MicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback.

Positive feedback driven by transcriptional regulation has long been considered a key mechanism underlying cell lineage segregation during embryogenesis. Using the developing spinal cord as a paradigm, we found that canonical, transcription-driven feedback cannot explain robust lineage segregation of motor neuron subtypes marked by two cardinal factors, Hoxa5 and Hoxc8. We propose a feedback mechanism involving elementary microRNA-mRNA reaction circuits that differ from known feedback loop-like structures. Strikingly, we show that a wide range of biologically plausible post-transcriptional regulatory parameters are sufficient to generate bistable switches, a hallmark of positive feedback. Through mathematical analysis, we explain intuitively the hidden source of this feedback. Using embryonic stem cell differentiation and mouse genetics, we corroborate that microRNA-mRNA circuits govern tissue boundaries and hysteresis upon motor neuron differentiation with respect to transient morphogen signals. Our findings reveal a previously underappreciated feedback mechanism that may have widespread functions in cell fate decisions and tissue patterning.

Inducible RasGEF1B circular RNA is a positive regulator of ICAM-1 in the TLR4/LPS pathway.

Circular RNAs (circRNAs) constitute a large class of RNA species formed by the back-splicing of co-linear exons, often within protein-coding transcripts. Despite much progress in the field, it remains elusive whether the majority of circRNAs are merely aberrant splicing by-products with unknown functions, or their production is spatially and temporally regulated to carry out specific biological functions. To date, the majority of circRNAs have been cataloged in resting cells. Here, we identify an LPS-inducible circRNA: mcircRasGEF1B, which is predominantly localized in cytoplasm, shows cell-type specific expression, and has a human homolog with similar properties, hcircRasGEF1B. We show that knockdown of the expression of mcircRasGEF1B reduces LPS-induced ICAM-1 expression. Additionally, we demonstrate that mcircRasGEF1B regulates the stability of mature ICAM-1 mRNAs. These findings expand the inventory of functionally characterized circRNAs with a novel RNA species that may play a critical role in fine-tuning immune responses and protecting cells against microbial infection.

Single-cell transcriptomic analysis reveals diversity within mammalian spinal motor neurons.

Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate movements. In vertebrates, the molecular identities of the cardinal MN types such as those innervating limb versus trunk muscles are well elucidated. Yet the identities of finer subtypes within these cell populations that innervate individual muscle groups remain enigmatic. Here we investigate heterogeneity in mouse MNs using single-cell transcriptomics. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. Additionally, we uncover that axial MNs are subdivided into three molecularly distinct subtypes, defined by mediolaterally-biased Satb2, Nr2f2 or Bcl11b expression patterns with different axon guidance signatures. These three subtypes are present in chicken and human embryos, suggesting a conserved axial MN expression pattern across higher vertebrates. Overall, our study provides a molecular resource of spinal MN types and paves the way towards deciphering how neuronal subtypes evolved to accommodate vertebrate motor behaviors.

Identification and characterization of a novel Epstein-Barr Virus-encoded circular RNA from LMP-2 Gene.

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity.

The mammalian imprinted Dlk1-Dio3 locus produces multiple long non-coding RNAs (lncRNAs) from the maternally inherited allele, including Meg3 (i.e., Gtl2) in the mammalian genome. Although this locus has well-characterized functions in stem cell and tumor contexts, its role during neural development is unknown. By profiling cell types at each stage of embryonic stem cell-derived motor neurons (ESC~MNs) that recapitulate spinal cord development, we uncovered that lncRNAs expressed from the Dlk1-Dio3 locus are predominantly and gradually enriched in rostral motor neurons (MNs). Mechanistically, Meg3 and other Dlk1-Dio3 locus-derived lncRNAs facilitate Ezh2/Jarid2 interactions. Loss of these lncRNAs compromises the H3K27me3 landscape, leading to aberrant expression of progenitor and caudal Hox genes in postmitotic MNs. Our data thus illustrate that these lncRNAs in the Dlk1-Dio3 locus, particularly Meg3, play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes and they shape MN subtype identity by regulating Hox genes.

When a gene is active, its DNA sequence is 'transcribed' to form a molecule of RNA. Many of these RNAs act as templates for making proteins. But for some genes, the protein molecules are not their final destinations. Their RNA molecules instead help to control gene activity, which can alter the behaviour or the identity of a cell. For example, experiments performed in individual cells suggest that so-called long non-coding RNAs (or lncRNAs for short) guide how stem cells develop into different types of mature cells. However, it is not clear whether lncRNAs play the same critical role in embryos.Yen et al. used embryonic stem cells to model how motor neurons develop in the spinal cord of mouse embryos. This revealed that motor neurons produce large amounts of a specific group of lncRNAs, particularly one called Meg3. Further experiments showed that motor neurons in mouse embryos that lack Meg3 do not correctly silence a set of genes called the Hox genes, which are crucial for laying out the body plans of many different animal embryos. These neurons also incorrectly continue to express genes that are normally active in an early phase of the stem-like cells that make motor neurons.There is wide interest in how lncRNAs help to regulate embryonic development. With this new knowledge of how Meg3 regulates the activity of Hox genes in motor neurons, research could now be directed toward investigating whether lncRNAs help other tissues to develop in a similar way.