851 Introduction Dissecting genetic cascades responsible for the development of the nervous system has helped to understand some of the molecular mechanisms involved in the genesis of distinct neuronal subtypes. The best-known molecular network implicated in neural cell fate decisions involves proneural and differentiation genes, which encode bHLH and other transcription factors, as well as neurogenic genes belonging to the Notch/Delta signalling pathway (Bertrand et al., 2002). The retina has been used as a model to study the role of these genes during retinal histogenesis because of its simple organisation and the limited number of neuronal types that it contains (Cepko, 1999; Perron and Harris, 2000; Vetter and Brown, 2001). The order of expression of several proneural and neurogenic genes has been studied in the retina, focusing on the proliferative ciliary marginal zone (CMZ). The CMZ is a region at the peripheral edge of the retina where cells are spatially ordered with respect to their development, with stem cells closest to the periphery, retinoblasts in the middle and differentiating precursors at the central edge (Wetts et al., 1989; Dorsky et al., 1995). In the CMZ, neurogenic and proneural genes are activated first in retinoblasts, followed by differentiation genes in differentiating precursors, reflecting a genetic hierarchy among these genes (Perron et al., 1998; Perron et al., 1999b). Overexpression or loss-of-function experiments of differentiation genes in the retina affects retinal cell type distribution (Perron and Harris, 2000; Cepko, 1999). For example, loss of ath5 (atoh7 – Zebrafish Information Network; Atoh7 – Mouse Genome Informatics) function in zebrafish and mouse prevents the differentiation of ganglion cells. Conversely, overexpression experiments show that ath5 promotes ganglion cell production at the expense of other cell types in Xenopus. It has therefore been proposed that ath5 is essential for retinal ganglion cell differentiation (Kanekar et al., 1997; Kay et al., 2001; Morrow et al., 1999; Brown et al., 2001). Recently, several lines of evidence have converged to propose that a combinatorial code of bHLH and homeobox proteins is responsible for the specification of the correct neuronal subtypes. For example, co-expression of the mouse bHLH genes Math3 (Neurod4 – Mouse Genome Informatics) and NeuroD (Neurod1 – Mouse Genome Informatics) with the homeobox genes Pax6 or Six3 significantly increases their ability to promote amacrine cell genesis (Inoue et al., 2002). As the various retinal cell types are born in a sequential order, it has also been proposed that the Notch/Delta pathway could RNA-binding proteins play key roles in the post- transcriptional regulation of gene expression but so far they have not been studied extensively in the context of developmental processes. We report on the molecular cloning and spatio-temporal expression of a novel RNA- binding protein, XSEB4R, which is strongly expressed in the nervous system. This study is focused on the analysis of Xseb4R in the context of primary neurogenesis and retinogenesis. To study Xseb4R function during eye development, we set up a new protocol allowing in vivo lipofection of antisense morpholino oligonucleotides into the retina. The resulting XSEB4R knockdown causes an impairment of neuronal differentiation, with an increase in the number of glial cells. By contrast, our gain-of-function analysis demonstrates that Xseb4R strongly promotes neural differentiation. We also showed a similar function during primary neurogenesis. Consistent with this proneural effect, we found that in the open neural plate Xseb4R expression is upregulated by the proneural gene XNgnr1, as well as by the differentiation gene XNeuroD, but is inhibited by the Notch/Delta pathway. Altogether, our results suggest for the first time a proneural effect for a RNA-binding protein involved in the genetic network of retinogenesis. Key words: Retina, RNA binding proteins, Morpholinos, Proneural genes, Xenopus Summary XSEB4R, a novel RNA-binding protein involved in retinal cell differentiation downstream of bHLH proneural genes Sébastien Boy 1, *, Jacob Souopgui 2,3, *, Marcos A. Amato 1 , Maurice Wegnez 1 , Tomas Pieler 2 and Muriel Perron 1,† 1 Laboratoire d’Embryologie Moléculaire et Expérimentale, UMR CNRS 8080, Bâtiment 445, Université Paris XI, 91405 Orsay, France 2 Universität Göttingen, Institut für Biochemie und Molekulare Zellbiologie, Abteilung Entwicklungsbiochemie, Justus von Liebig Weg 11, 37077 Göttingen, Germany. 3 Department of Biochemistry, Faculty of Science, University of Yaoundé I, PO Box 812, Yaoundé, Cameroon *These two authors have contributed equally to this work † Author for correspondence (e-mail: muriel.perron@emex.u-psud.fr) Accepted 12 November 2003 Development 131, 851-862 Published by The Company of Biologists 2004 doi:10.1242/dev.00983 Research article