The transcription factor Fezf2 directs the differentiation of neural stem cells in the subventricular zone toward a cortical phenotype

Abstract

Postnatal neurogenesis in mammals is confined to restricted brain regions, including the subventricular zone (SVZ). In rodents, the SVZ is a lifelong source of new neurons fated to migrate to the olfactory bulb (OB), where the majority become GABAergic interneurons. The plastic capacity of neonatal and adult SVZ stem/progenitor cells is still largely unknown. By overexpressing the transcription factor Fezf2, a powerful master gene specifying the phenotype of glutamatergic subcerebral projecting neurons, we investigated whether the fate of postnatally generated SVZ neurons can be altered. Following lentiviral delivery of Fezf2 in the neonatal and adult SVZ niche, we showed that ectopic Fezf2 expression is sufficient to redirect the fate of SVZ stem cells. Thus, based on in vivo and in vitro experiments, we provide evidence that numerous Fezf2-positive OB neurons expressed glutamatergic pyramidal cell molecular markers instead of developing a GABAergic identity. Overexpression of Fezf2 had no effect on transit-amplifying progenitors or neuroblasts but was restricted to neural stem cells. Fezf2-respecified neurons bore features of pyramidal cells, exhibiting a larger cell body and a more elaborate dendritic tree, compared with OB granule cells. Patch-clamp recordings further indicated that Fezf2-respecified neurons had synaptic properties and a firing pattern reminiscent of a pyramidal cell-like phenotype. Together, the results demonstrate that neonatal and adult SVZ stem cells retain neuronal fate plasticity.

Keywords: adult stem cell plasticity; respecification.

Fig. 1.

Fezf2-expressing cells have a larger cell body and are not GABAergic. (A) Lentiviruses expressing tdTomato and Fezf2-EGFP were mixed and injected into the SVZ of P4 mice and analyzed 4 wpi (n = 10 mice). (B) Frequency distribution of the cell body size of control and Fezf2 cells [bin width = 0.5 μm; **P = 0.0018, Kolmogorov–Smirnov (K-S) test]. (C) Whiskers graph for control and Fezf2 neurons subdivided into GAD67⁺ and GAD67⁻ (one-way ANOVA; ***P < 0.001, Bonferroni multiple comparison post hoc test). n.s., not significant. (D) Stacked bar graph showing that most oversized (os) Fezf2⁺ cells lost their GABAergic phenotype (P < 0.0001, Fisher’s exact test).

Fig. 2.

Altered phenotype of Fezf2-expressing cells in adults. (A) Immunohistochemistry for Fezf2-EGFP, tdTomato, and GAD67 analyzed 4 wpi of the viral mix in adult SVZ (n = 10 mice). Open arrowheads indicate control tdTomato⁺/GAD67⁺ cells. The filled arrowhead indicates a typical oversized Fezf2⁺/GAD67⁻ cell. (Scale bars: 20 μm.) (B) High magnification of a Fezf2 oversized neuron with a larger body size (14.45 μm) than control granule cells (9.78 μm). (Scale bars: 20 μm.) (C) Frequency distribution of cell body size of control and Fezf2 cells (bin width = 0.5 μm; ***P < 0.001, K-S test). (D) Whiskers graph for control and Fezf2 neurons subdivided in GAD67⁺ and GAD67⁻ (one-way ANOVA; ***P < 0.001, Bonferroni multiple comparison post hoc test). (E) Stacked bar graph showing that most oversized (os) Fezf2⁺ cells lost their GABAergic phenotype (P < 0.0001, Fisher’s exact test).

Fig. 3.

Oversized Fezf2⁺ cells display a more elaborate dendritic morphology. Representative camera lucida drawings of biocytin-filled control bulbar granule cells (A), small Fezf2⁺ cells (B), and respecified Fezf2⁺ cells (C) more than 3 wpi of the viral mix in P4 mice. (Scale bar: 100 μm.) Sholl analysis of the cell process length shows significant differences in the length of dendrites (D) and intersections of cell processes (E) (two-way ANOVA with a Bonferroni multiple comparison test). (F) Number of dendritic spines normalized to the corresponding cell process length (spine density) (two-way ANOVA with a Bonferroni multiple comparison test). Error bars were omitted for clarity, and data points indicate means (n = 14/6 cells/mice for control (ctrl), n = 19/9 cells/mice for Fezf2 small (sm), and n = 8/8 cells/mice for Fezf2 os. *P < 0.05; **P <0.01; ****P < 0.0001; n.s., not significant.

Fig. 4.

Fezf2 induces a glutamatergic phenotype. (A) Single-cell RT-PCR detection of VGlut1 (expected size = 311 bp) in Fezf2 oversized (n = 8/3 cells/mice) but not in TdTomato control (n = 5/2 cells/mice) cells. −, negative control (i.e., water). Neurosphere-derived control neurons (tdTomato, red) do not express VGlut1 (B; green), whereas Fezf2⁺ neurons (green) express VGlut1 (D; red). (Scale bars: B and D, 10 μm). βIII Tubulin (βIII-Tub; white) was used as a neuronal marker. (D′) Orthogonal projections show VGlut1⁺ particles (red) in neuronal processes (white). (Scale bar: 2 μm). (C) Total of 88.40 ± 3.003% of Fezf2⁺ neurons and 14.16 ± 3.136% of control neurons are VGlut1⁺ (n = 5 neurosphere preparations, n = 98 Fezf2⁺ cells vs. n = 90 control neurons, t test, ***P < 0.001). (E and F) Voltage steps (1 ms) elicited inward currents blocked by gabazine (10 μM) but not CNQX (10 μM) in control neurons 3 wk after differentiation (five of five neurons). In Fezf2⁺ neurons, inward currents were always blocked by either gabazine (five of five neurons) or CNQX (seven of seven neurons), suggesting that Fezf2⁺ neurons form glutamatergic synapses with neighboring GABAergic cells, which, in turn, releases GABA onto the patch-clamped neuron as shown in the schematic drawing (F).

Fig. 5.

Passive and active physiological properties are altered in oversized Fezf2⁺ cells. Electrophysiological recordings of ctrl granule cells (n = 20/6 cells/mice), sm Fezf2⁺ cells (n = 19/7 cells/mice), and os Fezf2⁺ cells (n = 15/14 cells/mice) were obtained 20–71 d after injection in P4 mice. Resting membrane potential (A) was similar among cell populations, whereas membrane decay τ (B), input resistance (C), and membrane capacitance (D) were significantly different between control and Fezf2 oversized neurons (A, one-way ANOVA followed by Tukey’s test: P = 0.7277; B–D, Kruskal–Wallis test followed by Dunn’s test: **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 6.

Fezf2-redirected neurons exhibit a pyramidal cell-like firing pattern. Action potential firing patterns of a representative control granule cell (A) and an oversized Fezf2⁺ cell (B) in response to a depolarizing current injection (Lower). (C) Spike number during 1-s current pulse injections in granule cells (ctrl, n = 20/6 cells/mice), sm Fezf2⁺ cells (n = 19/7 cells/mice), and os Fezf2⁺ cells (n = 15/14 cells/mice). Current pulses were injected with increments of 10 pA. Oversized Fezf2⁺ cells fire action potentials at significantly higher depolarizations than ctrl and sm Fezf2⁺ cells (two-way ANOVA followed by Bonferroni test: *P < 0.05; **P < 0.01; ****P < 0.0001). (D) Spike ratios show significant different spiking behaviors between the three cell subpopulations (Kruskal–Wallis test followed by Dunn’s test: ***P < 0.001; ****P < 0.0001). Recordings for spike ratios were obtained from the same cells as in B.

Fig. 7.

Synaptic input onto oversized Fezf2⁺ granule cells is altered. (A) Representative mEPSCs recorded from a control cell (Upper) and a Fezf2⁺ oversized cell (Lower). mEPSC amplitude (B) and frequency (C) are not significantly changed in Fezf2 os cells, but a cumulative distribution (D) shows that interevent intervals are significantly shorter in Fezf2⁺ os cells (***P < 0.001, K-S test). (E) Decay time constant of mEPSCs is slower in Fezf2⁺ os cells (**P = 0.0051, t test). (F) mIPSCs recorded from a control cell (Upper) and a Fezf2⁺ os cell (Lower). Mean mIPSC amplitude is increased (G; *P = 0.017) and mIPSC frequency is decreased in Fezf2⁺ os cells (H; *P = 0.033). (I) Cumulative histogram showing that interevent intervals are significantly shorter in Fezf2⁺ os cells (****P < 0.0001, K-S test). (J) Decay time constant of mIPSCs is faster in Fezf2⁺ os cells (**P = 0.0033, t test) (mEPSC ctrl, n = 19/4 cells/mice; mEPSC Fezf2 os, n = 9/7 cells/mice; mIPSC ctrl, n = 16/4 cells/mice; mIPSC Fezf2 os, n = 6/5 cells/mice).