Effects of posture on the respiratory mechanics of the chick embryo

In the chicken embryo, pulmonary ventilation and pulmonary gas exchange begin approximately one day before the completion of hatching. We asked to what extent the posture inside the egg, and the presence of the eggshell and membranes, may alter the mechanical behaviour of the respiratory system. The passive mechanical properties of the respiratory system were studied in chicken embryos during the internal pipping phase (rupture of the air cell) or the external pipping phase (hole in the eggshell). Tracheal pressure and changes in lung volume were recorded during mechanical ventilation, first, with the embryo curled up inside the egg, then again after exteriorization from the eggshell. In the internal pippers, respiratory system compliance increased and expiratory resistance decreased after exteriorization, whereas the mean inspiratory impedance did not change. In the external pippers, exteriorization had no significant effects on respiratory compliance, resistance, or impedance, and the values were similar to those of newly hatched chicks. We conclude that, in the chicken embryo, at a time when pulmonary ventilation becomes an important mechanism for gas exchange, the curled up posture inside the egg does not provide any significant mechanical constraint to breathing.     

Studies on the mechanics of development of the visual pathways in the chick embryo

Silver-stained whole mounts of the tectal surface were used to study the developing retinotectal fiber pathway in the chick embryo. The growing front of optic fibers appeared more disordered than fiber bundles to the rear. The fiber pattern as a whole appeared more orderly with age. Some fibers projected sparsely ahead of the growing front in a pattern suggesting the existence of preneural guidance channels. Fiber branching was rarely seen. An orthogonal gridwork of two layers of fibers, running roughly anteroposteriorly and dorsoventrally, was found on the developing tectal surface after removal of both optic vesicles. In unoperated specimens, fibers in the optic fiber layer followed these grid lines.Retinal quadrants from embryos 4–5 days old were transplanted to the optic tecta of embryos 6–7 days old. The graft fibers subsequently did not show specific orientation toward their appropriate tectal quadrant. Rather, the fibers followed the same straight courses taken by carbon particles implanted in comparable positions in controls.After the production of quadrantal retinal lesions in 4–5 day-old embryos, no evidence was found for specific tectal innervation defects at 12–13 days. Lesions, irrespective of retinal quadrant, resulted in a relative lack of innervation in the posterodorsal aspect of the 12–13 day-old tectum. This was probably due to a delay in the advancement of the growing front of fibers across the tectal surface. The results weaken previous support for specificity in the guidance of developing optic axons.     

Cardiac jelly arrangement during the formation of the tubular heart of the chick embryo.

The hearts of chick embryos from stages 9 to 11 according to Hamburger and Hamilton are studied by means of the electron microscopy, and the arrangement of the cardiac jelly (CJ) during the different steps of the fusion of the paired heart anlage is described. It is shown that two different areas can be distinguished in CJ: the CJ located between the endocardium and the myocardium (MECJ) and the CJ located between the endocardium and the ventral foregut endoderm (EECJ). MECJ is a zone poor on ultrastructural components most of which are unbanded filamentous material and low-density amorphous material, and its arrangement remains constant during the whole fusion process; EECJ, on the contrary, is very rich in ultrastructural components containing greater amounts of high-density amorphous material, collagen fibrils and cellular, detritus, and the arragement of this area of the CJ undergoes changes during the different fusion steps. Alcian blue staining does not show difference between both areas of CJ. It is suggested that the CJ can play a role during the fusion of the heart. In addition, some observations are reported which suggest that all the epithelial tissues surrounding the CJ can take part in the elaboration of that extracellular material.     

Investigating Murray's law in the chick embryo.

According to the optimization principle known as Murray's law, the blood vessel geometry at a bifurcation satisfies the relation alpha = (D3(1) + D3(2))/D3(0) = 1, where D0, D1, and D2 are the diameters of the parent and two daughter vessels, respectively. Previous investigations have shown that mature blood vessels adhere to this law fairly closely. The purpose of this study was to test Murray's law in the developing extraembryonic blood vessels of 2-4 day-old chick embryos. Vessel diameters were measured manually using image analysis software. The measurements for the group of all vessels at all studied stages (n = 449) gave alpha = 1.01+/-0.34 (mean +/- SD), and the value of alpha is similar at all stages. These results indicate that Murray's law holds in the chick embryo, even before medial smooth muscle becomes functional, suggesting that blood vessels follow the same basic morphogenetic rules throughout life.     

Glycogen metabolism in the prelaid chick embryo.

