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Paracrine regulation of epithelial cell proliferation in developing lung
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364 TIME-LAPSE CINEMICROGRAPHY DEMONSTRATES ACTIVE MIGRATION BY MESODERM CELLS IN PRIMITIVE-STREAK-STAGE MOUSE EMBRYOS. N.NAKATSUJI. Meiji Institute of Health Science, Naruta, Odawara 250, Japan, and MRC Mammalian Development Unit, University College London, London NWI 2HE, UK. The process of mesoderm formation in the primitive streak stage mouse embryo was recorded and analysed by using in vitro culture of the whole embryo and time-lapse cinemicrography. Seven-daysold mouse embryos were freed from the uterus and decidua and cultured in a small chamber on a heated stage. Use of differential-interference-contrast optics and relative transparency of the embryo enable direct observation of the individual mesoderm cells that are actively migrating between the epiblast and primary endoderm layers. They move by extending active cell processes of slender or broad shapes. They translocate at a speed of 0.5 - 1.0 micron/min, predominantly in the direction away from the primitive streak and toward the anterior and lateral part of the embryo. Mitosis occurred frequently, preceded by the bleb formation and rounding up of the cell body, and succeeded by the bleb formation and separation of the daughter cells.
OBSTRUCTION OF URINE DRAINAGE FACILITATES BUT DOES NOT CAUSE RENAL DYSPLASIA. M. MAIZELS and S.B. SIMPSON, Jr. Division of Urology, Children's Memorial Hospital and Department of Human Biology, Northwestern University, Chicago, Illinois, 60614, USA. Isolated kidney blastemas of the chick embryo were treated by: ligation of the ureter (79), tissue culture for 2-3 days (14) to reduce the condensed metanephrogenic mesenchyme, or ligation of the ureter + tissue culture (6). The blastemas were then permitted to develop as grafts on the chorio-allantoic membrane. Dysplasia, as diagnosed by the presence of primitive ducts was seen in kidneys which had been treated by: ligation of the ureter (0), tissue culture (14%), and ligation of the ureter + tissue culture (83%). Ligation of the ureter alone does not lead to dysplasia; obstruction of urine drainage in blastemas placed in culture for 2-3 days facilitates the induction of dysplasia ( p ~ . 0 5 ) .
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PARACRINE REGULATION OF ~PLTHELIAL CELL PROLIFERATION IN DEVELOPING LUNG. M.Post, J.S.Torday, A.Stiles & B.T.Smith. Dept'. Pediatrics, Harvard Medical School, Boston, MA 02115. Epithelial surface area increases markedly during fetal lung morphogenesis. Demand for new surface area is met by Type II cell proliferation followed by transition of some cuboidal Type II cells into flattened Type I cells. Epithelial cell growth appears to be regulated by mesenchyme, i. Conditioned medium of fetal lung fibroblasts stimulated growth of fetal rat lung Type II cells in primary cultures. Control experiments showed mitogenic activity is due to protein (trypsin t r e a t m e n t ) t h a t is heat labile (5min lO0°C). Gelfiltration studies revealed mitogen has molecular weight of approx. 30,000 daltons. Production of mitogen by fetal rat lung fibroblasts varied as function of gestational age (peak production at 19 days of gestation). 2. Conditioned medium f r o m fetal rat skin, kidney and liver fibroblasts did not stimulate Type II cell growth indicating mitogen is organ specific. 3. Conditioned medium from fetal lung fibrobloasts exposed to cortisol did not show mitogenic activity suggesting synthesis is hormone sensitive.
EVIDENCE OF INCOMPLETE TISSUE SEPARATION WHEN USING ETHYLENEDIAMNETETRAACETIC
ACID J. Richman and E.J. Kollar, Department of Oral Biology, University of Connecticut Health Center, Farmington, Connecticut, 06032 Two principle substances used for epithelial-mesenchymal tissue separation are trypsin and ethylenediaminetetraacetic acid (EDTA). 15-17 -day CD-I fetal mice provided molar tooth germs. Molar were treated in one of 3 ways: I% crude trypsin at 4°C for 2 hrs; mM E D T A for 1.5 hrs at 4°C, followed by 1.5 hrs at 37°C; 10 mM EDTA for 15 min at 37°C. Several papillae were fixed immediately after stripping the epithelium, others were cultured for 2 days in vitro, and the majority were grafted to the anterior chamber of the eyes of adult mice for 10-14 days of in vivo growth. After culture, trypsin separated papillae did not demonstrate epithelial growth. However, EDTA treated papillae contained islands of epithelial cells when examined after in vitro and intraocular culture. Trypsin was shown to provide more complete separation of enamel organ from dental papilla in the murine tooth germ.
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364 TIME-LAPSE CINEMICROGRAPHY DEMONSTRATES ACTIVE MIGRATION BY MESODERM CELLS IN PRIMITIVE-STREAK-STAGE MOUSE EMBRYOS. N.NAKATSUJI. Meiji Institute of Health Science, Naruta, Odawara 250, Japan, and MRC Mammalian Development Unit, University College London, London NWI 2HE, UK. The process of mesoderm formation in the primitive streak stage mouse embryo was recorded and analysed by using in vitro culture of the whole embryo and time-lapse cinemicrography. Seven-daysold mouse embryos were freed from the uterus and decidua and cultured in a small chamber on a heated stage. Use of differential-interference-contrast optics and relative transparency of the embryo enable direct observation of the individual mesoderm cells that are actively migrating between the epiblast and primary endoderm layers. They move by extending active cell processes of slender or broad shapes. They translocate at a speed of 0.5 - 1.0 micron/min, predominantly in the direction away from the primitive streak and toward the anterior and lateral part of the embryo. Mitosis occurred frequently, preceded by the bleb formation and rounding up of the cell body, and succeeded by the bleb formation and separation of the daughter cells.
OBSTRUCTION OF URINE DRAINAGE FACILITATES BUT DOES NOT CAUSE RENAL DYSPLASIA. M. MAIZELS and S.B. SIMPSON, Jr. Division of Urology, Children's Memorial Hospital and Department of Human Biology, Northwestern University, Chicago, Illinois, 60614, USA. Isolated kidney blastemas of the chick embryo were treated by: ligation of the ureter (79), tissue culture for 2-3 days (14) to reduce the condensed metanephrogenic mesenchyme, or ligation of the ureter + tissue culture (6). The blastemas were then permitted to develop as grafts on the chorio-allantoic membrane. Dysplasia, as diagnosed by the presence of primitive ducts was seen in kidneys which had been treated by: ligation of the ureter (0), tissue culture (14%), and ligation of the ureter + tissue culture (83%). Ligation of the ureter alone does not lead to dysplasia; obstruction of urine drainage in blastemas placed in culture for 2-3 days facilitates the induction of dysplasia ( p ~ . 0 5 ) .
365
366
PARACRINE REGULATION OF ~PLTHELIAL CELL PROLIFERATION IN DEVELOPING LUNG. M.Post, J.S.Torday, A.Stiles & B.T.Smith. Dept'. Pediatrics, Harvard Medical School, Boston, MA 02115. Epithelial surface area increases markedly during fetal lung morphogenesis. Demand for new surface area is met by Type II cell proliferation followed by transition of some cuboidal Type II cells into flattened Type I cells. Epithelial cell growth appears to be regulated by mesenchyme, i. Conditioned medium of fetal lung fibroblasts stimulated growth of fetal rat lung Type II cells in primary cultures. Control experiments showed mitogenic activity is due to protein (trypsin t r e a t m e n t ) t h a t is heat labile (5min lO0°C). Gelfiltration studies revealed mitogen has molecular weight of approx. 30,000 daltons. Production of mitogen by fetal rat lung fibroblasts varied as function of gestational age (peak production at 19 days of gestation). 2. Conditioned medium f r o m fetal rat skin, kidney and liver fibroblasts did not stimulate Type II cell growth indicating mitogen is organ specific. 3. Conditioned medium from fetal lung fibrobloasts exposed to cortisol did not show mitogenic activity suggesting synthesis is hormone sensitive.
EVIDENCE OF INCOMPLETE TISSUE SEPARATION WHEN USING ETHYLENEDIAMNETETRAACETIC
ACID J. Richman and E.J. Kollar, Department of Oral Biology, University of Connecticut Health Center, Farmington, Connecticut, 06032 Two principle substances used for epithelial-mesenchymal tissue separation are trypsin and ethylenediaminetetraacetic acid (EDTA). 15-17 -day CD-I fetal mice provided molar tooth germs. Molar were treated in one of 3 ways: I% crude trypsin at 4°C for 2 hrs; mM E D T A for 1.5 hrs at 4°C, followed by 1.5 hrs at 37°C; 10 mM EDTA for 15 min at 37°C. Several papillae were fixed immediately after stripping the epithelium, others were cultured for 2 days in vitro, and the majority were grafted to the anterior chamber of the eyes of adult mice for 10-14 days of in vivo growth. After culture, trypsin separated papillae did not demonstrate epithelial growth. However, EDTA treated papillae contained islands of epithelial cells when examined after in vitro and intraocular culture. Trypsin was shown to provide more complete separation of enamel organ from dental papilla in the murine tooth germ.
142S