Correction

NEUROSCIENCE Correction for “Liver X β and thyroid hormone re- ceptor α in brain cortical layering,” by Xin-jie Tan, Xiao-tang Fan, Hyun-jin Kim, Ryan Butler, Paul Webb, Margaret Warner, and Jan-Åke Gustafsson, which appeared in issue 27, July 6, 2010, of Proc Natl Acad Sci USA (107:12305–12310; first pub- lished June 21, 2010; 10.1073/pnas.1006162107). The authors note that Fig. 1 appeared incorrectly. The cor- rected figure and its legend appear below.

E6316–E6317 | PNAS | October 11, 2016 | vol. 113 | no. 41 www.pnas.org Downloaded by guest on October 2, 2021 CORRECTION

Fig. 1. Morphological alteration of embryonic and early postnatal cortex in LXRβ−/− mice. (A–I) Sagittal sections of the E15.5 and coronal sections − − of P2 and P14 stained with cresyl violet. (A and B) At E15.5, the CP is thinner and density of in the IZ is higher in LXRβ / mice (B) than in WT controls − − − − (A). (C and D) At P2, the layers II/III in LXRβ / mice (D) are thinner than in WT controls (C) and the neuronal density in layers IV and V of LXRβ / mice (D)is − − higher than that of WT controls (C). (E and F) No overall morphological difference can be observed in the cortex in LXRβ / mice (F) compared with WT controls (E) at P14. (A–F) There is no visible difference in the MZ, SVZ, and VZ at E15.5 (A and B) and layer I at P2 (C and D) or P14 (E and F) between WT and LXRβ−/− mice. (G and I) The thickness of cortical layers at E15.5 (G)(*P = 0.002 for CP; **P = 0.012 for IZ of LXRβ−/− mice compared with WT controls, by Student’s t test), and the thickness of layers II/III at P2 and P14 (I)(*P = 0.01 at P2 for LXRβ−/− mice compared with WT controls). (H) The density of cells/0.1 mm2. − − In LXRβ / mouse brain, there is a significant increase in the IZ at E15.5 (*P = 0.001), layer IV (**P = 0.032), and V (***P = 0.008) at P2. All of the results were expressed as mean ± SD. Three brains were used for each genotype. (Scale bars: A–F,50μm.)

www.pnas.org/cgi/doi/10.1073/pnas.1614988113

PNAS | October 11, 2016 | vol. 113 | no. 41 | E6317 Downloaded by guest on October 2, 2021 β and thyroid α in brain cortical layering

Xin-jie Tana, Xiao-tang Fanb, Hyun-jin Kima, Ryan Butlera, Paul Webbc, Margaret Warnera, and Jan-Åke Gustafssona,d,1 aCenter for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX 77204; bDepartment of Neurobiology, Third Military Medical University, Chongqing 400038, China; cMethodist Hospital Research Institute, Houston, TX 77030; and dDivision of Medical Nutrition, Department of Biosciences and Nutrition, Karolinska Institute, Novum, 141 86 Stockholm, Sweden

Contributed by Jan-Åke Gustafsson, May 4, 2010 (sent for review February 27, 2010) − − In the past year, two members of the family, liver Cortical abnormalities observed with LXRβ / mice are strik- X receptor β (LXRβ) and α (TRα), have ingly similar to defects produced by mutations of apolipoprotein E been found to be essential for correct migration of neurons in the receptor 2 (ApoER2) (11), originally termed the LDL receptor- developing cortex in mouse . TRα and LXRβ bind to iden- related CD91 (12). ApoER2 and its ligand are es- tical response elements on DNA and sometimes regulate the same sential for migration of developing cortical neurons to find their − − . The reason for the migration defect in the LXRβ / mouse place in the correct layer of the cortex (11, 13). Reelin is normal in − − and the possibility that TRα may be involved are the subjects of LXRβ / mice (9), but the fact that there are defects in neuronal the present study. At E15.5, expression of reelin and VLDLR was migration in the cortex raises the question of whether there may similar but expression of apolipoprotein E receptor 2 (ApoER2) (the be defects in ApoER2. − − reelin receptor) was much lower in LXRβ / than in WT mice. Many members of the nuclear hormone receptor family play Knockout of ApoER2 is known to lead to abnormal cortical lami- important roles in brain development. Thyroid hormone (TH) nation. Surprisingly, by postnatal day 14 (P14), no morphological regulates brain development and hypothyroidism can lead to − − abnormalities were detectable in the cortex of LXRβ / mice and mental retardation, anxiety, and even psychosis (14, 15). TH ApoER2 expression was much stronger than in WT controls. Thus, signaling is mediated by two closely related thyroid hormone a postnatal mechanism leads to increase in ApoER2 expression by receptors (TRs), with the TRα subtype strongly expressed in the − − P14. TRα also regulates ApoER2. In both WT and LXRβ / mice, brain (16). Like LXRs, the TRs are mostly localized in nuclei expression of TRα was high at postnatal day 2. By P14 it was re- and associate with DNA in the presence and absence of their duced to low levels in WT mice but was still abundantly expressed cognate ligand. TH, whose circulating levels increase sharply − − in the cortex of LXRβ / mice. Based on the present data we hy- after birth, regulates expression by altering receptor con- pothesize that reduction in the level of ApoER2 is the reason for formation and changing the complement of coregulators that are − − the retarded migration of later-born neurons in LXRβ / mice but recruited to TR on promoters of target genes (17–19). Recent that as thyroid hormone (TH) increases after birth the neurons do evidence suggests that TRs can also relocate to the cytoplasm, find their correct place in the cortex. where they may trigger rapid second messenger signaling events (20). The role of cytoplasmic TRs is not clear. apolipoprotein E receptor 2 | | development | LXRs bind to the same response element on DNA as TRs and sometimes regulate the same genes (21–23). The defects in − − iver X receptor (LXR) is a subfamily of the nuclear receptor cholesterol homeostasis seen in the livers of LXRα / mice are Lfamily of transcription factors. The two members of this not compensated for by TRs (24), but interactions of LXRs and subfamily are LXRα (1), which plays a key role in cholesterol TRs in the CNS have not been investigated. homeostasis and LXRβ (2), which has irreplaceable functions in In the present study, we have analyzed the architecture of the β−/− the central nervous system (3–6). cerebral cortex in embryonic and neonatal LXR mice. Re- We have previously shown that LXRβ regulates brain choles- markably, morphological abnormalities of the cortex seen at terol levels and that LXRβ expression is essential for maintenance E15.5 are normalized between postnatal day 2 (P2) and postnatal of motor neurons in the spinal cord and dopaminergic neurons in day 14 (P14). We now present evidence for the role of ApoER2 and TR in the abnormalities and reparation of the defects and the substantia nigra, suggesting that there are important roles for propose that TRα compensates for the lack of LXRβ in cortical LXR action in brain development and, possibly, also in neuro- development. logical disease (7, 8). LXRβ is widely expressed in mouse brain at later embryonic stages and is localized in the upper layers of the Results cerebral cortex in normal postnatal mice (9). Our analysis of brain Abnormal Cortical Layers in LXRβ−/− Mice at a Late Embryonic Stage −/− development in LXRβ mice revealed smaller brain size, which and Early Postnatal Stages. The laminated structure of the cerebral was caused by a reduction in the number of neurons in superficial cortex was examined at later embryonic and early postnatal stages − − cortical layers (9). Mammalian corticogenesis involves layering of and a comparison was made between LXRβ / mice and WT neurons in an “inside-out” fashion, with earliest generated neurons controls with cresyl violet Nissl staining. At E15.5, the cortical plate − − positioned in the deepest layers and later generated neurons mi- (CP) appeared thinner in the LXRβ / mice than in WT controls, grating beyond previously established layers to adopt progressively whereas the intermediate zone (IZ) was thicker in the mutant mice more superficial levels (9, 10). This process is essential for cortical than in WT controls (Fig. 1 A, B,andG). There was no overall NEUROSCIENCE structure and establishment of correct neural connections. We − − have shown that the defect in cortical development in LXRβ / mice is a direct consequence of an inability of later-born neurons Author contributions: X.T., M.W., and J.-Å.G. designed research; X.T., X.F., H.K., R.B., and fi M.W. performed research; X.T., P.W., M.W., and J.-Å.G. analyzed data; and X.T., M.W., to migrate to super cial layers and that this, in turn, is a conse- and J.-Å.G. wrote the paper. quence of abnormalities in vertical processes of radial glial cells The authors declare no conflict of interest. β along which migrating neurons travel (9). Thus, LXR plays 1To whom correspondence should be addressed. E-mail: [email protected]. fi aspeci c role in cortex lamination and is essential for radial mi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gration of later-generated neocortical neurons in embryonic mice. 1073/pnas.1006162107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1006162107 PNAS | July 6, 2010 | vol. 107 | no. 27 | 12305–12310 difference in the appearance or thickness of the marginal zone (MZ), subventricular zone (SVZ), or ventricular zone (VZ). − − However, the density of neurons occupying the IZ of LXRβ / mice was higher than in WT controls (Fig. 1 A, B,andH). This indicates that migration of many neurons was arrested at the IZ. To determine the consequences of the halt in neuronal mi- gration during embryogenesis on newborn mice, the cortex was examined at an early (P2) and a later postnatal stage (P14). At P2, the layers II/III occupied by the later-born neurons was − − thinner in LXRβ / mice and the neuronal density in layers IV and V was greater than that of WT controls (Fig. 1 C, D, H, and I). Surprisingly, however, no overall morphological differences in − − the cortex were observed between WT and LXRβ / mice at P14 (Fig. 1 E, F, H, and I). There were no visible differences in layer I (Fig. 1 C and D) and the at P2 (Fig. S1 A and B)or − − P14 (Fig. S1 C and D) between WT and LXRβ / mice. Thus, the early defects in cerebral cortex architecture are corrected in the postnatal period.

Important Role of LXRβ for Early and Later-Generated Neuronal Migration. To know which stage of neuronal migration was influenced by LXRβ, the transcription factors T box brain 1 (TBR1) and Brn2 were used as early-generated and later- generated neuronal labels, respectively (9, 10). At E15.5, TBR1 was highly expressed at the lower CP. Lower levels were observed at the IZ/SVZ, and it was not detectable in the VZ (Fig. 2 A and − − B). More TBR1-positive neurons were located at IZ in LXRβ / mice than WT controls (Fig. 2 A, B, and S). No statistical differ- ence for TBR1-positive neuronal density was observed in the CP − − between LXRβ / mice and WT controls at E15.5 (Fig. 2 A, B, − − and S). A few focal CP dysplasias were observed in LXRβ / mice at E15.5 (Fig.2 B), but at P2 (Fig. 2 C and D) and P14 (Fig. 2 E and F) the focal CP dysplasias were no longer present. At P2 in the − − LXRβ / mouse brain, the density of TBR1-positive neurons in layer VI was higher than that in WT mice (Fig. 2 C, D, and S). No such difference was detectable at P14 (Fig. 2 E, F, and S). At E15.5, there were fewer Brn2-positive neurons located in the su- − − perficial CP of LXRβ / mouse brain than in the WT controls (Fig. 2 G, H, and T). These differences indicate that the later- generated neurons migrate to the superficial CP to form the future layers IV, III, and II according to the right “inside-out” sequence − − in WT controls (9) but that in LXRβ / mice, Brn2-positive neurons could not migrate to the superficial CP. At P2, there was − − no difference between WT and LXRβ / mice in the density of Brn2-positive neurons in layers II/III measured as cells/0.1 mm2 (Fig. 2 I, J, and T). However, the total number of Brn2-positive − − neurons in layers II/III of LXRβ / mice was lower than in WT − − controls because this layer was thinner in LXRβ / mice (Fig. 2 I − − and J). At P14 there was no visible difference between LXRβ / mice and WT controls in the thickness of the Brn2-positive layers II/III and density of Brn2-positive neurons in layers II/III (Fig. 2 K, Fig. 1. Morphological alteration of embryonic and early postnatal cortex in − − L, and T) underscoring the idea that there is full recovery of LXRβ / mice. (A–I) Sagittal sections of the E15.5 neocortex and coronal sections of P2 and P14 stained with cresyl violet. (A and B) At E15.5, the CP is cortical architecture between P2 and P14. − − fi thinner and density of neurons in the IZ is higher in LXRβ / mice (B) than in Immunohistochemical staining with -speci c class III − − WT controls (A). (C and D) At P2, the layers II/III in LXRβ / mice (D)are beta-tubulin (TuJ1), a marker of immature neurons and Neuronal thinner than in WT controls (C) and the neuronal density in layers IV and V Nuclei (NeuN), a marker of postmitotic neurons, were used in this of LXRβ−/− mice (D) is higher than that of WT controls (C). (E and F)No study (25). TuJ1 immunofluorescent staining showed fewer posi- − − − − overall morphological difference can be observed in the cortex in LXRβ / tive neurons in the CP in LXRβ / mice than WT controls at E15.5 mice (F) compared with WT controls (E) at P14. (A–F) There is no visible (Fig. 2 M and N). At P2, most of the NeuN-positive neurons were difference in the MZ, SVZ, and VZ at E15.5 (A and B) and layer I at P2 (C and D E F β−/− G I located at the lower parts of layers II/III but more neurons ) or P14 ( and ) between WT and LXR mice. ( and ) The thickness of β−/− G P P β−/− remained at the layers IV and V in the LXR mice than the WT cortical layers at E15.5 ( )(* = 0.002 for CP; ** = 0.012 for IZ of LXR O, P U mice compared with WT controls, by Student’s t test), and the thickness of controls (Fig. 2 ,and ). Many immature neurons occupying layers II/III at P2 and P14 (I)(*P = 0.01 at P2 for LXRβ−/− mice compared with the superficial parts of layers II/III at P2 were NeuN-negative (Fig. − − −/− WT controls). (H) The density of cells/0.1 mm2.InLXRβ / mouse brain, there 2 O and P), and these neurons became mature at P14 in LXRβ is a significant increase in the IZ at E15.5 (*P = 0.001), layer IV (**P = 0.032), mouse cortex and WT controls (Fig. 2 Q and R) so that there was − − and V (***P = 0.008) at P2. All of the results were expressed as mean ± SD. no visible difference in NeuN distribution at P14 between LXRβ / A–F μ Three brains were used for each genotype. (Scale bars: ,50 m.) mouse cortex and WT controls (Fig. 2 Q, R,andU).

