Nuclear receptor NR5A2 negatively regulates cell proliferation and tumor growth in nervous system malignancies.

Nervous system malignancies are characterized by rapid progression and poor survival rates. These clinical observations underscore the need for novel therapeutic insights and pharmacological targets. To this end, here, we identify the orphan nuclear receptor NR5A2/LRH1 as a negative regulator of cancer cell proliferation and promising pharmacological target for nervous system-related tumors. In particular, clinical data from publicly available databases suggest that high expression levels of NR5A2 are associated with favorable prognosis in patients with glioblastoma and neuroblastoma tumors. Consistently, we experimentally show that NR5A2 is sufficient to strongly suppress proliferation of both human and mouse glioblastoma and neuroblastoma cells without inducing apoptosis. Moreover, short hairpin RNA-mediated knockdown of the basal expression levels of NR5A2 in glioblastoma cells promotes their cell cycle progression. The antiproliferative effect of NR5A2 is mediated by the transcriptional induction of negative regulators of the cell cycle, CDKN1A (encoding for p21cip1), CDKN1B (encoding for p27kip1) and Prox1 Interestingly, two well-established agonists of NR5A2, dilauroyl phosphatidylcholine (DLPC) and diundecanoyl phosphatidylcholine, are able to mimic the antiproliferative action of NR5A2 in human glioblastoma cells via the induction of the same critical genes. Most importantly, treatment with DLPC inhibits glioblastoma tumor growth in vivo in heterotopic and orthotopic xenograft mouse models. These data indicate a tumor suppressor role of NR5A2 in the nervous system and render this nuclear receptor a potential pharmacological target for the treatment of nervous tissue-related tumors.

Down-regulation of human long non-coding RNA LINC01187 is associated with nephropathies.

Chronic kidney diseases affect a substantial percentage of the adult population worldwide. This observation emphasizes the need for novel insights into the molecular mechanisms that control the onset and progression of renal diseases. Recent advances in genomics have uncovered a previously unanticipated link between the non-coding genome and human kidney diseases. Here we screened and analysed long non-coding RNAs (lncRNAs) previously identified in mouse kidneys by genome-wide transcriptomic analysis, for conservation in humans and differential expression in renal tissue from healthy and diseased individuals. Our data suggest that LINC01187 is strongly down-regulated in human kidney tissues of patients with diabetic nephropathy and rapidly progressive glomerulonephritis, as well as in murine models of kidney diseases, including unilateral ureteral obstruction, nephrotoxic serum-induced glomerulonephritis and ischemia/reperfusion. Interestingly, LINC01187 overexpression in human kidney cells in vitro inhibits cell death indicating an anti-apoptotic function. Collectively, these data suggest a negative association of LINC01187 expression with renal diseases implying a potential protective role.

The Neurodevelopmental Disorders Associated Gene Rnf113a Regulates Survival and Differentiation Properties of Neural Stem Cells.

RNF113A (Ring Finger Protein 113A) is genetically associated with autism spectrum disorders and X-linked trichothiodystrophy (TTD) syndrome. Loss-of-function mutations in human RNF113A are causally linked to TTD, which is characterized by abnormal development of the central nervous system (CNS) and mental retardation. How the loss of RNF113A activity affects brain development is not known. Here we identify Rnf113a1 as a critical regulator of cell death and neurogenesis during mouse brain development. Rnf113a1 gene exhibits widespread expression in the embryonic CNS. Knockdown studies in embryonic cortical neural stem/progenitor cells (NSCs) and the mouse cortex suggest that Rnf113a1 controls the survival, proliferation, and differentiation properties of progenitor cells. Importantly, Rnf113a1 deficiency triggers cell apoptosis via a combined action on essential regulators of cell survival, including p53, Nupr1, and Rad51. Collectively, these observations establish Rnf113a1 as a regulatory factor in CNS development and provide insights into its role in neurodevelopmental defects associated with TTD and autism.

Calreticulin in renal fibrosis: A short review.

Fibrosis is a common denominator of several pathological conditions. Over the last decade, Calreticulin has emerged as a critical player in the fibrotic processes in many tissues and organs. Here we review the recent advances in our understanding of the regulatory roles of Calreticulin in renal fibrosis. In particular, a proteomic screen that we performed more than 15 years ago, for the identification of novel components involved in the mechanisms of renal fibrosis, led to the observation that Calreticulin is associated with the initiation and progression of kidney fibrosis in a rodent model. We also showed that altered expression levels of Calreticulin in vitro and in vivo are significantly affecting the fibrotic phenotype in cellular systems and animal models, respectively. We also identified an upstream regulatory mechanism that mediates the transcriptional control of Calreticulin expression during the progression of renal fibrosis, by showing that the druggable orphan nuclear receptor NR5A2 and its SUMOylation is involved in this action. These data provide novel targets for future pharmacological interventions against fibrosis. In addition, further proteomic analysis uncovered a correlation between the up-regulation of Calreticulin and that of 14-3-3σ protein. Collectively, our previous observations suggest that Calreticulin is a central node in a regulatory axis that controls the initiation and progression of renal fibrosis.

Prox1 inhibits neurite outgrowth during central nervous system development.

During central nervous system (CNS) development, proper and timely induction of neurite elongation is critical for generating functional, mature neurons, and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here, we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting neurite elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying neurite outgrowth in newly formed neurons. Consistently, we show that Prox1 is sufficient to inhibit neurite extension in mouse and human neuroblastoma cell lines. More importantly, Prox1 overexpression suppresses neurite elongation in primary neuronal cultures as well as in the developing mouse brain, while Prox1 knock-down promotes neurite outgrowth. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca2+ signaling pathway, an important mediator of neurite extension and neuronal maturation. In accordance, Prox1 represses Ca2+ entry upon KCl-mediated depolarization and reduces CREB phosphorylation. These observations suggest that Prox1 acts as a potent suppressor of neurite outgrowth by inhibiting Ca2+ signaling pathway. This action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of neurites and axon elongation.

GemC1 governs multiciliogenesis through direct interaction with and transcriptional regulation of p73.

A distinct combination of transcription factors elicits the acquisition of a specific fate and the initiation of a differentiation program. Multiciliated cells (MCCs) are a specialized type of epithelial cells that possess dozens of motile cilia on their apical surface. Defects in cilia function have been associated with ciliopathies that affect many organs, including brain and airway epithelium. Here we show that the geminin coiled-coil domain-containing protein 1 GemC1 (also known as Lynkeas) regulates the transcriptional activation of p73, a transcription factor central to multiciliogenesis. Moreover, we show that GemC1 acts in a trimeric complex with transcription factor E2F5 and tumor protein p73 (officially known as TP73), and that this complex is important for the activation of the p73 promoter. We also provide in vivo evidence that GemC1 is necessary for p73 expression in different multiciliated epithelia. We further show that GemC1 regulates multiciliogenesis through the control of chromatin organization, and the epigenetic marks/tags of p73 and Foxj1. Our results highlight novel signaling cues involved in the commitment program of MCCs across species and tissues.This article has an associated First Person interview with the first author of the paper.

Synthesis of benzoxazole-based vorinostat analogs and their antiproliferative activity.

Hydroxamic acid derivatives constitute an interesting novel class of antitumor agents. Three of them, including vorinostat, are approved drugs for the treatment of malignancies, while several others are currently under clinical trials. In this work, we present new vorinostat analogs containing the benzoxazole ring as the cap group and various linkers. The benzoxazole-based analogs were synthesized starting either from 2-aminobenzoxazole, through conventional coupling, or from benzoxazole, through a metal-free oxidative amination. All the synthesized compounds were evaluated for their antiproliferative activity on three diverse human cancer cell lines (A549, Caco-2 and SF268), in comparison to vorinostat. Compound 12 (GK601), carrying a benzoxazole ring replacement for the phenyl ring of vorinostat, was the most potent inhibitor of the growth of three cell lines (IC50 1.2-2.1 µΜ), similar in potency to vorinostat. Compound 12 also inhibited human HDAC1, HDAC2 and HDAC6 like vorinostat. This new analog also showed antiproliferative activity against two colon cancer cell lines genetically resembling pseudomyxoma peritonei (PMP), namely HCT116 GNAS R201C/+ and LS174T (IC50 0.6 and 1.4 µΜ, respectively) with potency comparable to vorinostat (IC50 1.1 and 2.1 µΜ, respectively).

