1. Volume 2, Issue 2, Pages 345-357 (August 2012)
CTCF Is Required for Neural Development and Stochastic Expression of Clustered Pcdh Genes in Neurons 
Teruyoshi Hirayama, Etsuko Tarusawa, Yumiko Yoshimura, Niels Galjart, Takeshi Yagi 
Cell Reports 
Volume 2, Issue 2, Pages (August 2012)
DOI: /j.celrep
Copyright © 2012 The Authors Terms and Conditions

2. Cell Reports 2012 2, 345-357DOI: (10.1016/j.celrep.2012.06.014)
Copyright © 2012 The Authors Terms and Conditions

3. Figure 1 Phenotypes of the Nex-Cre-Mediated CTCF cKO Mice
(A) Strategy for the Cre-loxP-mediated gene targeting of Ctcf.
(B) Southern blots for control (fl/fl;+/+) and CTCF-cKO (fl/fl;+/Cre) cortex and hippocampus at P1. About 70% of the genomic DNA was deleted in the CTCF-cKO.
(C) Anti-CTCF immunostaining of the sagittal section of P0 brain. The CTCF deletion was found mostly in the dorsal telencephalon, including the cortex (ctx) and hippocampus (hip), but less in olfactory bulb (ob), striatum (st), thalamus (th), midbrain (md), and cerebellum (cb). High-magnification images of the cortex and hippocampus are shown in Figures 4A and 4B. Scale bar, 500 μm.
(D) Survival curves of control (n = 42) and CTCF-cKO (n = 33) mice.
(E) Body weight over time. The CTCF-cKO mice exhibited significant growth retardation by 7 days after birth. n > 10 for each group. ∗p < Error bars represent SEM.
(F) CTCF-cKO mice showed an abnormal limb-clasping reflex when suspended by the tail.
See also Figures S1, S2, and S3 and Table S1.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

4. Figure 2 Lack of Somatosensory Barrel Formation in CTCF-cKO Cortex
(A–C) Staining to detect the barrel structures in coronal cortical sections at P7. Nissl staining (A), CO staining (B), and 5-HTT immunostaining (C).
(D) Immunostaining for 5-HTT in tangential cortical sections at P7. A correct sensory body map was observed in the CTCF-cKO mice compared with control mice, but the barrel structures in the primary somatosensory map were deficient. v, visual cortex; a, auditory cortex; lw, large whisker; sw, small whisker; ll, lower lip; hp, hind paw; fp fore paw.
(E) Schematic diagram for the analysis of topographic projections from the thalamus to somatosensory cortex. DiI and DiA were placed at different sites in the somatosensory cortex (SC) at P0. After 4 weeks, both dyes were retrogradely transported to the ventroposterior nucleus (VP) in the thalamus.
(F) Neurons labeled with DiI (red) and DiA (green) were separated from each other in control and CTCF-cKO VP.
Scale bars, 100 μm (A–C and F) and 500 μm (D).
See also Figure S4.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

5. Figure 3
Severe Downregulation of the Stochastically Expressed Isoforms of the Clustered Pcdh Genes in the CTCF-cKO Cortex and Hippocampus
(A) Chromosomal location of up- and downregulated genes and the frequency of expression changes identified by microarray analysis. The transcriptional start sites of altered genes are plotted on each chromosome with their frequency. The highest frequency was observed in the clustered Pcdh locus in chromosome 18 (asterisk). See also Tables S2, S3, S4, S5, and S6.
(B) Genomic structure of the clustered Pcdh gene locus and binding sites for CTCF. Red triangles indicate CTCF-binding sites. Rhombuses indicate enhancer regions (HS5–HS1 enhances α3–α12 and αc1; CCR enhances β1–β22).
(C and D) Relative gene expression of the clustered Pcdh gene locus in the CTCF-cKO mouse in the P7 cortex (C) and hippocampus (D) compared to control. Asterisks indicate that quantitative reverse-transcription PCR validation was performed.
(E) Validation of microarray results by quantitative reverse-transcription PCR analysis. The stochastically expressed isoforms are shown as red characters. ∗p < 0.05. ∗∗p < Error bars represent SEM. αCR and γCR represent constant region exons of the Pcdhα and Pcdhγ clusters, respectively.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

6. Figure 4
In Situ Hybridization Analysis of Clustered Pcdh Expression in the Cortical and Hippocampal Regions in the CTCF-cKO Mouse
(A and B) Anti-CTCF immunostaining patterns in the cerebral cortex (A) and hippocampus (B) at P0 were compared between CTCF-cKO and control mice. In CTCF-cKO mice, only subpopulation of cells, which are presumably inhibitory neurons and glial cells, were immunopositive. th, thalamus.
(C–F) Expression patterns of the clustered Pcdh genes were examined by in situ hybridization at P7. (C and D) Example of the expression patterns of a stochastically expressed isoform, α4 (red), and a constitutively expressed C-type isoform, αc2, in the cortex (C) and hippocampus (D). In the CTCF-cKO mice the αc2 expression was constitutive and higher than in control mice. In contrast the α4 expression was dramatically downregulated in both the cortex and hippocampus of the CTCF-cKO mice. (E and F) High-magnification images of the boxed regions in (C) and (D), and other stochastically expressed isoforms, β16 and γa7. (See additional results in Figures S5 and S6.) The stochastically expressed isoforms are shown as red characters. Scale bars, 100 μm.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

