Nuclear receptors from the ctenophore
Mnemiopsis leidyi lack a zinc-finger DNA-binding
domain: lineage-specific loss or ancestral
condition in the emergence of the nuclear
Adam M Reitzel1, Kevin Pang2, Joseph F Ryan3, James C Mullikin3, Mark Q Martindale2, Andreas D Baxevanis3,
Ann M Tarrant1*
Background: Nuclear receptors (NRs) are an ancient superfamily of metazoan transcription factors that play critical
roles in regulation of reproduction, development, and energetic homeostasis. Although the evolutionary
relationships among NRs are well-described in two prominent clades of animals (deuterostomes and protostomes),
comparatively little information has been reported on the diversity of NRs in early diverging metazoans. Here, we
identified NRs from the phylum Ctenophora and used a phylogenomic approach to explore the emergence of the
NR superfamily in the animal kingdom. In addition, to gain insight into conserved or novel functions, we examined
NR expression during ctenophore development.
Results: We report the first described NRs from the phylum Ctenophora: two from Mnemiopsis leidyi and one from
Pleurobrachia pileus. All ctenophore NRs contained a ligand-binding domain and grouped with NRs from the
subfamily NR2A (HNF4). Surprisingly, all the ctenophore NRs lacked the highly conserved DNA-binding domain (DBD).
NRs from Mnemiopsis were expressed in different regions of developing ctenophores. One was broadly expressed in
the endoderm during gastrulation. The second was initially expressed in the ectoderm during gastrulation, in regions
corresponding to the future tentacles; subsequent expression was restricted to the apical organ. Phylogenetic
analyses of NRs from ctenophores, sponges, cnidarians, and a placozoan support the hypothesis that expansion of
the superfamily occurred in a step-wise fashion, with initial radiations in NR family 2, followed by representatives of
NR families 3, 6, and 1/4 originating prior to the appearance of the bilaterian ancestor.
Conclusions: Our study provides the first description of NRs from ctenophores, including the full complement
from Mnemiopsis. Ctenophores have the least diverse NR complement of any animal phylum with representatives
that cluster with only one subfamily (NR2A). Ctenophores and sponges have a similarly restricted NR complement
supporting the hypothesis that the original NR was HNF4-like and that these lineages are the first two branches
from the animal tree. The absence of a zinc-finger DNA-binding domain in the two ctenophore species suggests
two hypotheses: this domain may have been secondarily lost within the ctenophore lineage or, if ctenophores are
the first branch off the animal tree, the original NR may have lacked the canonical DBD. Phylogenomic analyses
and categorization of NRs from all four early diverging animal phyla compared with the complement from
bilaterians suggest the rate of NR diversification prior to the cnidarian-bilaterian split was relatively modest, with
independent radiations of several NR subfamilies within the cnidarian lineage.
* Correspondence: email@example.com
1Biology Department, Woods Hole Oceanographic Institution, Woods Hole,
Full list of author information is available at the end of the article
Reitzel et al. EvoDevo 2011, 2:3
© 2011 Reitzel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Nuclear receptors (NRs) are a class of transcription fac-
tors that regulate diverse developmental and physiologi-
cal processes in animals. The characteristic domains
of NRs are the DNA-binding domain (DBD), which
includes a Cys-Cys zinc coordinating region, and the
ligand-binding domain (LBD), a carboxy-terminal
domain that binds ligands and coactivators for modula-
tion of NR function, facilitates receptor dimerization,
and contains an activation function. Current evidence
has shown that NRs are restricted to animals because
they are present in all metazoan phyla that have been
surveyed but not in plants or fungi [1-6]. NR-like tran-
scription factors have been identified in some fungi
based on a region that has low conservation with the
LBD of animal NRs; however, whether these are evolu-
tionary related to NRs in animals is not known .
Genes in the NR superfamily are classified into six
major families (NR 1 through NR 6; Nuclear Receptor
Nomenclature Committee ). All six families (and
many subfamilies) are represented in protostomes and
deuterostomes, supporting the conclusion that NRs had
diversified prior to the bilaterian ancestor . Thus,
characterizing NRs in species representative of the early
diverging animal phyla would provide critical informa-
tion that would allow for a better understanding of the
evolutionary history and emergence of this superfamily.
NRs have been reported from three of the four classes
of cnidarians (Anthozoa [3,5], Cubozoa , and Hydro-
zoa ), including the full complement from the sea
anemone Nematostella vectensis , two sponges
[4,6,10], and one placozoan [11,12]. Phylogenetic ana-
lyses of these NRs have shown that most of these genes
belong to NR family 2. The exceptions are: (1) one NR
family 3 member from the placozoan Trichoplax adhae-
rens, (2) one ortholog to NR family 6 from the cnidarian
Nematostella, and (3) three genes from Nematostella
that form an outgroup to NR families 1 and 4. Taken
together, these data suggest that the evolution of NRs
may be complex and, more importantly, that early diver-
ging metazoans are the appropriate groups to study in
order to understand how and when the NR superfamily
has evolved and diversified.
Among the early branching animal lineages, NRs have
not been identified or described in any species from
Phylum Ctenophora. The apparent phylogenetic position
of ctenophores in relation to other animal phyla has var-
ied among studies, with recent analyses placing them at
the base of the animal tree [13,14], as a sister clade with
other non-bilaterians to the Bilateria , in one clade
with cnidarians , or as an outgroup to a clade of
the Placozoa, Cnidaria, and Bilateria [17,18] (see 
for a summary). Determining the correct phylogenetic
position of ctenophores and other metazoan phyla is
critical for accurately reconstructing the evolutionary
history of individual gene families. Reciprocally, accumu-
lated analyses comparing the diversity of multiple gene
families among early diverging phyla may help investiga-
tors to select among the particular phylogenetic hypoth-
eses. Characterizing the NR complement from a
ctenophore species is a constructive step towards under-
standing the evolution of the superfamily and, at the
same time, it provides some insight into the relationship
of ctenophores to other metazoans. Similarly, comparing
the expression of NRs in ctenophores with other species
would provide data needed to assess potential conserva-
tion of the spatial and temporal expression of these
Here, we describe NRs from two ctenophores, the
lobate Mnemiopsis leidyi and the tentaculate Pleurobra-
chia pileus. These two species are from different orders
and, thus, represent distant lineages within the Cteno-
phora . Additionally, for Mnemiopsis, we describe
the intron-exon structure and the spatio-temporal devel-
opmental expression of these NRs using in situ hybridi-
zation. We then applied phylogenetic methods to
characterize NR diversity from 12 early diverging
metazoans (two sponges, two ctenophores, one pla-
cozoan, and seven cnidarians) and developed a hypoth-
esis for the early diversification of the NR superfamily.
Identification and annotation of Mnemiopsis leidyi
We identified candidate NRs through BLAST queries of
the assembled genome and gene models for Mnemiopsis
leidyi (physical coverage of genome approximately 50×,
see ). For these searches we used a diverse set of full-
length NRs from the sponge Amphimedon queenslandica,
the anemone Nematostella vectensis, Homo sapiens, and
Drosophila melanogaster. Through these similarity
searches we identified two matches that were reciprocal
best BLAST hits with other animal NRs, showing strong
similarity to the LBD. No BLAST queries returned any
ctenophore gene model or region of the genome with
similarity to the DBD. TBLASTN searches of the Mne-
miopsis genome and BLASTP searches of the proteome
using only the DBD from sponge and anemone HNF4
resulted in weak matches that were on separate contigs
from the two predicted genes from Mnemiopsis with high
similarity to the LBD. The top ctenophore protein match
using the DBD from sponge HNF4 (E-value = 0.027) was
BLASTed to human refseq and exhibited greatest,
although low, similarity to members of the LIM family.
HMM searches of the ctenophore proteome failed to
identify any additional matches.
Reitzel et al. EvoDevo 2011, 2:3
Page 2 of 12
For the two candidate NRs, we performed 5’- and
3’-RACE with gene specific primers, using cDNA pre-
pared from RNA pooled from diverse developmental
stages (see Additional file 1 for all primers). RACE pro-
ducts were cloned into pGEMT (Promega, Madison,
WI, USA) and sequenced. Overlapping fragments were
assembled in silico to produce the complete transcript.
To confirm the two gene products, we amplified and
sequenced the entire open reading frame for each NR.
Each primer pair yielded a single product that matched
the conceptually assembled fragment. We characterized
gene structure for each Mnemiopsis NR by aligning the
full length transcripts to the assembled genome.
We also identified a NR in a second species of cteno-
phore, Pleurobrachia pileus, through BLAST queries of
ESTs in GenBank. We assembled a full-length transcript
for a single Pleurobrachia NR by assembling overlap-
ping ESTs (GenBank accession numbers FP998505,
FP993827, FP993707, and FP997412).
Developmental expression of Mnemiopsis nuclear
Whole-mount in situ hybridizations were performed as
previously described . A RACE product for MlNR1
(approximately 1,000 bp) and the full open reading
frame for MlNR2 (1,137 bp) were used as templates to
transcribe digoxigenin-labeled antisense RNA probes
(Megascript Kit, Ambion, Austin, TX, USA). Develop-
mental stages from gastrulation through the early cydip-
pid were probed for spatial expression.
Phylogenetic analyses of the nuclear receptor superfamily
Through a combination of literature searches and
BLAST queries, we assembled a dataset of 54 NRs from
early diverging animals in the phyla Ctenophora, Pori-
fera, Placozoa, and Cnidaria (Accession numbers given
in Additional file 2). We identified three additional NRs
from the coral Acropora millepora [data from 23] which
were combined with previously described NRs from this
species . NRs from two cnidarians (Metridium senile
(n = 2), Hydra magnipapillata (n = 6)) were identified
through BLAST queries against GenBank. Sequences
from Homo sapiens, Drosophila melanogaster, and
Caenorhabditis elegans from Bertrand et al.  were
used as representative bilaterian sequences.
