Visualization of corticofugal projections during early cortical development in a τ-GFP-transgenic...

Visualization of corticofugal projections during early corticaldevelopment in a s-GFP-transgenic mouse

Erin C. Jacobs, Celia Campagnoni, Kathy Kampf, Samuel D. Reyes, Vikram Kalra, Vance Handley, Yuan-Yun Xie,*Yan Hong-Hu, Vilma Spreur, Robin S. Fisher and Anthony T. CampagnoniUCLA Semel Institute for Neuroscience, 635 Charles E Young Drive South, Los Angeles, CA 90095–7332, USA

Keywords: cortex, pioneer axons, preplate, subplate, thalamus

Abstract

The first postmitotic neurons in the developing neocortex establish the preplate layer. These early-born neurons have a significantinfluence on the circuitry of the developing cortex. However, the exact timing and trajectory of their projections, between corticalhemispheres and intra- and extra-cortical regions, remain unresolved. Here, we describe the creation of a transgenic mouse using a1.3 kb golli promoter element of the myelin basic protein gene to target expression of a s–green fluorescent protein (GFP) fusionprotein in the cell bodies and processes of pioneer cortical neurons. During embryonic and early neonatal development, the timingand patterning of process extension from these neurons was examined. Analysis of s-GFP fluorescent fibers revealed thatprogression of early labeled projections was interrupted unexpectedly by transient pauses at the corticostriatal and telencephalic–diencephalic boundaries before invading the thalamus just prior to birth. After birth the pioneering projections differentially invaded thethalamus, excluding some nuclei, e.g. medial and lateral geniculate, until postnatal days 10–14. Early labeled projections were alsofound to cross to the contralateral hemisphere as well as to the superior colliculus. These results indicate that early corticothalamicprojections appear to pause before invading specific subcortical regions during development, that there is developmental regulationof innervation of individual thalamic nuclei, and that these early-generated neurons also establish early projections to commissuraland subcortical targets.

Introduction

During embryogenesis, the first cells generated in the neocortex arethe Cajal–Retzius and subplate neurons. These early postmitotic cellsleave the proliferative neuroepithelium to form the primordialplexiform (Marin-Padilla, 1971, 1978) or preplate (Stewart &Pearlman, 1987) layer. As subsequent generations of cortical neuronsare born they migrate into position in an ‘inside-out’ gradient withinthe preplate, essentially splitting this layer into a superficial marginalzone and a deep subplate (Marin-Padilla, 1971; Luskin & Shatz,1985a,b). Preplate neurons mature and differentiate prior to the cells inthe intervening cortical plate. Because of this distinction and by virtueof their position, preplate neurons appear to play key roles in theformation of cortical laminar structure and connectivity.

Subplate neurons pioneer the first pathways from the cerebral cortex(Allendoerfer & Shatz, 1994), serving as a scaffold for subsequentcorticofugal projections and incoming thalamocortical axons (Blake-more & Molnar, 1990; Molnar & Blakemore, 1995). Areas that havebeen proposed to receive pioneering projections from the subplate arethe dorsal thalamus (via the internal capsule; McConnell et al., 1989;De Carlos & O’Leary, 1992), the superior colliculus (Antonini &

Shatz, 1990) and the contralateral hemisphere (Antonini & Shatz,1990). Although it has been the focus of numerous studies, theliterature is still divided about the exact timing and extent of thesubplate projections. For example, while many studies indicate thatsubplate neurons project through the incipient internal capsule, howfast the fibers migrate through this region and when they reach thethalamus, if at all, is not clear (Molnar & Cordery, 1999; Hevner et al.,2002). It also remains unclear whether areas such as the contralateralhemisphere and superior colliculus are targets of subplate axons(Antonini & Shatz, 1990; Koester & O’Leary, 1994). Two key factorslimiting the interpretation of these data are the lack of selectivesubplate markers and the absence of exclusive anterograde axonaltracers. For example, many of the studies described used carbocyaninedyes such as DiI. However, as soon as thalamocortical and corticof-ugal fibers begin to commingle, around embryonic day (E)15 in themouse, application of DiI can also result in retrograde labeling of bothsets of projections thereby obscuring the process of corticofugalinvasion of the thalamus.In this study, we generated a transgenic mouse using the golli

promoter of the myelin basic protein (MBP) gene to drive expressionof a bovine microtubule-binding protein, s–enhanced green fluores-cent protein (GFP) fusion protein. Previously, we showed that thispromoter element was capable of targeting transgenes to corticalpioneer neurons as early as E11 (Landry et al., 1998; Xie et al., 2002).In this new transgenic mouse, the same promoter element was used totarget s-GFP to axonal projections during cortical embryonic andpostnatal development, made visible by either direct illumination ofthe GFP protein or immunohistochemical analysis with anti-GFP. With

Correspondence: Dr Erin C. Jacobs, Neuroscience Research Building, Rm #315, UCLASemel Institute for Neuroscience, as above.E-mail: [email protected]

*Present address: International Collaboration On Repair Discoveries (ICORD), 2469–6270 University Boulevard, University of British Columbia, Vancouver, BC, Canada V6T1Z4

Received 17 August 2006, revised 23 October 2006, accepted 26 October 2006

European Journal of Neuroscience, Vol. 25, pp. 17–30, 2007 doi:10.1111/j.1460-9568.2006.05258.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

this golli-s-GFP mouse, we studied the timing and patterning ofneurite extension from s-GFP-labeled cells in the embryonic and earlypostnatal brains.

Materials and methods

Generation of the golli-s-GFP construct

The golli-s-GFP plasmid was constructed by replacing the Not Ib-galactosidase cassette in the pNASS 1.3 kb golli promoter-lacZconstruct (as described in Landry et al., 1998) with a cassette encodingthe first 383 amino acids of a bovine s that contains four microtubulebinding sites (from the s-lacZ plasmid; the kind gift of Dr T. Callahan)fused in frame with GFP (pEGFP; Clontech Laboratories, Palo Alto,CA, USA). Briefly, the primer CCCGGTGAACAGGCAGCCGCT-CAGG and site-directed mutagenesis was used to eliminate the Not Isite within bovine s without changing the amino acid sequence. The1.25 kb s insert was then excised from ps-lacZ and inserted into theHind III-Bam HI site of pEGFP. This clone was linearized withHind III, Not I linkers were added and the final cassette was releasedfrom pEGFP with Not I and inserted into our pNASS clone.For production of the transgenic founders by the University of

California Transgenic Core Facility (Los Angeles, CA, USA), thepNASS plasmid was digested with Sca I and Sph I and the golli-s-GFP construct was isolated by gel electrophoresis. This fragmentcontained 901 bp pNASS, 1.3 kb Eco RI-Bam HI fragment of thegolli promoter cloned into the Eco RI-Xho I site of pNASS, 180 bp ofSV 40 splice donor ⁄ splice acceptor sequence, 1.25 kb of s, 777 bp ofpEGFP, 200 bp of SV 40 poly A signal and 300 bp of pNASS. Thebasic pNASS plasmid was produced by Clontech.

