The evolutionary emergence of cell type-specific genes inferred from the gene expression analysis of Hydra
Jung Shan Hwang*, Hajime Ohyanagi*†, Shiho Hayakawa*, Naoki Osato*, Chiemi Nishimiya-Fujisawa‡, Kazuho Ikeo*, Charles N. David§, Toshitaka Fujisawa‡, and Takashi Gojobori*¶
*Center for Information Biology and DNA Data Bank of Japan, ‡Department of Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540 Japan; †Tsukuba Division, Mitsubishi Space Software Co., Ltd., 1-6-1 Takezono, Tsukuba, Ibaraki 305-0032, Japan; and §Department Biologie II, Ludwig Maximilians University, Grosshadernerstrasse 2, D-82152 Planegg/Martinsried, Germany
Edited by Stanley Shostak, University of Pittsburgh, Pittsburgh, PA, and accepted by the Editorial Board July 18, 2007 (received for review April 13, 2007) Cell lineages of cnidarians including Hydra represent the funda- Previous results have shown that Hydra do not require the I-cell mental cell types of metazoans and provides us a unique oppor- lineage because, when hand fed, Hydra can carry out metabolic tunity to study the evolutionary diversification of cell type in the activities and grow after removal of the I-cell lineage (17, 18). Such animal kingdom. Hydra contains epithelial cells as well as a mul- epithelial Hydra contain only two epithelial layers and a limited tipotent interstitial cell (I-cell) that gives rise to nematocytes, nerve number of gland cells. These animals are unable to move and catch cells, gland cells, and germ-line cells. We used cDNA microarrays to or ingest food but can be force-fed by inserting food through the identify cell type-specific genes by comparing gene expression in mouth into the gastric cavity (19). Such epithelial Hydra can be normal Hydra with animals lacking the I-cell lineage, so-called maintained in the laboratory for years and remain capable of epithelial Hydra. We then performed in situ hybridization to normal growth, budding, and regeneration. localize expression to specific cell types. Eighty-six genes were In the present study, we identified genes that are expressed shown to be expressed in specific cell types of the I-cell lineage. An specifically in the I-cell lineage of Hydra by comparing gene additional 29 genes were expressed in epithelial cells and were expression in intact and I-cell-free Hydra. We constructed a 6.6 down-regulated in epithelial animals lacking I-cells. Based on the thousand cDNA microarray and performed competitive hybridiza- above information, we constructed a database (http://hydra.lab. tion by using probes from epithelial Hydra and normal Hydra to nig.ac.jp/hydra/), which describes the expression patterns of cell identify genes that are expressed in normal animals but lacking in type-specific genes in Hydra. Most genes expressed specifically in epithelial Hydra. We identified 151 genes expressed differentially in either I-cells or epithelial cells have homologues in higher meta- normal Hydra. Studies of these genes indicate that many encode zoans. By comparison, most nematocyte-specific genes and ap- ‘‘novel’’ proteins. Thus, the evolutionary diversification of differ- proximately half of the gland cell- and nerve cell-specific genes are entiated cell types in Hydra was accompanied by the evolution of unique to the cnidarian lineage. Because nematocytes, gland cells, lineage-specific genes. and nerve cells appeared along with the emergence of cnidarians, this suggests that lineage-specific genes arose in cnidarians in Results and Discussion conjunction with the evolution of new cell types required by the cDNA Microarray Analysis. The cDNA microarray that we con- cnidarians. structed in this study includes 6,528 individual genes derived from ͉ ͉ ͉ ͉ a normal Hydra strain, H. magnipapillata 105. This array was cnidarian database epithelial hydra microarray nematocyte screened with RNA probes from epithelial and normal animals. The differential gene expression between normal and epithelial ydra is a member of the phylum Cnidaria, which branched Hydra was evaluated by using a statistical algorithm, SMA (Statis- HϾ500 million years ago from the main stem leading to all tical Microarray Analysis; http://stat-www.berkeley.edu/users/terry/ bilaterian animals. Nevertheless, all Cnidaria including Hydra con- zarray/Software/smacode.html). Genes whose expression was Ͼ2- tain multiple cell types that represent the fundamental building fold higher in normal Hydra than in epithelial Hydra were blocks of all multicellular animals. Early investigations of Hydra cell provisionally identified as I-cell specific. By using this criterion, 151 morphology have revealed seven basic cell types (1, 2) including genes were identified from five independent arrays as candidates for interstitial cells (I-cells) and ectodermal and endodermal epithelial I-cell-specific genes. An additional 19 genes were identified that had cells that are capable of proliferation and self-renewal. I-cells are 2-fold greater expression in epithelial Hydra than in normal Hydra. multipotent stem cells, which proliferate and continuously differ- These genes will be described elsewhere. entiate into nematocytes, nerve cells, gland cells, and germ-line We examined the expression patterns of all 151 selected genes by cells. In Hydra, big I-cells occur as single cells, in pairs or in clusters whole-mount in situ hybridization. One hundred twenty-four genes of four, whereas small I-cells are found in clusters of eight or more cells (1). I-cells are a continuously proliferating, self-renewing population in the body column (3, 4, 5). Nematocytes and nerve Author contributions: J.S.H., K.I., T.F., and T.G. designed research; J.S.H., H.O., and S.H. cells continuously differentiate from this population (6, 7). At a late performed research; H.O., C.N.