Dafe Uwanogho

Neuronatin Promotes Neural Lineage in ESCs via Ca2+ Signaling

Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underling neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca2+ ([Ca2+]i) by antagonizing Ca2+‐ATPase isoform 2 (sarco/endoplasmic reticulum Ca2+‐ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway. STEM CELLS 2010;28:1950–1960

TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones.

p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter‐gene assays, we demonstrate that C‐promoter binding factor‐1 (CBF‐1)‐dependent gene transcription driven by the Notch‐1 intracellular domain (N1ICD) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH‐SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c‐Abl or cisplatin treatment also potently antagonized N1ICD/CBF‐1‐dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split‐1 (HES‐1) protein levels and antagonized the increase in HES‐1 protein induced by exogenous N1ICD expression. Evidence of a direct physical interaction between N1ICD and TAp73α was demonstrated by co‐immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73α binds the N1ICD in a region C‐terminal of aa 2094. Interestingly, ΔNp73α and TAp73αR292H also co‐purified with N1ICD, but neither inhibited N1ICD/CBF‐1‐dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73α reversed the N1ICD‐mediated repression of retinoic acid‐induced differentiation of SH‐SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73α on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis.

The utility of patient specific induced pluripotent stem cells for the modelling of Autistic Spectrum Disorders

Until now, models of psychiatric diseases have typically been animal models. Whether they were to be used to further understand the pathophysiology of the disorder, or as drug discovery tools, animal models have been the choice of preference in mimicking psychiatric disorders in an experimental setting. While there have been cellular models, they have generally been lacking in validity. This situation is changing with the advent of patient-specific induced pluripotent stem cells (iPSCs). In this article, we give a methodological evaluation of the current state of the iPS technology with reference to our own work in generating patient-specific iPSCs for the study of autistic spectrum disorder (ASD). In addition, we will give a broader perspective on the validity of this technology and to what extent it can be expected to complement animal models of ASD in the coming years.

PTP1B Is an Effector of Activin Signaling and Regulates Neural Specification of Embryonic Stem Cells

During embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.

The Notch intracellular domain represses CRE-dependent transcription

Members of the cyclic-AMP response-element binding protein (CREB) transcription factor family regulate the expression of genes needed for long-term memory formation. Loss of Notch impairs long-term, but not short-term, memory in flies and mammals. We investigated if the Notch-1 (N1) exerts an effect on CREB-dependent gene transcription. We observed that N1 inhibits CREB mediated activation of cyclic-AMP response element (CRE) containing promoters in a γ-secretase-dependent manner. We went on to find that the γ-cleaved N1 intracellular domain (N1ICD) sequesters nuclear CREB1α, inhibits cAMP/PKA-mediated neurite outgrowth and represses the expression of specific CREB regulated genes associated with learning and memory in primary cortical neurons. Similar transcriptional effects were observed with the N2ICD, N3ICD and N4ICDs. Together, these observations indicate that the effects of Notch on learning and memory are, at least in part, via an effect on CREB-regulated gene expression.

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming.

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter.