Retinoid Machinery in Distinct Neural Stem Cell Populations with Different Retinoid Responsiveness

Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Raralpha,beta,gamma, Rxralpha,beta,gamma). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation

Radial glia-like cell clones.

<p>Radial glia-like cell clones.</p

PCR primer sequences.

<p>PCR primer sequences.</p

Different neuronal phenotypes developed from radial glia-like cells.

<p>Neurons with different neurotransmitter-phenotypes developed from cloned populations of RGl-cells. Genes indicating noradrenergic (<i>Dbh</i>), serotonergic (<i>Tph2</i>) and cholinergic (<i>Chat</i>) neurotransmitter phenotypes were not expressed in neuron-rich cultures of any RGl-cells regardless of fetal or adult origin (<b>a</b>). GABA- and VGAT-immunopositive GABAergic (<b>b</b>, <b>c</b> respectively) and VGlut2-immunopositive glutamatergic (<b>d</b>) neurons developed in embryo-derived clones upon EGF-withdrawal (cells from clone C4 are shown). All adult-derived clones generated GABAergic neurons. Hippocampus- derived HC_A cells produced <i>VGlut1</i>-expressing (<b>a</b>) neurons, and all SVZ-clones gave rise to tyrosine-hydroxylase (TH)-positive (<b>e, e’</b>) but <i>dbh</i>-negative (<b>a</b>), neurons (<b>e</b>, <b>e’</b>: neurons from SVZ_M clone are shown). The pictures were taken on the 11^th day after EGF-withdrawal.</p

Characteristics of radial glia-like cell clones derived from adult mouse brain.

<p>Adult brain-derived radial glia-like cells (after the first passage) showed elongated cell shape (<b>a</b>), nestin- (<b>b</b>) RC2- (<b>d</b>) and Pax6- (<b>c</b>) immunoreactivity, like those derived from fetal forebrains, but in contrast to embryonic clones, they displayed GFAP-immunoreactivity (<b>c</b>). Cells of adult-derived RGl clones expressed genes characteristic to radial glial cells and many of the investigated positional genes (<b>e</b>).</p

Electrophysiological characteristics of radial glia-like cells.

<p>Electrophysiological characteristics of cloned RGl-1 cells (<b>a</b>, <b>b</b>) and RGl-1 derived neurons (<b>c</b>, <b>d</b>) were detected by whole-cell patch-clamp recording. Large passive conductance together with K_DR current (<b>a</b>) and current/voltage (I-V) relationship (<b>b</b>) are shown from a representative RGl-1 cell. Voltage-dependent inward Na-currents with small amplitude (<b>c</b>) were detected from primitive, differentiating RGl-derived neurons (n = 8). A representative current-profile and its current/voltage (I-V) relationship (<b>d</b>) are shown. The current traces were obtained by clamping the cell membrane from a -70 mV holding potential to values ranging from -160 mV to +20 mV, at 10 mV intervals.</p

Neural differentiation of radial glia-like cells.

<p>Withdrawal of EGF resulted in neuron formation in a 6-day period in both, embryo- (<b>a</b>, <b>b</b>) and adult- (<b>e</b>) derived RGl cell cultures. βIII-tubulin-positive neurons appeared on the top of flat substrate-attached cells which were RC2-positive and GFAP-negative in embryo-derived cultures (<b>b</b>), but displayed GFAP-immunoreactivity in adult-derived cultures (<b>e</b>). GFAP-positive astrocytes appeared in the cultures of embryo-derived RGl cells only in response to supplementation with FCS (<b>c</b>). A 4+4-day induction period (Glaser et al, 2007) evoked the appearance of O4-immunopositive oligodendrocyte-precursors in each investigated RGl clones (<b>d</b>).</p

Characteristics of fetal radial glia-like cell clones.

<p>Cultured radial glia-like cells display nestin- (<b>a, c</b>) RC2- (<b>b</b>) and Sox2-immunoreactivity (<b>c</b>). Cloned radial glia-like (RGl-1) cells contain euploid number (n = 40) of chromosomes (<b>d</b>). Cell viability was determined by MTT-assay in cultures maintained with EGF (20 ng/ml), with the EGF receptor antagonist AG 1478 (10⁻⁷ M) or with both (<b>e</b>). Averages and standard deviations were calculated from 6-8 identically treated sister-cultures; OD: optical density. Radial glia- and/or neural stem cell-specific genes were active in cloned RGl-1 cells, while “pluripotency markers” (<i>Oct4, Nanog</i>) and the neuron-specific gene (<i>Math2</i>) were not transcribed (<b>f</b>). <i>GFAP</i> was present at the mRNA-level, but the protein could not be detected. From the investigated region-specific genes, only <i>Ngn2</i> showed alteration between RGl-clones derived from the ventral (RGl-GFP-A2) and dorsal (RGl-GFP-C4) regions of the embryonic (E14.5) forebrain (<b>g</b>).</p

Rate of neuron- and oligodendrocyte production.

<p>The rate of neuron and oligodendrocyte production by adult-derived clones showed marked differences. After six days of EGF-withdrawal, adult cortex-derived RGl cells (CTX) gave rise to significantly less neurons than any other clones (<b>a</b>). RGl cells derived from the fetal ventral forebrain (clone A2) produced almost twofold more oligodendrocytes than those of dorsal origin (clone C4) (<b>b</b>). Adult RGl cells with hippocampal (HC; clone HC_A) and cortical (CTX; clone CTX_H) origin generated significant amount of oligodendrocytes (O4-immunopositive cells; <b>c</b>), while those of SVZ- (clone: SVZ_M) and midbrain (MID; clone MES_D)-origin produced significantly less O4-positive cells (∼1% of total cells).</p