© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
RESEARCH ARTICLE
A dynamic regulatory network explains ParaHox gene control of
gut patterning in the sea urchin
ABSTRACT
The anteroposterior patterning of the embryonic gut represents one of
the most intriguing biological processes in development. A dynamic
control of gene transcription regulation and cell movement is perfectly
orchestrated to shape a functional gut in distinct specialized parts. Two
ParaHox genes, Xlox and Cdx, play key roles in vertebrate and sea
urchin gut patterning through molecular mechanisms that are still mostly
unclear. Here, we have combined functional analysis methodologies
with high-resolution imaging and RNA-seq to investigate Xlox and Cdx
regulation and function. We reveal part of the regulatory machinery
responsible for the onset of Xlox and Cdx transcription, uncover a
Wnt10 signal that mediates Xlox repression in the intestinal cells, and
provide evidence of Xlox- and Cdx-mediated control of stomach and
intestine differentiation, respectively. Our findings offer a novel
mechanistic explanation of how the control of transcription is linked to
cell differentiation and morphogenesis for the development of a
perfectly organized biological system such as the sea urchin larval gut.
KEY WORDS: Stomach, Intestine, Gene regulatory network
INTRODUCTION
Epithelial cell sheet formation has been a crucial step in the evolution
of multicellularity. In particular, the development of an internalized
gastrointestinal system contributed to the increase of animal
morphological complexity conferring the capacity to perform
extracellular digestion and releasing organisms from body size
constrains, which very likely allowed the development of specialized
structures. Accordingly, the molecular system orchestrating digestive
tube development can arguably be considered to be one of the first
evolved developmental patterning systems and may involve genes that
were later co-opted for the development and patterning of new body
parts (Nielsen, 2008; Roberts, 2000; Wolpert, 1994), thus providing a
model with which to investigate widely employed developmental
processes such as differentiation, morphogenesis and axis formation.
The embryonic gut of most deuterostomes forms through common
developmental processes during gastrulation: endodermal cells are
internalized by invagination through the blastopore of sea urchin and
frog embryos and, in a very similar way, ingress with combined
involution and migration movements through the primitive streak of
the avian and mammalian epiblast. In all cases, the first endodermal
cells to exit either the blastopore or the primitive streak will
contribute to the anterior definitive endoderm, whereas the later
moving cells will contribute to the posterior endoderm. After
Stazione Zoologica Anton Dohrn, Cellular and Developmental Biology, Villa
Comunale, Napoli 80121, Italy.
*Present address: UMR 7238 CNRS-UPMC, Les Cordeliers 15, Rue de l’Ecole de
Mé decine, Paris 75006, France.
‡
Author for correspondence ([email protected])
Received 6 November 2013; Accepted 14 April 2014
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gastrulation, the primitive gut of most animals seems a tube with no
apparent differentiation at the morphological level. However, several
distinct or partially overlapping transcription factor and signaling
molecule expression territories can be detected along the whole
length of the archenteron (Jacobs et al., 2012; Lengyel and Iwaki,
2002; McGhee, 2007; Sherwood et al., 2011; van den Brink, 2007;
Zorn and Wells, 2009). The interaction between molecular regulators
leads, eventually, to the establishment of fine boundaries of gene
expression subdividing the gut into distinct domains, precursors of
the different digestive organs.
Two ParaHox transcription factors, Xlox and Cdx, have been
proposed as central actors in the gut development in several animals
(Brooke et al., 1998; Holland, 2013). Cdx is required for posterior
gut development in many cases examined, and Xlox is crucial for
more anterior gut domains and pancreas formation. In addition, a
number of signaling molecules have been shown to regulate different
aspects of vertebrate gut formation (Feng et al., 2012; Jacobs et al.,
2012; Spence et al., 2011). However, characterizing the molecular
interactions responsible for gut patterning has proved to be a difficult
task in vertebrate models such as mouse or chick, in which the
generation of transgenic lines is often required and tandem genome
duplications occurred (Donoghue and Purnell, 2005).
Most sea urchins are indirect developers and go through a bilateral
swimming (and feeding) pelagic larval stage (the pluteus), before
metamorphosis leads to the formation of the adult form with its
characteristic pentameric body plan. The pluteus has a functional
tripartite gut composed of a muscular esophagus, a large spherical
stomach and a small tubular intestine (Burke, 1981). A cardiac
sphincter between the esophagus and the stomach, and a pyloric
sphincter between the stomach and the intestine separate the gut
compartments. The small number of cells and the detailed description
of cell movements during gastrulation (McClay, 2011), the extensive
molecular understanding of the early endoderm specification
mechanisms (Croce and McClay, 2010; Davidson et al., 2002;
Peter and Davidson, 2010) and the reduced number of gene
duplications compared with vertebrates, all make the sea urchin
embryo an excellent but so far largely unexploited model to
investigate the molecular dynamics of gut development.
ParaHox gene involvement in the sea urchin gut patterning has
been already demonstrated (Arnone et al., 2006; Cole et al., 2009).
Although Xlox is required for the formation of the pyloric sphincter,
Cdx is dedicated to setting up the posterior border of Xlox
expression within the intestine.
In this study, we combine the use of functional analysis
methodologies with high resolution imaging, RNAseq and qPCR to
show that sea urchin larval gut patterning is achieved through the
integration of autonomous and conditional mechanisms of cell fate
specification. First, we demonstrate that the early active endodermal
transcription factors Hox11/13b, FoxA, Bra and Blimp1a (ArenasMena et al., 2006; Livi and Davidson, 2006; Oliveri et al., 2006; Rast
et al., 2002) synergistically regulate ParaHox gene expression. Then,
DEVELOPMENT
Rossella Annunziata* and Maria Ina Arnone‡
we uncover a Wnt10 signaling event mediating Cdx-controlled
repression of Xlox transcription in posterior gut cells, and provide
evidence of a positive auto-regulatory input of Cdx on its own
transcription. Moreover, we show that Xlox is required for the
activation of stomach terminal differentiation genes and also for the
transcription of the muscle-specific myosin heavy chain gene in the
pyloric sphincter cells. Finally, we identify a number of additional
developmental processes potentially controlled by Xlox and we do
that through a genome-wide comparative transcriptome analysis on
Xlox morphant gastrulae and plutei. From all the above studies, Xlox
clearly emerges as a crucial player of cell differentiation in the sea
urchin embryo, whereas Cdx acts as major regulator of intestinal cell
identity.
To our knowledge this is the first study, in a non-chordate
deuterostome (with the experimental advantages that this implies),
to have investigated the gut patterning process at the functional level
and offers a great contribution to our general understanding of the
molecular mechanisms involved in the development of a complex
and highly organized structure such as the embryonic digestive
apparatus.