12306 | www.pnas.org/cgi/doi/10.1073/pnas.1006162107 Tan et al. Fig. 2. Distribution of cortical layer markers TBR1 and Brn2 and neuronal specific markers. (A and B) TBR1 is located at the CP and IZ/SVZ, especially at the − − − − lower CP, but is absent in the VZ in WT controls (A) and LXRβ / mice (B) at E15.5. (B) A few focal CP dysplasias can be found in LXRβ / mice. (A–F)Inthe LXRβ−/− mouse brain, there is a significant increase in TBR1-positive neuronal density in the IZ at E15.5 (B) and in layer VI at P2 (D) compared with WT controls (A and C), but there is no difference at P14 (E and F). (G–L) Brn2-positive neurons locating in the superficial CP at E15.5 (H) and layers II/III at P2 (J) of LXRβ−/− mouse brain are fewer than in WT controls (G and I), but there is no difference at P14 (K and L). (M and N) TuJ1 staining shows that there are fewer positive − − neurons in the CP in LXRβ / mice (N) than in WT controls (M) at E15.5. (O and P) Most of the NeuN-positive neurons are located in the lower part of layers II/III − − at P2 but more NeuN-positive neurons stay at the layers IV and V in LXRβ / mice (P) than in WT controls (O). (Q and R) NeuN staining shows no overall − − − − difference between LXRβ / and WT cortex at P14. (S) The density of TBR1-positive cells in the CP and IZ at E15.5 (n =3;*P = 0.004 for LXRβ / mice compared −/− with WT controls) and layer VI at P2 (n =3;**P = 0.006 for LXRβ mice compared with WT controls) and P14 is shown. (T) The density of Brn2-positive NEUROSCIENCE neurons in the CP at E15.5 and layers II/III at P2 and P14 (n =3;*,P = 0.007 for LXRβ−/− mice compared with WT controls). (U) The density of NeuN-positive neurons in cortical layers at P2 and P14. In the LXRβ−/− mouse brain there is a significant increase in NeuN-positive neuronal density in the layers IV and V at P2 (n =3;*P = 0.008; **P = 0.001). (Scale bars: A, B, G, H, M, N, 100 μm; C–F, I–L, O–R,50μm.)

