The Genetic Toolkit for Development

The foremost challenge for embryology has been to identify the genes and proteins that control the development of animals from an egg into an adult. Early embryologists discovered that localized regions of embryos and tissues possess properties that have long-range effects on the formation and patterning of the primary body axes and appendages. Based on these discoveries, they postulated the existence of substances responsible for these activities. However, the search for such molecules proved fruitless until the relatively recent advent of genetic and molecular biological technologies. The most successful approach to understanding normal development has involved the isolation of single gene mutations that have discrete and often large-scale effects on body pattern.

In this chapter, we take an inventory of the essential genetic toolkit for animal development. We concentrate on genes first discovered in insects, where systematic screens for developmental genes were pioneered. Importantly, however, it turns out that related genes are present in many other animals. We describe how members of the genetic toolkit were identified and what kinds of gene products they encode. In addition, we illustrate the general correlation between these genes' patterns of expression with the development of the morphological features they affect. Finally, we briefly survey their distribution and function in other animals.

Only a small fraction of all genes in any given animal constitute the toolkit that is devoted to the formation and patterning of the body plan and body parts. Two classes of gene products with the most global effects on development are of special interest: families of proteins called transcription factors that regulate the expression of many other genes during development, and members of signaling pathways that mediate short- and long-range interactions between cells. The expression of specific transcription factors and signaling proteins marks the location of many classically defined regions within the embryo. These proteins control the formation, identity, and patterning of most major features of animal design and diversity.

The only way in which we may hope to get at the truth is by the organization of systematic experiments in breeding, a class of research that calls perhaps for more patience and more resources than any other form of biological inquiry. Sooner or later such investigation will be undertaken and then we shall begin to know.

—W. Bateson Material for the Study of Variation (1894)

. . . if the mystery that surrounds embryology is ever to come within our comprehension, we must . . . have recourse to other means than description of the passing show.

—T.H. Morgan Experimental Embryology (1927)


Long before any genes or proteins affecting animal development were characterized, embry-ologists sought to identify the basic principles governing animal design. In their search, they focused on the large-scale organization of the primary body axes, the differentiation of various germ layers (ectoderm, mesoderm, and endoderm), and the polarity of structures such as appendages and insect segments. By manipulating embryos and embryonic tissues, primarily by transplantation and ablation, researchers discovered many important properties of developing embryos and tissues. Much of the fascination of embryology stems from the remarkable activities of discrete regions within developing embryos in organizing the formation of body axes and body parts. Furthermore, these classical concepts of embryonic organization present a very useful framework for considering how that organization can change during evolution. We will briefly review some of these experiments and ideas before addressing their genetic and molecular manifestations.

The first demonstration of organizers—regions of embryos or tissues that have longrange effects on the fate of surrounding tissues—was achieved by Mangold and Spemann in 1924. They transplanted the lip of the blastopore, the invagination where mesoderm and endoderm move inside the amphibian embryo, of a newt gastrula into another newt embryo and found that the transplanted tissue could induce a second complete body axis (Fig. 2.1a). The additional embryo induced was partly derived from the transplanted graft and partly derived from the host. The equivalent of the "Spemann organizer" in amphibians has been found in chick and mouse embryos, and it is now recognized to be a structure characteristic of all chordate embryos.

Other organizers with long-range effects on surrounding tissues have been identified in the developing vertebrate limb bud. Transplantation of a discrete patch of posterior tissue to an ectopic anterior site induces the formation of limb structures (digits, tendons, muscles) with mirror-image polarity to the normal anteroposterior order (Fig. 2.1b). By contrast, transplantation or removal of anterior tissue has no effect on limb development, suggesting that this posterior region of the limb bud, dubbed the zone of polarizing activity (ZPA), organizes anteroposterior (that is, the thumb-to-pinkie axis) polarity and limb formation.

