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 dlx3band 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.

Specification, maintenance and differentiation of neural crest (NC) cells depend on multiple signaling pathways. In Xenopus and zebrafish, low levels of Bmp and high levels of Wnt signaling cooperate in NC induction (Saint-Jeannet et al., 1997;Wilson et al., 1997;Dorsky et al., 1998;Lewis et al., 2004). As Wnt signaling regulates anterior-posterior (A-P) patterning of the neural plate (Kim et al., 2000;McGrew et al., 1997;McGrew et al., 1995), Wnt signals might induce NC through posteriorization. However, neural A-P patterning and NC induction are separable events (Wu et al., 2005). A recent study indicates that Wnt-mediated posteriorization of the neural plate border (NPB) rather than the neural plate is crucial in NC induction . Signaling pathways active in NC specification are modulated by activators and inhibitors to regulate their strength and spatial distribution (Hong and SaintJeannet, 2007;Sauka-Spengler and Bronner-Fraser, 2008;Zhao et al., 2008). Here we introduce a factor, Potassium channel tetramerization domain containing 15 (Kctd15), that has a profound influence on NC formation in zebrafish and Xenopus embryos. KCTD15 was identified in humans (Hotta et al., 2009;Willer et al., 2009) as a BTB domain-containing protein of unknown function. We show that zebrafish kctd15a and kctd15b are expressed at the NPB at the end of gastrulation. Ectopic expression of Kctd15 inhibits NC specification, whereas knockdown leads to expansion of NC markers. Simultaneous attenuation of Wnt and Kctd15 expression rescues NC specification in zebrafish embryos. We suggest that Kctd15 restricts the NC domain by interfering with the functioning or output of the Wnt/b-catenin signaling pathway. MATERIALS AND METHODS Animal maintenance and embryonic stagingZebrafish (Danio rerio) embryos from AB/TL and TOP-dGFP (Dorsky et al., 2002) lines were obtained (Westerfield, 2000), and stages are indicated as hours post fertilization (hpf) (Kimmel et al., 1995). Xenopus laevis were staged according to (Nieuwkoop and Faber, 1967). Plasmids and DNA constructsThe open reading frames (ORFs) of zebrafish kctd15a (BC083478), zebrafish kctd15b (BC078294) and X. laevis kctd15 (BC077862) were subcloned into pCS2 + . The zebrafish b-cat* construct has four point mutations; S33A, S37A, T41A and S45A. MO and mRNA injectionMorpholino oligonucleotides (MO) were targeted to 5ЈUTR regions of zebrafish kctd15a (5Ј-TCCTTCCCTCCTTGGAAGACATAGC-3Ј) and zebrafish kctd15b (5Ј-AGCTCTCCTTCCCCCTCTTGATCTT-3Ј) (Gene Tools). Embryos at 1-2 cells were injected with 1.5 ng Kctd15a plus 0.5 ng Kctd15b MO; 2 ng Wnt8.1a MO (Lewis et al., 2004); or 2-4 ng standard control MO (5Ј-CCTC TTACCCTCAGTTACAATTTATA-3Ј) per embryo. 5Ј-capped mRNAs were prepared using the mMESSAGE mMACHINE Kit (Ambion). Each zebrafish embryo was injected with 50 pg kctd15a, kctd15b, DNkctd15a, DCkctd15a, kctd10, kctd6 or kctd13 mRNA, or 5 pg b-cat* mRNA. One blastomere of 2-cell Xenopus embryos was injected with 250 pg of Xkctd15 mRNA. For rescue experiments, 1-10 pg of mRNA was injected into fish...

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. Here, we profile global gene expression during optic fissure closure using laser capture microdissected (LCM) tissue from the margins of the fissure. From these data, we identify a unique role for the C 2H2 zinc finger proteins Nlz1 and Nlz2 in normal fissure closure. Gene knockdown of nlz1 and/or nlz2 in zebrafish leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. We also identify misregulation of pax2 in the developing eye of morphant fish, suggesting that Nlz1 and Nlz2 act upstream of the Pax2 pathway in directing proper closure of the optic fissure.coloboma ͉ eye ͉ pax2 ͉ zinc finger protein ͉ zebrafish T he mammalian eye begins as an evagination of forebrain neuroepithelium, the optic vesicle. As the optic vesicle approaches the surface ectoderm, it invaginates upon itself, forming a double-layered optic cup attached to the brain via the optic stalk. The asymmetric nature of this invagination leads to the formation of a gap along the ventral optic cup and optic stalk (the optic fissure) that remains open for hours to days, depending on the species. To form a spherical globe the margins of the optic fissure must meet and fuse ventrally during the 5th-7th

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.