A photographic study of development in the living embryo of Drosophila melanogaster

The changes which can be seen occurring during the development of a living embryo of Drosophila melanogaster are described in detail, and represented photographically as a series of developmental stages. This provides an easy, but accurate technique for selecting eggs at precise developmental stages for experiments.     

Effects of carbohydrate and lipid on resting energy expenditure, heart rate, sleepiness, and mood

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

Sequence-specific DNA alkylation of novel tallimustine derivatives

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.     

Postneurulation rapid brain growth represents a critical time for encephalocele formation: a chick model

Current theory regarding the pathogenesis of encephaloceles suggests that the defect occurs after neurulation is completed in which brain tissue herniates through defective mesodermal elements necessary for skull modeling. To better delineate the mechanisms of encephalocele development, we performed a variety of microsurgical manipulations in the developing cranium of the chick embryo during the postneurulation period of early rapid brain growth. The results of the study revealed that encephaloceles could be induced in 78% of chick embryos manipulated at stage 26 that had evidence of marked decompression of the primitive ventricle. On the other hand, control embryos or embryos manipulated without ventricular decompression at stage 26 did not develop encephaloceles (0 of 32). Embryos with decompression of the primitive ventricle treated at earlier stages (20-24) showed only a 5% incidence of encephaloceles. These findings suggest that there is both a critical time point during postneurulation rapid brain growth when encephaloceles are prone to occur and a mesenchymal defect associated with decompression of the primitive ventricle that must be present to induce encephaloceles.     

Embryo cell surfaces--lectin binding and cell proliferation

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Concanavalin A decrease cell number and proportion of cells incorporating [3H] thymidine in case of 8- and 10-day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-day-old embryo cells. No effect was noticed in 12-day cells. These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8th to the 12th day of development, and slowly increases on the 16-day cells, the affinity constant remaining rather constant. The results indicate that the age-dependent effect of lectin on embryo cells could not be directly related to the number of lectin-binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of concanavalin A. Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.     

HIV-1 disease progression and AIDS-defining disorders in rural Uganda

BACKGROUND: The majority of people infected with HIV-1 live in Africa, yet little is known about the natural history of the disease in that continent. We studied survival times, disease progression, and AIDS-defining disorders, according to the proposed WHO staging system, in a population-based, rural cohort in Uganda. METHODS: In 1990 we recruited a random sample of people already infected with HIV-1 (as prevalent cases) detected during the initial survey round of a general-population study to form a natural-history cohort. Individuals from the general-population cohort who seroconverted between 1990 and 1995 (incident cases) were also invited to enroll. Participants were seen routinely every 3 months and when they were III. FINDINGS: By the end of 1995, 93 prevalent cases and 86 incident cases had been enrolled. Four patients in the prevalent group were in stage 4 (AIDS) at the initial visit. During the next 5 years, 37 prevalent cases progressed to AIDS. Seven incident cases progressed to AIDS and the cumulative progression to AIDS at 1, 3, and 5 years after seroconversion was 2%, 6%, and 22%, respectively. The cumulative probability of AIDS at 4 years from entering stages 1, 2, and 3 was 11%, 33%, and 58%, respectively. There were 47 deaths among prevalent cases and seven among incident cases during follow-up. The cumulative mortality 4 years after patients entered stages 1, 2, 3, and 4 was 9%, 33%, 56%, and 86%, respectively. The median survival after the onset of AIDS was 9.3 months. INTERPRETATION: Our results are important for the setting of priorities and rationalisation of treatment availability in countries with poor resources. We found that progression rates to AIDS are similar to those in developed countries for homosexual cohorts and greater than for cohorts infected by other modes of transmission. However, we have found that the rates of all-cause mortality are much higher and the progression times to death are shorter than in developed countries.     

Actin gene expression in developing sea urchin embryos

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.     

BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo

At E4.0 the inner cell mass of the mouse blastocyst consists of a core of embryonic ectoderm cells surrounded by an outer layer of primitive (extraembryonic) endoderm, which subsequently gives rise to both visceral endoderm and parietal endoderm. Shortly after blastocyst implantation, the solid mass of ectoderm cells is converted by a process known as cavitation into a pseudostratified columnar epithelium surrounding a central cavity. We have previously used two cell lines, which form embryoid bodies that do (PSA1) or do not (S2) cavitate, as an in vitro model system for studying the mechanism of cavitation in the early embryo. We provided evidence that cavitation is the result of both programmed cell death and selective cell survival, and that the process depends on signals from visceral endoderm (Coucouvanis, E. and Martin, G. R. (1995) Cell 83, 279-287). Here we show that Bmp2 and Bmp4 are expressed in PSA1 embryoid bodies and embryos at the stages when visceral endoderm differentiation and cavitation are occurring, and that blocking BMP signaling via expression of a transgene encoding a dominant negative mutant form of BMP receptor IB inhibits expression of the visceral endoderm marker, Hnf4, and prevents cavitation in PSA1 embryoid bodies. Furthermore, we show that addition of BMP protein to cultures of S2 embryoid bodies induces expression of Hnf4 and other visceral endoderm markers and also cavitation. Taken together, these data indicate that BMP signaling is both capable of promoting, and required for differentiation of, visceral endoderm and cavitation of embryoid bodies. Based on these and other data, we propose a model for the role of BMP signaling during peri-implantation stages of mouse embryo development.     

