Origin of metazoan cadherin diversity and the antiquity
of the classical cadherin/β-catenin complex
Scott Anthony Nicholsa, Brock William Robertsb, Daniel Joseph Richterb, Stephen Robert Faircloughb,
and Nicole Kingb,1
a
Department of Biological Sciences, University of Denver, Denver, CO 80208; and bDepartment of Molecular and Cell Biology, University of California,
Berkeley, CA 94720
The evolution of cadherins, which are essential for metazoan multicellularity and restricted to metazoans and their closest relatives,
has special relevance for understanding metazoan origins. To reconstruct the ancestry and evolution of cadherin gene families, we
analyzed the genomes of the choanoflagellate Salpingoeca rosetta,
the unicellular outgroup of choanoflagellates and metazoans Capsaspora owczarzaki, and a draft genome assembly from the homoscleromorph sponge Oscarella carmela. Our finding of a cadherin
gene in C. owczarzaki reveals that cadherins predate the divergence of the C. owczarzaki, choanoflagellate, and metazoan lineages. Data from these analyses also suggest that the last common
ancestor of metazoans and choanoflagellates contained representatives of at least three cadherin families, lefftyrin, coherin, and
hedgling. Additionally, we find that an O. carmela classical cadherin
has predicted structural features that, in bilaterian classical cadherins, facilitate binding to the cytoplasmic protein β-catenin and,
thereby, promote cadherin-mediated cell adhesion. In contrast with
premetazoan cadherin families (i.e., those conserved between
choanoflagellates and metazoans), the later appearance of classical
cadherins coincides with metazoan origins.
T
he cadherin gene family is hypothesized to have had special
importance for metazoan origins (1–5). Cadherins are cellsurface receptors that function in cell adhesion, cell polarity, and
tissue morphogenesis (6–8). Moreover, cadherins are found in
the genomes of all sequenced metazoans, including diverse
bilaterians, cnidarians, and sponges, and are apparently lacking
from multicellular lineages such as plants, fungi, and Dictyostelium (9). Although it once seemed likely that cadherins were
unique to metazoans, 23 genes encoding the diagnostic extracellular cadherin (EC) domain (10) have since been discovered
in the genome of the unicellular choanoflagellate Monosiga
brevicollis, one of the closest living relatives of Metazoa (1, 11).
Proteins in the cadherin family are characterized by the
presence of one or more tandem copies of the EC domain, an
∼100-aa protein domain that mediates adhesion with EC
domains in other cadherins (10, 12–14). Cadherins are further
assigned to different subfamilies based on the number and arrangement of additional, non-EC protein domains and sequence
motifs that refine cadherin function and suggest shared ancestry
(2, 3). For example, classical cadherins are distinguished by the
presence of a cytoplasmic cadherin domain (CCD) at the C
terminus that regulates interactions with the cytoplasmic protein
β-catenin (2, 3, 12, 15). When bound to β-catenin, classical
cadherins on neighboring cells interact homophilically and,
thereby, promote cell-cell adhesion (16). When not bound to
β-catenin, classical cadherins are rapidly degraded (17, 18). The
regulation of classical cadherin function by β-catenin thereby
forms the foundation of adherens junctions and is crucial for cell
adhesion in all studied bilaterian tissues, including epithelia,
neurons, muscles, and bones (3, 19).
The classical cadherins are one of six cadherin families (including fat, dachsous, fat-like, CELSR/flamingo, and protocadherins) that are found in most metazoans. In contrast with
the cell adhesion functions of classical cadherins, CELSR/
www.pnas.org/cgi/doi/10.1073/pnas.1120685109
flamingo, dachsous, fat, and fat-like cadherins regulate planar
cell polarity in organisms as disparate as Drosophila and mouse
(20–22). Members of the protocadherin family have diverse
functions that include mechanosensation in stereocilia and regulation of nervous system development (23, 24). It is not known
whether the bilaterian roles of these cadherin families had already evolved in the last common ancestor of metazoans, and it
is not clear how these cadherin families themselves originated.
To date, only one cadherin family—the hedgling family—is
inferred to have been present in the last common ancestor of
choanoflagellates and metazoans. Hedgling family members are
defined by the presence of an N-terminal hedgehog signal domain
(Hh-N) and are absent from Bilateria (25, 26). Differences in the
cadherin repertoire of choanoflagellates and metazoans have led
to the proposal that cadherins in these two lineages may have
largely independent histories—that is, one or a few ancestral
cadherins may have undergone independent evolutionary radiations in each lineage (2). To reconstruct the evolutionary history
of cadherin families before and after the transition to metazoan
multicellularity, we have analyzed the diversity of cadherins in the
newly sequenced genomes of phylogenetically relevant taxa: the
colony forming choanoflagellate Salpingoeca rosetta, the close
choanoflagellate/metazoan outgroup Capsaspora owczarzaki, and
the homoscleromorph sponge Oscarella carmela.
Results
Reconstructing the Ancestry of Cadherin Diversity. By searching the
S. rosetta genome using BLAST analyses (27) and hidden Markov model (HMM)-based searches (28–30) for the EC domain
(Fig. 1), we identified at least 29 predicted cadherin genes (Fig. 1
and SI Appendix, Figs. S1 and S2), all of which were verified
through deep sequencing of the transcriptome (SI Appendix,
Table S1). The number of cadherin genes in S. rosetta, like that in
M. brevicollis (1), rivals that of most metazoans (Fig. 1), whereas
the C. owczarzaki genome assembly was found to contain only a
single cadherin gene.
To increase the taxonomic breadth of genomes available from
early branching metazoan lineages, we also sequenced the
Author contributions: S.A.N., B.W.R., and N.K. designed research; S.A.N., B.W.R., and D.J.R.
performed research; S.A.N., D.J.R., and S.R.F. contributed new reagents/analytic tools; S.A.N.,
B.W.R., and S.R.F. analyzed data; and S.A.N. and N.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. PRJNA20341 (Capsaspora genome); PRJNA37927S (Salpingoeca genome); EGD72656, EGD73963, EGD74518, EGD74667, EGD74707, EGD74783,
EGD75074, EGD75359, EGD75381, EGD75404, EGD75405, EGD75586, EGD75710,
EGD76846, EGD77346, EGD78086, EGD78170, GD78171, EGD78831, EGD78839,
EGD78969, EGD78970, EGD79002, EGD79017, EGD79249, EGD80879, EGD80917,
EGD81200, EGD82245, and EGD82557 (S. rosetta cadherins); EFW44034 (Capsaspora owczarzaki cadherins), JN197609 (Oscarella carmela lefftyrin), AEC12441 (Oscarella carmela
cadherin 1), and HQ234356 (Oscarella carmela β-catenin)].
