Nuclear factor one B regulates neural stem cell differentiation and axonal projection of corticofugal neurons

Abstract

During development of the cerebral cortex, neural stem cells divide to expand the progenitor pool and generate basal progenitors, outer radial glia, and cortical neurons. As these newly born neurons differentiate, they must properly migrate toward their final destination in the cortical plate, project axons to appropriate targets, and develop dendrites. However, a complete understanding of the precise genetic mechanisms regulating these steps is lacking. Here we show that a member of the nuclear factor one (NFI) family of transcription factors, NFIB, is essential for many of these processes in mice. We performed a detailed analysis of NFIB expression during cortical development, and investigated defects in cortical neurogenesis, neuronal migration, and differentiation in NfiB(-/-) brains. We found that NFIB is strongly expressed in radial glia and corticofugal neurons throughout cortical development. However, in NfiB(-/-) cortices, radial glia failed to generate outer radial glia, subsequently resulting in a loss of late basal progenitors. In addition, corticofugal neurons showed a severe loss of axonal projections, whereas late-born cortical neurons displayed defects in migration and ectopically expressed the early-born neuronal marker CTIP2. Furthermore, gene expression analysis, by RNA sequencing, revealed a misexpression of genes that regulate the cell cycle, neuronal differentiation and migration in NfiB(-/-) brains. Together these results demonstrate the critical functions of NFIB in regulating cortical development.

Keywords: NFIB; basal progenitors; neurogenesis; neuronal migration; outer radial glia; radial glia.

Figure 1

βGAL expression recapitulates NFIB expression in the cerebral cortex. Immunohistochemistry and confocal microscopy were used to determine expression of βGAL and NFIB expression in coronal sections of E18.5 NfiB^+/+ (A–F) and NfiB^+/− (G–L) brains. NFIB was expressed in deep cortical layers, 5 and 6, and proliferative zone of NfiB^+/+ (A, D) and NfiB^+/− (G, J) cortices. βGAL was not expressed in NfiB^+/+ (B, E) cortices, and therefore did not coincide with NFIB expression (C, F). In NfiB^+/− cortices, βGAL expression was restricted to nearly all NFIB-expressing cells in deep cortical layers and proliferative zones (H, K; Merge I, L), thus validating the use of GAL immunostaining in identifying NFIB-expressing cells in NfiB^+/− cortices. A–C, G–I Low magnification; scale bar, 500μm. D–F, J–L show boxed regions in C and I, respectively; scale bar, 100μm.

Figure 2

NFIB is expressed in neural progenitors and deep layer neurons throughout cortical development. Immunohistochemistry and confocal microscopy were used to determine cell-type specific expression of NFIB and βGAL in coronal sections of NfiB^+/+ (A–E, P–T, U–Y) and NfiB^+/− (F–O, Z–II) brains, respectively. A–O, E12.5; P–T, E13.5; U–II, E15.5. A, F, K, P, U, Z, EE Low magnification; scale bars, 500μm. B–E, G–J, L–O, Q–T, V–Y, AA–DD, EE–II show boxed regions (A, F, K, P, U, Z, EE, respectively); scale bars, 100μm.

Figure 2

NFIB is expressed in neural progenitors and deep layer neurons throughout cortical development. Immunohistochemistry and confocal microscopy were used to determine cell-type specific expression of NFIB and βGAL in coronal sections of NfiB^+/+ (A–E, P–T, U–Y) and NfiB^+/− (F–O, Z–II) brains, respectively. A–O, E12.5; P–T, E13.5; U–II, E15.5. A, F, K, P, U, Z, EE Low magnification; scale bars, 500μm. B–E, G–J, L–O, Q–T, V–Y, AA–DD, EE–II show boxed regions (A, F, K, P, U, Z, EE, respectively); scale bars, 100μm.