Abnormal Expression of ApoER2 and Abnormal Scaffold of Radial Glia ined whether the abnormalities observed could be caused by Fibers in E15.5 Neocortex of LXRβ−/− Mice. There was no visible a defect in the reelin receptors, ApoER2 and VLDLR (26). In the difference in the reelin expression in MZ between WT and WT mouse cortex at E15.5, ApoER2 was distributed mostly in the − − LXRβ / mice at E15.5 (Fig. 3 A and B). Therefore, we exam- MZ, lower CP, and subplate (SP) (Fig. 3 C, E, and M). However, in

Tan et al. PNAS | July 6, 2010 | vol. 107 | no. 27 | 12307 and more processes were distributed throughout the CP than in − − LXRβ / mouse cortex (Fig. 3 G and H). VLDLR, a stop signal for neuronal migration, was examined, but there was no staining at E15.5 or P2.

Up-Regulation of Thyroid Hormone Receptor α. The focal CP dys- − − plasia in LXRβ / mice observed at E15.5 disappeared after birth, and no morphological differences in the cortex could be − − discerned at P14 between WT and LXRβ / mice regarding the Nissl and layer-marker staining. Thus, some mechanism must compensate for the absence of LXRβ in the time between P2 and P14. Because LXRs and TRs sometimes regulate similar genes, we tested the possibility that alterations in TR signaling pathways could play a compensatory role and repair the neuronal deficit caused by loss of LXRβ (21–23). At P2, there was strong nuclear staining for TRα in layers II/III (Fig. 4 A and B) and the density

Fig. 3. Abnormal expression of ApoER2 and abnormal scaffold of radial glia fibers in E15.5 neocortex of LXRβ−/− mice. (A and B) There is no visible dif- ference in the reelin expression (Red) in the MZ between WT and LXRβ−/− mice at E15.5. Nuclei were counterstained with DAPI (Blue). (C, E, and M) ApoER2 is distributed mostly in the MZ, lower CP and SP in WT controls at − − E15.5. (D, F, and Q) ApoER2 is decreased sharply in the SP in LXRβ / mouse brain. (E and F) Higher-power views of the boxed areas in C and D. (I, J, and M–T) ApoER2 is expressed on neuronal projections and membrane during E15.5 (M–T) but only expressed on the neuronal membrane and cytoplasm at P14 (I and J). (I–L) The expression of ApoER2 (J) and PCSK9 (L) appears − − stronger in LXRβ / mouse brain than in WT controls (I and K) at P14. (G and H) The scaffold of radial glial fibers is more intact and more processes are − − distributed in the CP in WT controls (G) than in LXRβ / mice (H) at E15.5. (Scale bars: A, B, and E–T, 100 μm; C, D,50μm.)