1
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1120685109/-/DCSupplemental.
PNAS Early Edition | 1 of 6
EVOLUTION
Edited by Masatoshi Takeichi, RIKEN, Kobe, Japan, and approved June 20, 2012 (received for review December 19, 2011)
Holozoa
29
Number
of
Cadherins
23
17
119
Placozoa
Choano-
Porifera (sponge)
Metazoa
Cnidaria
18
17
16
12
15
8
3
1
0
0
0
*
*
Pult Plants Ddis Fungi Cowc Mbre Sros Aque Tadh Hmag Nvec Dmel Cele Cint Mmus
≥8
≥1
?
≥3
≥5
≥8
Functionally
characterized
cadherins
Fig. 1. Phylogenetic distribution and abundance of cadherins in the genomes of diverse eukaryotes. Once thought to be restricted to metazoans, cadherins
are abundant in choanoflagellates and evolved before the divergence of Capsaspora owczarzaki, choanoflagellates, and metazoans (1). EC domains detected
in the genome of the oomycte Pythium ultimum likely evolved through convergence or lateral gene transfer (9). The number of cadherin families inferred at
ancestral nodes (determined based upon their shared domain composition and organization) is indicated (open circles). The dashed lineage of Trichoplax
adhaerens reflects its uncertain phylogenetic placement. *All fungal and plant species represented in the Pfam v24.0 database (29) were analyzed. Aque,
A. queenslandica; Cele, Caenorhabditis elegans; Cint, Ciona intestinalis; Cowc, C. owczarzaki; Ddis, Dictyostelium discoideum; Dmel, D. melanogaster; Hmag,
Hydra magnipapillata; Mbre, M. brevicollis; Mmus, Mus musculus; Nvec, N. vectensis; Pult, P. ultimum; Sros, S. rosetta; Tadh, T. adhaerens.
genome of the sponge O. carmela by using massively parallel
sequencing (Illumina). Although the genome assembly is fragmented relative to traditional Sanger assemblies (SI Appendix),
multiple cadherin-domain encoding sequences were detected
and two cadherin genes assembled in near entirety (GenBank
accession nos. JN197609 and AEC12441). The value of this draft
genome for providing unique insights into cadherin evolution is
demonstrated by the fact that one of the two assembled cadherins, JN197609, has homologs in choanoflagellates, despite
being absent from the genome of the only other sequenced
sponge, Amphimedon queenslandica, which encodes at least 17
cadherins (Fig. 2 and ref. 31).
To reconstruct the evolutionary relationships among cadherins
from nonmetazoans and early branching metazoans, we grouped
cadherins from C. owczarzaki, choanoflagellates, and sponges
according to shared structural features (i.e., domain composition
and arrangement). Mapping of the phylogenetic distribution of
cadherin families reveals that they have origins that predate the
evolution of Metazoa. Although the earliest branching lineage
to contain a predicted cadherin (Owcz_Cdh1) is C. owczarzaki,
the evolutionary connection between this and cadherin families
from choanoflagellate and metazoans is uncertain (Fig. 2A).
Owcz_Cdh1 has at least 10 predicted EC domains, two membrane-proximal epidermal growth factor (EGF) domains, and
a transmembrane (TM) domain. This domain organization
resembles that of cadherins in the choanoflagellates M. brevicollis
(accession no. MBCDH14) and S. rosetta (accession nos.
EGD82557 and EGD79002) but is not sufficiently complex to
definitively indicate that these proteins are orthologous.
In contrast, two cadherin families are clearly shared by choanoflagellates and sponges to the exclusion of all other lineages
analyzed in this study. The first, lefftyrins, are defined by the
presence of an amino-terminal “LEF” cassette [containing a
Laminin N-terminal (Lam-N) domain, four EGF domains, and
a Furin domain] and a carboxyl-terminus “FTY” cassette [containing one or two Fibronectin 3 (FN3) domains, a TM domain
and a cytoplasmic protein tyrosine phosphatase (PTPase) domain; Fig. 2B]. The M. brevicollis lefftyrin family member,
MBCDH21, also has an N-terminal Laminin G (Lam-G) domain
that has prompted previous comparisons with metazoan classical
cadherins and fat cadherins (1, 4). Cadherins in the second
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1120685109
family, the coherins (Fig. 2C), are united by the presence of at
least one cohesin domain (not to be confused with the eukaryotic
cohesin protein that regulates sister chromatid separation). The
presence of cohesin domains (SI Appendix, Fig. S3) in coherins is
diagnostic because they are otherwise found only in bacteria and
archaea (32).
Members of the remaining premetazoan family of cadherins,
the hedglings (Fig. 2D), are found in choanoflagellates, sponges,
and the cnidarian Nematostella vectensis (1, 25, 26), but are absent from C. owczarzaki and bilaterians. Hedglings contain an
amino-terminal Hedgehog signal domain (Hh-N; ref. 33) that
was thought to be exclusive to the secreted signaling portion of
the metazoan-specific Hedgehog protein. The amino-terminal
Hh-N domain in all hedglings is adjacent to a von Willebrand
factor A (VWA) domain and, with the exception of one
M. brevicollis hedgling (accession no. MBCDH3), all hedglings
have a carboxyl-terminal cassette with between one and eight
extracellular EGF domains positioned proximal to the TM region. Although the first identified choanoflagellate hedgling,
MBCDH11 from M. brevicollis, contains additional domains
(including TNFR, Furin, and 9-cystein GPCR), all other choanoflagellate hedglings detected in this study and all known
metazoan hedglings lack these domains. Thus, hedgling in the
last common ancestor of metazoans more likely resembled hedglings from metazoans (e.g., Aque_hedgling and Nvec_hedgling)
and S. rosetta (accession no. EGD79017) than MBCDH11. The
inference that the last common ancestor of choanoflagellates and
metazoans contained lefftyrins, cohesins, and hedgling cadherins
reveals the evolutionary foundations for the subsequent origin of
metazoan-specific cadherins.
Metazoan Classical Cadherin/β-Catenin Adhesion Complex. Among
the cadherins that evolved along the metazoan stem lineage,
classical cadherins have the clearest potential link to metazoan
origins, both because of their ubiquity in modern metazoan lineages and because of their central roles in bilaterian cell adhesion
(4). To investigate whether the adhesive functions of classical
cadherins might extend to the earliest branching lineages of
metazoans, we examined the possibility that the regulatory interaction between classical cadherins and β-catenin is conserved
in sponges. The single detected classical cadherin homolog in
Nichols et al.
