(A, B) After whole limb β-gal staining, Adamtsl2 expression (blue) was observed in developing forelimb tendon at E14.5 (A) and E17.5 (B). (C-F) At E17.5, Adamtsl2 was expressed in prospective articular cartilage (C, D; tibiofemoral joint. Panel D represents higher magnification of boxed area in C), and skeletal muscle (E, F). Arrows in panel F indicate focal expression in muscle. (G-O) At PI2, Adamtsl2 was expressed in tendons (G, H), dermis (I; arrows indicate panniculus carnosus), skeletal muscle (I, J), prospective articular cartilage in the hind foot (K) and knee joints (L; arrows indicate joint lining cells, arrowheads indicate outer meniscal cell layer), shoulder joint (M), and the inner annulus fibrosus layer, but not the nucleus pulposus of the intervertebral disc (N, O; N is a tangential section that does not contain the nucleus pulposus). Images A-O are representative of n=2-3. Scale bars in A-O are 50 μm. AT, Achilles tendon, C, calcaneus; F, femur; H, humerus; NP, nucleus pulposus; T, tibia. (P) Gene targeting strategy for limb- and tendon-specific Adamtsl2 deletion. The lacZ-Δneo cassette was removed with FlpE (Adamtsl2fl) and Adamtsl2 was inactivated by excision of exon 5 with Prx1-Cre or Scx-Cre (Adamtsl2-Prx, Adamtsl2-Scx, respectively). (Q) PCR from toe and tail genomic DNA (gDNA). The band shift with primer pair P1-P3 from toe but not tail gDNA indicates excision of exon 5 (red). (R) qRT-PCR of Achilles tendon RNA shows reduction in Adamtsl2 expression after gene deletion (n=3). P-value was calculated with a 2-sided Student t-test.