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For topographical analysis of developing embryos, investigators typically rely on scanning electron microscopy (SEM) to provide the surface detail not attainable with light microscopy. SEM is an expensive and time-consuming technique, however, and the preparation procedure may alter morphology and leave the specimen friable. We report that by using a high-resolution compound epifluorescence microscope with inexpensive low-power objectives and the fluorochrome acridine orange, we were able to obtain surface images of fixed or fresh whole rat embryos and fetal palates of considerably greater topographical detail than those obtained using routine light microscopy. Indeed the resulting high-resolution images afford not only superior qualitative documentation of morphological observations, but the capability for detailed morphometry via digitization and computer-assisted image analysis.

Mechanistically based dose-response models for developmental toxicity require elucidation of critical biological events that intervene between maternal exposure and adverse developmental outcome. We have examined some of the major events in the rat embryo/fetus following a subcutaneous injection of 5-fluorouracil (5-FU; 0-40 mg/kg) to the dam on Day 14 of gestation. This treatment resulted in reduced fetal weight that was significant at doses of 20 mg/kg and higher, generalized reduced ossification at doses above 25 mg/kg, and wavy ribs at doses of 30 mg/kg and higher. Numerous malformations including cleft palate and hindlimb defects were substantially increased at doses of 35 and 40 mg/kg. 5-FU inhibits thymidylate synthetase (TS), resulting in inhibited growth of rapidly proliferating tissues. To identify early events in the pathogenesis of hindlimb defects, we examined the effects of 5-FU on TS activity, cell cycle, growth, and morphology in the developing hindlimb as a function of dose and time. The rate of decline of TS activity following 5-FU exposure was dose related, although maximal inhibition and recovery were similar at doses within (20 and 40 mg/kg) and below (10 mg/kg) the range of detectable developmental toxicity. Flow cytometric analysis of nuclei from embryonic hindlimbs revealed a transient increase in the percentage of cells in S phase and decrease in G0/G1 phase 8 hr after maternal injection of 20-40 mg 5-FU/kg, but not at lower doses. Reduction in growth and morphometric changes of hindlimbs were observed only after maternal exposure to 40 mg/kg. The tissue specificity of these effects was examined by comparing the hindlimb with other embryonic tissues. There was also a dose-related decline of TS activity in the embryonic liver. However, the pattern of recovery of TS activity and cell cycle alterations were different in the liver than in the hindlimb, probably reflecting the higher cell proliferative rate in the liver at this stage. We have derived a quantitative, empirical model for induction of hindlimb defects based on TS inhibition and subsequent cellular events following 5-FU exposure. The model predicted a dose response similar to that of the observed data although the predicted curve was shifted toward lower doses. These results suggest that while this model may not capture all of the critical events involved in the induction of hindlimb defects following maternal exposure to 5-FU, it does reflect a central mechanism of its developmental toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell-cycle analysis of nuclei obtained from the circulating erythroblasts (gestational day [GD] 11-16), livers (GD 14-19), and whole embryos (GD 10-13) or remaining (extrahepatic) tissues (GD 14-16) of rat embryos/fetuses revealed age- and tissue-dependent variations in the relative percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle. With development, the rate of cell proliferation declined resulting in decreases in the relative percentage of S-phase cells and increases in the G0/G1 percentage, while the percentage of G2/M-phase cells remained relatively constant. Comparing tissue cell-cycle profiles during development, erythroblasts exhibited the most rapid age-dependent decline in S-phase percentage (from 75% at GD 11 to 8% by GD 14), embryos/extrahepatic tissues exhibited a more gradual reduction (from 55% at GD 10 to 14% by GD 15), while the hepatic isolates exhibited a relatively constant S-phase percentage of approximately 40% from GD 14 to GD 18 before decreasing to 23% at GD 19. These age-dependent variations suggest that cell-cycle distribution may be useful in staging embryogenesis and in detecting abnormal development. To determine how these developmental and organ-specific cell-cycle variations affect toxic response, we sampled GD 11-13 embryos 6 hr after maternal administration of a teratogenic dose of 5-fluorouracil (5-FU), a thymidylate synthetase inhibitor that induces S-phase accumulation. The results indicate that, on a relative basis, the amount of induced S-phase accumulation in erythroblasts and whole embryos 6 hr postdosing increased with development.(ABSTRACT TRUNCATED AT 250 WORDS)

This study describes the timecourse of expression of low-affinity serotonin uptake sites in the developing craniofacial region of the mouse embryo. Whole mouse embryos were incubated in the presence of various serotonergic compounds followed by immunocytochemical localization of serotonin (5-HT) and its binding protein. In the gestational day 9 embryo (3-5 somites), 5-HT uptake was observed in the myocardium of the heart, the visceral yolk sac and foregut. A specific and transient pattern of 5-HT uptake was observed in the hindbrain neuroepithelium from day 9.5-11, where it was localized in rhombomeres 2-5 in the day 9.5 embryo. By day 10, when rhombomeres were no longer evident, uptake was present in the dorso-lateral neuroepithelium surrounding the fourth ventricle (rhombic lip; cerebellar anlage). Uptake of 5-HT was initially observed in the surface epithelium of the craniofacial region at day 10 (20-25 somites) and was greatly increased at day 11. The invaginating lens, nasal placode epithelium and otocyst also took up 5-HT at day 11. During these stages a 45 kD serotonin-binding protein (SBP) was expressed in craniofacial mesenchyme, and became progressively restricted to regions subjacent to epithelial uptake sites. These staining patterns were shown to be specific for 5-HT and SBP by their absence in embryos stained using preabsorbed antisera. The timecourse of these patterns are correlated with critical events in craniofacial morphogenesis including (1) onset of inductive epithelial-mesenchymal interactions, (2) invagination and fusion of placodal structures, (3) presence of rhombomeres, and (4) regions of low proliferative activity.