[Study of an embryonic heterograft: graft of mouse embryo limb bud on chick embryo].
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A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo.
In the chick embryo, two secreted factors have recently be shown to cooperate in inducing the first axial structure, the primitive streak: cWnt8C (normally expressed around the circumference of the embryo, in the marginal zone) and the TGF beta superfamily member cVg1 (expressed in the posterior part of the marginal zone) (Development 128 (2001) 2915). Misexpression of Vg1 in the anterior marginal zone induces an ectopic primitive streak and recapitulates the morphological changes associated with normal primitive streak formation. Here, we analyse the time-course of appearance and disappearance of expression of 12 genes (cVg1, Lef1, Nodal, FGF8, cWnt8C, cBra, cNot1, goosecoid, HNF3 beta, Chordin, Otx2 and Sox3, whose normal expression is also polarized at early stages of development) in response to cVg1 misexpression in the anterior marginal zone. We show that a hierarchy of gene expression accompanies induction of the ectopic axis, reminiscent of the order in which the same genes begin to be expressed in the normal embryo.
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Hapten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo.
The early chick blastoderm expresses two endogenous galactose-binding lectins of 14 kDa and 16 kDa. We have studied the effect the lectin hapten inhibitors thiodigalactoside and the synthetic neoglycoprotein lactosyl-bovine serum albumin as well as polyclonal anti-lectin antibodies on the development of early chick embryos cultured in a defined medium. Controls consisted of maltose, maltosyl bovine serum albumin and rabbit IgG. Embryos treated at the onset of cell migration during early gastrulation underwent blastoderm retraction with decrease in surface area. In addition, they exhibited a lack of demarcation between the presumptive embryonic area (area pellucida) and the presumptive extraembryonic area (area opaca). These blastoderms also lacked a primitive streak, that is, the structure that forms in the area pellucida during gastrulation as cell migrate to form the endodermal and mesodermal layers of the embryo. Embryos treated at later stages of gastrulation showed development similar to that of controls in that they were able to undergo early organogenesis. The results suggest that lectin mediated mechanisms are essential for the migratory movements of early gastrulation and that, at late gastrulation, other mechanisms exist in the embryo to compensate for lectin function.
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Ankle restraint modifies motility at E12 in chick embryos.
The chick's relationship to its environment changes dramatically over 21 days of embryonic development. At early ages embryos are buoyant; their posture and movements are relatively unconstrained. As embryos grow and fluid level in ovo decreases, movements are increasingly constrained by gravitational forces and reactive forces due to body contact with the shell wall. The issue of how age-related changes in the constraints on movement in ovo may affect embryonic motility is addressed in this paper. Our long-term goal is to determine whether experience imposed by these conditions contributes to development of posthatching motor behaviors. Because previous work indicated that parameters of motility can be modified by a reduction in buoyancy at embryonic day (E) 9, we sought to determine whether a restraint localized to a single joint could also alter either the episodic distribution of activity or the spatiotemporal patterns of limb movement at either E9 or E12. Thus a restraint was applied to the right ankle of embryos prepared for kinematic recordings. Video and kinematic analyses indicated that the restraint had minimal effect at E9, but significantly modified several motility parameters in both the wing and leg at E12. Ankle restraint decreased episode duration. Restraint also decreased most joint excursion parameters, including excursion range, cycles per sequence, and excursion velocity. Restraint increased cycle period duration and signal frequency content under 1.0 Hz. Parameters of intralimb and interlimb coordination exhibited small mixed effects. Results provide support for the hypothesis that environmental conditions contribute to features of embryonic motility. Further, significant modifications of wing excursions in ankle restrained embryos suggest that sensory feedback arising from mechanical perturbations of leg movements may entrain rostral spinal circuits for preservation of interlimb coordination at E12. Potential mechanisms and implications are discussed.
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Use of retinoic acid for the analysis of dermal-epidermal interactions in the tarsometatarsal skin of the chick embryo.
Feet of chicks are normally covered with scales. Injection of retinoic acid into the amniotic cavity of 10-day chick embryos causes the formation of feathers on the foot scales. To elucidate whether retinoic acid affects primarily the epidermis or the dermis, heterotypic dermal-epidermal recombinants of tarsometatarsal skin were tested as to their morphogenetic capacity, when grafted to the chick chorioallantoic membrane. Recombinants involving treated epidermis and untreated dermis formed feathered scales, while the reverse recombinants of untreated epidermis and treated dermis led to the formation of scales only. Likewise the association of treated tarsometatarsal dermis with untreated epidermis from a non-appendage-forming region (the midventral apterium) resulted in the formation of scales only. These results show that retinoic acid affects primarily the epidermis. Further insight into the mechanism of dermal-epidermal interaction was gained by heterotopic recombinations of early (8.5- and 10-day) untreated tarsometatarsal dermis with epidermis from the midventral apterium. These recombinants formed scales, proving that tarsometatarsal dermis is endowed with scale-forming properties as early as 8.5 days of incubation. Finally, it is concluded that retinoic acid acts on the chick foot epidermal cells by temporarily inhibiting their scale placode-forming properties, allowing their latent feather placode-forming properties to be expressed.
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Rubella virus-induced interference in chick embryo cells.
Primary chick embryo cells inoculated with rubella virus were found to develop interference with the multiplication of Sindbis virus. Substantial quantities of interferon (IFN) were detectable in the cell cultures indicating that the resistance induced was due to IFN production.
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Evidence for the synthesis of a somatomedin similar to insulin-like growth factor I by chick embryo liver cells.
Chick embryo liver cells were incubated in serum-free and hormone-free medium and shown to produce a mitogen starting after the first day in culture. This mitogen was purified by gel filtration and ion-exchange chromatography and identified by several assay systems as a somatomedin similar to human insulin-like growth factor I (IGF-I). This somatomedin stimulates thymidine incorporation into DNA of chick fibroblasts, it reacts in an IGF-specific protein binding assay and it cross-reacts immunologically with antibodies prepared against human IGF-I, but not with those against IGF-II. Its behaviour on ion-exchange chromatography and on sodium dodecylsulfate/polyacrylamide gel electrophoresis is indistinguishable from that of authentic human IGF-I. Concomitantly with the mitogenic activity, chick liver cells produce IGF-binding protein. Addition of purified human IGF-I to the liver cells inhibits production of the binding protein. Chick liver cells may thus be used to study synthesis and regulation of IGF and its binding protein.
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An experimental and morphological analysis of the tail bud mesenchyme of the chick embryo.
In the chick embryo, the tail bud reaches its maximum length at about stage 22 of Hamburger and Hamilton, after which it starts to regress. By this stage the neural tube and notochord extend right to the tip of the tail, but the somites do not do so, the terminal tail bud mesoderm never becoming segmented. The investigation is concerned with analysing why this mesoderm fails to segment. When tail buds were explanted to the chorio-allantoic membrane, they continued to form somites only until the "correct" number had segmented, i.e., the tail bud formed no more somites when isolated from the embryo than it would have formed if undisturbed. Morphological studies suggest that in the normal embryo massive cell death overtakes the tail bud mesoderm before it can segment. It is suggested therefore that cell death may be a contributory factor in preventing segmentation.
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Transition from non-muscle to muscle myosin light chains in chick embryo development.
In chick embryo blastoderm the electrophoretic pattern of myosin light chains changes between the 4-somite and the 19-somite stages (stages 8-13 of Hamburger and Hamilton) from that of non-muscle to muscle myosin. This transition seems to follow the differentiation of the myotomes and to be developmentally regulated.
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Vitamin A economy of the developing chick embryo and of the freshly hatched chick.
1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.
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Influence of progesterone on protein and RNA synthesis in cultured chick embryo liver cells.
Chick embryo liver primary cultures, when added with progesterone, exhibit, in comparison with the controls, a normal growth, a decline of both 3H-uridine uptake and incorporation into total RNA, a decline of 3H-leucine and 14C serine incorporation into the proteins. Progesterone is not able to stimulate phosvitin synthesis induction.
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Actinomycin D: inhibition of phospholipid synthesis in chick embryo cells.
When chick embryo fibroblasts are exposed to actinomycin D for 30 minutes to 3 hours, there is progressive inhibition of phospholipid synthesis, so that by 3 hours the inhibition is 40 percent. All phospholipids are affected. Since this inhibition is twice as great as the inhibition of protein synthesis, some effects of actinomycin D previously ascribed to decreased protein synthesis must be reevaluated.
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[The effect of the testicular mullerian-inhibiting hormone on ovarian development in the chick embryo].
In female chick embryos grafted with embryonic testis, it is a correlation between the effects of the graft on the mullerian ducts and on the ovary. Indeed, when the graft causes retrogression of the ducts, it induces an ovarian atrophy also. These facts lead to think that these two activities are due to the same substance, the mullerian inhibiting hormone of the testis.
