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. Early during development, neural progenitor cells (NPCs) can either proliferate or differentiate into neurons. Thus, generation of the correct number of neurons is governed by a tightly regulated balance between proliferation and differentiation, and disruption of this balance can result in severe developmental deficits, malformations, or cancers. Notch1 is a member of the Notch family of receptors, which make up a highly conserved cell signaling system. Notch1 signaling has been shown to inhibit NPC differentiation and to promote self-renewal, thereby allowing NPCs to divide and progressively generate the enormous number of neurons present in the central nervous system. The molecular mechanism by which NPCs overcome Notch1-mediated inhibition in order to differentiate into neurons, however, is not completely understood. In this study, we show that Prox1, a homeobox transcriptional repressor, plays a fundamental role in the switch to differentiation by suppressing the expression of Notch1 receptor, thereby preventing newly produced neuronal precursors from receiving inhibitory signals from Notch ligands present in neighboring cells. This transcriptional repression may regulate cell cycle exit and differentiation of NPCs as they migrate towards different regions and adopt their final cell fates. We suggest that Prox1 may exert its known influence on embryonic development, organ morphogenesis, and cancer through its ability to counteract Notch1 signaling.

Over the last decades, adult neurogenesis in the central nervous system (CNS) has emerged as a fundamental process underlying physiology and disease. Recent evidence indicates that the homeobox transcription factor Prox1 is a critical intrinsic regulator of neurogenesis in the embryonic CNS and adult dentate gyrus (DG) of the hippocampus, acting in multiple ways and instructed by extrinsic cues and intrinsic factors. In the embryonic CNS, Prox1 is mechanistically involved in the regulation of proliferation versus differentiation decisions of NSCs, promoting cell cycle exit and neuronal differentiation, while inhibits astrogliogenesis. During the complex differentiation events in adult hippocampal neurogenesis, Prox1 is required for maintenance of intermediate progenitors (IPs), differentiation and maturation of glutamatergic interneurons, as well as specification of DG cell identity over CA3 pyramidal fate. The mechanism by which Prox1 exerts multiple functions involves distinct signaling pathways currently not fully highlighted. In this mini-review, we thoroughly discuss the Prox1-dependent phenotypes and molecular pathways in adult neurogenesis in relation to different upstream signaling cues and cell fate determinants. In addition, we discuss the possibility that Prox1 may act as a cross-talk point between diverse signaling cascades to achieve specific outcomes during adult neurogenesis.

The enormous complexity of mammalian central nervous system (CNS) is generated by highly synchronized actions of diverse factors and signalling molecules in neural stem/progenitor cells (NSCs). However, the molecular mechanisms that integrate extrinsic and intrinsic signals to control proliferation versus differentiation decisions of NSCs are not well-understood. Here we identify nuclear receptor NR5A2 as a central node in these regulatory networks and key player in neural development. Overexpression and loss-of-function experiments in primary NSCs and mouse embryos suggest that NR5A2 synchronizes cell-cycle exit with induction of neurogenesis and inhibition of astrogliogenesis by direct regulatory effects on Ink4/Arf locus, Prox1, a downstream target of proneural genes, as well as Notch1 and JAK/STAT signalling pathways. Upstream of NR5a2, proneural genes, as well as Notch1 and JAK/STAT pathways control NR5a2 endogenous expression. Collectively, these observations render NR5A2 a critical regulator of neural development and target gene for NSC-based treatments of CNS-related diseases.

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 controlOlig2gene transcription are not well understood. Here we demonstrate that Prox1, a transcription repressor and downstream target of proneural genes, suppressesOlig2expression 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 ofOlig2expression and proper control of MN versus V2 interneuron identity. Mechanistically, Prox1 interacts with the regulatory elements ofOlig2gene locusin vivoand it is critical for properOlig2transcription regulation. Specifically, chromatin immunoprecipitation analysis in the mouse neural tube showed that endogenous Prox1 directly binds to the proximal promoter of theOlig2gene locus, as well as to the K23 enhancer, which drivesOlig2expression in the pMN domain. Moreover, plasmid-based transcriptional assays in mouse NPCs suggest that Prox1 suppresses the activity ofOlig2gene promoter and K23 enhancer. These observations indicate that Prox1 controls binary fate decisions between MNs and V2 interneurons in NPCs via direct repression ofOlig2gene regulatory elements.