Evaluation of temperature- and ethanol-related developmental degree variations by a new scoring system (FETAX-score) applicable to Frog Embryo Teratogenicity Assay: Xenopus.

The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2 % v/v. Gross malformations were observed only in tadpoles from the Eth 2 % group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies.

Unveiling vertebrate development dynamics in frog Xenopus laevis using micro-CT imaging.

BACKGROUND: Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism in various biological disciplines, prominently in developmental biology to study body plan reorganization during metamorphosis. However, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus' late developmental stages. FINDINGS: This study utilized micro-computed tomography (micro-CT), a noninvasive 3-dimensional (3D) imaging technique with micrometer-scale resolution, to explore the developmental dynamics and morphological changes in Xenopus laevis. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adults. This dataset enhances our understanding of vertebrate development and supports various analyses. We conducted a careful examination, analyzing body size, shape, and morphological features, focusing on skeletogenesis, teeth, and organs like the brain and gut at different stages. Our analysis yielded valuable insights into 3D morphological changes during Xenopus' development, documenting details previously unrecorded. These datasets hold the solid potential for further morphological and morphometric analyses, including segmentation of hard and soft tissues. CONCLUSIONS: Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus' development and the associated morphological changes in the future. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans can be used in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

Hepatic enzyme induction and its potential effect on thyroid hormone metabolism in the metamorphosing tadpole of Xenopus laevis (African clawed frog).

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-ß-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species.

Identification and functional characterization of a long-type peptidoglycan recognition protein, PGRP-L in amphibian Xenopus laevis.

Peptidoglycan recognition proteins (PGRPs) are a family of multifunctional proteins playing vital roles in PGN metabolism and antibacterial defense, and their functions have been well-characterized in mammals, bony fishes, and insects. However, the information about the functions of amphibian long-type PGRP is rather limited. Here, we identified and cloned a long-type PGRP gene (named Xl-PGRP-L) from African clawed frog, Xenopus laevis. Xl-PGRP-L gene was detected in all orangs/tissues examined, and was rapidly induced in intestine, liver, and lung following the stimulation of PGN. Sequence analysis showed that Xl-PGRP-L possesses four Zn2+-binding residues (His358, Tyr395, His470, and Cys478) required for amidase activity of catalytic PGRPs, and assays for amidase activity revealed that recombinant Xl-PGRP-L cloud degrade PGN in a Zn2+-dependent manner, indicating that Xl-PGRP-L is belonging to catalytic PGRPs. In addition, Xl-PGRP-L have antibacterial activity against Gram-negative bacteria Edwardsiella tarda and Gram-positive bacteria Streptococcus agalactiae. The present investigation represents the first characterization regarding the biological activities of amphibian long-type PGRPs, thus contributes to a better understanding of the functions of tetrapod PGRPs and the molecular mechanisms of amphibian antibacterial defense.

Rho-Associated Protein Kinase Activity Is Required for Tissue Homeostasis in the Xenopus laevis Ciliated Epithelium.

Lung epithelial development relies on the proper balance of cell proliferation and differentiation to maintain homeostasis. When this balance is disturbed, it can lead to diseases like cancer, where cells undergo hyperproliferation and then can undergo migration and metastasis. Lung cancer is one of the deadliest cancers, and even though there are a variety of therapeutic approaches, there are cases where treatment remains elusive. The rho-associated protein kinase (ROCK) has been thought to be an ideal molecular target due to its role in activating oncogenic signaling pathways. However, in a variety of cases, inhibition of ROCK has been shown to have the opposite outcome. Here, we show that ROCK inhibition with y-27632 causes abnormal epithelial tissue development in Xenopus laevis embryonic skin, which is an ideal model for studying lung cancer development. We found that treatment with y-27632 caused an increase in proliferation and the formation of ciliated epithelial outgrowths along the tail edge. Our results suggest that, in certain cases, ROCK inhibition can disturb tissue homeostasis. We anticipate that these findings could provide insight into possible mechanisms to overcome instances when ROCK inhibition results in heightened proliferation. Also, these findings are significant because y-27632 is a common pharmacological inhibitor used to study ROCK signaling, so it is important to know that in certain in vivo developmental models and conditions, this treatment can enhance proliferation rather than lead to cell cycle suppression.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Femoral bone structure and mechanics at the edge and core of an expanding population of the invasive frog Xenopus laevis.

