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First published online January 25, 2008
doi: 10.1242/10.1242/dev.013276

Development 135, 729-741 (2008)
Published by The Company of Biologists 2008
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Cux2 (Cutl2) integrates neural progenitor development with cell-cycle progression during spinal cord neurogenesis

Angelo Iulianella1, Madhulika Sharma2, Michael Durnin1, Greg B. Vanden Heuvel2 and Paul A. Trainor1,2,*

1 Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA.
2 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.


Figure 1
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  		Fig. 1. Cux2 protein localizations. (A) In E10.5 ventral spinal cords Cux2 (green) immunostaining is localized to the vz and iz (arrowhead). (B) Cux2 is observed in BrdU-labeled (red) proliferating spinal cord progenitors at E10.5 (arrows). (C-K) Immunostaining of E11.5 spinal cords demonstrating overlapping activity of Cux2 (green) and P27Kip1 (red) in the rp, vz and a 2-4-cell-layer-thick region outlining the iz (arrowhead) as well as the fp. Scale bar: 500 µm in C.

 

Figure 2
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  		Fig. 2. Aberrant neuronal differentiation in Cux2 mutants. (A) Schematic of gene trap insertion mutation in intron 3 (in3) of murine Cux2/Cutl2 gene containing a promoterless neomycin (neo) selectable cassette splice acceptor (SA) and donor (SD) sites, and a long terminal repeat (LTR). (B) Western blot of two individual Cux2+/+ (+/+), Cux2neo/+ (+/-) and Cux2neo/neo (-/-) E12.5 embryo lysates. Cux2 protein migrated near 110 kDa. Loading control westerns for the embryo lysates showed anti-alpha Tubulin levels migrate near 50 kDa. (C,D) Hematoxylin and Eosin staining of the neural tubes of E11.5 wild-type control (C) and Cux2neo/neo mutant (D) embryos, which exhibit hypoplastic neural tubes and reductions in the mz, drg and ventral commissure (arrowhead). (E,J) Ki67 (green) and TuJ1 (red) staining in Cux2neo/+ (E) and Cux2neo/neo (J) E11.5 spinal cords. (F-L) P27Kip1 staining in Cux2neo/+ (F,G) and Cux2neo/neo mutants (K,L) demonstrating reduced p27Kip1 expression levels in the iz at E11.5. (G,L) High magnification of F,K, respectively, detailing p27Kip1 activity in the iz (arrowhead). (J,M) Enlarged lumen (double-ended arrows) and hypoplastic drg (blue arrows). (H-N) Ki67 (green) and NeuN (red) expression in control Cux2neo/+ (H,I) and Cux2neo/neo mutant (M,N) spinal cords. (I,N) High magnification of motoneuron domains of E11.5 Cux2neo/+ and Cux2neo/neo spinal cords showing NeuN expression (red) in post-mitotic motoneurons (arrowhead). Scale bars: 250 µm in E; 500 µm in G.

 

Figure 3
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  		Fig. 3. Cux2 regulates neural progenitors and neuroblasts. (A-D) Pax6 expression in spinal cords of E11.5 wild-type (A), Cux2neo/neo (B) and Nestin-Cux2-ires-EGFP (C,D) embryos. (D) GFP (green) overlay on anti-Pax6 immmunohistochemistry (red) revealed a mediolateral expansion of Pax6-labeled vz cells following Cux2 overexpression (arrows). (E-H) Neurod immunostaining of neuroblasts exiting the vz and also cells in the drg in E11.5 control (E), Cux2neo/neo (F) and Nestin-Cux2-ires-EGFP (G,H) spinal cords. (I,J) Anti-Neurod1 (green) labeling of neuroblasts in the iz (arrow) adjacent to anti-p27Kip1 (red) immunohistochemistry in E10.5 ventral spinal cords of control and Cux2neo/neo embryos. (K,L) Enhanced Neurod1 (red) activity (arrows) in an E10.5 Cux2 transgenic embryo overexpressing Cux2-ires-EGFP (green). Scale bars: 250 µm in A,E; 500 µm in I.

 

Figure 4
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  		Fig. 4. Reduced cell cycle progression in Cux2 mutants. (A,B) Detection of M phase by IDU (green), S phase by BrdU (red) and G2/S phase by IDU/BrdU (yellow/orange) labeling in E10.5 forelimb bud level neural tubes of control Cux2neo/+ (A) and Cux2neo/neo mutant (B) littermates. (C) Schematic of IDU and BrdU pulsing regime of E10.5 pregnant dams from Cux2+/- intercrosses. (D,E) G2/S- and M-phase labeling by BrdU (fuchsia) and pH3 (green) immunohistochemistry, respectively, in neural tubes of E10.5 control (D) and Cux2neo/neo mutants. (F) Bar chart summarizing pH3 counts in forelimb level neural tubes at E10.5 and 11.5. Cux2neo/neo mutants displayed normal levels of pH3 staining at E10.5, but showed significant decreases in pH3 counts at E11.5 (P=0.009).

