The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells - PubMed (original) (raw)
Comparative Study
The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells
Tara L Walker et al. J Neurosci. 2007.
Abstract
Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently, these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic, early postnatal, and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However, the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus, which expressed low levels of DCX (DCX(low)), was enriched for neurosphere-forming cells, with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly, the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells, which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells, with some cells displaying characteristics of immature or mature neurons, whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX(low) cells retain their multipotentiality.
Figures
Figure 1.
DCX and GFP expression colocalize in DCX–GFP transgenic mouse lines. A–K, In transgenic DCX–GFP/2 kb brain sections GFP+ cells are found in the E14 cortex and thalamus (A–C) and in the cortex (D–F) in a pattern that colocalized with DCX staining. In the adult DCX–GFP/BAC line, GFP expression was observed in the rostral migratory stream (RMS) and olfactory bulb (OB) (H, K) in a pattern that coincides with native DCX staining (G, I, J). Scale bars: A–C, J, K, 1 mm; D–I, 100 μm.
Figure 2.
GFP relative fluorescence intensity correlates with DCX expression levels in individual cells. Cells from adult DCX–GFP/BAC SVZ were isolated and stained with a DCX-specific antibody. Flow cytometry analysis revealed a strong correlation between the GFP relative fluorescence intensity and the level of DCX immunostaining in individual cells. In all cases, cells that expressed GFP were also DCX+, with no false positives being observed.
Figure 3.
FACS isolation and neurosphere activity of E14 DCX–GFP/2 kb populations. A, Single viable cells were isolated from E14 GFP+ brains using FACS and were sorted into four populations (high, mid, low, and negative) based on DCX expression. B, Unsorted cells from a wild-type littermate were collected as a control. C, The DCXlow population was enriched for neurosphere formation, whereas the number of neurospheres grown from the DCXmid population was significantly reduced. Virtually no neurospheres were detected in the DCXhigh population (***p < 0.001; n = 4 when compared with unsorted control). FSC, Forward scatter (cell size).
Figure 4.
Neurosphere activity of P2 DCX+ cells. A, Higher neurosphere-forming activity was observed in the DCX+ population from whole P2 DCX–GFP/2 kb brains compared with DCX− and unsorted cells (n = 3). B, Within this DCX+ population, however, the higher expressing cells (DCXmid) had a lower neurosphere-forming frequency compared with the cells with lower (DCXlow) expression (n = 2). C, Overall, the P2 cerebellum had relatively high levels of DCX expression and was the only region in which a significantly higher number of neurospheres was observed in the DCXlow cells compared with the DCX− population (*p < 0.05; n = 3) OB, Olfactory bulb.
Figure 5.
Neurosphere activity of P2 cerebellum cells. The DCXhigh cells in the P2 DCX–GFP/2 kb cerebellum had a significantly lower neurosphere-forming frequency then the DCX− cells in this region (***p < 0.001; n = 4), whereas the DCXlow population was enriched for neurosphere-forming cells.
Figure 6.
A, B, Precursor properties of DCX+ cells from the adult SVZ (A) and hippocampus (B) of the DCX–GFP/BAC strain. A, Neurosphere assays revealed that neurosphere-forming frequency of the DCXlow population in the adult SVZ was similar to that of the DCX− population (n = 4). B, The DCXlow population in the adult hippocampus had a significantly higher neurosphere-forming frequency than the DCX− and unsorted populations (*p < 0.05; n = 4).
Figure 7.
Precursor properties of DCX+ cells in the adult DCX–GFP/BAC olfactory bulb. The olfactory bulb had the highest levels of DCX expression in the adult brain. A, This DCX+ population had a lower neurosphere-forming frequency than the DCX− population. B, Of this DCX+ population, the DCXhigh cells had no neurosphere activity, whereas the DCXlow cells were enriched for neurosphere formation.
Figure 8.
Electrical properties of cortical DCX+ cells in acute forebrain slices of E14 and P2 DCX–GFP/2 kb mice. A, Scatter plots of RMP and _R_in of cortical E14 (n = 18) and P2 (n = 24) DCX+ cells. Asterisks denote a significant difference between E14 and P2 groups (**p < 0.01). B, Representative traces of voltage responses to current injection in two different DCX+ cells. Examples of cells in which an action potential (AP) was either absent (E14) or evoked (P2) in response to depolarizing current pulses. C, Bar graphs of normalized populations of E14 (n = 18) and P2 (n = 24) cells in which an action potential (exhibiting an overshoot positive to 0 mV) either could or could not be evoked.
Figure 9.
Depolarization-activated whole-cell currents recorded from cortical DCX+ cells of E14 and P2 DCX–GFP/2 kb mice. A, Representative family of membrane currents recorded from a P2 DCXmid cell in response to depolarizing voltage steps. Holding potential, −60 mV. Inset, Transient inward currents displayed at higher gain. B, Membrane currents recorded from a P2 DCXmid cell in response to a voltage step from −60 to 0 mV in the absence (i) and presence (ii) of 300 n
m
TTX and 300 n
m
TTX plus 10 m
m
TEA (iii). A TTX-sensitive Na+ channel current trace (iv) was obtained by subtracting traces (i) and (ii). Similarly, a tetraethylammonium (TEA)-sensitive K+ channel current trace (v) was obtained by subtracting traces (ii) and (iii). C, D, Current–voltage (I–V) relationships obtained for transient inward Na+ current and persistent outward K+ current in cortical E14 and P2 DCX+ cells. Cells were divided into two groups based on their maximum inward current densities: those exhibiting currents <10 pA/pF (•) and those with transient inward currents >10 pA/pF (○).
Figure 10.
Passive and active electrical properties of SVZ and dentate gyrus DCXlow cells in adult DCX–GFP/BAC brain slices. A, Scatter plots of RMP and _R_in of DCXlow cells in the SVZ (n = 10) and the dentate gyrus (DG) (n = 12) and DCX− cells in the dentate gyrus (n = 6). Asterisks denote a significant difference between SVZ and DG DCXlow cells (**p < 0.01). **_B_**, Representative traces of action potentials (AP) induced by current injection in DCXlow and DCX− cells obtained from the dentate gyrus. Examples of cells that exhibited either a single (DCXlow) or repetitive (DCX−) AP. **_C_**, Bar graphs of normalized populations of SVZ (_n_ = 10) and dentate gyrus (_n_ = 12) DCXlow cells and dentate gyrus (_n_ = 6) DCX− cells in which an action potential (exhibiting an overshoot positive to 0 mV) either was or was not evoked. **_D_**, **_E_**, Current–voltage relationships obtained for transient inward Na+ current and persistent outward K+ current at a holding potential of −80 mV in DCXlow cells in the SVZ (_n_ = 5) and the dentate gyrus (_n_ = 11). Cells were divided into two groups: those exhibiting maximum inward current densities <10 pA/pF (•) and those exhibiting current densities >10 pA/pF (○).
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