Life Sciences Commons^™

The Giant Axolotl Genome Uncovers The Evolution, Scaling, And Transcriptional Control Of Complex Gene Loci, Siegfried Schloissnig, Akane Kawaguchi, Sergej Nowoshilow, Francisco Falcon, Leo Otsuki, Pietro Tardivo, Nataliya Timoshevskaya, Melissa C. Keinath, Jeramiah J. Smith, S. Randal Voss, Elly M. Tanaka

Biology Faculty Publications

Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth …

A Chromosome-Scale Assembly Of The Axolotl Genome, Jeramiah J. Smith, Nataliya Y. Timoshevskaya, Vladimir A. Timoshevskiy, Melissa C. Keinath, Drew Hardy, S. Randal Voss

Biology Faculty Publications

The axolotl (Ambystoma mexicanum) provides critical models for studying regeneration, evolution, and development. However, its large genome (∼32 Gb) presents a formidable barrier to genetic analyses. Recent efforts have yielded genome assemblies consisting of thousands of unordered scaffolds that resolve gene structures, but do not yet permit large-scale analyses of genome structure and function. We adapted an established mapping approach to leverage dense SNP typing information and for the first time assemble the axolotl genome into 14 chromosomes. Moreover, we used fluorescence in situ hybridization to verify the structure of these 14 scaffolds and assign each to its …

Tgf-Β, Wnt, And Fgf Signaling Pathways During Axolotl Tail Regeneration And Forelimb Bud Development, Qingchao Qiu

Theses and Dissertations--Neuroscience

Tgf-β, Wnt, and Fgf signaling pathways are required for many developmental processes. Here, I investigated the requirement of these signaling pathways during tail regeneration and limb development in the Mexican axolotl (Ambystoma mexicanum).

Using small chemical inhibitors during tail regeneration, I found that the Tgf-β signaling pathway was required from 0-24 and 48-72 hours post tail amputation (hpa), the Wnt signaling pathway was required from 0-120 hpa, and the Fgf signaling pathway was required from 0-12hpa. Tgf-β1 was upregulated after amputation and thus may mediate Tgf-β signaling pathway during tail regeneration. Both Smad-mediated and non-Smad mediated Tgf-β signaling …

Using Transcriptomics To Enable A Plethodontid Salamander (Bolitoglossa Ramosi) For Limb Regeneration Research, Claudia M. Arenas Gómez, Ryan M. Woodcock, Jeramiah J. Smith, Randal S. Voss, Jean Paul Delgado

Biology Faculty Publications

Background: Tissue regeneration is widely distributed across the tree of life. Among vertebrates, salamanders possess an exceptional ability to regenerate amputated limbs and other complex structures. Thus far, molecular insights about limb regeneration have come from a relatively limited number of species from two closely related salamander families. To gain a broader perspective on the molecular basis of limb regeneration and enhance the molecular toolkit of an emerging plethodontid salamander (Bolitoglossa ramosi), we used RNA-Seq to generate a de novo reference transcriptome and identify differentially expressed genes during limb regeneration.

Results: Using paired-end Illumina sequencing technology and Trinity …

Ion Channel Signaling Influences Cellular Proliferation And Phagocyte Activity During Axolotl Tail Regeneration, Brandon M. Franklin, S. Randal Voss, Jeffrey L. Osborn

Biology Faculty Publications

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K_V2.1, K_V2.2, L-type CaV channels and H/K ATPases) or completely (GlyR, GABA_AR, K_V1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in …

Gene Expression During The First 28 Days Of Axolotl Limb Regeneration I: Experimental Design And Global Analysis Of Gene Expression, S. Randal Voss, Alex Palumbo, Radha Nagarajan, David M. Gardiner, Ken Muneoka, Arnold J. Stromberg, Antony T. Athippozhy

Biology Faculty Publications

While it is appreciated that global gene expression analyses can provide novel insights about complex biological processes, experiments are generally insufficiently powered to achieve this goal. Here we report the results of a robust microarray experiment of axolotl forelimb regeneration. At each of 20 post-amputation time points, we estimated gene expression for 10 replicate RNA samples that were isolated from 1 mm of heterogeneous tissue collected from the distal limb tip. We show that the limb transcription program diverges progressively with time from the non-injured state, and divergence among time adjacent samples is mostly gradual. However, punctuated episodes of transcription …

Tissue Specific Reactions To Positional Discontinuities In The Regenerating Axolotl Limb, Malcolm Maden, Daima Avila, Molly Roy, Ashley W. Seifert

Biology Faculty Publications

We investigated cellular contributions to intercalary regenerates and 180^o supernumerary limbs during axolotl limb regeneration using the cell autonomous green fluorescent protein marker and exchanged blastemas between white and green fluorescent protein animals. After distal blastemas were grafted to proximal levels tissues of the intercalary regenerate behaved independently with regard to the law of distal transformation; graft epidermis was replaced by stump epidermis, muscle-derived cells, blood vessels, and Schwann cells of the distal blastema moved proximally to the stylopodium and cartilage and dermal cells conformed to the law. After 180^o rotation, blastemas showed contributions from stump tissues which …

Probability Of Regenerating A Normal Limb After Bite Injury In The Mexican Axolotl (Ambystoma Mexicanum), Sierra Thompson, Laura Muzinic, Christopher Muzinic, Matthew L. Niemiller, S. Randal Voss

Biology Faculty Publications

Multiple factors are thought to cause limb abnormalities in amphibian populations by altering processes of limb development and regeneration. We examined adult and juvenile axolotls (Ambystoma mexicanum) in the Ambystoma Genetic Stock Center (AGSC) for limb and digit abnormalities to investigate the probability of normal regeneration after bite injury. We observed that 80% of larval salamanders show evidence of bite injury at the time of transition from group housing to solitary housing. Among 717 adult axolotls that were surveyed, which included solitary-housed males and group-housed females, approximately half presented abnormalities, including examples of extra or missing digits and …

Characterization Of In Vitro Transcriptional Responses Of Dorsal Root Ganglia Cultured In The Presence And Absence Of Blastema Cells From Regenerating Salamander Limbs, Antony Athippozhy, Jeffrey Lehrberg, James R. Monaghan, David M. Gardiner, S. Randal Voss

Biology Faculty Publications

During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema-dorsal root ganglia (DRG) co-culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for five days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1,541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function …

Gene Expression Patterns Specific To The Regenerating Limb Of The Mexican Axolotl, James R. Monaghan, Antony Athippozhy, Ashley W. Seifert, Sri Putta, Arnold J. Stromberg, Malcolm Maden, David M. Gardiner, Stephen R. Voss

Biology Faculty Publications

Salamander limb regeneration is dependent upon tissue interactions that are local to the amputation site. Communication among limb epidermis, peripheral nerves, and mesenchyme coordinate cell migration, cell proliferation, and tissue patterning to generate a blastema, which will form missing limb structures. An outstanding question is how cross-talk between these tissues gives rise to the regeneration blastema. To identify genes associated with epidermis-nerve-mesenchymal interactions during limb regeneration, we examined histological and transcriptional changes during the first week following injury in the wound epidermis and subjacent cells between three injury types; 1) a flank wound on the side of the animal that …