PART Principles of development in biology Developmental biology: The anatomical tradition The Questions of Developmental Biology Anatomical Approaches to Developmental Biology Comparative Embryology Evolutionary Embryology Medical Embryology and Teratology Mathematical Modeling of Development Principles of Development: Developmental Anatomy References Life cycles and the evolution of developmental patterns The Circle of Life: The Stages of Animal Development The Frog Life Cycle The Evolution of Developmental Patterns in Unicellular Protists Multicellularity: The Evolution of Differentiation Developmental Patterns among the Metazoa Principles of Development: Life Cycles and Developmental Patterns References Principles of experimental embryology Environmental Developmental Biology The Developmental Mechanics of Cell Specification Morphogenesis and Cell Adhesion Principles of Development: Experimental Embryology References Genes and development: Techniques and ethical issues The Embryological Origins of the Gene Theory Evidence for Genomic Equivalence Differential Gene Expression RNA Localization Techniques Determining the Function of Genes during Development Identifying the Genes for Human Developmental Anomalies Principles of Development: Genes and Development References The genetic core of development: Differential gene expression Differential Gene Transcription Methylation Pattern and the Control of Transcription Transcriptional Regulation of an Entire Chromosome: Dosage Compensation Differential RNA Processing Control of Gene Expression at the Level of Translation Epilogue: Posttranslational Gene Regulation Principles of Development: Developmental Genetics References Cell-cell communication in development Induction and Competence Paracrine Factors Cell Surface Receptors and Their Signal Transduction Pathways The Cell Death Pathways Juxtacrine Signaling Cross-Talk between Pathways Coda Principles of Development:Cell-Cell Communication References PART 2: Early embryonic development Fertilization: Beginning a new organism Structure of the Gametes Recognition of Egg and Sperm Gamete Fusion and the Prevention of Polyspermy The Activation of Egg Metabolism Fusion of the Genetic Material Rearrangement of the Egg Cytoplasm Snapshot Summary: Fertilization References Early development in selected invertebrates An Introduction to Early Developmental Processes The Early Development of Sea Urchins The Early Development of Snails Early Development in Tunicates Early Development of the Nematode Caenorhabditis elegans References The genetics of axis specification in Drosophila Early Drosophila Development The Origins of Anterior-Posterior Polarity The Generation of Dorsal-Ventral Polarity References 10 Early development and axis formation in amphibians Early Amphibian Development Axis Formation in Amphibians: The Phenomenon of the Organizer References 11 The early development of vertebrates: Fish, birds, and mammals Early Development in Fish Early Development in Birds Early Mammalian Development References PART 3: Later embryonic development 12 The central nervous system and the epidermis Formation of the Neural Tube Differentiation of the Neural Tube Tissue Architecture of the Central Nervous System Neuronal Types Development of the Vertebrate Eye The Epidermis and the Origin of Cutaneous Structures Snapshot Summary: Central Nervous System and Epidermis References 13 Neural crest cells and axonal specificity The Neural Crest Neuronal Specification and Axonal Specificity References 14 Paraxial and intermediate mesoderm Paraxial Mesoderm: The Somites and Their Derivatives Myogenesis: The Development of Muscle Osteogenesis: The Development of Bones Intermediate Mesoderm Snapshot Summary: Paraxial and Intermediate Mesoderm References 15 Lateral plate mesoderm and endoderm Lateral Plate Mesoderm Endoderm References 16 Development of the tetrapod limb Formation of the Limb Bud Generating the Proximal-Distal Axis of the Limb Specification of the Anterior-Posterior Limb Axis The Generation of the Dorsal-Ventral Axis Coordination among the Three Axes Cell Death and the Formation of Digits and Joints Snapshot Summary: The Tetrapod Limb References 17 Sex determination Chromosomal Sex Determination in Mammals Chromosomal Sex Determination in Drosophila Environmental Sex Determination Snapshot Summary: Sex Determination References 18 Metamorphosis, regeneration, and aging Metamorphosis: The Hormonal Reactivation of Development Regeneration Aging: The Biology of Senescence References 19 The saga of the germ line Germ Plasm and the Determination of the Primordial Germ Cells Germ Cell Migration Meiosis Spermatogenesis Oogenesis Snapshot Summary: The Germ Line References PART 4: Ramifications of developmental biology 20 An overview of plant development Plant Life Cycles Gamete Production in Angiosperms Pollination Fertilization Embryonic Development Dormancy Germination Vegetative Growth The Vegetative-to-Reproductive Transition Senescence Snapshot Summary: Plant Development References 21 Environmental regulation of animal development Environmental Regulation of Normal Development Environmental Disruption of Normal Development References 22 Developmental mechanisms of evolutionary change "Unity of Type" and "Conditions of Existence" Hox Genes: Descent with Modification Homologous Pathways of Development Modularity: The Prerequisite for Evolution through Development Developmental Correlation Developmental Constraints A New Evolutionary Synthesis Snapshot Summary: Evolutionary Developmental Biology References Appendix PARTE Principles of development in biology Developmental biology: The anatomical tradition The Questions of Developmental Biology According to Aristotle, the first embryologist known to history, science begins with wonder: "It is owing to wonder that people began to philosophize, and wonder remains the beginning of knowledge." The development of an animal from an egg has been a source of wonder throughout history The simple procedure of cracking open a chick egg on each successive day of its 3-week incubation provides a remarkable experience as a thin band of cells is seen to give rise to an entire bird Aristotle performed this procedure and noted the formation of the major organs Anyone can wonder at this remarkable yet commonplace phenomenon, but the scientist seeks to discover how development actually occurs And rather than dissipating wonder, new understanding increases it Multicellular organisms not spring forth fully formed Rather, they arise by a relatively slow process of progressive change that we call development In nearly all cases, the development of a multicellular organism begins with a single cell the fertilized egg, or zygote, which divides mitotically to produce all the cells of the body The study of animal development has traditionally been called embryology, from that stage of an organism that exists between fertilization and birth But development does not stop at birth, or even at adulthood Most organisms never stop developing Each day we replace more than a gram of skin cells (the older cells being sloughed off as we move), and our bone marrow sustains the development of millions of new red blood cells every minute of our lives In addition, some animals can regenerate severed parts, and many species undero metamorphosis (such as the transformation of a tadpole into a frog, or a caterpillar into a butterfly) Therefore, in recent years it has become customary to speak of developmental biology as the discipline that studies embryonic and other developmental processes Development accomplishes two major objectives: it generates cellular diversity and order within each generation, and it ensures the continuity of life from one generation to the next Thus, there are two fundamental questions in developmental biology: How does the fertilized egg give rise to the adult body, and how does that adult body produce yet another body? These two huge questions have been subdivided into six general questions scrutinized by developmental biologists: The question of differentiation A single cell, the fertilized egg, gives rise to hundreds of different cell types muscle cells, epidermal cells, neurons, lens cells, lymphocytes, blood cells, fat cells, and so on (Figure 1.1) This generation of cellular diversity is called differentiation Since each cell of the body (with very few exceptions) contains the same set of genes, we need to understand how this same set of genetic instructions can produce different types of cells How can the fertilized egg generate so many different cell types? The question of morphogenesis Our differentiated cells are not randomly distributed Rather, they are organized into intricate tissues and organs These organs are arranged in a given way: the fingers are always at the tips of our hands, never in the middle; the eyes are always in our heads, not in our toes or gut This creation of ordered form is called morphogenesis How can the cells form such ordered structures? The question of growth How our cells know when to stop dividing? If each cell in our face were to undergo just one more cell division, we would be considered horribly malformed If each cell in our arms underwent just one more round of cell division, we could tie our shoelaces without bending over Our arms are generally the same size on both sides of the body How is cell division so tightly regulated? The question of reproduction The sperm and egg are very specialized cells Only they can transmit the instructions for making an organism from one generation to the next How are these cells set apart to form the next generation, and what are the instructions in the nucleus and cytoplasm that allow them to function this way? The question of evolution Evolution involves inherited changes in development When we say that today's one-toed horse had a five-toed ancestor, we are saying that changes in the development of cartilage and muscles occurred over many generations in the embryos of the horse's ancestors How changes in development create new body forms? Which heritable changes are possible, given the constraints imposed by the necessity of the organism to survive as it develops? The question of environmental integration The development of many organisms is influenced by cues from the environment Certain butterflies, for instance, inherit the ability to produce different wing colors based on the temperature or the amount of daylight experienced by the caterpillar before it undergoes metamorphosis How is the development of an organism integrated into the larger context of its habitat? Anatomical Approaches to Developmental Biology A field of science is defined by the questions it seeks to answer, and most of the questions in developmental biology have been bequeathed to it through its embryological heritage There are numerous strands of embryology, each predominating during a different era Sometimes they are very distinct traditions, and sometimes they blend We can identify three major ways of studying embryology: Anatomical approaches Experimental approaches Genetic approaches While it is true that anatomical approaches gave rise to experimental approaches, and that genetic approaches built on the foundations of the earlier two approaches, all three traditions persist to this day and continue to play a major role in developmental biology Chapter of this text discusses experimental approaches, and Chapters and examine the genetic approaches in greater depth In recent years, each of these traditions has become joined with molecular genetics to produce a vigorous and multifaceted science of developmental biology But the basis of all research in developmental biology is the changing anatomy of the organism What parts of the embryo form the heart? How the cells that form the retina position themselves the proper distance from the cells that form the lens? How the tissues that form the bird wing relate to the tissues that form the fish fin or the human hand? There are several strands that weave together to form the anatomical approaches to development The first strand is comparative embryology, the study of how anatomy changes during the development of different organisms For instance, a comparative embryologist may study which tissues form the nervous system in the fly or in the frog The second strand, based on the first, is evolutionary embryology, the study of how changes in development may cause evolutionary changes and of how an organism's ancestry may constrain the types of changes that are possible The third anatomical approach to developmental biology is teratology, the study of birth defects These anatomical abnormalities may be caused by mutant genes or by substances in the environment that interfere with development The study of abnormalities is often used to discover how normal development occurs The fourth anatomical approach is mathematical modeling, which seeks to describe developmental phenomena in terms of equations Certain patterns of growth and differentiation can be explained by interactions whose results are mathematically predictable The revolution in graphics technology has enabled scientists to model certain types of development on the computer and to identify mathematical principles upon which those developmental processes are based Evolutionary Embryology Charles Darwin's theory of evolution restructured comparative embryology and gave it a new focus After reading Johannes Müller's summary of von Baer's laws in 1842, Darwin saw that embryonic resemblances would be a very strong argument in favor of the genetic connectedness of different animal groups "Community of embryonic structure reveals community of descent," he would conclude in On the Origin of Species in 1859 Larval forms had been used for taxonomic classification even before Darwin J V Thompson, for instance, had demonstrated that larval barnacles were almost identical to larval crabs, and he therefore counted barnacles as arthropods, not molluscs (Figure 1.12; Winsor 1969) Darwin, an expert on barnacle taxonomy, celebrated this finding: "Even the illustrious Cuvier did not perceive that a barnacle is a crustacean, but a glance at the larva shows this in an unmistakable manner." Darwin's evolutionary interpretation of von Baer's laws established a paradigm that was to be followed for many decades, namely, that relationships between groups can be discovered by finding common embryonic or larval forms Kowalevsky (1871) would soon make a similar type of discovery (publicized in Darwin's Descent of Man) that tunicate larvae have notochords and form their neural tubes and other organs in a manner very similar to that of the primitive chordate Amphioxus The tunicates, another enigma of classification schemes (formerly placed, along with barnacles, among the molluscs), thereby found a home with the chordates Darwin also noted that embryonic organisms sometimes make structures that are inappropriate for their adult form but that show their relatedness to other animals He pointed out the existence of eyes in embryonic moles, pelvic rudiments in embryonic snakes, and teeth in embryonic baleen whales Darwin also argued that adaptations that depart from the "type" and allow an organism to survive in its particular environment develop late in the embryo.