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Reference request - A complete step-by-step explanation of meiosis, inheritance in humans and epigenetics

Reference request - A complete step-by-step explanation of meiosis, inheritance in humans and epigenetics


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I am a medical student and as such had contact with lots of superficial and bad biology/biochemistry literature. I have searched the internet for introductory articles, searched youtube for video lectures, they all fail at various stages!

What I want: I want a readable, logically-coherent and precise description/explanation of meiosis and a description of human inheritance, preferably in book-form and in one book. I want definitional precision, lots of my textbooks cannot even distinguish coherently between chromosomes and chromatides. Because of that, it took me quite some time to understand that "same" in "the same genes on homologous chromosomes" refers to sameness on a type-level not token-level. I have heard that Bruce Albert's book would be the right choice, but the price is hefty and I have grown skeptical of student literature.

Why I do not trust wikipedia but ask here? This supposed example of epigenetics:

"In a groundbreaking 2003 report, Caspi and colleagues demonstrated that in a robust cohort of over one-thousand subjects assessed multiple times from preschool to adulthood, subjects who carried one or two copies of the short allele of the serotonin transporter promoter polymorphism exhibited higher rates of adult depression and suicidality when exposed to childhood maltreatment when compared to long allele homozygotes with equal ELS exposure."

Call me stupid, but that sounds more like an example for a disposition to develope depression rather than an example of epigenetic inheritance. And for this reason I do not trust wikipedia on that matter.

Questions I want to have answered (by the book/requested reference):

  1. In diploid organisms, if both alleles are different but neither is dominant over the other, what determines at what rate each protein is produced? What if the two alleles are co-dominant? Is it always fifty-fifty?

  2. How do dominant genes repress the gene products of the same, recessive gene of the homologous chromosome?

  3. A precise description of epigenetics! What it is and what it isn't! Listening to my lecturers (physicians,medical school!) I get the feeling that with epigenetics "anything is possible" which I don't buy. How does

  4. How does genomic imprinting work? By that I mean not so much the underlying molecular mechanism but its consequences on phenotypes of future generation. From my lectures one could conclude that Lamarckism is back, and I doubt that! I read somewhere (Jerry Coyne's blog) that genomic imprinting only lasts a few generations, otherwise it would seriously undermine natural selection as a mechanism of evolution and I can see that.

  5. (A question I would like to have answered in this thread): According to my superficial lectures and German wikipedia, genomic imprinting can be understood as parent-specific expression of genes. Furthermore, there are certain alleles that under genomic imprinting can be either recessive, if inherited from the mother, or dominant, if coming from the father. Now, what if such an allele is located on the father's y-chromosome? Under the assumption that the father has a healthy son and that son has a son and that son has a son, and so on, it seems to me that there is no end to this kind of inheritance and genomic imprinting, contra Jery Coyne, can last indefinitely. Where am I wrong?

I hope you can guide a frustrated med student to some sort of understanding!

All the best!


The Cell Cycle, Mitosis and Meiosis

Actively dividing eukaryote cells pass through a series of stages known collectively as the cell cycle: two gap phases (G1 and G2) an S (for synthesis) phase, in which the genetic material is duplicated and an M phase, in which mitosis partitions the genetic material and the cell divides.

  • G1 phase. Metabolic changes prepare the cell for division. At a certain point - the restriction point - the cell is committed to division and moves into the S phase.
  • S phase. DNA synthesis replicates the genetic material. Each chromosome now consists of two sister chromatids.
  • G2 phase. Metabolic changes assemble the cytoplasmic materials necessary for mitosis and cytokinesis.
  • M phase. A nuclear division (mitosis) followed by a cell division (cytokinesis).

The period between mitotic divisions - that is, G1, S and G2 - is known as interphase.


Chromosomes

Meiosis Label – look at cells in various stages of meiosis, identify and order
Meiosis Internet Lesson – look at animations of meiosis and answer questions
Meiosis Powerpoint – slideshow covers meiosis, homologous chromosomes, crossing over…

Modeling Chromosomal Inheritance – use pipe cleaners to show how genes are inherited independent assortment, segregation, sex-linkage

Linkage Group Simulation – uses pipe cleaners and beads, students construct chromosomes with alleles and perform crosses, predicting outcomes (advanced)
Karyotyping Online – use a website simulator to learn how to pair chromosomes and diagnose abnormalities
Karyotyping Online II – another simulation on how to construct a karyotype
Chromosome Study – cut out chromosomes and tape them in pairs to construct a “paper” karyotype

Gender and Sex Determination – NOVA explores how sex is determined, and social issues of gender

DNA Powerpoint Presentation – covers the basics for a freshman level class

DNA Coloring – basic image of DNA and RNA
DNA Crossword – basic terms

How Can DNA Replication Be Modeled – students use colored paperclips to model how one side of the DNA serves as a template during replication (semi-conservative)

Transcription & Translation Coloring – shows structures involved, nucleotides, base pair rules, amino acids

DNA Analysis: Recombination – simulate DNA recombination using paper slips and sequences
DNA Extraction – instructions for extracting DNA from a strawberry, very simple, works every time!
DNA in Snorks – analyze and transcribe DNA sequences, construct a creature based on that sequence

How DNA Controls the Workings of a Cell – examine a DNA sequence, transcribe and translate
DNA Sequencing in Bacteria – website simulates the sequencing of bacterial DNA, PCR techniques
Ramalian DNA – imagine an alien species that has triple-stranded DNA, base pair rules still apply
Who Ate The Cheese – simulate gel electrophoresis to solve a crime
HIV Coloring – shows how viral DNA enters and infects a cell

Genetic Science Ethics – survey as a group ethical questions involved genetics (cloning, gene therapy..)
Your Genes Your Choices – this is a more involved group assignment where groups read scenarios about genetic testing and ethics involved.
Genetic Engineering Concept Map – Complete this graphic organizer on various techniques used in genetics, such as selective breeding and manipulating DNA

Virtual Labs and Resources

Genetic Engineering – presentation on cloning, recombinant DNA, and gel electrophoresis
Biotechnology Web Lesson – students explore genetic science learning center (https://learn.genetics.utah.edu/) and discover how clones are made, and how DNA is extracted and sequenced
Genetic Science Learning Center – explore website with animations and tutorials, answer questions

DNA From the Beginning -step by step tutorial on the discovery of genes, DNA, and how they control traits, site by Dolan DNA Learning Center
DNA Fingerprinting – another simulation, this one from PBS, that walks you through the steps of creating a DNA Fingerprint
Cloning – Click and Clone at GSLC where you can read about how clones are made and clone your own virtual mouse


Portions of DNA Sequence Are Transcribed into RNA

The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence𠅊 gene—into an RNA nucleotide sequence. The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name transcription.

Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds (Figure 6-4). It differs from DNA chemically in two respects: (1) the nucleotides in RNA are ribonucleotides—that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose (2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA. Since U, like T, can base-pair by hydrogen-bonding with A (Figure 6-5), the complementary base-pairing properties described for DNA in Chapters 4 and 5 apply also to RNA (in RNA, G pairs with C, and A pairs with U). It is not uncommon, however, to find other types of base pairs in RNA: for example, G pairing with U occasionally.

Figure 6-4

The chemical structure of RNA. (A) RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA, by the presence of an additional -OH group. (B) RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, (more. )

Figure 6-5

Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing thus, U-A base pairs closely resemble T-A base pairs (see Figure 4-4).

Despite these small chemical differences, DNA and RNA differ quite dramatically in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. RNA chains therefore fold up into a variety of shapes, just as a polypeptide chain folds up to form the final shape of a protein (Figure 6-6). As we see later in this chapter, the ability to fold into complex three-dimensional shapes allows some RNA molecules to have structural and catalytic functions.

Figure 6-6

RNA can fold into specific structures. RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base-pairs with complementary sequences found elsewhere on the same molecule. These interactions, along (more. )


Five Misconceptions in Genetics

Students may bring a variety of misconceptions with them when they enter a study of genetics. Watch your classroom for the 5 common misconceptions listed below. If you find any of them, just use the simple explanations𠅊lso provided below—to dispel your students’ incorrect notions.

  1. One set of alleles is responsible for determining each trait, and there are only 2 different alleles (dominant and recessive) for each gene. After learning about simple Mendelian inheritance and sex-linked traits, students often think that it is possible to model all traits so easily and predictably. In humans, at least 3 different genes are associated with eye color. Coat color in cats is controlled by at least 6 genes. Furthermore, the number of particular alleles inherited determines the expression of some characteristics for example, the number of alleles—that you inherit from each parent—that code for production of melanin may partially determine your hair color. Inheritance of more of the alleles may lead to darker hair, while inheritance of fewer may lead to lighter hair. For traits that show a Mendelian pattern of inheritance, students often assume that there are only 2 possible alleles for a trait. This is true in some cases, but in many cases, there are more alleles for a trait. In cat-coat-color genetics, 3 different alleles of 1 gene determine the position of pigmentation on the body.
  2. Your genes determine all of your characteristics, and cloned organisms are exact copies of the original. While genes play a huge role in how an organism develops, environmental factors also play a role. Epigenetics is the study of heritable changes that occur without changes in the genome. The gene expression in identical mice has shown changes from factors such as diet and exposure to toxins. Further studies with identical twins have suggested that these changes can accumulate over the life of the organism. The cloning of Rainbow, a domestic cat, demonstrated 1 striking example of epigenetics. Rainbow’s coat showed calico coloration, while the coat of the clone, named Copycat, is a tabby pattern. Because Copycat and Rainbow had identical genomes, the differences must be due to epigenetic factors.
  3. All mutations are harmful. A mutation is a change in the genetic code of an organism. Many mutations are harmful and cause the organism not to develop properly. However, many mutations are silent and some prove beneficial. In the case of a silent mutation, the change in the genome does not change the production of the amino acid sequence and subsequent protein (remember that multiple codons may code for the same amino acid, so a change in 1 nucleotide does not necessarily change the gene product). If an organism does live with a mutation, then often the environment will determine whether the mutation is beneficial or harmful. Production of 1 protein vs. another may confer a characteristic such as a difference in coloration or in the ability to digest a resource (e.g., the ability to digest lactose or maltose instead of sucrose). The phenotypic outcome may be selected, for or against, depending on environmental factors.
  4. A dominant trait is the most likely to be found in the population. The term 𠇍ominant allele” sometimes conveys to students the impression that the allele is the one that exists in the greatest proportion in a population however, 𠇍ominant” refers only to the allele’s expression over another allele. Human genetics includes examples of dominant traits that do not affect the majority of the population. In fact, achondroplasia, a type of dwarfism caused by the presence of a dominant allele, is found in fewer than 1 in 10,000 live births. Huntington’s disease, a degenerative disease caused by the presence of a dominant allele, occurs at a rate of about 3 to 7 cases per 100,000 people of European descent.
  5. Genetics terms are often confused. Many students understand the basic ideas of genetics but need more familiarity with the terms. For example, students often struggle with the difference between a chromosome, a gene, and an allele. Chromosomes are organized structures containing proteins and a single coiled strand of DNA chromosomes are visible with a microscope only during parts of the cell cycle. Genes are units of heredity—specific sequences of DNA or RNA that create proteins with particular functions in an organism. Alleles are variants of a gene. Making sure that students have a strong foundation in the terminology can greatly improve their understanding of genetics and prevent misconceptions.

Dispelling these 5 misconceptions will help students better understand genetics information and activities that you plan for both the classroom and the lab. They will also realize there are many influences on the way living things develop genetically over time.

