During embryogenesis, cells become progressively committed to different fates through a two-step process: specification followed by determination. Specification is demonstrated by removing a segment of an early embryo, “neutrally” culturing the tissue in vitro—for example, in a petri dish with simple medium—and then observing the derivatives. If the cultured region gives rise to cell types that it would normally generate in the embryo, this means that it is specified. In contrast, determination occurs if a region of the embryo is removed and placed in a “non-neutral” environment—such as in a dish containing complex medium supplemented with a variety of proteins, or even a different area of the embryo itself—and it still generates the expected derivatives. Specification and determination are two sequential steps in the developmental pathway of a cell, which precede the final stage of differentiation, during which mature tissues with unique morphologies and functions are produced.

In Vitro Demonstration of Specification

To study specification, researchers must first understand the normal derivatives of different regions of an embryo. To accomplish this, fate maps are often used, which are generated by dyeing or labeling cells early in embryonic development, culturing whole embryos and monitoring where the marked cells end up. For example, such techniques employed in the chicken have demonstrated that distinct regions located off-center in the embryo (at approximately the 9’ o’clock and 3 o’clock positions) give rise to neural crest cells, which are capable of migrating and generating the peripheral nervous system.

Importantly, these neural crest-destined areas can be excised during the early stages of gastrulation (when the embryo is transformed into a three-layered structure)—before they begin to express protein markers or any distinct features of the cell type. When any underlying tissue that may be a source of signaling factors is scraped away, and these explants are cultured within collagen drops in a simple medium, they generate cells expressing typical neural crest transcription factors. Interestingly, some cells are even observed migrating away from the body of the tissue fragment, which is another feature of this cell fate. These experiments have demonstrated that specification of the neural crest occurs early during embryonic development, and while much of this work has been performed in the chicken, more recent evidence suggests a similar pattern of specification in the rabbit; both of these organisms are used as models for human embryonic development.

Understanding the Signaling Behind Specification

Once specification of a region of an embryo has been demonstrated, researchers are also interested in determining how protein signals—and the position of the tissue in the embryo itself—result in a cell being sent down a particular developmental pathway. For the neural crest, researchers have determined that a combination of proteins, among them bone morphogenetic proteins and fibroblast growth factors, emanating from tissue abutting or underlying the prospective neural crest induce this cell fate. Such signals, in turn, elicit the expression of specifier proteins in these cells, which launch them into the neural crest pathway.