CFAP45, a heterotaxy and congenital heart disease gene, affects cilia stability.

Congenital heart disease (CHD) is the most common and lethal birth defect, affecting 1.3 million individuals worldwide. During early embryogenesis, errors in Left-Right (LR) patterning called Heterotaxy (Htx) can lead to severe CHD. Many of the genetic underpinnings of Htx/CHD remain unknown. In analyzing a family with Htx/CHD using whole-exome sequencing, we identified a homozygous recessive missense mutation in CFAP45 in two affected siblings. CFAP45 belongs to the coiled-coil domain-containing protein family, and its role in development is emerging. When we depleted Cfap45 in frog embryos, we detected abnormalities in cardiac looping and global markers of LR patterning, recapitulating the patient's heterotaxy phenotype. In vertebrates, laterality is broken at the Left-Right Organizer (LRO) by motile monocilia that generate leftward fluid flow. When we analyzed the LRO in embryos depleted of Cfap45, we discovered "bulges" within the cilia of these monociliated cells. In addition, epidermal multiciliated cells lost cilia with Cfap45 depletion. Via live confocal imaging, we found that Cfap45 localizes in a punctate but static position within the ciliary axoneme, and depletion leads to loss of cilia stability and eventual detachment from the cell's apical surface. This work demonstrates that in Xenopus, Cfap45 is required to sustain cilia stability in multiciliated and monociliated cells, providing a plausible mechanism for its role in heterotaxy and congenital heart disease.

Phthalates modulate steroid 5-reductase transcripts in the Western clawed frog embryo.

Phthalates are used worldwide in the manufacturing of plastics, added to cosmetic products, personal care products, pharmaceuticals, medical devices, and paints; and are widely detected in soil, surface water, and organism tissues. Phthalate esters have been previously shown to interfere with the endocrine system in vertebrates. However, few studies have investigated the effects of phthalates on testosterone-converting enzymes that affect hormone levels and reproduction. In the present study, we exposed the Western clawed frog (Silurana tropicalis) to 0.1, 1, and 10 µM diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), and diethyl phthalate (DEP) during early amphibian embryonic development. Additional DBP exposures were conducted ex vivo using mature frog testes. Malformations and mRNA levels of genes associated to reproduction and oxidative stress were evaluated. 0.1 µM DEHP, DBP, and DEP induced an array of malformations, including incomplete gut coiling, edemas, and eye malformations. Moreover, all three phthalates increased the expression of androgen-related genes, such as steroid-5α-reductase 1, 2, 3, steroid-5ß-reductase, and androgen receptor at concentrations ranging from 0.1 to 10 µM depending on the phthalate and gene. Data suggest that the phthalate esters tested are teratogens to the amphibian embryo and that these phthalates exhibit an androgenic activity in amphibians.

Analysis of Craniocardiac Malformations in Xenopus using Optical Coherence Tomography.

Birth defects affect 3% of children in the United States. Among the birth defects, congenital heart disease and craniofacial malformations are major causes of mortality and morbidity. Unfortunately, the genetic mechanisms underlying craniocardiac malformations remain largely uncharacterized. To address this, human genomic studies are identifying sequence variations in patients, resulting in numerous candidate genes. However, the molecular mechanisms of pathogenesis for most candidate genes are unknown. Therefore, there is a need for functional analyses in rapid and efficient animal models of human disease. Here, we coupled the frog Xenopus tropicalis with Optical Coherence Tomography (OCT) to create a fast and efficient system for testing craniocardiac candidate genes. OCT can image cross-sections of microscopic structures in vivo at resolutions approaching histology. Here, we identify optimal OCT imaging planes to visualize and quantitate Xenopus heart and facial structures establishing normative data. Next we evaluate known human congenital heart diseases: cardiomyopathy and heterotaxy. Finally, we examine craniofacial defects by a known human teratogen, cyclopamine. We recapitulate human phenotypes readily and quantify the functional and structural defects. Using this approach, we can quickly test human craniocardiac candidate genes for phenocopy as a critical first step towards understanding disease mechanisms of the candidate genes.

Effects of antagonist of retinoid X receptor (UVI3003) on morphology and gene profile of Xenopus tropicalis embryos.

We exposed Xenopus tropicalis embryos to a selective antagonist of retinoid X receptor (UVI3003). UVI3003 induced multiple malformations at the concentrations of 200-1000 µg/L after 48 h exposure. The most prominent malformations affected brains, eyes, cement gland and fins. UVI3003 also induced variable and divergent malformations at 250-1500 µg/L after 0-24 and 24-48 h exposure. Microarray analysis showed that seven genes (rps15, serp2, fmr1, cyp2e1, lrrc9, ugtla6 and LOC100490188) were differentially regulated in all three treatment groups after 0-24h exposure. The most significantly affected pathway was galactose metabolism. In 24-48 h exposure groups, 18 genes were differentially regulated, mainly comprising components of the PPAR signaling pathway. These results suggested that UVI3003 is teratogenic in amphibian embryos. Differential gene expression suggests that galactose metabolism and PPAR signaling pathways may provide underlying mechanistic detail accounting for the observed malformations.

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.

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

Xenopus tropicalis embryos were exposed for 48 h 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 1-30 µg/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 15 µg/L LGD1069 and 1-10 µg Sn/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.

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

Tributyltin (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.

Loss of REEP4 causes paralysis of the Xenopus embryo.

Members of the REEP (Receptor expression enhancing protein) family contain a TB2/DP1, HVA22 domain that is involved in intracellular trafficking and secretion. Consistent with the presence of this domain, REEP1 and REEP3 enhance the expression of odorant and taste receptors in mammals, while mutation of these genes causes defects in neural development. REEP4 was identified in the course of a functional antisense morpholino oligonucleotide screen searching for genes involved in the early development of Xenopus tropicalis: although over-expression of the gene causes no phenotype, embryos lacking REEP4 develop a slightly kinked body axis and are paralysed. At tailbud stages of development, REEP4 is expressed in the somites and neural tube. The paralysis observed in embryos lacking REEP4 might therefore be caused by defects in the nervous system or in muscle. To address this point, we examined the expression of various neural and muscle markers and found that although all are expressed normally at early stages of development, many are down regulated by the tailbud stage. This suggests that REEP4 plays a role in the maintenance of both the nervous system and the musculature.

Overexpression of receptor-type protein tyrosine phosphatase beta causes abnormal development of the cranial nerve in Xenopus embryos.

Roles of receptor-type protein tyrosine phosphatase beta (RPTPbeta, also called PTPzeta) were investigated in the nervous system development of Xenopus embryos. We previously showed that Xenopus embryos express mRNAs for 11 receptor-type (XRPTPbeta.1-XRPTPbeta.11) and two secretory (sXRPTPbeta.1 and sXRPTPbeta.2) variants generated by alternative RNA splicing. Whole-mount in situ hybridization analyzes demonstrated central nervous system-specific gene transcription in tailbud embryos. Distributions of mRNAs for receptor-type and secretory variants partially differ in the hindbrain. Overexpression of XRPTPbeta.4 or sXRPTPbeta.2, which was brought about by microinjection of the recombinant plasmid vectors, caused abnormal development of the cranial nerve X. Deletion of the cytoplasmic segment from XRPTPbeta.4 did not affect the ability to cause the abnormality, but deletion of the extracellular segment abolished it. These results suggest that normal development of the cranial nerve X requires regulated expression of the XRPTPbeta gene products.

Generation of body plan phenotypes in early embryogenesis.

We have presented a number of simple methods that can be used to interfere in the normal establishment and subsequent development of dorsal axial structures in Xenopus. It should be emphasized that, despite the striking similarity in phenotypes which result from these treatments, different developmental processes are being affected at the different windows of sensitivity. For example, UV light, known to damage RNA (for review, see Kalthoff, 1979), also disrupts microtubule polymerization. These activities may be important at different developmental times, relating to the distribution of maternal determinants (in the oocyte) or to the coordinated assembly of cortical microtubules (in the just-fertilized egg). The ventralizing and dorsalizing effects of the various late-acting agents (e.g., TB, suramin, RA, GV sap) undoubtedly stem from their interference with cellular behaviors during the critical morphogenetic period of gastrulation.