Full-length sequences for all taxa were aligned with
Muscle 3.6  and edited manually in the case of clear
errors. For some taxa, only partial sequences were avail-
able, containing only the DBD in many cases. Due
to the absence of a well-conserved DBD in the three
ctenophore sequences, these proteins were manually
corrected in an effort to optimize the alignment.
Maximum likelihood analyses were conducted with
RAxML (version 7.0.4, ) and Bayesian analyses with
MrBayes v.3.1.2  using a JTT+G+F matrix (model
determined by RAxML model picker) and a trimmed
alignment containing the DBD and most of the LBD,
beginning in helix 3. Support for particular nodes for
maximum likelihood analyses was assessed with 1,000
bootstraps. For the Bayesian analysis, two independent
analyses were performed with five chains run for five
million generations and sampled every 500 generations.
The first 1.25 million generations were discarded as
burn-in. Log likelihood values were plotted and found
to be asymptotic well before the burn-in fraction. Trees
were visualized and illustrated with FigTree v1.1.2
Nuclear receptors from the ctenophores Mnemiopsis
leidyi and Pleurobrachia pileus
BLAST queries of the Mnemiopsis genome and gene
models resulted in two hits with significant similarity to
the LBD of NRs from diverse metazoans. Assembled
RACE products contained the complete open reading
frame of each NR, as well as 5’- and 3’-UTR sequence
(protein sequences are given in Additional file 3).
MlNR1 is encoded by a transcript 1,408 bp long with an
open reading frame of 268 amino acids. MlNR1 is com-
posed of eight exons spanning almost 10 kb of genomic
sequence, with the coding sequence on seven exons
(Figure 1A). We identified a polyA tail 508 bp down-
stream from the stop codon and 156 bp of 5’-UTR.
MlNR2 is encoded by a transcript 1,650 bp long with an
open reading frame of 379 amino acids. MlNR2 is a sin-
gle-exon gene with no intervening introns. The 3’-RACE
sequence of MlNR2 contained a polyA tail 357 bp
downstream of the stop codon. The 5’-RACE sequence
contained 123 bp of UTR. By comparison, the
assembled NR from Pleurobrachia ESTs had an open
reading frame of 484 amino acids. We did not identify a
polyA tail in the assembled transcript.
Neither of the two NRs from Mnemiopsis nor the single
NR from Pleurobrachia contained a conserved zinc-fin-
ger DNA-binding domain (DBD) typical for NRs across
the superfamily. The optimized alignment of the amino-
terminal of the ctenophore NRs with the DBD from
HNF4 from other animals suggests that this region in
the ctenophore proteins contains very little conservation
and is interrupted by insertions (Figure 1B). It was not
possible to identify the conserved cysteines characteristic
of zinc-finger motifs within the ctenophore sequences.
The amino-terminal region of the ctenophore NRs failed
to consistently match any protein or domain when
BLASTed at NCBI. Despite the absence of a conserved
DBD, each of the ctenophore NRs contains an LBD that
aligned reasonably well with LBDs from other NRs
(Figure 1C). Two of the ctenophore NRs (MlNR2 and
Reitzel et al. EvoDevo 2011, 2:3
Page 3 of 12
PpNR1) have the NR signature motif (boxed region in
Figure 1C), whereas MlNR1 has retained only a portion
of this sequence. The relative conservation of the LBD
in the ctenophore NRs was similar to that of NRs pre-
viously reported from two sponges [4,6,10] when com-
pared with bilaterian HNF4 (approximately 30% identity,
approximately 50% similarity).
Developmental expression of Mnemiopsis nuclear
Using in situ hybridization, we documented the develop-
mental expression of MlNR1 and MlNR2. MlNR1 was
first expressed during midgastrulation in the ectoderm
in regions of future tentacle development (Figure 2A, B,
F, G). Later in embryogenesis, expression continued in
B. DNA-binding domain
C. Ligand-binding domain
A. Intron-exon structure
Figure 1 Nuclear receptors from the ctenophores Mnemiopsis leidyi and Pleurobrachia pileus. (A) Intron-exon structure of the two nuclear
receptors from Mnemiopsis. MlNR2 is a single exon gene. MlNR1 has a more complex intron-exon structure with eight exons, seven of which
code for the inferred open-reading frame. (B) Alignment of the amino-terminal region of the ctenophore NRs with the DNA-binding domains of
NRs from two sponges (Amphimedon queenslandica (Aq) and Suberites domuncula (Sd)), and HNF4 from Trichoplax adhaerens (Ta), Nematostella
vectensis (Nv), Drosophila melanogaster (Dm), and Homo sapiens (Hs). The ctenophore proteins align poorly, including an absence of the
conserved cysteines (indicated by black circles), and an optimized alignment contains insertions and deletions relative to the DBD of other
animals. (C) Alignment of the ligand-binding domain from the same taxa as in (B). The ctenophore LBD is well-conserved, particularly the
nuclear receptor signature motif spanning helix 3 and 4 (boxed).
Reitzel et al. EvoDevo 2011, 2:3
Page 4 of 12
the same location, as well as in an additional adoral
domain along the sagittal plane between the adpharyn-
geal comb rows (Figure 2C, D). In the cydippid stage,
expression of MlNR1 was restricted to four discreet
domains of the floor of the apical organ flanking the
sagittal plane (Figure 2E, J). These were not associated
with the balancing cilia that support the mineral con-
taining lithocytes. MlNR2 was expressed broadly in the
endoderm and parts of the ectoderm after gastrulation
and in the cydippid (Figure 2K-N). Expression of
MlNR2 was not observed earlier in gastrulation.
Phylogenetic position of ctenophore NRs and distribution
of NRs from early diverging metazoans
Maximum likelihood analyses of the NRs from the
selected taxa spanning the animal kingdom reproduced
monophyletic relationships for all six recognized NR
families (Figure 3). The bootstrap support for these
families varied from 97% for NR family 1 to 52% for NR
family 6. Previous studies have shown similarly low sup-
port for NR family 6 and for the node including this family
and its sister family, NR family 5 [5,27,28]. Bayesian ana-
lyses resulted in a largely identical topology as the maxi-
mum likelihood analysis, with high posterior probabilities
(0.93 to 1) for each NR family (Additional file 4).
The two NRs from Mnemiopsis and the one from Pleuro-
brachia cluster with the HNF4 subfamily (NR2A) and
share greatest sequence similarity with HNF4s from other
animals including Trichoplax, diverse cnidarians, and bila-
terians. This cluster also contains a NR recently identified
in the sponge Amphimedon . The sponges Amphime-
don and Suberites also contain a second NR-type identified
by Larroux et al.  and Wiens et al. , respectively, that
forms a sponge-specific NR cluster that groups between
the HNF4 subfamily and all other nuclear receptors.
These results suggest that ctenophores and sponges have
representatives that most closely group with only one NR
subfamily (NR2A) but that sponges contain an additional
type of NR not present in any other species sampled
to-date. Thus, ctenophores contain the most restricted
NR complement of any species yet described.
For the placozoan Trichoplax adhaerens, we identified
four NRs, consistent with results published earlier (Sup-
plemental Figure S9.13 in ). Trichoplax NR1
grouped in NR family 2, with weak support in subfamily
NR2F. Two additional NRs were strongly supported as
orthologs to HNF4 (NR2A) and RXR (NR2B). Tricho-
plax NR4 was a strongly supported member of NR
family 3. This result supports a previous report showing
a NR 3 member from Trichoplax . This earlier study
concluded that this NR was likely an ERR ortholog. In
our analysis, we found that this Trichoplax NR is a
member of NR family 3, but with no particular relation-
ship to any of the NR 3 subfamilies.
Figure 2 Expression of MlNR1 (A-J) and MlNR2 (K-N) in embryos and juveniles of Mnemiopsis. A-E show aboral views of MlNR1, with F-J
showing lateral views of the corresponding embryo. (A-B, F-G) MlNR1 expression in ectoderm of the future tentacle bulb (tb) in the gastrula and
late gastrula stage. (C-D, H-I) Expression continues in ectoderm of future tentacle bulb as well as in additional domain along the sagittal plane
(arrowhead) in the postgastrula. (E, J) MlNR1 expression is restricted to the apical organ (ao) in the cydippid stage. (K-N) MlNR2 is broadly
detected in the endoderm (en) in developmental stages after gastrulation and in the cydippid. It is only in the most aboral part of the pharynx
(ph). There are also additional domains in the ectoderm (white arrowheads).
Reitzel et al. EvoDevo 2011, 2:3
Page 5 of 12
NR 5 family
NR 6 family
NR 3 family
NR 4 family
NR 1 family
NR 2 family
Figure 3 Maximum likelihood tree of nuclear receptor superfamily. Clades are annotated to family and subfamily based on current
nomenclature for the NR superfamily . Tree was rooted with the cluster containing the ctenophore sequences plus HNF4 from diverse
animals. Values above nodes indicate percent of 1,000 bootstraps. Bootstrap values below 40 were removed.
Reitzel et al. EvoDevo 2011, 2:3
Page 6 of 12
For the cnidarians, we analyzed NRs from five anthozo-
ans, one cubozoan, and one hydrozoan. From the
anthozoans, a previous study reported that the NR com-
plement of Nematostella vectensis contained 17 NRs .
While most of the Nematostella NRs share well-
supported orthologs with Acropora, orthologs of several
of the Nematostella NRs have not yet been identified in
Acropora or other corals. Nematostella sequences without
clear Acropora counterparts are all sequences in subfami-
lies with coral orthologs, likely representing either para-
logs potentially resulting from lineage-specific gene
duplication or genes not yet sequenced from the coral.
Three previously published NRs from the coral Pocillo-
pora damicornis  grouped with tailless (NR2E),
COUP-TF (NR2F), and the cnidarian specific NR1/4
clade. We identified two NRs from an EST collection for
the anemone Metridium. These NRs grouped within
family NR2E and 2F; one was orthologous to bilaterian
COUP-TFs, and the other clustered with a cnidarian-spe-
cific NR radiation within family NR2E. We also included
two previously published sequences from Anemonia 
that grouped well with the anthozoan radiation of NR
family 1/4 and a GCNF, despite their previous annotation
as an FTZ and RXR, respectively. Together, the combined
NR data show that anthozoans contain representatives of
four of the five NR 2 subfamilies, with no ortholog of
RXR (NR2B) in any species. Additionally, we identified
three species (Nematostella, Acropora, and Anemonia)
having an NR that groups most closely with GCNF
(NR6), consistent with previous studies [5,10]. However,
the support for these cnidarian genes with NR6 was
modest and these genes may instead represent NR5/6
orthologs. Finally, in four anthozoan species, we observed
at least one NR that groups in a cnidarian-specific cluster
that was positioned as an outgroup to NR families 1 and
4, which only contain NRs from bilaterians. We originally
identified three NRs from this group in Nematostella,
leaving open the question as to whether these genes were
unique to this anemone as a result of a species-specific
duplication and divergence, or if these were more broadly
represented in anthozoans. By including additional
anthozoan genes, we identified well-supported coral
orthologs to each Nematostella gene, bolstering the
conclusion that this group is diverse within the class
We identified six NRs from the recently completed
genome of the hydrozoan Hydra magnipapillata .
One of these NRs (HmNR6) grouped strongly with one
previously reported COUP-TF (NR2F) gene from the
congener H. vulgaris . These two NRs group in a sepa-
rate cluster (with other cnidarian members of subfamily
NR2F), but more distantly than with COUP-TFs from
bilaterians and more closely related homologs from cni-
darians, including an NR from H. magnipapillata
(HmNR3). This suggests that these two NRs from two
Hydra species are retained duplicates from an earlier cni-
darian radiation in this family, a result consistent with
that found by Escriva et al. . The other NRs from H.
magnipapillata were all supported as members of the
NR2 family. HmNR2 was supported as an HNF4 family
member. HmNR1 and HmNR4 group as members of the
NR2E subfamily, although the position of HmNR4 varied
within the NR2 family among analyses. The last NR from
H. magnipapillata (HmNR5) grouped with strong sup-
port as an ortholog to RXR, grouping with RXRs from
bilaterians and the placozoan Trichoplax. This NR is only
the second RXR identified from the Cnidaria, with a pre-
vious report of a RXR from the cubozoan jellyfish Tripe-
dalia . Thus, RXR orthologs have only been reported
from the medusozoan clade within the phylum Cnidaria.
We did not identify an ortholog to NR family 1/4 or 6 in
H. magnipapillata. Finally, we found no NR from H.
magnipapillata or any anthozoan with support as a
member of the NR family 3.
By characterizing the first NRs from the phylum Cteno-
phora and assembling NR sequences from diverse early
diverging animals, we have gleaned new insights into
the evolution of this superfamily of transcription factors.
The ctenophore sequences in particular provoke new
hypotheses about the origin of this gene superfamily
and the evolution of the classical NR gene structure.
Ctenophore NRs lacking a DBD
The zinc-finger DBD is typically the mostly conserved
feature of NRs throughout the superfamily. The two
NRs from Mnemiopsis and the one from Pleurobrachia
have a conserved LBD, but all of these lacked a DBD
typical of NRs. The two NRs from Mnemiopsis are sup-
ported as paralogs due to a duplication event some-
where in the ctenophore lineage. One of the
Mnemiopsis NRs (MlNR2) is coded by a single exon;
this is evidence of a potential retroposition event .
The ctenophore genes are the first NRs lacking a DBD
from any non-bilaterian animal. We failed to identify
any matches to a DBD elsewhere in the genome. The
lack of a DBD and the potential position of ctenophores
as the first branch from the animal tree suggest two
competing hypotheses: the DBD was lost within the cte-
nophore lineage from an ancestral NR with both the
DBD and LBD, or the ancestral NR contained only the
LBD and the zinc-finger DBD was added later in animal
If the absence of the DBD is due to domain loss, it is
likely to be broadly represented in ctenophores because
Mnemiopsis and Pleurobrachia are members of separate
orders - the Lobata and Cydippida, respectively .
Reitzel et al. EvoDevo 2011, 2:3
Page 7 of 12
The NRs reported from the three other early diverging
phyla (Porifera, Placozoa, and Cnidaria) all contain both
a DBD and an LBD. Within the bilaterians, NR domain
structure has been modified in particular lineages,
including duplication of the DBD in a novel protostome
NR subfamily , loss of the DBD from vertebrate
DAX1 (NR0B1) and SHP (NR0B2) [34,35], and loss of a
conserved LBD in some NRs from insects (for example,
knirps ) and nematodes (for example, odr-7 ).
Thus, although loss of the DBD in ctenophores would
be unique among early diverging animals, similar modi-
fications of NR domains have occurred in other animals.