Figure 3

The molecular identity of NfiB mutant cells is similar to NFIB-expressing cells. Immunohistochemistry and fluorescent microscopy were used to compare the molecular marker identities of NFIB or βGAL expressing cells in coronal sections of NfiB^−/− and NfiB^+/+ or NfiB^+/− cortices at E18.5. A–B Expression of NFIB (magenta, A) or βGAL (magenta, B) and SOX2 (green) in NfiB^+/+ (A) and NfiB^−/− (B) cortices, respectively. C–L Expression of βGAL (magenta) and PAX6 (B–C), TBR2 (D–E), NFIA (G–H), DARPP32 (denoted as DP32, I–J) or SATB2 (K–L) (all in green) in NfiB^+/− (C, E, G, I and K) and NfiB^−/− (D, F, H, J, L) cortices. Scale bars, 25μm.

Figure 4

Severe thinning of upper cortical layers in NfiB^−/− mice. Immunohistochemistry and confocal microscopy were used to compare overall cortical lamination and expression of specific neuronal makers in NfiB^+/+ (A–D, I–L, Q–R) versus NfiB^−/− (E–H, M–R) brains at E18.5. A–H Expression of CTIP2 and SATB2 in NfiB^+/+ and NfiB^−/− cortices. I–P Expression of BETA3 and TBR1 in NfiB^+/+ and NfiB^−/− cortices. Arrows (in P) point to BETA3-expressing cells in layer 6. A–C, E–G, I–K, M–O scale bar, 100μm. D, H, L and P show boxed region in C, G, K and O, respectively; dashed lines indicate estimated boundaries between cortical layers; scale bar, 50μm. Q Schematic shows definitions of regions containing upper layer SATB2⁺ neurons (UL-S) and deep layer CTIP2⁺ neurons (DL-C); see materials and methods for details. R Graph showing comparison of UL-S and DL-C thickness (μm) between NfiB^+/+ and NfiB^−/− mice at E16.5 and E18.5. *p = 0.0077, ***p ≤ 0.0001 by t-test; n = 12 matched sections/age, 4 mice/genotype; error bars indicate SEM.

Figure 5

NfiB^−/− mice display a significant loss of corticofugal axonal projections. A–H PLAP-staining and bright field microscopy were used to identify PLAP-labeled (black) corticofugal axon tracts in NfiB^+/+; Fezf2^+/PLAP (A–D) and NfiB^−/−; Fezf2^+/PLAP (E–H) brains at E18.5. Sagittal sections (A, E) with dashed lines indicating approximate anterior-posterior location of coronal images displayed below (B–D, F–H, respectively). I–T Immunohistochemistry and fluorescent microscopy were used to identify GFP-labeled (white) corticofugal axon tracts in NfiB^+/+; Golli-τ-GFP⁺ (I–N) and NfiB^−/−; Golli-τ-GFP⁺ (O–T) brains at E18.5. I–N Sagittal (I, J, K, M) and coronal (L, N) views of subcerebral (I and J, N) and corticothalamic (K and M, L) tracts. Dashed lines in K and Q indicate approximate anterior-posterior location of coronal images (L and N, R and T, respectively). Black arrows (A, C, D, E, G, H) point to cerebral peduncle, black arrowheads (B, F) point to the thalamus. White arrows point to corticospinal tract through pons (I, J) or reduction thereof (O, P); open arrows point to corticothalamic tract (K–M) or reduction thereof (Q–S); asterisks (B, F, K, L, Q and R) indicate internal capsule; white arrowheads point to corticospinal tract through cerebral peduncle (N) or reduction thereof (T). A–H, I, K, L, N, O, Q, R, T Low magnification; scale bars, 1000μm. J, M, P, S show boxed region (I, K, O, Q, respectively); scale bars, 500μm. po, Pons; th, thalamus; cpd, cerebral peduncle Scale bars, 1000μm.

