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.

中文翻译：

---

三维器官培养系统中大鼠肢芽的全局 DNA 甲基化和软骨形成。

尽管 DNA 甲基化在表观遗传上调节发育，但有关肢芽 (LB) 发育过程中全球 DNA 甲基化的数据很少。我们的目的是研究在补充血清 (SS) 和化学定义的无血清和无蛋白质 (SF) 三维器官培养物中培养的大鼠 LB 中的全球 DNA 甲基化发育动态。在 SF 和 SS 条件下，在 Eagle 的最小必需培养基 (MEM) 或含有 50% 大鼠血清的 MEM 中，在气液界面处培养第 13 和第 14 妊娠天 (GD) 的 Fischer 大鼠前后 LBs 14 天，分别。通过焦磷酸测序评估重复 DNA 序列（SINE 大鼠 ID 元件）的甲基化。通过光学显微镜和番红 O 染色细胞外基质糖胺聚糖评估发育。分离后，弱番红 O 染色仅存在于更发达的 GD14 前 LBs 中。直到第 14 天，所有培养物中的软骨形成都进展顺利，除了 SF 培养的 GD13 后-LBs，其中第 3 天几乎不存在番红 O 染色。这与比 SF 培养的 GD13 前 - 更高百分比的 DNA 甲基化有关。第三天的LB。在 SF 培养的前 LBs 中，在第 3 天和第 14 天之间检测到显着的甲基化增加。在 SS 培养的 GD13 front-LBs 中，甲基化在第三天显着增加，然后下降。在较老的 GD14 SS 培养的 LBs 中，DNA 甲基化没有增加，但在第 3 天和第 14 天，它们相对于 SS 培养的 GD13 显着低甲基化。