www.sciencemag.org/cgi/content/full/340/6137/1239/DC1
Supplementary Materials for Geniculocortical Input Drives Genetic Distinctions Between Primary and Higher-Order Visual Areas Shen-Ju Chou, Zoila Babot, Axel Leingärtner, Michele Studer, Yasushi Nakagawa, Dennis D. M. O’Leary* *Corresponding author. E-mail:
[email protected] Published 7 June 2013, Science 340, 1239 (2013) DOI: 10.1126/science.1232806 This PDF file includes: Materials and Methods Figs. S1 to S9 References (28–31)
Materials and Methods Gene targeting and mice. A DNA cassette containing IRES/Cre/FRT/neo/FRT (from K-F. Lee) was inserted at the XbaI site 20bp downstream of the stop codon in the 3’UTR of the RORα gene. The resulting targeting vector in pBluescript SK(II) was linearized and electroporated into R1 ES cells (from A. Nagy) and G418-resistant clones were picked for Southern blot analysis. Southern blot analysis was employed to screen for the targeted ES cell clones. We identified 3 clones out of 144 screened. All of these clones were injected into blastocysts at the Salk Transgenic Core Facility, one of the lines resulted in two 100% male chimeras that consistently generated germ line transmitted offspring. COUP-TF1fl/fl mice were mated to RORα-IRES-Cre mice. Double heterozygous COUPTF1fl/+; RORαCre/+ mice were viable and fertile. Conditional (COUP-TF1fl/fl; RORαCre/+) mice were obtained by mating double heterozygous (COUP-TF1fl/+; RORαCre/+) to COUP-TF1 homozygous mice (COUP-TF1fl/fl). Midday of the day of the vaginal plug was embryonic day 0.5 (E0.5). Reporter lines used were the Ai14 line, in which a LoxP flanked Stop cassette in front of a CAG promoter driven tdTomato gene was knocked-in to the Rosa26 locus (Jackson lab strain name: B6;129S6Gt(ROSA)26Sortm14(CAT-tdTomato)Hze/J), the R26R (28) and the R26-GAP43-eGFP line (from M. Goulding). All experiments were conducted following guidelines of the Institutional Animal Care and Use Committee at the Salk Institute. In situ hybridization, histochemistry, immunohistochemistry and axonal tracing. For in situ hybridization, antisense RNA probes were labeled using a DIG-RNA labeling kit. In situ hybridization on 16–20-mm cryostat sections and on whole-mount in situ hybridization were carried out as previously described (29). Nissl staining and immunostaining were carried out as described (6,10). The following primary antibodies were used: rabbit anti-COUP-TF1 (1:500, (30)), rabbit anti-serotonin (1:50,000; Immunostar), rat a-m2AChR (1:500, Chemicon Int.), mouse monoclonal anti-neurofilament H non-phosphorylated (SMI-32) (1: 5000, Covance). DiI and DiD axonal tracing was carried out as described (5). In short, crystals of the fluorescent carbocyanide dyes were inserted in the locations described in the text and brains were incubated for 2–4 weeks in 4% paraformaldehyde at 37oC. After incubation, the brains were embedded in 5% low-melting agarose (wt/vol), cut into 100-µm-thick coronal sections on a vibratome, counterstained with DAPI (4′-6-diamidino-2-phenylindole), mounted in 0.1 M phosphate buffer and photographed under fluorescent light. Each tracing experiment was repeated at least three times and the results were reproducible. RORβ densitometry. Flattened cortices were sectioned at a thickness of 40 µm and in situ hybridization with RORβ probe was performed in all sections. All the sections containing layer IV were superposed and a Z-projection image was generated using ImageJ software. The image generated was oriented so that the caudal end of the PMBSF would be in the vertical axes and a rectangle was drawn from the B1 barrel to the caudal end of the cortex. The intensity profile was plot in that rectangular area and in order to compare different brains it was normalized to its own maximum (maximum intensity in B1 barrel = 100) and minimum (background = 0). The values obtained from 4 different brains for WT and cKO were averaged. For statistical analysis the normalized pixel intensity values were averaged in a 32 pixel-wide window in VHOs and V1. Difference between two means was analyzed by the unpaired Student’s t-test using GraphPad Prism version 4.0c for Macintosh. P < 0.05 was considered statistically significant. Authors’ contributions All authors have read and approved the manuscript, and contributed to design and discussions of the study. S-J.C. and Z.B. performed the experiments and are equal contributors; A.L. helped perform the axon tracing experiments. S-J.C., Z.B. and D.O’L. analyzed the findings, prepared figures and wrote the paper. Y.N. and D.O’L. designed and generated the RORα-IRES-Cre mice described here, and M.S. the COUP-TF1 floxed mice.
