Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development.

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

Differential Retinoic Acid Signaling in the Hippocampus of Aged Rats with and without Memory Impairment.

Retinoic acid (RA), a metabolite of vitamin A, has many physiological functions, and mounting evidence points to important roles in cognition. In vitro experiments indicate that RA is involved in homeostatic synaptic scaling in the hippocampus, which supports overall network stability during learning. It has been previously determined that disrupted RA signaling in the hippocampus causes deterioration of memory, that RA signaling declines with age in brain, and that application of RA reverses this decline. Here, we explore whether RA signaling is altered in an animal model of neurocognitive aging. We used a Morris water maze protocol to study cognitive decline in aged rats, which assesses hippocampus-dependent spatial memory and reveals substantial interindividual differences in aged animals. Aged unimpaired (AU) rats perform on par with young (Y), while aged impaired (AI) animals exhibit spatial memory deficits. We show that the major substrate for RA, retinol binding protein 4 (RBP4), is decreased in AU rats, and retinol cell surface receptor declines with chronological age. Other affected components of RA signaling include selective increases in AI animals in hippocampal synthesis (RALDH1) and catabolism of RA (CYP26B1), RA receptor α, the RA regulated ionotropic glutamate receptor (GluR1), as well as fragile X mental retardation protein (FMRP). The results support the conclusion that, surprisingly, increased RA signaling in the aged hippocampus is associated with poor cognitive outcome.

Highly Sensitive Quantitative Determination of Retinoic Acid Levels, Retinoic Acid Synthesis, and Catabolism in Embryonic Tissue Using a Reporter Cell-Based Method.

The effect of all-trans retinoic acid (RA) on embryogenesis is tissue specific and highly concentration dependent. Using a liquid chromatography/mass spectrometry-based method to quantify trace amounts of RA in embryonic tissue requires expensive specialist facilities. Here, we describe the use of a RA response element (RARE)-lacZ reporter cell-based method, which is simple and cost effective, to measure RA levels in small pieces of tissue from the embryo. We further apply this method to quantitatively assay activities of RA-synthesizing and RA-catabolizing enzymes, the key regulators of RA bioavailability in tissues and developing organs of the embryo.

Perturbation of Retinoid Homeostasis Increases Malformation Risk in Embryos Exposed to Pregestational Diabetes.

Pregestational diabetes is highly associated with an increased risk of birth defects. However, factors that can increase or reduce the expressivity and penetrance of malformations in pregnancies in women with diabetes remain poorly identified. All-trans retinoic acid (RA) plays crucial roles in embryogenesis. Here, we find that Cyp26a1, which encodes a key enzyme for catabolic inactivation of RA required for tight control of local RA concentrations, is significantly downregulated in embryos of diabetic mice. Embryonic tissues expressing Cyp26a1 show reduced efficiency of RA clearance. Embryos exposed to diabetes are thus sensitized to RA and more vulnerable to the deleterious effects of increased RA signaling. Susceptibility to RA teratogenesis is further potentiated in embryos with a preexisting genetic defect of RA metabolism. Increasing RA clearance efficiency using a preconditioning approach can counteract the increased susceptibility to RA teratogenesis in embryos of diabetic mice. Our findings provide new insight into gene-environment interactions that influence individual risk in the manifestation of diabetes-related birth defects and shed light on environmental risk factors and genetic variants for a stratified medicine approach to screening women with diabetes who are of childbearing age and assessing the risk of birth defects during pregnancy.

A neuroendocrine role for chemerin in hypothalamic remodelling and photoperiodic control of energy balance.

Long-term and reversible changes in body weight are typical of seasonal animals. Thyroid hormone (TH) and retinoic acid (RA) within the tanycytes and ependymal cells of the hypothalamus have been implicated in the photoperiodic response. We investigated signalling downstream of RA and how this links to the control of body weight and food intake in photoperiodic F344 rats. Chemerin, an inflammatory chemokine, with a known role in energy metabolism, was identified as a target of RA. Gene expression of chemerin (Rarres2) and its receptors were localised within the tanycytes and ependymal cells, with higher expression under long (LD) versus short (SD) photoperiod, pointing to a physiological role. The SD to LD transition (increased food intake) was mimicked by 2 weeks of ICV infusion of chemerin into rats. Chemerin also increased expression of the cytoskeletal protein vimentin, implicating hypothalamic remodelling in this response. By contrast, acute ICV bolus injection of chemerin on a 12 h:12 h photoperiod inhibited food intake and decreased body weight with associated changes in hypothalamic neuropeptides involved in growth and feeding after 24 hr. We describe the hypothalamic ventricular zone as a key site of neuroendocrine regulation, where the inflammatory signal, chemerin, links TH and RA signaling to hypothalamic remodeling.

Targeting Retinoid Receptors to Treat Schizophrenia: Rationale and Progress to Date.

This review provides the rationale and reports on the progress to date regarding the targeting of retinoid receptors for the treatment of schizophrenia and schizoaffective disorder and the role of retinoic acid in functions of the normal brain, and in psychotic states. After a brief introduction, we describe the normal function of retinoic acid in the brain. We then examine the evidence regarding retinoid dysregulation in schizophrenia. Finally, findings from two add-on clinical trials with a retinoid (bexarotene) are discussed. The authors of this review suggest that targeting retinoid receptors may be a novel approach to treat schizophrenia and schizoaffective disorder. Further studies are warranted.

Expression of the retinoic acid catabolic enzyme CYP26B1 in the human brain to maintain signaling homeostasis.

Retinoic acid (RA) is a potent regulator of gene transcription via its activation of a set of nuclear receptors controlling transcriptional activation. Precise maintenance of where and when RA is generated is essential and achieved by local expression of synthetic and catabolic enzymes. The catabolic enzymes Cyp26a1 and Cyp26b1 have been studied in detail in the embryo, where they limit gradients of RA that form patterns of gene expression, crucial for morphogenesis. This paracrine role of RA has been assumed to occur in most tissues and that the RA synthetic enzymes release RA at a site distant from the catabolic enzymes. In contrast to the embryonic CNS, relatively little is known about RA metabolism in the adult brain. This study investigated the distribution of Cyp26a1 and Cyp26b1 transcripts in the rat brain, identifying several novel regions of expression, including the cerebral cortex for both enzymes and striatum for Cyp26b1. In vivo use of a new and potent inhibitor of the Cyp26 enzymes, ser 2-7, demonstrated a function for endogenous Cyp26 in the brain and that hippocampal RA levels can be raised by ser 2-7, altering the effect of RA on differential patterning of cell proliferation in the hippocampal region of neurogenesis, the subgranular zone. The expression of CYP26A1 and CYP26B1 was also investigated in the adult human brain and colocalization of CYP26A1 and the RA synthetic enzyme RALDH2 indicated a different, autocrine role for RA in human hippocampal neurons. Studies with the SH-SY5Y human neuroblastoma cell line implied that the co-expression of RA synthetic and catabolic enzymes maintains retinoid homeostasis within neurons. This presents a novel view of RA in human neurons as part of an autocrine, intracellular signaling system.

Fenretinide prevents obesity in aged female mice in association with increased retinoid and estrogen signaling.

OBJECTIVE: The synthetic retinoid fenretinide (FEN) inhibits adiposity in male mice fed a high-fat diet (HFD) in association with alterations in retinoic acid (RA) signaling. Young female mice are protected from obesity via estrogen signaling. We, therefore, investigated whether FEN also influences adiposity in aged female mice differing in parity and whether such effects are mediated by retinoid and estrogen signaling. METHODS: Aged nulliparous and parous female mice were maintained on HFD ± FEN, and adiposity was assessed. Quantitative polymerase chain reaction was performed on white adipose tissue (WAT), liver, and 3T3-L1 adipocytes treated with RA or FEN ± estrogen. RESULTS: Parous females were more obese than nulliparous mice independent of age. FEN-HFD prevented the HFD-induced increase in adiposity and leptin levels independently of parity. FEN-HFD induced retinoid-responsive genes in WAT and liver. Parous females had reduced expression of hepatic estrogen-responsive genes, but FEN-HFD up-regulated WAT Cyp19a1 and Esr2 in parous mice. Estrogen and RA acted synergistically to increase RA receptor-mediated gene expression in 3T3-L1 adipocytes. FEN increased Cyp19a1 and Esr2, similar to our findings in vivo. CONCLUSIONS: The prevention of adiposity by FEN in response to HFD in female mice seems to involve increased retinoid signaling in association with induction of local estrogen production and estrogen signaling in WAT.

The evidence for a beneficial role of vitamin A in multiple sclerosis.

Vitamin A is an essential nutrient with important roles in immunological responses and in brain development. Its main metabolite is retinoic acid (RA), which is responsible for the neuroimmunological functions related to vitamin A. In the brain, RA is known to have interactions with other nuclear receptor-mediated signalling pathways. RA is involved in plasticity, regeneration, cognition and behaviour. In the peripheral blood, RA plays a major role both in increasing tolerance and in decreasing inflammation, through balancing T-lymphocyte populations. It is likely that RA synthesis may be manipulated by complex cross-talk among cells during infection and inflammation. The role of vitamin A in multiple sclerosis (MS) could be dual: at the same time as it decreases inflammation and increases tolerance of autoimmunity, it may also help in brain protection. The present review discusses the beneficial effects that vitamin A might have for controlling MS, although it must be clearly stated that, at the present time, there is no clear indication for using vitamin A as a treatment for MS. However, the results from the present review should encourage clinical trials with vitamin supplementation as a potential treatment or as an add-on option. Vitamin A acts in synergy with vitamin D, and the immunological homeostasis ensured by these vitamins should not be unbalanced in favour of only one of them.

The rhythm of retinoids in the brain.

The retinoids are a family of compounds that in nature are derived from vitamin A or pro-vitamin A carotenoids. An essential part of the diet for mammals, vitamin A has long been known to be essential for many organ systems in the adult. More recently, however, they have been shown to be necessary for function of the brain and new discoveries point to a central role in processes ranging from neuroplasticity to neurogenesis. Acting in several regions of the central nervous system including the eye, hippocampus and hypothalamus, one common factor in its action is control of biological rhythms. This review summarizes the role of vitamin A in the brain; its action through the metabolite retinoic acid via specific nuclear receptors, and the regulation of its concentration through controlled synthesis and catabolism. The action of retinoic acid to regulate several rhythms in the brain and body, from circadian to seasonal, is then discussed to finish with the importance of retinoic acid in the regular pattern of sleep. We review the role of vitamin A and retinoic acid (RA) as mediators of rhythm in the brain. In the suprachiasmatic nucleus and hippocampus they control expression of circadian clock genes while in the cortex retinoic acid is required for delta oscillations of sleep. Retinoic acid is also central to a second rhythm that keeps pace with the seasons, regulating function in the hypothalamus and pineal gland.

Fenretinide treatment prevents diet-induced obesity in association with major alterations in retinoid homeostatic gene expression in adipose, liver, and hypothalamus.

The synthetic retinoid, Fenretinide (FEN), inhibits obesity and insulin resistance in mice and is in early clinical trials for treatment of insulin resistance in obese humans. We aimed to determine whether alterations in retinoic acid (RA)-responsive genes contribute to the beneficial effects of FEN. We examined the effect of FEN on 3T3-L1 adipocyte differentiation and alterations in gene expression in C57Bl/6 and retinaldehyde dehydrogenase (RALDH) 1 knockout (KO) mice fed a high-fat (HF) diet. FEN completely inhibited adipocyte differentiation by blocking CCAAT/enhancer-binding protein (C/EBP) α/peroxisome proliferator-activated receptor (PPAR) γ-mediated induction of downstream genes and upregulating RA-responsive genes like cellular retinol-binding protein-1. In mice fed an HF diet, RA-responsive genes were markedly increased in adipose, liver, and hypothalamus, with short-term and long-term FEN treatment. In adipose, FEN inhibited the downregulation of PPARγ and improved insulin sensitivity and the levels of adiponectin, resistin, and serum RBP (RBP4). FEN inhibited hyperleptinemia in vivo and leptin expression in adipocytes. Surprisingly, hypothalamic neuropeptide Y expression was completely suppressed, suggesting a central effect of FEN to normalize hyperglycemia. Moreover, FEN induced RA-responsive genes in RALDH1 KO mice, demonstrating that FEN can augment RA signaling when RA synthesis is impaired. We show that FEN-mediated beneficial effects are through alterations in retinoid homeostasis genes, and these are strong candidates as therapeutic targets for the treatment of obesity and insulin resistance.

