1 12/10/2017 Origin of Animals

2 Evolution of Development: Evolution of Animal Body Plans as an Example

3 Or, another way to conceptualize today’s lecture:

4 Evolution of Gene Regulatory Networks: Evolution of Development as an Example

5 What is an Animal? What makes them different from other organisms? When did they Evolve? How did they Evolve?

6 What is an Animal? Multicellular (metazoan)12/10/2017 What is an Animal? Multicellular (metazoan) Heterotrophic (eat, not photo or chemosynthetic) Eukaryote No Cell Walls, have collagen Nervous tissue, muscle tissue Particular Life History-developmental patterns (this lecture)

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8 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 Polyploidy is tolerated more readily and common in plants

9 Outline Today: Bigger picture on how radical changes in body plan 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

10 Outline Next Lectures: Human Evolution… a great example of Evolution of Development Most differences between humans and other primates are due to evolutionary changes at a few developmental genes

11 Review concepts from previous lectures:cis- and trans-regulation Transcription factors Pleiotropy Cambrian Explosion Phylogeny

12 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)?

13 Ontogeny 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 embryos of vertebrates developed, they passed through stages that resembled the adult phase of more ancestral (“primitive”) organisms. For example, at one point each human embryo 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

14 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 hold for the evolution of developmental genes.

15 Romanes's 1892 copy of Ernst Haeckel’s embryonic drawings

16 The Cambrian Explosion12/10/2017 The Cambrian Explosion

17 230 mya: Permian Extinction12/10/2017 65 mya: Cretaceous Extinction (dinosaurs go extinct) 230 mya: Permian Extinction 570 mya: Cambrian Explosion

18 12/10/2017

19 Evolution of Animal Body Plans12/10/2017 Evolution of Animal Body Plans True Tissues Tissue Layers (Diplo vs Triploblasts) Body Symmetry Evolution of body cavity (Coelom) Evolution of Development

20 12/10/2017 Cambrian Explosion

21 12/10/2017

22 How could this happen? (genetic mechanism?)12/10/2017 How could this happen? (genetic mechanism?)

23 The Evolution of Development (Freeman& Herron, Chapter 19)The tremendous increase in diversity during the Cambrian explosion 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

24 The discovery of Hox genesNot the “most important” dev 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. Examples of Homeotic genes: Hox genes, paraHox genes, MADS-box containing genes, etc.

25 Changes in a few regulatory genes could have big impacts12/10/2017 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.

26 For example, small changes in gene regulation could cause changes in timing of developmental events (heterochrony), which could then lead to dramatic changes in morphology Stephan Jay Gould in 1977 proposed this as a mechanism for evolutionary change

27 So, what happened during the Cambrian Explosion?

28 Cambrian Explosion (1) Precambrian-Paleozoic Boundary (~570 MYA)12/10/2017 (1) Precambrian-Paleozoic Boundary (~570 MYA) All major Animal Phyla (different body plans) evolved within a relatively narrow window of time Cambrian Explosion

29 Precambrian Annelida Arthropoda Mollusca Agnatha Gnathostomata12/10/2017 Annelida Mollusca Million Years Ago Arthropoda Agnatha Echinodermata Gnathostomata 200 Cambrian “Cambrian Explosion” 600 Based on phylogeny of animals based on DNA sequence data, the radiation of animals predates the geological record of the Cambrian Explosion 800 1000 Precambrian 1200 Wray et al. 1996 1400

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

31 The Grand Mystery Why has there has been so little change in animal body plans since the Cambrian Explosion???

32 Davidson & Erwin Gene Regulatory Networks and the Evolution of Animal Body Plans. Science. 311:

33 Big phylogeny “Kernels” “Gene Batteries”

34 Different Hierarchical Components of Gene Regulatory Networks‘‘Kernels’’ of the GRN: Evolutionarily inflexible subcircuits (of regulatory genes) that perform essential upstream functions in building given body parts  main differences among phyla ‘‘Plug-ins’’ of the GRN: Certain small subcircuits (of regulatory genes), that have been repeatedly co-opted for diverse developmental purposes 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) Differentiation Gene Batteries: Consist of groups of protein-coding genes under common regulatory control, the products of which execute cell type–specific functions  Species differences

35 First, Basics on Developmental Gene Regulatory Networks

36 Developmental Gene Regulatory NetworkThe 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.

37 S. Sinha

38 Developmental Gene Regulatory NetworkThe 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.

39 Developmental Gene Regulatory NetworkExample shown for neural development

40 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

41 What kind of evolutionary changes (i. eWhat kind of evolutionary changes (i.e. mutations) lead to the evolution of Gene Regulatory Networks?

42 Evolutionary Changes within the Gene Regulatory NetworksDevelopmental Biologists have hypothesized that most changes within regulatory networks would be cis-regulatory (e.g. promoter, enhancer 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 occur, they have profound effects. So, developmental evolutionary changes have been assumed to be mostly cis-regulatory.

43 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.

44 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 morphogenesis are at the periphery.

45 Developmental Gene Regulatory NetworkExample shown for neural development

46 The fundamental differences“Kernels” “Gene Batteries”

47 Development occurs through a sequence of eventsDuring Development, regulation of gene expression is critical for determining the differential fate of genetically identical cells Morphological patterning during the course of development: General  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:

48 Christiane Nüsslein-Volhard and Sean Carroll12/10/2017 Christiane Nüsslein-Volhard and Sean Carroll

49 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.

50 Christiane Nüsslein-Volhard and Sean Carroll12/10/2017 Christiane Nüsslein-Volhard and Sean Carroll

51 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.

52 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.

53 Different Hierarchical Components of Gene Regulatory Networks‘‘Kernels’’ of the GRN: Evolutionarily inflexible subcircuits (of regulatory genes) that perform essential upstream functions in building given body parts  main differences among phyla ‘‘Plug-ins’’ of the GRN: Certain small subcircuits (of regulatory genes), that have been repeatedly co-opted for diverse developmental purposes 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) Differentiation Gene Batteries: Consist of groups of protein-coding genes under common regulatory control, the products of which execute cell type–specific functions  Species differences

54 Different Components of Gene Regulatory Networks‘‘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 Bilateria); eyefield specification [in arthropods]; gut regionalization [in chordates]; development of immune systems [across Bilateria]; and regionalization of the hindbrain and specification of neural crest [in chordates]

55 ‘‘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 pattern formation 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.

56 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 Fig. 2. Examples of putative GRN kernels. Networks were constructed and portrayed using BioTapestry software (55). (A) Endomesoderm specification kernel, common to sea urchin and starfish, the last common ancestor of which lived about half a billion years ago. The relevant area of the sea urchin network is shown at the top [(1, 9, 16); for currently updated version, details, and supporting data, see (56)]; the corresponding starfish network (14) is shown in the middle; and the network architecture, which has been exactly conserved since divergence—i.e., the kernel—is shown at the bottom. Horizontal lines denote cis-regulatory modules responsible for the pregastrular phase of expression considered, in endoderm (yellow), mesoderm (gray), or both endoderm and mesoderm (striped gray and yellow). The inputs into the cis-regulatory modules are denoted by vertical arrows and bars. The gray box surrounding the foxa input indicates that this repression occurs exclusively in mesoderm. (

57 Possible heart specification kernels; assembled from many literature sources. Dashed lines show possible interactions. (B) Possible heart specification kernels; assembled from many literature sources (15). Dashed lines show possible interactions. Some aspects of the GRN that may underlie heart specification in Drosophila are shown at the top; the approximately corresponding vertebrate relationships are shown in the middle; and shared linkages are shown at the bottom. Absence of a linkage simply means that this linkage is not known to exist, not that it is known not to exist. Many regulatory genes participate in vertebrate heart formation for which orthologous Drosophila functions have not been discovered, and the hearts themselves are of very different structure. However, as pointed out by many authors [see (7, 8, 57) for reviews of earlier references], a core set of regulatory genes are used in common and are now known to be linked in a similar way in a conserved subcircuit of the gene network architecture, as shown. The gray boxes represent in each case different ways that the same two nodes of the network are linked in Drosophila and vertebrates. These networks are also highly recursive

