Tailbud Embryos Research Articles

Expression and possible functions of a horizontally transferred glycosyl hydrolase gene, GH6-1, in Ciona embryogenesis

BackgroundThe Tunicata or Urochordata is the only animal group with the ability to synthesize cellulose directly and cellulose is a component of the tunic that covers the entire tunicate body. The genome of Ciona intestinalis type A contains a cellulose synthase gene, CesA, that it acquired via an ancient, horizontal gene transfer. CesA is expressed in embryonic epidermal cells and functions in cellulose production. Ciona CesA is composed of both a glycosyltransferase domain, GT2, and a glycosyl hydrolase domain, GH6, which shows a mutation at a key position and seems functionless. Interestingly, the Ciona genome contains a glycosyl hydrolase gene, GH6-1, in which the GH6 domain seems intact. This suggests expression and possible functions of GH6-1 during Ciona embryogenesis. Is GH6-1 expressed during embryogenesis? If so, in what tissues is the gene expressed? Does GH6-1 serve a function? If so, what is it? Answers to these questions may advance our understanding of evolution of this unique animal group.ResultsQuantitative reverse transcription PCR and in situ hybridization revealed that GH6-1 is expressed in epidermis of tailbud embryos and in early swimming larvae, a pattern similar to that of CesA. Expression is downregulated at later stages and becomes undetectable in metamorphosed juveniles. The GH6-1 expression level is higher in the anterior-trunk region and caudal-tip regions of late embryos. Single-cell RNA sequencing analysis of the late tailbud stage showed that cells of three clusters with epidermal identity express GH6-1, and that some of them co-express CesA. TALEN-mediated genome editing was used to generate GH6-1 knockout Ciona larvae. Around half of TALEN-electroporated larvae showed abnormal development of adhesive papillae and altered distribution of surface cellulose. In addition, three-fourths of TALEN-electroporated animals failed to complete larval metamorphosis.ConclusionsThis study showed that tunicate GH6-1, a gene that originated by horizontal gene transfer of a prokaryote gene, is recruited into the ascidian genome, and that it is expressed and functions in epidermal cells of ascidian embryos. Although further research is required, this observation demonstrates that both CesA and GH6-1 are involved in tunicate cellulose metabolism, impacting tunicate morphology and ecology.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4–6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

V-ATPase Regulates Retinal Progenitor Cell Proliferation During Eye Regrowth in Xenopus.

Purpose: The induction of retinal progenitor cell (RPC) proliferation is a strategy that holds promise for alleviating retinal degeneration. However, the mechanisms that can stimulate RPC proliferation during repair remain unclear. Xenopus tailbud embryos successfully regrow functional eyes within 5 days after ablation, and this process requires increased RPC proliferation. This model facilitates identification of mechanisms that can drive in vivo reparative RPC proliferation. This study assesses the role of the essential H+ pump, V-ATPase, in promoting stem cell proliferation. Methods: Pharmacological and molecular loss of function studies were performed to determine the requirement for V-ATPase during embryonic eye regrowth. The resultant eye phenotypes were examined using histology and antibody markers. Misexpression of a yeast H+ pump was used to test whether the requirement for V-ATPase in regrowth is dependent on its H+ pump function. Results: V-ATPase inhibition blocked eye regrowth. Regrowth-incompetent eyes resulting from V-ATPase inhibition contained the normal complement of tissues but were much smaller. V-ATPase inhibition caused a significant reduction in reparative RPC proliferation but did not alter differentiation and patterning. Modulation of V-ATPase activity did not affect apoptosis, a process known to be required for eye regrowth. Finally, increasing H+ pump activity was sufficient to induce regrowth. Conclusions: V-ATPase is required for eye regrowth. These results reveal a key role for V-ATPase in activating regenerative RPC proliferation and expansion during successful eye regrowth.

Searching for the Origin and the Differentiation of Haemocytes before and after Larval Settlement of the Colonial Ascidian Botryllus schlosseri: An Ultrastructural Viewpoint

