Preimplantation genetic testing using comprehensive genomic copy number analysis is beneficial for balanced translocation carriers.

Balanced chromosomal translocation is one of chromosomal variations. Carriers of balanced chromosomal translocations have an increased risk of spontaneous miscarriage. To avoid the risk, preimplantation genetic testing (PGT) using comprehensive genomic copy number analysis has been developed. This study aimed to verify whether and how embryos from couples in which one partner is a balanced translocation carrier have a higher ratio of chromosomal abnormalities. A total of 894 biopsied trophectoderms (TEs) were obtained from 130 couples in which one partner was a balanced translocation carrier (Robertsonian translocation, reciprocal translocation, or intrachromosomal inversion) and grouped as PGT-SR. Conversely, 3269 TEs from 697 couples who experienced recurrent implantation failure or recurrent pregnancy loss were included in the PGT-A group. The transferable blastocyst ratio was significantly lower in the PGT-SR group, even when bias related to the sample number and patient age was corrected. Subgroup analysis of the PGT-SR group revealed that the transferable blastocyst ratio was higher in the Robertsonian translocation group. Because the PGT-SR group had a higher proportion of untransferable embryos than the PGT-A group, PGT using comprehensive genomic copy number analysis was more beneficial for balanced translocation carriers than for infertility patients without chromosomal translocations. The frequencies of de novo aneuploidies were further analyzed, and the frequency in the PGT-SR group was lower than that in the PGT-A group. Therefore, we could not confirm the existence of interchromosomal effects in this study.

Preimplantation genetic testing for aneuploidy and chromosomal structural rearrangement: A summary of a nationwide study by the Japan Society of Obstetrics and Gynecology.

Purpose: The Japan Society of Obstetrics and Gynecology conducted a nationwide clinical study to evaluate the pregnancy outcomes of preimplantation genetic testing for aneuploidy or chromosomal structural rearrangement (PGT-A/SR). Methods: Patients that had experienced recurrent implantation failure, recurrent pregnancy loss, or chromosomal structural rearrangement were recruited from 200 fertility centers in Japan. For patients in whom one or more blastocysts were classified as euploid or euploid with suspected mosaicism, a frozen-thawed single embryo transfer (ET) was performed. Results: A total of 10 602 cycles, maternal age 28-50 years, were enrolled in this study. 42 529 blastocysts were biopsied, and 25.5%, 11.7%, and 61.7% of embryos exhibited euploidy, mosaicism, and aneuploidy, respectively. At least one euploid blastocyst was obtained in 38.3% of egg retrieval cycles with embryo biopsy. A total of 6080 ETs were carried out, and the clinical pregnancy rate per ET, ongoing pregnancy rate per ET, and miscarriage rate per pregnancy were 68.8%, 56.3%, and 10.4%, respectively. The rates of clinical pregnancy and miscarriage remained relatively constant across all maternal ages. Conclusions: Preimplantation genetic testing for aneuploidy or chromosomal structural rearrangement may improve the pregnancy rate per ET and reduce the miscarriage rate per pregnancy, especially in patients of advanced maternal age.

Broad applicability of a streamlined ethyl cinnamate-based clearing procedure.

Turbidity and opaqueness are inherent properties of tissues that limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here, we describe 2Eci (2nd generation ethyl cinnamate-based clearing), which can be used to clear a wide range of tissues in several species, including human organoids, Drosophila melanogaster, zebrafish, axolotl and Xenopus laevis, in as little as 1-5 days, while preserving a broad range of fluorescent proteins, including GFP, mCherry, Brainbow and Alexa-conjugated fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method opens up tissue clearing to a much broader group of researchers due to its ease of use, the non-toxic nature of ethyl cinnamate and broad applicability.

Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

The posterior neural plate in axolotl gives rise to neural tube or turns anteriorly to form somites of the tail and posterior trunk.