Genotyping founders by Southern blotting

Transgenic founders were identified by Southern blot analysis ofBam HI digests of isolated tail DNA probed with the Sca I-Sph Ifragment of the golli-s-GFP plasmid. The native golli promoter wasdetected at 6.2 kb. Bam HI sites within the transgene yielded a 1.4 kbs fragment, a 0.82 kb GFP fragment and a 2.4 kb band containingsome pNASS sequence plus the 1.3 kb portion of the golli promoter.Homozygous animals were identified by comparing the value obtainedwhen the transgene bands were corrected for loading variations bynormalising them against the wild-type (Wt) promoter. Once founderlines were established, homozygous animals were selected and used inall subsequent studies. Genotyping of additional progeny was notnecessary as the brains of the transgenic mice fluoresce green underillumination of blue (488 nm) light.

Animals and tissue preparation

In this study, 75 embryonic day (E) 12–18 and 35 postnatal day (P)0–14 mice, homozygous for the s-GFP transgene, were analysed. Forembryonic time points, timed-pregnant females (midday of concep-tion ¼ E0.5) were anaesthetized with CO2 and killed by cervicaldislocation. Embryos were removed and fixed overnight in 4%paraformaldehyde in 0.1 m phosphate buffer (PB), pH 7.4. Postnatalanimals were deeply anaesthetized with Halothane (HalocarbonLaboratories, River Edge, NJ, USA). They were then perfusedintracardially with 4% buffered paraformaldehyde, and the brainsremoved from the skull and postfixed overnight in the same fixative.After postfixation, brains were cryoprotected in 20% sucrose,embedded in OCT (TissueTek) and sectioned at 10–20 lm on a

cryostat (Leica 3050S). For immunohistochemistry experiments onfree-floating sections, brains were embedded in 4% agarose andsectioned coronally at 60 lm on a vibratome (Leica VT1000S). Theanimals used in this study were housed at the UCLA School ofMedicine vivarium and all procedures were approved by UCLA’sAnimal Care and Use Committee and conducted in accordance withthe guidelines in ‘NIH Guide for the Care and Use of LaboratoryAnimals’.

Immunohistochemistry and antibodies

To characterise the expression of the endogenous s-GFP transgene,serial cryostat sections in either the coronal or the sagittal plane weretaken at 20 lm from the rostral to caudal extent of homozygous golli-s-GFP mice. The sections were collected in 0.1 m PB, counterstainedfor 10 min in a 10 lg ⁄ mL solution of the nuclear dye bisbenzimide(Hoechst 33342, Invitrogen, Carlsbad, CA, USA) and mounted in PBand glycerol onto slides. The s-GFP-labeled cells and their processeswere then visualized by fluorescence microscopy. In a parallel series ofexperiments, free-floating vibratome sections were collected asdescribed above and rinsed in 0.1 m PB; endogenous peroxidaseactivity was quenched in a solution containing 0.3% H202 and 10%methanol and the sections were rinsed, incubated in primary antibodyovernight at 4 �C, rinsed and then incubated with secondary antibodyfor 1 h at room temperature. The secondary antibody was visualizedusing the avidin–biotin–peroxidase method (Vectastain Elite ABC) asrecommended by the manufacturer (Vector Laboratories, Burlingame,CA, USA) with 3, 3¢-diaminobenzidine (Roche, Indianapolis, IN,USA) as the chromogen. Sections were mounted onto Superfrost PlusGold slides (Fisher, Pittsburgh, PA, USA), dehydrated and cover-slipped in Permount (Fisher). For cytoarchitectural comparisons,adjacent vibratome sections were stained with Cresyl Violet. Fordouble-label immunofluorescence, cryostat sections were warmed to37 �C, rinsed in 0.1 m PB, incubated with the primary antibodyovernight at 4 �C, rinsed, incubated with the fluorophore-conjugatedsecondary antibody (1 : 400 rhodamine red-X; Jackson ImmunoResearch, West Grove, PA, USA) and counterstained with bisbenzi-mide.Primary antibodies used in this study were: rabbit polyclonal

anti-GFP (1 : 4000; Chemicon, Temecula, CA, USA), mousemonoclonal anti-microtubule-associated protein 2 (MAP2; 1 : 200;HM2 clone, Sigma, St. Louis, MO, USA), mouse monoclonal anti-Synapsin1 (1 : 100; Chemicon) and mouse monoclonal anti-5-bromo-2-deoxyuridine (BrdU; 1 : 100; BD PharMingen, San Diego,CA, USA).

RNA in situ hybridization

Digoxygenin-labeled sense and antisense riboprobes corresponding tothe full-length GFP sequence were synthesized using reagents fromRoche Applied Science from pEGFP-N3 (Clontech Laboratories, PaloAlto, CA, USA) subcloned into pBluescript SK vector (Stratagene, LaJolla, CA, USA). Frozen sections 20 lm in thickness were hybridizedessentially as described in Landry et al. (1994) with the followingmodifications. Thawed sections were rinsed in phosphate-bufferedsaline and pretreated with only 4% paraformaldehyde with trietha-nolamine and acetic anhydride prior to hybridization with theriboprobe at 55 �C. Post-hybridization washes were for 20 min in5 · SSC at 50 �C, 1 h in 2 · SSC at room temperature and 1 h in0.2 · SSC at 60 �C.

18 E. C. Jacobs et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 17–30

BrdU birth-dating and detection

Timed-pregnant females were given a single intraperitoneal injectionof 100 mg ⁄ kg body weight of BrdU in sterile phosphate-bufferedsaline on E11.5 and again on E12.5. After the BrdU injections,embryos were collected at E18.5 (n ¼ 5) and the brains fixed andfrozen sections prepared as described above. To detect and visualizethe BrdU epitope, cryostat sections were treated with BD PharMingenRetreivagen solution at 95 �C for 10 min. The sections were thenprocessed for immunohistochemistry using BrdU In-Situ Detection Kitreagents and peroxidase substrate according to the manufacturer’sinstructions (BD PharMingen, San Diego, CA, USA). In double-labelexperiments with GFP, sections were first incubated with anti-GFPwhich was visualized using the immunoperoxidase method using AEC(3-amino-9-ethylcarbazole) as the chromogen per manufacturer’sinstructions (BD PharMingen).

Microscopy and image processing

Microscopic analysis was completed using either a Leica uprightDMRXA microscope for both light and fluorescence microscopy or alaser scanning confocal microscope (Leica TCS SP). Images werecaptured with a SPOT CCD camera (Diagnostic Instruments, Inc.,Sterling Heights, MI, USA) and assembled into figures using AdobePhotoshop 6.0 software.

GFP cell counting and analysis

To determine the distribution of s-GFP-labeled cells in the neocortexat E15.5 and P6.5, confocal sections through the somatosensory cortexwere analysed. For quantification, z stacks of confocal images werecaptured with a 40· objective with filters for both GFP and thebisbenzimide counterstain. To count the cells, a grid of equal-sizedhorizontal bins (3750 lm2) from the pial surface–marginal zone to theintermediate zone (for example, see Fig. 4) was placed over theconfocal images and the number of s-GFP+ somas within each bin wascounted. If a soma fell across two bins, that cell was assigned to thelower bin. A total of eight sections (using one hemisphere) from threelittermates was used for quantification at each age. At E15.5, oneimage spanned the entire distance of the cortex and bin 1 was assignedto the intermediate zone and bin 10 to the marginal zone. At P6.5,three or four overlapping confocal images were required to cover theentire cortical thickness. For both ages, bins corresponding to aspecific cortical layer (as verified by the nuclear counterstain) werecollapsed and the density of the cells was calculated as the totalnumber of cells within the bins divided by the total area covered.

Results

Targeting of s-GFP to cell bodies and processes of corticalpioneer neurons

Previously, we identified a 1.3 kb promoter element in the MBP genethat was capable of targeting transgene expression [e.g. b-galactosi-dase (Landry et al., 1998) and herpes simplex virus–thymidine kinase(HSV-TK; Xie et al., 2002)] to early cortical preplate and subplateneurons. This promoter has also been shown to correlate well withanother marker of early-born cortical neurons, Tbr1 (Hevner et al.,2001; Kolk et al., 2005). In the cortex, expression of the 1.3 kbpromoter was restricted primarily to these cell populations andpersisted in the deep layers of the neocortex well into adulthood. In thepresent study we used this promoter element to target a third reporter

gene to these cells in order to follow establishment of their processesduring embryonic and early postnatal development. For this purposewe generated a plasmid in which the golli promoter element targeted as-GFP fusion protein to the cell bodies and processes of these corticalpioneer neurons (see Fig. 1). We began with a construct developed byCallahan & Thomas (1994) that fused a cDNA encoding the bovinemicrotubule-binding protein, s, to lacZ. This construct had been usedto effectively target a b-galactosidase fusion protein to the cell bodiesand axons of Drosophila neurons (Callahan & Thomas, 1994) and tovisualize neuronal processes in the mouse olfactory system (Mom-baerts et al., 1996). In this study, the s-lacZ plasmid was modified (seeMaterials and methods) by fusing the bovine s to GFP, and theexpression of this fusion protein was placed under the control of the1.3 kb fragment of the golli promoter (Fig. 1A). This construct wasthen used to generate the golli-s-GFP-transgenic mice. A founder linewas established and bred to homozygosity. Southern blots of genomicDNA from two homozygous transgenic (Tg) mice and one Wt, probedwith labeled construct cDNA, are shown in Fig. 1B. A comparison ofthe hybridization signal from the Wt promoter (a single copy gene) tothat of the Tg promoter band indicated that the s-GFP mice containapproximately five copies of the transgene per allele.During the embryonic and postnatal period examined, expression of

the golli promoter-driven s-GFP was highest in the telencephalon,specifically the dorsal pallium, and portions of the olfactory system(Fig. 1C–E). The pattern of expression of the s-GFP reporter in theneocortex was nearly identical to that reported previously for the lacZand HSV-TK transgenes (Landry et al., 1998; Xie et al., 2002; seeSupplementary material, Fig. S1). Expression of the golli promoter inpreplate neurons was further supported by BrdU birth-dating analyses,which confirmed that most of the cortical s-GFP+ cells were bornbetween E11 and E12, the expected birth date of the earliest borncortical neurons (Wood et al., 1992 and Price et al., 1997; seeSupplementary Fig. S2).

Expression of s-GFP in subplate neurons

The subplate layer is comprised of a heterogeneous population of cellsthat have a number of morphological features in common. Thesefeatures include a polymorphic-shaped cell body positioned in thedeepest cortical layers with an ascending projection to the marginalzone and ⁄ or a descending projection to subcortical targets (Bayer &Altman, 1990; Allendoerfer & Shatz, 1994). One of the reasons forcreating the golli-s-GFP transgenic mouse was to visualize theseprojections.

Position and morphology of s-GFP+ neurons

In Fig. 2A, the morphology of the s-GFP+ cells and their positionwithin the dorsal pallium is shown in a coronal section through theneocortex of an E15.5 golli-s-GFP mouse brain. While many of thelabeled cell bodies at this early stage of development were cleaved andsettled deep to the cortical plate (future layer 6b or subplate), thedistribution of s-GFP+ cells extended from the upper intermediatezone into and intercalated with cells in the deep layers of the corticalplate. In our birth-dating experiments, these s-GFP+ cells were born onE11 and E12.Many of the s-GFP-labeled cells in the neocortex appeared to have

processes that extended up to the marginal zone or down into theintermediate zone and the arrangement of the labeled cell bodiesappeared to define a double layer. The cells in the deepest layer justabove the intermediate zone generally exhibit a horizontal polarity

Formation of early cortical circuitry 19


Fig. 3. Distribution of s-GFP in the neocortex. (A and B) Fluorescent microscopy of coronal sections stained with bisbenzimide nuclear dye at E15.5 and P6.5,respectively. Red boxes indicate area of somatosensory cortex where the number of s-GFP+ cells was measured. Bin analysis of the number of s-GFP+ cells in eachcortical layer was completed for (C) E15.5 and (D) P6.5 and the distribution of the density of s-GFP+ cells plotted in (E) and (F), respectively. Scale bars,300 lm (A), 600 lm (B), 25 lm (C and D).

