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. 2021 Jan 27;13(3):evab008. doi: 10.1093/gbe/evab008

Transcription Profiling of Cultured Acropora digitifera Adult Cells Reveals the Existence of Ancestral Genome Regulatory Modules Underlying Pluripotency and Cell Differentiation in Cnidaria

Alejandro Reyes-Bermudez 1,✉, Michio Hidaka 2, Alexander Mikheyev 3,4
Editor: Maria Costantini
PMCID: PMC7936024  PMID: 33501945

Abstract

Due to their pluripotent nature and unlimited cell renewal, stem cells have been proposed as an ideal material for establishing long-term cnidarian cell cultures. However, the lack of unifying principles associated with “stemness” across the phylum complicates stem cells’ identification and isolation. Here, we for the first time report gene expression profiles for cultured coral cells, focusing on regulatory gene networks underlying pluripotency and differentiation. Cultures were initiated from Acropora digitifera tip fragments, the fastest growing tissue in Acropora. Overall, in vitro transcription resembled early larvae, overexpressing orthologs of premetazoan and Hydra stem cell markers, and transcripts with roles in cell division, migration, and differentiation. Our results suggest the presence of pluripotent cell types in cultures and indicate the existence of ancestral genome regulatory modules underlying pluripotency and cell differentiation in cnidaria. Cultured cells appear to be synthesizing protein, differentiating, and proliferating.

Keywords: coral cell culture, RNA-seq, coral stem cells, transcription regulation, cell differentiation

Significance

The inability to establish permanent cnidarian cell lines has directed attention to tissues with a high abundance of stem cells as an ideal material for establishing long-term cultures. Despite this, the lack of unifying principles associated with “stemness” across cnidaria complicates stem cells’ identification and isolation. Here, we report gene expression profiles for cultured coral cells. Our results revealed pluripotent cell types in cultures, identifying coral orthologs of stem cell markers that could be used for further isolation and characterization.

Introduction

Cell–cell interactions are fundamental for body plan establishment and function as they integrate cell type–specific genome regulation during animal development (Peter and Davidson 2011). Coordination and assembly of cell-specific transcription profiles generate interconnected transcriptional networks that in cnidaria generate organismal complexity by deploying morphogenetic borders along an oral–aboral axis (Hayward et al. 2015). In cnidaria, tissue morphogenesis and homeostasis mechanisms are very diverse. The phylum exhibits complex life cycles and diverse developmental mechanisms, spanning planula (P), polyp, and medusa morphologies (Daly et al. 2007). Anthozoans, in particular, display complex polyp morphologies and less regenerative potential, requiring signaling from an organizer region to reconstruct their tissues (Hayward et al. 2015).

At the cellular level, although hydrozoans maintain body structure via the integration of three distinct stem cell lineages established during gastrulation—two epithelial and one interstitial (Hemmrich et al. 2012)—nonhydrozoans cnidarians present two epithelial stem cell lineages lacking the interstitial cell type (i-cells) (review in Gold and Jacobs 2013). Despite this, it is accepted that transdifferentiation of epithelial cells and dedifferentiation of “committed” cell types predates the evolution of cnidarian stem cell systems (Gold and Jacobs 2013). In animals, precursor cells in vivo maintain “stemness” and control differentiation via complex cell–cell interactions within a tissue grade microenvironment known as “stem-cell niche” (Fuchs et al. 2004; Gattazzo et al. 2014). Within the niche, differential deployment of core networks regulates cellular identity by promoting the expression of “pluripotency” genes while repressing developmental signaling pathways (Orkin and Hochedlinger 2011).

In metazoa, stem cells have been reported as early as porifera, with archeocytes and choanocytes identified as the oldest animal stem cell system (Funayama 2010). In cnidaria, undifferentiated stem cells have been reported in a variety of adult (Mydlarz et al. 2008) and larval (Martin and Chia 1982) tissues, with transcriptional profiles of precursor cell types characterized for Hydra (Siebert et al. 2019) and Nematostella (Sebé-Pedrós et al. 2018). Due to the high abundance of stem cells in adult stages, adult tissues have been suggested as an ideal material for the establishment of long-term cnidarian cultures (Rinkevich 2011). However, the lack of unifying principles associated with stemness across the phylum complicates identification and isolation of cnidarian stem cells for culture.

In cnidaria, attempts to establish permanent cnidarian cell lines have failed due to decreasing in vitro viability and proliferation (review in Rinkevich 2011). Despite this, short-term primary cultures have been used to understand the cellular mechanisms underlying fundamental cnidarian processes, such as symbiosis (Barnay-Verdier et al. 2013), calcification (Domart-Coulon et al. 2001; Mass et al. 2012), thermal stress (Nesa and Hidaka 2009), and regeneration (Schmid and Alder 1984). These studies reported inconsistent results regarding survival, proliferation, and viability, reflecting the lack of standardized protocols (reviewed by Rinkevich 2005, 2011). Nonetheless, as primary cell cultures are taken directly from in vivo tissues, they represent a powerful tool to study cellular and physiological processes unattainable using whole organisms.

This paper, reports for the first time gene expression profiles for cultured coral cells, focusing on regulatory gene networks underlying pluripotency and differentiation. We initiated primary cell cultures from Acropora digitifera tip fragments, the fastest growing tissue in Acropora corals (Lirman et al. 2014). A. digitifera has become an emerging model to study coral responses to environmental change (Shinzato et al. 2011) and due to its basal phylogenetic position in cnidaria (Bridge et al. 1992), the species is also an ideal model to study the evolution of animal developmental mechanisms.

Overall, after 4 weeks of culture, we conducted quantitative RNA-seq analysis and compared the in vitro transcriptome with a previously published A. digitifera developmental time series (Reyes-Bermudez et al. 2016). In vitro transcription closely resembled gene expression used in early larvae during the establishment of larval cellular phenotypes. Likewise, we identified upregulation in vitro of orthologs of premetazoan and Hydra stem cell markers (HM) and transcripts with roles in DNA replication, cell division, migration, and differentiation. Our results suggest the existence of 1) pluripotent cell types in cultures and 2) ancestral genome regulatory modules underlying pluripotency and cell differentiation in cnidaria.

Results and Discussion

Cultures Consist of Multicellular Aggregates Actively Dividing and Differentiating

After 4 weeks, cultures consisted of cellular aggregates formed by cells displaying a previously reported unique and nonspecific small round morphology (Reyes-Bermudez and Miller 2009). Cells did not attach to the substrate, and signs of CaCO3 precipitation were not observed. Contrasting with previous results (Lecointe et al. 2013), cultures did not show evidence of decreased proliferation. The fact that one round of subculturing was necessary for 2 weeks after initiation, suggests that cells were actively dividing. Although zooxanthellae were present abundantly at initiation, after 4 weeks, chlorophyll fluorescence was absent. Our results support the idea that the capacity to maintain or reconstruct cell signaling between epitheliums in vitro is critical for the establishment of “long-term” successful primary cultures (reviewed by Rinkevich 2005, 2011).

Moreover, upregulation in vitro of genes with roles in protein synthesis, proliferation, and differentiation indicate that, at least after 4 weeks, a subset of cells was proliferating and differentiating in cultures (table 1). The fact that cultures were initiated from branch tips, the fastest growing tissue in Acropora corals (Lirman et al. 2014), suggest that undifferentiated cell populations in founder tissues might be responsible for in vitro enrichment of corals cells that is a crucial factor for culture viability (reviewed by Rinkevich 2005, 2011). Reduced proliferation in primary cnidarian cultures has been linked to a decrease in the proportion of animal cells as result of culture contamination (Frank et al. 1994).

Table 1.

Gene Ontology (GO) Enrichment Summary in DEGs Only Upregulated in Cells

GO ID Node Size Sample Match P Adj Term Ontology
GO:0000302 990 421 2.02E−09 Response to reactive oxygen species BP
GO:0006415 152 92 2.20E−09 Translational termination BP
GO:0006414 369 173 6.60E−06 Translational elongation BP
GO:0042755 258 125 0.000134999 Eating behavior BP
GO:0043462 293 131 0.018485676 Regulation of ATPase activity BP
GO:0000302 990 421 2.02E−09 Response to reactive oxygen species BP
GO:0006415 152 92 2.20E−09 Translational termination BP
GO:0006414 369 173 6.60E−06 Translational elongation BP
GO:0042755 258 125 0.000134999 Eating behavior BP
GO:0043462 293 131 0.018485676 Regulation of ATPase activity BP
GO:0009725 2914 1,050 0.000676117 Response to hormone stimulus BP
GO:0030865 350 181 4.75E−11 Cortical cytoskeleton organization BP
GO:0003012 826 364 2.70E−10 Muscle system process BP
GO:0050905 485 202 0.020936066 Neuromuscular process BP
GO:0002026 161 86 8.57E−05 Regulation of the force of heart contraction BP
GO:0060327 118 69 1.68E−05 Cytoplasmic actin-based contraction involved in cell motility BP
GO:0002027 337 158 3.54E−05 Regulation of heart rate BP
GO:0000916 184 95 0.000144077 Actomyosin contractile ring contraction BP
GO:0007109 148 82 1.95E−05 Cytokinesis, completion of separation BP
GO:0032060 158 81 0.002337013 Bleb assembly BP
GO:0048739 153 80 0.000935569 Cardiac muscle fiber development BP
GO:0035277 134 72 0.000968262 Spiracle morphogenesis, open tracheal system BP
GO:0007427 218 105 0.00265387 Epithelial cell migration, open tracheal system BP
GO:0046664 128 70 0.000572738 Dorsal closure, amnioserosa morphology change BP
GO:0007395 123 69 0.000180261 Dorsal closure, spreading of leading edge cells BP
GO:0007496 115 66 0.000106495 Anterior midgut development BP
GO:0021549 341 169 4.16E−08 Cerebellum development BP
GO:0030224 166 95 9.18E−08 Monocyte differentiation BP
GO:0002119 798 341 2.08E−07 Nematode larval development BP
GO:0030220 127 72 4.86E−05 Platelet formation BP
GO:0007527 125 71 5.55E−05 Adult somatic muscle development BP
GO:0045200 141 77 0.000140176 Establishment of neuroblast polarity BP
GO:0048147 35 25 0.014716604 Negative regulation of fibroblast proliferation BP
GO:0001767 116 66 0.000170584 Establishment of lymphocyte polarity BP
GO:0006930 130 74 2.35E−05 Substrate-dependent cell migration, cell extension BP
GO:0033057 527 224 0.000940241 Multicellular organismal reproductive behavior BP
GO:0051015 403 203 1.23E−11 Actin filament binding MF
GO:0008307 210 109 2.95E−06 Structural constituent of muscle MF
GO:0019829 394 179 2.23E−05 Cation-transporting ATPase activity MF
GO:0031762 119 68 2.73E−05 Follicle-stimulating hormone receptor binding MF
GO:0008013 155 80 0.000637174 Beta–catenin binding MF
GO:0043531 316 143 0.000915446 ADP binding MF
GO:0005167 47 30 0.017776487 Neurotrophin TRK receptor binding MF
GO:0005516 616 286 2.94E−11 Calmodulin binding MF
GO:0051020 651 279 3.30E−06 GTPase binding MF
GO:0005083 586 245 0.000425907 Small GTPase regulator activity MF
GO:0019901 1,428 583 1.59E−10 Protein kinase binding MF
GO:0004725 363 157 0.007252685 Protein tyrosine phosphatase activity MF
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Note.—Node size = total number of GO terms in node. Sample match = number of transcripts with GO terms associated to specific nodes.

Cultured Cells Express More Genes in Common with Early Larvae Than with Adult, P, or Embryonic Stages

In vitro transcription profile resulted in 14,286 transcripts compared with 11,926 observed in adult tissue (fig. 1A). Transcriptome comparison between cultured cells (C) and data on previously published A. digitifera stages (Reyes-Bermudez et al. 2016), showed that in vitro gene expression is closer to early larvae (sphere[S]) than to adult polyps (adult [A]) (fig. 1B). These results suggest that the abolishment of morphogenetic borders following disassociation of coral polyps resulted in in vitro overexpression of transcriptional networks that resemble those used by S (Reyes-Bermudez et al. 2016). Differential expression analysis supported the observation, showing that C cells expressed more genes in common with S than with other stages (fig. 1C). Clustering of embryonic transcriptomes (blastula—prawnchip-like blastula [PC] and gastrula—G) as a distinct group that differs from the remaining stages, indicates that C cells are most likely cellular lineages originated after gastrulation (fig. 1B).

Fig. 1.

Fig. 1.

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In vitro transcriptome characterization. Cells’ specific transcription profile resulted in 14,286 transcripts compared with 11,926 reported for adult tissue (A). Transcriptome comparison between cultured cells and Acropora digitifera developmental stages showed that although cultures were initiated from adult tissues, their expression profile was closer to early larvae (S) than to any other in vivo stage (B). Differential gene expression analysis revealed a lower number of DEGs in the SvsC comparison (C). Only a fraction of DEGs were identified as orthologs of HM. The lowest percentage was observed in AvsC and the highest in PCvsC (D). Blastula, PC; gastrula, G; early larvae, S; planula, P; adult, A; upregulated in C, UpC; upregulated in vivo, UpX.

Moreover, enrichment in the subset of differentially expressed genes (DEGs) upregulated in vitro with molecules involved in diverse morphogenetic and differentiation processes (table 1) indicates uncoordinated overexpression of genome regulatory programs following the loss of morphogenetic borders (Beloussov 2015). Results revealed a transcriptionally heterogenous C cell population that differed transcriptionally from the in vivo system. Significant transcriptional changes that reflect the emergence of more active and proliferative subgroups have been reported in cell cultures over time (Januszyk et al. 2015).

Cells Expressed Orthologs of Premetazoan and HM

Consistent with 500 Myr of independent cnidarian evolution (Steele et al. 2011), only a small fraction of DEGs were identified as orthologs of HM. We observed over expression of a higher number of HM in Blastula (PC) (fig. 1D), which is consistent with the predominant pluripotent cellular phenotypes present at the stage. Interestingly, Acropora HM orthologs upregulated in vitro were enriched with markers overexpressed by Hydra’s i-cells (nanos) (fig. 2A), which, in a strict sense, is the only true characterized cnidarian stem cell population (Frank et al. 2009). These results may not indicate the presence in coral tissues of stem cell populations homologous to Hydra’s i-cells but most likely indicate the utilization of conserved molecules underlying pluripotency and cell differentiation in Acropora.

Fig. 2.

Fig. 2.

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Orthologs of HM and coexpression networks. The HM fraction expressed in vitro was enriched by both endodermal and i-cell HM (A). Transcripts (18,264) were assigned to 38 different gene modules that ranged from 38 to 3,413 transcripts and grouped in two main coexpression clusters (C1 and C2). Eigengenes were calculated for each module and although we were able to identify discrete gene expression patterns, in most cases. significant module–trait correlations were observed in a stage-specific fashion. *P value ≤0.05, **P value ≤0.01, ***P value ≤0.01. Blastula, PC; gastrula, G; early larvae, S; planula, P; adult, A; cultured cells, C.

Similarly, we observed enrichment in cultures of orthologs of endodermal markers such as Brachyury (Yasuoka et al. 2016) and Hedgehog (Matus et al. 2008) (supplementary material S1–S4, Supplementary Material online). Whether this reflects endodermal enrichment in vitro or utilization of conserved transcriptional networks by different lineages, is not clear. For example, although i-cells are thought to be a “recent” cnidarian innovation, their transcriptome is phylogenetically older than those of Hydra’s epithelial lineages (Hemmrich et al. 2012), suggesting recruitment of ancestral regulatory networks during Hydra’s i-cell evolution. Our results support this idea and suggest that regulatory networks associated with maintenance of pluripotency and differentiation are not fixed entities linked to specific cell types, but dynamic modules recruited and modified multiple times by natural selection during cnidarian diversification.

Likewise, identification of premetazoan and metazoan stem cell markers in the subset of DEGs exclusively upregulated in vitro (table 2) is consistent with the idea that animal stem cell systems were built upon ancestral regulatory gene networks present in the last common metazoan ancestor (Alié et al. 2015). Upregulation in vitro of Acropora orthologs with roles in cell cycle, replication, chromosome maintenance, stress response, DNA repair, as well as diverse transcripts coding RNA-binding proteins, such as a Musashi-1 ortholog (table 2), imply that components of regulatory gene networks associated to stemness are being expressed in cultures.

Table 2.

DEGs with Putative Roles in Stem Cell Homeostasis

ID Annotation KEGG Marker Function
Ancestral genes
adi_v1.04467 Nuclear factor NF-kappa-B p105 subunit—Metazoa K02580 Nanos_3529 Cell growth and differentiation
adi_v1.12580 Heterogeneous nuclear ribonucleoprotein K—Metazoa K12886 Nanos_2219 Pre-mRNA splicing
adi_v1.12078 ATP-dependent RNA helicase DDX56/DBP9 - Eukariota K14810 Nanos_1725 RNA-Helicases
adi_v1.19896 DNA polymerase alpha subunit A—Eukariota K02320 Nanos_3477 DNA replication
adi_v1.00008 Replication factor C subunit 3/5—Eukariota K10756 Nanos_2523 DNA repair
adi_v1.17163 Flap endonuclease-1—Pre-Eukariota K04799 Nanos_Ecto_497 DNA repair
Replication
adi_v1.13235 Replication factor C subunit 2/4 K10755 Nanos_2940 Replication
adi_v1.00008 Replication factor C subunit 3/5 K10756 Nanos_2523 Replication
adi_v1.19896 DNA polymerase alpha subunit A (EC:2.7.7.7) K02320 Nanos_3477 Replication
adi_v1.02983 DNA polymerase sigma (EC:2.7.7.7) K03514 Nanos_74 Replication
adi_v1.10030 DNA polymerase zeta (EC:2.7.7.7) K02350 Nanos_4979 Replication
adi_v1.24240 ATP-binding protein involved in chromosome partitioning K03593 Nanos_1409 Replication
adi_v1.13671 DNA polymerase zeta (EC:2.7.7.7) K02350 Endo_Nanos_16 Replication
adi_v1.12265 DNA topoisomerase VI subunit B (EC:5.99.1.3) K03167 Endo_Nanos_1274 Replication
adi_v1.19036 DNA polymerase sigma (EC:2.7.7.7) K03514 Endo_Nanos_861 Replication
Cell cycle
adi_v1.05785 Cell cycle arrest protein BUB3 K02180 Nanos_5104 Cell cycle
adi_v1.04546 Cell cycle checkpoint protein K06662 Nanos_2749 Cell cycle
XLOC_000501 Cell division cycle 20-like protein 1, cofactor of APC complex K03364 Nanos_7115 Cell cycle
XLOC_019254 Cell division cycle 20-like protein 1, cofactor of APC complex K03364 Nanos_1396 Cell cycle
adi_v1.03932 Cell division cycle 20-like protein 1, cofactor of APC complex K03364 Nanos_10963 Cell cycle
adi_v1.06900 G1-/S-specific cyclin PLC1 K06656 Nanos_1058 Cell cycle
adi_v1.13930 Centromere protein B K11496 Nanos_2602 Cell cycle
adi_v1.09992 Cell division protein ZapA K09888 Endo_Nanos_2012 Cell cycle
adi_v1.24600 Signal-induced proliferation-associated gene 1 K08013 Endo_Nanos_1253 Cell cycle
adi_v1.24600 Signal-induced proliferation-associated gene 1 K08013 Endo_Nanos_1253 Cell cycle
XLOC_020915 Cell division cycle 20-like protein 1, cofactor of APC complex K03364 Ecto_3004 Cell cycle
Helicases
adi_v1.01424 Chromodomain–helicase–DNA-binding protein 7 (EC:3.6.4.12) K14437 Nanos_422 Chromatin remodeling
adi_v1.01424 Chromodomain–helicase–DNA-binding protein 7 (EC:3.6.4.12) K14437 Nanos_422 Chromatin remodeling
adi_v1.13340 RNAi-mediated heterochromatin assembly 1 (EC:3.6.4.13) K11701 Endo_Ecto_43 Chromatin remodeling
adi_v1.23884 ATP-dependent RNA helicase DHX8/PRP22 (EC:3.6.4.13) K12818 Nanos_1508 Splicing/transcription
adi_v1.08670 ATP-dependent RNA helicase DHX15/PRP43 (EC:3.6.4.13) K12820 Nanos_806 Splicing/transcription
adi_v1.23237 ATP-dependent RNA helicase DDX1 (EC:3.6.4.13) K13177 Nanos_1406 Splicing/transcription
adi_v1.12078 ATP-dependent RNA helicase DDX56/DBP9 (EC:3.6.4.13) K14810 Nanos_1725 Ribosome biogenesis
Chromosome maintenance
adi_v1.24240 ATP-binding protein involved in chromosome partitioning K03593 Nanos_1409 Chromosome maintenance
adi_v1.17702 Structural maintenance of chromosome 1 K06636 Nanos_Ecto_213 Chromosome maintenance
adi_v1.15153 Structural maintenance of chromosome 4 K06675 Nanos_Ecto_50 Chromosome maintenance
adi_v1.01018 Structural maintenance of chromosome 1 K06636 Nanos_653 Chromosome maintenance
adi_v1.14806 Structural maintenance of chromosome 4 K06675 Nanos_3734 Chromosome maintenance
adi_v1.11026 Structural maintenance of chromosome 4 K06675 Nanos_6620 Chromosome maintenance
adi_v1.04706 Chromosome segregation protein K03529 Endo_Nanos_897 Chromosome maintenance
adi_v1.01300 Chromosome transmission fidelity protein 1 (EC:3.6.4.13) K11273 Ecto_1702 Chromosome maintenance
adi_v1.21696 Chromosome segregation protein K03529 Endo_Ecto_309 Chromosome maintenance
DNA repair/stress response
adi_v1.23838 Three prime repair exonuclease 2 (EC:3.1.11.2) K10791 Nanos_3244 DNA repair
adi_v1.03868 DNA damage-inducible protein 1 K11885 Nanos_1310 DNA repair
adi_v1.02191 DNA excision repair protein ERCC-2 (EC:3.6.4.12) K10844 Nanos_1749 DNA repair
adi_v1.22737 DNA excision repair protein ERCC-3 (EC:3.6.4.12) K10843 Nanos_3028 DNA repair
adi_v1.22267 DNA excision repair protein ERCC-4 (EC:3.1.-.-) K10848 Nanos_1886 DNA repair
adi_v1.11724 DNA excision repair protein ERCC-8 K10570 Nanos_5126 DNA repair
adi_v1.03203 DNA excision repair protein ERCC-8 K10570 Nanos_6011 DNA repair
adi_v1.11542 DNA repair protein RAD16 K15083 Nanos_9086 DNA repair
adi_v1.06342 DNA repair protein RAD50 (EC:3.6.-.-) K10866 Nanos_2601 DNA repair
adi_v1.19161 DnaJ homolog subfamily A member 5 K09506 Nanos_1092 Stress response
XLOC_001068 DnaJ homolog subfamily B member 9 K09515 Nanos_4628 Stress response
adi_v1.04788 Heat shock 70 kDa protein 1/8 K03283 Nanos_2410 Stress response
adi_v1.02262 Heat shock 70 kDa protein 1/8 K03283 Nanos_2410 Stress response
adi_v1.04284 Stress-induced-phosphoprotein 1 K09553 Nanos_2665 Stress response
RNA-binding proteins
adi_v1.16723 Multiple RNA-binding domain-containing protein 1 K14787 Nanos_527 RNA-binding
adi_v1.13400 oo18 RNA-binding protein K02602 Nanos_1849 RNA-binding
adi_v1.10220 RNA-binding protein 15 K13190 Nanos_1931 RNA-binding
adi_v1.03795 RNA-binding protein 39 K13091 Nanos_357 RNA-binding
adi_v1.05305 RNA-binding protein Musashi K14411 Nanos_2716 RNA-binding
adi_v1.03308 RNA-binding protein 26 K13192 Endo_7052 RNA-binding
adi_v1.15031 U1 small nuclear ribonucleoprotein A K11091 Nanos_334 RNA-binding
adi_v1.00705 U3 small nucleolar ribonucleoprotein protein IMP4 K14561 Nanos_985 RNA-binding
adi_v1.06360 U3 small nucleolar RNA-associated protein 20 K14772 Nanos_283 RNA-binding
adi_v1.19916 U3 small nucleolar RNA-associated protein 21 K14554 Nanos_2230 RNA-binding
adi_v1.13965 U3 small nucleolar RNA-associated protein 24 K14566 Nanos_1277 RNA-binding
adi_v1.12414 U3 small nucleolar RNA-associated protein 5 K14546 Nanos_200 RNA-binding
adi_v1.11136 U3 small nucleolar RNA-associated protein 6 K14557 Nanos_1283 RNA-binding
adi_v1.07136 U3 small nucleolar RNA-associated protein 6 K14557 Nanos_1283 RNA-binding
XLOC_015243 U3 small nucleolar RNA-associated protein 7 K14768 Nanos_1007 RNA-binding
adi_v1.17859 Heterogeneous nuclear ribonucleoprotein K K12886 Nanos_2219 RNA-binding
adi_v1.09619 Heterogeneous nuclear ribonucleoprotein M K12887 Nanos_141 RNA-binding
adi_v1.23861 U4/U6 small nuclear ribonucleoprotein SNU13 K12845 Nanos_Ecto_54 RNA-binding
adi_v1.21303 RNA-binding protein 15 K13190 Endo_Nanos_332 RNA-binding
adi_v1.12580 RNA-binding protein 5/10 K13094 Endo_Nanos_738 RNA-binding
adi_v1.07694 Heterogeneous nuclear ribonucleoprotein L K13159 Endo_Nanos_24 RNA-binding
adi_v1.21839 Small nuclear ribonucleoprotein B and B' K11086 Endo_1503 RNA-binding
Ribosome biogenesis
adi_v1.12527 rRNA biogenesis protein RRP5 K14792 Nanos_669 Ribosome biogenesis
adi_v1.10921 rRNA biogenesis protein RRP5 K14792 Nanos_669 Ribosome biogenesis
adi_v1.20333 Regulator of ribosome biosynthesis K14852 Nanos_751 Ribosome biogenesis
adi_v1.09262 Ribosome assembly protein 4 K14855 Nanos_1608 Ribosome biogenesis
adi_v1.05788 Ribosome biogenesis protein BMS1 K14569 Nanos_481 Ribosome biogenesis
adi_v1.01894 Ribosome biogenesis protein MAK21 K14832 Nanos_2059 Ribosome biogenesis
adi_v1.05433 Ribosome biogenesis protein NSA2 K14842 Nanos_425 Ribosome biogenesis
XLOC_014614 Ribosome production factor 1 K14846 Nanos_1610 Ribosome biogenesis
adi_v1.04696 Ribosome biogenesis GTPase A K14540 Endo_Nanos_387 Ribosome biogenesis
adi_v1.02489 Ribosome biogenesis protein MAK21 K14832 Endo_Nanos_4397 Ribosome biogenesis
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Coexpression Modules Reveal Distinct G-/P-Specific Genome Regulatory Programs

