Dissecting early regulatory relationships in the
lamprey neural crest gene network
Natalya Nikitina1, Tatjana Sauka-Spengler1,2, and Marianne Bronner-Fraser2
Division of Biology, California Institute of Technology, Pasadena, CA 81125
Edited by Michael S. Levine, University of California, Berkeley, CA, and approved October 21, 2008 (received for review June 20, 2008)
The neural crest, a multipotent embryonic cell type, originates at the
border between neural and nonneural ectoderm. After neural tube
closure, these cells undergo an epithelial–mesenchymal transition,
migrate to precise, often distant locations, and differentiate into
diverse derivatives. Analyses of expression and function of signaling
that a neural crest gene regulatory network (NC-GRN) orchestrates
neural crest formation. Here, we interrogate the NC-GRN in the
lamprey, taking advantage of its slow development and basal phy-
logenetic position to resolve early inductive events, 1 regulatory step
at the time. To establish regulatory relationships at the neural plate
and effects of individually perturbing each on the remaining 5. The
results refine an upstream portion of the NC-GRN and reveal unex-
pected order and linkages therein; e.g., lamprey AP-2 appears to
function early as a neural plate border rather than a neural crest
specifier and in a pathway linked to MsxA but independent of ZicA.
These findings provide an ancestral framework for performing com-
more difficult to resolve because of their rapid development.
neural plate border ? transcription factor ? agnathan
border between neural and nonneural ectoderm, extensive migra-
tory ability, and capacity to differentiate into multiple, diverse
derivatives (1, 2). Neural crest cells are first morphologically rec-
ognizable when they begin migration, which occurs in most species
at the time of neural tube closure. As a consequence, neural crest
just before the onset of migration. However, recent data suggest
that this process initiates much earlier, during gastrulation (3–5).
Several signaling molecules (e.g., Wnts, FGFs, BMPs), and
transcription factors (e.g., Msx, Pax3/7, Snail, SoxE genes), have
been implicated in neural crest formation. Cumulative analysis
of their expression patterns and functions, assembled from
several model jawed vertebrates, has led to formulation of a
neural crest gene regulatory network (NC-GRN) proposed to
underlie neural crest formation (6).
The function of NC-GRN genes typically has been examined by
looking at 1 or 2 genes at a time in studies in Xenopus, zebrafish, or
chick. Recently, a more comprehensive functional analysis of
multiple components with the network was systematically tested in
a single species (7). The results suggested that much of the
NC-GRN is highly conserved across vertebrates (7), contrasting
with nonvertebrate chordates (6, 8–15), which appear to lack a
module involved in neural crest specification (16). However, prox-
imal portions of this network appear conserved to the base of the
chordate lineage (17).
network genes remain poorly understood. This is partially because
early interactions are difficult to ascribe in rapidly developing
higher vertebrates like Xenopus and zebrafish, because of the
address the connections between network genes involved in spec-
vertebrate innovation, the neural crest is a multipotent
embryonic cell type characterized by its site of origin at the
ification of neural crest progenitors at the border, we turned to the
basal vertebrate, lamprey, whose slow development makes it pos-
sible to resolve early events in neural crest induction, 1 regulatory
step at a time.
The basal-most extant vertebrates, lamprey and hagfish, are both
agnathans (jawless vertebrates) that have migrating neural crest
cells and most of the neural crest derivatives (7, 17–20). Only
lampreys reliably produce embryos that are accessible to experi-
mental manipulation in the laboratory. Because of its parasitic
lifestyle, the lamprey body plan may not reflect that of the early
chordate ancestor. However, it is interesting to note that modern
lampreys closely resemble lamprey fossils ?360 million years old
(21), suggesting that their body plan has remained fixed. This raises
the intriguing possibility that the core modules of the NC-GRN of
neural crest is unique to vertebrates, analysis of this network in a
basal vertebrate holds the promise of informing on the generic
architecture of a prototypic network.
In a previous study, we identified lamprey homologues of nu-
merous neural crest network genes and examined their effects on
the appearance of neural crest cells (7). However, the interactions
among neural plate border specifiers were not clear because of the
relatively late stage of analysis. Here, we interrogate the lamprey
NC-GRN by focusing on early stages during the most proximal
events in neural crest formation. Specifically, we test the effects of
establish their order and interrelationships, and their connection to
an early acting subset of neural crest specifiers that are also present
at the neural plate border. Our results allow refinement of the
NC-GRN and reveal some unexpected order and linkages therein.