Altered SERCA Expression in Breast Cancer.

Background and Objectives: Calcium (Ca2+) signaling is critical for the normal functioning of various cellular activities. However, abnormal changes in cellular Ca2+ can contribute to pathological conditions, including various types of cancer. The maintenance of intracellular Ca2+ levels is achieved through tightly regulated processes that help maintain Ca2+ homeostasis. Several types of regulatory proteins are involved in controlling intracellular Ca2+ levels, including the sarco/endoplasmic reticulum (SR/ER) Ca2+ ATPase pump (SERCA), which maintains Ca2+ levels released from the SR/ER. In total, three ATPase SR/ER Ca2+-transporting (ATP2A) 1-3 genes exist, which encode for several isoforms whose expression profiles are tissue-specific. Recently, it has become clear that abnormal SERCA expression and activity are associated with various types of cancer, including breast cancer. Breast carcinomas represent 40% of all cancer types that affect women, with a wide variety of pathological and clinical conditions. Materials and methods: Using cBioPortal breast cancer patient data, Kaplan-Meier plots demonstrated that high ATP2A1 and ATP2A3 expression was associated with reduced patient survival. Results: The present study found significantly different SERCA specific-type expressions in a series of breast cancer cell lines. Moreover, bioinformatics analysis indicated that ATP2A1 and ATP2A3 expression was highly altered in patients with breast cancer. Conclusion: Overall, the present data suggest that SERCA gene-specific expressioncan possibly be considered as a crucial target for the control of breast cancer development and progression.

Defective synaptic connectivity and axonal neuropathology in a human iPSC-based model of familial Parkinson's disease.

α-Synuclein (αSyn) is the major gene linked to sporadic Parkinson's disease (PD), whereas the G209A (p.A53T) αSyn mutation causes a familial form of PD characterized by early onset and a generally severe phenotype, including nonmotor manifestations. Here we generated de novo induced pluripotent stem cells (iPSCs) from patients harboring the p.A53T mutation and developed a robust model that captures PD pathogenic processes under basal conditions. iPSC-derived mutant neurons displayed novel disease-relevant phenotypes, including protein aggregation, compromised neuritic outgrowth, and contorted or fragmented axons with swollen varicosities containing αSyn and Tau. The identified neuropathological features closely resembled those in brains of p.A53T patients. Small molecules targeting αSyn reverted the degenerative phenotype under both basal and induced stress conditions, indicating a treatment strategy for PD and other synucleinopathies. Furthermore, mutant neurons showed disrupted synaptic connectivity and widespread transcriptional alterations in genes involved in synaptic signaling, a number of which have been previously linked to mental disorders, raising intriguing implications for potentially converging disease mechanisms.

Nuclear receptors in neural stem/progenitor cell homeostasis.

In the central nervous system, embryonic and adult neural stem/progenitor cells (NSCs) generate the enormous variety and huge numbers of neuronal and glial cells that provide structural and functional support in the brain and spinal cord. Over the last decades, nuclear receptors and their natural ligands have emerged as critical regulators of NSC homeostasis during embryonic development and adult life. Furthermore, substantial progress has been achieved towards elucidating the molecular mechanisms of nuclear receptors action in proliferative and differentiation capacities of NSCs. Aberrant expression or function of nuclear receptors in NSCs also contributes to the pathogenesis of various nervous system diseases. Here, we review recent advances in our understanding of the regulatory roles of steroid, non-steroid, and orphan nuclear receptors in NSC fate decisions. These studies establish nuclear receptors as key therapeutic targets in brain diseases.

Complex Effects of the ZSCAN21 Transcription Factor on Transcriptional Regulation of α-Synuclein in Primary Neuronal Cultures and in Vivo.

