The Thick Veins Gene of Drosophila Is Required for Dorsoventral Polarity of the Embryo

We have discovered a new member of the class of genes controlling embryonic dorsoventral patterning. Mutants of the thick veins (tkv) gene have been described previously (as slater alleles) as embryonic lethal, lacking dorsal epidermis, but not as showing a recognizable dorsoventral phenotype. We show here that maternal alteration of function coupled with zygotic reduction of function of tkv is strongly ventralizing. In addition, in double heterozygous combinations in the mother, tkv mutations increase the ventralizing effect of dominant, weakly ventralizing alleles of the maternal effect, dorsoventral genes easter and cactus. An interaction is also seen with zygotic dorsoventral genes: tkv interacts maternally and zygotically in double heterozygotes with decapentaplegic and zygotically with screw in double homozygotes. We conclude that both maternally and zygotically supplied wild-type tkv product can play a role in dorsoventral patterning of the early embryo. On the basis of the phenotype of trans-heterozygous adult escapers, we propose that tkv might act by potentiating the activity of the zygotically acting decapentaplegic gene.     

Maternal and zygotic activity of the zebrafish ogon locus antagonizes BMP signaling.

The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mercedes(tm305), ogon(m60), and short tail(b180) mutations produce ventralized phenotypes, including expanded bmp2b/4 expression domains. We find that the three mutations are allelic and that the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo(m60) and ogo(b180) mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo(tm305) retains partial function. Aspects of the ogo(m60) and ogo(tm305) mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl(tc300), a null mutation in bmp2b, is epistatic to ogo(m60) mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. We have ruled out zebrafish noggin homologues as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity.     

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.     

Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.     

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.