Fig. 2. Morphology and position of neocortical s-GFP+ cells. (A) Coronal section from an E15.5 brain illustrating the distribution of s-GFP+ cells in theneocortex. Two populations of labeled cells are observed in the lower cortical layers. The layer marked sp (subplate) is comprised of cells with primarily horizontalpolarity (red arrows). The labeled cells in the more superficial layer (spu; upper subplate) have a more vertical orientation (white arrows). Labeled ascendingprojections (blue arrowheads) terminating in the marginal zone (mz; yellow arrowheads) and descending projections to the intermediate zone (iz) were also observed.(B) Coronal section through the cortex of an E15.5 embryo showing mRNA expression for GFP in cells in the sp and spu. Diffuse label in the cortical plate (cp) andiz is presumably due to translocation of the GFP mRNA to the processes. Scale bars, 60 lm (A), 90 lm (B).

Fig. 1. Generation of the golli-s-GFP construct and transgenic mouse. (Panel A) Schematic diagram of how the golli-s-GFP construct was generated. Starting withthe s-lacZ construct from Callahan & Thomas (1994), the s fragment was isolated by digestion with Hind III (h)–Bam HI (b) and inserted into the Hind III–Bam HIsite of pEGFP, putting s in frame with GFP. This clone was then linearized with Hind III, Not I (n) linkers were added, and the Not I cassette was used to replace theb-galactosidase cassette in our original golli-lacZ construct in the pNASS vector (R, EcoRI). (Panel B) Southern blot of genomic DNA from Tg and Wt micedigested with Bam HI (b). The blot, probed with labeled construct cDNA, revealed additional bands of the appropriate size and number for the golli-s-GFP constructin the transgenic mice that were not present in the Wt. (C–E) Pattern of s-GFP expression in the E15.5 embryo under (C) normal and (D) fluorescent illumination.(E) Sagittal view of an E15.5 golli-s-GFP brain showing high levels of s-GFP expression in the telencephalon (T). M, midbrain; SC, spinal cord. Scale bars, 2 mm(in C for C and D).



Fig. 2.

Fig. 3.



(red arrows; Fig. 2A) and will probably form the future layer 6b orsubplate. Above the subplate, in the deep layers of the cortical plate,many labeled cells were detected with a more radial polarity (whitearrows; Fig. 2A) and were often arranged in vertical clusters. Theselabeled cells could represent what (in cats) Luskin & Shatz (1985b)and (in primates) Kostovic & Rakic (1990) referred to as the ‘uppersubplate’ (spu), and we have used this terminology in Figs 2 and 3. Inrodents, this layer is thought to be transient (Bayer & Altman, 1991).Accordingly, we found in the golli-s-GFP mouse that the number ofcells in this upper layer declined dramatically by early postnatal ages[see Fig. 3E (spu) and Fig. 3F (layers 5 and 6a)]. However, it is not yetclear whether the labeled cells in this ‘upper subplate layer’ arecomposed of true subplate neurons in rodents or are a mixture ofsubplate and cortical plate cells.Superficial to this double layer of heavily labeled s-GFP+ cells is a

region of relatively sparse and only lightly labeled s-GFP+ cells, but aregion rich in processes that appear to be derived from cells in thedeeper layers (blue arrowheads; Fig. 2A). As shown in Fig. 2B, in situhybridization studies confirmed the position of the majority of thes-GFP-expressing cells to the upper subplate and subplate. However,punctate labeling was observed throughout the cortical plate and intothe intermediate zone. As s is believed to be synthesized locally onribosomes in the axon or primary dendrites (Goldberg, 2003), thisdiffuse pattern of label is likely to represent s-GFP mRNA that wastranslocated to the ascending and descending processes of the s-GFP+

cells (Landry & Campagnoni, 1998).To analyse the distribution of labeled cells within the cortical

lamina, binning analysis was used to quantify the number of s-GFP+

cells within a defined area of the somatosensory cortex at E15.5 andP6.5 (see Fig. 3A and B). As shown in Fig. 3C and D, a grid of equal-sized horizontal bins was placed over confocal images of the cortex(from the pial surface to the intermediate zone) and the number ofs-GFP+ cells per bin was counted. While the thickness of theintermediate zone varied among sections, in general at E15.5 one gridof 10 bins covered the depth of the entire neocortex. At P6.5, thenumber of bins required to span the entire distance of the cortexranged from 30 to 40, requiring three or four overlapping images (anexample of one image is presented in Fig. 3D). As s-GFP+ cells werenot observed in the ventricular zone at either age, data from this areawere omitted. To analyse the density of cells in specific cortical layers,corresponding bins (as verified by the nuclear counterstain) werecollapsed and the density was calculated as the total number of cellswithin the bins divided by the total area covered. Between E15.5 andP6.5, the total number of cells counted within this region of neocortexdeclined by 62% (n ¼ 3389, mean ¼ 141.2 cells per section at E15.5;n ¼ 1290, mean ¼ 53.75 at P6.5). Quantification of the density ofs-GFP+ cells confirmed that at E15.5 labeled cells were most abundantin the upper subplate (spu; 60%) and subplate (23%; Fig. 3E). By P6.5,with the cortical layers better defined, labeled cells were foundprimarily in the subplate (52%) and layer 6a (42%; Fig. 3F). A fews-GFP+ cells were detected in the bottom of ‘immature’ layer 5 but themajority of the GFP fluorescence in this layer and the entire label inlayers 1–4 were associated with the ascending projections to themarginal zone.

Other features of the cortical s-GFP+ neurons

Another characteristic of subplate neurons is that they expressmature neuronal markers, such as microtubule-associated protein 2(MAP2), prior to cells in the overlying cortical plate. Frozen sectionswere taken at various embryonic ages and stained with an antibodyagainst MAP2. At E15.5, MAP2+ cells were detected primarily in

the subplate and they colocalized with cells expressing the s-GFPtransgene (Fig. 4A). Subplate neurons have also been shown to receivefunctional synaptic inputs before cortical plate neurons (Chun & Shatz,1988; Friauf et al., 1990). For example, synapsin 1, a synaptic vesicle-associated phosphoprotein present in presynaptic nerve terminals, wasfound to be restricted to the marginal zone and subplate in the fetal andneonatal cat (Chun & Shatz, 1989). This pattern of staining was alsoobserved in the s-GFP mice at E15.5 (Fig. 4B). The distribution ofsynapsin 1 immunoreactivity overlapped the s-GFP labeling through-out the subplate and to a lesser extent in the marginal zone (Fig. 4B).The difference in labeling between synapsin 1 and GFP in themarginal zone may reflect our observation that the golli 1.3 kbpromoter appears to label a subset of reelin-positive cells in thiscortical layer (Landry et al., 1998). The overlap in the distribution ofcells expressing s-GFP and those immunoreactive for MAP2 andsynapsin 1 provides further support that s-GFP is a marker for earlycortical preplate and subplate neurons.

