Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

Abstract

Although DNA methylation epigenetically regulates development, data on global DNA methylation during development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle's Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O. Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3. That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.

FIGURE 1

The anterior (A) and posterior (B) GD13 limb bud. x4. Immature epithelium (arrows); ray-like thickening of the mesenchyme (circle); future interdigital region (dashed line); future cartilage of wrist bones (asterisk); progression zone (plus); the base of the limb bud (B), HE. (C) Expression of cleaved caspase-3 in the mesenchymal cells of the GD14 anterior front limb bud; future interdigital region (arrows); future interphalangeal joint (arrowhead). Violet, ×200. (D) Expression of PCNA (brown) in mesenchymal cells of a GD14 hindlimb bud. Note negative internal control (blue). DAB, counterstained with hematoxylin. (E) No expression of GAGs in a GD14 hindlimb bud. Safranin O. (F) Expression of GAGs in a GD14 front limb bud. Safranin O. GD: Gestation day; PCNA: Proliferating cell nuclear antigen; GAGs: Glycosaminoglycans.

FIGURE 2

(A) Radial finger primordia (circle) in the anterior GD13 limb bud cultivated in vitro in the SS medium for 3 days. Interdigital region (dashed line), epithelium (arrow), central zone of decay (asterisk), mesenchyme (cross); Azan. (B) PCNA expression in the GD13 front limb bud cultivated for 14 days in vitro. PCNA signal in the mesenchymal cell (arrow); PCNA signal in the chondrocytes contained in the lacuna (thin arrow); direction of formation of the future interphalangeal joint (hollow arrow). GD: Gestation day; SS: Serum supplemented; PCNA: Proliferating cell nuclear antigen.

FIGURE 3

Progress of GD13 limb bud development/chondrogenesis in the SS medium. Note abundant, red-colored GAGs on the 3^rd day of culture in condensed mesenchyme, and intensely red, well-differentiated cartilage with lacunae after 14 days of cultivation. Safranin O. GD: Gestation day; SS: Serum supplemented; GAGs: Glycosaminoglycans.

FIGURE 4

Progress of GD14 limb bud development/chondrogenesis in the SS medium. Note abundant, red-colored GAGs on the 3^rd day of culture in condensed mesenchyme, and intensely red well-differentiated cartilage with lacunae after 14 days of cultivation. Safranin O. GD: Gestation day; SS: Serum supplemented; GAGs: Glycosaminoglycans.

FIGURE 5

Progress of GD13 limb bud development/chondrogenesis in the chemically defined SF medium. Note that after 3 days in culture of hindlimb buds, red-colored GAGs are almost nonexistent in comparison to 3 days cultivated front limb buds. After 14 days of culture, such difference is not seen, and GAGs are abundantly expressed also in the cartilage with lacunae of hindlimb cultures. Safranin O. GD: Gestation day; SF: Serum and protein free; GAGs: Glycosaminoglycans.

FIGURE 6

Progress of GD14 limb bud development/chondrogenesis in the chemically defined SF medium. Note abundant, red-colored GAGs on the 3^rd day of culture in condensed mesenchyme, and intensely red well-differentiated cartilage with lacunae after 14 days of cultivation. Safranin O. GD: Gestation day; SF: Serum and protein free; GAGs: Glycosaminoglycans.

FIGURE 7

Dynamics of global DNA methylation in GD13 limb buds during in vitro cultivation. N=6 samples per group. Kruskal–Wallis test with Dunn’s post hoc test was used for multiple comparisons. Mann–Whitney U-test was used for comparisons of two values. p < 0.05, ** p < 0.001. GD: Gestation day; MEM: Minimal essential medium.

FIGURE 8

Dynamics of global DNA methylation in GD14 limb buds during in vitro cultivation. N=6 samples per group. Kruskal–Wallis test with Dunn’s post hoc test was used for multiple comparisons. Mann–Whitney U-test was used for comparisons of two values. * p < 0.05. GD: Gestation day; MEM: Minimal essential medium.

FIGURE 9

Comparison of global DNA methylation between two stages of limb buds (GD13 and GD14) cultivated in the SS medium. N=6 samples per group. Mann–Whitney U-test was used for comparisons of two values. * p < 0.05, ** p < 0.001. GD: Gestation day; SS: Serum supplemented; MEM: Minimal essential medium.