Pax2-cre-mediated deletion of Lgl1 causes abnormal development of the midbrain

Abstract

Lgl1 protein plays a critical role in neurodevelopment, including hippocampus, olfactory bulb, and Purkinje cell. However, the specific mechanism of LGL1 function in the midbrain remains elusive. In this study, we generated Lgl1 conditional knockout mice using Pax2-Cre, which is expressed in the midbrain, and examined the functions of Lgl1 in the midbrain. Histological analysis exhibited abnormal midbrain development characterized by enlarged ventricular aqueduct and thinning tectum cortex. Lgl1 deletion caused excessive proliferation and heightened apoptosis of neural progenitor cells in the tectum of LP cko mice. BrdU labeling studies demonstrated abnormal neuronal migration. Immunofluorescence analysis of Nestin demonstrated an irregular and clustered distribution of glial cell fibers, with the adhesion junction marker N-cadherin employed for immunofluorescent labeling, unveiling abnormal epithelial connections within the tectum of LP cko mice. The current findings suggest that the deletion of Lgl1 leads to the disruption of the expression pattern of N-cadherin, resulting in abnormal development of the midbrain.

Keywords: Lgl1; Midbrain; Migration; N-Cadherin.

Fig. 1

Generation of LP cko mice mediated by Pax2-Cre transgenic mutation. (A) The overall appearance of WT and LP-cKO mice. (B) Weight curves of the control and LP-cKO mice. Data are expressed as the mean ± SEM, ***P < 0.001. (C–E) Immunofluorescence staining was performed on sagittal frozen sections of the midbrain of E13.5 WT and HOMO using anti-Lgl1 antibody. Nuclear marker DAPI was used for counterstaining (blue). (C′-E′) Lgl1 was efficiently deleted in the midbrain of HOMO mice. Scale bar represents 100 μm.

Fig. 2

Abnormal development of the midbrain in LP cko mice. (A, A′) General appearance of E12.5 embryos. Note dome-like appearance of the head of the LP cko embryo. Magnification, × 1.5 (B, B′) Sagittal section of the midbrain of E12.5 embryo. Red arrows indicate the disordered arrangement of cells in the aqueduct of the midbrain and expanding over the cells that maintain cell polarity. (C–G′) Histologic appearance of midbrain from control (C–G) and LP cko (C′–G′) at different age. Scale bar represents 500 μm. (H) Line graph of the midbrain length in control and LP-cKO mice. (I) Line graph of the cerebral cortex thickness of midbrain in control and LP-cKO mice. Data are expressed as the mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3

Abnormalities of cell proliferation and apoptosis in the tectum of Lgl1 cKO mice. (A, A′) Immunostaining of sagittal sections from E14.5 WT tectum with anti-PH3 antibodies (red) reveals increase in mitotic cells in the tectum of LP cko mice. (B, B′) Immunofluorescence staining with Cyclin-D (green) on coronal sections of the midbrain from E13.5 mice demonstrated a significant increase in the number of proliferating cells in LP cko mice compared to their WT counterparts. (C, C′) Increased number of apoptosis cells in the tectum of LP cko mice. HOMO have more TUNEL-positive (green) cells than the WT mice. DNA is counterstained by DAPI (blue). The inset in C′ shows prominent localization of TUNEL⁺ cells (green) to the Nestin positive rosettes (red) in the LP cko tectum. (D) Statistically significant differences are indicated. ***P < 0.001. Scale bar represents 100 μm.

Fig. 4

Impaired neuronal migration in LP cko mice during midbrain development. Sagittal sections of tectum immunostained with anti-BrdU antibody. BrdU was labeled at E12.5 (A, A′), and examined at E13.5. In control mice, most cells labeled with BrdU E13.5 migrated away from the VZ and the SVZ and reached the CP. Disrupted radial glial scaffold in LP cko. (D-F′) Nestin immunostaining (green) in radial glial cells at E12.5. DAPI (blue) was used to counterstain nuclei. Nestin was regularly radially arranged in WT mice (white arrows in F). The radial glial fibers show a chaotic pattern and gather in clusters in LP cko mice (white arrows in F′). (G-H′) Immunofluorescence co-staining was performed using adhesion marker antibodies N-cadherin (red) and DAPI (blue). The adhesion pattern of WT mice on the aqueduct surface was characterized by a linear and smooth configuration. However, the adhesion integrity of specific aqueduct regions started to deteriorate in E13.5 LP cko mice. H and H′ correspond to the highly magnified sections of areas in dotted lines in G and G’. Scale bar represents 100 μm (A-C′), 50 μm (D-F′), 200 μm (G-H′). (I) Quantification of the experiments shown in C and C’. (J) Quantification of the experiments shown in D and D’. Analysis of relative fluorescence intensity revealed an increased level of Nestin. (K) Quantification of the experiments shown in E and E’. Analysis of relative fluorescence intensity revealed a decreased level of N-cadherin. ***P < 0.001.