-F., and T.F. contributed new reagents/analytic tools; J.S.H., stage of differentiation, most precursors migrate to the hypostome/ N.O., and C.N.D. analyzed data; and J.S.H., C.N.D., and T.G. wrote the paper. EVOLUTION tentacles and the basal disk, whereas only a few remain in the body The authors declare no conflict of interest. column (8). Gland cells and mucous cells differentiate from I-cells, This article is a PNAS Direct Submission. S.S. is a guest editor invited by the Editorial Board. which migrate from the ectoderm into the endoderm and give rise Freely available online through the PNAS open access option. to at least two major types of secretory cells interspersed among the Data deposition: The sequences reported in this paper have been deposited in the GenBank endodermal epithelial cells (9, 10). Mucous cells are localized in the database (accession nos. are listed in SI Table 8). hypostome and upper body column. Gland cells or zymogen cells ¶To whom correspondence should be addressed. E-mail: [email protected]. are distributed throughout the body column (11). I-cells also give This article contains supporting information online at www.pnas.org/cgi/content/full/ rise to germ-line cells, which differentiate to sperm or egg during 0703331104/DC1. sexual reproduction (12–16). © 2007 by The National Academy of Sciences of the USA
www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703331104 PNAS ͉ September 11, 2007 ͉ vol. 104 ͉ no. 37 ͉ 14735–14740 Downloaded by guest on October 2, 2021 gave significant signals above background. By searching the Hydra the spines, the toxin proteins, and poly-␥-glutamate required for the genome assembly that has been annotated by the DOE Joint explosive extrusion as well as genes encoding the sensory apparatus Genome Institute (Berkeley, CA), we were able to identify full- (cnidocil) of mature nematocytes. Genes involved in capsule for- length or nearly full-length coding sequences for 115 genes. In mation are expressed in nests of differentiating nematocytes during summary, 51 were nematocyte-specific, 21 were gland cell-specific, the first half of the differentiation process. In addition to genes 9 were nerve cell-specific, and 5 genes were I-cell/germ-line cell- for known minicollagens and spinalin (25, 26), our screen identi- specific. Surprisingly, 29 of the selected genes were expressed fied nine minicollagen genes (hmp01875, hmp07679, specifically in epithelial cells (see below). All data including gene hydmg008secrev016, hmp18133, hmp11623, hmp01703, sequences and expression patterns can be viewed in the database as hmp17396, hmp02656, and hm02844; Fig. 1A and Table 1). All a primary resource for the biological community to study the these genes had signal peptides and short nematocyte sorting genetic characteristics of different cell types in Hydra (see Molec- motifs. We identified a protein disulfide isomerase homologue ular Database of Hydra Cells, http://hydra.lab.nig.ac.jp/hydra/). (hm02181) with a signal peptide and nematocyte sorting motif that may be required for disulfide bond isomerization during hardening Nematocyte-Specific Genes. Hydra contains four types of nemato- of the capsule wall (Fig. 1B) (27, 28). Finally, we identified a cytes (stenotele, holotrichous isorhiza, atrichous isorhiza, and des- ␥-glutamyltranspeptidase (GGT) homologue (hm02634), which moneme) (20). In general, the differentiation of nematocytes may be involved in synthesis of poly-␥-glutamate in the capsule proceeds as follows: growth (capsule wall and tubule formation), matrix (29). maturation (tubule invagination, spine assembly, capsule wall hard- Toxins are commonly found in the nematocysts of cnidarians. We ening and poly-␥-glutamate polymerization), migration (cnidocil found two homologues (hm04121, hmp11024) of equinotoxin apparatus formation), and deployment (septate junction formation (30), a member of the actinoporin family from sea anemones as well and cyst positioning) (21, 22). Using the array technique and in situ as a pathogen-related protein (hmp21268). One toxin (hm04121) hybridization, we have identified 51 nematocyte-specific genes that is localized in the isorhizas, which were previously thought to are expressed at distinct differentiation stages or in a particular type contain no toxins at all (Fig. 1C and Table 1). A second toxin of nematocyte (Table 1). It is not surprising that a large proportion (hmp11024) is present in stenoteles. Both toxins are believed to be of the genes identified here are nematocyte-specific, because nema- cytolysins and have hemolytic activity (31). A third gene tocytes comprise approximately half of the total cell population in (hmp21268) encodes a well conserved SCP domain and