(TN) Systems Biology Articles

Prospects & Overviews

The molecular and mathematical basis
of Waddingtons epigenetic landscape:
A framework for post-Darwinian biology?
Sui Huang
The Neo-Darwinian concept of natural selection is
plausible when one assumes a straightforward causation
of phenotype by genotype. However, such simple 1:1
mapping must now give place to the modern concepts
of gene regulatory networks and gene expression noise.
Both can, in the absence of genetic mutations, jointly
generate a diversity of inheritable randomly occupied
phenotypic states that could also serve as a substrate
for natural selection. This form of epigenetic dynamics
challenges Neo-Darwinism. It needs to incorporate the
non-linear, stochastic dynamics of gene networks. A first
step is to consider the mathematical correspondence
between gene regulatory networks and Waddingtons
metaphoric epigenetic landscape, which actually
represents the quasi-potential function of global network
dynamics. It explains the coexistence of multiple stable
phenotypes within one genotype. The landscapes
topography with its attractors is shaped by evolution
through mutational re-wiring of regulatory
interactions offering a link between genetic mutation
and sudden, broad evolutionary changes.

Keywords:
attractor; epigenetics; gene regulatory network;
Neo-Darwinism; systems biology

DOI 10.1002/bies.201100031
Institute for Systems Biology, Seattle, WA, USA
Corresponding author:
Sui Huang
E-mail: sui.huang@systemsbiology.org
Abbreviation:
GRN, gene regulatory network.
Supporting information online

Bioessays 34: 149157, 2011 WILEY Periodicals, Inc.

Introduction
In the 1940s and 1950s the Modern Synthesis in biology
reconciled Darwins theory of natural selection of random,
gradual variants with the Mendelian concept of discrete genes
as the vehicles of inheritance [1]. This synthesis, which we owe
to quantitative population genetics [24], gave Darwins
theories a mechanistic basis and led to what has become
known, loosely, as Neo-Darwinism (Fig. 1). A decade later,
the central dogma of molecular biology [5], now remembered
by its compact scheme gene ! mRNA ! protein (trait),
established the molecular basis of genetic inheritance. This
linear scheme of causation (one gene - one trait) further
cemented the Neo-Darwinian principle that implicitly
assumes a straight and deterministic genotype-to-phenotype
mapping. Such a linear causation scheme is necessary for
the enrichment of a gene by selection of a phenotype to be
plausible.
But what if a genotype does not translate in an obvious
manner into a corresponding phenotype or worse, if genes
are not even the sole basis of inheritance? This is of course not
a new proposal. It was first articulated by C. Waddington when
he coined the term epigenetics, at roughly the same time as
the consolidation of the Modern Synthesis. However, the idea
of non-genetic contributions to determination and inheritance
of traits has been sidelined as Neo-Darwinism and the central
dogma has risen to biological orthodoxy. But the arrival of
genome-wide analyses of genes and gene expression and the
entry of systems dynamics thinking into molecular biology
have now exposed, with inescapable clarity, the complexity in
the relationship between genotype and phenotype. It has also
brought the role of higher level organization that lies beyond
direct genetic instruction into sight. Specifically, experimental
characterization of physical interactions between genes and
the emergence of the concept of gene regulatory networks
(GRNs) [68], through which genes act not as soloists
but in concert, have begun to shed light on the population
geneticists black box where gene-gene interactions were
acknowledged, at best, in the form of the phenomenological
concept of epistasis [9].

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Prospects & Overviews

1900s

1940s

PHENOTYPE

Mendelian
Genetics

Protein: trait
mRNA: transcript

Soma-Germ plasm
separation (Weismann)
Population
Genetics

DNA: gene

Epigenetics (Waddington)

GENOTYPE

Development
1950s

1970s

MODERN
SYNTHESIS
NEO-DARWINISM
Gene
regulatory networks
(Kauffman)

Molecular Biology
central dogma

NOISE

Systems Biology

Genomics
TODAY

PHENOME

E
Ph pige
en ne
om tic
en
a

Biocomplexity

See Fig. 2

Problems & Paradigms

Darwins
Natural Selection

....

NETWORK
See
Box 1

POST-MODERN
SYNTHESIS
???
GENOME

Figure 1. Historical overview of the Modern Synthesis of evolutionary biology, Neo-Darwinism and the possibility of a similar future synthesis of the latter with Systems Biology in post-Darwinian biology
(Post-Modern Synthesis). Small arrows on the side indicate
theories, concepts or observations that influenced the development
of Darwinism. The proposed new synthesis would have to explain
findings and incorporate theories (blue arrows) that are not considered in the central dogma of molecular biology and appear to
defy Neo-Darwinism.

Both central pillars in the edifice of Neo-Darwinism, natural

selection and the linear mono-causal genotype-phenotype
mapping, more recently referred to as genetic determinism
[10], have received ample criticism ever since they were
erected, long before the rise of the modern-day formal notions
of complexity [1116]. Among the diverse alternative or complementary ideas, the old notion of constraints epitomizes a
departure from Neo-Darwinian thinking that is relevant here.
Waddington was one of the first to point to developmental
constraints in the physical implementation of the instructions
of the genes in forming an organism. He captured the idea of
constraints in the now famous metaphor of epigenetic landscape [1012], to which I turn my prime attention later. But it
is perhaps owing to the work of Gould and Lewontin [17] as
well as Goodwin [18, 19] that many contemporary biologists
are now aware of the fallacy of pure selectionism, according
to which natural selection is the sole, almighty sculptor of all
phenotypic traits. Natural constraints in organismal design,
emanating from the inescapable laws of chemistry, physics
and even mathematics, as well as from history [20], present
prefabricated modules of high complexity for natural selection
to choose from. But the complexity itself is not the work of

