Figure 3. Mouse En1 Functional Experiments.

a, Left: Quantitative expression of En1 and its temporal pattern (RNA-seq) in cultured calvarial murine osteoblasts (n=3 per time point). Right: Confirmation of the expression of En1 in a separate RT-PCR experiment of cultured calvarial murine osteoblasts and lack of expression in osteoclasts matured from bone marrow derived precursor cells (Positive controls for osteoblasts (osteocalcin) and osteoclast (RANK) are also shown).

b, Representative sections from lumbar vertebra 2 show the growth plate and bone marrow (GP and BM, left), cortical bone (CB, middle), and trabecular bone (TB, right) at 40x magnification from En1^lacZ/+ adult mice (n = 2) stained for β-gal activity (LacZ blue, En1+ cells) and alkaline phosphatase (AP, red late chondrocytes and actively calcifying tissues). In the periosteum (PO), all the LacZ+ cells were AP+; some AP- BM cells expressed LacZ. Some AP- proliferative chondrocytes in the GP expressed lacZ+, whereas most AP+ hypertrophic chondrocytes expressed LacZ. Some AP- osteocytes (Ocy) in CB and TB were LacZ+.

c, Left: Histomorphometry images of lumbar vertebrae 5 show decreased trabecular bone volume and increased bone surface area occupied by osteoclast cells when comparing En1^Cre/flox (self-deleted En1, sdEn1) mutants and En1^flox/+ control mice. Right: Reconstructed μCT images show the mineral density in a control and an sdEn1 animal (right panels).

d, Micro-CT (μCT) and histomorphometry measures within sdEn1 (n = 5) and controls (En1^lox/+, n = 6). By μCT, sdEn1 mutants exhibit decreased L5 trabecular number (Tb.N) and thickness (Tb.Th), as well as deceased bone volume fraction (BV/TV). Using histomorphometry, sdEn1 mutants exhibit increased osteoclastic area (TRAP/BS). Average for each measure denoted by solid horizontal line. P-value computed using paired t-test.