Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more ...rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation-sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type-specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, ...and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS “interaction hub” protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.
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•Rbfox proteins control splicing of cytoskeletal, membrane, and synaptic genes•Rbfox tKO neurons retain an immature splicing program and electrophysiology•The AIS is perturbed in Rbfox tKO neurons due to defects in AnkG localization•A developmental splicing switch in AnkG is critical for AIS assembly
Jacko et al. identified a comprehensive neuronal Rbfox splicing program, enriched for cytoskeletal, membrane, and synaptic genes. Rbfox1/Rbfox2/Rbfox3 triple knockout motor neurons exhibit defects in excitability and axon initial segment assembly, regulated by a developmental splicing switch in ankyrin G.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has ...called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for ...intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1, and column-specific markers that mirror those observed in vivo in human embryonic spinal cord. They also exhibited spontaneous and induced activity, and projected axons toward muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1(+)/LHX3(-)). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.
The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this ...region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1+ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.
Given its high penetrance, clearly delineated and evolutionary conserved genomic structure, mouse models of the 22q11.2 deletion provide an ideal organism-based and cell-based model of this ...well-established disease mutation for schizophrenia. In this study we examined the development of changes in intrinsic properties, action potential firing and synaptic transmission using whole-cell patch-clamp recordings of cultured embryonic cortical neurons from Df(16)A
and WT mice at DIV7 and DIV14, respectively. Compared to neurons from the WT littermates, significantly increased input resistance and decreased rising rate of action potential was observed in Df(16)A
mice at DIV7 but not at DIV14 indicative of delayed neuronal maturation. Neurons from Df(16)A
mice also showed significantly higher cellular excitability at both DIV7 and DIV14. Evaluation of Ca
homeostasis perturbation caused by 22q11.2 deletion using calcium imaging revealed a significantly lower amplitude of calcium elevation and a smaller area under the curve after depolarization in neurons from Df(16)A
mice at both DIV7 and DIV14. Furthermore, the properties of inhibitory synaptic events were significantly altered in Df(16)A
mice. We identified changes in mRNA expression profiles, especially in ion channels, receptors, and transporters that may underlie the neurophysiological effects of this mutation. Overall, we show a number of alterations in electrophysiological and calcium homeostatic properties of embryonic cortical neurons from a 22q11.2 deletion mouse model at different culture times and provide valuable insights towards revealing disease mechanisms and discovery of new therapeutic compounds.
Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate ...the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.
To assess cardiovascular magnetic resonance (CMR) measured myocardial perfusion reserve (MPR) and exercise testing in asymptomatic patients with moderate-severe AS.
Multi-centre, prospective, ...observational study, with blinded analysis of CMR data. Patients underwent adenosine stress CMR, symptom-limited exercise testing (ETT) and echocardiography and were followed up for 12-30 months. The primary outcome was a composite of: typical AS symptoms necessitating referral for AVR, cardiovascular death and major adverse cardiovascular events. 174 patients were recruited: mean age 66.2 ± 13.34 years, 76% male, peak velocity 3.86 ± 0.56 m/s and aortic valve area index 0.57 ± 0.14 cm2/m2. A primary outcome occurred in 47 (27%) patients over a median follow-up of 374 (IQR 351-498) days. The mean MPR in those with and without a primary outcome was 2.06 ± 0.65 and 2.34 ± 0.70 (P = 0.022), while the incidence of a symptom-limited ETT was 45.7% and 27.0% (P = 0.020), respectively. MPR showed moderate association with outcome area under curve (AUC) = 0.61 (0.52-0.71, P = 0.020), as did exercise testing (AUC = 0.59 (0.51-0.68, P = 0.027), with no significant difference between the two.
MPR was associated with symptom-onset in initially asymptomatic patients with AS, but with moderate accuracy and was not superior to symptom-limited exercise testing. ClinicalTrials.gov (NCT01658345).
The Deep Propagating Gravity Wave Experiment (DEEPWAVE) was designed to quantify gravity wave (GW) dynamics and effects from orographic and other sources to regions of dissipation at high altitudes. ...The core DEEPWAVE field phase took place from May through July 2014 using a comprehensive suite of airborne and ground-based instruments providing measurements from Earth’s surface to ∼100 km. Austral winter was chosen to observe deep GW propagation to high altitudes. DEEPWAVE was based on South Island, New Zealand, to provide access to the New Zealand and Tasmanian “hotspots” of GW activity and additional GW sources over the Southern Ocean and Tasman Sea. To observe GWs up to ∼100 km, DEEPWAVE utilized three new instruments built specifically for the National Science Foundation (NSF)/National Center for Atmospheric Research (NCAR) Gulfstream V (GV): a Rayleigh lidar, a sodium resonance lidar, and an advanced mesosphere temperature mapper. These measurements were supplemented by in situ probes, dropsondes, and a microwave temperature profiler on the GV and by in situ probes and a Doppler lidar aboard the German DLR Falcon. Extensive ground-based instrumentation and radiosondes were deployed on South Island, Tasmania, and Southern Ocean islands. Deep orographic GWs were a primary target but multiple f lights also observed deep GWs arising from deep convection, jet streams, and frontal systems. Highlights include the following: 1) strong orographic GW forcing accompanying strong cross-mountain flows, 2) strong high-altitude responses even when orographic forcing was weak, 3) large-scale GWs at high altitudes arising from jet stream sources, and 4) significant flight-level energy fluxes and often very large momentum fluxes at high altitudes.
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