A
M. brevicollis
(MBCDH14)
S. rosetta
(EGD82557)
S. rosetta
(EGD79002)
C. owczarzaki
(EFW44034)
B
Lam-G
M. brevicollis
(MBCDH21)
*
Candida ALS
S. rosetta
(EGD79249)
*
Dockerin 1
PKD
Lefftyrin
Family
PKD
O. carmela
(JN197609)
M. brevicollis
(MBCDH8)
Coherin
Family
S. rosetta
(EGD82245)
EVOLUTION
C
A. queenslandica
(Aqu1.221884)
D
M. brevicollis
(MBCDH11)
TNFR/FU
TNFR
9-cystein GPCR
M. brevicollis
(MBCDH3)
M. brevicollis
(MBCDH15)
Hedgling
Family
S. rosetta
(EGD79017)
Epidermal Growth Factor
(EGF)
Extracellular
Cadherin (EC)
Transmembrane (TM)
Cohesin Domain
N-terminal Hedgehog
Signal Domain (Hh-N)
von Willebrand A
(VWA)
Laminin N-terminal
Domain (Lam-N)
Furin
Protein Tyrosine
IG I-set
(PTPase),
* Phosphatase
inactive
A. queenslandica
(ABX90059)
IG I-set
SH2
Protein Tyrosine
Phosphatase (PTPase)
Fibronectin 3
Domain (FN3)
1000aa
N. vectensis
(ABX84114)
Fig. 2. Predicted domain architecture of modern representatives of premetazoan cadherins. At least three cadherin families evolved before the origin of
metazoans. (A) The single cadherin discovered in the genome of C. owczarzaki has a cassette of EGF repeats positioned proximal to a single transmembrane
domain (blue box) that is also found in choanoflagellate and sponge cadherins. The phylogenetic relationships among cadherins with this feature are not yet
clear. The lefftyrin (B) and coherin (C) families are present only in choanoflagellates and sponges. Lefftyrins are distinguished by an N-terminal “LEF” cassette
(orange box) with a Lam-N domain, four EGF repeats, and a Furin repeat and a C-terminal “FTY” cassette (purple box) with one or two Fibronectin 3 domains,
a transmembrane domain, and a tyrosine phosphatase domain. Coherins contain a diagnostic bacterial/archaeal-like cohesin (50) domain. (D) The hedgling
family (1, 26) is present in choanoflagellates, sponges and cnidarians and is absent from bilaterians. All hedglings contain an N-terminal Hedgehog signal
domain linked to a von Willebrand A domain (green box) and most contain a series of EGF repeats proximal to the transmembrane domain (blue box).
Candida ALS, Candida Agglutinin-like sequence; IG I-set, Ig I-set; KU, BPTI/Kunitz family of serine protease inhibitors; Lam-G, Laminin G domain; 9-cystein
GPCR, 9-cystein G protein coupled receptor; PKD, polycystic kidney disease; SH2, src homogy domain 2; TNFR, tumor necrosis factor receptor.
O. carmela, OcCdh1 (GenBank accession no. AEC12441), encodes at least seven EC domains and a CCD domain, as well as
multiple EGF and Lam-G domains that are typical of classical
cadherins in invertebrates (e.g., Drosophila melanogaster N-cadherin and Shotgun; Fig. 3A and refs. 3 and 34). By aligning the
amino acid sequence of the CCD of OcCdh1 with those of other
classical cadherins, we found that two residues (D675 and E682)
necessary for binding and modulating interactions with β-catenin
(35) in bilaterians are conserved (Fig. 3B).
Nichols et al.
We next investigated whether O. carmela β-catenin (Oc_bcat;
GenBank accession no. HQ234356) has diagnostic protein
domains and residues indicative of the ability to interact with
classical cadherins. Oc_bcat contains at least 11 of the 12 conserved armadillo (arm) repeats (36, 37) that are typical of
eumetazoan β-catenin proteins (Fig. 3C) and shows 66.4% amino
acid sequence identity with human β-catenin over the conserved
arm-repeat region. Furthermore, Oc_bcat has two lysine residues
(homologous to positions K312 and K435 in mouse) required for
PNAS Early Edition | 3 of 6
A
TM
EC domains
mouse
E-cadherin
CCD
Drosophila
Shotgun
EC domains
EGF LamG TM
CCD
O. carmela
cadherin1
EGF
EC domains
LamG
EGF TM
CCD
B
674
|
mouse D S L L V F D Y E G S G S E A A S L S S L - N S S E
Drosophila D D V R H Y A Y E G D G N S D G S L S S L A S C T D
O. carmela D E L L H F E D E G I L S E G A S L S S L S I A S E
C
armadillo repeats
zebrafish
Drosophila
O. carmela
helix-C
1 2
3
4 5
6 7
8
9
10 11 12
1
3
4
5
6
8
9
10
11 12
3
4
5 6
7 8
9
10
11 12
1
2
7
700
|
S D
D D
S S
D
zebrafish
K435
K312
O. carmela
K358
E
K482
312
435
|
|
mouse Y G N Q E S K L I I L A S . . . C N N Y K N K M M V C Q V
zebrafish Y G N Q E S K L I I L A S . . . C N N Y K N K M M V C Q V
O. carmela Y G N Q E S K L I I L A S . . . C N N Q Q N K V I V C Q C
|
|
358
482
Fig. 3. A conserved β-catenin/classical cadherin protein complex in a
sponge. (A) The genome of the sponge O. carmela encodes a classical cadherin, Oc_cdh1, identified by the presence of the diagnostic cadherin cytoplasmic domain (CCD). Oc_cdh1 also has EGF and Lam-G domains in
a membrane-proximal position that is typical of invertebrate classical cadherins (4). The dashed line at the N terminus of Oc_cdh1 indicates that the
gene model is incomplete because of the draft nature of the genome assembly. (B) An alignment of a portion of the Oc_cdh1 CCD with bilaterian
CCDs demonstrates the conservation of two residues (Aspartate and Glutamate, highlighted in green) required for binding to β-catenin (SI Appendix,
Fig. S4 depicts the full alignment and includes the only known CCD from the
demosponge A. queenslandica, in which critical β-catenin binding residues
are also conserved). Conserved residues are shaded gray and Casein Kinase II
and Glycogen Synthase Kinase 3b phosphorylation sites essential for the
regulation of adhesion dynamics are indicated by filled or open circles, respectively (35, 38, 39). (C) The O. carmela genome also encodes a single
β-catenin ortholog (Oc_bcat) with 11 predicted armadillo (arm) repeats and
a helix-C domain; each arm repeat is numbered according to its similarity
(determined by best-reciprocal Blast) with the 12 arm repeats from other
metazoan β-catenin homologs (SI Appendix, Fig. S4). (D) Through comparison of a surface representation of the 3D structure of zebrafish β-catenin
(37) with a structural model of Oc_bcat, we predict the conservation of
a positively charged groove lined by the third helix (blue) of each arm repeat. Within this groove there are two lysine residues whose orientation
resembles that of conserved lysines from zebrafish β-catenin. (E) These
lysines align with Lysine-312 and Lysine-435 of mouse β-catenin, each of
which are required for binding to mouse E-cadherin (35, 38, 39) at Aspartate647 and Glutamate-682 (highlighted in B). Ocar_cdh1 was initially discovered
from a yeast two-hybrid screen using full-length Ocar_bcat as bait (SI Appendix, Table S2; see SI Appendix for further discussion). CCD, cadherin cytoplasmic domain; EC, extracellular cadherin; EGF, epidermal growth factor
domain; Lam-G, Laminin G domain; TM, transmembrane domain.
