Fig. 3. Partition of gene expression and cell identity within callus. (A) Mounds of small, dividing cells marked by the pCUC2::3XVENUS-N7 reporter (green) formed and (B) gave rise to new shoot meristems (arrowheads), often observed in clusters. Chlorophyll autofluorescence is in red. (C,D) Scanning electron micrographs of early regenerating meristems (arrowheads, C), and (D) a late stage regenerated shoot emerging from callus. (E) The pWUS::mGFP-ER reporter (green) was expressed in callus cells poorly stained by FM4-64 dye (red) following 5 days induction on SIM. (F,G) Shoot progenitors (F, arrowhead, 12 days on SIM) were labeled with FM4-64 dye, and emerged from regions with peripheral pWUS::mGFP-ER expression, and formed mature shoot meristems (G), also strongly stained by FM4-64 dye. The pWUS::mGFP-ER reporter was upregulated in the center of the developing meristem (arrowhead). (H) pCUC2::3XVENUS-N7 (green) and pWUS::DsRed-N7 (red) reporters were active in opposing domains of cells, sometimes in gradients, shown after 10 days on SIM. (I) Higher magnification after 11 days on SIM, showing clusters of cells expressing the CUC2 reporter (arrowheads) surrounded by pWUS::DsRed-N7 expressing cells. (J) At later stages, WUS::DsRed-N7 expression was initiated in the center of the mound of shoot progenitors while pCUC2::3XVENUS-N7 was restricted to the future peripheral zone, shown here after 12 days on SIM. Scale bars: 50 µm (A,C,I,J); 100 µm (B,E-H); 300 µm (D). P_n, primordia.