Cell type complexity in the basal metazoan Hydra is maintained by both stem cell based mechanisms and transdifferentiation

Understanding the mechanisms controlling the stability of the differentiated cell state is a fundamental problem in biology. To characterize the critical regulatory events that control stem cell behavior and cell plasticity in vivo in an organism at the base of animal evolution, we have generated transgenic Hydra lines [Wittlieb, J., Khalturin, K., Lohmann, J., Anton-Erxleben, F., Bosch, T.C.G., 2006. Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis. Proc. Natl. Acad. Sci. U. S. A. 103, 6208-6211] which express eGFP in one of the differentiated cell types. Here we present a novel line which expresses eGFP specifically in zymogen gland cells. These cells are derivatives of the interstitial stem cell lineage and have previously been found to express two Dickkopf related genes [Augustin, R., Franke, A., Khalturin, K., Kiko, R., Siebert, S. Hemmrich, G., Bosch, T.C.G., 2006. Dickkopf related genes are components of the positional value gradient in Hydra. Dev. Biol. 296 (1), 62-70]. In the present study we have generated transgenic Hydra in which eGFP expression is under control of the promoter of one of them, HyDkk1/2/4 C. Transgenic Hydra recapitulate faithfully the previously described graded activation of HyDkk1/2/4 C expression along the body column, indicating that the promoter contains all elements essential for spatial and temporal control mechanisms. By in vivo monitoring of eGFP+ gland cells, we provide direct evidence for continuous transdifferentiation of zymogen cells into granular mucous cells in the head region. We also show that in this tissue a subpopulation of mucous gland cells directly derives from interstitial stem cells. These findings indicate that both stem cell-based mechanisms and transdifferentiation are involved in normal development and maintenance of cell type complexity in Hydra. The results demonstrate a remarkable plasticity in the differentiation capacity of cells in an organism which diverged before the origin of bilaterian animals.     

Identification of caspases and apoptosis in the simple metazoan Hydra.

Apoptosis is a normal process by which cells die and are eliminated from tissue by phagocytosis [1]. It is involved in regulating cell numbers in adult tissues and in eliminating 'excess' cells during embryogenesis and development. Apoptosis is mediated by activation of caspases, which then cleave a variety of cellular substrates and thereby cause the characteristic morphology of apoptotic cells (rounded cells, condensed chromatin, susceptibility to phagocytosis) [2]. Although apoptosis has been well documented in nematodes, insects and mammals, it is not yet clear how early in evolution apoptosis or its component enzymes arose. In the simple metazoan Hydra vulgaris, cell death regulates cell numbers [3] [4] [5]. In starved animals, for example, epithelial cell proliferation continues at a nearly normal rate although the tissue does not increase in size; the excess cells produced are eliminated by phagocytosis. Cell death can also be induced in wild-type hydra by treatment with colchicine [6] or in a mutant strain (sf-1) by temperature shock [7]. Here, we show that cell death in hydra is morphologically indistinguishable from apoptosis in higher animals, that hydra polyps express two genes with strong homology to members of the caspase 3 family, and that caspase-3-specific enzyme activity accompanies apoptosis in hydra. The occurrence of apoptosis and caspases in a member of the ancient metazoan phylum Cnidaria supports the idea that the invention of apoptosis was an essential feature of the evolution of multicellular animals.     

An IQGAP-related gene is activated during tentacle formation in the simple metazoan Hydra

Differentiation of body column epithelial cells into tentacle epithelial cells in Hydra is accompanied by changes in both cell shape and cell-cell contact. The molecular mechanism by which epithelial cells acquire tentacle cell characteristics is unknown. Here we report that expression of a Hydra homologue of the mammalian IQGAP1 protein is strongly upregulated during tentacle formation. Like mammalian IQGAP, Hydra IQGAP1 contains an N-terminal calponin-homology domain, IQ repeats and a conserved C terminus. In adult polyps a high level of Hydra IQGAP1 mRNA is detected at the basis of tentacles. Consistent with a role in tentacle formation, IQGAP1 expression is activated during head regeneration and budding at a time when tentacles are emerging. The observations support the previous hypothesis that IQGAP proteins are involved in cytoskeletal as well as cell-cell contact rearrangements. Received: 25 January 2000 / Accepted: 2 May 2000     

Disturbing epithelial homeostasis in the metazoan Hydra leads to drastic changes in associated microbiota

Microbes have profound influence on the biology of host tissue. Imbalances in host–microbe interaction underlie many human diseases. Little, however, is known about how epithelial homeostasis affects associated microbial community structure. In Hydra , the epithelium actively shapes its microbial community indicating distinct selective pressures imposed on the epithelium. Here, using a mutant strain of Hydra magnipapillata we eliminated all derivatives of the interstitial stem cell lineage while leaving both epithelial cell lineages intact. By bacterial 16S rRNA gene analysis we observed that removing gland cells and neurones from the epithelium causes significant changes in hydra's microbial community. Absence of interstitial stem cells and nematocytes had no affect on the microbiota. When compared with controls, animals lacking neurones and gland cells showed reduced abundance of β-Proteobacteria accompanied by a significantly increased abundance of a Bacteroidetes bacterium. This previously unrecognized link between cellular tissue composition and microbiota may be applicable to understanding mechanisms controlling host–microbe interaction in other epithelial systems.     

