1
Symbiosis-driven development in
an early branching metazoan
Authors:
Aki H. Ohdera
1,13*
, Justin Darymple
2
, Viridiana Avila-Magaña
1,14
, Victoria Sharp
1
,
Kelly Watson
1
,
Mark McCauley
3
, Bailey Steinworth
4
, Erika M. Diaz-Almeyda
5
, Sheila A.
Kitchen
6
, Angela Z. Poole
7
, Anthony Bellantuono
2
, Sajeet Haridas
8
, Igor V. Grigoriev
8,9
, Lea
Goentoro
6
, Elizabeth Vallen
10
, David M. Baker
11
, Todd C. LaJeunesse
1
, Sandra Loesgen
3
, Mark
Q. Martindale
4
, Matthew DeGennaro
2
, William K. Fitt
12
, Mónica Medina
1*
Affiliations:
1
Department of Biology, Pennsylvania State University, University Park, PA, 16802
2
Department of Biological Sciences & Biomolecular Sciences Institute, Florida International
University, Miami, FL 33199
3
Whitney Laboratory for Marine Bioscience, Department of Chemistry, University of Florida,
St. Augustine, FL, 32080
4
Whitney Laboratory for Marine Bioscience, Department of Biology, University of Florida, St.
Augustine, FL, 32080
5
Department of Biology and Environmental Studies, New College of Florida, Sarasota, FL,
34243
6
Division of Biology and Biological Engineering, California Ins
titute of Technology, Pasadena,
CA, 91125
7
Department of Biology, Berry College, Mount Berry, GA, 30149
8
U.S. Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720
9
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720
10
Biology Department, Swarthmore College, Swarthmore, PA, 19081
11
School of Biological Science, The University of Hong Kong, Hong Kong, PRC
12
Odum School of Ecology, University of Georgia, Athens, GA, 30602
13
National Museum of Natural History, Smithsonian Institute, Washington, D.C., 20560
14
Ecology and Evolutionary Biology Department, University of Colorado Boulder, CO, 80309
*Correspondence to: momedinamunoz@gmail.com
Abstract:
Microbes can initiate developmental gene regulatory cascades in animals. The
molecular mechanisms underlying microbe-induced animal development and the evolutionary
steps to integrate microbial signals into regulatory programs remain poorly understood. In the
upside-down jellyfish
Cassiopea xamachana
, a dinoflagellate endosymbiont initiates the life
stage transition from the sessile polyp to the sexual medusa. We found that metabolic products
derived from symbiont carotenoids may be important to initiate
C. xamachana
development, in
addition to expression of conserved
genes involved in medusa development of non-symbiotic
.
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jellyfish. We also revealed the transcription factor COUP is expressed during metamorphosis,
potentially as a co-regulator of nuclear receptor RXR.
These data suggest relatively few steps
may be necessary to integrate symbiont signals into gene regulatory networks and cements the
role of the symbiont as a key trigger for life history transition in
C. xamachana
.
Main Text:
The importance of microbes as regulators of animal physiology and life history is
increasingly evident, from drivers of evolutionary adaptations with implications beyond
individual organisms to supporting ecosystems,
e.g.,
coral reefs (
1-4
). In particular, induction of
developmental processes
such as metamorphosis and organogenesis by microbes is common
across metazoans, and may have facilitated the evolution of multicellularity in animals (
5, 6
).
Despite the importance of microbes in shaping animal development and evolution, the molecular
and evolutionary steps leading to the coupling of microbial signaling with animal developmental
pathways remain largely unknown.
We used an emerging model system, the upside-down jellyfish
Cassiopea xamachana
(Cnidaria:
Scyphozoa), to investigate how a photosynthetic dinoflagellate endosymbiont is integrated as a
developmental cue to initiate metamorphosis (Fig. 1A). In non-symbiotic jellyfish,
metamorphosis is triggered by environmental factors such as temperature (
7
) (Fig. 1B). For
C.
xamachana
, the metamorphic transition in jellyfish known as strobilation begins approximately
10 to 17 days after acquisition of its endosymbiont
Symbiodinium microadriaticum
by the sessile
polyp (scyphistoma) stage
(Dinoflagellata: Symbiodiniaceae) (
8
). If symbionts are not acquired
from the environment,
C. xamachana
will remain in the asexual scyphistoma stage indefinitely.
We performed a differential mRNA expression analysis across time at 0, 3, and 8 days post-
colonization (d.p.c) and mid-strobilation (~ 17 d.p.c) of
C. xamachana
with ImpulseDE (
9
). We
aligned the RNAseq reads to our new chromosome level
C. xamachana
genome assembly (NCBI
accession OLMO00000000) composed of 20 pseudo-chromosomes (N
50
=17.9 Mb) that captured
99% of the original assembly (Fig. 1C), and contained 29,645 predicted protein coding genes
(annotated genome available from
https://phycocosm.jgi.doe.gov/Casxa1
). We identified 5,414
genes (p-adjusted < 0.05) exhibiting an impulse-like (time-associated) expression pattern that
grouped into 11 clusters (Fig. 1C). In clusters where expression increased with strobilation,
genes involved in developmental regulation were enriched. Wnt, Ras, and cAMP signaling
pathways were enriched in cluster 7, and Notch signaling and cell cycle pathways were enriched
in cluster 9 (Fig. 1D,E; fig. S1, Supplementary File 1). Multiple pathways associated with
animal-microbe interactions were also found in cluster 1 (626 DEGs, Fig. 1E, fig. S1),
characterized by genes that remained stable after a downregulation at 3 d.p.c, and likely
reflecting a transcriptomic response coinciding with the immune suppression characteristic of
symbiosis maintenance in cnidarians (
10
).
We took advantage of the characterized molecular components of strobilation in non-symbiotic
jellyfishes
Aurelia aurita
and
Rhopilema esculentum
to investigate their contribution to
metamorphosis in
C. xamachana
. In non-symbiotic jellyfish,
strobilation is regulated by an
ortholog of
RXR
(
11, 12
), a nuclear receptor with a conserved function in metamorphosis across
diverse metazoan lineages including vertebrates (
11-15
). In
A. aurita
and
R. esculentum,
expression of
RXR
is steadily up-regulated up to and through strobilation. Additionally,
retinol
.
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dehydrogenase
(
RDH
), a gene involved in the biosynthesis of the RXR ligand 9-cis retinoic acid
(RA), also regulates metamorphosis in these non-symbiotic jellyfish (Fig. S2) (
11, 16
). In
C.
xamachana
, we found
CxRXR
expression to be relatively stable across all time-points (Fig. 2A)
and orthologs of
RDH1
and
RDH2
were either down-regulated 2-fold (
CxRADHa
, Fig. 3A) or
did not show an impulse-like behavior (
CxRADHb
) compared to aposymbiotic scyphistomae. An
additional gene,
CxCL112
, was recovered from a homology search of three hormone-like genes
linked to strobilation in
A. aurita
(
CL112, CL390, CL631
; fig. S4) (
11
). In contrast to the gradual
upregulation of
CL112
in
A. aurita
,
CxCL112
was down-regulated in the strobilating
scyphistoma (strobila) stage relative to the aposymbiotic scyphistoma in
C. xamachana
(Fig. 2A,
p-adjusted < 0.05). The only gene in which
C. xamachana
expression was similar to
A. aurita
during strobilation was of
DNA methyltransferase 1
(
DNMT1
; ~ 1 log
2
FC, p-adj = 0.009; Fig.
S3), consistent with a conserved role of this gene in development of animals (
17
). Thus, an initial
interrogation of gene expression in
C. xamachana
during strobilation appears to show
transcriptomic divergence between symbiotic and non-symbiotic species.
When
CxRXR
and
CxCL112
mRNA were visualized by
in situ
hybridization, both genes were
expressed orally in the aposymbiotic scyphistoma and mid-strobila stages, marking the region of
the scyphistoma body that would undergo metamorphosis (Fig. 2B, fig. S5A,B). A decrease in
expression of both genes coincided with progression of strobilation (late strobila), whereby
mRNA became restricted to the retracted tentacles and the central oral appendage. Surprisingly,
the diminished expression of
CxRXR
and
CxCL112
coinciding with completion of strobilation
resembles the spatial pattern observed in
A. aurita
(fig. S5C). The expression data suggest much
of the regulatory machinery identified in non-symbiotic jellyfish remains intact and may be
important for
C. xamachana
development, albeit with several modifications.