RESULTS
Dynamic control of gene transcription in the developing sea
urchin gut
The first step in the study of the functional regulation of cell
differentiation is the detailed analysis in time and space of the
actively transcribed factors putatively involved in the process. Since
late gastrula stage, the archenteron of the embryo can be subdivided
in three regions, foregut, midgut and hindgut that will, respectively,
form esophagus, stomach and intestine of the pluteus larva. We
analyzed Xlox and Cdx protein localization and gut gene expression
relative to Xlox and Cdx transcript localization, from the post
gastrular embryo to the pluteus larval stages, including active
transcription factors and terminal differentiation genes (Fig. 1).
The expression dynamics of SpLox and SpCdx proteins have been
analyzed by immunolocalization, using specific antibodies developed
in our laboratory, and the protein versus mRNA spatial localization in
the embryos and larvae have been compared. Xlox protein localization
at late gastrula stage matched the transcript pattern, being detected in
around 30 cells of the posterior gut (Fig. 1A,D,N,Q). However,
whereas Xlox transcripts are strictly confined to around 10 cells of the
larval pyloric sphincter, we detected the protein in about 30 cells
covering the pyloric sphincter and part of the stomach posterior
hemisphere, mostly on the aboral side (Fig. 1B,E,O,R). We detected
Xlox protein in the stomach domain until the 3-week larval stage. We
propose that, late in gastrulation, the most anterior cells expressing
Xlox mRNA stop transcribing the gene but retain the protein and
develop to form part of the larval stomach. Cdx protein localization
exactly matched the corresponding intestinal mRNA expression at all
stages (Fig. 1C-I,U,V).
We then analyzed Cdx mRNA spatial expression relative to Xlox,
Hox11/13b and Bra at both late gastrula (48 h) and pluteus stages.
Experiments at earlier gastrula stage (40-44 h), demonstrate that the
first cells expressing Cdx also express Xlox, Hox11/13b and Bra
(data not shown), thus suggesting that Cdx, Xlox, Hox11/13b and
Bra together define a specific cellular regulatory state, exclusive to
this most posterior part of the late gastrula archenteron, and hinting
to a possible regulatory interaction among them. Moreover,
considering the fact that Xlox, Hox11/13b and Bra expression in
those cells starts before Cdx transcription activation (Arenas-Mena
et al., 2006; Arnone et al., 2006; Rast et al., 2002), we contemplated
and investigated a possible role for Xlox, Hox11/13b and Bra in Cdx
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
regulation. At the late gastrula stage, the domain of Cdx expression
expands, overlapping anteriorly only with Xlox and posteriorly only
with Hox11/13b and Bra (Fig. 1D-I). The expression domains of
Cdx, Xlox, Hox11/13b and Bra in the pluteus show a different
relative pattern reflecting the significant changes occurred in the
morphology of the gut during the transition from late gastrula to
pluteus stages: Cdx is now expressed in the whole intestine, Xlox is
transcribed only in the pyloric sphincter cells, and Hox11/13b and
Bra in the most posterior cells of the intestine. Although no sign of
co-expression is observable at pluteus stage between Xlox and Cdx
(Fig. 1E), a partial overlapping between Cdx and Hox11/13b, and
between Cdx and Bra is still detectable in the posterior cells of the
gut (see arrowheads in Fig. 1G,I).
A very remarkable discovery that we present in this work is the
finding of a Wnt10 signal in the posterior gut cells of the embryo. We
found that Wnt10 transcription starts a few hours after the activation of
Cdx, and is localized in the posterior cells of the gut at late gastrula
(Fig. 1J), prism (Fig. 1K) and pluteus stages (Fig. 1L,M). The cells
expressing Wnt10 apparently represent a subset of the posterior-most
Cdx positive cells. Interestingly, from the moment these cells start
expressing Wnt10, Xlox transcripts become undetectable in them
(Fig. 1N) and when the gut attains its full length, all the intestinal cells
are depleted from Xlox transcripts (Fig. 1O).
Finally, we found that the stomach-specific ManrC1A and ChP
genes are already co-expressed at mid gastrula stage in the cells that
will give rise to the larval stomach (Fig. 1P) and are never expressed
in Xlox- (Fig. 1Q,R), Cdx- (data not shown) or Brn1/2/4-positive
cells (Fig. 1S,T). A third stomach marker, Endo-16, transcribed in
both midgut and hindgut cells at late gastrula (Ransick et al., 1993),
is progressively cleared from the intestinal cells in parallel with the
activation of Cdx, and becomes confined to the stomach larval cells.
No signs of co-expression of Endo-16 and Cdx have been detected
either at prism or at pluteus stages (Fig. 1U,V).
Xlox and Cdx transcription regulation in the developing gut
After the high resolution gene expression analysis, we investigated
the regulatory mechanisms of Xlox and Cdx transcription. We
perturbed protein translation by injecting morpholino antisense
oligonucleotides (MOs), then analyzed Xlox and Cdx mRNAs and,
in some cases, proteins, in the 72 h morphant larvae.
The results of the gene perturbation experiments are depicted in
Fig. 2, and supplementary material Figs S1 and S2. A description of
the postulated gene regulatory dynamics is provided below. In
control larvae, Cdx transcripts are localized in the intestinal cells,
whereas Xlox transcripts are detectable in the cells of the pyloric
sphincter. Interestingly, Cdx transcripts were undetectable in the
intestinal cells of Hox11/13b, Bra, FoxA, Cdx and Blimp-1a
morphant larvae (Fig. 2A-F), suggesting that they are involved in
Cdx regulation. Crucially, in Hox11/13b, Bra, FoxA and Cdx
knockdown larvae (in which Cdx expression is lost), Xlox transcripts
accumulated ectopically in the intestinal cells (Fig. 2G-L), providing
additional support to Cdx repressive role on Xlox transcription (Cole
et al., 2009). Moreover, Xlox transcripts were not detectable in
Blimp-1a morphants (Fig. 2L), suggesting that Blimp-1a might be
required for Xlox transcription, which also explained the absence of
Cdx from Blimp-1a morphants, as Cdx transcription requires Xlox
input (Cole et al., 2009). However, Blimp-1a transcription is active
in a broader domain compared with Xlox (Livi and Davidson, 2006),
suggesting that other unknown factors are expressed in prospective
Xlox-positive cells and recruited for its activation.
An intriguing finding has been the impairment of Cdx
transcription when its own protein formation was blocked
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DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
Fig. 1. Dynamics of gene expression
in the developing sea urchin gut.
