Regulatory elements of Xenopus col2a1 drive cartilaginous gene expression in transgenic frogs.
This study characterizes regulatory elements of collagen 2 alpha 1 (col2a1) in Xenopus that enable transgene expression in cartilage-forming chondrocytes. The reporters described in this study drive strong cartilage-specific gene expression, which will be a valuable tool for further investigations of Xenopus skeletal development. While endogenous col2a1 mRNA is expressed in many embryonic tissues, its expression becomes restricted to tadpole and adult chondrocytes. This chondrocyte-specific expression is recapitulated by col2a1 reporter constructs, which were tested through I-SceI meganuclease-mediated transgenesis. These constructs contain a portion of the Xenopus tropicalis col2a1 intron, which aligns to a cartilage-specific intronic enhancer that has been well characterized in mammals. Two overlapping regions of the first intron that are 1.5-Kb and 665-bp long, both of which contain this enhancer sequence, drove EGFP expression in both larval and adult chondrocytes when connected to an upstream promoter. However, neither a truncated 155-bp region that also contains the enhancer, nor a separate 347-bp intronic region that lacks it, was able to drive cartilaginous transgene expression. The two cartilage-specific transgenes are heritable in F1 progeny, which exhibit none of the background expression observed in the injected founders. This study is the first to use the I-SceI technique to characterize an enhancer element in Xenopus, and the first to generate chondrocyte-specific gene expression in a non-mammalian vertebrate. The creation of novel cartilage-specific gene expression provides a new tool for further studies of anuran skeletal development.
PubMed ID: 19757383
Article link: Int J Dev Biol.
Genes referenced: col2a1 gnl3 gtf2a1l pc.1 sftpc sox9
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|Fig. 1. In situ hybridizations during embryonic (A,B), larval (C), metamorphic (E,F) and post-metamorphic stages (D). (A,B) Whole-mount col2a1 in situ hybridization of a Nieuwkoop- Faber (NF) stage-33/34 embryo. (A) Lateral view, anterior is to the right. There is strong expression of col2a1 in the notochord (NO), otic vesicle (OV) and branchial arches (BA). Black line points to level of section depicted in (B). (B) Histological cross section through the trunk. Note diffuse staining in the somites (SO) and floor plate of the neural tube (FP). (C) Whole-mount col2a1 in situ hybridization; stage-46 tadpole head. Ventral view, anterior is to the top. There is strong expression in early larval cartilages, including the ceratobranchials (CB), but not in the interhyoideus muscle (IH). (D) Sectioned in situ hybridization of a stage-64 froglet nasal capsule. Coronal section, anterior is to the top. Col2a1 is expressed again in the adult cranial cartilages, including the post-metamorphic nasal septum (NS), planum terminale (PT) planum antorbitale (PA), and alary cartilage (AC). (E,F) Mid- metamorphic, stage-57 tadpole. Coronal histological sections of the nasal capsule, stained by in situ hybridizations with col2a1 and sox9 probes. (E) Col2a1 expression increases in the alary (AC) and superior prenasal (SPC) cartilages as they form during metamor- phosis. (F) Up-regulation of col2a1 corresponds with heightened expression of sox9 in these cartilages during metamorphic chon- drogenesis. Additional abbreviations: NC, nasal capsule; EY, eye. Scale bar, 0.5 mm.|
|Fig. 3. Distribution of EGFP in transgenic Xenopus laevis tadpoles. (A) Ventral views, anterior to the top. (H) Dorsal view, anterior to the top. (A) Cleared-and-stained tadpole showing Meckel (MC), ceratohyal (CH) and ceratobranchial (CB) cartilages stained with Alcian blue (stage 42). All other images show EGFP/GFP expression in living tadpoles. (B) Transient transgenic tadpole with a reporter carrying the intronic region A (stage 48). EGFP is not seen in the cartilaginous skeleton of any tadpole carrying reporter construct A. GFP expression is apparent in the lens of the left eye (EY), under the control of the gamma-crystallin promoter. (C) Transient alf-transgenictadpole with a reporter carrying the intronic region B (stage 42). Unilateral transgene expression is apparent in the same cartilages labeled in (A), along with GFP expression in the eye. (D) An F1 transgenic tadpole (stage 48), bred from a half-transgenic mother carrying construct B. (E) Transient half-transgenic tadpole carrying a reporter with the C construct (stage 42) also exhibits unilateral cartilagi- nous expression, although expression is weaker than that seen in tadpoles carrying the B construct. (F) An F1 transgenic tadpole (stage 40), bred from a half-transgenic mother carrying construct C. Early expression of EGFP is strongest as the cartilages are forming. (G) Stage 48 of the same tadpole in (F). None of the tadpole cartilages continues to express EGFP under construct C during the later larval stages. However, cartilagi- nous expression of EGFP increases again during the metamorphic formation of adult cartilages (Fig. 4). (H) Merged bright-field and fluores- cent image of a transgenic tadpole carrying a reporter with the smaller intronic region D (stage 48). This region does not drive cartilaginous EGFP expression during any stage in any injected tadpole. Auto-fluorescence is visible in the yolk of stage 402 individuals (asterisks in C,E,F) and in the gallbladder of stage-48 tadpoles (asterisks in B,D,G). Scale bar, 1 mm.|
|Fig. 4. Chondrocyte expression of reporter constructs in histological sections. (A,B) Tadpole injected with a reporter construct that contains intronic region B. Coronal sections through a single larval ceratobranchial cartilage stained with anti-collagen II antibody (red in A) or anti-GFP antibody (green in B) with DAPI nuclear counter stain (blue). (C,E,G) Trichrome and (D,F,H) merged bright-field and fluorescent images of anti-GFP antibody stained sections. (C,D) Coronal sections of the nasal capsule of a post-metamorphic froglet that carries the reporter construct with intronic region B. (D) EGFP is expressed in the alary cartilage (AC), nasal septum (NS) and planum terminale (PT). (E,F) Longitudinal sections through the leg of a froglet that carries a reporter construct with intronic region B. Cartilaginous expression is seen in the humerus (HU) and proliferating chondrocytes (PC) of the radio-ulna (RU). EGFP is not expressed in the hypertrophic (HC) or articular (AR) chondrocytes of the radio-ulna. (G,H) Serial coronal sections through the ethmoidal region of a froglet that carries the C.1 reporter. Cartilaginous expression is seen in the same post-metamorphic cartilages shown in D. Scale bar, 0.25 mm.|