Response to insulin and the expression pattern of a gene encoding an insulin receptor homologue suggest a role for an insulin-like molecule in regulating growth and patterning in Hydra

 A gene encoding a receptor protein-tyrosine kinase closely related to the vertebrate insulin receptor has been identified in the Cnidarian Hydra vulgaris. The gene is expressed in both epithelial layers of the adult polyp. A particularly high level of expression is seen in the ectoderm of the proximal portions of the tentacles and in a ring of ectodermal cells at the border between the foot basal disk and body column. The expression pattern of the gene in asexual buds is dynamic; expression is high throughout the newly emerging bud but the area of high expression becomes restricted to the apex as the bud lengthens. When the bud begins hypostome and tentacle formation, a high level of expression appears at the bases of the emerging tentacles. Finally, a ring of high expression appears just above the foot of the bud, completing the pattern seen in the adult polyp. The presence of this receptor and its pattern of expression suggested that an endogenous molecule related to insulin plays a role in regulating cell division in the body column and in differentiation of the tentacle and foot cells in Hydra, with the switch between the two being determined by the level of the receptor. Treatment of Hydra polyps with mammalian insulin caused an increase in the number of ectodermal and endodermal cells undergoing DNA synthesis. Received: 19 April 1996 / Accepted: 5 July 1996     

Totipotent migratory stem cells in a hydroid

Hydroids, members of the most ancient eumetazoan phylum, the Cnidaria, harbor multipotent, migratory stem cells lodged in interstitial spaces of epithelial cells and are therefore referred to as interstitial cells or i-cells. According to traditional understanding, based on studies in Hydra, these i-cells give rise to several cell types such as stinging cells, nerve cells, and germ cells, but not to ectodermal and endodermal epithelial cells; these are considered to constitute separate cell lineages. We show here that, in Hydractinia, the developmental potential of these migratory stem cells is wider than previously anticipated. We eliminated the i-cells from subcloned wild-type animals and subsequently introduced i-cells from mutant clones and vice versa. The mutant donors and the wild-type recipients differed in their sex, growth pattern, and morphology. With time, the recipient underwent a complete conversion into the phenotype and genotype of the donor. Thus, under these experimental conditions the interstitial stem cells of Hydractinia exhibit totipotency.     

Regulation of interstitial cell differentiation in Hydra attenuata. I. Homeostatic control of interstitial cell population size.

Mechanisms regulating the population size of the multipotent interstitial cell (i-cell) in Hydra attenuata were investigated. Treatment of animals with 3 cycles of a regime of 24 h in 10-2 M hydroxyurea (HU) alternated with 12 h in culture medium selectively killed 95-99% of the i-cells, but had little effect on the epithelial cells. The i-cell population recovered to the normal i-cell:epithelial cell ratio of I:I within 35 days. Continuous labelling experiments with [3H]thymidine indicate that the recovery of the i-cell population is not due to a change in the length of the cell cycle of either the epithelial cells or the interstitial cells. In control animals 60% of the i-cell population undergo division daily while 40% undergo differentiation. Quantification of the cell types of HU-treated animals indicates that a greater fraction of the i-cells were dividing and fewer differentiating into nematocytes during the first 2 weeks of the recovery after HU treatment. Therefore, the mechanism for recovery involves a shift of the 60:40 division:differentiation ratio of i-cells towards a higher fraction in division until the normal population size of the i-cells is regained. This homeostatic mechanism represents one of the influences affecting i-cell differentiation.     

In the multiheaded strain (mh-1) of Hydra magnipapillata the ectodermal epithelial cells are responsible for the formation of additional heads and the endodermal epithelial cells for the reduced ability to regenerate a foot

Hydra consist of three self-renewing cell lineages: the ectodermal epithelial, endodermal epithelial and interstitial cell lineages. The role of these cell lineages in head formation and foot regeneration in Hydra magnipapillata was studied by comparing the multiheaded strain mh-1 with the wild-type. Adult polyps of this strain show a reduced ability to regenerate a foot in the apical body half several days before additional heads are formed there. Cell lineage chimeras were produced, and it was found that in mh-1, the ectodermal epithelial cell lineage is responsible for the formation of additional heads, whereas the endodermal epithelial cell lineage and, to a lesser extent, the derivatives of the interstitial cell lineage, are responsible for the reduced ability of foot regeneration.     