* He noted that the differences between species within genera become greater as development persists, as predicted by von Baer's laws Thus, Darwin recognized two ways of looking at "descent with modification." One could emphasize the common descent by pointing out embryonic similarities between two or more groups of animals, or one could emphasize the modifications by showing how development was altered to produce structures that enabled animals to adapt to particular conditions Scientists are looking at co-option in the formation of novel evolutionary structures For instance, the carapace (dorsal shell) of the turtle is an evolutionary novelty that appears to form in a manner reminiscent of limbs There is even a carapacial ridge that organizes the mesenchyme much like the apical ectodermal ridge of the limb bud (Figure 22.22; Burke 1989a) The bones themselves appear to form in the manner of skull bones It is possible that certain developmental pathways (those used to form the limbs and those used to form the skull bones) have been recruited to form this new structure The existence of discrete developmental modules allows the principles of dissociation, duplication and divergence, and co-option to form new types of organisms *The lack of such transitional forms is often cited by creationists as evidence against evolution For instance, in the transition from reptiles to mammals, three of the bones of the reptilian jaw became the incus and malleus, leaving only one bone (the dentary) in the lower jaw (see Chapter and below) Gish (1973), a creationist, says that this is an impossible situation, since no fossil has been discovered showing two or three jaw bones and two or three ear ossicles Such an animal, he claims, would have dragged its jaw on the ground However, such a specific transitional form (and there are over a dozen documented transitional forms between reptilian and mammalian skulls) need never have existed Hopson (1966) has shown on embryological grounds how the bones of the jaw could have divided and been used for different functions, and Romer (1970) has found reptilian fossils wherein the new jaw articulation was already functional while the older bones were becoming useless There are several species of therapsid reptiles that had two jaw articulations, with the stapes brought into close proximity with the upper portion of the quadrate bone (which would become the incus) Developmental Correlation Correlated progression The modular nature of development also expects that modules will aggregate to form larger modules One evolutionary consequence of this phenomenon is correlated progression, in which changes in one part of the embryo induce changes in another Skeletal cartilage informs the placement of muscles, and muscles induce the placement of nerve axons In such cases, if one structure changes, it will induce other structures to change with it (Thomson 1988) The dramatic changes in bone arrangement from agnathans to jawed fishes, from jawed fishes to amphibians, and from reptiles to mammals were coordinated with changes in jaw structure, jaw musculature, tooth deposition and shape, and the structure of the cranial vault and ear (Kemp 1982; Thomson 1988; Fischman 1995) The mechanism through which the jaw apparatus has maintained its integrity from agnathans to amniotes is a remarkable example of embryonic modularity The neural crestderived structures of the vertebrate head include the pharyngeal arches (the precursors of the jaw, middle ear, tongue skeleton, etc.) as well as the dermal bones of the face and the facial musculature (see Chapter 13) The braincase is produced from mesodermal tissues Köntges and Lumsden (1996) were able to map the fates of the neural crest cells associated with particular rhombomeres by replacing individual chick rhombomeres with those of quail (Figure 22.23) Antibody staining of the quail neural crest cells showed that each rhombomere gives rise to particular skeletal elements and to the muscles attached to them Moreover, the muscle-andskeleton modules from each rhombomere were found to be innervated by a particular cranial nerve For instance, the neural crest cells from rhombomere generated four skeletal tissues: the retroarticular process of the lower jaw (found in birds, but not mammals), a portion of the tongue skeleton, the stapes bone of the middle ear, and, surprisingly, the small portion of the braincase where the jaw-opening muscle attaches to the otherwise mesodermally derived skull The muscles connecting these four skeletal elements also came from the r4 neural crest cells These muscles are all innervated by the seventh cranial nerve Thus, this rhombomere forms a modular unit, comprising the pharyngeal arch skeletal elements, the muscles that move them, the attachment site of the muscles to the braincase, and the nerves that innervate the muscles Because these muscles and bones are formed from the same cells, their relationships can be maintained despite the dramatic changes in position and function that these elements might undergo over time One can also see correlated progression over a shorter time in domesticated animals Humans have a great talent for selecting hereditary variants in domestic animals that involve those neural crest cells forming the frontonasal and mandibular processes In some cases, such as that of bulldogs, the breed is selected for a wide face with very little angle between head and jaw Other breeds, such as the collie, are selected for a narrow snout with a long jaw protruding away from the head All breeds of dogs can move their jaws, shake their heads, and bark, despite the differences in the way their bones are shaped or positioned Each variation is genetically determined, and it is important to note that each represents a harmonious rearrangement of the different bones with each other and with their muscular attachments As the skeletal elements were selected, so were the muscles that moved them, the nerves that controlled their movements, and the blood vessels that fed them.* Correlated progression has also been shown experimentally Repeating the earlier experiments of Hampé (1959), Gerd Müller (1989) inserted barriers of gold foil into the prechondrogenic hindlimb buds of a 3.5-day chick embryo This barrier separated the regions of tibia formation and fibula formation The results of these experiments were twofold First, the tibia is shortened, and the fibula bows and retains its connection to the fibulare (the distal portion of the tibia) Such relationships between the tibia and fibula are not usually seen in birds, but they are characteristic of reptiles (Figure 22.24) Second, the musculature of the hindlimb undergoes changes in parallel with the bones Three of the muscles that attach to these bones now show characteristic reptilian patterns of insertion It seems, therefore, that experimental manipulations that alter the development of one part of the mesodermal limb-forming field also alter the development of other mesodermal components This was crucial in the evolution of the bird hindlimb from the reptile hindlimb As with the correlated progression seen in facial development, these changes all appear to be due to interactions within a module, in this case, the chick hindlimb field These changes are not global effects and can occur independently of the other portions of the body Coevolution of ligand and receptor Another example of developmental correlation involves the ability of one tissue to interact with another In development, things have to fit together if the organism is to survive Ligands have to fit with receptors, and they have to be expressed at the right place and at the right time Changes in the ligand must be accommodated by complementary changes in the receptor if the receptor is to function If a mutation in a gene encoding ligand (or receptor) produces too great a change, it will not bind to its complementary receptor (or ligand), and development will stop When duplications of ligand and receptor genes occur, they can diverge and acquire new functions This is seen in the evolution of hormone families and their receptors (Moyle et al 1994) Such separation of functions can cause reproductive isolation and the separation of species when the receptor and ligand are proteins on the sperm and egg While most proteins of closely related marine species are very similar, the proteins responsible for fertilization are often extremely different (Metz et al 1994) In sea urchins, the bindin of the sperm and the complementary receptors of the egg have coevolved such that the bindin of one species often does not recognize the bindin receptors on the oocytes of other species Hofmann and Glabe (1994) have proposed a model whereby there are several distinct recognition sites on bindin and its receptor Mutations would cause some of these sites to be altered, and these alterations would select for complementary alterations on the opposite gamete There would be a stage wherein some unaltered sperm could bind, albeit weakly, to altered eggs, but eventually, this process of alteration and accommodation would produce two reproductively isolated groups within the species (Figure 22.25) In abalones, mutations of a small region of the lysin protein and its corresponding receptor appear to be responsible for the species specificity of fertilization Moreover, these changes in lysin and bindin proteins appear to be rapid and correlate with speciation (Shaw et al 1994; Metz and Palumbi 1996; Lyon and Vacquier 1999) *This coordination is not quite universal, however In dogs with greatly shortened faces (such as bulldogs), the skin has not coordinated its development with the bones and therefore hangs in folds from the head (Stockard 1941) Another example of a developmental mutation causing reproductive isolation involves a more mechanical function The snail shell coiling mutations discussed in Chapter are mutations that act during early development to change the position of the mesodermal organs Mating between left-coiling and right-coiling snails is mechanically very difficult, if not impossible, in some species (Clark and Murray 1969) Because this mutation is inherited as a maternal effect gene, a group of related snails would emerge that could mate with one another but not with other members of the original population These reproductively isolated snails could expand their range and, by the accumulation of new mutations, form a new species (Alexandrov and Sergievsky 1984) Developmental Constraints Another consequence of interacting modules is that these interactions limit the possible phenotypes that can be created, and they also allow change to occur in certain directions more easily than in others Collectively, these restraints on phenotype production are called developmental constraints Physical constraints There are only about three dozen animal phyla, constituting the major body plans of the animal kingdom One can easily imagine other types of body plans and animals that not exist (Science fiction writers it all the time.) Why aren't there more major body types among the animals? To answer this, we have to consider the constraints that development imposes on evolution There are three major classes of constraints on morphogenetic evolution First, there are physical constraints on the construction of the organism The laws of diffusion, hydraulics, and physical support allow only certain mechanisms of development to occur One cannot have a vertebrate on wheeled appendages (of the sort that Dorothy saw in Oz) because blood cannot circulate to a rotating organ; this entire possibility of evolution has been closed off Similarly, structural parameters and fluid dynamics forbid the existence of 5-foot-tall mosquitoes The elasticity and tensile strengths of tissues is also a physical constraint The six cell behaviors used in morphogenisis (cell division, growth, shape change, migration, death, and matrix secretion) are each limited by physical parameters, and thereby provide limits on what structures animals can form Interactions between different sets of tissues involves coordinating the behaviors of cell sheets, rods, and tubes in a limited number of ways (Larsen 1992) Morphogenetic constraints There are also constraints involving morphogenetic construction rules (Oster et al 1988) Bateson (1894) and Alberch (1989) noted that when organisms depart from their normal development, they so in only a limited number of ways Some of the best examples of these types of constraints come from the analysis of limb formation in vertebrates Holder (1983) pointed out that although there have been many modifications of the vertebrate limb over 300 million years, some modifications (such as a middle digit shorter than its surrounding digits) are not found Moreover, analyses of natural populations suggest that there is a relatively small number of ways in which limb changes can occur (Wake and Larson 1987) If a longer limb is favorable in a given environment, the humerus may become elongated, but one never sees two smaller humeri joined together in tandem, although one could imagine the selective advantages that such an arrangement might have This observation indicates a construction scheme that has certain rules The rules governing the architecture of the limb may be the rules of the reaction-diffusion model (outlined in Chapter 1; Newman and Frisch 1979) Oster and colleagues (1988) found that the reaction-diffusion model can explain the known morphologies of the limb and can explain why other morphologies are forbidden The reaction-diffusion equations predict the observed succession of bones from stylopod (humerus/femur) to zeugopod (ulna-radius/tibia-fibula) to autopod (hand/foot) If limb morphology is indeed determined by the reaction-diffusion mechanism, then spatial features that cannot be generated by reaction-diffusion kinetics will not occur Evidence for this mathematical model comes from experimental manipulations, comparative anatomy and cell biology When an axolotl limb bud is treated with the anti-mitotic drug colchicine, the dimensions of the limb are reduced In these experimental limbs, there is not only a reduction in the number of digits, but a loss of certain digits in a certain order, as predicted by the mathematical model and from the "forbidden" morphologies Moreover, these losses of specific digits produce limbs very similar to those of certain salamanders whose limbs develop from particularly small limb buds (Figure 22.26; Alberch and Gale 1983, 1985) The self-organization of chondrocytes into nodules can be modelled by the Turing equations, and TGF-β2 appears to have the properties of the activator molecule postulated by this hypothesis (Miura and Shiota 2000a,Miura and shiota 2000b) Thus, the use of reaction-diffusion mechanisms to construct limbs may constrain the possibilities that can be generated during development, because