Tsutomu Sugiyama

GENETIC ANALYSIS OF DEVELOPMENTAL MECHANISMS IN HYDRA I. SEXUAL REPRODUCTION OF HYDRA MAGNIPAPILLATA AND ISOLATION OF MUTANTS

Hydra magnipapillata strains collected from various localities in Japan were induced to reproduce sexually.

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and se-quenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Identification of a Hydra homologue of the β-catenin/plakoglobin/armadillo gene family

The beta-catenin/plakoglobin/armadillo gene family encodes a group of highly conserved proteins which play important roles in cadherin-mediated cell adhesion and in signal transduction mechanisms involved in regulating development. This gene family previously had been isolated only from higher metazoans. Here, we describe the isolation and characterization of a beta-catenin (beta Ctn) homologue from Hydra magnipapillata, a diploblastic lower metazoan. Comparison of the putative amino acid (aa) sequence of Hydra beta Ctn, with its homologues in higher metazoans, shows that a repeating 42-aa motif present in its central domain is highly conserved throughout the metazoa. This suggests that beta Ctn appeared very early in metazoan evolution, possibly when primitive multicellular animals started to form epithelial cell layers.

Isolation and characterization of a mini-collagen gene encoding a nematocyst capsule protein from a reef-building coral, acropora donei

Genomic and cDNA clones of a mcol gene encoding mini-collagen (MCOL), a nematocyst capsule protein, have been isolated from a reef-building coral, Acropora donei (Anthozoa). The gene and its flanking regions, comprising 5382 bp and covering three exons and two introns, were sequenced. Exons 2 and 3 together have an open reading frame which can encode a MCOL of 176 amino acids (aa). The coral MCOL has all the characteristic regions present in the four hydra MCOL specified by the four mcol cDNA clones previously isolated from Hydra magnipapillata (Hydrozoa) by Kurz et al. [J. Cell Biol. 115 (1991) 1159-1169], including a central Gly-Xaa-Yaa region and flanking Pro-rich and Cys-repeat regions. This observation suggests that a mcol family is highly conserved in Anthozoa and Hydrozoa, and also that the characteristic regions present in MCOL are essential for the structure and function of these peptides.

Genetic analysis of developmental mechanisms in hydra. X. Morphogenetic potentials of a regeneration-deficient strain (reg-16).

Morphogenetic potentials of hydra tissue involved in head or foot formation were examined in a standard wild-type strain (105) and a mutant strain (reg-16) which has a very low head regenerative but a nearly normal foot regenerative capacity (T. Sugiyama and T. Fujisawa, 1977, J. Embryol. Exp. Morphol. 42, 65-77). Hydra tissue has two types of morphogenetic potentials to control head formation: the potential to form head structure (head-activation potential) and the potential to inhibit head formation (head-inhibition potential). It also has two types of morphogenetic potentials to control foot formation: foot-activation and foot-inhibition potentials. A lateral tissue grafting procedure (G. Webster and L. Wolpert, 1966, J. Embryol. Exp. Morphol. 16, 91-104), was used to examine and compare the relative levels of these potentials in the normal and the mutant strains. The potential levels were examined along the body axis of the intact animals and also in the regenerating animals after head removal. The results obtained show that the potentials involved in head formation are highly abnormal, whereas the potentials involved in foot formation are apparently normal in the mutant strain (reg-16). This suggests that the abnormal potentials are related in some way to, and may be responsible for, the reduced head regenerative capacity in the mutant strain reg-16.

Control of planula migration by LWamide and RFamide neuropeptides in Hydractinia echinata.

SUMMARY Planula larvae of Hydractinia echinata (Cnidaria) settled on a substratum migrate toward light. We observed that planula migration is not a continuous process. Instead, it consists of repeating cycles of active migration (about 8 min on average) and inactive resting periods (about 26 min on average). This pattern of periodic migration is regulated by LWamide and RFamide neuropeptides. LWamide (10-8 mol l-1) stimulates migration primarily by making the active periods longer, whereas RFamide (10-7 mol l-1) inhibits migration by blocking the initiation and also shortening the length of the active periods. Since sensory neurons containing LWamides and RFamides are present in planula larvae, it appears likely that planula migration is regulated by the release of endogenous neuropeptides in response to environmental cues.