− − the LXRβ / mouse brain there was remarkably weak staining in the SP (Fig. 3 D, F, and Q). ApoER2 was expressed on the neu- ronal projections and membrane at the embryonic stage (Fig. 3 M– T) and only on the neuronal membrane and cytoplasm at P14 (Fig. 3 I and J). At P2, the ApoER2 staining was not detectable, but at −/− P14, ApoER2 expression in LXRβ mice was very strong, − − Fig. 4. Expression of TRα and proliferation in LXRβ / mouse cortex. (A and B) whereas it was barely detectable in layers II/III in WT controls At P2, layers II/III are occupied by TRα-positive cells and all of the cells have I J − − (Fig. 3 and ). nuclear staining in WT controls (A) and LXRβ / mice (B). (C and D) By contrast, − − To examine whether higher expression of ApoER2 in the at P14, TRα-positive cells distributing throughout the LXRβ / mouse cortex −/− LXRβ mouse brain at P14 is due to a difference in degradation (D) are far more abundant than in WT controls (C). (A and DInsets) Higher of the protein, we examined expression of the protein normally magnification image of area in box; note strictly nuclear staining in A Inset involved in its degradation, i.e., PSCK9 (27). Immunohistochem- and cytoplasmic staining in B Inset.(E–H) At P2, more Ki67-positive cells are −/− ical staining with a PCSK9 antibody showed no staining at E15.5, observed in the WT control cortex (E and G) than in LXRβ mice (F and H). β−/− (G and H) Higher-power views of the boxed areas in E and F.(K) The density of but at P14 expression of PSCK9 in LXR mice was stronger −/− K L TRα-positive cells in layers II/III in LXRβ mice at P2 (n =3;*P = 0.002 compared than in WT controls (Fig. 3 and ), indicating that decreased with WT controls) and P14 (n =3;**P = 0.005 compared with WT controls). (L) − − degradation by PCSK9 is not responsible for the accumulation of The density of Ki67-positive cells in the cortex at P2 and P14. In the LXRβ / −/− ApoER2 in the LXRβ mouse brain in the postnatal period. mouse cortex the Ki67-positive neuronal density is less than in WT controls at At E15.5, the scaffolds of radial glial fibers, detected with P2 (n =3;*P = 0.016) but no difference is seen at P14. (Scale bars: A–D, G,and antibodies against Rat401 (28) in WT controls, were more intact H,100μm; E and F,50μm.)

12308 | www.pnas.org/cgi/doi/10.1073/pnas.1006162107 Tan et al. − − − − of TRα-positive cells in WT controls was higher than in LXRβ / observed between LXRβ / mice and WT controls at P14. Thus the mice at P2 (Fig. 4 K). However, at P14, there were far more immature neurons located in the superficial layers in both mouse − − TRα-positive cells distributed throughout the LXRβ / mouse lines at P2 became mature at P14, and by this time, the abnormalities − − cortex than in the WT controls (Fig.4 C, D and K). In contrast to in LXRβ / mice had normalized. − − P2, however, almost all of the TRα-positive cells at day 14 We propose that the deficit in neuronal migration in LXRβ / exhibited cytoplasmic staining (Fig. 4 A and D Insets). TRα is mice is related to reduced expression of ApoER2, the reelin implicated in neuroblast proliferation (29) so we tested the receptor. Reelin has functions in the developing brain (31–35). possibility that TRα-dependent proliferation accounts for re- In cortical development, reelin is crucial for correct positioning covery of cortical morphology. Ki67 is a proliferation marker of radially migrating neuronal precursors via its binding to that is expressed by proliferating cells in all phases of the active ApoER2 and VLDLR on neuronal precursors (11, 36, 37). In cell cycle (G1, S, G2, and M phase) but not in resting (G0) cells ApoER2 mutants, many later-born neurons fail to migrate to (30). At P2, more proliferation was observed in the WT control their destinations in superficial cortical layers, and some neurons −/− cortex than in the LXRβ mice (Fig. 4 E–H and L). At P14 no invade the marginal zone in VLDLR mutants (10). Thus, the two −/− difference in Ki67 expression was detectable between LXRβ reelin receptors, VLDLR and ApoER2, play divergent roles, mice and WT controls. Thus, changes in TRα expression and with VLDLR mediating a stop signal for migrating neurons and subcellular localization are not related to changes in cortical ApoER2 as an essential signal for the migration of later-born neuronal proliferation. neurons (10). In the present study, we did not find any abnor- malities of the number of Cajal-Retzius neurons nor the intensity Discussion − − of their immunostaining for reelin in LXRβ / mice (9). How- β − − LXR is expressed ubiquitously in the brain and appears in the ever, expression of ApoER2 was different in LXRβ / mice. β neurons as early as E14.5 (9). Knockout of LXR gives rise to Later-born neurons destined to superficial cortical layers abnormal cortex lamination because of the retardation in the strongly express both LXRβ (9) and ApoER2 (10), suggesting migration of later-born neurons. The inability to migrate appears that ApoER2 may be regulated by LXRβ. In the WT mouse to be due to truncation of the vertical processes of radial glia (9) cortex at E15.5, ApoER2 was distributed mostly in the MZ, upon which the migrating neurons depend for their direction. β−/− β−/− lower CP, and subplate (SP) (10). In the LXR mouse brain, In the present study, we demonstrate that in LXR mice, there was weak staining for ApoER2 in the SP. Costaining of the cortical development is aberrant at later embryonic and early TuJ1 and ApoER2 showed that ApoER2 was expressed more postnatal stage but recovers at later postnatal stages. At E15.5, β−/− strongly on the neuronal projections and membrane during the cortical plate was thinner in LXR mice brains than in WT embryonic development. − − mice and there was an accumulation of neurons at the IZ. At P2, At P14, ApoER2 expression in LXRβ / mice was very strong the cortex developed from the cortical plate was still thinner in − − in layers II/III, whereas it was barely detectable in WT controls. LXRβ / mice with fewer neurons in layers II/III and in contrast, Therefore, ApoER2 appears to have a different regulation mode more neurons at layers IV and V. Surprisingly however, the and role during the embryonic and postnatal stages. To examine cortex appeared normal by P14. This normalization suggested − − whether the higher expression of ApoER2 in the LXRβ / brain that those neurons that were arrested at layers IV and V at P2 in − − at P14 is due to a difference in degradation of the protein, the LXRβ / mice migrated to the superficial layers later. Although enzyme—PSCK9—responsible for degradation of ApoER2 (27, loss of LXRβ arrested cortical neuronal migration, the lamination and morphology of the hippocampus were normal at P2 and P14. 38, 39) was examined. Immunohistochemical staining for PCSK9 Analysis of early and late-born neuronal markers supports showed that decreased degradation was not a likely cause for the the idea that there is an early deficit in neuronal migration in accumulation of ApoER2 at P14. β−/− fi LXRs bind to the same response element on DNA as does TR LXR mice but that the brain compensates for this de cit be- – tween P2 and P14. At E15.5, there were fewer TBR1-positive and regulates many of the same genes (21 23). Although TR − − neurons (early-born neurons) (9, 10) in CP in LXRβ / mice, al- does not compensate for the function of LXRs in LXR knockout mice in cholesterol homeostasis (24), it is possible that TR can though the cell density was not different. However in the IZ, as was β evident with Nissl staining, there were more TBR1-positive neu- compensate for some of the defects due to loss of LXR in the − − α rons in LXRβ / mice than in WT controls. At P14, no differences CNS. TR plays a key role in postnatal development where it β in the distribution of TBR1-positive neurons were observed be- presumably mediates most of the thyroid hormone effects. TR − − tween WT and LXRβ / mice. In addition, there were a few focal is detected in few areas of the brain (16, 40, 41). We found strong − − α β−/− cortical plate dysplasias in LXRβ / mice at E15.5, but none were nuclear staining for TR in WT and LXR mice at P2. α detectable after birth. These results suggest that some neurons However, at P14, there were far more TR -positive cells dis- β−/− recovered the ability to migrate after birth. Brn2 is a marker of tributed throughout the LXR mouse cortex than in the WT fi later-born neurons destined to superficial CP to form the future controls. This nding raises the possibility that increased ex- cortical layers II/III (9, 10). At E15.5 and P2, there were fewer pression of TRα may compensate for loss of LXRβ and stimulate − − Brn2-positive neurons in CP in the LXRβ / mice than in WT the retarded neurons to migrate to their correct position. It is controls. However, at P14 there were no differences in the number noteworthy that almost all of the TRα staining was cytoplasmic − − or location of Brn2-positive neurons between WT and LXRβ / at P14. This is in marked contrast to P2, where TRα was strictly mice. Clearly, as described previously (9), migration of later-born nuclear. From Ki67 staining, it appears that there was no in- neurons was arrested when LXRβ was not expressed but some crease in neuronal proliferation in the postnatal period in fi β−/− mechanism compensated for this de cit between P2 and P14. LXR mice. Therefore, a role of TR in normalizing the cortex NEUROSCIENCE Immunofluorescent staining showed that at E15.5, there were is in migration, not in proliferation. − − fewer TuJ1-positive neurons (25) in the CP of the LXRβ / mice. We suggest that there may be two possible effects of TRα on This is consistent with the Nissl and TBR1 staining results. At P2, LXR signaling in cortical development. First, unliganded TRs most of the NeuN-positive neurons in WT mice were located at the suppress gene transcription. Thus, migration of TRα from a nu- lower parts of layers II/III but more neurons remained in the layers clear to cytoplasmic location could derepress ApoER2 gene − − IV and V in the LXRβ / mice than in the WT controls. Almost all of expression by removing inhibitory influences at possible TRE/ − − the superficial neurons in layers II/III of LXRβ / mice were NeuN- LXRE elements (42). Alternatively, plasma levels of TH increase negative indicating that these neurons were latest-born and imma- sharply during neonatal and postnatal periods (42–44) and in the ture. No difference of NeuN-positive (mature neurons) staining was presence of its ligand, TR leaves the nucleus and is cytoplasmic.