α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is strongly implicated in Parkinson disease (PD). PD pathogenesis is linked to increased SNCA levels; however, the transcriptional elements that control SNCA expression are still elusive. Previous experiments in PC12 cells demonstrated that the transcription factor zinc finger and SCAN domain containing 21 (ZSCAN21) plays an important regulatory role in SNCA transcription. Currently, we characterized the role of ZSCAN21 in SNCA transcription in primary neuronal cultures and in vivo We found that ZSCAN21 is developmentally expressed in neurons in different rat brain regions. We confirmed its binding in the intron 1 region of SNCA in rat cortical cultures. Lentivirus-mediated silencing of ZSCAN21 increased significantly SNCA promoter activity, mRNA, and protein levels in such cultures. In contrast, ZSCAN21 silencing reduced SNCA in neurosphere cultures. Interestingly, ZSCAN21 overexpression in cortical neurons led to robust mRNA but negligible protein expression, suggesting that ZSCAN21 protein levels are tightly regulated post-transcriptionally and/or post-translationally in primary neurons. Efficient adeno-associated virus-mediated knockdown of ZSCAN21 in the postnatal and adult hippocampus, an area linked with non-motor PD symptoms, revealed no significant alterations in SNCA levels. Overall, our study demonstrates that ZSCAN21 is involved in the transcriptional regulation of SNCA in primary neuronal cultures, but the direction of the effect is variable, likely depending on neuronal maturation. However, the unaltered SNCA levels observed following ZSCAN21 down-regulation in the rat brain, possibly due to compensatory mechanisms, imply that ZSCAN21 is not a master regulator of SNCA in vivo.

Nuclear receptor NR5A2 is involved in the calreticulin gene regulation during renal fibrosis.

BACKGROUND: Renal fibrosis is a common histological finding present in many pathologies; however, key signaling pathways and molecular determinants involved in the development of fibrosis are not fully known yet. Previous findings have established a causative role of calreticulin's up-regulation during the development of renal fibrosis while its down-regulation exhibited a protective effect against fibrosis. Therefore, the mechanism of its up-regulation needs to be explored. METHODS: Bioinformatics analyses of the calreticulin gene promoter combined with transcriptional assays and in vivo chromatin immunoprecipitation experiments in the Unilateral Ureteric Obstruction (UUO) model of renal fibrosis, indicated that NR5A2 is a critical regulator of calreticulin expression. To confirm this finding, and further study post-translational modifications of NR5A2, real time RT-qPCR, immunohistochemistry and Western blotting experiments were performed. RESULTS: NR5A2 is up-regulated at both mRNA and protein level during kidney fibrosis in the UUO model. The post-translational modification of SUMOylation was identified as a critical parameter in this phenomenon and SUMOylation was observed to be up-regulated during the development of renal fibrosis. The enzyme Ubc9, critical for the process of SUMOylation was also upregulated at mRNA and protein level during the process. CONCLUSION: These data establish for the first time a role for NR5A2 and its SUMOylation on the transcriptional regulation of the calreticulin gene in a rodent model of renal fibrosis and raise the possibility that NR5A2 might be a novel target for future anti-fibrotic interventions.

Prox1 Is Required for Oligodendrocyte Cell Identity in Adult Neural Stem Cells of the Subventricular Zone.

Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. Stem Cells 2016;34:2115-2129.

Prox1 regulates Olig2 expression to modulate binary fate decisions in spinal cord neurons.

Specification of spinal cord neurons depends on gene regulation networks that impose distinct fates in neural progenitor cells (NPCs). Olig2 is a key transcription factor in these networks by inducing motor neuron (MN) specification and inhibiting interneuron identity. Despite the critical role of Olig2 in nervous system development and cancer progression, the upstream molecular mechanisms that control Olig2 gene transcription are not well understood. Here we demonstrate that Prox1, a transcription repressor and downstream target of proneural genes, suppresses Olig2 expression and therefore controls ventral spinal cord patterning. In particular, Prox1 is strongly expressed in V2 interneuron progenitors and largely excluded from Olig2+ MN progenitors (pMN). Gain- and loss-of-function studies in mouse NPCs and chick neural tube show that Prox1 is sufficient and necessary for the suppression of Olig2 expression and proper control of MN versus V2 interneuron identity. Mechanistically, Prox1 interacts with the regulatory elements of Olig2 gene locus in vivo and it is critical for proper Olig2 transcription regulation. Specifically, chromatin immunoprecipitation analysis in the mouse neural tube showed that endogenous Prox1 directly binds to the proximal promoter of the Olig2 gene locus, as well as to the K23 enhancer, which drives Olig2 expression in the pMN domain. Moreover, plasmid-based transcriptional assays in mouse NPCs suggest that Prox1 suppresses the activity of Olig2 gene promoter and K23 enhancer. These observations indicate that Prox1 controls binary fate decisions between MNs and V2 interneurons in NPCs via direct repression of Olig2 gene regulatory elements.

