Review: Regeneration of the tail in lizards appears regulated by a balanced expression of oncogenes and tumor suppressors.

BACKGROUND: Tail regeneration in lizards is the only case of large multi-tissue organ regeneration in amniotes. METHODS: The present Review summarizes numerous immunolocalization and gene-expression studies indicating that after tail amputation in lizards the stump is covered in 7-10 days by the migration of keratinocytes. This allows the accumulation of mesenchymal-fibroblasts underneath the wound epidermis and forms a regenerative blastema and a new tail. RESULTS: During migration keratinocytes transit from a compact epidermis into relatively free keratinocytes in a process of "Epithelial Mesenchymal Transition" (EMT). While EMT has been implicated in carcinogenesis no malignant transformation is observed during these cell movements in the regenerative blastema. Immunolabeling for E-cadherin and snail shows that these proteins are present in the cytoplasm and nuclei of migrating keratinocytes. The basal layer of the wound epithelium of the apical blastema express onco-proteins (wnt2b, egfr, c-myc, fgfs, fgfr, rhov, etc.) and tumor suppressors (p53/63, fat2, ephr, apc, retinoblastoma, arhgap28 etc.). This suggests that their balanced action regulates proliferation of the blastema. CONCLUSIONS: While apical epidermis and mesenchyme are kept under a tight proliferative control, in more proximal regions of the regenerating tail the expression of tumor-suppressors triggers the differentiation of numerous tissues, forming the large myomeres, axial cartilage, simple spinal cord and nerves, new scales, arteries and veins, fat deposits, dermis and other connective tissues. Understanding gene expression patterns of developmental pathways activated during tail regeneration in lizards is useful for cancer research and for future attempts to induce organ regeneration in other amniotes including humans.

Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

A 37†kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.

The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.

Trends towards revealing the genetic architecture of sheep tail patterning: Promising genes and investigatory pathways.

Different sheep breeds have evolved after initial domestication, generating various tail phenotypic patterns. The phenotypic diversity of sheep tail patterns offers ideal materials for comparative analysis of its genetic basis. Evolutionary biologists, animal geneticists, breeders, and producers have been curious to clearly understand the underlying genetics behind phenotypic differences in sheep tails. Understanding the causal gene(s) and mutation(s) underlying these differences will help probe an evolutionary riddle, improve animal production performance, promote animal welfare, and provide lessons that help comprehend human diseases related to fat deposition (i.e., obesity). Historically, fat tails have served as an adaptive response to aridification and climate change. However, the fat tail is currently associated with compromised mating and animal locomotion, fat distribution in the animal body, increased raising costs, reduced consumer preference, and other animal welfare issues such as tail docking. The developing genomic approaches provide unprecedented opportunities to determine causal variants underlying phenotypic differences among populations. In the last decade, researchers have performed several genomic investigations to assess the genomic causality underlying phenotypic variations in sheep tails. Various genes have been suggested with the prominence of several potentially significant causatives, including the BMP2 and PDGFD genes associated with the fat tail phenotype and the TBXT gene linked with the caudal vertebrae number and tail length. Although the potential genes related to sheep tail characteristics have been revealed, the causal variant(s) and mutation(s) of these high-ranking candidate genes are still elusive and need further investigation. The review discusses the potential genes, sheds light on a knowledge gap, and provides possible investigative approaches that could help determine the specific genomic causatives of sheep tail patterns. Besides, characterizing and revealing the genetic determinism of sheep tails will help solve issues compromising sheep breeding and welfare in the future.

Comparative Aspects of Annelid Regeneration: Towards Understanding the Mechanisms of Regeneration.

The question of why animals vary in their ability to regenerate remains one of the most intriguing questions in biology. Annelids are a large and diverse phylum, many members of which are capable of extensive regeneration such as regrowth of a complete head or tail and whole-body regeneration, even from few segments. On the other hand, some representatives of both of the two major annelid clades show very limited tissue regeneration and are completely incapable of segmental regeneration. Here we review experimental and descriptive data on annelid regeneration, obtained at different levels of organization, from data on organs and tissues to intracellular and transcriptomic data. Understanding the variety of the cellular and molecular basis of regeneration in annelids can help one to address important questions about the role of stem/dedifferentiated cells and "molecular morphallaxis" in annelid regeneration as well as the evolution of regeneration in general.