58 General Model for Heart Specification KernelA 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)

59 Zebrafish endoderm kernel (subcircuit)Photo shows gene expression of 4 transcription factors that are part of this kernel Genomes and Developmental Control An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish Wen-Fang Tsenga, Te-Hsuan Janga, Chang-Ben Huanga, Chiou-Hwa Yuh Fig. 1. Temporal and spatial expression profiles of gata5, gata6, otx2 and prdm1a. (A) Quantitative expression profiles of gata5, gata6, otx2 and prdm1a determined by using Q-PCR. The X-axis represents RNA molecules per embryo defined by Q-PCR. RNA was isolated from embryos at different time points, and the absolute molecular numbers per embryo are shown as a purple line for gata5, a blue line for gata6, a red line for otx2, and a green line for prdm1a. The experiments were conducted in triplicate, and the standard deviation is shown as a bar extending to both sides of the mean. (B–E) In situ hybridization of gata5, gata6, otx2 and prdm1a in wild-type embryos at 5 hpf. gata5 and gata6 are expressed in the mesendoderm and the YSL at 5 hpf. otx2 is expressed in the blastoderm at 5 hpf, and prdm1a is expressed in the blastoderm and ectoderm. Arrow indicates the location of mesendoderm lineage at the margin of blastoderm. (F–K) Co-localization of otx2, gata5 and gata6 at 5 hpf double in situ hybridization for gata6 and otx2 (F, G, H) and gata5 and gata6 (I, J, K) in wild-type embryos at 5 hpf. (F) gata6 mRNA is indicated by green fluorescence. (G) otx2 mRNA is shown by red fluorescence. (H) Merged image of F and G, indicating the co-localization of gata6 and otx2 in the mesendoderm. (I) gata5 mRNA is indicated by green fluorescence. (J) gata6 mRNA is shown by red fluorescence. (K) Merged image of I and J indicating the co-localization of gata5 and gata6 in the mesendoderm. Arrow indicates the location of mesendoderm lineage at the margin of the blastoderm. Tseng et al. 2011

60 Different Hierarchical Components of Gene Regulatory Networksblank ‘‘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: signal transduction systems, Wnt, transforming growth factor–b (TGF-b), fibroblast growth factor, Hedgehog, Notch, and epidermal growth factor

61 Sonic Hedgehog signaling pathway:Key role in regulating vertebrate organogenesis, such as in the growth of digits on limbs and organization of the brain. Sonic Hedgehog signaling controls neuronal identity in the developing spinal cord. Illustration shows the expression of the sonic hedghog protein (yellow) in floor plate cells and its influence on cell pattern in the spinal cord and hindbrain. Sonic hedghog induces expression of the homeodomain protein Nkx2.2 (red) in the most ventral progenitor cells and restricts expression of Pax6 (green) to more dorsal progenitor cells. Motor neurons are defined by expression of the homeodomain protein Isl1 (blue) and derive both from Nkx2.2 and ventral Pax6 progenitor cells. Image by J.Ericson. Ericson J and Jessell, T. et al (1997) Cell 90, Sonic Hedgehog (yellow) signaling controlling neuronal identity in the developing spinal cord

62 Different Hierarchical Components of Gene Regulatory Networks3. 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

63 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

64 Hox Clusters Gene family formed by gene duplication events12/10/2017 Hox Clusters Gene family formed by gene duplication events Hox gene products are transcription factors, regulatory proteins 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 embryonic development (e.g. legs, antennae, and wings in fruit flies or the different vertebrate ribs in humans)

65 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

66 Mutations in a Hox gene causing legs to grow out of the headIn this case, the identity of one head segment has been changed to that of a thoracic segment. Figure 19.2b Homeotic mutants in Drosophila (b) Mutations in the antp gene can produce adult flies with legs growing from the head. In this case, the identity of one head segment has been changed to that of a thoracic segment.

67 Hox genes in DrosophilaHox genes tend to be clustered along a chromosome in the order that they are expressed in many taxa (flies and vertebrates), but not all taxa Figure 19.1 Hox genes in Drosophila In these diagrams,T1-T3 and A1-A8 indicate the three thoracic and eight abdominal segments, respectively. The int, mx, and 1a regions of the embryo form head structures. The bottom part of the figure shows the relative locations of genes in the HOM cluster. Each gene is color coded to indicate where it is expressed. Expression of pb, for example, influences the identity of cells that make the proboscis or mouthparts of the fly; both are shaded green. From Gerhart and Kirschner (1997).

68 Confocal image of septuple in situ hybridization exhibiting the spatial expression of Hox gene transcripts in a developing Drosophila embryo. Stage 11 germband extended embryo (anterior to the left) is stained for labial (lab), Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), Abdominal-B (Abd-B). Their orthologous relationships to vertebrate Hox homology groups are indicated below each gene. P.Z. Myers

69 Evolution of Hox clusters12/10/2017 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

70 Animal body plans 12/10/2017

71 Evolutionary changes in Hox GenesNew 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

72 Hox gene 6 Hox gene 7 Hox gene 8 Ubx About 400 mya Drosophila ArtemiaFigure Origin of the insect body plan Drosophila Artemia

73 Figure 19.4 The arthropod radiationNote the diversity of form and function in the segments and limbs of these representative taxa. Onychophorans are the closest living relative of arthropods.

