No abstract

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.

Reptiles are the only amniotes that maintain the capacity to regenerate appendages. This study presents the first anatomical and histological evidence of tail repair with regrowth in an archosaur, the American alligator. The regrown alligator tails constituted approximately 6–18% of the total body length and were morphologically distinct from original tail segments. Gross dissection, radiographs, and magnetic resonance imaging revealed that caudal vertebrae were replaced by a ventrally-positioned, unsegmented endoskeleton. This contrasts with lepidosaurs, where the regenerated tail is radially organized around a central endoskeleton. Furthermore, the regrown alligator tail lacked skeletal muscle and instead consisted of fibrous connective tissue composed of type I and type III collagen fibers. The overproduction of connective tissue shares features with mammalian wound healing or fibrosis. The lack of skeletal muscle contrasts with lizards, but shares similarities with regenerated tails in the tuatara and regenerated limbs in Xenopus adult frogs, which have a cartilaginous endoskeleton surrounded by connective tissue, but lack skeletal muscle. Overall, this study of wild-caught, juvenile American alligator tails identifies a distinct pattern of wound repair in mammals while exhibiting features in common with regeneration in lepidosaurs and amphibia.

The immune system of ectotherms, particularly non-avian reptiles, remains poorly characterized regarding the genes involved in immune function, and their function in wild populations. We used RNA-Seq to explore the systemic response of Mojave desert tortoise (Gopherus agassizii) gene expression to three levels of Mycoplasma infection to better understand the host response to this bacterial pathogen. We found over an order of magnitude more genes differentially expressed between male and female tortoises (1,037 genes) than differentially expressed among immune groups (40 genes). There were 8 genes differentially expressed among both variables that can be considered sex-biased immune genes in this tortoise. Among experimental immune groups we find enriched GO biological processes for cysteine catabolism, regulation of type 1 interferon production, and regulation of cytokine production involved in immune response. Sex-biased transcription involves iron ion transport, iron ion homeostasis, and regulation of interferon-beta production to be enriched. More detailed work is needed to assess the seasonal response of the candidate genes found here. How seasonal fluctuation of testosterone and corticosterone modulate the immunosuppression of males and their susceptibility to Mycoplasma infection also warrants further investigation, as well as the importance of iron in the immune function and sexbiased differences of this species. Finally, future transcriptional studies should avoid drawing blood from tortoises via subcarapacial venipuncture as the variable aspiration of lymphatic fluid will confound the differential expression of genes.

Among amniote vertebrates, reptiles display the greatest variation in axial skeleton morphology. Only recently have they been used in gene expression studies of somitogenesis , challenging previous assumptions about the segmentation clock and axial patterning. An increasing number of reptile genomes and transcriptomes are becoming available as next-generation sequencing becomes more affordable. Information regarding gene sequence and structure can be used to design and synthesize labeled riboprobes by in vitro transcription for gene expression analysis by in situ hybridization, thus, enabling the characterization of spatial and temporal expression patterns of genes involved in somitogenesis, a topic of great interest within evolutionary developmental studies of vertebrates.

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Species with multimodal communication integrate information from social cues in different modalities into behavioral responses that are mediated by changes in gene expression in the brain. Differences in patterns of gene expression between signal modalities may shed light on the neuromolecular mechanisms underlying multisensory processing. Here, we use RNA-Seq to analyze brain transcriptome responses to either chemical or visual social signals in a territorial lizard with multimodal communication. Using an intruder challenge paradigm, we exposed 18 wild-caught, adult, male Sceloporus jarrovii to either male conspecific scents (femoral gland secretions placed on a small pebble), the species-specific push-up display (a programmed robotic model), or a control (an unscented pebble). We conducted differential expression analysis with both a de novo S. jarrovii transcriptome assembly and the reference genome of a closely related species, Sceloporus undulatus. Despite some interindividual variation, we found significant differences in gene expression in the brain across signal modalities and the control in both analyses. The most notable differences occurred between chemical and visual stimulus treatments, closely followed by visual stimulus versus the control. Altered expression profiles could explain documented aggression differences in the immediate behavioral response to conspecific signals from different sensory modalities. Shared differentially expressed genes between visually-or chemically-stimulated males are involved in neural activity and neurodevelopment and several other differentially expressed genes in stimuluschallenged males are involved in conserved signal-transduction pathways associated with the social stress response, aggression and the response to territory intruders across vertebrates.

Reptiles are the only amniotes that maintain the capacity to lose and regrow entire appendages. Among reptiles, tail regeneration has been extensively studied in Lepidosauria, the subclass that includes lizards, but this study presents the first anatomical and histological evidence of tail regeneration in an archosaur, the American alligator (Alligator mississippiensis). All animals were wild caught juveniles or sub‐adults with an average total body length of 133.4±29.2cm (n=3). Although the duration of tail regrowth is unknown, the average length of the regenerated tail was 15.7±7.3cm. The regenerated tail was externally distinct from the original tail. The scales were compacted and lacked organization as well as dorsal scutes. Gross dissection, radiographs, and magnetic resonance imaging revealed that the caudal vertebrae were replaced by a ventrally‐positioned, unsegmented endoskeleton. This contrasts with lepidosaurs, where the regenerated tail is radially organized around a central endoskeleton, but shares commonality with urodele amphibians that regrow a tail with a clear dorsal‐ventral axis. Immunohistochemistry confirmed that this alligator structure was composed of cartilage, which formed a hollow rod with foramina distributed along the proximal‐distal axis. Furthermore, in contrast to the original tail, the regenerated tail lacked skeletal muscle and instead featured adipose and fibrous connective tissue supplied by a dense network of newly established axons and blood vessels. Despite substantial regrowth of the tail, the deposition of extensive connective tissue is reminiscent of mammalian wound healing or fibrosis. Using Herovici’s stain, it was shown that the regenerated tail is composed of both type I and type III collagen fibers. The lack of muscle contrasts with the lepidosaurs, which regrow functional skeletal muscle groups, but shares similarities with regenerated limbs in Xenopus adult frogs, which have a cartilaginous endoskeleton surrounded by connective tissue, but lacking skeletal muscle. Overall, this study of regeneration in the alligator identifies a distinct pattern of regrowth with features in common with lepidosaurs, amphibia, and wound repair in mammals. Further analysis will help to shed light on the conservation of regeneration in amniote vertebrates. Support or Funding Information This work was funded by the College of Liberal Arts and Sciences at Arizona State University to KK.