Moreover, this putative network in lamprey provides an experi-
mental scaffold that can be compared, translated, and tested in
higher, more rapidly developing vertebrates.
Results and Discussion
Temporal Sequence of Neural Plate Border and Early Neural Crest
Specifier Genes Expression from Early Gastrula to Early Neurula
[Embryonic Day (E) 3.5 to E4.5]. To resolve events surrounding the
time of neural plate formation and their temporal and regulatory
sequence, we first examined the dynamic spatial and temporal
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: N.N., T.S.-S., and M.B.-F. designed research; N.N. and T.S.-S. per-
formed research; T.S.-S. contributed new reagents/analytic tools; N.N. and T.S.-S. analyzed
data; and N.N., T.S.-S., and M.B.-F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1N.N. and T.S.-S. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com and
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 ?
no. 51 ?
expression pattern and function of selected lamprey neural plate
border and neural crest specifier genes, focusing on the time points
between E3 and E4.5. At E3, the embryos have just initiated
gastrulation, whereas at E3.5, they are actively gastrulating and
resemble round balls of cells, comparable to stage 10.5 Xenopus
embryos, but with a smaller blastopore. By E4, the prospective
neural plate develops anteriorly by thickening and flattening of the
ectoderm, although it is difficult to distinguish in the absence of
molecular markers. However, by E4.5, the neural plate is morpho-
logically visible but has yet to condense into a neural rod.
We analyzed expression of 3 neural plate border specifier genes
(MsxA, Pax3/7, ZicA) and 3 early neural crest specifier genes
(n-Myc, Id, AP-2) by in situ hybridization and quantitative PCR
(QPCR) (Fig. 1). All transcripts are present at E3, although MsxA
is only expressed in a few cells. MsxA levels progressively increase
to peak approximately E3.5, when transcripts are in anterior-half
rapidly decline at approximately E3.75 to peak again between E4
and E4.5, predominantly within the neural plate border and non-
neural ectoderm (Fig. 1 M and data not shown). ZicA and Pax3/7
hybridized embryos show early expression at E3 in the embryonic
ectoderm (ZicA) and future mesoderm (Pax3/7), but their levels
decrease by E3.5 (Fig. 1 B, C, H, and I) to reappear by E4 in the
future neural plate border, neural (ZicA; Fig. 1N) and nonneural
ectoderm (Pax3/7; Fig. 1O). In contrast, all 3 early neural crest
specifiers show high levels of expression during gastrulation, with
1 D and E) and Id transcripts found mainly in the future posterior
n-Myc transcripts are present quasi-ubiquitously in the ectoderm,
whereas AP-2 and Id are expressed in the anterior portion of the
embryo but transcript-negative posteriorly, close to the site of
invagination (Fig. 1 J–L). As the neural plate thickens at E4, Pax3/7
transcripts appear, mostly confined to the broad medioposterior
border territory between the neural plate, labeled by ZicA, and
nonneural ectoderm, labeled by AP-2 and MsxA (Fig. 1 M–O).
Whereas MsxA, ZicA, Pax3/7, and Id transcripts encompass the
more posterior border territory, n-Myc and AP-2 are evenly dis-
tributed throughout the anterior–posterior extent of the neural
plate border (Fig. 1 M–R).
QPCR was used to examine dynamic changes in gene expression
between E3 and E4.5, using 7 different time points, each 6 h apart
(Fig. 1S). Changes in transcript levels of each gene are presented in
onset of zygotic transcription), set arbitrarily at 1 for each gene.
Conversely, the detailed in situ hybridization analysis (Fig. 1 A–R
and data not shown) allows comparison of transcript levels among
different genes. Interestingly the in situ patterns closely follow the
changes quantified by QPCR. In the early embryo, all of the
transcription factors examined are initially present as maternal
transcripts. QPCR data reveal that AP-2, ZicA, Id, and Pax3/7
exhibit 2 peaks of expression during early embryonic stages (Fig.
