SDHI Fungicide Toxicity and Associated Adverse Outcome Pathways: What Can Zebrafish Tell Us?

Succinate dehydrogenase inhibitor (SDHI) fungicides are increasingly used in agriculture to combat molds and fungi, two major threats to both food supply and public health. However, the essential requirement for the succinate dehydrogenase (SDH) complex-the molecular target of SDHIs-in energy metabolism for almost all extant eukaryotes and the lack of species specificity of these fungicides raise concerns about their toxicity toward off-target organisms and, more generally, toward the environment. Herein we review the current knowledge on the toxicity toward zebrafish (Brachydanio rerio) of nine commonly used SDHI fungicides: bixafen, boscalid, fluxapyroxad, flutolanil, isoflucypram, isopyrazam, penthiopyrad, sedaxane, and thifluzamide. The results indicate that these SDHIs cause multiple adverse effects in embryos, larvae/juveniles, and/or adults, sometimes at developmentally relevant concentrations. Adverse effects include developmental toxicity, cardiovascular abnormalities, liver and kidney damage, oxidative stress, energy deficits, changes in metabolism, microcephaly, axon growth defects, apoptosis, and transcriptome changes, suggesting that glycometabolism deficit, oxidative stress, and apoptosis are critical in the toxicity of most of these SDHIs. However, other adverse outcome pathways, possibly involving unsuspected molecular targets, are also suggested. Lastly, we note that because of their recent arrival on the market, the number of studies addressing the toxicity of these compounds is still scant, emphasizing the need to further investigate the toxicity of all SDHIs currently used and to identify their adverse effects and associated modes of action, both alone and in combination with other pesticides.

Thifluzamide exposure induced neuro-endocrine disrupting effects in zebrafish (Danio rerio).

Thifluzamide is widely used fungicide and frequently detected in aquatic system. In this study, the toxicity of fungicide thifluzamide to non-targeted aquatic organisms was investigated for neuroendocrine disruption potentials. Here, zebrafish embryos were exposed to a series of concentrations of thifluzamide for 6 days. The results showed that both the development of embryos/larvae and the behavior of hatched larvae were significantly affected by thifluzamide. Importantly, the decreased activity of acetylcholinesterase (AchE) and the increased contents of neurotransmitters such as serotonin (5-HT) and norepinephrine (NE), along with transcriptional changes of nervous system related genes were observed following 4 days exposure to thifluzamide. Besides, the decreased contents of triiodothyronine (T3) and thyroxine (T4) in whole body, as well as significant expression alteration in hypothalamic-pituitary-thyroid (HPT) axis associated genes were discovered in zebrafish embryos after 4 days of exposure to thifluzamide. Our results clearly demonstrated that zebrafish embryos exposed to thifluzamide could disrupt neuroendocrine, compromise behavior and induce developmental abnormality, suggesting impact of this fungicide on developmental programming in zebrafish.

Xenopus laevis tadpoles exposed to metamifop: Changes in growth, behavioral endpoints, neurotransmitters, antioxidant system and thyroid development.

Pesticides are a major cause of the reduction in the global amphibian population. In this study, the acute toxicity and chronic effects of metamifop on Xenopus laevis (X. laevis) tadpoles were investigated. The 96 h-LC50 value of metamifop on X. laevis tadpoles was 0.634 mg/L, which indicated that metamifop was highly toxic to tadpoles. In the chronic toxicity study, tadpoles were exposed to 0.063 mg/L of metamifop. After 14, 21 and 35 d of exposure, metamifop significantly inhibited the body weight and neurotransmitter synthesis of tadpoles, caused abnormal behavior and interfered with fat metabolism. According to the results of antioxidant enzymes and malondialdehyde (MDA), tadpoles exposed to 0.063 mg/L metamifop suffered severe lipid oxidative damage. Compared with the control group, the thyroid hormone (TH) levels and related gene expression in tadpoles in the treatment group were affected, reflecting the endocrine interference effect of metamifop. The data of this study can enrich our knowledge of the effects of aryloxyphenoxy propionate pesticides on amphibians and highlight the role of metamifop and other pesticides play in global decline of amphibians.

Next generation risk assessment of human exposure to anti-androgens using newly defined comparator compound values.