The colonial ascidian Botryllus schlosseri possesses an innate immunity, which plays fundamental roles in its survival, adaptability, worldwide spread and ecological success. Three lines of differentiation pathways of circulating haemocytes are known to be present in the haemolymph, starting from undifferentiated haemoblasts: (i) the phagocytic line (hyaline amoebocytes and macrophage-like cells), (ii) the cytotoxic line (granular amoebocytes and morula cells) and (iii) the storage cell line (pigment cells and nephrocytes). Many questions remain about their origin, and thus, observations during various stages of development were undertaken in this study. Haemocytes were detected beginning from the early tailbud embryo stage. Haemoblasts were always present and morula cells were the first differentiated haemocytes detected. In both the next stage, just before hatching, and the swimming tadpole larva stage, hyaline amoebocytes and pigment cells were also recognisable. Some morula cells containing active phenoloxidase migrated from the haemolymph into the tunic after having crossed the epidermis, and this behaviour could be related to the preparation of a defensive function for spatial competition. During larval metamorphosis, macrophage-like cells appeared with their phagosomes positive to acid phosphatase activity and containing apoptotic cells from tail tissue degeneration. After metamorphosis, in the filter-feeding oozoid stage, nephrocytes involved in nitrogen catabolism finally appeared. In both the subendostylar sinus and the peripheral blind-sac vessels (ampullae), clusters of haemoblasts were recognisable, some of which showed incipient specialisations, considering the hypothesis of the presence of putative niches of haemolymph stem cells.

Tbx15/18/22 shares a binding site with Tbx6-r.b to maintain expression of a muscle structural gene in ascidian late embryos

The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b.

Evolution of Developmental Programs for the Midline Structures in Chordates: Insights From Gene Regulation in the Floor Plate and Hypochord Homologues of Ciona Embryos.

In vertebrate embryos, dorsal midline tissues, including the notochord, the prechordal plate, and the floor plate, play important roles in patterning of the central nervous system, somites, and endodermal tissues by producing extracellular signaling molecules, such as Sonic hedgehog (Shh). In Ciona, hedgehog.b, one of the two hedgehog genes, is expressed in the floor plate of the embryonic neural tube, while none of the hedgehog genes are expressed in the notochord. We have identified a cis-regulatory region of hedgehog.b that was sufficient to drive a reporter gene expression in the floor plate. The hedgehog.b cis-regulatory region also drove ectopic expression of the reporter gene in the endodermal strand, suggesting that the floor plate and the endodermal strand share a part of their gene regulatory programs. The endodermal strand occupies the same topographic position of the embryo as does the vertebrate hypochord, which consists of a row of single cells lined up immediately ventral to the notochord. The hypochord shares expression of several genes with the floor plate, including Shh and FoxA, and play a role in dorsal aorta development. Whole-embryo single-cell transcriptome analysis identified a number of genes specifically expressed in both the floor plate and the endodermal strand in Ciona tailbud embryos. A Ciona FoxA ortholog FoxA.a is shown to be a candidate transcriptional activator for the midline gene battery. The present findings suggest an ancient evolutionary origin of a common developmental program for the midline structures in Olfactores.

Dynein-Mediated Regional Cell Division Reorientation Shapes a Tailbud Embryo.

SummaryRegulation of cell division orientation controls the spatial distribution of cells during development and is essential for one-directional tissue transformation, such as elongation. However, little is known about whether it plays a role in other types of tissue morphogenesis. Using an ascidian Halocynthia roretzi, we found that differently oriented cell divisions in the epidermis of the future trunk (anterior) and tail (posterior) regions create an hourglass-like epithelial bending between the two regions to shape the tailbud embryo. Our results show that posterior epidermal cells are polarized with dynein protein anteriorly localized, undergo dynein-dependent spindle rotation, and divide along the anteroposterior axis. This cell division facilitates constriction around the embryo's circumference only in the posterior region and epithelial bending formation. Our findings, therefore, provide an important insight into the role of oriented cell division in tissue morphogenesis.

Different strategies for tissue scaling in dwarf tailbud embryos revealed by single-cell analysis

The tailbud stage is part of the organogenesis period—an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 ​μm (correspond to ​> ​40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.

Using the Xenopus Developmental Eye Regrowth System to Distinguish the Role of Developmental Versus Regenerative Mechanisms.