Classical grafting experiments in the Mexican axolotl had shown that the posterior neural plate of the neurula is no specified neuroectoderm but gives rise to somites of the tail and posterior trunk. The bipotentiality of this region with neuromesodermal progenitor cell populations was revealed more recently also in zebrafish, chick, and mouse. We reinvestigated the potency of the posterior plate in axolotl using grafts from transgenic embryos, immunohistochemistry, and in situ hybridization. The posterior plate is brachyury-positive except for its more anterior parts which express sox2. Between anterior and posterior regions of the posterior plate a small domain with sox2+ and bra+ cells exists. Lineage analysis of grafted GFP-labeled posterior plate tissue revealed that posterior GFP+ cells move from dorsal to ventral, form the posterior wall, turn anterior bilaterally, and join the gastrulated paraxial presomitic mesoderm. More anterior sox2+/GFP+ cells, however, are integrated into the developing spinal cord. Tail notochord is formed from axial mesoderm involuted already during gastrulation. Thus the posterior neural plate is a postgastrula source of paraxial mesoderm, which performs an anterior turn, a novel morphogenetic movement. More anterior plate cells, in contrast, do not turn anteriorly but become specified to form tail spinal cord.

Development-related changes in the expression of the ovarian Kiss1 and Kiss1r genes and their sensitivity to human chorionic gonadotropin in prepubertal female rats.

Kisspeptin, which is encoded by the Kiss1 gene, and its receptor, the G protein-coupled receptor 54 (Kiss1r), play important roles in the regulation of reproductive functions in mammals. Several studies have shown that the Kiss1 and Kiss1r genes are expressed in the rat, primate, and human ovaries, and that the ovarian kisspeptin system plays a pivotal role in ovulation at the proestrous stage in adulthood. The purpose of this study was to evaluate development-related changes in the expression of ovarian Kiss1 and Kiss1r genes and in kisspeptin levels, and to identify the regulatory factors for these genes during the prepubertal period. The serum kisspeptin level was also measured to examine whether ovarian kisspeptin affects serum kisspeptin levels. Variations in the ovarian Kiss1 and Kiss1r mRNA levels were observed during the prepubertal period in female rats, with levels peaking around postnatal days 20 and 15, respectively. Nevertheless, the ovarian kisspeptin content per total protein level was stably maintained. Serum kisspeptin levels at postnatal days 30 and 35 were higher than those at earlier postnatal days. The pattern of the ovarian Kiss1 mRNA levels was similar to that of the serum luteinizing hormone (LH) levels, and the ovarian Kiss1 mRNA level increased after injection with human chorionic gonadotropin (HCG) on postnatal day 20, but not on postnatal days 10 and 30. These data indicate that ovarian Kiss1 and Kiss1r mRNA levels are increased on postnatal days 20 and 15, respectively, and that changes in the serum LH level and the ovarian sensitivity to LH may be involved in the alteration of ovarian Kiss1 mRNA levels.

Expression of cytokine-induced neutrophil chemoattractant suppresses tumor necrosis factor alpha expression and thereby prevents the follicles from undergoing atresia and apoptosis.

Aim: Cytokine-induced neutrophil chemoattractant (CINC/gro) is a CXC family chemokine, similar to interleukin-8 in rats, and is one of the factors that regulates ovulation. However, the mechanism that regulates atresia of the ovaries postovulation is not clearly defined. Methods: Whether antibody-blocking of CINC/gro can alter the number of ovulated oocytes and modulate neutrophil infiltration was investigated. The effect of the antibody on the level of inflammatory cytokine production and follicular atresia was examined. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and via analysis of the messenger RNA expression of Bcl-2 and Bcl2-associated X (Bax). Results: The anti-CINC/gro antibody treatment decreased the number of ovulated oocytes. The messenger RNA levels of cyclooxygenase-2 and interleukin-1 beta were decreased by the antibody treatment, whereas that of tumor necrosis factor (TNF) alpha was increased. The TUNEL analysis revealed a larger number of apoptotic cells in the antibody group, compared with those in the control group, as well as a significant increase in the Bax/Bcl-2 ratio 24 hours after human chorionic gonadotropin administration. Conclusion: These findings suggest that ovulation is accelerated by neutrophil infiltration into the theca layer. The CINC/gro appears to synergize with interleukin-1 beta for ovulation. By contrast, the data suggest that CINC/gro expression suppresses TNF alpha expression and that CINC/gro expression therefore prevents the follicles from undergoing atresia and apoptosis.