7. Figure 5
Decrease in Dendritic Arborization and Spine Number in the Cortical Projection Neurons
(A and B) Golgi staining of the cerebral cortex in control (A) and CTCF-cKO (B) at P14. Scale bars, 50 μm.
(C) Example of traced pyramidal neurons of cortical layer II/III stained by the Golgi method. Scale bars, 50 μm.
(D) Quantitative analysis of the apical and basal total dendritic length. n > 30 neurons from five mice for each group.
(E) Sholl analysis for dendritic complexity at P7.
(F) Sholl analysis for dendritic complexity at P14.
(G) Total branch points of dendrites.
(H) Number of primary dendrites.
(I) Apical dendritic spines of cortical pyramidal neurons (layer II/III). Scale bar, 5 μm.
(J) Quantitative analysis of spine density at P14. n > 30 neurons from five mice for each group.
∗p < Error bars represent SEM.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

8. Figure 6
Decrease in Dendritic Arborization and Spine Number in the Hippocampal Neurons
(A and B) Golgi staining of the hippocampus in control (A) and CTCF-cKO (B) at P14. Scale bar, 50 μm.
(C) Example of traced pyramidal neurons of the hippocampal CA1 at P14. Scale bar, 50 μm.
(D) Quantitative analysis of dendrite length.
(E) Sholl analysis for dendritic complexity.
(F) Apical dendritic spines of a CA1 pyramidal neuron. Scale bar, 5 μm.
(G) Quantitative analysis of the spine density. n > 30 neurons from five mice for each group.
∗p < Error bars show SEM.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

9. Figure 7
Comparison of Electrophysiological Properties between Control and CTCF-cKO Neurons
(A) Current maps show representative superimposed traces of NMDA-mediated EPSCs (positive) and AMPA-mediated EPSCs (negative) (average of 20 sweeps). Top, control; bottom, CTCF-cKO.
(B) The amplitude ratio (mean ± SEM) of AMPA-EPSCs to NMDA-EPSCs (top) and decay time constant of NMDA-EPSCs (bottom). Number of cells is nine (control) and ten (CTCF-cKO).
(C) Representative traces of mEPSCs.
(D) Mean frequency, amplitude, rise time, and half width of mEPSCs. Number of cells is 9 (control) and 11 (cKO).
∗p < Error bars represent SEM.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

10. Figure S1
Brain Structure and Nerve Fiber Tracts in the CTCF-cKO Mouse at P0, Related to Figure 1
(A) Nissl staining of coronal brain sections of CTCF-cKO mice showed a normal brain structure. Scale bar = 500 μm.
(B) Immunostaining with the 2H3 anti-neurofilament antibody indicated no obvious changes in the fiber tracts. Scale bar = 500 μm.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

11. Figure S2 Structure of the Cortical Layers, Related to Figure 1
(A) Nissl staining of the cerebral cortex at P7.
(B and C) In situ hybridization with cortical layer-specific probes. (B) Tbr1, a marker of layer II/III, VI and the subplate (SP). (C) RorB, a marker of layer IV. Scale bar = 100 μm.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

12. Figure S3
Evaluation of Cell Survival of the CTCF-cKO Mice at P0, Related to Figure 1
Single-strand DNA (ssDNA) immunostaining (red) with DAPI counterstaining (blue). ssDNA-positive cells exhibited similar distributions in the cortex and hippocampus of both genotypes. Arrowheads indicate anti-ssDNA-positive cells. Scale bar = 100 μm.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

13. Figure S4
Topographic Maps in the Central Relay Stations of the Somatosensory Pathway, Related to Figure 2
(A–D) CO staining of the somatosensory pathway at P7. (A and B) The principal nucleus of the trigeminal nerve (PrV) and the spinal nucleus of the trigeminal nerve (SpV) of the brain stem. (C) Ventral posteromedial (VPM) and posterolateral (VPL) nucleus of the thalamus. (D) Layer IV of the primary somatosensory cortex. Scale bar = 100 μm (A–C), 500 μm (D).
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

14. Figure S5
In Situ Hybridization Analysis of Clustered Pcdh Expression in the Cortical Region, Related to Figure 4
(A) Expression pattern of stochastically and combinatorially expressed isoforms, Pcdh-α12, -β22, and C-type isoforms.
(B) High-magnification images of the boxed regions in the left panel (A).
(C) Expression pattern of Pcdh-β16 and –γa7. High-magnification images of the boxed regions are shown in Figure 4E. The stochastically expressed isoforms showed as red characters. Scale bar = 100 μm.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

15. Figure S6
In Situ Hybridization Analysis of Clustered Pcdh Expression in the Hippocampal Region, Related to Figure 4
(A) Expression pattern of stochastically and combinatorially expressed isoforms, Pcdh-α12, -β22, and C-type isoforms.
(B) High-magnification images of the boxed regions in the left panel (A).
(C) Expression pattern of Pcdh-β16 and –γa7. High-magnification images of the boxed regions are shown in Figure 4F. The stochastically expressed isoforms showed as red characters. Scale bar = 100 μm.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions