Comparative Genomics and the Evolution of Ani

mal Diversity

Chapter 19

• There are 25 different animal phyla;each phylum represents a basic type of animal (f19-1).Where did all this evolutionary diversity come from?The systematic comparison of different animal genmes offers the promise of identifying the genetic basis for diversity.

TOPIC 1:• MOST ANIMALS HAVE E

SSENTIALLY THE SAME GENES

• Comparion of the currently avaible genomes reveals one particularly striking feature:different animals share essentially the same genens. With very few exceptions, just about every human gene has a clear counterpart in the mouse genime. In other words, no new genes were "invented"during the 50 million years of evolutionary divergence that separate mice and humans from their last share ancestor over 400million years ago. Yet,the two genomes contain the same number of genes, and most of these genes-more than three quarters-can be unambiguously alignes.

• The genetic conservation seen among vertebrates extends to the humble sea squirt, Ciona intestinalis. It contains half the number of genes group more than 500 million years ago. Nonetheless, nearly twothirds of the protein coding genes in sea squirts contain a clear, recognizable counterpart in vertebrates. Moreover the increase in gene number seen in vertebrates is primarily due to the duplicationof genes already present in the sea squirt.

• The genetic conservation seen among chordates appears to extend to other phyla. As seen for the sea squirt, increase in gene number in vertebrates is primarily due to the duplication of genes already present ub the ecdyszoans rather than the invertion of entirsly new genes.

How Does Gene Duplication Give Rise to Biological Diversity?

• The increace in gene number seen in vertebrates is largely due to gene duplication.

• There are two ways this can happen.

• First, the conventional view is that an ancestral gene produces nultiple genes via duplication, and the coding regions of the new genes undergo mutation.

• The second way that duplicated genes can generate diversity has been rather neglected until very recently. According to this model, the duplicated genes do not necessarily take on new functions, but instead acquire new regulatory DNA sequences.

• Thus, we have two models for how duplicated genes can create diversity. According to one scenario, the function of the gene is modified, through mutation of the coding sequence. According to the other scenario, the two genes are exoressed in different patterns within the organism. In some cases both mechanisms operate.

TOPIC 2:

•THREE WAYS GENE EXPRESSION IS CHANGED DURING EVOLUTION

• Regulatory genes encode proteins that control the expression of other genes. Most often these proteins are transcription factors, but some influence other steps of gene expression instead. Of particular interest form the perspective of the current discussion seem to cause significant changes in anmal morphology. The ds\istinguishing characteristic of pattern determining genes is that they cause the correct structures to develop, but in the wrong place,when they are misexpressed during development.

• The major focus of this chapter is to describe how changes in the deployment or activities of these pattern determining genes produce diversity during evolution.

• there are three major strategies for altering the activites of pattern determining genes.

• 1.A given pattern detemining gene can itself be expressed in a new pattern. This, in turn, will cause those genes whose expressed it control(so-called target genes)to acquire new patterns of expression.

• 2.The regulatory protein encoded by a pattern determining gene can acquire new functions, for example, a transcriptinal activation domain can be converted into a repression domain. Thus,a regulatory protein that was an activator of a set of genes might now repress them. note that, although this strategy involves a change in protein function, the evolutionary consequence is a result of changes in expression pattern of target genes.

• 3.Target genes of a given parttern determining gene can acquire new regulaory DNA sequences, and thus come under the control of a different regulatory gene. In this way, their pattern of expression is altered.

TOPIC 3:• EXPERIMENTAL MENIPULATIONS THAT ALTER ANIMAL MORPHOLOGY

• The first pattern determining gene was identified in Drosophila in the Morgan fly lab. A mutation called bxd cause a partial transformation of halteres into wings.

• Abnomal morphologies are obtained through each of the three mechanisms descibed above: altering the expression, function, and target of pattern determining genes.

Changes in Pax6 Expreesion Create Ectopic Eyes

• The most notorious pattern determining gene is Pax6, which controls eye development in most or all animal.

• Pax6 is normally expressed within developing eyes; but, when miseyes in the wrong tissues.

• Changes in the Pax6 expressiong pattern during evolutuion probably account for differences in the positioning of eyes in different animals.

• Evolutonary charges in the regulation of Pax6 expression have been more important for the creation of morphologically diverse eyes than have changes in Pax6 protein function. Thus, Pax6 genes from other animals also produce ectopic eyes when misexpressed in Drosophila.

• For example, fruit flies were engineered to misexpress the squid Pax6 gene. Extra eyes were obtained in the wings and legs, similar to those obtained when the Drosophila Pax6 was misexpressed. The flyand squid Pax6 protein share only 30%overall amino acid sequence indentity, yet they mediate similar activities in transgenic flies.

Changes in Antp Expression Transform Antennae into legs

• Asecond Drosophila pattern determining gene, Antp, controls the development of the middle segement of the thorax, the masothorax.

• Antp encodes a homeodomain regulatory protein that is nomally expressed in the mesothorax of the developing embryo.

• But, a dominant Antp mutation, cause by a chromosome inversion, brings the Antp protein coding sequence under the crotrol of a “foreign” regulatory DNA that mediates gene expression in head tissues, including the antennae.

• When misexpressed in the head, Antp cause a striking change in morphology:legs develop instead of antennae.

Importance of Protein Function: Interconversion of ftz and A

ntp• A second mechanism for evolutionary di

versity is changes in the sequence and functiong of the regulator proteins encoded by pattern determining genes.

• Consider two related pattern detemining genes in Drosophila, the segmentation gene ftz and the homeotic gene Antp.

• These genes are linked and arose from an ancient dupication event that predated the divergence of crustaceans and insects more than 400million years ago.

• The two cnoded proteins are related and contain very similar DNA-binding domains(homeodomains).

• Ftz-FtzF1 dimers recognize DNA sequences that are distinct from those bound by Antp-Exd dimers.

Subtle Changes in an Enhancer Sequence Can Produce New Patter

ns of Gene Expression• The third mechanism for evolutionary diver

sity is changes in the target enhancers that are regulated by pattern determining genes.In this case neither the pcpression pattern nor the functiong of the encoded regulatory protein is altered.

• The principle that changes in enchancers can rapidlly evolve new patterns of gene expression stems from the experimental manipulation of a 200 bp tissue specific enchancer that is activated only in the mesoderm.

• Sigle nucleotide substitutions that convert each site into an primal Dorsal binding site cause the modified enhancer to be activated in a broader pattern.

• Dorsal functons synergistically with another transcription factor Twist to activate gene expression in the neurogenic ectoderm.

• The modified enhancer now directs a broad pattern of gene expression in both the mesoderm and neurogenic ectoderm.

• Afew additional necleotide changes create binding siters for a zinc finger repressor,Snail.

• A modified enhancer contains optimal Dorsal sites, Twist activator, and Snail repressor sites,

The Misexpression of Ubx Changes the Morphology of

the Fruit Fly• The analysis of a Drosophila pattern determ

ining gene called Ubx illustrates all three principles of evolutiongary change: new patterns of gene expression are produced by changing the Ubx expressiong pattern, the encoded regulator protein, or its target enhancers.

• Ubx encodes a homeodomain regulatory protein that controls the development of the third thoracic segment, the metathorax.

• Ubx specifically represses the expression of genes that are requires for the development of the second thoracic segement, or mesothorax.

• Indeed, Antp is one of the genes that it regulates. This misexpression of Antp causes a transformation of the metathorax into a duplicated mesothorax.

• The expression of Ubx in the different tissues of the metathorax depends on regulatory sequences that encompass more than 80kb of genomic DNA.

• Amutation called Cbx disrupts this Ubx regulatory DNA without changing the Ubx protein coding region.

Changes in Ubx Function Modify the Morphology of Fruit Fly Embryos

• It is not currently known how Ubx functions as a repressor. Howover, the Ubx protein contains specific peptide squences that recruit repression complexes. One such peptide is composed of a stretch of alanine residues.

• Ubx normally functions as a repressor. Ii can be converted into an activator by fusing the Ubx DNA binding domainto the potent activation domain from the viral VP16 protein.

• The misexepression of he mesothoracic segments, not metathoracic segments an seen when the normal Ubx protein is misexepressed in engineered embryos.

• Thus, rather than behaving like the normal Ubx protein, the Ubx-VP16 fusion protein produces the same phenotype as that obtained with Antp.

