Molecular mechanisms controlling brain development: an overview of neuroepithelial secondary organizers CLAUDIA VIEIRA, ANA POMBERO, RAQUEL GARC��A���LOPEZ, LOURDES GIMENO, DIEGO ECHEVARRIA and SALVADOR MART��NEZ* Alicante Neuroscience Institute, Miguel Hernandez University, CSIC, San Juan de Alicante, Alicante, Spain ABSTRACT The vertebrate Central Nervous System (CNS) originates from the embryonic dorsal ectoderm. Differentiation of the neural epithelium from the ectoderm and the formation of the neural plate constitute the first phase of a complex process called neurulation which culminates in the formation of the neural tube, the anlage of the CNS in sauropsids and mammals (for review see Smith and Schoenwolf, 1997 Colas and Schoenwolf, 2001). At neural plate and neural tube stages, local signaling centers in the neuroepithelium, known as secondary organizers, refine the antero-posterior specification of different neural territories (for review see Echevarria et al., 2003 Stern et al., 2006 Woltering and Durston, 2008). In this review, we will describe the principle aspects of CNS development in birds and mammals, starting from early stages of embryogenesis (gastrulation and neurulation) and culminating with the formation of a variety of different regions which contribute to the structural complexity of the brain (regionalization and morphogenesis). We will pay special attention to the cellular and molecular mechanisms involved in neural tube regionalization and the key role played by localized secondary organizers in the patterning of neural primordia. KEY WORDS: patterning, neural plate, neural tube, gastrulation, neurulation, secondary organizer, anterior neural ridge, zona limitans intrathalamica, isthmic organizer Neural plate and neural tube formation A fundamental early step in neural development is the allocation of a group of ectodermal cells as precursors of the entire nervous system (Hemmati-Brivanlou and Melton, 1997). This process involves an inductive interaction first demon- strated in amphibian embryos by Spemann and Mangold in the 1920���s (see Spemann and Mangold, 2001). Their experiments which involved the grafting of differently pigmented species of newt established the concept of neural induction as an instruc- tive interaction between the dorsal lip of the blastopore (the ���organizer���) and the neighboring ectoderm. The discovery of a neural organization center for the amphibian gastrula initiated a search for homologous structures in other vertebrates. Soon thereafter, the equivalent region was discovered in most verte- brate species, including the shield of teleosts. In birds and mammals, the region was named ���Hensen���s node��� and ���the node���, respectively. When C.H. Waddington transplanted the Hensen node of a chick embryo, he observed the induction of Int. J. Dev. Biol. 54: 7-20 (2010) doi: 10.1387/ijdb.092853cv BIOLOGYDEVELOPMENTALOFJOURNALINTERNATIONALTHE www.intjdevbiol.com *Address correspondence to: Salvador Martinez. Institute of Neuroscience Alicante. Miguel Hernandez University (CSIC), Ctra. de Valencia Km 18, E-03550 San Juan de Alicante, Alicante, Spain. Fax. +34-965-919-555. e-mail: smartinez@umh.es Tel:+34-965-919-556. Web: http://www.ina.umh.es/grupos-detalle.aspx?grupo=6 Accepted: 13 March 2009. Final author-corrected PDF published online: 25 September 2009. ISSN: Online 1696-3547, Print 0214-6282 �� 2009 UBC Press Printed in Spain Abbreviations used in this paper: ANR, anterior neural ridge AP, antero- posterior BMP, bone morphogenetic protein DV, dorso-ventral FGF, fibroblast growth factor IsO, Isthmic organizer ML, medio-lateral TGF, transforming growth factor ZLI, zona limitans intrathalamica. an ectopic neural plate or the formation of a partial new embryonic axis containing neural tube, notochord and somites (Waddington, 1933 Waddington, 1936). This demonstration provided the first evidence that in chick embryos, the nervous system is induced by signals from non-neural cells. Recent works demonstrated that the capacity of ectodermal cells to undergo neural differentiation represents their default state. In fact, neural differentiation must be suppressed in the lateral ectoderm by signals transmitted between neighboring cells, in order to develop as epidermis. These molecular signals are members of the bone morphogenetic protein (BMP) subclass of transforming growth factor �� (TGF-��)-related proteins (for re- view see Wittler and Kessel, 2004).