Saccharification and alcohol fermentation in starch solution of steam-exploded potato

Methylglyoxal (2-oxopropanal) is a cytotoxic compound that can be formed endogenously as a by-product of glycolytic pathway; so its concentration is expected to increase when glycolysis activity increases such as during embryo development. In this work we study the effect of exogenous methylglyoxal on development and embryo viability during Bufo Bufo development and on the enzymes and cofactors involved in its detoxication process (glyoxalase I and II, reduced glutathione and glyceraldehyde 3-phosphate dehydrogenase). The results show that exogenous methylglyoxal does not affect the enzymatic pattern until stage 20, while it induces a significant activity decrease of the tested enzymes at stage 25. On the contrary methylglyoxal positively influences the reduced glutathione concentration at all the considered stages. At morphological and histological levels methylglyoxal causes a strong retardation of cell division in the early stages, that results in various abnormalities in the late development. In conclusion, methylglyoxal enters the embryo and is antiproliferative and teratogenic: the data further supports the hypothesis of the importance of the glyoxalase system in the process of cell growth and division.     

Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification

Caenorhabditis elegans is renowned for its invariant embryogenesis. This pattern of development is in apparent contrast to other organisms from Drosophila to higher vertebrates. With the aid of a 4D microscope system (multifocal, time-lapse video recording system) which permits the extensive documentation and analysis of cell divisions, cell positions, and migrations in single embryos we have analyzed normal embryogenesis of C. elegans. The instrumentation reveals a naturally occurring variability in cell division timing, cell positioning, and cell-cell contacts which could not have been detected by the direct observation used earlier (Sulston et al., 1983, Dev. Biol. 100, 64-119). Embryos are very flexible and produce an essentially invariant premorphogenetic stage from variable earlier stages. An analysis of the distribution of the descendants of the early founder blastomeres at the premorphogenetic stage shows that these establish discrete regions in the embryo, a process involving a considerable amount of cell movement, which again varies in different embryos. Only cell fate assignment remains invariant. However, as shown earlier, this is not due to an autonomous invariant specification of cell fates but due to the fact that cell-cell interactions occur very early when the topology of blastomeres in the embryo is still sufficiently precise to ensure reproducible patterns of inductions. A new concept that founder blastomeres produce embryonic regions in the embryo can explain the striking complexity of the lineage per se and also the complicated asymmetric lineage patterns by which the bilateral symmetry of the embryo is established. Many cells, including bilateral homologs, were apparently chosen for a specific fate solely by their position in the embryo, irrespectively of the lineage descent by which the cells are created. We postulate that the production of regions by cell-cell interactions is the pivotal principle guiding the embryogenesis of C. elegans and that the embryogenesis of the worm follows the same basic principles as embryogenesis in other organisms.     

Modified mRNA rescue of maternal CK1/8 mRNA depletion in Xenopus oocytes

This work addresses two issues, the use of antisense oligodeoxynucleotides to deplete specific mRNAs in Xenopus oocytes to analyze their functions during development and the role of cytokeratin filaments in cells of the early Xenopus embryo. We have shown previously that depletion of cytokeratin CK1/8 mRNA causes defects in the early embryo. In this study, we show that the oligos, modified with phosphoramidate linkages to improve stability, are capable of degrading exogenous mRNA up to 27 hours after injection in the oocyte. For this reason, the phenotype could not be rescued by injection of a synthetic CK1/8 mRNA. However, modification of the synthetic CK1/8 mRNA, which prevents annealing of the antisense oligonucleotide used for depleting the endogenous CK1/8 mRNA, did result in the rescue of the CK1/8 depletion phenotype. These results demonstrate that the phenotype observed after depletion of the CK1/8 mRNA is truly caused by the lack of CK1/8 protein. Injection of the closely related type II cytokeratin (CK55) did not result in the same level of rescue of the CK1/8 depletion phenotype, suggesting that structurally similar members of the cytokeratin family, expressed at different stages of development, cannot substitute for each other in the early embryo.     

Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis

Postembryonic development in higher plants is marked by repetitive organ formation via a self-perpetuating stem cell system, the shoot meristem. Organs are initiated at the shoot meristem periphery, while a central zone harbors the stem cells. Here we show by genetic and molecular analyses that the ZWILLE (ZLL) gene is specifically required to establish the central-peripheral organization of the embryo apex and that this step is critical for shoot meristem self-perpetuation. zll mutants correctly initiate expression of the shoot meristem-specific gene SHOOT MERISTEMLESS in early embryos, but fail to regulate its spatial expression pattern at later embryo stages and initiate differentiated structures in place of stem cells. We isolated the ZLL gene by map-based cloning. It encodes a novel protein, and related sequences are highly conserved in multicellular plants and animals but are absent from bacteria and yeast. We propose that ZLL relays positional information required to maintain stem cells of the developing shoot meristem in an undifferentiated state during the transition from embryonic development to repetitive postembryonic organ formation.     

Thoracoscopic microsurgical excision of herniated thoracic discs

Optokinetic Nystagmus (OKN) exhibit different responses depending on the direction of gravity, with respect to head and body. In 13 normal healthy volunteers (10 experimental subjects and 3 controls), we studied the OKN evoked by horizontal and vertical stimuli, in upright, side-lateral, and repeated upright positions during a long time course of six test stages. Stage 1 was an upright sitting position. Stage 2 was a 90-degree recumbent lateral position immediately after Stage 1. Stage 3 was the same position as in Stage 2, but 2h later. Stage 4 was the same position, another 2h later (total of 4h). Stage 5 was a return to the upright position (the same as Stage 1) immediately after Stage 4. Stage 6 was a return to the same upright position, 2h after Stage 5. The results showed continuous modifications of OKN. A lateral tilt produced different effects on the horizontal and vertical optokinetic oculomotor systems. The gain in horizontal OKN (HOKN) decreased during Stages 2-4 with time, and returned to normal in Stages 5 and 6. As for vertical OKN (VOKN), the gain of upward (the direction by slow phase) OKN increased during Stages 2-4, and returned to normal in Stages 5 and 6. But the gain of downward OKN did not show any clear modulation. We showed that the change in gravity direction has a clear effect on OKN, and that it takes some time to reach the maximal and desirable level of modification. The difference in time-dependent modifications between HOKN and VOKN should be due to the difference in OKN-producing mechanisms and velocity storage systems.     

Patterned acquisition of skin barrier function during development

Skin barrier function is conferred by the outer layer of epidermis, the stratum corneum, and is essential for terrestrial life. Quantitative trans-epidermal water loss assays show that barrier forms late in embryogenesis, permitting the foetus to survive a terrestrial environment at birth. Using qualitative in situ assays for skin permeability, we show that barrier forms in a patterned manner late in mouse gestation. Barrier forms at specific epidermal sites, then spreads around the embryo as a moving front. The moving front of permeability change is accompanied by multiple changes in the outer, stratum corneum-precursor cells. We use the permeability assays to show that final stages of cornified envelope assembly are coordinated with initial stages of barrier formation. Hence the whole-mount permeability assays record developmental acquisition of a known, essential component of the adult barrier. We demonstrate the authenticity of the whole-mount assays after maternal glucocorticoid therapy (known to accelerate barrier formation) and in additional species including the rat where barrier formation is well characterized by TEWL assay (Aszterbaum, M., Menon, G. K., Feingold, K. R. and Williams, M. L. Pediatr. Res. 31, 308-317). The demonstration of patterned barrier formation in other species suggests patterned change as the universal mode of embryonic barrier acquisition. These results highlight the importance of patterning as a mode of epidermal maturation during development.     

Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food

Magnetic resonance (MR) microscopy was used to noninvasively investigate the development of live rat embryos in utero. The difficulty in making sequential observations of a developing mammalian embryo has frustrated developmental biologists for many years. Most current technologies analyze normal and abnormal development by observing end point phenotypes (in fixed specimens) rather than investigating the live embryo. MR microscopy was adapted to allow rat litters to be scanned three times each (at 1- to 3-day intervals) and has produced images of live developing embryos. It was demonstrated that repeated anesthesia and imaging protocols produced no gross malformations in the rat pups that were subsequently delivered and observed. Three-dimensional projection encoding with phase rewinders produced isotropic [256(3)] image data sets in about 30 minutes with excellent tissue contrast arising from steady-state effects in the amniotic fluid.     

Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins

We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation.