A unique subpopulation of Tbr1-expressing deep layer neurons in the developing cerebral cortex
Introduction
Remarkable cellular complexity is a hallmark of the cerebral cortex and is requisite for both the speed and precision of cortical information processing. This complexity, however, creates difficulties in understanding the contributions of particular populations of cells to cortical organization. The first cells generated in corticogenesis, pioneer neurons, initially populate the preplate (PP) and then reside within a superficial marginal zone (MZ) and a deep subplate zone (SP) with later-born cells of the cortical plate (CP) accumulating in between (Allendoerfer and Shatz, 1994, Bayer and Altman, 1990, Luskin and Shatz, 1985). Populations of MZ and SP neurons have features in common with one another. For example, cells of both the MZ and SP express the Golli protein and a Golli-regulated transgene (Landry et al., 1997, Landry et al., 1998). Pioneer neurons are also heterogeneous in nature. Yet, identifying cellular and molecular markers of discrete populations of early-born cortical neurons has been a challenge (Hevner et al., 2001, Landry et al., 1998, Shatz et al., 1990).
SP and deep CP cells guide the formation of appropriate connections between the thalamus and the cortex (Ghosh et al., 1990) and shape cortical organization (Allendoerfer and Shatz, 1994, Antonini and Shatz, 1990, Chun and Shatz, 1989a, Friauf et al., 1990, McConnell et al., 1989, Meinecke and Rakic, 1992). The SP and deep CP, however, consist of several cellularly and functionally diverse populations. For example, connectional distinctions exist: some SP cells project subcortically (De Carlos and O'Leary, 1992, Friauf et al., 1990, McConnell et al., 1994, Shatz et al., 1990), while others project into the overlying cortical plate (Finney et al., 1998, Friauf et al., 1990), and still others contribute to local circuit neuronal populations (Antonini and Shatz, 1990). SP cells also vary in the expression of neurotransmitters, neuromodulators, and other cellular proteins, such as calcium binding proteins (Antonini and Shatz, 1990, Chun and Shatz, 1989a, Finney et al., 1998, McConnell et al., 1989). Developmental paths can also be distinct; some SP cells die postnatally (Chun and Shatz, 1989a, Price et al., 1997), while others persist, with some proportion becoming interstitial cells (Chun and Shatz, 1989b, Kostovic and Rakic, 1980, Meyer et al., 1991, Valverde and Facal-Valverde, 1988). Given this heterogeneity, analyses of SP and deep CP function are likely to have characterized phenotypes that corresponded to effects generated by several different cell types (Ghosh and Shatz, 1992, Ghosh et al., 1990, Hevner et al., 2001, Xie et al., 2002). Much of what we know about the SP and deep CP is derived from studies in cat, ferret, and nonhuman primates because of the relative accessibility of this cortical domain in these species. As a result, little is known about the rodent SP and deep CP.
Transgenic analysis has helped to illuminate the properties and functions of discrete cell types that were previously unrecognized in the rodent cerebral cortex (Cohen-Tannoudji et al., 1994, Feng et al., 2000, Monuki et al., 2001). Moreover, transgenic perturbations of gene expression in cortical cells elucidated roles for particular molecules (Borg and London, 2002, Chenn and Walsh, 2003) and select cortical cell types (Campsall et al., 2002, Xie et al., 2002). The success of transgenic approaches to the study of cellular diversity within the cortex, however, relies upon the existence of cell-type-specific regulatory elements. A variety of transcriptional elements have been identified that drive spatial and temporal patterns of gene expression in distinct populations of cells, such as neuroepithelium (Zimmerman et al., 1994), post-mitotic neurons (Forss-Petter et al., 1990, Gloster et al., 1994, Landry et al., 1998), and glia (Brenner et al., 1994, Mallon et al., 2002). To date, few transcriptional elements selective for gene expression in the developing cerebral cortex have been identified.
In this study, genomic fragments of the Tbrain-1 (Tbr1) locus, a cortex selective developmentally regulated gene (Bulfone et al., 1995, Hevner et al., 2001), were isolated, characterized, and used to generate reporter constructs that were incorporated into transgenic mice. The Tbr1-driven transgene is expressed in cortical neurons expressing the native Tbr1 gene in a temporal pattern that mimicked the endogenous gene's expression. The Tbr1-driven transgene, however, was selectively expressed by just a subset of Tbr1-positive cortical cells: some of the neurons of the deep CP and SP. Transgene-positive neurons are generated early in corticogenesis, are morphologically complex in development, and persisted into adulthood. This transgene expression highlights a hitherto unrecognized subset of pioneer neurons and provides a means to characterize this unique population of cells.
