corticogenesis is no longer tenable. Recent observations suggest that there are dynamic changes of cell populations in the marginal zone during corticogenesis, with new neurons being continuously added, while others die after having fulfilled their developmental role. The Reln-producing members of the Cajal– Retzius family are an important part of these populations. While some of them may derive from the preplate, as it has been universally accepted, others invade the MZ by tangential, subpial migration. Retzius cells correspond more closely to what is commonly understood as the prototype of the human Cajal– Retzius cell, and they are transient, restricted to the period of cortical migration. Cajal cells mature later in development, and may persist into adult life. In addition to Reln-positive cells, the developing MZ, from its earliest developmental stage, contains Reln-negative pioneer neurons that establish the first efferent projections of the cortex, and may play a significant role in the territorialization of the early cortical vesicle.


    Acknowledgments
 
We thank Victoria Bello, Lidia Ruiz and Antonio Arnedo for technical help. A.F. wishes to thank the members of his laboratory for discussion. We also wish to thank J.G. Parnavelas for sharing unpublished results and J.G. Nicholls for reading the manuscript. Supported by grants DGICYT PB94–0582 (G.M.), FRSM 3.4533.95 and ARC 186 (A.M.G.) and DGICYT PB94–0219-CO2–01 (A.F.).


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