Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

Summary

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

Abstract

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

Introduction

Breakthroughs in the molecular mechanisms that control gene expression during embryonic development have allowed us to understand how cell fate is determined. Commitment to different cell lineages occurs once cells begin the molecular expression of transcription factors¹. This expression pattern is highly coordinated in space and time and thereby directs the shaping, positioning, and patterning of cells, tissues, and organs^1,2,3,4,5. Embryonic induction is the process by which cells ....

Protocol

This research was reviewed and approved by the Institutional Review Board for the Care and Use of Laboratory Animals of the Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM, Mexico City, Mexico).

1. Egg incubation and embryo staging

NOTE: Fertilized hen eggs can be obtained from local farms. Fertilized White Leghorn chicken eggs are most commonly used. Store the freshly fertilized chicken eggs at 15 °C for up to 1 week prior to incubation.

1. Incubate the fertilized chicken eggs vertically with the pointy side down in a humidified....

Results

Using soaked beads to evaluate cell behavior in the embryonic chick limb
To assure the efficacy of this assay, the bead must be placed consistently and precisely in the correct location; in this case, the distal-most of the third interdigit beneath the apical ectodermal ridge AER (Figure 1A). This positioning permits the molecule in question to spread equally throughout the interdigital tissue. Moreover, the zone beneath the AER contains undifferentiated cells that are r.......

Discussion

The main advantage of the experimental tool detailed in this protocol is being able to control the time and location of the exposure to beads soaked in a given experimental molecule. Combining the correct positioning with precise developmental timing provides enormous possibilities to study cell differentiation processes. Performing these experiments in undifferentiated tissue enables investigating the first crucial events in cellular lineage. For example, placing a TGFß-soaked bead in the interdigital tissue of emb.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Affi-Gel Blue Gel beads	Bio-Rad	153-7302
AG1-X2 beads	Bio-Rad	1400123
Egg incubator	Incumatic de Mexico	Incumatic 1000
Fine surgical forceps	Fine Science Tools	9115-10
Heparine Sepharose beads	Abcam	ab193268
Petri dish	Nest	705001
Stereomicroscope	Zeiss	Stemi DV4
Tape	NA	NA
Tungsten needle	GoodFellow	E74-15096/01