影响大脑皮质神经细胞生成的信号通路

doi:10.3969/j.issn.1673-5374.2013.01.003

摘要/Abstract

摘要：

Notch是大脑皮质发育过程中决定神经干细胞/前体细胞命运的重要分子开关，而整联蛋白金属蛋白酶10很可能是Notch限速步骤的S2酶。在小鼠大脑皮质早期发育阶段敲除整联蛋白金属蛋白酶10可以造成神经干细胞数量的下降，然而整联蛋白金属蛋白酶10在大脑皮质发育晚期的表达和作用目前依然不清楚。我们研究的目的是鉴定整联蛋白金属蛋白酶10和Notch胞内段在小鼠大脑胚胎发育晚期是否有时空上表达的一致性。我们用原位杂交和免疫荧光组化的方法鉴定了整联蛋白金属蛋白酶10及Notch胞内段在胚胎晚期到出生后P1天内的小鼠大脑皮质中的表达；并将整联蛋白金属蛋白酶10的表达模式与多种标记物的免疫荧光组化结果进行对比，包括标记神经干细胞的Nestin，标记成熟神经元的Tuj1标记，标记胶质细胞的S100beta。结果显示整联蛋白金属蛋白酶10在孕15.5d到生后1d的皮质板和脑室下层区域有表达，并与Notch胞内段有很好的共标区域。整联蛋白金属蛋白酶10的表达区域不仅与Tuj1有部分重叠，还与S100beta标记的区间有较好的重叠。这些结果表明整联蛋白金属蛋白酶10-Notch信号通路可能在大脑皮质发育过程中的神经元成熟与胶质细胞生成中发挥一定作用。

关键词: 神经再生, 神经发生, 整联蛋白金属蛋白酶, Notch胞内段, 大脑皮质, 神经发育, 神经元成熟, 胶质细胞, 基金资助文章, 图片文章

Abstract:

The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a molecular switch for neural stem/progenitor cell fate during cortex development but the mechanism remains unclear. Biochemical and cellular studies showed that Notch receptor activation induces several proteases to release the Notch intracellular domain (NICD). A Disintegrin and Metalloprotease 10 (ADAM10) might be a physiological rate-limiting S2 enzyme for Notch activation. Nestin-driven conditional ADAM10 knockout in mouse cortex showed that ADAM10 is critical for maintenance of the neural stem cell population during early embryonic cortex development. However, the expression pattern and function of ADAM10 during later cerebral cortex development remains poorly understood. We performed in situ hybridization for ADAM10 mRNA and immunofluorescent analysis to determine the expression of ADAM10 and NICD in mouse cortex from embryonic day 9 (E14.5) to postnatal day 1 (P1). ADAM10 and NICD were highly co-localized in the cortex of E16.5 to P1 mice. Comparisons of expression patterns of ADAM10 with Nestin (neural stem cell marker), Tuj1 (mature neuron marker), and S100β (glia marker) showed that ADAM10 expression highly matched that of S100β and partially matched that of Tuj1 at later embryonic to early postnatal cortex developmental stages. Such expression patterns indicated that ADAM10-Notch signaling might have a critical function in neuronal maturation and gliogenesis during cortex development.

Key words: neural regeneration, neurogenesis, ADAM10, A Disintegrin and Metalloprotease, Notch, Notch intracellular domain, Tuj1, S100β, Nestin, cerebral cortex, development, neuronal maturation, glial cell, grants-supported paper, photographs-containing paper, neuroregeneration

. 影响大脑皮质神经细胞生成的信号通路[J]. 中国神经再生研究(英文版), 2013, 8(1): 24-30.

Zhixing Ma, Qingyu Li, Zhengyu Zhang, Yufang Zheng. A Disintegrin and Metalloprotease 10 in neuronal maturation and gliogenesis during cortex development[J]. Neural Regeneration Research, 2013, 8(1): 24-30.

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引言

材料方法

Design

A basic study of neural development.

Time and setting

The experiments were performed at the School of Life Sciences, Fudan University, Shanghai, China from 2009 to 2012.

Materials

Thirty time-pregnant C57BL/6 female mice (mated pregnant mice) and 20 newborn C57BL/6 mice were used. All C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Inc, Shanghai, China.

Methods

Constructs for in situ hybridization

In situ hybridization probe for mouse ADAM10 (NM_007399; bases 208–730 bp, amino acids 68–240) was cloned by PCR and the obtained cDNA fragments were subcloned into pSPT19 vector (Roche Diagnostics (Shanghai) Limited, Shanghai, China) between EcoRI and HindIII sites.

In situ hybridization

Frozen mouse brains were sectioned coronally at 15 μm using a cryostat (Leica CM1900, Solms, Germany). Frozen sections were air dried and fixed in 4% paraformaldehyde/PBS for 10 minutes. The slices were digested with proteinase K. Prehybridization and hybridization were performed with a 5 × SSC–50% formamide solution to avoid evaporation. After incubation, the sections were washed in 2 × SSC (room temperature) for 30 minutes, in 2 × SSC (50°C) for 1 hour, in 0.1 × SSC (50°C) for 1 hour, and equilibrated in Buffer 1 (100 mM Tris-HCl and 150 mM NaCl, pH 7.5) for 5 minutes. The sections were then incubated at room temperature with alkaline phosphatase-coupled anti-digoxigenin antibody (Roche Diagnostics (Shanghai) Limited, Shanghai, China) diluted 1:5 000 in Buffer 1 containing 0.5% blocking reagent for 2 hours. Excess antibody was removed by two 15-minute washes in Buffer 1, and the sections were equilibrated in Buffer 2 (100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl₂, pH 9.5) for 5 minutes. Color development was performed at room temperature (30 minutes to 3 days, depending on the amount of transcripts to be detected) in Buffer 2 containing 1.875 mg/mL nitro blue tetrazolium chloride and 0.94 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate. ADAM10 (208–730 bp) was cloned into pSPT19 vector and antisense RNA was transcripted using T7 promoter with Roche DIG RNA labeling Kit. Bright field images were taken by using Axiovert-200 microscope (Carl Zeiss, Oberkochen, Germany).

Immunofluorescent staining

Embryonic brains were fixed in 4% paraformaldehyde in PBS overnight and placed in 15% sucrose/PBS for 12 hours and then 30% sucrose/PBS for 24 hours at 4°C. Brains were then embedded in optimal cutting temperature mounting medium and frozen before coronal sectioning at 10 or 20 μm using a cryostat (Leica CM1900, Germany). The following primary antibodies were used: rabbit polyclonal anti-ADAM10 antibody, rabbit polyclonal anti-NICD antibody, rabbit polyclonal anti-S100β antibody, mouse monoclonal anti-Nestin antibody, mouse monoclonal anti-Tuj1 (beta III-tubulin) antibody (all purchased from Abcam, Cambridge, UK) at 1:200 dilution. Sections were then incubated with secondary Cy3-labeled goat antibody (anti-rabbit or anti-mouse, purchased from Beyotime, Suzhou, China) at 1:500 dilution. The negative control was performed using a secondary antibody alone on sections. Fluorescent images were taken using an Olympus IX71 microscope (Olympus, Tokyo, Japan).