Tan et al. PNAS | July 6, 2010 | vol. 107 | no. 27 | 12309 Cytoplasmic TR may modulate cell behavior and gene expression and Methods. Immunohistochemistry and immunofluorescence staining were by triggering second messenger pathways (17–19). performed as previously described (7, 9). Detailed procedures are described in We conclude that reduction in the level of ApoER2 is the likely SI Materials and Methods. For layer thickness measuring and cell counting, − − reason for the retarded migration of later born neurons in LXRβ / MicroSuite Basic Edition and Image Pro Plus 6.0 were used, respectively. The embryonic mice but that postnatal changes in TR signaling path- number of mice in each experiment was at least three per genotype. Data are presented as mean ± SD. The statistical significance of differences between fi − − ways permit neurons to nd their correct place in the cortex (45). LXRβ / and control samples was assessed by using Student’s t test. Materials and Methods ACKNOWLEDGMENTS. This work was supported by a grant from the β−/− The generation of LXR mice has been described (3). Heterozygous mice were Swedish Science Council and the European Integrated Project on Nuclear used for breeding. The day of vaginal plug detection was designated as E0.5. Receptors, Chemicals as contaminants in the food chain: an NoE for research, Embryonic and P2 and P14 mice brains were obtained as described in SI Materials risk assessment and education (CASCADE).

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