Fig. 2.

KGN promotes multiple limb developmental processes. E12.5 Forelimb (FL) and E13.5 hindlimb (HL) pairs isolated from mouse embryos were maintained in explant culture in control medium containing DMSO solution (10μl/mL) or 1 μM KGN. (A-D) Microscopic images of limbs at time of isolation (A-B) and at 96 hrs of culture in control (C) or KGN (D) medium. Note that KGN markedly increased overall limb growth, phalangeal elongation and interdigit mesenchymal invagination (arrowheads). Extent of growth stimulation was calculated by measuring length of each phalangeal element (E) and summation (F) using ImageJ software. Data are shown as average total digit length (pixels) + S.E.M. of 5 limbs at 96h (*p<0.05). (A, G-H) Images of E13.5 hindlimb explants at time of isolation (A) and maintained in control (G) or KGN (H) medium for 96 hrs and stained with Alcian blue. Note the significant growth of proximal phalanges (pp, arrows) caused by KGN. (I-J) Bright and dark field images of sections from 96 hrs forelimb specimens processed for analysis of proliferation marker H4C by in situ hybridization. Note the clearly higher hybridization signal over chondrocytes in KGN sample (J) versus control (I). (K-L) Fluorescence images of live forelimbs from E12.5 ScxGFP embryos at 96 hrs in control (K) or KGN (L) conditions. Tendon development and maturation were stimulated by KGN as indicated by stronger and sharper fluorescence signal and intense concentrated signal at joint locations (arrowheads). Bar for panels I-J, 100 μm. mc, metacarpal; mt, matarsal; pp, proximal phalange; and mp, medial phalange.