4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1120685109
the interaction of mouse β-catenin with E-cadherin (Fig. 3 D and
E and refs. 35, 38, and 39).
By threading the full-length sequence of Oc_bcat onto the
crystal structure of zebrafish β-catenin (Fig. 3D), we predict that
the third helix of each arm repeat in Oc_bcat orients along the
surface of a positively charged groove that has been shown to
contact E-cadherin directly in mouse (35, 38, 39). Moreover, the
conserved lysines of β-catenin that are required to mediate interactions with E-cadherin are oriented similarly in the 3D models
of the full-length zebrafish (37) and Oc_bcat. Furthermore, an
unbiased yeast two-hybrid screen of O. carmela proteins using
Oc_bcat as the “bait” recovered OcCdh1 as a binding partner (SI
Appendix). Further study is required to determine whether OcCdh1
and Oc_bcat have the capacity to bind to each other directly in vivo
and, thereby, contribute to cell adhesion in O. carmela.
Discussion
Cadherins represent a compelling case study for how large
metazoan gene families evolve. Like members of most metazoan
signaling and adhesion protein families, cadherins are typically
large, multidomain proteins. Such protein families evolve
through duplication and divergence and through the shuffling of
protein domains among different protein families (40, 41). By
using a phylogenetically informed comparative genomic approach, we were able to reconstruct a concrete portrait of the
minimal cadherin diversity in the metazoan stem lineage. Furthermore, by reconstructing the ancestral domain composition of
early-evolving cadherin families, we have been able to predict
their evolutionary relationships with other, later-evolving modern protein families.
Premetazoan Cadherin Diversity. An initially surprising result from
the genome of M. brevicollis was that the genomes of choanoflagellates and most metazoans have comparable numbers of
cadherin genes (1), despite vast differences in their biology. This
result is further supported by our analysis of the S. rosetta genome,
which has at least 29 predicted cadherin genes. In contrast, our
analyses of cadherin relationships among metazoans, choanoflagellates, and C. owczarzaki suggest that as few as three modern
cadherin families were present in the last common ancestor of
choanoflagellates and metazoans, and that potentially only one
cadherin was present in the last common ancestor of C. owczarzaki,
choanoflagellates, and metazoans (Fig. 4A). However, these
inferences may represent an underestimate because of limited
available data. For example, C. owczarzaki is the only known
member of its lineage, it diverged from choanoflagellates and
metazoans more than 650 Mya, and it is a symbiont (42) that is
likely to have evolved from a free-living ancestor; hence, aspects of
its biology and genome content may be reduced.
The contrast between the large number of cadherins in modern
lineages and the low diversity of cadherins inferred in the metazoan-stem lineage raises the intriguing possibility that modern
cadherin diversity arose from a handful of ancestral cadherin
families that still exist today (however, it is notable that all of the
premetazoan cadherin families detected are absent from Bilateria). Alternatively, although future studies of a broader diversity
of choanoflagellates and early branching metazoans may reveal
additional members of the premetazoan cadherin repertoire, it is
also possible that cadherins present in the ancestors of metazoans
and choanoflagellates were subsequently lost (or evolved beyond
recognition) in both lineages.
Radiation of Cadherins in Choanoflagellate and Metazoan Lineages.
The study of cadherin families conserved in choanoflagellates
and metazoans promises to provide an unprecedented perspective on cadherin function before the evolution of metazoan
multicellularity. Three cadherin families—lefftyrins, coherins,
and hedglings—were present in the last common ancestor of
Nichols et al.
coherin family
lefftyrin family
hedgling family
CELSR/flamingo
Cowc_Cdh1
Fungi
classical cadherins
Capsaspora Choanoflagellates Sponges
Cnidaria
Bilateria
Holozoa
Metazoa
B
C-terminal
cassette of
FN3-TM-PTPase
domains
Receptor Protein Tyrosine
Phosphatases
LamNT
Usherin, Laminin
and Netrin
coherins
Cohesin
domain
Bacterial Cellulosome
hedglings
HhN
domain
Hedgehog
lefftyrins
Fig. 4. An emerging model of cadherin evolution. (A) At least five modern
families of cadherins—hedglings, coherins, lefftyrins, CELSR/flamingo and
classical cadherins—evolved before the diversification of modern metazoans.
Of these families, only the CELSR/flamingo and classical cadherin families are
clearly conserved in all metazoan lineages (2, 4, 31). In contrast, among
metazoans, hedgling is restricted to sponges and cnidarians. All of the cadherin families that evolved before the divergence of choanoflagellates and
metazoans (“premetazoan” cadherin families) have been lost or have
evolved beyond recognition in bilaterians. The relationships among the single cadherin detected in the genome of C. owczarzaki (Cowc_Cdh1) and
other modern cadherin families are uncertain (indicated by dotted circle, also
see Fig. 2A). (B) In addition to having EC domains, members of many cadherin
families contain domains that provide clues to their evolutionary origins and
to their relationships with other modern protein families (see Discussion).
metazoans and choanoflagellates and seem to have evolutionary
connections to diverse metazoan signaling and adhesion gene
families (Fig. 4B). For example, lefftyrins, so far known only
from choanoflagellates and the sponge O. carmela, contain
a Lam-N domain that is otherwise found in the proteins laminin,
netrin, and usherin. These proteins are united by the fact that
they function in the extracellular matrix (43–46). Furthermore,
the carboxyl-terminal FTY cassette of lefftyrins is diagnostic of
metazoan receptor PTPases, which help regulate cellular
responses to interactions with neighboring cells and the extracellular matrix (47–49). C. owczarzaki is the most divergent
outgroup of metazoa that has cadherins, and we have discovered
that its genome also encodes a metazoan-like receptor PTPase
that lacks EC domains (GenBank accession no. EFW39745).