A more provocative but equally supported hypothesis
for the absence of a canonical DBD in ctenophore NRs
is that the ancestral NR contained only an LBD and the
DBD was added later in animal evolution. This hypoth-
esis is contingent on (1) the phylogenetic placement of
ctenophores as the first branch of the animal tree,
which has been shown in previous phylogenomic ana-
lyses [13,14] yet remains controversial [16,18], and (2)
NRs from other ctenophore species lacking a zinc-finger
DBD, like the distantly related species reported here.
NRs are unique to animals, but several recent studies
have shown “NR-like” genes in fungi . These pro-
teins (for example, Oaf1 and Pip2 from the yeast Sac-
charomyces cerevisiae ) have regions of structural
similarity to animal NR LBDs but not zinc-finger DBDs,
function as protein dimers, and bind fatty acids as
ligands which regulate their function.
Potential function of ctenophore nuclear receptors
Expression patterns of NRs from Mnemiopsis were con-
sistent with NRs having a role in development. MlNR1
was expressed in spatially restricted portions of the ecto-
derm during gastrulation, corresponding to the position
of the future tentacles. Subsequent expression in the
cydippid stage was confined to the apical organ.
However, MlNR2 was expressed broadly in the endo-
derm and portions of the ectoderm after gastrulation
and in the cydippid stage. Previous studies with HNF4
in the sponge Amphimedon showed ubiquitous expres-
sion throughout development . Spatial expression of
HNF4 genes has not been reported from any cnidarian
or the placozoan Trichoplax. However, HNF4 from the
anemone Nematostella was expressed at high levels
throughout developmental and adult stages, suggesting
potential roles in both development and in normal cell
Nuclear receptors regulate the transcription of down-
stream genes, primarily through binding to specific
DNA-responsive elements . Due to the absence of a
conserved DBD, the ctenophore NRs are unlikely to act
as transcription factors by binding DNA-responsive
elements typical of other NRs. The amino-terminal
regions of these ctenophore NRs contain no conserved
domains similar to other transcription factors; thus, we
have no a priori expectation for what DNA motifs these
proteins may bind (if any). Despite lacking a zinc-finger
DBD, the mammalian NR DAX-1 binds DNA by recog-
nizing hairpin structures  rather than through bind-
ing to specific sequence motifs. These ctenophore NRs
may similarly regulate transcription of target genes in
alternative ways by binding to non-canonical DNA
sequences or secondary structures.
DAX-1 and SHP from vertebrates both function in
molecular pathways as repressors. These NRs form
dimers with other NRs, modulate the recruitment of
cofactors, and repress transcriptional activity [41-43].
Whether the ctenophore NRs interact with other pro-
teins or function as repressors requires additional
Early divergence of the nuclear receptor superfamily
Previous reports on the evolution of the nuclear recep-
tor superfamily have concluded that the diversification
of NRs in bilaterians occurred through two major radia-
tion events [1,44]. The first wave occurred prior to the
bilaterian ancestor, due to the presence of all six NR
families in both representative deuterostomes and proto-
stomes (, Figure 4). A second stage of diversification
occurred within the vertebrate lineage, with the expan-
sion of particular subfamilies due to gene or genome
duplication events (or example, the steroidogenic family
NR3C). However, the early diversification of the NR
superfamily prior to the bilaterian ancestor has not been
clarified despite the critical importance of these events
to the understanding of the emergence of the six NR
families present at the divergence of protostomes and
This new classification of nuclear receptors, which is
based on the full complement of sequences from four
early branching phyla as well as bilaterian sequences,
allows us to begin reconstructing the evolutionary
events that led to the diversification of this superfamily
(Figure 4). The first two branches of the animal tree
(sponge and ctenophore) both contain NRs that cluster
with subfamily NR2A (HNF4) and are most similar to
these sequences. Sponges also have a second NR posi-
tioned between HNF4 and the rest of the NR superfam-
ily. Thus, these genes suggest that HNF4 represents our
best expectation for which NR was present in the ances-
tral metazoan. Reciprocally, the limited diversity of NRs
from sponges and ctenophores provides additional
evidence that these two phyla represent the first two
branches from the animal tree. The next branch in the
metazoan tree is represented by the placozoan Tricho-
plax and suggests a partial radiation in NR family 2
with the addition of NR2B (RXR) and NR2F (COUP-TF)
Reitzel et al. EvoDevo 2011, 2:3
Page 8 of 12
and the evolution of the first member of NR family 3 (as
previously reported by ). The phylum Cnidaria is the
closest sister group to the Bilateria from which NR
sequences are currently available. From the two cnidar-
ian species with complete genomes available (Nematos-
tella vectensis and Hydra magnipapillata), we can infer
that the cnidarian-bilaterian ancestor had a completely
differentiated NR family 2, with NR2B (RXR) secondarily
lost within the anthozoan lineage (see  for discus-
sion). The class Anthozoa also has NRs that are best
supported as NR family 6 (GCNF) and three class-specific
paralogs that form an outgroup to NR families 1 and 4 in
bilaterians. We hypothesize that the corresponding NRs
were lost from Hydra, similar to a variety of other tran-
scription factors (see ). Future characterization of
NRs from other medusozoans, particularly members of
the other two cnidarians classes Scyphozoa and Cubozoa,
will allow additional testing of this hypothesis. Our data
also support the hypothesis that the NR family 3 was lost
early in the cnidarian lineage due to its presence in both
Trichoplax and bilaterians. However, some phylogenetic
analyses have suggested that Placozoa are the sister
group to the Bilateria with the Cnidaria diverging from
the stem earlier [for example, [18,45]]. If these are the
correct phylogenetic relationships for these phyla, we
would infer substantial NR loss from Trichoplax, includ-
ing various members of NR family 2.
