18 Genetic Control of Development

Embryonic development (embryogenesis) is the process by which the embryo forms and develops. In mammals, the term refers chiefly to early stages of prenatal development, whereas the terms fetus and fetal development describe later stages.

Embryonic development starts with the fertilization of the egg cell (ovum) by a sperm cell, (spermatozoon). Once fertilized, the ovum is referred to as a zygote, a single diploid cell. The zygote undergoes mitotic divisions with no significant growth (a process known as cleavage) and cellular differentiation, leading to development of a multicellular embryo.

Evolutionary developmental biology (informally, evo-devo) is a field of biological research that compares the developmental processes of different organisms to infer the ancestral relationships between them and how developmental processes evolved.

The field grew from 19th-century beginnings, where embryology faced a mystery: zoologists did not know how embryonic development was controlled at the molecular level. Charles Darwin noted that having similar embryos implied common ancestry, but little progress was made until the 1970s. Then, recombinant DNA technology at last brought embryology together with molecular genetics. A key early discovery was of homeotic genes that regulate development in a wide range of eukaryotes.

The field is characterised by some key concepts which took evolutionary biologists by surprise. One is deep homology, the finding that dissimilar organs such as the eyes of insects, vertebrates and cephalopod molluscs, long thought to have evolved separately, are controlled by similar genes such as pax-6, from the evo-devo gene toolkit. These genes are ancient, being highly conserved among phyla; they generate the patterns in time and space which shape the embryo, and ultimately form the body plan of the organism. Another is that species do not differ much in their structural genes, such as those coding for enzymes; what does differ is the way that gene expression is regulated by the toolkit genes. These genes are reused, unchanged, many times in different parts of the embryo and at different stages of development, forming a complex cascade of control, switching other regulatory genes as well as structural genes on and off in a precise pattern. This multiple pleiotropic reuse explains why these genes are highly conserved, as any change would have many adverse consequences which natural selection would oppose.

New morphological features and ultimately new species are produced by variations in the toolkit, either when genes are expressed in a new pattern, or when toolkit genes acquire additional functions. Another possibility is the Neo-Lamarckian theory that epigenetic changes are later consolidated at gene level, something that may have been important early in the history of multicellular life.

18.1 The control of body structure

18.1.1 Deep homology

Roughly spherical eggs of different animals give rise to extremely different bodies, from jellyfish to lobsters, butterflies to elephants. Many of these organisms share the same structural genes for body-building proteins like collagen and enzymes, but biologists had expected that each group of animals would have its own rules of development. The surprise of evo-devo is that the shaping of bodies is controlled by a rather small percentage of genes, and that these regulatory genes are ancient, shared by all animals. The giraffe does not have a gene for a long neck, any more than the elephant has a gene for a big body. Their bodies are patterned by a system of switching which causes development of different features to begin earlier or later, to occur in this or that part of the embryo, and to continue for more or less time.

The puzzle of how embryonic development was controlled began to be solved using the fruit fly Drosophila melanogaster as a model organism. The step-by-step control of its embryogenesis was visualized by attaching fluorescent dyes of different colours to specific types of protein made by genes expressed in the embryo. A dye such as green fluorescent protein, originally from a jellyfish, was typically attached to an antibody specific to a fruit fly protein, forming a precise indicator of where and when that protein appeared in the living embryo.

Using such a technique, in 1994 Walter Gehring found that the pax-6 gene, vital for forming the eyes of fruit flies, exactly matches an eye-forming gene in mice and humans. The same gene was quickly found in many other groups of animals, such as squid, a cephalopod mollusc. Biologists including Ernst Mayr had believed that eyes had arisen in the animal kingdom at least 40 times, as the anatomy of different types of eye varies widely. For example, the fruit fly’s compound eye is made of hundreds of small lensed structures (ommatidia); the human eye has a blind spot where the optic nerve enters the eye, and the nerve fibres run over the surface of the retina,so light has to pass through a layer of nerve fibres before reaching the detector cells in the retina, so the structure is effectively “upside-down”; in contrast, the cephalopod eye has the retina, then a layer of nerve fibres, then the wall of the eye “the right way around”. The evidence of pax-6, however, was that the same genes controlled the development of the eyes of all these animals, suggesting that they all evolved from a common ancestor. Ancient genes had been conserved through millions of years of evolution to create dissimilar structures for similar functions, demonstrating deep homology between structures once thought to be purely analogous. This has caused a radical revision of the meaning of homology in evolutionary biology.

18.2 Gene toolkit

A small fraction of the genes in an organism’s genome control the organism’s development. These genes are called the developmental-genetic toolkit. They are highly conserved among phyla, meaning that they are ancient and very similar in widely separated groups of animals. Differences in deployment of toolkit genes affect the body plan and the number, identity, and pattern of body parts. Most toolkit genes are parts of signalling pathways: they encode transcription factors, cell adhesion proteins, cell surface receptor proteins and signalling ligands that bind to them, and secreted morphogens that diffuse through the embryo. All of these help to define the fate of undifferentiated cells in the embryo. Together, they generate the patterns in time and space which shape the embryo, and ultimately form the body plan of the organism. Among the most important toolkit genes are the Hox genes. These transcription factors contain the homeobox protein-binding DNA motif, also found in other toolkit genes, and create the basic pattern of the body along its front-to-back axis. Hox genes determine where repeating parts, such as the many vertebrae of snakes, will grow in a developing embryo or larva. Pax-6, already mentioned, is a classic toolkit gene. Homeobox genes are also found in plants, implying they are common to all eukaryotes.

The protein products of the regulatory toolkit are reused not by duplication and modification, but by a complex mosaic of pleiotropy, being applied unchanged in many independent developmental processes, giving pattern to many dissimilar body structures. The loci of these pleiotropic toolkit genes have large, complicated and modular cis-regulatory elements. For example, while a non-pleiotropic rhodopsin gene in the fruit fly has a cis-regulatory element just a few hundred base pairs long, the pleiotropic eyeless cis-regulatory region contains 6 cis-regulatory elements in over 7000 base pairs. The regulatory networks involved are often very large. Each regulatory protein controls “scores to hundreds” of cis-regulatory elements. For instance, 67 fruit fly transcription factors controlled on average 124 target genes each. All this complexity enables genes involved in the development of the embryo to be switched on and off at exactly the right times and in exactly the right places. Some of these genes are structural, directly forming enzymes, tissues and organs of the embryo. But many others are themselves regulatory genes, so what is switched on is often a precisely-timed cascade of switching, involving turning on one developmental process after another in the developing embryo.

Such a cascading regulatory network has been studied in detail in the development of the fruit fly embryo. The young embryo is oval in shape, like a rugby ball. A small number of genes produce messenger RNAs that set up concentration gradients along the long axis of the embryo. In the early embryo, the bicoid and hunchback genes are at high concentration near the anterior end, and give pattern to the future head and thorax; the caudal and nanos genes are at high concentration near the posterior end, and give pattern to the hindmost abdominal segments. The effects of these genes interact; for instance, the Bicoid protein blocks the translation of caudal’s messeger RNA, so the Caudal protein concentration becomes low at the anterior end. Caudal later switches on genes which create the fly’s hindmost segments, but only at the posterior end where it is most concentrated.

The Bicoid, Hunchback and Caudal proteins in turn regulate the transcription of gap genes such as giant, knirps, Krüppel, and tailless in a striped pattern, creating the first level of structures that will become segments. The proteins from these in turn control the pair-rule genes, which in the next stage set up 7 bands across the embryo’s long axis. Finally, the segment polarity genes such as engrailed split each of the 7 bands into two, creating 14 future segments.

This process explains the accurate conservation of toolkit gene sequences, which has resulted in deep homology and functional equivalence of toolkit proteins in dissimilar animals (seen, for example, when a mouse protein controls fruit fly development). The interactions of transcription factors and cis-regulatory elements, or of signalling proteins and receptors, become locked in through multiple usages, making almost any mutation deleterious and hence eliminated by natural selection.

18.3 Hox genes

Hox genes, a subset of homeobox genes, are a group of related genes that specify regions of the body plan of an embryo along the head-tail axis of animals. Hox proteins encode and specify the characteristics of ‘position’, ensuring that the correct structures form in the correct places of the body. For example, Hox genes in insects specify which appendages form on a segment (e.g. legs, antennae, and wings in fruit flies), and Hox genes in vertebrates specify the types and shape of vertebrae that will form. In segmented animals, Hox proteins thus confer segmental or positional identity, but do not form the actual segments themselves.

The Hox genes are so named because mutations in them cause homeotic transformations (the transformation of one organ into another). Homeotic transformations were first identified and studied by William Bateson in 1894, who coined the term “homeosis”. After the rediscovery of Mendel’s genetic principles, Bateson and others realized that some examples of homeosis in floral organs and animal skeletons could be attributed to variation in genes.

Definitive evidence for a genetic basis of some homeotic transformations was obtained by isolating homeotic mutants. The first homeotic mutant was found by Calvin Bridges in Thomas Hunt Morgan’s laboratory in 1915. This mutant shows a partial duplication of the thorax and was therefore named Bithorax (bx). It transforms the third thoracic segment (T3) toward the second (T2). Bithorax arose spontaneously in the laboratory and has been maintained continuously as a laboratory stock ever since.

The genetic studies by Morgan and others provided the foundation for the systematic analyses of Edward B. Lewis and Thomas Kaufman, which provided preliminary definitions of the many homeotic genes of the Bithorax and Antennapedia complexes, and also showed that the mutant phenotypes for most of these genes could be traced back to patterning defects in the embryonic body plan.

Ed Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus identified and classified 15 genes of key importance in determining the body plan and the formation of body segments of the fruit fly D. melanogaster in 1980. For their work, Lewis, Nüsslein-Volhard, and Wieschaus were awarded the Nobel Prize in Physiology or Medicine in 1995.

Drosophila melanogaster is an important model for understanding body plan generation and evolution. The general principles of Hox gene function and logic elucidated in flies applies to all bilaterian organisms, including humans. Drosophila, like all insects, has eight Hox genes. These are clustered into two complexes, both of which are located on chromosome 3. The Antennapedia complex (not to be confused with the Antp gene) consists of five genes: labial (lab), proboscipedia (pb), deformed (Dfd), sex combs reduced (Scr), and Antennapedia (Antp). The Bithorax complex, named after the Ultrabithorax gene, consists of the remaining three genes: Ultrabithorax (Ubx), abdominal-A (abd-A) and abdominal-B (abd-B).

(ref:hox)Homeobox (Hox) gene expression in the fruit fly Drosophila melanogaster.

Figure 18.1: (ref:hox)

An analogy for the Hox genes can be made to the role of a play director that calls which scene the actors should carry out next. If the play director calls the scenes in the wrong order, the overall play will be presented in the wrong order. Similarly, mutations in the Hox genes can result in body parts and limbs in the wrong place along the body. Like a play director, the Hox genes do not act in the play or participate in limb formation themselves.

Hox genes act at many levels within developmental gene hierarchies: at the “executive” level they regulate genes that in turn regulate large networks of other genes (like the gene pathway that forms an appendage). They also directly regulate what are called realisator genes or effector genes that act at the bottom of such hierarchies to ultimately form the tissues, structures, and organs of each segment. Segmentation involves such processes as morphogenesis (differentiation of precursor cells into their terminal specialized cells), the tight association of groups of cells with similar fates, the sculpting of structures and segment boundaries via programmed cell death, and the movement of cells from where they are first born to where they will ultimately function, so it is not surprising that the target genes of Hox genes promote cell division, cell adhesion, apoptosis, and cell migration.

The products of Hox genes are Hox proteins. Hox proteins are a subset of transcription factors, which are proteins that are capable of binding to specific nucleotide sequences on DNA called enhancers through which they either activate or repress hundreds of other genes. The same Hox protein can act as a repressor at one gene and an activator at another. The ability of Hox proteins to bind DNA is conferred by a part of the protein referred to as the homeodomain. The homeodomain is a 60-amino-acid-long DNA-binding domain (encoded by its corresponding 180-base-pair DNA sequence, the homeobox). This amino acid sequence folds into a “helix-turn-helix” (i.e. homeodomain fold) motif that is stabilized by a third helix. The consensus polypeptide chain is shown below:. Hox proteins often act in partnership with co-factors, such as PBC and Meis proteins encoded by very different types of homeobox gene .

Homeobox genes, and thus the homeodomain protein motif, are found in most eukaryotes. Hox genes, being a subset of homeobox genes, arose more recently in evolution within the animal kingdom or Metazoa. Within the animal kingdom, Hox genes are present across the bilateria (animals with a clear head-to-tail axis), and have also been found in Cnidaria such as sea anemones . This implies that Hox genes arose over 550 million years ago. In bilateria, Hox genes are often arranged in gene clusters, although there are many exceptions where the genes have been separated by chromosomal rearrangements . Comparing homeodomain sequences between Hox proteins often reveals greater similarity between species than within a species; this observation led to the conclusion that Hox gene clusters evolved early in animal evolution from a single Hox gene via tandem duplication and subsequent divergence, and that a prototypic Hox gene cluster containing at least seven different Hox genes was present in the common ancestor of all bilaterian animals.

In most bilaterian animals, Hox genes are expressed in staggered domains along the head-to-tail axis of the embryo, suggesting that their role in specifying position is a shared, ancient feature . The functional conservation of Hox proteins can be demonstrated by the fact that a fly can function to a large degree with a chicken Hox protein in place of its own. So, despite having a last common ancestor that lived over 550 million years ago, the chicken and fly version of the same Hox gene are similar enough to target the same downstream genes in flies.