Thus, it seems that lefftyrins may have evolved through a domain-shuffling event that brought PTPase and EC domains
together in the choanoflagellate/metazoan stem lineage.
Whereas lefftyrins may represent a case of protein family
evolution through the process of domain shuffling, the newly
discovered coherin family may have evolved through horizontal
gene transfer. Coherins, which are restricted to choanoflagellates
and sponges, are defined by the presence of EC domains and the
cohesin domain. The cohesin domain is otherwise known only
from archaea and bacteria. In the bacterial genus Clostridium,
the cohesin domain functions in the assembly of the cellulosome,
Nichols et al.
a complex of enzymes used to degrade plant cell walls (50).
The possible evolutionary connection between coherins and
the prokaryotic cohesin domain-containing proteins highlights
the complexities of the evolutionary processes that shaped cadherin evolution during the early ancestry of Metazoa. Unless the
cohesin domain of coherins evolved by convergent evolution with
its prokaryotic counterpart, then it must have been acquired by
horizontal gene transfer (32); this explanation seems quite
plausible when considering that the earliest metazoan ancestors
likely were bacterivorous (51). Either way, the presence of
a cohesin domain in coherins is compelling evidence of the homology of these proteins between sponges and choanoflagellates.
Premetazoan Cadherin Functions. Our understanding of the scope
of cadherin function derives from their study in morphologically
complex bilaterians, but C. owczarzaki is unicellular (42) and
choanoflagellates exist as either single cells or simple undifferentiated colonies (52–54). Cadherins in these organisms may
have functions that are unrelated to cadherin functions known
from bilaterians. For example, even in colony-forming S. rosetta,
adjacent cells are linked by cytoplasmic bridges and lack structures that resemble the cadherin-based adherens junctions of
metazoans (53). However, it is possible to identify some analogous functions that might be served by cadherins in nonmetazoans. For example, cadherins in unicellular lineages could
have adhesive functions other than the regulation of stable cellcell adhesion, such as during bacterial prey capture, attachment
to ECM, attachment to environmental substrates, or gamete
recognition (although sex is undocumented in choanoflagellates).
One biological context in which cadherin function may be
conserved between choanoflagellates and metazoans is in the
collar cells of sponges. Like choanoflagellates, sponge collar cells
have a motile flagellum used to generate water flow for the capture
of bacterial prey on a surrounding microvillar collar where they
are phagocytosed. It is reasonable to hypothesize that cadherin
families restricted to sponges and choanoflagellates (i.e., lefftyrins
and coherins), in particular, may have functions specific to the
biology of collar cells. Such functions may include roles in the
regulation of microvillar collar integrity or bacterial prey capture.
Indeed, one cadherin (MBCDH1) has been shown to localize to
the microvillar collar of M. brevicollis (1). Furthermore, there is
precedent for a physiologically important interaction between
bacteria and cadherins in metazoans: Some pathogenic bacteria
interact with classical cadherins in gut epithelia, thereby stimulating the host cells to phagocytose the invading pathogen (55–57).
Linking Cadherin Evolution to the Origin of Metazoa. A challenge for
relating cadherin gene family evolution to metazoan morphological evolution is that, until now, none of the functionally
characterized cadherin families of bilaterians have been studied
in nonbilaterians. Of all of the modern cadherin families, the
classical cadherin family is perhaps the strongest candidate for
having played a role in the evolution of metazoan multicellularity
(2, 4). The CCD of classical cadherins binds to β-catenin to
regulate cell-cell adhesion in all studied bilaterian tissues. Here,
we show that the genome of the sponge O. carmela encodes a
typical nonchordate classical cadherin with a CCD domaincontaining cytoplasmic tail that is predicted to be capable of
binding to O. carmela β-catenin. Thus, it is plausible that an
evolutionarily conserved classical cadherin/β-catenin adhesion
complex was a feature of the cell biology of the last common
ancestor of all modern metazoans.
The ubiquity of certain cadherin families in lineages that diverged more than 600 Mya indicates that these protein families
have conserved (and essential) roles in organisms with vastly
different biology. As we learn about their functions, we stand
to gain insight into ancestral features of metazoans and their
PNAS Early Edition | 5 of 6
EVOLUTION
A
single-celled relatives—similarities that are fundamental to their
basic cell biology.
Materials and Methods
The genomes of C. owczarzaki and S. rosetta were sequenced and assembled
by the Broad Institute (Massachusetts Institute of Technology/Harvard;
http://www.broadinstitute.org/annotation/genome/multicellularity_project/
MultiHome.html), and the S. rosetta gene models were refined by using
Illumina RNA-seq data. The O. carmela genome was sequenced by using
paired-end Illumina reads at the Vincent J. Coates Genomic Sequencing
Laboratory at the University of California, Berkeley and an early draft was
assembled in-house. To identify new cadherins in these genomes, we
performed protein homology-based searches (i.e., Blast; ref. 27) and domain-based searches (e.g., Pfam; ref. 29 and Smart; ref. 30). Any protein
containing an EC domain was defined as a cadherin, and most of these
also had a transmembrane domain. Cadherin families were identified
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For detailed experimental procedures, see SI Appendix.
ACKNOWLEDGMENTS. We thank M. Abedin, S. Brenner, A. Brooks, M. Eisen,
W. J. Nelson, M. Paris, D. Scannell, B. Steele, S. Q. Schneider, L. Tonkin, and
Q. Zhou for technical support, advice, and helpful discussions. This work was
supported in part by funding from an American Cancer Society Postdoctoral
Fellowship (to S.A.N.), American Cancer Society Research Scholar Grant
116795-RSG-09-044-01-DDC (to N.K.), the National Aeronautics and Space
Administration Astrobiology program (to N.K., S.A.N., and D.J.R.), the Hellman Family Fund (to N.K.), and a National Defense Science and Engineering
Graduate fellowship from the Department of Defense (to D.J.R.). N.K. is
a Fellow in the Integrated Microbial Biodiversity program of the Canadian
Institute for Advanced Research.
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Nichols et al. Supplemental Information
Detailed Experimental Procedures:
O. carmela, Illumina library construction
A paired-end genomic library for Illumina sequencing was constructed using
Oscarella carmela DNA prepared by whole genome amplification (WGA, (1)). To
reduce contamination and polymorphism that could complicate genome
assembly and analysis, a single sponge larva was isolated, washed five times in
sterile-filtered seawater and lysed using the REPLI-g Mini kit for WGA (Qiagen,
Valencia, CA). The lysate was divided and used to conduct four separate WGA
reactions that were pooled to reduce the effects of stochastic amplification bias.
