Live imaging of avian epiblast and anterior mesendoderm grafting reveals the complexity of cell dynamics during early brain development

Abstract

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.

Epiblast cells gather onto the anterior mesendoderm and initiate brain development without the direct involvement of the node in avian embryos: Insights from broad-field live imaging

“…However, the same map holds for early st. 5, as anterior epiblast cell migration is minimal until early st. 5. The brain precursor map was revised as shown in Figure 3 using the trajectory analysis of single-cell resolution in our study ( Yoshihi et al, 2022 ). The anterior side is directed upward.…”

“…Scale bar, 500 µm. This panel was adapted from Yoshihi et al (2022) Supplementary Figure S1 . (B) A translucent image of a cultured chicken embryo of the stage analogous to (A) , taken from a frame of Supplementary Movie S1 .…”

“…Time-lapse bright-field recordings of cultured embryos starting from st. 4 ( Figure 1B and Supplementary Movie S1 ) indicated that a cloudy cell mass converged toward the midline before the brain tissue developed, suggesting the occurrence of the anterior epiblast cells’ massive migration. To corroborate this model and to analyze cell migration leading to brain development in detail, we devised a technique to sparsely label epiblast cells with EGFP using electroporation of the “Supernova” vector cocktail ( Yoshihi et al, 2022 ). An additional advantage of this cell-labeling technique was that the fluorescence intensity increased only modestly over time, allowing an 18 h live recording with a constant photosensitivity setting.…”

“…Node formation marks the start of st. 4 ( Hamburger and Hamilton, 1951 ). At the earliest stage of st. 4, some node cells develop into the endoderm ( Yoshihi et al, 2022 ). After this moment, the cells around the node remain static.…”

“…The node tissue marked at st. 4 develops only PNC at st. 5 as the posterior component. In contrast, the node tissue marked at st. 5 also generates the median portions of the somites ( Yoshihi et al, 2022 ), indicating that the PNC component of st. 5 node tissue is carried over from st. 4; however, the somitogenetic components are due to a new addition to the node, possibly from the neighboring epiblast. The extending posterior end of the PNC is the neurocaudal hinge, which accounts for the observation of the common origin of the notochord and the floor plate, both derived from the node ( Catala et al, 1996 ; Teillet et al, 1998 ).…”