Extended Data Figure 4 : Histological assessment of En1cre‐expressing cells in skeletal cells of the vertebra.

From: Whole‐genome sequencing identifies EN1 as a determinant of bone density and fracture

Extended Data Figure 4

a, Lineage history of En1cre‐expressing cells in skeletal cells of the vertebra. The En1cre allele was combined with the R26LSL‐YFP 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 (En1) at some time in their history. In subpanel A, control animals lacking the R26LSL‐YFP reporter show low background YFP signal (green). In subpanel B, En1cre/+; R26LSL‐YFP/+ 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. 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 En1lacZ/+ mice (see 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.