Session 8 – Moderated by Prayag Murawala

Stephen Randal Voss (Online)

14.00- 14.45

Leukocyte Tyrosine Kinase is the Mendelian Determinant of the Axolotl Melanoid Color Variant

The great diversity of color patterns observed among animals is largely explained by the differentiation of relatively few pigment cells during development. Mexican axolotls present a variety of color phenotypes that span the continuum of leucitic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, relatively fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single origin hypothesis of pigment cells from a common stem cell and the potential role of pigment metabolites in directing the development of pigment organelles that define different pigment cell types. Specifically, these studies identified XDH activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used Bulked Segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and clone the causative locus. Significantly different frequencies of single nucleotide polymorphisms were identified between pooled RNA samples of wildtype and melanoid sibs for a chromosome 14q genomic region. This region harbored Gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and Leucocyte Tyrosine Kinase (Ltk), a phosphatase that is required for iridophore differentiation in zebrafish. Wildtype Ltk crispants exactly phenocopied the melanoid phenotype, thus confirming Ltk as the melanoid locus. We discuss these results within the contexts of the single origin of pigment cells hypothesis and current developmental genetic models of pigment cell differentiation.

Katharina Lust


Single-cell analyses of cell type diversity, neurogenesis and regeneration in the axolotl telencephalon

Salamanders, such as the axolotl (Ambystoma mexicanum), play a role in the study of tetrapod-conserved traits. Cell type diversity in salamander brains and their relation to other vertebrate brains has until now been studied mainly histologically. Axolotl brains grow during postembryonic life and also regenerate after injury through the neurogenic activity of ependymoglia cells. It is still unclear how similar postembryonic and regenerative neurogenesis programs of ependymoglia cells are and whether neuronal connections are accurately recovered after regeneration is completed.

Here, we delineate the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-nuclei genomic methods and spatial profiling and define their similarities to amniote telencephalic cell types. Among these, we identify a population of glutamatergic excitatory neurons with transcriptional similarity to amniote olfactory cortical neurons. These neurons receive input projections from the olfactory bulb, indicating a conserved role in olfactory processing.

After targeted brain injury, removing olfactory cortex-like glutamatergic neurons, we uncover that they are efficiently regenerated through a transcriptional program highly similar to homeostatic neurogenesis. Finally, we find that regenerated olfactory cortex-like neurons receive input projections from the olfactory bulb, suggesting re-establishment of the circuit.

Turan Demircan


The Effects of Yap1 Downregulation During Early Phase of Limb Regeneration

The Hippo pathway is crucial to regulating vital cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Activation of the transcription coregulator component of this pathway, YAP1, promotes gene expression to drive proliferation, migration, differentiation, and suppress apoptosis. Although YAP1 functions in mammalian organ regeneration are well-established, its role in axolotl limb regeneration is understudied. The axolotl is an astonishing model organism to explore the molecular basis of organ and extremity regeneration due to its exceptional functional restoration capacity. This study examined Yap1 function in the early phase of limb regeneration by combining molecular, histological, and proteomics methods. Knock-down of Yap1 resulted in impaired regeneration fidelity, evident by bone formation defects. The altered expression level of proteases, ECM components, immune system elements, and osteogenesis-chondrogenesis regulators may explain the diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. Further functional studies aiming to modulate the expression level or activity of the candidate proteins described in this study would provide new insights into axolotl limb regeneration regulation.

Henrik Lauridsen


Sleeping beauty: The effect of long-term anesthesia on the regenerative ability of the axolotl

a) Aim Whole animal experiments in the regenerative field often require repeated use of anesthesia during injury procedures and at follow up examinations. Usually, anesthesia lasts only from minutes to a few hours before animals are reawakened. This limits the level of acceptable invasiveness of procedures, and it makes it difficult to untangle behavioral changes caused by injury to physiological processes involved in the regenerative response. We aimed to uncouple behavior from the regenerative response by developing a model of continuous anesthesia in the axolotl for 60 days spanning the majority of a full regenerative cycle following limb amputation or cryoinjury to the heart.

b) M&M Extensive piloting was performed to optimize the type of anesthetic, dose, route of delivery and osmolality of the housing medium. Subsequently, a study was conducted in which 18 age-matched axolotls (BM=20.4±3.4 g) were separated into five groups: three groups (each n=4) of continuously anesthetized animals receiving either no injury, limb amputation or cryoinjury to the heart, respectively, and two groups (each n=3) of animals receiving the same injuries, but maintained awake after the initial injury. Benzocaine (ethyl 4‐aminobenzoate, anesthetic and analgesic) was used during surgery and propofol (2,6-diisopropylphenol, anesthetic) was used for long-term anesthesia. During the regenerative progression both metabolic rate (respirometry), limb regeneration rate (photo), heart function and regeneration rate (echocardiography), and kidney function (creatinine) were repeatedly measured. Sixty days post injury, heart injury animal were sacrificed for quantitative histology, whereas animals with regenerated limbs were reawakened and subjected to behavioral analysis.

c) Results A total of 75% of continuously anesthetized animals survived until experimental end. Limb and heart regeneration progression was not significantly different between anesthetized and awake control groups. Reawakened animals did not show behavioral changes. d) Conclusions Axolotls can tolerate prolonged exposure to propofol anesthesia (60 days) and regenerate both limb and heart at the same rate in a comatose state as control animals. Dosage must be carefully monitored to avoid propofol toxicity.