Effects of serotonin and serotonin antagonists on chick embryogenesis.

Serotonin and some selected substances known to interfere with its formation (diethyldithiocarbamate) and function (Catron®, 5-methyltryptamine, promethazine) were tested for their ability to affect chick embryo morphogenesis during the first 48 h of development. To detect possible differences in sensitivity between the successive morphogenetic events taking place during this period, the treatment was begun at successively more advanced stages corresponding to embryo ages of between 4 and 30 h incubation. In all cases, the treatment was terminated at an embryo age of 48 h incubation. The treatment was performed both in ovo and in vitro.With some exceptions, the substances induced malformations of the same characteristic types. The developmental processes subjected to disturbances included blastoderm expansion, primitive streak formation, neurulation with brain formation, and somitogenesis. At the cellular level, the malformations can be traced to delayed yolk degradation, impaired formation and function of microvilli, and impaired ability of the embryo cells to change shape.All of the tested chemicals can be expected to interfere with intracellular levels of serotonin. They obviously interfered with decomposition of the yolk granules, recognized centres for intracellular serotonin formation and we therefore conclude that the observed morphogenetical disturbances are ultimately due to impairment of the endogenous serotonin formation. We suggest that, in morphogenesis, serotonin primarily promotes the activity of microtubules and microfilaments.

Membrane production and yolk degradation in the early fly embryo (Calliphora erythrocephala meig.): An ultrastructural analysis.

Formation of nuclear envelopes during the last cleavage mitosis and the formation of the cell membranes during the cellularization of the blastoderm have been studied ultrastructurally in the blowfly egg. Dense bodies arising from yolk granules by budding could contain membrane material destined to be incorporated into the new membranes of the blastoderm. The presence of transitional structures indicates that these bodies can be converted into dark multivesicular bodies. Large amounts of endoplasmic reticulum are found around the mitotic nuclei. Clusters or branched chains of vesicles associated with this are interpreted as evidence for the formation of endoplasmic reticulum by the breakdown of dark multivesicular bodies. Nuclear envelopes of mitotic daughter nuclei probably originate from endoplasmic reticulum. The egg contains both intranuclear and extranuclear annulate lamellae. The main events of cytokinesis are furrow initiation and cell membrane growth during the slow first phase, but probably only cytokinetic movement during the rapid second phase. On the assumption that cell membrane growth occurs by incorporation of complete membrane pieces, the addition of coated vesicles and/or light multivesicular bodies is definitely most probable. Some intermediate profiles indicate that light and dark multivesicular bodies are related. The membrane needed for second phase cytokinesis could well be provided by the unfolding of surface microvilli and protuberances of the furrow canal.

Effects of L-tryptophan on morphogenesis and growth in the early chick blastoderm.

Chick blastoderms, suppliedin vitro andin ovo with L-tryptophan at the primitive streak stage, showed in their continued development typical retardation of brain formation and somitogenesis in the embryo, whereas heart formation remained unaffected. In contrast to an overall reduction in size observed at the higher L-tryptophan concentrations, a moderate enlargement of the area opaca, compared with the controls, was found at the lower concentrations. This enlargement was combined with an increased flattening of the ectodermal area opaca cells and a reduction of the number of microvilli covering these cells. As a simultaneous supply of glucose could reduce, to some extent, the morphogenetic disturbances, these might partly be ascribed to a blocking of gluconeogenesis from L-tryptophan, but the overall reduction in size mentioned, together with the observation of a reduced decomposition of intracellular yolk granules in the L-tryptophan-treated blastoderms, indicates that impairment of intracellular yolk granule decomposition was the principal disturbance. The possible role of serotonin-probably formed from the L-tryptophan supplied-is suggested as a regulating factor in this connexion.

Localization of serotonin in cleavage embryos of Ophryotrocha labronica La Greca and Bacci.

Serotonin distribution in early Ophryotrocha embryos was investigated with fluorescence microscopy based on formaldehyde gas treatment of the embryos, and with light- and electron-microscopic autoradiography after the embryos had been treated with3H-5-hydroxytryptophan.Sections of early cleavage embryos showed serotonin-specific fluorescence all over the blastomeres, but it was mainly concentrated on yolk granules, and to a lesser degree on lipid drops and vacuoles. In 2-8 cell embryos, marked regional concentration of serotonin fluorescence was noticeable along the completed cleavage furrows.The autoradiographs confirmed the picture of the yolk granules as the principal site of serotonin formation and serotonin accumulation; considerable amounts were also associated with their decomposition products, i.e. lipid drops, vacuoles, and vesicles, whereas major cell organelles, e.g. mitochondria, were almost totally lacking. Of cytoplasmic structures in the blastomeres without apparent yolk granule origin, only microfilaments, particularly those amassed along the cleavage furrow, showed consistent and significant association with formed serotonin. This suggests a connexion between serotonin and microfilaments and might imply that in early embryo cells the fundamental contractile machinery is controlled by serotonin gradually released from the yolk granules.Within the blastomere nuclei, moderate amounts of serotonin were demonstrated with both fluorescence microscopy and autoradiography.The monoamine oxidase (MAO) inhibitor catron® (phenylisopropylhydrazine), used to intensify the autoradiographic picture of serotonin in the Ophryotrocha embryos, markedly increased intragranular serotonin accumulation, but also retarded yolk granule disintegration and delayed the cell cleavage process. In embryos barely able to cleave after treatment with catron®, ultrastructural analysis demonstrated that membrane formation at cell cleavage depends on influx of material from the nearby disintegrating yolk granules.

Karyomeres in early cleavage embryos of ophryotrocha labronica lagreca and bacci.

Karyomeres or chromosome vesicles occur regularly at all cell divisions in cleavage embryos ofOphryotrocha labronica up to the 16-cell stage. They are formed as separate units, containing one or several nucleolus-like bodies (NLB) as well as intranuclear annulate lamellae (IAL), but coalesce later into a compound nucleus, in connection with copious blebbing and simultaneous appearance of cytoplasmic annulate lamellae (CAL). Labelling of the early embryos with3H-thymidine revealed marked localization of the synthesized DNA to the karyomere envelope region, whereas3H-uridine incorporation, indicating RNA synthesis, was sparse and notably absent in the NLB. On the other hand the latter structure like the envelopes preferentially incorporated3H-myoinositol, and displayed considerable labelling with3H-leucine. The mechanism and general significance of karyomere formation is discussed with particular attention to the NLB and their possible involvement in nuclear membrane formation.

Effects of lithium chloride on embryos ofOphryotrocha labronica LaGreca and Bacci.

Cleavage embryos of the polychaeteOphryotrocha labronica were exposed to LiCl solutions of various strength for up to 4 days. High concentrations strongly inhibited development and led to endodermal protrusion, whereas moderate concentrations permitted development of larvae, although with characteristic disturbances as a consequence. Ultrastructural analyses of embryos exposed to moderate concentrations demonstrated manifest changes already during the first 24 hours, viz. of lipid droplets and yolk granules, the latter being ruptured and the contents dispersed to an extent far surpassing that at normal development. The premature widespread splitting of yolk granules by LiCl was confirmed by quantitative measurements on embryos which during oogenesis had their yolk granule DNA selectively labelled with3H-thymidine.