Understanding how living tissues respond to changes in their mechanical environment is a key question in evolutionary biology. Invasive species provide an ideal model for this as they are often transplanted between environments that differ drastically in their ecological and environmental context. Spatial sorting, the name given to the phenomenon driving differences between individuals at the core and edge of an expanding range, has been demonstrated to impact the morphology and physiology of Xenopus laevis from the invasive French population. Here, we combined a structural analysis using micro-CT scanning and a functional analysis by testing the mechanical properties of the femur to test whether the increased dispersal at the range edge drives differences in bone morphology and function. Our results show significant differences in the inner structure of the femur as well as bone material properties, with frogs from the centre of the range having more robust and resistant bones. This is suggestive of an energy allocation trade-off between locomotion and investment in bone formation, or alternatively, may point to selection for fast locomotion at the range edge. Overall, our results provide insights on the growth of the long bones and the formation of trabecular bone in frogs.

Actin depolymerizing factor destrin governs cell migration in neural development during Xenopus embryogenesis.

The actin-based cytoskeleton is considered a fundamental driving force for cell differentiation and development. Destrin (Dstn), a member of the actin-depolymerizing factor family, regulates actin dynamics by treadmilling actin filaments and increasing globular actin pools. However, the specific developmental roles of dstn have yet to be fully elucidated. Here, we investigated the physiological functions of dstn during early embryonic development using Xenopus laevis as an experimental model organism. dstn is expressed in anterior neural tissue and neural plate during Xenopus embryogenesis. Depleting dstn promoted morphants with short body axes and small heads. Moreover, dstn inhibition extended the neural plate region, impairing cell migration and distribution during neurulation. In addition to the neural plate, dstn knockdown perturbed neural crest cell migration. Our data suggest new insights for understanding the roles of actin dynamics in embryonic neural development, simultaneously presenting a new challenge for studying the complex networks governing cell migration involving actin dynamics.

Ingestion of polyethylene terephthalate microplastic water contaminants by Xenopus laevis tadpoles negatively affects their resistance to ranavirus infection and antiviral immunity.

Small plastic debris (0.1 µm-5 mm) or microplastics (MPs) have become major pollutants of aquatic ecosystems worldwide and studies suggest that MPs exposure can pose serious threats to human and wildlife health. However, to date the potential biological impacts of MPs accumulating in low amount in tissues during early life remains unclear. Here, for a more realistic assessment, we have used environmentally representative, mildly weathered, polyethylene terephthalate microplastics (PET MPs), cryomilled (1-100 µm) and fluorescently labelled. We leveraged the amphibian Xenopus laevis tadpoles as an animal model to define the biodistribution of PET MPs and determine whether exposure to PET MPs induce perturbations of antiviral immunity. Exposure to PET MPs for 1-14 days resulted in detectable PET MPs biodistribution in intestine, gills, liver, and kidney as determined by fluorescence microscopy on whole mount tissues. PET MPs accumulation rate in tissues was further evaluated via a novel in situ enzymatic digestion and subsequent filtration using silicon nanomembranes, which shows that PET MPs rapidly accumulate in tadpole intestine, liver and kidneys and persist over a week. Longer exposure (1 month) of tadpoles to relatively low concentration of PET MPs (25 µg/ml) significantly increased susceptibility to viral infection and altered innate antiviral immunity without inducing overt inflammation. This study provides evidence that exposure to MPs negatively impact immune defenses of aquatic vertebrates.

Voltage Clamp Fluorometry: Illuminating the Dynamics of Ion Channels.

