Abstract
The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.
Highlights
Classical embryologists traditionally employed physical techniques to interrogate the mechanisms controlling embryonic development
Using a steel micro knife make an incision through the vitelline membrane and lateral plate mesoderm (LPM) adjacent to somites 26 to 32 (Figure 1C) on the right side of the embryo
We describe the use of impermeable barriers to prevent limb bud formation in the chick embryo
Summary
Classical embryologists traditionally employed physical techniques to interrogate the mechanisms controlling embryonic development. Surgical grafting techniques demonstrate that close association with the somites is essential if limb bud initiation is to occur in the LPM 34. In the 80s and 90s developmental biologists discovered diffusible signaling molecules that are essential in controlling developmental processes Beads soaked with these signaling molecules can be implanted in specific regions of the embryo and produce alterations to embryonic development. We wanted to exploit the accessibility and experimental robustness of the chick embryo to dissect the effect of barrier implantation on gene expression in the LPM around the time of limb bud initiation. Questions remain regarding the signals that control vertebrate limb bud induction and initiation and the following protocols are suitable to address them. We are the first to develop a protocol for the insertion of barriers to prevent chick limb bud outgrowth at a stage earlier than. All experiments were performed in accordance with King's College London, UK and the UK Home Office animal care guidelines
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