A quantitative method for maceration of hydra tissue

A method is described for the maceration (dissociation) of hydra tissue into single cells. The cells have characteristic morphology such that all basic types — epithelial, gland, mucous, interstitial, nematoblast, and nerve — can be distinguished. Criteria are given for identifying each cell type by phase contrast microscopy. It is shown that maceration quantitatively recovers cells from hydra tissue

Ferritin in the fungus Phycomyces

The iron-protein ferritin has been purified from mycelium, sporangiophores, and spores of the fungus Phycomyces blakesleeanus. It has a protein-to-iron ratio of 5, a sedimentation coefficient of 55S, a buoyant density in CsCl of 1.82 g/cm3, and the characteristic morphology of ferritin in the electron microscope. Apoferritin prepared from Phycomyces ferritin has a sedimentation coefficient of 18S and consists of subunits of molecular weight 25,000. In the cytoplasm of Phycomyces, ferritin is located on the surface of lipid droplets (0.5–2.0 µ in diameter) where it forms crystalline monolayers which are conspicuous in electron micrographs of sporangiophore thin-sections. Ferritin is found in all developmental stages of Phycomyces but is concentrated in spores. The level of ferritin iron is regulated by the iron level in the growth medium, a 50-fold increase occurring on iron-supplemented medium

LOSS OF DIFFERENTIATING NEMATOCYTES INDUCED BY REGENERATION AND WOUND HEALING IN HYDRA

Cell death was observed in the nematocyte differentiation pathway in Hydra during head and foot regeneration. This death occurs throughout the regenerating piece, is transient in nature and is selective for committed stenotele and desmoneme precursors. Proliferating nematoblasts are unaffected. Cell death appears to be caused by release of a toxic factor rather than loss of a hormone required for differentiation, since regenerating pieces released a factor that inactivated differentiating nematocytes, and injured animals that had intact head and foot tissue also lost differentiating nematocytes. The inactivated nematocytes are removed by phagocytosis by epitheliomuscular cells

COMMITMENT DURING NEMATOCYTE DIFFERENTIATION IN HYDRA

Nematocytes in Hydra differentiate from interstitial stem cells. Desmonemes differentiate mainly in the distal half of the body column while stenoteles differentiate predominantly in the proximal half. This difference was used to determine the timing of nematocyte-type commitment in the differentiation pathway. Cells were transferred from distal or proximal regions to all positions in the body column to test when the proportion of stenotele and desmoneme differentiation changed to reflect the new environment. In the first experiment, the distal region of the body column was isolated and permitted to regenerate a whole Hydra. In the second experiment, dissociated cells from distal or proximal regions were transplanted into regenerating aggregates of Hydra tissue. Both experiments effectively transferred cells from distal or proximal positions to positions throughout the body column. By comparing the kinetics of stenotele and differentiation with the time required for distal or proximal cells to differentiate stenoteles and desmonemes in accord with their new environment, it was possible to conclude that stenotele and desmoneme commitment occurs during the terminal cell cycle prior to nematocyte differentiation and not at the stem cell. Additional experiments indicated that the number of rounds of cell division preceding differentiation is fixed at the time stem cells enter the nematocyte pathway

Distribution of Interstitial Cells and Differentiating Nematocytes in Nests in Hydra attenuata

We have used tissue maceration to quantitate interstitial cell nests in Hydra attenuata and thiolacetic acid-lead nitrate staining to quantitate differentiating nematocytes. Most I-cell nests have 2, 4, 8, or 16 cells. Differentiating nematocytes occur in nests of 4, 8, 16, and 32. All classes of I-cell nests and nests of dilferentiating nematocytes are abundant in the gastric region. In contrast, the hypostome, basal disk and the regions immediately adjacent to them have no nests of differentiating nematocytes, virtually no nests of 4, 8, or 16 I-cells and reduced numbers of 1 and 2 I-cell nesls. Since nests of 4 or more I-cells are found only where nematocyte differentiation takes place, these nest classes are probably nematocyte precursors. Since differentiation of nerves occurs in the hypostome and basal disk where only single or paired I-cells are found, these size classes include nerve precursors. The disproportionately large number of I-cell nests of 2 suggests that these include the autoreproductive stem cells responsible for maintaining the populations of I-cell derivatives in growing hydra