This represents the first study to classify NRs from
each early animal lineage in a single analysis. From our
analyses, a number of NR families and subfamilies
differentiated in the time between the divergence of the
cnidarian lineage and the origin of the bilaterians. Most
striking is the diversification of the NR 1 family into
numerous subfamilies, at least eight of which were pre-
sent in the bilaterian ancestor. Gaining insight into the
diversification that occurred between these ancestors
(Inferred Bilaterian Ancestor)
NR 1 FamilyNR 2 Family
NR 3 Family
NR 4 Family NR 5 FamilyNR 6 Family
1A 1B 1C 1D 1E/G 1F 1H 1I/J/K 2A 2B 2C/D 2E 2F 3A 3B 3C 4A 5A 5B 6A
2A2C/D 2E 2F 6A
2A 2B 2E 2F
2A 2B 2F3A/B/C
1D 1E/G 1F 1H 1I/J/K 2A 2B 2C/D 2E 2F
3B 4A 5A 5B 6A
1A 1B 1C 1D 1E/G 1F 1H 1I/J/K 2A 2B 2C/D 2E 2F 3A 3B 3C 4A 5A
Figure 4 Evolutionary diversification of the NR superfamily in animals. At left is a cartoon of the metazoan tree showing the evolutionary
relationships between ctenophores, sponges, placozoan, cnidarians, and bilaterians (that is, protostomes and deuterostomes). Due to current
controversy about the branching order of the early diverging metazoans (see main text), the placement of lineages differs depending on
particular analyses and thus an inference for the timing of origin and lineage-specific loss of particular NR families would vary. Colored boxes
indicate a NR subfamily is represented by one or more genes for that species. The “inferred bilaterian ancestor” is based largely on a
phylogenetic analysis conducted by Bertrand et al. . However, two of these subfamilies are restricted to either protostomes (5B) or
deuterostomes (3C) and we have shaded these and used black text to reflect a lack of conclusive support for the presence of these subfamilies
in the bilaterian ancestor. Despite the absence of some genes in the D. melanogaster genome, studies of NRs from other protostomes (NR1A
from Schistosoma mansoni  and NR3A from mollusks and annelids [51,52]) indicate that these subfamilies were present in the bilaterian
ancestor and secondarily lost from Drosophila. Similarly, members of NR1B and 1C have been reported in mollusks and annelids , and thus
are not restricted to deuterostomes. * The sponge Amphimedon queenslandica has two NRs, one that is supported as an ortholog to HNF4
(NR2A) and a second that groups between subfamily NR2A and the rest of the NR superfamily [see also ].
Reitzel et al. EvoDevo 2011, 2:3
Page 9 of 12
will depend on characterizing the gene complement of
species that diverged during this time period. The best
current candidates are the acoelomorphs, which are sup-
ported as a closer sister taxa to the bilaterians than the
cnidarians [13,46]. Indeed, studies of the Hox comple-
ment from these species have provided insightful inter-
mediates for characterizing the emergence of the
homeobox family of transcription factors [47,48].
By identifying NRs from these early branching
metazoans with full genome sequences, we have addi-
tional power in characterizing the evolution of gene
families, particularly since we are not inadvertently
omitting genes missed in PCR-based surveys. For exam-
ple, the original report of NRs from the coral Acropora
identified ten NRs . We queried newly published EST
data from this species  and identified additional NRs
that group in three families: the cnidarian-specific NR
1/4 family, NR2E, and NR2F. Because the Acropora
sequences group with strong support with NRs from
Nematostella, we expect that up to five more NRs are
still not identified in this coral species.
With the additional cnidarian sequences and surveyed
species, phylogenetic analyses suggest that NR evolution
within the Cnidaria appears to have been a dynamic
process with both gains (for example, duplication of
TR2/4, COUP-TF anthozoans) and losses (NR family 6
and NR family 1/4 from either hydrozoans or ancestral
lineage leading to the medusozoans, RXR from anthozo-
ans). Two subfamilies in NR family 2 (NR2E and F)
have independently radiated at some point in this phy-
lum’s history due to the presence of multiple cnidarian
paralogs. TR2/4 (NR2C/D) most likely duplicated within
the anthozoans due to the presence of two homologs in
the anemone Nematostella and the coral Acropora. The
most dramatic cnidarian-specific radiation is represented
by the duplications of the group of NRs sister to NR
families 1 and 4 (see above). Together, these data sup-
port a hypothesis that NRs have undergone a handful of
independent radiations within the Cnidaria. For one
class of cnidarians, the Cubozoa, there is only one
published NR sequence  and for another class, the
Scyphozoa, there are no published NRs. Identifying
additional NRs in these two classes will provide much
needed data for assessing when particular NRs were
duplicated and lost within the cnidarians. Finally, we
have no evidence that similar radiations have occurred
in ctenophores, sponges, or placozoans. This supports
the conclusion that the NR radiations in the Cnidaria
are unique among the early diverging animal lineages.