an ShK Hydra (23). Many of the encoded proteins have N-terminal signal toxin domain that was first found as a short polypeptide in sea peptides, suggesting that they are synthesized on the endoplasmic anemones (Fig. 1D and SI Table 2) (32). reticulum (ER) and transported to the post-Golgi vacuole in which In addition to these genes with known functions, we identified 14 capsules are formed. Some of these, e.g., minicollagens and toxin more genes that had signal peptides and were expressed in differ- proteins, also contain a short ‘‘nematocyte sorting signal’’ that is entiating nematocytes, but which had no significant homologue in removed from the mature protein in capsules (24). Some of the the nr protein database and the genomic databases (Table 1 and SI signal-peptide-containing genes may not be incorporated into Table 3). These appear to be ‘‘novel’’ capsule proteins, although capsules but sorted to the cell membrane. Finally, there are some could represent nematocyte membrane proteins. Several had nematocyte-specific genes, which lack N-terminal sorting signals short proline-rich stretches, which are also present in minicollagens, and appear to encode cytoplasmic proteins specifically expressed in and thus may constitute novel structural proteins in the capsule wall. nematocytes. Two genes were expressed specifically in stenoteles, four genes were A striking feature of the list of nematocyte-specific genes is that expressed in isorhizas (Fig. 1E), and the rest were expressed in all only 18% (9 of 51) of nematocyte-specific genes have orthologues types of nematocytes. This indicates that each type of nematocyte in other species based on BLASTP (Table 1). These include has some unique proteins, which presumably contribute to the putative capsule-specific enzymes like protein disulfide isomerase particular function of that nematocyte type, as well as some proteins (PDI) and ␥-glutamyltranspeptidase as well as known cytoplasmic that are shared with other nematocyte types. proteins such as two calcium-binding EFh domain proteins, tubulin In addition to the proteins listed above, all of which contain signal and the centrosomal protein [supporting information (SI) Table 2]. peptides and, hence, can be incorporated into nematocyte capsules, The great majority of nematocyte-specific genes (42 of 51) lacked we also identified 17 genes (sensory apparatus and unknown) orthologues in other metazoan phyla, although in some cases, they encoding proteins that lack signal peptides but which are nemato- contained sequence motifs found in known proteins. For example, cyte specific (Table 1). These proteins are presumably cytoplasmic. minicollagens contain GlyXY collagen tripeptide domains, Several are expressed in migrating nematocytes after capsule hm02450 encodes a galactose lectin domain and a GlyXY collagen formation is complete and also in mature nematocytes mounted in tripeptide domain, and hmp21268 encodes an SCP domain. Re- the tentacles. Formation of the cnidocil apparatus takes place at this peating these searches with more sensitive PSI-BLAST (SI Table 2) stage of differentiation (22). The cnidocil is a mechanosensory did not identify a significant number of additional orthologues in organelle extending from the apical surface of nematocytes the protein database. Because not all protein-coding genes are mounted in the ectoderm. The two lamin-like genes (hmp08523 compiled in the nr proteins database, we also carried out additional and hm04087) (Fig. 1F) and the -tubulin gene (hmp00406) BLAST searches on the completed genomes of two fungi (Aspergil- encode proteins that participate in cnidocil formation. The centro- lus and Candida), the ciliate Tetrahymena, the parasitic protozoa somal protein homologue (hydmg019bw14) may be involved in Trypanosoma, the slime mold Dictyosterium, the choanoflagellate formation of the basal body at the base of the cnidocil. Finally, two Monosiga, the sponge Reneira, the flatworm Schmidtea, the mollusk genes (hmp08051 and hmp10958) encode small calcium-binding Lottia, the annelid Capitella, and two deuterosomes (Strongylocen- proteins, which may be involved in regulating intracellular calcium trotus and Branchiostoma). These searches (summarized in SI Table levels and exocytosis of the mature nematocyte (Fig. 1G). Inter- 3) identified one additional homologue not yet compiled in the nr estingly, all BLAST matches of hmp10958 occurred as proteins in protein database. This, however, does not significantly alter the protists (SI Table 3), possibly suggesting that either this gene is conclusion: the great majority (41 of 51) of nematocyte-specific originated from protist or that the orthologues of hmp10958 have genes identified in the array appear to be cnidarian-specific. Of evolved divergently in other metazoans. Most of the remaining 11 these, 17 lack a clear homologue in Nematostella and thus appear nematocyte-specific genes had no homologues in nr protein data- to be Hydra-specific. base or in other phyla, and we are unable to determine their Nematocyte-specific genes encode structural proteins that are functional role in nematocyte differentiation (Fig. 1H, Table 1, and essential for assembling the rigid capsule wall, the internal tubule, SI Table 3).
14736 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703331104 Hwang et al. Downloaded by guest on October 2, 2021 Table 1. Nematocyte-specific genes of hydra identified by cDNA microarray and whole-mount in situ hybridization ͉ Accession no. ͉ homologous Cnidarian- Clone name UniGene gene of BLASTP search E-value SP* specific† Expression localization
Capsule gene hmp09910 Hma.940 ͉ AAC39121 ͉ spinalin [Hydra magnipapillata]0.0ϩϩDifferentiating nematocyte hm03768 Hma.6702 ͉ CAA43379 ͉ mini-collagen [H. magnipapillata] 1e-11 ϩϩDifferentiating nematocyte hm02081 — ͉ CAA43380 ͉ collagen-related protein 2 2e-10 ϩϩDifferentiating nematocyte (fragment) [H. magnipapillata] hydmg014 secrev095 — ͉ CAA43381 ͉ collagen-related protein 3 precursor 4e-12 ϩϩDifferentiating nematocyte [H. magnipapillata] hmp01875 Hma.5797 ϩϩDifferentiating nematocyte hmp07679 Hma.1712 ͉ BAA06407 ͉ mini-collagen [Acropora donei] 8e-18 ϩϩDifferentiating nematocyte hydmg008 secrev016 Hma.2229 ϩϩDifferentiating nematocyte hmp18133 Hma.5361 ϩϩDifferentiating nematocyte hmp11623 Hma.933 ϩϩDifferentiating nematocyte hmp01703 — ͉ CAA43380 ͉ collagen-related protein 2 2e-14 ϩϩDifferentiating nematocyte (fragment) [H. magnipapillata] hmp17396 — ϩϩDifferentiating nematocyte hmp02656 Hma.8906 ϩϩDifferentiating nematocyte hm02844 Hma.1803 ϩϩDifferentiating nematocyte hm02181 Hma.3707 ͉ AAF78087 ͉ protein disulfide isomerase ER-60 1e-16 ϩϪDifferentiating nematocyte [Takifugu rubripes] hm02634 Hma.3432 ͉ AAH75461.1 ͉ ␥-glutamyltranspeptidase 1 9e-29 ϩϪDifferentiating nematocyte [Xenopus tropicalis] hydmg006 secrev019 Hma.1619 ͉ NP801012.1 ͉ carbonic anhydrase [Vibrio 7e-22 ϩϪDifferentiating stenotele parahaemolyticus RIMD 2210633] hm02290 Hma.1831 ͉ XP548098.2 ͉ similar to FK506-binding protein 8e-34 ϩϪDifferentiating nematocyte and epithelial 10 precursor (peptidyl-prolyl cis-trans isomerase) cells around tentacle base [Canis familiaris] hm04121 Hma.4708 ͉ AAV41095 ͉ physcomitrin [Physcomitrella patens] 3e-06 ϩϩDifferentiating isorhiza hmp11024 — ͉ BAC45007 ͉ PsTX-20A [Phyllodiscus semoni] 9e-08 ϩϩDifferentiating stenotele hmp21268 Hma.1520 ͉ BAB85217 ͉ PR-1 like protein [Volvox carteri 6e-15 ϩϩLate differentiating nematocyte f.nagariensis] Novel capsule gene hmp08654 Hma.1833 ϩϩNematoblast hm04447 Hma.8919 ϩϩNematoblast hmp15565 Hma.1043 ϩϩNematoblast hmp08480 Hma.4149 ͉ BG924950.1 ͉ HyTSRp1 protein [Hydra vulgaris] 1e-10 ϩϩDifferentiating nematocyte hmp22241 Hma.4697 ϩϩDifferentiating nematocyte hmp02020 Hma.4931 ϩϩDifferentiating nematocyte hydmg002bw25 — ϩϩDifferentiating nematocyte hmp09895 Hma.1654 ϩϩDifferentiating stenotele hm02137 Hma.1900 ͉ ABK35136.1 ͉ rendezvin [Lytechinus variegatus] 3e-10 ϩϩDifferentiating isorhiza hm02450 Hma.3102 ͉ XP971465.1 ͉ similar to tubulin tyrosine 4e-18 ϩϩDifferentiating isorhiza ligase-like protein 2 [Ribolium castaneum] hmp08269 Hma.4045 ϩϩLate differentiating nematocyte hmp07910 Hma.9335 ϩϩStenotele hmp17528 Hma.9348 ϩϩHolotrichous isorhiza hmp20434 Hma.3901 ϩϩHolotrichous isorhiza Sensory apparatus hmp08523 Hma.4022 ͉ NP523742.2 ͉ lamin C CG10119-PA [Drosophila 2e-21 ϪϩNematocyte melanogaster] hm04087 Hma.8965 ͉ NP523742.2 ͉ lamin C CG10119-PA [D. 2e-20 ϪϩNematocyte melanogaster] hmp00406 Hma.1413 ͉ AAG15328.1 ͉ -tubulin [Chionodraco e-100 ϪϪMigrating nematocyte rastrospinosus] hydmg019bw14 — ͉ NP055771 ͉ centrosomal protein 164kDa [Homo 2e-35 ϪϪLate differentiating nematocyte sapiens] hmp08051 Hma.1566 ͉ AAD00259.1 ͉ calbindin D28k [Xenopus laevis] 2e-20 ϪϪMigrating stenotele and desmoneme hmp10958 Hma.1341 ͉ Q27052 ͉ flagellar calcium-binding protein 8e-22 ϪϪMigrating nematocyte [Trypanosoma rangeli] Unknown hm03857 Hma.624 ͉ AAA65698.1 ͉ ADP-ribosyl cyclase precursor 1e-10 ϪϩSubpopulation of nematoblast [Aplysia californica] hmp20753 Hma.7753 ϪϩSubpopulation of nematoblast hmp07718 — ϪϩNematoblast at early stage hmp13584 — ϪϩNematoblast at early stage
hm02549 — ϪϩDifferentiating nematocyte EVOLUTION hmp06864 Hma.4958 ͉ ZP01459247 ͉ inositol 2-dehydrogenase 1e-62 ϪϪDifferentiating nematocyte [Stigmatella aurantiaca DW4/3–1] hmp16781 — ϪϩDifferentiating nematocyte hmp02578 — Ϫ (ϩ) Differentiating nematocyte hm04093 — ϪϩStenotele and differentiating desmoneme hmp07578 Hma.3700 ϪϩStenotele at body column hm02330 — ϪϩHolotrichous isorhiza
*Signal peptide (SP): ϩ, gene encoding a signal peptide; Ϫ, gene without a signal peptide. †Cnidarian-specific: ϩ, gene with homologues (E-value Ͻ1e-10) only in cnidarian species; Ϫ, gene with homologues (E-value Ͻ1e-10) in other phyla. (ϩ), homologue is not present in the current nr protein database but is detected in invertebrate genomes.
