Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo

Chenjun Shi; Chenchen Handler; Haden Florn; Jitao Zhang

doi:10.3791/66117

JoVE Journal > Developmental Biology

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발생학

监测雏鸡胚胎神经管闭合过程中组织的机械进化

Published: November 10, 2023

doi:

10.3791/66117

Chenjun Shi, Chenchen Handler, Haden Florn, Jitao Zhang

¹Department of Biomedical Engineering, College of Engineering,Wayne State University, ²Fischell Department of Bioengineering,University of Maryland

Summary

该协议旨在纵向监测雏鸡胚胎神经形成过程中神经板组织的机械性能。它基于布里渊显微镜和载物台孵化系统的集成，能够对卵培养雏鸡胚胎中的神经板组织进行实时机械成像。

Abstract

神经管闭合（NTC）是胚胎发育过程中的关键过程。这一过程的失败会导致神经管缺陷，导致先天性畸形甚至死亡。NTC涉及遗传、分子和机械水平的一系列机制。虽然近年来机械调节已成为一个越来越有吸引力的话题，但由于缺乏合适的技术来对原位进行 3D 胚胎组织的机械测试，它在很大程度上仍未得到探索。作为回应，我们开发了一种以非接触式和非侵入性方式量化鸡胚胎组织的机械性能的方案。这是通过将共聚焦布里渊显微镜与载物台孵育系统集成来实现的。为了探究组织力学，收集预培养的胚胎并将其转移到载物台培养箱中进行卵外培养。同时，布里渊显微镜在发育过程中的不同时间点采集神经板组织的机械图像。该协议包括样品制备的详细说明、布里渊显微镜实验的实施以及数据后处理和分析。通过遵循该协议，研究人员可以纵向研究胚胎组织在发育过程中的机械进化。

Introduction

神经管缺陷（NTD）是胚胎发育过程中神经管闭合（NTC）失败引起的严重中枢神经系统出生缺陷¹。被忽视的热带病的病因很复杂。研究表明，NTC 涉及一系列形态发生过程，包括收敛延伸、神经板弯曲（例如，顶端收缩）、神经皱襞抬高以及最终神经襞的粘附。这些过程受多种分子和遗传机制^的调节^2,3，^这些过程中的任何故障都可能导致NTDs4,5,6。随着越来越多的证据表明，机械线索在 NTC³^、⁷^、⁸^、⁹^、¹⁰^、¹¹ 中也起着至关重要的作用，并且已经发现了基因和机械线索^{之间的关系} 12,13,14，因此研究神经形成过程中的组织生物力学变得势在必行。

已经开发了几种用于测量胚胎组织机械性能的技术，包括激光消融（LA）¹⁵、组织解剖和松弛（TDR）^16,17、微量移液器抽吸（MA）¹⁸、基于原子力显微镜（AFM）的纳米压痕¹⁹、微压痕器（MI）和微孔板（MP）²⁰、使用光学/磁镊的微流变学（MR）²¹^、²²^、²³和基于液滴的传感器²⁴.现有方法可以在从亚细胞到组织尺度的空间分辨率下测量机械性能。然而，这些方法大多是侵入性的，因为它们需要与样品接触（例如，MA、AFM、MI 和 MP）、外部材料注射（例如，MR 和基于液滴的传感器）或组织解剖（例如，LA 和 TDR）。因此，现有方法在原位监测神经板组织的机械演化具有挑战性²⁵。最近，混响光学相干弹性成像在具有高空间分辨率的非接触式机械映射方面显示出前景²⁶。

共聚焦布里渊显微镜是一种新兴的光学模式，能够以²⁷^、²⁸^、²⁹^、³⁰ 亚细胞分辨率对组织生物力学进行非接触式定量。布里渊显微镜基于自发布里渊光散射原理，即入射激光与材料内部热波动引起的声波之间的相互作用。因此，散射光会经历频率偏移，称为布里渊频移 ω_R，公式为³¹：

