Ar Matsuyama City may need during the breeding season, and are in sea water aquaria supplied with circulating. Gametes were prepared using standard methods. The embryos were IkB Signaling incubated at 24 C. Millipore filtered seawater, erg Complements with antibiotics was used in the course of observations and experiments. The observation of the fluorescent cells to observe autofluorescence, the embryos with 10% formalin in MFL gel St were attached. The fixative was prepared just before use to obtain intense intrinsic fluorescence. Fixed embryos

, and examined under an epifluorescence microscope. Sometimes the embryos between strips were enclosed by parentheses. The observation chamber was placed on a ger Objekttr, And the embryo was recognized by the c Oral and aboral teas observed.
Images of fluorescence were ltlich by exposure to ultraviolet light or green embryos obtained. Z Select fluorescent cells to the Z To facilitate select of fluorescent cells were treated the embryos with a double concentration of free Ca 2 artificial Phloridzin seawater for 20 min. Treatedembryos then with 10% formalin and sea water on a Objekttr hunter. After the coverslip, the embryos were to some absorption by L Flattened schpapier fixer. The samples were irradiated with UV light or green and the fluorescence images were photographed. Fluorescent cells were quantified from these images. Halving embryo swam before handling, embryos treated briefly with hypertonic sea water, to remove the cilia.
Use of a glass needle, the embryos were at Equator under an inverted microscope and isolated fragments of the H Half of the animals were halved transferred to a 24 well tissue culture plate. Fixed 22-20 h after fertilization, embryo half fragments were exposed to UV light and green and examined. Treatment with DAPT and DAPT SB431542 and SB431542 were purchased from Sigma and dissolved to give St in dimethyl sulfoxide to 10 mmol Stamml L. solutions DAPT was diluted with MFL at 10 L mol L, w While SB431542 was diluted to 5 L mol The use before. Treatment was initiated by suspending embryos in diluted inhibitors. Untreated embryos were used for the controlled On. at the end of treatment, the embryos were rinsed three times with MFL. Measuring the diameter of cell embryos were briefly rinsed with the sea water free Ca2, and to 1 mol of glycine with 1 mmol L L MgCl2.
2 mg is important to the pigment-containing cells to protect lysis. The embryos were incubated for 10 min in the medium, and incubated in individual cells by gentle pipetting. The sample was then set at 10% seawater formalin and a small amount of this cell suspension was mounted on a Objekttr hunter and photographed under a field, a green light and UV illumination. After determining the type of cell by comparing two images of fluorescence, diameter of dissociated cells were prepared using the bright-field images. Whole embryos from embryonic cells were quantified with Carnois, fixer and stained with 1% aceto orcein fixed for a few days. A drop of the sample was mounted on a Objekttr hunter and an equal volume of lactic Acid added. After mixing solutions of L, The embryos were flattened by pressing with a glass coverslip. The distribution of these embryos were photographed, and the number of cells, which was marked received by Z Select nuclei.