Neuron, Vol. 27, 251–263, August, 2000, Copyright 2000 by Cell Press Midline Signals Regulate Retinal Neurogenesis in Zebrafish et al., 1999). This suggests either that there is redun- dancy in this pathway in fish or that other factors contrib- ute to the splitting of the retinal primordia. In support Ichiro Masai,* ‡§ Derek L. Stemple, † Hitoshi Okamoto,* and Stephen W. Wilson ‡§ *Laboratory for Developmental Gene Regulation of a role for Hh signaling in patterning the optic vesicles Brain Science Institute in fish, there is the observation that overexpression of RIKEN (The Institute of Physical and Hh proteins suppresses retina and expands optic stalk, Chemical Research) a phenotype in some ways opposite to cyclopia (Ekker 2-1 Hirosawa, Wako-shi et al., 1995; Macdonald et al., 1995). Saitama, 351-0198 The importance of Nodal signaling in early eye pat- Japan terning is evident from mutations in this pathway in ze- † National Institute for Medical Research brafish. cyclops ( cyc), squint ( sqt), and one-eyed pin- The Ridgeway head ( oep) mutant embryos all show varying degrees of Mill Hill cyclopia (Hatta et al., 1991; Heisenberg and Nu ¨sslein- London NW7 1AA Volhard, 1997; Schier et al., 1997). cyc and sqt encode ‡ Department of Anatomy and Developmental Nodal ligands that have both discrete and partially re- Biology dundant functions (Feldman et al., 1998; Rebagliati et University College London al., 1998; Sampath et al., 1998). Cyc is required for speci- fication of ventral midline CNS tissue, and a failure in Gower Street migration of this tissue contributes to the failure of bilat- London WC1E 6BT eral separation of the eyes in cyc -/ - embryos (Varga et United Kingdom al., 1999). Sqt appears to act earlier in development than Cyc and is required for prechordal plate development (Feldman et al., 1998). Indeed, defects in signaling from Summary the prechordal plate may contribute to the eye defects of sqt -/ - embryos (Heisenberg and Nu ¨sslein-Volhard, In zebrafish, neuronal differentiation progresses across 1997). Embryos lacking both Sqt and Cyc lack mesen- the retina in a pattern that is reminiscent of the neuro- doderm, revealing a partially redundant function for each genic wave that sweeps across the developing eye in gene in mesoderm induction (Feldman et al., 1998, Drosophila. We show that expression of a zebrafish 2000). The oep gene encodes a EGF-CFC protein (Zhang homolog of Drosophila atonal, ath5, sweeps across et al., 1998) required for Nodal signaling activity and the eye predicting the wave of neuronal differentiation. embryos missing zygotic Oep function lack prechordal By analyzing the regulation of ath5 expression, we plate and exhibit a cyclopic phenotype similar to cyc have elucidated the mechanisms that regulate initia- (Schier et al., 1997). Removal of both maternal and zy- tion and spread of neurogenesis in the retina. ath5 gotic Oep function generates the same phenotype as expression is lost in Nodal pathway mutant embryos cyc;sqt double mutants, confirming that Nodal signaling requires Oep function (Gritsman et al., 1999). lacking axial tissues that include the prechordal plate. After formation of the optic cup, multipotent retinal A likely role for axial tissue is to induce optic stalk precursors give rise to all major cell types in the retina cells that subsequently regulate ath5 expression. Our (Harris, 1997). Neuronal differentiation is usually initiated results suggest that a series of inductive events, initi- in the central retina and subsequently spreads peripher- ated from the prechordal plate and progressing from ally (McCabe et al., 1999, and references within). In ze- the optic stalks, regulates the spread of neuronal dif- brafish, differentiation is initiated in ventronasal rather ferentiation across the zebrafish retina. than central retina, but, as in other vertebrates, neuronal differentiation spreads as a wave from this position Introduction (Raymond et al., 1995; Laessing and Stuermer, 1996; Schmitt and Dowling, 1996; Hu and Easter, 1999). The During early forebrain development, the eye field initially mechanisms underlying the progression of neurogen- occupies a single domain in the anterior neural plate esis across the vertebrate retina are unknown but are (Woo and Fraser, 1995). Subsequent to this, cell move- reminiscent of the neurogenic wave that sweeps across ments within the midline of the neural plate, coupled the Drosophila eye imaginal disc generating ommatidia with signals derived from axial midline tissue lead to the behind the advancing morphogenetic furrow (Heberlein formation of two bilateral retinal primordia (Rubenstein and Moses, 1995). In this process, Hh secreted from and Beachy, 1998; Varga et al., 1999). These primordia mature ommatidial neurons initiates neurogenesis in ad- evaginate from the brain, form optic cups, and differenti- jacent undifferentiated cells through induction of the ate as neural retina and pigment epithelium. proneural bHLH transcription factor encoding gene The Hedgehog (Hh) and Nodal families of secreted atonal. Newly differentiated neurons in turn secrete Hh signaling molecules are involved in splitting of the retinal that initiates further neuronal differentiation and pro- field. shh mutations cause severe cyclopia in mouse gression of the morphogenetic furrow. In this way, the and human (Chiang et al., 1996; Roessler et al., 1996) cycle of Hh secretion and Atonal activation underlies but not in zebrafish (Schauerte et al., 1998; Karlstrom the progression of neurogenesis across the eye disc. In this study, we describe the expression and regula- tion of ath5, a zebrafish homolog of Drosophila atonal § To whom correspondence should be addressed (e-mail: imasai@ brain.riken.go.jp [I. M.], s.wilson@ucl.ac.uk [S. W. W.]). that is exclusively transcribed in the developing retina