Next Generation Risk Assessment (NGRA) can use the so-called Dietary Comparator Ratio (DCR) to evaluate the safety of a defined exposure to a compound of interest. The DCR compares the Exposure Activity Ratio (EAR) for the compound of interest, to the EAR of an established safe level of human exposure to a comparator compound with the same putative mode of action. A DCR ≤ 1 indicates the exposure evaluated is safe. The present study aimed at defining adequate and safe comparator compound exposures for evaluation of anti-androgenic effects, using 3,3-diindolylmethane (DIM), from cruciferous vegetables, and the anti-androgenic drug bicalutamide (BIC). EAR values for these comparator compounds were defined using the AR-CALUX assay. The adequacy of the new comparator EAR values was evaluated using PBK modelling and by comparing the generated DCRs of a series of test compound exposures to actual knowledge on their safety regarding in vivo anti-androgenicity. Results obtained supported the use of AR-CALUX-based comparator EARs for DCR-based NGRA for putative anti-androgenic compounds. This further validates the DCR approach as an animal free in silico/in vitro 3R compliant method in NGRA.

Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bones.

In childhood medulloblastoma patients, the hedgehog antagonist vismodegib is an effective anti-cancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients. We hypothesized that radial shock wave treatment (rSWT) may protect drug-induced growth impairment owing to its osteogenic effects. Fetal rat metatarsal bones were exposed to vismodegib (day 0-5; 100 nM) and/or rSWT (single session); other bones from day 1 were continuously exposed to a Gli1 antagonist (GANT61; 10 µM) and/or rSWT (single session). Control bones were untreated. The bone length was measured at intervals; histomorphometric analysis and immunostaining for PCNA, Gli1, and Ihh were performed on the sectioned bones. Bones treated with vismodegib showed impaired bone growth, reduced height of the resting-proliferative zone and reduced hypertrophic cell size compared to control. In vismodegib treated bones, a single session of rSWT partially rescued bone growth, increased the growth velocity, hypertrophic cell size, and restored growth plate morphology. Bones exposed to GANT61 showed impaired bone growth and disorganized growth plate while when combined with rSWT these effects were partially prevented. Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegib.

Chronic exposure of zebrafish (Danio rerio) to flutolanil leads to endocrine disruption and reproductive disorders.

The pesticide flutolanil has been detected in fish and aquatic environments, but its potential impact on the endocrine function is unknown. In this study, two-month zebrafish were exposed to the environmentally relevant concentrations of flutolanil for 60 days to examine the reproductive endpoints on the gonad endocrine system. Increased 17 beta-estradiol (17ß-E2) content and 17ß-E2/T ratio and decreased testosterone (T) in male suggested that flutolanil produces the estrogenic effect. In support of this view, vitellogene (vtg1, vtg2) and cytochrome P450 aromatase 19a (cyp19a) expression were up-regulated in the male liver. The levels of global DNA methylation were increased in ovary. Parental zebrafish exposure to different concentrations of flutolanil affected the offspring development as shown by short body length, and increased mortality. Thus, these results demonstrate that flutolanil exposure results in gonad endocrine disruption, decreased reproduction, and developmental toxicity in F1, highlighting the importance of assessing the potential environmental risks of flutolanil application.

Effects of fertilization on the triafamone photodegradation in aqueous solution: Kinetic, identification of photoproducts and degradation pathway.

Triafamone is a highly effective, low toxicity sulfonamide herbicide widely used for weeding paddy fields. The triafamone photodegradation in water environment must be explored for its ecological risk assessment. In this work, the effects of chemical fertilizer (urea, diammonium phosphate, potassium chloride, and potassium sulfate), urea metabolites (CO32- and HCO3-), and organic fertilizers (unfermented organic fertilizer [UOF] and fermented organic fertilizer [FOF]) on the triafamone photodegradation in aqueous solution under simulated sunlight were evaluated. Results showed that the triafamone photodegradation rate was unaffected by urea. The half-life of triafamone decreased from 106.8 h to 68.4 h with increasing diammonium phosphate concentration. Potassium chloride, potassium sulfate, CO32-, and HCO3- could accelerate the triafamone photodegradation at all concentrations, whereas the degradation rate of triafamone decreased when the concentration of potassium sulfate or CO32- was 2000 mg/L. Triafamone photodegradation was promoted by 20-200 mg/L UOF and FOF but decreased to 236.6 and 142.3 h when the concentration reached 2000 mg/L. Twenty-three transformation products were isolated and identified from triafamone by using ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry under simulated sunlight irradiation, and the kinetic evolution of these products was explored. Five possible degradation pathways were inferred, including the cleavage of C-N, C-C, and C-O bonds; CO bond hydrogenation; the cleavage of triazine ring; the cleavage of the sulfonamide bridge; hydroxylation; hydroxyl substitution; methylation; demethylation; amination; and rearrangement. In summary, these results are important for elucidating the environmental fate of triafamone in aquatic systems and further assessing environmental risks.