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Fig. S1. Generation of the RORα-IRES-Cre mouse line and Cre-mediated recombination in principal sensory thalamic nuclei but not cortex. A. Strategy for targeting the RORα gene. The cassette containing IRES-Cre-FRT-pgkneo-FRT was targeted by “knock-in” to the 3’ UTR of the RORα gene. B. Southern hybridization using the probe shown in A; two bands (10.5 kb for WT and 7 kb for KI) were detected from targeted ES cells genomic DNA digested with NcoI. C-K. The expression of Cre recombinase in the RORαCre mice and efficacy at recombination was revealed by the pattern of β-Gal reporter activation in RORαCre/+:R26R mice. We focus here on reporter activation relevant to the present studies on thalamocortical projections and cortical patterning. C-E. β-Gal reporter activation in dorsal thalamus (dTh) was first detected at E14.5 (C, D) in the same pattern as the expression of the endogenous RORα gene (E) in postmitotic thalamic neurons. At P0 (F-H) and P7 (I-K), RORαCre induced recombination and reporter activation in dTh, predominantly in the principal sensory nuclei, including dorsal lateral geniculate (dLG), ventral posterior (VP) and medial geniculate (MG) but little or no recombination was detected in the neocortex (Nctx). Reporter activation was also seen in dorsal midbrain (i.e. superior colliculus; SC). C,F are dorsal views of whole mounts (rostral to the top); D,E,G,H,I,J are coronal sections (lateral to the left, dorsal to the top) and K is a sagittal section (rostral to the left, dorsal to the top). Sections G and H were counterstained with Nuclear Fast Red. Abbreviations: M, motor areas, S, somatosensory areas, V, visual areas. Scale bars: in C is 0.5 mm for C, F and I-K and 0.25mm for D, E, G, H.
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Fig. S2. RORα-IRES-Cre deletes COUP-TF1 from dLG and leads to virtual loss of dLG. A. In situ hybridization of RORα and COUP-TF1 on coronal sections of E15.5 WT (COUP-TF1fl/+; RORαCre/+) and cKO (COUP-TF1fl/fl; RORαCre/+) brains. RORα and COUP-TF1 were robustly expressed in dLG and VP nuclei of dTh in the WT. COUP-TF1 expression was strongly diminished in dLG and VP, but remained robust in the neocortex (Nctx) in cKO mice. B. COUP-TF1 immunohistochemistry on coronal sections of P0 forebrain. In cKO mice, COUP-TF1 protein evident in WT dLG and VP was strongly diminished, whereas COUP-TF1 protein levels in neocortex remained robust. C. Nissl staining, immunostaining for 5HT and β-gal staining on coronal sections of P0 forebrain of WT (COUP-TF1fl/+; RORαCre/+; R26R) and cKO (COUP-TF1fl/fl; RORαCre/+; R26R). Size of dLG is modestly reduced in P0 cKO compared to WT. D. Nissl, CO and β-gal staining on coronal sections of P7 forebrain of WT (COUP-TF1fl/+; RORαCre/+; R26R) and cKO (COUP-TF1fl/fl; RORαCre/+; R26R). Size of dLG was substantially reduced in cKO compared to WT. Cell number and dLG volume were measured on coronal sections of P7 brains (n=3 for both WT and cKO). Cell number was decreased by 85% ± 2% (mean ± SEM; p < 0.001) and volume by 84% ± 7% (mean ± SEM; p < 0.001) in P7 cKO compared to WT. Interestingly, VP size in the cKO and WT were similar at both P0 (C) and P7 (D), despite expression of both RORα and COUP-TF1 in VP, and diminished expression in VP of cKO evident at earlier ages (A, B). Scale bars: 0.5 mm.