Network analysis assembled DEGs in 38 modules within two main coexpression groups, consisting of distinct and diverse stage-specific coexpression clusters (fig. 2B). Coexpression units usually reflect common functionality and regulation (review in Peter and Davidson 2011). Interestingly, most in vitro upregulated DEG’s were coexpressed in PC, S, and A but were significantly downregulated in G and P stages (fig. 2B). Differential usage of enhancers between G and P stages have been reported for Nematostella (Schwaiger et al. 2014), suggesting the existence of distinct G-/P-specific genome regulatory programs in cnidaria. More research is necessary to test this idea as transcriptional networks underlying early morphogenetic transitions in metazoans are variable and, in some cases, taxa-specific (Erwin and Davidson 2009; Davidson 2010).

Finally, transcriptome comparisons using coexpression networks and transcript composition showed slightly different results. Although the topology built using complete transcriptomes clustered S and C as a sister group to A, leaving P as the most dissimilar stage and PC and G as a separate group, the topology based on coexpression data, resolved P and A as a sister group to PC and G (fig. 1D). In both cases, the similarity between C and S was clear, indicating the usage in the two stages of shared genome regulatory programs based on similar transcript composition. On the other hand, differences between topologies reveal that in vivo complexity is strongly dependent on network interactions and supports the idea that body plan morphogenesis and evolution is a “system-level problem” that cannot be understood by looking at developmental conserved genes in isolation (Peter and Davidson 2011).

Conclusion

Our study demonstrated that primary coral cultures are valuable tools for studying genome regulatory programs and revealed the existence of ancestral genome regulatory modules underlying pluripotency and cell differentiation in cnidaria. Caution must be taken to interpret in vitro experiments as C populations are heterogeneous cell types that drastically differ transcriptionally from the in vivo system.

Materials and Methods

Collection of Samples and Tissue Culture

Tip fragments (∼3 cm) from six different colonies were kept in 50 ml falcon tubes containing 0.2 μm filtered seawater with antibiotics (FSWA) (1% Pen/Strep/L-Glu-solution, Sigma–Aldrich and 0.1% Fungizone, Invitrogen) prior the initiation of cultures. Samples were washed 3× with FSWA and then incubated at 32 °C for 4 h to induce bleaching (Desalvo et al. 2010). Fragments were washed (3×) with FSWA and further incubated (2 h/gentle shaking) in calcium-free FSWA (Marshall and Clode 2004). Following tissue dissociation, naked skeletons were removed, and detached tissue centrifuged (1,500 rpm/10 min) and resuspended in 5 ml of FSWA. Cell pellets were gently washed 3× with 5 ml of cell culture media (30% DMEM Gibco, 10% FBS Gibco, 1% Pen/Strep-solution, 0.1% Fungizone, 1% Glutamax, Gibco, 25 mM HEPES pH 8.0, and 55% 0.2 μm filtered seawater) and resuspended in 5 ml of fresh media. Founding cultures were kept individually in 6-well cultured plates and incubated at 23 °C in the dark for 48 h. After that, 1 ml of the original cultures were used to inoculate 4 ml of fresh media in 6-well cultured plates and returned to incubation conditions. Cultures were monitored daily on a standard inverted light microscope fitted with a color digital camera. Media was changed when cultures reached 60% confluence. After 4 weeks, cells from three wells were harvested for RNA extractions.

Sequencing and Data Analysis

Library preparation and data analysis were conducted as reported in Reyes-Bermudez et al. (2016). To identify Acropora HM, we download the T-CDS data set from Hydra vulgaris strain AEP from http://www.compagen.org/datasets.html. Orthologs were determined using OrthoMCL v.1.4 with a BLASTp E value cut-off of 1e−5, a minimum coverage of 70% and an inflation index of 1.5 (Li et al. 2003).

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online.

Supplementary Material

evab008_Supplementary_Data
Click here for additional data file. (2.5MB, xlsx)

Acknowledgments

We would like to thank all members of the Sesoko Station for their assistance during coral spawning. We are also thankful to members of the Hidaka and Mikheyev laboratories for field assistance and technical support, respectively. Sequencing was performed by the OIST DNA-sequencing section. This work was supported by a postdoctoral fellowship awarded to A.R.B. from the Japanese Society for the Promotion of Science and internal funds from the Okinawa Institute of Science and Technology Graduate University awarded to A.S.M.

Data Availability

Raw data for cell cultures can be found under bio-project ID PRJDB9497: BioSamples: SAMD00210801, SAMD00210803, and SAMD00210805. Experiment: DRX207005–DRX207007.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

evab008_Supplementary_Data
Click here for additional data file. (2.5MB, xlsx)

Data Availability Statement

Raw data for cell cultures can be found under bio-project ID PRJDB9497: BioSamples: SAMD00210801, SAMD00210803, and SAMD00210805. Experiment: DRX207005–DRX207007.

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