Nicholas Gaiano

Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis

In sprouting angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. VEGF-A is also essential for the induction of endothelial tip cells, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined. Here we present evidence that delta-like 4 (Dll4)–Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. We show that inhibition of Notch signalling using γ-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches. Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics. Together, our results suggest that Dll4–Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene, and indicates that modulators of Dll4 or Notch signalling, such as γ-secretase inhibitors developed for Alzheimer’s disease, might find usage as pharmacological regulators of angiogenesis.

Semaphorin 3E–Plexin-D1 signaling regulates VEGF function in developmental angiogenesis via a feedback mechanism

Blood vessel networks are typically formed by angiogenesis, a process in which new vessels form by sprouting of endothelial cells from pre-existing vessels. This process is initiated by vascular endothelial growth factor (VEGF)-mediated tip cell selection and subsequent angiogenic sprouting. Surprisingly, we found that VEGF directly controls the expression of Plexin-D1, the receptor for the traditional repulsive axon guidance cue, semaphorin 3E (Sema3E). Sema3E-Plexin-D1 signaling then negatively regulates the activity of the VEGF-induced Delta-like 4 (Dll4)-Notch signaling pathway, which controls the cell fate decision between tip and stalk cells. Using the mouse retina as a model system, we show that Plexin-D1 is selectively expressed in endothelial cells at the front of actively sprouting blood vessels and its expression is tightly controlled by VEGF secreted by surrounding tissues. Therefore, although the Sema3E secreted by retinal neurons is evenly distributed throughout the retina, Sema3E-Plexin-D1 signaling is spatially controlled by VEGF through its regulation of Plexin-D1. Moreover, we show that gain and loss of function of Sema3E and Plexin-D1 disrupts normal Dll4 expression, Notch activity, and tip/stalk cell distribution in the retinal vasculature. Finally, the retinal vasculature of mice lacking sema3E or plexin-D1 has an uneven growing front, a less-branched vascular network, and abnormal distribution of dll4-positive cells. Lowering Notch activity in the mutant mice can reverse this defect, solidifying the observation that Dll4-Notch signaling is regulated by Sema3E-Plexin-D1 and is required for its function in vivo. Together, these data reveal a novel role of Sema3E-Plexin-D1 function in modulating angiogenesis via a VEGF-induced feedback mechanism.

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.

A Dynamic Notch Injury Response Activates Epicardium and Contributes to Fibrosis Repair

Rationale: Transgenic Notch reporter mice express enhanced green fluorescent protein in cells with C-promoter binding factor-1 response element transcriptional activity (CBF1-REx4-EGFP), providing a unique and powerful tool for identifying and isolating “Notch-activated” progenitors. Objective: We asked whether, as in other tissues of this mouse, EGFP localized and functionally tagged adult cardiac tissue progenitors, and, if so, whether this cell-based signal could serve as a quantitative and qualitative biosensor of the injury repair response of the heart. Methods and Results: In addition to scattered endothelial and interstitial cells, Notch-activated (EGFP+) cells unexpectedly richly populated the adult epicardium. We used fluorescence-activated cell sorting to isolate EGFP+ cells and excluded hematopoietic (CD45+) and endothelial (CD31+) subsets. We analyzed EGFP+/CD45−/CD31− cells, a small (<2%) but distinct subpopulation, by gene expression profiling and functional analyses. We called this mixed cell pool, which had dual multipotent stromal cell and epicardial lineage signatures, Notch-activated epicardial-derived cells (NECs). Myocardial infarction and thoracic aortic banding amplified the NEC pool, increasing fibroblast differentiation. Validating the functional vitality of clonal NEC lines, serum growth factors triggered epithelial–mesenchymal transition and the immobilized Notch ligand Delta-like 1–activated downstream target genes. Moreover, cardiomyocyte coculture and engraftment in NOD-SCID (nonobese diabetic–severe combined immunodeficiency) mouse myocardium increased cardiac gene expression in NECs. Conclusions: A dynamic Notch injury response activates adult epicardium, producing a multipotent cell population that contributes to fibrosis repair.

The Notch Target Hes1 Directly Modulates Gli1 Expression and Hedgehog Signaling: A Potential Mechanism of Therapeutic Resistance

Purpose: Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy. Experimental Design: We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures. Results: GBM cells are at least partially resistant to long-term MRK-003 treatment, despite ongoing Notch pathway suppression, and show concomitant upregulation of Wnt and Hedgehog activity. The Notch target Hes1, a repressive transcription factor, bound the Gli1 first intron, and may inhibit its expression. Similar results were observed in a melanoma-derived cell line. Targeting Notch and Hedgehog simultaneously induced apoptosis, decreased cell growth, and inhibited colony-forming ability more dramatically than monotherapy. Low-passage neurospheres isolated from freshly resected human GBMs were also highly susceptible to coinhibition of the two pathways, indicating that targeting multiple developmental pathways can be more effective than monotherapy at eliminating GBM-derived cells. Conclusions: Notch may directly suppress Hedgehog via Hes1 mediated inhibition of Gli1 transcription, and targeting both pathways simultaneously may be more effective at eliminating GBMs cells. Clin Cancer Res; 16(24); 6060–70. ©2010 AACR.

Notch3 signaling initiates choroid plexus tumor formation

Notch3 has been studied in the context of brain development, but whether it plays a role in the formation of brain tumors is unclear. We demonstrate that the introduction of constitutively active Notch3 into periventricular cells of embryonic day 9.5 mice causes the formation of choroid plexus tumors (CPTs). Tumors arose in the fourth ventricles in 83% of animals and were associated with hydrocephalus. They were microscopically highly similar to choroid plexus papillomas in humans, with an ongoing proliferation rate of 4–6%. Signs of Notch pathway activity were also present in human choroid plexus lesions, and receptor mRNA levels in papillomas were elevated over those in non-neoplastic choroid plexus. Notch2 was overexpressed approximately 500-fold in one case, suggesting that the role of this pathway in CPTs may not be specific to Notch3. Our findings indicate that activated Notch3 can function as an oncogene in the developing brain, and link the Notch pathway to human CPT pathogenesis.

Lunatic Fringe Deficiency Cooperates with the Met/Caveolin Gene Amplicon to Induce Basal-like Breast Cancer

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC.

Notch, Neural Stem Cells, and Brain Tumors

The Notch pathway has a fundamental role during cell-fate specification in the developing mammalian nervous system. During neocortical development, Notch signaling inhibits neuronal differentiation and maintains the neural stem/progenitor cell pool to permit successive waves of neurogenesis, which are followed by gliogenesis. In addition, recent evidence suggests that Notch signaling is not uniformly used among distinct proliferative neural cells types, with the canonical cascade functional in neural stem cells but attenuated in neurogenic progenitors. Although the role of Notch in neural development is increasingly well understood, it has recently become evident that Notch also has a role in brain tumor biology. Notch receptors are overexpressed in many different brain tumor types, and they may have an initiating role in some. Stem-like cells in brain tumors share many similarities with neural stem/progenitor cells and may require Notch for their survival and growth. Understanding the role of Notch signaling in neoplastic and non-neoplastic stem/progenitor populations will advance our understanding of basic principles regulating developmental and stem cell biology and may also lead to more effective therapies for brain tumors.

Notch3 Signaling Promotes Radial Glial/Progenitor Character in the Mammalian Telencephalon

The Notch signaling pathway is known to influence cell fate in the developing mammalian nervous system. Previous work in the mouse telencephalon has shown that activated Notch1 promotes radial glial and astrocytic character in vivo, and fibroblast growth factor (FGF)-responsive neural progenitor character in vitro. In light of studies suggesting that Notch3 can antagonize Notch1, we tested the effects of activated Notch3 (NICD3) in the mouse telencephalon. Infection of embryonic day 9.5 telencephalic progenitors in vivo with NICD3 promoted radial glial/progenitor character embryonically and astrocyte fate postnatally. In addition, expression of NICD3 in telencephalic progenitors in vitro increased neurosphere frequency in FGF2, but was incompatible with neurosphere growth in epidermal growth factor (EGF). Thus, in the developing telencephalon, Notch1 and Notch3 function similarly, and may activate similar signaling cascades. Consistent with this notion, expression of an activated form of the Notch effector CBF1 (CBF1-VP16), or of the pathway target Hes5 promoted radial glial/progenitor character in vivo. Interestingly, unlike NICD1 and NICD3, CBF1-VP16 and Hes5 did not inhibit neurosphere growth in EGF, suggesting that this effect may be mediated at least in part by CBF1/Hes-independent signaling.

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.