Ion channels comprise one of the largest targets for drug development and treatment and have been a subject of enduring fascination since first discovered in the 1950s. Over the past decades, thousands of publications have explored the cellular biology and molecular physiology of these proteins, and many channel structures have been determined since the late 1990s. Trying to connect the dots between ion channel function and structure, voltage clamp fluorometry (VCF) emerges as a powerful tool because it allows monitoring of the conformational rearrangements underlying the different functional states of the channel. This technique represents an elegant harmonization of molecular biology, electrophysiology, and fluorescence. In the following chapter, we will provide a concise guide to performing VCF on Xenopus laevis oocytes using the two-electrode voltage clamp (TEVC) modality. This is the most widely used configuration on Xenopus oocytes for its relative simplicity and demonstrated success in a number of different ion channels utilizing a variety of attached labels.

Comparing the effects of three neonicotinoids on embryogenesis of the South African clawed frog Xenopus laevis.

Neonicotinoids (NEOs) are widely used insecticides that are ubiquitous in agricultural use. Since NEOs are found in natural waters as well as in tap water and human urine in regions where NEOs are widely used, NEOs pose a potential hazard to non-target organisms such as animals and humans. Some of the commonly detected NEOs are imidacloprid (IMD), thiamethoxam (TMX), and its metabolite clothianidin (CLO). Although previously published scientific information, including an assessment of the environmental risks, particularly for bees, had resulted in a ban on the outdoor use of these three NEOs in the EU - their use is now only permitted in closed greenhouses - these NEOs continue to be used in agriculture in many other parts of the world. Therefore, a detailed study and comparison of the effects of NEOs on the embryonic development of non-target organisms is needed to further define the risk profiles. Embryos of the South African clawed frog Xenopus laevis, a well-established aquatic model, were exposed to different concentrations of IMD, TMX, or CLO (0.1-100 mg/L) to study and compare the possible effects of a single contaminant in natural water bodies on early embryogenesis. The results included a reduced body length, a smaller orbital space, impaired cranial cartilage and nerves, and an altered heart structure and function. At the molecular level, NEO exposure partially resulted in an altered expression of tissue-specific factors, which are involved in eye, cranial placode, and heart development. Our results suggest that the NEOs studied negatively affect the embryonic development of the non-target organism X. laevis. Since pesticides, especially NEOs, pollute the environment worldwide, it is suggested that they are strictly controlled and monitored in the areas where they are used. In addition, the question arises as to whether pesticide metabolites also pose a risk to the environment and need to be investigated further so that they can be taken into account when registering ingredients.

Ionizing radiation induces neurotoxicity in Xenopus laevis embryos through neuroactive ligand-receptor interaction pathway.

Ionizing radiation (IR) poses a significant threat to both the natural environment and biological health. Exposure to specific doses of ionizing radiation early in an organism's development can lead to developmental toxicity, particularly neurotoxicity. Through experimentation with Xenopus laevis (X. laevis), we examined the effects of radiation on early developmental stage. Our findings revealed that radiation led to developmental abnormalities and mortality in X. laevis embryos in a dose-dependent manner, disrupting redox homeostasis and inducing cell apoptosis. Additionally, radiation caused neurotoxic effects, resulting in abnormal behavior and neuron damage in the embryos. Further investigation into the underlying mechanisms of radiation-induced neurotoxicity indicated the potential involvement of the neuroactive ligand-receptor interaction pathway, which was supported by RNA-Seq analysis. Validation of gene expression associated with this pathway and analysis of neurotransmitter levels confirmed our hypothesis. In addition, we further validated the important role of this signaling pathway in radiation-induced neurotoxicity through edaravone rescue experiments. This research establishes a valuable model for radiation damage studying and provides some insight into radiation-induced neurotoxicity mechanisms.

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the ß-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

Electrochemical degradation of diclofenac generates unexpected thyroidogenic transformation products: Implications for environmental risk assessment.

