1579 Introduction The pituitary gland is a key regulator of hormone secretion in vertebrates. The anterior lobe of the pituitary contains several cell types distinguished by their morphologies, locations and secreted hormones (Bentley, 1998; Dasen and Rosenfeld, 1999a; Dasen and Rosenfeld, 1999b). In mammals and birds, the anterior pituitary forms from Rathke’s pouch, an invagination of oral ectoderm beneath the ventral forebrain, whereas in zebrafish, the anterior pituitary derives from a placode that forms between the anterior hypothalamus and ventral ectoderm (Glasgow et al., 1997). Although several studies analyzed anterior pituitary precursor cells (Houart et al., 1998; Kozlowski et al., 1997; Whitlock and Westerfield, 2000), the precise origin of precursor cells is not well understood. Most studies suggest a dual origin for anterior pituitary; it derives either from the anterior neural ridge or from the non-neural ectoderm (Couly and Le Douarin, 1988; Dasen and Rosenfeld, 1999b; Eagleson et al., 1995; Eagleson and Harris, 1990; Kawamura and Kikuyama, 1992; Kondoh et al., 2000; Sheng and Westphal, 1999). Several transcription factors demarcate the presumptive pituitary and lens placodal field. Presumptive lens ectoderm in Xenopus expresses Otx2, Pax6 and Sox3, and in zebrafish, expression of dlx3b demarcates olfactory (Whitlock and Westerfield, 2000; Zygar et al., 1998) and otic placode precursor cells (Akimenko et al., 1994; Liu et al., 2003; Solomon and Fritz, 2002). We recently showed that dlx3b, dlx4b, sox9a and fibroblast growth factor (Fgf) signaling interact to specify the zebrafish otic placode (Liu et al., 2003). Because dlx3b and dlx4b are also expressed around the anterior border of the neural plate, these factors might also play a role in formation of the anterior pituitary. Analysis of zebrafish Hedgehog pathway mutants, you-too (gli2) (Karlstrom et al., 1999), iguana (Kondoh et al., 2000) and smu (smoothened) (Varga et al., 2001), suggests that Hedgehog signaling is required for anterior pituitary placode specification. The observations that zebrafish Hedgehog pathway mutants form ectopic lenses at the expense of pituitary and that early Rathke’s pouch expresses lens Delta-crystallin in chick suggest that these apparently unrelated tissues might arise from common precursors and that Hedgehog may be required to specify the pituitary lineage (Karlstrom et al., 1999; Kondoh et al., 2000; Varga et al., 2001). Other studies suggest that pituitary and lens cells ‘transdifferentiate’ in Hedgehog pathway mutants (Kondoh et al., 2000). In zebrafish, overexpression of Shh suggests that Hedgehog signaling is sufficient to pattern the anterior pituitary and induce pituitary Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3- expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non- Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types. Key words: Adenohypophysis, Anterior neural plate border, Cell fate specification, Cell lineage, Distal-less, Patched, Smoothened, Sonic hedgehog, Ventral ectoderm, Zebrafish Summary pitx3 defines an equivalence domain for lens and anterior pituitary placode Sunit Dutta 1 , Jens-Erik Dietrich 1 , Gudrun Aspöck 1 , Rebecca D. Burdine 2 , Alexander Schier 3 , Monte Westerfield 4 and Zoltán M. Varga 1, * 1 Department of Developmental Biology, University Freiburg, Germany 2 Department of Molecular Biology, Princeton University, NJ 08544-1014, USA 3 Skirball Institute, New York University School of Medicine, NY 10016, USA 4 Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA *Author for correspondence (e-mail: zoltan@zfin.org) Accepted 25 January 2005 Development 132, 1579-1590 Published by The Company of Biologists 2005 doi:10.1242/dev.01723 Research article Development