The Genetic Toolkit

Animal genomes contain thousands of genes. Many of these genes encode proteins that function in essential processes in all cells in the body (for example, metabolism, biosynthesis of macromolecules) and are often referred to as "housekeeping genes." Other genes encode proteins that carry out specialized functions in particular cells or tissues within the body (for example, oxygen transport, immune defense) or, to extend the housekeeping metaphor, in specific "rooms" in the "house." But here we are interested in a different set of genes, those whose products govern the construction of the house—the toolkit that determines the overall body plan and the number, identity, and pattern of body parts.

Toolkit genes have generally first been identified based on the catastrophes or monstrosities that arise when they are mutated. Two sources of toolkit gene mutations exist. The first source comprises rare, spontaneous mutations that arise in laboratory populations of model animals (for example, fruit flies, mice). The second source consists of mutations induced at random by treatment with mutagens (such as chemicals or radiation) that greatly increase the frequency of damaged genes throughout the genome. Elegant refinements of the latter approach, particularly in Drosophila melanogaster, have enabled systematic searches for members of the genetic toolkit for animal development.

Intensive screens for genes that affect the formation of the insect embryonic and adult body patterns and analysis of the structure, function, and expression of the proteins they encode have revealed several critical features of the genetic toolkit for development:

1. The toolkit is composed of a small fraction of all genes Only a small subset of the entire complement of genes in the genome affects development in discrete ways.

2. Most toolkit genes encode either transcription factors or components of signaling pathways Therefore, toolkit genes generally act, directly or indirectly, to control the expression of other genes.

3. The spatial and temporal expression of toolkit genes is often closely correlated with the regions of the animal in which the genes function.

4. Toolkit genes can be classified according to the phenotypes caused by their mutation Similar mutant phenotypes often reflect genes that function in a single developmental pathway. Distinct pathways exist for the generation of body axes, for example, and for the formation and identity of fields.

5. Many toolkit genes are widely conserved among different animal phyla.

Because the discovery of the insect toolkit has offered a direct path to identifying developmental genes in other animals, we will begin our inventory of the genetic toolkit for animal development by considering Drosophila melanogaster.

The Drosophila toolkit

Classifying genes according to their developmental function

Many mutations have been isolated that alter the embryonic and/or adult body pattern of Drosophila. It has proved very useful to group the genes affected by these mutations into several categories based on the nature of mutant phenotypes. Most toolkit genes can be classified according to their function in controling the identity of fields (for example, different segments and appendages), the formation of fields (for example, organs and appendages), the formation of cell types (for example, muscle and neural cells), and the specification of the primary body axes.

We begin by considering the genes that control the identity of segments and appendages. This choice is made partly for historical reasons and partly to follow a hierarchical approach. The genes controlling field identity were among the very first toolkit members discovered, and their identification inspired much of the genetic and molecular biological innovations that catalyzed the discovery of the rest of the toolkit. In addition, they are among the most globally acting developmental genes that affect animal form. Next, we discuss genes that control the patterning of fields at progressively finer scales, from the formation of entire fields, to compartments within fields, and then to differentiated cell types.

Homeotic genes and segmental identity

Among the most fascinating kinds of abnormalities to be described in animals are those in which one normal body part is replaced with another. Bateson catalogued several oddities of this nature, coining the term homeotic to describe such transformations. Among the most common homeotic variants noted by Bateson were arthropods in which one type of appendage formed in the position of another, such as a leg in place of an antenna (Fig. 2.2a), and vertebrates in which one type of vertebra or rib replaced another, such as a thoracic vertebra in place of a cervical vertebra (Fig. 2.2b).

Figure 2.2

Homeotic transformations in an arthropod and a vertebrate

(a) Homeosis in the insect Cimbexaxillaris, with the left antenna being transformed toward leg identity. (b) Homeosis in a frog. The middle specimen is normal. The specimen on the left has processes emanating from the atlas (top of vertebral column). The specimen on the right has an extra set of vertebrae.

Source: Bateson W. Materials for the study of variation. London: Macmillan, 1894.

Figure 2.2

Homeotic transformations in an arthropod and a vertebrate

(a) Homeosis in the insect Cimbexaxillaris, with the left antenna being transformed toward leg identity. (b) Homeosis in a frog. The middle specimen is normal. The specimen on the left has processes emanating from the atlas (top of vertebral column). The specimen on the right has an extra set of vertebrae.

Source: Bateson W. Materials for the study of variation. London: Macmillan, 1894.