8 C. Vieira et al. Recent studies using chick embryos have shown that neural induction really begins prior to the formation of the organizer region and thus must be initiated by signals derived from other cellular areas. Members of other families of signaling mol- ecules, notably the fibroblast growth factors (FGFs), have now been proposed as early-acting factors, which initiate neural induction by a progressive sequence of molecular interactions. First, the presumptive neural plate area is established by Fgf8 activity coming from the primary endoderm. Subsequently, the suppression of BMP signaling maintains rather than initiates the process of neural differentiation (Linker et al. 2009 for review see Stern, 2005). These molecular interactions together with the participation of Hox genes (Woltering and Durston, 2008 Hooiveld et al., 1999) during the process of gastrulation regulate cellular inductive events lead- ing to the definition of the antero-posterior and dorso-ventral axes of the embryo and to the gen- eration of the three blastodermal layers: ectoderm, mesoderm and endoderm (Stern et al.,2006). Thus, in the central area of the embryo (at its prospective dorsal region), ectoderm cells are induced to de- velop as neural plate cells as a result of these progressive cellular and molecular interactions, act- ing via planar and vertical induction (Fig. 1A). In- deed, formation of the neural plate involves apico- basal thickening and pseudostratification of the ectoderm, resulting in the formation of a flat but thickened epithelial region which expresses a unique pattern of molecular markers (Smith and Schoenwolf, 1989 Keller et al., 1992). Subsequently, the process of neurulation in- volves cell shape changes and epithelial rearrange- ment which result in the bending of the neural plate and apposition of its latent edges to form the neural tube. The neural plate lengthens along the antero- posterior axis and becomes narrower, so that sub- sequent bending will form a tube. Full antero-poste- rior formation and extension of the neural tube requires normal gastrulation movements and in particular, regression of the primitive streak (Voiculescu et al., 2007). Bending of the neural plate involves the forma- tion of hinge regions where the neural tube contacts surrounding tissues (for review see Colas and Schoenwolf, 2001). Elevation of the neural folds establishes a trough-like space called the neural groove, which becomes the lumen of the primitive neural tube after closure of the neural groove. In addition, the neural folds will generate the special- ized cells of the neural crest. The neural tube closes as the paired neural folds are brought together at the dorsal midline (Fig. 1B). During this stage, the epidermal ectoderm from each fold detaches from its ipsilateral neuroepithelial partner and fuses with the epidermal ectoderm of the contralateral neural fold, contributing to the dorsal skin of the embryo. Similarly the detached neuroepithelial layers from both sides fuse together below the epidermal ecto- Fig. 1. The neurulation process. (A) At neural plate stage, vertical induction (green arrows) from the underlying axial mesendoderm (notochord and prechordal plate), together with planar induction from Hensen���s node (orange arrows) and ectoderm (yellow arrows) regulate dorsoventral polarity and the initial steps of antero-posterior regionalization in the neuroepithelium. (B) During neurulation, neural folds close at the dorsal midline. Neural crests cells delaminate and migrate from the neural folds before closure and the neural grove become the lumen of the neural tube. Planar information from the ventral midline (floor plate FP yellow arrow) and dorsal midline (roof plate RP red arrow) plays a fundamental role in the establishment of definitive dorsoventral regionalization, using sonic hedgehog (SHH) and bone morphogenetic proteins (BMP) as signaling molecules. As a consequence of these inductive events, the lateral wall of the neural tube is subdivided into two columnar domains: the basal plate (close to the floor plate) and the alar plate (close to the roof plate). AP, alar plate BP, basal plate. B A derm, establishing the roof plate of the neural tube. Cellular patterns and molecular regionalization of the neu- ral plate Experiments involving the labeling of individual cells or small groups of them, and analysis of their fate during gastrulation and neurulation have been performed in different species. The resulting fate maps showed that the generation of the neural plate and tube involves similar morphogenetic programs in

