Meeting report: Salamander models in cross-disciplinary biological research meeting.

The third annual meeting on "Salamander Models in Cross-disciplinary Biological Research" took place online on August 2021, bringing together over 200 international researchers using salamanders as research models and encompassing diverse fields, ranging from Development and Regeneration through to Immunology, Pathogenesis, and Evolution. The event was organized by Maximina H. Yun (Center for Regenerative Therapies Dresden, Germany) and Tatiana Sandoval-Guzmán (TU Dresden, Germany) with the generous support of the Deutsche Forschungsgemeinschaft, the Center for Regenerative Therapies Dresden, Technische Universität Dresden, and the Company of Biologists. Showcasing a number of emerging salamander models, innovative techniques and resources, and providing a platform for sharing both published and ongoing research, this meeting proved to be an excellent forum for exchanging ideas and moving research forwards. Here, we discuss the highlights stemming from this exciting scientific event.

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.

Conserved and novel functions of programmed cellular senescence during vertebrate development.

Cellular senescence, a form of stable cell cycle arrest that is traditionally associated with tumour suppression, has been recently found to occur during mammalian development. Here, we show that cell senescence is an intrinsic part of the developmental programme in amphibians. Programmed senescence occurs in specific structures during defined time windows during amphibian development. It contributes to the physiological degeneration of the amphibian pronephros and to the development of the cement gland and oral cavity. In both contexts, senescence depends on TGFß but is independent of ERK/MAPK activation. Furthermore, elimination of senescent cells through temporary TGFß inhibition leads to developmental defects. Our findings uncover conserved and new roles of senescence in vertebrate organogenesis and support the view that cellular senescence may have arisen in evolution as a developmental mechanism.

Regulation of p53 is critical for vertebrate limb regeneration.

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration.

CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle.

The repair of DNA double-strand breaks (DSBs) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSBs occurs through non-homologous end-joining or microhomology-mediated end-joining (MMEJ). These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional, there is an increase in repair of DSBs by homologous recombination, which is mostly error-free. Consequently, the relative contribution of these different pathways to DSB repair in the cell cycle has a large influence on the maintenance of genetic integrity. It has remained unknown how DSBs are directed for repair by different, potentially competing, repair pathways. Here we identify a role for CtIP (also known as RBBP8) in this process in the avian B-cell line DT40. We establish that CtIP is required not only for repair of DSBs by homologous recombination in S/G2 phase but also for MMEJ in G1. The function of CtIP in homologous recombination, but not MMEJ, is dependent on the phosphorylation of serine residue 327 and recruitment of BRCA1. Cells expressing CtIP protein that cannot be phosphorylated at serine 327 are specifically defective in homologous recombination and have a decreased level of single-stranded DNA after DNA damage, whereas MMEJ remains unaffected. Our data support a model in which phosphorylation of serine 327 of CtIP as cells enter S phase and the recruitment of BRCA1 functions as a molecular switch to shift the balance of DSB repair from error-prone DNA end-joining to error-free homologous recombination.

Changes in Regenerative Capacity through Lifespan.

Most organisms experience changes in regenerative abilities through their lifespan. During aging, numerous tissues exhibit a progressive decline in homeostasis and regeneration that results in tissue degeneration, malfunction and pathology. The mechanisms responsible for this decay are both cell intrinsic, such as cellular senescence, as well as cell-extrinsic, such as changes in the regenerative environment. Understanding how these mechanisms impact on regenerative processes is essential to devise therapeutic approaches to improve tissue regeneration and extend healthspan. This review offers an overview of how regenerative abilities change through lifespan in various organisms, the factors that underlie such changes and the avenues for therapeutic intervention. It focuses on established models of mammalian regeneration as well as on models in which regenerative abilities do not decline with age, as these can deliver valuable insights for our understanding of the interplay between regeneration and aging.

Macrophages modulate fibrosis during newt lens regeneration.

BACKGROUND: Previous studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated. METHODS: Here we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery. RESULTS: Macrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process. CONCLUSIONS: Together, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Induction and Characterization of Cellular Senescence in Salamanders.

Cellular senescence is a permanent proliferation arrest mechanism induced following the detection of genotoxic stress. Mounting evidence has causally linked the accumulation of senescent cells to a growing number of age-related pathologies in mammals. However, recent data have also highlighted senescent cells as important mediators of tissue remodeling during organismal development, tissue repair, and regeneration. As powerful model organisms for studying such processes, salamanders constitute a system in which to probe the characteristics, physiological functions, and evolutionary facets of cellular senescence. In this chapter, we outline methods for the generation, identification, and characterization of salamander senescent cells in vitro and in vivo.

Senescent cells enhance newt limb regeneration by promoting muscle dedifferentiation.

Salamanders are able to regenerate their entire limbs throughout lifespan, through a process that involves significant modulation of cellular plasticity. Limb regeneration is accompanied by the endogenous induction of cellular senescence, a state of irreversible cell cycle arrest associated with profound non-cell-autonomous consequences. While traditionally associated with detrimental physiological effects, here, we show that senescent cells can enhance newt limb regeneration. Through a lineage tracing approach, we demonstrate that exogenously derived senescent cells promote dedifferentiation of mature muscle tissue to generate regenerative progenitors. In a paradigm of newt myotube dedifferentiation, we uncover that senescent cells promote myotube cell cycle re-entry and reversal of muscle identity via secreted factors. Transcriptomic profiling and loss of function approaches identify the FGF-ERK signalling axis as a critical mediator of senescence-induced muscle dedifferentiation. While chronic senescence constrains muscle regeneration in physiological mammalian contexts, we thus highlight a beneficial role for cellular senescence as an important modulator of dedifferentiation, a key mechanism for regeneration of complex structures.

Baculovirus Production and Infection in Axolotls.

Salamanders have served as an excellent model for developmental and tissue regeneration studies. While transgenic approaches are available for various salamander species, their long generation time and expensive maintenance have driven the development of alternative gene delivery methods for functional studies. We have previously developed pseudotyped baculovirus (BV) as a tool for gene delivery in the axolotl (Oliveira et al. Dev Biol 433(2):262-275, 2018). Since its initial conception, we have refined our protocol of BV production and usage in salamander models. In this chapter, we describe a detailed and versatile protocol for BV-mediated transduction in urodeles.

Obatoclax Rescues FUS-ALS Phenotypes in iPSC-Derived Neurons by Inducing Autophagy.

Aging is associated with the disruption of protein homeostasis and causally contributes to multiple diseases, including amyotrophic lateral sclerosis (ALS). One strategy for restoring protein homeostasis and protecting neurons against age-dependent diseases such as ALS is to de-repress autophagy. BECN1 is a master regulator of autophagy; however, is repressed by BCL2 via a BH3 domain-mediated interaction. We used an induced pluripotent stem cell model of ALS caused by mutant FUS to identify a small molecule BH3 mimetic that disrupts the BECN1-BCL2 interaction. We identified obatoclax as a brain-penetrant drug candidate that rescued neurons at nanomolar concentrations by reducing cytoplasmic FUS levels, restoring protein homeostasis, and reducing degeneration. Proteomics data suggest that obatoclax protects neurons via multiple mechanisms. Thus, obatoclax is a candidate for repurposing as a possible ALS therapeutic and, potentially, for other age-associated disorders linked to defects in protein homeostasis.

Macrophages modulate fibrosis during newt lens regeneration.

Previous studies indicated that macrophages play a role during lens regeneration in newts, but their function has not been tested experimentally. Here we generated a transgenic newt reporter line in which macrophages can be visualized in vivo. Using this new tool, we analyzed the location of macrophages during lens regeneration. We uncovered early gene expression changes using bulk RNAseq in two newt species, Notophthalmus viridescens and Pleurodeles waltl. Next, we used clodronate liposomes to deplete macrophages, which inhibited lens regeneration in both newt species. Macrophage depletion induced the formation of scar-like tissue, an increased and sustained inflammatory response, an early decrease in iris pigment epithelial cell (iPEC) proliferation and a late increase in apoptosis. Some of these phenotypes persisted for at least 100 days and could be rescued by exogenous FGF2. Re-injury alleviated the effects of macrophage depletion and re-started the regeneration process. Together, our findings highlight the importance of macrophages in facilitating a pro-regenerative environment in the newt eye, helping to resolve fibrosis, modulating the overall inflammatory landscape and maintaining the proper balance of early proliferation and late apoptosis.