150

natural selection. The fractal, optimally space-filling structure

of branching tissues, such as the lung, or the appealing stripe
and spot patterns of animal coats are the most lucid examples
of the creative force of self-organization that can be reduced to
the laws of physics [21] and whose genesis does not require
natural selection. More likely, it is the incorporation and
polishing, but not the initial design, of such modules in
building complex organisms that benefited from selection.
Constraints through self-organization offer Darwinian selection order for free, as Kauffman, the pioneer of biocomplexity, aptly put it [22]. I show that constraints play a central role
in how GRNs produce, almost for free, the stable gene expression patterns needed to govern coherent cellular behaviours.

Towards a theory of epigenetics based

on GRNs
A recurring theme in the voices cautioning against unfettered
Neo-Darwinism and genetic determinism, while calling for
more holistic approaches that consider complexity theory,
is a set of observations that I summarize here under epigenetic
phenomena. Since they pertain to all that is beyond (epi) the
genetic mechanisms of phenotype specification and inheritance, such phenomena most prosaically defy the paradigm
of genetic determination. (The confusing semantics of epigenetic is discussed in the online Supporting Information, Text I)
But what epigenetic apparatus can universally provide
information encoding and storage without altering the DNA
sequence? The answer is: the GRN ([6], or briefly, gene network) the network established by the fact that genes influences the expression of other genes via a web of molecular

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Prospects & Overviews

S. Huang

Problems & Paradigms

Figure 2. The unequivocal correspondence (unique mapping) between

genome (A) and associated network architecture and the epigenetic
landscape (D) via the dynamics of the expression patterns (B) in state
space (C) controlled by the GRN. The schematic representation is for
a 9-gene GRN. The central concept to understanding the landscape is
that each network state S (gene expression pattern, hence cell
state blue discs in B and C) maps into a point (blue balls in D) on
the landscape. The position of the point (network state) S, is determined as follows: The N gene expression values defining a given state
S act as the coordinates in defining its position in that N-dimensional
space, where each dimension (axis) represents the expression level of
a gene. Each step (orange arrow) in entering a new (more abstract)
conceptual domain (boxes A, B, C, D) can be formalized in terms of
mathematical principles [8, 45]. For A, see also Box 1. In B the two
time points t1 and t2 represent the dynamics and the constrained
change of gene expression pattern. Note that the quasi-potential is not
a true potential energy since the gene network dynamics is a nonequilibrium, typically non-integrable system. The value of U can be
intuitively (but formally not correctly) approximated by the negative
logarithm of the steady-state probability P(S) to find the network in
state S, i.e. U
ln [P(S)], or by decomposing the vector field that
contains the forces F(S) that drive S into two perpendicular components, one of which is a gradient of some quasi-potential function
U [53]. Red circles genes; blue axes state space coordinates after
hypothetical dimension-reduction to two dimensions, permitting the
projection of the state space into an XY-plane (light blue in C, D), so
that it can be used to display U as a third dimension.

Bioessays 34: 149157, 2011 WILEY Periodicals, Inc.

regulatory interactions encoded in the genome (Fig. 2A). The

GRN concept is the pea under the mattress of the comfortable
paradigm of a direct genotype-phenotype mapping that has
served Neo-Darwinian thinking so well. But commentators
have begun to explicitly articulate the need to integrate gene
networks in evolution theory [9, 23, 24].
Since the 1990s the amount of literature on gene networks has exploded. It covers a broad range of forms: from
the use as graphical representation of regulatory interactions
between genes collected by high-throughput experiments or
as toy models in computer simulations of in silico evolution
[2529] to the analysis of the static structure of gene networks
and its evolution [3034]. However, the dynamic behaviour of
networks is rarely considered as the object per se of evolution.

Structure and dynamics of gene

networks a critical distinction
For a fruitful discussion of the dynamics of networks and its
role in the genotype-phenotype mapping, a set of shared,
unambiguous definitions of terms is critical. But sadly, many
biologists are not explicitly aware of the ontological dichotomy between network structure (topology) and network

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Prospects & Overviews

I argue that viewpoint (A) has a more solid semasiological foundation. Its consistency is illustrated in the
bottom panel example, which displays the behaviour (network dynamics) of the very same 3-gene network: In
condition a where Gene 1 is not expressed (due to inhibitory signals, symbolized by the wiggling arrow) the
Genes 2 and 3 balance each other and are expressed
at intermediate levels. Once Gene 1 is expressed (b) this
balance of mutual check is tilted in favour of expression of
Gene 3 as prescribed by the molecularly specified
(genomically hard-wired) interactions. The existing
law that Gene 1 suppresses Gene 2 but activates
Gene 3 now is applied because Gene 1 is expressed.
This constitutes a change of conditions (external inputs
allowing the presence of Gene 1 activity) that triggers a
change of the network state (now Gene 3 is high, Gene 2 is
low) and not a change of the network wiring (the law).
The network states in b, c illustrate the behaviour of a
toggle switch. The very same network architecture of
mutual inhibition between Genes 2 and 3 allows the
network (genome) to produce two alternative stable
expression patterns, either [Gene 2] >> [Gene 3] or vice
versa, depending on the perturbation of the gene
expression levels that could even have occurred transiently in the past [35, 64]. Thus, the very same genome can
generate multiple enduring network states (phenotypes)
herein lies its flexibility and not in the change of its actual
wiring.
I recommend adhering to the network definition
of (A) as it is conceptually clean and consistent with
that used by pioneers of GRN long before the idea of
networks entered mainstream biology [8, 63] and it is
also the basis for the increasing mathematical treatment
of networks as dynamical systems. The usage (B) is
an ad hoc descriptive term, warranted in some
context, but it may perpetuate confusion and hamper
communication.

On the terminology of gene regulatory network:

A tacit dualism in meaning
A widespread but unarticulated misunderstanding of the
concept of gene regulatory network (GRN) warrants a
clarifying remark. In current discussions there is a dichotomy of notions of network among biologists.
One group (A) holds that the term network refers to a
hard-wired (i.e. time-invariant) architecture. Here, the links
in the network represent gene regulatory relationships,
which are akin to the law: carved in stone and thus invariant, but applied to situations only when relevant. In this
definition the GRN is fully specified by the genome
sequence (top), which through encoding protein structure
and cis-regulatory sequences defines which gene locus
regulates which one. This definition also implies that the
network does not change in a life time (except as a consequence of mutations). What changes is the expression
level for the genes, which collectively manifest as the
change of the gene expression pattern (bottom panel)
and is referred to as network dynamics.
In the view of a second group of biologists (B) the
network is not fixed. Instead, regulatory interactions
change with time. For instance, Gene 2 may inhibit
Gene 3 (bottom panel b and c) or vice versa depending
on the condition (e.g. cell type). Consequently, the network is not hard-wired in the genome but continuously
changes its connections during life time [71]. This use of
the term network captures the loose idea of a web of
instantaneous causal relationships. Some authors go as
far as equating an entire gene expression pattern, as
measured by microarrays, to a network to imply the
massively parallel, correlated behaviour. In this view
any two genes that exhibit highly correlated changes over
time or across conditions are connected by a line in a
diagram giving rise to the impression of a network [72].
LOCUS 1

... the specific

molecular interactions
between the gene loci ...

LOCUS 2

C
Protein 2

Protein 1
Gene 12
Gene

Gene 11
Gene

US 3

2
1

1
3

common short hand

notation of a GRN
(as used in Fig. 2A)

Gene 3

2
1

C
LO

Protein 3

There is one
network architecture
per genome.
It is genome-specific
and invariant.

152

2
US

S1
CU

. . . which in turn
form a hardwired
gene regulatory network
(GRN).

What varies is
the network state
- defined by the gene
expression pattern
that reflects the
network interactions.

LOCUS 3

LO

LO

Problems & Paradigms

Box 1

The genome sequence

encodes...

....

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Prospects & Overviews

(i) The dynamics of the GRN can generate a multitude of

stable (persisting) states of the system and, thus, act as

Bioessays 34: 149157, 2011 WILEY Periodicals, Inc.

a source of variability and as a medium of information

storage both capabilities traditionally attributed to
mutations and genes [8];
(ii) Gene expression noise, i.e. the random fluctuations of
gene expression levels that propagate through the GRN,
permits the random occupation of the various stable
states, which results in (non-genetic) phenotypic heterogeneity of (even clonal) cell populations and, thus, is a
source of phenotypic randomness [40, 41].
In brief, networks and noise are non-genetic sources
of diversity and stochasticity the chief ingredients of
Darwinian evolution. They may claim a spot on the pedestal
of evolutionary mechanisms that has long been the uncontested domain of genetic mutations. If properly incorporated
into Neo-Darwinian thinking, so to speak in a Post-Modern
Synthesis, these two epigenetic phenomena will not
threaten its fundament but broaden it, reconciling natural
selection with observations that have stimulated quite
imaginative stretching of the narrative of natural selection.
Here I present a concise, permissively simplified explanation of the formal concepts of gene network dynamics. This
will hopefully provide a more tangible notion of network
behaviour, hence allowing us to study its evolution, and clear
the view on the obscure term epigenetics, the loose usage of
which has stifled communication in biology and made its
integration with Neo-Darwinism difficult (see Supporting
Information, Text I).

From molecular interaction to gene

networks to systems dynamics
How can a network of regulatory interactions between genes
create distinct phenotypic states with memory of themselves
and which can be occupied in response to a signal or just by
chance? What Waddington called developmental constraints
and epigenetics [4244] can now be identified as the layers of
molecular regulatory networks and cell-cell communication
networks a web of interactions through which genomic
information must percolate to produce the macroscopic phenotype. Viewing through the lens of dynamical systems
theory, this blurry layer of dense biological interactions will
appear in our focal plane with new clarity. Here I discuss the
layer of GRNs to illustrate the basic principles.
The key ideas of GRN dynamics are summarized in Fig. 2
(for more details see [8, 45]). Let us start here by declaring,
for the sake of argument, the premise that a given phenotypic
state of a cell can be viewed, in a first approximation, as
encoded by its momentary genome-wide gene expression pattern. (Expression is here understood as the active state of a
gene locus manifested as the presence of the protein that it
encodes.) A given gene expression pattern is one among the
hyperastronomically vast number of combinatorial configurations of the expression status of the N gene loci in the entire
genome. Now, the expression of an individual gene is not
independent of that of other genes, precisely because of
the presence of the GRN, through which the gene loci
(in a locus-specific manner) influence each others expression
and, hence, contribution to the phenotype. This inter-loci

