Fig. 3. EGFP expression profile during differentiation and neural specification. The expression was prominent in progenitors and downregulated in differentiating neurons. Localized EGFP expression in the EB (A,B; d7+2) and in outgrowths (arrow) of the EB (C; d7+4) post-plating. (C) Arrowhead, central part of EB. (D) EBs (d7+7) show differentiating neurons (arrow) at the periphery with EGFP expression (see inset); arrowhead shows the central part of the EB. (E) d7+14; three clusters of neurons (1,2,3) with short and long processes. (F) (d7+20); with further plating time cluster 2 shows an extension of neurite outgrowths from the central cell mass. (G,H) Magnified view of the same cluster, where some neurites are still EGFP positive. The arrowhead indicates the presence of EGFP-positive neural progenitors in the vicinity of mature neurons, and the arrow indicates the lengthening of neurite processes with longer plating time from d7+14 to d7+20 (E-H). (J,K) Almost all of the EGFP-expressing cells (J) in EBs (d7+7) are nestin positive (K); the yellow colour indicates the superimposed pattern between EGFP and Cy3. (L,M) In the differentiated neurons labelled with MAP2 (M, double filter), a weak or no EGFP signal (L, d7+7) was observed. Combined transmission and fluorescent light: A,C,D-F,G. Fluorescent light alone: B,H,J,L,inset D. Bars, 50 µM (A,B,inset D); 100 µM (C,D,G,H,J-M); 250 µM (E,F).