A longstanding challenge in regeneration biology is to understand the role of developmental mechanisms in restoring lost or damaged tissues and organs. As these body structures were built during embryogenesis, it is not surprising that a number of developmental mechanisms are also active during regeneration. However, it remains unclear whether developmental mechanisms act similarly or differently during regeneration as compared to development. Since regeneration is studied in the context of mature, differentiated tissues, it is difficult to evaluate comparative studies with developmental processes due to the latter’s highly proliferative environment. We have taken a more direct approach to study regeneration in a developmental context (regrowth). Xenopus laevis, the African clawed frog, is a well-established model for both embryology and regeneration studies, especially for the eye. Xenopus eye development is well-defined. Xenopus is also an established model for retinal and lens regeneration studies. Previously, we demonstrated that Xenopus tailbud embryo can successfully regrow a functional eye that is morphologically indistinguishable from an age-matched control eye. In this study, we assessed the temporal regulation of retinal differentiation and patterning restoration during eye regrowth. Our findings showed that during regrowth, cellular patterning and retinal layer formation was delayed by approximately 1 day but was restored by 3 days when compared to eye development. An assessment of the differentiation of ganglion cells, photoreceptor cells, and Müller glia indicated that the retinal birth order generated during regrowth was consistent with that observed for eye development. Thus, retina differentiation and patterning during regrowth is similar to endogenous eye development. We used this eye regrowth model to assess the role of known mechanisms in development versus regrowth. Loss-of-function studies showed that Pax6 was required for both eye development and regrowth whereas apoptosis was only required for regrowth. Together, these results revealed that the mechanisms required for both development and regrowth can be distinguished from regrowth-specific ones. Our study highlights this developmental model of eye regrowth as a robust platform to systematically and efficiently define the molecular mechanisms that are required for regeneration versus development.

Axis elongation during Xenopus tail-bud stage is regulated by GABA expressed in the anterior-to-mid neural tube.

The receptors of gamma-aminobutyric acid (GABA), which is a well-known neurotransmitter, are expressed in the anterior-to-mid neural tube at an early stage of Xenopus development, but there has been no report on the role of GABA in the presumptive central nervous system. Therefore, we tried to reveal the function of GABA for Xenopus early embryogenesis. We first confirmed that the region expressing a gene encoding glutamate decarboxylase 1 (gad1), which is an enzyme that catalyzes the decarboxylation of L-glutamate to GABA, overlapped with that of several genes encoding GABA receptors (gabr) in the neural tube. Metabolome analysis of culture medium of dorsal tail-bud explants containing the neural tube region of tail-bud stage embryos also revealed that GABA was expressed at this stage. Then, we examined the treatment of pentylenetetrazole (PTZ) and picrotoxin (PTX), which are known as inhibitors of GABA receptors (GABA-R), on the early stages of Xenopus embryogenesis, and found that axis elongation in the tail-bud was inhibited by both treatments, and these phenotypic effects were rescued by co-treatment with GABA. Moreover, our spatial- and temporal-specific inhibitor treatments revealed that the gabr- and gad1-overlapped region, which presents at the anterior-to-mid neural tube during the tail-bud stages, was much more sensitive to PTZ and thus caused severe inhibition of axis elongation. Taken together, our results indicate that the small ligand molecule GABA functions as a regulator to induce the axis elongation event in the tail-bud during early embryogenesis via direct stimulation of the neural tube and indirect stimulation of the surrounding area.

Development of a multi-point indentation tester on the section of Xenopus laevis tailbud embryo

Development of a multi-point indentation tester on the section of Xenopus laevis tailbud embryo

Stress-generating tissue deformations in Xenopus embryos: Long-range gradients and local cell displacements

BackgroundAlthough the role of endogenous mechanical stresses in regulating morphogenetic movements and cell differentiation is now well established, many aspects of mechanical stress generation and transmission in developing embryos remain unclear and require quantitative studies. ResultsBy measuring stress-bearing linear deformations (caused by differences in cell movement rates) in the outer cell layer of blastula – early tail-bud Xenopus embryos, we revealed a set of long-term tension-generating gradients of cell movement rates, modulated by short-term cell-cell displacements much increasing the rates of local deformations. Experimental relaxation of tensions distorted the gradients but preserved and even enhanced local cell-cell displacements. During development, an incoherent mode of cell behavior, characterized by extensive cell-cell displacements and poorly correlated cell trajectories, was exchanged for a more coherent regime with the opposite characteristics. In particular, cell shifts became more synchronous and acquired a periodicity of several dozen minutes. ConclusionsMorphogenetic movements in Xenopus embryos are mediated by mechanically stressed dynamic structures of two different levels: extended gradients and short-term cell-cell displacements. As development proceeds, the latter component decreases and cell trajectories become more correlated. In particular, they acquire common periodicities, making morphogenesis more coherent.

Cynoglossus semilaevis Rspo3 Regulates Embryo Development by Inhibiting the Wnt/β-Catenin Signaling Pathway.

