Gene regulatory network subcircuit controlling
a dynamic spatial pattern of signaling in the
sea urchin embryo
Joel Smith1and Eric H. Davidson1
Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX and approved August 28, 2008 (received for review July 7, 2008)
We dissect the transcriptional regulatory relationships coordinat-
ing the dynamic expression patterns of two signaling genes, wnt8
and delta, which are central to specification of the sea urchin
embryo endomesoderm. cis-Regulatory analysis shows that tran-
scription of the gene encoding the Notch ligand Delta is activated
by the widely expressed Runx transcription factor, but spatially
restricted by HesC-mediated repression through a site in the delta
5?UTR. Spatial transcription of the hesC gene, however, is con-
delta, thereby permitting its transcription. The blimp1 gene is itself
linked into a feedback circuit that includes the wnt8 signaling
ligand gene, and we showed earlier that this circuit generates an
expanding torus of blimp1 and wnt8 expression. The finding that
delta expression is also controlled at the cis-regulatory level by the
blimp1-wnt8 torus-generating subcircuit now explains the pro-
gression of Notch signaling from the mesoderm to the endoderm
of the developing embryo. Thus the specific cis-regulatory linkages
of the gene regulatory network encode the coordinated spatial
expression of Wnt and Notch signaling as they sweep outward
across the vegetal plate of the embryo.
Blimp1 ? Delta ? HesC ? Notch ? Wnt
interactions underlying specification of the embryonic territories,
ing of the Strongylocentrotus purpuratus genome permitted the
systematic identification by homology of all transcription factor
genes encoded in this genome, and their developmental character-
ization (2–7). By incorporating all (or nearly all) of the relevant
regulatory players, some portions of the GRN have achieved
sufficient maturity to allow an overall causal understanding of
development. This was shown recently for the network underlying
specification and differentiation of the skeletogenic micromere
lineages or skeletogenic mesoderm (SM) of this embryo (8). In this
article we consider another major aspect of endomesodermal
specification, a concentrically expanding progression of Notch and
Wnt signaling that is initiated in the SM but then sweeps outward
across the vegetal domains of the embryo (see fate map in Fig. 1).
This signaling is required first for specification of the nonskeleto-
genic mesoderm (NSM), and it then participates in endoderm
specification. We ask whether the static genomic regulatory code
can also provide an explanation for this dynamic progression of
intercellular signal transmission.
The Notch receptor is initially expressed maternally and is
globally distributed, although by early blastula stage it is region-
ally localized (9, 10). Before this, the location of intracellular
Notch signaling is determined entirely by spatial control of
transcriptional expression of the delta gene, encoding a Notch
ligand. The delta gene is initially activated as an output of the SM
GRN (8). The early SM Delta signal is critical for specification
of the adjacent ring of cells as NSM beginning at the early
blastula stage. In the NSM, reception of the Delta signal causes
represents the genomic code that specifies the regulatory
the essential transcriptional regulatory gene gcm to receive a
direct Notch-mediated Su(H) input (9, 11–13), initiating NSM
specification. When the SM cells ingress into the blastocoel at
late (mesenchyme) blastula stage, the delta gene is turned off in
these cells, but at this time it is activated in the NSM. In
which become gut endoderm, now receive Notch signaling, and
this is required for endoderm specification (14). Expression of
wnt8 is also essential for specification of both NSM and
endoderm. Thus, blocking Wnt8 translation with morpholino
antisense oligonucleotides (MASO), or blocking nuclearization
of its downstream effector, ?-catenin, by overexpression of an
intracellular fragment of Cadherin (15, 6), prevents both NSM
and endoderm specification. These treatments interfere with
activation of a wide number of endomesdoderm genes depen-
dent on a ?-catenin/Tcf input (1, 7).
Recent studies have revealed a dynamic sequence of regulatory
a set of genes in an expanding-torus pattern of expression (Fig. 1).
We showed that the regulatory circuitry underlying this phenom-
enon is a double-feedback loop linking the wnt8 and the blimp1
regulatory genes in a causal embrace (18). cis-Regulatory studies
show that the wnt8 gene requires inputs from both ?-catenin/Tcf
(i.e., from the same signal transduction system that it activates) and
from Blimp1 factor for expression, and conversely, the blimp1 gene
requires inputs from the same Wnt8/Tcf signaling system as well as
from another then ubiquitous factor, Otx (16, 18). However, the
blimp1 gene also contains autorepression sites which, after some
hours when the Blimp1 factor attains a high concentration, shut
activated the feedback circuit there. The consequence is that
following their initial expression in the centrally located SM terri-
tory, expression of these genes disappears from the SM and is
activated in the NSM; but again after some hours, it is shut off in
the NSM and activated in the next outer rings of prospective
endoderm cells, producing the expanding-torus pattern of gene
expression seen in Fig. 1 (18). Additional cis-regulatory experi-
ments showed that the hox11/13b and evenskipped genes are also
directly controlled by this subcircuit (20). We noticed in the course
This paper results from the Arthur M. Sackler Colloquium of the National Academy of
Sciences, ‘‘Gene Networks in Animal Development and Evolution,’’ held February 15–16,
2008, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and
Engineering in Irvine, CA. The complete program and audio files of most presentations are
available on the NAS web site at http://www.nasonline.org/SACKLER_Gene_Networks.
Author contributions: J.S. and E.H.D. designed research; J.S. performed research; J.S. and
E.H.D. analyzed data; and J.S. and E.H.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
whom correspondencemay beaddressed.E-mail:firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
December 23, 2008 ?
vol. 105 ?
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of these studies that the dynamically changing locations of wnt8
as the patterns expand outward: at the eighth cleavage, delta is
expressed in the SM lineage, and wnt8 is expressed in the neigh-
boring NSM; later, during skeletogenic cell ingression, wnt8 turns
off in the NSM cells and is activated in the adjacent endoderm,
while delta becomes expressed in the NSM. As a result, Wnt8 and
Delta signaling domains remain aligned side by side. Here we show
by direct cis-regulatory manipulation that the GRN subcircuit
controlling the expanding-torus pattern of gene expression also
explains the dynamic alignment of Delta and Wnt8 expression and,
more generally, the progression of Delta-Notch signaling first in
mesoderm and then in endoderm specification.
Exclusive Expression of Delta and Its Repressor, HesC. Expression of
the delta gene begins between 8 h and 8 h 40 min postfertilization
(i.e., late fifth cleavage). A high-resolution quantitative PCR
(QPCR) time course is shown in Fig. 2A. Whole-mount in situ
hybridization (WMISH) reveals that delta is expressed exclu-
sively in the SM lineage until swimming blastula stage (19–20
hpf) (21). The delta gene then begins to be expressed in the
adjacent NSM territory (Figs. 2B and 2C). Although not the
focus of this study, delta is activated as well in the apical plate at
thus occurs in three territories: the SM from fifth cleavage to
ingression, the NSM from just before skeletogenic cell ingres-
sion, and at this time also the apical plate.
In its initial phase of expression in the skeletogenic lineage,
delta is activated by ubiquitous factors and repressed by HesC
(21). S. purpuratus HesC (5) belongs to a well-known class of
transcriptional repressors (22–24) encoding group E bHLH and
Orange domains and a C-terminal WRPW motif thought to
recruit the Groucho/TLE corepressor (22, 25). The hesC gene is
expressed zygotically, initially throughout the embryo, except for
the SM lineage (21). There, its transcription is repressed by the
product of the pmar1 gene (21). pmar1 is transcribed exclusively
of hatching (26). The initial phase of delta expression in the SM
therefore follows that of pmar1, and it is controlled by a
double-negative gate in which HesC repression of delta is
relieved by Pmar1 repression of hesC (8, 21). Importantly for
what follows, expression of the hesC gene turns off in the NSM
and apical plate before delta expression in both of these terri-
tories (Figs. 2D and 2E), just as earlier it is turned off in the SM
(i.e., by Pmar1) before expression of delta there. The expression
domains of hesC and delta are thus specifically exclusive in all
Delta cis-Regulation. Previous work identified a delta cis-
regulatory module (CRM), the R11 CRM, located ?16 kb
downstream of the last delta exon, which when coupled to a
heterologous basal promoter is sufficient to drive reporter
expression in the SM (21). In response to pmar1 overexpression,
an R11 reporter expresses throughout the embryo, as explicitly
required by the double-negative gate. Three putative Ets1
binding sites were identified in the R11 CRM within a 75 bp
stretch of sequence; when these Ets1 target sites were disrupted,
sharply decreased reporter activity was observed (27). The R11
CRM was additionally found to respond to treatment with hesC
MASO by producing global ectopic expression (21). However,
the mode of HesC repression remained undetermined, as func-
tional HesC binding sites were not identified in the R11 CRM.
We constructed a recombinant delta BAC clone that contains
the GFP gene knocked into the delta coding sequence, the
shown (also shown are similar patterns seen for several transcription factors).
As illustrated in the bottom panel, the innermost cells (excluding small mi-
cromeres, white) are fated to become skeletogenic mesoderm (SM, brown);
prospective nonskeletogenic mesoderm cells (NSM, green; endoderm,
brown). At SM ingression, expression of delta moves from the SM to the NSM
as wnt8 transitions out of the NSM to the endoderm.
Dynamic expression of wnt8 and delta signaling genes. Schematic
Copies of delta
Time post-fertilization, h
05 10 15 20
mRNA accumulation determined by QPCR. Embryos were harvested at 20 min
intervals. Expression of the delta gene in the SM begins between 8 h and 8 h
40 min PF (i.e., late fifth cleavage). (B and C) Detection of delta transcripts in
21 h mesenchyme blastulae by WMISH; vv, vegetal view; lv, lateral view. delta
transcripts are present in NSM (red arrows) and apical plate (black arrow) but
not in small micromeres (asterisk) or ingressed SM cells. (D and E) WMISH
of this gene are present everywhere except in NSM, apical plate, and SM;
symbols as in (B) and (C).
www.pnas.org?cgi?doi?10.1073?pnas.0806442105 Smith and Davidson
remainder of the gene, plus 66 kb of upstream and 60 kb of
downstream noncoding sequence. This reporter construct faith-
fully expresses in the same domains in which endogenous delta
transcripts are found (e.g., Fig. 3B). Using a series of deletion
constructs, we identified a second functional delta CRM. This
consisted of noncoding sequence from ?90 to ?484 into the
S1A. This proximal CRM, in contrast to the R11 CRM, drives
expression in the SM, the NSM, and the apical plate territories,
thereby reproducing the temporal and spatial pattern of pregas-
trular delta expression in its entirety (Table 1 and Figs. 3A and
3C). In contrast to R11, the activity of the proximal CRM is not
decreased by ets1 MASO perturbation (data not shown). How-
ever, like the R11 CRM, it does respond to hesC MASO,
displaying ectopic expression throughout the embryo (Fig. 3D).
A putative Runx binding site is present at ?365 in the delta
proximal CRM—namely, 5?-TGTGGGA-3? (28). Mutation of
the 5?-TGGGA-3? of this Runx site caused a decrease in
normalized reporter output to 12% ? 9% of control values,
demonstrating potent activation mediated by Runx (Table 1).
Sea urchin Runx is broadly expressed before gastrulation (29),
including in the NSM, and these results implicate Runx as the
major activator of delta in the NSM. However, this in turn
requires repression outside of the NSM to explain why delta is
expressed only there at mesenchyme blastula stage, rather than
everywhere Runx is present. As we see in Figs. 2D and 2E, hesC
is a prime candidate to be the repressor executing this function,
because of its perfectly complementary expression. A possible
HesC binding site, 5?-CACGCGTG-3?, is located at ?359 within
the delta proximal CRM (Fig. S1A). This site is an 8 bp
palindrome, including an inverted 6 bp repeat of the class C,
E-box binding site 5?-CACGCG-3? (30, 31). Disruption of the
sion (Fig. 3E and Table 1), whereas mutation of a class B E-box,
or of N-box sites within the module (Fig. S1A), had no effects.
These results indicate that the expression of delta in the NSM
results from activation by Runx and repression by HesC through
sites in the proximal CRM. Fig. 3F summarizes these relation-
ships: Runx, broadly expressed at stages considered, activates
delta transcription, while repression by HesC confines delta
expression to the domain where HesC is not present, here, the
NSM at mesenchyme blastula stage. Earlier, Pmar1 represses
hesC in the SM, but the question now arises: What is the
repressor of hesC in the NSM?
HesC cis-Regulation and the Role of Blimp1. In the expanding-torus
gene expression subcircuit reviewed briefly herein, the autore-
pression of blimp1 occurs in the NSM at about the same time as
hesC expression is extinguished there. Thus Blimp1 could play
A candidate Blimp1 binding site is indeed present in the first
intron of the hesC gene (Fig. S1B), the sequence of which,
5?-TACTTTCAACT-3?, conforms to the consensus target site
for Blimp1, 5?(A/C/T)(A/G)(G/T)NGAAAG(G/T)(A/G/T)-3?
(18, 32, 33). This sequence is expected less than once per 8 kb
of random sequence, and includes the invariant core CTTTC.
Mutation of this site in a hesC reporter construct caused
expression to remain strong in the NSM territory, in contrast to
controls where, as we have seen, HesC expression is extinguished
(Table 2 and Fig. 4A and B and Fig. S4). Parallel experiments
showed that hesC reporter activity also continues in the NSM of
embryos treated with blimp1 MASO (Figs. S2A, S2C, and S2D);
these same embryos contained ?3-fold lower levels of endoge-
nous delta mRNA at 21 h by QPCR measurement, evidently
reflecting the continued, abnormal presence of the HesC re-
pressor in the NSM. Overexpression of other candidate genes
(foxA, gataE, hox11/13b, gcm, soxC, z13) produced no effect on
hesC expression (Table S2), whereas blimp1 mRNA overexpres-
sion nearly abolished expression of the hesC BAC-GFP reporter
(Figs. S2B and S2E–J). These experiments also revealed that
Blimp1 is responsible for keeping hesC off in the skeletogenic
mesoderm once pmar1 transcription ceases, because disruption
of the Blimp1 site, or introduction of blimp1 MASO, resulted in
return of hesC reporter activity to the skeletogenic mesenchyme
after ingression, a result never observed with control constructs
(Figs. 4 A and B, Table 2, and Fig. S2 C and D and Fig. S4).
Blimp1-mediated repression in other organisms entails hetero-
construct consisting of delta proximal module driving GFP in early mesen-
chyme blastula stage NSM. (B) Same pattern of expression generated by
Delta:GFP knockin BAC. (C) Expression of proximal module reporter construct
in apical plate of 24 h mesenchyme blastula embryo. (D) Same construct,
expressing ectopically in presence of 400 ?M morpholino antisense oligonu-
cleotide against hesC. (E) Ectopic expression of same construct but with HesC
target site mutated, in normal embryos. Quantitative data for experiments
illustrated in (A–C) and (E) are in Table 1. (F) Summary network subcircuit
showing delta and hesC genes: delta receives widespread activation input
from the Runx transcription factor and dominant repression by HesC; hesC is
repressed in the NSM (NSM R of HesC; see text).
cis-Regulatory analysis of the delta gene. (A) Expression of reporter
Table 1. Activity of Delta reporter constructs
ReporterNo. injectedCorrect GFP?/total GFP?*
5 kb reporter
Proximal module only
w/Hes site disruption
w/Runx site disruption
of endogenous delta expression and in no ectopic territory at 18 h and 27 h
Table 2. Activity of HesC reporter constructs
10 kb reporter
w/? Blimp site
w/? Su(H) site
10 kb constructs (Fig. 5A) at 27 h postfertilization—namely, weak activity in
zone 1, strong activity zone 2, intermediate activity zone 3 (NSM), and no
activity zone 4 (ingressed skeletogenic mesoderm).
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chromatin induction and long-term gene silencing, suggesting a
mechanism for the persistent repression we observe (34–36).
Note also that members of the Blimp transcription factor family
are well known to act as activators, as we have shown that Blimp1
does in the wnt8 cis-regulatory system, and also as repressors, as
in its own cis-regulatory system (18). Blimp1, in addition to
shutting down its own transcription, is responsible for doing the
same to hesC. Because HesC is the dominant repressor of delta,
this demonstrates a causal chain linking Blimp1 with delta:
Blimp1 represses hesC and HesC represses delta.
Delta-Notch Signaling Input into the hesC Gene. Awild-typereporter
assay for hesC at mesenchyme blastula stage revealed a complex
pattern of expression. There are four distinct domains of activity
of the apical plate and extending into the presumptive Veg1
endoderm; (2) an extraordinarily high level of activity in the Veg2
tier of endoderm cells (also noted in analyses of hesC expression by
WMISH); (3) a low level in the NSM; and (4) no wild-type
expression in the (ingressed) SM. The cells receiving Notch signal-
ing at this stage are those in the Veg2 endoderm adjacent to the
NSM; the previous phase of Notch signaling was received by the
NSM. Because high expression of the HesC reporter first in NSM
and then in Veg2 endoderm is coincident with Notch signaling first
within the NSM and then the Veg2 endoderm, the hesC gene itself
could be a target of activation by Notch signaling. In fact, a
consensus Su(H) target site exists at ?2 from the start of tran-
scription in the hesC gene (Fig. S1B). Mutation of this site led to
sharp decreases in reporter activity in the Veg2 endoderm cells to
the basal levels seen in the ectoderm, where no Delta-Notch
signaling at this stage occurs (Figs. 4A and 4C). The mutation
furthermore abolished activity entirely in NSM cells. The Delta-
Notch signal therefore accounts for the unusually high level of
fluorescence in the Veg2 endoderm cells receiving the signal at this
reporter activity seen in NSM cells, which previously received the
Delta-Notch signal, reflects residual reporter protein and not
continued expression. Thus Fig. 4 shows that the hesC cis-
regulatory system responds negatively to Blimp1 repression and
for the dynamic pattern of spatial expression of the potent HesC
To close the circle, so to speak, we also found that the gene
of the Blimp1 repressor, just as is the hesC gene. Following the
gradual disappearance of maternal Notch protein (9), the notch
gene is zygotically expressed like hesC in the Veg2 endoderm
ring (10), where it is responsible for transducing the NSM Delta
(Fig. S1C) causes massive vegetal expansion of expression of a
Notch:GFP reporter construct into the NSM domain (Fig. S3).
The activator of the notch gene remains unidentified.
In brief summary, the new results presented here show the
following cis-regulatory linkages: the delta gene is repressed
directly by HesC and activated directly by Runx; the HesC gene
is repressed by Blimp1 and activated by Notch signaling; and the
notch gene is also subject to direct Blimp1 repression. As we now
discuss, these regulatory linkages, combined with those earlier
discovered in the expanding-torus GRN subcircuit, suffice to
explain the dynamic pregastrular progression of Notch signaling
states across the endomesodermal domains of the embryo.
The cis-regulatory target sites functionally analyzed in this work
are structural features of the genomic sequence, but the regu-
latory linkages they specify produce a dynamic sequence of
spatial signaling interactions. The motivating engine of this
dynamic system is the wnt8-blimp1 expanding-torus subcircuit
(18). As reviewed previously, this is a feedback subcircuit
composed of the wnt8 and blimp1 genes, expression of which
progresses concentrically outward from the SM to the NSM and
hesC GFP reporter reveals four domains at mesenchyme blastula stage: (1) low
endoderm; (2) high activity in the preendoderm cells proximal to the NSM; (3)
medium-low activity in NSM; and (4) no expression in the already ingressed SM
cells (exposure time: 375 msec). The embryos were oriented identically and
for embryos displaying each variant of the expression patterns in these experi-
1 of the hesC locus. Strong expression of hesC reporter now occurs both in NSM
sible for the persistent extinction of hesC transcription in both mesodermal
territories (exposure time: 268 msec). (C) Effect of mutating Su(H) binding site in
activity in the endoderm of zone 2 (exposure time: 920 msec). (D) Subcircuit
diagram showing Blimp1 repression input into both itself (18) and hesC (this
work). (E) Subcircuit showing Delta-Notch signaling input (chevron) from neigh-
boring cell into hesC gene and HesC repressive input back into delta.
cis-Regulatory analysis of the hesC gene. (A) Expression pattern of the
www.pnas.org?cgi?doi?10.1073?pnas.0806442105 Smith and Davidson
then to the surrounding endoderm. At each stage, after some
hours of transcription, the blimp1 gene represses itself, causing
of downstream regulatory genes (20) in what now becomes the
silenced center of a torus of gene expression. Meanwhile, Wnt8
diffusion causes expansion of the subcircuit expression torus to
the adjacent cells of the next domain. But we now see that the
functional significance of this system extends even beyond the
sweep of regulatory states across the vegetal plate: it specifically
controls the activities of the Notch signaling system as well as
maintaining an exact Boolean complementarity between Wnt
and Notch signaling as the domains of each shift outward.
The sequence of regulatory transactions is summarized in Fig.
5. The overall subcircuit is shown in Fig. 5A, from results
presented here and elsewhere (18, 20). The subcircuit thus
portrayed indicates the relevant cis-regulatory linkages encoded
in the genomic DNA (the ‘‘view from the genome’’; ref. 1). The
lynchpin of this subcircuit is the blimp1 gene and its autorepres-
sive cis-regulatory module. The present study shows that blimp1
executes two crucial transitive repressions in addition to autore-
pression, the targets of which are the notch and hesC genes. Thus
when blimp1 shuts itself down in the center of the torus it does
the same to these genes. But HesC in turn controls transcription
of the Notch ligand Delta; and Delta presentation, plus the
zygotic expression of the Notch receptor, determines the chang-
ing locus of Notch signaling. The state of the subcircuit in the
eighth cleavage NSM is shown in the ‘‘view from the nucleus’’ (1)
of Fig. 5B. Here the torus subcircuit genes, including wnt8, are
active in the NSM, as is hesC, driven by Notch signal input in
response to the Delta ligand, then being expressed in the inner
SM domain. Because HesC is present, delta is inactive. By early
mesenchyme blastula stage in the NSM (VFN in Fig. 5C), the
state of the subcircuit has changed. Blimp1 autorepression has
eliminated blimp1 transcription in the NSM, and therefore wnt8
transcription, and Blimp1-mediated repression has extinguished
hesC expression on the same schedule. Thus expression of delta,
driven by the widespread activator Runx, is now allowed to occur
for the first time in the NSM, but because maternal Notch is no
longer available, the NSM cannot itself respond to Delta because
the zygotic notch gene is also clamped silent by Blimp1 repres-
sion. At the same stage the state of the subcircuit is again
the cells receiving the Delta ligand are enabled to use it for
Notch signaling, becaise zygotic notch is actively expressed, as is
hesC, which silences delta. An important Notch target in the
endoderm is the gatae gene (38). Meanwhile, the blimp1/wnt8
subcircuit is active in the endoderm, where their various targets
are important for specification of the preendodermal state.
The structure of the subcircuit in Fig. 5A, particularly the causal
link between blimp1 and hesC genes, thus ordains the two signaling
delta expression in the wake of the expanding torus. The essential
developmental result is that Delta and Wnt8 domains remain
exclusive and adjacent in the NSM and endoderm (Figs. 5C and
5D), respectively, just as they were during the previous stage when
Delta was expressed in the SM and Wnt8 in the NSM (Fig. 5B).
Although biochemical constants of protein/mRNA synthesis and
turnover, and protein-DNA association and transcriptional re-
sponse must determine the actual kinetics of these events (39), the
basis for the coordination of the signaling systems rests solely in the
cis-regulatory logic. The genomic sequences mediating Blimp1
autorepression, hesC, and notch repression; the AND logic depen-
dence of wnt8 on Blimp1; and dominant HesC repression of delta
permit only this sequential outcome. A general feature of this
subcircuit is the extremely important role in its logic played by
repression: Blimp1 represses the hesC gene, HesC in turn represses
the delta gene, and Blimp1 represses the blimp1 gene as well as
activating the wnt8 gene.
In summary, we here show experimentally a gene regulatory
network subcircuit that (1) causes Delta-Notch signaling first in
the NSM and then in the endoderm, (2) prevents delta and notch
transcription in the same cells, and (3) ensures exclusive adjacent
From skel. meso.
signaling with the wnt8-blimp1 expanding-torus subcircuit. (A) Fate map and
summary network subcircuit showing all linkages. Expression of delta is
input and repressed by Blimp1. For clarity the Wnt signaling pathway is
simplified to show only the end result, nuclearized ?-catenin interacting with
the transcription factor Tcf (nb-Tcf); NICD, Notch intracellular domain; chev-
ron, intercellular input. Fate map at left shows the future skeletal mesoderm
(SM), purple; NSM, green; and endoderm, brown. (B–D) Depiction of linkages
that are active in given stages and domains (i.e., views from the nuclei, VFNs).
Note that NSM cells migrate toward vegetal pole as a consequence of skel-
etogenic cell ingression, and thus cells marked green in (B) and (C) are of the
same lineage. Components shown in gray are inactive, and in color are active.
(B) VFN for eighth cleavage NSM precursors. The blimp1/wnt8 subcircuit is
active, whereas delta is not transcribed due to the presence of the dominant-
acting HesC repressor. (C) VFN for NSM at early mesenchyme blastula. Expres-
sion of delta expression begins as hesC transcription ceases due to repression
extinguished. The onset of delta expression in the NSMs is thereby synchro-
nized with the disappearance of wnt8 from those same cells in the center of
the moving torus. (D) VFN for endoderm at early mesenchyme blastula. The
blimp1/wnt8 subcircuit is now active as a result of previous Wnt8 diffusion
ligand, further enhanced by transcription of the Notch receptor, which in this
domain is not subject to Blimp1 repression; however, delta expression is
reciprocally forbidden by Notch-driven HesC expression.
The gene regulatory network linkages that coordinate Delta-Notch
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domains of Wnt8 and Delta signaling. The sea urchin endome-
sodermal GRN displays many functions mediated by these signal
systems in both NSM and endoderm, and now we know how they
come to be alternatively and successively expressed.
Materials and Methods
Microinjection and QPCR Measurement of GFP mRNA in Eggs Expressing GFP
Constructs. PCR products were purified with the Qiagen Qiaquick PCR purifi-
cation kit and microinjected into fertilized S. purpuratus eggs as described
(40). Linearized BAC constructs were desalted by drop dialysis into TE buffer
on a 0.025 ?m VSWP filter (Millipore). Approximately 1500 molecules of the
desired reporter construct were injected, along with a 6-fold molar excess of
the BAC per 4 pl and no carrier DNA. Embryos were collected at different
stages for observation by fluorescence microscopy for qualitative assessment
of spatial activity, or for quantitative analysis of transcript prevalence by
real-time PCR (QPCR). For high-density cDNA time-course experiments, ga-
metes were harvested from three females and three males, pooled, and
cultured at 14.5°C. Three separate samples were removed at 20 min intervals
for independent processing and QPCR analysis. Data points represent the
average of the three samples. All experimental and control constructs were
tested in multiple batches of eggs. Microinjection and measurement of GFP
mRNA by QPCR was performed as described (41).
mRNA for microinjection was prepared using full-length cDNA with either
Sp6 or T7 polymerase sequences at the 5? end. The Sp6 or T7 mMessage
mMachine kit (Ambion) were used to generate mRNA in a standard reaction
Products were assessed by gel electrophoresis and quantified by spectropho-
tometry before the tailing reaction. Samples were desalted by use of the
Qiagen Micro RNeasy columns and stored at ?70°C. ??Ct was computed by
taking the change in cycle number of an internal standard (ubiquitin) mRNA
ubiquitin mRNA and target gene in experimental condition. A ?1.6 cycle
difference was considered significant.
Constructs. Standard PCR and fusion PCR techniques using the High Fidelity
PCR Kit (Roche) were used to build constructs. PCR products for all significant
reporter constructs were subsequently cloned using the EPICENTRE Copy
Vector System from Promega, and confirmed by sequencing.
Binding-site sequences were mutated by PCR, and the resulting constructs
were checked by sequencing. The PCR primers were designed with tailed non-
priming sequences, including the mutant form of the candidate transcription
ACKNOWLEDGMENTS. We are grateful to Roger Revilla-i-Domingo for
whole-mount in situ hybridizations, Christina Theodoris for high-resolution
recombinants. J.S. is a Fellow of the California Institute for Regenerative
and Human Development Grant HD-037105 and National Institute of General
Medical Sciences Grants GM-075089 and GM-061005.
1. Davidson EH (2006) The Regulatory Genome: Gene Regulatory Networks in Develop-
ment and Evolution (Academic, San Diego).
2. Sodergren E, et al. (2006) The genome of the sea urchin Strongylocentrotus purpura-
tus. Science 314:941–952.
of Strongylocentrotus purpuratus and their expression in embryonic development.
Dev Biol 300:108–120.
4. Howard-Ashby M, et al. (2006) Identification and characterization of homeobox
transcription factor genes in Strongylocentrotus purpuratus, and their expression in
embryonic development. Dev Biol 300:74–89.
5. Howard-Ashby M, et al. (2006) Gene families encoding transcription factors expressed
in early development of Strongylocentrotus purpuratus. Dev Biol 300:90–107.
6. Tu Q, Brown CT, Davidson EH, Oliveri P (2006) Sea urchin Forkhead gene family:
Phylogeny and embryonic expression. Dev Biol 300:49–62.
7. Rizzo F, Fernandez-Serra M, Squarzoni P, Archimandritis A, Arnone MI (2006) Identi-
fication and developmental expression of the ets gene family in the sea urchin
Strongylocentrotus purpuratus. Dev Biol 300:35–48.
8. Oliveri PO, Tu Q, Davidson EH (2008) Global regulatory logic for specification of an
embryonic cell lineage. Proc Natl Acad Sci USA 105:5955–5962.
10. Walton KD, Croce JC, Glenn TD, Wu SY, McClay DR (2006) Genomics and expression
profiles of the Hedgehog and Notch signaling pathways in sea urchin development.
Dev Biol 300:153–164.
11. McClay DR, Peterson RE, Range RC, Winter-Vann AM, Ferkowicz MJ (2000) A micromere
12. Sweet HC, Gehring M, Ettensohn CA (2002) LvDelta is a mesoderm-inducing signal in
the sea urchin and can endow blastomeres with organizer-like properties. Develop-
urchin glial cells missing gene during mesoderm specification. Dev Biol 297:587–602.