Genetic analysis of developmental mechanisms in hydra: XVIII. Mechanism for elimination of the interstitial cell lineage in the mutant strain sf-1

The interstitial cell lineage in mutant strain sf-1 of hydra is temperature sensitive and is lost rapidly from tissue when the animal is cultured at a restrictive temperature of 23 degrees C or higher. The mechanism responsible for this cell elimination process was investigated. Sf-1 polyps were treated at a restrictive temperature of 27 degrees C for varying lengths of time, their tissues were macerated, and the resultant dissociated cells were examined for evidence of phagocytosis after Feulgen staining. It was found that large phagocytic vacuoles were present in the cytoplasm of some epithelial cells. These vacuoles contained partially degraded cells, whose nuclei had highly-condensed and intensely Feulgen-positive chromatin granules. This indicated that, as in colchicine-treated (Campbell, 1976) or starved (Bosch and David, 1984) wild-type hydra, the epithelial cells in strain sf-1 engulfed and disintegrated other cells in the phagocytic vacuoles. The incidence of phagocytosis was higher in sf-1 tissue maintained at elevated temperature than in sf-1 tissue maintained at normal temperature. However, the observed incidence was relatively low (maximally 0.14 phagocytosed cells per epithelial cell) and appeared to be too low to account for the very rapid interstitial cell loss occurring in this strain. We concluded that elimination of the interstitial cell lineage at a restrictive temperature in strain sf-1 takes place in part by phagocytosis and in part by other yet-unidentified mechanisms (cf., Marcum et al., 1980).

Genetic analysis of developmental mechanisms in hydra: XIV. Identification of the cell lineages responsible for the altered developmental gradients in a mutant strain, reg-16

Abstract The developmental gradients of six chimeric strains of hydra produced from a normal strain (105) and a regeneration-deficient strain (reg-16) were analyzed. The reg-16 mutant has been shown to have a lower gradient of head-activation potential and a higher gradient of head-inhibition potential than the normal 105 strain. The chimeric animals consisted of different combinations of the three self-renewing cell lineages found in hydra (the ectodermal and endodermal epithelial cell lineages and the interstitial cell lineage) from each of the parental origins. To identify the cell lineages responsible for the abnormal gradients in reg-16, the head-activation and head-inhibition potentials of these cell lineage chimeras were assayed by lateral transplantation of tissue. The results obtained have provided evidence which indicates that the defect responsible for the low head-activation potential in reg-16 resides in its ectodermal and endodermal epithelial cell lineages, whereas the defect responsible for its high head-inhibition potential resides in its endodermal epithelial and interstitial cell lineages. The cellular localization of these defects is not identical but very similar to the cellular localization of the regenerative defects in reg-16. This finding is consistent with and supports the view that the abnormalities of the developmental gradients are correlated to the reduced head regenerative capacity in reg-16.

Nematocyte differentiation in hydra

Nematocyte differentiation from interstitial stem cells in hydra occurs in a highly position-dependent manner along the body axis. The results of the studies summarized here have shown that the morphogenetic factors involved in head formation are probably not responsible for this. Whether the morphogenetic factors involved in foot formation are responsible has not been determined. A new factor, presumably unrelated to any of the known morphogens, has been identified which specifically inhibits the developing nematoblasts to differentiate into stenoteles. This factor is present in a gradient along the body column, and appears to be responsible, at least in part, for producing position-dependent nematocyte differentiation. Nematoblasts which normally differentiate into one nematocyte type can be altered to differentiate into another by means of regeneration or treatment with stenotele inhibitor. This alteration occurs near the S/G2 boundary in the terminal cell cycle in the nematocyte differentiation pathway. It appears that either the nematoblasts are not committed to any specific nematocyte pathway until this critical time, or the nematoblasts committed to differentiate into a specific type can transdifferentiate into another type at this step.

Genetic analysis of developmental mechanisms in hydra. XIII: Identification of the cell lineages responsible for the reduced regenerative capacity in a mutant strain, reg-16

Abstract Chimeric hydra strains were constructed from a normal strain (105) and a mutant strain (reg-16) which has a reduced regenerative capacity and altered developmental gradients along its body column. Hydra tissue consists of three self-proliferating cell lineages: the ectodermal epithelial, the endodermal epithelial, and the interstitial cell lineages. By using the interstitial cell elimination and reintroduction method of B. A. Marcum and R. D. Campbell (1978b, J. Cell. Sci. 32, 233–247) and the epithelial migration method of N. Wanek and R. D. Campbell (1982, J. Exp. Zool. 221, 37–47) it was possible to produce six different combinations of the three cell lineages from 105 and reg-16 in chimeric animals. Several developmental properties (population growth rate, budding rate, bud developmental time, tentacle number per polyp, and polyp size) of the chimeric strains were examined and compared to those of each other and the two parental strains. The results provided evidence which suggests that, in general, the cell lineages from the two parental strains are able to work in harmony in the various chimeric combinations. The chimeric strains did not show signs of any major cell lineage incompatibilities. The head regeneration ability of the chimeric strains was determined to more fully characterize the regeneration deficiency of the reg-16 mutant strain, and in turn to gain insight into the controlling mechanisms underlying normal head regeneration in hydra. Two defects were identified. One affects the initiation of head regeneration, and it appears to be located in the ectodermal and endodermal epithelial cell lineages. The other defect affects the number of tentacles regenerated and is located in the endodermal epithelial cell lineage.