, 794 (2007); 318
Dynamic Pattern of Gene Expression
A Gene Regulatory Network Subcircuit Drives a
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raneous divergence of ancestral lineages dur-
ing the Cretaceous, LBA, and limited taxon
and gene sampling (25). The results from our
expanded data set (table S2) contrast with pre-
vious studies supporting Sundatheria. When
Ptilocercus lowii and both colugo genera are in-
cluded, the ML and Bayesian trees become con-
sistent with rare genomic changes. The importance
of P. lowii was evident when it was removed
from the data set; ML trees lacked significant
bootstrap support for the divergence between
primates, treeshrews, and colugos (fig. S13).
A Bayesian relaxed molecular clock approach
with eight fossil constraints estimated the origin
of Euarchontoglires at 88.8 million years ago (My),
Euarchonta at 87.9 My, and Primatomorpha
at 86.2 My (see Fig. 2 and table S4 for 95%
credibility intervals). Our divergence dates for
Hominoidea/Cercopithicoidea (26.8 My), Anthro-
poidea (41.7 My), Lemur/Microcebus (40.4 My),
Strepsirhini (62.1 My), and Primates (79.6 My)
were very similar to those estimated from an in-
dependent 59.7-kb alignment of the CFTR gene
region (26) (table S4). The rapid divergence
across the basal euarchontan nodes explains why,
despite the seven indels and high bootstrap and
Bayesian support for Primatomorpha, we were
not able to reject the Sundatheria hypothesis on
the basis of sequence data alone (Shimodaira-
Hasegawa test, P = 0.065) (23). We did reject an
alliance of treeshrews and primates (P = 0.047),
despite the single discrepant indel supporting
primates + tree shrews. This observation is sim-
ilar to other findings of incomplete lineage sorting
in the common ancestor of rapidly diversifying
eutherian clades (27, 28).
The inclusion of nuclear gene sequences
from ptilocercid treeshrews allowed us to date
the origin of extant treeshrews (Scandentia) to
~63.4 My (Fig. 2 and table S4), near the
Cretaceous-Tertiary boundary, concomitant with
divergence estimates of many eutherian orders
and consistent with the long-fuse model of eu-
therian diversification (25). This deep divergence
between Ptilocercus and other scandentians
complements profound anatomical and behav-
ioral distinctions that have been documented be-
tween these groups (2, 13, 21, 29) and vindicates
recent classifications that have separated Ptilo-
cercus in a unique family, Ptilocercidae (21, 22).
As the sole living representative of a eutherian
lineage that diverged in the early Tertiary along
with many modern mammalian orders, we sug-
gest that the phylogenetic uniqueness of Ptilo-
cercus, combined with its restriction to lowland
forest habitats within a relatively limited global
range, should render it an important conservation
priority in global context.
Because our conclusions imply that colugos,
rather than treeshrews, are the most appro-
priate outgroup for Primates in studying the
evolution of adaptive traits, these results may
affect the placement of euarchontan fossils and
our understanding of primate genomic evolution
(3–5). For example, a recent morphological anal-
ysis supporting Sundatheria placed extinct
plesiadapiforms in a monophyletic clade with
Primates (3), in contrast to Beard (12), who
identified plesiadapiforms as members of Der-
moptera, within Primatomorpha. Our reanal-
ysis of the data set from (3) that constrains the
monophyly of Euprimates and Dermoptera
agrees with the placement of plesiadapiforms
as the sister group to Euprimates, though this
result is only weakly supported (3) (fig. S14).
Finally, our results indicate that a draft genome
sequence from a colugo is a necessary prerequisite
to accurately reconstruct the ancestral primate
References and Notes
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U.S.A. 103, 9929 (2006).
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Shrews (Univ. of California Press, Berkeley, CA, 2000).
30. This work was supported in part by NSF (grants EF0629849
to W.J.M. and EF0629860 to M.S.S.) and the National
Institutes of Health (grant HG02238 to W.M.). We thank
A. Jambhekar, T. Crider, A. Wilkerson, V. David, K. Durkin,
D. Wilson, L. Grassman Jr., and A. Wilting for technical
advice and support and the Broad Institute/Massachusetts
Institute of Technology, Baylor College of Medicine–Human
Genome Sequencing Center, and Washington University
Genome Sequencing Center for access to unpublished
sequence data. Sequences from this study have been
deposited in GenBank with accession numbers EU142140-
EU142251 and EU213052-EU213059.
