Time-lapse imaging of ingression and cellular contacts by trigeminal placodal neurons in the mesenchyme. (A) Sequence of images showing actively ingressing placodal cells labeled by membrane GFP in green (also shown separately in bottom gray panels) and nuclear H2B-RFP in red. Ingressing cells delaminate as a short string of cells (arrowhead) through a common exit point on the basal side of the epithelium to enter the mesenchyme (mes). Solid white lines delineate the basal surface of ectoderm with dotted lines marking the site of placodal ejection. Newly ingressing cells (asterisk) remain in close contact with one another while also making contacts (arrows in bottom panels) with the placodal cells which have already reached the ganglionic anlage (g) and have begun to assemble into a ganglion mass. The full sequence of images are shown as time-lapse videos in additional files 7 and 8. (B) Highly dynamic axonal contacts are observed between placodal cells in the ganglionic anlage. Changes to axonal morphology, including irregular cytoplasmic protrusions, were found (arrows) and growth cones collapse at time of connection (arrowhead). Movie is shown in additional file 9. (C) Fluorescence images are superimposed on the bright-field image at one time point showing the view of the entire cranial slice that can be broken down to show different steps of placode development: ingression as shown in A and cell-cell interactions as shown in B. Yellow dotted line marks the edge of the ectoderm. (D) Image showing a volume rendering of the cranial slice over time using Imaris. Trajectories of migrating placodal cells are shown by colored lines that change from blue to red to white with progressing time, which was used to calculate average speed (n = 5). Displacements of placodal cell movement are indicated by the solid white arrows, which was used to calculate average velocity (n = 5). Scale bar: 10 um. ecto, ectoderm; mes, mesenchyme; nt, neural tube; g, ganglionic anlage.