(A-C) Xlox and Cdx protein
accumulation visualized by
immunostaining. (D-V) mRNA
localization determined by two-color
fluorescent in situ hybridization. Nuclei
are stained with DAPI and depicted in
blue. The antibodies and probes used,
the age and the orientation of the
embryos are reported in each panel:
h, hours post fertilization; ov, view from
the oral side; lv, lateral view; bv, view
from the blastopore. Overlapping
domains of expression are highlighted
by arrowheads. Arrows indicate the
position of the pyloric sphincter in the
pluteus larvae. Full projections of
confocal z-series (E,N,O,Q-V) or single
slices (A-D,F-M,P) are shown.
(W,X) Schematic of a late gastrula in
frontal view (W) and a pluteus larva in
lateral view (X): endodermal domains
are depicted with different colors
according to gene expression.
Overlapping domains of expression are
represented with colored lines.
ManrC1A name has been shortened to
ManR for clarity. fg, foregut; mg,
midgut; hg, hindgut; es, esophagus; st,
stomach; in, intestine; ps, pyloric
sphincter; cs, cardiac sphincter.
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A Wnt10 signaling mediates Cdx repressive action on Xlox in
intestinal cells
As described above, we found that Cdx activation requires Hox11/13b,
FoxA, Bra and Xlox, and that after the translation of Cdx protein, Xlox
transcription stops in the developing intestine. In order to explore
the possible involvement of intercellular signaling in a Xlox-Cdx
positive-negative feedback loop, we screened by qPCR (see
Materials and methods for details) a variety of signaling molecules
expressed during sea urchin embryonic development. The screening
revealed that SpWnt10 transcript number was strongly reduced
in Cdx knockdown larvae, persuading us of its involvement in Cdxregulated processes. We then tested Wnt10 function through
the embryonic injection of a morpholino designed to block its
translation. Wnt10 knockdown larvae presented an almost normal
gut characterized by a significant reduction of the pyloric sphincter
constriction when compared with control larvae (for the phenotype
of the morphants, see the full projection of confocal z-series in
Fig. 3). Interestingly, Xlox and Cdx double in situ hybridization
revealed that the two genes were co-expressed in the intestinal cells
of Wnt10 knockdown larvae. Immunolocalization experiments
confirmed that Xlox protein ectopically accumulates in Wnt10
knockdown larval intestines (supplementary material Fig. S2K).
These results significantly contributed to the understanding of Xlox
and Cdx regulation, indicating that Cdx-negative control of
Xlox transcription is indirect and occurs via a Wnt10 signaling
event (Fig. 3A-D). Very importantly, Wnt10 transcripts were not
DEVELOPMENT
(Fig. 2E), with the consequent accumulation of Xlox transcripts in
the posterior larval gut cells (Fig. 2K). We suggest that Hox11/13b,
Bra and FoxA, together with Xlox, all cooperate for the activation of
Cdx in the subset of gut cells destined to form the intestine.
Subsequently, because in the larval intestine only some Cdxpositive cells still express the factors responsible for its initial
activation (see double in situ hybridization in Fig. 1), Cdx becomes
recruited for its own transcription, ‘locking-in’ the regulatory state
of intestinal cells.
The injection of morpholinos blocking the translation of GataE
and TgiF (Howard-Ashby et al., 2006; Lee and Davidson, 2004) did
not produce any change in the Xlox and Cdx transcript distribution
(supplementary material Fig. S1), allowing us to exclude their
involvement in Xlox and Cdx regulation. Most of the results
presented in this section and the demonstration of morpholino
functionality have been confirmed through the use of anti- Lox, Cdx
and Bra antibodies (supplementary material Fig. S2).
Taking all the data into account, we propose a model that places
Hox11/13b, Bra, FoxA and Xlox together as members of the
regulatory machinery activating Cdx transcription. As expression of
the first three genes starts many hours before Cdx activation, we
hypothesize that Xlox functions as a switch controlling the exact time
at which Cdx transcription is initiated. Finally, Blimp1a results as one
of the activators of Xlox transcription: its expression starts many hours
before the beginning of gastrulation, thus suggesting the presence of
additional factors involved in Xlox transcription activation.
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Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
Fig. 2. Xlox and Cdx transcriptional regulators. (A-L) Xlox and Cdx mRNA localization determined by chromogenic in situ hybridization in control embryos
(A,G) and in embryos injected with MOs directed against the translation of Hox11/13b (B,H), Bra (C,I), FoxA (D,J), Cdx (E,K) and Blimp1b (F,L) RNAs. All
the embryos were fixed at 72 h. The orientation of the larvae is reported for each panel: ov, view from the oral side; av, view from the aboral side; lv, lateral view.
detectable in Cdx knockdown larvae by in situ hybridization
(Fig. 3E,F), confirming the role of Cdx in the activation of
Wnt10 transcription. We propose that Cdx participates in Wnt10
transcription in the posterior cells of the intestine and that
Wnt10 ligands diffuse towards the anterior side of the gut, clearing
the intestinal cells of Xlox transcripts through the activation of an
effector molecule with repressive functions.
and ManrC1A transcription (Fig. 4C,F,I), Endo-16 ectopic expression
was observed in the intestine of both Xlox and Cdx morphants
(Fig. 4J-L). We propose that Cdx is responsible for Endo-16
repression from the intestinal cells, acting either directly on its
transcription or through the repression in the intestine of the Endo-16
midgut-hindgut activators, and that Xlox executes a repressive
function on Endo16 transcription (Cole et al., 2009) in the cells where
Cdx is never active and that will eventually form the pyloric sphincter.
Xlox and Cdx control the AP patterning of the larval gut
Xlox: a crucial player of cell differentiation in the sea urchin
embryo
The most obvious morphological defect in Xlox knockdown larvae is
the absence of the pyloric sphincter (e.g. compare confocal images in
Fig. 4G,H), the cells of which express the muscle-specific terminal
differentiation gene, Myosin heavy chain (SpMHC) (Andrikou et al.,
2013; Venuti et al., 1993). In control larvae, MHC is expressed in the
four muscular structures of the gut, the esophageal muscles and
the cardiac, pyloric and anal sphincters. The esophageal muscles are of
mesodermal origin whereas sphincter muscles are endodermal
(Gustafson and Wolpert, 1967), suggesting the existence of different
regulatory mechanisms involved in the acquisition of muscle cell
identity in each type. We found that MHC transcripts were specifically
absent from the cells forming the pyloric sphincter in Xlox morphants
Fig. 3. A Wnt10 signaling event mediates Cdx repressive
action on Xlox in the presumptive intestine. (A-D) Twocolor fluorescent in situ hybridization coupled with nuclear
staining (blue). In E and F, single-color fluorescent in situ
hybridization (magenta) and larval cilia staining (green,
acetylated tubulin immunostaining). In A-C, a single slice of a
confocal stack shows Xlox and Cdx co-expression in the
intestine of a Wnt10 knockdown larva. Xlox ectopic
expression in the intestinal cells is indicated by arrowheads.