Transcriptomic and proteomic analysis of Hemidactylus frenatus during initial stages of tail regeneration.

Epimorphic regeneration of appendages is a complex and complete phenomenon found in selected animals. Hemidactylus frenatus, house gecko has the remarkable ability to regenerate the tail tissue upon autotomy involving epimorphic regeneration mechanism. This study has identified and evaluated the molecular changes at gene and protein level during the initial stages, i.e., during the wound healing and repair mechanism initiation stage of tail regeneration. Based on next generation transcriptomics and De novo analysis the transcriptome library of the gecko tail tissue was generated. A total of 254 genes and 128 proteins were found to be associated with the regeneration of gecko tail tissue upon amputation at 1, 2 and 5-day post amputation (dpa) against control, 0-dpa through differential transcriptomic and proteomic analysis. To authenticate the expression analysis, 50 genes were further validated involving RTPCR. 327 genes/proteins identified and mapped from the study showed association for Protein kinase A signaling, Telomerase BAG2 signaling, paxillin signaling, VEGF signaling network pathways based on network pathway analysis. This study empanelled list of transcriptome, proteome and the list of genes/proteins associated with the tail regeneration.

Tail regeneration alters the digestive performance of lizards.

Tissue regeneration is a fundamental evolutionary adaptation, which is well known in lizards that can regenerate their entire tail. However, numerous parameters of this process remain poorly understood. Lizard tail serves many functions. Thus, tail autotomy comes with many disadvantages and the need for quick regeneration is imperative. To provide the required energy and materials for caudal tissue building, lizards are expected to undergo a number of physiological and biochemical adjustments. Previous research showed that tail regeneration induces changes in the digestive process. Here, we investigated if and how tail regeneration affects the digestive performance in five wall lizard species deriving from mainland and island sites and questioned whether the association of tail regeneration and digestion is affected by species relationships or environmental features, including predation pressure. We expected that lizards from high predation environments would regenerate their tail faster and modify accordingly their digestive efficiency, prioritizing the digestion of proteins; the main building blocks for tissue repair. Second, we anticipated that the general food shortage on islands would inhibit the process. Our findings showed that all species shifted their digestive efficiency, as predicted. Elongation rate was higher in sites with stronger predation regime and this was also applied to the rate with which protein digestion raised. Gut passage time increases during regeneration so as to improve the nutrient absorbance, but among the islanders, the pace was more intense. The deviations between species should be attributed to the different ecological conditions prevailing on islands rather than to their phylogenetic relationships.

Genetic effects of FTO gene insertion/deletion (InDel) on fat-tail measurements and growth traits in Tong sheep.

Tong sheep is a kind of famous fat-tailed sheep in China, which no longer meets market demands because of the large amount of fat deposition in tail. Fat mass and obesity associated (FTO) gene regulates fatty acid transport and fat metabolism to affect obesity and is also reported to regulate phenotypic traits in healthy animals. To identify the insertion/deletion (InDel) variations of the FTO gene and evaluate their effects on fat-tail measurements and growth traits, 166 healthy individuals from Tong sheep were identified and analyzed. Herein, 10 novel InDel polymorphisms were founded in the Tong sheep FTO gene, which displayed intermediate polymorphism (0.25 < PIC < 0.5) and were in Hardy-Weinberg equilibrium (p > .05). Correlation analysis of 78 Tong sheep phenotypic traits data and InDel polymorphisms showed that eight InDel loci were significantly associated with partial growth traits (p < .05), four InDel loci were significantly correlated with fat-tail measurements (p < .05). In particular, individuals with genotype DD showed better phenotypic traits than individuals with other genotypes at male sheep InDel 5 and InDel 8 loci, which had small tail-fat dimensions while having good growth traits. These results confirmed potential usefulness of FTO gene in marker-assisted selection programs of Tong sheep breeding.

Gene expression in regenerating and scarring tails of lizard evidences three main key genes (wnt2b, egfl6, and arhgap28) activated during the regulated process of tail regeneration.

We have analyzed the expression of key genes orchestrating tail regeneration in lizard under normal and scarring conditions after cauterization. At 1-day post-cauterization (1 dpc), the injured blastema contains degenerating epithelial and mesenchymal cells, numerous mast cells, and immune cells. At 3 and 7 dpc, a stratified wound epidermis is forming while fibrocytes give rise to a scarring connective tissue. Oncogenes such as wnt2b, egfl6, wnt6, and mycn and the tumor suppressor arhgap28 are much more expressed than other oncogenes (hmga2, rhov, fgf8, fgfr4, tert, shh) and tumor suppressors (apcdd1, p63, rb, fat2, bcl11b) in the normal blastema and at 7 dpc. Blastemas at 3 dpc feature the lowest upregulation of most genes, likely derived from damage after cauterization. Immunomodulator genes nfatc4 and lef1 are more expressed at 7 dpc than in normal blastema and 3 dpc suggesting the induction of immune response favoring scarring. Balanced over-expression of oncogenes, tumor suppressor genes, and immune modulator genes determines regulation of cell proliferation (anti-oncogenic), of movement (anti-metastatic), and immunosuppression in the normal blastema. Significant higher expression of oncogenes wnt2b and egfl6 in normal blastema and higher expression of the tumor suppressor arhgap28 in the 7 dpc blastema indicate that they are among the key/master genes that determine the regulated regeneration of the tail.

Ciona embryonic tail bending is driven by asymmetrical notochord contractility and coordinated by epithelial proliferation.

Ventral bending of the embryonic tail within the chorion is an evolutionarily conserved morphogenetic event in both invertebrates and vertebrates. However, the complexity of the anatomical structure of vertebrate embryos makes it difficult to experimentally identify the mechanisms underlying embryonic folding. This study investigated the mechanisms underlying embryonic tail bending in chordates. To further understand the mechanical role of each tissue, we also developed a physical model with experimentally measured parameters to simulate embryonic tail bending. Actomyosin asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis was faster than that in the ventral counterpart during embryonic tail bending. Genetic disruption of actomyosin activity and inhibition of cell proliferation dorsally caused abnormal tail bending, indicating that both asymmetrical actomyosin contractility in the notochord and the discrepancy of epidermis cell proliferation are required for tail bending. In addition, asymmetrical notochord contractility was sufficient to drive embryonic tail bending, whereas differential epidermis proliferation was a passive response to mechanical forces. These findings showed that asymmetrical notochord contractility coordinates with differential epidermis proliferation mechanisms to drive embryonic tail bending.This article has an associated 'The people behind the papers' interview.

Abnormal Vasculature Development in Zebrafish Embryos with Reduced Expression of Pantothenate Kinase 2 Gene.

Mutations in pank2 gene encoding pantothenate kinase 2 determine a pantothenate kinase-associated neurodegeneration, a rare disorder characterized by iron deposition in the globus pallidus. To extend our previous work, we performed microinjections of a new pank2-specific morpholino to zebrafish embryos and thoroughly analyzed vasculature development. Vessels development was severely perturbed in the head, trunk, and tail, where blood accumulation was remarkable and associated with dilation of the posterior cardinal vein. This phenotype was specific as confirmed by p53 expression analysis and injection of the same morpholino in pank2-mutant embryos. We can conclude that pank2 gene is involved in vasculature development in zebrafish embryos. The comprehension of the underlining mechanisms could be of relevance for understanding of pantothenate kinase-associated neurodegeneration.

Studies on tail regeneration and homeotic transformation in anuran tadpoles.

Anuran tadpoles are excellent models for regeneration studies. The tail, an organ essential for swimming for the aquatic tadpole, regenerates completely following injury or amputation. However, treatment with the morphogen, vitamin A or retinoic acid inhibits normal tail regeneration and induces homeotic transformation of tail to limbs. This phenomenon was discovered for the first time in the Indian marbled balloon frog Uperodon systoma in the Developmental Biology laboratory of Utkal University (Odisha, India) in the year 1992. In this paper, we present the results of morphological, histological, biochemical and molecular (immonohistochemistry) investigations of vitamin A induced homeotic transformation in different anuran species. In addition, we discuss the putative role of fibroblast growth factor 1 during spinal cord regeneration in the tadpoles of the Indian tree frog, Polypedates maculatus, an ideal model for regeneration studies in an Indian context.

Distal spinal nerve development and divergence of avian groups.

The avian transition from long to short, distally fused tails during the Mesozoic ushered in the Pygostylian group, which includes modern birds. The avian tail embodies a bipartite anatomy, with the proximal separate caudal vertebrae region, and the distal pygostyle, formed by vertebral fusion. This study investigates developmental features of the two tail domains in different bird groups, and analyzes them in reference to evolutionary origins. We first defined the early developmental boundary between the two tail halves in the chicken, then followed major developmental structures from early embryo to post-hatching stages. Differences between regions were observed in sclerotome anterior/posterior polarity and peripheral nervous system development, and these were consistent in other neognathous birds. However, in the paleognathous emu, the neognathous pattern was not observed, such that spinal nerve development extends through the pygostyle region. Disparities between the neognaths and paleognaths studied were also reflected in the morphology of their pygostyles. The ancestral long-tailed spinal nerve configuration was hypothesized from brown anole and alligator, which unexpectedly more resembles the neognathous birds. This study shows that tail anatomy is not universal in avians, and suggests several possible scenarios regarding bird evolution, including an independent paleognathous long-tailed ancestor.

Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors.

Vertebrate appendage regeneration requires precisely coordinated remodeling of the transcriptional landscape to enable the growth and differentiation of new tissue, a process executed over multiple days and across dozens of cell types. The heterogeneity of tissues and temporally-sensitive fate decisions involved has made it difficult to articulate the gene regulatory programs enabling regeneration of individual cell types. To better understand how a regenerative program is fulfilled by neural progenitor cells (NPCs) of the spinal cord, we analyzed pax6-expressing NPCs isolated from regenerating Xenopus tropicalis tails. By intersecting chromatin accessibility data with single-cell transcriptomics, we find that NPCs place an early priority on neuronal differentiation. Late in regeneration, the priority returns to proliferation. Our analyses identify Pbx3 and Meis1 as critical regulators of tail regeneration and axon organization. Overall, we use transcriptional regulatory dynamics to present a new model for cell fate decisions and their regulators in NPCs during regeneration.

Comprehensive RNA-Seq analysis of notochord-enriched genes induced during Xenopus tropicalis tail resorption.

Anuran metamorphosis is perhaps the most dramatic developmental process regulated by thyroid hormone (TH). One of the unique processes that occur during metamorphosis is the complete resorption of the tail, including the notochord. Interestingly, recent gene knockout studies have shown that of the two known vertebrate TH receptors, TRα and TRß, TRß appears to be critical for notochord regression during tail resorption in Xenopus tropicalis. To determine the mechanisms underlying notochord regression, we carried out a comprehensive gene expression analysis in the notochord during metamorphosis by using RNA-Seq analyses of whole tail at stage 60 before any noticeable tail length reduction, whole tail at stage 63 when the tail length is reduced by about one half, and the rest of the tail at stage 63 after removing the notochord. This allowed us to identify many notochord-enriched, metamorphosis-induced genes at stage 63. Future studies on these genes should help to determine if they are regulated by TRß and play any roles in notochord regression.

Amputation-induced reactive oxygen species signaling is required for axolotl tail regeneration.

BACKGROUND: Among vertebrates, salamanders are unparalleled in their ability to regenerate appendages throughput life. However, little is known about early signals that initiate regeneration in salamanders. RESULTS: Ambystoma mexicanum embryos were administered tail amputations to investigate the timing of reactive oxygen species (ROS) production and the requirement of ROS for regeneration. ROS detected by dihydroethidium increased within minutes of axolotl tail amputation and levels remained high for 24 hr. Pharmacological inhibition of ROS producing enzymes with diphenyleneiodonium chloride (DPI) and VAS2870 reduced ROS levels. Furthermore, DPI treatment reduced cellular proliferation and inhibited tail outgrowth. CONCLUSIONS: The results show that ROS levels increase in response to injury and are required for tail regeneration. These findings suggest that ROS provide instructive, if not initiating cues, for salamander tail regeneration. Developmental Dynamics 248:189-196, 2019. © 2018 Wiley Periodicals, Inc.

Homeotic transformation of tails into limbs in anurans.

Anuran tadpoles can regenerate their tails after amputation. However, they occasionally form ectopic limbs instead of the lost tail part after vitamin A treatment. This is regarded as an example of a homeotic transformation. In this phenomenon, the developmental fate of the tail blastema is apparently altered from that of a tail to that of limbs, indicating a realignment of positional information in the blastema. Morphological observations and analyses of the development of skeletal elements during the process suggest that positional information in the blastema is rewritten from tail to trunk specification under the influence of vitamin A, resulting in limb formation. Despite the extensive information gained from morphological observations, a comprehensive understanding of this phenomenon also requires molecular data. We review previous studies related to anuran homeotic transformation. The findings of these studies provide a basis for evaluating major hypotheses and identifying molecular data that should be prioritized in future studies. Finally, we argue that positional information for the tail blastema changes to that for a part of the trunk, leading to homeotic transformations. To suggest this hypothesis, we present published data that favor the rewriting of positional information.

Hedgehog and Wnt Signaling Pathways Regulate Tail Regeneration.

Urodele amphibians have a tremendous capacity for the regeneration of appendages, including limb and tail, following injury. While studies have focused on the cellular and morphological changes during appendicular regeneration, the signaling mechanisms that govern these cytoarchitectural changes during the regenerative response are unclear. In this study, we describe the essential role of hedgehog (Hh) and Wnt signaling pathways following tail amputation in the newt. Quantitative PCR studies revealed that members of both the Hh and Wnt signaling pathways, including the following: shh, ihh, ptc-1, wnt-3a, ß-catenin, axin2, frizzled (frzd)-1, and frzd-2 transcripts, were induced following injury. Continuous pharmacological-mediated inhibition of Hh signaling resulted in spike-like regenerates with no evidence of tissue patterning, whereas activation of Hh signaling enhanced the regenerative process. Pharmacological-mediated temporal inhibition experiments demonstrated that the Hh-mediated patterning of the regenerating tail occurs early during regeneration and Hh signals are continuously required for proliferation of the blastemal progenitors. BrdU incorporation and PCNA immunohistochemical studies demonstrated that Hh signaling regulates the cellular proliferation of the blastemal cells following amputation. Similarly, Wnt inhibition resulted in perturbed regeneration, whereas its activation promoted tail regeneration. Using an inhibitor-activator strategy, we demonstrated that the Wnt pathway is likely to be upstream of the Hh pathway and together these signaling pathways function in a coordinated manner to facilitate tail regeneration. Mechanistically, the Wnt signaling pathway activated the Hh signaling pathway that included ihh and ptc-1 during the tail regenerative process. Collectively, our results demonstrate the absolute requirement of signaling pathways that are essential in the regulation of tail regeneration.

Avian tail ontogeny, pygostyle formation, and interpretation of juvenile Mesozoic specimens.

The avian tail played a critical role in the evolutionary transition from long- to short-tailed birds, yet its ontogeny in extant birds has largely been ignored. This deficit has hampered efforts to effectively identify intermediate species during the Mesozoic transition to short tails. Here we show that fusion of distal vertebrae into the pygostyle structure does not occur in extant birds until near skeletal maturity, and mineralization of vertebral processes also occurs long after hatching. Evidence for post-hatching pygostyle formation is also demonstrated in two Cretaceous specimens, a juvenile enantiornithine and a subadult basal ornithuromorph. These findings call for reinterpretations of Zhongornis haoae, a Cretaceous bird hypothesized to be an intermediate in the long- to short-tailed bird transition, and of the recently discovered coelurosaur tail embedded in amber. Zhongornis, as a juvenile, may not yet have formed a pygostyle, and the amber-embedded tail specimen is reinterpreted as possibly avian. Analyses of relative pygostyle lengths in extant and Cretaceous birds suggests the number of vertebrae incorporated into the pygostyle has varied considerably, further complicating the interpretation of potential transitional species. In addition, this analysis of avian tail development reveals the generation and loss of intervertebral discs in the pygostyle, vertebral bodies derived from different kinds of cartilage, and alternative modes of caudal vertebral process morphogenesis in birds. These findings demonstrate that avian tail ontogeny is a crucial parameter specifically for the interpretation of Mesozoic specimens, and generally for insights into vertebrae formation.

Cellular Processes of Notochord Formation.

This review covers recent advances in our understanding of the cell biology and morphogenesis of the ascidian notochord. In its development, the ascidian notochord undergoes a rapid series of cellular and morphogenic events that transform a group of 40 loosely packed cells in the neurula embryo into a tubular column with central lumen in the larva. The ascidian notochord has been a subject of intensive research in recent years, and particular focus in this review will be on events associated with the development and function of polarized cell properties, and the mechanism of lumen formation.