1S). However, QPCR analysis alone fails to demonstrate the timely
which are evident from our in situ data. The initially very high
concentration of MsxA transcripts accumulated in the mesoderm
and the embryonic ectoderm during gastrulation likely masks the
increase in transcript levels in the newly forming neural plate
border. The second peak only becomes evident after E4.25. The
first peak in expression of MsxA, AP-2, ZicA, Id, and Pax3/7 most
likely corresponds to different roles these genes play during gas-
trulation (i.e., prominent presence of MsxA activity in the lateral
mesoderm, as shown by in situ analysis). The second peak of
genes in the midgastrula to early neurula.
(A–R) Dorsal view of in situ hybridization
border (MsxA, Pax3/7, and ZicA) and early
neural crest specifiers (AP-2, n-Myc, and Id)
in lamprey at early gastrula (E3; A–F), mid-
gastrula (E3.5; G–L), and early neurula
All of the transcripts, except MsxA, are
clearly present at the onset of gastrulation
(E3). (G and J–L) At midgastrula, MsxA is
expressed in the ventral ectoderm, but not
in the prospective neural plate (G), whereas
AP-2, n-Myc and Id are expressed ubiqui-
tously throughout the ectoderm (J–L). (H
by E3.5. (N and O) At E4 ZicA is seen in the
neural plate (N), whereas Pax3/7 is confined
to the neural plate border (O). (M–R) By E4,
MsxA, AP-2, and Id are expressed in the
ectoderm and neural plate border, whereas
n-Myc is present throughout the ectoderm
including the neural plate. (S) Quantitative
sion between E3 and E4.5 by QPCR. The
dynamic changes in transcription level of
individual genes are depicted as fold-
changes relative to their levels at E3. (Mag-
nification: A–R, 20?.)
Expression of neural crest network
www.pnas.org?cgi?doi?10.1073?pnas.0806009105Nikitina et al.
expression at E4–4.5 of the above-mentioned genes corresponds to
their function in the neural plate border. n-Myc appears to be
present at relatively high concentrations in dorsal ectoderm and its
levels remain approximately constant during this period with a
slight decline at approximately E3.75, as it becomes confined to the
neural plate border. This finding suggests that with respect to
border function, E4 is the time of maximal developmental activity
of their gene products, consistent with the possibility that this time
point corresponds to ongoing inductive events (Fig. 1S).
based on their previous classification as ‘‘neural crest specifiers.’’ In
fact, our more detailed analysis suggests that they may act concom-
itant with neural plate border genes. Interestingly, other neural
plate border specifiers, ZicA and Pax3/7, are induced slightly later,
suggesting they may be either downstream of AP-2 and MsxA or in
a parallel pathway but up-regulated slightly later. The transcrip-
tional regulators, Id and n-Myc, appear to be in a separate regula-
tory module that acts early in gastrulation, likely involved in cell
cycle control, and later also in neural plate border progenitors.
Although the precise temporal sequence of neural plate border
specifiers has yet to be precisely resolved in frog, several reports
suggest similarities to our observations in lamprey. For example, it
is clear that induction of Msx1 and Zic1/Pax3/7 represent indepen-
dent events in the frog (3, 4, 22). Although classified as neural crest
specifier genes, c-Myc, Id, and AP-2 are first deployed at the neural
cells (23–29). Because recent studies suggest that onset of neural
crest formation and specifier expression in both frog and chick may
occur in the gastrula, these genes may act earlier in neural crest
specification than formerly assumed. The coregulation of neural
plate border and neural crest specifiers at the border may reflect a
previously unrecognized pan-vertebrate characteristic of the NC-
GRN, illustrating how information from lamprey may translate
because of its tight conservation and ancient origin.
Effects of Knockdown of Neural Crest Genes with Antisense Morpho-
linos (MOs). To unravel regulatory relationships among early neural
crest transcription factors MsxA, Pax3/7, ZicA, n-Myc, Id, and AP-2,
we tested the effects of loss of each gene on expression of the other
5 (Fig. 2 and supporting information (SI) Fig. S1). To this end, 1
blastomere of 2-cell stage embryos was injected with MO antisense
oligonucleotides, and embryos were collected for analysis at E4.5.
This time point was chosen to address interactions between these
early regulatory factors at the neural plate border itself rather than
at the time by which the neural crest proper has formed. This
represents the earliest analysis in any model organism, largely,
aimed to separate events at the neural plate border from neural
By E4.5, the neural plate border is clearly distinguishable in
lamprey embryos, as characterized by increased expression of
border and early crest specifier genes (Fig. 2, Control). To analyze
in situ hybridization (Fig. 2 and Fig. S1), whereas QPCR analysis
was used to quantify the fold change in gene expression (Fig. S2).
Because mosaic incorporation of MO can sometimes lead to subtle
and difficult to interpret phenotypes, we selected only the embryos
that showed strong unilateral incorporation of the MO.
Below, we describe the effects of MO-mediated knockdown of 3
neural border specifiers (MsxA, ZicA, Pax3/7) and 3 early neural
ral plate border specifiers and 3 neural crest specifiers
on neural plate border formation and gene expres-
sion. Dorsal view of stage E4.5 lamprey neurula, an-
terior is to the top. MO-injected side, marked with the
light blue stain after anti-FITC antibody staining, is to
the left (MsxA, ZicA, Ap-2, and n-Myc columns). Id MO
was coinjected with rhodamine-dextran as a tracer (Id
column). MsxA MO injection caused loss of MsxA,
Pax3/7, and AP-2 expression from the neural plate
border, expansion of ZicA expression (caused by the
expression from the posterior neural plate border
(MsxA column). Injection of ZicA MO had a similar
effect on MsxA, ZicA, and Pax3/7 expression, whereas
AP-2 was lost from the neural plate border specifi-
cally, and n-Myc and Id were lost or decreased in the
entire neural plate border (ZicA column). Phenotypes
of AP-2 injected embryos were very similar to those
seen in MsxA MO-injected embryos (compare AP-2
column to MsxA column). N-Myc MO resulted in the
down-regulation of MsxA and Pax3/7, whereas AP-2
and Id expression appeared unaffected. Id MO caused
loss of MsxA, Pax3/7, and AP-2 expression, without
affecting ZicA or n-Myc. Black vertical lines indicate
the midline. Arrowheads indicate loss of gene expres-
sion in the neural plate border. (Magnification: 18?.)
Effect of MO-mediated knockdown of 3 neu-
Nikitina et al.
December 23, 2008 ?
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crest specifiers (AP-2, n-Myc, Id), on each other in the border
territory (Fig. 1; for cumulative results see Table S1).
The specificity of MOs was demonstrated by using a Xenopus
laevis oocyte in vitro translation system (Fig. S3). Furthermore, the
was confirmed in a separate set of rescue experiments (7).
MsxA Knockdowns. MsxA MO-injected embryos exhibited loss or
decreased expression of Pax3/7 (66%, n ? 15), ZicA (59%, n ? 17),
and AP-2 (54%, n ? 22) at all axial levels, whereas Id and n-Myc
expression was lost only in the posterior region on the injected side.
As N-myc and Id are expressed in a large ectodermal domain
encompassing the future neural plate border at stage E3.5 (Fig. 1
stage, but could be controlling their expression in the posterior
neural plate border at E4. Conversely, the effect of MO-mediated
knockdown of MsxA protein on expression of 2 other border
specifiers, ZicA and Pax3/7, is different; they are lost at all axial
phases of border specification at E3.5 when MsxA is widely distrib-
uted along the anterior/posterior axis, and before its restriction to
the posterior border at E4. Interestingly, the effect of MsxA
knockdown on AP-2 expression mirrors that on border specifiers,
with AP-2 depleted at all axial levels. This finding suggests that
direct or indirect autoregulation (30). These results demonstrate
that MsxA plays a critical proximal role in neural crest formation,
because it profoundly affects other neural plate border genes and
early neural crest specifiers. Intriguingly, its effects on AP-2 (i.e.,
crest specifiers, suggesting that AP-2 may be controlled by MsxA.
