Deciphering the role of retinoic acid in hepatic patterning and induction in the mouse.

Retinoic acid (RA), a metabolite of vitamin A, is a small molecule and morphogen that is required for embryonic development. While normal RA signals are required for hepatic development in a variety of vertebrates, a role for RA during mammalian hepatic specification has yet to be defined. To examine the requirement for RA in murine liver induction, we performed whole embryo culture with the small molecule RA inhibitor, BMS493, to attenuate RA signaling immediately prior to hepatic induction and through liver bud formation. BMS493 treated embryos demonstrated a significant loss of hepatic specification that was confined to the prospective dorsal anterior liver bud. Examination of RA attenuated embryos demonstrates that while the liver bud displays normal expression of foregut endoderm markers and the hepato-pancreatobiliary domain marker, PROX1, the dorsal/anterior liver bud excludes the critical hepatic marker, HNF4α, indicating that RA signals are required for dorsal/anterior hepatic induction. These results were confirmed and extended by careful examination of Rdh10^trex/trex embryos, which carry a genetic perturbation in RA synthesis. At E9.5 Rdh10^trex/trex embryos display a similar yet more significant loss of the anterior/dorsal liver bud. Notably the anterior/dorsal liver bud loss correlates with the known dorsal-ventral gradient of the RA synthesis enzyme, Aldh1a2. In addition to altered hepatic specification, the mesoderm surrounding the liver bud is disorganized in RA abrogated embryos. Analysis of E10.5 Rdh10^trex/trex embryos reveals small livers that appear to lack the dorsal/caudal lobes. Finally, addition of exogenous RA prior to hepatic induction results in a liver bud that has failed to thicken and is largely unspecified. Taken together our ex vivo and in vivo evidence demonstrate that the generation of normal RA gradients is required for hepatic patterning, specification, and growth.

Synergistic role of retinoic acid signaling and Gata3 during primitive choanae formation.

Developmental defects of primitive choanae, an anatomical path to connect the embryonic nasal and oral cavity, result in disorders called choanal atresia (CA), which are associated with many congenital diseases and require immediate clinical intervention after birth. Previous studies revealed that reduced retinoid signaling underlies the etiology of CA. In the present study, by using multiple mouse models which conditionally deleted Rdh10 and Gata3 during embryogenesis, we showed that Gata3 expression is regulated by retinoid signaling during embryonic craniofacial development and plays crucial roles for development of the primitive choanae. Interestingly, Gata3 loss of function is known to cause hypoparathyroidism, sensorineural deafness and renal disease (HDR) syndrome, which exhibits CA as one of the phenotypes in humans. Our model partially phenocopies HDR syndrome with CA, and is thus a useful tool for investigating the molecular and cellular mechanisms of HDR syndrome. We further uncovered critical synergy of Gata3 and retinoid signaling during embryonic development, which will shed light on novel molecular and cellular etiology of congenital defects in primitive choanae formation.

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

Retinoid signaling regulates angiogenesis and blood-retinal barrier integrity in neonatal mouse retina.

OBJECTIVE: The neonatal mouse retina is a well-characterized experimental model for investigating factors impacting retinal angiogenesis and inner blood-retinal barrier (BRB) integrity. Retinoic acid (RA) is an essential signaling molecule. RA is needed for vasculogenic development in embryos and endothelial barrier integrity in zebrafish retina and adult mouse brain; however, the function of this signaling molecule in developing mammalian retinal vasculature remains unknown. This study aims to investigate the role of RA signaling in angiogenesis and inner BRB integrity in mouse neonatal retina. METHODS: RA distribution in the developing neurovascular retina was assessed in mice carrying an RA-responsive transgene. RA function in retinal angiogenesis was determined by treating C57BL/6 neonatal pups with a pharmacological inhibitor of RA signaling BMS493 or control vehicle. BRB integrity assessed by monitoring leakage of injected tracer into extravascular retinal tissue. RESULTS: RA signaling activity is present in peripheral astrocytes in domains corresponding to RA activity of the underlying neural retina. RA inhibition impaired retinal angiogenesis and reduced endothelial cell proliferation. RA inhibition also compromised BRB integrity. Vascular leakage was not associated with altered expression of CLDN5, PLVAP, LEF1, or VEcad. CONCLUSIONS: RA signaling is needed for angiogenesis and integrity of the BRB in the neonatal mouse retina.

