Research report
Cajal–Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin

https://doi.org/10.1016/S0165-3806(02)00641-7Get rights and content

Abstract

Cajal–Retzius cells are reelin-secreting neurons found in the marginal zone of the mammalian cortex during development. Recently, it has been proposed that Cajal–Retzius cells may be generated both early and late in corticogenesis, and may migrate into the cortex from proliferative zones in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) or cortical hem. In the present study, we used reelin as a marker to study the properties of Cajal–Retzius cells, including their likely origins, neurotransmitters, and birthdates. In double labeling experiments, Cajal–Retzius cells (reelin+) expressed transcription factors characteristic of pallial neurons (Tbr1 and Emx2), contained high levels of glutamate, were usually calretinin+, and were born early in corticogenesis, on embryonic days (E)10.5 and E11.5. Tbr1+ cells in the marginal zone were almost always reelin+. The first Cajal–Retzius cells (Tbr1+/reelin+) appeared in the preplate on E10.5. In contrast, interneurons expressed a subpallial transcription factor (Dlx), contained high levels of GABA, were frequently calbindin+, and were born throughout corticogenesis (from E10.5 to E16.5). Interneurons (Dlx+) first appeared in the cortex on E12.5. Our results suggest that the marginal zone contains two main types of neurons: Cajal–Retzius cells derived from the pallium, and migrating interneurons derived from the subpallium.

Introduction

Cajal–Retzius (C–R) cells play a critical role in the development of the neocortex and hippocampus. Their foremost function is to regulate the formation of cortical layers, by the constitutive secretion of reelin [9], [17], [32], [36]. C–R cells have also been implicated in maintenance of the radial glia phenotype [48], and may regulate neural circuit formation, in part via reelin signaling as observed in the hippocampus [15], and through electrical activity and neurotransmitter secretion [32]. Reelin is a large glycoprotein that acts as a signal in the marginal zone, to regulate the migrations of cortical plate neurons, which express the receptor molecules VLDLR and ApoER2 [12], [13], [23], [40]. Loss of reelin activity, as occurs in the mouse mutant strain reeler and in rare human neurological cases, causes severe malformations of the cortex as well as the cerebellum [10], [11], [24]. C–R cells have no apparent functions in the mature cortex, and most undergo apoptosis after the cortical layers have formed [16], [43], [51].

Despite many decades of study, some fundamental properties of C–R cells remain in question, including their birthdays and their embryonic origins. C–R cells were thought to rank as the earliest-born cells in the cortex [6], [44], but this view has been challenged by Meyer et al., who proposed that C–R neurons may be produced and added to the marginal zone continuously throughout neurogenesis [36]. Underlying the uncertainty, most previous studies have not used markers to identify C–R cells for birthdating, but have instead relied on the less specific criteria of cell size and shape, in addition to location in layer 1.

With regard to the embryonic origins of C–R cells, many sources have been proposed, including the pallium (cortical neuroepithelium) [1], [20], [22], [32], the subpallium (medial ganglionic eminence) [27], the retrobulbar cortex [33], [34], [35], and the cortical hem region [38]. The precedent that some cortical neurons may originate from extrapallial sources was established with the discovery that most cortical interneurons are born in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) and migrate tangentially into the cortex [2], [3], [4], [27]. C–R cells have recently been considered as possibly another immigrant cell type. Lavdas et al. [27] found that in rat forebrain slices, some cells migrated from the medial ganglionic eminence into the marginal zone of the neocortex, where they displayed a bipolar horizontal morphology and appeared (by epifluorescence microscopy) to express reelin [27]. They thus proposed that some C–R cells originate in the medial ganglionic eminence [27]. On the other hand, Meyer and co-workers [34], [35], [36], [37], [38] have found gradients in the distribution of C–R cells, which seem to originate in the retrobulbar cortex and in the cortical hem. They have proposed that C–R cells may have multiple pallial and extrapallial origins [34], [35], [36], [37], [38].

The neurochemical and molecular properties of C–R cells have been controversial as well. The two primary neurotransmitters used by cortical neurons are glutamate and GABA, which are secreted by projection neurons and interneurons, respectively [4], [22], [26], [31]. C–R cells have been variously reported to contain high levels of glutamate and/or GABA [1], [14], [25], [32], [35]. Equally confusing, C–R cells have been reported to express developmental transcription factors characteristic of both the pallium (Emx2, Tbr1) and the subpallium (Lhx6) [22], [27], [29]. The accumulated data would seem to indicate that C–R cells are an extremely heterogeneous group, with a diversity of origins, and neurochemical and molecular properties.

