Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments.

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high. This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

Amniotic fluid stem cells increase embryo survival following injury.

Although amniotic fluid cells can differentiate into several mesenchymal lineages and have been proposed as a valuable therapeutic cell source, their ability to undergo terminal neuronal differentiation remains a cause of controversy. The aim of this study was to investigate the neuronal differentiation ability of the c-Kit-positive population from GFP-transgenic rat amniotic fluid, amniotic fluid stem (AFS) cells, and to assess how they affected injury response in avian embryos. AFS cells were found to express several neural stem/progenitor cell markers. However, no overt neuronal differentiation was apparent after either treatment with small molecules known to stimulate neuronal differentiation, attempts to differentiate AFS cell-derived embryoid body-like structures, or grafting AFS cells into environments known to support neuronal differentiation (organotypic rat hippocampal cultures, embryonic chick nervous system). Nonetheless, AFS cells significantly reduced hemorrhage and increased survival when grafted at the site of an extensive thoracic crush injury in E2.5 chick embryos. Increased embryo survival was induced neither by desmopressin treatment, which also reduced hemorrhage, nor by grafting other mesenchymal or neural cells, indicating a specific effect of AFS cells. This was found to be mediated by soluble factors in a transwell coculture model. Altogether, this study shows that AFS cells reduce tissue damage and increase survival in injured embryos, providing a potentially valuable tool as therapeutic agents for tissue repair, particularly prenatal/perinatal repair of defects diagnosed during gestation, but this effect is mediated via paracrine mechanisms rather than the ability of AFS cells to fully differentiate into neuronal cells.

Optimisation of in ovo electroporation of the chick neural tube.

Chick in ovo neural tube electroporation has become a widely used method for assaying gene function during embryonic development. Since its first description, many variants of this technique have been described, with varying values for specific parameters such as electrode type and spacing, voltage, pulse duration and plasmid DNA concentration. Here we examine the influence of some of these variables and derive a detailed and optimal protocol for electroporating the caudal neural tube during the third day of embryonic development. Our findings highlight the importance of electrode placement and DNA dilution buffer for optimal expression and absence of electroporation artifacts.

An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

Transplantation of human embryonic stem cells and derivatives to the chick embryo.

Traditional methods of studying the differentiation of human embryonic stem cells (hESCs) include generation of embryoid bodies, induced differentiation in vitro, and transplantation to immune-deficient mice. The chick embryo is a well-studied and accessible experimental system that has been used for many years as a xenograft host for mammalian cells. Several years ago, we performed experiments transplanting colonies of hESC into organogenesis-stage chick embryos to establish a novel system for studying the developmental programs and decisions of pluripotent human cells. Fluorescent hESC were used, in order to permit identification of the hESC in living embryos. We transplanted hESC into the trunk of chick embryos, both into and instead of developing somites. Our results showed that hESC survive, migrate, and integrate into the tissues of the chick embryo. Some of the hESC differentiated and the type of embryonic microenvironment that the implanted cells were exposed to modified their differentiation. Several other laboratories have subsequently xenografted hESC-derived cells to chick embryos for evaluating their differentiation in vivo. Therefore, the hESC-chick embryo system is a useful xenograft system complementing studies in rodents and in vitro, as well as uniquely shedding light on early processes in the development of human cells in the embryonic context.

An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival.

We have investigated the role of Sonic hedgehog (Shh) in the development of facial structures by depriving chicken embryos of the most anterior sources of this morphogen, including the prechordal plate and the anterior ventral endoderm of the foregut, before the onset of neural crest cell (NCC) migration to the first branchial arch (BA1). The entire forehead, including the foregut endoderm, was removed at 5- to 10-somite stage (ss), which led to the absence of the lower jaw when the operation was performed before 7-ss. If the embryos were deprived of their forehead at 8- to 10-ss, they were later on endowed with a lower beak. In embryos that were operated on early, the NCCs migrated normally to BA1 but were subjected to massive apoptosis a few hours later. Cell death did not occur when forehead excision was performed at a later stage. In this case, onward expression of Shh in the ventral foregut endoderm extended caudally over the excision limit, and we hypothesized that absence of Shh production by the endoderm in embryos that were operated on early could be responsible for the NCC apoptosis and the failure of BA1 development. We thus provided exogenous Shh to the embryos that were operated on before 7-ss. In this case, the development of the lower jaw was rescued. Therefore, Shh derived from the ventral foregut endoderm ensures the survival of NCCs at a critical stage of BA1 development.

Cues from neuroepithelium and surface ectoderm maintain neural crest-free regions within cranial mesenchyme of the developing chick.

Within the developing vertebrate head, neural crest cells (NCCs) migrate from the dorsal surface of the hindbrain into the mesenchyme adjacent to rhombomeres (r)1 plus r2, r4 and r6 in three segregated streams. NCCs do not enter the intervening mesenchyme adjacent to r3 or r5, suggesting that these regions contain a NCC-repulsive activity. We have used surgical manipulations in the chick to demonstrate that r3 neuroepithelium and its overlying surface ectoderm independently help maintain the NCC-free zone within r3 mesenchyme. In the absence of r3, subpopulations of NCCs enter r3 mesenchyme in a dorsolateral stream and an ectopic cranial nerve forms between the trigeminal and facial ganglia. The NCC-repulsive activity dissipates/degrades within 5-10 hours of r3 removal. Initially, r4 NCCs more readily enter the altered mesenchyme than r2 NCCs, irrespective of their maturational stage. Following surface ectoderm removal, mainly r4 NCCs enter r3 mesenchyme within 5 hours, but after 20 hours the proportions of r2 NCCs and r4 NCCs ectopically within r3 mesenchyme appear similar.

Interruption of cardiac output does not affect short-term growth and metabolic rate in day 3 and 4 chick embryos.

The heart beat of vertebrate embryos has been assumed to begin when convective bulk transport by blood takes over from transport by simple diffusion. To test this hypothesis, we measured eye growth, cervical flexure and rates of oxygen consumption ( V(O2)) in day 3-4 chick embryos denied cardiac output by ligation of the outflow tract and compared them with those of embryos with an intact cardiovascular system. Eye diameter, used as the index for embryonic growth, increased at a rate of approximately 4.5-5 % h(-)(1) during the observation period. There was no significant difference (P>0.1) in the rate of increase in eye diameter between control (egg opened), sham-ligated (ligature present but not tied) and ligated embryos. Similarly, the normal progression of cervical flexure was not significantly altered by ligation (P>0.1). V(O2) (ml O(2 )g(-)(1 )h(-)(1)) at 38 degrees C, measured by closed respirometry, was not significantly different (P>0.1) on day 3 in sham-ligated (14.5+/-1.9 ml O(2 )g(-)(1 )h(-)(1)) and ligated 17.6+/-1.8 ml O(2 )g(-)(1 )h(-)(1)) embryos. Similarly, on day 4, V(O2) in sham-ligated and ligated embryos was statistically the same (sham-ligated 10. 5+/-2.9 ml O(2 )g(-)(1 )h(-)(1); ligated 9.7+/-2.9 ml O(2 )g(-)(1 )h(-)(1)). Expressed as a linear function of body mass (M), V(O2) in sham-ligated embryos was described by the equation V(O2)=-0.48M+24.06 (r(2)=0.36, N=18, P<0.01), while V(O2) in ligated embryos was described by the equation V(O2)=-0.53M+23.32 (r(2)=0.38, N=16, P<0.01). The regression line describing the relationship between body mass and V(O2) for pooled sham-ligated and ligated embryos (the two populations being statistically identical) was V(O2)=-0.47M+23.24. The slope of this regression line, which was significantly different from zero (r(2)=0.30, N=34, P<0.01), was similar to slopes calculated from previous studies over the same range of body mass.Collectively, these data indicate that growth and V(O2) are not dependent upon cardiac output and the convective blood flow it generates. Thus, early chick embryos join those of the zebrafish, clawed frog and axolotl in developing a heart beat and blood flow hours or days before required for convective oxygen and nutrient transport. We speculate that angiogenesis is the most likely role for the early development of a heart beat in vertebrate embryos.

Cirugía fetal experimental / Experimental fetal surgery

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Development of maternal IgG-free chick obtained from surgically bursectomized hen.

IgG-free eggs and chicks were developed, so as to study the role of maternal IgG in the development of the immune system. Surgical bursectomy on the 18th day of incubation deprived chickens of B cells and eliminated IgG synthesis. Bursectomized chickens are usually dead before sexual maturity under conventional conditions. When surgically bursectomized chickens were housed in an isolated clean room and antibiotics were administered to them, they could survive to sexual maturity. Finally, we succeeded in obtaining IgG-free fertilized eggs and maternal IgG-free chicks from surgically bursectomized hens. The amount of yolk IgG in IgG-free eggs was one-ten thousandth less than that in normal eggs. The level of IgM in the serum of maternal IgG-free chicks reached six times higher than that of normal chicks 5 days after hatching.

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast.

In the amniotes, two unique layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage. All the tissues of the adult will derive from the epiblast, whereas hypoblast cells will form extraembryonic yolk sac endoderm. During gastrulation, the endoderm and the mesoderm of the embryo arise from the primitive streak, which is an epiblast structure through which cells enter the interior. Previous investigations by others have led to the conclusion that the avian hypoblast, when rotated with regard to the epiblast, has inductive properties that can change the fate of competent cells in the epiblast to form an ectopic embryonic axis. Thus, it has been suggested that the hypoblast normally induces the epiblast to form a primitive streak at a specific locus. In the work reported here, an attempt was made to reexamine the issue of induction. In contrast to previous reports, it was found that the rotated hypoblast of the chicken embryo does not initiate formation of an ectopic axis in the epiblast. The embryonic axis always initiates and develops according to the basic polarity of the epiblast layer. These results provoke a reinterpretation of the issues of mesoderm induction and primitive streak initiation in the avian embryo.

Pattern regulation in the chick autopodium at advanced stages of embryonic development.

In previous studies we have observed that the interdigital tissue of the chick embryo leg bud during the stages previous to interdigital cell death exhibits a considerable chondrogenic potentiality both in vivo and in vitro. In the present investigation we have carried out a variety of experimental manipulations of the chick leg bud at stage 29 to discover possible mechanisms accounting for interdigital ectopic chondrogenesis and extradigit formation. Our results show that the interdigital tissue is capable of forming an extradigit when temporarily isolated microsurgically and regrafted in its original location and after deletion of one of the adjacent digital primordia, suggesting that developing phalangeal cartilages exercise an inhibitory effect on chondrification in adjoining tissues. Furthermore, and of greater importance, ablation of the primordium of a digit is followed by normal development of the definitive digit if the wound surfaces are suitably apposed. These results reveal a considerable regulatory potential in the autopodium at advanced stages of development.

Action of estradiol and tamoxifen on the Müllero-regressive activity of the chick embryonic testis assayed in vivo by organotypic grafting.