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Curriculum Content

Overview

Content (from A Level)
The content from the specification that is covered by this delivery guide is:
5.1.2 Population genetics and epigenetics
(a)the role of natural selection in changing allele frequencies within populationsTo include the link between malaria and the frequency of the sickle cell allele including the effect on the phenotype of each of the three possible genotypes for the normal and sickle cell allele.
M1.4, M1.5, M2.2, M2.3, M2.4
(b)the link between the changes in the amino acid sequence to the change in structure and properties of proteins (e.g. haemoglobin)HSW1, HSW5, HSW6
(c)the use of Hardy-Weinberg equations to analyse changes in allele frequencies in populationsHSW3, HSW8
M2.1, M2.2, M2.3, M2.4
(d)factors other than natural selection that contribute to genetic biodiversityTo include the role of the founder effect and genetic bottlenecks in creating genetic differences between human populations. Examples to include blood group distribution and Ellis-van Creveld syndrome distribution.
HSW1, HSW5, HSW6, HSW9, HSW10, HSW12
(e)the role of geographical and reproductive isolation in the formation of new speciesTo include a consideration of the implications for speciation of primates, including humans.
(f)epigenetics in terms of the effect of environment on gene expressionTo include theories of the role of DNA methylation and histones in gene expression AND a review of some human epigenetic studies (such as the Norrbotten studies, studies on the effect of the Dutch Hunger Winter and twin studies) and possible implications from these studies.
M1.7
HSW1, HSW2, HSW5, HSW6, HSW8, HSW9, HSW10, HSW11, HSW12
Charles Darwin is credited with coining the term Natural Selection after his publication of the book The Origin Of Species but it was actually a joint presentation by Alfred Russel Wallace and Charles Darwin that brought the theory of Natural Selection to the public. Many advances in our understanding of inheritance has lead to a branch of biology known as epigenetics.

This topic provides a number of specific challenges for teachers. These include:

  • being aware of misconceptions students may have towards natural selection and speciation, particularly in relation to geographical and reproductive isolation mechanisms
  • helping students to perceive natural selection and the consequences it has on population genetics
  • overcoming students aversion to mathematical calculations such as Hardy-Weinberg equations
  • making students aware of the factors other than natural selection that contribute to genetic diversity.

Examples of evolution

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8 examples of evolution in action

Genetic drift

McGraw Hill provide a short animation showing genetic drift within a population. This can be used in conjunction with the webpage by Nectunt who provide a range of resources aimed at helping students understand evolution with a particular focus on genetic drift, bottle necking and the founder effect amongst other topics.

Summarising the resources in a flow diagram or similar representation would be of use to the students.

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Genetic drift simulation
Nectunt

Causes of genetic variation

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Nature.com

Hardy-Weinberg equation

A brief overview of the calculation is available from Nature.com. This can be used with the animation from Intergrative Biology showing the factors that must be controlled in completing the Hardy-Weinberg equation for the more visual learner.

These resources could be used by the students to research and complete examples posed by the teacher at the start of the lesson.

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Nature.com: Overview
Animation

Charles Darwin game

The Science Channel have a Charles Darwin game ‘who wants to live a million years’ showing how mutations can increase the chance of survival during particular environmental changes.

Would make an ideal plenary.

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Charles Darwin game

For more information about cell growth and division:

The National Human Genome Research Institute's Talking Glossary provides information about the cell cycle.

The National Cancer Institute's fact sheet What is Cancer? explains the growth of cancerous tumors.


Evolutionary biology today and the call for an extended synthesis

Evolutionary theory has been extended almost continually since the evolutionary synthesis (ES), but except for the much greater importance afforded genetic drift, the principal tenets of the ES have been strongly supported. Adaptations are attributable to the sorting of genetic variation by natural selection, which remains the only known cause of increase in fitness. Mutations are not adaptively directed, but as principal authors of the ES recognized, the material (structural) bases of biochemistry and development affect the variety of phenotypic variations that arise by mutation and recombination. Against this historical background, I analyse major propositions in the movement for an ‘extended evolutionary synthesis’. ‘Niche construction' is a new label for a wide variety of well-known phenomena, many of which have been extensively studied, but (as with every topic in evolutionary biology) some aspects may have been understudied. There is no reason to consider it a neglected ‘process’ of evolution. The proposition that phenotypic plasticity may engender new adaptive phenotypes that are later genetically assimilated or accommodated is theoretically plausible it may be most likely when the new phenotype is not truly novel, but is instead a slight extension of a reaction norm already shaped by natural selection in similar environments. However, evolution in new environments often compensates for maladaptive plastic phenotypic responses. The union of population genetic theory with mechanistic understanding of developmental processes enables more complete understanding by joining ultimate and proximate causation but the latter does not replace or invalidate the former. Newly discovered molecular phenomena have been easily accommodated in the past by elaborating orthodox evolutionary theory, and it appears that the same holds today for phenomena such as epigenetic inheritance. In several of these areas, empirical evidence is needed to evaluate enthusiastic speculation. Evolutionary theory will continue to be extended, but there is no sign that it requires emendation.

1. Introduction

The current framework of evolutionary theory grew out of the evolutionary synthesis (ES), or the modern synthesis, as Huxley [1] called it. In any discussion of extending or revising current theory, some understanding of the history of the ES and the subsequent development of the subject will be useful. My impression of the history of biology, and of evolutionary biology in particular, is one of generally gradual, rather than paradigm-shaking, development that builds successively on previous accomplishments. For example, soon after the discovery and canonization of Mendel's ‘laws’ in the earliest twentieth century, the ‘law' of independent assortment had to be modified to account for linkage. The ‘gene' went from a particulate ‘factor' to a trinity of recon, muton and cistron (unit of recombination or mutation or function), thence to a protein-signifying code, and recently to an increasingly ambiguous functional part of a genome. Nevertheless, genetics has not cast out the old to accommodate the revolutionary new. Quite the opposite: classical Mendelian segregation, meiosis, linkage mapping and mutation are still important foundations of today's immensely more complex genetics.

The same holds for the evolutionary theory that has developed since the late 1920s. The ES [2] remains, mutatis mutandis, the core of modern evolutionary biology. The ES included both the formulation of population genetic theory by Fisher [3], Haldane [4] and Wright [5], and the interpretation of variation within species [6] and of diverse information in zoology [7–9], botany [10] and palaeontology [11].

Since the 1930s and 1940s, there has been a steady incorporation of new information, ranging from phylogeny and field studies of natural selection to evolutionary genomics and the panoply of genetic phenomena that could not have been imagined in the 1940s or even the 1960s—information that has informed (and sometimes been predicted by) a steady expansion of theory. Modern evolutionary biology recognizes and studies transposable elements, exon shuffling and chimeric genes, gene duplication and gene families, whole-genome duplication, de novo genes, gene regulatory networks, intragenomic conflict, kin selection, multilevel selection, phenotypic plasticity, maternal effects, morphological integration, evolvability, coevolution and more—some of these being phenomena and concepts unknown or dimly perceived a few decades ago.

— The basic process of biological evolution is a population-level, not an individual-level, process that entails change not of the individual organism, but of the frequency of heritable variations within populations, from generation to generation. Dobzhansky defined evolution as change of allele frequencies, but some organism-focused evolutionary biologists, such as Rensch, Simpson and Mayr, had a more comprehensive conception of evolution, including phenotypic evolution, speciation and differential proliferation of clades, while recognizing that phenotypic evolution and speciation occur by changes in allele frequencies. When Rensch wrote of ‘Evolution above the species level' and Simpson wrote about ‘Tempo and mode in evolution', they were not talking about allele frequencies—although they recognized this as the elementary, generation by generation process of change.

— Heredity is based on ‘genes’, now understood to be DNA or RNA. DNA sequences transmitted in eukaryotes' gametes are not affected by an individual organism's experiences. Cultural inheritance has long been recognized, but insofar as it affects biological evolution, it does so by affecting natural selection. Some authors prefer to limit the term ‘inheritance' to genetic transmission. For the sake of using a common language in this discourse, I will use the term ‘inclusive inheritance' to include several forms of non-genetic ‘inheritance’, recognizing that this terminology may be disputed.

— Inherited variation arises by individually infrequent mutations they are random in that their phenotypic effects, if any, are not directed towards ‘need'. ‘Random' should always be qualified by ‘with respect to' randomness of mutation has never meant that all possible alterations are equally likely, or that all genes mutate at the same rate, or that rates of mutation are immune from environmental factors (such as radiation and mutagens). Claims of ‘directed mutation' have been shown to be groundless [12]. The great majority of mutations that affect fitness are deleterious [13]. Likewise, the direct effects of novel environments are more often harmful than beneficial: that is why they engender natural selection and adaptive change. These facts imply that we should be sceptical of the view that organisms are so constructed as to have well integrated, functional responses to mutations or novel environments (as proposed in [14, p. 2]). Without question, organisms have diverse homeostatic properties that buffer fitness against many environmental or genetic destabilizing events but the maintenance of function depends on stabilizing or purifying natural selection.

— The frequencies of hereditary variants are altered by mutation (very slightly), gene flow, genetic drift, and natural selection. Directional or positive natural selection is the only known cause of adaptive change. Natural selection is not an agent, but a name for a consistent (biased, non-random) difference in the production of offspring by different classes of reproducing entities. The entities that were the focus of the ES were mostly phenotypically different individual organisms, but they can also be genes (as already recognized by Fisher, Haldane and Wright), populations or species.

— Species of sexually reproducing organisms are reproductively isolated groups of populations that arise by evolutionary divergence of geographically isolated (allopatric) populations. Species evolve gradually, so not all populations can be classified into discrete species. Non-allopatric speciation is now recognized as possible, although its frequency is unknown.

— Large phenotypic changes of the kind that distinguish higher taxa and occur over long periods of time evolve gradually, as Darwin proposed, i.e. by the cumulation of relatively small incremental changes.

It is important to recognize that in population genetics theory, ‘mutation’ means any new alteration of the hereditary material that is stably transmitted across generations. The discovery of the molecular basis of heredity after the ES led to a greatly amplified understanding of evolutionary process and history, but the core theory of population genetics remained intact. For example, the core theory does not specify whether a mutation is a single base pair substitution, an insertion of a transposable element in a regulatory sequence, a gene duplication or a doubling of the entire genome. The framework of population genetics has incorporated new kinds of mutations, such as transposable elements, as they have been discovered.

Natural selection commonly was, and often still is, thought of as stemming from the ecological environment, but the forgers of the ES were well aware that selection had a far broader basis. Fisher described the evolution by selection of sex ratio, selfing and outcrossing, and he provided a genetic interpretation of Darwin's idea of sexual selection Wright (who influenced Dobzhansky, who influenced Mayr) emphasized epistasis for fitness, in which prevalent alleles at one locus affect the selective value of alleles at another locus. Schmalhausen described ‘internal selection' mutations can have environment-independent effects on the function of physiological and developmental processes, and in turn on viability and reproduction. A causal account of any instance of selection requires different kinds of data—molecular, behavioural, ecological or other—but showing the existence of selection on a gene locus or a trait requires only data on components of fitness, such as rates of survival, fecundity or mating success.

Thus, the broad concepts of mutation and natural selection lack material content, in the sense that empirical data are needed to describe real instances of evolution, by identifying the agents of selection and the molecular and developmental basis of phenotypic variants. The conception of causes of evolution embodied in the synthetic theory, i.e. allele frequency change, differs from the ‘structuralist' view of the causes of differences in morphology, physiology or behaviour that are commonly envisioned by mechanistic developmental biologists, physiologists or neurobiologists (cf. [15]). A ‘structuralist' approach to biology is cast in terms of the physical and chemical features of organisms, such as cell types and organs, and a ‘structuralist' explanation of a morphological difference among species would be expressed in terms of signalling cascades, gene regulation and assembly of proteins into features that distinguish cell types [16, pp. 7–38]. A complete account of any evolutionary change in phenotype would combine the two kinds of information: population genetic processes (causes of allele frequency change) together with the specific agents of selection and the structural and developmental basis of the altered phenotype. Part of the great power of the population genetic theory of evolutionary change lies in its generalization across diverse kinds of mutations, selective causes and phenotypic structures.