Distribution of neurons expressing s-GFP during embryonic andearly postnatal development

Neocortical expression

From the earliest embryonic age examined (E12.5) through to earlypostnatal development (P14.5), s-GFP-labeled cells were consistentlydetected in the developing cortex. An overview of the distribution oflabeled cells can be seen in Fig. 5, in which coronal sections examinedunder fluorescent microscopy to detect the s-GFP-expressing cells(green) are counterstained with the nuclear dye bisbenzimide (blue).The first labeled cells were detected in the preplate. At E14.5, whenthe cortical plate was beginning to form in the more lateral aspects ofthe neocortex, s-GFP+ cells were observed in both the upper marginalzone and lower subplate (Fig. 5A and B). GFP expression continued inthese layers as they extended ventrally in the pyriform cortex. Duringmid to late embryonic development (Fig. 5C and D), corticalexpression of the s-GFP transgene remained strong in the subplateneurons. Labeled cells were observed in the primitive amygdala andclaustrum at this time (not shown). This pattern of labeled cellspersisted in the early postnatal cortex (Fig. 5E–H). As the corticalplate expanded and developed into a mature six-layered cortex,ascending processes to layer 4 became evident along with thoseprojecting to layer 1. Labeled corticofugal fibers were evident as earlyas E13.5. The timing and progression of these subcortical projectionsis described below. Other areas of telencephalic GFP expressionincluded the hippocampal formation (Fig. 5B and D) and olfactorysystem (Fig. 5D) along with their respective fiber tracts, the fimbriaand the lateral olfactory tract (arrowhead).

Additional areas of s-GFP expression in the nervous system

Although the focus of this study was to characterise the s-GFPexpression in the neocortex, Table 1 lists the developmental expres-sion of the golli 1.3kb promoter in other areas of nervous system.

Developmental progression of s-GFP fibers from the cortex tothe thalamus

To determine the timing of the labeled corticofugal fibers as theyprogressed towards the thalamus we examined s-GFP expression fromE13.5 to P14.5 in serial sections from both thin (20 lm) fluorescentsections and thick (60 lm) sections immunostained with anti-GFP(see Table 2). Bisbenzimide-counterstained and Nissl-stained adjacentsections were used for cytoarchitectural comparisons. Similar results



Fig. 4. Cortical s-GFP+ neurons expressed markers of a mature neuronal phenotype. Immunofluorescence of the s-GFP-expressing cells (green) in the subplate (sp)colocalizes with the mature neuronal marker MAP2 (red; A) and synapsin 1 (red; B) at E15 indicates a mature neuronal phenotype. Other abbreviations: cp,cortical plate; mz, marginal zone. Scale bar, 30 lm.

Fig. 5. Pattern of s-GFP expression during embryonic and early postnatal development. Coronal sections from E14.5 to P14.5 are shown under (B, D, F and H)fluorescece microscopy to illustrate the expression of s-GFP in neocortical neurons and the progression of their processes through the internal capsule (ic) andultimately to the thalamus (th). (A, C, E and G) The same sections are shown under uv illumination to reveal the bisbenzimide (bisb) counterstain forcytoarchitectural comparisons. Other abbreviations: am, amygdaloid complex; ctx, cortex; fi, fimbria; fx, fornix; lot, lateral olfactory tract; pc, pyriform cortex. Scalebars, 1 mm (each also applies to right-adjacent fluorescence section).



were seen with the two methods; however, the majority of the dataanalysed and presented here (see Figs 6–8) are from the GFP-immunostained sections, due to the increased sensitivity and perma-nent labeling of the immunoperoxidase method. For each ageanalysed, serial coronal sections were made from the rostral-to-caudalextents of the brain and the data presented in Figs 6 and 7 representthe farthest growth of the labeled axons observed.

E13–15: s-GFP fibers innervated and waited in the lateral internalcapsule

At E13.5, prior to the formation of the cortical plate, preplate neuronsbegin to extend processes, forming the first corticofugal fibers. At thisdevelopmental stage we observed s-GFP-labeled fibers descendingobliquely into the intermediate zone (presumptive corona radiata),passing the corticostriatal junction (see dashed line in Fig. 6B) andterminating in growth cones at the more lateral aspects of the internalcapsule (see red dashed line in Fig. 6A). Labeled fibers appeared towait in this area as a diffuse group of individual processes until E15.5(Fig. 6C–F). At all ages examined, the s-GFP fibers in the internalcapsule were distinct from the bundle of fibers present more mediallyin the stria terminalis.

E16.5–17.5: labeled fibers extended towards and paused near theventral thalamus and cerebral peduncle

BetweenE15.5andE16.5 thes-GFPfiberschangedsignificantly in termsof both the distance traveled and their topography. At E16.5, s-GFP+

fibers had progressed � 400 lm over a 24-h period, descending in theinternal capsule well into the presumptive neostriatum and pallidum(Fig. 6G and H). In addition, labeled corticofugal fibers began to formdiscrete bundles as they exited the presumptive white matter and enteredthe neostriatum whereas, prior to E16.5, s-GFP+ fibers entered theinternal capsule as a loose plexus of smaller fascicles (Fig. 6A, C and E).Between E16.5 and E17.5 s-GFP fibers continued in the internal

capsule as a tight bundle sweeping towards the diencephalon andapproaching the lateral border of the ventral thalamus (Fig. 6G and H).At this stage, labeled axons again appeared to pause (or slow theirprogression) and gather at the border between the telencephalon anddiencephalon (see dotted line in Fig. 6H, J and K), just lateral to thethalamic reticular nucleus. Additionally, at E17.5 labeled fibers in thedeveloping cerebral peduncle became apparent as they separatedventrally from labeled corticothalamic fibers (Fig. 6I). Sagittal sectionsat this same age (data not shown) indicate that s-GFP+ cerebralpeduncle fibers extended as far as the midbrain, presumably indicativeof labeled corticobulbar tracts. Labeled fibers in the cerebral pedunclecontinued to be present as late as P14 (see Table 1). It is unclear at thistime whether labeled fibers in the corticospinal tract were present.However, labeled fibers were detected in the pons by E13.5 whichcould indicate that these early corticofugal fibers may assist inestablishing initial corticospinal pathways.