RDH10-mediated retinol metabolism and RARα-mediated retinoic acid signaling are required for submandibular salivary gland initiation.

In mammals, the epithelial tissues of major salivary glands generate saliva and drain it into the oral cavity. For submandibular salivary glands (SMGs), the epithelial tissues arise during embryogenesis from naïve oral ectoderm adjacent to the base of the tongue, which begins to thicken, express SOX9 and invaginate into underlying mesenchyme. The developmental mechanisms initiating salivary gland development remain unexplored. In this study, we show that retinoic acid (RA) signaling activity at the site of gland initiation is colocalized with expression of retinol metabolic genes Rdh10 and Aldh1a2 in the underlying SMG mesenchyme. Utilizing a novel ex vivo assay for SMG initiation developed for this study, we show that RDH10 and RA are required for salivary gland initiation. Moreover, we show that the requirement for RA in gland initiation involves canonical signaling through retinoic acid receptors (RAR). Finally, we show that RA signaling essential for gland initiation is transduced specifically through RARα, with no contribution from other RAR isoforms. This is the first study to identify a molecular signal regulating mammalian salivary gland initiation.

Foxc2 is required for proper cardiac neural crest cell migration, outflow tract septation, and ventricle expansion.

BACKGROUND: Proper development of the great vessels of the heart and septation of the cardiac outflow tract requires cardiac neural crest cells. These cells give rise to the parasympathetic cardiac ganglia, the smooth muscle layer of the great vessels, some cardiomyocytes, and the conotruncal cushions and aorticopulmonary septum of the outflow tract. Ablation of cardiac neural crest cells results in defective patterning of each of these structures. Previous studies have shown that targeted deletion of the forkhead transcription factor C2 (Foxc2), results in cardiac phenotypes similar to that derived from cardiac neural crest cell ablation. RESULTS: We report that Foxc2-/- embryos on the 129s6/SvEv inbred genetic background display persistent truncus arteriosus and hypoplastic ventricles before embryonic lethality. Foxc2 loss-of-function resulted in perturbed cardiac neural crest cell migration and their reduced contribution to the outflow tract as evidenced by lineage tracing analyses together with perturbed expression of the neural crest cell markers Sox10 and Crabp1. Foxc2 loss-of-function also resulted in alterations in PlexinD1, Twist1, PECAM1, and Hand1/2 expression in association with vascular and ventricular defects. CONCLUSIONS: Our data indicate Foxc2 is required for proper migration of cardiac neural crest cells, septation of the outflow tract, and development of the ventricles. Developmental Dynamics 247:1286-1296, 2018. © 2018 Wiley Periodicals, Inc.

Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium.

BACKGROUND: Retinoic acid (RA), the active metabolite of vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. RESULTS: Here, we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and nonneuronal mesenchyme. By culturing epithelium explants in isolation from other tissues, we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. CONCLUSIONS: RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. Developmental Dynamics 246:135-147, 2017. © 2016 Wiley Periodicals, Inc.

Retinoic acid regulates embryonic development of mammalian submandibular salivary glands.

Organogenesis is orchestrated by cell and tissue interactions mediated by molecular signals. Identification of relevant signals, and the tissues that generate and receive them, are important goals of developmental research. Here, we demonstrate that Retinoic Acid (RA) is a critical signaling molecule important for morphogenesis of mammalian submandibular salivary glands (SMG). By examining late stage RA deficient embryos of Rdh10 mutant mice we show that SMG development requires RA in a dose-dependent manner. Additionally, we find that active RA signaling occurs in SMG tissues, arising earlier than any other known marker of SMG development and persisting throughout gland morphogenesis. At the initial bud stage of development, we find RA production occurs in SMG mesenchyme, while RA signaling occurs in epithelium. We also demonstrate active RA signaling occurs in glands cultured ex vivo, and treatment with an inhibitor of RA signaling blocks growth and branching. Together these data identify RA signaling as a direct regulator of SMG organogenesis.

Mef2c-F10N enhancer driven ß-galactosidase (LacZ) and Cre recombinase mice facilitate analyses of gene function and lineage fate in neural crest cells.

Neural crest cells (NCC) comprise a multipotent, migratory stem cell and progenitor population that gives rise to numerous cell and tissue types within a developing embryo, including craniofacial bone and cartilage, neurons and glia of the peripheral nervous system, and melanocytes within the skin. Here we describe two novel stable transgenic mouse lines suitable for lineage tracing and analysis of gene function in NCC. Firstly, using the F10N enhancer of the Mef2c gene (Mef2c-F10N) linked to LacZ, we generated transgenic mice (Mef2c-F10N-LacZ) that express LacZ in the majority, if not all migrating NCC that delaminate from the neural tube. Mef2c-F10N-LacZ then continues to be expressed primarily in neurogenic, gliogenic and melanocytic NCC and their derivatives, but not in ectomesenchymal derivatives. Secondly, we used the same Mef2c-F10N enhancer together with Cre recombinase to generate transgenic mice (Mef2c-F10N-Cre) that can be used to indelibly label, or alter gene function in, migrating NCC and their derivatives. At early stages of development, Mef2c-F10N-LacZ and Mef2c-F10N-Cre label NCC in a pattern similar to Wnt1-Cre mice, with the exception that Mef2c-F10N-LacZ and Mef2c-F10N-Cre specifically label NCC that have delaminated from the neural plate, while premigratory NCC are not labeled. Thus, our Mef2c-F10N-LacZ and Mef2c-F10N-Cre transgenic mice provide new resources for tracing migratory NCC and analyzing gene function in migrating and differentiating NCC independently of NCC formation.

Cochleovestibular nerve development is integrated with migratory neural crest cells.

The cochleovestibular (CV) nerve, which connects the inner ear to the brain, is the nerve that enables the senses of hearing and balance. The aim of this study was to document the morphological development of the mouse CV nerve with respect to the two embryonic cells types that produce it, specifically, the otic vesicle-derived progenitors that give rise to neurons, and the neural crest cell (NCC) progenitors that give rise to glia. Otic tissues of mouse embryos carrying NCC lineage reporter transgenes were whole mount immunostained to identify neurons and NCC. Serial optical sections were collected by confocal microscopy and were compiled to render the three dimensional (3D) structure of the developing CV nerve. Spatial organization of the NCC and developing neurons suggest that neuronal and glial populations of the CV nerve develop in tandem from early stages of nerve formation. NCC form a sheath surrounding the CV ganglia and central axons. NCC are also closely associated with neurites projecting peripherally during formation of the vestibular and cochlear nerves. Physical ablation of NCC in chick embryos demonstrates that survival or regeneration of even a few individual NCC from ectopic positions in the hindbrain results in central projection of axons precisely following ectopic pathways made by regenerating NCC.

Alcohol and aldehyde dehydrogenases: retinoid metabolic effects in mouse knockout models.

Retinoic acid (RA) is the active metabolite of vitamin A (retinol) that controls growth and development. The first step of RA synthesis is controlled by enzymes of the alcohol dehydrogenase (ADH) and retinol dehydrogenase (RDH) families that catalyze oxidation of retinol to retinaldehyde. The second step of RA synthesis is controlled by members of the aldehyde dehydrogenase (ALDH) family also known as retinaldehyde dehydrogenase (RALDH) that further oxidize retinaldehyde to produce RA. RA functions as a ligand for DNA-binding RA receptors that directly regulate transcription of specific target genes. Elucidation of the vitamin A metabolic pathway and investigation of the endogenous function of vitamin A metabolites has been greatly improved by development of mouse ADH, RDH, and RALDH loss-of-function models. ADH knockouts have demonstrated a postnatal role for this enzyme family in clearance of excess retinol to prevent vitamin A toxicity and in generation of RA for postnatal survival during vitamin A deficiency. A point mutation in Rdh10 generated by ethylnitrosourea has demonstrated that RDH10 generates much of the retinaldehyde needed for RA synthesis during embryonic development. Raldh1, Raldh2, and Raldh3 knockouts have demonstrated that RALDH1, RALDH2, and RALDH3 generate most of the RA needed during embryogenesis. These mouse models serve as instrumental tools for providing new insight into retinoid function. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays.

RDH10 is the primary enzyme responsible for the first step of embryonic Vitamin A metabolism and retinoic acid synthesis.

Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10(trex)-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.

Rdh10 mutants deficient in limb field retinoic acid signaling exhibit normal limb patterning but display interdigital webbing.

Retinoic acid (RA) is purported to be required for expression of genes controlling proximodistal (Meis2) or anteroposterior (Shh) limb patterning. Embryos lacking RDH10, the primary enzyme synthesizing retinaldehyde during mouse development, survive until E14.5 with stunted forelimbs but apparently normal hindlimbs. Using embryos carrying the RARE-lacZ RA-reporter transgene, we show that endogenous RA activity in Rdh10(trex/trex) mutants is detected in neuroectoderm but not limbs during initiation and patterning. Treatment of Rdh10 mutants with 25 nM RA restores RARE-lacZ activity to limb mesoderm, validating RARE-lacZ and verifying that RA is absent in mutant limbs. In Rdh10 mutants, hindlimbs exhibit normal Meis2/Shh expression and skeletal patterning, and although forelimbs are growth-retarded their Meis2 expression remains normal. Later in development, Rdh10 mutants lack interdigital RA activity and accordingly fail to exhibit normal loss of interdigital mesenchyme. These findings demonstrate that RA is unnecessary for limb patterning but required later for interdigital tissue loss.

A phenotype-driven ENU mutagenesis screen identifies novel alleles with functional roles in early mouse craniofacial development.

Proper craniofacial development begins during gastrulation and requires the coordinated integration of each germ layer tissue (ectoderm, mesoderm, and endoderm) and its derivatives in concert with the precise regulation of cell proliferation, migration, and differentiation. Neural crest cells, which are derived from ectoderm, are a migratory progenitor cell population that generates most of the cartilage, bone, and connective tissue of the head and face. Neural crest cell development is regulated by a combination of intrinsic cell autonomous signals acquired during their formation, balanced with extrinsic signals from tissues with which the neural crest cells interact during their migration and differentiation. Although craniofacial anomalies are typically attributed to defects in neural crest cell development, the cause may be intrinsic or extrinsic. Therefore, we performed a phenotype-driven ENU mutagenesis screen in mice with the aim of identifying novel alleles in an unbiased manner, that are critically required for early craniofacial development. Here we describe 10 new mutant lines, which exhibit phenotypes affecting frontonasal and pharyngeal arch patterning, neural and vascular development as well as sensory organ morphogenesis. Interestingly, our data imply that neural crest cells and endothelial cells may employ similar developmental programs and be interdependent during early embryogenesis, which collectively is critical for normal craniofacial morphogenesis. Furthermore our novel mutants that model human conditions such as exencephaly, craniorachischisis, DiGeorge, and Velocardiofacial sydnromes could be very useful in furthering our understanding of the complexities of specific human diseases.

Branchiomeric Muscle Development Requires Proper Retinoic Acid Signaling.

The first and second branchiomeric (branchial arch) muscles are craniofacial muscles that derive from branchial arch mesoderm. In mammals, this set of muscles is indispensable for jaw movement and facial expression. Defects during embryonic development that result in congenital partial absence of these muscles can have significant impact on patients' quality of life. However, the detailed molecular and cellular mechanisms that regulate branchiomeric muscle development remains poorly understood. Herein we investigated the role of retinoic acid (RA) signaling in developing branchiomeric muscles using mice as a model. We administered all-trans RA (25 mg/kg body weight) to Institute of Cancer Research (ICR) pregnant mice by gastric intubation from E8.5 to E10.5. In their embryos at E13.5, we found that muscles derived from the first branchial arch (temporalis, masseter) and second branchial arch (frontalis, orbicularis oculi) were severely affected or undetectable, while other craniofacial muscles were hypoplastic. We detected elevated cell death in the branchial arch mesoderm cells in RA-treated embryos, suggesting that excessive RA signaling reduces the survival of precursor cells of branchiomeric muscles, resulting in the development of hypoplastic craniofacial muscles. In order to uncover the signaling pathway(s) underlying this etiology, we focused on Pitx2, Tbx1, and MyoD1, which are critical for cranial muscle development. Noticeably reduced expression of all these genes was detected in the first and second branchial arch of RA-treated embryos. Moreover, elevated RA signaling resulted in a reduction in Dlx5 and Dlx6 expression in cranial neural crest cells (CNCCs), which disturbed their interactions with branchiomeric mesoderm cells. Altogether, we discovered that embryonic craniofacial muscle defects caused by excessive RA signaling were associated with the downregulation of Pitx2, Tbx1, MyoD1, and Dlx5/6, and reduced survival of cranial myogenic precursor cells.

The Mediator Subunit, Med23 Is Required for Embryonic Survival and Regulation of Canonical WNT Signaling During Cranial Ganglia Development.

