Differentiation trajectories of the Hydra nervous system reveal transcriptional regulators of neuronal fate.

The small freshwater cnidarian polyp Hydra vulgaris uses adult stem cells (interstitial stem cells) to continually replace neurons throughout its life. This feature, combined with the ability to image the entire nervous system (Badhiwala et al., 2021; Dupre & Yuste, 2017) and availability of gene knockdown techniques (Juliano, Reich, et al., 2014; Lohmann et al., 1999; Vogg et al., 2022), makes Hydra a tractable model for studying nervous system development and regeneration at the whole-organism level. In this study, we use single-cell RNA sequencing and trajectory inference to provide a comprehensive molecular description of the adult nervous system. This includes the most detailed transcriptional characterization of the adult Hydra nervous system to date. We identified eleven unique neuron subtypes together with the transcriptional changes that occur as the interstitial stem cells differentiate into each subtype. Towards the goal of building gene regulatory networks to describe Hydra neuron differentiation, we identified 48 transcription factors expressed specifically in the Hydra nervous system, including many that are conserved regulators of neurogenesis in bilaterians. We also performed ATAC-seq on sorted neurons to uncover previously unidentified putative regulatory regions near neuron-specific genes. Finally, we provide evidence to support the existence of transdifferentiation between mature neuron subtypes and we identify previously unknown transition states in these pathways. All together, we provide a comprehensive transcriptional description of an entire adult nervous system, including differentiation and transdifferentiation pathways, which provides a significant advance towards understanding mechanisms that underlie nervous system regeneration.

Control of osteoblast regeneration by a train of Erk activity waves.

Regeneration is a complex chain of events that restores a tissue to its original size and shape. The tissue-wide coordination of cellular dynamics that is needed for proper morphogenesis is challenged by the large dimensions of regenerating body parts. Feedback mechanisms in biochemical pathways can provide effective communication across great distances1-5, but how they might regulate growth during tissue regeneration is unresolved6,7. Here we report that rhythmic travelling waves of Erk activity control the growth of bone in time and space in regenerating zebrafish scales, millimetre-sized discs of protective body armour. We find that waves of Erk activity travel across the osteoblast population as expanding concentric rings that are broadcast from a central source, inducing ring-like patterns of tissue growth. Using a combination of theoretical and experimental analyses, we show that Erk activity propagates as excitable trigger waves that are able to traverse the entire scale in approximately two days and that the frequency of wave generation controls the rate of scale regeneration. Furthermore, the periodic induction of synchronous, tissue-wide activation of Erk in place of travelling waves impairs tissue growth, which indicates that wave-distributed Erk activation is key to regeneration. Our findings reveal trigger waves as a regulatory strategy to coordinate cell behaviour and instruct tissue form during regeneration.

Can laboratory model systems instruct human limb regeneration?

Regeneration has fascinated scientists since well before the 20th century revolutions in genetics and molecular biology. The field of regenerative biology has grown steadily over the past decade, incorporating advances in imaging, genomics and genome editing to identify key cell types and molecules involved across many model organisms. Yet for many or most tissues, it can be difficult to predict when and how findings from these studies will advance regenerative medicine. Establishing technologies to stimulate regrowth of a lost or amputated limb with a patterned replicate, as salamanders do routinely, is one of the most challenging directives of tissue regeneration research. Here, we speculate upon what research avenues the field must explore to move closer to this capstone achievement.

Vitamin D Stimulates Cardiomyocyte Proliferation and Controls Organ Size and Regeneration in Zebrafish.

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.

In Toto Imaging of Dynamic Osteoblast Behaviors in Regenerating Skeletal Bone.

Osteoblasts are matrix-depositing cells that can divide and heal bone injuries. Their deep-tissue location and the slow progression of bone regeneration challenge attempts to capture osteoblast behaviors in live tissue at high spatiotemporal resolution. Here, we have developed an imaging platform to monitor and quantify individual and collective behaviors of osteoblasts in adult zebrafish scales, skeletal body armor discs that regenerate rapidly after loss. Using a panel of transgenic lines that visualize and manipulate osteoblasts, we find that a founder pool of osteoblasts emerges through de novo differentiation within one day of scale plucking. These osteoblasts undergo division events that are largely uniform in frequency and orientation to establish a primordium. Osteoblast proliferation dynamics diversify across the primordium by two days after injury, with cell divisions focused near, and with orientations parallel to, the scale periphery, occurring coincident with dynamic localization of fgf20a gene expression. In posterior scale regions, cell elongation events initiate in areas soon occupied by mineralized grooves called radii, beginning approximately 2 days post injury, with patterned osteoblast death events accompanying maturation of these radii. By imaging at single-cell resolution, we detail acquisition of spatiotemporally distinct cell division, motility, and death dynamics within a founder osteoblast pool as bone regenerates.

Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue.

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.

Multicolor Cell Barcoding Technology for Long-Term Surveillance of Epithelial Regeneration in Zebrafish.

Current fate mapping and imaging platforms are limited in their ability to capture dynamic behaviors of epithelial cells. To deconstruct regenerating adult epithelial tissue at single-cell resolution, we created a multicolor system, skinbow, that barcodes the superficial epithelial cell (SEC) population of zebrafish skin with dozens of distinguishable tags. With image analysis to directly segment and simultaneously track hundreds of SECs in vivo over entire surface lifetimes, we readily quantified the orchestration of cell emergence, growth, repositioning, and loss under homeostatic conditions and after exfoliation or appendage amputation. We employed skinbow-based imaging in conjunction with a live reporter of epithelial stem cell cycle activity and as an instrument to evaluate the effects of reactive oxygen species on SEC behavior during epithelial regeneration. Our findings introduce a platform for large-scale, quantitative in vivo imaging of regenerating skin and reveal unanticipated collective dynamism in epithelial cell size, mobility, and interactions.

Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration.

In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.

An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas.

PURPOSE: To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. METHODS: A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. RESULTS: A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta(1), identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) mutant embryos. CONCLUSIONS: We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.