A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells

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.

Advancements to the Axolotl Model for Regeneration and Aging

“…The regeneration permissive microenvironment of the axolotl is composed of dynamic acellular and cellular components that share striking similarities with tumors. Blastema cells, for example, exhibit typical cancer cell phenotypes including, but not limited to, increased proliferation [55], oncogene expression [56], activation of line-1 elements [57], and large-scale chromatin modifications [58] relative to mature stump tissue. These highly proliferative, undifferentiated cells have short, unpolarized, disordered actin fibers constituting their cytoskeletons and are surrounded by a disorganized ECM [55], a characteristic feature of the tumor microenvironment (as reviewed in [59]).…”

Gene expression modulation by the linker of nucleoskeleton and cytoskeleton complex contributes to proteostasis

“…One possible explanation to the differential gene expression due to anc‐1 knockdown suggests that this treatment leads to chromatin reorganization. To test this possibility, we visualized DAPI‐stained nuclei of untreated and anc‐1 RNAi‐treated day 6 adult worms, and computationally measured their chromatin condensation (Sosnik, Vieira, Webster, Siegfried, & McCusker, ). Our results show a potential difference in chromatin organization upon the knockdown of anc‐1 (Figure a,b) .…”

Transcriptome dynamics along axolotl regenerative development are consistent with an extensive reduction in gene expression heterogeneity in dedifferentiated cells

“…Since TE activation is known to occur as a consequence of the extensive chromatin relaxation in un/dedifferentiated cells ( Feher, 2015 ; Grafi & Barak, 2015 ; Macia, Blanco-Jimenez & Garcia-Perez, 2015 ; Wang & Wang, 2012 ), TE activations detected in axolotl regenerative development responses might be consistent with the possibility that the formation of the blastema required, at least partially, dedifferentiating cells that undergo extensive chromatin relaxation. Furthermore, Sosnik et al (2017) recently developed a new method to quantify the nuclear condensation using microscopic images and demonstrated that blastemal cells show a significant reduction in chromatin condensation when compared with somatic dermal cells. Thus, the use of transcriptomic analyses for axolotl regenerative development models seems like an adequate place to seek preliminary support for the hypothesized extensive decrease in gene expression heterogeneity for processes that concur with cell dedifferentiation ( Díaz-Castillo, 2017b ).…”

“…On the other hand, based on the analysis of chromatin markers and the activation of genetic elements usually silent because of their location in chromosome regions with highly compacted chromatin, it has been suggested that both naturally-occurring and artificially-induced cell dedifferentiation is characterized by an extensive relaxation of chromatin throughout the nucleus ( El-Badawy & El-Badri, 2015 ; Feher, 2015 ; Grafi & Barak, 2015 ; Jiang, Zhu & Liu, 2013 ; Krause, Sancho-Martinez & Izpisua Belmonte, 2015 ; Lee et al, 2015 ; Macia, Blanco-Jimenez & Garcia-Perez, 2015 ; Sosnik et al, 2017 ; Wang & Wang, 2012 ; Zhu et al, 2012a ). Ultimately, chromosome regions with high chromatin compaction are supposed to promote gene expression heterogeneity because of the slow dynamics of the chromatin remodeling needed to grant DNA accessibility to the transcription machinery ( Liu, Francois & Capp, 2016 ; Symmons & Raj, 2016 ).…”