セミナー情報

The limb bud progenitors (LPs), which originate from the somatopleural layer of the lateral plate mesoderm, give rise to the majority of tissues within the mature patterned limb including bones, cartilage and tendons. While LPs have such differentiation and patterning potentials, lateral plate-derived cells outside of the limb buds do not exhibit these properties. To identify factors involved in establishing limb progenitor properties in lateral plate mesoderm, we took a reprogramming approach, reasoning that the full set of the factors specifying “limbness” might be sufficient to convert non-limb fibroblasts into LP-like cells. First, we attempted to establish a robust culture system for endogenous mouse LPs (mLPs) that would underpin reprogramming assays. By taking advantage of hyaluronan-based hydrogels, we have succeeded in maintaining LP marker expression in cultured mLPs for 10 days. To screen a set of potential candidates, we generated a list of transcription factors expressed specifically in the early limb fields by using RNAseq. As a result, 18 factors were deemed as candidates, and overexpression of them was indeed sufficient to induce the expression of LP marker genes such as Sall4 and Lhx9 in 3D-cultured mouse embryonic fibroblasts (MEFs). We further narrowed down a minimum set of factors by examining the effect of withdrawal of individual factors, and identified that the combination of Prdm16, Plzf and Lin28a greatly upregulated the expression levels of LP genes. Moreover, single-cell RNAseq analysis revealed that a fraction of the reprogrammed cells had quite similar transcriptome profiles to legitimate mLPs. Strikingly, when placed in high-density “micromass” culture, the cells differentiated into alcian blue-positive cartilage as mLPs do. Together, these results suggest that the three factors could convert MEFs into LP-like cells, and they may play pivotal roles in cell specification of LPs during limb development.