Metal embryotoxicity from urban particles in Sao Paulo city: An experimental study in chicken embryos

Chicken eggs were inoculated with suspensions of ambient air particles (<= 10 mu m, PM(10)) from Sao Paulo city in 3, 0.3 or 0.03 mu g doses on one of the four early days of embryo development. On the eleventh day of development alterations were observed on embryos inoculated with PM(10) 3 mu g on the third day. Particles analysis showed high content of metals. Hence, embryos were also inoculated with PM(10) (3 mu g) combined with metal chelating EDTA. PM(10) (3 mu g) embryos presented underdevelopment (stage 29.44 +/- 11.4) compared to vehicle and positive controls (stage 36.44 +/- 0.51 Saline and stage 31.20 +/- 9.7 Cyclophosphamide, p <= 0.05); higher (47%) mortality rate (23% Saline and 42% Cyclophosphamide) and low (68%) viability (100% Saline and 70% Cyclophosphamide, p = 0.04). Effects were attenuated when embryos received PM(10) + EDTA (stage 33.63 +/- 0.94, 18.9% mortality rate and 82% viability). PM(10) from Sao Paulo city is embryotoxic and metal may be implicated in the toxic mechanism. (C) 2010 Elsevier Inc. All rights reserved.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Influence of the neural tube/notochord complex on MyoD expression and cellular proliferation in chicken embryos

Important advances have been made in understanding the genetic processes that control skeletal muscle formation. Studies conducted on quails detected a delay in the myogenic program of animals selected for high growth rates. These studies have led to the hypothesis that a delay in myogenesis would allow somitic cells to proliferate longer and consequently increase the number of embryonic myoblasts. To test this hypothesis, recently segmented somites and part of the unsegmented paraxial mesoderm were separated from the neural tube/notochord complex in HH12 chicken embryos. In situ hybridization and competitive RT-PCR revealed that MyoD transcripts, which are responsible for myoblast determination, were absent in somites separated from neural tube/notochord (1.06 and 0.06 10-3 attomol MyoD/1 attomol ß-actin for control and separated somites, respectively; P<0.01). However, reapproximation of these structures allowed MyoD to be expressed in somites. Cellular proliferation was analyzed by immunohistochemical detection of incorporated BrdU, a thymidine analogue. A smaller but not significant (P = 0.27) number of proliferating cells was observed in somites that had been separated from neural tube/notochord (27 and 18 for control and separated somites, respectively). These results confirm the influence of the axial structures on MyoD activation but do not support the hypothesis that in the absence of MyoD transcripts the cellular proliferation would be maintained for a longer period of time.

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts

The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26-HH28/E5-E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

Heat or cold chronic stress affects organ weights and Hsp70 levels in chicken embryos

This study was carried out with the objective of evaluating the effect of heat (38.8 degreesC) or cold (35.8 degreesC) stress on chicken embryo development and tissues Hsp70 levels, after the 13th day of incubation. Embryo weight (percent egg weight), organ weight (percent embryo weight) and Hsp70 levels (ng Hsp70 mug(-1) total protein) in different tissues (liver, breast muscle, heart, lungs, brain and kidney) were studied at the end of incubation. Cold stress induced a lower embryo weight and lower kidney and lungs weights, whereas heart and liver were lighter in heat-stressed embryos. An interaction between temperature and age was obtained only for Hsp70 levels in kidney and heart. Cold-stressed embryos showed higher Hsp70 levels in the brain, lungs and liver; a decrease in brain and breast muscle Hsp70 levels was seen from the 19th to 20th days in control embryos. Hsp70 levels increased with age in kidneys of control embryos and in heart of heat- and cold-stressed embryos. In conclusion, this study showed that chicken embryo organ weights are affected by incubation temperature, and that Hsp70 expression is tissue dependent (higher levels being seen in the brain) being cold-stress more effective in increasing Hsp70 levels in most studied tissues.

Expression of MyoD, myogenin, myostatin and Hsp70 transcripts in chicken embryos submitted to mild cold or heat

The expression of the MyoD, myogenin, myostatin and Hsp70 genes was estimated in chicken embryos submitted to mild cold (36 +/- 0.5degreesC) or heat (44 +/- 0.5degreesC) for 1 h. 2. Marked decreases in MyoD, myogenin and myostatin transcript levels were observed in embryos exposed to high temperature, contrasting to the higher expression of the Hsp70 mRNA detected in heat-stressed embryos. 3. The exposure of chicken embryos to low temperature significantly affected only the abundance of myogenin mRNA. 4. These findings suggest that myogenic proliferation and differentiation events are compromised by variations in environmental temperature during avian embryogenesis. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Breeder age and bone development in broiler chicken embryos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Abundance of MyoD and myostatin transcripts in chicken embryos submitted to distinct incubation temperatures and timing exposures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.

Role of Notch signaling on the differentiation of early lymphoid progenitors cells: a view throughout the development of the embryonic chicken thymus and spleen

Dissertação para a obtenção do grau de Mestre em Genética Molecular e Biomedicina

Thermogenesis, vocalization, and temperature preference of 1-day-old chicken hatchlings after cold-exposure in late embryogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

5-Methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.