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

Abstract

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.

Paraxis Is a Basic Helix-Loop-Helix Protein That Positively Regulates Transcription through Binding to Specific E-box Elements

“…Among the E-boxes tested was the right E-box of the muscle creatine kinase gene (RMCK), this E-box is bound by E12 homodimers and E12/scleraxis heterodimers (59). Scleraxis will also bind to an E-box (Scx) from its own promoter as a heterodimer with E12 (59,60), and rhomboid promoters are bound by E12/Twist heterodimers (58). An E-box from the promoter of the insulin gene (Ins-1) binds to an E12/ Beta1 heterodimer (48).…”

“…The ability of paraxis to bind to an E-box from the scleraxis promoter that is required for transcription of this gene during embryogenesis (60) suggests that paraxis may play a role in chondrogenesis. Further, paraxis is expressed in the region of the somite fated to become the chondrogenic lineage (41,42), and this led us to examine the expression of Pax-1, another sclerotomal transcriptional regulator.…”

Expression analysis of limb element markers during mouse embryonic development

“…Nerve fascicles, originating from the brachial plexus and the spinal lumbosacral plexus, are present in the proximal mouse forelimb and hindlimb, respectively, at E11.5 (Martin 1990). Tendon progenitor cells in the limb condense and differentiate into tenocytes, with pre‐tendon mesenchymal masses detected at E13.5 (Perez et al, 2003; Birch et al, 2013). Tenocytes then produce extracellular matrix molecules, including type I and III collagen, and organize them into tendon fibrils (Birch et al, 2013; Liu et al, 2015).…”

“…Tenocytes then produce extracellular matrix molecules, including type I and III collagen, and organize them into tendon fibrils (Birch et al, 2013; Liu et al, 2015). Fibrils become further organized into tendon fibers, and by E14.5 tendons are visible in the digits (Perez et al, 2003). By E14.5, distal muscles are also visible, the nerves have formed dense plexuses, and the epidermis is thickening.…”

Expression of scleraxis and tenascin C in equine adipose and umbilical cord blood derived stem cells is dependent upon substrata and FGF supplementation

“…Scleraxis (scx) is a class II basic helix-loop-helix transcription factor expressed early during mouse embryogenesis in the syndetome, a derivative of the somitic sclerotome compartment (Brent et al 2003;Cserjesi et al 1995). Expression is associated with connective tissue and skeleton structures during prenatal development and with periodontal ligaments, force generating tendons, brain, lung and Sertoli cells in adult rodents (Liu et al 1996;Muir et al 2005;Murchison et al 2007;Perez et al 2003;Pryce et al 2007). Expression of scleraxis is largely confined to tendons early in postnatal development and absent in the structures as they become increasingly acellular (Pryce et al 2007).…”