Section snippets
Tbr1 expression in the cerebral cortex
The Tbr1 gene was the focus of our studies because of its distinctive expression within a select population of post-mitotic neurons in the developing cerebral cortex. To better characterize transgene expression in our experiments, we briefly examined the characteristics of endogenous Tbr1 expression. In situ hybridization demonstrated that Tbr1 expression was expressed in the cerebral cortex, concentrated in cells of the superficial cortex early in corticogenesis (Fig. 1A). Later in development
Correspondence and divergence in transgene and native gene expression profiles
Tbr1 expression is dynamic during cortical development, broadly expressed by post-mitotic neurons populating the CP early in development but restricted to a subset of MZ and deep CP cells later in development (Bulfone et al., 1995, Hevner et al., 2001, Hevner et al., 2003). Many aspects of Tbr1+5-GFP expression were similar to endogenous Tbr1 expression patterns. The expression of both genes was observed exclusively in neurons, was present in the cortex, and displayed similar temporal
Preparation of embryonic and postnatal mouse brains
All animal use and care was in accordance with institutional guidelines, under Yale IACUC protocol # 2002-10098.
Sodium pentobartital (60 mg/kg) was administered to euthanize pregnant mice. The abdominal wall was then opened, the uterine horn exposed, and the embryos (E12.5–18.5) were dissected into phosphate-buffered saline (PBS). Alternatively, P0–5 mice were euthanized by hypothermia, and their brains were removed from their skulls into PBS. Tissue was fixed by submersion for 1–3 h (h) in 4%
Acknowledgments
We thank Jeff Miner for providing the genomic library, Andrea Gocke for preliminary characterization of transgene expression, Mladen-Roko Rasin for sharing cortical neuronal cultures, and Quynh Chu for tissue preparation, and the following people for sharing their reagents: Robert Hevner for α-Tbr1, M. Ogawa for α-Reelin, and Celia Campagnoni for α-Golli. We also thank Nenad Sestan for his insightful comments and Kate Miller for reading the manuscript. This work was supported by start-up funds
References (55)
- et al.
Development of layer I and the subplate in the rat neocortex
Exp. Neurol.
(1990) - et al.
T-brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex
Neuron
(1995) - et al.
The Eph kinase ligand AL-1 is expressed by rostral muscles and inhibits outgrowth from caudal neurons
Mol. Cell. Neurosci.
(1996) - et al.
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
(2000) - et al.
Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control
Neuron
(1990) - et al.
Tbr1 regulates differentiation of the preplate and layer 6
Neuron
(2001) - et al.
How do thalamic axons find their way to the cortex?
Trends Neurosci.
(1995) - et al.
Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-lhx2 pathway
Neuron
(2001) - et al.
Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors [published erratum appears in Neuron 1994 Jun; 12 (6): following 1388]
Neuron
(1994) - et al.
The subplate, a transient neocortical structure: its role in the development of connections between thalamus and cortex
Annu. Rev. Neurosci.
(1994)
Relation between putative transmitter phenotypes and connectivity of subplate neurons during cerebral cortical development
Eur. J. Neurosci.
Copper/zinc superoxide dismutase overexpression promotes survival of cortical neurons exposed to neurotoxins in vitro
J. Neurosci. Res.
GFAP promoter directs astrocyte-specific expression in transgenic mice
J. Neurosci.
Characterization of transgene expression and Cre recombinase activity in a panel of Thy-1 promoter-Cre transgenic mice
Dev. Dyn.
Increased neuronal production, enlarged forebrains and cytoarchitectural distortions in beta-catenin overexpressing transgenic mice
Cereb. Cortex
A 3′ enhancer is required for temporal and tissue-specific transcriptional activation of the chicken adult beta-globin gene
Nature
The earliest-generated neurons of the cat cerebral cortex: characterization by MAP2 and neurotransmitter immunohistochemistry during fetal life
J. Neurosci.
Interstitial cells of the adult neocortical white matter are the remnant of the early generated subplate neuron population
J. Comp. Neurol.
Early determination of a mouse somatosensory cortex marker
Nature
Growth and targeting of subplate axons and establishment of major cortical pathways [published erratum appears in J. Neurosci. 1993 Mar; 13 (3): following table of contents]
J. Neurosci.
A muscle-specific enhancer is located at the 3′ end of the myosin light-chain 1/3 gene locus
Genes Dev.
Major glutamatergic projection from subplate into visual cortex during development
J. Comp. Neurol.
Functional synaptic circuits in the subplate during fetal and early postnatal development of cat visual cortex
J. Neurosci.
Involvement of subplate neurons in the formation of ocular dominance columns
Science
Requirement for subplate neurons in the formation of thalamocortical connections
Nature
The T alpha 1 alpha-tubulin promoter specifies gene expression as a function of neuronal growth and regeneration in transgenic mice
J. Neurosci.
A gene expression atlas of the central nervous system based on bacterial artificial chromosomes
Nature
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Current address: Department of Pharmacology and Anatomy, Subsection Neurodevelopment, Rudolf Magnus Institute of Neuroscience, University of Utrecht, UMC, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.