Arrestin-mediated desensitization enables intraneuronal olfactory discrimination in Caenorhabditis elegans.

In the mammalian olfactory system, cross-talk between olfactory signals is minimized through physical isolation: individual neurons express one or few olfactory receptors among those encoded in the genome. Physical isolation allows for segregation of stimuli during signal transduction; however, in the nematode worm Caenorhabditis elegans, ∼1,300 olfactory receptors are primarily expressed in only 32 neurons, precluding this strategy. Here, we report genetic and behavioral evidence that ß-arrestin-mediated desensitization of olfactory receptors, working downstream of the kinase GRK-1, enables discrimination between intraneuronal olfactory stimuli. Our findings suggest that C. elegans exploits ß-arrestin desensitization to maximize responsiveness to novel odors, allowing for behaviorally appropriate responses to olfactory stimuli despite the large number of olfactory receptors signaling in single cells. This represents a fundamentally different solution to the problem of olfactory discrimination than that which evolved in mammals, allowing for economical use of a limited number of sensory neurons.

Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.

Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR) repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system.

Constraint-Induced Movement Therapy (CIMT) and Neural Precursor Cell (NPC) Transplantation Synergistically Promote Anatomical and Functional Recovery in a Hypoxic-Ischemic Mouse Model.

Cerebral palsy (CP) is a common neurodevelopmental disorder characterized by pronounced motor dysfunction and resulting in physical disability. Neural precursor cells (NPCs) have shown therapeutic promise in mouse models of hypoxic-ischemic (HI) perinatal brain injury, which mirror hemiplegic CP. Constraint-induced movement therapy (CIMT) enhances the functional use of the impaired limb and has emerged as a beneficial intervention for hemiplegic CP. However, the precise mechanisms and optimal application of CIMT remain poorly understood. The potential synergy between a regenerative approach using NPCs and a rehabilitation strategy using CIMT has not been explored. We employed the Rice-Vannucci HI model on C57Bl/6 mice at postnatal day (PND) 7, effectively replicating the clinical and neuroanatomical characteristics of hemiplegic CP. NPCs were transplanted in the corpus callosum (CC) at PND21, which is the age corresponding to a 2-year-old child from a developmental perspective and until which CP is often not formally diagnosed, followed or not by Botulinum toxin injections in the unaffected forelimb muscles at PND23, 26, 29 and 32 to apply CIMT. Both interventions led to enhanced CC myelination and significant functional recovery (as shown by rearing and gait analysis testing), through the recruitment of endogenous oligodendrocytes. The combinatorial treatment indicated a synergistic effect, as shown by newly recruited oligodendrocytes and functional recovery. This work demonstrates the mechanistic effects of CIMT and NPC transplantation and advocates for their combined therapeutic potential in addressing hemiplegic CP.

Small-Molecule-Directed Endogenous Regeneration of Visual Function in a Mammalian Retinal Degeneration Model.

Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.

Administration of BDNF in the ventral tegmental area produces a switch from a nicotine-non-dependent D1R-mediated motivational state to a nicotine-dependent-like D2R-mediated motivational state.

Brain-derived neurotrophic factor (BDNF) has been implicated in the transition from a non-dependent motivational state to a drug-dependent and drug-withdrawn motivational state. Chronic nicotine can increase BDNF in the rodent brain and is associated with smoking severity in humans; however, it is unknown whether this increased BDNF is linked functionally to the switch from a nicotine-non-dependent to a nicotine-dependent state. We used a place conditioning paradigm to measure the conditioned responses to nicotine, showing that a dose of acute nicotine that non-dependent male mice find aversive is found rewarding in chronic nicotine-treated mice experiencing withdrawal. A single BDNF injection in the ventral tegmental area (in the absence of chronic nicotine treatment) caused mice to behave as if they were nicotine dependent and in withdrawal, switching the neurobiological substrate mediating the conditioned motivational effects from dopamine D1 receptors to D2 receptors. Quantification of gene expression of BDNF and its receptor, tropomyosin-receptor-kinase B (TrkB), revealed an increase in TrkB mRNA but not BDNF mRNA in the VTA in nicotine-dependent and nicotine-withdrawn mice. These results suggest that BDNF signalling in the VTA is a critical neurobiological substrate for the transition to nicotine dependence. The modulation of BDNF signalling may be a promising new pharmacological avenue for the treatment of addictive behaviour.

ß2* nAChRs on VTA dopamine and GABA neurons separately mediate nicotine aversion and reward.