RACK1, a PKC targeting protein, is exclusively localized to basal airway epithelial cells.

The novel isoform of protein kinase C (PKC), PKCepsilon, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCepsilon signaling in the airways and the potential role of the PKCepsilon-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, beta-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCepsilon signaling in the basal airway cell may involve RACK1; however, PKCepsilon regulation in ciliated cells uses RACK1-independent pathways.     

A novel neuropeptide (FRamide) family identified by a peptidomic approach in Hydra magnipapillata

In the course of systematic identification of peptide signaling molecules combined with the expressed sequence tag database from Hydra, we have identified a novel neuropeptide family that consists of two members with FRamide at the C-terminus; FRamide-1 (IPTGTLIFRamide) and FRamide-2 (APGSLLFRamide). The precursor sequence deduced from cDNA contained a single copy each of FRamide-1 and FRamide-2 precursor sequences. Expression analysis by whole-mount in situ hybridization showed that the gene was expressed in a subpopulation of neurons that were distributed throughout the body from tentacles to basal disk. Double in situ hybridization analysis showed that the expressing cell population was further subdivided into one population consisting of neurons expressing both the FRamide and Hym176 (neuropeptide) genes and the other consisting of neurons expressing only the FRamide gene. FRamide-1 evoked elongation of the body column of 'epithelial' Hydra that was composed of epithelial cells and gland cells but lacked all the cells in the interstitial stem cell lineage, including neurons. In contrast, FRamide-2 evoked body column contraction. These results suggest that both of the neuropeptides directly act on epithelial cells as neurotransmitters and regulate body movement in an axial direction.     

Upregulation of a Hydra vulgaris cPKC gene is tightly coupled to the differentiation of head structures

 Two different cDNA clones from Hydra (HvPKC1a and HvPKC1b) were characterized, which encode members of the cPKC family of protein kinase Cs (PKCs). The two predicted proteins differ only in their amino-terminal sequences and thus probably represent the products of alternatively spliced mRNAs from a single gene. In situ hybridization with a probe recognizing sequences in common between the two mRNAs detects HvPKC1 RNA in all parts of the adult polyp except the foot. The mRNA is contained in ecto- and endodermal epithelial cells as well as a certain subset of gland cells and pairs of interstitial cells. During head and foot formation, induced by either regeneration, budding, lithium treatment or repeated application of a diacylglycerol, HvPKC1 expression is upregulated immediately prior to the evagination of tentacles and downregulated by foot formation. Although PKC activity is clearly inducible in vitro by diacylglycerol and a tumour promoting phorbol ester, structural features detected in the regulatory domains of HvPKC1a and 1b indicate that endogenous activators for Hydra PKC might differ from those of other organisms. The results corroborate the hypothesis that signal transduction systems using protein kinase C are key elements controlling the formation of head structures in Hydra. Received: 2 May 1997 / Accepted: 4 December 1997     

Oogenesis in hydra. II. Cytophotometric determination of DNA concentration in the nuclei of somatic and sex cells

During sexual reproduction in Hydra, interstitial cells in the female sex zone of the body (i-cells) undergo mitotic division and form a thickening in the epiderm. The proliferation of i-cells is accompanied by the increase of cytoplasm volume and by the appearance in the cytoplasm of a great number of membranous structures (rough endoplasmic reticulum, Golgi apparatus and mitochondria), enzymatic granules, lipid inclusions and glycogen. All cells of the epidermal thickening soon (in approximately twenty four hours) acquire the characteristics of typical phagocytes. However it is the cell situated inside the group of syncytially connected ones and adjacent to mesogloea that begins to grow rapidly and phagocytize surrounding cells. The cells of the epidermal thickening, though they are often given the name of oogonia, were found to have a tetraploid DNA content in their nuclei. The presence of four unseparated centrioles of equal size suggests that all preparatory processes for division were completed. A conclusion was drawn that cells of the epidermal thickening undergo premeiotic DNA synthesis prior to their phagocytizing by the growing oocyte and, thus, are oocytes themselves. The oogonial stage in Hydra coincides with the early period of mitotic reproduction of i-cells. The data obtained are discussed from the viewpoint of the formation of the accessory gonad apparatus.     

