Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor.

Abstract

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:

Maternal induction of ventral fate by zebrafish radar

“…3 M and N), indicating that rdr atgMOmediated dorsalization persists when Chordin is removed. This result is consistent with MRdr acting on early bmp2b expression in a step prior to initial BMP2b activity that is normally up-regulated in din homozygous mutants (8,9). Altogether, these experiments clearly place rdr upstream of the bmp pathway.…”

“…As a consequence, ich mutants fail to form a dorsal organizer and develop as ventralized embryos in which dorsal structures are lost whereas ventral tissues expand (6). Because boz and din synergistically interact to inhibit zygotic ventralizing morphogens, namely bone morphogenetic proteins (BMPs), the ich phenotype is likely due in part to bmps gain-of-function (7)(8)(9)(10)(11). Indeed, the expression of bmp2b and bmp4 ectopically expands from ventrolateral regions into dorsal regions of ich gastrulae, thereby promoting ventral fates in cells that would normally give rise to dorsal mesoderm (6).…”

Ubc9 Regulates Mitosis and Cell Survival during Zebrafish Development

“…For enzymatic detection (see Figure 5), biotinylated secondary antibodies and the Vectastain ABC kit (Axxora, Gruenberg, Germany) were used, as previously described (Hammerschmidt et al, 1996). For fluorescent detection (see Figure 6), the following secondary antibodies were used: AlexaFluor 488 -conjugated chicken anti-goat IgG, AlexaFluor 555-conjugated donkey anti-rabbit IgG, AlexaFluor 647-conjugated chicken anti-mouse IgG (Molecular Probes, Invitrogen, Karlsruhe, Germany).…”

“…Whole mount in situ hybridizations and immunohistochemistry were carried out as previously described (Hammerschmidt et al, 1996). For riboprobe synthesis, plasmid pSport1-ubc9.1 containing the full ubc9.1 cDNA (1.3 kb) was linearized with SalI and transcribed with Sp6 RNA polymerase.…”

Secreted bone morphogenetic protein antagonists of the Chordin family

“…In the mutant embryos, the tail is enlarged at the expense of the head and the anterior region of the trunk. The phenotype suggests that dino is required for the establishment of dorsal fates in both the marginal and the animal zone of early gastrula embryos (43). The dino phenotype is caused by a mutation in Chrodin (44).…”