Jeffrey S. Nye

Radial Glial Identity Is Promoted by Notch1 Signaling in the Murine Forebrain

In vertebrates, Notch signaling is generally thought to inhibit neural differentiation. However, whether Notch can also promote specific early cell fates in this context is unknown. We introduced activated Notch1 (NIC) into the mouse forebrain, before the onset of neurogenesis, using a retroviral vector and ultrasound imaging. During embryogenesis, NIC-infected cells became radial glia, the first specialized cell type evident in the forebrain. Thus, rather than simply inhibiting differentiation, Notch1 signaling promoted the acquisition of an early cellular phenotype. Postnatally, many NIC-infected cells became periventricular astrocytes, cells previously shown to be neural stem cells in the adult. These results suggest that Notch1 promotes radial glial identity during embryogenesis, and that radial glia may be lineally related to stem cells in the adult nervous system.

Glycogen Synthase Kinase-3β Modulates Notch Signaling and Stability

Notch receptors modulate transcriptional targets following the proteolytic release of the Notch intracellular domain (NotchIC). Phosphorylated forms of NotchIC have been identified within the nucleus and have been associated with CSL members, as well as correlated with regions of the receptor that are required for activity. Genetic studies have suggested that the Drosophila homolog of glycogen synthase kinase-3beta (GSK3beta), Shaggy, may act as a positive modulator of the Notch signaling. GSK3beta is a serine/threonine kinase and is a component of the Wnt/wingless signaling cascade. Here, we observed that GSK3beta was able to bind and phosphorylate Notch1IC in vitro, and attenuation of GSK3beta activity reduced phosphorylation of NotchIC in vivo. Functionally, ligand-activated signaling through the endogenous Notch1 receptor was reduced in GSK3beta null fibroblasts, implying a positive role for GSK3beta in mammalian Notch signaling. As a possible mechanistic explanation of the effect of GSK3beta on Notch signaling, we observed that inhibition of GSK3beta shortened the half-life of Notch1IC. Conversely, activated GSK3beta reduced the quantity of Notch1IC that was degraded by the proteasome. These studies reveal that GSK3beta modulates Notch1 signaling, possibly through direct phosphorylation of the intracellular domain of Notch, and that the activity of GSK3beta protects the intracellular domain from proteasome degradation.

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.

Developmental Signalling: Vertebrate ligands for Notch

Abstract Newly discovered potential ligands for the Notch family of cell–cell signaling receptors illuminate the importance of lateral signaling during development and promise to help unravel signal transduction by Notch.

Genetic studies in neural tube defects

Neural tube defects (NTD) are one of the most common birth defects and are caused by both environmental and genetic factors. The approach to identifying the genes predisposing to NTD, through linkage analysis and candidate gene analysis, is reviewed along with characteristics of a large, nationally ascertained cohort of families. Results from specific assessments of p53, PAX3 and MTHFR failed to suggest that these genes play a major role in NTD development in these families. Advances in genetic laboratory and statistical techniques have made this a prime opportunity for investigation into the causes of complex disorders, such as NTD. However, traditional approaches may prove to be challenging due to the difficulty of ascertaining samplable multiplex families.

Myelocystocele-cloacal exstrophy in a pedigree with a mitochondrial 12S rRNA mutation, aminoglycoside-induced deafness, pigmentary disturbances, and spinal anomalies

A large Filipino-American family with progressive matrilineal hearing loss, premature graying, depigmented patches, and digital anomalies was ascertained through a survey of a spina bifida clinic for neural crest disorders. Deafness followed a matrilineal pattern of inheritance and was associated with the A1555G mutation in the 12S rRNA gene (MTRNR1) in affected individuals as well as unaffected maternal relatives. Several other malformations were found in carriers of the mutation. The proband had a myelocystocele, Arnold-Chiari type I malformation, cloacal exstrophy, and severe early-onset hearing loss. Several family members had premature graying, white forelock, congenital leukoderma with or without telecanthus, somewhat suggestive of a Waardenburg syndrome variant. In addition to the patient with myelocystocele, two individuals had scoliosis and one had segmentation defects of spinal vertebrae. The syndromic characteristics reported here are novel for the mitochondrial A1555G substitution, and may result from dysfunction of mitochondrial genes during early development. However, the mitochondrial A1555G mutation is only rarely associated with neural tube defects as it was not found in a screen of 218 additional individuals with spina bifida, four of whom had congenital hearing loss.