Paired-end library construction was performed using the Illumina PE Adapter
Oligo Mix and PCR primers (Illumina Inc., San Diego, CA) in combination with
protocol modifications suggested by Quail and colleagues (2). Additionally,
during each spin-column purification step, residual ethanol was pipetted out of
the column prior to elution to prevent ethanol carry-over. Library quality was
determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) to
confirm fragment size and concentration.
O. carmela Illumina sequencing and draft genome assembly
A total of 388,627,652 reads were generated from two separate paired-end
Illumina runs on the same library: 39,460,320 reads from 2 lanes of 76 cycle
sequencing (hereafter called “run 1”), and 349,167,332 reads from 7 lanes of 101
cycle sequencing (“run 2”). Before assembly, low frequency “noise” k-mers were
corrected in the reads using the Corrector tool version 1.00 from the Beijing
Genomics Institute [http://soap.genomics.org.cn/down/correction.tar.gz] with
default parameter values. The two lanes from run 1 were corrected together
using a frequency cutoff of 5 per k-mer, and each lane from run 2 was corrected
individually using a frequency cutoff of 10 per k-mer. After correction, 33,249,809
reads from run 1 and 298,166,837 reads from run 2 remained, for a total of
331,416,646 reads. Genome assembly was performed iteratively using
1 Nichols et al. SOAPdenovo version 1.04 (3) with default parameter values (unless otherwise
noted), as follows: an initial assembly was created using a k-mer size of 31, with
both runs used for building contigs and only run 1 used for building scaffolds. To
close gaps in the initial assembly, we ran GapCloser version 1.10 (4) with default
parameter values using only reads from run 1. We found that the processes of
building scaffolds and gap closing were more successful using fewer reads, and
thus we chose run 1 for both tasks; using the reads from any single lane of run 2
produced similar results. After running SOAPdenovo and GapCloser, we mapped
all corrected reads back to the assembly using Bowtie version 0.12.1 (5) with
default parameter values. We then created a final assembly using only the reads
that mapped to the initial gap-closed assembly. We ran SOAPdenovo followed by
GapCloser, repeating the initial assembly process but instead using at each step
the set of reads mapping to the initial assembly. Assembly statistics are shown in
Tables S1-S3.
O. carmela gene prediction
Gene prediction was performed de novo on the final assembly using Augustus
version 2.3 (6) with the autoAug script and the 6,235 assembled Sanger ESTs
(7) as prediction aids. Gene prediction was only performed on sequences with a
minimum length of 500 (9,823 genes were predicted).
O. carmela genome: assembly statistics for scaffolds
Assemblies
Total
Number of Assembly
Scaffolds Size (bp)
Number of
Scaffolds +
Contigs
Longest
(bp)
N50 (bp)
N90 (bp)
Pilot assembly
29,148
57,006,393
70,595
49,630
3,324
416
Initial assembly
22,699
60,727,654
77,270
84,460
4,699
351
Final assembly
17,451
56,386,309
67,767
108,178
5,897
368
2 Nichols et al. O. carmela genome: assembly statistics for contigs
Assemblies
Number of
Reads
Total Assembly
Size (bp)
Longest
(bp)
N50
(bp)
N90
(bp)
Average
Coverage (x)
Pilot assembly
39,460,320
46,779,956
6,153
339
124
22
Initial assembly 331,416,646
54,313,237
28,111
890
132
568
Final assembly 239,209,057
54,193,990
43,946
1,158
142
562
O. carmela genome: scaffold GC content, paired end insert size, and gap
information
Assemblies
Estimated
Total Size
Insert
Number of of Gaps
Size
Number of Total Size of
Gaps
Remaining
GC
Estimated Standard
Gaps
Gaps Before Remaining
After
Content
Insert
Deviation
Before
GapCloser
After
GapCloser
(percent) Size (bp)
(bp)
GapCloser
(bp)
GapCloser
(bp)
Pilot assembly
43.7
390
78
85,376
13,410,978
-
-
Initial assembly
43.5
397
71
55,768
7,991,639
21,580
5,733,376
Final assembly
43.5
395
79
39,994
4,765,458
8,105
2,452,188
Discovery and annotation of novel cadherins The stand-alone BLAST search algorithm was used to search the best predicted
protein set from the draft genomes of S. rosetta, C. owczarzaki, and O. carmela
using the 23 predicted cadherins from the M. brevicollis genome (8) as a query.
As a complement to this approach, Pfam (9), SMART (10) and Phobius (11)
domain prediction programs were run on all predicted S. rosetta proteins. Every
protein predicted to have at least one extracellular cadherin (EC) domain was
annotated and categorized according to whether its overall domain composition
and architecture matched known cadherins from M. brevicollis or any metazoan.
The S. rosetta gene models are supported by 33-fold sequence coverage
suggesting that we have identified most, if not all cadherins in the genome (12).
Accurate abundance data for O. carmela could not be determined due to the
3 Nichols et al. early draft status of the genome. Therefore, cadherin abundance in sponges was
determined from the genome of Amphimedon queenslandica (11). Cadherin
abundance estimates for eumetazoans were derived from Hulpiau and van Roy
(13) and references therein. Taxonomic data from SMART were used to
conclude that no EC domains are present in any annotated plant or fungus.
HMM searches for Hh-N domain-containing proteins
We used the HMMER 3.0 suite of tools (14) to build custom models of the Hh-N
signaling domain in order to increase sensitivity for searches of choanoflagellates
and other opisthokonts. We used hmmsearch (14) with the Pfam domain
Hh_signal [PF01085, Pfam version 24.0 (9)] to detect Hh-N domains in the
predicted protein sets from the genomes of the sponge A. queenslandica (15),
the sea anemone N. vectensis (16), and the choanoflagellates S. rosetta (12) and
M. brevicollis (17). Using the sequences of all domains predicted by hmmsearch
with an E value below the gathering threshold for the model in Pfam, we built a
multiple alignment using the FSA web server version 1.15.2 (18). We used the
resulting alignment to build a custom model with hmmbuild (14), and ran
hmmsearch with the custom model against the predicted protein sets from O.
carmela, S. rosetta and M. brevicollis in order to detect previously unidentified
instances of the Hh-N domain.