Expression and developmental regulation of the Hydra-RFamide and Hydra-LWamide preprohormone genes in Hydra: evidence for transient phases of head formation

Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, we found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head showed that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. Our head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, which they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra.     

Somatic tissue–male germ cell barrier in Hydra viridis (hydrozoa,coelenterata)

In Hydra viridis, cordons of male germ cells lie in gonadal compartments, which are enlarged spaces between the elongated and “spongy” epidermal cells. The germ cells are surrounded by these cells, except for small areas where the interstitial cells and spermatogonia are in direct contact with the mesoglea. Cells from both epidermis and gastrodermis project cytoplasm into the mesoglea, where they contact each other and form trans-mesogleal bridges. The latter exhibit gap junctions, which are particularly abundant at the spermary region. Here, the mesoglea is thinner then elsewhere in the body. Both epithelia are joined by septate junctions toward their apical ends, which are totally impermeable to horseradish peroxidase (HRP). HRP gained entry to the cells of both epithelia by pinocytosis. Incorporation into the cells was high at the basal disk, in the tentacles, and in the mesoglea in the lower part of the body stalk. The tracer was never found within the gonadal space of the testis during spermatogenesis. In mature spermaries during spermiation, tracer-filled intracellular vacuoles fused with the gonadal spaces as the thin cytoplasmic columns of the epidermal cells ruptured; HRP thus gained access to the germ cells. During spermatogenesis, germ cells of Hydra viridis are in a closed compartment. The barrier that controls the access of metabolites to the germ cells is formed by epidermal cells, thinned-out mesoglea, and numerous transmesogleal interepithelial bridges. The presumed role of the barrier is the control of the environment (1) where interstitial cells are differentiating into spermatogonia and meiosis occurs and (2) in which ripe spermatozoa are kept immotile until spermiation.     

CnNK-2,an NK-2 Homeobox Gene, Has a Role in Patterning the Basal End of the Axis in Hydra

NK-2-class homeobox genes have been identified in a variety of metazoans, from sponges to arthropods and vertebrates, and have been shown to play roles in a variety of cell and tissue specifications. Here we describe the characterization of the NK-2 homologCnNK-2fromHydra vulgaris,a freshwater cnidarian.CnNK-2expression is restricted to the endodermal epithelial cells of hydra and is primarily in the peduncle, the lower end of the body column. In some species it is graded along the apical–basal axis with a maximum in the basal tissue of the lower peduncle, adjacent to the foot.CnNK-2expression invariably precedes foot formation as part of the normal tissue dynamics of the adult as well as during asexual reproduction by budding, foot regeneration, or ectopic foot formation. Manipulations which alter the gradient of positional value along this axis affectCnNK-2expression in a manner which indicates that expression of this gene is closely linked to the gradient. The normal and altered patterns of expression of this gene extend the understanding of the regulation of foot formation in hydra.     

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.     