Intra-follicular kisspeptin levels are related to oocyte maturation and gonadal hormones in patients who are undergoing assisted reproductive technology.

Purpose: To assess the kisspeptin concentrations in follicular fluid and their relationship with clinical outcomes during assisted reproductive technology. Methods: Thirty-nine patients who were aged 24-40 years and underwent oocyte retrieval for in vitro fertilization/intracytoplasmic sperm injection participated in this study. In 65 follicular fluid samples that had been obtained from 30 patients and their blood samples, the kisspeptin levels were measured in order to investigate the correlations with their gonadal hormone levels. Venous blood samples were collected from 14 patients to investigate their plasma kisspeptin levels across different phases of assisted reproductive technology. Results: The follicular fluid kisspeptin level was significantly higher than that of the plasma level and was positively associated with the follicular fluid estradiol concentration and with the serum estradiol and number of mature oocytes. In the plasma, the maximum concentration of kisspeptin was observed on the day of ovum pick-up and on the day of embryo transfer during ovarian stimulation for assisted reproductive technology. Conclusion: Kisspeptin was present in the follicular fluid and the plasma kisspeptin concentration was affected by ovarian stimulation. Kisspeptin appears to affect oocyte maturation and ovulation.

Axolotls with an under- or oversupply of neural crest can regulate the sizes of their dorsal root ganglia to normal levels.

How animals adjust the size of their organs is a fundamental, enduring question in biology. Here we manipulate the amount of neural crest (NC) precursors for the dorsal root ganglia (DRG) in axolotl. We produce embryos with an under- or over-supply of pre-migratory NC in order to find out if DRG can regulate their sizes during development. Axolotl embryos are perfectly suitable for this research. Firstly, they are optimal for microsurgical manipulations and tissue repair. Secondly, they possess, unlike most other vertebrates, only one neural crest string located on top of the neural tube. This condition and position enables NC cells to migrate to either side of the embryo and participate in the regulation of NC cell distribution. We show that size compensation of DRG in axolotl occurs in 2 cm juveniles after undersupply of NC (up-regulation) and in 5 cm juveniles after oversupply of NC (down-regulation). The size of DRG is likely to be regulated locally within the DRG and not via adaptations of the pre-migratory NC or during NC cell migration. Ipsi- and contralateral NC cell migration occurs both in embryos with one and two neural folds, and contralateral migration of NC is the only source for contralateral DRG formation in embryos with only one neural fold. Compensatory size increase is accompanied by an increase in cell division of a DRG precursor pool (PCNA+/SOX2-), rather than by DRG neurons or glial cells. During compensatory size decrease, increased apoptosis and reduced proliferation of DRG cells are observed.

Tumor necrosis factor alpha inhibits ovulation and induces granulosa cell death in rat ovaries.

PURPOSE: We evaluated the role of tumor necrosis factor alpha (TNFα) in rat ovulation and granulosa cell death of ovarian follicles during the periovulatory stage. METHODS: Immature rats primed with pregnant mare serum gonadotropin were injected intraperitoneally with human chorionic gonadotropin (hCG), and TNFα was injected into the bursa 48 h later. The total number of released oocytes was counted. Apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and the expression of cleaved caspase 3 and Bax/Bcl-2. Autophagy was assessed by the expression of light chain protein 3 (LC3) and autophagosomes under transmission electron microscopy. RESULTS: TNFα significantly decreased the number of released oocytes, and many unruptured follicles were observed. TUNEL analysis revealed a larger number of apoptotic cells, and the cleaved caspase 3 and Bax/Bcl-2 increased more than that of the control 12 h after hCG administration. Furthermore, the expression of LC3 wwas significantly higher than that of the control, and autophagosomes were observed in the cytoplasm. CONCLUSIONS: Our data indicated that TNFα is an important mediator of ovulation in terms of decreasing the number of released oocytes and inducing granulosa cell death of unruptured follicles via apoptosis and autophagy for remodeling ovarian tissues.

