BackgroundNotophthalmus viridescens, an urodelian amphibian, represents an excellent model organism to study regenerative processes, but mechanistic insights into molecular processes driving regeneration have been hindered by a paucity and poor annotation of coding nucleotide sequences. The enormous genome size and the lack of a closely related reference genome have so far prevented assembly of the urodelian genome.ResultsWe describe the de novo assembly of the transcriptome of the newt Notophthalmus viridescens and its experimental validation. RNA pools covering embryonic and larval development, different stages of heart, appendage and lens regeneration, as well as a collection of different undamaged tissues were used to generate sequencing datasets on Sanger, Illumina and 454 platforms. Through a sequential de novo assembly strategy, hybrid datasets were converged into one comprehensive transcriptome comprising 120,922 non-redundant transcripts with a N50 of 975. From this, 38,384 putative transcripts were annotated and around 15,000 transcripts were experimentally validated as protein coding by mass spectrometry-based proteomics. Bioinformatical analysis of coding transcripts identified 826 proteins specific for urodeles. Several newly identified proteins establish novel protein families based on the presence of new sequence motifs without counterparts in public databases, while others containing known protein domains extend already existing families and also constitute new ones.ConclusionsWe demonstrate that our multistep assembly approach allows de novo assembly of the newt transcriptome with an annotation grade comparable to well characterized organisms. Our data provide the groundwork for mechanistic experiments to answer the question whether urodeles utilize proprietary sets of genes for tissue regeneration.

A number of organs have the intrinsic ability to regenerate, a distinctive feature that varies among organisms. Organ regeneration is a process not fully yet understood. However, when its underlying mechanisms are unraveled, it holds tremendous therapeutic potential for humans. In this review, we chose to summarize the repair and regenerative potential of the following organs and organ systems: thymus, adrenal gland, thyroid gland, intestine, lungs, heart, liver, blood vessels, germ cells, nervous system, eye tissues, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage. For each organ, a review of the following is presented: (a) factors, pathways, and cells that are involved in the organ's intrinsic regenerative ability, (b) contribution of exogenous cells – such as progenitor cells, embryonic stem cells, induced pluripotent stem cells, and bone marrow‐, adipose‐ and umbilical cord blood‐derived stem cells – in repairing and regenerating organs in the absence of an innate intrinsic regenerative capability, (c) and the progress made in engineering bio‐artificial scaffolds, tissues, and organs. Organ regeneration is a promising therapy that can alleviate humans from diseases that have not been yet cured. It is also superior to already existing treatments that utilize exogenous sources to substitute for the organ's lost structure and/or function(s). (Part C) 96:1–29, 2012. © 2012 Wiley Periodicals, Inc.

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Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl’s ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls’ ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.

Matching appendage size to body size is fundamental to animal function. Generating an appropriately-sized appendage is a robust process executed during development which is also critical for regeneration. When challenged, larger animals are programmed to regenerate larger limbs than smaller animals within a single species. Understanding this process has important implications for regenerative medicine. To approach this complex question, models with altered appendage size:body size ratios are required. We hypothesized that repeatedly challenging axolotls to regrow limb buds would affect their developmental program resulting in altered target morphology. We discovered that after 10 months following this experimental procedure, limbs that developed were permanently miniaturized. This altered target morphology was preserved upon amputation and regeneration. Future experiments using this platform should provide critical information about how target limb size is encoded within limb progenitors.

Regeneration of the lens in newts is quite a unique process. The lens is removed in its entirety and regeneration ensues from the pigment epithelial cells of the dorsal iris via transdifferentiation. The same type of cells from the ventral iris are not capable of regenerating a lens. It is, thus, expected that differences between dorsal and ventral iris during the process of regeneration might provide important clues pertaining to the mechanism of regeneration. In this paper, we employed next generation RNA-seq to determine gene expression patterns during lens regeneration in Notophthalmus viridescens. The expression of more than 38,000 transcripts was compared between dorsal and ventral iris. Although very few genes were found to be dorsal- or ventral-specific, certain groups of genes were up-regulated specifically in the dorsal iris. These genes are involved in cell cycle, gene regulation, cytoskeleton and immune response. In addition, the expression of six highly regulated genes, TBX5, FGF10, UNC5B, VAX2, NR2F5, and NTN1, was verified using qRT-PCR. These graded gene expression patterns provide insight into the mechanism of lens regeneration, the markers that are specific to dorsal or ventral iris, and layout a map for future studies in the field.

Coleoid cephalopods have an elaborate camera eye whereas nautiloids have primitive pinhole eye without lens and cornea. The Nautilus pinhole eye provides a unique example to explore the module of lens formation and its evolutionary mechanism. Here, we conducted an RNA-seq study of developing eyes of Nautilus and pygmy squid. First, we found that evolutionary distances from the common ancestor to Nautilus or squid are almost the same. Although most upstream eye development controlling genes were expressed in both species, six3/6 that are required for lens formation in vertebrates was not expressed in Nautilus. Furthermore, many downstream target genes of six3/6 including crystallin genes and other lens protein related genes were not expressed in Nautilus. As six3/6 and its controlling pathways are widely conserved among molluscs other than Nautilus, the present data suggest that deregulation of the six3/6 pathway led to the pinhole eye evolution in Nautilus.

Newts have the ability to repeatedly regenerate their lens even during ageing. However, it is unclear whether this regeneration reflects an undisturbed genetic activity. To answer this question, we compared the transcriptomes of lenses, irises and tails from aged newts that had undergone lens regeneration 19 times with the equivalent tissues from young newts that had never experienced lens regeneration. Our analysis indicates that repeatedly regenerated lenses showed a robust transcriptional program comparable to young never-regenerated lenses. In contrast, the tail, which was never regenerated, showed gene expression signatures of ageing. Our analysis strongly suggests that, with respect to gene expression, the regenerated lenses have not deviated from a robust transcriptional program even after multiple events of regeneration throughout the life of the newt. In addition, our study provides a new paradigm in biology, and establishes the newt as a key model for the study of regeneration in relation to ageing.DOI: http://dx.doi.org/10.7554/eLife.09594.001