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.

Broad applicability of a streamlined ethyl cinnamate-based clearing procedure.

Turbidity and opaqueness are inherent properties of tissues that limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here, we describe 2Eci (2nd generation ethyl cinnamate-based clearing), which can be used to clear a wide range of tissues in several species, including human organoids, Drosophila melanogaster, zebrafish, axolotl and Xenopus laevis, in as little as 1-5 days, while preserving a broad range of fluorescent proteins, including GFP, mCherry, Brainbow and Alexa-conjugated fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method opens up tissue clearing to a much broader group of researchers due to its ease of use, the non-toxic nature of ethyl cinnamate and broad applicability.

A somitic contribution to the apical ectodermal ridge is essential for fin formation.

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

Toward whole tissue imaging of axolotl regeneration.

The axolotl is a highly regenerative organism and has been studied in laboratories for over 150 years. Despite a long-standing fascination with regeneration in general and axolotl specifically, we are still scratching the surface trying to visualize and understand the complex cellular behavior that underlies axolotl regeneration. In this review, we will discuss the progress that has been made in visualizing these processes focusing on four major aspects: cell labeling approaches, the removal of pigmentation, reductionist approaches to perform live cell imaging, and finally recent developments applying tissue clearing strategies to visualize the processes that underly regeneration. We also provide several suggestions that the community could consider exploring, notably the generation of novel alleles that further reduce pigmentation as well as improvements in tissue clearing strategies.

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

Cellular dynamics underlying regeneration of appropriate segment number during axolotl tail regeneration.

BACKGROUND: Salamanders regenerate their tails after amputation anywhere along their length. How the system faithfully reconstitutes the original number of segments and length is not yet known. METHODS: To gain quantitative insight into how the system regenerates the appropriate length, we amputated tails at 4 or 16 myotomes post-cloaca and measured blastema size, cell cycle kinetics via cumulative Bromodeoxyuridine (BrdU) incorporation and the method of Nowakowski, and myotome differentiation rate. RESULTS: In early stages until day 15, blastema cells were all proliferative and divided at the same rate at both amputation levels. A larger blastema was formed in 4th versus 16th myotome amputations indicating a larger founding population. Myotome differentiation started at the same timepoint in the 4th and 16 th level blastemas. The rate of myotome formation was more rapid in 4th myotome blastemas so that by day 21 the residual blastema from the two amputation levels achieved equivalent size. At that time point, only a fraction of blastema cells remain in cycle, with the 4th myotome blastema harboring double the number of cycling cells as the 16th myotome blastema allowing it to grow faster and further reconstitute the larger number of missing myotomes. CONCLUSIONS: These data suggest that there are two separable phases of blastema growth. The first is level-independent, with cells displaying unrestrained proliferation. In the second phase, the level-specific growth is revealed, where differing fractions of cells remain in the cell cycle over time.

A chromatin code for limb segment identity in axolotl limb regeneration.

The salamander limb correctly regenerates missing limb segments because connective tissue cells have segment-specific identities, termed "positional information". How positional information is molecularly encoded at the chromatin level has been unknown. Here, we performed genome-wide chromatin profiling in mature and regenerating axolotl limb connective tissue cells. We find segment-specific levels of histone H3K27me3 as the major positional mark, especially at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. In the hand, the permissive chromatin state of the homeoprotein gene HoxA13 engages with the regeneration program bypassing the upper limb program. Comparison of regeneration regulatory elements with those found in other regenerative animals identified a core shared set of transcription factors, supporting an ancient, conserved regeneration program.

Ethyl Cinnamate-Based Tissue Clearing Strategies.

Tissue clearing turns otherwise turbid and opaque tissue transparent, enabling imaging deep within tissues. The nontransparent nature of most tissues is due to the refractive index mismatch between its three major constituent components (lipids, proteins, and water). All tissue clearing methods rectify this mismatch by homogenizing the refractive index within the tissue and carefully matching it to the surrounding media. Here we describe a detailed protocol to clear a wide range of salamander tissues. We also include several optional steps such as depigmentation, antibody staining, and tissue mounting. These steps are optional, and do not change anything in the steps needed for tissue clearing. Depending on the fluorescent signal and optics employed, images up to several millimeters inside of the tissue can be acquired.

A safe and reusable imaging chamber compatible with organic solvents.

High refractive index organic solvents are commonly used as an imaging medium in tissue clearing approaches. While effective, such solvents provide serious concerns for the safety of users and the equipment, especially in a central microscopy unit. To overcome these concerns, we have developed a large and reusable imaging chamber compatible with the universal mounting frame AK (PeCon GmbH). This chamber is easy to assemble and significantly improves the working environment in a central microscopy unit, where hazardous chemicals could negatively affect equipment and people. To encourage the uptake of these chambers, the design is made publicly available for download.

Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

Low-cost silicone imaging casts for zebrafish embryos and larvae.

Due to their size and optical clarity, zebrafish embryos have long been appreciated for their usefulness in time-lapse confocal microscopy. Current methods of mounting zebrafish embryos and larvae for imaging consist mainly of mounting in low percentage, low melting temperature agarose in a Petri dish. Whereas imaging methods have advanced greatly over the last two decades, the methods for mounting embryos have not changed significantly. In this article, we describe the development and use of 3D printed plastic molds. These molds can be used to create silicone casts and allow embryos and larvae to be mounted with a consistent and reproducible angle, and position in X, Y, and Z. These molds are made on a 3D printer and can be easily and cheaply reproduced by anyone with access to a 3D printer, making this method accessible to the entire zebrafish community. Molds can be reused to create additional casts, which can be reused after imaging. These casts are compatible with any upright microscope and can be adapted for use on an inverted microscope, taking the working distance of the objective used into account. This technique should prove to be useful to any researcher imaging zebrafish embryos.

Pair-wise regulation of convergence and extension cell movements by four phosphatases via RhoA.

Various signaling pathways regulate shaping of the main body axis during early vertebrate development. Here, we focused on the role of protein-tyrosine phosphatase signaling in convergence and extension cell movements. We identified Ptpn20 as a structural paralogue of PTP-BL and both phosphatases were required for normal gastrulation cell movements. Interestingly, knockdowns of PTP-BL and Ptpn20 evoked similar developmental defects as knockdown of RPTPα and PTPε. Co-knockdown of RPTPα and PTP-BL, but not Ptpn20, had synergistic effects and conversely, PTPε and Ptpn20, but not PTP-BL, cooperated, demonstrating the specificity of our approach. RPTPα and PTPε knockdowns were rescued by constitutively active RhoA, whereas PTP-BL and Ptpn20 knockdowns were rescued by dominant negative RhoA. Consistently, RPTPα and PTP-BL had opposite effects on RhoA activation, both in a PTP-dependent manner. Downstream of the PTPs, we identified NGEF and Arhgap29, regulating RhoA activation and inactivation, respectively, in convergence and extension cell movements. We propose a model in which two phosphatases activate RhoA and two phosphatases inhibit RhoA, resulting in proper cell polarization and normal convergence and extension cell movements.