Another organizer operates from the most distal tip of the limb bud, the apical ectodermal ridge (AER). Removal of this region truncates the limb and deletes distal elements (digits), whereas transplantation of the AER to an early limb bud can induce outgrowth of a duplicate limb (Fig. 2.1b).

One explanation for the long-range polarizing and inductive effects of the Spemann organizer, ZPA, and AER is that these tissues are sources of inducer molecules, or morphogens —that is, substances whose concentrations vary within a tissue and to which surrounding cells and tissues respond in a concentration-dependent manner. The response to a morphogen depends, then, on the distance of the responding tissue from the source. For example, if the ZPA is a source of a morphogen, then diffusion of this substance can establish a gradient of inducer concentration. Induction of different digit types depends on the morphogen concentration, with low levels of morphogen inducing anterior digits (thumb) and high levels inducing posterior digits (pinkie) (Fig. 2.1b).

Organizers have been demonstrated and morphogens postulated in insects as well as vertebrates. Ligature and cytoplasmic transplantation experiments first suggested that the anteroposterior axis of certain insect embryos is influenced by two organizing centers, one at

Figure 2.1

Organizers in animal embryos

Transplantation and ablation experiments have been used to investigate the long-range organizing activities of embryonic tissues. (a) The Spemann organizer. The dorsal blastopore lip of an early amphibian embryo can induce a second embryonic axis and embryo when transplanted to the ventral region of a recipient embryo. ( b) Limb organizers. The apical ectodermal ridge (AER) is required for formation of distal limb elements. Removal leads to loss of structures; transplantation to specific ectopic sites induces extra elements. The zone of polarizing activity (ZPA) organizes the anteroposterior pattern; transplantation to an ectopic site induces extra digits with reverse polarity. (c) Insect egg organizer. Ligation of the insect Euscelis embryo (marked by the gray line) early in development deletes the thorax and abdomen; later ligations leave more segments intact. However, transplantation of the posterior pole cytoplasm (marked by the black dot) into the anterior of a ligated embryo induces the formation of a complete embryo. This result demonstrates that the posterior cytoplasm has organizer activity. (d) Within insect segments, epithelial polarity is organized by signaling sources. Ablation of a segment boundary (indicated by the interruption of the black line) reorganizes segment polarity (indicated by the orientation of small black hairs).

Source: Parts a-c redrawn from Gilbert S. Developmental biology, 5th edn. Sunderland: Sinauer Associates, 1997; part redrawn d from Lawrence PA. The making of a fly. Oxford, UK: Blackwell Science, 1992.

each pole of the egg (Fig. 2.1c), that behave as sources of morphogens. Similarly, the polarity of cells within insect segments appears to be organized by signals that produce a graded pattern (Fig. 2.1d).

One difficulty with this picture of morphogen-producing organizers arises when we attempt to explain the boundaries of their range of influence. All of the cells in a growing embryo are in contact with other cells, so how is it that some parts respond and others do not? One explanation involves the concept of the morphogenetic field. Early embryologists demonstrated that some parts of developing animals, such as the forelimb field, could be transplanted to another site and still differentiate properly— that is, into a forelimb. In addition, if undetermined cells were introduced into the field, they could become incorporated into the limb. These transplantable, self-regulating fields are discrete physical units or modules of embryonic development. They form bounded domains within which specific programs of morphogenesis occur. The term "primary field" applies to the entire embryo before the axes are determined; the limbs, eyes, and other organs are termed "secondary fields," or organ primordia.

Secondary fields may be further subdivided into "tertiary fields," defined by physical or developmental boundaries. Compartments are one special type of subdivision. First demonstrated within the wing imaginal disc of the fruit fly Drosophila melanogaster, compartments are composed of populations of cells that do not intermix with cells outside the compartment.

Further progress in understanding the nature of organizers, morphogens, and fields stalled after their discovery and description in the first half of the 1900s. The impasse was ultimately broken by the discovery of genes whose products governed the activity of organizers, behaved as morphogens, and controlled the formation and identity of embryonic fields. These genes make up the "toolkit" for animal development.

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