Mechanotransduction and hyperpolarization-activated currents contribute to spontaneous activity in mouse vestibular ganglion neurons

The hyperpolarization-activated, cyclic nucleotide–sensitive current, Ih, is present in vestibular hair cells and vestibular ganglion neurons, and is required for normal balance function. We sought to identify the molecular correlates and functional relevance of Ih in vestibular ganglion neurons. Ih is carried by channels consisting of homo- or heteromeric assemblies of four protein subunits from the Hcn gene family. The relative expression of Hcn1–4 mRNA was examined using a quantitative reverse transcription PCR (RT-PCR) screen. Hcn2 was the most highly expressed subunit in vestibular neuron cell bodies. Immunolocalization of HCN2 revealed robust expression in cell bodies of all vestibular ganglion neurons. To characterize Ih in vestibular neuron cell bodies and at hair cell–afferent synapses, we developed an intact, ex vivo preparation. We found robust physiological expression of Ih in 89% of cell bodies and 100% of calyx terminals. Ih was significantly larger in calyx terminals than in cell bodies; however, other biophysical characteristics were similar. Ih was absent in calyces lacking Hcn1 and Hcn2, but small Ih was still present in cell bodies, which suggests expression of an additional subunit, perhaps Hcn4. To determine the contributions of hair cell mechanotransduction and Ih to the firing patterns of calyx terminals, we recorded action potentials in current-clamp mode. Mechanotransduction currents were modulated by hair bundle defection and application of calcium chelators to disrupt tip links. Ih activity was modulated using ZD7288 and cAMP. We found that both hair cell transduction and Ih contribute to the rate and regularity of spontaneous action potentials in the vestibular afferent neurons. We propose that modulation of Ih in vestibular ganglion neurons may provide a mechanism for modulation of spontaneous activity in the vestibular periphery

RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells

Electrophysiological Properties of Embryonic Stem Cell-Derived Neurons

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP+ neurons in culture display functional neuronal properties even at early stages of differentiation

Voltage-gated sodium currents from individual GFP⁺ TK23 cells.

<p><b>A,</b> Representative example of fast-activating, fast-inactivating inward current (upper panel) evoked by depolarizing current steps (lower panel; steps range from −80 mV to +15 mV; pre-pulse to −120 mV; Vm = −60 mV). <b>B</b>, Peak current-voltage plot for the cell shown in <b>A</b>. <b>C</b>, Average (±sem) activation curve for all neurons (n = 36).</p

TK23 embryonic stem cells differentiate into neurons expressing GFP.

<p><b>A</b>, TK23 cell cultures containing GFP⁺ neurons. <b>B</b>, Higher magnification image of a single GFP positive neuron, showing extensive processes. <b>C</b>, Glass microelectrode recording from a single neuron in the whole-cell configuration. DIC  =  differential image contrast; GFP  =  green fluorescent protein.</p

Whole-cell recordings from individual GFP⁺ TK23 cells in current-clamp mode.

<p><b>A, B</b>, Examples of spontaneous action potential activity and averaged waveforms from two different neurons. <b>C</b>, Example of an evoked single action potential and the same spike at higher temporal resolution. <b>D</b>, Example of multiple evoked action potentials and higher temporal resolution of the first action potential. <b>E</b>, A hyperpolarizing pulse showing a depolarizing sag followed by a single rebound action potential and the same action potential at higher temporal resolution.</p

Voltage-gated potassium currents from individual GFP⁺ TK23 cells.

<p><b>A,</b> Representative example of delayed rectifier current (upper panel) evoked by depolarizing current steps (lower panel; steps range from −120 mV to +80 mV; Vm = −60 mV). <b>B</b>, Peak (open square) and steady-state (filled square) current-voltage plot for the cell shown in <b>A</b>. <b>C,</b> Representative example of the protocol used to determine K⁺ reversal potential. K⁺ current in upper panel, protocol in lower panel (steps range from −100 mV to −20 mV; pre-pulse to +20 mV; Vm = −60 mV). <b>D</b>, Current-voltage relationship from data in <b>C</b>, illustrating values taken at 2 ms (filled circle) and 20 ms (open circle) after the end of the conditioning pulse to +20 mV. Reversal potential is noted as the point at which the two lines intersect (dashed gray line). <b>E,</b> Another example of delayed rectifier current (upper panel) evoked by short depolarizing current steps followed by a step to −50 mV for use in calculating K⁺ activation (lower panel; steps range from −120 mV to +100 mV; tail current step to −50 mV (arrow); Vm = −60 mV). <b>F</b>, Average (±sem) activation curve for all neurons (n = 35). Data obtained from the tail current (arrow in <b>E</b>), were used for the calculation of K⁺ channel activation.</p

DLK1 promotes neurogenesis of human and mouse pluripotent stem cell-derived neural progenitors via modulating notch and BMP signalling

A better understanding of the control of stem cell maintenance and differentiation fate choice is fundamental to effectively realising the potential of human pluripotent stem cells in disease modelling, drug screening and cell therapy. Dlk1 is a Notch related transmembrane protein that has been reportedly expressed in several neurogenic regions in the developing brain. In this study, we investigated the ability of Dlk1 in modulating the maintenance and differentiation of human and mouse ESC-derived neural progenitors. We found that DLK1, either employed as an extrinsic factor, or via transgene expression, consistently promoted the generation of neurons in both the mouse and human ESC-derived neural progenitors. DLK1 exerts this function by inducing cell cycle exit of the progenitors, as evidenced by an increase in the number of young neurons retaining BrdU labelling and cells expressing the cycling inhibitor P57Kip2. DLK1 antagonised the cell proliferation activity of Notch ligands Delta 1 and Jagged and inhibited Hes1-mediated Notch signaling as demonstrated by a luciferase reporter assay. Interestingly, we found that DLK1 promotes the neurogenic potential of human neural progenitor cells via suppression of Smad activation when they are challenged with BMP. Together, our data demonstrate for the first time a regulatory role for DLK1 in human and mouse neural progenitor differentiation and establish an interaction between DLK1 and Hes1-mediated Notch signaling in these cells. Furthermore, this study identifies DLK1 as a novel modulator of BMP/Smad signalling

Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells

Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson’s disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. In this study, we systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. We show that a brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, we provide evidence that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells