developmental mechanisms of

evolutionary change

When Charles Darwin consulted his friend Thomas Huxley concerning the origins of variation, Huxley told him that the differences between

organisms could be traced to differences in their development. He said these differences “result not so much of the development of new parts

as of modification of parts already existing and common to both the divergent types”.

This is a major tent of evolutionary developmental biology also known as “evo-devo” which views evolution as result of changes in

development.

If development is the change of gene expression and cell position over time evolution is the change of development over time.

The two major views on the origin of species in the nineteenth century

1. Conditions of existence: This view championed by Georges Cuvier and Charles Bell focussed on the differences between species that allowed each to adapt to its environment. Thus they believed that the hand of the human, flipper of the seal and the wings of the birds and bats were marvellous contrivances, each fashioned by the creator to adapt these animals to their “conditions of existence”.

2. Unity of type: This view was championed by Etienne Geoffrey Saint-Hilaire and Richard Owen was that “unity of type” (similarity of organisms, which Owens called “homologies”)is critical. The hand of the human, flipper of the seal and the wings of the birds and bats were all modifications of the same basic plan. In discovering the plan, one could find the form upon which the creator designed these animals. The adaptations were secondary.

Darwin acknowledged his debt to these earlier debates when he wrote “ it is genrally acknowledged that all organic beings have been formed on two great laws-Unity of type and the conditions of

existence. Darwin went on to explain that his theory would explain unity of type by descent from a common ancestor, while adaptations to the conditions of existence could be explained by natural

selection. Darwin called this concept descent with modification.

Preconditions of evolution: the developmental structure of the genome

If natural selection can only operate on existing variants, where does all the variation come from?

If variation arose from changes in development then how could development which is such a complex and fine tuned process undergo changes without destroying the entire organism?

Evolutionary developmental biologists showed that large morphological changes could arise during development because of two conditions that underlie development of all multicellular organisms: modularity and molecular parsimony.

Modularity: Divergence through dissociationEven early stages of development can be altered to produce evolutionary novelties because development occurs through a series of discrete and interacting modules.

Examples of developmental modules are: Morphogenetic fields (heart, limb or eye) Signal transduction pathways (Wnt or BMP cascades) Imaginal discs Cell lineages (Inner cell mass or trophoblast) Insect parasegments Vertebrate organ rudiments.

The ability of one module to develop differently from the other is often called dissociation.

An important discovery of evolutionary developmental biologists is that there are not only anatomical units which are modular but DNA regions that form enhancers are also modular.

This modularity of enhancers e.g. there can be multiple enhancers for each gene allows particular sets of genes to be activated together as well as allowing a particular gene to be expressed at different places.

Thus mutations that lead to loss or gain of a modular enhancer element allows differential expression of genes possibly resulting in adoption of different anatomical or physiological morphologies.

Duffy blood group substancePlasmodium vivax is one species of protozoan that causes malaria.

Some African populations are immune to P. vivax because their RBCs lack the Duffy glycoprotein that the P. vivax uses to attach to the RBC.

Duffy glycoprotein is a receptor for IL8 and is found on cerebellar purkinje neurons, blood veins and RBCs.

The people immune to P. vivax express Duffy on Purkinje neurons and blood vessels but not in the RBCs.

A mutation in the RBC specific enhancer for Duffy has allowed these people to retain expression of Duffy in other cells while losing it in the RBC.

Thus the modular nature of the Duffy enhancer has made it possible for these populations to be resistant to P. vivax infection.

Pitx1 and stickleback evolution There are two populations of threespine stickleback

fishes, one found in freshwater and the other is marine. Bony plates and pelvic spines characterize the marine

populations which serve as protection against predators. The freshwater population does not have the pelvic spines

because they do not have other fish preying on them rather invertebrate predators could use the spines to grasp on to them.

By crossing spined varieties with spineless varieties and using molecular markers to identify various regions of chromosomes the major region which determines development of pelvic spines was narrowed to distal end of chromosome 7.

Examining the candidate genes in this region known to be expressed in the hindlimb then zeroed on to Pitx1.

Found that the hindlimb specific enhancer of Pitx1 was non-functional in the spineless variety.

When this 2.5 kb DNA fragment from marine varieties was fused to GFP and inserted into stickleback eggs, GFP was expressed in the pelvis.