Development of the vertebrate head is a complex and dynamic process, which requires integration of all three germ layers and their derivatives. Of special importance are ectoderm-derived cells that form the cranial placodes, which then differentiate into the cranial ganglia and sensory organs. Critical to a fully functioning head, defects in cranial placode and sensory organ development can result in congenital craniofacial anomalies. In a forward genetic screen aimed at identifying novel regulators of craniofacial development, we discovered an embryonically lethal mouse mutant, snouty, which exhibits malformation of the facial prominences, cranial nerves and vasculature. The snouty mutation was mapped to a single nucleotide change in a ubiquitously expressed gene, Med23, which encodes a subunit of the global transcription co-factor complex, Mediator. Phenotypic analyses revealed that the craniofacial anomalies, particularly of the cranial ganglia, were caused by a failure in the proper specification of cranial placode neuronal precursors. Molecular analyses determined that defects in cranial placode neuronal differentiation in Med23 sn/sn mutants were associated with elevated WNT/ß-catenin signaling, which can be partially rescued through combined Lrp6 and Wise loss-of-function. Our work therefore reveals a surprisingly tissue specific role for the ubiquitously expressed mediator complex protein Med23 in placode differentiation during cranial ganglia development. This highlights the importance of coupling general transcription to the regulation of WNT signaling during embryogenesis.

RDH10 function is necessary for spontaneous fetal mouth movement that facilitates palate shelf elevation.

Cleft palate is a common birth defect, occurring in approximately 1 in 1000 live births worldwide. Known etiological mechanisms of cleft palate include defects within developing palate shelf tissues, defects in mandibular growth and defects in spontaneous fetal mouth movement. Until now, experimental studies directly documenting fetal mouth immobility as an underlying cause of cleft palate have been limited to models lacking neurotransmission. This study extends the range of anomalies directly demonstrated to have fetal mouth movement defects correlated with cleft palate. Here, we show that mouse embryos deficient in retinoic acid (RA) have mispatterned pharyngeal nerves and skeletal elements that block spontaneous fetal mouth movement in utero Using X-ray microtomography, in utero ultrasound video, ex vivo culture and tissue staining, we demonstrate that proper retinoid signaling and pharyngeal patterning are crucial for the fetal mouth movement needed for palate formation. Embryos with deficient retinoid signaling were generated by stage-specific inactivation of retinol dehydrogenase 10 (Rdh10), a gene crucial for the production of RA during embryogenesis. The finding that cleft palate in retinoid deficiency results from a lack of fetal mouth movement might help elucidate cleft palate etiology and improve early diagnosis in human disorders involving defects of pharyngeal development.

Oxidative stress during diabetic pregnancy disrupts cardiac neural crest migration and causes outflow tract defects.

BACKGROUND: Maternal diabetes increases risk for congenital malformations, particularly cardiac outflow tract defects. Maternal diabetes inhibits expression of Pax3 in neuroepithelium through hyperglycemia-induced oxidative stress. The neuroepithelium gives rise to the neural crest, and Pax3 expression in cardiac neural crest (CNC) is required for CNC migration to the heart and for outflow tract septation. Here we tested whether maternal diabetes, through hyperglycemia-induced oxidative stress, before the onset of CNC delamination, impairs CNC migration and cardiac outflow tract septation. METHODS: CNC migration was mapped in mouse embryos whose mothers were diabetic, or transiently hyperglycemic, or in which oxidative stress was transiently induced, using reporters linked to Pax3 expression. CNC apoptosis was examined by TUNEL assay. Outflow tract septation was examined histologically and by gross inspection. RESULTS: Few, if any, migrating CNC cells were observed in embryos of diabetic mice, and this was associated with increased apoptosis along the path of CNC migration. Outflow tract defects were significantly increased in fetuses of diabetic mice. Notably, induction of hyperglycemia or oxidative stress on the day prior to the onset of Pax3 expression and CNC migration also impaired CNC migration, increased apoptosis, and caused outflow tract defects. However, antioxidants administered on the day prior to the onset of Pax3 expression and CNC migration prevented these effects of hyperglycemia or oxidative stress. CONCLUSIONS: In diabetic pregnancy, oxidative stress, which inhibits expression of genes required for CNC viability, causes subsequent CNC depletion by apoptosis during migration, which leads to outflow tract defects.