No abstract

Background: Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. Results: Unlike the blastema-based response used during appendage regeneration, lung amputation led to organ-wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post-amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. Conclusion:Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ-wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.

Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell cycle transcripts were expressed more highly. These and other expression patterns suggest upper arms undergo more robust tissue remodeling and cell proliferation responses after amputation, and thus provide an explanation for why the overall time to complete regeneration is similar for proximal and distal amputations. Additionally, we identified candidate positional memory genes that were expressed differently between fore and upper arms that encode a surprising number of epithelial proteins and a variety of cell surface, cell adhesion, and extracellular matrix molecules. Also, genes were discovered that exhibited different, bivariate patterns of gene expression between fore and upper arms, implicating dynamic transcriptional regulation for the first time in limb regeneration. Finally, 43 genes expressed differently between fore and upper arm samples showed similar transcriptional patterns during retinoic acid-induced reprogramming of fore arm blastema cells into upper arm cells. Our study provides new insights about the basis of positional information in regenerating axolotl limbs.

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease, but the factors regulating chromatin topology are incompletely understood and currently there are no technologies to efficiently screen for new regulators of long-range chromatin organization. Here, we developed an image-based high-content CRISPR screening platform that combines a new FISH-based barcode readout method (BARC-FISH) with chromatin tracing. We performed a pooled loss-of-function screen of 137 selected genes (420 sgRNAs) in human cells and visualized alterations to their genome organization alongside sgRNA-paired barcode readout by BARC-FISH in the same single cells. Using 1.4 million 3D positions along chromosome traces, we identified 26 regulators of chromatin architectures at different length scales. We found that the ATP-dependent helicase CHD7, the loss of which causes the congenital syndrome CHARGE, cooperates with CTCF to promote large-scale chromatin compaction. Altogether, our method enables scalable, high-throughput identification of chromatin topology regulators that will stimulate new insights into the 3D genome.

The induction of new lung tissue after disease or trauma has the potential to save lives and transform patient outcomes. Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration, primarily after limb amputation. While it is hypothesized that axolotls regenerate all of their tissues, exploration of lung regeneration has not been performed until now. Proliferation after lung injury was observed to be a global response, suggesting that regeneration utilizes a compensatory mechanism, in contrast to limb regeneration’s epimorphic response. ErbB signaling is crucial for the proliferative response during lung regeneration, likely through the ErbB2:ErbB4 receptor heterodimer. ErbB4 mRNA was found to be highly upregulated at both one and three weeks post amputation. Neuregulin-1p (NRG1) can induce proliferation in the lung and likely exerts molecular control over lung regeneration. Inhibition of ErbB2 was sufficient to both block regeneration and the proliferative response observed after NRG1 treatment.