A vitamin for the brain.

In the central nervous system (CNS) the function of retinoic acid, the active metabolite of vitamin A, is best understood from its action in guiding embryonic development; as development comes to completion, retinoic acid signaling declines. However, it is increasingly recognized that this signaling mechanism does not disappear in the adult brain but becomes more regionally focused and takes on new roles. These functions are often tied to processes of neural plasticity whether in the hippocampus, through homeostatic neural plasticity, the olfactory bulb or the hypothalamus. The role of retinoic acid in the control of plastic processes has led to suggestions of its involvement in neural disorders, both degenerative and psychiatric. This review presents a snapshot of developments in these areas over recent years.

Astrocytes as a regulated source of retinoic acid for the brain.

Retinaldehyde dehydrogenases (RALDH) catalyze the synthesis of the regulatory factor retinoic acid (RA). Cultured astrocytes express several of the RALDH enzyme family, and it has been assumed that this can be extrapolated to astrocytes in vivo. However, this study finds that few astrocytes in the rodent brain express detectable RALDH enzymes, and only when these cells are grown in culture are these enzymes upregulated. Factors controlling the expression of the RALDHs in cultured astrocytes were explored to determine possible reasons for differences between in vitro versus in vivo expression. Retinoids were found to feedback to suppress several of the RALDHs, and physiological levels of retinoids may be one route by which astrocytic RALDHs are maintained at low levels. In the case of RALDH2, in vivo reduction of vitamin A levels in rats resulted in an increase in astrocyte RALDH2 expression in the hippocampus. Other factors though are likely to control RALDH expression. A shift in astrocytic RALDH subcellular localization is a potential mechanism for regulating RA signaling. Under conditions of vitamin A deficiency, RALDH2 protein moved from the cytoplasm to the nucleus where it may synthesize RA at the site of the nuclear RA receptors. Similarly, in conditions of oxidative stress RALDH1 and RALDH2 moved from the cytoplasm to a predominantly nuclear position. Thus, the RALDHs have been revealed to be dynamic in their expression in astrocytes where they may maintain retinoid homeostasis in the brain.

A paradoxical teratogenic mechanism for retinoic acid.

Retinoic acid, an active metabolite of vitamin A, plays essential signaling roles in mammalian embryogenesis. Nevertheless, it has long been recognized that overexposure to vitamin A or retinoic acid causes widespread teratogenesis in rodents as well as humans. Although it has a short half-life, exposure to high levels of retinoic acid can disrupt development of yet-to-be formed organs, including the metanephros, the embryonic organ which normally differentiates into the mature kidney. Paradoxically, it is known that either an excess or a deficiency of retinoic acid results in similar malformations in some organs, including the mammalian kidney. Accordingly, we hypothesized that excess retinoic acid is teratogenic by inducing a longer lasting, local retinoic acid deficiency. This idea was tested in an established in vivo mouse model in which exposure to excess retinoic acid well before metanephric rudiments exist leads to failure of kidney formation several days later. Results showed that teratogen exposure was followed by decreased levels of Raldh transcripts encoding retinoic acid-synthesizing enzymes and increased levels of Cyp26a1 and Cyp26b1 mRNAs encoding enzymes that catabolize retinoic acid. Concomitantly, there was significant reduction in retinoic acid levels in whole embryos and kidney rudiments. Restoration of retinoic acid levels by maternal supplementation with low doses of retinoic acid following the teratogenic insult rescued metanephric kidney development and abrogated several extrarenal developmental defects. This previously undescribed and unsuspected mechanism provides insight into the molecular pathway of retinoic acid-induced teratogenesis.

Photoperiod regulates vitamin A and Wnt/ß-catenin signaling in F344 rats.

In seasonal mammals, growth, energy balance, and reproductive status are regulated by the neuroendocrine effects of photoperiod. Thyroid hormone (TH) is a key player in this response in a number of species. A neuroendocrine role for the nutritional factor vitamin A has not been considered, although its metabolic product retinoic acid (RA) regulates transcription via the same nuclear receptor family as TH. We hypothesized that vitamin A/RA plays a role in the neuroendocrine hypothalamus alongside TH signaling. Using a reporter assay to measure RA activity, we demonstrate that RA activity levels in the hypothalamus of photoperiod-sensitive F344 rats are reduced in short-day relative to long-day conditions. These lower RA activity levels can be explained by reduced expression of a whole network of RA signaling genes in the ependymal cells around the third ventricle and in the arcuate nucleus of the hypothalamus. These include genes required for uptake (Ttr, Stra6, and Crbp1), synthesis (Raldh1), receptor response (RAR), and ligand clearance (Crapb1 and Cyp26B1). Using melatonin injections into long-day rats, we show that the probable trigger of the fall in RA is melatonin. Surprisingly we also found RPE65 expression in the mammalian hypothalamus for the first time. Similar to RA signaling genes, members of the Wnt/ß-catenin pathway and NMU and its receptor NMUR2 are also under photoperiodic control. Our data provide strong evidence for a novel endocrine axis, involving the nutrient vitamin A regulated by photoperiod and melatonin and suggest a role for several new players in the photoperiodic neuroendocrine response.

Retinoic acid and affective disorders: the evidence for an association.

OBJECTIVE: Isotretinoin (13-cis-retinoic acid), approved by the US Food and Drug Administration for the treatment of acne, carries a black box warning related to the risk of depression, suicide, and psychosis. Retinoic acid, the active form of vitamin A, regulates gene expression in the brain, and isotretinoin is its 13-cis isomer. Retinoids represent a group of compounds derived from vitamin A that perform a large variety of functions in many systems, in particular the central nervous system, and abnormal retinoid levels can have neurologic effects. Although infrequent, proper recognition and treatment of psychiatric side effects in acne patients is critical given the risk of death and disability. This article reviews the evidence for isotretinoin's relationships with depression and suicidality. DATA SOURCES: The PsycINFO, MEDLINE, and PubMed searchable database indexes were searched for articles published in the English language from 1960 to June 2010 using the key words isotretinoin, retinoids, retinoic acid, depression, depressive disorders, and vitamin A. Evidence examined includes (1) case reports; (2) temporal association between onset of depression and exposure to the drug; (3) challenge-rechallenge cases; (4) class effect (other compounds in the same class, like vitamin A, having similar neuropsychiatric effects); (5) dose response; and (6) biologically plausible mechanisms. STUDY SELECTION: All articles in the literature related to isotretinoin, depression, and suicide were reviewed, as well as articles related to class effect, dose response, and biologic plausibility. DATA EXTRACTION: Information from individual articles in the literature was extracted, including number of episodes of depression, suicidality, suicide, psychosis, violence and aggression, past psychiatric history, time of onset in relation to isotretinoin usage, medication dosage, duration of treatment, and dechallenge and challenge history. RESULTS: The literature reviewed is consistent with associations of isotretinoin administration with depression and with suicide in a subgroup of vulnerable individuals. CONCLUSIONS: The relationship between isotretinoin and depression may have implications for a greater understanding of the neurobiology of affective disorders.

Influence of isotretinoin on hippocampal-based learning in human subjects.

RATIONALE: The acne drug isotretinoin has 13-cis retinoic acid as its active agent. Adverse effects that have been described include severe depression. Animal studies indicate that the hippocampus is particularly sensitive to retinoic acid. Changes induced by isotretinoin to hippocampal function could contribute to depression but may be more evident in altered visuospatial learning and memory, the primary function of the hippocampus. OBJECTIVES: We aimed to test the hypothesis that a course of oral isotretinoin therapy would result in declining visuospatial learning and memory. METHODS: CANTAB tasks designed to assess visuospatial memory were performed repeatedly on 14 males and 3 females in an open prospective observational study of patients with severe acne undergoing isotretinoin therapy. Beck's Depression Inventory and Global Acne Grade were also administered. RESULTS: Performance stayed unchanged for DMS, SRM and PRM tasks, while surprisingly participants improved their speed on the PRM task. Performance improved across sessions on the PAL task, and moreover the dose of isotretinoin correlated with improvement in the total trial score, reduction in total error rate and stage completed at the first trial. CONCLUSION: Isotretinoin does not reduce learning and memory and our study suggests that it may instead lead to a dose-related improvement in specific aspects of hippocampal learning and memory. Retinoic acid functions in the hippocampus as the active metabolite of vitamin A, suggesting that this may be a limiting factor in the human hippocampus and addition of exogenous retinoic acid brings levels closer to an optimal state.