(1)

这里，是材料的折射率，λ是入射光的波长，M’是纵向模量，ρ是质量密度，θ是入射光与散射光之间的角度。对于同类型的生物材料，折射率和密度之比近似恒^定28,32,33,34,35,36。因此，布里渊位移可以直接用于估计生理过程中的相对机械变化。布里渊显微镜的可行性已在各种生物样品中得到验证29,37,38。最近，通过将布里渊显微镜与载物台孵化系统相结合，演示了活雏鸡胚胎的延时机械成像³⁹。该协议提供了样品制备、实验实施以及数据后处理和分析的详细说明。我们希望这项工作将促进非接触式布里渊技术的广泛采用，以研究胚胎发育和出生缺陷中的生物力学调节。

Protocol

该协议已获得韦恩州立大学机构动物护理和使用委员会的批准。 1.实验准备使用70%乙醇溶液清洁和消毒剪刀和镊子。此外，准备一次性移液器和注射器。通过向 495 mL 去离子水中加入 3.595 g NaCl 来制备洗涤介质。然后，向培养基中加入5ml青霉素 – 链霉素（5U / mL）。用洗涤介质填充100毫米培养皿并将其加热至37°C。根据图 1</s…

Representative Results

图6 显示了布里渊显微镜的原理图。该系统采用660nm激光作为光源。在激光头的正后放置一个隔离器以抑制任何背向反射光，并使用中性密度（ND）滤光片来调节激光功率。一对焦距分别为 f1 = 16 mm 和 f2 = 100 mm 的透镜 L1 和 L2 用于扩展激光束。在偏振分束器（PBS）之后，使用半波板（HWP）和线性偏振器（偏振器1）来调节照射在样品或校准材料（即水和甲醇）上的光束功率。?…

Discussion

胚胎的早期发育很容易受到外界干扰的影响。因此，在样品提取和转移过程中需要格外小心。一个潜在的问题是胚胎从滤纸上脱落，这可能导致卵黄膜收缩，并导致布里渊成像中神经板的倾斜伪影。此外，这种收缩可能会阻止胚胎的发育。应注意防止分离的几个关键步骤。首先，在步骤2.4中，确保从膜表面彻底去除蛋白至关重要。任何残留的蛋白都会阻碍膜与滤纸^46,47<su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院（K25HD097288，R21HD112663）尤尼斯·肯尼迪·施莱弗国家儿童健康与人类发展研究所的支持。

Materials

100 mm Petri dish	Fisherbrand	FB0875713
2D motorized stage	Prior Scientific	H117E2
35 mm Petri dish	World Precision Instruments	FD35-100
Brillouin Microscope with on-stage incubator	N/A	N/A	This is a custom-built Brillouin Microscope system based on Ref. 30
Chicken eggs	University of Connecticut	N/A
CMOS camera	Thorlabs	CS2100M-USB
EMCCD camera	Andor	iXon
Ethanol	Decon Laboratories, Inc.	#2701
Filter paper	Whatman	1004-070
Incubator for in ovo culture	GQF Manufacturing Company Inc.	GQF 1502
Ring	Thorlabs	SM1RR
Microscope body	Olympus	IX73
NaCl	Sigma-Aldrich	S9888
On-stage incubator	Oko labs	OKO-H301-PRIOR-H117
Parafilm	Bemis	PM-996
Penicillin-Streptomycin	Gibco	15070-063
Pipettes	Fisherbrand	13-711-6M
Scissors	Artman instruments	N/A	3pc Micro Scissors 5
Syringe	BD	305482
Tissue paper	Kimwipes	N/A
Tube	Corning	430052
Tweezers	DR Instruments	N/A	Microdissection Forceps Set

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Shi, C., Handler, C., Florn, H., Zhang, J. Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo. J. Vis. Exp. (201), e66117, doi:10.3791/66117 (2023).