Figure 5

NfiB^−/− mice display a significant loss of corticofugal axonal projections. A–H PLAP-staining and bright field microscopy were used to identify PLAP-labeled (black) corticofugal axon tracts in NfiB^+/+; Fezf2^+/PLAP (A–D) and NfiB^−/−; Fezf2^+/PLAP (E–H) brains at E18.5. Sagittal sections (A, E) with dashed lines indicating approximate anterior-posterior location of coronal images displayed below (B–D, F–H, respectively). I–T Immunohistochemistry and fluorescent microscopy were used to identify GFP-labeled (white) corticofugal axon tracts in NfiB^+/+; Golli-τ-GFP⁺ (I–N) and NfiB^−/−; Golli-τ-GFP⁺ (O–T) brains at E18.5. I–N Sagittal (I, J, K, M) and coronal (L, N) views of subcerebral (I and J, N) and corticothalamic (K and M, L) tracts. Dashed lines in K and Q indicate approximate anterior-posterior location of coronal images (L and N, R and T, respectively). Black arrows (A, C, D, E, G, H) point to cerebral peduncle, black arrowheads (B, F) point to the thalamus. White arrows point to corticospinal tract through pons (I, J) or reduction thereof (O, P); open arrows point to corticothalamic tract (K–M) or reduction thereof (Q–S); asterisks (B, F, K, L, Q and R) indicate internal capsule; white arrowheads point to corticospinal tract through cerebral peduncle (N) or reduction thereof (T). A–H, I, K, L, N, O, Q, R, T Low magnification; scale bars, 1000μm. J, M, P, S show boxed region (I, K, O, Q, respectively); scale bars, 500μm. po, Pons; th, thalamus; cpd, cerebral peduncle Scale bars, 1000μm.

Figure 6

Loss of actively dividing neural progenitors during late corticogenesis. Immunohistochemistry and confocal microscopy were used to quantify and analyze the distribution of proliferating cells in coronal sections of NfiB^+/+ and NfiB^−/− brains throughout cortical development. A–D, Expression of TBR2 (green) and Ki67 (magenta) in NfiB^+/+ (A) and NfiB^−/− (B) cortices at E16.5. C Schematic showing definition of the proliferative zone (V + o/SVZ); see materials and methods for details. D Graph and table representing V + o/SVZ thickness as both a percentage of total cortical thickness and as absolute value (μm) in NfiB^+/+ and NfiB^−/− mice at E12.5, E14.5, E16.5 and E18.5. ***p = 0.0004 (E16.5), ***p < 0.0001 (E18.5). E–I Expression of CTIP2 (green) and PHH3 (magenta) in NfiB^+/+ (E, F) and NfiB^−/− (G, H) cortices at E16.5. I, Graph showing comparison of the number of PHH3-expressing cells/mm in VZ and o/SVZ (and total number) between NfiB^+/+ and NfiB^−/− mice. *p = 0.0263, **p = 0.0044, ***p = 0.0001. J–O BrdU-labeled cells (green) and DRAQ5 nuclear stain (magenta) in E18.5 NfiB^+/+ (J, K) and NfiB^−/− (L, M) cortices, 2 hours post BrdU-labeling. N, Schematic showing analysis of cortical distribution of BrdU-labeled cells by binning; see Materials and Methods for details. O Number of total BrdU-labeled cells/100μm and in bins 1–6, 7–9 and 10 was compared between E18.5 NfiB^+/+ and NfiB^−/− cortices. *p = 0.0267, ***p < 0.0001. Statistical analyses by t-test, error bars represent SEM; n = 12 matched sections/age, 4 mice/genotype (graphs D, I, O). A, B, E, G, J, L scale bars, 100μm. F, H, K, M show boxed regions in E, G, J, L, respectively; scale bar 50μm.

Figure 6

Loss of actively dividing neural progenitors during late corticogenesis. Immunohistochemistry and confocal microscopy were used to quantify and analyze the distribution of proliferating cells in coronal sections of NfiB^+/+ and NfiB^−/− brains throughout cortical development. A–D, Expression of TBR2 (green) and Ki67 (magenta) in NfiB^+/+ (A) and NfiB^−/− (B) cortices at E16.5. C Schematic showing definition of the proliferative zone (V + o/SVZ); see materials and methods for details. D Graph and table representing V + o/SVZ thickness as both a percentage of total cortical thickness and as absolute value (μm) in NfiB^+/+ and NfiB^−/− mice at E12.5, E14.5, E16.5 and E18.5. ***p = 0.0004 (E16.5), ***p < 0.0001 (E18.5). E–I Expression of CTIP2 (green) and PHH3 (magenta) in NfiB^+/+ (E, F) and NfiB^−/− (G, H) cortices at E16.5. I, Graph showing comparison of the number of PHH3-expressing cells/mm in VZ and o/SVZ (and total number) between NfiB^+/+ and NfiB^−/− mice. *p = 0.0263, **p = 0.0044, ***p = 0.0001. J–O BrdU-labeled cells (green) and DRAQ5 nuclear stain (magenta) in E18.5 NfiB^+/+ (J, K) and NfiB^−/− (L, M) cortices, 2 hours post BrdU-labeling. N, Schematic showing analysis of cortical distribution of BrdU-labeled cells by binning; see Materials and Methods for details. O Number of total BrdU-labeled cells/100μm and in bins 1–6, 7–9 and 10 was compared between E18.5 NfiB^+/+ and NfiB^−/− cortices. *p = 0.0267, ***p < 0.0001. Statistical analyses by t-test, error bars represent SEM; n = 12 matched sections/age, 4 mice/genotype (graphs D, I, O). A, B, E, G, J, L scale bars, 100μm. F, H, K, M show boxed regions in E, G, J, L, respectively; scale bar 50μm.

Figure 7

NfiB^−/− brains display a major loss of outer radial glia during late corticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the quantity and distribution of PAX6-expressing cells in NfiB^+/+ and NfiB^−/− mice throughout cortical development. A–F PAX6 (magenta) and Tuj1 (green) expression in E12.5 NfiB^+/+ (A–C) and NfiB^−/− (D–F) cortices. G–L PAX6 (magenta) and Tuj1 (green) expression in E14.5 NfiB^+/+ (G–I) and NfiB^−/− (J–L) cortices. M–V PAX6 (magenta) and CTIP2 (green) expression in E16.5 NfiB^+/+ (M–Q) and NfiB^−/− (R–V) cortices. Arrows (P, U) point to CTIP2⁺ cells with rounded morphology; arrowheads (U) point to CTIP2⁺ cells with elongated morphology; dashed lines (O, Q, T, V) indicate D–V boundaries of o/SVZ. W–DD PAX6 (magenta) and SOX2 (green) expression in E18.5 NfiB^+/+ (W–Z) and NfiB^−/− (AA–DD). Dashed lines (Y, Z, CC, DD) indicate approximate border between SVZ and oSVZ. A–O, R–T, W–Y, AA–CC scale bars, 100μm. P and U show boxed regions in layer 5 of O and T, respectively; scale bars, 10μm. Q and V show boxed regions in o/SVZ in O and T respectively; scale bars, 50μm. Z and DD show boxed regions in Y and CC, respectively; scale bars 25μm. EE, Total number of PAX6⁺ cells/100μm and in VZ, o/SVZ were compared between NfiB^+/+ and NfiB^−/− mice at E16.5, and E18.5. *p = 0.0461, ***p < 0.0001 by t-test, error bars represent SEM; n = 10 matched sections/age, 4 mice/genotype.

Figure 7

NfiB^−/− brains display a major loss of outer radial glia during late corticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the quantity and distribution of PAX6-expressing cells in NfiB^+/+ and NfiB^−/− mice throughout cortical development. A–F PAX6 (magenta) and Tuj1 (green) expression in E12.5 NfiB^+/+ (A–C) and NfiB^−/− (D–F) cortices. G–L PAX6 (magenta) and Tuj1 (green) expression in E14.5 NfiB^+/+ (G–I) and NfiB^−/− (J–L) cortices. M–V PAX6 (magenta) and CTIP2 (green) expression in E16.5 NfiB^+/+ (M–Q) and NfiB^−/− (R–V) cortices. Arrows (P, U) point to CTIP2⁺ cells with rounded morphology; arrowheads (U) point to CTIP2⁺ cells with elongated morphology; dashed lines (O, Q, T, V) indicate D–V boundaries of o/SVZ. W–DD PAX6 (magenta) and SOX2 (green) expression in E18.5 NfiB^+/+ (W–Z) and NfiB^−/− (AA–DD). Dashed lines (Y, Z, CC, DD) indicate approximate border between SVZ and oSVZ. A–O, R–T, W–Y, AA–CC scale bars, 100μm. P and U show boxed regions in layer 5 of O and T, respectively; scale bars, 10μm. Q and V show boxed regions in o/SVZ in O and T respectively; scale bars, 50μm. Z and DD show boxed regions in Y and CC, respectively; scale bars 25μm. EE, Total number of PAX6⁺ cells/100μm and in VZ, o/SVZ were compared between NfiB^+/+ and NfiB^−/− mice at E16.5, and E18.5. *p = 0.0461, ***p < 0.0001 by t-test, error bars represent SEM; n = 10 matched sections/age, 4 mice/genotype.

Figure 8

Loss of basal progenitors in NfiB^−/− brains during late coticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the number and distribution of TBR2-expressing cells in NfiB^+/+ and NfiB^−/− mice throughout cortical development. A–H SOX2 (red, A, E), TBR2 (green, B, F) and Tuj1 (blue, C, G) expression (Merge D, H) in NfiB^+/+ (A–D) and NfiB^−/− (E–H) cortices at E14.5. I–L TBR2 expression in NfiB^+/+ (I, J, M, N) and NfiB^−/− (K, L, O, P) at E16.5 (I–L) and E18.5 (M–P). White outlines (I, K, M, O) indicate apical and basal surfaces of the cortex, yellow dashed lines (J, L, N, P) indicate approximate boundary between SVZ and oSVZ. J, L, N, and P show boxed regions in I, K, M and O, respectively. Scale bars, 100μm. Q Total numbers of TBR2⁺ cells/100μm and in VZ and o/SVZ were compared between NfiB^+/+ and NfiB^−/− mice at E16.5, and E18.5. *p = 0.0095 (E16.5), *p = 0.0429 (E18.5), **p = 0.0011 (E18.5 oSVZ), **p = 0.0028 (E18.5 total number), ***p < 0.0001 by t-test, error bars indicate SEM; n = 9 matched sections/age, 4 mice/genotype.

Figure 8

Loss of basal progenitors in NfiB^−/− brains during late coticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the number and distribution of TBR2-expressing cells in NfiB^+/+ and NfiB^−/− mice throughout cortical development. A–H SOX2 (red, A, E), TBR2 (green, B, F) and Tuj1 (blue, C, G) expression (Merge D, H) in NfiB^+/+ (A–D) and NfiB^−/− (E–H) cortices at E14.5. I–L TBR2 expression in NfiB^+/+ (I, J, M, N) and NfiB^−/− (K, L, O, P) at E16.5 (I–L) and E18.5 (M–P). White outlines (I, K, M, O) indicate apical and basal surfaces of the cortex, yellow dashed lines (J, L, N, P) indicate approximate boundary between SVZ and oSVZ. J, L, N, and P show boxed regions in I, K, M and O, respectively. Scale bars, 100μm. Q Total numbers of TBR2⁺ cells/100μm and in VZ and o/SVZ were compared between NfiB^+/+ and NfiB^−/− mice at E16.5, and E18.5. *p = 0.0095 (E16.5), *p = 0.0429 (E18.5), **p = 0.0011 (E18.5 oSVZ), **p = 0.0028 (E18.5 total number), ***p < 0.0001 by t-test, error bars indicate SEM; n = 9 matched sections/age, 4 mice/genotype.