TOPIC 4:•MORPHOLOGICAL CHANGES IN CRUSTACEANS AND INSECTS

• We now discuss how the three strtegies for altering the activities of pattern detemining genes can explain examples of natural morphological diversity found among different arthropods. The first two machanisms,changes in the expression and function of pattern detemining genes, can account for changes in limb morphology seen in certain crustaceans and insect.The third mechanism is changes in regulatory sequences.

Arthropods Are Remarkably Diverse

• The success of the arthropods derives, in part, from their modular architecture.

• These organisms are composed of a series of repeating body segments that can be modified in seemingly limitless ways.

Changes in Ubx Expression Explain Modifications in Limbs amon

g the Crustaceans• Crustaceans include most, but not all, of the

arthropods that swim.

• Slightly different patterns of Ubx expression are observedin branchiopods and isopods. These different expression patterns are correlated with the modification of the swimming limbs on the first thoracic segment of isopods.

What is the basis for the different patterns of Ubx exoression in iso

pods and branchiopods?• There are several possible explanations, but

the most likely one is that the Ubx regilatory DNA of isopods acquired mutations.

• In fact, there is a tight correlation between the absence of Ubx expression in the throax and the developmet of the feeding appendages in the different crustaceans.

Why Insects Lack Abdominal Limbs

• In inscts, Ubx and abd-A repress the expression of a critical gene that is required for the development of limbs, called Distalless(Dll).

• IN crustaceans, there are high levels of both Ubx and Dll in all 11thoracic segments. The Ubx protein has diverged between insects and crustaceans. Thus, Ubx represses Dll expression in the abdominal sedments of insects, but nut crustaceans.

Modification of Flight Limbs Might Arise from the Evolution of R

egulatory DNA SEquences

• In Drosophila, Ubx is expressed in the developing halteres where it functions as a repressor of wing development.

• It is likely that Ubx functions as a repressor of wing development in all dipteras.

• For example. In butterflies, the loss of Ubx in pacthes of cells in the hindwing causes them to be transformed into forewing structures. This observation suggests that the butterfly Ubx protein functions as a expressor that suppresses the development of forewings. Whil not proven, it is possible that the regulatory DNAs of the wing patterning genes have lost the Ubx binding sites. As a result, they are no longer repressed by Ubx in the developing hindwing.

TOPIC 5:

•GENOME EVOLUTION AND HUMAN ORIGINS

Humans Contain Surprisingly Few Genes

• Based on the logic that we have introduces in this chapter, we anticipate that higher vertebrates, such as humans, contain sophisticated mechanisms fir gene regulation in order to produce many paterns of gene expression. In other words, organismal complexity is not correlated with gene number, but instead depends in the number of gene expression patterns.

The Human Genome is very Similar o that of the Mouse and Virtu

ally Identical to the Chimp

• Mice and humans contain roughly he same number of genes-about 28,000 protein coding genes.

• The chimp and human genomes are even more highly conserved.

• Between mice and human, approximately 80%of these genes possessa clear and unique one-to-one sequence alignment with one another between the two species.

• Between the chimp and human,they vary by an average of just 2%squence divergence.

• By comparison, two sea squirts in the same population differ by more than 1%sequence divergence, while individuals from different populations exhibit as much as 2.5% sequence variation.

• We have seen that regulatory Dna evolve more repidly than proeins. Perhaps the limited sequence divergence between chimps and humans is sufficient to alter the activities of several key regulatory DNAs.

The Evolutionary Origins of Human Speech•

Speech depends on the precise coordination of the small muscles in our larynx and mouth. Reduced levels of a regulatory protein called FOXP2 cause severe defects in speech.

• The human form of the protein is slightly different from those present in mice and the primates. In particular, there are two amino acid residues at positions 303and 325 that are unique to human: thr to asn(T to N) at position 303and asn to ser (N to S) at possition 325. Perhaps these changes have altered the function of the human FOXP2 protein.

• Alternatively, changes in the expression patternor changes in FOXP2 target genes might br responsible for the ability of FOXP2 to promote speech in humans.

How FOXP2 Fosters Speech in Humans

• A combination of all three mechanisms,changes in the FOXP2 expression pattern, changes in its amino acid sequence, and changes in FOXP2target genes micht explain its emergence as an imprtant meditor of human speech.

• FOXP2 is just one example of a regulatory gene that underlies human speech. However, we have seen that fewer than 100 pattern detemining genes are sufficient to account for the morphological diversification of different arthropod goups. Perhaps a significantly smaller set can account for the acquisition of the language.