Using this enhance to drive the expression of Pitx1 in spineless varieties led them to develop spines.

RecruitmentOften a new structure will form by recruitment

of existing modules (subroutines) into older ones.

The horns of dung beetles arose from co-option of leg patterning genes.

The developmental network co-ordinating the expression of Homothorax, Dachshund and Distal-less in limb formation is used to generate a novel structure, the horn in the dung beetle larva.

The placement of pigments on some Drosophila wings arise when the enhancer for the yellow locus (which makes black pigment) becomes responsive to transcription factors activated by Wingless.

Recruitment

Beetles differ from other insects in forming an elytron---a forewing encased in a hard exoskeleton. In beetles as in drosophila the Apterous gene is expressed in the dorsal compartment of the wing imaginal discs. Apterous transcription factor organizes the tissue to differentiate dorsal wing structures. However in beetles and no other known insects the Apterous also activates the exoskeleton genes in the

forewing while repressing them in the hindwing. Thus a new type of structure emerges from recruitment of one subroutine (exoskeleton development) into

another (dorsal forewing development).

Molecular parsimony: Gene duplication and divergence

The second precondition of macroevolution through developmental change is molecular parsimony sometimes called the “small toolkit”.

Although development differs enormously from lineage to lineage, development within all lineages use the same types of molecules.

The transcription factors, paracrine factors, cell adhesion molecules and signal transduction cascades are remarkably similar between different phyla

It appear that jellyfish and flatworms use the same major kit of transcription factors and paracrine factors as flies and vertebrates.

The small toolkit Certain transcription factors are (such as those of BMP, Hox and Pax groups) are found in all animal phyla including cnidarians, arthropods and chordates.

The BMP levels appear to be used throughout the animal kingdom to specifiy dorsal ventral axis. In sea anemone embryo the Bmp4 and Dpp ortholog is expressed asymmetrically at the edge of the blastopore (A).

The Wnt and Hox genes are used to specify A-P axis throughout all bilaterans. The Hox gene Anthox6 is expressed on the blastopore side of the larval sea anemone (B).

Pax6 appears to be involved in specifying light-sensing organs, irrespective of whether it is an eye of a mollusc, insect or a primate. Expression of mouse Pax6 in the fly leg can produce an ectopic eye structures (C).

Homologues of Otx2 specify head structures in both invertebrates and vertebrates.

Although insect and vertebrate hearts are very different both are formed by usinf tinman/Nks2-5.v

Duplication and divergenceOne common thread that runs through paracrine and transcription factor studies is that genes that encode them come in families. How do these families come into existence?

The answer is through duplication of an original gene and the subsequent mutation of the original duplicates. This creates families of genes that are related by common descent (and often next to each other).

Subsequent mutations in the copies can often lead to the subdivision of the gene’s original function, such that each duplicated gene is now expressed in a different cell type.

The Hox genes were generated by successive rounds of gene duplication

Possible scheme for the formation of the Hox paralogue clusters in metazoans by gene duplication and divergence

The duplication and divergence of human SRGAP2 The SRGAP2 gene is found in a single copy in genomes of all mammals except humans.

In lineage giving rise to humans duplications gave rise to four similar versions of the gene, designated A-D.

The SRGAP2A helps in maturation of dendrites and spine formation with some help from SRGAP2B and D.

The SRGAP2C is a partial duplication and actually inhibits spine formation thus delaying the process of matuaration providing human brains with greater fleixibility.

Deep homologyIn some instances homologous pathways made of homologous components are used for the same function in protostomes and deutrostomes. This has been called deep homology.

The same set of instructions form the nervous system in mice and flies.

In the fruit fly the TGFβ family member Dpp is expressed dorsally and opposed by Sog ventrally.

In the mouse the TGFβ family member BMP4 is expressed ventrally and countered by the Chordin dorsally.

The highest concentration of Chordin becomes the midline which is dorsal in vertebrates and ventral in invertebrates

The concentration gradient of Bmp4/Dpp then specify the regions of the nervous system in the same order Msh/Msx, ind/Gsx and vnd/Nkx2.