Absence of Wnt10 transcripts in Cdx knockdown larvae is
shown in F. (D-F) Full projections of confocal z-series. All
larvae have been fixed at pluteus stage and are shown in
lateral view, oral side right.
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DEVELOPMENT
Digestive functions in the stomach are inhibited in Xlox knockdown
larvae (Cole et al., 2009). We explored the expression of stomach
terminal differentiation genes, ManrC1A, ChP and Endo-16, in
Xlox and Cdx knockdown larvae. The expression of all three genes
in Xlox morphants was severely affected (Fig. 4). In particular, ChP
and ManrC1A transcription was dramatically reduced, to the
point that no transcripts could be detected by either colorimetric
(Fig. 4A,B,D,E) or fluorescent in situ hybridization (Fig. 4G-H).
Xlox is thus upstream of a cascade of regulatory events responsible for
the differentiation of the larval stomach. However, the localization of
Xlox mRNA and protein relative to ChP and ManrC1A transcripts
during gut development (see Fig. 1) advocates the involvement of a
signaling event in the induction of stomach cell differentiation.
Although Cdx knockdown did not have any noticeable effect on ChP
RESEARCH ARTICLE
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
(Fig. 4M-O) and suggest that a MHC-specific cis-regulatory module
active only in these cells is responsive, directly or indirectly, to an Xlox
regulatory input. This regulatory mechanism drives the differentiation
into muscles of a small population of endodermal gut cells, allowing
the formation of the constriction between stomach and intestine.
A differential transcriptomic analysis for Xlox morphants
In order to obtain a global vision of transcriptional responses to
Xlox perturbation, we performed a genome-wide comparative
transcriptome analysis (RNAseq) on Xlox knockdown versus
control 48 and 72 h embryonic RNA (Fig. 5 and Table 1). The
MA plots in Fig. 5 clearly show that the effect of Xlox perturbation
on gene expression was much stronger at 72 h, thus potentially
supporting our hypothesis of a signaling event depending on Xlox
and directing stomach differentiation. In particular, we found 5767
differentially expressed transcripts, 23.2% of them upregulated and
28.8% downregulated (by at least 1.5 fold). In Table 1 we provide a
list of genes whose expression was significantly affected in the
differential RNAseq analysis, grouping them based on their
functional category and providing the expression pattern in the
sea urchin embryo, when available. Among these, there are genes,
such as SpCdx, SpLox and Endo16, that have been previously
identified as SpLox targets through qPCR and in situ hybridization
analysis by Cole et al. (2009). Among the most strongly newly
identified downregulated genes are the enzymes involved in the
metabolism of proteins, carbohydrates and lipids, possibly
supporting a role for Xlox in the differentiation of a functional
digestive system. In addition, we found a substantial decrease in the
expression of Ache-13 (an acetylcholinesterase that functions in
neuromuscular junctions) that we correlated with the absence of
pyloric sphincter muscular fibers in Xlox morphant larvae (Fig. 4MO). Likewise, three transcription factors of the Forkhead family
(FoxY, FoxD and FoxP), mainly expressed in foregut and midgut
territories, were significantly downregulated, thus suggesting their
potential involvement in Xlox gut patterning control. NeuroD1, a
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regulator of insulin production in vertebrates (Babu et al., 2008;
Kaneto et al., 2009), was also strongly downregulated, indicating a
possible evolutionary conserved cooperative function for Xlox and
NeuroD1 in the sea urchin larva. Moreover, a GABA receptor and a
cholinergic receptor were highly downregulated and we associated
these results with the potential role of Xlox in the specification of
some neural cells of the ciliary band from a group of ectodermal
cells in which Xlox transcripts have been revealed (Cole and
Arnone, 2009).
The expression of several gut transcription factors, e.g. SpGataE
(Lee and Davidson, 2004), SpHnf1 and SpTgiF (Howard-Ashby et al.,
2006), and SpPtf1a (Annunziata et al., 2013b), was not significantly
affected in Xlox knockdown larvae. Finally, this analysis did not
reveal any significantly affected signaling molecule-encoding gene,
thus leaving unresolved the mechanism mediating Xlox function in
the activation of stomach differentiation genes and introducing new
possible hypotheses, such as Xlox requirement in the control of posttranslational events, something not testable with the approaches
followed so far.
Interestingly, the validation by qPCR of one of the most affected
genes, SpPpglcp (a phosphoglycolate phosphatase), with a 3 h time
resolution, from 44 h to 50 h (supplementary material Fig. S3), has
revealed a strong decrease in its expression since the first analyzed
stage (44 h), hinting a possible direct role for Xlox in the activation
of Ppglcp transcription. However, information about Ppglcp spatial
expression would be needed for a more complete interpretation of
the strong effect of Xlox knockdown on its transcription.
DISCUSSION
Xlox as a potential tissue fate organizer at the stomachintestine boundary
In the present study, we show that the Xlox transcription factor is
required to induce ChP and ManrC1A transcription in the stomach
cells. This, together with the reduction of digestive function and the
alteration of the stomach morphology in Xlox morphants (Cole et al.,
DEVELOPMENT
Fig. 4. Xlox controls the differentiation of stomach and pyloric muscles; Cdx represses Endo-16 in the intestinal cells. (A-F,J-N) mRNA localization
determined by chromogenic in situ hybridization, (G-I,O) full projections of confocal z-series of two-color (G-I) or single-color (O) in situ hybridization coupled with
nuclear staining (blue). All the larvae have been fixed at 72 h and are shown in lateral view, oral side right. The positions of the pyloric (arrowhead) and anal (arrow)
sphincters are indicated. In O, the full projection of a z-stack shows absence of pyloric sphincter.
RESEARCH ARTICLE
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
Fig. 5. Global effect of Xlox translation
perturbation on gene transcription.
(A,B) MA plots showing logarithmic fold changes
(on the y-axis) as a function of the mean of
normalized counts (on the x-axis), in A at 72 h, in B
at 48 h. Genes with a significant response at
adjusted P value <0.05 are in red. Genes
mentioned in Table 1 that display 1.5<FC<−1.5 at
72 h are in green, with the exception of Xlox which
is in blue and magenta in A and B, respectively.
we employed in this context was the genome-wide differential
transcriptomic analysis on Xlox morphants. This type of study has
provided a large amount of information and has high potential in
resolving Xlox patterning functions. Among the most strongly
affected genes were the ones encoding metabolic enzymes, muscleassociated proteins and neuronal factors, plus some transcription
factors expressed in the gut, strongly suggesting that Xlox is upstream
of a number of events leading to the gut AP differentiation.
Finally, we detected Xlox proteins in cells where the mRNA
was untraceable. Analyses of gene expression through transcript
detection are widely used in the construction of gene regulatory
networks. Our study highlights the importance of taking protein in
addition to transcript localization into account when defining
cellular regulatory states.
Cdx conserved function and regulation in intestine
differentiation
Whereas the foregut and midgut domains appear definitively
established at the late gastrula stage, undergoing mainly morphological
changes during the subsequent stages of development, the hindgut
domain remains a dynamic site of cell fate specification throughout
the late gastrula and early prism stages (see Fig. 6). A crucial event
taking place during hindgut development is the activation of Cdx
transcription in the most posterior cells of the late gastrula gut; we
found a cassette of regulatory factors, namely Xlox, Hox11/13b,
FoxA and Bra, involved in Cdx activation. These four transcription
factors are all necessary for Cdx transcription, although we do not
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DEVELOPMENT
2009), as well as the strong reduction in expression of digestive
enzymes observed in our transcriptomic analysis, provide a strong
support to Xlox key role in the development of a functional stomach.
However, the detailed expression dynamics analyzed in this work
imply the existence of a signaling event (S1 in Fig. 6) under the control
of Xlox that induces the expression of the two stomach-specific genes.
Moreover, the sharp boundary of gene expression between stomach
and esophagus suggests that the stomach precursor cells are already
committed to their future fate, but require an Xlox-induced signal in
order to differentiate. Additionally, Xlox is upstream of the entire
cascade that regulates intestine formation by acting as a decisive
activator of Cdx transcription in the intestine precursor cells (Cole
et al., 2009). Xlox also drives the formation of the pyloric sphincter
constriction, triggering the differentiation of muscles from a small
subset of endodermal cells, via the activation of MHC transcription.
Interestingly, the murine Xlox homolog Pdx-1 has a crucial role in
pancreas formation and differentiation, and is also involved in pyloric
sphincter morphogenesis (Offield et al., 1996). Tissue boundaries
keep physically separated neighboring groups of cells with distinct
differentiation fates and often act as organizing centers for
compartment patterning (for a review, see Dahmann et al., 2011).
We propose that Xlox-positive cells located at the stomach-intestine
boundary may function as an organizer of gut domains in both the
anterior and posterior sides. Thus, the pyloric sphincter represents a
very powerful model for investigating the gene regulatory interactions
acting at the level of morphological boundaries, and the sea urchin
embryo is well suited to this kind of study. A powerful approach
RESEARCH ARTICLE
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
Table 1. Main effects of Xlox translation perturbation on gene transcription
Gene
regulation
and signal
transduction
Cytoskeleton
and
membrane
Metabolism
and
respiration
Adjusted
Adjusted
P value
P value
at 48 h
FC at 72 h at 72 h
Gene
category
Main domain
of expression
at 48 h
Gene name
Gene ref*
FC at
48 h
Sp-Lox
Sp-FoxY
Sp-IrxB
Sp-Nkx3.2
Sp-Otx
Sp-Tbr
Sp-Ets4
Sp-Notchl5_1
Sp-FoxA
Sp-Gatae
SPU_020637
SPU_010403
SPU_011246
SPU_017837
SPU_010424
SPU_025584
SPU_008528
SPU_016016
SPU_006676
SPU_010635
1.20
2.17
1.87
1.84
−1.06
1.98
1.52
1.86
1.12
1.11
0.02
0.00
0.00
0.00
0.79
0.00
0.00
0.00
0.00
0.00
12.73
10.27
4.61
4.43
4.21
3.88
3.65
3.61
1.97
1.69
0.00E+00
7.02E−30
4.72E−55
2.75E−32
1.46E−21
3.79E−22
0.00E+00
2.44E−147
1.34E−269
1.10E-56
TF
TF
TF
TF
TF
TF
TF
Notch ligand
TF
TF
HG
SM
na
Endo+AO
Endo+Ecto
PMC
Endo+NSM
na
MG+HG+NSM
MG+HG+NSM
Sp-Tgif
SPU_018126
−1.07
0.08
1.40
3.89E−25
TF
Endo
Sp-Ptf1a
SPU_002677
1.20
0.06
−1.18
5.21E−02
TF
MG+SCBC
Sp-Cdx
Sp-Brn1-2-4
SPU_024715
SPU_016443
−1.18
−1.12
0.29
0.02
−1.53
−1.58
1.78E−07
1.86E−29
TF
TF
HG
FG+SCBC
Sp-FoxB
Sp-FoxM
Sp-Rx
Sp-SoxE
Sp-FoxD
Sp-Tbx6
SPU_004551
SPU_025590
SPU_014289
SPU_016881
SPU_014418
SPU_020346
1.07
−1.01
−1.15
1.06
−1.34
−1.49
0.94
1.00
0.57
0.73
0.00
0.14
−1.71
−1.90
−1.94
−2.15
−2.26
−2.74
6.86E−02
1.57E−18
1.75E−06
3.47E−83
6.31E−33
4.05E−12
TF
TF
TF
TF
TF
TF
Endo
Ubiq
Endo+AO
NSM
HG+AO
PMC+NSM
Sp-PaxC
SPU_000276
−1.00
1.00
−2.93
8.44E−62
TF
AO+CB
Sp-Egfrp
Sp-NeuroD1
Sp-Tgfbr3
Sp-Phox2
Sp-FoxP
Sp-Endo16
Sp-Gelsolin
Sp-Actb
Sp-Ache-13
Sp-Cyp3L12
Sp-MeprinAaL
Sp-Alpi
SPU_004845
SPU_024918
SPU_027380
SPU_013464
SPU_009876
SPU_011038
SPU_003985
SPU_006661
SPU_005168
SPU_027796.
