Bioinformatics Methods for Transcriptome Analysis on Teratogenesis Testing.

Teratogenesis testing can be challenging due to the limitations of both in vitro and in vivo models. Test-systems, based especially on human embryonic cells, have been helping to overcome the difficulties when allied to omics strategies, such as transcriptomics. In these test-systems, cells exposed to different compounds are then analyzed in microarray or RNA-seq platforms regarding the impacts of the potential teratogens in the gene expression. Nevertheless, microarray and RNA-seq dataset processing requires computational resources and bioinformatics knowledge. Here, a pipeline for microarray and RNA-seq processing is presented, aiming to help researchers from any field to interpret the main transcriptome results, such as differential gene expression, enrichment analysis, and statistical interpretation. This chapter also discusses the main difficulties that can be encountered in a transcriptome analysis and the better alternatives to overcome these issues, describing both programming codes and user-friendly tools. Finally, specific issues in the teratogenesis field, such as time-course analysis, are also described, demonstrating how the pipeline can be applied in these studies.

Teratogenesis and the epigenetic programming of congenital defects: Why paternal exposures matter.

Until recently, clinicians and researchers did not realize paternal exposures could impact child developmental outcomes. Indeed, although there is growing recognition that sperm carry a large amount of non-genomic information and that paternal stressors influence the health of the next generation, toxicologists are only now beginning to explore the role paternal exposures have in dysgenesis and the incidence of congenital malformations. In this commentary, I will briefly summarize the few studies describing congenital malformations resulting from preconception paternal stressors, argue for the theoretical expansion of teratogenic perspectives into the male preconception period, and discuss some of the challenges in this newly emerging branch of toxicology. I argue that we must consider gametes the same as any other malleable precursor cell type and recognize that environmentally-induced epigenetic changes acquired during the formation of the sperm and oocyte hold equal teratogenic potential as exposures during early development. Here, I propose the term epiteratogen to reference agents acting outside of pregnancy that, through epigenetic mechanisms, induce congenital malformations. Understanding the interactions between the environment, the essential epigenetic processes intrinsic to spermatogenesis, and their cumulative influences on embryo patterning is essential to addressing a significant blind spot in the field of developmental toxicology.

Contribution of miR-124 rs531564 polymorphism to the occurrence of congenital Zika syndrome.

Zika virus (ZIKV) cause Congenital Zika Syndrome (CZS) in individuals exposed during pregnancy. Studies have shown that ZIKV infection positively regulates the miR-124 expression in neural cells, which leads to a decrease of TFRC, a gene targeted of this miRNA. Both miR-124 and TFRC exhibit a pivotal role in nervous system development. Therefore, in this study we aimed to investigate whether genetic variants that affect the expression of these genes could act together with ZIKV to increase the risk of individuals developing CZS. TFRC rs406271 and MIR-124-1 rs531564 polymorphisms were genotyped, using TaqMan® Genotyping Assays, in a sample of children who were exposed to ZIKV during pregnancy, of whom 40 were born with CZS and 48 without congenital anomalies. We identified that individuals with CZS presented a higher frequency of CG genotype of rs531564 polymorphism in MIR-124-1 (p=0.048), which is associated with increased expression of miR-124. Since ZIKV also upregulates the expression of this miRNA, the presence of CG genotype in individuals exposed to the virus could lead to a scenario of overexpression of miR-124 in the brain. Since teratogenesis is a multifactorial event, this genetic finding could partly explain why such individuals are more susceptible to CZS, considering both the downregulation of important neurodevelopment genes, as well as deregulation of the neurogenesis process. Thus, we provide preliminary evidence about a possible genetic risk factor to CZS and highlight the importance of analyzing functional polymorphisms related to epigenetic modulators of neurodevelopment genes in the context of ZIKV teratogenesis.

Toxic and Teratogenic Effects of Prenatal Alcohol Exposure on Fetal Development, Adolescence, and Adulthood.

Prenatal alcohol exposure (PAE) can have immediate and long-lasting toxic and teratogenic effects on an individual's development and health. As a toxicant, alcohol can lead to a variety of physical and neurological anomalies in the fetus that can lead to behavioral and other impairments which may last a lifetime. Recent studies have focused on identifying mechanisms that mediate the immediate teratogenic effects of alcohol on fetal development and mechanisms that facilitate the persistent toxic effects of alcohol on health and predisposition to disease later in life. This review focuses on the contribution of epigenetic modifications and intercellular transporters like extracellular vesicles to the toxicity of PAE and to immediate and long-term consequences on an individual's health and risk of disease.

Teratogenic Toxicity Evaluation of Bladder Cancer-Specific Oncolytic Adenovirus on Mice.

BACKGROUND: In our previous studies, we had demonstrated the efficiency and specificity of constructed bladder tissue-specific adenovirus Ad-PSCAE-UPII-E1A-AR (APU-EIA-AR) on bladder cancer. The virus biodistribution and body toxicity in nude mice have also been investigated. However, the safety of the bladder cancer-specific oncolytic adenovirus on fetal mice and F1 mice should be under intense investigation. OBJECTIVE: In order to evaluate the teratogenic toxicity of bladder cancer-specific oncolytic adenovirus APU-EIA-AR on mice, in this study, we investigated the fetal mice weight, fetal body length and tail length, fetal skeleton development, as well as the F1 mice weight, growth curve, and major organ pathology. These teratogenic toxicity data of bladder tissue-specific adenovirus Ad-PSCAE- UPII-E1A-AR (AD) would provide safe information prior to embarking on clinical trials. METHODS: On the sixth day of being fertilized, the pregnant mice began to be intramuscularly administrated with AD (1×107VP, 1×108VP, 1×109VP) every other day for ten days. The pregnant mice were then divided into two groups. One group was euthanized on the seventeenth day; the fetal mice were taken out, and the bone structure of the infants was observed. The other group was observed until natural childbirth. The Filial Generation (F1) is fed for 30 days; the variations in the growth progress and development were assessed. The mice were then euthanized; The tissues from major organs were harvested and observed under the microscope. RESULTS: In the process of teratogenic toxicity test, the Placenta weight, fetal mice weight, body length, and a tail length of mice fetal in adenovirus treated group did not reveal any alteration. Meanwhile, comparing with the PBS group, there is no obvious change in the skeleton of fetal mice treated with adenovirus. During the development process of F1 mice treated with adenovirus, the changes in mice weight show statistical significance. However, in the progress of the growth curve, this difference is not very obvious. Furthermore, the pathological section showed no obvious alteration in major organs. CONCLUSION: Our study demonstrated that bladder cancer-specific adenovirus Ad-PSCAE-UPII- E1A-AR appears safe in pregnant mice without any discernable effects on fetal mice and F1 development. Hence, it is relatively safe for tumor gene therapy.

Elimination of Teratogenic Human Induced Pluripotent Stem Cells by Bee Venom via Calcium-Calpain Pathway.

Induced pluripotent stem cells (iPSCs) are regarded as a promising option for cell-based regenerative medicine. To obtain safe and efficient iPSC-based cell products, it is necessary to selectively eliminate the residual iPSCs prior to in vivo implantation due to the risk of teratoma formation. Bee venom (BV) has long been used in traditional Chinese medicine to treat inflammatory diseases and relieve pain, and has been shown to exhibit anti-cancer, anti-mutagenic, anti-nociceptive, and radioprotective activities. However, the potential benefits of BV in iPSC therapy, particularly its anti-teratoma activity, have not been examined. In this study, we found that BV selectively induced cell death in iPSCs, but not in iPSC-derived differentiated cells (iPSCs-Diff). BV rapidly disrupted cell membrane integrity and focal adhesions, followed by induction of apoptosis and necroptosis in iPSCs. We also found that BV remarkably enhanced intracellular calcium levels, calpain activation, and reactive oxygen speciesgeneration in iPSCs. BV treatment before in ovo grafting efficiently prevented iPSC-derived teratoma formation. In contrast, no DNA damage was observed in iPSCs-Diff following BV treatment, further demonstrating the safety of BV for use with iPSCs-Diff. Taken together, these findings show that BV has potent anti-teratoma activity by eliminating residual iPSCs, and can be used for the development of effective and safe iPSC-based cell therapies.

The role of ESCO2, SALL4 and TBX5 genes in the susceptibility to thalidomide teratogenesis.

Thalidomide is widely used for several diseases; however, it causes malformations in embryos exposed during pregnancy. The complete understanding of the mechanisms by which thalidomide affects the embryo development has not yet been obtained. The phenotypic similarity makes TE a phenocopy of syndromes caused by mutations in ESCO2, SALL4 and TBX5 genes. Recently, SALL4 and TBX5 were demonstrated to be thalidomide targets. To understand if these genes act in the TE development, we sequenced them in 27 individuals with TE; we verified how thalidomide affect them in human pluripotent stem cells (hPSCs) through a differential gene expression (DGE) analysis from GSE63935; and we evaluated how these genes are functionally related through an interaction network analysis. We identified 8 variants in ESCO2, 15 in SALL4 and 15 in TBX5. We compared allelic frequencies with data from ExAC, 1000 Genomes and ABraOM databases; eight variants were significantly different (p < 0.05). Eleven variants in SALL4 and TBX5 were previously associated with cardiac diseases or malformations; however, in TE sample there was no association. Variant effect prediction tools showed 97% of the variants with potential to influence in these genes regulation. DGE analysis showed a significant reduction of ESCO2 in hPSCs after thalidomide exposure.

Hox Genes in Reptile Development, Epigenetic Regulation, and Teratogenesis.

Reptiles are ancestral organisms presenting a variety of shapes, from the elongated vertebral column of the snake to the turtle dorsalized ribs or retractile neck. Body plans are specified by a conserved group of homeobox-containing genes (Hox genes), which encode transcription factors important in cell fate and vertebral architecture along the anteroposterior axis during embryonic development; thus, dysregulation of these genes may cause congenital malformations, from mild-sublethal to embryonic-lethal. The genetic pool, maternal transfer, and environmental conditions during egg incubation affect development; environmental factors such as temperature, moisture, oxygen, and pollution may alter gene expression by epigenetic mechanisms. Thus, in this review, we present information regarding Hox genes and development in reptiles, including sex determination and teratogenesis. We also present some evidence of epigenetic regulation of Hox genes and the role of the environment in epigenetic modulation of gene expression. So far, the evidence suggests that the molecular instructions encoded by Hox genes to build a snake, a lizard, or a turtle represent the interplay between genome and epigenome after years of evolution, with occasional environmentally induced molecular mistakes leading to abnormal body shapes.

Geometric Morphometrics as a Tool to Evaluate Teratogenic Effects in Zebrafish (Danio rerio).

Geometric morphometrics allows for the characterization of shape using Cartesian geometric coordinates rather than linear or volumetric measurements, which are dependent upon size and are insufficient to capture geometric shape. By using landmarks on specimens, variations in position, orientation and scale between specimens can be removed to better compare variations in shape. This method has primarily been used in the fields of evolutionary biology and taxonomy. Here we describe how geometric morphometrics can be used to delineate variations in shape caused by teratogenic compounds in zebrafish.

Omics in Zebrafish Teratogenesis.

The genome revolution represents a complete change on our view of biological systems. The quantitative determination of changes in all major molecular components of the living cells, the "omics" approach, opened whole new fields for all health sciences. Genomics, transcriptomics, proteomics, metabolomics, and others, together with appropriate prediction and modeling tools, will mark the future of developmental toxicity assessment both for wildlife and humans. This is especially true for disciplines, like teratology, which rely on studies in model organisms, as studies at lower levels of organization are difficult to implement. Rodents and frogs have been the favorite models for studying human reproductive and developmental disorders for decades. Recently, the study of the development of zebrafish embryos (ZE) is becoming a major alternative tool to adult animal testing. ZE intrinsic characteristics makes this model a unique system to analyze in vivo developmental alterations that only can be studied applying in toto approaches. Moreover, under actual legislations, ZE is considered as a replacement model (and therefore, excluded from animal welfare regulations) during the first 5 days after fertilization. Here we review the most important components of the zebrafish toolbox available for analyzing early stages of embryotoxic events that could eventually lead to teratogenesis.

Valproic acid increases NF-κB transcriptional activation despite decreasing DNA binding ability in P19 cells, which may play a role in VPA-initiated teratogenesis.

The nuclear factor-kappa B (NF-κB) family of transcription factors regulate gene expression in response to diverse stimuli. We previously demonstrated that valproic acid (VPA) exposure in utero decreases total cellular protein expression of the NF-κB subunit p65 in CD-1 mouse embryos with a neural tube defect but not in phenotypically normal littermates. This study evaluated p65 mRNA and protein expression in P19 cells and determined the impact on DNA binding ability and activity. Exposure to 5mM VPA decreased p65 mRNA and total cellular protein expression however, nuclear p65 protein expression was unchanged. VPA reduced NF-κB DNA binding and nuclear protein of the p65 DNA-binding partner, p50. NF-κB transcriptional activity was increased with VPA alone, despite decreased phosphorylation of p65 at Ser276, and when combined with tissue necrosis factor α. These results demonstrate that VPA increases NF-κB transcriptional activity despite decreasing DNA binding, which may play a role in VPA-initiated teratogenesis.

Arrested Development: Infantile Hemangioma and the Stem Cell Teratogenic Hypothesis.

BACKGROUND: Early-life programming is defined by the adaptive changes made by the fetus in response to an adverse in utero environment. Infantile hemangioma (IH), a vascular anomaly, is the most common tumor of infancy. Here we take IH as the tumor model to propose the stem cell teratogenic hypothesis of tumorigenesis and the potential involvement of the immune system. OBJECTIVES: Teratogenic agents include chemicals, heavy metals, pathogens, and ionizing radiation. To investigate the etiology and pathogenesis of IH, we hypothesized that they result from a teratogenic mechanism. Immature, incompletely differentiated, dysregulated progenitor cells (multipotential stem cells) are arrested in development with vasculogenic, angiogenic, and tumorigenic potential due to exposure to teratogenic agents such as extrinsic factors that disrupt intrinsic factors via molecular mimicry. During the critical period of immunological tolerance, environmental exposure to immunotoxic agents may harness the teratogenic potential in the developing embryo or fetus and modify the early-life programming algorithm by altering normal fetal development, causing malformations, and inducing tumorigenesis. Specifically, exposure to environmental agents may interfere with physiological signaling pathways and contribute to the generation of IH, by several mechanisms. DISCUSSION: An adverse in utero environment no longer serves as a sustainable environment for proper embryogenesis and normal development. Targeted disruption of stem cells by extrinsic factors can alter the genetic program. CONCLUSIONS: This article offers new perspectives to stimulate discussion, explore novel experimental approaches (such as immunotoxicity/vasculotoxicity assays and novel isogenic models), and to address the questions raised to convert the hypotheses into nontoxic, noninvasive treatments.

Peroxisome Proliferator-Activated Receptor α Activation Is Not the Main Contributor to Teratogenesis Elicited by Polar Compounds from Oxidized Frying Oil.

We previously reported that polar compounds (PO) in cooking oil are teratogenic and perturbed retinoic acid (RA) metabolism. Considering PO as a potent peroxisome proliferator-activated receptor α (PPARα) activator, this study aimed to investigate the role of PPARα in PO-induced teratogenesis and disturbance of RA metabolism. Female PPARα knockout or wild type mice were mated with males of the same genotype. Pregnant mice were fed a diet containing 10% fat from either fresh oil (FO) or PO from gestational day1 to day18, and killed at day18. The PO diet significantly increased the incidence of teratogenesis and fetal RA concentrations, regardless of genotype. Though PPARα deficiency disturbed maternal RA homeostasis, itself did not contribute to teratogenesis as long as FO diet was given. The mRNA profile of genes involved in RA metabolism was differentially affected by diet or genotype in mothers and fetuses. Based on hepatic mRNA levels of genes involved in xenobiotic metabolism, we inferred that PO not only activated PPARα, but also altered transactivity of other xenobiotic receptors. We concluded that PO-induced fetal anomalies and RA accumulation were independent of PPARα activation.

Angiogenesis-related genes and thalidomide teratogenesis in humans: an approach on genetic variation and review of past in vitro studies.

Thalidomide embryopathy (TE) has affected more than 10,000 babies worldwide. The hypothesis of antiangiogenesis as the teratogenic mechanism of thalidomide has been investigated in several experimental models; but, in humans, it has only been accessed by in vitro studies. Here, we hypothesized the effect of thalidomide upon angiogenesis-related molecules or proteins, previously identified in human embryonic cells, through the in silico STRING-tool. We also investigated ten polymorphisms in angiogenesis-related genes in 38 Brazilian TE individuals and 136 non-affected Brazilians. NOS2, PTGS2, and VEGFA polymorphisms were chosen for genotyping. The STRING-tool suggested nitric oxide and ß-catenin as the central angiogenesis-related molecules affected by thalidomide's antiangiogenic property. We did not identify a significant difference of allelic, genotypic or haplotypic frequencies between the groups. We could not predict a risk allele or a protective one for TE in NOS2, PTGS2, or VEGFA, although other genes should be analyzed in larger samples. The role of nitric oxide and ß-catenin must be further evaluated, regarding thalidomide teratogenesis complex etiology.

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis.

Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells' vulnerability. Gallus gallus embryos having 3-6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10-17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10-12, 60 genes), RNA Polymerase (P = 2.2 x 10-3, 15 genes) and Spliceosome (P = 2.6 x 10-2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work suggests ribosome biogenesis may be a novel target mediating alcohol's damage to developing neural crest. Our findings are consistent with observations that gene-environment interactions contribute to vulnerability in FASD.

