No abstract

The mammalian embryonic lethal abnormal vision (ELAV)-like protein HuD is a neuronal RNA-binding protein implicated in neuronal development, plasticity, and diseases. Although HuD has long been associated with neuronal development, the functions of HuD in neural stem cell differentiation and the underlying mechanisms have gone largely unexplored. Here we show that HuD promotes neuronal differentiation of neural stem/progenitor cells (NSCs) in the adult subventricular zone by stabilizing the mRNA of special adenine-thymine (AT)-rich DNA-binding protein 1 (SATB1), a critical transcriptional regulator in neurodevelopment. We find that SATB1 deficiency impairs the neuronal differentiation of NSCs, whereas SATB1 overexpression rescues the neuronal differentiation phenotypes resulting from HuD deficiency. Interestingly, we also discover that SATB1 is a transcriptional activator of HuD during NSC neuronal differentiation. In addition, we demonstrate that NeuroD1, a neuronal master regulator, is a direct downstream target of SATB1. Therefore, HuD and SATB1 form a positive regulatory loop that enhances NeuroD1 transcription and subsequent neuronal differentiation. Our results here reveal a novel positive feedback network between an RNA-binding protein and a transcription factor that plays critical regulatory roles in neurogenesis.HuD | neural stem cells | NeuroD1 | neurogenesis | SATB1

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Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.

Human patient-derived induced pluripotent stem cells (hiPSCs) provide unique opportunities for disease modeling and drug development. However, adapting hiPSCs or their differentiated progenies to high throughput assays for phenotyping or drug screening has been challenging. Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and a major genetic cause of autism. FXS is caused by mutational trinucleotide expansion in the FMR1 gene leading to hypermethylation and gene silencing. One potential therapeutic strategy is to reactivate the silenced FMR1 gene, which has been attempted using both candidate chemicals and cell-based screening. However, molecules that effectively reactivate the silenced FMR1 gene are yet to be identified; therefore, a high throughput unbiased screen is needed. Here we demonstrate the creation of a robust FMR1-Nluc reporter hiPSC line by knocking in a Nano luciferase (Nluc) gene into the endogenous human FMR1 gene using the CRISPR/Cas9 genome editing method. We confirmed that luciferase activities faithfully report FMR1 gene expression levels and showed that neural progenitor cells derived from this line could be optimized for high throughput screening. The FMR1-Nluc reporter line is a good resource for drug screening as well as for testing potential genetic reactivation strategies. In addition, our data provide valuable information for the generation of knock-in human iPSC reporter lines for disease modeling, drug screening, and mechanistic studies.