Figure 9

Number of apoptotic cells is similar in NfiB^+/+ and NfiB^−/− cortices at E15.5. Immunohistochemistry and confocal microscopy of coronal brain sections were used to quantify CC3⁺ cells in NfiB^+/+ and NfiB^−/− mice at E15.5. A, E Low magnification view of CTIP2 (blue), SOX2 (red) and CC3 (green) expression in NfiB^+/+ (A) and NfiB^−/− (E) cortices; arrows point to CC3-expressing cells. B–D, F–H Higher magnification view of CC3 (green) and DAPI nuclear stain (magenta) expression (B, F and C, G, respectively, Merge D, F) in NfiB^+/+ (B–D) and NfiB^−/− (F–H) cortices; CC3⁺ cells are indicated by dashed outlines. A, E scale bars, 500μm; B–D, F–H scale bars, 10μm. I Schematics showing regions of CC3⁺ cell quantification. Coronal sections within anterior (A) to posterior (P) region indicated by dashed lines (top panel) were used; CC3⁺ cells were quantified on the medial (M) to lateral (L) axis within region indicated by dashed lines (bottom panel); ctx, cortex. J Number of CC3⁺ cells was compared between NfiB^+/+ and NfiB^−/− cortices at E15.5. p = 0.379 by t-test, error bars indicate SEM; n=10 matched sections, 4 mice/genotype.

Figure 10

Increase in neurogenesis at E15.5 and defects in neuronal migration. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the quit fractions throughout neurogenesis and molecular identities of neurons born at E15.5 in NfiB^+/+ and NfiB^−/− cortices. A Quit fractions in NfiB^+/+ and NfiB^−/− mice at E11.5–E15.5. **p = 0.0045 by t-test, error bars indicate SEM; n = 9 matched sections/age, 3 mice/genotype. B–L BrdU labeling was used to quantify and analyze the distribution of neurons generated at E15.5 in NfiB^+/+ and NfiB^−/− cortices; combining with SATB2 and CTIP2 immunostaining (B-– and L) allowed for analysis of neuronal marker expression. B–I E15.5 BrdU⁺ cells (B, F) and expression of CTIP2 and SATB2 (C, G and D, H, respectively, Merge E, I) in E18.5 NfiB^+/+ (B–E) and NfiB^−/− (F–I) cortices; scale bar, 100μm. J Total number of E15.5 BrdU⁺ cells/100μm was compared between NfiB^+/+ and NfiB^−/− cortices; ***p = 0.0001. K The cortical distribution of E15.5 BrdU⁺ cells was analyzed by binning; see Materials and Methods for details. E15.5 BrdU⁺ cells in bins 1–2, 3–6, 7–9 and 10 as a percentage of total number were compared between NfiB^+/+ and NfiB^−/− cortices. *p = 0.038, *p = 0.0287, *p = 0.0066 (bins 1–2, 7–9 and 10, respectively). L Percentages of total E15.5 BrdU⁺ cells that expressed either SATB2 or CTIP2 were compared between NfiB^+/+ and NfiB^−/− cortices. ***p <0.0001. J–L p-values calculated by t-test, error bars indicate SEM; n = 12 matched sections, 4 brains/genotype.

Figure 11

mRNA expression of Notch signaling pathway members is upregulated in NfiB^−/− mice. In situ hybridization and bright field microscopy were used to compare mRNA expression levels (black) of Notch1 (A–D), Hes1 (E–H) and Hes5 (I–L) in E18.5 NfiB^+/+ (A, B, E, F, I, J) and NfiB^−/− (C, D, G, H, K, L) brains. A, C, E, G, I, K Low magnification views; scale bar, 1000μm. B, D, F, H, J and L show boxed regions in A, C, E, G, I and K, respectively. Scale bar 500μm.