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.

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.

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.

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.

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.

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.

Yorkie is required to restrict the injury responses in planarians.

Regeneration requires the precise integration of cues that initiate proliferation, direct differentiation, and ultimately re-pattern tissues to the proper size and scale. Yet how these processes are integrated with wounding responses remains relatively unknown. The freshwater planarian, Schmidtea mediterranea, is an ideal model to study the stereotyped proliferative and transcriptional responses to injury due to its high capacity for regeneration. Here, we characterize the effector of the Hippo signalling cascade, yorkie, during planarian regeneration and its role in restricting early injury responses. In yki(RNAi) regenerating animals, wound responses are hyper-activated such that both stem cell proliferation and the transcriptional wound response program are heighted and prolonged. Using this observation, we also uncovered novel wound-induced genes by RNAseq that were de-repressed in yki(RNAi) animals compared with controls. Additionally, we show that yki(RNAi) animals have expanded epidermal and muscle cell populations, which we hypothesize are the increased sources of wound-induced genes. Finally, we show that in yki(RNAi) animals, the sensing of the size of an injury by eyes or the pharynx is not appropriate, and the brain, gut, and midline cannot remodel or scale correctly to the size of the regenerating fragment. Taken together, our results suggest that yki functions as a key molecule that can integrate multiple aspects of the injury response including proliferation, apoptosis, injury-induced transcription, and patterning.

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.

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.

Reactivating head regrowth in a regeneration-deficient planarian species.

Species capable of regenerating lost body parts occur throughout the animal kingdom, yet close relatives are often regeneration incompetent. Why in the face of 'survival of the fittest' some animals regenerate but others do not remains a fascinating question. Planarian flatworms are well known and studied for their ability to regenerate from minute tissue pieces, yet species with limited regeneration abilities have been described even amongst planarians. Here we report the characterization of the regeneration defect in the planarian Dendrocoelum lacteum and its successful rescue. Tissue fragments cut from the posterior half of the body of this species are unable to regenerate a head and ultimately die. We find that this defect originates during the early stages of head specification, which require inhibition of canonical Wnt signalling in other planarian species. Notably, RNA interference (RNAi)-mediated knockdown of Dlac-ß-catenin-1, the Wnt signal transducer, restored the regeneration of fully functional heads on tail pieces, rescuing D. lacteum's regeneration defect. Our results demonstrate the utility of comparative studies towards the reactivation of regenerative abilities in regeneration-deficient animals. Furthermore, the availability of D. lacteum as a regeneration-impaired planarian model species provides a first step towards elucidating the evolutionary mechanisms that ultimately determine why some animals regenerate and others do not.

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.

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.

Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae.

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 µm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H2O2) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H2O2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H2O2 degradation rates from intravital H2O2-biosensor imaging data. Our results indicate that at high tissue H2O2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H2O2 degradation stalls or ceases. Although the wound H2O2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 µm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H2O2 diffusion.

Transcriptional dynamics of tail regeneration in Xenopus tropicalis.

In contrast to humans, many amphibians are able to rapidly and completely regenerate complex tissues, including entire appendages. Following tail amputation, Xenopus tropicalis tadpoles quickly regenerate muscle, spinal cord, cartilage, vasculature and skin, all properly patterned in three dimensions. To better understand the molecular basis of this regenerative competence, we performed a transcriptional analysis of the first 72 h of tail regeneration using RNA-Seq. Our analysis refines the windows during which many key biological signaling processes act in regeneration, including embryonic patterning signals, immune responses, bioelectrical signaling and apoptosis. Our work provides a deep database for researchers interested in appendage regeneration, and points to new avenues for further study.

Tail regeneration after autotomy revives survival: a case from a long-term monitored lizard population under avian predation.

Caudal autotomy in lizards has intrigued scientists for more than 100 years. Because of the relative lack of literature under natural conditions, the complicated association among field autotomy rate, real predation pressure, the long-term cost of tail loss, and the benefit of regeneration remains equivocal. In this study, we conducted a 7-year capture-mark-recapture (CMR) programme with a wild population of a sexually dichromatic lizard, Takydromus viridipunctatus We used autotomy indexes and a contemporary bird census mega-dataset of four predatory birds as predictors to examine the association between tail loss and predation pressure. We further estimated the survival cost of tail loss and alleviation by regeneration under natural conditions through CMR modelling. We found that large and small avian predators affect lizard survival through the following two routes: the larger-sized cattle egret causes direct mortality while the smaller shrikes and kestrels are the major causes of autotomy. Following autotomy, the survival rate of tailless individuals over the next month was significantly lower than that of tailed individuals, especially males during the breeding season, which showed a decline of greater than 30%. This sex-related difference further demonstrated the importance of reproductive costs for males in this sexually dichromatic species. However, the risk of mortality returned to baseline after the tails were fully grown. This study indicates the benefit of tail regeneration under natural conditions, which increases our understanding of the cost-benefit dynamics of caudal autotomy and further explains the maintenance of this trait as an evolutionarily beneficial adaption to long-term predator-prey interactions.

zic-1 Expression in Planarian neoblasts after injury controls anterior pole regeneration.