In the chick, the implantation of a testis graft from a 13-day-old male donor embryo into the extra-embryonic coelom of 3-day-old female embryos induces the total regression of their Müllerian ducts because of the anti-Müllerian hormone (AMH or MIS) secreted by the implant. Pre-treatment of the donors with estradiol (E2), between day 12 and day 13, counteracts in a significant way the Müllero-regressive activity of the implant. Co-treatment of donors at the same stage with both Tamoxifen (TAM) and E2 restores the initially observed activity, thus demonstrating the presence of Tamoxifen-sensitive estrogen receptors at the late stage of treatment in the Sertoli cells responsible for AMH secretion. The treatment of 3-day-old male donor embryos with E2 causes the differentiation of their left gonad into an ovotestis which provides implants totally devoid of Müllero-regressive activity. The additional treatment with TAM of the grafted host embryos, does not modify the results obtained when E2-treated male gonads are grafted to host embryos not treated with TAM. This shows that the lack of Müllero-regressive activity exhibited by the E2-treated male gonads does not depend on the estrogens they may secrete during the time of the assay, i.e., it cannot be attributed to a protecting action of estrogens on the MDs of the host. Our results therefore favor the idea that E2 down-regulates AMH. The relevance of such a regulation to the phenomenon of Müllerian duct maintenance, either in the E2-feminized male or in the female chick embryo, is discussed.

[The chick embryo as a model of fetal surgery: intestinal atresia]. / El embrión de pollo como modelo de cirugía fetal: atresia intestinal.

Aiming at depicting the advantages of chick embryo as a model of fetal surgery we report on our experience of 402 operations carried out in an attempt to induce fetal intestinal obstruction. We have used fertile domestic hen (gallus gallus) eggs incubated at 37.5 degrees with 80% humidity and turned every hour. At the 12th day of incubation we coagulated, after exposing the umbilical cord, a small bowel loop in the physiologic umbilical hernia through a 1 cm-wide opening in the shell which was then sealed with plastic sterile adhesive. After sacrifice at the 15th day of the 76 surviving embryos (19%) we found type I intestinal atresia with localized meconium peritonitis and dilated proximal loop in 46 cases. In 3 out of them there was, like in some human newborns with intestinal obstruction, increased amount of bile-stained amniotic fluid. Both macroscopic and microscopic findings were identical to those usually found in human atresia and in that experimentally induced in fetus of other animal species. We firmly believe that our model is as good as any other for this purpose and, at the same time, it is cheaper and easier to handle.

A method to increase the survival rate of early chick embryos in experiments involving surgical intervention.

A method is presented by which the survival rate of early chick embryos following surgical procedure can be greatly improved. It was discovered that the embryos were dying at the stage of the operating procedure at which ink was being introduced under the embryo. Ink was used to visualize the embryonic features more easily. The use of a contrast medium in which the ink has been previously mixed with yolk in the ratio of 1:30 solved the problem.

Total thymectomy in the early chick embryo.

A new microsurgical procedure is described through which the thymus can be completely removed bilaterally prior to its seeding by lymphoid precursor cells in the chick embryo. 53% of the embryos operated at 5 days of incubation and sacrificed either before or after hatching were totally thymectomized as controlled on serial sections of the neck. Hatchability is low, as it normally is when operations are performed on embryos in ovo. However totally thymectomized viable chicken could be recovered. In certain series of experiments, the graft of a 5-day quail embryonic thymus was performed into the site of excision of the chick thymus. The quail thymic rudiment then became colonized by chick lymphoid precursor cells and developed normally. The possibility of using this excision-graft technique is suggested to study the role of MHC gene products in T-cell differentiation.

Isotopic labeling of embryo, yolk sac, and intracellular parasites of the embryonated hen's egg by yolk replacement.

A technique is described which allows the replacement of 50% of the yolk of the embryonated hen's egg with large volumes of diverse but chemically defined solutions. By using an electrosurgical unit and a polyethylene tunnel, the procedure was performed on eggs from days 3 through 7 with greater than 90% surgical success and viability for the short term. More than 50% of the eggs replaced showed viability for 2 weeks, and a significant proportion went full term. (32)PO(4) and amino acids ((3)H and (14)C) added to the replaced eggs were incorporated into the macromolecules of the embryo and yolk sac as well as into parasitic rickettsiae cultivated in the replaced eggs. The incorporated (32)PO(4) was shown to be assimilated into a variety of biochemical species.