RESUMO
The specification of the embryonic central nervous system (CNS) into future brain (forebrain, midbrain, or hindbrain) and spinal cord (SC) regions is a critical step of CNS development. A previous chicken embryo study indicated that anterior epiblast cells marked by Sox2 N2 enhancer activity are specified to the respective brain regions during the transition phase of the epiblast to the neural plate-forming neural primordium. The present study showed that the SC precursors positioned posterior to the hindbrain precursors in the anterior epiblast migrated posteriorly in contrast to the anterior migration of brain precursors. The anteroposterior specification of the CNS precursors occurs at an analogous time (â¼E7.5) in mouse embryos, in which an anterior-to-posterior incremental gradient of Wnt signal strength was observed. To examine the possible Wnt signal contribution to the anteroposterior CNS primordium specification, we utilized mouse epiblast stem cell (EpiSC)-derived neurogenesis in culture. EpiSCs maintained in an activin- and FGF2-containing medium start neural development after the removal of activin, following a day in a transitory state. We placed activin-free EpiSCs in EGF- and FGF2-containing medium to arrest neural development and expand the cells into neural stem cells (NSCs). Simultaneously, a Wnt antagonist or agonist was added to the culture, with the anticipation that different levels of Wnt signals would act on the transitory cells to specify CNS regionality; then, the Wnt-treated cells were expanded as NSCs. Gene expression profiles of six NSC lines were analyzed using microarrays and single-cell RNA-seq. The NSC lines demonstrated anteroposterior regional specification in response to increasing Wnt signal input levels: forebrain-midbrain-, hindbrain-, cervical SC-, and thoracic SC-like lines. The regional coverage of these NSC lines had a range; for instance, the XN1 line expressed Otx2 and En2, indicating midbrain characteristics, but additionally expressed the SC-characteristic Hoxa5. The ranges in the anteroposterior specification of neural primordia may be narrowed as neural development proceeds. The thoracic SC is presumably the posterior limit of the contribution by anterior epiblast-derived neural progenitors, as the characteristics of more posterior SC regions were not displayed.
RESUMO
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.
RESUMO
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 has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110â µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published 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 towards 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 is proposed, also accounting for previous observations.
