Tropicalis Embryos Research Articles

Toxicity of the azo dyes Acid Red 97 and Bismarck Brown Y to Western clawed frog (Silurana tropicalis)

Azo compounds are used in a variety of industrial applications, such as textile colorant. Azo dyes have been found to contaminate aquatic environments and it has been shown that these compounds could potentially be toxic or induce endocrine disruption in aquatic organisms. However, there are few data available on the toxicity of these dyes, specifically Acid Red 97 (AR97) and Bismarck Brown Y (BBY). The aim of this study was to determine the toxicity and the endocrine-disrupting properties of AR97 and BBY in frogs. As fugacity modeling predicted that both compounds would sorb to sediment, sediment exposures were performed using a geometric range of concentrations (0, 1, 10, 100 and 1,000 ppm). Both AR97 and BBY dyes were not lethal to Silurana tropicalis embryos; however, BBY significantly induced malformations. Gene expression analysis of oxidative stress and mutagen-related genes was performed in BBY-treated larvae. There were significant two-fold increases of the tumor-suppressing protein p53 and heat shock protein 70 mRNA at 1,000 ppm suggesting that BBY induces cellular stress in early S. tropicalis development. Transcripts of the heat shock protein 90 did not change. Furthermore, reproductive-related genes were assessed and a 2.1-fold change was observed in the mRNA of the steroidogenic acute regulatory protein while steroid 5 alpha-reductase type 2 and androgen receptor transcript levels did not vary among treatments. In conclusion, high concentrations of BBY lead to increased developmental defects in frog embryogenesis and early larval development.

Small RNA profiling of Xenopus embryos reveals novel miRNAs and a new class of small RNAs derived from intronic transposable elements

Small RNA control of gene expression is critical for developmental processes in vertebrate embryos. To determine the dynamics of small RNA expression and to uncover novel small RNAs in the early vertebrate embryo, we performed high-throughput sequencing of all small RNAs in Xenopus tropicalis embryos at three developmental time points and in dissected halves of gastrula embryos. This analysis allowed us to identify novel microRNAs and we show that microRNA expression is highly dynamic and spatially localized in early embryos. In addition, we have developed a microRNA prediction pipeline and demonstrate that it has the power to predict new miRNAs that are experimentally detectable in frogs, mice, and humans. By combining the small RNA sequencing with mRNA profiling at the different developmental stages, we identify a new class of small noncoding RNAs that we name siteRNAs, which align in clusters to introns of protein-coding genes. We show that siteRNAs are derived from remnants of transposable elements present in the introns. We find that genes containing clusters of siteRNAs are transcriptionally repressed as compared with all genes. Furthermore, we show that this is true for individual genes containing siteRNA clusters, and that these genes are enriched in specific repressive histone modifications. Our data thus suggest a new mechanism of siteRNA-mediated gene silencing in vertebrates, and provide an example of how mobile elements can affect gene regulation.

Targeted Gene Disruption in theXenopus tropicalisGenome using Designed TALE Nucleases

Transcription activator-like effector nucleases (TALENs) are attractive and powerful molecular tools for targeted gene disruption because of their simple design and quick assembly. To evaluate the utility of TALENs in genome editing in Xenopus tropicalis, we prepared nine pairs of TALENs for the tyrosinase, noggin and MMP-9TH genes. All of the TALENs had some activity in a single-strand annealing assay using a cultured frog cell line, suggesting double-stranded DNA cleavage activity by the TALENs at their target site. The injection of mRNAs encoding TALENs into fertilized X. tropicalis embryos resulted in Cel-1 cleavage of the PCR fragment containing the target site amplified from embryo genomic DNA, indicating that a mutation in the target gene had occurred during embryogenesis. These mutations were confirmed by the sequencing of clones derived from the PCR fragments of genomic DNA. Patches of vitiligo were observed in tadpoles raised from fertilized eggs that had been injected with mRNAs of TALENs for the tyrosinase gene. TALENs containing the repeat variable di-residue (RVD) NN appeared to show more activity than TALENs containing RVD NK, although both RVD NN and NK preferentially associate with a G nucleotide.

Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos

Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

Embryonic exposure to propylthiouracil disrupts left-right patterning in Xenopus embryos.

Antithyroid medications are the preferred therapy for the treatment of Graves' disease during pregnancy. Propylthiouracil (PTU) is favored over methimazole (MMI) due to potential teratogenic concerns with MMI. This study was to determine the teratogenic potential of MMI and PTU using a validated Xenopus tropicalis embryo model. Embryos were exposed to 1 mM PTU (EC(50)=0.88 mM), 1 mM MMI, or vehicle control (water) from stages 2 to 45. Treated embryos were examined for gross morphological defects, ciliary function, and gene expression by in situ hybridization. Exposure to PTU, but not MMI, led to cardiac and gut looping defects and shortening along the anterior-posterior axis. PTU exposure during gastrulation (stage 8-12.5) was identified as the critical period of exposure leading to left-right (LR) patterning defects. Abnormal cilia polarization, abnormal cilia-driven leftward flow at the gastrocoel roof plate (GRP), and aberrant expression of both Coco and Pitx2c were associated with abnormal LR symmetry observed following PTU exposure. PTU is teratogenic during late blastula, gastrulation, and neurulation; whereas MMI is not. PTU alters ciliary-driven flow and disrupts the normal genetic program involved in LR axis determination. These studies have important implications for women taking PTU during early pregnancy.

Interaction of triphenyltin and an agonist of retinoid X receptor (LGD1069) in embryos of Xenopus tropicalis

Xenopus tropicalis embryos were exposed for 48h to mixtures of triphenyltin and LGD1069 (an agonist of the retinoid X receptor). The index of fin deficiency (IFD) of the embryos increased in the triphenyltin-treated groups, and the index of axis deficiency (IAD) increased in LGD1069-treated groups in a concentration-dependent manner. When embryos were exposed to mixtures of 5μgSn/L triphenyltin and 1–30μg/L LGD1069, IFD decreased from 2.9 to 0.6 and IAD increased from 0.1 to 2.4 with increasing LGD1069 concentrations. Conversely, when embryos were exposed to mixtures of 15μg/L LGD1069 and 1–10μgSn/L triphenyltin, IFD increased from 0.1 to 3.0 with increasing triphenyltin concentrations. Co-exposure induced some new phenotypes, such as posteriorized anus. These results suggest that LGD1069 suppressed the teratogenicity of triphenyltin and that the retinoid X receptor was involved in triphenyltin-induced teratogenicity. Histological observations indicate that co-exposure inhibited the invagination of the yolk plug.

The teratogenic effects of sediments from the Yangtze Estuary and adjacent bay, China, on frog embryos

In recent decades, the Yangtze (Changjiang) Estuary has suffered great environmental changes due to intensive human activities in the Yangtze River basin. This study assessed the ecotoxicity of sediments from the Yangtze Estuary with the frog embryo teratogenesis assay—Xenopus (FETAX). The results showed that the sediment extracts induced multiple malformations in embryos of Xenopus tropicalis. In the embryos treated with nearshore extracts, abnormal eyes, narrow fins and hypopigmentation were the dominant phenotypes, followed by enlarged proctodeums. In these embryos, the percentages of total malformations were greater than 50 % at five sampling sites and less than 40 % at the other seven sites. However, in the embryos treated with the offshore extracts, elongation of the proctodeums was the dominant phenotype, followed by bent tails. The percentages of total malformations ranged from 25 to 45 %. Comparatively, the embryos treated with extracts from the nearshore zone showed a greater variety of phenotypes than those treated with extracts from the offshore zone. These results suggest that the sediments showed high teratogenicity to the amphibian embryos. The results also indicate that the teratogencity to X. tropicalis embryos is a useful indicator of the pollution of sediments.

