13.1 The Genetic Approach to Development

For many decades, the study of embryonic development largely entailed the physical manipulation of embryos, cells, and tissues. Several key concepts were established about the properties of developing embryos through experiments in which one part of an embryo was transplanted into another part of the embryo. For example, the transplantation of a part of a developing amphibian embryo to another site in a recipient embryo was shown to induce the surrounding tissue to form a second complete body axis (Figure 13-2a). Similarly, transplantation of the posterior part of a developing chick limb bud to the anterior could induce extra digits, but with reversed polarity with respect to the normal digits (Figure 13-2b). These transplanted regions of the amphibian embryo and chick limb bud were termed organizers because of their remarkable ability to organize the development of surrounding tissues. The cells in the organizers were postulated to produce morphogens, molecules that induced various responses in surrounding tissue in a concentration-dependent manner.

Figure 13-2: Organizers in animal embryos
Figure 13-2: Transplantation experiments played a central role in early embryology and demonstrated the long-range organizing activity 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) In the developing chick vertebral limb bud, the zone of polarizing activity (ZPA) organizes pattern along the anteroposterior axis. Transplantation of the ZPA from a posterior to anterior position induces extra digits with reverse polarity.

Although these experimental results were spectacular and fascinating, further progress in understanding the nature of organizers and morphogens stalled after their discovery in the first half of the 1900s. It was essentially impossible to isolate the molecules responsible for these activities by using biochemical separation techniques. Embryonic cells make thousands of substances—proteins, glycolipids, hormones, and so forth. A morphogen could be any one of these molecules but would be present in minuscule quantities—one needle in a haystack of cellular products.

The long impasse in defining embryology in molecular terms was broken by genetic approaches—mainly the systematic isolation of mutants with discrete defects in development and the subsequent characterization and study of the gene products that they encoded. The genetic approach to studying development presented many advantages over alternative, biochemical strategies. First, the geneticist need not make any assumptions about the number or nature of molecules required for a process. Second, the (limited) quantity of a gene product is no impediment: all genes can be mutated regardless of the amount of product made by a gene. And, third, the genetic approach can uncover phenomena for which there is no biochemical or other bioassay.

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Drosophila

Mutational Analysis of Early Drosophila Development

The initial insights into the genetic control of pattern formation emerged from studies of the fruit fly Drosophila melanogaster. Drosophila development has proved to be a gold mine to researchers because developmental problems can be approached by the use of genetic and molecular techniques simultaneously.

The Drosophila embryo has been especially important in understanding the formation of the basic animal body plan. One important reason is that an abnormality in the body plan of a mutant is easily identified in the larval exoskeleton in the Drosophila embryo. The larval exoskeleton is a noncellular structure, made of a polysaccharide polymer called chitin that is produced as a secretion of the epidermal cells of the embryo. Each structure of the exoskeleton is formed from epidermal cells or cells immediately underlying that structure. With its intricate pattern of hairs, indentations, and other structures, the exoskeleton provides numerous landmarks to serve as indicators of the fates assigned to the many epidermal cells. In particular, there are many distinct anatomical structures along the anteroposterior (A–P) and dorsoventral (D–V) axes. Furthermore, because all the nutrients necessary to develop to the larval stage are prepackaged in the egg, mutant embryos in which the A–P or D–V cell fates are drastically altered can nonetheless develop to the end of embryogenesis and produce a mutant larva in about 1 day (see diagram). The exoskeleton of such a mutant larva mirrors the mutant fates assigned to subsets of the epidermal cells and can thus identify genes worthy of detailed analysis.

The development of the Drosophila adult body pattern takes a little more than a week (see diagram). Small populations of cells set aside during embryogenesis proliferate during three larval stages (instars) and differentiate in the pupal stage into adult structures. These set-aside cells include the imaginal disks, which are disk-shaped regions that give rise to specific appendages and tissues in each segment as the leg, wing, eye, and antennal disks. Imaginal disks are easy to remove for analysis of gene expression (see Figure 13-7).

Genes that contribute to the Drosophila body plan can be cloned and characterized at the molecular level with ease. The analysis of the cloned genes often provides valuable information on the function of the protein product—usually by identifying close relatives in amino acid sequence of the encoded polypeptide through comparisons with all the protein sequences stored in public databases. In addition, one can investigate the spatial and temporal patterns of expression of (1) an mRNA, by using histochemically tagged single-stranded DNA sequences complementary to the mRNA to perform RNA in situ hybridization, or (2) a protein, by using histochemically tagged antibodies that bind specifically to that protein.

Using Knowledge from One Model Organism to Fast-Track Developmental Gene Discovery in Others

With the discovery that there are numerous homeobox genes within the Drosophila genome, similarities among the DNA sequences of these genes could be exploited in treasure hunts for other members of the homeotic-gene family. These hunts depend on DNA base-pair complementarity. For this purpose, DNA hybridizations were carried out under moderate stringency conditions, in which there could be some mismatch of bases between the hybridizing strands without disrupting the proper hydrogen bonding of nearby base pairs. Some of these treasure hunts were carried out in the Drosophila genome itself, in looking for more family members. Others searched for homeobox genes in other animals, by means of zoo blots (Southern blots of restriction-enzyme-digested DNA from different animals), by using radioactive Drosophila homeobox DNA as the probe. This approach led to the discovery of homologous homeobox sequences in many different animals, including humans and mice. (Indeed, it is a very powerful approach for “fishing” for relatives of almost any gene in your favorite organism.) Now homologous genes are typically identified by computational searches of genome sequences (Chapter 14).

Overview of Drosophila development. The larva forms in 1 day and then undergoes several stages of growth during which the imaginal disks and other precursors of adult structures proliferate. These structures differentiate during pupation, and the adult fly hatches (eclosion) and begins the cycle again.

From the genetic viewpoint, there are four key questions concerning the number, identity, and function of genes taking part in development:

  1. Which genes are important in development?

  2. Where in the developing animal and at what times are these genes active?

  3. How is the expression of developmental genes regulated?

  4. Through what molecular mechanisms do gene products affect development?

To address these questions, strategies had to be devised to identify, catalog, and analyze genes that control development. One of the first considerations in the genetic analysis of animal development was which animal to study. Of the millions of living species, which offered the most promise? The fruit fly Drosophila melanogaster emerged as the leading genetic model of animal development because its ease of rearing, rapid life cycle, cytogenetics, and decades of classical genetic analysis (including the isolation of many very dramatic mutants) provided important experimental advantages (see the Model Organism box on Drosophila above). The nematode worm Caenorhabditis elegans also presented many attractive features, most particularly its simple construction and well-studied cell lineages. Among vertebrates, the development of targeted gene disruption techniques opened up the laboratory mouse to more systematic genetic study, and the zebrafish Danio rerio has recently become a favorite model owing to the transparency of the embryo and to advances in its genetic study. Among plants, Arabidopsis thaliana has played a similar role as Drosophila in illuminating fundamental mechanisms in plant development.

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Through systematic and targeted genetic analysis, as well as comparative genomic studies, much of the genetic toolkit for the development of the bodies, body parts, and cell types of several different animal species has been defined. We will first focus on the genetic toolkit of Drosophila melanogaster because its identification was a source of major insights into the genetic control of development; its discovery catalyzed the identification of the genetic toolkit of other animals, including humans.

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