Evidence shows that the neurotransmitter dopamine mediates the rewarding effects of nicotine and other drugs of abuse, while nondopaminergic neural substrates mediate the negative motivational effects. ß2* nicotinic acetylcholine receptors (nAChR) are necessary and sufficient for the experience of both nicotine reward and aversion in an intra-VTA (ventral tegmental area) self-administration paradigm. We selectively reexpressed ß2* nAChRs in VTA dopamine or VTA γ-amino-butyric acid (GABA) neurons in ß2-/- mice to double-dissociate the aversive and rewarding conditioned responses to nicotine in nondependent mice, revealing that ß2* nAChRs on VTA dopamine neurons mediate nicotine's conditioned aversive effects, while ß2* nAChRs on VTA GABA neurons mediate the conditioned rewarding effects in place-conditioning paradigms. These results stand in contrast to a purely dopaminergic reward theory, leading to a better understanding of the neurobiology of nicotine motivation and possibly to improved therapeutic treatments for smoking cessation.

Constraint-induced movement therapy promotes motor recovery after neonatal stroke in the absence of neural precursor activation.

Neonatal stroke is a leading cause of long-term disability and currently available rehabilitation treatments are insufficient to promote recovery. Activating neural precursor cells (NPCs) in adult rodents, in combination with rehabilitation, can accelerate functional recovery following stroke. Here, we describe a novel method of constraint-induced movement therapy (CIMT) in a rodent model of neonatal stroke that leads to improved functional outcomes, and we asked whether the recovery was correlated with expansion of NPCs. A hypoxia/ischemia (H/I) injury was induced on postnatal day 8 (PND8) via unilateral carotid artery ligation followed by systemic hypoxia. One week and two weeks post-H/I, CIMT was administered in the form of 3 botulinum toxin (Botox) injections, which induced temporary paralysis in the unaffected limb. Functional recovery was assessed using the foot fault task. NPC proliferation was assessed using the neurosphere assay and EdU immunohistochemistry. We found that neonatal H/I injury alone expands the NPC pool by >2.5-fold relative to controls. We determined that using Botox injections as a method to provide CIMT results in significant functional motor recovery after H/I. However, CIMT does not lead to enhanced NPC activation or migration into the injured parenchyma in vivo. At the time of functional recovery, increased numbers of proliferating inflammatory cells were found within the injured motor cortex. Together, these findings suggest that NPC activation following CIMT does not account for the observed functional improvement and suggests that CIMT-mediated modification of the CNS inflammatory response may play a role in the motor recovery.

Dual embryonic origin of the mammalian enteric nervous system.

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.

Segregation of caffeine reward and aversion in the rat nucleus accumbens shell versus core.

Caffeine, the most commonly consumed psychoactive drug in the world, is readily available in dietary sources, including soft drinks, chocolate, tea and coffee. However, little is known about the neural substrates that underlie caffeine's rewarding and aversive properties and what ultimately leads us to seek or avoid caffeine consumption. Using male Wistar rats in a place conditioning procedure, we show that systemic caffeine at a low intraperitoneal dose of 2 mg/kg (or 100 µM injected directly into the rostral, but not caudal, portion of the ventral tegmental area) produced conditioned place preferences. By contrast, high doses of systemic caffeine at 10 and 30 mg/kg produced conditioned place aversions. These aversions were not recapitulated by a caffeine analog restricted to the periphery. Both caffeine reward and aversion were blocked by systemic D1-like receptor antagonism using SCH23390, while systemic D2-like receptor antagonism with eticlopride had smaller effects on caffeine motivation. Most important, we demonstrated that pharmacological blockade of dopamine receptors using α-flupenthixol injected into the nucleus accumbens shell, but not core, blocked caffeine-conditioned place preferences. Conversely, α-flupenthixol injected into the nucleus accumbens core, but not shell, blocked caffeine-conditioned place aversions. Thus, our findings reveal two dopamine-dependent and functionally dissociable mechanisms for processing caffeine motivation, which are segregated between nucleus accumbens subregions. These data provide novel evidence for the roles of the nucleus accumbens subregions in mediating approach and avoidance behaviours for caffeine.

Induction of Rod and Cone Photoreceptor-Specific Progenitors from Stem Cells.

Retinal degeneration includes a variety of diseases for which there is no regenerative therapy. Cellular transplantation is one potential approach for future therapy for retinal degeneration, and stem cells have emerged as a promising source for future cell therapeutics. One major barrier to therapy is the ability to specify individual photoreceptor lineages from a variety of stem cell sources. In this review, we focus on photoreceptor genesis from progenitor populations in the developing embryo and how this understanding has given us the tools to manipulate cultures to specific unique rod and cone lineages from adult stem cell populations. We discuss experiments and evidence uncovering the lineage mechanisms at play in the establishment of fate-specific rod and cone photoreceptor progenitors. This may lead to an improved understanding of retinal development in vivo, as well as new cell sources for transplantation.

Mutations in the guanylate cyclase gcy-28 neuronally dissociate naïve attraction and memory retrieval.

The molecules and mechanisms that are involved in the acquisition, storage, and retrieval of memories in many organisms are unclear. To investigate these processes, we use the nematode worm Caenorhabditis elegans, which is attracted naïvely to the odorant benzaldehyde but learns to avoid it after paired exposure with starvation. Mutations in the receptor-like guanylate cyclase GCY-28 have previously been thought to result in a behavioral switch in the primary chemosensory neuron AWCON , from an attractive state to an aversive (already-learned) state. Here, we offer a different interpretation and show that GCY-28 functions in distinct neurons to modulate two independent processes: naïve attraction to AWCON -sensed odors in the AWCON neuron, and associative learning of benzaldehyde and starvation in the AIA interneurons. Consequently, mutants that lack gcy-28 do not approach AWCON -sensed odors and cannot associate benzaldehyde with starvation. We further show that this learning deficit lies in memory retrieval, not in its acquisition or storage, and that GCY-28 is required in AIA for sensory integration only when both AWC neurons (ON and OFF) are activated by chemical stimuli. Our results reveal a novel role of GCY-28 in the retrieval of associative memories and may have wide implications for the neural machineries of learning and memory in general.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP)- Oct4+ cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP+ NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4fl/fl ;Sox1Cre (Oct4CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP+ dNSCs, in these Oct4CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine ß-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP+ dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082.

A single administration of the hallucinogen, 4-acetoxy-dimethyltryptamine, prevents the shift to a drug-dependent state and the expression of withdrawal aversions in rodents.

Despite several studies suggesting the therapeutic use of 5-hydroxytryptamine receptors type 2A (5-HT2A ) agonists in the treatment of substance use disorders, the neurobiological basis accounting for such effects are still unknown. It has been observed that chronic exposure to drugs of abuse produces molecular and cellular adaptations in ventral tegmental area (VTA) neurons, mediated by brain-derived neurotrophic factor (BDNF). These BDNF-induced adaptations in the VTA are associated with the establishment of aversive withdrawal motivation that leads to a drug-dependent state. Growing evidence suggests that 5-HT2A receptor signaling can regulate the expression of BDNF in the brain. In this study, we observed that a single systemic or intra-VTA administration of a 5-HT2A agonist in rats and mice blocks both the aversive conditioned response to drug withdrawal and the mechanism responsible for switching from a drug-naive to a drug-dependent motivational system. Our results suggest that 5-HT2A agonists could be used as therapeutic agents to reverse a drug dependent state, as well as inhibiting the aversive effects produced by drug withdrawal.

Deletion of α5 nicotine receptor subunits abolishes nicotinic aversive motivational effects in a manner that phenocopies dopamine receptor antagonism.

Nicotine addiction is a worldwide epidemic that claims millions of lives each year. Genetic deletion of α5 nicotinic acetylcholine receptor (nAChR) subunits has been associated with increased nicotine intake, however, it remains unclear whether acute nicotine is less aversive or more rewarding, and whether mice lacking the α5 nAChR subunit can experience withdrawal from chronic nicotine. We used place conditioning and conditioned taste avoidance paradigms to examine the effect of α5 subunit-containing nAChR deletion (α5 -/-) on conditioned approach and avoidance behaviour in nondependent and nicotine-dependent and -withdrawn mice, and compared these motivational effects with those elicited after dopamine receptor antagonism. We show that nondependent α5 -/- mice find low, non-motivational doses of nicotine rewarding, and do not show an aversive conditioned response or taste avoidance to higher aversive doses of nicotine. Furthermore, nicotine-dependent α5 -/- mice do not show a conditioned aversive motivational response to withdrawal from chronic nicotine, although they continue to exhibit a somatic withdrawal syndrome. These effects phenocopy those observed after dopamine receptor antagonism, but are not additive, suggesting that α5 nAChR subunits act in the same pathway as dopamine and are critical for the experience of nicotine's aversive, but not rewarding motivational effects in both a nondependent and nicotine-dependent and -withdrawn motivational state. Genetic deletion of α5 nAChR subunits leads to a behavioural phenotype that exactly matches that observed after antagonizing dopamine receptors, thus we suggest that modulation of nicotinic receptors containing α5 subunits may modify dopaminergic signalling, suggesting novel therapeutic treatments for smoking cessation.