Genome sizes of spiders

Spiders represent a diverse and familiar group of animals, but to date no information has been made available regarding their genome sizes. Arachnids in general have been almost entirely overlooked, and are currently represented by a single tick in the animal genome size data set. The present study provides new genome size estimates for 115 species of spiders from 19 families (all from the infraorder Araneomorphae), thereby adding considerably to the coverage of these arthropods. No clear-cut patterns of variation were detectable even with this relatively large data set, but some interesting avenues for future research have been illuminated by this preliminary survey.     

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.     

Histological and ultrastructural studies of the basal disk of Hydra

Summary The glandulomuscular cells of Hydra are located exclusively in the basal disk. They are derived from epithelio-muscular cells which have been forced proximally. Light and electron microscopical studies show that prior to their destruction and elimination, the transformed epithelio-muscular cells (i.e. the glandulomuscular cells) undergo certain striking morphological and physiological changes. Golgi complexes and elements of rough E. R. increase remarkably in activity, and individually or jointly produce at least six types of morphologically different droplets. One additional type of droplet is thought to originate from neighboring digestive cells. Although the chemical nature of the individual droplets is uncertain, it is known that some are Alcian blue and PAS positive and contain hyaluronic acid. These evidences suggest the presence of an acid mucopolysaccharide material, the adhesive agent which attaches the animal to a substrate. The myonemes contain thick (200 Å in diameter) and thin (60 Å in diameter) filaments as in epithelio-muscular cells. There are also filaments of intermediate sizes and large fibers (770 Å in diameter). The myonemes are oriented radially with respect to the aboral pore and therefore in addition to contributing to the contraction and relaxation of the body column, they apparently regulate the opening and closing of the aboral pore. Although there is no evidence to substantiate the mechanism for transformation of epithelio-muscular cells to glandulomuscular cells as well as cell death of the latter cell types, these problems are discussed briefly.This investigation was supported by The National Science Foundation, Grant Number GB-27395.With the technical assistance of Linda M. Bookman.     

Histological and ultrastructural studies of the basal disk of Hydra

Summary The gastrodermis and mesoglea of the basal disk of Hydra were investigated to conclude a three-part series of papers. The gastrodermis is composed of digestive cells (most predominant cell type), mucous and nerve cells (both immature and fully differentiated). The principal function of the digestive cells appears to be storage of protein, lipid and glycogen reserves which are utilized by neighboring cells. Mucous cells apparently use some of the reserves to synthesize their secretions which lubricate cells and prevent cell damage during egestion of waste through the aboral pore. The function of the gastrodermal nerve cells is uncertain.The mesoglea of the basal disk, contains the same structural components as seen in other regions of the polyp. It is reasonable to assume that it maintains the same function of cell adhesion and migration. As the mesoglea converges on the aboral pore, it loses its structural integrity and cells are sloughed off the column.This investigation was supported by The National Science Foundation, Grant Number GB-27395.     

Structure and expression of STK, a src-related gene in the simple metazoan Hydra attenuata.

Both cDNA clones and a genomic DNA clone encoding a 509-amino-acid protein that is 64% similar to chicken pp60c-src were isolated from the simple metazoan Hydra attenuata. We have designated this gene STK, for src-type kinase. Features of the amino acid sequence of the protein encoded by the STK gene suggest that it is likely to be myristoylated and regulated by phosphorylation in a manner similar to that found for pp60c-src. The genomic sequence encoding the protein was found to be interrupted by at least two introns, one of which was located in a position identical to that of one of the introns in the chicken src gene. The STK gene was expressed during early development of H. attenuata and at high levels in the epithelial cells of adult polyps. Probing of Hydra proteins with an antibody to phosphotyrosine indicated that the major phosphotyrosine-containing protein in H. attenuata may be the STK protein itself. H. attenuata is the simplest organism from which a protein-tyrosine kinase gene has been isolated. The presence of such a gene in the evolutionarily ancient phylum Cnidaria suggests that protein-tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms.     

Genome sizes of mammalian and avian Ureaplasmas

The sizes of the genomes of 12 Ureaplasma strains isolated from six mammalian and one avian species, as determined by pulsed-field gel electrophoresis, varied from 760 to 1,170 kilobase pairs (470 to 723 megadaltons); and these values encompassed the values for the genomes of Ureaplasma urealyticum strains. Variation of at least 54% in the genome sizes restricts the usefulness of this taxonomic criterion for the genus Ureaplasma.     

Expression pattern of the homeobox gene Not in the basal metazoan Trichoplax adhaerens

The homeobox gene Not is highly conserved in Xenopus, chicken and zebrafish with an apparent role in notochord formation, which inspired the name of this distinct subfamily. Interestingly, Not genes are also well conserved in animals without notochord such as sea urchins, Drosophila or even Hydra, but appear to be highly derived in mammals. A search for homeobox genes in the placozoan Trichoplax adhaerens, one of the simplest organisms available today, revealed only two homeobox genes: a Not homologue and the previously described gene Trox-2, which is most similar to the Gsx subfamily of the Hox/ParaHox cluster genes. Not has a unique expression profile in Trichoplax. It is highly expressed in folds of intact animals and in the wounds of regenerating animals. The dynamic expression pattern of Trichoplax Not is discussed in comparison with the invariable expression pattern of Trox-2 and the putative secreted protein Secp1. The high sequence conservation of Not from Trichoplax to lower vertebrates, but not to mammals, represents a rare example of an apparent gene decay in the lineage leading to humans.     

Genome sizes for all genera of Cycadales

Nuclear DNA content (2C) is reported for all genera of the Cycadales, using flow cytometry with propidium iodide. Nuclear DNA content ranges from 24 to 64 pg in cycads. This implies that the largest genome contains roughly 40 × 10(9) more base pairs than the smallest genome. The narrow range in nuclear DNA content within a genus is remarkable for such an old group. Furthermore, 42 of the 58 plants measured, covering five genera, have 18 chromosomes. They vary from 36.1 to 64.7 pg, covering the whole range of genome sizes (excluding the genome of Cycas). Hence, their does not seem to be a correlation between genome size and the number of chromosomes.     

Inductive interactions and embryonic equivalence groups in a basal metazoan, the ctenophore Mnemiopsis leidyi

Ctenophores undergo locomotion via the metachronal beating of eight longitudinally arrayed rows of comb plate cilia. These cilia are normally derived from two embryonic lineages, which include both daughters of the four e1 micromeres (e11 and e12) and a single daughter of the four m1 micromeres (the m12 micromeres). Although the e1 lineage is established autonomously, the m1 lineage requires an inductive interaction from the e1 lineage to contribute to comb plate formation. Successive removal of the e1 progeny at later stages of development indicates that this interaction takes place after the 32-cell stage and likely proceeds over a prolonged period of development. Normally, the e1, cell lies in closest proximity to the m12 cell that generates comb plate cilia; however, either of the e1 daughters (e11 or e12) is capable of emitting the signal required for m1 descendants to form comb plates. Previous cell lineage analyses indicate that the two e1 daughters generate the same suite of cell fates. On the other hand, the m1 daughters (m11 and m12) normally give rise to different cell fates. Reciprocal m1 daughter deletions show that in the absence of one daughter, the other cell can generate all the cell types normally formed by the missing cell. Together, these findings demonstrate that the two m1 daughters (m11 and m12) represent an embryonic equivalence group or field and that differences in the fates of the two m1 daughters are normally controlled by cell-cell interactions. These combined properties of ctenophore development, including the utilization of deterministic cleavage divisions, inductive interactions, and the establishment of embryonic fields or equivalence groups, are remarkably similar to those present in the development of various bilaterian metazoans.     

Sex chromosomes of basal placental mammals

Placental (eutherian) mammals are currently classified into four superordinal clades (Afrotheria, Xenarthra, Laurasiatheria and Supraprimates) of which one, the Afrotheria (a unique lineage of African origin), is generally considered to be basal. Therefore, Afrotheria provide a pivotal evolutionary link for studying fundamental differences between the sex chromosomes of human/mouse (both representatives of Supraprimates and the index species for studies of sex chromosomes) and those of the distantly related marsupials. In this study, we use female fibroblasts to investigate classical features of X chromosome inactivation including replication timing of the X chromosomes and Barr body formation. We also examine LINE-1 accumulation on the X chromosomes of representative afrotherians and look for evidence of a pseudoautosomal region (PAR). Our results demonstrate that asynchronous replication of the X chromosomes is common to Afrotheria, as with other mammals, and Barr body formation is observed across all Placentalia, suggesting that mechanisms controlling this evolved before their radiation. Finally, we provide evidence of a PAR (which marsupials lack) and demonstrate that LINE1 is accumulated on the afrotherian and xenarthran X, although this is probably not due to transposition events in a common ancestor, but rather ongoing selection to retain recently inserted LINE1 on the X.     

The replicon revisited: an old model learns new tricks in metazoan chromosomes

The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are com- posed of non-redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.     

Genome sizes in afrotheria, xenarthra, euarchontoglires, and laurasiatheria

Topical literature and Web site databases provide genome sizes for approximately 4,000 animal species, invertebrates and vertebrates, 330 of which are mammals. We provide the genome size for 67 mammalian species, including 51 never reported before. Knowledge of genome size facilitates sequencing projects. The data presented here encompassed 5 Metatheria (order Didelphimorphia) and 62 Eutheria: 15 Xenarthra, 24 Euarchontoglires (Rodentia), as well as 23 Laurasiatheria (22 Chiroptera and 1 species from Perissodactyla). Already available karyotypes supplement the haploid nuclear DNA contents of the respective species. Thus, we established the first comprehensive set of genome size measurements for 15 Xenarthra species (armadillos) and for 12 house-mouse species; each group was previously represented by only one species. The Xenarthra exhibited much larger genomes than the modal 3 pg DNA known for mammals. Within the genus Mus, genome sizes varied between 2.98 pg and 3.68 pg. The 22 bat species we measured support the low 2.63 pg modal value for Chiroptera. In general, the genomes of Euarchontoglires and Laurasiatheria were found being smaller than those of (Afrotheria and) Xenarthra. Interspecific variation in genome sizes is discussed with particular attention to repetitive elements, which probably promoted the adaptation of extant mammals to their environment.     

Quantitative analysis of epithelial cell aggregation in the simple metazoan Hydra reveals a switch from homotypic to heterotypic cell interactions

Hydra, a member of the diploblastic phylum Cnidaria, exhibits the most basic type of organized metazoan tissues. Two unicellular sheets of polarized epithelial cells - ectoderm and endoderm - form a double layer throughout the body column. The double layer can be reestablished from single-cell suspensions by tissue-specific cell-sorting processes. However, the underlying pattern of interactions between ectodermal and endodermal epithelial cells responsible for double-layer formation is unclear. By analyzing cell interactions in a quantitative adhesion assay using mechanically dissociated Hydra epithelial cells, we show that aggregation proceeds in two steps. First, homotypic interactions within ectodermal epithelial cells (ecto-ecto) and within endodermal epithelial cells (endo-endo) form homotypic cell clusters. Second, at an aggregate size of about ten epithelial cells/cluster, ectodermal and endodermal clusters start to form heterotypic aggregates. Homotypic ecto-ecto interactions are inhibited by a polyclonal anti-Hydra membrane antiserum, and under these conditions homotypic endo-endo interactions do not proceed beyond a size of about ten epithelial cells/cluster. These data suggest that homotypic cell clusters reduce their initial homotypic affinity and acquire a new heterotypic affinity. A link between cell adhesion and cell signaling in early Hydra aggregates is discussed.     

Sequence analysis of a nuclear pore complex protein in a lower metazoan: nucleoporin p62 of the coelenterate Hydra vulgaris

We have isolated cDNA clones and polymerase chain reaction products containing the entire coding region for nuclear pore complex protein p62 of a lower metazoan, the freshwater polyp Hydra vulgaris (Hv), and compared the deduced amino acid (aa) sequence with those of the vertebrate and yeast homologues. The open reading frame defines a protein of 534 aa, corresponding to a molecular mass of 56 072 Da and an isoelectric point (pI) of 5.0. Secondary structure predictions revealed the division into two domains, as previously observed in vertebrate p62: the N-terminal domain (aa 1–338) with a pI of 10.7 contains the evolutionarily conserved repeated pentapeptide motif, XFXFG, known from several nucleoporins, a low content of charged aa (3.25%) and a high degree of hydroxy aa (40.2%). Otherwise, sequence identity between the N-terminal domains of p62 from Hv and various vertebrates is rather low (28–34%). By contrast, the C-terminal domain with a pI of 4.6 is richer in charged aa (36.7%), exhibits heptad repeats typical for α-helices organized in coiled-coils and shows a high sequence identity with amphibian (53%) and mammalian p62 (55%). Differences and similarities between p62 of Hv and vertebrates, and between Hv p62 and its putative homologues from the budding yeast, Saccharomyces cerivisiae, and the fission yeast, Schizosaccharomyces pombe, are discussed.