E18.5–P2.5: s-GFP projections entered the dorsal thalamus just priorto birth

At E18.5, the leading edge of labeled corticofugal axons had passedthrough the thalamic reticular nucleus, crossed the external medullarylamina and entered the dorsal thalamus (Fig. 6K and L). At this age theextent of thalamic innervation appeared to be restricted to the morelateral aspects of those nuclei involved in processing motor (ventrallateral and ventral anterior) and somatosensory (ventrobasal complex;VB) processing. On P0.5, labeled fibers had progressed to the moremedial aspects of these nuclei (e.g. VB shown in Fig. 7A and B).During the developmental period from E18.5 to P2.5, labeled fiberswere not detected in any of the midline nuclei nor did they innervatethe lateral or medial geniculate nuclei, LGN (see * in Fig. 7A–D) andMGN, respectively.

P4.5–14.5: differential innervation of thalamic nuclei by s-GFP+

corticofugal fibers

At older ages, labeled projections spread into the midline nuclei. AtP4.5, we detected the first fibers in the dorsal LGN (Fig. 7E and F) and

Table 1. Areas of golli-s-GFP expression in developing brain and spinal cord

E13 E15 E17 P6 P14

TelencephalonNeocortex +++ +++ +++ +++ ++Anterior commissure +++ +++ +++ +++ ++Corpus callosum nd +++ +++ ++ +Internal capsule nd ++ +++ +++ ++Cerebral peduncle nd nd + ++ ++Piriform cortex + ++ ++ ++ +

Olfactory bulbMitral cells ++ +++ +++ ++ ++Granule cells + + + + +Lateral olfactory tract ++ ++ ++ + +

Hippocampal formationCA 1, 2 +++ +++ +++ +++ ++Subiculum +++ +++ +++ ++ ++Fimbria ⁄ fornix +++ +++ +++ ++ +Amygdala + ++ ++ ++ ++

DiencephalonPosterior hypothalamus ++ ++ ++ ++ ++Mammillary bodies ++ ++ ++ ++ ++

MesencephalonSuperior colliculus ++ ++ + nm nmInferior colliculus ++ ++ + nm nmIsthmus +++ ++ ++ nm nm

RhombencephalonPonsTectopontine fibers ++ ++ + nm nmMedullaSpinal trigeminal nucleus ++ ++ ++ nm nm

Spinal cordLateral motor column ++ ++ ++ nm nmDorsal root ganglia + + + nm nm

Qualitative comparison of levels of expression: +++, high; ++, medium; +, low;nd, not detected; nm, not measured.

Table 2. Numbers of animals studied to determine the timing of progressionof cortical s-GFP fibers to subcortical targets

Age Coronal 20 lm Sagittal 20 lm Coronal 60 lm Total

E13.5 4 3 3 10E14.5 6 – 4 10E15.5 6 3 4 13E16.5 4 – 2 6E17.5 4 3 4 11E18.5 4 – 2 6P0.5 – – 2 2P1.5 1 1 – 2P2.5 – – 2 2P3.5 1 1 – 2P4.5 – – 2 2P5.5 2 2 – 4P6.5 – – 2 2P7.5 2 2 – 4P10.5 1 1 2 4P14.5 – – 2 2



MGN (data not shown). The expansion of s-GFP fibers towards themidline continued during the first and second postnatal weeks. Duringthis time the fiber density in the thalamus increased such thatindividual fibers were no longer distinguishable. The LGN was the lastthalamic nucleus to be completely innervated. Labeled fibers extendedinto the ventral LGN by P6.5 (see *, Fig. 7G and H) but they did notcompletely innervate the nucleus until P14.5 (see *, Fig. 7I–L). TheMGN also showed a similar delay in its pattern of innervation.However, the MGN appeared to be completely innervated by P10.5(data not shown).

Evidence that s-GFP fibers established early cortical projectionsto the contralateral cortex and other subcortical targets

During embryonic development s-GFP+ fibers were observed project-ing to target nuclei in the thalamus. We also found labeled fibers along

the first formed commissural pathways, including the anteriorcommissure and the corpus callosum. In the anterior commissure,s-GFP+ fibers were seen as early as E13.5 and persisted until at leastP6.5. By E14.5, labeled fibers appeared to cross the midsagittal planeas shown in Fig. 8A and its adjacent Nissl-stained section in Fig. 8B.In the corpus callosum, labeled processes were first detected on E16.5in the more lateral aspects of the fiber tract. Within 24 h, s-GFP+ fiberswere observed to extend across the midline to the contralateralhemisphere (Fig. 8E and F). The presence of s-GFP+ fibers in theseearly projections to the contralateral hemisphere supports the hypo-thesis that these early-generated neurons pioneer commissural as wellas corticofugal pathways.In addition to the cortical commissural projections, s-GFP+

processes were also observed projecting to the superior colliculusduring embryonic development. In lateral sagittal sections, labeledfibers were detected in the brachium of the superior colliculus,

Fig. 6. Labeled corticofugal fibers transiently paused in the striatum and near the telencephalic–diencephalic border. Serial coronal sections were stained with anti-GFP and the farthest growth of the labeled axons for each age is presented in A, C, E, G, I and K. As early as E13.5, labeled fibers were detected in the intermediatezone. As these fibers progressed through the internal capsule (ic) to the diencephalon, the leading edge appeared to slow first in the striatum and then near thetelencephalon–diencephalon border (red dashed lines in A, C and E and in G and I, respectively). (K) Just prior to birth, labeled fibers were observed to enter thelateral aspects of the ventrobasal nucleus (vb) of the thalamus (th). s-GFP+-expressing cells were not detected in the thalamus at any age examined. (B, D, F, H, J andL) Adjacent Nissl-stained sections shown for cytoarchitectural comparisons. Other abbreviations: cp, cerebral peduncle; ctx, cortex; fx, fornix; hp, hippocampus; st,stria terminalis; trn, thalamic reticular nucleus. Scale bar in L, 500 lm (A and B), 530 lm (C and D), 550 lm (E and F), 580 lm (G and H), 620 lm (I and J),700 lm (K and L).



terminating in the more rostral aspects of the tectum as early as E13.5.This projection was also observed at E17.5 although the size of theprojection was much reduced by this age (see Table 1). Labeledprojections were also detected within the lateral olfactory tract(Fig. 8C and D); the hippocampal efferent pathway, the fimbria(Fig. 8C and D); the hippocampal commissure (data not shown); andthe stria terminalis (Fig. 8C and D). These pathways are believed tooriginate from s-GFP-labeled neurons in the anterior olfactorynucleus, hippocampus and amygdala, respectively. Projections fromthese nuclei were observed as early as E13.5. However, the number oflabeled fibers in all of these pathways declined significantly during thefirst week postnatal, such that by P10.5 only a few fibers in theanterior commissure and the fornix were still visible. The earlyappearance and temporary nature of these pathways suggests that thepopulations of cells expressing the s-GFP transgene are important inpioneering many of the early efferent pathways in both allocortex andneocortex.

