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Notes: Summary 1. Naked germ-anlagen and germ-bands during segmentation from univoltine strains ofBombyx mori L. were used without HCl-treatment for culturein vitro. With improved methods germs could develop without their prospective diapause until they had finished organogenesis and if kept in hanging drops nearly one month. 5 culture mediums contained defined extracts: (1) eggs with prospective diapause germs, (2) eggs with nondiapausing germs (3) eggs with germbands in eudiapause, (4) eggs with embryos after diapause and (5) eggs with nondiapausing embryos. In every case explanted germs developed at least to fully segmented germbands (stage of dormancyin ovo) but often further. 2. In culture medium (1), (2), (4), (5) 64–73% of germ-anlagen develop to embryos with articulate limbs and open backs, but in medium (3) only 55%. On the contrary germbands develop to this stage and to shortened embryos in medium (1) only to 38%, in (2) to 5%, in (3) to 12% but in (4) to 75% and in (5) even to 84%. In culture medium (3) there occur abnormal differentiations. 3. A store of yolk or of yolk and serosa separately from the tested germ allows development in many more cases without dormancy and to small larvae with closed backs, with eye pigmentation and muscle contractions. Embryos with appendage formation grow to 100% in culture medium (4) with depositum. Therefore it is the most efficient medium for experiments with fragmented parts of a germ. 4. The different rate of development without dormancy with germbands in culture medium (1) to (5) points to their competence for determining factors of diapause. One day diapausing germbands inovo arein vitro with yolk stores able to develop further. But 2 days old germs in dormancy also with stores cannot any further develop. The germband is definitively determined to eudiapause. 5. Also the material in the stores can develop. Yolkcells may aggregate and will be enclosed by the serosa. Together they form bubbles which pulsate and yolk inside. This material could include factors for diapause, but also for basal metabolism and for syntheses of cell differentiation. Possibly it couldclean also the drop. 6. The results invite discussions aboutin vitro methods, about the ability of the explanted germ for formation and about factors determining dormancy or organogenesis. The selected hypothesis requires experiments with a medium without egg-extract but with stores from extraembryonal egg-materials and it needs investigations of regulation in cell differentiation.

Notes: Summary Eggs ofCalliphora were operated (I) at the undifferentiated blastoderm stage, (II) after blastoderm differentiation and in the stages showing border furrows and the beginnings of gastrulation, and (III) during forward extension of the posterior germ band region along the dorsal egg surface. The operation consisted of cutting off up to 1/3 of the egg anteriorly and removing the posterior egg fragment from the vitelline membrane for culture in a hanging drop. As control experiments for the “explanted” egg fragments, anteriorly pricked eggs and corresponding posterior fragments not removed from the vitelline membrane were cultured in hanging drops. Different technical prerequisites for the sterile culture ofCalliphora embryo fragmentsin vitro were tested. Development of the living embryo fragments was followed, and representative embryos fixed and microscopically examined for comparison with normally developing embryos. In explants I cell formation in the blastoderm did not reach completion and the blastoderm nuclei did not divide further. Slight contractions of parts of the preblastoderm did occur, but no morphogenetic movements. —In control fragments cell formation mostly proceeded further, but gastrulation was abortive except in large fragments. In explants II, irregular immigration of mesoderm cells was possible in fragments explanted before mesoderm groove initiation, but the explants did not develop further. Invagination of mesoderm through a groove, followed by segmentation of the germ band and some differentiation, was possible only in explants operated after initiation of groove formation. The germ band did not extend antero-dorsally, but became folded laterally, these folds foreshadowing the intersegmental boundaries formed later. Organogenesis and histological differentiation were in many respects abnormal. — In contrast, mesoderm invagination and initiation of germ band extension were possiblein control fragments operated before mesoderm groove initiation, although abnormalities in gastrulation were common. Explants III, despite cessation of germ band extension after explantation, often underwent further development resulting in histologically differentiated partial embryos, showing various abnormalities in organogenesis. The dorso-lateral furrows often persisted until segmentation and may correspond to primitive intersegmental furrows. The hind-gut and part of the posterior mid-gut-rudiment often evaginated during germ band contraction. Only when avitelline membrane was present did the germ band continue to extend antero-dorsally and, although this extension was rarely complete, further development with normal organogenesis and histological differentiation took place. Of the three germ layers, the ectoderm most closely approached normal differentiation in both explants and control fragments, with differentiation of nerve ganglia, tracheae, salivary glands, and a hind-gut epithelium with Malpighian tubules. Differentiation of the mesoderm in the explants was confined to muscle fibres situated near the ganglia. The endoderm remained undifferentiated in explants but formed a mid-gut epithelium in some control fragments, when splanchnic mesoderm was also present. The bearings of these results on morphogenetic problems in the insect egg is discussed.