Neural tube defects and the 13q deletion syndrome: Evidence for a critical region in 13q33‐34

Neural tube defects (NTD) are common findings in the 13q deletion syndrome, but the relationship between the 13q- syndrome and NTDs is poorly understood. We present a child with a 13q deletion and lumbosacral myelomeningocele. This was a boy with microcephaly, telecanthus, minor facial anomalies, and ambiguous genitalia. Cytogenetic and fluorescence in situ hybridization analysis showed a de novo 46,XY,del(13)(q33.2-->qter) with no visible translocation. By using microsatellite markers, the deletion breakpoint was mapped to a 350-kb region between D13S274 and D13S1311 and was paternal in origin. An analysis of 13q deletions with NTDs, including the present case, suggests that a deletion in 13q33-34 is sufficient to cause an NTD. The deletions associated with NTDs are distal to and nonoverlapping with the previously defined critical region in 13q32 for the major malformation syndrome [Brown et al., 1999: Am J Hum Genet 57: 859-866]. Our analysis also suggests that one or more genes in 13q33-34 produces NTDs by haploinsufficiency.

Possible interaction of genotypes at cystathionine β‐synthase and methylenetetrahydrofolate reductase (MTHFR) in neural tube defects

Neural tube defects are a common, complex disorder with genetic and environmental components to risk. We investigated the previously reported interaction between homozygosity for the thermolabile variant at the methylenetetrahydrofolate reductase and heterozygosity for the 844ins68 allele at the cystathionine β‐synthase loci in cases with lumbosacral myelomeningocele and their parents. Using control allele frequencies from our sample pooled with those published in the literature, we confirm a marginally significant interaction at these two loci. This finding suggests that additional, larger studies are warranted to investigate this possible interaction in more detail.

Lack of requirement for presenilin1 in Notch1 signaling

Studies in invertebrates have indicated a functional requirement for presenilin (PS) genes in the Notch pathway [1-5]. One model of Notch signal transduction suggests that proteolysis releases an activated Notch fragment that migrates to the nucleus and regulates gene transcription in concert with CBF1/Su(H)/lag1 (CSL) proteins [6-9]. Recent studies suggest that PS genes control the proteolysis and nuclear access of the Notch intracellular domain [3,4,10,11], offering a basis for the functional interaction of PS and Notch genes [12]. Here, we report that Notch1 signaling elicited by the ligand Delta1 was quantitatively unchanged in PS1-deficient primary embryonic fibroblasts (PEFs). Notch1 signals were measured by both the activation of the hairy/enhancer of split (HES1) promoter and by the antagonism of MyoD-induced muscle creatine kinase (MCK) promoter activity. A membrane-tethered ligand-independent Notch1 construct also showed full efficacy in both assays, despite its presumed requirement for cleavage. Although signaling through Notch1 persisted in PS1-deficient cells, we found a marked reduction in the appearance of a complex of a cleaved, intracellular Notch fragment (NICD) and a CSL protein, as previously reported [6] [10]. These studies reveal that PS1 is not required for ligand-dependent Notch signaling, and that PS1 and PS2 may be redundant. Our data also suggest that the identified NICD fragment may not be necessary for Notch signal transduction [9].

Updated investigations of the role of methylenetetrahydrofolate reductase in human neural tube defects

Folate supplementation appears to reduce the risk for neural tube defects (NTDs). Methylenetetrahydrofolate reductase (MTHFR) is a candidate gene in the folate metabolism pathway that has been extensively studied in different human populations. We examined the risk associated with having the thermolabile variant (TT) of MTHFR in a study of 175 American Caucasians with NTDs and their families. We found a significant association in patients compared with 195 unrelated controls [odds ratio (OR) = 2.13, 95% confidence interval (95% CI) = 1.11–4.09)], but not in mothers (OR = 1.29, 95% CI = 0.622–2.67) or in fathers (OR = 1.45, 95% CI = 0.681–3.09). We found no evidence for unequal transmission from parents to an affected child (p > 0.10). We failed to find a previously reported association for a combined haplotype for MTHFR and cystathionine β‐synthase, except in subjects with NTDs compared with 559 pooled controls (OR = 2.87, 95% CI = 1.03–8.03). We found no evidence for an association for a novel CA‐repeat polymorphism identified in a gene closely linked to MTHFR (p > 0.10). Our studies continue to suggest that additional candidate genes other than MTHFR may be responsible for an increased risk to NTD in some American Caucasian families.