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H OX GENES AND THEIR ROLE IN THE EVOLUTION OF MORPHOLOGY

Development is the process through which an egg becomes an adult organism.

During this process, an organism’s genotype is expressed as a phenotype, and the latter is exposed to the action of natural selection. Studies of development are important to evolutionary biology for several reasons. First, changes in the genes controlling development can have major effects on the adult’s morphology, and thus it is thought that changes in developmental genes have driven large-scale evolutionary transformations.

These genes should thus explain how some hoofed mammals invaded the ocean, or how small, armored invertebrates evolved wings. Because of their major effects on morphology, developmental processes may also constrain evolutionary change, possibly preventing certain characters from evolving in certain lineages. Thus, for example, development may explain why there are no six-fingered tetrapods. And finally, an organism’s development may also contain clues about its evolutionary history, which can be used to disentangle relationships among different lineages. Then, comparisons among different lineages should provide answers to general questions such as: Does morphological evolution occur gradually or in big steps? Are there trends in evolution?

Why are some clades very diverse and some unusually sparse? How does evolution produce morphological novelties? Which are the genes and gene networks involved in morphological evolution? Are they shared among species? These and other questions are within the scope of the relatively new discipline Evolutionary Developmental biology (Evo-Devo) (GILBERT 2003).

1.5.1.ORIGIN OF THE BODY PLAN IN ANIMALS

Life during the first 3 billion years on the Earth consisted of single-celled organisms only. Multicellular animals arose from one of these single-celled organisms related to choanoflagellates, a group that originated ∼1 billion years ago. The most primitive living animal phyla are the sponges, forms of which have been found in Neoproterozoic fossils dating back 565 MYA. The Earth is now populated by 1-20 million animal species, probably <1% of all animal species that have ever existed, but strikingly all of their diversity was originated >540 MYA from a common bilaterally

symmetrical ancestor. These are indeed the most important milestones in early animal history: (i) the evolution of bilaterally symmetric animals, and (ii) the explosive radiation of these forms in the Cambrian period >500 MYA. The ‘Cambrian explosion’ signified a burst of biological creativity unprecedented in the Earth’s history. Many of these animals are now extinct, but those that remained established all of the basic body plans we see today. As a consequence of this ancient origin of today’s phyla, all living animals belong to a limited number of basic designs, referred to as Bauplan or ‘body plans’ (ERWIN et al.

1997).

1.5.2.HOMEOTIC GENES AND THE DISCOVERY OF THE HOMEOBOX

Early interest in the development of body pattern was largely motivated by curiosity on the origin of the diversity of living species. As early as 1859, Darwin noticed a common feature of many creatures: the repetition of elements along the length of the body, today known as segmentation (DARWIN 1859). Some years later, in 1894, W. Bateson described one of the most extraordinary phenotypes ever described in animals that affected indeed the patterning of the body plan and body parts. He catalogued several cases in nature in which one normal body part was replaced with another, such as a leg in place of an antenna in arthropods, or a thoracic vertebra in place of a cervical vertebra in vertebrates, and termed this phenomenon homeosis (BATESON 1894). In 1923, C. B.

Bridges and T. H. Morgan showed that homeosis was heritable in flies, and that whatever was responsible for such inheritance was coded in the fly’s third chromosome (BRIDGES

and MORGAN 1923). But it was not until half a century later that the genetic basis of homeosis could be unveiled (GARCIA-BELLIDO 1975; LEWIS 1978). E. B. Lewis studied the relationship genotype-phenotype of homeotic mutations at the fly’s Bithorax Complex of genes (BX-C), and reported that this cluster consisted of various genetic elements and that mutations mapped in an order that corresponded to the anteroposterior (A/P) body axis of the embryo (spatial collinearity). He already predicted that the identity of an individual body segment was produced by a combination of different BX-C genes, and that these were activated in response to an A/P gradient. Shortly later, T. C. Kaufman’s lab described a second homeotic complex affecting anterior regions of the fly’s body, the Antennapedia Complex (ANT-C), and made similar predictions to those by Lewis (KAUFMAN et al. 1980; LEWIS et al. 1980a; LEWIS et al. 1980b).

Two teams, one led by M. P. Scott and the other including W. McGinnis, M. S.

Levine and W. J. Gehring, showed in 1984 that genes involved in homeotic mutations — called homeotic genes— share a highly conserved sequence of 180 nucleotides (LAUGHON

and SCOTT 1984; MCGINNIS et al. 1984; SCOTT and WEINER 1984). This sequence — called homeotic box or homeobox— codes for a 60 amino acid protein domain —the homeodomain— that binds particular sequences in the DNA through a ‘helix-turn-helix’

structure (Figure 11). This highly conserved sequence, which is not exclusive of homeotic genes, was soon used in homology searches to pull out more homeobox-containing genes, which could be easily identified in such disparate organisms as hydra (SCHUMMER et al.

1992; GAUCHAT et al. 2000), nematodes (WANG et al. 1993), leech (NARDELLI -HAEFLIGER and SHANKLAND 1992), amphioxus (HOLLAND et al. 1992), zebrafish

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Helix 1 Helix 2 Helix 3

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(A) (B)

(C)

Homeodomain

Figure 11

Structure and conservation of the homeodomain

(A,B) The structure of the homeodomain bound to DNA is shown as ribbon models. (C) Sequence logo of the homeodomain and surrounding amino acids for a set of ortholog and paralog Hox genes in several species of vertebrates. The overall height of the stacked amino acids indicates sequence conservation at each position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. [Figure modified from LYNCH et al. (2006).]

(NJOLSTAD and FJOSE 1988; NJOLSTAD et al. 1988) or humans (ACAMPORA et al. 1989).

Indeed, this motif has been found in >200 non-homeotic genes in vertebrates and ∼100 in invertebrates, all coding for DNA-binding proteins often involved in different aspects of animal development (NAM and NEI 2005).

1.5.3.THE HOX GENE COMPLEX

One of the most important biological discoveries of the past two decades is that most animals, no matter how divergent in form, share specific families of genes that regulate major aspects of body pattern, such as the determination of anterior versus posterior, or dorsal versus ventral (MCGINNIS 1994; ERWIN et al. 1997). The discovery of this common genetic ‘toolkit’ for animal development unveiled conserved molecular, cellular and developmental processes that were previously hidden by disparate anatomies.

It also focused the study of the genetic basis of animal diversity on how the number, regulation and function of genes within the toolkit have changed over the course of animal history (DE ROSA et al. 1999; CARROLL et al. 2001).

Hox genes are an essential class of homeobox-containing genes involved in the specification of regional identities along the A/P body axis of the developing embryo (LEWIS 1978; KAUFMAN et al. 1980; MCGINNIS and KRUMLAUF 1992). They play as transcription factors (TFs) that modulate levels of expression of other genes located downstream in the regulatory cascade of development. Additionally, Hox genes have the following particularities: (i) they are usually clustered together in complexes (LEWIS 1978;

KAUFMAN et al. 1980) (but see NEGRE and RUIZ (2007)), (ii) they are arranged in the chromosome in an order that corresponds to the A/P body axis of the embryo (spatial collinearity) (MCGINNIS and KRUMLAUF 1992) (Figure 12), (iii) they are expressed also in a temporal order that match their physical order on the chromosome (temporal collinearity) (DUBOULE 1994; KMITA and DUBOULE 2003), and (iv) they are universal in animals, suggesting that they are evolutionarily related (MCGINNIS and KRUMLAUF 1992; SLACK et al. 1993; GARCIA-FERNANDEZ 2005) (Box 3).

1.5.4.NEW FUNCTIONS FOR SOME INSECT HOX GENES

The fact that all animal species share the basic Hox gene content suggests that

Figure 12

Conservation of the genomic structure and expression patterns of Hox genes

Hox gene complexes and expression patterns of Drosophila (top) and mammals (bottom).

The hypothetical gene complement of the ancestral Hox cluster is shown in the middle.

[Figure from VERAKSA et al.

(2000).]

Box 3 Origin of the Hox gene complex

The reconstruction of the evolutionary history of the Hox gene family is a key issue to understand the evolution of body plans in bilateria and the relationships between genetic complexity and morphology. The Hox gene complex probably arose by tandem duplications and posterior divergence from an ancestral Hox gene (DE ROSA et al. 1999; FERRIER and MINGUILLON 2003;

GARCIA-FERNANDEZ 2005). These duplicated genes have usually remained together in the genome; now, all metazoans show different configurations of the ancestral Hox gene cluster (Figure 13). Cnidarians, the most ancient animal phyla, have only one anterior and one posterior

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Hox genes. Before the bilaterian radiation, the Hox groups Hox1-Hox5 were established and fixed.

Then, early expansion of the Hox complex at the base of the bilaterian lineage generated many of the central Hox genes: Hox6-Hox13 in deuterostomes, Ubx and Abd-B in ecdysozoans, and Lox5, Lox2, Lox4, Post1 and Post2 in lophotrochozoans. During early vertebrate evolution, the entire complex was duplicated; e.g. tetrapods have 4 complexes summing a total of 39 Hox genes. Teleost fish have undergone an additional round of tetraploidization, creating the seven Hox complexes found in zebrafish and at least five in Fugu.

Box 3 (continued)

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Figure 13

Evolution of metazoan Hox genes

The relative timing of Hox duplication events is mapped onto a phylogenetic tree of Metazoan phyla (left), as deduced from the distribution of Hox genes in the different species (right). Common ancestors: M, metazoan; B, bilaterian; D, deuterostome; E, stem ecdysozoan; L, stem lophotrochozoan; P, protostome. Striped colors indicate fast-evolving Hox-derived genes. [Figure modified from CARROLL et al. (2001).]

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evolutionary changes in gene regulation —and not gene content— might have been the key in shaping large-scale changes in animal body plans and body parts. In particular, differences in the spatial and temporal regulation of Hox genes have been correlated to changes in axial morphology in many comparative analyses of Hox gene expression in arthropods, annelids and vertebrates (CASTELLI-GAIR et al. 1994; MANN 1994; CASTELLI -GAIR and AKAM 1995). Such differences in Hox expression domains during evolution are most probably be explained by changes in the cis-regulatory regions of Hox genes and/or changes in the expression of their trans-acting regulators (BELTING et al. 1998; DOEBLEY

and LUKENS 1998; WEATHERBEE and CARROLL 1999). The logic behind this statement is related to the pleiotropy of mutations. In general, it is expected that mutations with far-reaching effects will have more deleterious consequences on organismal fitness and will be a less common source of variation than mutations with less widespread effects. While mutations in a single cis-regulatory element affect gene expression only in the domain governed by that element, changes in the coding region of a TF may directly affect all of the genes it regulates, and thus, have broad effects in the developing organism (CARROLL

2005). On these grounds, any modification in a Hox gene pathway might produce changes in animal morphology, and the extent of these morphological changes may correlate with the pleiotropy of the modification (GELLON and MCGINNIS 1998). Notably, the genome tetraploidization events at the base of vertebrates and further genome duplications in fish might have been responsible for the huge morphological diversity in these lineages. Yet, the basic components and the biochemical functions of the encoded proteins are surprisingly conserved across hundreds of millions of years.

However, some members of the insect Hox complex have shown a relaxation in their constraint and have evolved new functions. In winged insects, including Drosophila, Hox3 (STAUBER et al. 1999; STAUBER et al. 2002; BONNETON 2003) and fushi tarazu (ftz) (TELFORD 2000) have lost their Hox-like role in regulating regional identity along the A/P body axis and acquired new functions in animal development (HUGHES et al. 2004). Hox3 gained a novel extraembryonic function, and underwent two consecutive duplications that gave rise to bicoid (bcd), zerknüllt (zen) and zerknüllt-related (zen2) (hereafter called Hox-derived genes) (Figure 14). The first duplication took place in the cyclorrhaphan fly lineage and gave rise to zen and bcd (STAUBER et al. 1999; STAUBER et al. 2002).

Afterwards, but before the Drosophila radiation, zen went through a second duplication

that gave birth to zen2 (NEGRE et al. 2005). The gene zen is expressed in extraembryonic tissue during early development in several different insects, indicating that the shift in zen function occurred fairly early in insect evolution (PANFILIO and AKAM 2007). The gene bcd codes for an important morphogen that establishes A/P polarity during oogenesis (BERLETH et al. 1988). zen2 has the same expression pattern of zen, although its function is unknown. These duplicated copies of Hox3 may have experienced a period of accelerated evolution following duplication during which they may have adopted part of the functions of their parental gene (subfunctionalization) and/or acquired new functions (neofunctionalization) (LYNCH and CONERY 2000; LYNCH and FORCE 2000; LONG et al.

2003; ZHANG 2003). This rapid evolution has already been demonstrated for the homeodomains of all of these genes (and especially that of zen2) (DE ROSA et al. 1999), which might have facilitated the rapid functional evolution of these genes in the development of insects.

Diptera Brachycera

Eremoneura Cyclorrhapha

Nematocera

Schizophora

Drosophilasps.

Aschiza

Megaselia abdita

Empidoidea

Empis livida

lower Brachycera

Haemotopota pluvialis

Nematocera

Clogmia albipunctata

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1

*

2

Hox3-zen zen bcd zen2 zen bcd

Hox3

Figure 14

Origin of bcd, zen and zen2 from Hox3

The composition of Hox3-related genes is shown for the major groups of diptera and for their inferred ancestor. Duplications are shown as asterisks. See text for details. (Note that the image for the Aschiza group corresponds to Megaselia scalaris).