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Problems & Paradigms

dynamics [8]. This indifference has so far prevented meaningful discourse. (The problem surrounding the very concept
of gene network is explained in Box 1.) Whereas the structure of a network is a static entity, a collection of interconnected nodes representing the genes Xi that warrants the oft
used analogy of a wiring diagram, the dynamics of a network pertains to the collective behaviour of genes that
is relevant only if one considers that each gene, Xi, changes
its level of expression, xi(t), over time. Network dynamics is,
then, the coordinated change of the expression levels, x1(t),
x2(t), x3(t) . . ., of all the genes in a GRN as a consequence of the
entirety of the regulatory interactions displayed as connections (wiring) in the fixed wiring diagram. In other words:
strictly speaking, when talking about GRNs, there are no
dynamic networks. Instead, network dynamics is a legitimate term indicating the characteristic dynamic behaviour
exhibited by a given network.
Thus, it is crucial to distinguish between two separate
timescales: network dynamics (the coordinated change of
the expression levels xi(t) of the genes of a given network)
takes place within one organisms lifetime, during which the
wiring diagram of that network does not change. By contrast, a
change of the structure, or rewiring of the network, is the
result of a genomic mutation (which may, for instance, affect
how a regulatory gene controls its target gene) and occurs at
the evolutionary time scale. Obviously, a change in the structure of the network will affect its dynamics in a particular way.
Precisely herein lies the central principle for comprehending
how a mutation maps, not directly but via the network, into a
new phenotype.
Perhaps network dynamics and its characteristics are
rarely considered as a phenotype in evolution biology [7]
because their description is too abstract, although the concept
dates back to the 1960s when Monod and Jacob found that
genes can regulate each others expression [35], which
inspired Kauffman to build the first computational gene
networks [36] more than 40 years ago to study the global
dynamics of complex GRNs.
Network dynamics has recently gained popularity among
molecular biologists who realize that the phenotype cannot be
understood as a linear superposition of the isolated action of
individual genetic pathways. This insight, accompanied by the
arrival of new technologies, was a major driving force behind
the rise of systems biology more than a decade ago [37]. But
systems biology should aspire to more than being the exhaustive categorization and characterization of the molecular parts
list of the genome and the static interactions between these
parts. To understand how the phenotype (the whole) is more
than the sum of the genes (the parts) [38], entirety of analysis
must be followed by analysis of entirety [39]. This entails
embracing the collective dynamics of the genes coordinated
by the GRN.
In the Neo-Darwinian scheme, if the genes provide the
molecular medium for inheritance, and mutations deliver the
random variability required for natural selection, the following two epigenetic phenomena may rival genes and mutations
as the substrate of natural selection (Fig. 1):

S. Huang

Problems & Paradigms

S. Huang

communication is best known to occur via transcriptional

control of gene expression and via protein-protein interactions. However, one can also include nucleosome and chromatin conformation [4648] as well as chromosome-mediated
interaction between distant loci as exerting regulatory effects
[4952].
The GRN is thus the inter-gene loci control network and is
commonly plotted as a wiring diagram indicating which gene
has the potential to regulate which other, under what conditions and by what modality (inhibition or induction of
expression). Here I reiterate the fundamental notion that
the structure of the GRN diagram (Fig. 2A) is fully encoded
(hard-wired) in the genome because each single interaction is
ultimately molecular in nature and its specificity and effect is
determined by the structure of proteins and by the cisregulatory DNA sequence. Therefore, the network structure
has been wired by evolution and hard-coded into the genomic
sequence. It is intrinsic to and, in the absence of mutations,
invariant for a given genome (organism). Thus, each cell in
metazoa has the same GRN.
Here, I also emphasize again that the GRN structure
changes in phylogenetic time scale only. This central property
is often misrepresented in expressions like the changes in the
GRN during development (see Box 1 for elaboration of this
misunderstanding). There is ONE, and only ONE, static network topology (structure wiring diagram) in a given
genome. What changes in development and physiology is
not the network structure but the gene expression patterns,
i.e. the configuration of the expression states xi(t) of the
individual genes in the very same (invariant) genome. This
configuration constitutes a network state S. A change of the
network states S takes place in ontogenetic time scale. This is
what is commonly referred to by the term network dynamics.
In summary, a change in network state (i.e. network
dynamics) is caused by the coordinated alteration in the
expression status of the genes within the same genome,
whereas a change in network structure is caused by a
genetic mutation thus, naturally separating ontogeny from
phylogeny. This dichotomy is central for a formal understanding of the following explanation.

Network dynamics reflects constraints on

the realization of expression patterns
As a consequence of the inter-dependence of expression of
individual gene loci, the genome is not free to choose any
possible gene expression pattern. Each of the zillions of combinatorially possible configurations of expression status at all
the gene loci across the genome establish a gene expression
pattern but they are not created equal (Fig. 2B): some gene
expression patterns S are much more stable than others.
Some are so unstable as to be barely realizable because doing
so would violate the inter-dependencies of gene expression
determined by the regulatory relationships. For instance, if a
gene A (unconditionally) inhibits the expression of the genes B
and C, as determined by the network structure, then any gene
expression pattern in which gene A is highly active but genes B
and C are also highly expressed would be forbidden by the
network. Such a pattern would spontaneously adopt a more

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Prospects & Overviews

....

stable configuration (e.g. by downregulating genes B and C)

that complies with the interaction rules.
Thus, gene-gene regulatory interactions introduce massive
constraints on the realization of possible configurations of
gene activities and this is manifest in the distinct stabilities
of gene expression patterns. The coordinated change of gene
expression underlies the change of the network state as an
integrated entity. Mathematically, the network state S of a
network with N genes can be represented by a point at a
particular position S, whose coordinates are defined by the
N values of the gene expression levels in S, within the space of
all possible gene expression patterns. This abstract space is
referred to as the N-dimensional state space (Fig. 2C). When
the gene expression pattern changes, the position of S changes
accordingly and the state S moves along a predestined path
(trajectory, Fig. 2C). This movement is driven by the networks search for more stable expression patterns or network
states.

Attractors as quasi-potential wells:

multistability
Since network states have distinct stabilities, one needs to
know the relative stability of each state S, which is a consequence of the particular wiring of the regulatory interactions; this will allow one to predict a phenotype change.
The relative stabilities can indeed be computed (with some
fundamental restrictions see legend of Fig. 2) and
represented as a quasi-potential energy U of each state S
[53]. Thus, the specific network interactions impart on each
state S the urge to move on a specified trajectory towards a
more stable state until all regulatory influences are satisfied.
In such a balanced, force-free state S the gene expression
pattern is stationary and also locally stable. Such stability
implies that it is surrounded by less stable states thus, it will
re-establish its characteristic gene expression pattern when
slightly perturbed and displaced to a close neighbourhood.
A stable state S is called attractor state as it attracts the
nearby, less-stable states that are in its basin of attraction.
Trajectories within a basin of attraction converge to the attractor state of that respective basin the state where the quasipotential U exhibits a local minimum akin to being at the
bottom of a potential energy well in classical physics [53, 54].
If complex regulatory networks are rather sparse and
contain non-linear regulatory relationships and positive feedback loops [8], as is typically the case with GRNs, it turns out
that they readily give rise to a multitude attractor states within
one network. This coexistence of multiple attractors (multistability) is the chief reason why a quasi-potential U offers
additional information not available in traditional dynamical
systems theory that examines only local stabilities; the landscape affords a comparison of the relative stabilities (depths)
of attractors [5355].
Since a stable attractor state S defines a particular stable
expression pattern of all the N genes of a network, it also,
according to the premise, represents an observable cellular
phenotype [8, 45]. It is now accepted that the distinct cell types
in metazoa, which are characterized by specific, self-stabilizing gene expression profiles, are attractor states a general

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Prospects & Overviews

Towards the epigenetic (quasi-potential)

landscape
To achieve the familiar three-dimensional potential landscape picture accessible to human imagination, the N dimensions of the gene expression state space is compressed
(projected) into a two-dimensional XY plane (Fig. 2d), in which
the neighbourhood relationship is preserved, and in which
two nearby points (states) represent similar expression patterns. The third, Z-dimension then can be utilized to depict the
quasi-potential energy U of each gene expression pattern at
the given XY position (state S) as an elevation that reflects the
stability of respective state S. A higher elevation, by convention, represents higher quasi-potential and, hence, lower
stability. The resulting quasi-potential landscape can be
depicted as a geographical landscape with a characteristic
topography precisely as Waddington envisioned in his epigenetic landscape [9, 43, 44, 61]. The cell state S is defined by a
position on that landscape, marked by Waddingtons marble
which rolls down the valleys as S changes, seeking the lowest
points available (local minimum of U), which corresponds to
the most stable gene expression pattern in a given region. The
stable gene expression pattern of the attractor state that the
cell adopts appears to self-organize across the genome, when
in reality it merely seeks stability of the network state. Again,
order for free. By contrast, hills represent energy barriers
that constrain the course of the marble to the trajectory of S in
state space. This trajectory represents the succession of gene
expression patterns and, hence, represents a developmental
path that Waddington called chreods.
In summary, the specific landscape topography (Fig. 2D) is
a mathematical manifestation of the constraints imposed by
the particular network architecture (Fig. 2A) on the global
dynamics of a GRN. From GRN studies in silico it has been
shown that a network of the architecture type found in evolved
GRNs almost inevitably generates a rugged landscape with
multiple attractor states [8, 22, 28]. Thus, one genome specifies
one particular GRN wiring diagram, which in turn specifies
one particular landscape that captures that genomes entire
developmental potential. It is through the landscape topog-

Bioessays 34: 149157, 2011 WILEY Periodicals, Inc.

raphy, as an extended genotype, that the genome determines

developmental trajectories and terminal gene expression profiles of cell types and thus, ultimately, the organismal phenotype. This principle is in line with the idea that Davidson
has long championed: that embryonic development is hardwired in the genome [62, 63]. But there is room for some
malleability as discussed next.

Epigenetic landscape and network

perturbations
Beyond the intrinsic downward force imposed by the interactions hard-wired in the network, external control or perturbation of the expression of individual gene loci also affects the
dynamics of the state S. An alteration of the expression levels
of genes will, by definition, displace a state S, thus changing
its position on the landscape, by working against the intrinsic
topography-driven movement. Perturbations, akin to a wind
gust blowing onto Waddingtons descending marble, can
derail the marble from its fated trajectory and, if large enough,
cause it to switch valleys and hence destination phenotype
[53, 64]. Such an attractor transition represents an externally
induced, discrete phenotype switch. Once in an attractor, the
network (or cell) will stay there long after the external perturbation has vanished. Thus, attractor states convey a
memory allowing the cell to remember a particular gene
expression profile induced by a previously encountered signal.
It stores information of past perturbations (which can be a
random event) but without the need for mutations. The
genome and, hence, the topography (specifically, heights of
separating hills) controls the susceptibility to such perturbation-induced transitions.
A perturbation of a particular kind is caused by gene
expression noise, due to continuous intrinsic thermal fluctuations of the expression activity of each gene locus or to
random external events that impinge of the expression mechanism [41]. Because the amplitudes of the fluctuations in the
former case are small, they are typically seen as a wiggling
movement of S within the valleys, which usually does not
deviate much from its course because of the attracting
forces around attractor states. Its trajectory is buffered as
Waddington said. But the intrinsic noise or, more likely,
random external perturbations, may occasionally allow a cell
to jump out of a basin of attraction over a sufficiently low hill
into a neighbouring valley (attractor).
It is important to note that the landscape is not a true
energy potential as in classical physics, which gives rise to
path independence (see legend of Fig. 2). It arises from the
mathematical analysis of the global stability of states of the
GRN treated as a dynamic system.
To sum up, the landscape manifests the forces emanating
from the intrinsic regulatory constraints of the network that
extrinsic perturbations need to work against [54]. For all
practical purposes, it has to be remembered that each genome,
which uniquely determines which gene regulates which one
and how, uniquely maps into a particular landscape topography. This purist definition of the endogenous, networkdetermined constraints allows us to erect a clean conceptual
separation between nature and nurture and facilitates the

155

Problems & Paradigms

ck [56] and by Monod and Jacob

idea first proposed by Delbru
[35] more than half a century ago, and in its modern form by
Kauffman in the 1970s [22, 57]. Experimental support has been
obtained by measuring the convergence of trajectories of
genome-wide transcript profile [58, 59], although a nominal
cell type may actually be a heterogeneous mixture of sub-cell
types represented accordingly by multiple small adjacent
attractors [60]. Accordingly, a coherent developmental change
of cell phenotype, equivalent to the coordinated alteration of
the expression status of genes across the genome, is a movement in state space towards a more stable state (among
multiple alternative choices) a journey along a path predestined by the wiring of the GRN.
The broader biological significance of the attractor concept
is that one single GRN can produce a diversity of attractor
states discretely distinct, stable gene expression profiles,
hence, phenotypes. This represents a first departure from the
one-to-one genotype-to-phenotype mapping.

S. Huang

Problems & Paradigms

S. Huang

discussion of epigenetics and its reconciliation with NeoDarwinism. With the above narrow formalism, it is now plausible that mutations change the network architecture and
hence reshape the landscape topography, whereas non-mutagenic perturbations change the gene expression pattern and
hence displace the marble on the landscape.

Conceptual implications for evolutionary

biology
The formalism for mapping a GRN into an epigenetic, quasipotential landscape sheds new light on the black box of
the genotype-phenotype correspondence. (The Supporting
Information Text II lists the most salient corollaries of this
concept.) The quasi-potential offers a solid conceptualization
of the much-sought departure from the one-to-one mapping
implied by the central dogma (Fig. 1) that fails to capture
complex, non-linear relationships, and opens a new perspective for examining the evolution of network behaviour itself.
A single-gene mutation will only alter a phenotype in a
predictable manner if the phenotype is directly encoded by the
mutated gene as is the case for peripheral effector genes (e.g.
encoding haemoglobin chain or enzymes involved in melanin
synthesis) that do not control expression of other loci and
hence do not contribute to network dynamics [65]. Mutations
in such effector genes are plausibly apparent as monogenetic
inherited disease (e.g. albinism, thalassemia). If, in contrast,
the gene subjected to a mutation is a regulator embedded in
the GRN, the network will be rewired and the phenotypic
consequences are indirect and non-obvious [66]. But the landscape concept now affords some general explanatory principles (if the entire network architecture is known) [28]. As
explained above, the mutation will alter the landscape, either
by inflicting a graduated change, such as distorting the contours of a valley (basin of attraction) and altering the height of
separating hills (hence modulating accessibility), or, more
dramatically, by destroying or creating an attractor [67].
The latter type of mutation could explain phenomena in
evolution characterized by sudden, rare and discontinuous
events that have been difficult to reconcile with the gradual
nature of evolution. When a new attractor suddenly becomes
accessible and occupied following a mutational landscape
distortion (a rather rare event [28]), not only does it open
an entire pre-organized stable, but previously inaccessible,
gene expression configuration (possibly, a new cell type) but
also a spurt of evolutionary progress could be triggered. This is
because the gene expression patterns encoded by the newly
occupied attractors in uncharted land have not been subjected
to evolution and thus, have not undergone optimization for
contributing to organismal fitness. Therefore, these still possess much optimization potential allowing for a sudden
rapid climb to a new fitness peak (e.g. the evolution of the
effector functions of a new cell type). This formal explanation
obviously differs fundamentally from that of the NeoDarwinian school that depends on a linear genotype-phenotype mapping and has struggled with the question of how
small mutations in the conserved homeotic genes cause large,
discrete jumps in phenotype space [68].

156

Prospects & Overviews

....

Conclusions
Many of the conceptual difficulties of Neo-Darwinian theory can
be traced to its failure to embrace the dynamic consequences
of gene regulatory interactions in their entirety a much
neglected source of self-organizing constraints, variability
and randomness with persistent effects. The quasi-potential
landscape with attractors a mathematical entity that has a
molecular basis and is not a mere metaphor must be considered as a key intermediate layer in the genotype-to-phenotype correspondence that underlies Neo-Darwinian theory.
Gene network dynamics readily accounts for Waddingtons
genetic assimilation, the related Baldwin effect [69], NeoLamarckism and other epigenetic phenomena [70] presented
by critics of Neo-Darwinism. It is plausible that these nongenetic mechanisms could accelerate evolutionary change by
allowing the temporary selection of a metastable, epigenetically encoded phenotype while it waits for the appropriate
genetic mutation to occur (by chance) as if acting as a
lubricant for Darwinian natural selection. This would help
to explain observations that hitherto required faith in some
unlimited power of natural selection of random mutants.
Ironically, both critics as well as defenders of NeoDarwinian orthodoxy carry out their arguments often with
a temperament of well-articulated, colourful metaphoric
ideas instead of using the formal, inescapable principles of
complex dynamic systems. But both groups would benefit
from incorporating them in their thinking.

Acknowledgments
The author would like to thank Stuart Kauffman for inspiring
discussions over the past decade and Adam Wilkins for
thoughtful comments, and to acknowledge the following
agencies for funding: iCore/Alberta Innovates the Future
(AITF); Canadian Institutes of Health Research (CIHR), the
Natural Sciences and Engineering Research Council of Canada
(NSERC).

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SUPPLEMENTARY TEXT (online)

For article in Bioessays, 2011

THE MOLECULAR AND MATHEMATICAL BASIS OF WADDINGTONs

EPIGENETIC LANDSCAPE: A FRAMEWORK FOR POSTDARWINIAN BIOLOGY?
Sui Huang
Institute for Systems Biology
Seattle, WA

I. Semasiology: The Many Meanings of Epigenetics

The following is a discussion of the longstanding semasiological issue regarding the term epigenetics
seen through the optics of the theory of genomewide network dynamics. C. Waddingtons ideas of
epigenetics were initially proposed in a rather loose, metaphoric fashion, motivated by his unease
with genetic determinism and natural selection [1] and inspired by a series of careful perturbation
experiments [2]. They have become iconized and (mis)used in modern days of stem cell biology through
the picture of his Epigenetic Landscape. Yet, it now turns out that Waddingtons qualitative concepts
are beautifully aligned with the mathematical principles of network dynamics that in turn is grounded in
molecular biology of gene regulation. Fortunately, he lived long enough to learn about the connection
between attractors and the valleys in his epigenetic landscape, as he conveyed to S. Kauffman at the 3rd
Waddington Conference (S. Kauffman, personal communication). This late, deep insight shines through
in the writing of his last (unfinished) posthumously published book, Tools for Thought [3].
The new concepts of stable attractors as distinct epigenetic states is also in line with the less
well known epigenetics concept proposed by D. Nanney in the 1960s [4,5] to describe a broad set of
extrachromosomal, extranuclear heredity or functional states.
Alas, this historical unification of originally unrelated concepts is currently threatened by the
hijacking of the term epigenetics for use in a completely separate realm of thought among cell and
molecular biologists unaware of Waddingtons work. They employ epigenetics to describe a distinct
and narrow set of phenomena that is only loosely related to the original meaning intended by
Waddington [6 ]: the methylation of DNA and histones as well as other covalent modifications of
histones. Such modifications are thought to represent epigenetic marks that form an epigenetic
code and to store information without altering the DNA sequence [7]. Epigenetic marks alter
nucleosomes and the chromatin structure, which in turn controls the access of transcriptional regulators
to ciselements on the DNA, and thereby modulate the regulation of gene expression [8]. Since
epigenetic marks can be conserved across cell divisions [9] and, due to incomplete genome rebooting
(erasure of epigenetic marks in germ line cells), could even be transmitted to progenies, they are the
prime candidate for explaining the inheritance of gene expression patterns across generations [10].
Such molecular epigenetics has been implicated in the transgenerational effects of environmental
influences (e.g., toxins, famine) in the absence of genetic mutations [11], although research on the
molecular mechanisms of such epigenetic transgenerational inheritance is still in its infancy [12].

Supplementary Text

Sui Huang

Bioessays (2011)

Aside from opening a potential backdoor for inviting back some kind of NeoLamarckian
mechanisms, as discussed in detail by Jablonka and Lamb [13], changes in epigenetic marks, or
epimutations, are actually the conceptual cousins of genetic mutations and thus, share with the central
dogma the limitations of linear thinking: they affect individual gene loci and thus, by invoking them in
reasoning about inheritance one implicitly follows the scheme of a monocausal mapping from
(epi)genotype to phenotype of the central dogma.
Therefore, epimutations cannot come to the rescue of the NeoDarwinian framework in view of
the nonlinear genotypephenotype relationship. But another difficulty is that, as it turned out, covalent
histone and DNA modifications are highly dynamic and reversible [1416] and thus, cannot engender
new persistent traits by themselves. Moreover, the enzymes that write or erase epigenetic marks (in
mammals) are devoid of sequence specificity and this lack of locus awareness prevents them from
coordinating gene expression across the genome to generate stable patterns of epigenetic marks.
Coordination is achieved only if gene loci talk to each other, i.e. via the GRN, since only the promoter
specific transcription factors possess locusawareness the sine qua non for orchestrating gene
expression among genes. In fact, as we now learn, for the few cases carefully examined some
transcription factors can recognize their target DNA even in compacted chromatin and recruit chromatin
modifying enzymes to open specific loci thus they are the primum movens in changing gene expression
patterns [17]. Indeed, the cellular signaling context is important so that the chromatin state of the loci
alone does not constitute an autonomous code [18]. Thus, ultimately it is the stable gene expression
pattern imposed by a network of transactivating effects (the attractor state) that establishes epigenetic
memory and not the reversible, locusagnostic epigenetic marks. The latter may perhaps confer
additional stability and durability to the expression status of individual gene loci. Therefore, the
patterns of activating and silencing epigenetic marks may just mirror the dynamical state of the GRN.
Then, the burgeoning analyses of genomewide DNA methylation or histone modification patterns
(epigenomics) [19] may be seen as a merely another genomewide readout for positioning the cell state
on the epigenetic landscape in the Waddingtonian sense, joining the other layers of dynamic genome
wide determinants of the GRN state embodied by transcriptomes, proteomes, metabolomes, etc no
more and no less.

