Lineage tracing of Shh+ floor plate cells and dynamics of dorsal-ventral gene expression in the regenerating axolotl spinal cord.

Both development and regeneration depend on signaling centers, which are sources of locally secreted tissue-patterning molecules. As many signaling centers are decommissioned before the end of embryogenesis, a fundamental question is how signaling centers can be re-induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration.

Egr1 regulates regenerative senescence and cardiac repair.

Senescence plays a key role in various physiological and pathological processes. We reported that injury-induced transient senescence correlates with heart regeneration, yet the multi-omics profile and molecular underpinnings of regenerative senescence remain obscure. Using proteomics and single-cell RNA sequencing, here we report the regenerative senescence multi-omic signature in the adult mouse heart and establish its role in neonatal heart regeneration and agrin-mediated cardiac repair in adult mice.

Hallmarks of regeneration.

Regeneration is a heroic biological process that restores tissue architecture and function in the face of day-to-day cell loss or the aftershock of injury. Capacities and mechanisms for regeneration can vary widely among species, organs, and injury contexts. Here, we describe "hallmarks" of regeneration found in diverse settings of the animal kingdom, including activation of a cell source, initiation of regenerative programs in the source, interplay with supporting cell types, and control of tissue size and function.

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.

Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition.

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs.

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum.

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union.

Transposon waves at the water-to-land transition.

The major transitions in vertebrate evolution are associated with significant genomic reorganizations. In contrast to the evolutionary processes that occurred at the origin of vertebrates or prior to the radiation of teleost fishes, no whole-genome duplication events occurred during the water-to-land transition, and it remains an open question how did genome dynamics contribute to this prominent evolutionary event. Indeed, the recent sequencing of sarcopterygian and amphibian genomes has revealed that the extant lineages immediately preceding and succeeding this transition harbor an exceptional number of transposable elements and it is tempting to speculate that these sequences might have catalyzed the adaptations that enabled vertebrates to venture into land.

Topological morphogenesis of neuroepithelial organoids.

Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function. However, how topology emerges during the development of organ shape, or morphogenesis, remains elusive. Here we combine tissue reconstitution and quantitative microscopy to show that tissue topology and shape is governed by two distinct modes of topological transitions.

Developmental cell fate choice in neural tube progenitors employs two distinct cis-regulatory strategies.

In many developing tissues, the patterns of gene expression that assign cell fate are organized by graded secreted signals. Cis-regulatory elements (CREs) interpret these signals to control gene expression, but how this is accomplished remains poorly understood. In the neural tube, a gradient of the morphogen sonic hedgehog (Shh) patterns neural progenitors.

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration.

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts.

Muscles are barely required for the patterning and cell dynamics in axolotl limb regeneration.

Regeneration of a complex appendage structure such as limb requires upstream and downstream coordination of multiple types of cells. Given type of cell may sit at higher upstream position to control the activities of other cells. Muscles are one of the major cell masses in limbs.

Now that We Got There, What Next?

As seen in the protocols in this book, the opportunities to pursue work at the cellular and molecular work in salamanders have considerably broadened over the last years. The availability of genomic information and genome editing, and the possibility to image tissues live and other methods enhance the spectrum of biological questions accessible to all researchers. Here I provide a personal perspective on what I consider exciting future questions open for investigation.

Artificial Insemination in Axolotl.

In axolotls (Ambystoma mexicanum), fertilization takes place internally. After courtship, the male axolotl deposits spermatophores, which the female takes up into her cloaca in order to fertilize eggs internally. The success of axolotl breedings is subject to several poorly understood factors including age, pairing, and genotype.

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.

Chromosome Conformation Capture for Large Genomes.

The gigantic 32Gb Axolotl genome inspires fascinating questions such as: how such a big genome is organized and packed in nuclei and how regulation of gene transcription can happen over such large genomic distances. Currently, there are many technical challenges when we investigate chromatin architecture in axolotl. For example, probing promoter-enhancer interactions in such a large genome.

Navigation and Use of Custom Tracks within the Axolotl Genome Browser.

The availability of the chromosome-scale axolotl genome sequences has made it possible to explore genome evolution, perform cross-species comparisons, and use additional sequencing data to analyze both genome-wide features and individual genes. Here, we will focus on the UCSC genome browser and demonstrate in a step-by-step manner how to use it to integrate different data to approach a broad question of the Fgf8 locus evolution and analyze the neighborhood of a gene that was reported missing in axolotl - Pax3.

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.

Single-cell analyses of axolotl telencephalon organization, neurogenesis, and regeneration.

Salamanders are tetrapod models to study brain organization and regeneration; however, the identity and evolutionary conservation of brain cell types are largely unknown. We delineated the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-cell genomic profiling. We identified glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved γ-aminobutyric acid-releasing (GABAergic) neuron classes.

Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in axolotl limb development and regeneration.

The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, , , , and are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme.

Inducible and tissue-specific cell labeling in Cre-ER transgenic Xenopus lines.

Investigating cell lineage requires genetic tools that label cells in a temporal and tissue-specific manner. The bacteriophage-derived Cre-ER /loxP system has been developed as a genetic tool for lineage tracing in many organisms. We recently reported a stable transgenic Xenopus line with a Cre-ER /loxP system driven by the mouse Prrx1 (mPrrx1) enhancer to trace limb fibroblasts during the regeneration process (Prrx1:CreER line).

The Axolotl's journey to the modern molecular era.

The salamander Ambystoma mexicanum, commonly called "the axolotl" has a long, illustrious history as a model organism, perhaps with one of the longest track records as a laboratory-bred vertebrate, yet it also holds a prominent place among the emerging model organisms. Or rather it is by now an "emerged" model organism, boasting a full cohort molecular genetic tools that allows an expanding community of researchers in the field to explore the remarkable traits of this animal including regeneration, at cellular and molecular precision-which had been a dream for researchers over the years. This chapter describes the journey to this status, that could be helpful for those developing their respective animal or plant models.

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.

Positional Memory in Vertebrate Regeneration: A Century's Insights from the Salamander Limb.

Salamanders, such as axolotls and newts, can regenerate complex tissues including entire limbs. But what mechanisms ensure that an amputated limb regenerates a limb, and not a tail or unpatterned tissue? An important concept in regeneration is positional memory-the notion that adult cells "remember" spatial identities assigned to them during embryogenesis (e.g.

The cellular and signaling dynamics of salamander limb regeneration.

Limb amputation in salamanders yields a wound response that ultimately leads to replacement of the missing part. This unique-among-tetrapod trait involves the migration and recruitment of multiple cell types including epithelium, immune cells, axonal growth cones, and connective tissue cells to build the blastema which contains the proliferating stem and progenitor cells to rebuild the limb tissues. A number of the signaling and cell biological events have been defined.