The confusion surrounding C–R cells has been aggravated by three problems. Firstly, C–R cells may differ between species, not only in dendritic complexity [32], [36], but perhaps in other properties as well. Secondly, it has been unclear exactly how many distinct types of cells inhabit embryonic layer 1 (the marginal zone). Markers such as GABA, acetylcholinesterase, calretinin and calbindin have suggested that at least two cell types are present in the marginal zone, and possibly as many as four [1], [14], [35], [37]. The various cell types cannot be distinguished by morphological criteria alone, as different types of neurons can exhibit very similar appearances; C–R cells and migrating interneurons, for example, both exhibit bipolar horizontal morphology in the embryonic mouse marginal zone [1], [16], [17], [27], [35], [42]. The third problem is that C–R cells have been difficult to define. Calretinin has often been used as a marker for C–R cells in the embryonic mouse (e.g. Refs. [1], [14], [20], [22]), but not all C–R cells are calretinin+ in the mouse hippocampus [39], and calretinin is clearly not specific for C–R cells in the embryonic rat [35]. More recently, reelin has been characterized as a marker of C–R cells in the embryonic cortex of many species, leading to the provisional definition of C–R cells as reelin-expressing neurons in the marginal zone [36]. Nevertheless, it remains uncertain whether C–R cells are a single coherent type of cell, or a heterogeneous population as suggested by their diverse properties.

We previously found that reelin+ C–R cells in the newborn mouse neocortex and hippocampus expressed transcription factor Tbr1 [22], which is a pallial marker [8], [45]. On this basis, we hypothesized that C–R cells in the mouse neocortex are derived mainly or entirely from the pallium. Using double labeling immunofluorescence and confocal microscopy, we tested for co-localization of reelin with glutamate, GABA, Tbr1, Emx2, Dlx, calretinin and calbindin. We also determined the birthdays of C–R cells, by co-localizing reelin or calretinin with bromodeoxyuridine (BrdU), a birthdating tracer, administered on embryonic days from E10.5 to E16.5, covering the major period of corticogenesis [44].

Section snippets

Animals, bromodeoxyuridine (BrdU) injections, and tissue fixation

Mice were used according to a protocol approved by the Institutional Animal Care and Use Committee at the University of Washington, and NIH guidelines were followed. B6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and bred in a colony. Insemination was assessed by the presence of a vaginal plug. Noon of the vaginal plug day was designated embryonic day (E) 0.5.

For experiments using embryos, the dam was anesthetized with pentobarbital (90 mg/kg, i.p.) and killed by

Transcription factor expression

Many developmental transcription factors are expressed in region-specific patterns in the developing telencephalon, including some that are markers for the pallium, for subdivisions of the pallium, or for the subpallial telencephalon [45], [46]. To investigate the telencephalic origins of C–R neurons, two pallial transcription factors (Tbr1 and Emx2), and one subpallial transcription factor (Dlx) were studied.

Discussion

The properties of C–R cells have been controversial, in part due to inconsistent criteria for their identification. In the present study, we used the criteria of Meyer et al. [36], who defined C–R cells as reelin-immunoreactive cells in the marginal zone. We assessed the reelin+ C–R cells for transcription factor expression, neurotransmitters, calbindin and calretinin expression, and birthdays. These properties of C–R cells are important for testing the hypothesis that C–R cells may migrate

Acknowledgements

We thank Dr. Andre Goffinet for G10 anti-reelin, Dr. Jhumku Kohtz for anti-Dlx, and Dr. Morgan Sheng for anti-Tbr1. This work was supported by a grant from NIH/NINDS (K08 NS01973).

References (52)

  • E. Soriano et al.

    Cajal–Retzius cells regulate the radial glia phenotype in the adult and developing cerebellum and alter granule cell migration

    Neuron

    (1997)
  • J.G. Wood et al.

    Evidence that the earliest generated cells of the murine cerebral cortex form a transient population in the subplate and marginal zone

    Dev. Brain Res.

    (1992)
  • S. Alcántara et al.

    Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

    J. Neurosci.

    (1998)
  • S.A. Anderson et al.

    Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes

    Science

    (1997)
  • S.A. Anderson et al.

    Distinct cortical migrations from the medial and lateral ganglionic eminences

    Development

    (2001)
  • S.A. Anderson et al.

    Distinct origins of neocortical projection neurons and interneurons in vivo

    Cereb. Cortex

    (2002)
  • J.B. Angevine et al.

    Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse

    Nature

    (1961)
  • A. Bulfone et al.

    Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries

    J. Neurosci.

    (1993)
  • S.R. y Cajal

    Texture of the cerebral gyri of the lower mammals

  • V.S. Caviness et al.

    The reeler malformation. Implications for neocortical histogenesis

  • G. D’Arcangelo et al.

    A protein related to extracellular matrix proteins deleted in the mouse mutant reeler

    Nature

    (1995)
  • G. D’Arcangelo et al.

    Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody

    J. Neurosci.

    (1997)
  • J.A. Del Rı́o et al.

    Glutamate-like immunoreactivity and fate of Cajal–Retzius cells in the murine cortex as identified with calretinin antibody

    Cereb. Cortex

    (1995)
  • J. Del Rı́o et al.

    A role for Cajal–Retzius cells and reelin in the development of hippocampal connections

    Nature

    (1997)
  • P. Derer et al.

    Axonal secretion of reelin by Cajal–Retzius cells: evidence from comparison of normal and RelnOrl mutant mice

    J. Comp. Neurol.

    (2001)
  • D.D. Eisenstat et al.

    DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation

    J. Comp. Neurol.

    (1999)
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