High expression of retinoic acid receptors and synthetic enzymes in the human hippocampus.

Retinoic acid, the active form of the nutrient vitamin A, regulates several facets of neuronal plasticity in the hippocampus, including neurogenesis and synaptic strength, acting via specific retinoic acid receptors (RARs). Essential for conversion of vitamin A to retinoic acid is the enzyme retinaldehyde dehydrogenase (RALDH) and in the rodent hippocampus this is only present in the adjacent meninges where it must act as a locally released paracrine hormone. Little is known though about the expression of RALDHs and RARs in the human hippocampus. This study confirms that RALDH levels are very low in mouse neurons but, surprisingly, strong expression of RALDH protein is detected by immunohistochemistry in hippocampal neurons. The receptors RARα, ß and γ were also detected, each receptor exhibiting differing subcellular locations implying their potential regulation of both transcription and non-genomic actions. These results imply an essential function of retinoic acid in the human hippocampus likely to include regulation of neuronal plasticity.

Photoperiodic regulation of retinoic acid signaling in the hypothalamus.

Both retinoic acid (RA) and thyroid hormone (TH) regulate transcription via specific nuclear receptors. TH regulates hypothalamic homeostasis and active T3 is generated by deiodinase enzymes in tanycytes surrounding the third ventricle. However, RA has not been previously considered in such a role. Data presented here highlights novel parallels between the TH and RA synthetic pathways in the hypothalamus implying that RA also acts to regulate hypothalamic gene expression and function. Key elements of the RA cellular signaling pathway were shown to be regulated in the rodent hypothalamus. Retinoid synthetic enzymes and the retinol transport protein Stra6 were located in the cells lining the third ventricle allowing synthesis of RA from retinol present in the CNS to act via RA receptors and retinoid X receptors in the hypothalamus. Photoperiod manipulation was shown to alter the expression of synthetic enzymes and receptors with lengthening of photoperiod leading to enhanced RA signaling. In vitro RA can regulate the hypothalamic neuroendocrine peptide adrenocorticotrophic hormone. This work presents the new concept of controlled RA synthesis by hypothalamic tanycytes giving rise to possible involvement of this system in endocrine, and possibly vitamin A, homeostasis.

Defects in tangential neuronal migration of pontine nuclei neurons in the Largemyd mouse are associated with stalled migration in the ventrolateral hindbrain.

The LARGE gene encodes a putative glycosyltransferase that is required for normal glycosylation of dystroglycan, and defects in LARGE can cause abnormal neuronal migration in congenital muscular dystrophy (CMD). Previous studies have focused on radial migration, which is disrupted at least in part due to breaks in the basal lamina. Through analysis of precerebellar nuclei development in the Large(myd) mouse hindbrain, we show that tangential migration of a subgroup of hindbrain neurons may also be disrupted. Within the precerebellar nuclei, the pontine nuclei (PN) are severely disrupted, whereas the inferior olive (IO), external cuneate nuclei (ECN) and lateral reticular nuclei (LRN) appear unaffected. Large and dystroglycan are widely expressed in the hindbrain, including in the pontine neurons migrating in the anterior extramural migratory stream (AES). BrdU labeling and immunohistochemical studies suggest normal numbers of neurons begin their journey towards the ventral midline in the AES in the Large(myd) mouse. However, migration stalls and PN neurons fail to reach the midline, surviving as ectopic clusters of cells located under the pial surface dorsally and laterally to where they normally would finish their migration near the ventral midline. Stalling of PN neurons at this location is also observed in other migration disorders in mice. These observations suggest that glycan-dependent dystroglycan interactions are required for PN neurons to correctly respond to signals at this important migrational checkpoint.