Tropicalis Embryos Research Articles

Mechanistic study of transcription factor Sox18 during heart development

Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/β-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and β-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/β-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/β-Catenin signaling.

SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Developmental expression of peroxiredoxin gene family in early embryonic development of Xenopus tropicalis

Peroxidase genes (Prdx) encode a family of antioxidant proteins, which can protect cells from oxidative damage by reducing various cellular peroxides. This study investigated the spatiotemporal expression patterns of gene members in this family during the early development of Xenopus tropicalis. Real-time quantitative PCR showed that all members of this gene family have a distinct temporal expression pattern during the early development of X. tropicalis embryos. Additionally, whole mount in situ hybridization revealed that individual prdx genes display differential expression patterns, with overlapping expression in lymphatic vessels, pronephros, proximal tubule, and branchial arches. This study provides a basis for further study of the function of the prdx gene family.

Mucociliary Wnt signaling promotes cilia biogenesis and beating

It is widely thought that Wnt/Lrp6 signaling proceeds through the cytoplasm and that motile cilia are signaling-inert nanomotors. Contrasting both views, we here show in the mucociliary epidermis of X. tropicalis embryos that motile cilia transduce a ciliary Wnt signal that is distinct from canonical β-catenin signaling. Instead, it engages a Wnt-Gsk3-Ppp1r11-Pp1 signaling axis. Mucociliary Wnt signaling is essential for ciliogenesis and it engages Lrp6 co-receptors that localize to cilia via a VxP ciliary targeting sequence. Live-cell imaging using a ciliary Gsk3 biosensor reveals an immediate response of motile cilia to Wnt ligand. Wnt treatment stimulates ciliary beating in X. tropicalis embryos and primary human airway mucociliary epithelia. Moreover, Wnt treatment improves ciliary function in X. tropicalis ciliopathy models of male infertility and primary ciliary dyskinesia (ccdc108, gas2l2). We conclude that X. tropicalis motile cilia are Wnt signaling organelles that transduce a distinct Wnt-Pp1 response.

Issue Information

Cover Photograph:Upper left: Newt (P. waltl) juveniles and Xenopus froglets were subjected to agarose‐embedded heat‐shock (AeHS) method. GFP expression was induced specifically in the regenerating limbs (see Matsubara et al. 86–93).Upper right: Schematic diagram of the intermittent hypoxia exposure model. Pimonidazole‐positive cells were detected in the pulmonary tissues of Xenopus laevis during repeated dives, indicating tissue hypoxia (see Fujiyama, 94–99).Lower left: The intercalary element of Zhangixalus schlegelii (arrowheads). Such skeletal elements are seen between the terminal and subterminal phalanges of all the digits of arboreal frogs (see Nakanishi, 100–108).Lower right: Tetraspanin9 expressions in Xenopus laevis (above) and X. tropicalis embryo (below) hybridized in the same tube. (see Kuriyama and Tanaka, 109–119).Bottom right corner: A 48 hpf early four‐armed pluteus taken through a confocal microscope. Catecholamine‐positive cells are colored in yellow (see Kalachev and Tankovich, 120–131). image

Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis

BackgroundThe true diploid frog, Xenopus tropicalis (X. tropicalis) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation.ResultsThrough one-cell stage injection of Cas/gRNAs into X. tropicalis embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in X. tropicalis embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in X. tropicalis embryos.ConclusionSaCas9 and KKH SaCas9 are robust genome editing tools in X. tropicalis embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in frogs.

Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis.

The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

Exposure to the synthetic phenolic antioxidant 4,4′-thiobis(6-t-butyl-m-cresol) disrupts early development in the frog Silurana tropicalis

Many chemicals in commonly used household and industrial products are being released into the environment, yet their toxicity is poorly understood. The synthetic phenolic antioxidant, 4,4′-thiobis(6-t-butyl-m-cresol) (CAS 96-69-5; TBBC) is present in many common products made of rubber and plastic. Yet, this phenolic antioxidant has not been tested for potential toxicity and developmental disruption in amphibians, a sensitive and susceptible class. We investigated whether acute and chronic exposure to TBBC would interfere with thyroid hormone-dependent developmental processes in the frog Silurana tropicalis and thus affect its early life-stage development. We exposed S. tropicalis embryos at the Nieuwkoop-Faber (NF) 9–10 stage to TBBC at nominal concentrations (0, 25, 50, 75, 100, 200 and 400 μg/L) to determine the 96h lethal concentrations and sublethal effects. We conducted a chronic exposure starting at stage NF47-48 to three sublethal TBBC nominal concentrations (0, 0.002, 0.1 and 5 μg/L) for 48–52 days to evaluate effects on growth and metamorphosis. The 96h lethal and effective (malformations) TBBC concentrations (LC50 and EC50) were 70.5 and 76.5 μg/L, respectively. Acute exposure to all TBBC concentrations affected S. tropicalis growth and was lethal at 200 and 400 μg/L. Chronic exposure to sublethal TBBC concentrations reduced body size by 8% at 5 μg/L and body mass by 17% at 0.002 μg/L when metamorphosis was completed. This study demonstrates that TBBC is toxic, induces malformations and inhibits tadpole growth after acute and chronic exposures. These findings call for further investigations on the mode of actions of TBBC and related antioxidants for developmental disruption in amphibians.

Deep learning is widely applicable to phenotyping embryonic development and disease.

ABSTRACTGenome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated ‘The people behind the papers’ interview.

Three-dimensional folding dynamics of the Xenopus tropicalis genome

Animal interphase chromosomes are organized into topologically associating domains (TADs). How TADs are formed is not fully understood. Here, we combined high-throughput chromosome conformation capture and gene silencing to obtain insights into TAD dynamics in Xenopus tropicalis embryos. First, TAD establishment in X. tropicalis is similar to that in mice and flies and does not depend on zygotic genome transcriptional activation. This process is followed by further refinements in active and repressive chromatin compartments and the appearance of loops and stripes. Second, within TADs, higher self-interaction frequencies at one end of the boundary are associated with higher DNA occupancy of the architectural proteins CTCF and Rad21. Third, the chromatin remodeling factor ISWI is required for de novo TAD formation. Finally, TAD structures are variable in different tissues. Our work shows that X. tropicalis is a powerful model for chromosome architecture analysis and suggests that chromatin remodeling plays an essential role in de novo TAD establishment.

Obtaining Xenopus tropicalis Embryos by Natural Mating.

Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. Natural matings are a simple and common method to obtain embryos for injection or other experimental use or to raise to adulthood. This method does not require sacrificing a male as in vitro fertilization (IVF) does. Male and female frogs are injected with an ovulation hormone, placed together in a mating bucket, and left for 4-6 h or overnight to mate. Embryos are then collected, treated with cysteine to remove the sticky jelly coat, and used for injections and/or raised to later stages or adulthood. For embryos raised past free-swimming stages, the cysteine step can optionally be skipped, and tadpoles can be allowed to hatch naturally from the jelly coat. Although there are many similarities between natural mating protocols for Xenopus laevis and Xenopus tropicalis, there are key differences such as hormone dosage, timing of ovulation, and embryo incubation temperature. Here we provide a specific protocol for inducing natural matings in X. tropicalis.

4-Methylcyclohexane methanol (MCHM) affects viability, development, and movement of Xenopus embryos.

Following chemical spill disasters, it is important to estimate the effects of spilled chemicals on humans and the environment. Here we analyzed the toxicological effects of the coal cleaning chemical, 4-methylcyclohexane methanol (MCHM), which was spilled into the Elk River water supply in 2014. The viability of HEK293 T human cell line cultures and Xenopus tropicalis embryos was negatively affected, and the addition of the antioxidants alleviated toxicity with MCHM exposure. Additionally, X. tropicalis embryos suffered developmental defects as well as reversible non-responsiveness and melanization defects. The impact MCHM has on HEK293 T cells and X. tropicalis points to the importance of continued follow-up studies of this chemical.

Potential reproductive toxicity of multi-walled carbon nanotubes and their chronic exposure effects on the growth and development of Xenopus tropicalis

The increasing production and use of multi-walled carbon nanotubes (MWCNTs) will inevitably lead to discharge into the environment and exert negative effects on organisms. Many studies have focused on the toxicity of MWCNTs to aquatic animals, but little is known about their possible potential reproductive toxicity. In this study, 6 sexually mature Xenopus tropicalis were exposed to 0.5 and 2.5 mg/L MWCNTs suspensions for 56 days, and the toxicity of MWCNTs to the growth and reproduction of X. tropicalis were studied. The results showed that MWCNTs could inhibit the growth of body, including the testis, ovaries and fat of X. tropicalis. Histopathological section analysis showed that MWCNTs affected the formation of spermatogonia and oocytes, while had no notable effect on the heart or liver. MWCNTs would be accumulated in lungs of X. tropicalis inducing lung cannons. In addition, MWCNTs changed the microbial community structure and diversity of gut microbiota but did not change its abundance significantly. Moreover, MWCNTs could even decrease the fertilized and survival rate of X. tropicalis embryos. These results indicated that chronic exposure to MWCNTs would not only affect the growth and development of X. tropicalis, but also pose a potential risk on their reproduction.

RNA-Seq analysis on ets1 mutant embryos of Xenopus tropicalis identifies microseminoprotein beta gene 3 as an essential regulator of neural crest migration.

The proto-oncogene ets1 is highly expressed in the pre-migratory and migratory neural crest (NC), and has been implicated in the delamination and migration of the NC cells. To identify the downstream target genes of Ets1 in this process, we did RNA sequencing (RNA-Seq) on wild-type and ets1 mutant X. tropicalis embryos. A list of genes with significantly differential expression was obtained by analyzing the RNA-Seq data. We validated the RNA-Seq data by quantitative PCR, and examined the expression pattern of the genes identified from this assay with whole mount in situ hybridization. A majority of the identified genes showed expression in migrating NC. Among them, the expression of microseminoprotein beta gene 3 (msmb3) was positively regulated by Ets1 in both X. laevis and X. tropicalis. Knockdown of msmb3 with antisense morpholino oligonucleotides or disruption of msmb3 by CRISPR/Cas9 both impaired the migratory streams of NC. Our study identified msmb3 as an Ets1 target gene and uncovered its function in maintaining neural crest migration pattern.

Bixafen exposure induces developmental toxicity in zebrafish (Danio rerio) embryos

Bixafen (BIX), a new generation succinate dehydrogenase inhibitor (SDHI) fungicide commonly used in agriculture, is regarded as a potential aquatic pollutant because of its lethal and teratogenic effects on Xenopus tropicalis embryos. To evaluate the threat of BIX to aquatic environments, information concerning BIX's embryonic toxicity to aquatic organisms (especially fish) is important, yet such information remains scarce. The present study aimed to fill this knowledge gap by employing zebrafish embryos as model animals in exposure to 0.1, 0.3 and 0.9 μM BIX. Our results showed that BIX caused severe developmental abnormalities (hypopigmentation, tail deformity, spinal curvature and yolk sac absorption anomaly) and hatching delay in zebrafish embryos. The expression levels of early embryogenesis-related genes (gh, crx, sox2 and neuroD) were downregulated after BIX exposure, except for nkx2.4b, which was upregulated. Furthermore, transcriptome sequencing analysis showed that all the downregulated differentially expressed genes were enriched in cell cycle processes. Taken together, these results demonstrated that BIX has strong developmental toxicity to zebrafish that may be due to the downregulated expression of genes involved in embryonic development. These findings provide valuable reference for evaluating BIX's potential adverse effects on aquatic ecosystems.

Transcriptome Analysis Reveals That Naphthenic Acids Perturb Gene Networks Related to Metabolic Processes, Membrane Integrity, and Gut Function in Silurana (Xenopus) tropicalis Embryos

Naphthenic acids (NAs) are oil-derived mixtures of carboxylic acids and are considered emerging contaminants with the potential to disrupt development of aquatic species. In the Oil Sands Region of Canada, NAs are components of the water released following processing of the bitumen-containing sand. The aim of this research was to identify potential mechanisms of toxicity of NA mixtures. Silurana (Xenopus) tropicalis embryos were raised in water spiked with commercial oil-derived NA extracts (S1 and S2) at a sub-lethal concentration (2 mg/L). The transcriptomic responses of the whole 4-day old embryos following exposure were assessed using a custom oligonucleotide microarray. Both NA mixtures induced embryonic abnormalities that included edema, and cardiac and gut abnormalities. Exposure to NAs also affected morphometric parameters and decreased total length, tail length, and interorbital distance of the embryos. Gene ontology analysis revealed that 18 biological processes, 5 cellular components, and 19 molecular functions were significantly enriched after both S1 and S2 exposures. Sub-network enrichment analysis revealed pathways that were related to phenotypic abnormalities; these included gut function, edema, and cartilage differentiation. Other notable networks affected by NAs included metabolism and cell membrane integrity. In a separate dose-response experiment, the expression of key genes identified by microarray (cyp4b1, abcg2, slc26a6, eprs, and slc5a1) was determined by Real-Time qPCR in S. tropicalis embryos exposed to the commercial NAs and to acid-extractable organics (AEOs) prepared from Oil Sands Process-Affected Water. In general, the RT-qPCR data agreed with the microarray data. In S. tropicalis embryos exposed to the AEOs, the mRNA levels of eprs (bifunctional glutamate/proline-tRNA ligase) and slcs5a1 (sodium/glucose cotransporter 1) were significantly decreased compared to the controls. Such changes are likely indicative of increased edema and disrupted gut function, respectively. These data suggest that NAs have multiple modes of action to induce developmental toxicity in amphibians. Some modes of action may be shared between commercial NAs and AEOs.

Special Considerations for Making Explants and Transplants with Xenopus tropicalis.

Although Xenopus laevis is an important model organism for embryological experimentation, the smaller, more genetically tractable, and faster developing Xenopus tropicalis provides advantages for using genetic approaches to understand developmental mechanisms. Explant cultures and transplants of X. tropicalis embryonic tissues present unique opportunities to examine embryonic tissue determination in a simplified setting. Here we demonstrate preparation of explants and transplants of preplacodal head ectoderm in order to illustrate these approaches; however, these methods apply broadly to tissues throughout the embryo. We focus on technical adjustments to accommodate the differences in size, tissue character, and rate of development between X. laevis and X. tropicalis With only modest modifications, X. tropicalis embryos are quite amenable to the same kinds of experimental manipulations as X. laevis.

Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Naphthenic Acid Mixtures and Acid-Extractable Organics from Oil Sands Process-Affected Water Impair Embryonic Development of Silurana (Xenopus) tropicalis.

Naphthenic acids (NAs) are carboxylic acids naturally occurring in crude oils and bitumen and are suspected to be the primary toxic substances in wastewaters associated with oil refineries and mining of oil sands. Oil sands process-affected water (OSPW) generated by the extraction of bitumen from oil sands are a major source of NAs and are currently stored in tailings ponds. We report on the acute lethality and teratogenic effects of aquatic exposure of Silurana (Xenopus) tropicalis embryos to commercial NA extracts and from the acid extractable organics (AEOs) fraction of a Canadian OSPW. Using electrospray ionization-high resolution mass spectrometry, we determined that the O2 species proportion were 98.8, 98.9 and 58.6% for commercial mixtures Sigma 1 (S1M) and Sigma 2 (S2M), and AEOs, respectively. The 96h LC50 estimates were 10.4, 11.7, and 52.3 mg/L for S1M, S2M, and the AEOs, respectively. The 96h EC50 estimates based on frequencies of developmental abnormalities were 2.1, 2.6, and 14.2 mg/L for S1M, S2M, and the AEOs, respectively. The main effects observed were reduced body size, edema, and cranial, heart, gut and ocular abnormalities. Increasing concentrations of the mixtures resulted in increased severity and frequency of abnormalities ( p < 0.05). The rank-order potency was S1M > S2M > AEO based on LC50 and EC50 estimates. These data provide insight into the effects NAs in amphibian embryos and can contribute to the development of environmental guidelines for the management of OSPW.

Assay for Transposase-Accessible Chromatin-Sequencing Using Xenopus Embryos

The DNA of eukaryotic genomes is packaged into chromatin by nucleosomes. This not only compacts the DNA but also plays a central role in gene regulation and establishment of cellular identity during development. Because of this packaging, the DNA is relatively inaccessible to nucleoplasmic factors; however, regulatory elements such as promoters, enhancers, and insulators are largely kept nucleosome-free. The assay for transposase-accessible chromatin (ATAC-seq) can be used to identify genomic locations of "open" chromatin, footprints of DNA-binding proteins, and positioned nucleosomes. It therefore is a powerful tool for unraveling the dynamic regulatory landscape of chromatin. The method exploits the action of hyperactive prokaryotic Tn5-transposase, which preferentially cuts DNA in accessible chromatin and tags the sites with sequencing adaptors. Here we describe an ATAC-seq protocol for use with Xenopus tropicalis embryos.