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Fig. S3. The dLG is progressively decreased in size in cKO. β-gal staining with Nuclear Fast Red counterstaining (A) and CO staining (B) on coronal sections of forebrainsof WT (COUP-TF1fl/+; RORαCre/+; R26R) and cKO (COUP-TF1fl/fl; RORαCre/+; R26R) mice at E17.5, P0, P2, P5 and P7. CO is a marker that delineates dTh nuclei, including dLG and VP. The dLG nucleus is outlined in each panel with a dashed line. At E17.5, dLG size was similar between WT and cKO. The reduction in dLG size in cKO mice relative to WT occurred gradually and by P7 it was virtually absent. Scale bar: 0.2 mm
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Fig. S4. Geniculocortical TCA projection to V1 is substantially diminished at P0 and is absent by P7. A. Anterograde labeling from thalamus to cortex with fluorescent axon tracers in fixed brains from P7 WT (COUP-TF1fl/+; RORαCre/+) and cKO (COUP-TF1fl/fl; RORαCre/+) mice. Injections sites of DiI into dLG and DiD into VP are shown with brightfield microscopy in non-counterstained coronal sections through dTh to the left, and labeling at the level of V1 in WT and cKO is shown with fluorescence microscopy to the right in coronal sections counterstained with DAPI. A strong DiI labeled geniculocortical projection to V1 (red) was labeled in WT but absent in the cKO brain at P7. No ectopic DiD labeled VP TCAs were seen. B-D. Retrograde labeling from cortex to thalamus with fluorescent axon tracers in fixed brains at P7 (B), E17.5 (C) and P0 (D), shown in coronal sections at the level of dLG and VP nuclei in WT and cKO brains counterstained with DAPI. The axon tracer DiI (red) was placed in V1 at all ages, and DiD (green) was placed in S1 at P7 and P0. Similar injections of DiI localized to nascent V1 in occipital cortex at E17.5 retrogradely labeled a similar density and number of dLG neurons in WT and cKO mice. However, similar DiI injections localized to nascent V1 at P0 labeled very few dLG neurons in cKO mice compared to large number of densely packed neurons labeled in dLG of WT mice, and by P7, essentially no DiI labeled neurons were detected in the substantially reduced dLG of cKO mice whereas again high density of dLG neurons were well labeled in WT. No ectopic DiI labeled neurons were detected in WT or cKO. At P0 and P7, DiD injections in S1 labeled a similar number and density of neurons in the VP in WT and cKO. Scale bars, 0.2 mm (top); 0.4 mm (bottom).
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Fig. S5. Deletion of geniculocortical TCA input to V1 does not alter positioning of S1 or size of occipital cortex. A. Immunostaining for GFP on flattened tangential sections through cortex of P7 WT (COUP-TF1fl/+; RORαCre/+; R26-GAP43-eGFP) and cKO (COUP-TF1fl/fl; RORαCre/+; R26-GAP43eGFP) mice crossed to the GAP43-eGFP reporter line. The GFP labeled TCA projections from RORα-IRES-Cre expressing neurons in the principal sensory nuclei of dTh project to the primary sensory areas, S1 and A, and the geniculocortical TCA projection from dLG to V1. In cKO mice, the geniculocortical projection to ‘V1’ was absent. Anterior to left and medial to top. Scale bar, 0.5 mm. B. The overall anterior-posterior length of neocortex (T) and length of ‘occipital’ cortex (C, the length from the C1 barrel to the caudal end of the neocortex) were not different at P7 between WT (n=7) and cKO (n=7) (p
