Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging
Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging
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DOI：
10.1186/1471-213x-11-68
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发表时间：
2011-11-09
影响因子：
--
通讯作者：
Yamamura, Ken-ichi
中科院分区：
文献类型：
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作者：
Kawakami, Minoru;Umeda, Masafumi;Yamamura, Ken-ichi
背景：神经嵴细胞（NCCs）是胚胎多能干细胞。其最显著的特点之一是远距离和精确制导的迁移。结果：本研究将P0-Cre/CAG-CAT-EGFP双转基因小鼠E9.5胚胎包埋于胶原凝胶中，体外培养24 h以上，获得了完整的胚胎移植模型，并成功地将其移植到裸鼠体内。这种发展具有足够的NCC荧光信号强度，能够进行单细胞分辨率分析，伴随的NCC迁移潜力完整，并且保持了对细胞外信号的适当NCC响应。通过植入吸附血小板衍生生长因子-AA（PDGF-AA）的珠粒，我们证明了PDGF-AA在胚胎中作为NCC引诱剂。我们还在培养板上用从P0-Cre/CAG-CAT-EGFP胚胎分离的NCC进行测定。已知神经介质5-羟色胺（5-HT）调节NCC迁移。我们最近证明，多巴胺，除了5-HT，刺激NCC迁移在体外。两个NCC人群，具有不同的轴向水平的起源，表现出独特的分布模式，关于迁移速度和不同的剂量反应模式，以5-HT和多巴胺。虽然在NCC研究中鸟类物种占主导地位，但我们的新系统应该使我们能够使用小鼠来分析胚胎或培养板上NCC的许多不同方面，如迁移，分裂，分化，和凋亡。
Background: Neural crest cells (NCCs) are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs.Results: In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA), we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT) has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine.Conclusions: Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.