In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus a substantial improvement of the live-imaging of primary cell cultures, planarians, zebrafish and human cerebral organoids.
Data from: A tunable refractive index matching medium for live imaging cells, tissues and model organismsAvailable at Dryad Digital Repository under a CC0 Public Domain Dedication.
- Tobias Boothe
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Clare M Waterman, National Institutes of Health, United States
© 2017, Boothe et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
MAF1, a key repressor of RNA polymerase III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show that MAF1 plays a critical role in the regulation of osteoblast differentiation and bone mass. A high bone mass phenotype was noted in mice with a global deletion of Maf1 (Maf1-/- mice). However, osteoblasts isolated from Maf1-/- mice showed reduced osteoblastogenesis ex vivo. Therefore, we determined the effect of MAF1 overexpression specifically in cells from the mesenchymal lineage (Prx1-Cre;LSL-MAF1 mice). These mice showed increased bone mass. Ex vivo, cells from Prx1-Cre;LSL-MAF1 mice showed enhanced osteoblastogenesis concordant with their high bone mass phenotype. Thus, the high bone mass phenotype in Maf1-/- mice is likely due to the confounding effects of the global absence of Maf1 in Maf1-/- mice. MAF1 overexpression promoted osteoblast differentiation and shRNA-mediated Maf1 downregulation inhibited differentiation of ST2 cells, overall indicating MAF1 enhances osteoblast formation. We also found that, in contrast to MAF1 overexpression, other perturbations that repress RNA pol III transcription, including Brf1 knockdown and chemical inhibition of RNA pol III by ML-60218, inhibited osteoblast differentiation. All perturbations that decrease RNA pol III transcription, however, enhanced adipogenesis in ST2 cell cultures. RNA-seq was used to determine the basis for these opposing actions on osteoblast differentiation. The modalities used to perturb RNA pol III transcription resulted in distinct gene expression changes, indicating that this transcription process is highly sensitive and triggers diverse gene expression programs and phenotypic outcomes. Specifically, MAF1 induced genes in ST2 cells known to promote osteoblast differentiation. Furthermore, genes that are induced during osteoblast differentiation displayed codon bias. Together, these results reveal a novel role for MAF1 and RNA pol III-mediated transcription in osteoblast fate determination and differentiation and bone mass regulation.
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