Single-cell internalization during zebrafish gastrulation.

During gastrulation, germ layers are formed as prospective mesodermal and endodermal cells internalize and come to underlie the ectoderm [1-9]. Despite the pivotal role of gastrulation in animal development, the cellular interactions underlying this process are poorly understood. In zebrafish, mesoderm and endoderm formation requires the Nodal signals Cyclops and Squint and their cofactor One-eyed pinhead (Oep) [10-14]. We found that marginal cells in maternal-zygotic oep (MZoep) mutants do not internalize during gastrulation and acquire neural and tail fates at the expense of head and trunk mesendoderm. The lack of internalization in MZoep embryos and the cell-autonomous requirement for oep in Nodal signaling enabled us to test whether internalization can be achieved by individual cells or whether it depends on interactions within a group of cells. We found that individual MZoep mutant cells transplanted to the margin of wild-type blastula embryos initially internalize with their neighbors but are unable to contribute to the mesendoderm. In the reciprocal experiment, single wild-type cells transplanted to the margin of MZoep mutant embryos autonomously internalize and can express the mesendodermal markers axial/foxA2 and sox17. These results suggest that internalization and mesendoderm formation in zebrafish can be attained autonomously by single cells.

Head and trunk in zebrafish arise via coinhibition of BMP signaling by bozozok and chordino.

Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical determinant of dorsoanterior-ventroposterior pattern in vertebrate embryos. Whereas BMP overexpression abolishes both head and trunk development, known single and double loss-of-function mutations in BMP inhibitors have less dramatic effects. We report that combining mutations in the zebrafish genes bozozok and chordino causes a synergistic loss of head and trunk, whereas most cells express ventro-posterior markers and develop into a tail. Genetic inactivation of BMP signaling fully suppresses these defects. Thus, a remarkably simple genetic mechanism, involving a coinhibition of BMP function by the partially overlapping bozozok and chordino pathways is used to specify vertebrate head and trunk.

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.