Histological observation on unique phenotypes of malformation induced in Xenopus tropicalis larvae by tributyltin

AbstractTributyltin (TBT), a biocide used in antifouling paints, has shown strong teratogenic effects on Xenopus tropicalis embryos at environmentally relevant concentrations. X. tropicalis embryos were exposed to 50, 100 and 200 ng/L tributyltin chloride for 72 hr. The histological changes were further observed on abnormal eyes, enlarged trunks, enlarged proctodaeums and absence of fins induced by TBT. The lens and the retinal layers of abnormal eyes were slightly or barely differentiated, and that the pigment epithelium was neither continuous nor smooth. The abdomens were full of undifferentiated gut tissue with yolk-rich inclusions in the tadpoles with enlarged trunks. The proctodaeums formed a bump-like or columnar structure. The mass of yolk-rich cells occupied the lumen, blocked the opening and even turned inside out of the proctodaeum. Both the ventral and dorsal fins in trunks and tails became narrow or even disappeared totally. Our results suggest that great changes of histology took place corresponding to the unique phenotypes. The gut tissue was poorly differentiated, which led to the failed elongation of the guts and subsequently the enlarged trunks. The enlarged proctodaeums were due to the undifferentiation of inner layer, the expansion of outer epidermal part and the absence of fins around them. In brief, the histological observations provided insights into the reason of the unique external malformations in some degree.

Zebrafish obesogenic test: a tool for screening molecules that target adiposity

Dietary and xenobiotic compounds may alter endocrine signaling and lipid homeostasis, thus inducing obesity. We describe a short-term assay method, the zebrafish obesogenic (ZO) test, for examining the effects of diet, drugs, and environmental contaminants, singly or in combination, on white adipose tissue (WAT) dynamics in live larvae. The ZO test is an intermediate step in obesity research, between in vitro and rodent assays, and may be also used to study the effect of environmental toxicants on the adiposity of aquatic species. The procedure, using Nile Red (NR) fluorescent probe to reveal adipocyte lipid droplets, is suitable for pharmaceutical or toxicological screening. Larvae treated at an environmentally-relevant concentration of tributyltin chloride (TBT), an environmental obesogen, exhibited a remarkable increase in adiposity, irrespective of the lipid composition of the background diet. Exogenous compounds, e.g., rosiglitazone or TBT, known to increase adiposity in the fasting state, were classified as obesogenic. Anti-obesogenic compounds favored a decrease in adiposity in the fasting state. The ZO test, using adipocyte lipid droplet size and adiposity as its endpoints, is a whole-organism alternative testing assay for obesogenic and anti-obesogenic compounds and mixtures and provides relevant information for environmental and human risk assessments.

Stage-specific malformations and phenotypic changes induced in embryos of amphibian ( Xenopus tropicalis) by triphenyltin

Tributyltin (TBT) and triphenyltin (TPT) can induce unique malformations in embryos of amphibian ( Xenopus tropicalis). We further exposed X. tropicalis embryos to TPT during different stages. Severe malformations were observed in the embryos after exposure to 5–10 μgSn/L TPT during S32–40 and S40–46. Comparatively, the embryos showed slight or moderate malformations after exposure during S10–25, S25–32 or S46–47. The most characteristic malformations were narrow or no fins, followed by enlarged proctodaeums and skin hypopigmentation. The developed fins were still affected after TPT exposure, but the inhibited fins could not recover even after the removal of TPT exposure. In TPT treatment groups, the proctodaeums of embryos externally expanded, and the pigment cells of embryos were poor, small and dot like. Our results suggested that TPT-induced teratogenicity was highly stage-specific. The sensitive window of fin defects indicated that TPT affected the formation rather than induction or expansion of fins.

HEB and E2A function as SMAD/FOXH1 cofactors

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.