Hwang et al. PNAS ͉ September 11, 2007 ͉ vol. 104 ͉ no. 37 ͉ 14737 Downloaded by guest on October 2, 2021 A B AB
C D Fig. 2. Expression of gland cell-specific genes using whole-mount in situ hybridization. Whole animals are shown on the left, and high-magnification images showing the expression signal in individual cells are shown on the right. (A) hmp14705. (B)hm02146. All clone names refer to Table 2. (Scale bars, 50 m.)
The gland cell-specific genes can be further grouped into sub- F types according to their gene expression patterns. For instance, the E expression of hmp14705 (a homologue of metalloproteinase) was strong in the upper body column but gradually less toward the peduncle (Fig. 2A); whereas, hm02146 (a homologue of serine protease) was expressed primarily in the peduncle (Fig. 2B). This indicates that gland cells are not identical but establish a position- specific expression pattern along the body axis. Recent reports also showed similar expression patterns in gland cells (35, 36).
G H Nerve Cell-Specific Genes. Nine genes isolated from the microarray were nerve cell-specific. Six of them encoded neuropeptides and were expressed in ganglion cells (SI Table 5). Various neuropep- tides have been isolated from Hydra by using systematic screening for peptide signaling molecules (37–39), and four sequences re- ported in this study encode such known neuropeptides (SI Fig. 3D). However, additional paralogues of RFamide (hmp01824) and hym-176 (hmp10112) neuropeptides were identified here. So far, Fig. 1. Expression of nematocyte-specific genes using whole-mount in situ all nerve cell-specific genes isolated from Hydra encode neuropep- hybridization. Whole animals are shown on the left, and high-magnification tides, and more than half of the genes identified in this study also images showing the expression signal in individual cells are shown on the encode neuropeptides, indicating neuropeptides play a key role in right. (A) hmp11623, a previously uncharacterized minicollagen expressed in Hydra neurotransmission. A few of the neuropeptides in Hydra such differentiating nematocytes. (B)hm02181, a protein disulfide isomerase as RFamide and LWamide/MWamide have homologues in meta- expressed in differentiating nematocytes. (C)hm04121, a toxin protein ex- pressed in differentiating isorhizas. (D) hmp21268, a toxin protein expressed zoans. However, other neuropeptides such as hym-176 (KVamide) in differentiating nematocytes. (E) hmp17528, a cnidarian-specific capsule and hym-355 (RGamide) have no metazoan homologues so far. protein expressed in mature holotrichous isorhizas. (F) hmp08523, a lamin- Three additional nerve cell-specific genes, hmp09790, like protein expressed in all mature nematocytes. (G) hmp08051, a calcium- hmp11958, and hmp13646, were identified in this study (SI Table binding protein expressed in migrating nematocytes. (H) hmp07578, a 5). Expression of hmp09790 was localized in sensory cells, and the cnidarian-specific protein with unknown function expressed only in stenoteles tentacles of polyps were highly stained by whole-mount in situ and desmonemes. All clone names refer to Table 1. S, stenotele; HI, holotri- hybridization (SI Fig. 3A). No homologue for hmp09790 could be chous isorhiza; I, isorhiza; D, desmoneme; dS, differentiating stenotele; dI, identified in the protein databases, and a search against the genome differentiating isorhiza; dD, differentiating desmoneme. (Scale bars, 50 m.) databases also resulted in no match (SI Table 3). Hmp11958 was expressed in both sensory and ganglion cells (SI Fig. 3B). Its Gland Cell-Specific Genes. Gland cells and mucous cells are secretory predicted protein sequence can be cleaved at KR residues into cells located in the endoderm of Hydra. They secrete proteolytic putative peptide signaling molecules, although such peptides have enzymes and mucopolysacchrides (33) into the gastric cavity of not yet been identified in Hydra. We suspect that both genes above Hydra. In this study, a total of 21 genes were expressed in either may function as neurotransmitters involved in signal transduction or gland or mucous cells (SI Table 4). Almost all these genes had as neurohormones involved in developmental processes. clearly recognizable signal peptides, suggesting that they encode Hmp13646 encodes an unknown protein of 222 aa. The first 123 secreted proteins. Five gland cell-specific genes (hmp01803, aa show homology to the ligand-binding domain of the ligand-gated hmp03838, hmp17122, hm02146, and hmp14705) showed clear ion channel pHCl-C (AAX11177) of Drosophila