Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Summary

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Abstract

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., ^{1, 2, 3, 4}). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick ⁵ or mouse-chick ⁶ chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues ⁷. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras ⁸. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

Protocol

1. Preparing the Donor Tissue

1. Prepare culture media, sharpen glass pins, sharpen tungsten needles.
2. Collect embryo from shell, wash in ice-cold PBS.
   1. Using a 10 mL syringe and an 18 gauge needle remove 1.0 mL of albumin from the pointed end of the egg shell.
   2. Make a small hole on the top of the shell using the point of scissors, and then cut a circular opening to expose the embryo
3. Remove head and place into DMEM (serum free, room temperature)
4. Dissect Frontonasal Process from the embryo (or other donor site such as maxillary or mandibular process)
5. Digest in 2.4U/ mL dispase in PBS....

Discussion

Using this transplantation method has allowed us to determine that the ectoderm contains signaling information that regulates dorsoventral polarity and proximodistal extension of the upper jaw. The similarity of outcomes when using quail or mouse ectoderm, and the conservation of molecular signals in this tissue among many species ^6,11 indicates that this is a highly conserved signaling center among vertebrates. Furthermore, other investigators have used similar techniques to test the signaling properties of d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Tektronix, Inc.	TEKZR114
DMEM	University of California - San Francisco	CCFDA003
BSA	Sigma-Aldrich	A7906
Dispase	GIBCO, by Life Technologies	17105-041
35x10 mm Petri dish	Falcon BD	1008
No. 5 Dumont forceps	Fine Science Tools	11252-20
Scissors	Fine Science Tools	14058-11
Spring Scissors	Fine Science Tools	15010-11
Needle holder	Fine Science Tools	26016-12
Tungsten Needle	Fine Science Tools	26000
Microcapillary tube	Drummond Scientific	3-000-225-G
Pasteur Pipets	Fisher Scientific	13-678-6B
Spring scissors	Fine Science Tools	15010-11
Blade holder	Fine Science Tools	10052-11
Razor blade	Fine Science Tools	10050-00