only certain types of limbs are possible under these rules Phyletic constraints Phyletic constraints constitute the third set of constraints on the evolution of new types of structures (Gould and Lewontin 1979) These are historical restrictions based on the genetics of an organism's development For instance, once a structure comes to be generated by inductive interactions, it is difficult to start over again The notochord, for example, which is still functional in adult protochordates such as amphioxus (Berrill 1987), is considered vestigial in adult vertebrates Yet it is transiently necessary in vertebrate embryos, where it specifies the neural tube Similarly, Waddington (1938) noted that although the pronephric kidney of the chick embryo is considered vestigial (since it has no ability to concentrate urine), it is the source of the ureteric bud that induces the formation of a functional kidney during chick development (see Chapter 14) Until recently, it was thought that the earliest stages of development would be the hardest to change, because altering them would either destroy the embryo or generate a radically new phenotype But recent work (and the reappraisal of older work: Raff et al 1991) has shown that alterations can be made to early cleavage without upsetting the final form Evolutionary modifications of cytoplasmic determinants in mollusc embryos can give rise to new types of larvae that still metamorphose into molluscs, and changes in sea urchin cytoplasmic determinants can generate sea urchins that develop without larvae but still become sea urchins In fact, while all the vertebrates arrive at a particular stage of development called the pharyngula, they so by very different means (see Figure 1.5) Birds, reptiles, and fishes arrive there after meroblastic cleavages of different sorts; amphibians get to the pharyngula stage by way of radial holoblastic cleavage; and mammals reach the same stage after constructing a blastocyst, chorion, and amnion The earliest stages of development, then, appear to be extremely plastic Similarly, the later stages are very different, as the different phenotypes of mice, sunfish, snakes, and newts amply demonstrate There is something in the middle of development, however, that appears to be invariant Raff (1994) argues that the formation of new body plans (Baupläne) is inhibited by the need for global sequences of induction during the neurula stage (Figure 22.27) Before that stage, there are few inductive events After that stage, there are a great many inductive events, but almost all of them occur within discrete modules During early organogenesis, however, there are several inductive events occurring simultaneously that are global in nature At this stage, the modules overlap and interact with one another In vertebrates, to use von Baer's example, the earliest stages of development involve specifying axes and undergoing gastrulation Induction has not yet happened on a large scale Moreover, as Raff and colleagues have shown (Henry et al 1989), there is a great deal of regulative ability at these stages, so small changes in morphogen distributions or the position of cleavage planes can be accommodated After the major body plan is fixed, inductions occur all over the body, but are compartmentalized into discrete organforming systems The lens induces the cornea, but if it fails to so, only the eye is affected Similarly, there are inductions in the skin that form feathers, scales, or fur If they not occur, the skin or patch of skin may lack these structures, but the rest of the body is unchanged But during early organogenesis, the interactions are more global (Slack 1983) Failure to have the heart in a certain place can affect the induction of eyes (see Figure 6.4) Failure to induce the mesoderm in a certain region leads to malformations of the kidneys, limbs, and tail It is this stage that constrains evolution and that typifies the vertebrate phylum Thus, once a vertebrate, it is difficult to evolve into anything else Leibniz, probably the philosopher who most influenced Darwin, noted that existence must be limited not only to the possible but to the compossible That is, whereas numerous things can come into existence, only those that are mutually compatible will actually exist (see Lovejoy 1964) So although many developmental changes are possible, only those that can integrate into the rest of the organism (or which can cause a compensatory change in the rest of the organism) will be seen Canalization and the Release of Developmental Constraints Not all mutations produce mutant phenotypes Rather, development appears to be buffered so that slight abnormalities of genotype or slight perturbations of the environment will not lead to the formation of abnormal phenotypes (Waddington 1942) This phenomenon, called canalization, serves as an additional constraint on the evolution of new phenotypes It is difficult for a mutation to actually affect development (Nijhout and Paulsen 1997) It is the rare mutation that is 100% penetrant Stress, however, in the form of environmental factors such as temperature, can overpower the buffering systems of development and alter the phenotype Moreover, the altered phenotype then becomes subject to natural selection, and if selected, will evebtually appear without the stress that originally induced it Waddington called this phenomenon genetic assimilationChapter21) For instance, when Waddington subjected Drosophila larvae of a certain strain to high temperatures, they lost their wing crossveins After a few generations of repeated heat shock, the crossveinless phenotype continued to be expressed in this population even without the heat shock treatment While Waddington's results look like a case of "inheritance of acquired characteristics," there is no evidence for that view Certainly, the crossveinless phenotype was not an adaptive response to heat Nor did the heat shock cause the mutations Rather, the heat shock overcame the buffering systems, allowing preexisting mutations to result in mutant phenotypes rather than wild-type phenotypes In 1998, Suzanne Rutherford and Susan Lindquist showed that a major agent responsible for this buffering was the "heat shock protein" Hsp90 Hsp90 is a protein that binds to a set of signal transduction molecules that are inherently unstable When it binds to them, it stabilizes their tertiary structure so that they can respond to upstream signaling molecules Heat shock, however, causes other proteins in the cell to become unstable, and Hsp90 is diverted from its normal function (of stabilizing the signal transduction proteins) to the more general function of stabilizing any of the cell's now partially denatured peptides (Jakob et al 1995; Nathan et al 1997) Since Hsp90 was known to be involved with inherently unstable proteins and could be diverted by stress, it was possible that Hsp90 might be involved in buffering developmental pathways against environmental contingencies Evidence for the role of Hsp90 as a developmental buffer first came from mutations of Hsp83, the gene for Hsp90 Homozygous mutations of Hsp83 are lethal in Drosophila Heterozygous mutations increase the proportion of developmental abnormalities in the population into which they are introduced In populations of Drosophila heterozygous for Hsp83, deformed eyes, bristle duplications, and abnormalities of legs and wings appeared (Figure 22.28) When different mutant alleles of Hsp83 were brought together in the same flies, both the incidence and severity of the abnormalities increased Abnormalities were also seen when a specific inhibitor of Hsp90 (geldanamycin) was added to the food of wild-type flies, and the types of defects differed between different stocks of flies The abnormalities observed did not show simple Mendelian inheritance, but were the outcome of the interactions of several gene products Selective breeding of the flies with the abnormalities led over a few generations to populations in which 80 90% of the progeny had the mutant phenotype Moreover, these mutants did not keep the Hsp83 mutation In other words, once the mutation in Hsp83 had allowed the cryptic mutations to become expressed, selective matings could retain the abnormal phenotype even in the absence of abnormal Hsp90 Thus, Hsp90 is probably a major component of the buffering system that enables the canalization of development It provides a way to resist fluctuations due to slight mutations or slight environmental changes Hsp90 might also be responsible for allowing mutations to accumulate but keeping them from being expressed until the environment changes No individual mutation would change the phenotype, but mating would allow these mutations to be "collected" by members of the population An environmental change (anything that might stress the cells) would thereby release the hidden phenotypic possibilities of the population In other words, transient decreases in Hsp90 (resulting from its aiding stress-damaged proteins) would uncover preexisting genetic interactions that would produce morphological variations Most of these morphological variations would probably be deleterious, but some might be selected for in the new environment Such release of hidden morphological variation may be responsible for the many examples of rapid speciation found in the fossil record A New Evolutionary Synthesis In 1922, Walter Garstang declared that ontogeny (an individual's development) does not recapitulate phylogeny (evolutionary history); rather, it creates phylogeny Evolution is generated by heritable changes in development "The first bird," said Garstang, "was hatched from a reptile's egg." Thus, when we say that the contemporary one-toed horse evolved from a five-toed ancestor, we are saying that heritable changes occurred in the differentiation of the limb mesoderm into chondrocytes during embryogenesis in the horse lineage This view of evolution as the result of hereditary changes affecting development was lost during the 1940s, when the Modern Synthesis of population genetics and evolutionary biology formed a new framework for research in evolutionary biology The Modern Synthesis has been one of the greatest intellectual achievements of biology By merging the traditions of Darwin and Mendel, evolution within a species could be explained: Diversity within a population arose from the random production of mutations, and the environment acted to select the most fit phenotypes Those animals capable of reproducing would transmit the genes that gave them their advantage These genes included, for example, those encoding enzymes with better rates of synthesis and globins with better oxygen-carrying capacity It was assumed that the same kinds of changes (gene or chromosomal mutations) that caused evolution within a species also caused the evolution of new species There would need to be an accumulation of these mutations, and a mechanism of reproductive isolation to enable them to accumulate in new ways, if a new phenotype was to be produced Not only could the Modern Synthesis explain evolution within a species remarkably well, it also explained medically relevant questions such as why certain alleles that seem deleterious (the hemoglobin gene variant that can result in sickle cell anemia, for example) might be selected for in certain populations The population genetic approach to evolution was summed up by one of its foremost practitioners and theorists, Theodosius Dobzhansky, when he declared, "Evolution is a change in the genetic composition of populations The study of the mechanisms of evolution falls within the province of population genetics" (Dobzhansky (1951) The developmental approach to evolution was excluded from the Modern Synthesis (Hamburger 1980; Gottlieb 1992; Dietrich 1995; Gilbert et al 1996) It was thought that population genetics could explain evolution, so morphology and development were seen to play little role in modern evolutionary theory (Adams 1991) In other words, macroevolution (the large morphological changes seen between species, classes, and phyla) could be explained by the mechanisms of microevolution, the "differential adaptive values of genotypes or deviations from random mating or both these factors acting together" (Torrey and Feduccia 1979) The population genetics model contained some major assumptions that have now been called into question Gradualism The supposition that all evolutionary changes occur gradually was debated by Darwin and his friends Thomas Huxley, for instance, accepted evolution, but he felt that Darwin had burdened his theory with an unnecessary assumption of gradualism A century later, Eldredge and Gould (1972), Stanley (1979), and others postulated punctuated equilibrium as an alternative to the gradualism that characterized the Modern Synthesis According to this theory, species were characterized by their morphological stability Evolutionary changes tended to be rapid, not gradual At the same time, molecular studies (King and Wilson 1975) showed that 99% of the DNA of humans and chimpanzees was identical, demonstrating that a small change in DNA could cause large and important morphological changes New findings in paleontology and molecular biology prompted scientists to consider seriously the view that mutations in regulatory genes can create large changes in morphology in a relatively short time Extrapolation of microevolution to macroevolution The idea that accumulations of small mutations result in changes leading to new species has also been criticized Richard Goldschmidt (1940) began his book The Material Basis of Evolution by asking the population genetic evolutionary biologists to try to explain the evolution of the following features by accumulation and selection of small mutations: hair in mammals; feathers in birds; segmentation in arthropods and vertebrates; the transformation of the gill arches into structures including aortic arches, muscles, and nerves; teeth; shells of molluscs; compound eyes; and the poison apparatus of snakes Interestingly, both Goldschmidt and Waddington saw homeotic mutations as the kind of genetic change that could alter one structure into another and possibly create new structures or new combinations of structures These mutations would not be in the structural genes, but in the regulatory genes Few scientists paid attention to Goldschmidt or Waddington, however, because they were not working under the population genetics paradigm of the Modern Synthesis and because their scientific programs were suspect (Goldschmidt did not believe in Morgan's notion of the gene as a particulate entity, and Waddington's work was misinterpreted as supporting the inheritance of acquired traits: see Gilbert 1988; 1991; Dietrich 1995.) Specificity of phenotype from genotype Developmental biologists have found that life is more complicated than a 1:1 relationship between genotype and phenotype Chapter 21 documents numerous cases wherein the genotype can permit any of several phenotypes to form These cases include polyphenisms induced by predators, diet, day length, or antigenic or visual experience Moreover, development always mediates between genotype and phenotype The same gene can produce different phenotypes depending on the other genes that are present (Wolf 1995) A mutant gene that produces limblessness in one generation can produce only a mild thumb abnormality in the next (Freire-Maia 1975) That evolution is the result of heritable changes in development (Goldschmidt 1940) is as true for whether a fly has two or three bristles on its back as for whether an appendage is to become a fin or a limb One way of visualizing this is to use a mathematical analogy (Gilbert et al 1996): Functional biology = anatomy, physiology, cell biology, gene expression Developmental biology = Evalutionary biology = [functional biology]/ t /[developmental biology]/ t To go from functional biology to evolutionary biology without development is like going from displacement to acceleration without dealing with velocity Lack of genetic similarity in disparate organisms We have come a long way from when Ernst Mayr (1966) could state, concerning macroevolution: "Much that has been learned about gene physiology makes it evident that the search for homologous genes is quite futile except in very close relatives." Indeed, when one considers the Hox genes, the signal transduction cascades, and the families of paracrine factors, adhesion molecules, and transcription factors, the opposite has been seen to be the case Adult organisms may have dissimilar structures, but the genes instructing the formation of these structures are extremely similar The population genetics model was formulated to explain natural selection It is based on gene differences in adults competing for reproductive advantage The developmental genetics model is formulated to account for phylogeny evolution above the species level It is based on the similarities in regulatory genes that are active in embryos and larvae We are still approaching evolution in the two ways that Darwin recognized One can emphasize the similarities or the differences When the Modern Synthesis was formulated, developmental biology (and developmental genetics) were not even sciences Embryology was left out of the Modern Synthesis, as most evolutionary biologists and geneticists felt it had nothing to contribute However, we know now that it does The developmental genetics approach to evolution concerns more the arrival of the fittest than the survival of the fittest Even critics of the Modern Synthesis (including Goldschmidt and Gould) agree that macroevolutionary change is predicated upon mutation and recombination However, these macroevolutionary changes are in developmental regulatory genes, not the usual genes for enzymes and structural proteins; and these changes occur in embryos and larvae, not in adults competing for reproductive success (see Waddington 1953; Gilbert 1998) Developmental biology brings to evolutionary biology, first, a new understanding about the relationships between genotypes and phenotypes, and second, a new understanding about the close genetic relationships between organisms as diverse as flies and frogs In doing so, developmental biology complements the population genetics approach to evolutionary biology It also highlights new questions For instance, there can now be a population genetic approach to the regulatory genes (see Arthur 1997; Macdonald and Goldstein 1999; Zeng et al 1999) One can also look at how paracrine factors, signal transduction pathways, and transcription factors have changed during the evolution of various phyla Evolutionary developmental biology can also provide answers to classic evolutionary genetics questions such as these posed by mimicry and industrial melanism The genes involved in these processes are being identified so the mechanisms of these phenomena can be explained (Koch et al 1998; Brakefield 1998) To explain evolution, both the population genetics and the developmental genetics accounts are required Leaving developmental biology out of the population genetics model of evolution has left evolutionary biology open to attacks by creationists According to Behe (1996), population genetics cannot explain the origin of structures such as the eye, so Darwinism is false.* How could such a complicated structure have emerged by a collection of chance mutations? If a mutation caused a change in the lens, how could it be compensated for by changes in the retina? Mutations would serve only to destroy complex organs, not create them However, once one adds development to the evolutionary synthesis, one can see how the eye can develop through induction, and that the concepts of modularity and correlated progression can readily explain such a phenomenon (Waddington 1940; Gehring 1998) Moreover, when one sees that the formation of eyes in all known phyla is based on the same signal transduction pathway, using the Pax6 gene, it is not difficult to see descent with modification forming the various types of eyes This was much more difficult before the similarity of eye instructions had been discovered Indeed, one study based in population genetics claimed that photoreceptors or eyes arose independently over forty times during the history of the animal kingdom (Salvini-Plawen and Mayr 1977) In his review of evolution in 1953, J B S Haldane expressed his thoughts about evolution with the following developmental analogy: "The current instar of the evolutionary theory may be defined by such books as those of Huxley, Simpson, Dobzhansky, Mayr, and Stebbins [the founders of the Modern Synthesis] We are certainly not ready for a new moult, but signs of new organs are perhaps visible." This recognition of developmental ideas "points forward to a broader synthesis in the future." We have finally broken through the old pupal integument, and a new, broader, developmentally inclusive evolutionary synthesis is taking wing *Behe (1996) makes this point explicitly, using the example of the eye Although he attempts to disprove the theory of evolution by using the eye as an example, he never once mentions the studies on Pax6 Rather, Behe mentions theories from the 1980s (based solely on population genetics) and puts them forth as contemporary science Snapshot Summary: Evolutionary Developmental Biology Evolution is caused by the inheritance of changes in development Modifications of embryonic or larval development can create new phenotypes that can then be selected Darwin's concept of "descent with modification" explained both homologies and adaptations The similarities of structure were due to common ancestry (homology), while the modifications were due to natural selection (adaptation to the environmental circumstances) The Urbilaterian ancestor can be extrapolated by looking at the developmental genes common to invertebrates and vertebrates and which perform similar functions These include the Hox genes that specify body segments, the tinman gene that regulates heart development, the Pax6 gene that specifies those regions able to form eyes, and the genes that instruct head and tail formation Changes in the targets of Hox genes can alter what the Hox genes specify The Ubx protein, for instance, specifies halteres in flies and hindwings in butterflies Changes of Hox gene expression within a region can alter the structures formed by that region For instance, changes in the expression of Ubx and abdA in insects regulate the production of prolegs in the abdominal segments of the larvae Changes in Hox gene expression between body regions can alter the structures formed by that region In crustaceans, different Hox expression patterns enable the body to have or to lack maxillipeds on its thoracic segments Changes in Hox gene expression are correlated with the limbless phenotypes in snakes Changes in Hox gene number may allow Hox genes to take on new functions Large changes the numbers of Hox genes correlate with major transitions in evolution Duplications of genes may also enable these genes to become expressed in new places The formation of new cell types may result from duplicated genes whose regulation has diverged 10 In addition to structures being homologous, developmental pathways can be homologous Here, one has homologous proteins organized in homologous ways These pathways can be used for different developmental phenomena in different organisms and within the same organism 11 Deep homology results when the homologous pathway is utilized for the same function in greatly diverged organisms The instructions for forming the central nervous system and for forming limbs are possible examples of deep homology 12 Modularity allows for parts of the embryo to change without affecting other parts 13 The dissociation of one module from another is shown by heterochrony (changing in the timing of the development of one region with respect to another) and by allometry (when different parts of the organism grow at different rates) 14 Allometry can create new structures (such as the pocket gopher cheek pouch) by crossing a threshold 15 Duplication and divergence are important mechanisms of evolution On the gene level, the Hox genes, the Distal-less genes, the MyoD genes, and many other gene families started as single genes The diverged members can assume different functions 16 Co-option (recruitment) of existing genes and pathways for new functions is a fundamental mechanism for creating new phenotypes One such recruitment is the limb development pathway being used to form eyespots in butterfly wings 17 Developmental modules can include several tissue types such that correlated progression occurs here, a change in one portion of the module causes changes in the other portions When skeletal bones change, the nerves and muscles serving them also change 18 Tissue interactions have to be conserved, and if one component changes, the other must If a ligand changes, its receptor must change Reproductive isolation may result from changes in sperm or egg proteins 19 Developmental constraints prevent certain phenotypes from occurring Such restraints may be physical (no rotating limbs), morphogenetic (no middle finger smaller than its neighbors), or phyletic (no neural tube without a notochord) 20 The Hsp90 protein enables cells to accumulate genes that would otherwise give abnormal phenotypes When the organisms are stressed during development, these phenotypes can emerge 21 The merging of the population genetics model of evolution with the developmental genetics model of evolution is creating a new evolutionary synthesis that can account for macroevolutionary as well as microevolutionary phenomena A partial list of genes active in human development Gene I Transcription Factors Androgen receptor* AZF1* CBFA1* EMX2* Estrogen receptor* Forkhead-like15 GLI3 (Ci)* HOXA-13* HOXD-13* LMX1B* MITF* MSX2* PAX2 PAX3* Phenotype(s) caused by mutations of the gene Androgen insensitivity syndrome Azoospermia (male sterility due to low sperm number) Cleidocranial dysplasia (defects in skull and shoulders due to poor osteoblast differentiation) Schizencephaly (infolding of cortical neurons due to migration defect) Growth regulation problems, sterility Thyroid agenesis and cleft palate Grieg syndrome (digital malformations and skull shape) Hand-foot-genital syndrome Syndactyly (fused digits) or polysyndactyly (extra toe and webbed digits) Nail-patella syndrome (ventralization of limb) Waardenburg syndrome II (microphthalmia, deafness, pigment loss) Craniosynostoses (fusions of skull and limb bones) Renal-coloboma (iris) syndrome Waardenburg syndrome I (microphthalmia, deafness, pigment loss); cleiocranial dysplasia Aniridia Reiger's syndrome (dental, iris, and corneal defects) Congenital cataracts Deafness and dystonia (mitochondrial transport protein deficiency) Campomelic dysplasia (bowed legs) and male sex reversal Male sex reversal Schinzel syndrome (ulna, mammary, and sweat gland anomalies) Holt-Oram syndrome (hand and heart anomalies) Treacher-Collins syndrome (cranial facial dysplasia) Seathre-Chotzen syndrome (digit webbing, facial abnormalities) Various urogenital anomalies, depending on mutation PAX6* PITX2* PITX3 POU3F4 SOX9* SRY* TBX3 TBX5* TCOF TWIST* WT1* II Paracrine Factors and Their Signaling Pathways AMH* Persistence of Müllerian duct in males AMHR Persistance of Müllerian duct in males due to deficiency of AMH receptor DHCR7* Microcephaly or holoprosencephaly, mental retardation, hypotonia, male genital anomalies due to cholesterol deficiency EDA1* Anhydrotic ectodermal dysplasia (poor teeth and sweat gland formation) EDN3* Hirschspung's disease due to endothelin deficiency EDNRB* Hirschsprung's disease due to endothelin receptor deficiency FGFR1* Pfeiffer syndrome (digit and cranial abnormalities) FGFR2* Several skeletal syndromes involving digits, face, and skull, depending on type of mutation FGFR3* Dwarfisms: hypochondroplasia, achondroplasia, or thanatophoric KIT* LIS* RET* SHH* III Structural Proteins Enzymes Adenosine deaminase Arylsulfatase E COL1A1, 2A1 COL4A5 Connexin-32* Connexin-43* Elastin Fibrillin* Glypican-3 Jagged* KALIG-1* Ketosteroid reductase A2* L1CAM* Lysyl oxidase Myosin VIIA p57 (KIP2) Steroid sulfatase WRN * dysplasia, depending on the severity of the mutation Piebaldism Lissencephaly (mental retardation due to poor neuronal migration) Hirschsprung's disease (pigment and gut neuron deficiency) Holoprosencephaly due to Sonic hedgehog deficiency and Combined T and B cell immunodeficiency, short stature Chondrodysplasia punctata (mental retardation, deafness, cartilage nodules) Osteogenesis imperfecta (insufficient collagen) or Ehrler-Danlos syndromes, depending on severity of the mutation Alport syndrome due to insufficient renal collagen Degeneration of spinal nerve roots Cardiac malformations, abnormal spleen and lung development Aortic stenosis Marfan syndrome (tall stature, loose lenses, aortic fragility) Gigantism Alagille syndrome (jaundice, abnormal vertebrae, pointed mandible) Kallmann syndrome (anosmia and male hypogonadism) Male pseudohermaphroditism (1) MASA (mental retardation, aphasia, shuffling gait, adducted thumbs) or (2) hydrocephalus, depending on mutation Ehlers-Danlos syndrome VI (collagen defect) Usher syndrome (deafness, depigmented retina) Beckwith-Wiedemann syndrome (gigantism due to failure to regulate cell cycle) Ichthyosis (skin scales of cholesterol sulfate) Premature aging the gene probably encodes a helicase for DNA replication Indicates that the gene is discussed either in the text or on the website ... Determining the Function of Genes during Development Identifying the Genes for Human Developmental Anomalies Principles of Development: Genes and Development References The genetic core of development: ... Developmental Biology References Appendix PARTE Principles of development in biology Developmental biology: The anatomical tradition The Questions of Developmental Biology According to Aristotle,... References Early development in selected invertebrates An Introduction to Early Developmental Processes The Early Development of Sea Urchins The Early Development of Snails Early Development in Tunicates