SPU_030013
SPU_022639
−1.95
na
1.25
na
1.21
1.43
−1.74
1.73
na
5.76
3.13
−1.31
0.40
na
0.92
na
0.00
0.00
0.65
0.00
na
0.00
0.00
0.03
−3.67
−3.73
−6.11
−6.57
−7.12
8.24
−3.71
−6.50
−8.36
15.89
4.75
−2.85
5.28E−106
2.06E−16
1.57E−03
1.13E−03
5.04E−18
0.00E+00
4.19E−35
1.68E−62
5.54E−24
6.56E−91
1.69E−85
2.72E−63
na
na
na
na
FG
MG+HG
na
na
na
na
na
na
Sp-Mgl
Sp-Cpa3L_2
SPU_010842
SPU_017120
na
na
na
na
−3.94
−5.44
8.64E−04
5.61E−72
Notch or Notch ligand
TF
TGFβ receptor
TF
TF
Ca2+-binding protein
Actin-binding protein
Cytoskeleton actin
Acetylcholinesterase
Cytochrome p450
Protein metabolism
Intestinal AP
precursor
Methionine γ-lyase
Carboxypeptidase
Sp-Cub/
TolloidL
Sp-PlbL
WHL22.2178
1.00
1.00
−7.79
8.64E−04
na
SPU_015099
na
na
−9.91
6.11E−15
Cubilin, intestinal
factor
Phospholipase
SPU_007135
Sp-Aadac_1
Sp-SpdecL
SPU_013685
Sp-Ppglcp
SPU_007135
SPU_014449
SPU_002513
SPU_013685
SPU_017527
na
na
na
na
−5.45
0.77
na
na
na
0.00
−10.70
−10.74
−10.78
−16.02
−20.15
1.06E−59
3.56E−42
4.05E−22
4.01E−42
0.00E+00
Regucalcin
Lipase
Sphingomyelinase C
Lipase
Phosphoglycolate
Phosphatase
na
na
na
na
na
na
MG
MG
Reference
Arnone et al., 2006
Ransick et al., 2002
M.I.A., unpublished
Li et al., 1997
Oliveri et al., 2002
M.I.A., unpublished
Oliveri et al., 2006
Lee and Davidson,
2004
Howard-Ashby
et al., 2006
Annunziata et al.,
2013b
Arnone et al., 2006
Cole and Arnone,
2009
David et al., 1999
Tu et al., 2006
Burke et al., 2006
Luo and Su, 2013
Tu et al., 2006
Gene expression
database‡
Gene expression
database‡
Tu et al., 2006
Ransick et al., 1993
Annunziata et al.,
2013b
Annunziata et al.,
2013b
*Gene reference according to spbase (http://sugp.caltech.edu/SpBase/).
‡
http://goblet.molgen.mpg.de/cgi-bin/seaurchin-database.cgi
AO, apical organ; AP, alkaline phosphatase; CB, ciliary band; Ecto, ectoderm; Endo, endoderm; FG, fold change; FG, foregut; HG, hindgut; MG, midgut; na, not
available; NSM, non skeletogenic mesoderm; PMC, primary mesenchyme cell; SCBC, scattered ciliary band cells; SM, small micromere lineage; TF,
transcription factor; Ubiq, ubiquitous.
know whether they function directly on Cdx activation or recruit
intermediate factors. One possibility, as they are all expressed in the
cells where Cdx transcription starts, is that they might function in an
‘AND’ logic (Davidson, 2010), probably together with other
transcription factors and/or co-factors, to drive the specification of
intestinal cells. Cdx, however, has an ‘exclusion effect’ (Oliveri and
2468
Davidson, 2007), repressing a potential alternative cell regulatory
state available to the intestinal cells (its effect on Xlox and Endo-16).
We discovered that the role of Cdx in repressing Xlox in the intestine
is mediated by a Wnt10 ligand. In addition, we showed that Wnt10
ligands are actively transcribed in the most posterior cells of the gut at
the late gastrula-prism stage and propose that they diffuse towards
DEVELOPMENT
Functional
category
RESEARCH ARTICLE
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
the anterior side of the gut, progressively clearing the intestinal cells
from Xlox (and probably other) transcripts. We do not know whether
the newly identified Wnt signal works through β-catenin or activates
a non-canonical Wnt pathway. The identity of the receptor mediating
this Wnt10 signaling event is also unknown. Four Frizzled receptor
genes have been identified so far in the Strongylocentrotus
purpuratus genome but no information is available about the
expression dynamics of these genes during embryogenesis. The
expression of these receptors has been deeply investigated in another
sea urchin species, Paracentrotus lividus, indicating Fzd9/10 as
possible candidate for mediating Wnt10 signaling in the hindgut
(Croce et al., 2006; Lhomond et al., 2012; Robert et al., 2014). It
would be interesting to study the dynamics of expression of the four
Frizzled receptor genes in S. purpuratus in order to identify a
possible candidate for the transduction of the Wnt10 signal to the
intestinal cells. Interestingly, mechanisms of function for Cdx
transcription factors involving activation of Wnt signaling have been
previously observed in mouse, precisely in the uro-rectal mesoderm
development (van de Ven et al., 2011). Moreover, Cdx appears,
together with Bra, FoxA and Wnt, as part of a conserved cassette of
factors regulating posterior gut development. These genes are all
expressed in the blastopore or blastopore equivalent of frog,
zebrafish, Drosophila and mouse (Lengyel and Iwaki, 2002).
Furthermore, Cdx genes, Wnt signaling, Bra and posterior Hox
genes act together to control posterior morphogenesis in the different
murine embryonic germ layers (van de Ven et al., 2011). We found
Cdx, Wnt10, FoxA, Bra and Hox11/13b actively involved in the sea
urchin posterior gut development, suggesting conservation of a gene
network functioning for the differentiation of posterior structures.
The strong reduction of Bra expression in Cdx morphant embryos
observed by qPCR (supplementary material Fig. S4), together with
the very similar expression pattern observed for Bra and Wnt10 from
late gastrula stage until pluteus, allow us to propose two possible
scenarios: (1) Bra receives a fundamental regulatory input from Cdx
and participates, together with Cdx, in Wnt10 activation in the very
posterior cells of the post gastrular embryo; (2) Cdx activates Wnt10
expression indirectly, by activating Bra. Both scenarios would
provide an explanation for the fact that Wnt10 is expressed only in a
subset of Cdx-expressing cells, although we have no data so far to
support a role for Bra in Wnt10 activation. This would not be the first
case in which Bra activates Wnt signals: in chordates, Bra recruits
canonical Wnt signaling to sustain the posterior mesodermal
progenitors during the outgrowth of the body and to regulate the
mechanism of somitogenesis (Martin and Kimelman, 2009).
We also showed that Endo-16 transcripts accumulate ectopically in
the intestine of Cdx morphant larvae and that Hox11/13b is required
for activating Cdx transcription, thus providing a candidate for the
factor hypothesized to mediate the Hox11/13b knockdown phenotype
in the posterior gut (Arenas-Mena et al., 2006). Interestingly, Cdx
also functions as a repressor in the intestine of vertebrates: Cdx2
2469
DEVELOPMENT
Fig. 6. Regulatory states and the view from the nuclei of interactions in each compartment of the sea urchin developing gut. In the upper panels, three
stages of the sea urchin embryonic gut are depicted in lateral view. Different colors are used for compartments showing exclusive regulatory states. In the
lower part, the regulatory states and the interactions found within this study are summarized, using the same color code as the schematics of guts above. The
wiring between the genes is shown with solid lines for representation issues but none of them has been demonstrated to be direct. In the diagram, arrows
represent positive regulation, bars represent repression. The white bullets, together with the black dashed lines, indicate signaling events: S1 represents the
unknown signaling molecule under the control of Xlox and directing stomach genes activation through the action of an unknown effector gene (E1); E2 represents
the effector gene responsible for Xlox repression in the intestinal cells. Xlox and Cdx genes, together with their inputs (when active), have been colored green and
red, respectively. Most of the results obtained through in situ hybridization have been validated by qPCR (see supplementary material Fig. S4). The input of Cdx
on Bra transcription has been discovered by qPCR (supplementary material Fig. S4). The expression dynamics of GataE, Blimp1a and FoxA have been
extrapolated from published works (Lee and Davidson, 2004; Livi and Davidson, 2006; Oliveri et al., 2006). ManrC1A has been shortened to ManR for clarity.
conditional ablation from early murine endoderm results in a
posterior-to-anterior gut transformation, through the replacement of
the posterior intestinal epithelium with esophageal epithelia (Gao
et al., 2009). Furthermore, ectopic expression of Pdx1 (the ortholog of
the sea urchin Xlox gene) has been demonstrated in Cdx2 mouse
mutant intestine (Grainger et al., 2010). As already suggested by
Grainger et al. (2010), this would hint at conservation of the crossregulatory loop described in sea urchin between the two genes.
However, two phases of Cdx activation have been observed in
chordates (Chawengsaksophak et al., 2004; Osborne et al., 2009;
Reece-Hoyes et al., 2002), the first in the blastopore-primitive streak
regions, where it is required for axial elongation, at least in mouse
(Chawengsaksophak et al., 2004), and the second in the posterior gut,
where it is involved in intestinal patterning. In the sea urchin embryo,
Cdx transcription starts towards the end of gastrulation. Thus, in the
sea urchin, Cdx function in the early phases of gastrulation has been
lost and only its function in posterior gut patterning has been retained.
This condition is more likely to be an echinoid peculiarity rather than
a feature common to all ambulacrarians or to all echinoderms, as
Cdx ‘biphasic’ transcription has been described in sea stars and
hemichordates (Annunziata et al., 2013a; Ikuta et al., 2013). Very
importantly, the fact that Cdx is not required in the early steps of sea
urchin gastrulation offers a great advantage for the study of its
role in the intestine differentiation, facilitating the interpretation of
functional studies.
Conclusions and perspectives
The purpose of this work was to investigate the gut patterning process
in its entirety, exploring the dynamics of gene expression and
regulation in space and time, a feasible objective using the sea urchin
embryo as model system. The data we present here clearly show that
while the initial patterning of the gut tube relies mainly on the A-P
distribution of transcription factor territories of expression, the
subsequent compartmentalization depends, at least partially, on
signaling events under the control of the two ParaHox genes, Xlox
and Cdx (see Fig. 6). It is important to note that none of the interactions
found in this study has been demonstrated to be direct and that the
wiring diagram presented in Fig. 6 remains far from being complete,
missing many other levels of regulation such as the involvement of
small non coding RNAs, post translational modifications, proteinprotein interactions and chromatin remodeling.
Our findings suggest that Xlox ancestral function in the endoderm
was probably to pattern the digestive tube directing stomach cell
differentiation and pyloric sphincter formation, as supported by
conservation of its gut domain of expression in several protostomes
and non-vertebrate deuterostomes. The vertebrate homolog Pdx-1
has been possibly coopted for the development of the pancreas,
maintaining its role in the formation of the stomach-intestine
constriction. Similarly, the crossregulatory loop between Xlox and
Cdx firstly described in sea urchin (Cole et al., 2009) appears as a
conserved gut patterning mechanism among deuterostomes (Grainger
et al., 2010). We predict that many of the hereby demonstrated
regulatory interactions shaping the sea urchin gut, and the many others
we will find by the ongoing ChIPSeq and RNASeq analyses
downstream of SpLox and SpCdx, will be instrumental for the
discovery of yet unknown patterning mechanisms of the vertebrate gut.
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
Anton Dohrn of Naples, Italy. Gametes were obtained by standard methods
and embryos were cultured at 15°C in filtered sea water diluted 9:1 with deionized water.
RNA in situ hybridization
For single gene expression, we followed the protocol outlined previously
(Minokawa et al., 2004). Fluorescent double WM in situ hybridization were
performed as described previously (Cole et al., 2009). RNA probe
sequences for SpLox, SpCdx, SpBrn1/2/4, SpBra and SpEndo16 are as
previously published: SpLox and SpCdx (Arnone et al., 2006); SpBrn1/2/4
(Cole and Arnone, 2009); SpBra (Rast et al., 2002); and SpEndo16 (Ransick
et al., 1993). SpHox11/13b, SpChP, SpManrC1A, SpMHC bacterial clones
were picked from the S. purpuratus cDNA library available in the laboratory
(http://goblet.molgen.mpg.de/cgi-bin/seaurchin-database.cgi). SpWnt10 cDNA
was obtained by RT-PCR from 65 h embryo total RNA (using the following
primers: SpWnt10-F: 50 -AGACGATGGAATTGCTCCAG-30 ; SpWnt10-R:
50 -GGTTAACCCATTGCGAGCTA-30 ) and then cloned into the Topo-TA
cloning vector (Invitrogen).
Immunohistochemistry
For acetylated tubulin staining combined with in situ hybridization, a
1:250 dilution of the mouse monoclonal anti-acetylated tubulin antibody
(T7451; Sigma-Aldrich) was added to the blocking solution containing the
peroxidase-conjugated antibody and incubated overnight at 4°C. The next
day the embryos were processed for in situ hybridization, then incubated
for 1 h in blocking solution with a dilution 1:1000 of the AlexaFluor 488
goat anti-mouse immunoglobulin G (IgG) (A21202; Molecular Probes),
washed in MOPS (3-morpholinopropane-1-sulfonic acid) buffer and
mounted for imaging. For Xlox and Cdx immunostaining, larvae were
fixed in 2% PFA in phosphate-buffered saline (PBS) for 10 min at room
temperature, washed multiple times in PBS with 0.1% Tween-20 (PBST),
blocked in 5% goat serum in PBST and incubated overnight at 4°C with the
custom primary antibodies diluted at working concentration (1:500;
PRIMM) in 5% goat serum in PBST. For Bra immunostaining, embryos
and larvae were fixed in 2% PFA in PBS for 10 min, then for 1 min in
methanol, blocked in 5% goat serum in PBST and incubated overnight
with the custom antibody at working concentration (1:200; A11008;
PRIMM). Following primary antibody incubation, embryos and larvae
were washed several times in PBST, incubated for 1 h at room temperature
with the secondary antibody Alexa Fluor 488 goat anti-rabbit IgG
(Molecular Probes, Invitrogen) diluted 1:1000 in 5% goat serum in PBST,
washed in PBST and mounted for imaging with a confocal microscope
(Zeiss 510Meta).