Molecular brain development and secondary organizers 9 vertebrates (reviewed by Rubenstein et al., 1998 Cobos et al., 2001). During gastrulation, the bending of the neural plate, together with the intercalation of neuroectodermal precursors and with regional differences in proliferation, transform the initial medio-lateral arrangement of cells in the neural plate into the dorso-ventral organization of the neural tube (Fig. 1 Leikola 1976 Stern et al., 2006). Gene expression patterns provide insights into the location, onset and developmental consequences of inductive processes, which generate regional specification within the developing brain. They also help to identify the candidate molecules which regulate these processes. During the past two decades, re- searchers have identified many regulatory genes whose pat- terns of expression in the embryonic neural plate and tube have yielded important insights into brain regionalization (Shimamura et al., 1995 1997 Rubenstein et al. 1998 Crossley et al. 2001 Puelles and Rubenstein 2003 Echevarria et al. 2003 Aroca and Puelles, 2005). Interpretation of the expression patterns in terms of the topology of the neural plate axes provides a clearer picture of its molecular regionalization and also contributes to our understanding of how the expression of specific sets of genes in the neuroepithelium is related to brain histogenesis. Thus, longitudinal patterns reflect expression which extends along part or the entire antero-posterior axis and may mark the three primordia of the floor, basal and alar plates (Fig. 2). At the anterior pole of the neural plate, there is evidence indicating that medial (basal plate primordium) and lateral (alar plate primordium) domains form nested arcs which are concentric with the anterior neural ridge (Shimamura et al., 1995 Cobos et al., 2001 Echevarria et al., 2003). Other genes have expression domains which are restricted to transverse regions of the neural plate, showing molecular discontinuities along the antero-posterior axis (Fig. 2). Interest- ingly some transcription factors are expressed in nested do- mains of the neural plate, playing a fundamental role in the establishment of particular transverse territories which actively regulate the development of the neighboring neuroepithelium. These nested domains have been termed secondary organiz- ers (Fig. 2 Martinez, 2001 Echevarria et al., 2003 Aroca and Puelles 2005). Regionalization of the neural tube The early neural tube is, in most vertebrates, a straight structure. However, even before the posterior portion of the tube has formed, the most anterior portion of the neural tube is undergoing drastic changes. In this region, the tube balloons into three primary vesicles: the forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon) (Fig. 3 reviewed in Martinez and Puelles, 2000). By the time the posterior end of the neural tube closes, secondary bulges ��� the optic vesicles ��� have extended laterally from each side of the developing forebrain. At this early stage of development (three vesicle stage), the bending of the long axis, already observed at the late neural plate stages, increases considerably after neurulation, leading to the cephalic and cervical flexures of the neural tube (Fig. 3). Then, the prosencephalon becomes sub- divided into the anterior secondary prosencephalon (telen- cephalon and hypothalamus) and the more caudal diencepha- lon (Pombero and Martinez, 2009). The discovery that putative regulatory genes are expressed in regionally restricted patterns in the developing forebrain has Fig. 2. Molecular regionalization of the neural plate. Schematic (A) and realistic (B) representations of gene expression domains at the neural plate stage. Different colors represent differ- ent genes (gene symbol and color codes are identified in the schematic diagram). Medio-lateral (dorso-ventral) and antero- posterior (rostro-caudal) regionalization are identifiable by the limits between expression domains. The presumptive epithelia of different brain regions have been identified, as well as the pre- sumptive localization of the secondary organizers, in relation to precise bound- aries between gene expression do- mains. Secondary organizers: ANR: anterior neural ridge ZLI: zona limitans intrathalamica IsO: isthmic organizer. Longitudinal domains: RP, roof plate AP, alar plate: BP (bp), basal plate FP, floor plate. Abbreviations: Hypoth, hy- pothalamus prethal, prethalamus tg thal, thalamus prerub, prerubral pretect, pretectum MES, mesencepha- lon mes tg, mesencephalic tegmen- tum r1, rhombomere 1. A B