Abstract
The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC) formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.
Highlights
Changes to the nuclear landscape in cells are hallmark of many developmental processes occurring in embryos, adults, and diseases such as cancer
Epigenetic modifications that occur to these nucleosomal particles controls their spacing, which regulates the density of the chromatin compaction, and the ability of transcriptional machinery to access the DNA
Methodological improvements and algorithm efficiency As mentioned above, computational and methodological issues hindered the utilization of the algorithm developed by Irianto et al to quantify chromatin condensation in situ
Summary
Changes to the nuclear landscape in cells are hallmark of many developmental processes occurring in embryos, adults, and diseases such as cancer. Increased abundances of tri-methyl marks on lysine residue number 27 of the nucleosomal core protein histone 3 (H3K27-me3) results in gene silencing and can lead to large-scale compaction of the DNA in these regions [4,5,6] Examples of these large-scale changes occur frequently during cellular differentiation (as reviewed by [7], [8]), and have been largely characterized quantitatively in tissue culture cells [9]. A few techniques that quantitatively assess the changes in chromatin compaction in situ have been published These methods utilize tissue culture cells and evaluate nuclear architecture quantitatively through changes in DNA density within the nucleus, assessed through spatial differences in local intensities of DNA specific dyes using fluorescence microscopy or fluorescence lifetime microscopy [10,11]. The focus of the current project was to establish a rigorous method to quantify the nuclear architecture in situ, which could be applied to different species and developmental processes
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