Discussion

Early cortical neurons and their processes are thought to be importantin establishing both extrinsic and intrinsic cortical connections.However, the exact timing and extent of these projections is still amatter of debate. To address this issue, we have continued our studiesusing the golli promoter of the MBP gene to generate transgenic micethat can be of value in following the development and axonalprojections of the cortical pioneer neurons. In this study, the promoterelement was used to target expression of a s-GFP fusion protein tothese cells thereby permitting direct visualisation of the cell’s emergingprocesses. Our results support the following conclusions: (i) the golli-s-GFP transgenic mouse is a consistent marker of early cortical pioneerneurons; (ii) these pioneer neurons project to the thalamus but pausealong the way, entering the thalamus just prior to birth; (iii) uponentering the thalamus, early corticofugal fibers proceed in an orderedventrolateral-to-dorsomedial gradient; and (iv) early cortical preplate

Fig. 7. Differential innervation of the thalamus by s-GFP+-labeled corticofugal fibers. (A) After entering the thalamus just prior to birth, labeled axons continuedduring the first 2 weeks postnatal to invade the thalamus (th) in a ventrolateral-to-dorsomedial gradient. The ventrobasal nuclear complex (vb) was one of the firstthalamic nuclei to be innervated. (C, E, and G) During the first postnatal week, labeled fibers gradually expanded toward midline nuclei. The last thalamic nucleusto be completely innervated by the s-GFP-labeled fibers was the lateral geniculate nucleus (*). A, C, E, G, I and K are coronal sections stained with an antibody forGFP; B, D, F, H, J and L are adjacent Nissl-stained sections. Other abbreviations: cx, cortex; hp, hippocampus; ic, internal capsule. Scale bar in L, 1 mm (A–D),1.17 mm (E and F), 1.3 mm (G and H), 1.4 mm (I and J), 1.45 mm (K and L).



neurons may establish initial projections to the contralateral hemi-sphere and additional subcortical targets such as the superior colliculus.

Generation of the golli-s-GFP transgenic mouse

The 1.3 kb golli promoter element of the MBP gene has proven todirect the expression of various transgenes with a high degree offidelity. The specificity of the promoter was confirmed in this study bythe strong overlap in expression between s-GFP and that previouslyreported for the lacZ (Landry et al., 1998) and HSV-TK (Xie et al.,2002) transgenes. The s-GFP mice displayed essentially an identicalpattern of expression with the other two transgenic models. In thisreport we focused our analysis on the expression of the s-GFPtransgene within the developing telencephalon and in particular itspresence in the population of neocortical pioneer neurons.

s-GFP Transgene is expressed in subplate neurons

In this study we confirmed that cortical preplate and ⁄ or subplateneurons express the s-GFP transgene by birth-dating, phenotyping andmorphological analyses of these cells. Birth-dating experiments withBrdU determined that many of the s-GFP+ cells were generatedbetween E11 and E12, the period in which cortical pioneer neurons arethought to be born in the mouse (Wood et al., 1992). Interestingly,

during embryonic stages the GFP+–BrdU+ double-labeled cells werenot only located below the cortical plate in what is typically identifiedas the subplate layer or interstitial neurons of the white matter, butthese early-generated cells were also present in the lower corticalplate. The distribution of the s-GFP-labeled cells is very similar to thatobserved in the cat visual cortex by Luskin & Shatz (1985b) whocalled this area the ‘upper subplate’, a term also used by Kostovic &Rakic (1990) in their study of the primate neocortex. Likewise, using[3H] thymidine, the work of Bayer & Altman (1991) indicated that, inthe rat, subplate neurons first accumulate in the expanding corticalplate prior to settling into a more defined subplate at �E19–22. Ourbinning analysis supports this hypothesis in that the majority ofs-GFP+ cells shifted their position from the cortical plate at E15.5 tolayers 6a and 6b by P6.5. Furthermore, qualitative analyses showedthat the majority of s-GFP+ cells in the marginal zone and subplateexpressed immunoreactivity for mature neuronal markers, MAP2 andsynapsin 1 and had morphological characteristics consistent with thatof subplate neurons (Allendoerfer & Shatz, 1994). We cannot excludethe possibility that some of the labeled cells in the deep cortical plate(future layers 5 and 6a) represent true cortical plate neurons. In spiteof the potential ambiguity, the cortical neurons labeled in the golli-s-GFP mouse represent some of the earliest born cortical neurons andexpression of the s-GFP in their processes allows us to follow thedevelopment of the farthest extent of their pioneering projections.

Fig. 8. s-GFP-labeled fibers established early projections to the contralateral cortex. During embryonic development, s-GFP-labeled fibers were observed to projectto the contralateral hemisphere via (A) the anterior commissure (ac) and (E) the corpus callosum (cc). (C) At this same developmental stage, strong expression ofs-GFP was detected in other subcortical pathways including the lateral olfactory tract (lot), stria terminalis (st) and the fimbria (fi). A, C and E are coronal sectionsstained immunohistochemically for GFP and adjacent sections in B, D and F were Nissl stained with Cresyl Violet. Scale bars, 1 mm (A–F).



