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Essential roles of leucine-rich glioma inactivated 1 in the development of embryonic and postnatal cerebellum.

Xie YJ, Zhou L, Jiang N, Zhang N, Zou N, Zhou L, Wang Y, Cowell JK, Shen Y - Sci Rep (2015)

Bottom Line: The function of LGI1 in CNS development remains undefined.BrdU staining showed an inhibited proliferation of CGPs in KO embryos, which might be explained by the reduced Sonic hedgehog in embryos.Together, our results demonstrate that LGI1 is an essential player in the cerebellar development.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of Health, Zhejiang Province Key Laboratory of Neurobiology, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT
Leucine-rich glioma inactivated 1 (LGI1) is a secreted protein that interacts with ADAM transmembrane proteins, and its mutations are linked to human epilepsy. The function of LGI1 in CNS development remains undefined. Here, we report novel functions of LGI1 in the generation of cerebellar granule precursors (CGPs) and differentiation of radial glial cells (RGCs) in the cerebellum. A reduction in external granule layer thickness and defects in foliation were seen in embryonic and new-born LGI1 knockout (KO) mice. BrdU staining showed an inhibited proliferation of CGPs in KO embryos, which might be explained by the reduced Sonic hedgehog in embryos. In addition, the differentiation of RGCs into Bergmann glias was suppressed in KO mice. Enhanced Jagged1-Notch1 signaling in KO mice via reduced β-secretase proteolysis suggests that altered phenotype of RGCs is due to abnormal Notch1 signaling. Together, our results demonstrate that LGI1 is an essential player in the cerebellar development.

No MeSH data available.