Supporting Online Material
Materials and Methods
Figs. S1 to S14
Tables S1 to S5
9 July 2007; accepted 1 October 2007
A Gene Regulatory Network
Subcircuit Drives a Dynamic
Pattern of Gene Expression
Joel Smith, Christina Theodoris, Eric H. Davidson*
Early specification of endomesodermal territories in the sea urchin embryo depends on a moving torus
of regulatory gene expression. We show how this dynamic patterning function is encoded in a gene
regulatory network (GRN) subcircuit that includes the otx, wnt8, and blimp1 genes, the cis-regulatory
control systems of which have all been experimentally defined. A cis-regulatory reconstruction
experiment revealed that blimp1 autorepression accounts for progressive extinction of expression in the
center of the torus, whereas its outward expansion follows reception of the Wnt8 ligand by adjacent
cells. GRN circuitry thus controls not only static spatial assignment in development but also dynamic
mesodermal components of the sea urchin
embryo is embodied in a gene regulatory net-
he genomic regulatory code that controls
the specification of the future skeleto-
work (GRN). The GRN states the interactions of
about 50 genes encoding transcription factors, as
determined in an extensive perturbation analysis
along with other data (1, 2). The subcircuits of
this network control the establishment of tran-
2 NOVEMBER 2007VOL 318
on November 2, 2007
sient regulatory states in the spatial domains of
the developing embryo. Here we consider a
GRN subcircuit, the function of which is to
direct a dynamically expanding ring or torus of
regulatory gene transcription early in sea urchin
embryogenesis. Transcription of the torus
regulatory genes begins at the vegetal pole of
the egg in the newly born fourth-cleavage mi-
cromeres. These cells give rise to the skeleto-
genic lineages of the embryo. Transcription of
the earliest torus genes starts at about 6 hours
after fertilization, then extends to the adjacent
ring of mesodermal blastomeres in the early
blastula stage (12 hours), and finally encom-
passes the precursor cells that will generate the
gut just before mesenchyme blastula stage (>18
hours) (Fig. 1A). However, within a few hours
after these genes are first activated, their
expression is extinguished, first in the skeleto-
genic domain and then in the mesodermal
Determination of the GRN underlying en-
domesodermal development in the sea urchin
embryo (1, 2) has revealed that the key driver
of the dynamic torus pattern is the GRN sub-
circuit shown in Fig. 1B. To understand the
operation of this subcircuit, it is important to
note that the cis-regulatory control appa-
ratuses of both wnt8 and blimp1 function as
AND operators (3); that is, wnt8 expression
requires both b-catenin/TCF and Blimp1 in-
puts (4) and blimp1 expression requires both
b-catenin/TCF and Otx inputs (table S1).
Morpholino-substituted antisense oligonu-
cleotide (MASO) targeting blimp1 mRNA (5)
or wnt8 mRNA (4) blocks endomesoderm
Expression of the wnt8 gene illustrates the
canonical torus pattern and directly controls its
expansion. Several different wnt genes are ex-
pressed in the sea urchin embryo (6). Although
earlier evidence from sea urchin and Xenopus
indicated that Wnt8 is probably responsible for
driving progressive b-catenin nuclearization
during cleavage (7–9), we found that Wnt8 is
responsible for producing the b-catenin/TCF
input, which, according to cis-regulatory analy-
sis, is obligatory for blimp1 expression (table
S1). Thus, MASO repression of Wnt8 expres-
sion eliminates 80 to 98% of early blimp1
expression (fig. S1).
High-resolution measurements of blimp1
mRNA by quantitative real-time fluorescence
polymerase chain reaction (fig. S2, A and B)
show that a small amount of blimp1 mRNA is
present maternally; however, there is no ma-
ternal wnt8 mRNA. In the early-cleavage
embryo, b-catenin localizes to the nucleus,
and by fourth cleavage, b-catenin can be
visualized in the newly born micromere
nuclei (7). By fifth cleavage, the wnt8 gene
is activated in the micromeres (Fig. 1C). Be-
cause b-catenin/TCF and Blimp1 are the
required inputs into the wnt8 gene, maternal
Blimp1 factor must be available, consistent
with the evidence that this gene is maternally
expressed (fig. S2A). When the wnt8 gene
begins to be transcribed, its response to its
own signal transduction system produces a
positive feedback circuit between adjacent
endomesodermal cells that both produce and
receive Wnt8 (1, 4). Otx protein is also nu-
clearized initially in the micromeres early in
cleavage (10), hence it is available ab initio.
blimp1 transcription is activated one cleavage
after wnt8 transcription (Fig. 1C and fig. S2A).
Activation must depend on the enhanced level
of the b-catenin/TCF input driven by wnt8
transcription itself. Once both genes are tran-
scribed in the same cells (i.e., from sixth
cleavage on), the subcircuit architecture (Fig.
1B) indicates that the patterns of expression of
the mutual regulatory partners, blimp1 and
wnt8, should be similar. This was confirmed
by whole-mount in situ hybridization (WMISH)
(Fig. 1C), and their patterns of expression are
equally represented in Fig. 1A.
An essential design feature of the relevant
blimp1 cis-regulatory module is that it includes
autorepression sites (5).The architecture of the
subcircuit in Fig. 1B suggests that autorepres-
5th cleavage 6th cleavageHatched blastula
Fig. 1. Moving-torus gene expression pattern. (A) Representation of
expression pattern of blimp1 or wnt8 genes (red). The innermost cells are
skeletogenic micromeres; the red ring in the second drawing shows
mesoderm cells (prospective secondary mesenchyme); the outer ring is
definitive endoderm. Expression of the blimp1 gene begins in the
micromeres around 6 hours after fertilization and appears in the adjacent
tier of mesodermal cells by 12 hours. Soon after, expression disappears
from the micromeres. By 18 hours, expression of blimp1 begins in the
cells. (B) GRN subcircuit including otx, blimp1, and wnt8 genes; blimp1b
indicates the early isoform of the blimp1gene(5);nb-TCF,complex of nuclear
b-catenin clearance, the activity of which is inhibited as a consequence of
Groucho/TCF complex forms instead (12) and acts as a dominant repressor at
both the wnt8 and blimp1 loci (dark blue barred stems). nb-TCF is inhibited
from forming by GSK3, the biochemical mechanism of which is symbolized by
the solid circle. Positive inputs from Blimp1 and nb-TCF control wnt8
transcription (4), whereas both nb-TCF and Otx are required for blimp1
expression; blimp1 is subject to autorepression viatwo Blimp1 target sites.(C)
Expression of wnt8 and blimp1, visualized by WMISH. By fifth cleavage, wnt8
transcript is evident in the four micromeres at the vegetal pole. One cleavage
later (sixth), blimp1 transcripts are present in the micromeres. After this, wnt8
and blimp1 are expressed in the same territories.
Division of Biology, 156-29, California Institute of
Technology, Pasadena, CA 91125, USA.
*To whom correspondence should be addressed. E-mail:
VOL 3182 NOVEMBER 2007
on November 2, 2007
sion of the blimp1 gene, some hours after its
activation, could account for the progressive
clearance of both blimp1 and wnt8 transcripts
from the center of the moving torus of regu-
latory gene transcription. A series of cis-
regulatory reengineering experiments showed
that this is indeed the mechanism of clearance.
We used a blimp1 cDNA expression construct
that produces normal blimp1 mRNA under
control of the cis-regulatory module responsible
for early blimp1 expression (5) (Fig.2, A and B).
When the cis-regulatory autorepression sites
were mutated (Fig. 2B), the construct produced
patches of mesodermal blimp1 transcript lying
within the endogenous (mesodermal) blimp1
clearance zone, whereas the control generated
only the normal torus pattern of expression (Fig.
2C, first three columns). We used these con-
structs to test whether, as predicted, ectopic
redeployment of blimp1 mRNA was sufficient
to cause continued expression of wnt8 in
mesodermal territories. Endogenous wnt8 gene
expression monitored by WMISH, as well as a
coinjected wnt8 BAC-GFP (bacterial artificial
chromosome–green fluorescent protein) knock-in
reporter, produced persistent mesodermal expres-
sion in experimental embryos engineered to
express blimp1 in the mesoderm (Fig. 2C, fourth
and fifth columns). [See (11) and fig. S3 for
quantitative data from these experiments, includ-
ing an enhanced GFP mRNA output from the
wnt8 BAC-GFP construct.]
According to the architecture of the sub-
circuit in Fig. 1B, positive spatial input into the
blimp1 gene is provided by b-catenin/TCF (i.e.,
in response to Wnt8 signaling), because the
other positive input, Otx, is continuously
available throughout the whole region. To test
this, we used a blimp1 GFP reporter bearing
mutated Blimp1 target sites. This construct
cannot autorepress, but it displays a normal
pattern of expression if the location of blimp1
transcript is normal (Fig. 2C, sixth column, top).
Therefore, the restricted pattern of endogenous
b-catenin/TCF, due to the restricted domain of
Wnt8 expression, suffices for the restricted
spatial expression of the blimp1 gene. But when
the restriction of Wnt8 expression in the meso-
derm was relaxed by introduction of ectopic
blimp1 mRNA, the expression of the blimp1
GFP construct lacking Blimp1 target sites was
also relaxed (Fig. 2C, sixth column, bottom).
These experiments show that the cause of
progressive vegetal clearance of wnt8 expres-
sion is the restricted localization of the Blimp1
input, which is due entirely to blimp1 auto-
repression, as portrayed in the network sub-
circuit of Fig. 1B.
The Fig. 1B subcircuit architecture is directly
authenticated at the cis-regulatory level (3, 6, 7)
and in this work. Its design ordains its func-
tion. It consists of two partially overlapping
feedback loops, both of which are subject to
an autorepression function, one directly and
one indirectly. One loop is signal-mediated:
Reception of Wnt8 ligand in recipient cells
produces the active b-catenin/TCF transcription
factor complex that is required for expression of
the wnt8 gene itself. In the endomesoderm,
whether or not there is also an autocrine com-
ponent, adjacent cells are indeed linked through
this signal-driven transcription loop [the “com-
munity effect” (1)]. The cis-regulatory system
of the wnt8 gene operates as an AND proces-
sor, in the sense that it requires both the
Blimp1 and b-catenin/TCF inputs for function.
Thus, the requirement for Blimp1 links it
obligatorily to the second feedback loop. The
Fig. 2. Experimental
demonstration of spatial
(A) Genomic locus. Red
start of transcription;
light blue boxes, non-
coding sequence patches
displaying high conser-
vation between the sea
variegatus and Strongy-
Only the exons of the
isoform are shown (5).
(B) Expression constructs.
three conserved regions
that are sufficient to re-
produce correct blimp1
expression and to drive
expression of blimp1
cDNA in the normal pat-
tern of expression. In the
the two Blimp target sites
indicated are mutated,
overlapping TCF sites and
a third possible Blimp site
clusterremainintact.(C)Expression of genes and constructs indicated in upper
right corner of each panel in embryos bearing ExoBlimp (top row) or
mutExoBlimp (bottom row). LV, lateral view; VV, vegetal view; VLV, oblique
lateral-vegetal view. Stable incorporation of injected constructs is mosaic in
sea urchins (18); therefore, not all cells in the center of the ring are uniformly
stained in mutExoBlimp embryos. In ExoBlimp embryos, expression of blimp1
blimp1 expression. Wnt8→GFP denotes a BAC containing the wnt8 gene that
contains a GFP reporter sequence in place of exon 1 of the gene, produced by
in vitro recombination (11). Blimp→GFP denotes a construct similar to
mutExoBlimp [compare with (B)], except that the blimp1 cDNA has been
replaced by a GFP sequence.
LV VLV VV
2 NOVEMBER 2007VOL 318
on November 2, 2007
second loop consists of the requirement for
Blimp1 as a driver of wnt8 expression and the
reciprocal requirement of b-catenin/TCF for
blimp1 expression. The AND processor of the
blimp1 gene similarly links it into the first loop
by its obligatory requirement for the b-catenin/
TCF input, but the other partner here is the Otx
activator. If both cis-regulatory systems did not
include AND gates dependent on the b-
catenin/TCF input, the subcircuit would not
work. The subcircuit has conditional operating
features; that is, its behavior depends on the
particular inputs it sees. The TCF input can
function either negatively or positively, be-
cause (except in cells receiving Wnt8 signal) it
binds the transcriptional repressor Groucho
(12). This keeps the whole subcircuit quiet in
the ectoderm. Its second conditional “off”
function is the autorepression of the blimp1
gene, which from time-course data depends on a
certain accumulation of blimp1 mRNA (and
factor) (fig. S2C).
The subcircuit acts to produce the moving
torus of gene expression, as summarized in Fig.
3. Measurements of the transcript concentrations
indicate that during this phase the blimp1 gene
is producing about 50 transcripts per cell-hour
[this is only a few percent of the maximum
possible transcription rate (13)]. Blimp1 factor
eventually reaches a level where it acts to
repress its own transcription when there could
be as many as ~1500 molecules per nucleus,
given sea urchin translation rates (14) [more
than sufficient for target site occupancy by the
typical transcription factor (13), particularly in
the small-micromere nuclei]. The Blimp1 factor
then disappears from these cells and wnt8 gene
expression turns off as the reinforcing feedback
loop is broken. The half-life of blimp1 tran-
scripts is about 1.5 hours in the micromeres and
2.5 hours in the mesoderm, versus a default
average of 3 to 5 hours for all polysomal sea
urchin embryo mRNAs (15). Meanwhile, how-
ever, the Wnt8 ligand has diffused to the next
tier of cells, the future mesoderm (middle tier in
Fig. 3). The intercellular diffusion rate of Wnt8
plus the molecules to which it is bound is most
unlikely to be rate-limiting, given the very small
intercellular distance and the rates that have
been assumed for this process by others (16).
Upon receipt of the Wnt8 signal, the subcircuit
is thereby activated within the mesodermal
territory, and the same cycle of events runs its
course in this tier of cells, where it operates with
very similar kinetics (Fig. 1 and fig. S2C).
Subcircuit reactivation in the cells on the inside
of the torus of gene expression (by inward
signaling) is not observed: Once blimp1 tran-
scription goes off, it stays off. Blimp1, a SET
domain protein, could be silencing its own locus
by recruitment of factors such as histone meth-
yltransferases (17). The subsequent disap-
pearance of nuclear b-catenin from the cells
within the torus could also result in Groucho
The genomic regulatory code is a static
linear structure, whereas embryonic develop-
ment is intrinsically a process driven by dy-
namically changing regulatory states. Here, we
resolved a gene regulatory network subcircuit
that combines these aspects in one small ap-
paratus. The subcircuit’s kinetics depend on
the synthesis and turnover rates of the relevant
mRNAs and proteins, as well as on the af-
finities of the transcription factors for their cis-
regulatory target sites (13). What the apparatus
does, however, depends on the genomic cis-
regulatory sequence of the blimp1 and wnt8
genes, where its unique features, its feedback
loops, AND gates, and autorepression function
References and Notes
1. E. H. Davidson, The Regulatory Genome: Gene Regulatory
Networks in Development and Evolution (Academic Press,
San Diego, CA, 2006).
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102, 4954 (2005).
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Biol. 288, 545 (2005).
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13. H. Bolouri, E. H. Davidson, Proc. Natl. Acad. Sci. U.S.A.
100, 9371 (2003).
14. E. H. Davidson, Gene Activity in Early Development
(Academic Press, Orlando, FL, 1986).
15. C. V. Cabrera, J. J. Lee, J. W. Ellison, R. J. Britten,
E. H. Davidson, J. Mol. Biol. 174, 85 (1984).
16. S. Sick, S. Reinker, J. Timmer, T. Schlake, Science 314,
1447 (2006); published online 2 November 2006
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Immunol. 5, 299 (2004).
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E. H. Davidson, Development 113, 385 (1991).
19. Supported by a California Institute of Regenerative
Medicine fellowship (J.S.) and by NIH grant
Supporting Online Material
Materials and Methods
Figs. S1 to S3
15 June 2007; accepted 2 October 2007
i1Initial State: Groucho-Tcf
represses zygotic blimp1
and wnt8 expression
i2 Initial Input: maternal
nß-catenin at vegetal pole
i3Nuclear ß-catenin +
maternal Blimp initiate
nß-catenin + Otx drive
zygotic blimp1 (to step )
4 Wnt8 signaling: among
ligand diffuses to neighboring
tier (to step in next tier)
5 Blimp1 represses self
6 In absence of Blimp, wnt8 turns off
Wnt8 ligand diffuses from
inner tier to middle tier or
middle tier to outer tier
2 nß-catenin + Otx drive
3 nß-catenin + Blimp1
drive wnt8 expression
(to step )
Initial steps (inner tier only)
Initial steps (middle and outer tiers)
Common to all tiers
Fig. 3. Summary of mechanism by which the dynamic, concentrically expanding torus of wnt8
and blimp1 expression is generated. The drawing shows a seventh-cleavage embryo viewed from
the vegetal pole to illustrate the radial concentric organization. However, the events indicated in
the numbered key begin at fourth cleavage and extend out to mesenchyme blastula stage (18 to
VOL 3182 NOVEMBER 2007
on November 2, 2007