SummaryKupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut Research article Development 1248However, genes implicated in ciliated node cell function are also expressed in non-node cells, and mutations of these genes give rise to a variety of laterality phenotypes and more pleiotropic phenotypes, making it impossible to exclude other mechanisms for LR development during early embryogenesis (for reviews, see Tabin and Vogan, 2003;Wagner and Yost, 2000;Yost, 2003). Furthermore, it is not known whether nodal flow is a murine-specific mechanism. Cilia formation and expression of lrd homologues have recently been observed in structures analogous to the node in chick, frog (Xenopus laevis) and zebrafish embryos (Essner et al., 2002), but the existence of motile cilia and nodal flow has not been demonstrated in non-murine embryos. Further confounding the issue, there are molecular asymmetries that precede the appearance of lrd expression and monocilia in Xenopus Levin et al., 2002) and perhaps in chick (Levin et al., 1995;Stern et al., 1995). This raises the issue of whether ciliated cells in other vertebrate embryos generate fluid flow and have a conserved function for ciliogenesis genes in LR development. In zebrafish, ciliated cells arise in the tailbud at the end of gastrulation (Essner et al., 2002) in a transient spherical organ called Kupffer's vesicle (KV). KV, first described in 1868 (Kupffer, 1868), is a conserved structure among teleost fishes. Electron microscopy studies in the bait fish Fundulus heteroclitus have shown that a single cilium (i.e. monocilium) protrudes from each cell lining KV into the lumen (Brummett and Dumont, 1978). In zebrafish, KV is formed from a group of approximately two-dozen cells, known as dorsal forerunner cells (DFCs), that migrate at the leading edge of the embryonic shield (the zebrafish equivalent of the mouse node) during gastrulation. In contrast to other cells in this region, DFCs do not involute during gastrulation, but remain at the leading edge of epibolic movements. At the end of gastrulation, DFCs migrate deep into the embryo and organize to form KV (Cooper and D'Amico, 1996; D'Amico and Cooper, 1997;Melby et al., 1996). During subsequent somite stages, KV is found ventral to the forming notochord in the tailbud and adjacent to the yolk cell. Although KV was first described well over 100 years ago, it remains unknown whether DFCs and KV are mesodermal or endodermal in origin, and it is unclear what role they play during development, leading to the categorization of KV as an embryonic 'organ of ambiguity ' (Warga and Stainier, 2002).Recently, using a novel technique to knockdown gene expression specifically in DFCs, we reported the first evidence that DFCs and/or KV function in LR patterning (Amack and Yost, 2004). Here, we show that cilia that arise inside KV are motile and generate a consistent counterclockwise fluid flow. A combination of laser ablations, embryological manipul...

Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic; however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, we have identified two new Wnt targets, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, we show that zic2a and zic5 transcription is activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair.

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. We demonstrate that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. We show that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance our understanding of a key gene regulatory network that, when disrupted, causes HPE.