Diclofenac (DCF) is an environmentally persistent, nonsteroidal anti-inflammatory drug (NSAID) with thyroid disrupting properties. Electrochemical advanced oxidation processes (eAOPs) can efficiently remove NSAIDs from wastewater. However, eAOPs can generate transformation products (TPs) with unknown chemical and biological characteristics. In this study, DCF was electrochemically degraded using a boron-doped diamond anode. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was used to analyze the TPs of DCF and elucidate its potential degradation pathways. The biological impact of DCF and its TPs was evaluated using the Xenopus Eleutheroembryo Thyroid Assay, employing a transgenic amphibian model to assess thyroid axis activity. As DCF degradation progressed, in vivo thyroid activity transitioned from anti-thyroid in non-treated samples to pro-thyroid in intermediately treated samples, implying the emergence of thyroid-active TPs with distinct modes of action compared to DCF. Molecular docking analysis revealed that certain TPs bind to the thyroid receptor, potentially triggering thyroid hormone-like responses. Moreover, acute toxicity occurred in intermediately degraded samples, indicating the generation of TPs exhibiting higher toxicity than DCF. Both acute toxicity and thyroid effects were mitigated with a prolonged degradation time. This study highlights the importance of integrating in vivo bioassays in the environmental risk assessment of novel degradation processes.

Assessing the impact of antiviral drugs commonly utilized during the COVID-19 pandemic on the embryonic development of Xenopus laevis.

The antiviral drugs favipiravir and oseltamivir are widely used to treat viral infections, including coronavirus 2019 (COVID-19), and their levels are expected to increase in the aquatic environment. In this study, the potential toxic and teratogenic effects of these drugs were evaluated using the frog embryo teratogenesis assay Xenopus (FETAX). In addition, glutathione S-transferase (GST), glutathione reductase (GR), catalase, carboxylesterase (CaE), and acetylcholinesterase (AChE) enzyme activities and malondialdehyde levels were measured as biochemical markers in embryos and tadpoles for comparative assessment of the sublethal effects of the test compounds. Prior to embryo exposure, drug concentrations in the exposure medium were measured with high-performance liquid chromatography. The 96-h median lethal concentration (LC50) was 137.9 and 32.3 mg/L for favipiravir and oseltamivir, respectively. The teratogenic index for favipiravir was 4.67. Both favipiravir and oseltamivir inhibited GR, CaE, and AChE activities in embryos, while favipiravir increased the GST and CaE activities in tadpoles. In conclusion, favipiravir, for which teratogenicity data are available in mammalian test organisms and human teratogenicity is controversial, inhibited Xenopus laevis embryo development and was teratogenic. In addition, sublethal concentrations of both drugs altered the biochemical responses in embryos and tadpoles, with differences between the developmental stages.

Toxicity of water-soluble polymers polyethylene glycol and polyvinyl alcohol for fish and frog embryos.

Personal Care Products (PCPs) have been one of the most studied chemicals in the last twenty years since they were identified as pseudo-persistent pollutants by the European Union in the early 2000s. The accumulation of PCPs in the aquatic environment and their effects on non-target species make it necessary to find new, less harmful, substances. Polyethylene glycol (PEGs) and polyvinyl alcohol (PVAs) are two polymers that have increased their presence in the composition of PCPs in recent years, but little is known about the effect of their accumulation in the environment on non-target species. Through embryotoxicity tests on two common models of aquatic organisms (Danio rerio and Xenopus laevis), this work aims to increase the knowledge of PEGs and PVAs' effects on non-target species. Animals were exposed to the pollutant for 96 h. The main embryotoxicity endpoint (mortality, hatching, malformations, heartbeat rate) was recorded every 24 h. The most significant results were hatching delay in Danio rerio exposed to both chemicals, in malformations (oedema, body malformations, changes in pigmentation and deformations of spine and tail) in D. rerio and X. laevis and significant change in the heartbeat rate (decrease or increase in the rate) in both animals for all chemicals tested.

Spontaneous Overactivation of Xenopus Frog Eggs Triggers Necrotic Cell Death.