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Interferon induction by viruses. XX. Acid-labile interferon accounts for the antiviral effect induced by poly(rI).poly(rC) in primary chick embryo cells.
Primary chick embryo cells which were "aged" in vitro and exposed to poly(rI).poly(rC) developed a disproportionately high degree of resistance to virus challenge in relation to the amount of conventional acid-stable interferon (IFN) produced. Cells were often highly resistant after exposure to a few nanograms of poly(rI).poly(rC) which gave rise to little or no acid-stable IFN. Study of the mechanism(s) responsible showed that this antiviral state was not due to the double-stranded ribonuclease induced in parallel, nor to residual poly(rI).poly(rC) (which this ribonuclease destroyed), but to the production of a hitherto undescribed acid-labile chicken IFN.
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Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos.
In the chick embryo, neural cells acquire midbrain, hindbrain, and spinal cord character over a approximately 6 hr period during gastrulation. The convergent actions of four signals appear to specify caudal neural character. Fibroblast growth factors (FGFs) and a paraxial mesoderm-caudalizing (PMC) activity are involved, but neither signal is sufficient to induce any single region. FGFs act indirectly by inducing mesoderm that expresses PMC and retinoid activity and also directly on prospective neural cells, in combination with PMC activity and a rostralizing signal, to induce midbrain character. Hindbrain character emerges from cells that possess the potential to acquire midbrain character upon exposure to higher levels of PMC activity. Induction of spinal cord character appears to involve PMC and retinoid activities.
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[Feminizing action of dehydroepiandrosterone on the left testis of the chick embryo: ultrastructural aspect].
Chick embryos were injected with 4 X 0.5 mg of dehydroepiandrosterone from day 4 onwards and the ultrastructure of the 20-day old hormone-treated male gonads was studied. The cortex was well-developed and some oocytes had reached the meiotic prophase stage. The prefollicular cells did not seem to be stimulated by the androgenic hormone. We especially studied the seminiferous cords with Sertoli cells. Dense core granules, which might be related to secretory activity, were observed in the basal part of the Sertoli cells. The extensive development of smooth endoplasmic reticulum vesicles suggested that some Sertoli cells might have a steroidogenic function. The interstitial cells did not show an important steroidogenic synthesis. The action of dehydroepiandrosterone, which could induce the development of an ovarian-like cortex in intersexual gonads, is discussed.
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Effect of viruses and interferon on chick embryo otocyst cultures.
Chick embryo otocyst organ cultures were subjected to live vesicular stomatitis virus and rubella virus preparations, to interferon (IFN), and to a combination of both virus and IFN, and compared to control untreated otocysts. We observed morphologic and microscopic changes suggestive of individual cell death and delayed organ differentiation in the virus-treated groups, along with an appreciable decrease in size of the otocyst. Low-dose IFN treatment prior to virus inoculation appeared to partially prevent these effects. The addition of IFN alone did not seem to affect the differentiation process. Time-lapse videophotography further confirmed the above findings. This study suggests that the peripheral component of congenital deafness associated with viral infections is likely to be an effect of the virus itself, and not of the IFN. Interferon provides a partial protective effect against the insult from the virus in vitro and does not seem to be toxic to the developing otocyst.
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Ultrastructural observations on cytodifferentiation of thymic cystic cells of chick embryo.
Thymuses from chick embryos from the 13th-19th day of incubation were examined by light and electron microscopy. At the 13th day of incubation, the primitive medulla consists of undifferentiated, cystic, reticular and lymphoid cells interspersed with developing cysts. Undifferentiated cells contain indented nuclei, poorly developed endoplasmic reticulum and numerous free ribosomes. Synthesis of cytoskeletal material and assembly of microvilli denote their differentiation to cystic cells. Cystic cells possess rough- and smooth-surfaced endoplasmic reticulum, a Golgi complex, mitochondria and microvilli. They are organized individually or in clusters to form intra- and intercellular cysts respectively. By the 15th day of incubation, cystic structures are more apparent. Their lumina contain a low density flocculent or moderately dense granular material. At the 19th day, there is a further increase in number and complexity of the cysts. The results demonstrate the existence of undifferentiated cells and their differentiation to cystic cells. Morphological evidence suggests secretory and/or absorptive activities of the cystic cells in the prehatching period.
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