Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene

Abstract

Defects in tendon patterning and differentiation are seldom assessed in mouse mutants due to the difficulty in visualizing connective tissue structures. To facilitate tendon analysis, we have generated mouse lines harboring two different transgene reporters, alkaline phosphatase (AP) and green fluorescent protein (GFP), each expressed using regulatory elements derived from the endogenous Scleraxis (Scx) locus. Scx encodes a transcription factor expressed in all developing tendons and ligaments as well as in their progenitors. Both the ScxGFP and ScxAP transgenes are expressed in patterns recapitulating almost entirely the endogenous developmental expression of Scx including very robust expression in the tendons and ligaments. These reporter lines will facilitate isolation of tendon cells and phenotypic analysis of these tissues in a variety of genetic backgrounds.

Mechanical regulation of PTHrP expression in entheses

“…Another example of this phenomenon is the expression of PTHrP/ lacZ in the perichondrium of the costal cartilage [7,9,13]. These surrounding mesenchymal/ connective tissue structures arise from precursors present in the early limb buds and can be detected by the presence of the scleraxis gene from this stage through the life of the mouse (38). Many of these connective tissue structures express PTHrP at the junctions of the skeletal elements with each other and with the musculature, essentially inviting the hypothesis that the gene might be regulated by mechanical force in these sites.…”

Scleraxis is Required for Differentiation of the Stapedius and Tensor Tympani Tendons of the Middle Ear

“…Scx −/− (Scx-null) were generated by intercrossing Scx +/− mice which also carried the Scx-GFP allele. The validity of the Scx-GFP reporter expression in the middle and inner ears was confirmed by in situ hybridization with the full-length Scx probe (Brown et al 1999) in CD1 tissues since this probe also detects the Scx-GFP mRNA (Pryce et al 2007). The breeding paradigm described above and all mouse procedures and animal husbandry protocols described below were approved by the Oregon Health & Science University Institutional Animal Care and Use Committee.…”

“…Adult (P16-28) Scx-GFP mice were immersion fixed in 4% PFA overnight, and then dissected as described above. The Scx-GFP reporter is extremely intense (Pryce et al 2007), allowing simultaneous bright field (halogen) and epifluorescence (BF/EPI) illumination to record precise anatomical localization of GFP expression in whole mount middle ears without the need to merge separate bright field and epifluorescence images. BF/EPI-illuminated Scx-null and Scx-GFP middle ears were visualized with the Leica MZFLIII stereofluorescence dissecting microscope and imaged with a Retiga 1300 CCD camera.…”

“…Scx is a basic helix-loop-helix (bHLH) transcription factor with distinct expression in a number of tissues including the musculoskeletal system, epididymi, testes, adrenal gland, kidney, lung, heart, dorsal root ganglia, and body wall Schweitzer et al 2001;Pryce et al 2007). In the axial and appendicular skeleton, the Scx-GFP transgenic reporter labels tendon and ligament progenitor cells and all differentiated cell types within these connective tissues (Pryce et al 2007).…”

“…In the axial and appendicular skeleton, the Scx-GFP transgenic reporter labels tendon and ligament progenitor cells and all differentiated cell types within these connective tissues (Pryce et al 2007). We hypothesized that Scx-GFP would serve as a useful marker of middle ear tendon progenitors and permit analysis of middle ear tendon formation.…”