Intriguing as Bateson's specimens were, most were one-of-a kind museum pieces in which only one member of a bilateral pair of structures was affected. To carry out a thorough investigation of the phenomenon of homeosis and its genetics, researchers required mutants that would breed true in subsequent generations. In 1915, Calvin Bridges isolated a spontaneous mutation in Drosophila, dubbed bithorax, in which part of the haltere (the posterior flight appendage in flies) was transformed into wing tissue. The haltere and wing are serially homologous appendages, so the bithorax mutation causes the partial transformation of the identity of a structure on the third thoracic segment (the haltere) into its serial homolog found on the second thoracic segment (the wing). A more complete transformation of the entire haltere into a wing can occur if additional mutations are combined with bithorax, producing a four-winged fly (Fig. 2.3).

In the following decades, several more homeotic mutants were identified in Drosophila, and in other insects as well. All of these homeotic mutations transform the identities of segments and their associated structures into those of other segments. For example, certain Antennapedia mutations cause the transformation of antennae into legs (Fig. 2.3), which are

Figure 2.3

Homeotic mutants of Drosophila melanogaster

(top) Normal fly with one pair of wings on T2 and halteres on T3. (middle) Triple mutant for three mutations in the Ultrabithorax gene abolishes Ubx function in the posterior thorax and causes the appearance of an extra set of wings (transformation of T3 ^ T2 identity). (bottom) Antennapedia mutant in which the antennae are transformed into legs.

also serial homologs. The direction of the homeotic transformations depends on whether a mutation causes a loss of homeotic gene function where the gene normally acts, or a gain of homeotic gene function in places where the homeotic gene does not normally act. For example, Ultrabithorax (Ubx) acts in the haltere to promote haltere development and repress wing development. Loss-of-function mutations in Ubx transform the haltere into a wing. Dominant mutations that cause Ubx to gain function in the wing transform that structure into a haltere. Similarly, the antenna-to-leg transformations of Antennapedia mutants reflect a dominant gain of Antennapedia gene function in the antenna.

The fascination with homeotic mutants stems from two issues. First, it is startling that a single gene mutation could change entire developmental pathways so dramatically in a complex animal. Second, it is curious that the structure formed in the mutant is a well-developed likeness of another body part.

More detailed understanding of homeotic gene function was made possible by some particularly ingenious methods for analyzing the effects of mutations on the behavior of a group of cells in otherwise normal (or "wild-type") tissues. That is, rather than being limited to examining the effect of homeotic mutations on whole animals, the behavior of clones of mutant cells could be observed within otherwise normal animals (Fig. 2.4). This technique

Figure 2.4

Cell autonomy of homeotic mutations

The Drosophila wing and haltere have different pattern elements, such as the occurrence of sensory bristles at the leading edge of the wing (red). Clones lacking Ubx function in the haltere form wing structures (for example, the sensory bristles shown in red) in positions corresponding to those of the wing.

Source: Redrawn from Lawrence PA. The making of a fly. Oxford, UK: Blackwell Scientific, 1992.

Figure 2.4

Cell autonomy of homeotic mutations

The Drosophila wing and haltere have different pattern elements, such as the occurrence of sensory bristles at the leading edge of the wing (red). Clones lacking Ubx function in the haltere form wing structures (for example, the sensory bristles shown in red) in positions corresponding to those of the wing.

Source: Redrawn from Lawrence PA. The making of a fly. Oxford, UK: Blackwell Scientific, 1992.

was used to determine that the effects of homeotic mutations generally remain limited to cells with mutant genotypes; such behavior is termed cell autonomous. Thus a patch of cells in the haltere that lacks Ubx function forms wing tissue, even when it is surrounded by normal haltere cells (Fig. 2.4). This finding suggested that homeotic genes act within cells to select their developmental fate. Homeotic genes, and other genes with analogous functions in controlling cell fate, are therefore known as selector genes.

Although homeotic genes were first identified through spontaneous mutations affecting adult flies, they are required throughout most of Drosophila development to determine segmental identity. Systematic screening for homeotic genes led to the identification of eight linked genes, collectively referred to asHox genes, that affect the specification of particular segment identities in the developing Drosophila embryo, larva, and adult. In addition to Ultrabithorax (Ubx) and Antennapedia (Antp), they include labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), abdominal-A (abd-A), and Abdominal-B (Abd-B). Generally, the complete loss of any Hox gene function causes transformations of segmental identity and is lethal in early development. The spontaneous homeotic mutants found in viable adults are caused by partial loss of gene function or are dominant such that in heterozygotes normal gene function is provided by the wild-type allele.

One of the most intriguing features of these Hox genes is that they are linked in two gene complexes in Drosophila, the Bithorax and Antennapedia Complexes; each complex contains several distinct homeotic genes. Furthermore, the order of the genes on the chromosome and within the two complexes corresponds to the rostral (head) to caudal (rear) order of the segments that they influence, a relationship described as colinearity (Fig. 2.5).

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