Notes: Summary When extraembryonic egg material is placed apart from prospective diapause germ anlagen or early germ bands, it will stimulate their development without dormancy via the medium. This method of a bipartial systemin vitro (21° C) helps to analyze the regulatory mechanisms involved in the egg diapause ofBombyx mori. 1. In preliminary experiments, a culture medium free of egg extracts but containing foreign proteins (LYS) proved useful, since 100% of the test germs reached dormancy in the absence of stored egg material. Mitoses decrease and morphogenesis decelerates until the stage of the fully segmented germ-band is reached, which means the end of the prediapausal period. 2. When eggs were opened they developedin vitro without egg diapause. One may assume that the access of free oxygen activates some regulatory mechanisms permitting development without dormancy (nondormancy=Nd). In addition, the separate deposit of chorion, serosa and yolk cells (CDS Depot) will in any case prevent dormancy. Thus, the factors responsible for egg diapause must be sought in the extraembryonic egg system. A direct contact between the extraembryonic action-system and the embryonic reactionsystem is not a prerequisite. TheLYS medium without deposits offers sufficient oxygen to the test germ. Therefore the prospective diapause germ possesses a tendency to dormancy, according to its reaction norm. The potency to stimulateNd was tested with various depotmaterials (C, D, S, CS, DS, CDS) removed from eggs during prediapause (21° C), diapause (3° C) and post-diapause (returned to 21° C for 4 days). Each material produced a specificNd rate.In vitro, the test germs can progress in their organogenesis optimally to the stage of a small larva. The means of a collective effect in development are determined and related to one of the nine possibledegrees of organogenesis. 3. In comparison to serosa and chorion, yolk material has the highest mean both inNd rate (68 %,n=219) and degree of organogenesis. Surprisingly, cell-free chorionic material prevents dormancy development in 55% (n=296). As compared to theD Depot, the combination ofDS elicits a higherNd rate (79%,n=234), which is only surpassed by theCDS combination (100%,n=76). In comparison toS Depot(44%,n=294) theNd rate of aCS Depot reaches only 37% (n=161), presumably due to a restriction of the experiments to young material only. Probes, tested separately according to germ anlage or germ band, showed that there was no influence of the operational age of the test germ on theNd rate. 4. However, theNd-stimulating potency ofC, D, S, CS andDS depends on the operational age of the donator egg. Yolk material starts out having a highNd effect, decreasing with pre-diapausal age and staying relatively high in diapausal age. Similar changes are observed in the combination of yolk and serosa. TheNd rate of chorion starts low, increases steeply with the operational age and remains rather uniform. TheNd rate of serosa increases steeply in the stage critical to the beginning of egg diapause (dish-like germ anlage), decreases after pre-diapause and increases again after the minimal period for diapause (3 months at 3° C). HigherNd rates are observed whenS, D, andDS Depot were returned to 21° C for 4 days.D Depot has the maximal potency favouring organogenesis at the dish-like germ anlage stage. 5. The following subjects are discussed: the results of Chino (1957, 1958) on glycogen metabolismin ovo, the findings of Okada (1971) on the development of de-chorionized eggs under paraffin oil and our ownin ovo observations on the ultrastructural changes in the chorion, the mitotic activity before and after diapause and the distribution of glycogen in germ, yolk cells and serosa. These facts can be utilized to formulate a concept of the physiological phases of egg diapausein ovo: Egg diapause begins during a critical stage of the germ anlage with a reaction between serosa and chorionic material, which reduces the rate of oxygen consumption. Under these conditions, glycogen is metabolized into sorbitol and glyoerol. The physiologicalprophase of egg diapause is terminated, when the germ-band reaches dormancy. Diapause begins (e.g. at 3° C) with themesophase, during which the metabolism of glycogen continues decreasingly. Now glycogen is found only in the germ.Metaphase may begin with the re-uptake of oxygen, which starts the re-synthesis of glycogen from sorbitol and glycerol via oxydation and phosphorylation. However, the exposure to cold (3° C) will inhibit mitosis in the dormant germ band. In thetelophase of egg diapause, after terminated resynthesis, the dormant germ can remain in quiescence. When exposed to 21° C during the embryonic post-diapause period, it consumes the stored glycogen. If the high temperature starts prematurely during the mesophase, no embryo will hatch. However, when high temperatures set in during the metaphase, glycogen resynthesis and glycogen-breakdown in embryogenesis will compete and thus the hatching rate will be low. 6. Assuming that in the depot experimentsin vitro at 21° C and with free access of oxygen, glycogen metabolism can be considered one parameter of theNd rate, a satisfactory explanation of our experiments can be offered. With aCDS Depot,Nd stimulatory mechanism will always work satisfactorily, assuming a considerable resynthesis of glycogen of previously cold-exposed depot material.Nd rate ofD Depot will first follow the glycogen parameterin ovo; when removed from diapause, it may be capable of the resynthesis of glycogen. This will also explain the correspondingNd rates of theDS Depots. There is no correlation between theNd rates ofC Depots and the glycogen parameterin ovo. S Depots acquire a dependency on the glycogen parameter, which is independent of exposure to high temperature and oxygenin vitro. Further investigations on the glycogen metabolim of the depot materialin vitro are necessary to clarify these hypotheses. 7. The observations on the physiological phases inBombyx may also hold true for egg diapause of other insects. Various experiments with eggs of other strains ofBombyx with different reaction norms may substantiate our present conclusions. The enzymatic basis of the regulatory mechanisms with special regard to chorion should receive further clarification.