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.

Multi-species atlas resolves an axolotl limb development and regeneration paradox.

Humans and other tetrapods are considered to require apical-ectodermal-ridge (AER) cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, a primary model organism for regeneration, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first establish that axolotls contain cells with AER characteristics. Further analyses and spatial transcriptomics reveal that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.

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.

Characterizing the lens regeneration process in Pleurodeles waltl.

BACKGROUND: Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime. RESULTS: Here, we used Spectral-Domain Optical Coherence Tomography (SD-OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related change in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms. CONCLUSIONS: Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes associated with aging alter the kinetics of this process. By understanding how these changes affect lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.

In Vivo and Ex Vivo View of Newt Lens Regeneration.

Lens regeneration in the adult newt illustrates a unique example of naturally occurring cell transdifferentiation. During this process, iris pigmented epithelial cells (iPECs) reprogram into a lens, a tissue that is derived from a different embryonic source. Several methodologies both in vivo and in culture have been utilized over the years to observe this phenomenon. Most recently, Optical Coherence Tomography (OCT) has been identified as an effective tool to study the lens regeneration process in continuity through noninvasive, real-time imaging of the same animal. Described in this chapter are three different methodologies that can be used to observe the newt lens regeneration process both in vivo and ex vivo.

Isolation and Characterization of Peritoneal Macrophages from Salamanders.

Salamanders have been used as research models for centuries. While they exhibit a wide range of biological features not seen in mammals, none has captivated scientists like their ability to regenerate. Interestingly, axolotl macrophages have emerged as an essential cell population for tissue regeneration. Whether the same is true in other salamanders such as newt species Notophthalmus viridescens, Cynops pyrrhogaster, or Pleurodeles waltl remains to be seen. Unfortunately, regardless of the species, molecular tools to study macrophage function in salamanders are lacking. We propose that the readily available, terminally differentiated peritoneal macrophages from newts or axolotls could be used to validate molecular reagents in the study of macrophage function during tissue regeneration in salamanders.

In Vivo Imaging of Newt Lens Regeneration: Novel Insights Into the Regeneration Process.

Purpose: To establish optical coherence tomography (OCT) as an in vivo imaging modality for investigating the process of newt lens regeneration. Methods: Spectral-domain OCT was employed for in vivo imaging of the newt lens regeneration process. A total of 37 newts were lentectomized and followed by OCT imaging over the course of 60 to 80 days. Histological images were obtained at several time points to compare with the corresponding OCT images. Volume measurements were also acquired. Results: OCT can identify the key features observed in corresponding histological images based on the scattering differences from various eye tissues, such as the cornea, intact and regenerated lens, and the iris. Lens volume measurements from three-dimensional OCT images showed that the regenerating lens size increased linearly until 60 days post-lentectomy. Conclusions: Using OCT imaging, we were able to track the entire process of newt lens regeneration in vivo for the first time. Three-dimensional OCT images allowed us to volumetrically quantify and visualize the dynamic spatial relationships between tissues during the regeneration process. Our results establish OCT as an in vivo imaging modality to track/analyze the entire lens regeneration process from the same animal. Translational Relevance: Lens regeneration in newts represents a unique example of vertebrate tissue plasticity. Investigating the cellular and morphological events that govern this extraordinary process in vivo will advance our understanding and shed light on developing new therapies to treat blinding disorders in higher vertebrates.