The leaders of the ES affirmed Darwin's gradualist view of long-term evolution, and rejected the saltationism of Schindewolf, Goldschmidt and others who supposed that higher taxa evolve by macromutations. Still, what might qualify as a ‘large' mutational change was and is difficult to specify. Certainly some species differences and polymorphisms map to single loci with discretely different effects phenomena such as neoteny (e.g. paedomorphic salamanders) were recognized, and nobody seemed to worry that partially paedomorphic salamanders were unknown and functionally unlikely. The ‘instantaneous’ origin of reproductively isolated species by polyploidy was likewise well known, especially in plants—but the species produced by polyploidy closely resemble the parent species: they are not new higher taxa.

2. Extensions of the evolutionary synthesis

Evolutionary research in the 1950s and 1960s greatly increased information on genetic variation in natural populations, the seeming ubiquity of natural selection and speciation. In the 1960s, efforts to synthesize ecology with evolutionary biology were renewed as ‘population biology' [17,18], the beginning of a flourishing field of evolutionary ecology [19]. The development of kin selection theory and the distinction between individual selection and group selection gave rise to fields such as behavioural ecology and life-history theory. The abundant evidence of natural selection and the development of optimality models for characters that almost unquestionably affect fitness may have led to a broadly held view of selection as an almost exclusive factor of evolution. But the all-important role of selection was challenged by interpretations of molecular polymorphism and evolution in neutralist terms [20–22], and the ‘neutralist–selectionist' debate ultimately resolved itself into rendering unto Kimura and unto Darwin those provinces of variation that each best explains. In the 1980s and 1990s, the field of molecular evolution grew so massively as to warrant its own society and journal. This expansion was accompanied by the maturation of phylogenetic analysis and its long-deferred integration with the study of evolutionary processes [23–25]. Thus, evolutionary theory has undergone enormous expansion since the ES [26,27], with the neutral theory of molecular evolution its most radical extension.

Nevertheless, there have been undercurrents of discontent with the synthetic theory ever since the ES [28]. For example, Beyond neo-Darwinism [29] was a diverse collection of essays by developmental biologists, systematists, ecologists and others whose common characteristic seemed to be only an animus against the prevailing paradigm. Stephen Jay Gould's [30,31] calls for extension of the ES had rather more impact. Gould played a significant role in reviving development as a factor in the evolution of form [32], as well as the role of developmental or genetic constraints. Although Gould flirted with Goldschmidtian saltation, there is still no evidence that single mutations are responsible for the multiple character differences that typify most genera or other higher taxa. However, population geneticists found that many character differences between closely related species appear to be based on fewer gene differences, of larger effect, than previously supposed [33,34]. (Nonetheless, even the effects of a single gene can sometimes be ascribed to complementary effects of several mutations [35].) The model of punctuated equilibria introduced by Eldredge & Gould [36] was a different challenge to gradualism. They claimed that most fossil lineages display rapid shifts (punctuations) between one long-lasting, virtually constant (static) phenotype and another. Following Mayr [37,38] in large part, they postulated that populations cannot readily respond to natural selection because of genetic constraints that may be loosened when a population undergoes a bottleneck associated with founder-effect speciation. This model has been almost universally rejected, but Eldredge and Gould called attention to the important and still not fully explained pattern of stasis, and raised a possible role for speciation in fostering long-term character evolution, which is a topic of ongoing research [39–42]. The controversy unleashed by Eldredge & Gould [36] also contributed to a renaissance of palaeontology, in the form of palaeobiology. Possibly the current calls for an extended synthesis will similarly have some positive effects.

3. Proposed extensions of the evolutionary synthesis

Against this background, I will consider the major themes of the proposed extended evolutionary synthesis (EES), drawing largely on the position paper by Laland et al. [14] and the oral presentations at the discussion meeting sponsored by the Royal Society and the British Academy (November 2016). I will consider niche construction, phenotypic plasticity, inclusive inheritance and the role of development in the evolution of form.

3.1. Niche construction

The concept of niche construction emphasizes ways in which organisms actively modify their environment, such as burrowing by gophers and dam-building by beavers, but the broadest expression of the idea of niche construction is simply, as Lewontin [43, p. 280] wrote, that ‘organisms determine what is relevant'. The core idea is that the evolved properties of organisms make some aspects of the environment relevant sources of natural selection, and screen off others, thereby helping to shape and constrain likely paths of the population's evolution. Thus (although Lewontin did not explicate this point), properties of the organism that we think of as proximate mechanisms (e.g. biochemcal capabilities, tolerances, habitat selection, other behaviours of animals) can determine or even constitute Mayr's ‘ultimate' (i.e. selective) causes of organisms' characteristics. The proponents of niche construction [44] take this broad view, even if they stress examples in which species (especially of animals such as beavers) actively modify their environment—a theme that has also been developed by Dawkins [45].

I have been a naturalist since boyhood. I think I recognized niche construction even then, because Lewontin's principle is blindingly obvious to any naturalist. Even if a species does not literally construct its environment, like a beaver, it determines its environment by its behaviour and physiology. What is relevant to the life of an aerially foraging swift, to a foliage-gleaning warbler, and to a fish-eating loon (diver) is obviously very different. To a eucalyptus-feeding koala or a larval monarch butterfly that eats only milkweed (Asclepias), the defensive compounds of these few plants are highly relevant, but those of hundreds of other plant species in their habitat are not. Likewise, an understory herb experiences a very different environment from an epiphytic bromeliad in the same tropical forest. This principle was obvious to ES figures such as Mayr [46], who emphasized that behaviour (such as habitat choice) often frames selection on morphology and physiology. Jakob von Uexküll's [47] concept of an animal's Umwelt—its species-specific perceptual environment—has long been familiar to students of animal behaviour.

In my own research area, the evolution of herbivore–plant associations, the coevolution of behaviour and physiology has been a major topic for modelling and empirical study [48–50]. In simple two-locus models, one locus affects which species of plant an insect chooses this affects allele frequency dynamics at a second locus that determines fitness on these plants (and vice versa). The evolution of host choice will influence the subsequent evolution of other features, such as coloration that makes the insect cryptic by resembling part of the plant. This coevolution of habitat preference and other features is one of a rather long list of well-studied topics that Odling-Smee et al. include under niche construction, which seems to embrace much of behavioural ecology, evolutionary ecology and (by virtue of the many effects of niche construction) community ecology. So the phenomena gathered under the label ‘niche construction' are unquestionably important, and have long been the subjects of research, but the inclusiveness of the term ‘niche construction' has been cited as ‘precisely what weakens the value of the idea’, for ‘organismal influences on the environment with profoundly different evolutionary impacts are lumped together’ [51].

The related notion of ‘ecological inheritance' can likewise be criticized because of its imprecision and excessive breadth. Odling-Smee et al. [44, p. 45] define ecological inheritance as ‘any case in which organisms encounter a modified feature–factor relationship between themselves and their environment where the change in the selective pressures is a consequence of the prior niche construction by parents or other ancestral organisms’, including ‘the ancestors of other species in their communities’! Their examples include offspring inheritance of their parent's environment (e.g. burrow), occupation of an environment constructed by antecedent generations without reference to kinship, and simple parental care. (For example, because many insects lay eggs on a food plant or other food resource, the offspring ‘inherit from their mother the legacy of an appropriate source of larval food' [44, p. 65].)

‘Ecological inheritance' must differ profoundly from genetic inheritance if it is not transmitted down ancestor–descendant lineages. The critical distinction is whether or not there is covariance between niche-constructing behaviour and offspring fitness [51,52]. The literature of quantitative genetics has long recognized genotype–environment covariance [53]. Situations in which this is the case, including maternal effects and gene–culture coevolution, have been described by models based in traditional population genetics [54,55].

Niche construction may prove useful if it prompts questions and generates research on familiar aspects of biology (as has research on phenomena such as stasis and sex ratios). So far, studies that identify themselves with niche construction have been mostly theoretical, and mostly addressed to cultural niche construction, especially by humans. But these themes had already been addressed long before the term ‘niche construction' was introduced [52], and understudied phenomena are available to anyone who becomes familiar with enough biology (why do haploid chromosome numbers range from one to more than one hundred among insect species?) so associating a study with a term does not in itself show that the term or concept played a critical generative role. A great deal of research, on many topics as I have noted, has long used the concept of niche construction, without using the label.

Odling-Smee et al. [44, p. 2] proposed that niche construction ‘should be regarded, after natural selection, as a second major participant in evolution', and indeed as a ‘core evolutionary process’. In a valuable interchange [56], sceptics point out that niche construction can influence or even cause the evolutionary process of natural selection, but is not itself an evolutionary process, any more than a changing environment is. If niche construction shapes selection, so do the sources of ecological selection, internal selection, and sexual or social selection. The sources of these several forms of selection are not processes. We can identify many evolutionary processes (Red Queen evolution, kin selection, changes in linkage disequilibrium and more), i.e. ongoing series of events that constitute evolutionary change. Are they ‘core' evolutionary processes? I do not know what the criterion of a ‘core’ evolutionary process might be, but none of these seems to be as fundamental and comprehensive as mutation, genetic drift, gene flow and natural selection. Perhaps, a taxonomy of processes would be useful.

Professor Laland also suggests that the value of niche construction is that it provides a different point of view. Whether or not that will prove to be so will depend on whether or not it yields theoretical and empirical research that differs from what would otherwise be pursued [56]. What, exactly, as Gupta et al. [52] ask, has been neglected by standard evolutionary theory that niche construction theory proposes to supply? Will ‘niche construction' be merely a label or ‘brand' that advertises its advocates' research, or will it be uniquely productive of insight and understanding? So far, no new, general theoretical principles that promise to guide novel empirical research have been articulated by proponents of niche construction.

3.2. The role of phenotypic plasticity in evolution

Phenotypic plasticity refers to the expression of different phenotypic states (together forming a norm of reaction) by a single genotype under different environmental conditions. It takes many forms. It can be reversible, as are many behavioural reactions, physiological acclimation, upregulation or downregulation of an enzyme level, and some morphological states, such as seasonal changes in bird plumages. Or it may be irreversible, as are many alternative morphological phenotypes induced by environmental conditions during development familiar examples include height in some plants, the solitary versus gregarious phases of plague locusts (Schistocerca), and the castes of many eusocial insects. Plastic responses sensu lato include many environmentally induced phenotypes that are called developmental defects, such as skeletal aberrations in rickets, but most evolutionary literature is concerned with adaptive plasticity, such as the cases I have cited.

The concept of phenotypic plasticity, if not the term, is about as old as the distinction between genotype and phenotype. I learned about it in an undergraduate genetics course. As a graduate student, I learned that genotype × environment interaction was a staple in quantitative genetics [53,57]. Clausen et al. [58] and many other researchers described adaptive plasticity in plants and animals, and the evolution of adaptive reaction norms was a major topic in Schmalhausen's Factors of evolution [59]. Biologists agreed that some plastic responses are adaptive, and that others are harmful effects of environment. There is no doubt that plasticity can extend tolerance to some new environments and help prevent population extinction. By the 1990s, a large body of quantitative genetic theory on the evolution of adaptive phenotypic plasticity had been developed [60–63], and a large theoretical and empirical literature on the topic has developed since then. For example, young tiger snakes (Notechis scutatus) that are fed larger prey develop longer jaws, and the response is enhanced in an island population that normally feeds on larger prey [64]. This simply shows that reaction norms can evolve by natural selection. There is also an extensive literature on the benefits and costs of phenotypic plasticity. Thus, the phenomenon of phenotypic plasticity is widespread, is very well known and is understood to a considerable degree [65,66].

Under some conditions, the optimal reaction norm is ‘flat', i.e. a constitutive (constant) expression of the same phenotype in all normally encountered environments. The evolution of a constant (constitutive) phenotype from an ancestrally more plastic reaction norm often exemplifies what I will refer to as genetic assimilation, following Waddington [67]. Waddington observed that a phenotype that was induced by an environmental stimulus could become constitutive, and be expressed in the normal environment in the absence of the stimulus, after several generations of selection for individuals most prone to exhibit the phenotype when stimulated. He rightly postulated that selection had increased the frequency of alleles that enhanced the reliability and consistency of the new phenotype. Strong evidence for this hypothesis was provided by experiments in which selection yielded no genetic assimilation in inbred stocks that lacked genetic variation [68].

Genetic assimilation of a character state in a plastic reaction norm underlies a controversial hypothesis by Mary Jane West-Eberhard [69] that she calls genetic accommodation because it includes more genetic shaping of characters than in simple genetic assimilation—which, however, is the core of her hypothesis. West-Eberhard proposed that adaptation to a novel environment often proceeds first by inducing a phenotypic response that increases fitness (phenotypic plasticity), followed by allele frequency changes that assimilate and perhaps fine-tune the new character state, so that it becomes a novel, species-typical trait. She stated, provocatively, that ‘most phenotypic evolution begins with environmentally initiated phenotypic change… The leading event is a phenotypic change with particular, sometimes extensive, effects on development. Gene-frequency change follows, as a response to the developmental change. In this framework, most adaptive evolution is accommodation of developmental-phenotypic change. Genes are followers, not necessarily leaders, in phenotypic evolution' [69, pp. 157–158] The hypothesis has drawn some favourable attention as an important contribution of development to evolution [70].

West-Eberhard's hypothesis is very similar to what Simpson [71] called the Baldwin effect. Simpson said that the idea does not violate the standard theory of evolution by natural section. Indeed, Lande [72] (also Chevin et al. [73]) modelled the role of plasticity and genetic assimilation in adaptation to environmental change, using orthodox quantitative genetics. But Simpson noted that there were few real examples, and doubted they would prove to be common. Should his assessment be revised? Not greatly, at least based on currently available evidence.

West-Eberhard cites many cases in which a species constitutively exhibits a character state that is part of a more variable reaction norm in a related species. In almost none of these examples is there phylogenetic or fossil evidence on the direction of the change, so these tell us only that reaction norms can evolve, an issue not in doubt. Information on the direction of evolution (from plastic to constitutive) has been recognized as an important criterion for testing the hypothesis, and such information is available in a few recent cases which show that genetic assimilation can happen in natural populations [74,75]. For example, montane populations of Daphnia, recently faced with introduced fish that more easily detect melanized individuals, have lost a melanization reaction to ultraviolet light [76]. Anadromous marine sticklebacks (Gasterosteus aculeatus), when reared under lake-like conditions, display changes in body form that slightly resemble those that have evolved in derived lake-dwelling populations [77]. However, there is a long history of bidirectional gene flow between freshwater and marine populations [78], and the possibility cannot be ruled out that the reaction norm of the marine fishes is affected by alleles derived from freshwater populations. The experimental marine fishes do not show a plastic response of other characters, such as gill raker number, that have also evolved in freshwater populations.

More importantly, the genetically variable reaction norms in a population may or may not be oriented towards the character state that is optimal in the altered environment [79]. If it is directed towards the optimum, evolution towards and possible genetic fixation of the optimal character state may occur but as Pigliucci [80, p. 369] (who is sympathetic to West-Eberhard's hypothesis) has noted, the novel environment ‘will often not be novel at all, but will be some variant of the sort of environment that has been common in the history of the species’. In this case, the reaction norm has previously been shaped by natural selection acting on genetic variation genes are ‘followers’ only to the extent that genetic assimilation or accommodation ‘fine-tunes’ an adaptation that had already evolved by selection and genetic variation.

Therefore, phenotypic plasticity could be said to truly play a leading role (with genes as followers) if an advantageous phenotype were to be triggered by an environment that really is novel for the species lineage, an environment that its recent ancestors did not experience and which, therefore, had not exerted natural selection. Of course it is possible that a novel environment—a new pesticide, for example—could evoke a developmental effect that happens to improve fitness, just as it is possible that a random DNA mutation improves fitness. But no theory leads us to expect such an effect to be especially likely. On the empirical front, a few candidate instances have been described. Terrestrial tiger snakes that were raised in water for several months swam faster than land-reared siblings [81] but how does this differ from the performance of trained human athletes? Aside from the lack of inheritance of this phenotypic change, how would one show that this response played a role in the evolution of aquatic species, such as the confamilial sea snakes? Perhaps the best example I have found that fits the Baldwin effect or genetic accommodation is the study by Ledón-Rettig et al. [82] of the tadpoles of spadefoot toads in the genus Spea. Their diet is commonly algae and detritus, but if tadpoles eat animal prey when young, they develop into a carnivorous morph with very large jaw muscles and a shorter gut. Another genus, Scaphiopus, in which the carnivorous morph has never been recorded, feeds entirely on algae and detritus. This is almost surely the ancestral diet, so animal prey is a novel environmental stimulus. Young Scaphiopus tadpoles, experimentally fed shrimp, developed a shorter than normal gut (as in Spea), a point in favour of West-Eberhard's hypothesis. However, they did not develop the most conspicuous features of Spea's carnivorous morph, the greatly enlarged jaw muscles. The critical evidence, induction of an adaptive plastic response by a truly novel environment (to say nothing of subsequent genetic assimilation and accommodation) is supported by only tenuous evidence at this time.

Moreover, phenotypically plastic reactions to novel environments are often wholly or partly counteradaptive. In the well-known phenomenon of countergradient adaptation [83], genetic differences between populations are precisely opposite to the maladaptive direct effects of the different environments the populations inhabit, and compensate for the maladaptive plastic effect [84]. Human acclimation to high altitude improves performance, but at the cost of increased haematocrit, decreased affinity of haemoglobin for oxygen, and hypertensive pulmonary vessels—all features that differ from genetically adapted highland populations [85]. A guppy (Poecilia reticulata) population exhibited evidently maladaptive plastic changes in expression of many genes when reared in a novel environment (one that lacked predatory fish) descendants of this population adapted to a predator-free environment by precisely opposite evolutionary changes in gene regulation [86]. Deleterious phenotypic plasticity may be at least as effective in triggering adaptive genetic change as plasticity that enhances fitness [87].

I conclude that the evidence so far does not warrant much enthusiasm for the proposition that plasticity often paves the way for adaptation to novel selection pressures, much less for novel morphological or physiological adaptations. Abundant traditional theory, based in population genetics, describes how reaction norms evolve by selection on genetic variation, and there is abundant evidence of adaptation by natural selection on standing genetic variation [88,89]. Some conditions favour plasticity, some a fixed phenotype. The implication that development has inherent properties that are usually likely to generate new, adaptively directed phenotypes lacks any theoretical—or material, as far I can tell—foundation.

3.3. Inclusive inheritance

Several bases for non-genetic inheritance (meaning here inheritance that is not based on DNA sequence) have long been recognized, including culture, behavioural imprinting, parental environment including some maternal effects, and parental transmission of nucleic acids and diverse chemical compounds. Some, but by no means all, cases of niche construction also qualify [90]. As noted earlier, many of these phenomena have been modelled and empirically studied by evolutionary biologists for several decades [55,91–93], and it is not clear that the EES brings anything new to the topic.

More recently, novel molecular mechanisms of inheritance have proved to be widespread, such as inherited DNA methylation and other epigenetic ‘marks’. Some authors have placed a provocative Lamarckian interpretation on certain of these phenomena [94,95], while others have urged that they be viewed as ‘interpretive machinery' that can influence gene expression and development, and are inherited along with DNA [96]. These mechanisms can and should be studied, like other organismal and genomic features, in order to determine their evolutionary dynamics and their evolutionary effects. For example, some of these mechanisms can cause traits to continue to evolve after selection has ceased, and can even evolve in a direction opposite to selection [55]. Day & Bonduriansky [96] have developed a general model that they claim applies broadly across the various kinds of non-genetic inheritance, in which a key feature is that phenotypic change across generations can be decoupled from genetic change. Depending on the mechanism, non-genetic inheritance may be more transient (lasting for few generations) or more persistent, but in some cases, even transient inheritance can influence the direction of genetic evolution.

Some inherited epigenetic effects are influenced by environment, and have been described as vindicating Lamarckian inheritance. I think Haig [97] and Dickins & Rahman [98] are likely to be right: they do nothing of the kind. Transgenerational epigenetic inheritance does not intrinsically produce advantageous environmentally induced phenotypes. Epigenetic imprinting, whether inherited or not, can have both benefits and costs, which provide fuel for theoretical and empirical research [99]. Many epigenetic effects are deleterious [100], so population-typical advantageous instances are best interpreted as the result of natural selection of those genetic variants with epigenetic modification that enhance fitness. The capacity of DNA sites to be marked is genetically variable, and epigenetic variation responds to selection [98,100,101]. Therefore, most instances of adaptive epigenetic variation are best viewed as transgenerationally transmitted adaptive phenotypic plasticity [102,103] that has evolved by mutation and natural selection [104].

That is not to deny the possible importance of such effects in evolution, but the importance has yet to be determined. By contrast to a century's accumulated evidence that variation within and among species is based on genes, there is little evidence, so far, that ecologically adaptive features of whole populations or species have an epigenetic basis [105]. The frequency of certain methylated sites differs among some populations of both plants and animals, and in some instances suggests a correlation with environment [106,107], although in only a few cases have population samples been reared in a common environment, in order to exclude direct environmental induction [108]. By contrast, a massive literature provides evidence that character differences between species are based on DNA sequence differences in genes [109]. Epigenetic transmission seems to last for at most a few dozen generations, and usually much less. The peloria variant of the toadflax Linaria vulgaris that Linnaeus described (an epigenetically based reversion from a bilaterally symmetrical to a radially symmetrical flower) can be found in populations today, but there is no reason to think there has been transmission of a mutant lineage since the eighteenth century. At this time, ‘empirical evidence for epigenetic effects on adaptation has remained elusive' [101]. Charlesworth et al. [110], reviewing epigenetic and other sources of inherited variation, conclude that initially puzzling data have been consistent with standard evolutionary theory, and do not provide evidence for directed mutation or the inheritance of acquired characters.

Perhaps epigenetic inheritance will prove to have important effects in evolution, affecting the dynamics and direction of genetic adaptation [100]. However, just as evolutionary biology embraced the discovery of introns, transposable elements, and highly repetitive DNA, and easily adapted traditional population genetic models to describe their evolutionary behaviour, so it will be, I suspect, with epigenetic and other non-genetic inheritance. The basic framework of orthodox evolutionary theory has served well in evolutionary genomics thus far, and will almost certainly do so in this context, too. Evolutionary theory will be extended, just as it has been by other discoveries about genomes, but there is no sign that any of its components will have to be discarded.

3.4. Development and evolution

During and since the ES, relationships between developmental biologists and evolutionary biologists have at times been not entirely comfortable. It is sometimes said that development was excluded from the ES, but there is little ground for this accusation [15,111,112]. In the early twentieth century, experimental embryologists divorced themselves from what they viewed as a descriptive, speculative tradition of evolutionary embryology. The developmental biologist Viktor Hamburger [113] noted that during the period of the ES, books on experimental embryology did not treat evolution Mayr [114] claimed that developmental biologists ‘were not left out of the synthesis…they simply did not want to join'. The split between genetics and embryology, initiated by T. H. Morgan, probably affected the content of the synthetic theory, which built more on genetic than developmental foundations [115]. Nonetheless, development was not entirely ignored, as I note below. (My treatment of this topic draws on a rather lengthy essay on macroevolution [116] that at this time can be downloaded without cost at http://www.springer.com/us/book/9783319150444.)

Whatever the reasons may have been, development was not as effectively assimilated into the ES as it might have been as many authors have noted, the ES lacked a theory of the origin of phenotypic variation, and especially of phenotypic novelty. One may well wonder what kind of theory could have been developed when the mechanisms of development, and even the molecular nature of genes, were entirely unknown. Experimental embryologists used phenomenological descriptors such as induction and prepattern, just as comparative embryologists had descriptors such as heterochrony and allometry. Kirschner & Gerhardt [117, p. 276] write that the ‘Modern Synthesis did not and could not incorporate any understanding of how the phenotype is generated'.

Certainly some evolutionary biologists were sensitive to the significance of development. Huxley contributed an analysis of allometry to the ES, and speculated that it provided a non-adaptive (we might now say pleiotropic) explanation for some exaggerated features. Rensch [8,9] discussed allometry and other developmental phenomena at length, and speculated that parallel evolution, as in the wing patterns of Lepidoptera, may arise from similar genetic and developmental factors. Wright [118,119] provided a polygenic model for threshold traits (and Lande [120,121] later modelled how such traits evolve under natural selection). The idea that development can influence the direction of evolution was fully congenial to the architects of the ES. Mayr [38, pp. 607–610] wrote, in a passage on ‘Evolutionary potential and predisposition', that ‘Every group of animals is “predisposed” to vary in certain of its structures, and to be amazingly stable in others’, and that this is reflected in parallel evolution: ‘Only part of these differences can be explained by the differences in selection pressures to which the organisms are exposed the remainder are due to the developmental and evolutionary limitation set by the organisms' genotype and its epigenetic 1 system…The epigenotype sets severe limits to the phenotypic expression of …mutations it restricts the phenotypic potential'. Stebbins [10] wrote about evolutionary trends in plants, such as the repeated evolution of fused petals, which he analysed in terms of development: ‘while the process of “fusion” is begun by the initiation of a new type of growth center, the degree of union, like that of reduction, is determined chiefly by allometry'. Stebbins [10,122] noted that the evolution of different floral structures constrained the way in which seed number might evolve for example, by increasing the number of ovules per carpel in lilies (Liliaceae), by the number of carpels per flower in buttercups (Ranunculaceae) and by the number of flowers (florets) in the flowerheads of sunflowers (Asteraceae).

The first steps towards modern developmental biology, such as the Jacob & Monod [123] and the Britten–Davidson [124] models of gene regulation, were featured in textbooks by Dobzhansky et al. [125] and myself [126], both of which emphasized that evolutionary changes in gene regulation could underlie morphological evolution. The second edition of my textbook [127] included 14 pages on development and evolution, including discussions of prepatterns, the Turing model of pattern formation, the genetics of segment identity in Drosophila, developmental constraints and developmental integration. Today's leading textbooks of evolutionary biology all cover evolutionary developmental biology [128–131]. There are now journals that integrate developmental and evolutionary perspectives. Development is now well integrated into evolutionary thinking.

The rise of modern EDB (evolutionary developmental biology) is a valuable maturation of a dimension of evolutionary biology that has been present all along. It represents a structuralist approach that adds material mechanisms to the theory of allele frequency change [15]. It is part of a broader union of the theory of evolutionary dynamics with mechanistic biology—a conjunction of Mayr's [132] ‘ultimate' (evolutionary) explanation with ‘proximate' explanation at the level of organisms' structure and function.

These are complementary, not alternative explanations. For instance, one role of EDB may be to demonstrate and clarify the importance of developmental constraints on evolution [133,134]. For example, Brakefield's research group showed that the colours of two spots on the wing of a butterfly species could not be decoupled by artificial selection [135]. This well-known phenomenon is usually referred to as genetic correlation, which is typically ascribed to pleiotropic effects of genes that affect both characters, and is understood to act as a potential constraint on the direction of evolution [39,136,137]. Genetic constraints become developmental constraints when the mechanisms underlying genetic patterns are understood.

Developmental (or genetic) constraints of this kind clearly influence the direction of evolution, just as the direction of my travel is influenced when the police close a road. As the passages I cited from Mayr and Stebbins show, the idea that genetic or developmental ‘potential' biases variation and evolution is neither new nor a challenge to traditional theory. Where EDB can provide insight is by identifying mechanistic causes. The demonstration, early in the twentieth century, that some salamanders are paedomorphic because of reduced thyroxin production provided a mechanistic complement to an evolutionary explanation [32]. Alberch & Gale's [138] famous experiment provided a mechanistic understanding of the evolutionary sequence of digit loss in amphibians, following a long history of developmental and genetic studies of the mechanisms and evolution of the reduction and loss of limbs in tetrapods [120]. Likewise, Turing's model provided a possible mechanism for patterns (e.g. the distribution of hairs or coloured spots on an animal [139]), but it did not explain why an animal would evolve spots. If spots do evolve, there must be a physical mechanism for their production and distribution. This proximate mechanism should not be confused with the ultimate explanation.

EDB helps to explain some enigmatic evolutionary changes. For example, the pectoral girdle of turtles lies below the ribs, but above the ribs in all other tetrapods. Recent descriptions of development, together with a key fossil turtle, show how changes in the development of the rib primordia and in the antero-posterior orientation of the ribs enabled this remarkable change [140,141]. EDB addresses interesting and important questions, such as coordinated evolutionary changes in functionally integrated characters [142] and the origin of truly novel characters [16]. But the argument for the importance of EDB need not be weakened by unnecessary claims and speculations. Müller [143] provides a very interesting treatment of morphological evolutionary novelties, such as the turtle carapace and the mammalian patella (knee cap). He describes models of developmental mechanisms that produce changes that result from ‘activation–inhibition thresholds in geometrically confined spaces’. That may well be, but he goes on to postulate that the innovation may be produced as a phenotypically plastic effect that is later genetically assimilated. Thus ‘genetic evolution, while facilitating innovation, serves a consolidating role rather than a generative one, capturing and routinizing morphogenetic templates’.

I view this as an unnecessary concatenation of speculations. Why is so complex and unsupported a hypothesis needed to explain the origin of the patella, which is formed by existing cellular and molecular processes of osteogenesis in a phylogenetically novel location in the body? Changes in gene regulation are known to trigger expression of entire developmental pathways at different times in development (heterochrony) or different sites in the body (heterotopy). Why not simply suppose genetic variation in sites of gene expression, and natural selection for those particular sites in which novel sesamoid bones prove advantageous? The patella is one of many heterotopic bones (e.g. osteoderms of armadillos) that have clear selective value. There have undoubtedly been many disadvantageous mutations that produced heterotopic bones in places that were decidedly wrong. Simpler explanations are generally preferred over more complex (and vague) hypotheses, unless these are supported by evidence.

The EDB described by some adherents to an EES is oddly different from the research literature that has made the most substantial progress in evolutionary developmental biology. That literature is largely concerned with evolutionary changes in gene regulation, and identifying the nature and causes of changes in cis-regulatory regions and trans-regulatory factors [144–146]. To be sure, much of this literature describes what might be called genetic blueprints or algorithms for making organisms, and not the final molecular processes by which tissues and organs are produced. But models of developmental fields and activation thresholds generally do not achieve this level of mechanism either. Some developmental biologists have been downright anti-genetical in the past [147]. A disjunction of developmental process from the genetic-cum-environmental specification of development seems unlikely to enhance our understanding of evolution.

4. Core assumptions

(i) The ES assumes preeminence of natural selection, the EES ‘reciprocal causation' owing to developmental bias and niche construction. No advocates of the ES have long recognized (as did Darwin himself) that organisms determine, to a considerable extent, the environmental sources of selection, including by modifying their environment, and they recognized a role for developmental (or genetic) biases and constraints that can affect the directions of evolutionary change. But natural selection remains the only process that increases fitness and shapes adaptive characteristics.

(ii) The ES assumes random genetic variation, the EES non-random variation. No it has always been recognized that some phenotypic variants are more likely to arise by mutation (expressed via development) than others. The fact that most mutations that affect fitness are deleterious, and that most novel environments reduce fitness, contradicts the EES claim that developmental systems facilitate well-integrated, functional phenotypic responses to mutation or environmental induction.

(iii) The ES assumes gradualism, the EES variable rates of change. Of course, advocates of the ES, beginning with Simpson [11] and Rensch [8], have recognized that rates of evolution are highly variable evolutionary rates at both the phenotypic and genomic levels have been a focus of extensive research. This passage from Laland et al. [14] refers to the supposition in the EES that saltations are possible, and that the ES does not entertain saltations. I have explained that advocates of the ES have always accepted certain kinds of ‘large effect' mutations, as in paedomorphosis and polyploidy. There is little or no evidence that saltations occur in evolution via mutations in gene regulation.

(iv) The ES has a gene-centred perspective, the EES an organism-centred perspective. A reading of the seminal works of the ES will show that most of the authors had a deep interest in and knowledge of organisms—considerably greater, I venture, than many or even most practicing evolutionary biologists today. I have illustrated this with examples concerning niche construction, developmental processes and constraints. But the founders of the ES recognized that these and other properties of organisms evolve only if they are inherited across generations. Like Darwin, they recognized the common elements of a truly general theory of the evolution of phenotypes—including the phenotypic states that influence subsequent evolution—in variation, inheritance, and natural selection. (We would add genetic drift today.)

(v) The ES assumes genetic inheritance, the EES inclusive inheritance. Of course, the ES has always recognized cultural inheritance, but largely ignored it because it was considered taxonomically very restricted. As the prevalence of several widespread molecular mechanisms of inheritance other than by DNA sequence has come to light, evolutionary biology is provided with new phenomena to study and explain. Interesting theory is being developed as a consequence, and it is largely grounded in traditional population genetics. Documenting the frequency and distribution of non-genetic inheritance in natural populations, and its importance for evolved differences among populations and species, will be a task for empirical research.

(vi) Macroevolution: ES explains by microevolutionary processes, the EES by additional processes such as developmental bias and ecological inheritance. I have noted that developmental bias is not a new idea, and no evolutionary biologist who studies macroevolution would deny it. The role of ecological inheritance in macroevolution is speculative and unnecessary until shown otherwise.

5. Conclusion

Laland et al. [14] add that in the EES, evolution is ‘redefined as a transgenerational change in the distribution of heritable traits of a population'. That sounds equivalent to one traditional definition of evolution as change in gene frequencies, except that the EES redefinition would include non-genetic inheritance. Definitions are conventions, so does the definition of evolution matter? Perhaps, the philosophers of science Evelyn Fox Keller and Elizabeth Lloyd [148, pp. 2–3] noted that words ‘help to hold worldviews together' and that ‘the effort to “control and curtail the power of language” remains a significant feature of scientific activity. The very extent to which scientists…aim at a language of fixed and unambiguous meanings constitutes, in itself, one of the most distinctive features of their enterprise. And even though never quite realizable, this effort to control the vicissitudes of language, like the commitment to objectivity, reaps distinctive cognitive benefits'. Definitions, then, should not be altered lightly.

Are advocates of an EES engaged in an ‘effort to control the vicissitudes of language', and to what end? Some of the emphases in the proposed EES, such as niche construction, the supposed pioneering role of phenotypic plasticity in adaptation, and quasi-Lamarckian interpretations of epigenetic inheritance, are reminiscent of the rise of neo-Lamarckism in the early twentieth century, during the ‘eclipse of Darwinism'. Bowler [149, p. 258] writes that ‘Lamarckism allows life itself to be seen as purposeful and creative. Living things are in charge of their own evolution: they choose their response to each environmental challenge and thus direct evolution by their own efforts. With or without any religious implications, this is certainly a more hopeful vision than that derived from Darwinism. Life becomes an active force in nature, no longer merely responding in a passive manner to environmental pressures’. Welch [28] hears echoes of this theme in current critiques of standard evolutionary theory. He quotes neurobiologist Steven Rose [150] to that effect ‘redefining evolution as “a change of gene frequency in a population” is a reductionism too far, depriving living organisms of playing any part in their own destiny’, and recalls that Gregory Bateson [151] found in Waddington's genetic assimilation implications ‘for the battle between non-moral materialism and the more mystical view of the universe'. I do not think all advocates of an EES are impelled by emotional distaste for the utter lack of purpose and agency in evolution by natural selection, but it may be useful to ask if our views of evolutionary theory are affected by extrascientific values. As Welch [28] notes, ‘we do need to explain why ideas are so often hailed as important before they have done much scientific work'.

Some of the emphases in the proposed EES, especially non-genetic inheritance, may prove interesting, if developed both theoretically and empirically. Evolutionary developmental biology is an exciting field that can join a structuralist approach to the traditional emphasis on genetic variation — but it does not diminish the roles of genetic variation and selection. Modern versions of the Baldwin effect will need considerably more evidence before we can conclude that this kind of effect is important, and there are good reasons to doubt that it is. Overall, I have seen little evidential support for challenges to the basic tenets of the ES.

There have now been many essays on why a new, or supposedly new, viewpoint or approach is warranted. If advocates of an EES are to convince many biologists, they will need to provide empirical support. To remain vital, a field of science requires challengers who aim to topple traditional views but if it is not to be knocked about and smashed by unruly children (I am thinking of current politics in my country), the science also needs traditionalists. John Maynard Smith [152], one of the most broad-minded of great evolutionary biologists, wrote, ‘It is in the nature of science that once a position becomes orthodox it should be subjected to criticism…It does not follow that because a position is orthodox, it is wrong'.


Author response

Essential revisions:

1) The authors rightly describe the confusion in the field due to conflicting results in the literature concerning the distribution of the H3K9 mark relative to the nuclear periphery. They rightly attribute this to issues with regard to antibody specificity. But then they based their entire manuscript on exactly one antibody against H3K9me2. Given the importance of these results, and the existing conflicting data from many laboratories, it is important for the authors to validate their results with more than one antibody. The blocking peptide experiments are very valuable. But they just show that the antibody binds to the relevant peptide in solution. They do not address whether an epitope could be on a non-histone protein or if it recognizes the histone peptide sequence and modification but is also dependent on other properties surrounding the H3 histone in its native context.

Localization of H3K9me2-marked chromatin at the nuclear periphery was previously confirmed with several independent antibodies and methods in our earlier publication (Poleshko et al., 2017) as mentioned in the text. In brief, localization of the H3K9me2-marked chromatin at the nuclear periphery was confirmed by immuno-fluorescence (IF) and ChIP-seq (see Figure 1—figure supplement 1B) using multiple validated antibodies. Furthermore, the OligoPaint experiment using probes to H3K9me2-enriched genomic regions correlates with the H3K9me2 IF staining (see Figure 4—figure supplement 1 and Figure 6—figure supplement 2).

To further address the reviewer’s concerns about antibody specificity, we have added a new Figure 2—figure supplement 1 that includes histone peptide array data for two H3K9me2 antibodies (Figure 2—figure supplement 1A-B), IF staining with 3 anti-H3K9me2 antibodies (Figure 2—figure supplement 1C) and a Western blot (Figure 2—figure supplement 1D). Note that IF staining with all 3 antibodies confirms the peripheral localization of H3K9me2-marked chromatin. Also, the Western blot shows only a single band at the appropriate size for H3, making recognition of a non-histone protein unlikely. The histone peptide arrays show that the antibodies recognize H3K9me2 in combination with other neighboring histone modifications, except S10p or T11p. This observation is consistent with the “phospho-methyl switch” model presented in the manuscript.

The histone peptide array data for the H3K9me2 antibodies were published previously and were referenced in the original text although some of the graphical representations shown in the revised figures are new. Given the reviewers’ concerns about antibody specificity, we believe that the manuscript will benefit from the added supplementary figure depicting this previously published data, which we have referenced appropriately in the figure legend.

2) How similar is the reported H3K9me2 staining relative to the previously described "epichromatin" epitope displayed by another anti-histone antibody by Don and Ada Olins? A direct comparison would be valuable. The similarity in comparing the figures in this manuscript with published papers from Olins and Olins is striking! This epichromatin staining is clearly context and conformation dependent – although the antibody is specific for histones, it only stains in situ the chromatin at the periphery of mitotic plates and at the nuclear periphery in nuclei, but this specificity is not shown for purified nucleosomes or sonicated chromatin using ChIP.

For current and previous studies we used two anti-H3K9me2 antibodies: Active Motif pAb #39239 and Abcam mAb #ab1220. Both antibodies were tested for specificity and the influence of neighboring modifications using a peptide array, and both antibodies showed a single, specific band by Western blot (new Figure 2—figure supplement 1). Peripheral localization of the H3K9me2-marked chromatin was confirmed by IF with 3 different antibodies. Given the extensive characterization of these antibodies and the confirmation of the peripheral location of H3K9me2-marked chromatin by multiple methods, we do not believe it would advance our work substantively to further explore the relationship of our work to the anti-nucleosome PL2-6 antibody used by Don and Ada Olins. Indeed, the cryoEM structure of the single chain antibody fragment from this antibody bound to a CENP-A nucleosome was recently published (Zhou et al., Nature Communications, 2019). Perhaps future studies from our lab, the Olins’ or others could address whether any connection exists between our findings and the pattern of staining seen with PL2-6 that recognizes a distinct epitope.

3) The ability of H3S10 phosphorylation to block peripheral tethering of H3K9me2-modified chromatin is supported by the inability of a phosphomimetic mutant of H3S10 (GFP-tagged H3S10E, Figure 3) to enrich at the nuclear periphery and by the anti-correlation between H3K9me2/S10 phosphorylation and lamina assembly around H3K9me2 (Figure 4B, 4C). However, in some images (Figure 4A) it seems that H3K9me2 and H3K9me2S10p both seem quite peripherally enriched through all stages of mitosis. The "increased" separation from the lamina actually seems to be just the visualization of a lower level of staining within the body of the condensed prophase chromatids. Are the line scans in Figure 4C single examples from one image? This point would be more convincing if the extent of H3K9me2's peripheral enrichment through mitosis were quantified similarly to the quantification done in Figure 3C. This would be complicated by the lack of an intact lamina to use as a fiducial mark, but the centroid of the chromatin mass could be used instead. Indeed, how do the authors reconcile the apparent contradiction between the peripheral staining of mitotic plates throughout mitosis using the H3K9me2 antibody (Figure 4) with the loss of peripheral localization until telophase for the LAD FISH (Figure 6)? This again opens the possibility that they are looking at an "epichromatin" like staining pattern with their antibodies.

We did not intend to suggest that H3K9me2 remains peripheral during all stages of mitosis and indeed, we did not state this. H3K9me2-/H3K9me2S10p-marked chromatin detaches from the nuclear lamina in prophase and is packed into mitotic chromosomes until the telophase stage when H3K9me2-chromatin is restored at the nuclear periphery (Figure 4). H3K9me2-chromatin and euchromatin remains distributed throughout the chromosome arms but is excluded from the center of the mitotic plate during prometaphase-metaphase. The center of the mitotic plate contains centromeres and pericentromeric heterochromatin enriched for H3K9me3 (Figure 5). We agree with the reviewer that a single confocal plane might not be definitive. Therefore, we have included a new Figure 4—figure supplement 1 that shows 3D image reconstructions of the images presented in Figure 4A.

Confocal images displayed in Figure 4C are representative and line profiles show an analysis of a single line. To address the reviewer’s point, we have included additional images and line profiles as new Figure 4—figure supplement 3.

As mentioned above, H3K9me2 staining is distributed throughout the chromosome arms during prometaphase-anaphase (new Figure 4—figure supplement 1). OligoFISH data using LAD and non-LAD probes are consistent with this observation. We have included a new Figure 6—figure supplement 2 that displays the 3-dimensional distribution of H3K9me2-enriched LAD and non-LAD probes throughout the chromosome arms with exclusion from the center of the mitotic plate where pericentromeric heterochromatin/chromocenters localize. This correlates well with the distribution of the H3K9me2 mark as shown in Figure 4A and Figure 4—figure supplement 1. We also modified Video 2 to highlight the central position of chromocenters and non-central distribution of LAD and non-LAD probes.

4) These experiments use an antibody that recognizes the Histone H3 tail when dually modified with H3K9 dimethylation and H3S10 phosphorylation. This antibody is blocked by an H3K9me2S10p peptide but not by an H3K9me2 peptide. Is it blocked by an S10phospho peptide? If any S10phospho cross-reactivity exists, this may contribute central nucleoplasmic signal that may be more prominent especially as the specific antigen is removed (H3K9me2S10p). A different way to answer this question might be, how does S10phospho distribution compare to H3K9me2S10p distribution in mitotic cells? This may be an important point needed to support the argument that H3K9me2S10p must be de-phosphorylated for peripheral enrichment to resume.

The H3K9me2S10p antibody does not recognize H3S10p. We extended the peptide blocking experiment with addition of the H3S10p peptide as the reviewer suggested (Figure 4—figure supplement 2A). We also added histone peptide array data demonstrating antibody specificity of the H3K9me2S10p antibody (Figure 4—figure supplement 2B).

5) Similarly, the authors have demonstrated the specificity of the H3K9me2 antibody for H3K9me2 over other methylation states. However, the ability of the H3K9me2 antibody to detect the H3K9me2S10p dual modification is not conclusively proven. For example, can the H3K9me2 antibody also be blocked by an H3K9me2S10p peptide? If not, this would suggest that this antibody has a lower affinity for the dually modified H3 tail than the H3K9me2/unmodified Ser10 tail. This would then open up the alternative interpretation that "enrichment" of H3K9me2 at the nuclear periphery over H3K9me2S10p is due to a higher affinity of the H3K9me2 antibody for un-phosphorylated H3 tails.

The H3K9me2 antibody does not recognize the H3K9me2S10p epitope. We have added histone peptide array data (Figure 2—figure supplement 1B) demonstrating antibody specificity. The antibody cannot recognize H3K9me2 if neighboring S10 or T11 is phosphorylated. As the reviewer suggested, we also tested anti-H3K9me2 antibodies in IF assays to determine that binding is not blocked by H3K9me2S10p peptides (Figure 2—figure supplement 1E-F). Each antibody used is specific for its epitope.

We believe that any confusion is the result of partial colocalization of H3K9me2 and H3K9me2S10p staining in Figure 4. We interpret this finding to suggest that not every S10 adjacent to K9me2 is phosphorylated during mitosis. Note that during telophase the separation of the two epitopes becomes more clear (Figure 4D). To avoid confusion, we have modified the text to address this point (subsection “H3K9me2 persists through mitosis and associates with reassembling nuclear lamina in daughter cells at mitotic exit”):

“Our data suggest that not every histone H3 Ser10 adjacent to H3K9me2 is phosphorylated since we observe some overlap of staining with the H3K9me2 and H3K9me2S10p antibodies.”

6) The idea of a phosphorus-switch by which H3K9me2S10 phosphorylation leads to loss of lamina association during mitosis, being a major punchline of the manuscript, does not appear to be demonstrated by the current manuscript. The argument used by authors rests on the lack of GFP-H3 localization of certain deletion mutants and some line scans of mitotic cells which were not convincing of showing loss of H3K9me2S10 phosphorylation from the peripheral staining. Overall, the authors show that H3K9me2 modification of genomic loci correlates with lamina association, and that histone S10 phosphorylation is anti-correlated with lamina association. However, functional tests to link these elements together are lacking. The authors assert that S10 phosphorylation disrupts the interaction of H3K9me2 with its tether. This could be tested, for instance, with Cec-4 if Cec-4 interacts specifically with H3K9me2 and not with H3K9me2S10p, this would support this model.

