Fgf8-Related Secondary Organizers Exert Different Polarizing Planar Instructions along the Mouse Anterior Neural Tube Ivan Crespo-Enriquez1, Juha Partanen2, Salvador Martinez1, Diego Echevarria1* 1 Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Cient´ıficas–Universidad Miguel Hernandez, Alicante, Spain, 2 Department of Biosciences, Division of Genetics, University of Helsinki, Helsinki, Finland Abstract Early brain patterning depends on proper arrangement of positional information. This information is given by gradients of secreted signaling molecules (morphogens) detected by individual cells within the responding tissue, leading to specific fate decisions. Here we report that the morphogen FGF8 exerts initially a differential signal activity along the E9.5 mouse neural tube. We demonstrate that this polarizing activity codes by RAS-regulated ERK1/2 signaling and depends on the topographical location of the secondary organizers: the isthmic organizer (IsO) and the anterior neural ridge (anr) but not on zona limitans intrathalamica (zli). Our results suggest that Sprouty2, a negative modulator of RAS/ERK pathway, is important for regulating Fgf8 morphogenetic signal activity by controlling Fgf8-induced signaling pathways and positional information during early brain development. Citation: Crespo-Enriquez I, Partanen J, Martinez S, Echevarria D (2012) Fgf8-Related Secondary Organizers Exert Different Polarizing Planar Instructions along the Mouse Anterior Neural Tube. PLoS ONE 7(7): e39977. doi:10.1371/journal.pone.0039977 Editor: Alain Che´dotal, Institut de la Vision, France Received January 12, 2012; Accepted May 30, 2012; Published July 6, 2012 Copyright: 2012 Crespo-Enriquez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by national grants from the Spanish Government BFU2008-00588, SAF 2008-01004 and Consolider-Ingenio CSD2007-00023. ICE was supported by a Spanish Government Fellowship FPI-BES 2006-12420M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: diegoaza@umh.es Introduction Proper embryonic development requires an accurately orches- trated complex network of interactions between signaling and transcription factors. Secreted signaling molecules (morphogens) organize fields of surrounding cells into molecular patterns and are tightly associated to the concept of positional information. This concept implies that a cell reads its position and determines its developmental fate/response according to a concentration gradi- ent of these extracellular factors [1]. These morphogens form long- range concentration gradients emanating from discrete sources and diffusing across the target fields [2–5]. The process of neurulation in vertebrates implies a major morphogenetic step for the initiation of brain regionalization. Localized signaling centers along the tube (called secondary organizers) and the morphogens emanating from them have a key role in refining the subdivisions of the embryonic brain. Among other morphogens, Fibroblast Growth Factors (FGFs) are a family of structurally related polypeptides with pleiotropic activities and are involved in a signaling system conserved from insects to humans [6]. Most FGFs mediate their biological responses as extracellular proteins by binding to and activating cell surface tyrosine kinase receptors (FGFRs). Three receptors, FgfR1, 2 and 3, are expressed in the vertebrate neural tube [7,8], FgfR1 being the important for morphogenetic activity of FGF8. Out of the 22 known FGFS, FGF8 has been proven to be a crucial morphogen for early vertebrate brain patterning [9–12]. Fgf8 is expressed preferentially at the so-called secondary organizers [13–16]. For more than a decade, the Isthmic organizer (IsO) has been used as a model to understand the morphogenetic activity of FGF8 and the planar induction mechanisms during mes- and rhombencephalon development in vertebrates [17–22]. Inactivation of Fgf8 transcription at early neural plate stages causes death of the entire mesencephalic and cerebellar primordia revealing a requirement for FGF8 signal in survival of neural progenitors [22]. If FGF8 activity is only moderately reduced, the anterior midbrain appears normal, but posterior midbrain, isthmus and vermis are lost indicating concentration dependency of this signal activity [23,24]. Moreover, misexpression of Sprouty2 (one of the negative feedback modulators of FGF8 signaling; [25,26]) moderately reduces FGF8 signaling in the IsO causing cell death in the anterior mesencephalon and rostralization of the remaining caudal midbrain epithelium suggesting that cell survival and patterning are independent properties [27]. Eight FGF8 isoforms have been identified so far, but only FGF8a and FGF8b isoforms have been related with IsO activity [28,29]. They have different signaling activities over the neural tube depending on the signal concentration and receptor binging affinity [9,29,30]. Only a strong FGF signal mediated by FGF8b activates the Ras extracellular signal-regulated kinase (ERK) pathway, which is sufficient to induce cerebellar development [31]. In chick, ERK1/2 induction is afterwards downregulated by Sprouty2 [32]. On the other hand, a lower level of signaling by FGF8a, FGF17 and FGF18 induces exclusively midbrain devel- opment [29,31,33]. Numerous feedback loops are known to maintain appropriate mesencephalon/cerebellum development and gene expression profiles around the IsO [19,30,34]. In fact, PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e39977