Regulation in the numbers of tentacles of aggregated hydra cells

Head formation was investigated during regeneration of dissociated and aggregated cells of Hydra magnipapillata. The surface area measured at the hollowing stage was found to be a useful quantity for characterizing the size of an aggregate. Four kinds of aggregates were examined, using tissue originating from (1) whole animals, (2) apical halves, (3) decapitated animals, and (4) decapitated animals allowed to regenerate for several hours before dissociation. For aggregate types (1), (2), and (4), not all the tentacles observed at an intermediate stage of the regeneration process were localized around hypostomes: the number of such body tentacles at the intermediate stage was comparable to that of the hypostomal tentacles and was approximately proportional to the surface area. These results and others suggest that the formation of body tentacles takes place independently of hypostome formation. However, for aggregate type (3), most of the tentacles appearing at the intermediate stage were hypostomal. The correlation between the surface area and the number of tentacles at the steady state apparently resulted from a regulation process by which body tentacles decreased and hypostomal tentacles increased. It is considered that the number of body tentacles appearing at an intermediate stage of regeneration would depend on the initial level of head-activation potential and that body tentacles are formed by the local fluctuation of head-activation potential.     

Enhancement of foot formation in Hydra by a novel epitheliopeptide, Hym-323

During the course of a systematic screening of peptide signaling molecules in Hydra magnipapillata, a novel peptide, Hym-323, which enhances foot regeneration was identified. The peptide is 16 amino acids long, and is encoded in the precursor protein as a single copy. Northern blot analysis, in situ hybridization analysis and immunohistochemistry showed that it was expressed in both ectodermal and endodermal epithelial cells throughout the body, except for the basal disk and the head region. The peptide enhanced foot regeneration by acting on epithelial cells. Lateral transplantation experiments indicated that the foot activation potential was increased in the peptide-treated tissue. These results suggest that Hym-323 is a peptide involved in a foot-patterning process in Hydra.     

GFP expression in Hydra: lessons from the particle gun

The cnidarian Hydra is an important model organism to study pattern formation and tem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have hot been accessible to gene transfection studies, we have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.     

Patterning processes in aggregates of Hydra cells visualized with the monoclonal antibody, TS19

The monoclonal antibody, TS19, (Heimfeld et al., 1985), labels the apical surface of ectodermal epithelial cells of tentacles and lower peduncles in Hydra. To investigate the patterning process in a tissue whose original pattern was completely destroyed, the TS19 staining pattern was examined in developing aggregates of Hydra cells. Two types of aggregates were prepared. G-aggregates were made from tissue of the gastric portion of animals and RG-aggregates from gastric tissue allowed to regenerate for 24 hr before making aggregates. G-aggregates were initially TS19-negative, and later dim and uniformly TS19-positive. Thereafter, TS19 staining broke up into brightly stained and unstained regions. The brightly staining regions developed into head or foot structures. The TS19 pattern in RG-aggregates developed differently. Since the initial aggregates contained cells of regenerating tips, they started with TS19-positive cells as well as TS19-negative cells. The numbers of brightly staining TS19-positive cells increased with time. Some patches of these cells developed into head or foot structures, while others did not. These results and a simulation using a reaction-diffusion model suggest that the changes in activation levels affected the temporal changes in the pattern of TS19 staining, and that the de novo pattern formation in hydra can be explained in terms of a process involving activation and inhibition properties.     

Hym-301, a novel peptide, regulates the number of tentacles formed in hydra

Hym-301 is a peptide that was discovered as part of a project aimed at isolating novel peptides from hydra. We have isolated and characterized the gene Hym-301, which encodes this peptide. In an adult, the gene is expressed in the ectoderm of the tentacle zone and hypostome, but not in the tentacles. It is also expressed in the developing head during bud formation and head regeneration. Treatment of regenerating heads with the peptide resulted in an increase in the number of tentacles formed, while treatment with Hym-301 dsRNA resulted in a reduction of tentacles formed as the head developed during bud formation or head regeneration. The expression patterns plus these manipulations indicate the gene has a role in tentacle formation. Furthermore, treatment of epithelial animals indicates the gene directly affects the epithelial cells that form the tentacles. Raising the head activation gradient, a morphogenetic gradient that controls axial patterning in hydra, throughout the body column results in extending the range of Hym-301 expression down the body column. This indicates the range of expression of the gene appears to be controlled by this gradient. Thus, Hym-301 is involved in axial patterning in hydra, and specifically in the regulation of the number of tentacles formed.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.