melanogaster, homology to annotated metazoan genes. These encoded extracel- indicating hmp13646 may also function in neurotransmission. lular proteases. Hmp08169 encodes antistasin (34), a gland cell- Expression of hmp13646 is localized in ganglion cells (SI Fig. 3C). specific protease inhibitor. Three more genes encoded homologues The apparent enrichment of neuropeptide genes among nerve of nonenzymatic lysosomal proteins (hmp09905, hmp22174, and cell-specific genes suggests that neuropeptide signaling systems play hmp00993), yet they are relatively divergent from many others an important role in the neural network of Hydra. RFamide and including those from Nematostella (SI Table 3). Finally, 11 of 21 LWamide neuropeptides are known from several other metazoan gland- and mucous cell-specific genes encoded sequences with no phyla. The genes encoding the precursors to these neuropeptides known homologues in the nr protein database (SI Table 4). Half of are not well conserved, and, hence, no homologues in other phyla these genes also lacked a clear Nematostella homologue and thus are listed in SI Table 3.ThisisalsotruefortheNematostella appear to be Hydra-specific (SI Table 3). homologues. Although initially surprising, because both Hydra and
14738 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703331104 Hwang et al. Downloaded by guest on October 2, 2021 Nematostella are cnidarians, this is probably because of the fact that types in higher metazoans. This latter observation suggests that these two cnidarian lineages have been separated for Ͼ500 million these cell types in cnidarians have features that are unique to years. In addition to the evidence for neuropeptide signaling shown cnidarians, despite their morphological similarity to equivalent cell here, there is genetic evidence that Hydra possess glutamatergic and types in other phyla. GABAnergic systems signaling systems (40). The high proportion of cnidarian-specific genes in Table 1 raises the question of the origin of these genes. To answer this question, Interstitial Cell-Specific Genes. Five genes identified in the array we searched the complete genomes or genome reads for two fungi experiment were expressed specifically in the I-cells and germ-line (Aspergillus and Candida), four protists (Tetrahymena, Trypano- cells. They are hmp06154, hm03505, hmp06895, hmp22205, and soma, Dictyosterium, and Monosiga), and the sponge Reneira. For hydmg005secrev068 (SI Table 6 and SI Fig. 4). Hmp06154 is a most nematocyte genes (41 of 51), approximately half the gland cell putative homologue of cyclin C that is associated with RNA genes (11 of 21), and all of the nerve cells genes (9 of 9), no polymerase II and involved in the control of cell cycle (41). homologues could be found (SI Table 3). Thus, the origin of more Hm03505 is a nuclear localized protein containing a domain called than half the lineage-specific genes is presently not clear. nucleoplasmin (SI Fig. 4A). Human nucleoplasmin 2 is a chromatin It is important to note that the conclusions cited above are based decondensation protein that is required for nucleolar organization on highly expressed genes. The mRNA for the screen was isolated and embryonic development (42). Hmp06895 is related to another from whole animals. Weakly expressed transcripts are poorly nuclear protein, thymocyte nuclear protein 1, that is highly con- represented in this total mRNA population. Similarly, transcripts served among vertebrates, plants and bacteria (SI Fig. 4B). from less-abundant cell types such as nerve cells are also not well Hmp22205 (SI Fig. 4C) has no significant homologue in the current represented in total mRNA preparations. Hence, most genes database. Hydmg005secrev068 (SI Fig. 4D and SI Table 2)isa identified in the screen represent strongly expressed genes. This cytoskeletal protein that may be required for the elongation of may be the reason most of the identified genes encoded secretory germ-line cell during mitosis because it contains a spectrin domain proteins. Capsule proteins in nematocytes, secreted enzymes in and a growth-arrest-specific protein 2 domain (43). It is conserved gland and mucous cells, and neuropeptides in nerve cells are all throughout metazoans. The conservation of stem cell-specific genes secreted cell products, which are synthesized in large quantities in between Hydra and human suggests that they may play an important their respective cell types. role in metazoan stem cell behavior. Smaller screens for cell type-specific genes have been carried out previously in Hydra (25, 26, 34). The nematocyte-specific