Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

The interaction of PRC2 with RNA or chromatin is mutually antagonistic.

Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3' UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, the release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.

Mitochondrial mutations and ageing: can mitochondrial deletion mutants accumulate via a size based replication advantage?

The mitochondrial theory of ageing is one of the main contenders to explain the biochemical basis of the ageing process. An important line of support comes from the observation that mtDNA deletions accumulate over the life course in post-mitotic cells of many species. A single mutant expands clonally and finally replaces the wild-type population of a whole cell. One proposal to explain the driving force behind this accumulation states that the reduced size leads to a shorter replication time, which provides a selection advantage. However, this idea has been questioned on the grounds that the mitochondrial half-life is much longer than the replication time, so that the latter cannot be a rate limiting step. To clarify this question, we modelled this process mathematically and performed extensive deterministic and stochastic computer simulations to study the effects of replication time, mitochondrial half-life and deletion size. Our study shows that the shorter size does in principle provide a selection advantage, which can lead to an accumulation of the deletion mutant. However, this selection advantage diminishes the shorter is the replication time of wt mtDNA in relation to its half-life. Using generally accepted literature values, the resulting time frame for the accumulation of mutant mtDNAs is only compatible with the ageing process in very long lived species like humans, but could not reasonably explain ageing in short lived species like mice and rats.