Muscle and connective tissue progenitor populations show distinct Twist1 and Twist3 expression profiles during axolotl limb regeneration.

Limb regeneration involves re-establishing a limb development program from cells within adult tissues. Identifying molecular handles that provide insight into the relationship between cell differentiation status and cell lineage is an important step to study limb blastema cell formation. Here, using single cell PCR, focusing on newly isolated Twist1 sequences, we molecularly profile axolotl limb blastema cells using several progenitor cell markers. We link their molecular expression profile to their embryonic lineage via cell tracking experiments. We use in situ hybridization to determine the spatial localization and extent of overlap of different markers and cell types. Finally, we show by single cell PCR that the mature axolotl limb harbors a small but significant population of Twist1(+) cells.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

BACKGROUND: Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. RESULTS: In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. CONCLUSION: As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

Structural characteristic of folding/unfolding intermediate of pokeweed anti-viral protein revealed by time-resolved fluorescence.

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.

Reduction of ribosome biogenesis with activation of the mTOR pathway in denervated atrophic muscle.

Mammalian target of rapamycin (mTOR) pathway positively regulates the cell growth through ribosome biogenesis in many cell type. In general, myostatin is understood to repress skeletal muscle hypertrophy through inhibition of mTOR pathway and myogenesis. However, these relationships have not been clarified in skeletal muscle undergoing atrophy. Here, we observed a significant decrease of skeletal muscle mass at 2 weeks after denervation. Unexpectedly, however, mTOR pathway and the expression of genes related to myogenesis were markedly increased, and that of myostatin was decreased. However, de novo ribosomal RNA synthesis and the levels of ribosomal RNAs were dramatically decreased in denervated muscle. These results indicate that ribosome biogenesis is strongly controlled by factors other than the mTOR pathway in denervated atrophic muscle. Finally, we assessed rRNA transcription factors expression and observed that TAFIa was the only factor decreased. TAFIa might be a one of the limiting factor for rRNA synthesis in denervated muscle.

Application of a tungsten apron for occupational radiation exposure in nursing care of children with neuroblastoma during 131I-meta-iodo-benzyl-guanidine therapy.

The use of effective shielding materials against radiation is important among medical staff in nuclear medicine. Hence, the current study investigated the shielding effects of a commercially available tungsten apron using gamma ray measuring instruments. Further, the occupational radiation exposure of nurses during 131I-meta-iodo-benzyl-guanidine (131I-MIBG) therapy for children with high-risk neuroblastoma was evaluated. Attachable tungsten shields in commercial tungsten aprons were set on a surface-ray source with 131I, which emit gamma rays. The mean shielding rate value was 0.1 ± 0.006 for 131I. The shielding effects of tungsten and lead aprons were evaluated using a scintillation detector. The shielding effect rates of lead and tungsten aprons against 131I was 6.3% ± 0.3% and 42.1% ± 0.2% at 50 cm; 6.1% ± 0.5% and 43.3% ± 0.3% at 1 m; and 6.4% ± 0.9% and 42.6% ± 0.6% at 2 m, respectively. Next, we assessed the occupational radiation exposure during 131I-MIBG therapy (administration dose: 666 MBq/kg, median age: 4 years). The total occupational radiation exposure dose per patient care per 131I-MIBG therapy session among nurses was 0.12 ± 0.07 mSv. The average daily radiation exposure dose per patient care among nurses was 0.03 ± 0.03 mSv. Tungsten aprons had efficient shielding effects against gamma rays and would be beneficial to reduce radiation exposures per patient care per 131I-MIBG therapy session.

Radiation exposure in nurses during care of 131I-MIBG therapy for pediatric patients with high-risk neuroblastoma.

