Naturally occurring, rostrally conjoining chicken twins attempt to make a forebrain

Conjoined twinning is a special case of monozygotic, monoamniotic twinning. Human conjoined twinning, and vertebrate conjoined twinning in general, is a very rare phenomenon. It has been suggested that the risk of conjoined twinning increases with some medication and upon assisted reproduction. Survival rates are low. When conjoined twins occur in the chicken, they most often present with fused heads, anatomically unrecognisable brains and two normal bodies. Recent studies suggested that forebrain, midbrain and rostral hindbrain identities are established in the early epiblast before neural induction and independent from caudal hindbrain and spinal cord identities. Therefore, it is unclear whether in conjoined twins, the aberrant brain anatomy is a result of the rostral fusion, or whether the brains failed to develop in the first place. Here, we collected conjoined twins as they spontaneously appeared in eggs incubated for stages HH4 (late primitive steak stage) to HH13 (early pharyngula). The twinned embryos and stage-matched normal embryos were analysed for the expression of the rostral epiblast and forebrain-midbrain marker Otx2 and the ventral forebrain marker Six3. We found normal anatomy and marker gene expression that lasted up to stage HH9. By HH12-13, the brain anatomy had deteriorated, but marker genes remained expressed. This suggests that the fusing embryos attempted to generate a brain including the forebrain. Besides addressing forebrain development, our work for the first time provides a time frame to study the mechanisms underlying the interaction and fusion of conjoined twins, which will pave the way to a better understanding and management of risk factors in humans.