Microfluidic characterization of cilia-driven fluid flow using optical coherence tomography-based particle tracking velocimetry

Motile cilia are cellular organelles that generate directional fluid flow across various epithelial surfaces including the embryonic node and respiratory mucosa. The proper functioning of cilia is necessary for normal embryo development and, for the respiratory system, the clearance of mucus and potentially harmful particulate matter. Here we show that optical coherence tomography (OCT) is well-suited for quantitatively characterizing the microfluidic-scale flow generated by motile cilia. Our imaging focuses on the ciliated epithelium of Xenopus tropicalis embryos, a genetically manipulable and experimentally tractable animal model of human disease. We show qualitative flow profile characterization using OCT-based particle pathline imaging. We show quantitative, two-dimensional, two-component flow velocity field characterization using OCT-based particle tracking velocimetry. Quantitative imaging and phenotyping of cilia-driven fluid flow using OCT will enable more detailed research in ciliary biology and in respiratory medicine.

Temporal uncoupling of the DNA methylome and transcriptional repression during embryogenesis.

DNA methylation is a tightly regulated epigenetic mark associated with transcriptional repression. Next-generation sequencing of purified methylated DNA obtained from early Xenopus tropicalis embryos demonstrates that this genome is heavily methylated during blastula and gastrula stages. Although DNA methylation is largely absent from transcriptional start sites marked with histone H3 lysine 4 trimethylation (H3K4me3), we find both promoters and gene bodies of active genes robustly methylated. In contrast, DNA methylation is absent in large H3K27me3 domains, indicating that these two repression pathways have different roles. Comparison with chromatin state maps of human ES cells reveals strong conservation of epigenetic makeup and gene regulation between the two systems. Strikingly, genes that are highly expressed in pluripotent cells and in Xenopus embryos but not in differentiated cells exhibit relatively high DNA methylation. Therefore, we tested the repressive potential of DNA methylation using transient and transgenic approaches and show that methylated promoters are robustly transcribed in blastula- and gastrula-stage embryos, but not in oocytes or late embryos. These findings have implications for reprogramming and the epigenetic regulation of pluripotency and differentiation and suggest a relatively open, pliable chromatin state in early embryos followed by reestablished methylation-dependent transcriptional repression during organogenesis and differentiation.

Loss of Xenopus tropicalis EMSY causes impairment of gastrulation and upregulation of p53

EMSY interacts directly with BRCA2 and links the BRCA2 pathway to sporadic breast and ovarian cancer. It also interacts with BS69 and HP1b, both of which are involved in chromatin remodelling, and with NIF-1 and DBC-1 in the regulation of nuclear receptor-mediated transcription. Here we investigate the function of EMSY during amphibian development, and in doing so provide the first loss-of-function analysis of this protein. Injection of Xenopus tropicalis embryos with antisense morpholino oligonucleotides targeting XtEMSY impairs gastrulation movements, disrupts dorsal structures, and kills embryos by tailbud stages. Consistent with these observations, regional markers such as Xbra, Chd, Gsc, Shh, Sox3 and Sox17 are downregulated. In contrast to these regional markers, expression of p53 is upregulated in such embryos, and at later stages Bax expression is elevated and apoptotic cells can be detected. Our results demonstrate that EMSY has an essential role in development and they provide an in vivo loss-of-function model that might be used to explore the biochemical functions of this protein in more detail.

ADAM13 Induces Cranial Neural Crest by Cleaving Class B Ephrins and Regulating Wnt Signaling

The cranial neural crest (CNC) consists of multipotent embryonic cells that contribute to craniofacial structures and other cells and tissues of the vertebrate head. During embryogenesis, CNC is induced at the neural plate boundary through the interplay of several major signaling pathways. Here, we report that the metalloproteinase activity of ADAM13 is required for early induction of CNC in Xenopus. In both cultured cells and X. tropicalis embryos, membrane-bound Ephrins (Efns) B1 and B2 were identified as substrates for ADAM13. ADAM13 upregulates canonical Wnt signaling and early expression of the transcription factor snail2, whereas EfnB1 inhibits the canonical Wnt pathway and snail2 expression. We propose that by cleaving class B Efns, ADAM13 promotes canonical Wnt signaling and early CNC induction.

