Subpallial origin of a population of projecting pioneer neurons during corticogenesis Javier Morante-Oria*†‡, Alan Carleton*‡, Barbara Ortino†§, Eric J. Kremer¶, Alfonso Faire´ n†ʈ, and Pierre-Marie Lledo*ʈ** *Laboratory of Perception and Memory, Centre National de la Recherche Scientifique, Unite´de Recherche Associe´e 2182, Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris, France; †Instituto de Neurociencias, Consejo Superior de Investigaciones Cientı´ficas,Universidad Miguel Herna´ndez, 03550 San Juan de Alicante, Spain; §Dipartimento Neurofisiologia Sperimentale, Istituto ‘‘C. Besta’’, Via Celoria 11, 20133 Milan, Italy; and ¶Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 5535, 1919 Route du Monde, 34293 Montpellier, France Communicated by D. Carleton Gajdusek, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France, June 16, 2003 (received for review February 17, 2003) Pyramidal neurons of the mammalian cerebral cortex are gener- reliable markers of such neurons. We show that MZ pioneer ated in the ventricular zone of the pallium whereas the subpallium neurons migrate tangentially to the MZ from subpallial sources. provides the cortex with inhibitory interneurons. The marginal We demonstrate that such a migration is reelin-dependent and, zone contains a subpial stream of migratory interneurons and two in addition, that reelin controls the arrival of MGE-derived different classes of transient neurons, the pioneer neurons pro- interneurons to the CP during early corticogenesis. vided with corticofugal axons, and the reelin-expressing Cajal– Retzius cells. We found in cultured slices that the medial ganglionic Materials and Methods eminence provides the reelin-negative pioneer neurons of the Organotypic Slices. Brains were obtained from embryos of preg- marginal zone. Pioneer neurons sent long projection axons that nant OF1 mice at embryonic day 14 (E14) (Iffa Credo; n ϭ 64; went through the cortical plate and reached the subplate and the date of plug ϭ E0). All animals used in this study were lateral border of the lateral ganglionic eminence. In the cultured maintained and treated according to protocols approved by the slices, pioneer neurons were functionally mature: they displayed a authors’ institutions. Dams were killed by cervical dislocation voltage-gated sodium current, expressed functional ␣-amino-3- under Isoflurane anesthesia (Belamont, Neuilly-sur-Seine, hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, France). The uterus and embryos were removed and rapidly and showed ␥-aminobutyric acid type A (GABA ) postsynaptic A isolated in cold oxygenated (5% CO2 and 95% O2) artificial events that were modulated by presynaptic AMPA receptors. corticospinal fluid (in mM): 124 NaCl, 3 KCl, 1.3 MgSO4,26 Pioneer neurons expressed the adhesion molecules L1 and TAG-1; NaHCO3, 1.25 NaH2PO4, 10 glucose, and 2 CaCl2. Brains were the latter has been reported to control tangential migrations to the embedded in 4% agar and coronally sliced (300 ␮m) with a neocortex [Denaxa, M., Chan, C.-H., Schachner, M., Parnavelas, J. & vibrating microtome (VT-1000, Leica, Argenteuil, France). Karagogeos, D. (2001) Development (Cambridge, U.K.) 128, 4635– Slices were placed onto Millicell-CM membranes (Millipore, 4644], and we show here that the pioneer neurons of the marginal Bedford, MA) in 35-mm Petri dishes containing 1 ml of the zone are the cellular substrate of such a function. Finally, we show following medium (Life Technologies, Cergy Pontoise, France): that, in early corticogenesis, reelin controls both the tangential 50% basal medium with Eagle’s salts, 25% horse normal serum, migration of cortical interneurons toward the cortical plate and the 25% Hanks’ balanced salt solution, 4.5 mg͞ml D-glucose, and 0.1 tangential migration of pioneer neurons toward the marginal mM L-glutamine (25). The medium had been heated previously zone. at 56°C for 30 min to inactivate the complement. wo germinative zones of the telencephalon, the dorsal (pal- Adenovirus Vector. We previously described the construction, Tlial) and the ventral (subpallial) ventricular zones (VZ), purification, and storage of CAVGFP (canine adenovirus-GFP; provide new neurons to the cerebral cortex. Postmitotic cells refs. 26 and 27). CAVGFP is an E1-deleted vector derived from from the dorsal VZ differentiate into excitatory projection the canine adenovirus serotype 2 (CAV-2), and harbors a neurons after a radial migration whereas the ventral part of the cytomegalovirus GFP (eGFP, Clontech) expression cassette. VZ, including the lateral (LGE), medial (MGE), and caudal The stock concentration was 2.5 ϫ 1012 particles per ml with a ganglionic eminences, generates neurons of the basal ganglia and particle to infectious unit