Enhanced and Sustained Cutaneous Delivery of Vismodegib by Ablative Fractional Laser and Microemulsion Formulation.

Oral administration of vismodegib for basal cell carcinoma treatment is limited by significant class-specific systemic side effects. We investigated the approach of combining ablative fractional laser-assisted drug delivery with an extended-release microemulsion formulation of vismodegib to provide efficient cutaneous delivery in vivo. The developed formulation consisted of an oil-in-water microemulsion stabilized by Tween-80. Pig skin was exposed to ablative fractional laser followed by topical application of vismodegib microemulsion for 4 hours. At 4 hours, 2 days, 5 days, and 9 days, we evaluated vismodegib biodistribution in superficial, mid, and deep dermis and plasma (n = 189 measurements) and assessed local skin reactions. Sustained topical delivery of vismodegib was detected in all depths of ablative fractional laser-exposed skin over the course of the study, with peak concentrations found at 5 days and 9 days. The highest vismodegib concentrations reached 1,409.7 µmol/liter in superficial dermis and 62.3 µmol/liter in deep dermis, exceeding steady-state plasma concentrations previously reported for oral administration of vismodegib (5.5-56.0 µmol/liter). Ablative fractional laser increased vismodegib uptake up to 16.6-fold compared with intact skin. Only mild local skin responses to vismodegib were observed, and no vismodegib was detected in plasma. We report sustained topical delivery of vismodegib in vivo at high concentrations with favorable skin tolerability, suggesting a future safer vismodegib treatment.

Toxicity of thifluzamide in earthworm (Eisenia fetida).

An increase in the area treated with the fungicide thifluzamide has triggered concerns for soil ecosystem service providers such as earthworms. Here, we assessed effects of thifluzamide on earthworm (Eisenia fetida) biomarker indicators of stress responses and reproduction following exposure to 0, 0.1, 1.0, and 10.0 mg of thifluzamide kg-1 soil for 7, 14, 21, and 28 d (biomarker indicators) and 30 d (reproduction). Growth and reproduction were inhibited by exposure to thifluzamide at 10.0 mg/kg, and the activities of succinate dehydrogenase (SDH) and respiratory chain complex II were inhibited by exposure to 1.0 and 10.0 mg/kg thifluzamide for the majority of the 28-d experiment. Reactive oxygen species (ROS) increased across all thifluzamide treatments, and the activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) tended to be inhibited by thifluzamide. Upon exposure to thifluzamide, the activities of catalase (CAT) and guaiacol peroxidase (POD) initially increased and then decreased. Increased levels of malondialdehyde (MDA) were detected only at seven days after exposure, and genotoxicity increased as the thifluzamide concentration increased. The results suggest that thifluzamide presents a potential risk to earthworms at the concentration of 10.0 mg/kg, and its use should be moderated to reduce damage to soil ecosystem function.

Molecular basis for the resistance of American sloughgrass to aryloxyphenoxypropionic acid pesticides and its environmental relevance: A combined experimental and computational study.