Related in: MedlinePlus

CGP proliferation is reduced in KO mice.(a) Mid-sagittal sections of cerebella stained for Pax6. White and yellow arrowheads indicate Pax6 staining in the EGL and IGL, respectively. Pax6 intensity was reduced at E15.5 (n = 8 pairs), but unchanged at P1 (n = 7 pairs). Scale bars: 50 μm. (b) Total Pax6 expression was measured at E15.5, P1, P3, and P7, using GAPDH as the loading control. The percentage changes of Pax6 (KO/WT) were 36.3 ± 7.9% (E15.5; n = 5 pairs, p = 0.00021), 92.8 ± 9.9% (P1; n = 4 pairs, p = 0.35); 101.7 ± 6.9% (P3; n = 4 pairs, p = 0.32); 99.7 ± 8.9% (P7; n = 4 pairs, p = 0.43). Statistical analysis was done by one-sided Student's t-test. *** p < 0.001. n.s.: no significance. (c) Left panels show the staining of NeuN (red), Pax6 (green), and DAPI (blue) in cultured granule cells (DIV12). Higher magnification in white boxes, where the DAPI signal was removed, shows cells double-stained with NeuN and Pax6, indicating that 100% of NeuN-positive cells were Pax6-positive. Right panels: DIV12 granule cells labeled with TuJ1 (green) and NeuN (red). Nuclei were counterstained with DAPI (blue). Immunoreactivity for TuJ1 displayed long processes from differentiated neurons in both groups. Scale bars: 50 μm. (d) Numbers of NeuN+Pax6+ cells in cultured granule cells (DIV12) expressed as percentages of DAPI+stained cells (WT: 46.3 ± 2.8%; KO: 44.5 ± 5.5%; n = 6 pairs) Two-sided Student's t-test. p = 0.34. n.s.: no significance. (e) Mid-sagittal sections of P0 cerebella were stained with Pax6, BrdU, and DAPI. Dashed lines define the EGL. Higher magnifications in the white boxes, with the DAPI signal removed, show cells stained by BrdU and Pax6 (arrowheads). Scale bars: 50 μm. (f) Percentages of BrdU+Pax6+ cells/Pax6+ cells in a lobe. n = 5, *** p < 0.001 (p = 0.00012, two-sided Student's t-test). (g) Percentages of BrdU+Pax6+ cells/Pax6+ cells within the EGL. n = 5, * p < 0.001 (p = 0.00018, two-sided Student's t-test).
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f3: CGP proliferation is reduced in KO mice.(a) Mid-sagittal sections of cerebella stained for Pax6. White and yellow arrowheads indicate Pax6 staining in the EGL and IGL, respectively. Pax6 intensity was reduced at E15.5 (n = 8 pairs), but unchanged at P1 (n = 7 pairs). Scale bars: 50 μm. (b) Total Pax6 expression was measured at E15.5, P1, P3, and P7, using GAPDH as the loading control. The percentage changes of Pax6 (KO/WT) were 36.3 ± 7.9% (E15.5; n = 5 pairs, p = 0.00021), 92.8 ± 9.9% (P1; n = 4 pairs, p = 0.35); 101.7 ± 6.9% (P3; n = 4 pairs, p = 0.32); 99.7 ± 8.9% (P7; n = 4 pairs, p = 0.43). Statistical analysis was done by one-sided Student's t-test. *** p < 0.001. n.s.: no significance. (c) Left panels show the staining of NeuN (red), Pax6 (green), and DAPI (blue) in cultured granule cells (DIV12). Higher magnification in white boxes, where the DAPI signal was removed, shows cells double-stained with NeuN and Pax6, indicating that 100% of NeuN-positive cells were Pax6-positive. Right panels: DIV12 granule cells labeled with TuJ1 (green) and NeuN (red). Nuclei were counterstained with DAPI (blue). Immunoreactivity for TuJ1 displayed long processes from differentiated neurons in both groups. Scale bars: 50 μm. (d) Numbers of NeuN+Pax6+ cells in cultured granule cells (DIV12) expressed as percentages of DAPI+stained cells (WT: 46.3 ± 2.8%; KO: 44.5 ± 5.5%; n = 6 pairs) Two-sided Student's t-test. p = 0.34. n.s.: no significance. (e) Mid-sagittal sections of P0 cerebella were stained with Pax6, BrdU, and DAPI. Dashed lines define the EGL. Higher magnifications in the white boxes, with the DAPI signal removed, show cells stained by BrdU and Pax6 (arrowheads). Scale bars: 50 μm. (f) Percentages of BrdU+Pax6+ cells/Pax6+ cells in a lobe. n = 5, *** p < 0.001 (p = 0.00012, two-sided Student's t-test). (g) Percentages of BrdU+Pax6+ cells/Pax6+ cells within the EGL. n = 5, * p < 0.001 (p = 0.00018, two-sided Student's t-test).

Mentions: To investigate whether the reduction of EGL thickness was due to altered neuronal differentiation, we studied paired-box homeodomain transcription factor (Pax6) expression in the cerebellum. Pax6 expression begins ~E8.5 in the mouse brain24 and promotes neuronal differentiation, migration, and neurite extension2526. Immunocytochemical staining and western blotting analysis showed that Pax6 was robustly expressed at E15.5 and P1 in WT mice (Fig. 3a), representing CGPs in the EGL and migrating granule cells in the inner granular layer (IGL)2027. Pax6 immunoreactivity was much stronger at P1 and displayed a thickened appearance in the EGL (Fig. 3a), similar to previous descriptions2027. In the E15.5 KO cerebellum, however, there was a substantial decrease of Pax6 intensity throughout the cerebellum, including the EGL and IGL (Fig. 3a). Interestingly, this decrease in Pax6 immunolabeling was not seen after birth (P1, Fig. 3a), suggesting that the LGI1 deficiency only suppresses Pax6 expression in embryos. Western blotting analysis also confirmed that the total expression of Pax6 was substantially reduced in KO cerebella from E14.5-E16.5 (Fig. 3b, supplementary Fig. S2), but not in cerebella at E17.5 (supplementary Fig. S2) and postnatal P1 to P7 (Fig. 3b). These results implied that LGI1 influences the generation of CGPs during early embryogenesis.


Essential roles of leucine-rich glioma inactivated 1 in the development of embryonic and postnatal cerebellum.

Xie YJ, Zhou L, Jiang N, Zhang N, Zou N, Zhou L, Wang Y, Cowell JK, Shen Y - Sci Rep (2015)

CGP proliferation is reduced in KO mice.(a) Mid-sagittal sections of cerebella stained for Pax6. White and yellow arrowheads indicate Pax6 staining in the EGL and IGL, respectively. Pax6 intensity was reduced at E15.5 (n = 8 pairs), but unchanged at P1 (n = 7 pairs). Scale bars: 50 μm. (b) Total Pax6 expression was measured at E15.5, P1, P3, and P7, using GAPDH as the loading control. The percentage changes of Pax6 (KO/WT) were 36.3 ± 7.9% (E15.5; n = 5 pairs, p = 0.00021), 92.8 ± 9.9% (P1; n = 4 pairs, p = 0.35); 101.7 ± 6.9% (P3; n = 4 pairs, p = 0.32); 99.7 ± 8.9% (P7; n = 4 pairs, p = 0.43). Statistical analysis was done by one-sided Student's t-test. *** p < 0.001. n.s.: no significance. (c) Left panels show the staining of NeuN (red), Pax6 (green), and DAPI (blue) in cultured granule cells (DIV12). Higher magnification in white boxes, where the DAPI signal was removed, shows cells double-stained with NeuN and Pax6, indicating that 100% of NeuN-positive cells were Pax6-positive. Right panels: DIV12 granule cells labeled with TuJ1 (green) and NeuN (red). Nuclei were counterstained with DAPI (blue). Immunoreactivity for TuJ1 displayed long processes from differentiated neurons in both groups. Scale bars: 50 μm. (d) Numbers of NeuN+Pax6+ cells in cultured granule cells (DIV12) expressed as percentages of DAPI+stained cells (WT: 46.3 ± 2.8%; KO: 44.5 ± 5.5%; n = 6 pairs) Two-sided Student's t-test. p = 0.34. n.s.: no significance. (e) Mid-sagittal sections of P0 cerebella were stained with Pax6, BrdU, and DAPI. Dashed lines define the EGL. Higher magnifications in the white boxes, with the DAPI signal removed, show cells stained by BrdU and Pax6 (arrowheads). Scale bars: 50 μm. (f) Percentages of BrdU+Pax6+ cells/Pax6+ cells in a lobe. n = 5, *** p < 0.001 (p = 0.00012, two-sided Student's t-test). (g) Percentages of BrdU+Pax6+ cells/Pax6+ cells within the EGL. n = 5, * p < 0.001 (p = 0.00018, two-sided Student's t-test).
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f3: CGP proliferation is reduced in KO mice.(a) Mid-sagittal sections of cerebella stained for Pax6. White and yellow arrowheads indicate Pax6 staining in the EGL and IGL, respectively. Pax6 intensity was reduced at E15.5 (n = 8 pairs), but unchanged at P1 (n = 7 pairs). Scale bars: 50 μm. (b) Total Pax6 expression was measured at E15.5, P1, P3, and P7, using GAPDH as the loading control. The percentage changes of Pax6 (KO/WT) were 36.3 ± 7.9% (E15.5; n = 5 pairs, p = 0.00021), 92.8 ± 9.9% (P1; n = 4 pairs, p = 0.35); 101.7 ± 6.9% (P3; n = 4 pairs, p = 0.32); 99.7 ± 8.9% (P7; n = 4 pairs, p = 0.43). Statistical analysis was done by one-sided Student's t-test. *** p < 0.001. n.s.: no significance. (c) Left panels show the staining of NeuN (red), Pax6 (green), and DAPI (blue) in cultured granule cells (DIV12). Higher magnification in white boxes, where the DAPI signal was removed, shows cells double-stained with NeuN and Pax6, indicating that 100% of NeuN-positive cells were Pax6-positive. Right panels: DIV12 granule cells labeled with TuJ1 (green) and NeuN (red). Nuclei were counterstained with DAPI (blue). Immunoreactivity for TuJ1 displayed long processes from differentiated neurons in both groups. Scale bars: 50 μm. (d) Numbers of NeuN+Pax6+ cells in cultured granule cells (DIV12) expressed as percentages of DAPI+stained cells (WT: 46.3 ± 2.8%; KO: 44.5 ± 5.5%; n = 6 pairs) Two-sided Student's t-test. p = 0.34. n.s.: no significance. (e) Mid-sagittal sections of P0 cerebella were stained with Pax6, BrdU, and DAPI. Dashed lines define the EGL. Higher magnifications in the white boxes, with the DAPI signal removed, show cells stained by BrdU and Pax6 (arrowheads). Scale bars: 50 μm. (f) Percentages of BrdU+Pax6+ cells/Pax6+ cells in a lobe. n = 5, *** p < 0.001 (p = 0.00012, two-sided Student's t-test). (g) Percentages of BrdU+Pax6+ cells/Pax6+ cells within the EGL. n = 5, * p < 0.001 (p = 0.00018, two-sided Student's t-test).
Mentions: To investigate whether the reduction of EGL thickness was due to altered neuronal differentiation, we studied paired-box homeodomain transcription factor (Pax6) expression in the cerebellum. Pax6 expression begins ~E8.5 in the mouse brain24 and promotes neuronal differentiation, migration, and neurite extension2526. Immunocytochemical staining and western blotting analysis showed that Pax6 was robustly expressed at E15.5 and P1 in WT mice (Fig. 3a), representing CGPs in the EGL and migrating granule cells in the inner granular layer (IGL)2027. Pax6 immunoreactivity was much stronger at P1 and displayed a thickened appearance in the EGL (Fig. 3a), similar to previous descriptions2027. In the E15.5 KO cerebellum, however, there was a substantial decrease of Pax6 intensity throughout the cerebellum, including the EGL and IGL (Fig. 3a). Interestingly, this decrease in Pax6 immunolabeling was not seen after birth (P1, Fig. 3a), suggesting that the LGI1 deficiency only suppresses Pax6 expression in embryos. Western blotting analysis also confirmed that the total expression of Pax6 was substantially reduced in KO cerebella from E14.5-E16.5 (Fig. 3b, supplementary Fig. S2), but not in cerebella at E17.5 (supplementary Fig. S2) and postnatal P1 to P7 (Fig. 3b). These results implied that LGI1 influences the generation of CGPs during early embryogenesis.

