Scleraxis (Scx) directs lacZ expression in tendon of transgenic mice

Scleraxis is a transcription factor expressed during early periods of mouse tendon morphogenesis. We have determined that tendon is first clearly present in mouse limb at embryonic day 14.5 (E14.5) and, by in situ hybridization, that scleraxis is expressed in the mouse tendons at E14.5. We have also investigated the regulatory elements that direct scleraxis gene expression to the limb tendons. DNA constructs were engineered such that the lacZ reporter gene was expressed under the control of portions of scleraxis regulatory regions. Transgenic mice carrying these constructs were made and expression of the construct was monitored by staining for beta-galactosidase activity. A construct containing 7 Kbp of 5' flanking sequence, the intron, both exons and 1.8 Kbp of 3' flanking sequence was expressed in a pattern that closely resembled the endogenous scleraxis gene. Mouse embryos carrying this construct expressed lacZ in their limb flexor and extensor tendons at E14.5. The lacZ stain in tendon was readily distinguished from -muscle using an anti-myosin heavy chain antibody to visualize muscle. Deletion of the intron, exons and 3' flanking region did not affect the pattern of tendon expression in the limbs of E14.5 transgenic mice. Additional constructs which deleted 5' flanking sequences up to -355 bp from the published cDNA sequence, showed limb tendon expression that was similar to the endogenous gene. When an additional 160 bp were deleted so that only approximately 200 bp of 5' flanking region was directing lacZ expression, no beta-galactosidase activity was observed in the tendons.