Organic pesticides are one of the main environmental pollutants, and how to reduce their environmental risks is an important issue. In this contribution, we disclose the molecular basis for the resistance of American sloughgrass to aryloxyphenoxypropionic acid pesticides using site-directed mutagenesis and molecular modeling and then construct an effective screening model. The results indicated that the target-site mutation (Trp-1999-Leu) in acetyl-coenzyme A carboxylase (ACCase) can affect the effectiveness of the pesticides (clodinafop, fenoxaprop, cyhalofop, and metamifop), and the plant resistance to fenoxaprop, clodinafop, cyhalofop, and metamifop was found to be 564, 19.5, 10, and 0.19 times, respectively. The established computational models (i.e. wild-type/mutant ACCase models) could be used for rational screening and evaluation of the resistance to pesticides. The resistance induced by target gene mutation can markedly reduce the bioreactivity of the ACCase-clodinafop/fenoxaprop adducts, and the magnitudes are 10 and 102, respectively. Such event will seriously aggravate environmental pollution. However, the biological issue has no distinct effect on cyhalofop (RI＝10), and meanwhile it may markedly increase the bioefficacy of metamifop (RI＝0.19). We could selectively adopt the two chemicals so as to decrease the residual pesticides in the environment. Significantly, research findings from the computational screening models were found to be negatively correlated with the resistance level derived from the bioassay testing, suggesting that the screening models can be used to guide the usage of pesticides. Obviously, this story may shed novel insight on the reduction of environmental risks of pesticides and other organic pollutants.

Short-term developmental toxicity and potential mechanisms of the herbicide metamifop to zebrafish (Danio rerio) embryos.

Metamifop is a novel aryloxyphenoxy propionate (AOPP) herbicide that is widely applied in paddy fields, which will inevitably enter aquatic environments and pose a risk to aquatic organisms. However, the potential threat and toxicological mechanisms of metamifop in aquatic organisms are poorly understood. In this study, zebrafish embryos were used to investigate the potential developmental toxicity and mechanisms of metamifop. The results showed that metamifop exhibited high acute toxicity to zebrafish, with 96 h-LC50 values of 0.648 and 0.216 mg/L to embryos and larvae of 72 h post-hatching (hph), respectively. Decreased body lengths, heartbeat number, and hatching rates, and increased malformation rates of embryos were observed after 96 h of exposure to 0.38 mg/L or higher concentration of metamifop. Furthermore, oxidative stress was caused in embryos, with increased contents of reactive oxygen species (ROS) and malondialdehyde (MDA), and altered activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Metamifop exposure clearly triggered cell apoptosis in embryos, result in the increase of Caspase-3 and Caspase-9 activities and up-regulation of apoptosis-related genes (bax, p53, apaf1, caspase-3, and caspase-9). Additionally, the transcriptions of innate immune-related genes (il-8, il-1b, and ifn) were increased in the groups treated with 0.25 and 0.5 mg/L of metamifop. These results indicate that metamifop induced developmental toxicity in zebrafish, and the potential toxicological mechanisms were related to oxidative stress, cell apoptosis, and the innate immune responses in embryos.

Dysregulation of circadian rhythm in zebrafish (Danio rerio) by thifluzamide: Involvement of positive and negative regulators.

Thifluzamide as a fungicide is toxic to brain of zebrafish embryos. Brain can regulate biological rhythms. To clarify whether thifluzamide would influence circadian rhythms, zebrafish embryos were treated with thifluzamide (0, 0.19, 1.90 and 2.85 mg/L) for 4 days. Exposure to thifluzamide induced pronounced changes in embryo brain and melatonin levels. The mRNA levels of genes related to circadian rhythms were apparently altered. Among these, the transcripts of cry1ba and clock1 were extremely correlated with exposure concentrations. Importantly, the content of cry1 showed no apparent changes, but the clock level was dramatically increased. Moreover, consistent with the inhibition of development and behavior, the levels of GH and DA were significantly inhibited. In addition, the expression levels of genes related to development, behavior and reproduction were significantly changed by thifluzamide. Therefore, we speculated that circadian disruption due to thifluzamide exposure were primarily attributed to increases in expression of clock1a and contents of clock, which might be at least in part responsible for abnormal development and behavior of zebrafish. In addition, our research will provide important insights into the grouped assessment of SDHI pesticides in future.

Flutolanil affects circadian rhythm in zebrafish (Danio rerio) by disrupting the positive regulators.

