Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Sandra J Shefelbine
- Eun Kyung Jeon
- Eun Kyung Jeon
- James R Monaghan
- James R Monaghan
- Ester Comellas
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Axolotls (Ambystoma mexicanum: d/d RRID Catalog #101L) were either bred in captivity at Northeastern University or purchased from the Ambystoma Genetic Stock Center at the University of Kentucky. Experiments were performed in accordance with Northeastern University Institutional Animal Care and Use Committee. Animals were grown to 4-6cm (Mean 5.3cm, SD 0.36) and 1-1.5g (Mean 1.3g, SD 0.19g) for use in all studies. For all experiments, animals were anesthetized by treatment of 0.01% benzocaine until visually immobilized.
- Tatiana Sandoval-Guzman
© 2020, Duerr 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.
Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are, and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations collectively comprising the most uncommitted of all the pool, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Lastly, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.
Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify genomic regions with histone modifications consistent with enhancer activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high-confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Footprinting analyses provided further evidence that these potential TF binding sites are likely to be occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of TFs in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq datasets, providing further evidence that our work predicts active enhancers that regulate adult stem cells and regenerative mechanisms.