AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Summary

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

Abstract

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

Introduction

The physical principles underlying the biomechanical control of morphogenetic processes are largely undefined. While biomechanical studies at the molecular and cellular level are gathering considerable momentum, the exploration of biomechanical parameters at the tissue/organism level is at its infancy. Hydrodynamic Regression¹ or Video Force Microscopy² allow researchers to distinguish active and passive forces, while laser microsurgery³, or the less intrusive atomic force microscopy (AFM) of dissociated cells⁴ or in vivo⁵ or nanoparticle mi....

Protocol

All the protocol steps described below follow the animal care guidelines of our institutions.

1. Zebrafish Culture

1. Breed and maintain adult zebrafish under standard conditions.
   NOTE: AB and TL wild type embryos were used for this study.
2. Collect embryos and grow them at 28.5 °C in E3 embryo medium²⁴. Stage them according to morphology as previously described¹⁹.

2. Atomic Force Microscopy

1. Manually dechorionate staged zebrafish embryos (of different ages according to individual interests). Remove chorions with two th....

Results

Cortical tension measurements
For each measurement point, five force-displacement (F-z) curves were acquired by AFM by ramping the cantilever at 1 Hz with a peak-to-peak amplitude of 5 µm (velocity = 10 µm/s) up to a maximum indentation of ~2 µm. This procedure was taking less than 20 min and was not affected by epiboly progression. Each experimental condition was tested in at least 5 embryos.

The force-disp.......

Discussion

Here we show that the material properties and some biomechanical parameters of the zebrafish yolk cell during epiboly can be readily estimated by AFM and nanoparticles microrheology.

While AFM has been employed to retrieve rheological features of cells and tissues in physiological conditions^4,5,25,28, here we developed a protocol for applying AFM to intact developing.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Inverted optical microscope	Nikon	TE2000	Visualization of the sample and AFM cantilever
Atomic force microscope (AFM)	Custom made	-	Any commercialized AFM could be employed
Inverted Confocal microscope	Zeiss	LSM780	Nanorheology data collection
Agarose D1 Low EEO	Conda	8016.00	Casting embryos
Ultrapure LMP Agarose (low melting)	Invitrogen	16520-100	Securing embryos
Zebrafish Microinjection and Transplantation Molds	World Precision Instruments	Z-MOLDS	Casting embryos. Custom made with the original dimensions can also be employed
Dumont 5 Forceps	FST	11251-20	1.5 mm diameter
Si3N4 cantilever	Novascan	PT.GS	Measuring the embryo by AFM. Spherical tip: 4.5 µm diameter and k=0.01 N/m
Fluorescent nanoparticles	Life Technologies	FluoSpheres F8811,	For tracking nanorheology
Micropipette puller	Sutter Instruments	P-2000	Fabricating tailored microneedles
Borosilicate capillary glass	Warner Instruments	G100TF-4	Fabricating tailored microneedles
Micromanipulator	Narishige	MN-153	Manipulating the micropipette
Microinjector	Eppendorf	FemtoJet Express	Controlling injection time and pressure
Stereomicroscope	Leica	DFC365FX	Visualization of the embryos during injection
Analysis Software	MathWorks	Matlab	Analyzing AFM and particle tracking data
Zebrafish	AB and TL wild type	-	Strains employed for embryo collection
Glass Bottom Plates	Mat Tek	P35G-0.170-14-C	Mounting embryos for nanorheology data collection
Stage micrometer	FST	29025-02	Measuring injection volume