Cloning full-length Ocar_bcat
Tissue of O. carmela was flash frozen and ground to a powder using a mortar
and pestle containing liquid nitrogen. Messenger RNA was isolated using Trizol
Reagent (Invitrogen Corp., Carlsbad, CA) followed by the Oligotex mRNA Mini
Kit (Qiagen, Valencia, CA). The unknown 5’ sequence of Ocar_bcat was cloned
and sequenced using GeneRacer (Invitrogen Corp., Carlsbad, CA) in
combination with an antisense primer (SN33R: 5’
CCCAAGGGCAAGTCTTCGCTGGAT 3’) corresponding to the known 3’ EST
sequence (7). The full-length sequence is deposited in GenBank (HQ234356).
4 Nichols et al. Ocar_bcat structural predictions
The full-length sequence of Ocar_bcat was translated from the cloned mRNA
transcript using NCBI ORF Finder. The predicted protein was analyzed for its
homology to known beta-catenin sequences by comparing its primary sequence
to the non-redundant Genbank database (nr) via blastp (19) and by searching for
conserved structural domains (arm repeats) using Pfam (9) and SMART (10).
Each predicted arm repeat in beta-catenin-related proteins from human, O.
carmela, M. brevicollis, S. rosetta, Dictyostelium discoideum and Arabidopsis
thaliana was subjected to pair-wise reciprocal blast (9). For example, arm repeat
1 from Ocar_bcat was used to perform a Blastp (19) search against a database
of all arm repeats from all sampled proteins. We expected that orthologous
sequences from different species would exhibit a co-linear sequence of arm
repeat homology with human beta-catenin [Fig.S5; method modified from (20)].
In the example of O. carmela arm repeat 1, only a best-reciprocal blast with arm
repeat 1 from human beta-catenin would be interpreted support homology of
these two proteins.
To identify conserved functional residues and motifs within Ocar_bcat, multiple
sequence alignment was performed using MUSCLE (21). Additionally, the threedimensional structure of Ocar_bcat was analyzed using alignment-based foldprediction as implemented by LOOPP (22). Predicted structures were visualized
with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre,
Schrödinger, LLC.).
Yeast two-hybrid screen
A yeast two-hybrid screen was conducted to identify candidate binding-partners
of full-length Ocar_bcat. To construct a yeast expression library representative of
the expressed genes of O. carmela, mRNA was isolated from pooled adult and
embryonic tissues (from many individuals to maximize transcript diversity) and
cloned into pDONR222 using the CloneMiner cDNA Library Construction Kit
(Invitrogen Corp., Carlsbad, CA). Inserts from this library were shuttled into the
5 Nichols et al. yeast two-hybrid prey plasmid, pDEST22 using LR Clonase II enzyme mix
(Invitrogen Corp., Carlsbad, CA) and transformed for storage and amplification
into ElectroMAX DH10B T1 Phage Resistant Cells (Invitrogen Corp., Carlsbad,
CA). Likewise, full-length Ocar_bcat was modified using PCR to incorporate
Gateway compatible attB1/attB2 recombination sites and cloned into pDONR221
using BP Clonase II enzyme mix (Invitrogen Corp., Carlsbad, CA). This insert
was shuttled into the yeast two-hybrid bait-plasmid, pDEST32 using LR Clonase
II enzyme mix.
Yeast transformation and screening was performed at the yeast two-hybrid
facility at Indiana University (23). Full-length Ocar_bcat and positive clones were
tested for autoactivation on his- media. E-Amino-1,2,4-Triazol (3AT), which acts
as a quantitative inhibitor of the HIS3 reporter gene, was used to control
autoactivation by Ocar_bcat. After a <10 day screen, positive clones were
retested on his- media, ura- media, and in LacZ assays. Inserts from positive
clones were rescued and sequenced at the University of California DNA
sequencing facility. Insert sequences from positive clones were compared
against the draft assembly of the O. carmela genome using blastn (19) and
predicted proteins were annotated using blastp (19), Pfam (9) and SMART (10)
to test for homology with known proteins.
Seventeen unique candidate binding-partners of Oc_bcat were detected (Table
S2), including three clones encoding the CCD region of OcCdh1 and an
additional well-known beta-catenin binding protein, Axin. These detected
interactions could not be independently validated using in vitro binding assays
because recombinant forms of Ocar_bcat proved to be highly insoluble.
Nevertheless, the conserved structural features of Ocar_bcat and Ocar_Cdh1,
coupled with the fact that this is a widely conserved interaction in metazoans,
suggest that the yeast two-hybrid result represents a bona fide interaction.
6 Nichols et al. Fig. S1
7 Nichols et al. Fig. S1, continued.
B
M. brevicollis
(MBCDH12)
M. brevicollis
(MBCDH1)
M. brevicollis
(MBCDH2)
S. rosetta
(EGD72656)
PKD
S. rosetta
(EGD75710)
S. rosetta
(EGD74518)
C M. brevicollis
(MBCDH10)
S. rosetta
(EGD78831)
S. rosetta
(EGD78839)
D M. brevicollis
(MBCDH9)
M. brevicollis
(MBCDH13)
S. rosetta
(EGD75586)
E
M. brevicollis
(MBCDH7)
PbH1
PbH1
PbH1 PbH1
Candida ALS
PbH1
PbH1
S. rosetta
(EGD81200)
F
KU
M. brevicollis
(MBCDH18)
KU
S. rosetta
(EGD77346)
Fig. S1. Domain architecture of S. rosetta cadherins without orthologs in
Metazoa or C. owczarzaki. (A) 16 out of 29 predicted S. rosetta cadherin
proteins have no clear orthology to any cadherins known from other species,
8 Nichols et al. whereas five protein families (B-F) can be identified as shared between and
exclusive to S. rosetta and M. brevicollis based upon similarities in their domain
composition and arrangement. Of these, one family (E) has partial homology to
the lefftyrin family that is found in choanoflagellates and sponges. However,
genes in this family differ from choanoflagellate lefftyrins in that they are
predicted to have catalytically active cytoplasmic PTPase domains.
(Abbreviations: Candida ALS = Candida Agglutinin-like sequence; CCP = domain
abundant in complement control proteins; FN2 = fibronectin 2; HYR = Hyalin
Repeat; KU = BPTI/Kunitz family of serine protease inhibitors; LamG = laminin G
domain; P protein = Proprotein convertase P-domain; PbH1 = parallel beta-helix
repeats; PKD = polycystic kidney disease; TIG = transcription factor
immunoglobulin-like domain; TSPN = Thrombospondin N-terminal-like domain;
WAP = whey acidic protein; ZnF_c2h2 = zinc-finger, c2h2 type).