74 Differences in Hox gene expression distinguish the various arthropod segmentation patternsFigure 19.5 Differences in Hox gene expression distinguish the various arthropod segmentation patterns The Hox cluster for all arthropods is similar, represented by the color-coded boxes on the left. The evolutionary tree shows the relationships between some of the major arthropod groups and the Onychophora. The diagrams on the right show where the various Hox genes are expressed in representatives of these taxa. Appendages such as antennae, mouthparts, wings, and legs are shown for some segments. Each unique body plan is associated with a unique pattern of Hox gene expression. From Knoll and Carroll (1999).

75 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

76 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

77 Hypothetical vertebrate ancestor (invertebrate) with a single Hox cluster First Hox duplication Hypothetical early vertebrates (jawless) with two Hox clusters Second Hox duplication Figure Hox mutations and the origin of vertebrates Vertebrates (with jaws) with four Hox clusters

78 Different Hierarchical Components of Gene Regulatory Networks4. 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.

79 Different Hierarchical Components of Gene Regulatory Networks4. 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.

80 Evolution within Developmental Gene Regulatory NetworksSo.... Kernels of the network: Kernels specify the domain for each body part in the spatial coordinate system of the postgastrular embryo Highly pleiotropically constrained internal recursive wiring—many linkages 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.

81 Evolution within Developmental Gene Regulatory NetworksIn 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 adaptation to the environment

82 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)

83 So then, why did massive diversification of major body forms (evolutionary changes in the pleiotropic kernels) occur at the time of the “Cambrian Explosion” And why did such changes not occur after that?

84

85 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.

86 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.

87 Diverse kinds of change in GRNs and their diverse evolutionary consequencesFig. 3. The left column shows changes in network components; the right column shows evolutionary consequences expected, which differ in their taxonomic level (red).

88 Big phylogeny “Kernals” “Gene Batteries”

89 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 differences among animal phyla (e) They function by allowing or disallowing developmental subcircuits to function within segmental regions (like an "on/off" switch)

90 2. Which of the following would be most evolutionary constrained2. 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

91 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

92 4. What are hox genes within an individual animal4. What are hox genes within an individual animal? (a) Orthologs (b) Paralogs (c) Homologs (d) Xenologs (e) None of the above

93 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

94 1D 2B 3E 4B 5D

95 Optional Slides (for your own interest)

96 Different sub-circuits within Gene Regulatory NetworksDon’t need to know this, just showing as an example a | The control system of a typical differentiation gene battery. The output of the specification GRN is expression in given cells of a small set of 'differentiation driver' transcription factors. In combination, these drivers transcriptionally activate the protein-coding genes of the 'differentiation gene battery', because these genes are all controlled by cis-regulatory modules that include target sites for subsets of the drivers. The driver regulatory genes are often cross-regulated, as in the feedback circuit shown, and they provide the multiple inputs into the downstream differentiation genes. These are typically 'wired' in parallel, often in feed-forward circuitry1, 2, 65. b | The control system for a morphogenetic function, such as invagination or migration. Based on the few available examples (including Refs 22, 66), the output of the specification GRN is expression of transcription factors that activate (or repress) a few key checkpoint genes (so-called morphogenes), which are required to trigger the process. Most of the genes encoding proteins that contribute to the process (the effector support genes) are broadly expressed rather than directly controlled by the specification GRN (in contrast to the way differentiation gene batteries are controlled). c | The components and topology of a specification GRN (Box 1) and diverse types of evolutionary change that might occur therein1, 2. The GRN includes a kernel of highly conserved regulatory interactions that establish the progenitor field for regional specification of a developing structure (shown in grey). This kernel in turn provides key inputs into regionally active sub-circuits, the role of which is to establish regional regulatory states (green). The outputs of these activate or repress the activity of differentiation gene batteries at the periphery of the GRN (red). GRNs also encompass Input/Output (I/O) switches that permit or prohibit the operation of the regulatory sub-circuits (purple), and signals between the regulatory sub-circuits (dashed line). At the right of the figure, using the same colour code, are listed the different types of change that might occur as cis-regulatory modules evolve, according to where in the hierarchical GRN the affected cis-regulatory modules operate.

97 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 Single base-pair mutation can cause gain of new binding sites, loss of sites, or strengthening or weakening of binding to sites. 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. 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 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. 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 Regional duplications can result in subfunctionalization and neofunctionalization. Translocations can bring new genes into large regulatory domains.