MO microinjection
For each experiment and for each morpholino oligonucleotide (MO), around
400 embryos were injected with approximately 2-4 pl of oligonucleotide
solution at 0.1 mM and each experiment was repeated three times. In all
experiments, as a negative control, embryos were injected with 0.1 mM of the
standard control morpholino and compared side by side with uninjected and
knockdown embryos. The injection of the standard control morpholino
(GeneTools) did not have any effect on the development of embryos. MOs
against SpLox, SpHox11/13b, SpFoxA, SpBlimp1a, SpGataE, SpTgiF and
SpBra protein translation and the morpholino targeting the donor splice site
between the first and second SpCdx exons, were already available (http://sugp.
caltech.edu/endomes/) (Arenas-Mena et al., 2006; Cole et al., 2009; Livi and
Davidson, 2006; Oliveri et al., 2006; Rast et al., 2002). MOs against SpWnt10
and SpCdx translation were newly designed and acquired from Gene
Tools (Corvallis) (SpWnt10, 50 -AACTGCATCTGCTTACGATTCATAC-30 ;
SpCdxM, 50 -TGGGTGCAGATACTCTAGCGTCATC-30 ).
Screening of signaling molecules
MATERIALS AND METHODS
Animals, embryo cultures
Adult Strongylocentrotus purpuratus were obtained from Patrick Leahy
(Kerchoff Marine Laboratory, California Institute of Technology, Pasadena,
USA) and housed in circulating sea water aquaria in the Stazione Zoologica
2470
In order to identify the signaling molecule responsible for the midgut
differentiation process, 15 signaling ligand encoding genes have been
analyzed. The genes included in the analysis were selected from the
Strongylocentrotus purpuratus Signaling Ligand Page (available at http://
140.109.48.251/ICOBUserfile/SuYuLab/Su_and_Yu_Lab/Home.html), a
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
RNA extraction, RNA-seq and data analysis
Three biological replicas of 500 injected with SpLox MO and control (KClinjected) embryos were collected at 48 and 72 h. About 1 μg of RNA was
extracted from each sample using the RNAqueous-Micro Kit (Ambion,
Life Technologies-Invitrogen) and used for RNA-seq. cDNA libraries were
prepared with 1 µg of starting total RNA and using the Illumina TruSeq
RNA Sample Preparation Kit (Illumina), according to TruSeq protocol.
Library size and integrity were determined using the Agilent Bioanalyzer
2100. Each library was diluted to 2 nM and denaturated, 8 pM of each
library was loaded onto cBot (Illumina) for cluster generation with cBot
Paired End Cluster Generation Kit (Illumina) and sequenced using the
Illumina HiSeq 1500 with 100 bp paired-end reads in triplicate, obtaining
∼31-38 million reads for replicate. The sequencing service was provided by
the Laboratory of Molecular Medicine and Genomics (http://www.
labmedmolge.unisa.it) at the University of Salerno, Italy. Sequence read
quality was controlled using FastQC program (http://www.bioinformatics.
bbsrc.ac.uk/projects/fastqc/). Filtered reads were mapped to the Sea Urchin
genome Spur_3_1.LinearScaffold downloaded from SpBase (www.
spbase.org) using TopHat v2.0.8b (Trapnell et al., 2009), that runs on
Bowtie 2 version 2.1.0 (Langmead et al., 2009). The reads were counted
using the HTSeq-package (Anders and Huber, 2010). Normalization of
read numbers between samples and differential expression analysis was
performed using DEseq (Anders and Huber, 2010). The transcripts with an
adjusted P<0.05 were considered to be differentially expressed.
Differential expression is represented with MA-plots generated with the
DESeq software.
Acknowledgements
We acknowledge Patrick Leahy and the Southern California Sea Urchin Company
for animal supply; Davide Caramiello for taking care of the sea urchins; the SZN
Molecular Biology Service for technical assistance; the Laboratorio di Medicina
Molecolare e Genomica, Università degli Studi di Salerno, Italy, for the RNA-seq
experiment; Giorgio Giurato and Francesca Rizzo for the MA-plots; Eric Davidson
for Bra, FoxA, GataE and TgiF MOs; Yi-Hsien Su for the signaling molecule
database; Evelyn Houliston and Pedro Martinez for revising our manuscript;
and Claudia Cuomo for the kind experimental help.
Competing interests
The authors declare no competing financial interests.
Author contributions
R.A. performed all the experimental work with the exception of the microinjections
(performed by M.I.A.), contributed to the interpretation of the results and wrote the
manuscript. M.I.A. conceived and supervised the project, contributed to the analysis
of the data and prepared the figures.
Funding
This work was supported by the ‘Evonet’ ( project 215781) EU-Marie Curie Early
Training Network, and (a fellowship to R.A.) by POR Campania FSE 2007-2013
Project MODO, Model Organism.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.105775/-/DC1
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DEVELOPMENT
database created by Yi-Hsien Su’s laboratory (Institute of Cellular and
Organismic Biology, Academia Sinica, Taiwan), in which the temporal
expression pattern of all signaling ligands expressed during sea urchin
embryonic development is provided. The signaling ligand database was
screened using as selective criteria the windows of activation of the genes. In
particular, the ligands whose expression started or significantly increased a
few hours after 44 hpf (the developmental time when Cdx expression
begins) were considered to be eligible signaling molecules and screened by
qPCR. The differential quantitative expression of the selected genes in
control and Cdx knockdown embryos (data not shown) was analyzed. The
15 signaling molecules included in the analysis were: SpWnt8, SpIGF2,
SpHGF, SpAgrin3, SpVegF, SpBMP3, SpBMP5/8, SpNotch-lik2, SpNotchlig3, SpNotch-lid5, SpEphrin, SpSHH, SpWnt10, SpWnt4 and SpFGF9/16/
20. For the qPCR, the primers were designed based on the sequences
available in the signaling ligand database.
Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
RESEARCH ARTICLE
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Development (2014) 141, 2462-2472 doi:10.1242/dev.105775
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