minicol- Epithelial Cell-Specific Genes. Unexpectedly, some genes selected in lagens and spinalin were identified in such screens, as was the gland the screen were not expressed in the I-cell lineage but in epithelial cell-specific protease inhibitor antistasin. A large-scale screen for cells (see below). Close examination of the array data indicated that embryogenesis-specific genes also revealed large numbers of pre- these genes were indeed less strongly expressed in animals lacking viously unidentified genes (45) and many of these encoded secreted the I-cell lineage. This suggests that these genes are up-regulated in proteins as found here. animals containing the I-cell lineage and hence that they could play A second striking feature of the results was the isolation of genes a role in establishing a suitable environment for I-cells in such intact expressed specifically in epithelial cells. Although the screen was animals. These genes were expressed in either ectodermal or designed to identify genes specific to the I-cell lineage, 25% of the endodermal epithelial cells or both (SI Table 7). The expression selected genes turned out to be epithelial cell-specific when tested pattern in ectodermal epithelial cells was not uniform along the by in situ hybridization. Closer examination of the array results body column. For example, tyrosine kinase receptor homologues showed that expression of these genes was indeed up-regulated in exhibited various regional expression patterns: in one case expres- intact animals compared with epithelial Hydra. This suggests that sion was localized in the bottom half of body column and the expression of these genes is required in animals containing the I-cell peduncle (SI Fig. 5A), whereas in another case it was localized lineage. In this context, it is interesting to note that many of these around the base of tentacle and in the hypostome (SI Fig. 5B). epithelial cell-specific genes are involved in intracellular signaling Expression patterns in endodermal epithelial cells also were specific and signal transduction. Another example of such signaling is the and regional. For example, endodermal epithelial cells of the release from epithelial cells of peptide signals regulating nerve cell hypostome expressed hmp02857 and hmp06947 (SI Fig. 5C). differentiation (37). Both observations are consistent with the idea Similarly, the expression of hmp09678 was strongly localized to that, in multicellular animals, there is cross talk between different both the hypostome and the peduncle (SI Fig. 5D). Although the cell lineages to maintain constant proportions. This is particularly number of epithelial cell specific genes selected in the screen was important for Hydra tissue, which is constantly expanding during not large, there appears to be a bias for secreted and transmem- asexual growth and where it has been shown that cell type propor- brane proteins. Several of these are novel receptor tyrosine kinases tions remain constant during growth (2). Raff (46) has proposed that lack high sequence similarity to other metazoan receptor that cellular homeostasis in multicellular animals is mediated by tyrosine kinases. Interestingly, none of them were detected in the growth/survival factors secreted by one cell type, i.e., epithelial cells, genome of Nematostella, a cnidarian lacking the I-cell lineages (SI and required for survival by a second cell type, i.e., I-cells. Sugges- Table 3) (44). It is interesting to speculate that these receptors are tive evidence that signaling via receptor tyrosine kinases may play involved in cross talk between the I-cell lineage and epithelial cells a role in cellular homeostasis in Hydra has come from specific to coordinate the behavior of these two cell populations in intact inhibition experiments. Treatment of Hydra with wortmannin, an animals. inhibitor of PI-3-kinase, which blocks receptor tyrosine kinase signaling, leads to massive apoptosis (47). Emergence of Cnidarian-Specific Genes and Specialized Cell Types. A EVOLUTION striking feature of the present results is the high proportion of cell Materials and Methods type-specific genes, which appear to lack homologues in other Hydra Strains. Hydra magnipapillata strain 105 were cultured at 18°C metazoan phyla. Some of these genes may even be Hydra-specific and fed with Artemia larvae three times per week. Epithelial Hydra because no homologues were found in Nematostella. This suggests were derived from strain 105 by treatment with colchicine (48) and that the emergence of specialized cell types in Hydra was accom- have been kept in the laboratory for Ͼ10 years without changing panied by the evolution of ‘‘novel’’ proteins. Although this was the their phenotype. expected result in the case of nematocytes, which are a novel cell type unique to Cnidaria, it was not expected for gland cells and Construction of 6.6 Thousand cDNA Array. 6,528 clones were obtained nerve cells, which appear to be similar to the corresponding cells from nonredundant ESTs and subjected to PCR. One of the two
