Floor plate develops upon depletion of tiggy-winkle and sonic hedgehog

Hedgehog signaling is thought to play an important role in the differentiation of the vertebrate neural tube (Tanabe and Jessell, 1996). By injecting a morpholino (MO) directed against tiggy-winkle hedgehog (twhh) (Ekker et al., 1995) into zebrafish sonic-you (syu) embryos mutant for the sonic hedgehog (shh) gene (Schauerte et al., 1998), we examined Hedgehog-dependent and independent aspects of early neural patterning. The floor plate (FP) is a specialized row of cells located in the ventral midline of the vertebrate neural tube that influences axon guidance and neuronal differentiation (Placzek et al., 2000). Experiments performed on chick embryos indicated that the FP is specified by Shh signaling from the notochord to the overlying neural plate (Tanabe and Jessell, 1996). This model of a classic cellular induction was supported by studies showing that notochord-derived signals induce FP formation (Yamada et al., 1991), and that notochord removal results in loss of FP (Placzek et al., 1990). Analyses of chick-quail chimeric embryos and of zebrafish mutants with midline defects suggest that FP specification commences before notochord formation (Le Douarin and Halpern, 2000). When Hensen’s node of chick embryos is replaced by the quail counterpart, for example, the regressing node produces FP, notochord, and dorsal endoderm of quail origin (Charrier et al., 1999). Together with the finding that FP has a different origin than the rest of the neural plate (Catala et al., 1996), the data suggest that notochord and FP originate from a population of multipotential midline precursor cells that reside in Hensen’s node in the chick (Le Douarin and Halpern, 2000). Analogous cells are predicted to be found in the dorsal shield or early organizer region of the zebrafish gastrula (Halpern et al., 1997; Appel et al., 1999). Zebrafish floating head (flh) and no tail (ntl) mutants develop FP even though they lack an Shh-expressing notochord (Halpern et al., 1993, 1995; Talbot et al., 1995). In fact, ntl mutants develop a wider FP than wild-type (WT) embryos (Odenthal et al., 1996; Strahle et al., 1996; Halpern et al., 1997), suggesting that ntl regulates allocation of notochord and FP from the proposed common precursor population (Halpern et al., 1997). The Notch-Delta signaling pathway has been similarly implicated in mediating cell fate choices in the zebrafish gastrula midline (Appel et al., 1999). The role of Shh in FP formation has also come under question from the study of zebrafish syu/shh mutants. Homozygous null syu mutants develop the medial FP row, indicating that Shh signaling is not essential for induction of these cells (Schauerte et al., 1998; Odenthal et al., 2000). One explanation is that the shh paralogue twhh is also expressed in the dorsal shield and in the gastrula midline, where it could function redundantly with shh in FP specification (Fig. 1a–e). Later, during segmentation, shh is expressed in the developing notochord and FP (Krauss et al., 1993), whereas twhh expression is confined to the FP (Ekker et al., 1995). Expression of a third zebrafish gene, echidna hedgehog (ehh), is first detected at midgastrulation, following shh and twhh expression in the midline (Currie and Ingham, 1996), and presumably too late to be involved in the initial steps of FP specification. Recent experiments using the ehh-MO support this view (Lewis and Eisen, 2001). An argument in favor of the early specification of FP comes from the analysis of FP-specific gene expression, which is evident shortly after cells emerge from the dorsal shield during extension of the embryonic axis. We find that twhh and shh expression segregates into discrete cell layers during gastrulation (Fig. 1c–e), with shh transcripts colocalized to the Ntl immunoreactive cells of the presumptive notochord and twhh transcripts confined to overlying cells (Fig. 1c, d). These cells are presumed to develop as FP on the basis of their position and continuous and specific expression of twhh. Al-