We therefore tested whether treatment of aposymbiotic
C. xamachana
scyphistomae with
exogenous chemicals that induce strobilation of non-symbiotic jellyfish (9-cis RA, 5-methoxy-2-
methyl indole) would be as efficient in triggering metamorphosis compared to acquisition of
symbionts (
16
). The potent strobilation inducer 5-methoxy-2-methylindole, a molecule
containing a predicted pharmacophore of the
A. aurita
gene
CL390
, induced strobilation in
C.
xamachana
at similar rates to
A. aurita
(91.7 %, one-way ANOVA with
post-hoc
Tukey test, p-
value < 0.0005). While the target of the chemical inducer is not proposed hormone gene
CL390
,
both
A. aurita
and
C. xamachana
share an inducible strobilation regulator. Interestingly, 9-cis
RA at saturation (1 uM) failed to significantly induce strobilation relative to the vehicle control
(6.25 %, p-value > 0.05, Fig. 2C). We thus hypothesized that strobilation in
C. xamachana
may
require additional regulators and activation of RXR by 9-cis RA is insufficient to induce
complete strobilation.
To identify genes correlated with strobilation and symbiosis establishment, we next performed
weighted gene correlation network analysis (WGCNA) (
18
). We identified three modules (2,846
genes, Modules 1, 2, and 14) that were significantly correlated with symbiosis establishment (3
and 8 d.p.c), and four modules (3,714 genes, Modules 9,10,11, and 12) associated with
strobilation (p-value < 0.05) (fig. S6A, Supplementary File 2). Genes in the strobilation modules
were enriched in pathways responsible for energy modulation and metabolism (mTOR signaling,
insulin signaling, glucagon signaling, oxidative phosphorylation) coinciding with developmental
processes, as well as cell proliferation and morphogenesis (cell cycle, DNA replication, TGF-
β
signaling) (fig. S6B).
.
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We investigated whether genes in the strobilation module were associated with the conserved
RXR
response element (AGGTCA). If
RXR
regulates strobilation in
C. xamachana
, we expect
the response element to be enriched with genes linked to strobilation. We scanned the 5,000 bp
region flanking each gene for RXR half-sites separated by 1 to 5 nucleotides and found 1,676
genes of the strobilation module were proximal to a putative
RXR
response element
(Supplementary File 3). Of these, we found 83 genes with orthologs in related jellyfish species
with conserved proximity to an
RXR
response element (Supplementary File 4). Moreover, the
strobilation module M10 was enriched with genes associated with an
RXR
response element (Fig.
3A, p-value < 5.70e
-6
Chi-squared test). We next compared module membership (kME), high
values akin to high inter-connectedness within the network, to fold-change. It revealed several
“hub” genes including two
hox
genes and a chicken ovalbumin upstream promoter transcription
factor
(
COUP-TF,
nuclear receptor subfamily II) among those with high kME and expression
(Fig. 3B, fig. S6A). Developmental genes including
Doublesex and Mab3-related
(
DMRT
),
which is associated with strobilation in non-symbiotic jellyfish, were also among those with high
kME values (
11
). Genomic scans for additional motifs that were significantly enriched (p-value
< 1e
-10
) within the strobilation modules included binding targets of homeobox genes (Fig. 3C).
Although with marginally lower significance (p-value < 1e
-5
),
COUP
motifs were also identified
to be enriched (Supplementary File
5
), making them likely candidates to regulate strobilation in
C. xamachana
.
In further exploring
COUP-TF
we identified
CxCOUP-TFa
, which exhibited a stepwise pattern
of expression that increased during days 3 and 8 d.p.c., and
CxCOUP-TFb
, which showed
gradual up-regulation over time (fig. S7). A third closely related nuclear receptor (
CxCOUP-
like)
showed a rapid increase in expression during strobilation, similar to patterns observed for other
important players in strobilation (Fig. 3D). Members of the
COUP-TF
s exhibit dual roles as both
initiators and repressors of transcription, with potential regulatory roles in embryogenesis
through heterodimerization with the
RXR
gene (
19, 20
). Although it remains unknown whether
CxRXR
forms a heterodimer with other nuclear receptors in
C. xamachana
, the low induction of
strobilation under 9-cis RA treatment suggests
CxRXR
activation of strobilation requires a co-
regulator (
21
). To test the participation of a
COUP-TF
in
C. xamachana
strobilation, we treated
scyphistomae harboring
S. microadriaticum
with a chemical inhibitor of COUP transcription
factors,
4-methoxy-1-napthol
(MNol). We found strobilation to be significantly delayed (Fig
2D), thus implicating
COUP-TF
as a key regulator of strobilation.
As carotenoids are common ligands of nuclear receptors like RXR we further investigated our
datasets for genes involved in carotenoid metabolism. We found a
β
-carotene oxygenase
(CxBCOa
)
up-regulated approximately four-fold during strobilation (p-value = 6.27e
-5
, Fig. 4A,
fig. S6). BCO genes
perform symmetric or asymmetric cleavage of
β
-carotene.
β
-carotene
monooxygenase (BCMO) produces retinal, while
β
-carotene dioxygenase (BCDO2) generates
β
-
ionone and
β
-apo-carotenal. These molecules can be further processed by a dehydrogenase to
produce potential nuclear receptor ligands,
i.e.
retinoic acid (
22, 23
) (Fig. 4B). Two BCO-like
genes (
CxBCOLb, CxBCOLf
) were also found in the WGCNA symbiosis establishment modules
(M1, M14) (Fig. 4A, fig. S8). While the exact type of reaction performed by
CxBCOa
requires
further characterization, key residues responsible for catalytic activity were found in all genes
(fig. S9), suggesting cleavage activity is likely analogous with BCO orthologs in mammals. In
mammals, BCDO2 is thought to be predominantly active in the mitochondria where it functions
against oxidative damage caused by carotenoid accumulation, whereas BCMO is restricted to the
cytosol (
24, 25
). None of the
CxBCO
genes possessed a mitochondrial transport signal within the
.
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N-terminus. However, signaling peptide sequences were present in
CxBCOL
genes, suggestive of
their post-secretion enzymatic activity within the extracellular matrix (fig. S10) (
26
). Treatment
of symbiotic scyphistomae with the RDH inhibitor 4-diethylaminobenzaldehyde (DEAB) led to
significant delays to strobilation (Mantel-Cox test, p-value < 0.005) (Fig 2D). Treatment with a
single concentration of butylated hydroxytoluene (BHT), a BCO inhibitor, did not alter rates of
strobilation, but further experiments with additional concentrations may be useful (fig. S11).
These results suggest carotenoids are important for strobilation in
C. xamachana
, and potentially
sourced from the symbionts.
Symbiodinium
synthesizes multiple carotenoid products that can be potential substrates for
further processing (
27
). Genes required for carotenoid synthesis were confirmed to be expressed
by
S. microadriaticum in hospite,
including those responsible for production of
β
-carotene,
lycopene, peridinin, zeaxanthin, and dinoflagellate specific carotenoids (dinoxanthin,
diadinoxanthin) (Fig. 4B, fig. S12A) (
28
).
In silico
predictions of binding affinities for putative
carotenoid ligands suggests cleavage products other than 9-
cis
retinoic acid have similar binding
kinetics with CxRXR, including zeaxanthenoic acid, a metabolic equivalent of RA derived from
zeaxanthin (Fig. 4C, fig. S12B).
Taken together, our data suggest that the regulation of development in a symbiotic jellyfish
remains largely similar to non-symbiotic species with some notable exceptions.
We hypothesize
that after colonization of
C. xamachana
scyphistomae by
S. microadriaticum
, accumulated
carotenoids are processed by CxBCO, leading to the production of nuclear receptor ligands.
Subsequently, nuclear receptors act as a sensor by binding these carotenoid cleavage products to
trigger a downstream signaling cascade leading to the initiation of strobilation
(Fig. 4E). With
the exception of diadinoxanthin and peridinin, which generally comprise over 80% of the
produced carotenoids in
Symbiodinium
, minor pigments make up less than 5% of the total (
29
).
Consequently, the observed lag period to the start of strobilation following initial acquisition of
symbionts may reflect the time necessary to accumulate the minimum concentration of a minor
carotenoid (e.g.,
β
-carotene, zeaxanthin) required to activate RXR and irreversibly initiate
development. The difference in expression of key genes responsible for RA metabolism between
C. xamachana
and non-symbiotic jellyfish, including the constitutive expression of
RXR
, may
also accelerate the time to strobilation by priming the animal for the transition. We suggest the
integration of
Symbiodinium
as driver of development in
C. xamachana
likely resulted from a
compatibility of precursor molecules of symbiont origin with the RXR developmental signaling
pathway, accompanied by the modulation in expression of key genes. Although specific
pathways may vary, symbiont-driven development may evolve through relatively few changes to
existing programs.
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Acknowledgement
: The work conducted by the U.S. Department of Energy Joint Genome
Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the
U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
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Fig. 1.
Symbiont induced metamorphosis coincides with activation of canonical
developmental genes.
(A) Lifecycle of
C. xamachana
displaying in clockwise order a free-
swimming ephyra, aposymbiotic scyphistomae, symbiotic scyphistomae mid-strobilation
(strobila), symbiotic scyphistomae during late strobilation.
(B) Phylogenetic relationship of
scyphozoan (colored box) jellyfish species relative to other cnidarian lineages. Species that
metamorphosis under environmental factors are indicated in blue, while metamorphosis in
C.
xamachana
(orange) is regulated by its symbiont.
(C)
HiC plot of the
C. xamachana
genome
v2.0. Color bar indicates frequency of contact at each coordinate within the scaffolds. (D)
Differentially expressed genes identified with ImpulseDE2 were clustered (k-means, k=11)
according to co-expression patterns across the four experimental time points. Cluster centroids
are plotted with standardized expression (E) KEGG pathways enriched in cluster 9 of k-means
clustered genes exhibiting impulse-like expression. (F) KEGG pathways enriched in cluster 1 of
k-means clustered genes exhibiting impulse-like expression.
Fig. 2.
Regulation of retinoic acid pathway associated genes during strobilation in
Cassiopea xamachana
. A) Expression of genes implicated in non-symbiotic jellyfish strobilation
across all stages (0=aposymbiotic
scyphistomae
, 3 and 8 days post-colonization, S=strobila).
Values are log
2
transformed DESeq2 normalized read counts. Colors indicate experiment 1
(orange) and experiment 2 (grey). P-adjusted values from the ImpulseDE analysis are indicated
in black. P-adjusted values generated using DESeq2 are in orange (Exp. 1) and grey (Exp. 2). (B)
Whole mount
in situ
hybridization of
CxRXR
and
CxCL112
in aposymbiotic (0 days) and
strobila. Late-strobila were collected near completion of strobilation. (C) Strobilation rates of
C.
xamachana
scyphistomae after
17 days post-treatment / colonization. ***p-value < 0.005
ANOVA with
post-hoc
Tukey test. (E) Strobilation rates of scyphistomae treated with an
aldehyde dehydrogenase inhibitor (DEAB) and a COUP inhibitor (MNol) in symbiotic
scyphistomae (n=40 / treatment group). DEAB (p-value < 0.0005, Mantel-Cox test) treated and
MNol (p-value < 0.005, Mantel-Cox test) treated animals strobilated significantly slower than
controls.
Fig. 3. Developmental transcription factors are correlated with strobilation in
Cassiopea
xamachana
.