ZicA Knockdowns. ZicA morphant embryos have an expanded
itself (75%, n ? 20). ZicA levels were often visibly reduced on the
injected side, although the overall area of expression was larger
because of the expanded neural plate. There is loss of Pax3/7 (81%,
n ? 21), MsxA (85%, n ? 33), n-Myc (72%, n ? 18), and Id (72%,
with AP-2 in nonneural ectoderm (72%, n ? 25). These results are
consistent with our QPCR analysis, because transcript levels of
that ZicA only appears at E4 and therefore may not participate in
the earliest phases of border induction, the effects of its depletion
on MsxA and AP-2 in the border territory is likely to occur via a
feedback regulatory loop that maintains rather than activates their
Pax3/7 Knockdowns. Pax3/7 morphant embryos exhibited depletion
of all neural plate border and neural crest markers examined.
Quantification of the phenotypes by QPCR indicates a 5-fold
decrease in MsxA and ZicA levels and 2-fold decrease in expression
of all other factors (Fig. S2). The regulation of MsxA and AP-2 by
Pax3/7 is likely to occur via a feedback regulatory loop that
maintains their expression. In contrast, Pax3/7 may be an activator
of n-Myc and Id.
AP-2 Knockdowns. Our results suggest that AP-2 plays a more
upstream role in neural plate border formation than previously
stage, where it regulates expression of neural plate border specifi-
ers. Knockdown of AP-2 causes a decrease in MsxA (72%, n ? 43)
and Pax3/7 (87%, n ? 28), whereas there was no change in ZicA
expression (Fig. 2). Whereas AP-2 is expressed earlier than and
likely controls initial expression of Pax3/7, it is not clear whether
AP-2 activates MsxA expression at the border or vice versa,
although it is clearly involved in maintenance of its expression at
subsequent stages. In AP-2 morphants, MsxA transcripts are lost in
the neural plate border, but not in the nonneural ectoderm. In the
epidermis, AP-2 directly regulates keratin (31) where it may act
input (32). Conversely, in the neural plate border AP-2 could be
positioned upstream of MsxA, because the murine Msx1 promoter
contains a consensus AP-2-binding site (30). The effects of AP-2
depletion on n-Myc and Id expression are very similar to the effects
between neural and nonneural ectoderm (Fig. 2) in 62% of
Id-stained morphants (n ? 39) and 78% of n-Myc-stained mor-
phants (n ? 38). In contrast, the expression of these genes in the
MsxA, is unaffected. The observed similarity of these phenotypes
suggests that AP-2 and MsxA may act in the same pathway to
only in the posterior portion at E4) and AP-2 might activate the
transcription of Id and n-Myc in the posterior neural plate border
of the embryo at E4.
Id Knockdowns. MO-mediated Id knockdowns result in the loss of
both MsxA and AP-2 expression in the neural plate border and in
the ectoderm on the injected side (80%, n ? 25 and 75%, n ? 16,
respectively; Fig. 2). Pax3/7 expression is lost in the neural plate
of cases, loss of ZicA expression is seen on the injected side, which we
attribute to nonspecific cell death caused by depletion of Id.
N-Myc Knockdowns. Loss of n-Myc affects the spatial distribution of
ZicA (100%, n ? 18), AP-2 (100%, n ? 11), and Id (100%, n ? 14)
transcripts, although we often observed expansion of the neural
expression is significantly reduced in both the neural plate border
and ectoderm (53%, n ? 19), whereas Pax3/7 is reduced but never
absent from the neural plate border (53%, n ? 31). Quantitative
analysis of n-Myc morphants shows that whereas expression levels
of n-Myc and Id do not change, there is an increase in ZicA
expression (concomitant with neural plate expansion), but also in
AP-2 expression, likely corresponding to supplementary transcripts
mostly in the nonneural ectoderm (Fig. 2A and Fig. S2).
Proliferation/Cell Death Assays. ToascertainwhetherMO-mediated
knockdowns exert their effects via influencing cell cycle progres-
sion, we examined expression of the proliferation marker, phos-
phohistone H3, and the apoptotic marker, cleaved Caspase-3, on
the injected versus control, noninjected side (Fig. 3). Interestingly,
the results show that knockdown of MsxA and, to a lesser extent,
AP-2 protein leads to a prominent increase in cell proliferation
within the dorsal field on the MO-injected side versus control side
(Fig. 3A). The mean increase in numbers of proliferating cells for
MsxA was 104% (range: 38.5% to 243%; n ? 8/9 embryos) and
77.6% (range: 37.5% to 113% n ? 6/10) for AP-2 (Fig. 3F). N-myc
range of 6.3% to 14.6% in n ? 5/6 embryos), whereas ZicA (mean
?1.3%; range: ?9.1% to 4.5%; n ? 7/7; Fig. 3F) had no significant
differences from control embryos (mean ?1.7%; range: ?8.7% to
9.1%; n ? 7/7; Fig. 3 C and F).
WT embryos at the beginning of neurulation show few signs of
programmed cell death. Using cleaved Caspase-3 as a marker of
cells undergoing terminal apoptosis, we noted a slight increase in
the numbers of dying cells in the case of Id MO-treated embryos
(n ? 5/10; Fig. 3 D and E) and n-Myc treated embryos (n ? 3/7).
Taken together, these data indicate that transcription factors
present at the neural plate border extensively cross-regulate. As
www.pnas.org?cgi?doi?10.1073?pnas.0806009105Nikitina et al.
predicted, neural plate border specifiers are positioned upstream.
However, AP-2 appears to be aligned with them where it, together
with MsxA, appears to play a role activating initial induction. MsxA
and AP-2 may function in a common pathway to regulate the later
onset of Id and n-Myc in the neural plate border. Based on our cell
cycle progression data, an intriguing possibility is that MsxA and
exit the cell cycle and proceed with expression of the neural crest
where they may act independently in a separate pathway, involved
in maintaining expression of the neural plate border regulatory
module through feedback and self-regulatory loops.
AP-2 and ZicA Function Through Independent Pathways. To test
possible epistatic relationships within the early neural plate border
of AP-2 MO knockdown and vice versa. Embryos were incubated
until E6.5 and the emergence of neural crest was assessed by
FoxD-A staining. AP-2 MO completely abolishes the formation of
the neural crest (Fig. 4A), in a manner that is not abrogated by
coinjection with ZicA mRNA (100%, n ? 79) (Fig. 4B). Similarly,
in the reciprocal experiment, AP-2 mRNA was not capable of
rescuing the effects of Zic Mo (100%, n ? 56) (Fig. 4 C and D). In
contrast, AP-2 mRNA injection can rescue AP-2 MO-mediated
phenotype, as ZicA coexpression rescues the effects of ZicA MO
activation of their expression AP-2 and ZicA act in parallel rather
than common pathways.
Functional redundancy and parallel pathways are common
features of the few vertebrate gene regulatory networks studied
embryos. Because the neural crest is a particularly plastic and highly
self-regulating cell population, the complexity of the gene regulatory
network controlling its formation may reflect the robustness and
regulative ability of this cell population.
Long-Term Effects of AP-2 Knockdown on Formation of Neural Crest
Derivatives. All neural plate border specifiers examined to date are
essential for the formation of all neural crest derivatives and some
other neural plate border derivatives. To confirm that AP-2 is
long-term effects of its loss on neural crest derivatives at E10–15.
Morphant embryos exhibited a significant loss in the neural crest-
derived pigment cell population, with a decrease in Neurogenin
expression in the cranial ganglia and in SoxE1 expression in the
neural crest-derived portion of the branchial cartilage at E11, with
many fewer neural crest cells that failed to extend as far distally on
the MO-injected side of the embryo (Fig. S4). These observations
are consistent with AP-2 acting at the level of a neural plate border
specifier. However, we did not detect a significant change in
expression of SoxE1 downstream effector, Col2a1, at E14, possibly
because of the very subtle penetrance of the cartilage phenotype or
to a partial rescue caused by regulative compensation by another
factor or crest cells crossing from the contralateral side.
In mouse, loss of AP2 results in a perinatal lethal phenotype
characterized by the absence or severe reduction of cranial bones
or a combination of ZicA MO and AP-2 mRNA (B) or AP-2 MO and ZicA mRNA
the neural crest marker FoxD-A. Both AP-2 and ZicA MO abolish the marker
experiments suggest that these 2 genes act in parallel pathways. Black lines
indicate the midline. (Magnification: 40?.)
ZicA and AP-2 act in parallel pathways to bring about the formation
Fig. 5. Current status of the NC-GRN in the lamprey.
assayed by phosphohistone H3 expression (anti-PH3), after Msx MO treatment
(A), but not in Zic MO-treated (B) or control embryos (C). Black line indicates the
on the injected (D) compared with control (E) side, as shown by anti-cleaved
proliferating cells on the MO-injected versus control side. The MOs used are
indicated on the left. (Magnification: A–C, 25?; D and E, 12?.)