In this study, we have identified two NRs from the Mne-
miopsis leidyi genome and one NR from Pleurobrachia
pileus. All three ctenophore NRs contain the conserved
LBD but lack a conserved zinc-finger DBD, a domain
that is conserved across all reported NRs in animals
(with the exception of two genes unique to vertebrates).
By applying a phylogenomic approach using NR
sequences from organisms throughout the animal king-
dom, we showed that ctenophores and sponges contain
representatives of the same subfamily (NR2A), suggest-
ing that the original NR was most similar to HNF4. The
absence of the DBD from ctenophores may reflect an
ancestral NR domain structure or a lineage-specific loss
of this domain from an ancestral NR that contained
both the DBD and LBD. Through analysis of NR family
and subfamily representation in representative taxa, we
conclude that the rate of diversification for the NR
superfamily was fairly modest in the early diverging ani-
mals, similar to other gene families . Additionally,
several subfamilies underwent separate radiations in the
phylum Cnidaria. Future work aimed at characterizing
the function of the NRs from these early diverging phyla
will enable tests of hypotheses regarding conserved and
novel functions of members of this critical superfamily
of transcription factors.
Additional file 1: Primers used for 5- and 3-prime RACE of
Mnemiopsis leidyi NRs.
Additional file 2: Nuclear receptors from early diverging taxa used
for phylogenetic study of nuclear receptor superfamily. Table
showing gene names and accession numbers
Additional file 3: Protein sequences for NRs from Mnemiopsis leidyi
and Pleurobrachia pileus.
Additional file 4: Bayesian analysis of NR superfamily. Tree was
constructed using the identical alignment used for the maximum
likelihood analysis presented in Figure 3. Clades are annotated to family
and subfamily based on current nomenclature for the NR superfamily .
This tree is the consensus of four independent runs and was rooted with
the cluster containing the ctenophore sequences plus HNF4 from diverse
animals. Values above nodes indicate Bayesian support values. Posterior
probabilities below 0.7 were removed.
BLAST: basic local alignment search tool; cDNA: complementary DNA; COUP-
TF: chicken ovalbumin upstream promoter transcription factor; DAX-1:
dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on
the X chromosome; DBD: DNA-binding domain; DNA: deoxyribonucleic acid;
ERR: estrogen related receptor; EST: expressed sequence tag; FTZ: fushi
tarazu; GCNF: germ cell nuclear factor; HMM: hidden Markov model; HNF4:
hepatocyte nuclear factor 4; LBD: ligand-binding domain; NR: nuclear
receptor; PCR: polymerase chain reaction; RACE: rapid amplification of cDNA
ends; RNA: ribonucleic acid; RXR: retinoid “X” receptor; SHP: small
heterodimerization partner; UTR: untranslated region.
This research was supported by Award Number F32HD062178 from the
Eunice Kennedy Shriver National Institute of Child Health & Human
Development (NICHD) to AMR, an NSF Graduate Research Fellowship to KP,
grants from the NSF and NASA to MQM, the Tropical Research Initiative of
the Woods Hole Oceanographic Institution, and the Intramural Research
Reitzel et al. EvoDevo 2011, 2:3
Page 10 of 12
Program of the National Human Genome Research Institute (NHGRI),
National Institutes of Health. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the Eunice
Kennedy Shriver National Institute of Child Health and Human Development
or the National Institutes of Health.
1Biology Department, Woods Hole Oceanographic Institution, Woods Hole,
MA, USA.2Kewalo Marine Laboratory, Pacific Bioscience Research Center,
University of Hawaii, Honolulu, HI, USA.3Genome Technology Branch,
National Human Genome Research Institute, National Institutes of Health,
Bethesda, MD, USA.
AMR and AMT designed and conceived the study and drafted the
manuscript. AMR also isolated Mnemiopsis nuclear receptors, performed
gene annotation, and performed phylogenetic analyses. JFR identified
Mnemiopsis nuclear receptors from the genomic assembly and contributed
to phylogenetic analyses. KP isolated Mnemiopsis DNA and RNA and
performed in situ hybridizations. JCM assembled the Mnemiopsis genome.
ADB and MQM participated in the design of the study. All authors read and
approved the final manuscript.
The authors declare that they have no competing interests.
Received: 29 July 2010 Accepted: 3 February 2011
Published: 3 February 2011
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Cite this article as: Reitzel et al.: Nuclear receptors from the ctenophore
Mnemiopsis leidyi lack a zinc-finger DNA-binding domain: lineage-
specific loss or ancestral condition in the emergence of the nuclear
receptor superfamily? EvoDevo 2011 2:3.
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