Supporting InformationCoffinier et al. 10.1073/pnas.0908790107

Fig. S1. The Lmnb2 knockout mutation does not affect the level of lamin B3 transcripts (a testis-specific transcript of Lmnb2). (A) Schematic of lamin B2 andlamin B3 exon–intron organization. Testis-specific use of the alternative exon 1b in intron 4 results in the production of the lamin B3 transcript. Arrows indicatethe positions of the oligonucleotide primers used for qRT-PCR analysis. “Total” indicates primers in exons 9 and 10 (present in both lamin B2 and lamin B3transcripts); the “B2-only” primers correspond to sequences in exon 1 and exon 2; the “B3-only” primers correspond to sequences in exon 1b and exon 5. (B)Quantification of total Lmnb2 transcripts, lamin B2–only transcripts, and lamin B3–only transcripts in undifferentiated embryonic stem (ES) cells and in testes ofadult male mice (n = 3 WT mice and n = 4 Lmnb2+/− mice) relative to β2-microglobulin.

Fig. S2. Lamin B2 deficiency has no significant effect on steady-state levels or localization of several other nuclear envelope proteins. (A) Immunofluorescencemicroscopy of Lmnb2+/+ and Lmnb2−/− embryonic fibroblasts with antibodies against three nuclear lamins (lamin B2, lamin B1, lamin A); the nuclear envelopeproteins LAP2β, emerin, SUN1, and nesprin 3; and the nucleopore component Nup153. Except for lamin B2, no differences were observed between Lmnb2+/+ andmutant cells. (B) Western blots of mouse embryonic fibroblast extracts with antibodies against lamin B2, lamin B1, LAP2β, emerin, Nup153, SUN1, SUN2, nesprin2, and nesprin 3. Actin was used as a loading control. Except for lamin B2, no differences were observed. Data are presented for one pair of Lmnb2+/+ andLmnb2−/− embryonic fibroblasts isolated from sibling embryos; identical results were obtained for another pair of fibroblast cell lines from an independent litter.

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Fig. S3. Abnormal patterning of the cerebral cortex in Lmnb2−/− embryos at E18.5. Immunostaining on paraffin sections of E18.5 WT and Lmnb2−/− siblingembryos with antibodies specific for (A) nestin, an intermediate filament protein expressed in neuronal progenitors of the ventricular zone (VZ); (B) Reelin,expressed by Cajal-Retzius cells (arrows) in the marginal zone (MZ); (C), Ctip2, expressed in neurons of layers V and VI of the cortical plate (CP); and (D) β-IIItubulin, strongly expressed in newly specified neurons. All sections were counterstained with methyl green. The VZ of Lmnb2−/− brains was normal in size(relative to the thickness of the cerebral cortex), as shown by nestin staining (A). However, Lmnb2−/− brains lacked Reelin-positive cells in the marginal zone (B),and reduced numbers of Ctip2-positive cells were found in the cortical plate. Bin analysis indicated that Ctip2-positive cells within a 600 μm × 200 μm section ofthe cortical plate were reduced by 33% in the mutant mice (C). In addition, methyl green staining revealed reduced numbers of cell nuclei in the cortical plateof Lmnb2−/− embryos. However, β-III tubulin staining was similar in Lmnb2−/− and WT brains, and there was no evidence of ventricular heterotopia (i.e., anaccumulation of neurons in the ventricular zone) (D).

Fig. S4. Abnormalities in the marginal layer of the cerebral cortex of Lmnb2−/− embryos appear after E16.5. The neocortex of Lmnb2−/− embryos appearsnormal at E13.5. (A) Paraffin sections of E13.5 WT and Lmnb2−/− embryos stained with H&E and with an antibody against Reelin. No differences in histologywere observed between the WT and Lmnb2−/− neocortex at this stage. Reelin-positive Cajal-Retzius cells (arrows) were observed in both cases, consistent withthe initial formation of a normal marginal zone. (B and C) Immunostaining on sections of E16.5 and E18.5 WT and Lmnb2−/− embryos with an antibody againstReelin. Reelin-positive cells were detected in both WT and Lmnb2−/− embryos at E16.5 (B) but were not detected in Lmnb2−/− embryos at E18.5 (C and Fig. S3B).

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Fig. S5. The early stage of cortical plate formation in Lmnb2−/− embryos appears to be normal at E15.5. (A) Immunostaining of frozen sections of E15.5 WTand Lmnb2−/− embryos with an antibody against neuron-specific β-III tubulin (green). Nuclear DNA was stained with DAPI (blue). The presence of two layers ofneurons that stain for β-III tubulin, corresponding to the marginal zone (MZ) and the subplate (SP), indicates that the cortical plate (CP) is present in both WTand Lmnb2−/− embryos. This conclusion was supported by staining with CS56, a monoclonal antibody against the chondroitin sulfate proteoglycans of thesubplate (B). The subplate can be detected as a deep CS56-positive layer in both WT and Lmnb2−/− embryos. However, at later stages, CS56 staining was morediffuse in the cortex of Lmnb2−/− mice (C, E16.5) and extended into more superficial layers, compared with WT mice (D, E18.5).

Fig. S6. Both lamin B1 and lamin B2 are present in cells of the cerebral cortex. (A) Immunostaining of frozen sections of E17.5 WT brains, revealing lamin B2(red) in all cells and nestin (green) in neuronal progenitors. (B) Immunostaining of E17.5 WT cortex revealing a similar pattern of lamin B2 (red) and lamin B1(green) staining. A merged image is shown on the right. (C) Immunostaining on frozen sections of E17.5 WT and Lmnb2−/− embryos with antibodies specific forlamin B2 (red) and lamin B1 (green); DNA was stained with DAPI (blue). No lamin B2 protein is detected in Lmnb2−/− embryos.

Fig. S7. Neuronal birthdating experiments after injection of 5-bromo-2-deoxyuridine (BrdU) at different stages of gestation. BrdU labeling (detected with arat monoclonal antibody) is specific for cells that exited the cell cycle soon after the injection of BrdU. An injection of BrdU at E13.5 labels neurons in deeplayers of the cortical plate, as illustrated in the WT sections (brackets in A and B); injecting BrdU at E15.5 labels neurons in more superficial layers in the WTbrains (C). In Lmnb2−/− embryos, neurons labeled at E13.5 are found in superficial layers (A and B) because neurons born after this time point are defective inmoving up past the BrdU-positive cells. Supporting this view, Lmnb2−/− neurons labeled at E15.5 are found in deeper positions than WT neurons labeled at thesame time point (C).

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Fig. S8. Lmnb2 deficiency modestly reduces the population of neuronal progenitors in the cortex. (A) Frozen sections of WT (Left) and Lmnb2−/− (Right) E15.5embryos stained for the mitotic marker Ki-67 (green) and the neuronal progenitor marker nestin (red). Bin analysis of stained sections showed a 42% reductionin Ki67-positive cells in the ventricular zone of Lmnb2−/− mice (n = 428 Ki-67-positive cells for each 280 μm × 56 μm sector; total of 1929 cells counted) comparedwith WT mice (n = 738 Ki67-positive cells per identically sized sector; total of 2207 cells counted). (B) Frozen sections of WT (Left) and Lmnb2−/− (Right) E16.5embryos stained for the mitotic marker Ki-67 (green) and the intermediate progenitor marker TBR2 (red).

Fig. S9. Lmnb2 deficiency does not affect the ability of cultured embryonic fibroblasts to migrate and fill in a “wound” on a cell culture plate. (A) Wound-healing assay on WT and Lmnb2−/− mouse embryonic fibroblasts. (B) Wound closure with time, expressed as a percentage of the initial wound distance.Squares, WT (n = 3); circles, Lmnb2−/− (n = 2). Fibroblasts isolated from two independent litters were used (open and closed symbols, respectively). From 6 to 14independent sites were monitored per cell line.

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Fig. S10. Lmnb2 deficiency affects the layering of neurons in the hippocampus. Shown here are H&E-stained paraffin sections of brains from WT andLmnb2−/− embryos at E18.5 and E19.5 and in newborn mice (P0). The CA1 and CA3 layers of the hippocampus appeared to be fairly normal in Lmnb2−/−

embryos at E18.5, but the neurons were less compact at E19.5 and P0. Similar layering defects have been observed in the hippocampus in mice with mutationsin Reelin or defects in the lissencephaly pathway.

Table S1. List of primary antibodies used in the study, with dilutions used for Western blots,immunocytochemistry (ICC), and immunohistochemistry (IHC)

Antigen Antibody Species Company Western Blot ICC IHC

Actin (I-19) Polyclonal Goat Santa Cruz Biotechnology 1:1,000BrdU [BU1/75] Monoclonal Rat Abcam 1:200BrdU Polyclonal Sheep Abcam 1:200Calbindin [CL300] Monoclonal Mouse Abcam 1:100CDP/Cux1 Polyclonal Rabbit Santa Cruz Biotechnology 1:100Chondroitin Sulfate [CS56] Monoclonal Mouse Sigma 1:400CTIP2 [25B6] Monoclonal Rat Abca 1:500Emerin Monoclonal Mouse Novo Castra, Leica 1:200 1:200FoxP1 [JC12] Monoclonal Mouse Abcam 1:200FoxP2 Polyclonal Rabbit Abcam 1:1,000Ki-67 [MM1] Monoclonal Mouse Novo Castra 1:100Lamin A (H-102) Polyclonal Rabbit Santa Cruz Biotechnology 1:200Lamin B1 (M-20) Polyclonal Goat Santa Cruz Biotechnology 1:400 1:400 1:400Lamin B2 [E3] Monoclonal Mouse Zymed, Invitrogen 1:200 1:50 1:200LAP2β Monoclonal Mouse BD Transduction Lab. 1:200 1:400Nesprin 2 Monoclonal Mouse Abcam 1:1,000Nesprin 3 Polyclonal Rabbit Abcam 1:500 1:400Nestin Polyclonal Rabbit Abcam 1:1,000NeuN Monoclonal Mouse Millipore 1:500Nup153 [QE5] Monoclonal Mouse Abcam 1:1,000 1:500Reelin Monoclonal Mouse Chemicon, Millipore 1:1,000SUN1 Polyclonal Rabbit Abcam 1:1,000 1:400SUN2 Polyclonal Rabbit Sigma 1:500Tbr2/Eomes Polyclonal Rabbit Abcam 1:500βIII-Tubulin [TUJ1] Monoclonal Mouse Abcam 1:4,000

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