In order to address this point, we have provided additional antibody validation data which demonstrates that phosphorylation of S10 adjacent to H3K9me2 (H3K9me2S10p) blocks recognition of the epitope by the H3K9me2 antibodies (Figure 2—figure supplement 1B, E-F).

The functional experiment suggested by the reviewer has been published previously (Gonzalez-Sandoval et al., 2015) and we have referenced this result in the text. Briefly, the Gasser lab demonstrated that S10 or T11 phosphorylation reduces K9 methylation-dependent CEC-4 binding by 75 or 105 times, respectively. Combined, these results support the “phospho-methyl switch” model.

“Indeed, experimental results from the Gasser lab demonstrated that CEC-4 binds methylated H3K9 peptides and this binding is reduced by 2 orders of magnitude if the adjacent Ser10 is phosphorylated (Gonzalez-Sandoval et al., 2015).”

7) To what degree does the reduced z-resolution, projection through the depth of focus, and intensity scaling play a role in their conclusions of an exclusively peripheral localization of H3K9me2. Thus, in Results subsection “H3K9me2 is an evolutionarily conserved mark of peripheral heterochromatin”: "H3K9me2 marks only peripheral heterochromatin, whereas H3K9me3 and H3K27me3 co-localize with heterochromatin in the nuclear interior, or at both the interior and the periphery." However, the ratio of peripheral rim staining seems not that different for H3K9me2 and K3K9me3. If it is ignored the very intense staining over chromocenters in mouse cells that have large PCH, the ratio of the peripheral rim and internal foci staining does not seem that different for H3K9me2 and H3K9me3 staining. Eyeballing Figure 1A I see ratios of peripheral to interior foci intensities ranging from

80:25 to 80-10 for H3K9me2, versus

40:10 for H3K9me3 – a factor only of about 2-fold difference. Indeed, peripheral rim staining of chromatin in individual optical sections represents actually a z-projection through the z-depth of focus. Because of the finite thickness of the heterochromatin rim, this leads to a significant enhanced intensity due to this projection effect – as would be seen even for DNA staining. This effect is especially true for confocal imaging but also true for STORM imaging – both have much worse resolution in z. This effect needs to be compensated for when comparing the "enrichment" of signal at the periphery versus interior. The comparison could be with the corresponding measurement done for DNA staining such as DAPI for the peripheral rim and interior condensed foci (other than chromocenters) or between the intensity of internally stained foci with grazing sections of nuclei. Nuclei from cells growing flat in a monolayer will tend to have flat nuclear surfaces, particularly basal. These grazing sections will not have this superposition, projection effect. Finally, what is the actual comparison of intensity between the internal foci seen with H3K9me2 STORM staining and foci at the periphery. The beautiful STORM images in Figure 1B appear to show internal foci (spots and short fiber-like segments) at relatively the same brightness as foci at the periphery.

We do not rely solely on the immunofluorescence experiments to conclude that H3K9me2-marked chromatin is localized at the nuclear periphery. Localization of H3K9me2-marked chromatin was observed at the nuclear periphery by multiple methods presented in the manuscript and previously published, including genome-wide ChIP-seq results demonstrating high LB1-H3K9me2 co-occupancy (Poleshko et al., 2017) as mentioned in the original text.

As stated in the text, H3K9me3 and H3K27me3 heterochromatin are observed in multiple regions of the nucleus in addition to the nuclear periphery thus both are non-specific to the nuclear periphery. In contrast, the H3K9me2 predominantly localized at the nuclear periphery. These observations are consistent between IF and ChIP-seq data (see Poleshko et al., 2017).

The described effect has no influence on image analysis/quantifications or any conclusions presented in the manuscript. To address the reviewer’s concern, we have provided additional supplementary images to display XY, XZ and YZ-projections as well as 3D image reconstruction of the H3K9me2 staining (Figure 1—figure supplement 1A).

To address the nature of the internal H3K9me2 foci, a blocking peptide was used to distinguish specific from background signal of the H3K9me2 antibody (Figure 2, Figure 1—figure supplement 2 and Figure 2—figure supplement 1). As mentioned in the original text, the signal in the nuclear interior is largely background as confirmed by both confocal and STORM microscopies.

8) How exactly do the authors explain the GFP-H3 mutant results, given the documented low level of expression of the GFP-H3 variants? Can the authors elaborate on their logic? Thus, the H3K9me2 antibody rim staining appears unperturbed by any of the H3 mutants, suggesting that LAD distribution overall is unperturbed. Also, only a small fraction of the nucleosomes should contain the exogenous H3. But then why should this matter? Specifically, why should a 500-1000 kb LAD change position because a small percentage of the nucleosomes have the mutant H3? If there were actually some type of cooperative effect actually causing displacement, then why is the H3K9me2 staining unperturbed? Conversely, how would a modified nucleosome be able to localize 100s of nm or microns away from the nuclear periphery while the surrounding nucleosomes with wtH3 are localized at the periphery. These LADs contain condensed chromatin and its compaction and the known size of the nucleosome and linker DNA would seem to preclude such spatial separation.

As the reviewer points out, the GFP-H3 mutants are expressed at relatively low levels, and they do not appear to alter endogenous H3K9me2 staining. We therefore do not think that they are displacing LADs, i.e. LADs do not change position. We interpret the inability of the K9A (and other) mutants to partition to the periphery to suggest that lysine 9 dimethylation is required for either incorporation into peripheral nucleosomes, or for retention within nucleosomes at the periphery. Perhaps interaction with a dimethyl reader at the periphery stabilizes and incorporates a K9 dimethylated histone H3 protein. We have added our interpretation of these results to further address this point.

“We interpret the inability of the K9A and K9E mutants to partition to the periphery to suggest that lysine 9 dimethylation is required for either incorporation into peripheral nucleosomes, or for retention within nucleosomes at the periphery.”

[Editors' note: further revisions were requested prior to acceptance, as described below.]

[…] The manuscript has certainly been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The way the manuscript is now written seems to be more focused on the phospho-switch, the exclusive localization of the H3K9me2 to the periphery, and the absolute requirement for the H3K9me2 for peripheral localization in mammalian cells, the latter a conclusion entirely based on the GFP-H3 mutant localization data. However, the explanation of how the GFP-H3 mutants can be interpreted if they are only a small fraction of the H3 in the cell is not clear and that the authors should address this point with a detailed explanation in the paper itself – taking account of the low percentage of H3 that is GFP-tagged and mutated. The authors acknowledge that either biased incorporation or biased positioning of nucleosomes at/away from the nuclear periphery could explain their results. Anyhow, biased incorporation would represent a very different process than biased positioning, one that is completely different from the way that endogenous assembled nucleosomes are regulated by histone modifications. The authors should provide an explanation of how a low fraction of mutant H3 incorporation could mislocalize LADs from the periphery and explain the absence of any change to the endogenous H3K9me2 enrichment at the nuclear periphery.

We disagree that the focus of the manuscript has changed. In the revised manuscript, we haven’t changed any of our conclusions and we have not removed any data. We provided additional data as requested by the reviewers.

The conclusion that H3K9me2 acts as a 3D architectural mitotic guidepost is based on multiple experiments and observations and not solely on the GFP-H3 mutant localization.

Given that both wild-type GFP-tagged H3 and the S10A mutant GFP-tagged H3 proteins are incorporated and observed at the nuclear periphery, the most straight-forward conclusion is that only certain H3 mutants, namely those that preclude critical modifications, are not localized to the nuclear periphery. We clarify our reasoning in the text.

“Given that wild-type GFP-H3 is incorporated and observed at the nuclear periphery, we interpret the inability of the K9A and K9E mutants to partition to the periphery to suggest that lysine 9 dimethylation is required for either incorporation into peripheral nucleosomes, or for retention within nucleosomes at the periphery.”

Further, we do not observe any alteration of the endogenous H3K9me2 staining at the nuclear periphery upon expression of low levels of mutant H3 as we stated in the prior response, and we do not think that LADs are displaced from the nuclear lamina. The reviewer previously asked why we think LADs are being displaced in this experiment and we responded “We therefore do not think that they are displacing LADs, i.e. LADs do not change position”. We therefore do not understand why we are asked again why LADs are mislocalized. They are not.

With respect to the nonrandom binding of LADs after mitosis, the authors describe resolving a conflict in the literature regarding random shuffling of LADs to different heterochromatin compartments (i.e. nuclear lamina versus nucleoli) versus specific targeting of the same set of LADs to the periphery or interior (i.e. nucleolus) from mother to daughter cells, but it unclear they can do this by their current methodologies. It looks like that all can be concluded is that the preferred localization of

80% of cLADs to the nuclear periphery occurs early after reformation of the telophase/early G1 nucleus. Please revise and discuss in more detail.

In order to avoid any confusion that we were “resolving a conflict”, and to indicate that our data support (but do not prove) the model in which H3K9me2-marked LADs are specifically repositioned at the nuclear periphery, we have substituted the word “suggest” in the Discussion:

“Our results showing localization of H3K9me2-enriched lamina-associated chromatin, including those produced with LAD-specific oligopaints, suggest that H3K9me2-marked LADs which are re-established at the nuclear periphery at the end of mitosis concomitant with nuclear lamina re-assembly are likely distinct from the H3K9me3-marked NADs.”

Figure 4. Typical confocal microscope hardware/software often has the user define a "black" level which is the analog level which the analog to digital (A/D) converter sets as the 0 value. All analog values below this "black" level are truncated to zero. If the "black" level is set to a level that represents non-zero intensity then this introduces a nonlinearity which prevents measurement of relative intensity levels. The hallmark of this is spatial resolution higher than possible with the psf of the microscope due to this truncation effect (i.e. values going from high to zero in a short distance relative to the normal blurring predicted by the point spread function) and also zero values of intensities inside the stained region and/or immediately outside of it. There is no description of how the authors set the "black" level during their microscopy. The images and line-scans indicate a black and zero level of intensity immediately outside the lamin ring staining and even at locations inside the nucleus. This is weird, as nonspecific antibody staining, out of focus light, even the in-focus point-spread function, and the dark current and readout noise typically produces nonzero intensity values. Therefore, the authors should describe how the intensity levels were set and whether they allow actual linear measurements of intensities.

To address the reviewer’s concern, we extended the Methods section that describes image acquisition (see below). Confocal images were taken using the HyD detectors. Only DAPI staining was acquired with a PMT detector with a “black” level defined as an offset -0.1%. Images were taken using minimal laser power to ensure there was no signal saturation. The concerns raised by the reviewer are not relevant to images taken with HyD detectors.

“All confocal immunofluorescent images were taken using a Leica TCS SP8 3X STED confocal microscope using 63x/1.40 oil objective. DAPI staining (blue channel) was acquired using a PMT detector with offset -0.1%. All other staining (green, red and far red channels) were acquired using HyD detectors in the standard mode with 100% gain. All images were taken with minimal laser power to avoid saturation. 3D images were taken as Z-stacks with 0.05μm intervals with a range of 80-250 Z-planes per nucleus. Confocal 3D images were deconvoluted using Huygens Professional software using the microscope parameters, standard PSF and automatic settings for background estimation.”

Related to above, the predicted banding pattern of LADs versus iLADs could be better appreciated if they did chromosome spreads or looked at isolated chromosomes. This would tell if the unusual telomeric concentration of intensities towards the telomeres was real or not. If the staining of isolated chromosomes is different from the staining of the cells, it would point to a staining issue when staining whole cells.

Based on the proposed mitotic spread experiments, we assume that the reviewer refers to prometaphase-anaphase staining. We do not rely on these images to draw any conclusions. Note that we show markedly different patterns of staining in telophase for H3K9me2 and several other histone marks (Figure 5) making artifactual staining exceedingly unlikely. We do not feel that staining of isolated chromosomes would add substantially to our work.

Figure 4D. There is peripheral H3K9me2S10p staining although not a brighter ring of staining. It would be nice to see some type of aggregate analysis of a number of nuclei at each of several different stages of telophase to establish this temporal correlation.

The proposed additional analyses will not change the overall conclusions and we do not feel that it would add any clarity to our manuscript.


Practical Work for Learning

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LEARNING EXPERIENCE 1
The Search for Medicinal Plants: Unifying Characteristics of Life

Characteristics of life, plants in everyday life, nature of science and scientific investigations.

Students consider the various roles plants play in their everyday lives and read about ethnobotanist Mark Plotkin and his quest for medicinal plants. Students carry out an experiment to investigate the potential of certain herbs and spices in treating microbial infections then construct an argument based on their findings and readings about the promise of medicinal plants.

LEARNING EXPERIENCE 2
Simple Change, Unintended Consequences: Exploring Ecosystems

Connections among biological, physical, and chemical processes how interactions among the biotic components and between the biotic and abiotic component define the features of an ecosystem dynamic equilibrium in ecosystems impact of change in ecosystems.

Students examine the impact of a seemingly small change on the ecosystem of Lake Victoria. To learn about ecosystems, students first characterize and analyze soil and water samples as ecosystems and read about dynamic equilibrium in ecosystems. They then analyze the impact of a natural or human-made change in the ecosystem on the biological, chemical, and physical components and their interrelationships.

LEARNING EXPERIENCE 3
Changes in the Neighborhood: Ecological Succession

Factors involved in ecological succession.

Students read about the impact the eruption of Krakatoa had on surrounding ecosystems and explore ecological succession directly by investigating milk as it undergoes successive changes. Then they apply their understandings to explaining ecological succession in the recovery of Krakatoa.

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So Many Species, So Much Time: The Origins of Biodiversity

Nature of biomes, definition of species, origins of biodiversity, natural selection, vertical and divergent evolution.

Students analyze patterns in the distribution of species around the world and identify factors in the environment that determine the amount of biodiversity in an ecosystem. They identify criteria that define a species using the results of an activity and a reading by E.O. Wilson. Students model natural selection using different implements to pick up different foods, explain observations about finch evolution in the Galapagos Islands, and are challenged to explain the enormous diversity observed in the cichlids of Lake Victoria.

LEARNING EXPERIENCE 5
Go Forth and Populate: Population Dynamics

Communities and populations in ecosystems, population interactions and resource use, carrying capacity and limiting factors, patterns of population growth, biotic potential.

Students calculate an ecological footprint for a fictional person, then read about the possibility of a population crisis in the future. They construct growth curves for a model rabbit population, identify factors that affect the population growth using a model system of a rabbit population, and consider how carrying capacity determines the growth of populations. Then they apply their understandings about factors that affect the population growth to decide whether Earth can sustain a population of 9.5 billion people.

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Cycling Through the Ecosystem: Matter and Energy in Ecosystems

Interdependence of organisms, flow of energy in ecosystems, trophic levels, role of decomposers, biogeochemical cycles, recycling of matter through ecosystems and through organisms.

Students read about biomes and biogeochemical cycles, develop a presentation, and present their cycle to the class in a jigsaw learning activity. Students consider the foods they eat, the functions the foods have in their survival, and the need to recycle elements. They explore how energy flows through an ecosystem and the cycling of matter in biogeochemical cycles. Students then apply their understanding of the movement of energy and matter in ecosystems to designing a lunar biosphere that can sustain life for 2 years.

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Snake Oil or Science?

Plants make many chemical compounds that serve a purpose for the plant but may also be beneficial for humans not all sources of information are trustworthy, and that criteria can be used to identify reliable sources.

Students return to the topic of medicinal plants that they explored in Learning Experience 1 and research the biology and ecology of certain plant-based supplements credited with improving human health. Students use literacy skills such as reading for comprehension, selecting credible sources of information, evaluating information, and using evidence to support a claim.

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Corn to Milk: Metabolic Pathways

Universality of biomolecules and their functions, catabolic and anabolic pathways, metabolism as an indicator of common ancestry.

Students learn about biomolecules in a jigsaw learning experience. They conduct a chemical analysis to compare the biomolecules found in corn and milk. They then trace carbon atoms from starch molecules in corn to lactose in milk and read about the chemical reactions involved in capturing energy, the breakdown of nutrients, and the biosynthesis of new biomolecules. Students then trace a food item that they consumed through their own metabolic pathways.

LEARNING EXPERIENCE 8
Pernicious Poisons: Enzymes in Metabolic Pathways

Structure, function, and mechanisms of action of enzymes role of enzymes in metabolic pathways.

Students observe a demonstration of enzyme action and explore the mechanisms. They then determine the consequences to an organism if a specific metabolic pathway is blocked by an enzyme inhibitor.

LEARNING EXPERIENCE 9
Cell—At the Center of Excellence: Cell Structure and Function

The importance of being cellular, cell structure (including membranes) and function, cell specialization, differences between prokaryotes and eukaryotes.

Students read about scientists who are trying to create a living cell, then discuss why they think scientists might want to "build a cell in a test tube." Students build a model of a cell and describe the relationships between metabolic functions and cellular components.

LEARNING EXPERIENCE 10
The Unsolved Mystery: Exploring the Origins of Life on Earth

Scientific theories related to the origins of biomolecules, the coalescence, and the formation of cells.

Students read a story about the hypothesis that life did not originate on Earth but rather was brought by meteorites and comets. They create a timeline depicting the history of Earth using scientific information and evidence. They demonstrate how phospholipid membranes can be formed from biomolecules present in the environment and read about the conditions of Earth during the Archaean Eon. Students assume the role of scientists and research 1 hypothesis of the origins of life in preparation for a class panel discussion.

PROJECT 2 (optional available in Teacher's Guide only)
Creature Feature

Applying concepts in ecology, evolution, and cell biology.

Students imagine and describe an organism using concepts in ecology and cell biology they have explored so far in this course. Students prepare a species entry for the Encyclopedia of Life Web site, an online database for all of the species that are known on this planet.

LEARNING EXPERIENCE 11
Discovering the Nature of the Genetic Material: DNA—The Master Molecule

DNA structure and function DNA replication history of the discovery of DNA as the genetic material, science as a human endeavor, the nature of scientific research.

Students role-play scientists whose research contributed to the understanding of the structure and function of DNA. They build a model of DNA and use it to learn about DNA replication.

LEARNING EXPERIENCE 12
Translating Information into Action: Information Transfer from DNA to Protein

Nature of a gene, transcription, translation, protein structure, mutations, relationship between changes in DNA sequence and changes in traits.

Students read about a scientist's plan to use DNA as a way of sending secret messages. They decode the language of DNA, and model transcription and translation. Students explain how information moves from DNA to proteins to traits and analyze the impact of mutations on proteins and traits. They use their understandings to explain Griffith's classic experiment.

LEARNING EXPERIENCE 13
Of Proteins and Traits: The Molecular Basis of Traits

Relationships among DNA, protein, and traits biochemical basis of traits making genetically modified organisms.

Students read about genetically modified organisms and conduct an experiment in which they insert of new gene into bacteria, giving the bacteria a new trait. They learn how new traits are inserted into plants then decide whether they would eat a potato with a gene from a different organism, using evidence to explain their decision.

LEARNING EXPERIENCE 14
Home on the Chromosome: Structure and Function of Chromosomes

Chromosome structure, chromosomes as the genetic legacy, meiosis and gamete formation, recombination, the origins of trait variation, karyotypes.

Students assume the role of genetic counselors and analyze karyotypes for a couple expecting a baby. They build a model of a chromosome then model gamete formation and meiosis. Students explain how mistakes can occur during meiosis and the consequences of those mistakes.

LEARNING EXPERIENCE 15
The Sickling Cell: Dominant and Recessive Traits

Dominance and recessiveness, homozygosity and heterozygosity, relationship between genotype and phenotype.

Students read about sickle cell disease, use data to explain the biochemical and molecular basis of the disease, and explore patterns of inheritance. They explain why, from an evolutionary perspective, a mutated gene might be retained in a population.

LEARNING EXPERIENCE 16
Return of Martin Guerre: Exploring Simple Inheritance Patterns

Mendelian genetics, patterns of inheritance, Punnett squares, crossover predicting and explaining variations in offspring, DNA analysis, and RFLPs.

Students read about a man returning to a village claiming an identity. They analyze Mendel's data and determine how variation can occur using chromosome models. Students then analyze molecular genetic data and patterns of inheritance to determine the man's true identity.

LEARNING EXPERIENCE 17
So Many Traits, So Few Genes: Exploring Non-Mendelian Traits

Non-Mendelian patterns of gene expression principle of 1 gene more than 1 protein role of environment in trait variation.

Students consider patterns of height variation and speculate how this might occur. They read descriptions of various traits and identify the non-Mendelian mode demonstrated. Students then apply their understanding of non-Mendelian traits to the trait of height.

LEARNING EXPERIENCE 18
There's More to Life than Sequences: Exploring Epigenetics

Epigenetics: the effect of environment on gene expression.

Students read about changes that occur in identical twins as they age. Students build a model of chromatin and investigate how methylation affects gene expression. They use their understandings about epigenetics and the effects of environment to explain changes in gene expression.

LEARNING EXPERIENCE 19
One If by Land, Another If by Sea: Exploring the Process of Evolution

Meaning of the word "theory" in science change over time is the result of random variation in the genetics of a population and natural selection interaction between environment and evolution the role of population dynamics in evolution.

Students discuss the scientific meaning of theory and the kinds of evidence they might look for to support the theory of evolution. They read about the discovery of a fossil of a killer whale that lived 12 million years ago. Students are challenged to prepare an exhibit for the local library on the evolution of the whale and the biological factors involved that resulted in the change from the terrestrial to aquatic habitat of whales. They create a timeline of evolutionary history, read the evolutionary history of whales, order pictures of the evolutionary intermediates that led to the modern-day whale, and prepare their exhibit on whale evolution.

LEARNING EXPERIENCE 20
Marvelous Blunders: The Molecular Basis of Natural Selection

Mechanism of natural selection, role of variation in populations, changes in the gene pool of a species as the basis for all evolutionary changes.

Students read about mutating bacteria as a world health crisis. They conduct an experiment to determine how bacteria develop antibiotic resistance and discuss how the development of resistant strains of bacteria is the result of natural selection. Students discuss the importance of understanding evolution to life in the modern world.

LEARNING EXPERIENCE 21
Ancient Genes, Age-Old Processes: The Molecular Evidence for Evolution

The nature of scientific evidence similarities and differences among the biochemical and molecular structures and functions of organisms relationships between molecular and anatomical evidence for evolution.

Students identify similarities among seemingly very diverse organisms, interpret an experiment, and analyze data relating to gene homologies among different organisms. Students are challenged to create a model of evolution that accounts for the molecular and anatomical evidence. They determine how cladograms can provide information about the relatedness of organisms.

LEARNING EXPERIENCE 22
Fishing Expedition: Anatomical and Fossil Evidence for Evolution

Anatomical homologies nature of fossils and the significance of fossil evidence transitional organisms.

Students take on the role of paleontologists looking for transition animals between fish and amphibians. They make predictions about what to look for and where based on their understanding of evolution. Students revise their model of evolution based on new evidence and understandings.

PROJECT 3 (optional available in Teacher's Guide only)
Creature Feature Returns
Apply concepts in molecular biology, genetics, and evolution.

Students return to the organism they described for the Encyclopedia of Life Web page and add information about its molecular biology, genetics, and evolution.


Watch the video: Ο ρόλος της επιγενετικής και η εξέλιξη της γηριατρικής (January 2023).