Origin of

Carol E. Lee University of Wisconsin 1 Copyright©2020; do not upload without permission of Development: Evolution of Body Plans as an Example Or, another way to conceptualize today’s lecture: Evolution of Regulatory Networks: Evolution of Development as an Example C.H. Waddington --his resurgence • Largely dismissed during the Evolutionary Synthesisà attacked for being “Lamarckian” for his ideas on “” Conrad Hal Waddington • Father of – Introduced the concept of “Canalization” – Coined the term “”

Interested in the interplay between (response to stress) and selectionà “Genetic Assimilation” Why did Waddington become popular starting in the 1990’s?

• Evo-Devo: Evolution of Development as playing an important role in the evolution of – Evolution of developmental program could cause radical phenotypic change – The idea of evolution of canalization and decanalization of a developmental program – Genetic Assimilation: Stress could cause decanalization, and the phenotypes that are exposed could then be under selection à creation of “Hopeful Monsters” • What is an Animal? • What makes them different from other organisms?

• When did they Evolve? • How did they Evolve? What is an Animal?

Multicellular (metazoan) Heterotrophic (eat, not photo or chemosynthetic)

No Cell Walls, have collagen Nervous tissue, muscle tissue

Particular History-developmental patterns (this lecture) • Some animals (Plankton): • https://www.youtube.com/watch?v=xFQ_fO2 D7f0 Are there differences between plant and animal evolution? • Greater diversity in sexual systems in plants

– Abundant asexuality • More chemistry less behavior in plants • Development is less rigid and regulated in plants: perhaps allowing for more evolution by “hopeful monsters,” as developmental abnormalities are more tolerable in plants • is tolerated more readily and common in plants Outline • Today: Bigger picture on how radical changes in come about

• Evolution of Development • Evolution of Developmental Gene Regulatory Networks (GRNs) • Hierarchy in Evolution of GRNs • Evolution of GRNs leading to evolution of major phylogenetic breaks in Earth History Review concepts from previous lectures:

• cis- and trans-regulation • factors • • Cambrian “Explosion” • Phylogeny Evolution of Development:

• What is it?

• How can it lead to evolution of radical changes in body plan?

• How can different types of developmental changes (mutations at different developmental stages) lead to different hierarchical evolutionary changes (that distinguish phylum, class, order, family, genus, species)? Recapitulates Phylogeny Ernst Haeckel (1834-1919) • Ontogeny is the course of development of an organism from fertilized egg to adult; phylogeny is the evolutionary history of a group of organisms.

• Haeckel observed that as of vertebrates developed, they passed through stages that resembled the adult phase of more ancestral (“primitive”) organisms. For example, at one point each human has gills and resembles a tadpole.

• Haeckel’s idea was that a species’ biological development, or ontogeny, parallels and summarizes the species’ evolutionary history, or phylogeny Ontogeny Recapitulates Phylogeny Ernst Haeckel (1834-1919)

• Some of his analogies have been discredited (in favor of Von Baer’s ideas)

• However, Haeckel's general concept, that the developmental process reveals some clues about evolutionary history, appears to often hold for the evolution of developmental Romanes's 1892 copy of Ernst Haeckel’s embryonic drawings The Cambrian “Explosion” 65 mya: Cretaceous Extinction (dinosaurs go extinct)

230 mya: Permian Extinction

570 mya: Cambrian “Explosion”

Evolution of Animal Body Plans

• True Tissues • Tissue Layers (Diplo vs Triploblasts) • Body Symmetry • Evolution of body cavity (Coelom) • Evolution of Development Cambrian “Explosion”

Evolutionary and comparative physiology Zoology 611/612 Instructor: Scott Hartman 12:05-12:55 pm MWF (3 credits) / Optional capstone lab 1:20-5:20 M (2 credits)

This course is offered in the Spring and is designed for upper-level undergraduates interested in an integrated approach to animal physiology, ecology and evolution. We examine general physiological principles by comparing taxa from diverse evolutionary histories and ecological to see how evolutionary processes shaped life today and explore how the same processes are shaping the future in our rapidly changing world. We read key papers in evolution, physiology, and ecology that are further explored in student- driven discussion sections. The course also offers an optional 2 credit capstone research lab (Zoo 612), where students design, conduct and statistically analyze physiological experiments on invertebrates. How could this happen? (genetic mechanism?) The Evolution of Development (Freeman& Herron, Chapter 19)

• The tremendous increase in diversity during the Cambrian radiation appears to have been caused by evolution of developmental genes

• Changes in developmental genes can result in radically new morphological forms

• Developmental genes control the rate, timing, and spatial pattern of changes in an organism’s form as it develops into an adult • The discovery of Hox genes – Not the “most important” development genes – Not the only developmental genes – But, among the first studied

Hox genes are types of Homeotic genes, which are genes that control the patterns and order of development in plants and animals. For example, homeotic genes are involved in determining where, when, and how body segments develop in organisms. Hox genes encode transcription factors.

Examples of Homeotic genes: Hox genes, paraHox genes, MADS-box containing genes, etc. Changes in a few regulatory genes could have big impacts

• Most new features of multicellular organisms arise when preexisting cell types appear at new locations or new times in the embryo.

• Changes in the specification of cell fates are a major mechanism for the evolution of different organismal forms. • For example, small changes in gene regulation could cause changes in timing of developmental events (), which could then lead to dramatic changes in morphology

• Stephan Jay Gould in 1977 proposed this as a mechanism for evolutionary change So, what happened during the Cambrian “Explosion”? (1) Precambrian-Paleozoic Boundary (~570 MYA)

All major Animal Phyla (different body plans) evolved within a relatively narrow window of time Cambrian “Explosion” Million Million Years Ago Cambrian 1400 1200 1000 800 200 600 0

Precambrian

Mollusca

Annelida Arthropoda

Echinodermata

Agnatha

Gnathostomata Explosion record Cambrian of the predates geological the radiation of animals data,sequence the on DNA based animals on phylogenyBased of Wray et al. 1996 Explosion” “Cambrian The Grand Mystery

• How did all the animal phyla appear within a relatively short period of time?

• How can different types of developmental changes lead to different hierarchical evolutionary changes (over longer periods of time, that distinguish phylum, class, order, family, genus, species) The Grand Mystery

Why has there been so little change in major animal body plans since the Cambrian “Explosion”??? Davidson & Erwin. 2006. Gene Regulatory Networks and the Evolution of Animal Body Plans. Science. 311: 796-800. Big phylogeny

“Kernels”

“Gene Batteries” Different Hierarchical Components of Gene Regulatory Networks

1. ‘‘Kernels’’ of the GRN: Evolutionarily inflexible subcircuits (of regulatory genes) that perform essential upstream functions in building given body parts à main differences among phyla

2. ‘‘Plug-ins’’ of the GRN: Certain small subcircuits (of regulatory genes), that have been repeatedly co-opted for diverse developmental purposes

3. Input/Output (I/O) devices within the GRN: Switches that allow or disallow developmental subcircuits to function in a given context (e.g. Hox genes)

4. Differentiation Gene Batteries: Consist of groups of - coding genes under common regulatory control, the products of which execute cell type–specific functions à Species differences First, Basics on Developmental Gene Regulatory Networks Developmental

• The binding of transcription factors to regulatory DNA sequences controls the spatial and temporal expression of genes in the developing organism

• Because each regulates the expression of multiple genes, regulatory gene interactions form a network S. Sinha Developmental Gene Regulatory Network

• The binding of transcription factors to regulatory DNA sequences controls the spatial and temporal expression of genes in the developing organism

• Because each transcription factor regulates the expression of multiple genes, regulatory gene interactions form a network. Developmental Gene Regulatory Network

Example shown for neural development Developmental Gene Regulatory Networks (GRNs)

• Development is controlled directly by progressive changes in the regulatory state in the spatial domains of the developing organism.

• As regulatory genes regulate one another as well as other genes, and because every regulatory gene responds to multiple inputs while regulating multiple other genes, the total map of their interactions has the form of a network.

• Gene Regulatory Networks consist of: • Regulatory genes, which encode transcription factors • Signaling genes, which encode ligands and receptors for intercellular communication What kind of evolutionary changes (i.e. mutations) lead to the evolution of Gene Regulatory Networks? Evolutionary Changes within the Gene Regulatory Networks • Developmental Biologists have hypothesized that most changes within regulatory networks would be cis-regulatory (e.g. , at the gene)

• The reason is that cis-regulatory changes would only change the expression of one gene

• On the other hand, Trans-regulatory changes are often overly pleiotropic, and thus don’t occur as often. But, when they do occur, they have profound effects.

• So, developmental evolutionary changes have been assumed to be mostly cis-regulatory. But, trans effects have big effects when they do arise Developmental Gene Regulatory Networks (GRNs)

• Comparative developmental evidence indicates that reorganizations in developmental gene regulatory networks (GRNs) underlie evolutionary changes in animal morphology, including body plans.

• The nature of the evolutionary alterations that arise from regulatory changes depends on the hierarchical position of the change within a GRN. Developmental Gene Regulatory Networks (GRNs)

• GRNs are hierarchical, so that the portions controlling the initial stages of development are at the top of the hierarchy (early in development), the portions controlling intermediate processes of spatial subdivision or the formation of future morphological pattern are in the middle, and the portions controlling the detailed functions of cell differentiation and are at the periphery. Developmental Gene Regulatory Network

Example shown for neural development The fundamental differences

“Kernels”

“Gene Batteries” Development occurs through a sequence of events

• During Development, regulation of is critical for determining the differential fate of genetically identical cells

• Morphological patterning during the course of development: General à more detailed

• Developmental changes lead to divergence at different hierarchical levels from the more upstream “kernels” early in development, to the more peripheral “gene batteries”

• Ontogeny recapitulates phylogeny: Christiane Nüsslein-Volhard and Sean Carroll Ontogeny Recapitulates Phylogeny Ernst Haeckel (1834-1919)

• Haeckel’s idea was that a species’ biological development, or ontogeny, parallels and summarizes the species’ evolutionary history, or phylogeny

• Haeckel's general concept, that the developmental process reveals some clues about evolutionary history, might generally hold for the evolution of developmental genes. Christiane Nüsslein-Volhard and Sean Carroll Architectural changes in animal body plans might have been produced over the past 600 million years by changes in GRNs (gene regulatory networks) of multiple classes, with extremely different developmental consequences and rates of occurrence. Evolution of GRNs

• The modular sub-circuits of developmental GRNs differ in evolutionary lability ().

• The most slowly changing components — called kernels — consist of highly conserved regulatory interactions that establish the progenitor field of a developing structure.

• The evolutionary stability (constraint) of kernels contrasts with the lability (evolvability) of other GRN sub-circuits. Different Hierarchical Components of Gene Regulatory Networks (NOT comprehensive list) 1. ‘‘Kernels’’ of the GRN: Evolutionarily inflexible subcircuits (of regulatory genes) that perform essential upstream functions in building given body parts à main differences among phyla

2. ‘‘Plug-ins’’ of the GRN: Certain small subcircuits (of regulatory genes), that have been repeatedly co-opted for diverse developmental purposes

3. Input/Output (I/O) devices within the GRN: Switches that allow or disallow developmental subcircuits to function in a given context (e.g. Hox genes)

4. Differentiation Gene Batteries: Consist of groups of protein- coding genes under common regulatory control, the products of which execute cell type–specific functions à Species differences Different Components of Gene Regulatory Networks 1. ‘‘Kernels’’ of the GRN: Evolutionarily inflexible (constrained) subcircuits that perform essential upstream functions in building given body parts

• Often dedicated to major formation of body parts • Often sub-circuit of interacting transcription factors • Often highly constrained by pleiotropy • Often cannot undergo evolutionary change without catastrophic effects

• Examples in next four slides. Other possible Examples : anterior to posterior and midline to lateral specification of the nervous system (in deuterostomes and possibly across ); eyefield specification [in ]; gut regionalization [in chordates]; development of immune systems [across Bilateria]; and regionalization of the hindbrain and specification of [in chordates] Christiane Nüsslein-Volhard and Sean Carroll ‘‘Kernels’’ of the GRN • Kernels are sub-circuits composed of recursively wired regulatory genes (that is, they share inputs through multiple cis- regulatory interactions), which operate during the initial phase of regional for a particular body part.

• If any of the genes in the sub-circuit are prevented from functioning, the body part fails to develop.

• A kernel interacts with regional regulatory state sub-circuits, which in turn activate or repress the activity of differentiation gene batteries at the periphery of the GRN (next figures).

• The conserved structure of developmental GRN kernels might be responsible for the phenotypic stability of animal body plans that has persisted at least since the Early Cambrian period, 520 million years ago. Endomesoderm specification kernel, common to sea urchin and starfish, the last common ancestor of which lived about half a billion years ago.

Five of the six genes in the kernel (all except delta) encode DNA-recognizing transcription factors

The linkages are highly recursive. The cis-regulatory module of the otx gene receives input from three of the five genes; the foxa gene, from three of the five; and the gatae, foxa, and bra genes from two of the same five genes Possible heart specification kernels; assembled from many literature sources. Dashed lines show possible interactions.

These networks are also highly recursive General Model for Heart Specification Kernel

A core set of regulatory genes are used in common and are linked in a similar way in a conserved subcircuit of the gene network architecture (grey boxes) endoderm kernel (subcircuit)

Photo shows gene expression of 4 transcription factors that are part of this kernel

Tseng et al. 2011 Different Hierarchical Components of Gene Regulatory Networks 1. blank 2. ‘‘Plug-ins’’ of the GRN: Certain small subcircuits that have been repeatedly co-opted for diverse developmental purposes

• Not dedicated to formation of body parts. Instead, they are inserted in many different networks where they provide inputs into a great variety of regulatory apparatus.

• Often expressed differentially in the (species-specific) terminal phases of development

• Their connections into the network are evolutionarily very labile (evolvable)

• Examples: systems, Wnt, transforming growth factor–b (TGF-b), fibroblast growth factor, Hedgehog, Notch, and epidermal growth factor signaling pathway:

Key role in regulating vertebrate organogenesis, such as in the growth of digits on limbs and organization of the brain.

Sonic Hedgehog (yellow) signaling controlling neuronal identity in the developing spinal cord Different Hierarchical Components of Gene Regulatory Networks

3. Input/Output (I/O) devices within the GRN: Switches that allow or disallow developmental subcircuits to function in a given context

• Permit or prohibit the operation of the regulatory sub- circuits, and signals between the regulatory sub-circuits

• They can act to permit or prohibit patterning subcircuits from acting in given regions of an animal.

• Examples: regulation of size of homologous body parts. regulation of fate of segments in animals hox genes, Ubx, pitx2 Hox Genes

• Hox genes are examples of “Input/Output Devices”… that is, operate like “on/off” switches

• If they are “on” within an animal region, they will dictate the fate of that segment

• Hox genes are transcription factors, which regulate genes that in turn regulate large networks of other genes Hox Clusters

• Gene family formed by events

• products are transcription factors, regulatory that bind to DNA and control the transcription of other genes

• Hox genes determine the identity of segmental regions along the anterio-posterior axis of animals during early (e.g. legs, antennae, and wings in fruit flies or the different vertebrate ribs in humans) Hox Genes

• Hox genes are a class of homeotic genes that provide positional information during development

• If Hox genes are expressed in the wrong location, body parts can be produced in the wrong location

• For example, in crustaceans, a swimming appendage can be produced in a segment instead of a feeding appendage Mutations in a Hox gene causing legs to grow out of the head

In this case, the identity of one head segment has been changed to that of a thoracic segment. Hox genes in

Hox genes tend to be clustered along a in the order that they are expressed in many taxa (flies and vertebrates), but not all taxa P.Z. M ye rs Evolution of Hox clusters

• HOX-clusters undergo essential rearrangements in evolution of main taxa

• Duplication, deletion, divergence of the genes lead to differentiation in body plans

• Other regulatory genes/gene families are also important Animal body plans Evolutionary changes in Hox Genes

• New morphological forms likely come from gene duplication events that produce new developmental genes

• A possible mechanism for the evolution of six- legged insects from a many-legged crustacean ancestor has been demonstrated in lab experiments

• Specific changes in the Ubx gene have been identified that can “turn off” leg development Hox gene 6 Hox gene 7 Hox gene 8

Ubx

About 400 mya

Drosophila Artemia

Differences in Hox gene expression distinguish the various patterns Evolution of Vertebrates (Phylum Chordata)

• Evolution of vertebrates from invertebrate animals was associated with alterations in Hox genes

• Two duplications of Hox genes are thought to have occurred in the vertebrate lineage

• These duplications may have been important in the evolution of new vertebrate characteristics • Polyploidization is probably the single most important mechanism for the evolution of major lineages and for speciation in plants

Multiple rounds of polyploidization might have occurred during the early evolution of vertebrates Hypothetical vertebrate ancestor (invertebrate) with a single Hox cluster First Hox duplication

Hypothetical early vertebrates (jawless) with two Hox clusters Second Hox duplication

Vertebrates (with jaws) with four Hox clusters Different Hierarchical Components of Gene Regulatory Networks

4. Differentiation Gene Batteries:

Consist of groups of functionally linked protein-coding genes under common regulatory control, the products of which execute cell type–specific functions and are major determinant of cell specialization in metazoans

They are expressed in the final stages of given developmental processes. Different Hierarchical Components of Gene Regulatory Networks 4. Differentiation Gene Batteries: Consist of groups of functionally linked protein-coding genes under common regulatory control, the products of which execute cell type–specific functions and are major determinant of cell specialization in metazoans • Reside at the periphery of developmental GRNs, and are expressed in the final stages of given developmental processes • They do not regulate other genes (in contrast to kernels and plug-ins, which are entirely regulatory) • They do not control the progressive formation of spatial patterns of gene expression that underlies the building of the body plan; in short, they do not make body parts. • Differentiation gene batteries build muscle cells and make skeletal biominerals, skin, synaptic transmission systems, etc. Evolution within Developmental Gene Regulatory Networks

So.... Kernels of the network: • Kernels specify the domain for each body part in the spatial coordinate system of the postgastrular embryo • • Highly pleiotropically constrained o internal recursive wiring—many linkages o position high in the developmental network hierarchy

When sufficient comparative network data are available, it is likely that conserved network kernels will be found to program the initial stages of development of every phylum-specific body part and perhaps of superphylum and pan-bilaterian body parts as well. Evolution within Developmental Gene Regulatory Networks

In contrast, peripheral regions of the GRN (i.e. differentiation gene batteries) are less pleiotropically constrained, and more likely to evolve.

There are no downstream consequences in changes at this level.

Examples: many cases of speciation, many cases of to the environment So, not all mutations are equal:

Mutations that are retained that affect the earlier stages of development (e.g. kernels) will have more profound effects on animal body plans than mutations that affect the terminal steps of development (e.g. gene batteries) So then, why did massive diversification of major body forms (evolutionary changes in the pleiotropic kernels) occur at the time of the “”

And why did such changes not occur after that?

The kernels would have formed through the same processes of evolution that affect the other components (while new lineages were forming during the late Pre-Cambrian-early Cambrian),

But, once formed and operating to specify particular body parts, kernel structure would have become refractory (resistant) to subsequent change (because of the catastrophic costs of altering fundamental structures—because the developmental pathways had already been laid out).

Molecular phylogeny places this evolutionary stage in the late Neoproterozoic when Bilateria begin to appear in the fossil record, between the end of the Marinoan glaciation at about 630 million years ago and the beginning of the Cambrian. Therefore the mechanistic explanation for the surprising fact that essentially no major new phylum-level body parts have evolved since the Cambrian may lie in the internal structural and functional properties of GRN kernels: Once they were assembled, they could not be disassembled or basically rewired, only built upon. Diverse kinds of change in GRNs and their diverse evolutionary consequences

Fig. 3. The left column shows changes in network components; the right column shows evolutionary consequences expected, which differ in their taxonomic level (red). Big phylogeny

“Kernals”

“Gene Batteries” Sample Exam Questions

1. Which of the following is FALSE regarding hox Genes? (a) They serve the role of defining segmental regions along the anterior to posterior axis during development (b) Their functions have diversified through gene duplications followed by differentiation (e.g. subfunctionalization), leading to differentiation of segmental regions in animals (c) They encode transcription factors that perform trans- regulatory functions (d) They are responsible for the major body plan differences among animal phyla (e) They function by allowing or disallowing developmental subcircuits to function within segmental regions (like an "on/off" switch) 2. Which of the following would be most evolutionary constrained? (a) Plug-ins of the GRN (b) Kernels of the GRN (c) Input/Output devices (d) Gene batteries (e) Hox genes 3. Changes at which below are most likely to be responsible for the radiation of animal phyla? (a) Plug-ins of the GRN (b) Sonic Hedgehog (c) Input/Output devices (d) Gene batteries (e) Kernels of the GRN 4. What are hox genes within an individual animal? (a) Orthologs (b) Paralogs (c) Homologs (d) Xenologs (e) None of the above 5. Developmental evolutionary differences between humans and chimpanzees are most likely to be at the level of (a) Plug-ins of the GRN (b) hox genes (c) Input/Output devices (d) Gene batteries (e) Kernels • 1D • 2B • 3E • 4B • 5D • Optional Slides (for your own interest) Different sub- circuits within Gene Regulatory Networks

Don’t need to know this, just showing as an example Changes that can affect cis-regulatory modules (CRMs) (can review lecture notes on cis-regulatory evolution)

• Internal changes that affect the function of a pre-existing CRM o Single base-pair mutation can cause gain of new binding sites, loss of sites, or strengthening or weakening of binding to sites. o Insertions and deletions can change the distance between interacting sites, cause gain or loss of sites, or an increase in the copy number of given sites. o Insertion of mobile element carrying regulatory sequences can cause gain or potential loss of site, change in the distance between interacting sites and increase in copy number, as well as alter the strength of binding at the site.

• Changes that alter CRM repertoire of pre-existing genes o Insertion of CRMs from elsewhere: carried by mobile elements, by inversions, by translocations, or by intronic retrotranspositions can cause gain of developmental functions without loss of the gene. o Loss of a CRM: by translocation, large deletion, inversion breakage or insertion of mobile element can cause loss of specific developmental function without loss of gene.

• Large-scale rearrangements that produce novel gene–CRM complexes o Regional duplications can result in subfunctionalization and neofunctionalization. o Translocations can bring new genes into large regulatory domains.