a, Lineage history of En1‐expressing cells in skeletal cells of the vertebra. The cre En1 allele was combined with the cre R26 reporter allele and examined using frozen fluorescent immunohistochemistry and alkaline phosphatase (AP) staining. Cell nuclei were detected with DAPI. YFP‐expressing cells have expressed Cre ( LSL‐YFP En1) at some time in their history. In subpanel A, control animals lacking the R26 reporter show low background YFP signal (green). In subpanel B, LSL‐YFP En1; cre/+ R26 mice YFP‐expressing cells are detected in the growth plate chondrocytes of the vertebra (asterisk), trabecular bone lining cells (arrow) and osteocytes (arrowhead). Note, high fluorescent background staining in the marrow space. In subpanel C, the same section is shown stained for AP activity using the Fast Red substrate. Strong activity is present in the hypertrophic chondrocytes of the growth plate and trabecular bone lining cells (arrow). In subpanel D, alignment of the AP and YFP images shows that the trabecular lining cells co‐express AP and YFP. LSL‐YFP/+ b, Co‐localization of En1 and alkaline phosphatase expression. Images of lumbar vertebrae sections (growth plate and trabecular bone regions, ×40 magnification) from two‐month old En1 mice (see lacZ/+ Fig. 3b), stained for LacZ and alkaline phosphatase (AP), false‐coloured as indicated. Double‐positive cells are indicated by arrows, single‐positive cells are indicated by arrowheads (LacZ +) or asterisks (AP +). Except for some chondrocytes, most AP + cells are also LacZ +, that is, express En1. The bone marrow was digitally removed, as it contains no AP + cells.