Hwang et al. PNAS ͉ September 11, 2007 ͉ vol. 104 ͉ no. 37 ͉ 14739 Downloaded by guest on October 2, 2021 primers that were used to amplify the clones had an amino-link intensity and verify the integrity of the array, the self–self hybrid- modification at the 5Ј site. All PCR products were checked by ization was carried out by using labeled cDNAs of intact Hydra. agarose gel electrophoresis, and only those consisting of a single band were used for the array. PCR products were precipitated with Scanning of the Arrays. Cy3 and Cy5 intensity signals from five 0.3 M sodium acetate and 100% isopropanol and then resuspended independent arrays (in which two were performed by a dye- in 50% DMSO. Slides were printed in-house with an SPBIO spotter swapping experiment) were scanned by using ScanArray BS2000 (Hitachi Software Engineering, Yokohama, Japan) on ␥-amin- (GSi Lumonica, Wilmington, MA) and quantified by using Quan- opropyl silane-coated slides (CMT GAPS; Corning, Corning, NY). tarray (GSi Lumonica). To identify genes that were differentially After printing, all arrays were incubated in an oven at 80°C for 3 h. expressed in normal Hydra as compared with epithelial Hydra, all Four sets of 15 controls were printed at four locations on the slides. array data were normalized and processed by a Lowess print-tip Positive controls included Hydra collagen, 60s ribosomal RNA, normalization method computed by using the SMA (Statistics for elongation factor, choline transporter (weakly expressed gene), and Microarray Analysis) package (49). The final expression differences nicotinic acetylcholine receptor ␣-like-1 (weakly expressed gene). between intact and epithelial Hydra were denoted as log2 intensity ϭ Negative controls included poly(dA)45, luciferase gene, GFP gene, ratio, M log2(R/G), where R and G were the two different dyes. and water. In addition, two exogenous genes, Cab (photosystem I chlorophyll a/b-binding protein) and RCP1 (root cap 1) of Arabi- Sequence Homology Search and Signal Peptide Identification. One dopsis thaliana, were purchased from Stratagene (La Jolla, CA) and hundred fifteen Hydra ESTs (accession nos. listed in SI Table 8) used to monitor the signal intensity differences between Cy3 were queried by using BLASTN against the Hydra genome at the and Cy5. DOE Joint Genome Institute (Berkeley, CA). Either a full-length or longer sequence detected from the genomic sequence was cDNA Labeling and Hybridization. mRNAs of epithelial and intact translated into amino acid sequence. By using BLASTP (50), amino Hydra were extracted and amplified by using a MessageAmp II acid sequences were searched against the GenBank nr protein aRNA kit (Ambion, Austin, TX). By using random hexamers, 2 g database (February, 2007). Signal peptide of protein sequence was of amplified RNAs were labeled in a reverse-transcriptase reaction predicted by using SignalP 3.0 (www.cbs.dtu.dk/services/SignalP/). with Cy3-dUTP/Cy3-dCTP and Cy5-dUTP/Cy5-dCTP (CyDye; Amersham Biosciences, Piscataway, NJ), respectively, or vice versa. Whole-Mount in Situ Hybridization. The protocol used for all in situ The mRNAs of the external controls (1 ng of each), Cab, and RCP1 hybridization experiments was originally described by Grens et al. was also added to each Cy3- and Cy5-labeling reaction. After (51). The concentration of riboprobe used for the hybridization varied from 50 ng/ml to 200 ng/ml. reverse transcription, Cy3- and Cy5-labeled cDNAs were pooled, purified by using Microcom YM-30 (Amicon; Millipore, Billerica, We thank Chi-Chiu Wang and Hiroaki Ono for advice in the early stage MA), and denatured at 95°C for 2 min. Hybridization was carried ϫ of microarray design; Akemi Mizuguchi, Chie Iwamoto, and Ayako out in 5 SSC and 0.5% SDS at 65°C for 40 h. The array slide was Otake for excellent technical assistance with the preparation of microar- treated with a series of washes as follows: 2ϫ SSC, 0.1% SDS; 0.2ϫ ray and whole-mount in situ hybridization; and Kohji Hotta for kindly SSC, 0.1% SDS four times; 0.2ϫ SSC. To monitor the background providing an Apache web server to run the database.
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