Human pluripotent stem cell derived models that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signaling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells (NPCs) in the embryonic and adult nervous system1-3. This can be exploited to isolate distinct populations of human embryonic stem (ES) cell derived NPCs4. Here, we report the transcriptional and epigenomic analysis of six consecutive stages derived from a HES5-GFP reporter ES cell line5 differentiated along the neural trajectory aimed at modeling key cell fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect the regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell state transition based on the progressive remodeling of the epigenetic landscape and then validated these through a pooled shRNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and show here that they are mediated by combinations of core and stage- specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we have prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and midneurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage-specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modelling development in health and disease.

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.

MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft-mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures-spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL-laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein-protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

e Dengue virus (DENV) causes dengue fever, a major health concern worldwide. We identified an amphipathic helix (AH) in the N-terminal region of the viral nonstructural protein 4A (NS4A). Disruption of its amphipathic nature using mutagenesis reduced homo-oligomerization and abolished viral replication. These data emphasize the significance of NS4A in the life cycle of the dengue virus and demarcate it as a target for the design of novel antiviral therapy. Dengue virus (DENV) infection is a growing public health threat, with more than one-third of the world population at risk (1). DENV is a positive single-strand RNA virus. Its genome is translated into a single polyprotein, which is cleaved to produce structural (components of the mature virus) and nonstructural (NS) proteins. In addition, the NS proteins generate the viral replication complexes (RC) (2). DENV replicates its RNA genome in association with modified intracellular membranes; the details of the assembly of these complexes are incompletely understood.NS4A, a transmembrane endoplasmic reticulum (ER) resident protein, is thought to induce the host membrane modifications that harbor the viral RC (3). A similar function for NS4A was reported in other flaviviruses (4, 5). To further understand the role of NS4A, we analyzed its cytosolic N-terminal region (amino acids 1 to 48) using sequence alignment of the four DENV serotypes. Within this sequence, amino acids that differed in their identity maintained their biochemical properties, suggesting the presence of a conserved structural motif with a potential functional significance (Fig. 1A). Secondary structure algorithms (6) indicated that this segment is predicted to fold into an ␣-helix (Fig. 1B). Helical wheel projections of amino acids 3 to 20 indicated a conserved polar-nonpolar asymmetry indicative of an amphipathic helix (AH) (Fig. 1C). To experimentally examine the conformation of the NS4A N terminus, a recombinant peptide comprising amino acids 1 to 48 was prepared. Codon-optimized DENV2 NS4A 1-48 was cloned into pGEV2 (7) with an N-terminal fusion to the immunoglobulin binding domain of streptococcal protein G (GB1). A tobacco etch virus (TEV) protease cleavage site (ENLYFQ) was introduced into the beginning of the NS4A coding sequence. Due to difficulties in separating NS4A from GB1 after TEV protease cleavage, an N-terminal glutathione S-transferase (GST) affinity tag was added to GB1-TEV-NS4A(1-48) by cloning it into pGEX4T-2. Protein expression and purification of the GST-GB1-NS4A(1-48) fusion were performed as described previously (8), except that an on-column cleavage was performed by adding TEV protease. The flowthrough was concentrated and subjected to size exclusion chromatography (HiLoad 16/60 Superdex 75) yielding pure NS4A(1-48). The resulting peptide was characterized using far-UV circular dichroism (CD) (Fig. 1D). In aqueous buffer, NS4A(1-48) showed limited solubility and a CD spectrum typical of a random coil conformation with an ␣-helix content below 10%. Addition of various memb...