Mechanisms that enable injury responses to prompt regenerative outgrowth are not well understood. Planarians can regenerate essentially any tissue removed by wounding, even after decapitation, due to robust regulation of adult pluripotent stem cells of the neoblast population. Formation of pole signaling centers involving Wnt inhibitors or Wnt ligands promotes head or tail regeneration, respectively, and this process requires the use of neoblasts early after injury. We used expression profiling of purified neoblasts to identify factors needed for anterior pole formation. Using this approach, we identified zic-1, a Zic-family transcription factor, as transcriptionally activated in a subpopulation of neoblasts near wound sites early in head regeneration. As head regeneration proceeds, the Wnt inhibitor notum becomes expressed in the newly forming anterior pole in zic-1-expressing cells descended from neoblasts. Inhibition of zic-1 by RNAi resulted in a failure to express notum at the anterior pole and to regenerate a head, but did not affect tail regeneration. Both injury and canonical Wnt signaling inhibition are required for zic-1 expression, and double-RNAi experiments suggest zic-1 inhibits Wnt signaling to allow head regeneration. Analysis of neoblast fate determinants revealed that zic-1 controls specification of notum-expressing cells from foxD-expressing neoblasts to form the anterior pole, which organizes subsequent outgrowth. Specialized differentiation programs may in general underlie injury-dependent formation of tissue organizing centers used for regenerative outgrowth.

An energetic perspective on tissue regeneration: The costs of tail autotomy in growing geckos.

Tail autotomy is a crucial antipredatory lizard response, which greatly increases individual survival, but at the same time also compromises locomotor performance, sacrifices energy stores and induces a higher burden due to the ensuing response of regenerating the lost body part. The potential costs of tail autotomy include shifts in energy allocation and metabolic rates, especially in juveniles, which invest their energy primarily in somatic growth. We compared the metabolic rates and followed the growth of juvenile males with and without regenerating tails in the Madagascar ground gecko (Paroedura picta), a nocturnal ground-dwelling lizard. Geckos with intact tails and those that were regrowing them grew in snout-vent-length at similar rates for 22weeks after autotomy. Tail regeneration had a negligible influence on body mass-corrected metabolic rate measured at regular intervals throughout the regenerative process. We conclude that fast-growing juveniles under the conditions of unrestricted food can largely compensate for costs of tail loss and regeneration in their somatic growth without a significant impact on the total individual body mass-corrected metabolic rate.

Time course for tail regression during metamorphosis of the ascidian Ciona intestinalis.

In most ascidians, the tadpole-like swimming larvae dramatically change their body-plans during metamorphosis and develop into sessile adults. The mechanisms of ascidian metamorphosis have been researched and debated for many years. Until now information on the detailed time course of the initiation and completion of each metamorphic event has not been described. One dramatic and important event in ascidian metamorphosis is tail regression, in which ascidian larvae lose their tails to adjust themselves to sessile life. In the present study, we measured the time associated with tail regression in the ascidian Ciona intestinalis. Larvae are thought to acquire competency for each metamorphic event in certain developmental periods. We show that the timing with which the competence for tail regression is acquired is determined by the time since hatching, and this timing is not affected by the timing of post-hatching events such as adhesion. Because larvae need to adhere to substrates with their papillae to induce tail regression, we measured the duration for which larvae need to remain adhered in order to initiate tail regression and the time needed for the tail to regress. Larvae acquire the ability to adhere to substrates before they acquire tail regression competence. We found that when larvae adhered before they acquired tail regression competence, they were able to remember the experience of adhesion until they acquired the ability to undergo tail regression. The time course of the events associated with tail regression provides a valuable reference, upon which the cellular and molecular mechanisms of ascidian metamorphosis can be elucidated.

C. elegans PVF-1 inhibits permissive UNC-40 signalling through CED-10 GTPase to position the male ray 1 sensillum.

Graded distributions of netrin and semaphorin guidance cues convey instructive polarity information to migrating cells and growth cones, but also have permissive (i.e. non-polarity determining) functions in mammalian development and repair. The permissive functions of these cues are largely uncharacterised at a molecular level. We found previously that UNC-6 (netrin) signals permissively through UNC-40 (DCC) and UNC-5 receptors to prevent anterior displacement of the ray 1 sensillum in the C. elegans male tail. UNC-6/UNC-40 signalling functions in parallel with SMP-1 (semaporin 1)/PLX-1 (plexin) signalling to prevent this defect. Here, we report that a deletion allele of pvf-1, which encodes a VEGF-related protein, causes no ray 1 defects, but enhances ray 1 defects of a plx-1 mutant, and unexpectedly also suppresses unc-6(ev400)-null mutant ray 1 defects. These mutant ray 1 inductive and suppressive effects are mimicked by the ability of unc-40(+) and ced-10(gain-of-function) multi-copy transgene arrays to induce ray 1 defects or suppress unc-6 mutant ray 1 defects, depending on their dosage, suggesting the pvf-1 mutation causes UNC-40 overactivity that interferes with signalling but is partially sensitive to UNC-6. Additional data suggest PVF-1 functions through four VEGF receptor-related proteins and inhibits only CED-10 (a GTPase), but not MIG-2-dependent UNC-40 activity, even though UNC-40 functions through both GTPases to position ray 1. pvf-1 and receptor mutant ray 1 defects are rescued by transgenes expressing mouse VEGF164 and human VEGF receptors, respectively. These data report the first case of VEGF-induced inhibition of the netrin signalling and a molecular conservation of VEGF function from worms to humans.