Short Report Sfrp1 demarcates the anterior neural plate and promotes eye development in the chick embryo PILAR ESTEVE, FRANÇOISE TROUSSE, JOSANA RODRÍGUEZ-SANCHEZ and PAOLA BOVOLENTA* Departamento de Neurobiología del Desarrollo, Instituto Cajal, CSIC, Madrid, Spain Int. J. Dev. Biol. 45 (S1): S65-S66 (2001) *Address correspondence to: Paola Bovolenta. Departamento de Neurobiología del Desarrollo, Instituto Cajal, CSIC, Dr. Arce 37, Madrid 28002, Spain. e-mail: bovolenta@cajal.csic.es ABSTRACT Secreted frizzled related proteins (SFRPs) are a new class of signaling molecules that antagonize the activity of Wnt proteins. However, the full range of SFRP activities and mecha- nisms of action are not fully understood. In our search for molecules which may participate in the development of the eye, we have isolated the chick homologue of Sfrp1. Here we show that in chick, Sfrp1 has a widespread and dynamic expression in the most anterior portion of the embryo. At later stages of development, Sfrp1 continues to be expressed with a pattern that is consistent with a function in cell fate determination and differentiation in both the eye and the neural tube. On the basis of this expression pattern, we have analyzed possible roles of Sfrp1 in the development of chick embryos. With in vitro and in vivo experiments, we show here that Sfrp1 has a dual function in the development of the chick retina: it promotes axon outgrowth and increases neuronal differentiation, while inhibiting cell proliferation. WNT proteins regulate cell fate and cell behavior in a wide variety of biological processes. During nervous system develop- ment, Wnt signaling has been implicated in the induction of posterior neural tissue in both Xenopus and mammalian embryos. WNT proteins are also required for midbrain development and for controlling the proliferation of precursor cells of the neural crest, dorso-lateral neural tube and hippocampus. In both fly and verte- brate, WNT proteins have been further implicated in modulating growth cone behavior and during cerebellar development they act as retrograde synaptogenic factors (Patapoutian and Reichardt, 2000). Wnt signaling is initiated by binding of WNT proteins to transmembrane Frizzled (Fz) receptors and involves the cytoplas- mic accumulation and the consequent nuclear translocation of beta-catenin, a protein involved in the activation of Wnt target genes. In the extracellular space, Wnt signaling is further controlled by a number of soluble molecules, including the expanding family of Secreted Frizzled Related Proteins (SFRPs), a new class of molecules that, as their name indicates share structural homology to the extracellular cystein rich domain (CRD) of the Fz receptors (Wodarz and Nusse, 1998). In search for molecules that may participate in the development of the eye, we have isolated the chick homologue of Sfrp1. By means of whole mount in situ hybridization using a digoxigenin- labeled anti-sense probe generated from a 2Kb cDNA fragment (1040-3065 bp; Accession number: AJ404652), we have deter- mined that, in chick, Sfrp1 has a widespread and dynamic expres- sion since blastula stage. During early primitive streak formation, its expression is restricted to the anterior primitive streak with a pattern which overlaps with that of Otx2 and is complementary to that of cWnt8c (Esteve et al., 2000). During neural plate formation cSfrp1 mRNAs are abundantly localized only to the anterior domain of the neural tissue, including the prospective eye field (Fig. 1). As neural tube closes, the expression of Sfrp1 extends with a rostro-caudal gradient to the ventral neural tube. At later stages of development, Sfrp1 continues to be expressed with a pattern that is consistent with a function in cell fate determination and differentiation in both the eye and the neural tube. Based on this expression pattern, we have set out the hypoth- esis that Sfrp1 might be necessary for the establishment of the antero-posterior axis of the embryo, as already shown in Xenopus and mouse embryos for other members of this family (Borello et al., 1999; DeRobertis et al., 2001). In addition, we have addressed the question of whether Sfrp1 plays a role in the specification and differentiation of the neural retina. To address these issues, we have generated a source of soluble SFRP1 protein by transfecting MDCK cells with a pcDNA3.1 eukaryotic expression vector containing the full coding sequence of cSfrp1 or with the vector alone. Isolated clones with the highest yields of protein secretion into the culture medium were selected as source of soluble molecule. Conditioned medium containing soluble SFRP1 protein was added to either dissociated or explant cultures derived from E5 chick embryos. After one day of culture, the effects of SFRP1 addition were evaluated by immunocy- tochemical staining with cell proliferation (bromo-deoxy-uridine incorporation and phospho-histone H3 expression) and cell differ- entiation (Beta-tubulin-III, neurofilament and Islet1/2 expression) A St5 St5/6 St6 Fig. 1. Expression pattern of cSfrp1 during neural induction. Dorsal view of embryos of St 5-6 hybridized in toto with a digoxigenin labeled probe against Sfrp1. Note the strong expression in the anterior neural plate.