Teashirt 3 expression in the chick embryo reveals a remarkable association with tendon development

Drosophila teashirt (tsh) is involved in the patterning of the trunk identity together with the Hox genes. In addition, it is also a player in the Wingless and the Hedgehog pathways. In birds and mammals, three Tshz genes are identified and the expression patterns for mouse Tshz1 and Tshz2 have been reported during embryogenesis. Recently, we showed that all three mouse Tshz genes can rescue the Drosophila tsh loss-of-function phenotype, indicating that the function of the teashirt genes has been conserved during evolution. Here we describe the expression pattern of chick TSHZ3 during embryogenesis. Chick TSHZ3 is expressed in several tissues including mesodermal derivatives, the central and peripheral nervous systems. Emphasis is laid on the dynamic expression occurring in regions of the somites and limbs where tendons develop. We show that TSHZ3 is activated in the somites by FGF8, a known inducer of the tendon marker SCX.     

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart.

Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.     

Synthesis and degradation of collagen in the developing corium of the chick embryo

1. Indices of collagen synthesis and degradation were developed in a chick corium system in vitro. 2. These indices were determined by quantitatively measuring non-diffusible and diffusible hydroxy[(14)C]proline in the tissues after a standard period of incubation. 3. Under these incubation conditions collagen metabolism of the corium appears to be stable for at least 180min. 4. The indices of collagen synthesis and degradation seem to reflect the conditions of collagen metabolism in vivo. 5. A rapid collagen turnover occurs in the corium of the 13-14-day embryo owing to an accelerated collagen degradation at that period. 6. Epidermal elements may influence the synthesis as well as the degradation of collagen.     

Electron microscope study of the developing chick embryo heart

Summary Myofibrillar differentiation in heart myocells of the chick embryo was studied with aid of the electronmicroscope. At early stages (29 hrs.) thin myofibrillar bundles are seen already differentiated in the cytoplasm of the myoblast and a few hours later cross striation can be depicted as ill-defined densities regularly spaced along the myofilaments.At later stages it was seen that some myofibrillar bundles seem to arise from a Z-band belonging to a thicker bundle. This disposition was frequently seen but the number of bundles observed arising from a single Z-band is varied. Cases were found in which two or three bundles were interconnected by myofibrillar bridges. The disposition was nominated Z center and it is interpreted as favouring spreading of the contractil wave from bundle to bundle. Conduction of the contractil wave from cell to cell is insured through intercalated discs.Desmosomes and intercalated discs were observed during the course of this study, but no relationship between both components of the cell membranes nor between desmosomes and myofilaments were found.Fellow working under a local fellowship program supported by Grant No. 56030 of The Rockefeller Foundation.     

Regulation of the glycosylations of collagen hydroxylysine in chick embryo tendon and cartilage cells

The regulation of the glycosylations of hydroxylysine was studied in isolated chick-embryo cells by labelling with a [¹⁴C]lysine pulse. The course of the procollagen lysyl modifications was compared in tendon and cartilage cells, and the effect on the glycosylations of the degree of lysyl hydroxylation and the concentration of Mn²⁺ and Fe²⁺ were also studied, in tendon cells. Procollagen triple helix formation was inhibited in most experiments in order to eliminate the effect of this process on the continuation of the reactions.Both in the tendon and cartilage cells the intracellular lysyl modifications proceeded in a biphasic fashion. After an initial sharp linear increase, the reactions did not cease but were protracted at a slower but constant rate. Lysyl hydroxylation was followed by rapid galactosylation in both cell types and this was followed almost immediately by rapid glucosylation, suggesting a close association of the corresponding enzymes. The data further suggest that other factors must also exist, in addition to the differences in the timing of triple helix formation and the actual hydroxylysine content, which are responsible for the different amounts of galactose in the collagens synthesized by these cell types. The amount of glycosylgalactosylhydroxylysine nevertheless seemed to be determined by the available acceptor sites, i.e., the amount of galactosylhydroxylysine.In further experiments wiht tendon cells the oxygen participating in lysyl hydroxylation was displaced by nitrogen at various points in time. When the degree of lysyl hydroxylation was reduced to less than one-third of the original, the total amounts of glycosylated residues decreased correspondingly, but their proportion relative to total hydroxylysine remained unchanged.Extra Mn²⁺ increased the proportion of galactosylated hydroxylysine, suggesting that the activity of hydroxylysyl galactosyltransferase is not saturating in respect of the catalyzed reaction. Experiments on the addition of Fe²⁺ or its chelation by α, α′-dipyridyl gave indications that the presence of this co-factor is not required for either glycosylation reaction in isolated tendon cells.     

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.     

Claudin-5 expression in the vasculature of the developing chick embryo

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Studies in human, mouse, Xenopus, and zebrafish have shown that only a limited number of claudins are expressed in endothelial cells. Here, we report the expression pattern of Claudin-5 during chick development. Between HH stage 4 and 6 Claudin-5 expression was observed exclusively in extraembryonic tissue. Claudin-5 expression was not observed in the embryo until HH stage 8, coincident with the onset of embryonic vascularization. Claudin-5 expression was maintained in the developing vasculature in the embryonic and extraembryonic tissue throughout organogenesis (HH stage 19–35), including the vasculature of the ectoderm and of organs derived from the mesoderm and endoderm lineages. These data describe a conserved expression pattern for Claudin-5 in the endothelial tight junction barrier and is the first report of the onset of Claudin-5 expression in a vertebrate embryo.     

Alpha-fetoprotein expression in intra- and extraembryonic fluids of developing chick embryo

The ontogeny of alpha-fetoprotein (AFP) has been studied in the chicken (from 7 days of incubation until 2 days after hatching) using (1) the two-dimensional immunoelectrophoresis technique, (2) the polyacrylamide gel electrophoresis, and (3) the high resolution two-dimensional polyacrylamide gel electrophoresis. The molecular weight of AFP was estimated at 71,000. AFP was seen as a heterogeneous population composed of four isoforms which slightly differ by their isoelectric points. Up to the 18th day of development, qualitative changes in AFP heterogeneity do not occur. Only traces of the two alkaline isoforms were observed in plasma of 2 days post-hatching chickens. AFP has been identified in allantoic and cerebrospinal fluids but is not present in amniotic fluid. At 7 days of embryonic age, all the plasma AFP species are present in cerebrospinal fluid.     

Positive inotropic action of acetylcholine on the heart ventricles of the chick embryo

Acetylcholine increased twitch tension in the whole ventricle or in ventricular strips from 2-10-day chick embryos. The effect of acetylcholine was mediated by muscarinic receptors, since it was prevented by atropine, but not by tubocurarine or propranolol. Prostigmine significantly increased the sensitivity to acetylcholine in the strips from 7-day embryos, being almost ineffective in the strips from 3-day embryos. The decrease in acetylcholine sensitivity in the developing chick embryo is presumably associated with the increase in cholinesterase activity of the myocardial tissue.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on glycine and leucine accumulation

1. The accumulation of [1-(14)C]glycine and the uptake, accumulation, incorporation (into protein, lipid, glycogen) and oxidation of l-[1-(14)C]leucine in 5-day-old chick embryo hearts were investigated in vitro, and the effects of insulin, puromycin and 4-methyl-2-oxopentanoic acid on these processes were studied. 2. With glycine, the ratio of concentration of the labelled amino acid in the cell water to that in medium markedly exceeded unity. Insulin significantly increased this ratio. Puromycin did not prevent the insulin effect. 3. With leucine, the concentration ratio of the labelled amino acid between intracellular and extracellular water approached unity in the absence of puromycin and was doubled by its presence. In neither case did insulin substantially alter this ratio. The addition of 4-methyl-2-oxopentanoic acid had no effect in the absence of insulin, but produced a significant increase of the concentration ratio in the presence of the hormone. 4. Leucine uptake was increased slightly by insulin in all experimental conditions except in the presence of puromycin, where a more pronounced stimulation was observed. The hormone had no effect on the incorporation of the labelled amino acid into protein, but accelerated its oxidation to carbon dioxide; the latter effect was particularly evident in the presence of puromycin and disappeared after the addition of 4-methyl-2-oxopentanoic acid.     

Effects of taxol on endothelial cells of the developing semilunar heart valves in the chick embryo

Recent ultrastructural studies have revealed that there are differences in endothelial cell shape and cytoskeletal architecture between the arterial and ventricular faces of developing semilunar valves. In the present work we have analyzed valvular endothelial cell response to taxol in the chick embryo. Following taxol administration, abnormal mitotic cells appear in the endothelium of both faces of the cusps. Interphase cells show few structural alterations. No differential response of the valvular endothelium to the drug was observed in the arterial or ventricular faces of the cusps.