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. Here, we review the genome dynamics associated with the major transitions in vertebrate evolution and discuss how the highly repetitive genomic landscapes revealed by recent efforts to characterize the genomes of amphibians and sarcopterygians argue for turbulent genome dynamics occurring before the water-to-land transition and possibly enabling it.

Topological morphogenesis of neuroepithelial organoids.

Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function1-3. 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 transitions4,5. One mode involves the fusion of two separate epithelia and the other involves the fusion of two ends of the same epithelium. The morphological space is captured by a single control parameter that can be traced back to the relative rates of the two epithelial fusion modes. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of two modes of epithelial fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues6.

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.

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.

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. Chromatin capture methods (e.g., Chromatin Conformation Capture) have been used in a variety of species. The large size of the axolotl nuclei and its genome requires the adaptation of such methods. Here, we describe a detailed protocol for high-throughput genome-wide conformation capture (Hi-C) using axolotl limb cells. This Hi-C library preparation protocol can also be used to prepare libraries from other nonmodel organisms such as Lungfish and Cephalopods. We believe that our protocol could be useful for a variety of animal systems including other salamanders.

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.

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. In some cases, individuals are unable to breed naturally despite having significant scientific value. Assisted reproductive technologies represent one approach to maintaining stocks of such individuals, as well as supplementing natural breedings of laboratory stocks.Here, we describe a protocol for artificial insemination--an assisted reproductive technology in which sperm is extracted from a male and transferred into the female cloaca, thus mimicking natural fertilization in axolotls. We believe that this simple method can be applied to other salamander species that have internal fertilization and also help restore endangered wild populations.

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.

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. We identify two distinct ways in which CREs translate graded Shh into differential gene expression in mouse neural progenitors. In most progenitors, a common set of CREs control gene activity by integrating cell-type-specific inputs. By contrast, the most ventral progenitors use a unique set of CREs, established by the pioneer factor FOXA2. This parallels the role of FOXA2 in endoderm, where FOXA2 binds some of the same sites. Together, the data identify distinct cis-regulatory strategies for the interpretation of morphogen signaling and raise the possibility of an evolutionarily conserved role for FOXA2 across tissues.

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. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

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. However, the subtle functional relationship between muscle and other cells in vertebrate complex tissue regeneration are still not well established. Here, we use Pax7 mutant axolotls, in which the limb muscle is developmentally lost, to investigate limb regeneration in the absence of skeletal muscle. We find that the pattern of regenerated limbs is relative normal in Pax7 mutants compared to the controls, but the joint is malformed in the Pax7 mutants. Lack of muscles do not affect the early regeneration responses, specifically the recruitment of macrophages to the wound, as well as the proliferation of fibroblasts, another major population in limbs. Furthermore, using single cell RNA-sequencing, we show that, other than muscle lineage that is mostly missing in Pax7 mutants, the composition and the status of other cell types in completely regenerated limbs of Pax7 mutants are similar to that in the controls. Our study reveals skeletal muscle is barely required for the guidance of other cells, as well the patterning in complex tissue regeneration in axolotls, and provides refined views of the roles of muscle cell in vertebrate appendage regeneration.

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. We inferred transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific neurogenesis and unraveled conserved differentiation signatures. After brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield insights into the organization, evolution, and regeneration of a tetrapod nervous system.

Inducible and tissue-specific cell labeling in Cre-ERT2 transgenic Xenopus lines.

Investigating cell lineage requires genetic tools that label cells in a temporal and tissue-specific manner. The bacteriophage-derived Cre-ERT2 /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-ERT2 /loxP system driven by the mouse Prrx1 (mPrrx1) enhancer to trace limb fibroblasts during the regeneration process (Prrx1:CreER line). Here we describe the detailed technological development and characterization of such line. Transgenic lines carrying a CAG promoter-driven Cre-ERT2 /loxP system showed conditional labeling of muscle, epidermal, and interstitial cells in both the tadpole tail and the froglet leg upon 4-hydroxytamoxifen (4OHT) treatment. We further improved the labeling efficiency in the Prrx1:CreER lines from 12.0% to 32.9% using the optimized 4OHT treatment regime. Careful histological examination showed that Prrx1:CreER lines also sparsely labeled cells in the brain, spinal cord, head dermis, and fibroblasts in the tail. This work provides the first demonstration of conditional, tissue-specific cell labeling with the Cre-ERT2 /loxP system in stable transgenic Xenopus lines.

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, Fgf 4, 8, 9, and 17 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. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

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. 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.

Gene and transgenics nomenclature for the laboratory axolotl-Ambystoma mexicanum.

The laboratory axolotl (Ambystoma mexicanum) is widely used in biological research. Recent advancements in genetic and molecular toolkits are greatly accelerating the work using axolotl, especially in the area of tissue regeneration. At this juncture, there is a critical need to establish gene and transgenic nomenclature to ensure uniformity in axolotl research. Here, we propose guidelines for genetic nomenclature when working with the axolotl.

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., "head" or "hand") and use this information to restore the correct body parts after injury. Although positional memory is well documented at a phenomenological level, the underlying cellular and molecular bases are just beginning to be decoded. Herein, we review how major principles in positional memory were established in the salamander limb model, enabling the discovery of positional memory-encoding molecules, and advancing insights into their pattern-forming logic during regeneration. We explore findings in other amphibians, fish, reptiles, and mammals and speculate on conserved aspects of positional memory. We consider the possibility that manipulating positional memory in human cells could represent one route toward improved tissue repair or engineering of patterned tissues for therapeutic purposes.

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. They point to the intimate coordination of physical events such as osmotic pressure, cell migration, and cell-cell communication with changes in cell identity such as dedifferentiation into embryonic-like epithelial and mesenchymal cells.