Macrophages modulate fibrosis during newt lens regeneration.

Background: Previous studies indicated that macrophages play a role during lens regeneration in newts, but their function has not been tested experimentally. Methods: Here we generated a transgenic newt reporter line in which macrophages can be visualized in vivo. Using this new tool, we analyzed the location of macrophages during lens regeneration. We uncovered early gene expression changes using bulk RNAseq in two newt species, Notophthalmus viridescens and Pleurodeles waltl. Next, we used clodronate liposomes to deplete macrophages, which inhibited lens regeneration in both newt species. Results: Macrophage depletion induced the formation of scar-like tissue, an increased and sustained inflammatory response, an early decrease in iris pigment epithelial cell (iPEC) proliferation and a late increase in apoptosis. Some of these phenotypes persisted for at least 100 days and could be rescued by exogenous FGF2. Re-injury alleviated the effects of macrophage depletion and re-started the regeneration process. Conclusions: Together, our findings highlight the importance of macrophages in facilitating a pro-regenerative environment in the newt eye, helping to resolve fibrosis, modulating the overall inflammatory landscape and maintaining the proper balance of early proliferation and late apoptosis.

Cellular senescence promotes progenitor cell expansion during axolotl limb regeneration.

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure that nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact their microenvironment via Wnt pathway modulation. Further, we identify a link between Wnt signaling and senescence induction and propose that bSC-derived Wnt signals facilitate the proliferation of neighboring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in the regeneration of complex structures.

Tig1 regulates proximo-distal identity during salamander limb regeneration.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Salamander Insights Into Ageing and Rejuvenation.

Exhibiting extreme regenerative abilities which extend to complex organs and entire limbs, salamanders have long served as research models for understanding the basis of vertebrate regeneration. Yet these organisms display additional noteworthy traits, namely extraordinary longevity, indefinite regenerative potential and apparent lack of traditional signs of age-related decay or "negligible senescence." Here, I examine existing studies addressing these features, highlight outstanding questions, and argue that salamanders constitute valuable models for addressing the nature of organismal senescence and the interplay between regeneration and ageing.

Rising from the ashes: cellular senescence in regeneration.

Cellular senescence has recently become causally implicated in pathological ageing. Hence, a great deal of research is currently dedicated towards developing senolytic agents to selectively kill senescent cells. However, senescence also plays important roles in a range of physiological processes including during organismal development, providing a barrier to tumorigenesis and in limiting fibrosis. Recent evidence also suggests a role for senescence in coordinating tissue remodelling and in the regeneration of complex structures. Through its non-cell-autonomous effects, a transient induction of senescence may create a permissive environment for remodelling or regeneration through promoting local proliferation, cell plasticity, tissue patterning, balancing growth, or indirectly through finely tuned interactions with infiltrating immune mediators. A careful analysis of the beneficial roles of cellular senescence may provide insights into important physiological processes as well as informing strategies to counteract its detrimental consequences in ageing and disease.

Understanding the functions of BRCA1 in the DNA-damage response.

Inheritance of a mutation in BRCA1 (breast cancer 1 early-onset) results in predisposition to early-onset breast and ovarian cancer. Tumours in these individuals arise after somatic mutation or loss of the wild-type allele. Loss of BRCA1 function leads to a profound increase in genomic instability involving the accumulation of mutations, DNA breaks and gross chromosomal rearrangements. Accordingly, BRCA1 has been implicated as an important factor involved in both the repair of DNA lesions and in the regulation of cell-cycle checkpoints in response to DNA damage. However, the molecular mechanism through which BRCA1 functions to preserve genome stability remains unclear. In the present article, we examine the different ways in which BRCA1 might influence the repair of DNA damage and the preservation of genome integrity, taking into account what is currently known about its interactions with other proteins, its biochemical activity and its nuclear localization.

A conserved regulatory program initiates lateral plate mesoderm emergence across chordates.

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.