14. Peterson RE, McClay DR (2005) A Fringe-modified Notch signal affects specification of
mesoderm and endoderm in the sea urchin embryo. Dev Biol 282:126–137.
15. Wikramanayake AH, et al. (2004) Nuclear beta-catenin-dependent Wnt8 signaling in
vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation
of endoderm and mesodermal cell lineages. Genesis 39:194–205.
16. Minokawa T, Wikramanayake AH, Davidson EH (2005) cis-Regulatory inputs of the
wnt8 gene in the sea urchin endomesoderm network. Dev Biol 288:545–558.
17. Davidson EH, et al. (2002) A genomic regulatory network for development. Science
18. Smith J, Theodoris C, Davidson EH (2007) A gene regulatory network subcircuit drives
a dynamic pattern of gene expression. Science 318:794–797.
19. Livi CB, Davidson EH (2006) Expression and function of blimp1/krox, an alternatively tran-
scribed regulatory gene of the sea urchin endomesoderm network. Dev Biol 293:513–525.
20. Smith J, Kraemer E, Liu H, Theodoris C, Davidson EH (2008) A spatially dynamic cohort
of regulatory genes in the endomesodermal gene network of the sea urchin embyro.
Dev Biol 313:863–875.
21. Revilla-i-Domingo R, Oliveri P, Davidson EH (2007) A missing link in the sea urchin
embryo gene regulatory network: hesC and the double-negative specification of
micromeres. Proc Natl Acad Sci USA 104:12383–12388.
separable modes of transcriptional repression. Mol Cell Biol 15:6923–6931.
23. Alifragis P, Poortinga G, Parkhurst SM, Delidakis C (1997) A network of interacting
transcriptional regulators involved in Drosophila neural fate specification revealed by
the yeast two-hybrid system. Proc Natl Acad Sci USA 94:13099–13104.
24. Giagtzoglou N, Alifragis P, Koumbanakis KA, Delidakis C (2003) Two modes of recruit-
ment of E(spl) repressors onto target genes. Development 130:259–270.
25. Fisher AL, Caudy M (1998) Groucho proteins: transcriptional corepressors for specific
subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes
26. Oliveri P, Carrick DM, Davidson EH (2002) A regulatory gene network that directs
micromere specification in the sea urchin embryo. Dev Biol 246:209–228.
27. Revilla-i-Domingo R (2007) Cis-Regulatory Analysis of the Sea Urchin Delta Gene:
Validating the Architecture of the Gene Regulatory Network Model for Micromere
Lineage Specification. (California Institute of Technology, Pasadena, CA).
28. Thirunavukkarasu K, et al. (2006) Regulation of the human ADAMTS-4 promoter by
transcription factors and cytokines. Biochem Biophys Res Commun 345:197–204.
29. Robertson AJ, Dickey CE, McCarthy JJ, Coffman JA (2002) The expression of SpRunt
during sea urchin embryogenesis. Mech Dev 117:327–330.
30. Tietze K, Oellers N, Knust E (1992) Enhancer of splitD, a dominant mutation of
Proc Natl Acad Sci USA 89:6152–6156.
31. Oellers N, Dehio M, Knust E (1994) bHLH proteins encoded by the Enhancer of split
complex of Drosophila negatively interfere with transcriptional activation mediated
by proneural genes. Mol Gen Genet 244:465–473.
32. Kuo TC, Calame KL (2004) B lymphocyte-induced maturation protein (Blimp)-1, IFN
regulatory factor (IRF)-1, and IRF-2 can bind to the same regulatory sites. J Immunol
node in the sea urchin endomesoderm gene regulatory network. Dev Biol 269:536–551.
by blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase. Mol Cell Biol
35. Gyory I, Wu J, Feje ´r G, Seto E, Wright KL (2004) PRDI-BF1 recruits the histone H3
methyltransferase G9a in transcriptional silencing. Nat Immunol 5:299–308.
36. Osipovich O, et al. (2004) Targeted inhibition of V(D)J recombination by a histone
methyltransferase. Nat Immunol 5:309–316.
38. Lee PY, Nam J, Davidson EH (2007) Exclusive developmental functions of gatae cis-
40. Rast, JP (2000) Transgenic position of the sea urchin embryo. Developmental Biology
NJ), Vol 136, pp 365–373.
sea urchin embryo gene network that controls early expression of SpDelta in micro-
meres. Dev Biol 274:438–451.
www.pnas.org?cgi?doi?10.1073?pnas.0806442105 Smith and Davidson