Research reportCajal–Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin
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).
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