Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Summary

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Abstract

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Introduction

The mammalian brain is composed of a large number of various types of neurons and glial cells. All cells are generated from neural stem/progenitor cells (NPCs), which first proliferate to expand their numbers, then start to differentiate into neurons, and finally give rise to glial cells^1,2,3,4,5. Once cells have differentiated into neurons, they cannot proliferate or increase their numbers, and, therefore, the maintenance of NPCs until later stages is important. Notch signaling via cell-cell interactions p....

Protocol

All the procedure including animal subjects have been approved by Institutional Animal Care and Use Committee at the institute for Frontier Life and Medical Sciences, Kyoto University.

1. Bioluminescence reporters

NOTE: The luciferase reporter is suitable for measuring the rapid dynamics of promoter activity by fusing the degradation signal. Moreover, the luciferase fusion reporter enables monitoring of the protein dynamics in the single cell. Both types of reporters are available for mono-layer culture (dissociation culture) and tissue culture (slice culture) experiment.

1. Reporter for monitoring Dll1 ....

Results

Expressions of the genes Hes1/7 exhibit 2 h oscillation cycle in various cell lines and during somitogenesis. Furthermore, the period of oscillation is very short and both their mRNAs and proteins are extremely unstable with the half-lives of around 20 min. If using a slow response reporter, we cannot trace such rapid dynamics, and if using a stable reporter, it gradually accumulates while the gene expression oscillates. Thus, the reporter must be rapidly degraded to monitor the rapid turnover of such cyclically.......

Discussion

The components of Notch signaling show oscillatory expressions in synchrony during somitogenesis but out of synchrony during neurogenesis, leading to the difficulties in capturing the expression dynamics by static analysis in the latter case. Thus, real-time monitoring is required to reveal the expression dynamics of Notch signaling components, such as Hes1 and Dll1. Because the periods of the expressions of Hes1 and Dll1 oscillations are extremely short, approximately 2-3 h, rapid res.......

Materials

Name	Company	Catalog Number	Comments
Bioluminescence Imaging System
Chilled water circulator (chiller)	Julabo	Model: F12-ED
Cooled CCD camera	Andor Technology	Model: iKon-M 934
Incubator system	TOKAI HIT	Model: INU-ONICS
Inverted microscope	Olympus	Model: IX81
Inverted microscope	Olympus	Model: IX83
LED illumination device	CoolLED	Model: pE1
MetaMorph	MOLECULAR DEVICES	Model: 40000
Mix gas controller	Tokken	Model: TK-MIGM OLO2
Objective lens	Olympus	Model: UPLFLN 40X O
Preparations for Dissection
Dissection microscope	Nikon	Model: SMZ-2B
Fluorescence stereoscopic microscope	Leica	Model: MZ16FA
Fine forceps	DUMONT	INOX No.5
Scissors, Micro scissors
Forceps
Ring-shaped forceps
10-cm plastic petri dish	greiner	664160-013
35-mm plastic petri dish	greiner	627160
PBS	Nacalai Tesque	14249-24
DMEM/F12	invitrogen	11039-021
Reagents for NPC dissociation culture
B27 supplement	invitrogen	12587-010
bFGF	invitrogen	13256-029	Stock solution: 1 μg/ml in 0.1% BSA/PBS
D-luciferin	Nacalai Tesque	01493-85	Stock solution: 100mM in 0.9% saline
DNase	Worthington Biochemical Corporation	LK003172	Stock solution: 1000U/ml in EBSS
EBSS	Worthington Biochemical Corporation	LK003188
Glass bottom dish	IWAKI	3910-035
N2 supplement (100x)	invitrogen	17502-048
N-acetyl-cystein	Sigma	A-9165-25G
Papain	Worthington Biochemical Corporation	LK003178	Stock solution: 7U/ml in EBSS
Penicillin/Streptmycine	Nacalai Tesque	09367-34
Poly-L-lysine	Sigma	P-6281	40 mg/ml in DW
Preparations for in utero electroporation
50-ml syringe	TERUMO	181228T
Electrode	Neppagene	7-mm
Electroporator	Neppagene	CUY21 EDIT
Forceps
Gauzes	Kawamoto co.	7161
Micro capillary	Made in-house
PBS	Nacalai Tesque	14249-24
Pentbarbital	Kyoritsuseiyaku	Somnopentyl
Ring-shaped forceps
Scissors, Micro scissors
Suture needle	Akiyama MEDICAL MFG. CO	F17-40B2
Xylazine	Bayer	Seractal
Preparations for Slice culture
10-cm plastic petri dish	greiner	664160-013
35-mm plastic petri dish	greiner	627160
Culture insert	Millipore	PICM01250
DMEM/F12	invitrogen	11039-021
Fetal Bovine Serum	Sigma	172012-500ML
Fine forceps	DUMONT	INOX No.5
Forceps
Horse Serum	Gibco	16050-122
Micro surgical knife	Alcon	19 Gauge V-Lance
Multi-gas incubator	Panasonic	MCO-5MUV-PJ
N2/B27 media	Made in-house		ref. NPC dissociatioin culture
PBS	Nacalai Tesque	14249-24
Ring-shaped forceps
Scissors, Micro scissors
Silicon rubber cutting board	Made in-house