(A) Percentage of genes with an RXR response element (RE) within 5,000 bp of the
gene. RXR-RE associated genes with orthologs in
A. aurita
,
N. nomurai
, and
R. esculentum
are
shown in black. Non-conserved genes are shown in orange and genes lacking a proximal RXR-
RE are shown in grey. Module 10 had a statistically significant number of genes compared to
other modules (p-value = 5.70e
-6
chi-squared test). (B) Module membership (kME) plotted
against log
2
fold-change. Dotted lines indicate (kME) cutoff of 0.9, and log
2
fold-change cutoff
(5, -3.5). (C) Position weight matrix logos for the top 10 most significantly enriched eukaryote
motifs (p-value cutoff < 1 x 10
-10
) associated with genes in WGCNA modules correlating with
strobilation (D) ImpulseDE2 normalized read counts of
Hox1
and
COUP-TF
c across the four
time points, which showed a statistically significant impulse pattern during a time point.
Fig. 4. Potential RXR ligand interactions underlying regulation of symbiosis driven
strobilation.
(A) DESeq2 normalized read counts of
C. xamachana
β
-carotene oxygenases
found to be correlated with establishment / strobilation over the four time points. (B) Carotenoids
.
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9
produced by the dinoflagellate symbiont
Symbiodinium microadriaticum
. Retinol, retinal, and
rosafluene are cleavage products of
β
-carotene. (C) Binding probability of potential ligands
against their dissociation constant modeled with BindScope and Kdeep. 3HI = 3 hydroxy-beta-
ionone. 11HRL = 11-cis-3-hydroxyretinal. ATR = all-trans retinoic acid. AZN = apo-9
zeaxanthenone. BAC = beta-10-apocarotenal. BAN = 13-apo-beta-apocarotenone. HRA=9-cis
4hydroxyretinoic acid. LYC=lycopene. ZXL=all-trans zeaxanthenal. (D) Proposed model of
C.
xamachana
strobilation. Carotenoids produced by
Symbiodinium
are cleaved by
CxBCO
. Along
with a secondary nuclear receptor (
e.g.
,
COUP-TF
),
CxRXR
binds to response elements proximal
to developmental genes (e.g.,
Hox
,
DMRT
) to initiate the downstream developmental cascade
leading to strobilation.
.
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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;
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10
20
30
40
0.01
0.02
0.03
0.04
size
p.adjust
3
6
9
12
0.01
0.02
0.03
0.04
Nemopilema
nomurai
Cassiopea
xamachana
Cubozoa
Staurozoa
Anthozoa
Rhopilema
esculentum
A
−
1.5
−
1.0
−
0.5
0.0
0.5
1.0
1.5
6
Glycosaminoglycan
biosynthesis - haparan sulfate /
heparin
Protein processing in
endoplasmic reticulum
Antifolate resistance
Insulin resistance
Longevity regulating
pathway - worm
Autophagy
- animal
Shigellosis
Necroptosis
Endocytosis
RNA transport
Ribosome
Scy 3 dpc
8 dpc
Strob
Scy 3 dpc
8 dpc
Strob
Scy 3 dpc
8 dpc
Strob
Expression
Expression
Expression
Scy 3 dpc
8 dpc
Strob
1
2
3
−
1.5
−
1.0
−
0.5
0.0
0.5
1.0
1.5
4
5
7
8
9
10
11
D
E
F
C
B
1.
2.
3.
4
.
Scaffold Number
735
366,849,413
Total Size (bp)
N50 (bp)
17,884,241
L50 (scaffolds)
8
# of predicted genes
29,645
BUSCO
Complete
85.6% (817/954)
BUSCO
Complete+Fragmented
93.9% (896/954)
Uniquitin mediated
proteolysis
Oocyte meiosis
Cell cycle -
yeast
Progesterone-mediate
oocyte maturation
Hepatocellular
carcinoma
Human T-cell
leukemia virus
1 infection
Breast cancer
Wnt signaling
pathway
Notch signaling
pathway
size
p.adjust
mRNA
surveillance
pathway
RNA transport
TGF-
β
signaling
pathway
Cell cycle
−
1.5
−
1.0
−
0.5
0.0
0.5
1.0
1.5
Salmonella
infection
Pathogenic
Escherichia coli
infection
Fc gamma R-mediated
phagocytosis
Human immunodeficiency
virus 1 infection
Alzheimer disease
Legionellosis
Hydrozoa
Aurelia
aurita
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 22, 2022.
;
https://doi.org/10.1101/2022.07.21.500558
doi:
bioRxiv preprint