Cynoglossus semilaevis is an important economic fish species and has long been cultivated in China. Since the completion of its genome and transcriptome sequencing, genes relating to C. semilaevis development have been extensively studied. R-spondin 3 (Rspo3) is a member of the R-spondin family. It plays an important role in biological processes such as vascular development and oncogenesis. In this study, we cloned and characterized the expression patterns and functions of C. semilaevis Rspo3. Initial structural and phylogenetic analyses revealed a unique FU3 domain that exists only in ray-finned fish RSPO3. Subsequent embryonic expression profile analysis showed elevating expression of Rspo3 from gastrulation to the formation of the eye lens, while, in tail bud embryos, Rspo3 expression was significantly high in the diencephalon and mesencephalon. The overexpression of C. semilaevis Rspo3 in Danio rerio embryos resulted in a shortened rostral–caudal axis, edema of the pericardial cavity, stubby yolk extension, and ecchymosis. Vascular anomalies were also observed, which is consistent with Rspo3 role in vascular development. Drug treatment and a dual-luciferase reporter assay confirmed the inhibitory role of C. semilaevis Rspo3 in D. rerio Wnt/β-catenin signaling pathway. We further concluded that the FU2, FU3, and TSP1 domains regulate the maternal Wnt/β-catenin signaling pathway, while the FU1 domain regulates the zygotic Wnt/β-catenin signaling pathway. This study enriches Rspo3 research in non-model animals and serves as the basis for further research into the interactions between Rspo and the Wnt/β-catenin signaling pathway.

Effect of vitrification solutions on survival rate of cryopreserved Epinephelus moara embryos

Embryo cryopreservation is important for long-term preservation of germplasm and assisted reproduction. However, it is still very difficult to obtain viable embryos from cryopreserved fish embryos. In this study, embryos of Epinephelus moara were used to investigate the effects of various cryopreservation methods. Embryos in stages 10 pairs somite (10S), 18 pairs somite (18S), 22 pairs somite (22S), tail-bud (TB), embryo twitching (ET) and pre-hatch (PH) were treated with five-step equilibrium penetration in 40% PMG3T vitrification solution, which contained 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Trehalose. We found that 18S, 22S, TB and ET stage embryos had higher survival rates and were more tolerant to the vitrification solution. Five-step equilibrium treatments on the embryos at the tail-bud stage were performed using two vitrification solutions: 40% PMG3T and 40% PMG3S, which consisted of 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Sucrose. The embryonic survival rate under PMG3S treatment (63.36%) was significantly higher than PMG3T treatment (43.93%) (P < 0.05). PMG3S and PMG3T with concentrations of 35%, 40% and 45% were tested on tail-bud stage embryos. Higher concentration of the vitrification solution led to significantly lower embryonic survival rate (P < 0.05). The survival rate was 36.79–72.05% in PMG3S, and 37.11–55.18% in PMG3T, and there were non-significant differences in embryonic development and malformation rates among the groups treated with different concentrations. The embryonic normal development rates in PMG3S and PMG3T were 21.27% and 11.04%, and the malformation rates were 36.13% and 31.04%, respectively. The optimum treatment condition was 40 min using 40% PMG3S on embryos at the tail-bud stage. Both PMG3S and PMG3T were used for cryopreserving embryos at 16 pairs somite, tail-bud and ET stage in liquid nitrogen, where we obtained 190 surviving embryos, and 44 fishes underwent normal development and hatched. The survival rate of cryopreserved embryos was 5.15%, the normal development rate was 1.31%, and the malformation rate was 3.66%. We found that PMG3S and PMG3T were effective for cryopreservation of Epinephelus moara embryos. The results provide a foundation for further explorations of fish embryo cryopreservation techniques.

Identification and characterization of microRNAs involved in ascidian larval metamorphosis

BackgroundMetamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level. Large numbers of miRNAs have been demonstrated to be involved in many developmental and metamorphic processes. However, the identification of miRNAs in ascidian larval metamorphosis has not yet been investigated.ResultsTotally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway.ConclusionsTotally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.

Musashi and Plasticity of Xenopus and Axolotl Spinal Cord Ependymal Cells.

The differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog Xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander Ambystoma mexicanum, the Axolotl. Ependymal cells are epithelial in intact spinal cord of all vertebrates. After transection, body region ependymal epithelium in both Xenopus and the Axolotl disorganizes for regenerative outgrowth (gap replacement). Injury-reactive ependymal cells serve as a stem/progenitor cell population in regeneration and reconstruct the central canal. Expression patterns of mRNA and protein for the stem/progenitor cell-maintenance Notch signaling pathway mRNA-binding protein Musashi (msi) change with life stage and regeneration competence. Msi-1 is missing (immunohistochemistry), or at very low levels (polymerase chain reaction, PCR), in both intact regeneration-competent adult Axolotl cord and intact non-regeneration-competent Xenopus tadpole (Nieuwkoop and Faber stage 62+, NF 62+). The critical correlation for successful regeneration is msi-1 expression/upregulation after injury in the ependymal outgrowth and stump-region ependymal cells. msi-1 and msi-2 isoforms were cloned for the Axolotl as well as previously unknown isoforms of Xenopus msi-2. Intact Xenopus spinal cord ependymal cells show a loss of msi-1 expression between regeneration-competent (NF 50–53) and non-regenerating stages (NF 62+) and in post-metamorphosis froglets, while msi-2 displays a lower molecular weight isoform in non-regenerating cord. In the Axolotl, embryos and juveniles maintain Msi-1 expression in the intact cord. In the adult Axolotl, Msi-1 is absent, but upregulates after injury. Msi-2 levels are more variable among Axolotl life stages: rising between late tailbud embryos and juveniles and decreasing in adult cord. Cultures of regeneration-competent Xenopus tadpole cord and injury-responsive adult Axolotl cord ependymal cells showed an identical growth factor response. Epidermal growth factor (EGF) maintains mesenchymal outgrowth in vitro, the cells are proliferative and maintain msi-1 expression. Non-regeneration competent Xenopus ependymal cells, NF 62+, failed to attach or grow well in EGF+ medium. Ependymal Msi-1 expression in vivo and in vitro is a strong indicator of regeneration competence in the amphibian spinal cord.

A model for investigating developmental eye repair in Xenopus laevis

Vertebrate eye development is complex and requires early interactions between neuroectoderm and surface ectoderm during embryogenesis. In the African clawed frog, Xenopus laevis, individual eye tissues such as the retina and lens can undergo regeneration. However, it has been reported that removal of either the specified eye field at the neurula stage or the eye during tadpole stage does not induce replacement. Here we describe a model for investigating Xenopus developmental eye repair. We found that tailbud embryos can readily regrow eyes after surgical removal of over 83% of the specified eye and lens tissues. The regrown eye reached a comparable size to the contralateral control by 5 days and overall animal development was normal. It contained the expected complement of eye cell types (including the pigmented epithelium, retina and lens), and is connected to the brain. Our data also demonstrate that apoptosis, an early mechanism that regulates appendage regeneration, is also required for eye regrowth. Treatment with apoptosis inhibitors (M50054 or NS3694) blocked eye regrowth by inhibiting caspase activation. Together, our findings indicate that frog embryos can undergo successful eye repair after considerable tissue loss and reveals a required role for apoptosis in this process. Furthermore, this Xenopus model allows for rapid comparisons of productive eye repair and developmental pathways. It can also facilitate the molecular dissection of signaling mechanisms necessary for initiating repair.

Measurement of surface topography and stiffness distribution on cross-section of Xenopus laevis tailbud for estimation of mechanical environment in embryo.

The stress distribution inside a Xenopus laevis tailbud embryo was estimated to examine the cause of the straightening and elongation. The embryos were cut in the middle, yielding a cross-section perpendicular to the body axis. The section was not flat, owing to the residual stress relief. The stress needed to restore the flatness corresponded to the stress inside the embryo and was calculated using the surface topography and Young's-moduli in the section. We found the areas of the notochord (Nc), neural tube (NT), and abdominal tissue (AT) bulged in the cross-section, which revealed that compressive forces acted in these tissues. The moduli of the Nc, NT, and AT were in the order of several thousand, hundred, and tens of pascals, respectively. In the Nc, the compressive force was largest and increased with the development, suggesting Nc playing a central role in the elongation. The bending moment generated by the AT was 10 times higher than that by the Nc in the early stages of the tailbud formation, and the two were similar in the latter stages, suggesting that the compressive force in the AT was the major cause of the straightening during the early stage. The straightening and elongation could be orchestrated by changes in the compressive forces acting on the Nc, NT, and AT over time. For the sake of simplicity, we calculated the compressive force only and neglected the tensile force. Thus, it should be noted that the amount of the compressive force was somewhat overestimated.

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16–22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16–22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16–22 somite stage embryos survived when cooled at −25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at −25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at −140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (−196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.

Acute phase response in amputated tail stumps and neural tissue-preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene.

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the 'regeneration' period, as well as in the 'refractory' period, when tadpoles lose their tail regenerative ability. Within 10h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48h after amputation, xNP1 expression was sustained at the spinal cord of tails in the 'regeneration' period tadpoles, but not in the 'refractory' period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.