The abnormally shaped cranium in Lmnb1Δ/Δ
embryos (Vergnes et al., 2004
) led us to consider a potential role for lamin B1 in brain development. Histological analyses revealed that the brains of newborn Lmnb1Δ/Δ
mice were abnormal (). The layering of neurons in the cerebral cortex was absent, with reduced numbers of cells (); no lamination was observed in the hippocampus; and the cerebellum was reduced in size, with no foliation (). The number of cortical neurons was reduced, as judged by immunostaining for the neuronal marker NeuN (). The neurodevelopmental abnormalities in Lmnb1Δ/Δ
mice suggested that Lmnb1
is important during embryonic development. Indeed, β-galactosidase staining at E15.5 revealed Lmnb1
expression throughout the cerebral cortex (). At the same stage, Lmnb2
expression was prominent in the ventricular zone of the cortex (Coffinier et al., 2010a
), and Lmna
expression in the brain was minimal (although it was detected in the surrounding mesenchyme; ).
FIGURE 1: Brain abnormalities in newborn Lmnb1Δ/Δ mice. (A) Top, hematoxylin and eosin (H&E) staining of paraffin-embedded sections from newborn wild-type (WT) and Lmnb1Δ/Δ mice. ctx, cortex; hi, hippocampus; ob, olfactory (more ...)
At E15.5 and E17.5, the cortical plate was thinner in Lmnb1Δ/Δ
embryos than in wild-type (WT) embryos (). Immunostaining for TBR1 (a marker of cortical layer VI) revealed similar numbers of TBR1-positive (TBR1+
) cells in WT and Lmnb1Δ/Δ
embryos at E13.5; however, there were fewer TBR1+
cells in Lmnb1Δ/Δ
embryos by E15.5, and those neurons were located more superficially than in WT brains (). Immunohistochemical studies of E16.5 embryos with antibodies against Otx1 (a marker of cortical layers V–VI) and TBR1 revealed that neurons expressing those markers were located more superficially in Lmnb1Δ/Δ
brains (). The positions of the subplate and of the lateral projections, visualized by staining with the monoclonal antibodies CS56 and L1, respectively, were also more superficial than normal (). At E18.5, neurons expressing the layer V marker Ctip2 were also located more superficially in Lmnb1Δ/Δ
embryos (). The abnormal positioning of those deep-layer neurons suggested a defect of the neurons born later in forming the upper layers of the cortex. To test this hypothesis, we performed neuronal birthdating experiments; pregnant mice were injected with bromodeoxyuridine (BrdU) at E13.5, and BrdU-labeled neurons were examined in E18.5 embryos (). In WT brains, BrdU-positive neurons (i.e., cells born at E13.5) were found in layer V, whereas in the brain of Lmnb1Δ/Δ
mice, BrdU-positive neurons were scattered throughout the cortical plate (). Together, these studies demonstrated a defect in neuronal migration in Lmnb1Δ/Δ
embryos. We used immunohistochemistry to assess Reelin expression in the marginal zone, as Reelin deficiency is known to impair neuronal migration (Rice and Curran, 2001
). However, Reelin appeared to be expressed similarly in Lmnb1Δ/Δ
and control embryos at E12.5–E17.5 (Supplemental Figure S1).
FIGURE 2: Reduced size of the cortical plate and abnormal layering of cortical neurons in Lmnb1Δ/Δ embryos. (A, B) H&E staining on parasagittal sections of wild-type (WT) and Lmnb1Δ/Δ brains at E15.5 (A) and E17.5 (B). IZ, (more ...)
The small size of the cortical plate was due in part to reduced numbers of neuronal progenitors. At E13.5, similar numbers of Sox2+
progenitors were found in Lmnb1Δ/Δ
and WT brains, but by E14.5–E15.5 their numbers were clearly reduced in Lmnb1Δ/Δ
brains (167 ± 10 Sox2+
cells in E15.5 mutant embryos [n = 3] vs. 267 ± 72 in WT embryos [n = 3], per area of 430 × 470 μm; p = 0.06; ). At the same stage, the proportion of Sox2+
cells expressing the mitotic marker Ki67 was higher in Lmnb1Δ/Δ
brains, suggesting the possibility that neurons in Lmnb1Δ/Δ
embryos spend more time in the S–M phase (). At E16.5, the numbers of Ki67+
cells were ~20% higher in Lmnb1Δ/Δ
embryos than in WT embryos (47.8 ± 5.2% [n = 3] vs. 36.7 ± 4.6% [n = 4]; p = 0.03). In addition to producing cortical neurons, neuronal progenitors give rise to intermediate progenitors that accumulate in the subventricular zone and express TBR2 (Dehay and Kennedy, 2007
). Intermediate progenitors differentiate at later stages and contribute to layers II–III of the cortical plate. At E15.5 and E17.5, we observed fewer TBR2+
cells in the subventricular zone of Lmnb1Δ/Δ
embryos (). At E15.5, the number of intermediate progenitors was reduced by 50% in Lmnb1Δ/Δ
embryos (163 ± 52 TBR2+
cells in an area of 430 × 350 μm; at least three areas evaluated per embryo; n = 3 embryos) compared with WT embryos (322 ± 49 cells; n = 3 embryos; p = 0.018). Aside from reduced proliferation, we detected apoptotic cells in the cortex of E16.5 Lmnb1Δ/Δ
embryos by staining for active caspase 3 (). The brains of E16.5 Lmnb2–/–
embryos also stained positively for active caspase 3, but the apoptotic cells were fewer in number and confined to the cortical plate; in contrast, fewer than two positive cells were observed per slice of WT cortex ().
FIGURE 3: Reduced numbers of neuronal progenitors in Lmnb1Δ/Δ brains. (A) Immunostaining for the neuronal progenitor marker Sox2 (top, green) and the mitotic antigen Ki67 (middle, red) at E13.5, E14.5, and E17.5. Bottom, merged images with DAPI (more ...)
Defects in lamins A and C often lead to severe nuclear shape abnormalities in cultured fibroblasts (Muchir et al., 2004
), but misshapen nuclei are seldom found in mouse tissues. For example, we observed many nuclear blebs in fibroblasts cultured from “lamin A–only mice” (LmnaLAO/LAO
), but no misshapen nuclei were found in the tissues of those mice (Coffinier et al., 2010b
). Vergnes et al. (2004)
documented nuclear blebs in Lmnb1Δ/Δ
fibroblasts, but we were skeptical that we would find misshapen nuclei in tissues of Lmnb1Δ/Δ
mice. To our surprise, however, we observed severe nuclear shape abnormalities in the cerebral cortex of E16.5 Lmnb1Δ/Δ
embryos. In brain sections stained for the nuclear envelope protein Lap2β or for lamin B2, 24.8 ± 6.8% of cortical neurons from Lmnb1Δ/Δ
embryos (n > 350 cells evaluated per embryo, three different embryos) contained a solitary
nuclear bleb versus none in WT neurons (n > 123 cells evaluated per embryo, three different embryos; p = 0.003; ). Immunostaining for lamin B2 uncovered a second abnormality: 75 ± 6.1% of the cortical neurons in Lmnb1Δ/Δ
embryos exhibited an asymmetric distribution of lamin B2 at the nuclear rim (; compared with none in the WT samples; same numbers of cells evaluated; n = 3 embryos/group; p < 0.0001). The nuclear bleb was invariably found in the region of the nuclear rim enriched in lamin B2 ().
FIGURE 4: Lamin B1 and lamin B2 deficiencies yield distinct nuclear abnormalities in cortical neurons from E16.5 embryos. (A) Immunostaining of cortex from WT and Lmnb1Δ/Δ embryos with antibodies against Lap2β (red) or lamin B2 (green). (more ...) Lmnb2–/–
fibroblasts do not have nuclear blebs (Coffinier et al., 2010a
), but the finding of misshapen nuclei in Lmnb1Δ/Δ
neurons prompted us to investigate whether Lmnb2–/–
neurons might also have nuclear shape abnormalities (). At E16.5, cortical neurons of Lmnb2–/–
mice did not have nuclear blebs, but we found cells with an elongated nucleus in lamin B2–deficient brains (), and there was a significant increase in the length of the nucleus in lamin B2–deficient embryonic neurons in situ compared with WT neurons (p < 0.0001; Supplemental Figure S2A). Lamin B1 was evenly distributed at the nuclear rim of Lmnb2–/–
neurons—even in cells with elongated nuclei ().
Nuclear shape abnormalities were also observed in neurons grown from cortical explants of E13.5 embryos. Many Lmnb1Δ/Δ neurons had a single nuclear bleb, and some exhibited an asymmetric distribution of lamin B2 (Supplemental Figure S3). In the case of neurons from Lmnb2–/– embryos, we observed occasional comet-shaped nuclei with detached centrosomes (located >20 μm from the cell nucleus; Supplemental Figure S2B). Comet-shaped nuclei were never observed in neuronal progenitors from WT embryos.
mice die soon after birth. To assess postnatal phenotypes in the brain, we used Lmnb1
conditional knockout alleles (Yang et al., 2011
) and the Emx1-Cre
transgene (Gorski et al., 2002
) to generate forebrain-specific Lmnb1
- and Lmnb2
-knockout mice (Emx1-Cre Lmnb1fl/fl
, respectively). For both conditional knockout alleles, CRE-mediated recombination excises exon 2, creating a frameshift and yielding a null allele. Specific expression of the Emx1-Cre
transgene in the forebrain was documented with a CRE-activated lacZ
reporter (Supplemental Figure S4A; Soriano, 1999
), confirming results already in the literature (Gorski et al., 2002
). Efficient forebrain-specific gene inactivation during embryogenesis was achieved with both the Lmnb1
conditional alleles. Immunohistochemical studies on the brain from an E15.5 Emx1-Cre Lmnb1fl/fl
embryo revealed markedly reduced lamin B1 expression in the forebrain (Supplemental Figure S4B). Higher-magnification images of Emx1-Cre Lmnb1fl/fl
and Emx1-Cre Lmnb2fl/fl
brains revealed that ~90% of E15.5 cortical cells had no lamin B1 or lamin B2, respectively (Supplemental Figure S4C).
Emx1-Cre Lmnb1fl/fl and Emx1-Cre Lmnb2fl/fl mice were born at the expected Mendelian frequency, appeared to have normal longevity (mice were observed for >1 yr), and were grossly indistinguishable from WT mice. After removal of the skin, however, we observed that the cranium in both models was smaller, and the cerebral cortex was reduced in size (). At 4 mo of age, the average length of the cortex in Emx1-Cre Lmnb1fl/fl mice was 0.65 ± 0.03 cm versus 0.90 ± 0.03 cm in WT mice (n = 3 per group; p < 0.001). The length of the cortex in Emx1-Cre Lmnb2fl/fl mice was 0.80 ± 0.0 cm versus 1.02 ± 0.02 cm in WT mice (n = 2 per group). We also bred mice lacking both lamin B1 and lamin B2 in the forebrain (Emx1-Cre Lmnb1fl/fl Lmnb2fl/fl). The cortex of these “double-knockout” mice was significantly smaller than those of Emx1-Cre Lmnb1fl/fl and Emx1-Cre Lmnb2fl/fl mice (). Compared to WT siblings, the double-knockout mice had very similar body weights (16.20 ± 2.96 g vs. 16.10 ± 2.39 g) at 1 mo of age, but the brain weight of the double-knockout mice was significantly smaller than that of WT mice (0.27 ± 0.01 vs. 0.47 ± 0.02 g, respectively; n = 4 and 5 females; p < 0.0001). Brain sections of 1-mo-old double-knockout mice revealed atrophy of the cortex, which was reduced to a thin layer of tissue overlaying the striatum (). Coronal sections also showed a complete absence of the hippocampal structures (). Immunohistochemistry studies on E17.5 Emx1-Cre Lmnb1fl/fl Lmnb2fl/fl embryos revealed cells that lacked both lamin B1 and lamin B2 (Supplemental Figure S4D). Immunohistochemical studies on the thin layer of tissue above the striatum in adult Emx1-Cre Lmnb1fl/fl Lmnb2fl/fl mice failed to detect any neurons (Supplemental Figure S4E), and none of the remaining cells lacked expression of both lamin B1 and lamin B2 (Supplemental Figure S4F).
FIGURE 5: Forebrain-specific deletion of Lmnb1 and Lmnb2 results in small forebrains and abnormal layering of cortical neurons in adult mice. (A–C) Reduced size of the forebrain in Emx1-Cre Lmnb1fl/fl (A), Emx1-Cre Lmnb2fl/fl (B), and double-knockout Emx1-Cre (more ...)
We analyzed the effect of the forebrain-specific inactivation of Lmnb1 or Lmnb2 on the layering of neurons in the cortex. As expected, the layering of the cortical neurons in the adult brain was abnormal in both Emx1-Cre Lmnb1fl/fl mice and Emx1-Cre Lmnb2fl/fl mice. Immunostaining for the neuronal marker NeuN revealed reduced numbers of neurons in Emx1-Cre Lmnb1fl/fl mice, with most neurons expressing the layer V marker Ctip2 and very few neurons expressing the layer II–III marker Cux1 (). Emx1-Cre Lmnb2fl/fl mice had fewer cortical neurons than did WT mice ( and Supplemental Figure S4C), and neurons failed to organize into proper layers. However, there were significantly more neurons and more Cux1-positive cells in Emx1-Cre Lmnb2fl/fl brains than in Emx1-Cre Lmnb1fl/fl brains ().
Misshapen cell nuclei were easily detectable in both forebrain-specific knockout models. Many neurons of Emx1-Cre Lmnb1fl/fl embryos contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2 (Supplemental Figure S5A). In Emx1-Cre Lmnb2fl/fl brains, we observed an increased frequency of neurons with elongated nuclei, but lamin B1 was distributed evenly at the nuclear rim (Supplemental Figure S5B). Of interest, few neurons in the cerebral cortex of adult Emx1-Cre Lmnb1fl/fl and Emx1-Cre Lmnb2fl/fl mice exhibited abnormal nuclear morphology (). However, in the dentate gyrus of adult Emx1-Cre Lmnb1fl/fl mice, many neurons exhibited a markedly asymmetric distribution of lamin B2 (). In contrast, neurons in the dentate gyrus from Emx1-Cre Lmnb2fl/fl mice exhibited normal nuclear morphology ().
FIGURE 6: Nuclear morphology in the forebrain of adult Emx1-Cre Lmnb1fl/fl and Emx1-Cre Lmnb2fl/fl mice. (A) Immunohistochemical studies of the cortex in 1-mo-old mice with antibodies against lamin B1 (green) and lamin B2 (red); nuclear DNA is stained with DAPI (more ...)
Recently Yang et al. (2011)
showed that the absence of both lamin B1 and lamin B2 in skin keratinocytes had no effect on skin development or on keratinocyte proliferation and survival. In contrast, we found dramatic nuclear abnormalities in neurons and severe neurodevelopmental abnormalities in Lmnb1
- and Lmnb2
-deficient mice. In addition, no neurons were detected in the remnant of cortex found in adult Emx1-Cre Lmnb1fl/fl Lmnb2fl/fl
mice. We suspected that the different effects of lamin B deficiencies in skin and brain might relate to different levels of lamin A/C expression. Indeed, Lmna
expression is almost undetectable in the embryonic brain, but Lmna
expression in the skin of E15.5 and E19.5 embryos is robust (Supplemental Figure S6A). Immunostaining of embryos at E17.5 provided further evidence for robust lamin A/C expression in the skin; however, no lamin A/C expression could be detected in the cortical plate (Supplemental Figure S6B).
In contrast to the embryonic brain, which does not express significant amounts of lamin A/C (), all three lamin genes are expressed in the adult mouse brain (Supplemental Figure S6C). In the setting of lamin B1 deficiency, a robust signal for lamin A/C was associated with a normal distribution of lamin B2 at the nuclear rim (Supplemental Figure S6D). Of interest, some neurons of the dentate gyrus in adult mice—which retained an abnormal distribution of lamin B2 ( and Supplemental Figure S6E)—exhibited lower levels of lamin A/C expression (Supplemental Figure S6E).
The phenotypes of the conventional knockout mice and the forebrain-specific knockout mice are summarized in .
Summary of the phenotypes in the setting of lamin B1 deficiency or lamin B2 deficiency.