Bottom Line: The function of LGI1 in CNS development remains undefined.BrdU staining showed an inhibited proliferation of CGPs in KO embryos, which might be explained by the reduced Sonic hedgehog in embryos.Together, our results demonstrate that LGI1 is an essential player in the cerebellar development.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of Health, Zhejiang Province Key Laboratory of Neurobiology, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT
Leucine-rich glioma inactivated 1 (LGI1) is a secreted protein that interacts with ADAM transmembrane proteins, and its mutations are linked to human epilepsy. The function of LGI1 in CNS development remains undefined. Here, we report novel functions of LGI1 in the generation of cerebellar granule precursors (CGPs) and differentiation of radial glial cells (RGCs) in the cerebellum. A reduction in external granule layer thickness and defects in foliation were seen in embryonic and new-born LGI1 knockout (KO) mice. BrdU staining showed an inhibited proliferation of CGPs in KO embryos, which might be explained by the reduced Sonic hedgehog in embryos. In addition, the differentiation of RGCs into Bergmann glias was suppressed in KO mice. Enhanced Jagged1-Notch1 signaling in KO mice via reduced β-secretase proteolysis suggests that altered phenotype of RGCs is due to abnormal Notch1 signaling. Together, our results demonstrate that LGI1 is an essential player in the cerebellar development.

No MeSH data available.


Related in: MedlinePlus