Glycogen metabolism has been studied during the development of the early chick embryo, at the cytochemical and ultrastructural levels. Two waves of glycogen synthesis and breakdown have been found. In the first, free clusters of glycogen particles are synthesized at late oogenesis. These clusters are found later in invaginations of the membrane of vesicles containing a floccular material (FLOV). The glycogen clusters are degraded there during ovulation and the first hours in the oviduct. The second wave of glycogen synthesis begins before cleavage, reaching a maximum at mid-uterine age. This second wave occurs in another type of vesicle (GLYV), which eventually disintegrates releasing free clusters of glycogen granules. This glycogen is degraded in membranous structures containing a floccular material, as in the first wave of degradation. The degradation ends at the late uterine stages, and at the same time numerous ribosomes are formed. This period corresponds to area pellucida formation, which probably depends on the energy liberated during the second wave of glycogen degradation.     

Cardiac differentiation induced by dopamine in undifferentiated cells of early chick embryo

Evidence suggests that neurotransmitters can act as possible chemical signals involved in cell division and morphogenetic movements long before neurons appear in the embryo. However, whether they are playing a role in differentiation is now unknown. It was recently observed (M. Sarasa and S. Climent, 1987, J. Exp. Zool. 241, 181-190) that the neurotransmitter dopamine exerted a stimulating effect on cardiac differentiation in the chick in ovo. We show here that dopamine acts as a specific inducer of heart muscle differentiation in vitro. When cells of the gastrula of embryos treated with dopamine were dissociated and reaggregated, the aggregates obtained almost entirely underwent cardiac muscle differentiation. Also, when small postnodal pieces obtained from the most posterior region of the gastrula were cultivated in the presence of dopamine, they differentiated into myocardic tissue instead of following their fate map. Therefore, dopamine can trigger a process that both causes undifferentiated cells to differentiate into heart muscle and compels cells already determined to another way of differentiation to become myocardic tissue.     

Neural induction and regionalisation in the chick embryo.

Induction and regionalisation of the chick nervous system were investigated by transplanting Hensen's node into the extra-embryonic region (area opaca margin) of a host embryo. Chick/quail chimaeras were used to determine the contributions of host and donor tissue to the supernumerary axis, and three molecular markers, Engrailed, neurofilaments (antibody 3A10) and XlHbox1/Hox3.3 were used to aid the identification of particular regions of the ectopic axis. We find that the age of the node determines the regions of the nervous system that form: young nodes (stages 2-4) induced both anterior and posterior nervous system, while older nodes (stages 5-6) have reduced inducing ability and generate only posterior nervous system. By varying the age of the host embryo, we show that the competence of the epiblast to respond to neural induction declines after stage 4. We conclude that during normal development, the initial steps of neural induction take place before stage 4 and that anteroposterior regionalisation of the nervous system may be a later process, perhaps associated with the differentiating notochord. We also speculate that the mechanisms responsible for induction of head CNS differ from those that generate the spinal cord: the trunk CNS could arise by homeogenetic induction by anterior CNS or by elongation of neural primordia that are induced very early.     

Cell lineage restrictions in the chick embryo hindbrain.

During development of the chick embryo, early neuronal differentiation and axonogenesis in the hindbrain follow a segmented pattern in register with the segmented morphology of this region. Cell marking experiments have shown that the segments, or rhombomeres, are lineage-restriction units each constructing a defined piece of the hindbrain. This raises the interesting possibility that, as in the developing fly, metamerism is used to generate level-specific anatomical structures with great and reliable precision. In the hindbrain, as for many invertebrates, lineage ancestry may be important in the determination of cell fate. The segmentation seen in this body region could therefore reflect a similar condition once present in the ancestor common to vertebrates and invertebrates.     

Cardiac mechanics at the cellular level.

The active mechanical properties of heart muscle are load, length, and time-dependent. The capability for investigating cardiac mechanisms at the cellular level may help to distinguish between those properties of the myocardium which arise from myocardial cells and those which arise from the tissue architecture and extracellular matrix of connective fibers. We present here, for the first time, a general approach for subjecting single heart cells to isometric, isotonic, afterloaded, or physiological loading sequences, while obtaining on-line measures of cell force and length. This approach has been implemented and tested on freshly dissociated, adult frog ventricular myocytes. Examples are presented for each of the four loading sequences.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.     

Erythropoiesis in the developing chick embryo

The types of erythroid cells of chick embryos developing in ovo have been correlated with the hemoglobins of the embryos. Prior to 5 days, when primitive cells constitute the only erythroid cells, two hemoglobins can be resolved by polyacrylamide gel electrophoresis. The two adult hemoglobins and a minor hemoglobin found only in embryos and young chicks first appear simultaneously with initiation of definitive erythropoiesis.