OBJECTIVE: 131I-meta-iodo-benzyl-guanidine (131I-MIBG) therapy has been used in children with high-risk neuroblastoma, who, in Japan, are cared for by trained nurses. To determine the safety of occupational radiation exposure in nurses, we retrospectively examined radiation exposure during therapy. METHODS: Sixty-two nurses who received radiation exposure during 131I-MIBG therapy were assessed for the daily percentage of total radiation exposure received using the formula, daily radiation exposure/total radiation dose × 100; self-care score of children was evaluated. RESULTS: Fifty-four 131I-MIBG treatments (592 ± 111 MBq/kg) were performed in neuroblastoma patients (M/F; 27 /27, mean age at 131I-MIBG treatment; 7 ± 2 years), who were isolated for 5 ± 1 days. Average total (0.36 ± 0.18 mSv; range 0.09-0.97 mSv) and daily (0.07 ± 0.05 mSv/day; range 0.02-0.32 mSv/day) radiation exposure to nurses per patient care. The daily percentage of total radiation exposure significantly decreased in 3 days after 131I-MIBG treatment (days 0, 1, and 2 was 38.2 ± 14.7%, 26.9 ± 12.6%, and 15.3 ± 7.1%, respectively), and the average self-care score was 12.2 ± 3.5 (10-27) for all patients. Higher self-care score was significantly related to younger patients' age and higher daily radiation exposure in nurses. CONCLUSION: Individual exposure to radiation was well controlled. Nurses who care for pediatric patients needing daily assistance must be aware of the radiation exposure risks, which can be reduced by establishing a care system and monitoring radiation exposure.

Publisher Correction: Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper.

Spinal cord is required for proper regeneration of the tail in Xenopus tadpoles.

Tail regeneration in urodeles is dependent on the spinal cord (SC), but it is believed that anuran larvae regenerate normal tails without the SC. To evaluate the precise role of the SC in anuran tail regeneration, we developed a simple operation method to ablate the SC completely and minimize the damage to the tadpole using Xenopus laevis. The SC-ablated tadpole regenerated a twisted and smaller tail. These morphological abnormalities were attributed to defects in the notochord (NC), as the regenerated NC in the SC-ablated tail was short, slim and twisted. The SC ablation never affected the early steps of the regeneration, including closure of the amputated surface with epidermis and accumulation of the NC precursor cells. The proliferation rate of the NC precursor cells, however, was reduced, and NC cell maturation was retarded in the SC-ablated tail. These results show that the SC has an essential role in the normal tail regeneration of Xenopus larvae, especially in the proliferation and differentiation of the NC cells. Gene expression analysis and implantation of a bead soaked with growth factor showed that fibroblast growth factor-2 and -10 were involved in the signaling molecules, which were expressed in the SC and stimulated growth of the NC cells.

Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.

Mesodermal origin of median fin mesenchyme and tail muscle in amphibian larvae.

Mesenchyme is an embryonic precursor tissue that generates a range of structures in vertebrates including cartilage, bone, muscle, kidney, and the erythropoietic system. Mesenchyme originates from both mesoderm and the neural crest, an ectodermal cell population, via an epithelial to mesenchymal transition (EMT). Because ectodermal and mesodermal mesenchyme can form in close proximity and give rise to similar derivatives, the embryonic origin of many mesenchyme-derived tissues is still unclear. Recent work using genetic lineage tracing methods have upended classical ideas about the contributions of mesodermal mesenchyme and neural crest to particular structures. Using similar strategies in the Mexican axolotl (Ambystoma mexicanum), and the South African clawed toad (Xenopus laevis), we traced the origins of fin mesenchyme and tail muscle in amphibians. Here we present evidence that fin mesenchyme and striated tail muscle in both animals are derived solely from mesoderm and not from neural crest. In the context of recent work in zebrafish, our experiments suggest that trunk neural crest cells in the last common ancestor of tetrapods and ray-finned fish lacked the ability to form ectomesenchyme and its derivatives.