Mechanisms of evolutionary changeIn the 1940s, Richard Goldschmidt wrote that accumulation of small genetic changes was not sufficient to generate evolutionarily novel structures such as neural crest, turtle shells or feathers. He argued that such evolution could occur only though inheritable changes in genes that regulate development.

In 1977, this idea was extended by Francois Jacob. He said that evolution works with what it has by combining existing parts in new ways rather than creating new parts. He further stated that such “tinkering” would occur primarily in genes that construct the embryo, and not in genes that function in the adult.

Wallace Arthur (2004) argued the following four ways in which this “tinkering” can occur at the level of gene expression to generate phenotypic variation available for natural selection.

Heterotopy (change in location)Heterochrony (change in time)Heterometry (change in amount)Heterotypy (change in kind)

Heterotopy (change in location)

How the bat got its wings?The bat evolved its wings by changing the development of the of the forelimb such that the cells in the interdigital webbing did not die.

This very similar to how the duck retains webbing in the hindlimb by blocking the BMPs that would otherwise cause the interdigital cells to undergo apoptosis.

Both Gremlin and Fgf appear to block BMP functions in the bat wing. Unlike other mammals bata express Fgf8 in their interdigital mesenchyme. This also provides the mitotic signal for digit extension in bats, therby expanding the wing.

How the turtle got its shell? What distinguishes turtles from other

vertebrates are their ribs, which migrate laterally into the dermis instead of forming a rib cage.

Certain regions of the turtle dermis attract rib precursor cells and these regions differ from that of other vertebrates because they synthesize Fgf10.

Once inside the dermis the rib cells undergo endochondral ossification, wherein cartilage cells are replaced by bone and to do this they need BMP.

Since the ribs are embedded in the dermis the dermal cells also respond to the BMp and become bone.

A and B: bright field and autoradiographic staining for Fgf10.C: H&E staining of slightly later turtle embryo showing rib growing into dermis.D: Hatchling turtle stained with alizarin red to show bone.

Development of an Evolutionarily Novel Structure: Fibroblast Growth Factor Expression in the Carapacial Ridge of Turtle EmbryosLoredo et alJOURNAL OF GE.XAP. ELROIRMEEDNOT EATL AZLO.OLOGY (MOL DEV EVOL) 291:274–281 (2001)

How birds got their feathers? Although it was known that feathers were modified reptilian scales, the mechanism of how they formed has remained elusive. Harris and colleagues have provided a developmental mechanism for feather evolution.

Stage 0 shows the expression of Shh and Bmp2 in the scale bud where they are separated.

Stage 1 represents a tubular feather as evolved from an archosaurian scale. The Shh and Bmp expressions are postulated to be at the tip.

Stage 2 represents the evolution of a branched feather evolved by further changing the expression pattern of Shh and Bmp2 to form rows along the proximo-distal axis. The interaction between Bmp2 and Shh proteins causes each of these regions to form its own axis-the barbs of the feather

In stage 3a changes in feather morphology evolved by altering the pattern to produce a central rachis.Shh-Bmp2 Signaling Module and the Evolutionary Origin and

Diversification of Feathers.Harris et alJOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 294:160–176 (2002)

How snakes lost their limbs? Snakes evolved from four-limbed reptiles and appear to have lost their legs in a two step process.

Paleontological and embryological evidence suggest that snakes first lost their forelimbs then their hind limbs. Fossil snakes have found with no forelimbs but with hind limbs. The most derived snakes such as vipers are completely limbless while more primitive snake such as boas and pythons have pelvic girdles and rudimentary femurs.

The missing limbs can be explained by altered Hox gene expression. In most vertebrates the forelimbs form just anterior to the most anterior expression of Hoxc6. Caudal to this point Hoxc6 in combination with HoxC8 helps specify vertebrae to be thorasic and develop ribs.

During early python development Hoxc6 is not expressed in absence of Hoxc8, so no forelimbs develop. Rather there is continuous expression of Hoxc6 and Hoxc8 telling the vertebrae to form ribs throughout most of the body.

The loss of hindlimbs occur by a different mechanism, hindlimb buds do begin to form but do not produce anything more than a femur due to lack of Shh expression and no AER.

