Notch in the vertebrate nervous system: an old dog with new tricks.

The Notch pathway is prominent among those known to regulate neural development in vertebrates. Notch receptor activation can inhibit neurogenesis, maintain neural progenitor character, and in some contexts promote gliogenesis and drive binary fate choices. Recently, a wave of exciting studies has emerged, which has both solidified previously held assertions and expanded our understanding of Notch function during neurogenesis and in the adult brain. These studies have examined pathway regulators and interactions, as well as pathway dynamics, with respect to both gene expression and cell-cell signaling. Here, focusing primarily on vertebrates, we review the current literature on Notch signaling in the nervous system, and highlight numerous recent studies that have generated interesting and unexpected advances.

Notch3 activation promotes invasive glioma formation in a tissue site-specific manner.

Although Notch signaling has been widely implicated in neoplastic growth, direct evidence for in vivo initiation of neoplasia by the pathway in murine models has been limited to tumors of lymphoid, breast, and choroid plexus cells. To examine tumorigenic potential in the eye and brain, we injected retroviruses encoding activated forms of Notch1, Notch2, or Notch3 into embryonic mice. Interestingly, the majority of animals infected with active Notch3 developed proliferative lesions comprised of pigmented ocular choroid cells, retinal and optic nerve glia, and lens epithelium. Notch3-induced lesions in the choroid, retina, and optic nerve were capable of invading adjacent tissues, suggesting that they were malignant tumors. Although Notch3 activation induced choroidal tumors in up to 67% of eyes, Notch1 or Notch2 activation never resulted in such tumors. Active forms of Notch1 and Notch2 did generate a few small proliferative glial nodules in the retina and optic nerve, whereas Notch3 was 10-fold more efficient at generating growths, many of which were large invasive gliomas. Expression of active Notch1/Notch3 chimeric receptors implicated the RBPjk-association molecule and transactivation domains of Notch3 in generating choroidal and glial tumors, respectively. In contrast to our findings in the optic nerve and retina, introduction of active Notch receptors, including Notch3, into the brain never caused glial tumors. Our results highlight the differential ability of Notch receptor paralogs to initiate malignant tumor formation, and suggest that glial precursors of the optic nerve, but not the brain, are susceptible to transformation by Notch3.

Activity-induced Notch signaling in neurons requires Arc/Arg3.1 and is essential for synaptic plasticity in hippocampal networks.

Notch signaling in the nervous system has been most studied in the context of cell fate specification. However, numerous studies have suggested that Notch also regulates neuronal morphology, synaptic plasticity, learning, and memory. Here we show that Notch1 and its ligand Jagged1 are present at the synapse, and that Notch signaling in neurons occurs in response to synaptic activity. In addition, neuronal Notch signaling is positively regulated by Arc/Arg3.1, an activity-induced gene required for synaptic plasticity. In Arc/Arg3.1 mutant neurons, the proteolytic activation of Notch1 is disrupted both in vivo and in vitro. Conditional deletion of Notch1 in the postnatal hippocampus disrupted both long-term potentiation (LTP) and long-term depression (LTD), and led to deficits in learning and short-term memory. Thus, Notch signaling is dynamically regulated in response to neuronal activity, Arc/Arg3.1 is a context-dependent Notch regulator, and Notch1 is required for the synaptic plasticity that contributes to memory formation.

Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

The exon junction complex component Magoh controls brain size by regulating neural stem cell division.

Brain structure and size require precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, and mechanistic explanations of how aberrant NSC division causes the reduced brain size seen in microcephaly are lacking. Here we show that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly because of INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes, as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number and genomic stability. In utero rescue experiments showed that a key function of Magoh is to control levels of the microcephaly-associated protein Lis1 during neurogenesis. Our results uncover requirements for the EJC in brain development, NSC maintenance and mitosis, thereby implicating this complex in the pathogenesis of microcephaly.