Konstantinos Sousounis

A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration.

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.

Organ repair and regeneration: an overview.

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 organs 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 organs lost structure and/or function(s).

Aging and Regeneration in Vertebrates

Aging is marked by changes that affect organs and resident stem cell function. Shorting of telomeres, DNA damage, oxidative stress, deregulation of genes and proteins, impaired cell-cell communication, and an altered systemic environment cause the eventual demise of cells. At the same time, reparative activities also decline. It is intriguing to correlate aging with the decline of regenerative abilities. Animal models with strong regenerative capabilities imply that aging processes might not be affecting regeneration. In this review, we selectively present age-dependent changes in stem/progenitor cells that are vital for tissue homeostasis and repair. In addition, the aging effect on regeneration following injury in organs such as lung, skeletal muscle, heart, nervous system, cochlear hair, lens, and liver are discussed. These tissues are also known for diseases such as heart attack, stroke, cognitive impairment, cataract, and hearing loss that occur mostly during aging in humans. Conclusively, vertebrate regeneration declines with age with the loss of stem/progenitor cell function. Future studies on improving the function of stem cells, along with studies in fish and amphibians where regeneration does not decline with age, will undoubtedly provide insights into both processes.

Transcriptome Analysis of Newt Lens Regeneration Reveals Distinct Gradients in Gene Expression Patterns

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.

Loss of the six3/6 controlling pathways might have resulted in pinhole-eye evolution in Nautilus

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.

Plasticity for axolotl lens regeneration is associated with age-related changes in gene expression

Abstract Mexican axolotls lose potential for lens regeneration 2 weeks after hatching. We used microarrays to identify differently expressed genes before and after this critical time, using RNA isolated from iris. Over 3700 genes were identified as differentially expressed in response to lentectomy between young (7 days post‐hatching) and old (3 months post‐hatching) axolotl larvae. Strikingly, many of the genes were only expressed in the early or late iris. Genes that were highly expressed in young iris significantly enriched electron transport chain, transcription, metabolism, and cell cycle gene ontologies, all of which are associated with lens regeneration. In contrast, genes associated with cellular differentiation and tissue maturation were uniquely expressed in old iris. Many of these expression differences strongly suggest that young and old iris samples were collected before and after the spleen became developmentally competent to produce and secrete cells with humoral and innate immunity functions. Our study establishes the axolotl as a powerful model to investigate age‐related cellular differentiation and immune system ontogeny within the context of tissue regeneration.

A robust transcriptional program in newts undergoing multiple events of lens regeneration throughout their lifespan

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

Conservation of the three-dimensional structure in non-homologous or unrelated proteins.

In this review, we examine examples of conservation of protein structural motifs in unrelated or non-homologous proteins. For this, we have selected three DNA-binding motifs: the histone fold, the helix-turn-helix motif, and the zinc finger, as well as the globin-like fold. We show that indeed similar structures exist in unrelated proteins, strengthening the concept that three-dimensional conservation might be more important than the primary amino acid sequence.

Patterns of gene expression in microarrays and expressed sequence tags from normal and cataractous lenses

In this contribution, we have examined the patterns of gene expression in normal and cataractous lenses as presented in five different papers using microarrays and expressed sequence tags. The purpose was to evaluate unique and common patterns of gene expression during development, aging and cataracts.

Impaired Recall of Positional Memory following Chemogenetic Disruption of Place Field Stability

SUMMARY The neural network of the temporal lobe is thought to provide a cognitive map of our surroundings. Functional analysis of this network has been hampered by coarse tools that often result in collateral damage to other circuits. We developed a chemogenetic system to temporally control electrical input into the hippocampus. When entorhinal input to the perforant path was acutely silenced, hippocampal firing patterns became destabilized and underwent extensive remapping. We also found that spatial memory acquired prior to neural silencing was impaired by loss of input through the perforant path. Together, our experiments show that manipulation of entorhinal activity destabilizes spatial coding and disrupts spatial memory. Moreover, we introduce a chemogenetic model for non-invasive neuronal silencing that offers multiple advantages over existing strategies in this setting.