The excessive activation of frog eggs, referred to as overactivation, can be initiated by strong oxidative stress, leading to expedited calcium-dependent non-apoptotic cell death. Overactivation also occurs spontaneously, albeit at a low frequency, in natural populations of spawned frog eggs. Currently, the cytological and biochemical events of the spontaneous process have not been characterized. In the present study, we demonstrate that the spontaneous overactivation of Xenopus frog eggs, similarly to oxidative stress- and mechanical stress-induced overactivation, is characterized by the fast and irreversible contraction of the egg's cortical layer, an increase in egg size, the depletion of intracellular ATP, a drastic increase in the intracellular ADP/ATP ratio, and the degradation of M phase-specific cyclin B2. These events manifest in eggs in the absence of caspase activation within one hour of triggering overactivation. Importantly, substantial amounts of ATP and ADP leak from the overactivated eggs, indicating that plasma membrane integrity is compromised in these cells. The rupture of the plasma membrane and acute depletion of intracellular ATP explicitly define necrotic cell death. Finally, we report that egg overactivation can occur in the frog's genital tract. Our data suggest that mechanical stress may be a key factor promoting egg overactivation during oviposition in frogs.

Bioconcentration, oxidative stress and molecular mechanism of the toxic effect of acetamiprid exposure on Xenopus laevis tadpoles.

Acetamiprid is a neonicotinoid commonly detected in aquatic ecosystems, with residual concentrations of up to 0.41 mg/L in surface water, posing a threat to the health of nontarget aquatic organisms. However, studies on the potential toxicity and underlying mechanisms of action of acetamiprid on nontarget aquatic organisms are limited. This study investigated the acute and short-term toxicity of acetamiprid to Xenopus laevis tadpoles. A 96-h acute toxicity test determined the LC50 of acetamiprid to be 32.1 mg/L. After 28 days of exposure to 1/10 and 1/100 LC50 concentrations, tadpole samples were collected for bioconcentration elimination analysis, biochemical analyses, transcriptomics, and metabolomics studies to comprehensively evaluate the toxic effects of acetamiprid and its underlying mechanisms. The results, indicating bioconcentration factors (BCFs) < 1, suggest that acetamiprid has a low bioconcentration in tadpoles. Additionally, oxidative stress was observed in treated Xenopus laevis tadpoles. Transcriptomic and nontargeted metabolomic analyses identified 979 differentially expressed genes (DEGs) and 95 differentially metabolites in the 0.321 mg/L group. The integrated analysis revealed that disruption of purine and amino acid metabolic pathways potentially accounts for acetamiprid-induced toxic effects in tadpoles. The disruptive effects of acetamiprid on valine, leucine and isoleucine biosynthesis; and aminoacyl-tRNA biosynthesis metabolic pathways in tadpoles were validated through targeted metabolomics analysis. These findings are crucial for assessing the risk of acetamiprid to nontarget aquatic organisms.

Accelerated protein retention expansion microscopy using microwave radiation.

Protein retention expansion microscopy (ExM) retains genetically encoded fluorescent proteins or antibody-conjugated fluorescent probes in fixed tissue and isotropically expands the tissue through a swellable polymer network to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite numerous advantages ExM brings to biological studies, the full protocol is time-consuming and can take multiple days to complete. Here, we adapted the ExM protocol to the vibratome-sectioned brain tissue of Xenopus laevis tadpoles and implemented a microwave-assisted protocol to reduce the workflow from days to hours. In addition to the significantly accelerated processing time, our microwave-assisted ExM (M/WExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, the M/WExM protocol yields higher magnitude of expansion, suggesting that in addition to accelerating the process through increased diffusion rate of reagents, microwave radiation may also facilitate the expansion process. To demonstrate the applicability of this method to other specimens and protocols, we adapted the microwave-accelerated protocol to whole mount adult brain tissue of Drosophila melanogaster fruit flies, and successfully reduced the total processing time of a widely-used Drosophila IHC-ExM protocol from 6 days to 2 days. Our results demonstrate that with appropriate adjustment of the microwave parameters (wattage, pulse duration, interval, and number of cycles), this protocol can be readily adapted to different model organisms and tissue types to greatly increase the efficiency of ExM experiments.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.