Waddington's "Epigenetic Landscape"

(from his 1957 book: The Strategies of the Gene)

Supplementary Text

Sui Huang

Bioessays (2011)

II. Consequences of the gene regulatory network dynamics and

the quasipotential landscape for the NeoDarwinian paradigm
1. The highdimensional attractor states are capable to selforganize a specific genomewide gene
expression pattern following imprecise external signals or in response to perturbations by relaxing to
the same state (lowest point in the valley). Thus the associated gene expression programme is robust
and can be propagated across cell generations in the absence of the signal that originally caused the
cell to enter that state. Attractors thus constitute a genomic memory that classify and remember
past environments.
2. A complex GRN typically gives rise to a rugged epigenetic landscape that represent a multistable
system in a highdimensional state space in which multiple attractors (in the 1001000 for a gene
network with 10,000 genes) coexist. Each attractor offers a distinct, stable gene expression profile
that maps into a distinct cellular functionality and hence can be viewed as a genetic programme. A
cell can switch between the attractors by a change of gene expression at multiple gene loci, either
driven by gene expression noise or, more efficiently, triggered by specific regulatory signals that may
have evolved to coordinate the necessary expression changes. It has been suggested that early in the
evolution of new gene expression programmes no specific signal existed to direct a particular
attractor transition but that noiseinduced attractor transitions and selection for cells that are in the
adaptive attractor could underlie physiological adaptation of a cell population to a new
environment. The possibility of such a mutationindependent attractor selection has been
demonstrated in a synthetic network in E. Coli [20]. The signal transduction apparatus may have
evolved later to enhance the probability of specific attractor transitions and associate them to
particular conditions.
3. The central historical idea: Each highdimensional attractor represents a differentiated state, or a
cell type, as Kauffman has proposed four decades ago (and Waddington has indirectly implied more
than six decades ago). The correspondence between a distinct cell (phenol)type and attractor state is
now receiving increasing attention after recent transcriptome measurements lent empirical support
to it [21,22].
4. A critical corollary of the above: not all attractors on landscape are actually occupied by a cell type in
a given metazoan body (Fig 2d). A simple calculation will confirm that only a fraction of the entire
epigenetic landscape is normally occupied by cells of the adult organisms or traversed by the
maturing cells during development. The unoccupied attractors, lets imagine them as side valleys,
do not encode phenotypes that contribute to the organisms fitness. They are inevitable
mathematical byproducts of the network that has evolved to produce the set of useful (occupied)
attractors and to ensure proper development and robust homeostasis. Importantly, only those
attractors that are occupied during life are exposed to finetuning of their associated gene expression
programmes (including the effector gene they command that establish the phenotype) by natural
selection. There exists no selection pressure to rewire the network such as to fully eliminate those
attractors that are never occupied a task likely to meet insurmountable mathematical constraints.
3

Supplementary Text

Sui Huang

Bioessays (2011)

5. The landscape topography offers now an intermediate phenotype, perhaps in the Waddingtonian
sense of an epigenotype [23], to understand how natural selection operates to form a complex
traits if there is no simple onetoone mapping between genotype and phenotype. The landscape
topography is carved by evolution as follows: Mutations will typically alter gene regulatory
interactions (by affecting ciselements or interaction domains of proteins) and thus, rewire the
network. As a mathematical consequence, they change the landscape. Then, natural selection can
act: A landscape feature that contributes to streamlining developmental trajectories, preventing
cells to enter unused attractors where they can be trapped, will be selected.
6. It is tempting to speculate about the significance of the unused, normally inaccessible but
mathematically inevitable attractors, the forbidden sidevalleys on the landscape: They may lead to
tumorigenesis [24] (discussed in detail in [25]) when cells, with the help of somatic mutations that
make them accessible, are trapped in such attractors and stay immature. Furthermore, unused
attractors may harbour the potential for evolution: just few mutations may suffice to distort the
epigenetic landscape to make uncharted land with nonevolved attractor accessible, thus opening
entire sets of intrinsically stable gene expression programmes for exploration. (One may even
further speculate that cancer is the price we pay for the builtin potential to evolve new cell types).
7. Since mutations change the landscape topography they can delete, alter existing or create new
attractor states. Then, not just genes but attractors (if accessible and occupied) may be units of
selection. Hinshelwood first proposed such an idea by suggesting that stable reaction patterns in
the chemical networks of the cell could be the target of selection [26]. But to what extent do
random mutations in general affect the landscape topography? Intriguingly, computer simulations of
artificial gene networks have shown that (for the particular class of critical network structures
thought to be used by living systems [27,28]) most mutations do not destroy the overall landscape
topography but tend to preserve existing attractors while distorting their basins or they generating
new attractors [29].

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