Midline signaling by Hedgehog (Hh) family members has been implicated in patterning the vertebrate embryo. We have explored the potential regulatory role of cAMP-dependent protein kinase A (PKA) in these events. Zebrafish embryos injected with RNAs encoding Sonic hedgehog (Shh), Indian hedgehog (Ihh), or a dominant-negative regulatory subunit of PKA, PKI, have equivalent phenotypes. These include the expansion of proximal fates in the eye, ventral fates in the brain, and adaxial fates in somites and head mesenchyme. Moreover, ectopic expression of PKI partially rescues somite and optic stalk defects in no tail and cyclops mutants that lack midline structures that normally synthesize Shh. Conversely, all cell types promoted by ectopic expression of hhs and PKI are suppressed in embryos injected with RNA encoding a constitutively active catalytic subunit of PKA (PKA*). These results, together with epistasis studies on the block of ectopic Hh signaling by PKA*, indicate that PKA acts in target cells as a common negative regulator of Hedgehog signaling.[Key Words: Sonic hedgehog; Indian hedgehog; protein kinase A; midline signaling; zebrafish] Received December 20, 1995; revised version accepted February 2, 1996.Members of the hedgehog (hh) gene family encode signaling proteins involved in induction and patterning processes in vertebrate and invertebrate embryos. In vertebrates, hhs constitute a multigene family. Sonic hedgehog (Shh), which has received most of the experimental attention, has been isolated from mouse (Echelard et al. 1993; Chang et al. 1994}, chick (Riddle et al. 1993, rat (Roelink et al. 1994), Xenopus (Ekker et al. 1995), and zebrafish (Krauss et al. 1993). The early expression is limited to the notochord, the floor plate and its anterior extension in the brain, and the zone of polarizing activity (ZPA) in the posterior mesenchyme of the limb bud. All of these sites have been shown to act as organizing centers patterning neighboring tissues by the secretion of morphogenetic signals (e.g., Saunders and Gasseling 1983;Brand-Saberi et al. 1993; Pourquie et al. 1993;Yamada et al. 1993). Recent evidence indicates that Shh is involved in these interactions.For example, ectopically expressed Shh leads to a ventralization of large regions of the mid-and hindbrain in mouse and zebrafish, as revealed by the dorsally expanded expression pattern of HNF-3~ and its zebrafish homolog axial (Echelard et al. 1993;Krauss et al. 1993).In explants of intermediate neuroectoderm at spinal cord levels, Shh protein induces, in a dose-dependent fashion, floor plate, and motor neuron development (Marti et al. 1Corresponding author.1995a; Roelink et al. 1995;Tanabe et al. 1995). In explants taken at midbrain and forebrain levels, Shh also induces the appropriate ventrolateral neuronal cell types, dopaminergic (Heynes et al. 1995;Wang et al. 1995} and cholinergic (Ericson et al. 1995) precursors, respectively. In chick somites (Johnson et al. 1994) and mouse presomitic explants (Fan et al. 1995), Shh promotes the expression...

According to a model based on embryological studies in amphibia, dorsoventral patterning is regulated by the antagonizing function of ventralizing bone morphogenetic proteins (BMPs) and dorsalizing signals generated by Spemann's organizer. Large-scale mutant screens in the zebrafish, Danio rerio, have led to the isolation of two classes of recessive lethal mutations affecting early dorsoventral pattern formation, dino mutant embryos are ventralized, whereas swirl mutants are dorsalized. We show that at early gastrula stages, dino and swirl mutants display an expanded or reduced Bmp4 expression, respectively. The dino and swirl mutant phenotypes both can be phenocopied and rescued by the modulation of BMP signaling in wild-type and mutant embryos. By suppressing BMP signaling in dino mutants, adult fertile dino -/-fish were generated. These findings, together with results from the analysis of dino-swirl double mutants, indicate that dino fulfills its dorsalizing activity via a suppression of swirl-dependent, BMP-like ventralizing activities. Finally, cell transplantation experiments show that dino is required on the dorsal side of early gastrula embryos and acts in a non-cell-autonomous fashion. Together, these results provide genetic evidence in support of a mechanism of early dorsoventral patterning that is conserved among vertebrate and invertebrate embryos.[Key Words: Dorsoventral pattern formation; dino; swirl; BMP4; noggin; Spemann's organizer; zebrafish; Danio rerio]Received June 10, 1996; revised version accepted July 22, 1996.Our understanding of how the axes of the vertebrate embryo are patterned is dominated by studies of amphibia. Patterning of the anterior-posterior and dorsal-ventral axes of the amphibian embryo is thought to depend on discretely localized signals (for review, see Sive 1993). Maternal signals emanating from vegetal cells induce ventral mesoderm in most of the marginal zone and the dorsal mesoderm of Spemann's organizer in a rather small dorsal region at the site where the formation of the blastopore is initiated. Later, zygotic signals generated by the ventral and dorsal mesoderm themselves regulate the refinement of dorsoventral pattern within the mesoderm and the induction of neuroectoderm in the dorsal animal zone of the early gastrula embryo.Recent evidence, principally based on studies in Xenopus laevis, supports a simple model of opposing dorsal and ventral activities. Two members of the family of bone morphogenetic proteins (BMPs), members of the TGF[3 superfamily, BMP2 and BMP4, are expressed strongly on the ventral side of early gastrula and have strong ventralizing properties. Overexpression of either 1Corresponding author. Present address:

Three of the class I mutants show a change in the pattern of gene expression in the anlage of a brain structure prior to the onset of degeneration. These results suggest that focal cell death may be a useful clue for the detection of early patterning defects of the vertebrate nervous system in regions devoid of visible landmarks.

We describe two genes, dino and mercedes, which are required for the organization of the zebrafish body plan. In dino mutant embryos, the tail is enlarged at the expense of the head and the anterior region of the trunk. The altered expression patterns of various marker genes reveal that, with the exception of the dorsal most marginal zone, all regions of the early dino mutant embryo acquire more ventral fates. These alterations are already apparent before the onset of gastrulation. mercedes mutant embryos show a similar but weaker phenotype, suggesting a role in the same patterning processes. The phenotypes suggests that dino and mercedes are required for the establishment of dorsal fates in both the marginal and the animal zone of the early gastrula embryo. Their function in the patterning of the ventrolateral mesoderm and the induction of the neuroectoderm is similar to the function of the Spemann organizer in the amphibian embryo.

Early dorsoventral pattern formation in vertebrate embryos is regulated by opposing activities of ventralizing bone morphogenetic proteins (BMPs) and dorsal-specific BMP antagonists such as Chordin, Noggin and Follistatin. Specific defects in early dorsoventral patterning have been recently found in a number of zebrafish mutants, which exhibit either a ventralized or dorsalized phenotype. One of these, the ventralized mutant chordino (originally called dino) is caused by a mutation in the zebrafish chordin homologue and interacts genetically with the dorsalized mutant swirl. In swirl mutant embryos, dorsal structures such as notochord and somites are expanded while ventral structures such as blood and nephros are missing. Here we demonstrate that the swirl phenotype is caused by mutations in the zebrafish bmp2 gene (zbmp2). While injection of mRNAs encoded by the mutant alleles has no ventralizing effect, injection of wild-type zbmp2 mRNA leads to a complete rescue of the swirl mutant phenotype. Fertile adult mutant fish were obtained, showing that development after gastrulation is not dependent on zbmp2 function. In addition zBMP2 has no maternal role in mesoderm induction. Our analysis shows that swirl/BMP2, unlike mouse BMP2 but like mouse BMP4, is required for early dorsoventral patterning of the zebrafish embryo.