Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Summary

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

Abstract

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Introduction

The vertebrate limb is a formidable model for studying cell differentiation, cell proliferation, cell death, pattern formation, and morphogenesis^1,2. During development, limbs emerge as bulges from the cells derived from lateral plate mesoderm¹. Limb buds consist of a central core of mesodermal cells covered by an ectoderm. From this early structure, a whole and well-formed limb emerge. After the limb bud arises, three axes are recognized: (1) the proximo-distal axis ([PD] shoulder to fingers), (2) the dorso-ventral axis ([DV] from the back of the hand to palm), and (3) the anterior-pos....

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). A schematic flowchart of the general steps of this protocol is shown in Figure 1A.

1. Embryo incubation and determination of viability

1. Incubate fertilized chicken eggs at 38 °C and 60% relative humidity for about three and a half days until they reach the 22 HH stage (according to Hamilton and Hamburger, 1951)¹⁶

Results

Recognizing a well-performed recombinant limb
After grafting, the manipulated embryos were returned to the incubator to allow the RL to develop. The incubation time correlated with the requirements of the experiment. Nevertheless, the RL can be easily distinguished after 12 h of implantation. To determine whether the implantation was adequate, the RL was observed as a protuberance that was securely attached to the mesodermal wall of the donor embryo (Figure 2A). On the c.......

Discussion

In general, the RL protocol can be divided into five steps: (1) embryo incubation, (2) obtaining limb mesodermal cells to fill the ectoderms, (3) obtaining the ectoderms, (4) assembling mesodermal cells inside the ectodermal covers, and (5) transplantation of the filled ectoderms into the host embryos. The major limitation of the RL technique is the long, detailed protocol, which has many critical points that require patience to perform appropriately. To successfully complete the protocol, critical moments need to be ide.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alcian Blue 8GX	Sigma	A5268
Angled slit knife	Alcon	2.75mm DB
Blunt forceps	Fine Science Tools	11052-10
Collagenase type IV	Gibco	1704-019
DMEM-HG	Sigma	D5796
Egg incubator	Incumatic de Mexico	Incumatic 1000
Fetal Bovine Serum	Gibco	16000069
Fine surgical forceps	Fine Science Tools	9115-10
Hanks Balanced Salt Solution	Sigma	H6648
Microcentrifuge	Eppendorf	5417R
Micropipet	NA	NA
Palladium wire	GoodFellow	7440 05-3
Petri dish	Nest	705001
Pippette	crmglobe	PF1016
Stereomicroscope	Zeiss	Stemi DV4
Tape	NA	NA
Trypsin porcine	Merck	9002 07-7
Tungsten needle	GoodFellow	E74-15096/01