Versican G1 domain and V3 isoform overexpression results in increased chondrogenesis in the developing chick limb in ovo

Abstract

Previous work has shown that versican proteoglycan is highly expressed in the extracellular matrix of precartilage limb mesenchyme. Although much of versican's role in chondrogenesis has been attributed to its glycosaminoglycan complement, N- and C-terminal G1 and G3 domains of versican have been shown to possess distinct functions when expressed ectopically. This study was undertaken to test the hypothesis that overexpression of the versican G1 domain and short V3 isoform, comprised of only G1 and G3, in the chick wing in ovo would result in increased chondrogenesis, suggesting function for discrete versican domains in limb skeletal development. Recombinant adenoviruses encoding G1 and V3 proteins were microinjected into proximal HH19-25 chick wing buds which resulted in significant enlargement of humeral primordia at HH35. Enhanced cartilage deposition appeared due to increased chondrogenic aggregation as a result of recombinant G1 or V3 overexpression, further implicating versican in early stages of limb development.

Figure 1

Expression of endogenous versican V3 isoform transcripts in the chick limb. A) RT-PCR at Hamburger and Hamilton (HH) stages 25, 28, 34 shows the predicted 313 bp amplicon in the developing limb and heart. Heart cDNA was used to generate riboprobes for in situ hybridization. Position of the 298 base pair standard is marked by arrowhead and actin loading control for each lane at the bottom. B) In situ hybridization with antisense probe shows low level expression of V3 mRNA in the wing core at HH25 (asterisk). C) Sense control probe in an adjacent section shows lack of signal in the limb core. Note staining in the dorsal mesenchyme underlying the ectoderm represents non-specific signal in panels B and C.

Figure 2

Whole-mount alcian blue and alizarin red staining of the HH35 wing demonstrating effects of recombinant versican G1 domain and V3 isoform expression in wings injected with recombinant adenoviruses at HH20 (C), HH21 (A, B, E) and HH22 (D). Targeted adenoviral injection resulted in enlargement of skeletal primordia in the proximal (A, C), distal (B) or mid humerus (D). There was little or no enlargement of the skeletal anlagen observed with the LacZ adenovirus control (E). Humeral sites from which measurements in Table 2 were taken are shown in panel F. CLC indicates un-injected contralateral control limbs. Scale bar = 0.5 mm.

Figure 3

Whole mount β-galactosidase staining and hemaglutinin tag immunostaining shows localization of recombinant versican G1 domain and V3 isoform expression in enlarged humeral cartilages at HH35 in wings injected with recombinant adenoviruses at HH23 (C&D) and HH24 A&B). Whole mount β-galactosidase reactivity localized sites of adenoviral infection (arrow, A; arrowhead, C). Immunostaining of hemaglutinin tagged G1 (B) and V3 (D) constructs correlated well with β-galactosidase reactivity in enlarged areas of the mid (mh; A,) and proximal (ph; C) humeral template. Inset in panel A shows higher magnification image of β-galactosidase positive cells indicated by the arrow. Scale bars = 250μm (A, B and C, D); 125μm, inset in A.

Figure 4

Recombinant versican V3 isoform expression does not correlate with increased mitosis in humeral cross section of the HH31 wing following co-injection with recombinant V3/LacZ adenovirus at HH23. Change in overall number of anti-phosphohistone H3 (ser10) positive cells (B) did not show correlation with V3/LacZ infected areas as indicated by β-galactosidase staining (boxed area in A) in either pre- and early chondrogenic stages HH23-31 or in later stage cartilaginous tissue (HH35). Scale bar = 100μm.

Figure 5

Cross section through the proximal humeral element of the chick wing co-injected with recombinant versican G1/LacZ at HH23 displays pericellular hyaluronan localization in peripheral limb cartilages at HH35. A) G1-hemaglutinin tag localization (arrows) associated with individual hyaluronan-positive cells or uninfected hyaluronan-positive chondrocytes (B). C) Area of infection shown by β-galactosidase reactivity. asterisk, perichondrium. Scale bar = 25μm.

Figure 6

Recombinant hemaglutinin-tagged versican G1 and V3 proteins co-distribute with PNA-binding materials in sagittal sections through humeral cartilage condensations of the HH25 wing following injection with recombinant adenoviruses at HH19 (A-I). Double labeling with anti-hemaglutinin shows G1 (A) and V3 (D) overlapping with PNA in chondrogenic areas (B, E; overlay in C, F). Small clusters of cells co-labeled with both anti-hemaglutinin tag and PNA are noted at the edges of chondrogenic regions in both G1 and V3 infected limbs (arrowheads). Enlarged view of area indicated by arrowheads in F is shown in G. No hemaglutinin-tag immunoreactivity (H) is noted in the PNA-positive chondrogenic core (I) of non-injected contralateral control limbs (CLC)). Scale bars = 100μm in C, 25μm in G.