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Cardiac rhythms in chick embryos during hatching.

Avian embryos develop within a hard eggshell which permits the measurement of heart rate while maintaining an adequate gas exchange through the chorioallantoic membrane. Heart rate has been determined from cardiogenic signals detected either noninvasively, semi-invasively or invasively with various transducers. Firstly, we reviewed these previously-developed methods and experimental results on heart rate fluctuations in prenatal embryos. Secondly, we presented new findings on the development of heart rate fluctuations during the last stages of incubation, with emphasis on the perinatal period, which remained to be studied. Three patterns of acceleration of the instantaneous heart rate were unique to the external pipping period: irregular intermittent large accelerations, short-term repeated large accelerations and relatively long-lasting cyclic small accelerations. Besides these acceleration patterns, respiratory arrhythmia, which comprimised oscillating patterns with a period of 1-1.5 s, appeared during the external pipping period. Furthermore, additional oscillating patterns with a period of 10-15 min were found in some externally pipped embryos.

Animals↗

Regional hypoxia elicits regional changes in chorioallantoic membrane vascular density in alligator but not chicken embryos.

Hypoxic incubation increases vascularization in the chick chorioallantoic membrane (CAM). The effect of regional hypoxia on the vascular density of American alligator (Alligator mississippiensis) and chicken (Gallus gallus) CAMs was studied to determine if hypoxic proliferation of blood vessels is localized or global across the CAM. Eggs were incubated under normoxic conditions with a portion of the eggshell covered with non-toxic beeswax to induce external regional hypoxia. CAMs were examined under a microscope with a 'bulls eye' coverslip and a vascular density index (VDI) was determined. The hypoxic portions of the alligator CAMs were more vascular than the normoxic portions (VDI = 200.9 versus 157. 8, respectively). Presumably this response is maladaptive by causing increased blood flow to the poorly oxygenated portions, i.e. increased 'shunt'. Thus, we hypothesize increased vascularity due to hypoxic incubation is due to local release and subsequent rapid local breakdown or uptake of angiogenic factors. In contrast, the hypoxic and normoxic portions of the chick CAMs exhibited virtually no difference in VDI (VDI= 211.5 versus 217.9, respectively). We suggest the air cell and air space of the chicken eggs allows for circulation of gas in ovo, eliminating the possibility of regional internal hypoxia.

Allantois↗

Evolution of viviparity: what can Australian lizards tell us?

Historically, Australia has been important in the study of, and the development of hypotheses aimed at understanding, the evolution of viviparity in amniote vertebrates. Part of the importance of Australia in the field results from a rich fauna of skinks, including one of the broadest ranges of diversity of placental structures within one geographic region. During the last decade, we have focussed our studies on one lineage, the Eugongylus group of skinks of the subfamily Lygosominae because it contains oviparous species and some that exhibit complex placentae. Our specific objective has been to attempt to understand the fundamental steps required when viviparity, and ultimately complex placentae, evolve from oviparous ancestors. We have taken a three-prong approach: (1) detailed study of the morphology and ontogeny of the placentae of key species at the light microscope level; (2) study of changes in the uterus associated with pregnancy, or the plasma membrane transformation; and (3) measures of the net exchange of nutrients across the placenta or eggshell of key species. In turn, we have found that: (1) details of the morphology and ontogeny of placentae are more complex that originally envisaged, and that the early conclusions about a sequence in the evolution of complex placentae was naïve; (2) a plasma membrane transformation occurs in viviparous, but not oviparous lizards, and thus may be a fundamental feature of the evolution of viviparity in amniotes; and (3) species with more complex chorioallantoic placentae tend to transport more nutrients across the placenta during pregnancy than those with simpler chorioallantoic placentae but, because the correlation is not tight, the importance of the omphaloplacenta in transporting nutrients may have been overlooked. Also, the composition of yolk of highly matrotrophic species is broadly similar, but not identical, to the yolk of oviparous species. Some of the interpretation of our data within the context of our specific objective is not yet possible, pending the publication of a robust phylogeny of Eugongylus group skinks. Once such a phylogeny is available, we are in a position to propose specific hypotheses about the evolution of viviparity that can be tested using another lineage of amniotes, possibly Mabuya group skinks.

Animals↗

Differential biomarker gene and protein expressions in nonylphenol and estradiol-17beta treated juvenile rainbow trout (Oncorhynchus mykiss).

The time- and dose-dependent transcriptional and translational expression of biomarker genes in nonylphenol (NP) and estradiol-17beta (E(2)) treated juvenile rainbow trout is reported. Fish were exposed to NP (1, 5 and 25 mg/kg) and E(2) (5 mg/kg) and killed at 2, 6, 12, 24, 48 and 72 h after exposure. The estrogen receptor (ER), vitellogenin (Vtg) and eggshell zona radiata protein (Zr-protein) gene expressions were analyzed in total liver RNA using Northern and slot hybridization with specific cDNA probes. Plasma Vtg and Zr-protein levels were evaluated using indirect ELISA. While Zr-protein gene showed an induction only at 24 h post-exposure, the plasma protein levels showed a time-dependent increase in the 25-mg NP treated group. Vtg transcripts showed an apparent time-dependent increase without a concomitant increase in protein levels in the 25-mg NP treated fish. Time-dependent increases in Vtg and Zr-protein gene expressions without the corresponding increases in ER gene transcription was observed in E(2)-treated fish at 2, 6 and 12 h post-exposure. Induction of ER gene transcripts was observed from 24 h and did not change significantly at 48 and 72 h. In the E(2)-treated fish, induction of plasma Vtg levels was observed at 48 and 72 h, while plasma Zr-protein was induced at 24, 48 and 72 h, after exposure. We conclude that the E(2)- and NP-induced Vtg and Zr-protein gene expressions at the early time intervals after exposure are not dependent on increase in the transcriptional activity of the ER gene and that Vtg and Zr-protein gene transcriptions require only basal or minimal ER concentration, in addition to other mechanisms.

Animals↗

Gene amplification as a developmental strategy: isolation of two developmental amplicons in Drosophila.

Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, we identified two additional developmental amplicons in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression.

Animals↗

A genome analysis of endoreplication in the Drosophila ovary.

Gene amplification is used by follicle cells to increase the copy number of Drosophila chorion genes, which encode structural components of the eggshell. A new study by Claycomb et al. in this issue of Developmental Cell raises the possibility that gene amplification might also be used for the developmental patterning of the egg chamber and oocyte.

Animals↗

Immunocytochemical and ultrastructural studies on Dipetalonema viteae (Filarioidea).

The antigenic properties of adult male and female of Dipetalonema viteae were studied by immunocytochemistry. Using antisera of the rodents Meriones unguiculatus and Mastomys natalensis infected with D. viteae, the binding of antibodies to sections of filariae embedded in Epon was assayed by the peroxidase-antiperoxidase (PAP) technique and by electron microscopy. The optimal staining intensity was obtained with an antiserum dilution of 1:5000. Control sera were obtained from sex and age matched uninfected animals. Female D. viteae showed maximal antigen-antibody reactions within the uterus: in the inner uterus wall, in the "nutrient channels" between the maturing eggs and the differentiating microfilariae, on the eggshells, in the cuticula of microfilariae and in the spermatheca on the cell membrane of the mature spermatozoa. Male filariae showed binding of antibodies in the vesicula seminalis: in the nucleus and the nuclear membrane of primary spermatocytes and on maturing spermatids. Less pronounced antigen-antibody reactions in the cuticula, muscle and intestine were observed in both sexes. The PAP-technique offers significant improvements in comparison with other techniques, e.g., immunofluorescence, used to detect antigens on filariae: the PAP-technique has an increased sensitivity with a concomitant reduction in nonspecific background and can be used for both light and electron microscopy; moreover, PAP-treated tissues can be stored indefinitely at room temperature.

Animals↗

A histidine-rich protein from the vitellaria of the liver fluke Fasciola hepatica.

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Regulatory elements controlling chorion gene expression are conserved between flies and moths.

Flies and moths are approximately as distant phylogenetically as are mammals and birds. In terms of morphology, physiology and biochemistry, the complex proteinaceous eggshell or chorion differs substantially in these two insect groups, which are typified by Drosophila melanogaster and Bombyx mori. The major chorion proteins of moths are encoded by two families of genes, A and B, which have no obvious homologues in flies. Unlike Drosophila, where chorion genes are oriented in tandem, moths show mostly chorion gene pairs (A plus B) that are divergently transcribed and coordinately expressed. The 5' ends of the paired genes are separated by a DNA segment of only 300 +/- 50 base pairs, which may well include at least some of the cis-regulatory elements necessary for gene expression. Despite these differences, we have tested whether moth chorion genes might be expressed in flies. Cloned DNA fragments bearing moth chorion genes were introduced into the Drosophila germ line by P-element-mediated transformation. Analysis of RNAs from transformed lines revealed that the genes are expressed with correct sex, tissue and temporal specificity, resulting in the accumulation of abundant moth chorion transcripts in late fly follicles.

Animals↗

Specific expression of a silk-encoding gene of Bombyx in the anterior salivary gland of Drosophila.

Successful expression of genes transferred into distantly related species in which genetic functions have been maintained through evolution has been reported previously. In the case of the silkmoth Bombyx mori and the fruitfly Drosophila melanogaster, both of which produce chorions (eggshells), Bombyx chorion genes are correctly expressed in Drosophila despite their estimated 240-Myr phylogenetic divergence. Here we report that, although Drosophila does not produce silk, mechanisms regulating transcription have been conserved between the salivary gland of the fruitfly and the silk gland of the silkmoth larva.

Amino Acid Sequence↗

Palaeontology: pterosaur embryo from the Early Cretaceous.

Dinosaur embryos have been discovered all over the world, but so far no pterosaur embryos have been reported. Here we describe a Chinese fossil from the Early Cretaceous period containing an embryo that is unambiguously a pterosaur. The embryonic skeleton, which is exquisitely preserved in its egg, is associated with eggshell fragments, wing membranes and skin imprints. This discovery confirms that pterosaurs were egg-layers and sheds new light on our understanding of pterosaur development.

Animals↗

Palaeobiology: Argentinian unhatched pterosaur fossil.

Our knowledge of the eggs and embryos of pterosaurs, the Mesozoic flying reptiles, is sparse. Until now, the recent discovery of an ornithocheirid embryo from 121-million-year-old rocks in China constituted the only reliable evidence of an unhatched pterosaur. Here we describe an embryonic fossil of a different pterosaur from the Early Cretaceous lacustrine deposits of Loma del Pterodaustro (the Lagarcito Formation, which is about 100 million years old) in central Argentina. This new fossil provides insight into the eggshell morphology, early growth and nesting environments of pterosaurs.

Animals↗

Chromatin regulates origin activity in Drosophila follicle cells.

It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa.

Acetylation↗

Destruction of helminth eggs by photosensitized porphyrin.

Tunisian untreated wastewater exhibits an average of 30 human helminth eggs per litre. After treatment, the concentration decreases to one egg per litre, or more in some cases. The percentage removal cited for wastewater processes provides no real indication of the destruction of the organisms, but merely of their transfer to another medium. In this study, we report the use of an environmentally friendly photoactive compound for wastewater disinfection. Photosensitization involves the generation of very toxic short-lived species on absorption of light by porphyrin. Microorganism photosensitization is potentially useful for sterilization and for the treatment of certain bacterial diseases. Gram-positive bacteria can be photoinactivated by a range of photosensitizers, but Gram-negative bacteria are not usually susceptible to photosensitized destruction. Our findings clearly demonstrate that the cationic meso-substituted porphyrin, tetra-(4-N-methylpyridyl) porphin tetra-tosylate (T4MPYP), is an efficient photosensitizer of helminth eggs on visible light illumination. The microscopic observation of helminth eggs shows that many types of ultrastructural alterations are potentially associated with exposure to T4MPYP and an adequate intensity of light: morphological changes without breakage; small alterations of eggshells; complete destruction. The degree of egg alteration increases with both increasing T4MPYP concentration and irradiation time. Moreover, the dissolved oxygen concentration, water quality and the type of eggs can influence the sensitivity of helminth eggs to photosensitization. Indeed, suspended solids (turbidity) were the most influential solution parameter on the efficiency of the photochemical process.

Animals↗

Absorption spectroscopy and binding constants for first-row transition metal complexes of a DOPA-containing peptide.

A diverse array of biological systems incorporate 3,4-dihydroxyphenlyalanine (DOPA) into proteins and small molecules for cross-linking and material generation. Marine worm eggshells, sea squirt wound plugs, and marine mussel adhesives may all be formed by combining DOPA-containing molecules with high levels of metals. In order to provide model systems for characterizing these biomaterials, we carried out a study on metal binding to a DOPA-containing peptide. Ultraviolet-visible absorption spectra are presented for the AdopaTP peptide binding to Fe3+, V3+, VO2+, Mn3+, Ti4+, Cu2+, Co2+, and Ni2+ in mono, bis, and where applicable, tris coordination modes. Association constants were determined for selected metal ions binding to the peptide. In general, the spectroscopic and binding properties of this DOPA-containing peptide were found to be similar to those of catechol.

Animals↗

Detection and survival of Campylobacter in chicken eggs.

AIMS: Campylobacter jejuni, a food-borne human pathogen, is widespread in poultry; however, the sources of infection and modes of transmission of this organism on chicken farms are not well understood. The objective of this study was to determine if vertical transmission of C. jejuni occurs via eggs. METHODS AND RESULTS: Using a temperature differential method, it was shown that Campylobacter had limited ability to penetrate the eggshell. When C. jejuni was directly inoculated into the egg yolk and the eggs were stored at 18 degrees C, the organism was able to survive for up to 14 days. However, viability of C. jejuni was dramatically shortened when injected into the albumen or the air sac. When freshly laid eggs from Campylobacter-inoculated specific pathogen-free (SPF) layers were tested, C. jejuni-contamination was detected in three of 65 pooled whole eggs (5-10 eggs in each pool) via culture and PCR. However, the organism was not detected from any of the 800 eggs (80 pools), collected from the same SPF flock, but kept at 18 degrees C for 7 days before testing. Likewise, Campylobacter was not recovered from any of 500 fresh eggs obtained from commercial broiler-breeder flocks that were actively shedding Campylobacter in faeces. Also, none of the 1000 eggs from broiler breeders obtained from a commercial hatchery were positive for Campylobacter. CONCLUSIONS: These results suggest that vertical transmission of C. jejuni through the egg is probably a rare event and does not play a major role in the introduction of Campylobacter to chicken flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of Campylobacter transmission to chicken flocks should focus on sources of infection that are not related to eggs.

Animal Husbandry↗

In vivo electroporation: a new frontier for gene delivery and embryology.

One of the key techniques in developmental biology is introducing transgenes into tissues and analyzing their subsequent effects on morphogenesis and organogenesis. In mammals, the transgenic approach is a way to misexpress foreign genes in various tissues and organs. However, targeting expression to certain tissues is totally dependent on the availability of specific promoters. Hence, it is not an easy task to control transgene expression temporally and spatially during embryogenesis. Further, if the transgene is toxic, embryonic development can be disrupted, resulting in premature death before the desired stages of development. As alternative systems, Xenopus and zebrafish are used frequently. In these vertebrate models, overexpression of genes can be carried out by injecting synthetic RNAs into eggs. However, genetic techniques in these systems are limited only to early development, prohibiting the precise analysis of gene effects on organogenesis in later stages. In contrast, the chick embryo has long served as a powerful and useful model system, holding a unique position in the field of developmental biology. Although trials of transgenic chicks have never been successful, easy accessibility to the developing embryo through a window opened in an eggshell enables performance of a variety of techniques, such as time-lapse cinephotomatography, microsurgical manipulations (including chick/quail chimeras), transplantation of cells and tissues, New's in vitro culture, etc. (Bortier et al., 1996; Douarin et al., 1996; Selleck, 1996). In addition to these experimental advantages, retrovirus-mediated gene delivery, and recently, adenovirus-mediated misexpression have been employed routinely in chick embryos (Leber et al., 1996; Morgan and Fekete, 1996).

Animals↗

Ultrastructure of the eggs of Polymorphus magnus (Acanthocephala, Polymorphidae).

The ultrastructure of Polymorphus magnus acanthocephalan eggs has been studied. The eggshell consists of four envelopes that are morphologically similar to those of the eggs of other Palaeacanthocephala representatives. The studied acanthors are formed by the cortical syncytium and "central nuclear mass". In the anterior part of acanthors there is a penetration gland. Its secret may provide the larvae migration into intermediate host's organism. "Central nuclear mass" consists of several germinative nuclei and numerous fibrillar bodies, formed as a result of germinative nuclei degradation.

Acanthocephala↗