 

Figure 5
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  		Fig. 5. Neurod1 and p27Kip1 are direct Cux2 targets in the embryonic nervous system. Complexes isolated from E12.5 mouse brains were immunoprecipitated with either anti-Cux2 or anti-RNA polymerase II antibodies followed by reverse cross-linking of protein and DNA, and PCR amplification of a 248 bp fragment of the Neurod1 promoter spanning +48 to -201 (A), or a 227 bp fragment of the p27Kip1 promoter spanning -707 to -481 (C), relative to the transcription start sites. (A) Inverse image of ethidium-stained agarose gel showing PCR amplification of a 248 bp Neurod1 product from genomic DNA (lane 2), input DNA (lane 5), following ChIP with antibody directed against RNA polymerase II (lane 8), indicating that the Neurod1 gene is transcriptionally active, and following ChIP with 5 µl (lane 9) or 10 µl (lane 10) of anti-Cux2 antibodies, indicating that Cux2 interacts with the Neurod1 promoter in vivo. (B) To control for non-specific chromatin immunoprecipitation, a 250 bp product from -5557 to -5308 relative to the Neurod1 transcription start site was amplified from input DNA (lane 2), following ChIP with an anti-RNA polymerase II antibody (lane 6), and following ChIP with 10 µl (lane 7) of anti-Cux2 antibodies. (C) PCR amplification of a 227 bp p27Kip1 product from genomic DNA (lane 2), input DNA (lane 5), following ChIP with anti-RNA polymerase II antibodies (lane 8), indicating that p27Kip1 is transcriptionally active, and following ChIP with 10 ml (lane 9) or 5 ml (lane 10) of anti-Cux2 antibodies, indicating that Cux-2 interacts with the p27Kip1 promoter in vivo. (D) To control for non-specific chromatin immunoprecipitation, a 148 bp product from -5241 to -5094 relative to the p27Kip1 transcription start site was amplified from input DNA (lane 2), following ChIP with anti-RNA polymerase II antibodies (lane 6), and following ChIP with 10 µl (lane 7) of anti-Cux2 antibodies. In both A and C, controls were water (lane 3), no antibody (lane 4), normal mouse IgG (lane 6) and normal rabbit IgG (lane 7). In both B and D, controls were water (lane 3), no antibody (lane 4) and normal rabbit IgG (lane 5).

 

Figure 6
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  		Fig. 6. Cux2 overexpression. (A-C) P27Kip1 levels (red) in E11.5 wild-type (A) and Nestin-enhancer-driven Cux2-ires-EGFP (green) transgenic (B-C) spinal cords in which Cux2 enhances p27Kip1 in the vz and iz in a cell-autonomous manner (*). (D-F) NeuN immunohistochemistry on E11.5 control (D) and Cux2 transgenic (E,F) ventral spinal cords revealed that Cux2 increases NeuN-labeled post-mitotic neurons emerging from the vz (arrow). (G-I) Neurofilament immunohistochemistry labels axons growing towards the ventral midline in control embryos; however, in Cux2-ires-EGFP transgenic embryos, Cux2 overexpression induced ectopic disorganized and misdirected axons throughout the mz (*). (J,K) TuJ1 staining of HH stage 14-15 chick embryos electroporated with a murine Cux2-ires-EGFP vector. (L) Quantification of TuJ1+ and GFP+ double-positive cells following control vector or Cux2 overexpression. *P=0.05 by Student's t-test. Scale bars: 250 µm in A; 300 µm in G.

 

Figure 7
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  		Fig. 7. Dorsoventral patterning of the spinal cord. (A-D) Lhx1 (red) immunostaining of interneurons in the ventral spinal cords of E10.5 control (A), Cux2neo/neo mutants (B) and Nestin-enhancer-driven Cux2-ires-EGFP transgenic (C,D) embryos. (E-H) Isl1 (blue) and Olig2 (red) co-labeling of control (E), Cux2neo/neo mutant (F) and Cux2 transgenic embryos (G,H). Olig2 labels ventral motoneuron progenitors and Isl1 identifies post-mitotic motoneurons and v3 interneurons. (I,J) Nkx2.2 labeling of the ventral most progenitor domain in spinal cords from E10.5 control (I), Cux2neo/neo mutant (J) and Cux2 transgenic (K,L) embryos. (M) Quantification of Cux2 gain- and loss-of-function on Isl1-positive motoneuron formation at E10.5. Cux2neo/neo mutants (n=11) displayed a 32% increase (P=0.000003) in Isl1-positive motoneurons relative to controls (n=8), whereas Cux2 transgenic neural tubes showed a 20% decrease (n=4, P=0.053). A 61% increase in Isl1 numbers are observed when Cux2neo/neo mutants are compared with transgenic embryos (P=0.001). (N) Quantification of Cux2 gain- and loss-of-function on the formation of Lhx1-positve ventral interneurons at E10.5. Cux2neo/neo mutants displayed a 16% decrease (n=8, P=0.08) in Lhx1-positive cells relative to controls (n=4), while Cux2 transgenic neural tubes showed a 43% increase (n=4, P=0.006). A 70% decrease in Lhx1 numbers are observed when Cux2neo/neo mutants are compared with transgenic embryos (P=0.0007). Data are summarized in Table S2 in the supplementary material.

 

Figure 8
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  		Fig. 8. Cux2 and V2 interneuron formation. (A,B) Expression of homeodomain transcription factor Chx10 in V2 interneurons in the spinal cords of E10.5 wild-type (A) and Cux2neo/neo (B) embryos. (C) Cux2neo/neo mutants (n=8; 9.25±5.09) displayed a 45% decrease (P=0.002) in Chx10-positive V2 interneurons relative to controls (n=5; 20.40±5.03). The average values of Chx10-positive cells were plotted with error bars reflecting the standard deviation from the mean.

 

Figure 9
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  		Fig. 9. Model for the dual role of Cux2 in spinal cord neurogenesis. Cux2 activity stimulates the cell-cycle progression of neural progenitors in the spinal cord vz. In so doing, Cux2 initiates a neuroblast gene expression program that includes the bHLH factor Neurod, and subsequently directs these populations to exit the cell cycle by inducing p27Kip1 in the iz. Cux2 activity in neural progenitors also biases their fate to mature interneurons (IN) in the mz, thereby acting as a cell-fate selector in nascent neurons.

 

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