ratio of 3- to 10:1. cortical interneurons (1–7). The cortical neurons, born early to form the preplate, are thought to function as a scaffold for the MGE Injections. CAVGFP and the membrane-permeant dyes assembly of the cortical architecture (8–12). Other cortical CellTracker Green (CMFDA) and CellTracker Orange neurons, born later, migrate radially and insert themselves into (CMTMR) (Molecular Probes; see ref. 28) were both pressure the preplate to generate the cortical plate (CP), which splits the injected with a glass micropipette into the LGE or MGE of one preplate into the marginal zone (MZ) and the subplate (SP) hemisphere of E14 slices, and kept for two days (2 D.I.V.). Crystals (8, 11). of DiI (1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine Cajal–Retzius cells, which reside in the preplate and thereafter in the MZ, are considered to be the earliest neurons to differ- entiate in the developing neocortex (8). These cells secrete reelin Abbreviations: CAVGFP, canine adenovirus-GFP; CP, cortical plate; DiI, 1,1Ј-dioctadecyl- (13–19), an extracellular matrix glycoprotein that is crucial for 3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate; D.I.V., days in vitro;En, embryonic day ␥ cortical lamination (20, 21). Pioneer neurons are also preplate- n; GABA, -aminobutyric acid; LGE, lateral ganglionic eminence; IPSC, inhibitory post- synaptic current; MZ, marginal zone; MGE, medial ganglionic eminence; NBQX, 1,2,3,4- derived; they guide thalamocortical afferent axons into the tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide; mIPSC, miniature IPSC; cortex and cortical efferent axons to their subcortical targets (10, sIPSC, spontaneous IPSC; SP, subplate; VZ, ventricular zone; NMDA, N-methyl-D-aspartate; 11). Recently, we described a population of pioneer neurons in CMTMR, CellTracker orange; IZ, intermediate zone; PSA-NCAM, polysialic acid neural cell the MZ (17, 18, 22, 23), but their origins, their molecular adhesion molecule. properties, and their functions remained unexplored (12, 24). ‡J.M.-O. and A.C. contributed equally to this work. We here characterize the MZ pioneer neurons by combining cell ʈA.F. and P.-M.L. contributed equally to this work. tracers and cell-specific markers with patch-clamp recordings. **To whom correspondence should be addressed. E-mail: [email protected]. We found that the adhesion molecules TAG-1 and L1 are © 2003 by The National Academy of Sciences of the USA 12468–12473 ͉ PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1633692100 Downloaded by guest on October 1, 2021 perchlorate, Molecular Probes) were inserted in the MGE. Slices were then either used for electrophysiological experiments or fixed in 4% paraformaldehyde for immunohistochemistry. Immunohistochemistry. Brains of E12–E16 OF1 mice, E12 Nkx2.1 knockout mice, and E16 and early postnatal reeler Orleans mice (including control littermates) were fixed overnight in 4% paraformaldehyde (PFA). Forty-micrometer-thick vibratome sections were obtained. Cultured slices were also sectioned into 40-␮m-thick slices after PFA fixation. Primary antibodies in- cluded monoclonal antibodies CR-50 (1:500; ref. 20) and G10 (1:1,000; ref. 29) to reelin, 4D7͞TAG-1 (1:100; refs. 30 and 31) to TAG-1 and 12E3 (1:1,000; ref. 32) to PSA-NCAM (polysialic acid neural cell adhesion molecule; 1:100; T. Seki, Juntendo University School of Medicine, Tokyo), and polyclonal antibod- ies to TAG-1 (1:1,000; K. Takeuchi, Nagoya, Japan), L1 (1:1,000; F. G. Rathjen, Berlin), Dlx (1:40; ref. 33); calretinin (1:2,000; Swant, Bellinzona, Switzerland), and calbindin (1:5,000; Swant). Secondary antibodies were conjugated to Alexa Fluor 488 or Fig. 1. Morphology of pioneer neurons in the MZ labeled after CAV injec- Alexa Fluor 568 (Molecular Probes) (for details, see Supporting tions in the MGE and intracellular filling with Dextran-rhodamine. (A)A Text, which is published as supporting information on the PNAS neuron with a fusiform, vertically oriented perikaryon. The axon is descending and gives rise to a collateral branch that extends toward the lateral side of the web site, www.pnas.org.). slice (arrow) and some descending branches. Dendrites are not shown. (B)A large multipolar neuron endowed with a descending axon that collateralizes Electrophysiological Recordings. Whole-cell patch-clamp record- in the CP and the SP. (Scale bar ϭ 50 ␮m.) ings were performed by using an RK 300 patch-clamp amplifier (Biologic, Claix, France). GFP-expressing cells were identified under visual control by using an upright Zeiss Axioskop micro- processes descended into the CP with collaterals in the CP and scope equipped with fluorescence and infrared differential the SP. NEUROSCIENCE interference contrast (DIC) video-microscopy.