9 Nichols et al. Fig. S2.
Fig. S2. Additional detected Hh-N domain containing proteins from S.
rosetta and M. brevicollis. (A) In S. rosetta, two adjacent gene models on a
single scaffold have close homology to parts of M. brevicollis hedgling
(MBCDH11). Both gene models are supported by RNAseq expression data, but
there is a predicted stop codon between them and there are no RNAseq reads
that span the divide. We infer either that the stop codon that splits S. rosetta
hedgling evolved following the divergence of the M. brevicollis and S. rosetta
lineages, or that it is the result of a genome assembly error. Further interpretation
10 Nichols et al. will require experimental investigation of these gene models. (B) Using a custom
HMM created against the Hh-N domain of known hedgling proteins we also
identified five S. rosetta proteins and one M. brevicollis protein that have a
conserved Hh-N domain, but lack EC domains. In each case, as in all known
hedglings, the Hh-N domain is adjacent to a von Willebrand A domain. Therefore,
we hypothesize that the association of these two domains in diverse proteins and
in diverse organisms reflects an ancestral function that has been lost in
eumetazoans.
11 Nichols et al. Fig. S3
Fig. S3. Cohesin domains from Coherin family proteins aligned against the
Cohesin Hidden Markov Model from Pfam. Residues that exactly match Pfam
HMM (highlighted in blue) are indicated with black shading whereas residues that
are considered to be a conservative substitution with respect to what the model
expects are indicated with gray shading. Cohesin domains 1 and 2 from
Monosiga brevicollis (MBCDH8) are identical to each other. Protein identifiers
correspond to Fig. 2c. (Abbreviations: HMM: Hidden Markov Model).
12 Nichols et al. Fig. S4
Fig. S4. Annotated alignment of classical cadherin cytoplasmic tails. The
juxtamembrane domain (purple box) that constitutes the binding site for p120
catenin is partially conserved between human and Drosophila and Amphimedon,
but is divergent in Ocar_Cdh1. In contrast, the beta-catenin binding domain (light
green box) of the predicted CCD (light orange box) of Ocar_Cdh1 is conserved,
including at residues that are required for the interaction (dark green). The
sponge sequences are predicted to be longer than their bilaterian counterparts,
complicating alignment of all but the most highly conserved residues.
13 Nichols et al. Fig. S5.
Fig. S5. Domain organization and phylogenetic distribution of proteins with
homology to beta-catenin.
Protein diagrams are mapped onto a previously determined phylogenetic tree
(24) with arm domains colored to indicate their similarity. Repeats of the same
color are best-reciprocal Blast pairs. Arm repeats without close identity to any
other are uncolored and indicated with an asterisk. Linear conservation of
homologous arm repeats is restricted to metazoan beta-catenin orthologs,
suggesting that the metazoan roles of beta-catenin evolved in the metazoan
stem lineage and have been highly conserved throughout metazoan evolution.
14 Nichols et al. Tables.
Table S1. S. rosetta cadherin expression levels.
Genbank ID
Min FPKM1
Max FPKM
Mean FPKM
Median FPKM
EGD80879
27.617977
113.246096
56.93163613
48.3590645
EGD80917
2.25581
6.049739
3.860855875
3.533781
EGD78831
7.874201
40.03944
19.4444355
15.1492895
EGD78839
0.109114
26.26796
11.104367
9.8600525
EGD79002
1.87756
6.256101
3.839630625
3.370277
EGD79017
29.325694
128.553899
80.03320963
89.619573
EGD82245
3.403667
15.877104
9.775254375
10.434421
EGD82557
0.85664
8.627106
4.377121
3.1091385
EGD72656
168.694501
984.67225
624.6796178
621.626123
EGD73963
2.017099
8.457588
4.626551625
3.828202
EGD74518
46.138224
267.075716
159.4101904
161.7252545
EGD74707
1.962277
15.002993
8.222477875
8.487063
EGD75381
0.133699
51.162787
18.63580838
15.35265
EGD75404
3.990599
9.91731
7.319792125
7.684626
EGD75405
2.37142
9.804725
6.56574025
6.3694265
EGD75586
0.087914
6.21004
2.840604125
2.17229
EGD75074
2.197013
6.533185
4.66290875
4.722256
EGD74783
0.026136
3.799631
1.556376
1.0930275
EGD75710
71.962177
626.409101
259.4577603
220.060925
EGD76846
5.967787
85.11871
33.87954975
17.35544
EGD77346
7.357232
20.994633
12.16801713
10.4218215
EGD78086
0
7.934519
2.76326325
1.801815
EGD78170
18.746381
50.514396
28.3529975
26.7736315
EGD78171
23.099038
61.605291
35.97480775
33.790346
EGD81200
0.053023
20.10651
9.513880375
8.764376
EGD78969
9.214266
59.752132
31.85870863
33.0626255
EGD78970
5.89713
31.968329
15.953967
15.8754105
EGD74667
2.071066
14.728778
7.26199325
6.9755495
EGD75359
0.023944
7.275513
3.04185575
2.4945935
EGD79249
0.020866
3.374047
1.3963085
1.166545
1
The number of fragments per kilobase per million sequenced reads (FPKM)
mapping to each identified S. rosetta cadherin from RNA-seq of eight growth
conditions is summarized as evidence of gene expression.
15 Nichols et al. Table S2. O. carmela binding partners predicted from yeast two-hybrid screen of
beta-catenin.
gene ID
Tentative
Identification
Predicted domain
architecture (Pfam)
Predicted domain
architecture (Smart)
g4908.t1
none
none
none
g9583.t1
none
death
none
CP2
none
Ribosomal S17
none
g6098.t1
g8349.t1
g6246.t1
Tenascin
EGF 2 (x9); EGF Ca
(x2)
VWD; EGF like; EGF
(x10); EGF Ca (x2)
g6719.t1
none
EIF4E-T
coiled coil
g8701.t1
Transcription
factor AP-1/c-Jun
bZIP 1
BRLZ
g2054.t1
Calumenin
SPARC Ca bdg;
efhand (x2)
EFh (x2)
g6285.t1
E74-like factor
Ets
ETS
g10012.t1
Chromosomal
segregation
protein SMC
none
coiled-coil
g4744.t1
GTPase Rab2
Ras
RAB
BIR (x4)
BIR (x4); RING
Ribosomal L13e
none
g8915.t1
g6056.t1
Upstream binding
protein
40S ribosomal
protein S11
Baculoviral IAP
repeat-containing
protein 4
Ribosomal protein
L13
g2979.t1
Ral
Ras
RAS
g3724.t1
Choline-phosphate
cytidylyltransferase
none
coiled-coil
g6554.t1
Axin
RGS; DIX
RGS; DAX
AEC12441
Ocar_Cdh1
EC; EGF; Lam-G; CCD
EC; EGF; Lam-G
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