Rasl11b Knock Down in Zebrafish Suppresses One-Eyed- Pinhead Mutant Phenotype Guillaume Pe ´ zeron 1,2 , Guillaume Lambert 1,2 , Thomas Dickmeis 3 , Uwe Stra ¨hle 4 , Fre ´de ´ric M. Rosa 1,2 , Philippe Mourrain 1,2 * 1 INSERM, U784, Paris, France, 2 Ecole Normale Supe ´ rieure, Paris, France, 3 Max Planck Institute for Developmental Biology, Tu ¨ bingen, Germany, 4 Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany The EGF-CFC factor Oep/Cripto1/Frl1 has been implicated in embryogenesis and several human cancers. During vertebrate development, Oep/Cripto1/Frl1 has been shown to act as an essential coreceptor in the TGFb/Nodal pathway, which is crucial for germ layer formation. Although studies in cell cultures suggest that Oep/Cripto1/Frl1 is also implicated in other pathways, in vivo it is solely regarded as a Nodal coreceptor. We have found that Rasl11b, a small GTPase belonging to a Ras subfamily of putative tumor suppressor genes, modulates Oep function in zebrafish independently of the Nodal pathway. rasl11b down regulation partially rescues endodermal and prechordal plate defects of zygotic oep 2/2 mutants (Zoep). Rasl11b inhibitory action was only observed in oep-deficient backgrounds, suggesting that normal oep expression prevents Rasl11b function. Surprisingly, rasl11b down regulation does not rescue mesendodermal defects in other Nodal pathway mutants, nor does it influence the phosphorylation state of the downstream effector Smad2. Thus, Rasl11b modifies the effect of Oep on mesendoderm development independently of the main known Oep output: the Nodal signaling pathway. This data suggests a new branch of Oep signaling that has implications for germ layer development, as well as for studies of Oep/Frl1/Cripto1 dysfunction, such as that found in tumors. Citation: Pe ´zeron G, Lambert G, Dickmeis T, Stra ¨hle U, Rosa FM, et al (2008) Rasl11b Knock Down in Zebrafish Suppresses One-Eyed-Pinhead Mutant Phenotype. PLoS ONE 3(1): e1434. doi:10.1371/journal.pone.0001434 INTRODUCTION Endoderm and mesoderm germ layer formation in vertebrate embryos requires the activity of the conserved TGFb/Nodal signaling pathway [1,2,3,4,5,6]. Extensive studies in mouse, Xenopus and zebrafish have allowed the establishment of the current TGFb/Nodal pathway: Nodal signaling is activated by the interaction of Nodal ligands with activin (Alk4/ActR-IIB/Taram- A) receptors [7,8,9,10,11,12] and is regulated extracellularly by an EGF-CFC coreceptor (see below) and by antagonists belonging to the TGFb/Lefty family [13,14,15,16]. Nodal signaling is then transmitted intracellularly by phosphorylation of Smad2, and Smad2-P translocation in the nucleus in turn induces the expression of conserved mesendodermal transcription factors (see [17,18] for review). Mesendoderm induction by Nodal signaling requires a con- served EGF-CFC factor encoded by the gene Cripto1 in mouse [19,20], Frl1 (Fgf receptor ligand 1) in Xenopus [21] and one-eyed- pinhead (oep) in zebrafish [22,23]. The function of Oep/Cripto1/ Frl1 as a coreceptor of the Nodal pathway has been clearly demonstrated. In mouse, cripto mutants fail to form embryonic mesendoderm and resemble the mouse nodal mutant [10,19,24,25]. Similarly, in zebrafish, embryos lacking both maternal and zygotic oep function (MZoep, obtained by crossing rescued oep2/2 adult mutants [22]) display a phenotype similar to the zebrafish cyclops;squint (cyc;sqt) nodal double mutant [22]. They have an alterated antero-posterior axis and fail to develop endoderm, posterior mesoderm and prechordal plate (PP, a mesendodermal structure). More recently, biochemical analyses have shown that Oep/Cripto1/Frl1 is required for Nodal ligand binding to the Alk4/ActR-IIB/Taram-A receptor complex and subsequent Smad2 phosphorylation [10,26,27]. Other signaling pathways may also require the participation of EGF-CFC molecule either as a cofactor or as a ligand. For instance, in Xenopus and zebrafish, Frl1/Oep could function as antagonist of BMP [28] and more recently, in Xenopus, Frl1 has been implicated in the Wnt pathway and also been shown to be a coreceptor for Wnt11 [29]. Moreover, in cell culture, a soluble form of Cripto1 has been shown to bind the receptor Glypican1 and activate both the ras/raf/ERK/MAPK and PI3-K/AKT/GSK-3b intracellular sig- naling pathways, which are both well known for their involvement in oncogenic processes [30,31,32,33]. Interestingly, Cripto1 has been found in the conditioned medium of several human carcinoma cell lines suggesting that a naturally soluble isoform of Cripto1 could act as a diffusible ligand [34,35,36]. Thus, several reports suggest a multifunctional capability, both as ligand and as coreceptor of Oep/ Cripto1/Frl1. The role of Oep/Cripto1/Frl1 in pathways other than Nodal in vivo is still a matter of debate, certainly due in part to this complexity. The identification of additional elements which respond to Oep signaling stands to elucidate important signaling outputs of this protein. Here we provide genetic data which supports that oep can influence mesendoderm formation independently of Nodal signaling. We report the analysis of Rasl11b and the first developmental function for a member of the Rerg/Rasl11-12 subfamily [37,38,39,40,41]. We find that rasl11b down regulation acts as a specific suppressor of the oep phenotype and partially Academic Editor: Thomas Zwaka, Baylor College of Medicine, United States of America Received October 20, 2007; Accepted December 9, 2007; Published January 16, 2008 Copyright: ß 2008 Pe ´zeron 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: GP was supported by a fellowship from the Ministe ` re de l’Enseignement Supe ´rieur et de la Recherche and La ligue Nationale Contre le Cancer. FR was supported by grants ZF-MODELS (EU FP6 program) and Association pour le Recherche contre le Cancer. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: mourrain@wotan.ens. fr PLoS ONE | www.plosone.org 1 January 2008 | Issue 1 | e1434