Abstract
Although DNA methylation epigenetically regulates development, data on global DNA methylation during the 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 the 13th and 14th gestation days (GDs) 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 (short interspersed DNA repetitive element [SINE] rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by safranin O. On isolation, weak safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures toward 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 3. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th days was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day 3 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 toward differentiation in vitro, which is of importance for regenerative medicine strategies.
Highlights
Limb development is the consequence of inductive interaction between ectoderm and mesoderm, with regulatory inputs that are incredibly complex
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
We showed the dynamics of global DNA methylation changes in an in vitro 3D organ culture model of mammalian limb bud development by comparing two successive developmental stages and two different culture media, i.e., serum-supplemented (SS) and chemically defined serum- and protein-free (SF)
Summary
Limb development is the consequence of inductive interaction between ectoderm and mesoderm, with regulatory inputs that are incredibly complex It must be regarded as a fourdimensional process where the signals from the three axes change with the progress of development [1]. Deregulation of DNA methylation may lead to developmental anomalies of mammalian limbs in vivo [8] or changes in developmental parameters of limb buds grown in vitro [9]. It is implicated in the pathogenesis of osteoarthritis [10], while recently, DNA methylation profiling has been proposed for sarcoma classification [11]
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