Circadian rhythms are fundamental to behavior and physiology of organisms. Flutolanil as a fungicide is toxic to zebrafish embryos. The aims of this study were to determine whether flutolanil would influence circadian rhythms of zebrafish and the mechanism involved. Zebrafish embryos were exposed to flutolanil (0, 0.125, 0.5 and 2 mg/L) for 4 days. Here we report that flutolanil increased the melatonin levels of zebrafish. The mRNA levels of genes related to circadian rhythms were significantly altered. The clock level was significantly increased, but the content of cry1 showed no apparent changes. Moreover, our findings that the level of GH was significantly decreased were consistent with the abnormal development of zebrafish embryos. The expression levels of genes related to development, behavior and reproduction were significantly altered by flutolanil. These results indicate that flutolanil disturbed circadian rhythms of zebrafish primarily by affecting the positive elements, which were at least in partial responsible for abnormal development and behavior of zebrafish. And we speculate that flutolanil is toxic to zebrafish embryos at least in part via dysregulation of circadian rhythms involving clock.

Chronic Toxic Effects of Flutolanil on the Liver of Zebrafish ( Danio rerio).

Flutolanil is a broad-spectrum amide fungicide that is widely used to prevent fungal pathogens in agriculture. However, its usage may have a potential environmental impact on organisms. So far, few literatures have investigated the chronic toxicity of flutolanil at concentrations relevant to environmental conditions in the nontarget aquatic organisms. This study was aimed at evaluating whether the long-term exposure of flutolanil affects oxidative stress, immune response, and apoptosis in the liver of zebrafish ( Danio rerio). The results showed that the activity of catalase (CAT) was significantly decreased in the liver in all flutolanil-treated groups. Interestingly, the malondialdehyde (MDA) contents were remarkably increased following the flutolanil exposure. Deoxyribonucleic acid (DNA) damage was increased with a concentration-dependent manner. The transcription level of genes involved in apoptosis and the immune system were significantly altered following flutolanil chronic exposure in zebrafish liver. Furthermore, the caspase-3 enzyme activity was significantly increased. Taken together, this study demonstrated that the resulting effects on oxidative stress, immune toxicity, and apoptosis may be responsible for the pathological alterations in zebrafish liver after flutolanil exposure at concentrations relevant to environmental conditions, advancing the knowledge of pesticide environmental risk assessment.

Developmental toxicity by thifluzamide in zebrafish (Danio rerio): Involvement of leptin.

Although previous trials have indicated that thifluzamide induces developmental inhibition in zebrafish, understanding the distinct mechanism of thifluzamide in this process remains challenging. This study investigated the effect of thifluzamide on zebrafish development and the underlying related signaling pathway. Thifluzamide repressed glucagon (GC) levels but increased growth hormone (GH) levels, and changed the expression of the genes related to growth and development. Additionally, protein kinase A (PKA) and leptin levels were obviously decreased in zebrafish after exposure to thifluzamide for 28 days, but the phosphorylation of cAMP responsive element-binding protein (CREB) was increased. Our results suggested that the anti-developmental effects of thifluzamide in zebrafish are largely associated with alterations in expressions of genes related to growth and development through modulation of leptin.

Facile development, characterization, and evaluation of novel bicalutamide loaded pH-sensitive mesoporous silica nanoparticles for enhanced prostate cancer therapy.

It is a challenge to deliver therapeutics exclusively to cancer cells, while sparing the normal cells. However, pH-sensitive delivery systems have proved to be highly efficient in fulfilling this task due to their ability to provide on-demand and selective release of drug at acidic tumor sites. As a proof of concept, here pH responsive drug delivery system based on mesoporous core shell nanoparticles (NPs) surrounded with poly acrylic acid (PAA) layers were prepared employing a facile synthesis strategy. Bicalutamide (BIC) was encased into surface functionalized MCM-41 nanoparticles via electrostatic interactions. The synthesized NPs were characterized by nitrogen adsorption and desorption isotherms, SEM-EDS, TEM, LXRD, and WXRD. In vitro release studies demonstrated that BIC-MSN-PAA NPs exhibited a higher release in the acidic media which varied inversely with the increase in pH. Further, the results of cell cytotoxicity assay were evident that BICMSNs exhibited more potent killing of both PC-3 and LNCaP cells than free BIC. PAA-MSNs also exhibited an enhanced cellular uptake and prolonged circulation time in vivo. The results are suggestive of the fact that PAA functionalized MSNs can serve as an effective pH-responsive template and hold a great potential ahead in controlled release and effective cancer treatment.