Effects of tributyltin (TBT) on Xenopus tropicalis embryos at environmentally relevant concentrations

Tributyltin (TBT) has been widely used as a biocide in antifouling paints and is a known endocrine disrupting chemical. In this paper, we exposed embryos of Xenopus tropicalis to 50–400 ng L −1 tributyltin chloride. TBT significantly decreased the survival rate, reduced the body length and retarded the development of embryos after 24, 36 and 48 h of exposure. These effects of TBT were concentration- and time-dependent. Embryos treated with TBT showed multiple malformations. The most obvious alterations were abnormal eyes, enlarged proctodaeum, narrow fins, and skin hypopigmentation. Enlarged proctodaeum and narrow fins were mainly observed after 36 and 48 h of exposure. The loss of eye pigmentation or the absence of external eyes occurred after 24 and 36 h of exposure, while extended lenses or edemas of eyes were more commonly observed after 48 h of exposure. Additional malformations included: small anterior region of heads, pericardial edemas, enlarged trunks, and bent tails. These results suggested that TBT is very toxic to X. tropicalis embryos at environmentally relevant concentrations.

Teratogenic effects of tetrabromobisphenol A on Xenopus tropicalis embryos

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant and a known thyroid disruptor. We reported exposing Xenopus tropicalis embryos (NF10) to 0.01, 0.1 or 1 mg/L of TBBPA with or without 70 µg/L triiodothyronine (T 3). Compared with the controls, 1 mg/L of TBBPA significantly reduced the body length of embryos after 24, 36 and 48 h of exposure. Embryos treated with TBBPA showed multiple malformations, including: abnormal eyes, skin hypopigmentation, enlarged proctodaeum, narrow fins and pericardial edemas. The effect of abnormal eyes manifested itself in the loss of pigmentation, reduction in size, or absence of external eyes. The degree of eye malformations was quantified with the index of eye malformations (IEM) with 0 being normal and 3 being severe. In the 1 mg/L TBBPA treatment groups, the incidence of total malformations (ITM) was 68–93%, and IEM was 0.8–0.9. T 3 showed no teratogenic effects on embryos, but it significantly enhanced TBBPA-induced teratogenic effects. In the T 3 + 1 mg/L TBBPA treatment groups, ITM was 91–99%, and IEM was 1.8–1.9. Histological observations showed that the retinas were generally smaller, and the lenses were underdeveloped or even absent. These results indicate that TBBPA at relatively high concentration has teratogenic effects on X. tropicalis embryos. The results also suggest that thyroid hormone signaling might be involved in TBBPA induced-teratogenicity.

Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk-1 and Egr-1.

Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.

A Hierarchy of H3K4me3 and H3K27me3 Acquisition in Spatial Gene Regulation in Xenopus Embryos

Epigenetic mechanisms set apart the active and inactive regions in the genome of multicellular organisms to produce distinct cell fates during embryogenesis. Here, we report on the epigenetic and transcriptome genome-wide maps of gastrula-stage Xenopus tropicalis embryos using massive parallel sequencing of cDNA (RNA-seq) and DNA obtained by chromatin immunoprecipitation (ChIP-seq) of histone H3 K4 and K27 trimethylation and RNA Polymerase II (RNAPII). These maps identify promoters and transcribed regions. Strikingly, genomic regions featuring opposing histone modifications are mostly transcribed, reflecting spatially regulated expression rather than bivalency as determined by expression profile analyses, sequential ChIP, and ChIP-seq on dissected embryos. Spatial differences in H3K27me3 deposition are predictive of localized gene expression. Moreover, the appearance of H3K4me3 coincides with zygotic gene activation, whereas H3K27me3 is predominantly deposited upon subsequent spatial restriction or repression of transcriptional regulators. These results reveal a hierarchy in the spatial control of zygotic gene activation.

The Curious Case of Bivalent Marks

Bivalently marked chromatin, containing both histone H3 lysine 4 (H3K4) and H3K27 trimethylation, is a hallmark of developmentally regulated paused promoters in mammalian embryonic stem cells. In this issue of Developmental Cell, Akkers et al. report that Xenopus tropicalis embryos transition through early development without the requirement for bivalently marked promoters.