Timing of corticothalamic projections

McConnell et al. (1989) were the first to demonstrate that subplateneurons in carnivores pioneer the cortical projection to the thalamusthrough the use of lipophilic fluorescent tracers such as DiI.Subsequent studies using similar techniques in rodents have supportedthis finding (Blakemore & Molnar, 1990; De Carlos & O’Leary, 1992;Erzurumlu & Jhaveri, 1992; Molnar & Blakemore, 1995). De Carlos &O’Leary (1992) and Auladell et al. (2000) found that in rats theprogression of the early subplate axons was rapid and that by E16 (E15in the mouse) subplate cells could be retrogradely labeled from DiIinjections into the thalamus. However, findings by other investigators(Molnar et al., 1998 in mouse; Molnar & Cordery, 1999 and Milleret al., 1993 in rat; Sheng et al., 1991 in wallaby; and Clasca et al., 1995in ferret) have suggested that subplate projections grow over a moreprolonged period of time with thalamic innervation, in the case ofrodents, not occurring until after birth, if at all (see Molnar, 1998).Our results, summarised in Fig. 9, are in agreement with those of

Molnar et al. (1998) and Auladell et al. (2000). We found that

axons from early pioneer neurons exit the intermediate zone at thecorticostriatal junction and enter the internal capsule quickly duringearly cortical development, �E13.5 in mice. However, havingreached the middle of the internal capsule by E14.5, the labeledfibers did not progress rapidly to the thalamus but instead appearedto pause in the presumptive neostriatum for 24–48 h. By E16.5,labeled fibers had progressed rapidly (nearly 0.5 mm in 24 h) to themedial aspect of the pallidum (i.e. entopeduncular nucleus) near thelateral border of the ventral thalamus. At this point, theirprogression appeared to slow down between E16.5 and E17.5.Our results are very similar to the findings of Molnar & Cordery(1999), who used different carbocyanine dye injections in theembryonic rat cortex and thalamus to conclude that the growth ofaxons from the cortex to dorsal thalamus was delayed in tworegions. The first was at E14–15 (E13–14 mouse) within the lateralentrance of the internal capsule and the second was closer to thethalamus, possibly the thalamic reticular nucleus, at �E16 (E15 inmouse). While the purpose of this ‘waiting period’ is not clear, itcan be speculated that these early corticofugal fibers require time to

Fig. 9. Summary diagram of pioneering corticofugal projections in the developing golli-s-GFP mouse forebrain. Corticothalamic projections: (A) by E13.5, axonsfrom labeled cells in the neocortex (Ctx; green dots) leave the intermediate zone and extend past the corticostriatal boundary (CSB) into the striatum (STR). (B)Between E13.5 and E15.5, as the fibers progress through the internal capsule, the leading edge of the processes transiently pauses in the striatum. (C) Between E16.5and E17.5, the early corticofugal projections slow again as they reach the diencephalic–telencephalic boundary (DTB). (D) By birth, these projections have reachedthe thalamus where they continue to differentially innervate the various thalamic nuclei over the next 2 weeks. Commissural Projections: (E–H) labeled corticofugalaxons were also observed in the earliest forming commissural pathways including (E and F) the anterior commissure and (G and H) corpus callosum. Otherabbreviations: AC, anterior commissure; CC, corpus callosum; HP, hippocampus.



gather and organise in order to ‘match up’ and guide theircorresponding thalamocortical afferents.

Differential innervation of the thalamus

In contrast to the findings of Miller et al., 1993) and Molnar &Cordery (1999), our results indicate that subplate projections doinvade the dorsal thalamus prior to birth. We found that at E18.5 asmall number of fibers entered the lateral dorsal thalamus but theirdistribution and arborization appeared to be very narrow in both theirrostral-to-caudal and lateral-to-medial dimensions. This restrictedpattern of innervation may explain why studies using injections of DiIcrystals may not have detected these fibers. The timing of thisinnervation also suggests that initial corticothalamic axons areinnervating the thalamus at approximately the same time asthalamocortical projections are beginning to invade the cortical plate(De Carlos & O’Leary, 1992; Molnar & Cordery, 1999). This apparentsynchronization of thalamocortical and corticothalamic inputs mayhave important functional implications for proper innervation of theirrespective targets both spatially and temporally. Thus, our data supporta key role for these early-generated cortical neurons in the propertiming and topography of both corticothalamic and thalamocorticalconnections.

During early postnatal development, our findings indicate that, ingeneral, early corticofugal fibers innervate the thalamus in an ordered(lateral-to-medial and ventral-to-dorsal) fashion that mirrors theknown neurogenetic gradient of the thalamic nuclei, with ventrolateralneurons being older than dorsomedial neurons (for review see Bayer &Altman, 1991). However, the pattern of innervation was not uniform.As the early corticofugal fibers begin to invade the thalamus, the firstnuclei to be innervated were the motor (ventral lateral, ventralanterior) and somatosensory (ventrobasal complex) nuclei whereasother nuclei, in particular the LGN and MGN, were excluded andappeared not to be completely innervated until P10.5–14.5. There maybe functional significance to this pattern of innervation in that thesomatosensory and motor connections are established prior to theauditory and visual modalities, mimicking the order of functionalmaturation. Thus somatosensory data necessary for tactile sensation ofthe head, which is important for nursing, for example, appears toprecede the other sensory modalities that are required at older ages forlong-range sensation and independent living.

s-GFP neurons pioneer commissural pathways

In rodents, developing commissural fibers are believed to cross thetelencephalic midline a few days before birth (Valentino & Jones, 1982;Bayer & Altman, 1991; Ozaki & Wahlsten, 1992). In our experiments,labeled projections were seen in the anterior commissure and corpuscallosum from the earliest stages of commissural development. Thesedata indicate that, in addition to the corticofugal pathways, the early-generated cortical neurons may also pioneer projections to thecontralateral hemisphere; they are also supported by the earlier studiesof Antonini & Shatz (1990) and Koester & O’Leary (1994).

Subplate neurons have been shown to be critical in establishingearly cortical connectivity (Ghosh et al., 1990; Ghosh & Shatz, 1993),and a recent study by Kanold et al. (2003) indicates that they may alsoplay a key role in the maturation of functional cortical circuits. Giventhe importance of these cells in cortical development, we believe thatthe golli-s-GFP transgenic mouse offers an important murine model tohelp elucidate the mechanisms by which these pioneer neurons serveto establish proper cortical connectivity.

Supplementary material

The following supplementary material may be found onwww.blackwell-synergy.comFig. S1. Cortical s-GFP+ neurons are born on E11.5 and E12.Fig. S2. Similar patterns of expression in s and golli-lacZ transgenesin cerebral cortices at E13.5.

Abbreviations

BrdU, 5-bromo-2-deoxyuridine; E, embryonic day; GFP, enhanced greenfluorescent protein; HSV-TK, herpes simplex virus–thymidine kinase; LGN,lateral geniculate nucleus; MAP2, microtubule-associated protein 2; MBP,myelin basic protein; MGN, medial geniculate nucleus; P, postnatal day; PB,phosphate buffer; spu, upper subplate; Tg, transgenic; Wt, wild type.

Acknowledgements

We thank Dr Chris Callahan for providing us with the bovine s-lacZ construct.This work was supported by National Institutes of Health Grants NS23022 andNS33091.

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