Crosstalk of oxidative damage, apoptosis, and autophagy under endoplasmic reticulum (ER) stress involved in thifluzamide-induced liver damage in zebrafish (Danio rerio).

Although the hepatotoxicity of thifluzamide in zebrafish has been characterized, its toxic mechanisms have not been fully explored. The present study demonstrated that thifluzamide damaged the zebrafish liver and endoplasmic reticulum (ER). In addition, thifluzamide significantly changed the expression of genes encoding antioxidant proteins and increased the malondialdehyde (MDA) content, leading to oxidative damage in zebrafish liver. Additionally, the autophagic ultrastructure was observed by transmission electron microscopy (TEM), and LC3-I/LC3-II conversion was obviously upregulated under western blotting (WB) measurements, verifying that autophagy was induced by thifluzamide. Moreover, the activities of Caspase-3 and Caspase-9 were obviously decreased, indicating that apoptosis was inhibited in adult zebrafish exposed to a higher concentration of thifluzamide. In summary, oxidative damage and autophagy but not apoptosis under ER injury might lead to the hepatotoxicity of thifluzamide in zebrafish. Our findings provide a new mechanistic insight into the toxicity of thifluzamide in zebrafish.

Acute exposure of zebrafish embryo (Danio rerio) to flutolanil reveals its developmental mechanism of toxicity via disrupting the thyroid system and metabolism.

Flutolanil, an amide fungicide, had been detected frequently in aquatic environments; it is thus potentially a great risk to aquatic organisms and human health. Therefore, we investigated the developmental toxicity and the potential mechanism of thyroid endocrine disruption induced by flutolanil based on 1H NMR metabolomics analysis using a zebrafish model. Hatching of zebrafish embryo exposed to flutolanil was inhibited at 72 hpf (hour post-fertilization) and survival and body length at 96 hpf. In addition, increased teratogenic effects on embryos were observed, including pericardial edema, spine deformation, and tail malformation. Furthermore, flutolanil induced slower heartbeat and larger pericardial area in the treated groups than control group. Transcription levels of TRH, TSHR, TPO, Dio1, TRα, and UGT1ab were significantly altered after flutolanil exposure. Metabolomics analysis further indicated that flutolanil induced alterations of energy, amino acids, nucleotide, lipids, and fatty acid metabolism. Our study also indicated that flutolanil exposure led to alterations of endogenous metabolites, which induced the thyroid endocrine disruption in zebrafish. Ultimately, embryonic developmental toxicity was caused by flutolanil.

[Hedgehog pathway antagonist-induced oromandibular limb hypogenesis in mouse].

Objective: To analysis teratogenic effect of GDC-0449 to fetus and set up the animal model of GDC-0449 induced oromandibular limb hypogenesis in mouse for further research of its pathogenesis. Methods: Twenty-seven pregnant Institute of Cancer Research (ICR) mice were randomly divided into: control group, embryonic day 8.5 (E8.5) exposed groups, E9.5 exposed groups, E10.5 exposed groups, E11.5 exposed groups, E12.5 exposed groups, E13.5 exposed groups, E14.5 exposed groups and E15.5 exposed groups. Each group had 3 mice. Exposed groups were treated with the Hedgehog pathway antagonist GDC-0449 at a single dose 150 mg/kg by oral gavage from E8.5 to E15.5. At E16.5, embryonic phenotypes were analyzed in detail by stereo microscope and histology. After establish an optimal dysmorphogenic concentration, 6 pregnant ICR mice were randomly divided into control group and the optimal group, embryonic phenotypes were analyzed by whole-mount skeletal staining and micro-computed tomography at E18.5. Results: The mice were exposed to GDC-0449 on E11.5 and E12.5 had a high incidence of cleft palate. GDC-0449 exposed between E9.5 and E10.5 caused craniofacial and limb dysmorphology, including micrognathia, microglossia, ectrodactylia, partial anodontia and cleft palate. Most interestingly, these are extremely similar to oromandibular limb hypogenesis syndrome. Conclusions: The results of this study indicate that GDC-0449 can be used to induce micrognathia, microglossia, ectrodactylia, partial anodontia and cleft palate. This work established a novel mouse model for oromandibular limb hypogenesis.