Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. Connective tissue (CT) – skeleton, periskeleton, tendon, dermis, interstitial fibroblasts – contributes the vast majority of cells that populate the blastema, however, it is unclear how individual CT cells may reprogram their fate in order to rebuild the tetrapod limb. Here we use a combination of Cre-loxP reporter lineage tracking and single-cell (sc) RNA-seq to molecularly track, for the first time, adult CT cell heterogeneity and its transition to a limb blastema state. We uncover a multi-phasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that participates in inflammation and extracellular matrix disassembly prior to proliferation, establishment of positional information, and ultimately re-differentiation. While the early regeneration transcriptome states are unique to the blastema, the later stages recapitulate embryonic limb development. Notably, we do not find evidence of a pre-existing blastema-like precursor nor limb bud-like progenitors in the uninjured adult tissue. However, we find that distinct CT subpopulations in the adult limb differentially contribute to extending bone at the amputation plane versus regenerating new segments. Together, our data illuminates molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

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.

Turbidity and opaqueness are inherent properties of tissues which 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 method) which can be used to clear a wide range of tissues, including cerebral organoids, Drosophila melanogaster, zebrafish, axolotl, and Xenopus laevis in as little as 1-5 days while preserving a broad range of fluorescence proteins including GFP, mCherry, Brainbow, and alexa-fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method will open up clearing to a much broader group of researchers, due to its broad applicability, ease of use, and non-toxic nature of Ethyl cinnamate.Summary statementThe non-toxic, broadly applicable, and simplified protocol of 2Eci tissue clearing makes it possible for non-specialist labs to use clearing approaches on conventional inverted microscopes.

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.

BackgroundSalamanders 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.MethodsTo 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.ResultsIn 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.ConclusionsThese 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.

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.

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.

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.