Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development have remained uninvestigated. Upon the live imaging of epiblast cells sparsely labeled with green fluorescence protein, anterior epiblast cell migration to form individual brain portions was investigated. Anterior epiblast cells from a broad area migrated collectively toward the head axis during st. 5–7 at a rate of 70–110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. The analysis revised the head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration toward ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map was proposed, also accounting for previous observations.

We investigated the initial stages of head development using a new method to randomly label chicken epiblast cells with enhanced green fluorescent protein, and tracking the labeled cells. This analysis was combined with grafting mCherry-expressing quail nodes, or node-derived anterior mesendoderm (AME). These live imagings provided a new conception of the cellular mechanisms regulating brain and head ectoderm development. Virtually all anterior epiblast cells are bipotent for the development into the brain or head ectoderm. Their fate depends on the positioning after converging to the AME. When two AME tissues exist following the ectopic node graft, the epiblast cells converge to the two AME positions and develop into two brain tissues. The anterior epiblast cells bear gross regionalities that already correspond to the forebrain, midbrain, and hindbrain axial levels shortly after the node is formed. Therefore, brain portions that develop with the graft-derived AME are dependent on graft positioning.

Live imaging of migrating and interacting cells in developing embryos has opened a new means for deciphering fundamental principles in morphogenesis and patterning, which was not possible with classic approaches of experimental embryology. In our recent study, we devised a new genetic tool to sparsely label cells with a green-fluorescent protein in the broad field of chicken embryos, enabling the analysis of cell migration during the early stages of brain development. Trajectory analysis indicated that anterior epiblast cells from a broad area gather to the head axis to form the brain primordia or brain-abutting head ectoderm. Grafting the mCherry-labeled stage (st.) 4 node in an anterior embryonic region resulted in the anterior extension of the anterior mesendoderm (AME), the precursor for the prechordal plate and anterior notochord, from the node graft at st. 5. Grafting the st. 4 node or st. 5 AME at various epiblast positions that otherwise develop into the head ectoderm caused local cell gathering to the graft-derived AME. The node was not directly associated with this local epiblast-gathering activity. The gathered anterior epiblast cells developed into secondary brain tissue consisting of consecutive brain portions, e.g., forebrain and midbrain or midbrain and hindbrain, reflecting the brain portion specificities inherent to the epiblast cells. The observations indicated the bipotentiality of all anterior epiblast cells to develop into the brain or head ectoderm. Thus, a new epiblast brain field map is proposed, allowing the reinterpretation of classical node graft data, and the role of the AME is highlighted. The new model leads to the conclusion that the node does not directly participate in brain development.