Developmental basis of limblessness andaxial patterning in snakesMartin J .Cohn*† & Cheryll Tickle†‡NATURE |VOL 399 | 3 JUNE 1999

Heterochrony (change in time)In heterochrony one module changes its time of expression or growth rate relative to other modules of the embryo

Heterochrony is quite common in vertebrate evolution. In marsupials whose jaws and forelimbs develop at a

faster rate than those of placental mammals, allowing the marsupial newborn to climb into the maternal pouch and suckle.

The enormous numbers of vertebrae and ribs formed in snakes is due to heterochrony .

The elongated fingers of the dolphin flipper appear to be the result of heterochronic expression of Fgf8.

Molecular heterochrony is observed in the lizard Hemiergis which includes species with 3,4 or 5 digits per limb. The number of digits is regulated by the length of time Sonic hedgehog remains active in the limb bud’s zone of polarizing activity. The shorter the duration of Shh expression the fewer the number of digits.

In primates there is a shift in the pattern of transcription of a set of cerebral mRNAs such that the expression pattern in the adult human resembles that in the juvenile chimpanzee.

Heterometry (change in amount)

Correlation between beak shape and the expression of Bmp4 in five species of Darwin’s finches

Correlation between beak length and the amount of Calmodulin (CaM) gene expression in six species of Darwin’s finches

One of the best examples of heterometry involves Darwin’s finches, a set of closely related birds on Galapagos islands.

There are differences in beak morphology between cactus finches which have long narrow beaks, helpful for probing cactus flowers and ground finches with deep broad beaks which help them to crack open seeds.

Abzhanov et al found a correlation between beak shape and amount and timing of Bmp4 expression.

The same group also found through microarray analysis that Calmodulin is expressed fifteen fold higher in beaks of cactus finches.

Role of BMP4 and Calmodulin (CaM) in beak evolution among Darwin’s finchesBMP4 and Calmodulin represent two targets of natural selection, and together they explain the shape variations of Darwin’s finches. BMP4 is regulated heterochronically as well as heterometrically. Calmodulin is regulated heterometrically. While natural selection will allow certain morphologies to survive the generation of these morphologies depend on variations of developmentally regulatory genes such as those for BMP4 and calmodulin.

Allometry Another consequence of modularity associated with heterometry and heterochrony is allometry-changes which occur when different parts of the organism grow at different rates.

As animals develop their shape changes , a result of differences in timing and the duration of growth events.

Indeed morphological evolution specially within a phylum is due primarily to changes in body size and the relative sizes of body parts. Such differential changes in growth rate can involve altering a target cell’s sensitivity to growth factors or altering the amounts of growth factors produced.

A dramatic example of allometry comes from whale skulls. In the very young (4-5 mm long) whale embryo the nose is in the usual mammalian position. However the enormous growth of the maxilla and premaxilla (upper jaw) pushes over the frontal bone and forces the nose to the top of the skull.

This new position of the nose turns it into blowhole, allowing the whale to have a large and highly specialized jaw apparatus and to breathe while swimming at the water’s surface.

Allometric growth in the whale head

Heterotypy (change in type)In heterochrony, heterometry and heterotopy, mutations affect the regulatory region of the gene. The gene’s produc the protein remains the same. The changes in heterotypy affect the coding regions of the gene.

Ability of the mammalian Hoxa11 protein, in combination with Foxo1a, to promote expression of the uterine Prolactin enhancer

One amazing feature of mammals is the female uterus which can nurture and protect a developing fetus within the mother’s body

One of the key proteins enabling this is prolactin as it promotes differentiation of uterine epithelial cells, regulates trophoblast growth and downregulates immune and inflammatory responses.

The mammalian Hoxa11 appears to have undergone extensive mutation and selection in the lineage giving rise to placental mammals such that it now associates with Foxo1a. This association allows the expression of prolactin from a uterine specific enhancer.

Hoxa11 from opossum and platypus and from chicks does not upregulate prolactin.

How pregnancy may have evolved in mammals

Why insects have only six legs

Changes in Ubx protein associated with the insect clade in the evolution of arthropods

Of all arthropods only the insects have Ubx protein that is able to repress Distal-less expression and thereby inhibit abdominal legs. This ability to repress Distal-less is due to a mutation whereby the original 3’end of the protein-coding region was replaced by a group of nucelotides encoding a stretch of about 10 alanines. This polyalanine region represses Distal-less expression in abdominal segments.