Embryonic regeneration by relocalization of the Spemann organizer during twinning in .

The formation of identical twins from a single egg has fascinated developmental biologists for a very long time. Previous work had shown that blastulae bisected along the dorsal-ventral (D-V) midline (i.e., the sagittal plane) could generate twins but at very low frequencies. Here, we have improved this method by using an eyelash knife and changing saline solutions, reaching frequencies of twinning of 50% or more. This allowed mechanistic analysis of the twinning process. We unexpectedly observed that the epidermis of the resulting twins was asymmetrically pigmented at the tailbud stage of regenerating tadpoles. This pigment was entirely of maternal (oocyte) origin. Bisecting the embryo generated a large wound, which closed from all directions within 60 minutes, bringing cells normally fated to become Spemann organizer in direct contact with predicted ventral-most cells. Lineage-tracing analyses at the four-cell stage showed that in regenerating embryos midline tissues originated from the dorsal half, while the epidermis was entirely of ventral origin. Labeling of D-V segments at the 16-cell stage showed that the more pigmented epidermis originated from the ventral-most cells, while the less-pigmented epidermis arose from the adjoining ventral segment. This suggested a displacement of the organizer by 90°. Studies with the marker Chordin and phospho-Smad1/5/8 showed that in half embryos a new D-V gradient is intercalated at the site of the missing half. The displacement of self-organizing morphogen gradients uncovered here may help us understand not only twin formation in amphibians, but also rare cases of polyembryony.