Epithelial calreticulin up-regulation promotes profibrotic responses and tubulointerstitial fibrosis development.

Renal fibrosis is the common anatomical feature underlying the progression of chronic kidney disease, a leading cause of morbidity and mortality worldwide. In a previous study, we demonstrated that during development of renal fibrosis in a rat model of unilateral ureteric obstruction, calreticulin (CRT) is up-regulated in tubular epithelial cells (TECs). In the present study, we used in vitro and in vivo approaches to examine the role of CRT in TECs and its contribution to the progression of fibrosis. In cultured renal TECs, CRT overexpression induced acquisition of an altered, profibrotic cellular phenotype. Consistently, the opposite effects were observed for CRT knockdown. Subsequently, we confirmed that critical changes observed in vitro were also apparent in tubular cells in vivo in the animal model of unilateral ureteric obstruction. In agreement with these results, we demonstrate that substantial (50%) reduction in the expression of CRT reduced the development of tubulointerstitial fibrosis at a comparable level through regulation of inflammation, transcriptional activation, transforming growth factor ß1-associated effects, and apoptosis. In summary, our findings establish that CRT is critically involved in the molecular mechanisms that drive renal fibrosis progression and indicate that inhibition of CRT expression might be a therapeutic target for reduction of fibrosis and chronic kidney disease development.

Asymmetric Synthesis of Saturated and Unsaturated Hydroxy Fatty Acids (HFAs) and Study of Their Antiproliferative Activity.

Hydroxy fatty acids (HFAs) constitute a class of lipids, distinguished by the presence of a hydroxyl on a long aliphatic chain. This study aims to expand our insights into HFA bioactivities, while also introducing new methods for asymmetrically synthesizing unsaturated and saturated HFAs. Simultaneously, a procedure previously established by us was adapted to generate new HFA regioisomers. An organocatalytic step was employed for the synthesis of chiral terminal epoxides, which either by alkynylation or by Grignard reagents resulted in unsaturated or saturated chiral secondary alcohols and, ultimately, HFAs. 7-(S)-Hydroxyoleic acid (7SHOA), 7-(S)-hydroxypalmitoleic acid (7SHPOA) and 7-(R)- and (S)-hydroxymargaric acids (7HMAs) were synthesized for the first time and, together with regioisomers of (R)- and (S)-hydroxypalmitic acids (HPAs) and hydroxystearic acids (HSAs), whose biological activity has not been tested so far, were studied for their antiproliferative activities. The unsaturation of the long chain, as well as an odd-numbered (C17) fatty acid chain, led to reduced activity, while the new 6-(S)-HPA regioisomer was identified as exhibiting potent antiproliferative activity in A549 cells. 6SHPA induced acetylation of histone 3 in A549 cells, without affecting acetylated α-tubulin levels, suggesting the selective inhibition of histone deacetylase (HDAC) class I enzymes, and was found to inhibit signal transducer and activator of transcription 3 (STAT3) expression.

The Multi-temporal and Multi-dimensional Global Urban Centre Database to Delineate and Analyse World Cities.

Monitoring sustainable urban development requires comparable geospatial information on cities across several thematic domains. Here we present the first global database combining such information with city extents. The Global Human Settlement Urban Centre Database (GHS-UCDB) is produced by geospatial data integration to characterise more than 10,000 urban centres worldwide. The database is multi-dimensional and multi-temporal, containing 28 variables across five domains and having multitemporal attributes for one or more epochs when the UC are delineated (1975-1990-2000-2015). Delineation of urban centres for the year 2015 is performed via a logic of grid cell population density, population size, and grid cell contiguity defined by the Degree of Urbanisation method. Each of the urban centres has 160 attributes, including a validation assessment. The novel aspects of this database concern the thematic richness and temporal depth of the variables (across geography, socio-economic, environmental, disaster risk reduction, and sustainable development domains) and the type of geo-information provided (location and extent), featuring an overall consistency that allows comparative analyses across locations and time.

Multi-level profiling of the Fmr1 KO rat unveils altered behavioral traits along with aberrant glutamatergic function.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disabilities and the most prevalent monogenic cause of autism. Although the knockout (KO) of the Fmr1 gene homolog in mice is primarily used for elucidating the neurobiological substrate of FXS, there is limited association of the experimental data with the pathophysiological condition in humans. The use of Fmr1 KO rats offers additional translational validity in this regard. Therefore, we employed a multi-level approach to study the behavioral profile and the glutamatergic and GABAergic neurotransmission status in pathophysiology-associated brain structures of Fmr1 KO rats, including the recordings of evoked and spontaneous field potentials from hippocampal slices, paralleled with next-generation RNA sequencing (RNA-seq). We found that these rats exhibit hyperactivity and cognitive deficits, along with characteristic bidirectional glutamatergic and GABAergic alterations in the prefrontal cortex and the hippocampus. These results are coupled to affected excitability and local inhibitory processes in the hippocampus, along with a specific transcriptional profile, highlighting dysregulated hippocampal network activity in KO rats. Overall, our data provide novel insights concerning the biobehavioral profile of FmR1 KO rats and translationally upscales our understanding on pathophysiology and symptomatology of FXS syndrome.

The role of nuclear receptors in controlling the fine balance between proliferation and differentiation of neural stem cells.

In the central nervous system (CNS) of vertebrates a large variety of cell types are specified from a pool of highly plastic neural stem/progenitor cells (NSCs) via a combined action of extrinsic morphogenetic cues and intrinsic transcriptional regulatory networks. Nuclear receptors and their ligands are key regulators of fate decisions in NSCs during development and adulthood, through their ability to control transcription of downstream genes. In the last few years considerable progress has been made towards the understanding of the actions of nuclear receptors in NSCs as well as elucidating the mechanistic basis for these actions. Here we summarize recent progress in the role of nuclear receptors in the biology of NSCs. These studies highlight the importance of this family of transcriptional regulators in CNS development and function in health and disease. Furthermore, they raise the intriguing possibility of using nuclear receptors as therapeutic targets for nervous system related diseases and traumas.

Prox1 regulates the notch1-mediated inhibition of neurogenesis.

Activation of Notch1 signaling in neural progenitor cells (NPCs) induces self-renewal and inhibits neurogenesis. Upon neuronal differentiation, NPCs overcome this inhibition, express proneural genes to induce Notch ligands, and activate Notch1 in neighboring NPCs. The molecular mechanism that coordinates Notch1 inactivation with initiation of neurogenesis remains elusive. Here, we provide evidence that Prox1, a transcription repressor and downstream target of proneural genes, counteracts Notch1 signaling via direct suppression of Notch1 gene expression. By expression studies in the developing spinal cord of chick and mouse embryo, we showed that Prox1 is limited to neuronal precursors residing between the Notch1+ NPCs and post-mitotic neurons. Physiological levels of Prox1 in this tissue are sufficient to allow binding at Notch1 promoter and they are critical for proper Notch1 transcriptional regulation in vivo. Gain-of-function studies in the chick neural tube and mouse NPCs suggest that Prox1-mediated suppression of Notch1 relieves its inhibition on neurogenesis and allows NPCs to exit the cell cycle and differentiate. Moreover, loss-of-function in the chick neural tube shows that Prox1 is necessary for suppression of Notch1 outside the ventricular zone, inhibition of active Notch signaling, down-regulation of NPC markers, and completion of neuronal differentiation program. Together these data suggest that Prox1 inhibits Notch1 gene expression to control the balance between NPC self-renewal and neuronal differentiation.