Role of Fgf8 signalling in the specification of rostral Cajal-Retzius cells

Cline Zimmer ( 2 4 Jun Lee 1 Amlie Griveau 0 Silvia Arber 1 Alessandra Pierani 0 Sonia Garel 3 FranoisGuillemot 2 0 Institut Jacques Monod, Program in Development and Neurobiology, CNRS UMR 7592 and Universite Paris Diderot , Paris 75013 , France 1 Biozentrum, Department of Cell Biology, University of Basel, and Friedrich Miescher Institute for Biomedical Research , 4056 Basel , Switzerland 2 National Institute for Medical Research (NIMR), Medical Research Council (MRC), Department of Molecular Neurobiology , London NW7 1AA , UK 3 INSERM U784, Ecole Normale Superieure, Departement de Biologie , Paris 75005 , France 4 Present address: IBDML, CNRS UMR6216 , Marseille 13009 , France T N E M P O L E V E D - SUMMARY Cajal-Retzius (CR) cells play a key role in the formation of the cerebral cortex. These pioneer neurons are distributed throughout the cortical marginal zone in distinct graded distributions. Fate mapping and cell lineage tracing studies have recently shown that CR cells arise from restricted domains of the pallial ventricular zone, which are associated with signalling centres involved in the early regionalisation of the telencephalic vesicles. In this study, we identified a subpopulation of CR cells in the rostral telencephalon that expresses Er81, a downstream target of Fgf8 signalling. We investigated the role of the rostral telencephalic patterning centre, which secretes FGF molecules, in the specification of these cells. Using pharmacological inhibitors and genetic inactivation of Fgf8, we showed that production of Fgf8 by the rostral telencephalic signalling centre is required for the specification of the Er81+ CR cell population. Moreover, the analysis of Fgf8 gain-of-function in cultivated mouse embryos and of Emx2 and Gli3 mutant embryos revealed that ectopic Fgf8 signalling promotes the generation of CR cells with a rostral phenotype from the dorsal pallium. These data showed that Fgf8 signalling is both required and sufficient to induce rostral CR cells. Together, our results shed light on the mechanisms specifying rostral CR cells and further emphasise the crucial role of telencephalic signalling centres in the generation of distinct CR cell populations. INTRODUCTION The cerebral cortex contains different subclasses of excitatory projection neurons derived from the dorsal telencephalon or pallium, as well as inhibitory interneurons originating from the ventral telencephalon or subpallium (Marin and Rubenstein, 2003; Molyneaux et al., 2007). The cerebral cortex is subdivided into different regions with divergent evolutionary histories, i.e. the archicortex, neocortex and paleocortex, and into areas with distinct functions, i.e. the motor, somatosensory, visual and auditory cortical areas (OLeary et al., 2007; Rakic, 1988; Rash and Grove, 2006; Sur and Rubenstein, 2005). The cortex is also organised into six layers containing neurons with different morphological, molecular and physiological characteristics and unique patterns of connectivity. This cytoarchitecture is tightly regulated, with a defined number of neurons adopting specific laminar features in each zone, which is crucial for the proper activity of the cerebral cortex. Cajal-Retzius (CR) cells are among the first neurons to be generated between E10.5 and E13.5 in mouse (Hevner et al., 2001; Takiguchi-Hayashi et al., 2004), and they die during the first postnatal weeks (Abraham and Meyer, 2003; del Rio et al., 1995; Derer and Derer, 1990; Marin-Padilla, 1990; Marin-Padilla, 1992; Zecevic and Rakic, 2001). This transient pioneer neuronal population was discovered more than a century ago, in humans by G. Retzius and in lagomorphs by S. Ramon y Cajal, but the features and functions of these cells remain largely unknown. They appear to play a key role in the radial migration of cortical neurons and in the laminar organisation of the mouse and human cortex, largely through the production of the extracellular glycoprotein reelin (DArcangelo et al., 1995; Ogawa et al., 1995; Rice and Curran, 2001; Super et al., 2000). CR cells populate the marginal zone (MZ) of the cortex evenly in the prospective neocortex, but accumulate at distinct locations such as at the olfactory piriform cortex. Various CR cell subpopulations have been identified and shown to differentially express several molecular markers, including reelin, calretinin (calbindin 2 Mouse Genome Informatics) and p73a (Trp73 Mouse Genome Informatics) (Bielle et al., 2005; Meyer et al., 2002; Takiguchi-Hayashi et al., 2004). CR cells have long been assumed to arise from the whole pallial ventricular zone (VZ) and to migrate radially to the cortical surface, similarly to other glutamatergic cortical neurons (del Rio et al., 1995; Hevner et al., 2003; Marin-Padilla, 1998). However, Meyer and colleagues identified restricted sites of generation of CR cells based on the expression of p73a, a transcription factor that is expressed by different CR cell subpopulations (Meyer et al., 2002). Consistently, fate mapping and cell lineage tracing studies have shown that CR cells arise from specific locations along the rostrocaudal and dorsoventral axes of the pallial VZ (Bielle et al., 2005; GarciaMoreno et al., 2007; Imayoshi et al., 2008; Monuki et al., 2001; Takiguchi-Hayashi et al., 2004; Yoshida et al., 2006; Zhao et al., 2006). Four different sites of CR generation have been identified, comprising the pallial domain of the septum in the rostromedial (RM) pallium (Bielle et al., 2005), the ventral pallium (VP) laterally (Bielle et al., 2005), the prospective choroid plexus, and the cortical hem (CH) caudally (Garcia-Moreno et al., 2007; Imayoshi et al., 2008; Monuki et al., 2001; Takiguchi-Hayashi et al., 2004; Yoshida et al., 2006; Zhao et al., 2006). CR cells migrate tangentially from these focal sites to populate the entire cortical surface. Furthermore, fate mapping studies have shown that CR cells originating from different sources preferentially settle in distinct regions of the cortex (Bielle et al., 2005; Imayoshi et al., 2008; Takiguchi-Hayashi et al., 2004; Yoshida et al., 2006; Zhao et al., 2006). Indeed, ablation of the CH results in a substantial depletion of CR cells, except in the rostral cortex, thus demonstrating that CH-derived CR cells mainly populate the caudal cortex (Yoshida et al., 2006). Intriguingly, the neuroepithelial domains that generate CR cells are closely associated with signalling centres involved in the early regionalisation of the telencephalic vesicles. These signalling centres secrete morphogens that provide positional and proliferative cues to the surrounding telencephalic neuroepithelium (OLeary et al., 2007; Rash and Grove, 2006; Sur and Rubenstein, 2005). Recently, the signals produced by the CH in the caudomedial telencephalon, which include TGFb molecules, were shown to be necessary for the generation of caudal CR cells (Friedrichs et al., 2008; Hanashima et al., 2007; Siegenthaler and Miller, 2008; (...truncated)