Targeted activation of primitive neural stem cells in the mouse brain.

Primitive neural stem cells (pNSCs) are the earliest NSCs to appear in the developing forebrain. They persist into the adult forebrain where they can generate all cells in the neural lineage and therefore hold great potential for brain regeneration. Thus, pNSCs are an ideal population to target to promote endogenous NSC activation. pNSCs can be isolated from the periventricular region as leukaemia inhibitory factor-responsive cells, and comprise a rare population in the adult mouse brain. We hypothesized that the pup periventricular region gives rise to more clonal pNSC-derived neurospheres but that pup-derived pNSCs are otherwise comparable to adult-derived pNSCs, and can be used to identify selective markers and activators of endogenous pNSCs. We tested the self-renewal ability, differentiation capacity and gene expression profile of pup-derived pNSCs and found them each to be comparable to adult-derived pNSCs, including being GFAP(-) , nestin(mid) , Oct4(+) . Next, we used pup pNSCs to test pharmacological compounds to activate pNSCs to promote endogenous brain repair. We hypothesized that pNSCs could be activated by targeting the cell surface proteins C-Kit and ErbB2, which were enriched in pNSCs relative to definitive NSCs (dNSCs) in an in vitro screen. C-Kit and ErbB2 signalling inhibition had distinct effects on pNSCs and dNSCs in vitro, and when infused directly into the adult brain in vivo. Targeted activation of pNSCs with C-Kit and ErbB2 modulation is a valuable strategy to activate the earliest cell in the neural lineage to contribute to endogenous brain regeneration.

Oct4 is required ~E7.5 for proliferation in the primitive streak.

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

BDNF signaling in the VTA links the drug-dependent state to drug withdrawal aversions.

Drug administration to avoid unpleasant drug withdrawal symptoms has been hypothesized to be a crucial factor that leads to compulsive drug-taking behavior. However, the neural relationship between the aversive motivational state produced by drug withdrawal and the development of the drug-dependent state still remains elusive. It has been observed that chronic exposure to drugs of abuse increases brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. In particular, BDNF expression is dramatically increased during drug withdrawal, which would suggest a direct connection between the aversive state of withdrawal and BDNF-induced neuronal plasticity. Using lentivirus-mediated gene transfer to locally knock down the expression of the BDNF receptor tropomyosin-receptor-kinase type B in rats and mice, we observed that chronic opiate administration activates BDNF-related neuronal plasticity in the VTA that is necessary for both the establishment of an opiate-dependent state and aversive withdrawal motivation. Our findings highlight the importance of a bivalent, plastic mechanism that drives the negative reinforcement underlying addiction.

Surfaceome profiling reveals regulators of neural stem cell function.

The composition of cell-surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry-based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor-ß signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N-cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c-kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain-derived populations and identifies a host of other candidates that may regulate NSCs.

Critical evaluation of imprinted gene expression by RNA-Seq: a new perspective.

In contrast to existing estimates of approximately 200 murine imprinted genes, recent work based on transcriptome sequencing uncovered parent-of-origin allelic effects at more than 1,300 loci in the developing brain and two adult brain regions, including hundreds present in only males or females. Our independent replication of the embryonic brain stage, where the majority of novel imprinted genes were discovered and the majority of previously known imprinted genes confirmed, resulted in only 12.9% concordance among the novel imprinted loci. Further analysis and pyrosequencing-based validation revealed that the vast majority of the novel reported imprinted loci are false-positives explained by technical and biological variation of the experimental approach. We show that allele-specific expression (ASE) measured with RNA-Seq is not accurately modeled with statistical methods that assume random independent sampling and that systematic error must be accounted for to enable accurate identification of imprinted expression. Application of a robust approach that accounts for these effects revealed 50 candidate genes where allelic bias was predicted to be parent-of-origin-dependent. However, 11 independent validation attempts through a range of allelic expression biases confirmed only 6 of these novel cases. The results emphasize the importance of independent validation and suggest that the number of imprinted genes is much closer to the initial estimates.