Nikitina et al.
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ofneuralcrestorigin.InXenopus,MO-mediateddepletionofAP-2 Download full-text
leads to down-regulation of both Sox9 and Slug expression during
neural crest induction. Conversely, studies of AP-2 mutants in
zebrafish failed to demonstrate an early role for this gene in neural
crest specification, as various AP-2 mutants exhibit no change in
neural crest specifier expression during early steps of neural crest
formation. However, they do show defects in specific subsets of
neural crest derivatives (27, 29, 33). The absence of early defects could
be caused by compensation by redundant paralogs, endogenously
coexpressed with the mutated gene, suggesting all relevant copies of
AP-2 gene should be inactivated in zebrafish to replicate the AP-2
mutant phenotype observed in other vertebrates (34).
took advantage of its basal phylogenetic position to examine a
NC-GRN that likely bears many characteristics of an ancestral
network. Furthermore, lamprey’s slow development allows precise
temporal resolution at the neural plate border in a manner difficult
to accomplish in other model vertebrates. The results reveal some
surprising regulatory relationships in the network. Expression
analysis together with functional studies suggest that AP-2 and
MsxA are upstream of other transcription factors in the NC-GRN
(Fig. 5), despite the fact that AP-2 was previously considered to be
a neural crest specifier gene. Both have nearly identical spatial
distributions and the dynamics of their early expression are similar,
whereas Pax3/7 and ZicA are expressed slightly later. Furthermore,
regulators. However, n-Myc/Id have different expression dynamics
and phenotype. Accordingly, we propose that MsxA and AP-2 are
the most proximal regulators in the network and likely act in the
same pathway. Ultimate confirmation of direct interactions will
require identification and interrogation of neural crest enhancers
for AP-2 and MsxA and detailed epistasis experiments.
Materials and Methods
Animal Husbandry/Embryo Culture. Adult lampreys (Petromyzon marinus) at
different stages of sexual maturation were obtained from Hammond Bay Bio-
logical Station, Millersburg, MI. Eggs were fertilized, cultured and collected as
described (7, 35).
RNA and MO Injections. FITC-labeled MOs against Pax3/7, ZicA, MsxA, n-Myc, Id,
n-Myc, MsxA, and Id MO, and 40 ng/cell of ZicA and AP-2 MO. The injected
embryos were allowed to develop for 96 h, selected for unilateral incorporation
the protocol in ref. 7. For rescue experiments, 1 blastomere of 2-cell lamprey
ZicA transcript (7) and 10–20 ng of AP-2 or ZicA Mo.
In Situ Hybridization. In situ hybridization on lamprey embryos was performed
according to the protocol of Sauka-Spengler et al. (7) with several modifications
RNA Extractions and RT-QPCR. For QPCR assays, both blastomeres of 2-cell
MO integration and lysed at 96 hours after fertilization. For timeline of gene
the manufacturer’s instructions. Real-time PCR was performed on an ABI7000
using the iTaq SYBR green Mix with ROX (BioRad) in the presence of 7–50 pg of
cDNA and 450 nM of each primer. The standard curve method was used for
quantification, and fold change in expression was determined by dividing the
amount of the gene of interest, normalized to RPS9 in MO-injected embryos, by
the normalized transcript amount in control embryos (see SI Text).
Cell Proliferation/Cell Death Assays. Embryos were preselected for unilateral
incorporation on either the right or left side, collected at E4.5, fixed in MEMFA
(0.1 MMOPS,pH 7.4,2 mMEGTA,1 mMMgSO4,3.7%Formaldehyde),andkept
at 4 °C in PBS. Immunocytochemistry with anti-cleaved Caspase 3 (G7481; Pro-
mega) and antiphosphohistone H3 (06Ð570; Upstate Biotech) was performed as
described (35), except that PBT (PBS, 0.1% Triton X-100, 0.2% BSA) was used in
place of PTW (PBS, 0.1% Tween-20).
ACKNOWLEDGMENTS. This work was supported by the National Institutes of
Health Grant DE017911 (to M.B.F.).
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