Embryonic hypocellularity, blastogenetic malformations, and fetal growth restriction.

An association between congenital malformations and fetal growth restriction (FGR) can be largely explained by a relationship with early embryonic hypocellularity. The malformations include the VACTERL association, which is exceptional as a Mendelian syndrome, but is commonly associated with monozygotic twinning, maternal diabetes, and some forms of aneuploidy, all characterized by a small embryo early in development. Parsimony suggests that these different links to VACTERL are related to the hypocellularity as a single common factor, rather than as an expression of three independent pathogenetic processes. A distinct non-genetic pathogenesis is further supported by increased frequencies in the same conditions of a single umbilical artery (SUA), which is also unusual in Mendelian disorders. SUA often involves the atrophy of one artery, which may be facilitated by altered hemodynamics in a smaller embryo, providing a direct link to hypocellularity. Hypocellularity may also explain a possible connection between VACTERL and certain mitochondrial disorders, where reduced energy might slow early cell division and growth, reducing the size of the embryo. © 2016 Wiley Periodicals, Inc.

Immunomodulatory drugs disrupt the cereblon-CD147-MCT1 axis to exert antitumor activity and teratogenicity.

Immunomodulatory drugs (IMiDs), such as thalidomide and its derivatives lenalidomide and pomalidomide, are key treatment modalities for hematologic malignancies, particularly multiple myeloma (MM) and del(5q) myelodysplastic syndrome (MDS). Cereblon (CRBN), a substrate receptor of the CRL4 ubiquitin ligase complex, is the primary target by which IMiDs mediate anticancer and teratogenic effects. Here we identify a ubiquitin-independent physiological chaperone-like function of CRBN that promotes maturation of the basigin (BSG; also known as CD147) and solute carrier family 16 member 1 (SLC16A1; also known as MCT1) proteins. This process allows for the formation and activation of the CD147-MCT1 transmembrane complex, which promotes various biological functions, including angiogenesis, proliferation, invasion and lactate export. We found that IMiDs outcompete CRBN for binding to CD147 and MCT1, leading to destabilization of the CD147-MCT1 complex. Accordingly, IMiD-sensitive MM cells lose CD147 and MCT1 expression after being exposed to IMiDs, whereas IMiD-resistant cells retain their expression. Furthermore, del(5q) MDS cells have elevated CD147 expression, which is attenuated after IMiD treatment. Finally, we show that BSG (CD147) knockdown phenocopies the teratogenic effects of thalidomide exposure in zebrafish. These findings provide a common mechanistic framework to explain both the teratogenic and pleiotropic antitumor effects of IMiDs.

ASK1 mediates the teratogenicity of diabetes in the developing heart by inducing ER stress and inhibiting critical factors essential for cardiac development.

Maternal diabetes in mice induces heart defects similar to those observed in human diabetic pregnancies. Diabetes enhances apoptosis and suppresses cell proliferation in the developing heart, yet the underlying mechanism remains elusive. Apoptosis signal-regulating kinase 1 (ASK1) activates the proapoptotic c-Jun NH2-terminal kinase 1/2 (JNK1/2) leading to apoptosis, suggesting a possible role of ASK1 in diabetes-induced heart defects. We aimed to investigate whether ASK1 is activated in the heart and whether deleting the Ask1 gene blocks diabetes-induced adverse events and heart defect formation. The ASK1-JNK1/2 pathway was activated by diabetes. Deleting Ask1 gene significantly reduced the rate of heart defects, including ventricular septal defects (VSDs) and persistent truncus arteriosus (PTA). Additionally, Ask1 deletion diminished diabetes-induced JNK1/2 phosphorylation and its downstream transcription factors and endoplasmic reticulum (ER) stress markers. Consistent with this, caspase activation and apoptosis were blunted. Ask1 deletion blocked the increase in cell cycle inhibitors (p21 and p27) and the decrease in cyclin D1 and D3 and reversed diabetes-repressed cell proliferation. Ask1 deletion also restored the expression of BMP4, NKX2.5, and GATA5, Smad1/5/8 phosphorylation, whose mutations or deletion result in reduced cell proliferation, VSD, and PTA formation. We conclude that ASK1 may mediate the teratogenicity of diabetes through activating the JNK1/2-ER stress pathway and inhibiting cell cycle progression, thereby impeding the cardiogenesis pathways essential for ventricular septation and outflow tract development.

Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish.

Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.