Abstract-We compared the relative sensitivity of chicken embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) injected either into the yolk or into the air cell. The TCDD was injected at the start of incubation (embryonic day 0) and the embryos were sacrificed at multiple times during embryonic development. A subset of embryos were allowed to hatch undisturbed. The chick embryo was significantly more sensitive to TCDD when injected into the yolk than when injected into the air cell. The resultant median lethal dose (LD50) (122 pg/g egg, determined by probit analysis; 146 pg/g egg determined by interpolation) was 60% lower than the LD50 (297 pg/g egg by probit; 255 pg/g egg determined by interpolation) for air-cell-injected TCDD. A significant decrease in hatch weight of embryos exposed to high concentrations of TCDD compared to controls occurred, and this decrease was even more pronounced at a lower concentration in the yolk-injected birds. Interestingly, during the period of embryonic days 11 through 15, the mean weight of the yolk-injected embryos was smaller than the mean weight of the air-cell-injected embryos. This difference was not noticably evident just before or just after this developmental period. Embryos exposed to high concentrations of TCDD injected into either the yolk or the air cell tended to die within the first 2 weeks of incubation. A number of TCDD-exposed embryos survived the entire 21-d incubation period, but only air-cell-injected embryos were able to hatch successfully. Because the injection site varies in studies reported by different laboratories, the relative sensitivity must be considered when comparing results from different studies.

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Spatiotemporally regulated cell proliferation and differentiation are crucial for the successful completion of morphogenesis of the vertebrate secondary palate. An understanding of the mechanisms by which these cellular phenomena are regulated during palate development involves the identification of the various signal transduction pathways. In the present study, the presence and activation of mitogen-activated protein (MAP) kinases were investigated during the development of quail secondary palate. The palatal shelves were dissected on days 5-9 of incubation, homogenized, and centrifuged, after which the samples were separated by anion exchange fast protein liquid chromatography. The fractions were analyzed for myelin basic protein (MBP) phosphorylation. In addition, primary cultures of quail palate mesenchymal cells (QPMCs) were treated with epidermal growth factor (EGF) and prepared for MBP phosphorylation assays. A temporally regulated pattern of phosphotransferase activity, characterized by a three-fold increase in phosphotransferase activity toward MBP between days 5 and 8 of incubation, was observed during quail palate development. Western blotting, using MAP kinase antibodies, demonstrated the presence of a 42-kDa isoform between days 5 and 9 of incubation, during which the level of protein remained constant. Antityrosine immunoblotting with 4G10 also detected a 42-kDa protein. Phosphotransferase assays, using either a MAP kinase-specific substrate peptide (S5) or a protein kinase C inhibitor (R3), further confirmed the presence of a MAP kinase in the developing palate of quail. Because diverse biological processes occur concurrently during in vivo palate morphogenesis, the involvement of MAP kinase was explored further in primary cell culture. The data showed that EGF stimulated proliferation and activated 42-kDa MAP kinase in QPMCs. It is suggested that MAP kinase cascade may be involved in growth factor-regulated cell proliferation during morphogenesis of quail secondary palate.

Background During the progression of avian secondary palate morphogenesis, the rate of cell proliferation declines, whereas the production and accumulation of extracellular matrices increases. To investigate the regulation of these events, we examined the quail secondary palate for the activity of casein kinase 2 (CK 2), a pleiotropic serine/threonine second messenger independent enzyme implicated in cell growth and differentiation. Methods Quail palatal shelves were dissected between days 5 and 9 of incubation, which is the period of palate morphogenesis in quail, and prepared either for light microscopic observations or homogenized, cleared by ultracentrifugation, and then subjected to fractionation on a MonoQ column by fast protein liquid chromatography and Western immunoblotting. Results Histological examination showed that the palatal shelves appeared on day 5 of incubation and approximated by day 8 of incubation. Fractionation of palate extract using a Mono‐Q column revealed the presence of a major peak of phosvitin phosphotransferase activity which eluted with 0.5 M NaCl. This activity peak coincided with the presence of a 42 kDa subunit of CK 2 as determined by Western blotting with a CK 2 specific antibody. The CK 2 activity towards phosvitin was elevated on days 5 and 6 and then rapidly declined by day 9. The decrease in CK 2 activity did not correlate with a decrease in CK 2 protein during palate development indicating that the differential activity of the CK 2 enzyme observed during quail palate development may be due to post‐translational modifications of the enzyme. A high positive correlation was found between the CK 2 phosphotransferase activity and both the proliferation index and DNA synthesis during palate development. Conclusion On the basis of literature analysis and the results of the present study, it was suggested that the activity of CK 2 may be regulated along with protein kinase A to coordinate cell proliferation and the synthesis of extracellular matrices during palate development in quail. Anat. Rec. 247:102–108 © 1997 Wiley‐Liss, Inc.

Background Casein kinase 2 (CK 2) is a serine/threonine kinase that has been ubiquitously conserved in all eukaryotic cells. The exact functions of this enzyme have not yet been clarified; however, studies have repeatedly suggested that it may play crucial roles in the regulation of cell proliferation. During the formation of the secondary palate in the hamster, bursts of cell proliferation occur during the initial half of vertical shelf development, which decrease during the subsequent steps of palate morphogenesis, thus indicating that the cell cycle in the developing vertical palate may be tightly regulated. Methods In the present study, palatal shelves were dissected at 12‐hour intervals between days 10 and 12 of gestation, which is the period of vertical shelf development in the hamster. The palates were homogenized and cleared by ultracentrifugation and the resultant supernatants were fractionated on a Mono Q column by fast protein liquid chromatography. Results Using phosvitin as a substrate, the phosphotransferase activity in the fractionated samples decreased steadily from days 10 to 11, increased to a fivefold peak on day 11:12, and then decreased on day 12 of gestation. Western blot analysis using two CK 2 specific antibodies demonstrated that both the 42‐kDa (α) and the 38‐kDa (α′) subunits of the CK 2 holoenzyme were found throughout the formation of the vertical palatal shelves in the hamster. The amount of α and α′ subunits appears to remain constant, which suggested that the differential activity of the CK 2 enzyme may be due to posttranslational modifications. CK 2 activity correlated well with DNA synthesis (i.e., cell proliferation) rates from days 10 to 11, but not from days 11 to 12 of gestation. Conclusions It is proposed that the activity of CK 2 may regulate the rate of cell proliferation by stimulation of progression through G1 phase of the cell cycle and may also relate to the effects of various growth factors during the vertical development of mammalian palate. © 1996 Wiley‐Liss, Inc.