Vascular system in the developing wing bud of normal and talpid mutant chick embryos

The vascular limb mesoderm probably plays a prominent role in limb pattern formation in both normal and talpid chick embryos. The differential vascularization, or its consequences, is a factor controlling the initial stages of differentiation in the developing bud. The axial artery runs from the subclavian artery to the distal region of the normal limb bud, whereas in the talpid3 only secondary blood vessels develop. In the talpid, the gene permits the chondrogenic regions to grow and at the same time keeps the peripheral regions to a certain size. The mesenchyme tissue lies within the effective range of a metabolic gradient extending from either the ectodermal surface or the peripheral vessels to the limb axis.     

Cell generation times in normal and talpid3 mutant chick limb mesenchyme

A comparative study of the cell cycle and its parameters, and of limb pattern development, was carried out in normal and talpid3 wing buds. The relative duration of the G2 + M phase is the least variable. The change in the length of the G1 phase and the S phase is associated with the appearance of mucopolysaccharides in chondrogenic zones and of myosin in myogenic zones. The effect of the ta3 gene is failure of chondrogenesis-induced lengthening of the G1 phase to appear in the chondrogenic condensation in the limb mesenchyme. The data also raise the question of the possible role of the apical ectodermal ridge in control of the cell division rate in the sub-ridge region.     

Intercellular adhesion and formation of aggregates in normal and Talpid-3 mutant chick limb mesenchyme.

It is demonstrated, using the Couette viscometer method, that talpid-3 mutant chick wing mesenchyme cells are more adhesive to one another than are normal cells. The relation of this to differences in the size and shape, and the internal architecture, of aggregates produced in rotation cultures of these cells was investigated. Sequences of sections through aggregates in all stages of formation, from 2-cell aggregates up to those with large cell numbers, were prepared. These confirm the theoretically predicted relationships among adhering cells which would produce the observed small, spherical talpid-3 aggregates and the larger, unevenly shaped normal aggregates. The cell contacts are further analysed with electron micrographs.     

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D_Davis (Hb D_D), an autosomal codominant in chickens, the α^D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D_D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M_D), on high-resolution isoelectric focusing gels. Comparison of $\text{P1}\text{P2}$ ratios in the four phenotypes indicated that homozygous Hb D_D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D_D) than did red cells from corresponding adult birds. Heterozygous Hb D_D embroys and adults had both Hb D and Hb D_D. Hb D_D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D_D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D_D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D_D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.     

The differentiation of normal and muscle-free distal chick limb bud mesenchyme in micromass culture

Distal chick wing bud mesenchyme from stages 19 to 27 embryos has been grown in micromass culture. The behavior of cultures comprising mesenchyme located within 350 microns of the apical ectodermal ridge (distal zone mesenchyme) was compared to that of cultures of the immediately proximal mesenchyme (subdistal zone cultures). In cultures of the distal mesenchyme from stages 21-24 limbs, all of the cells stained immunocytochemically for type II collagen within 3 days, indicating ubiquitous chondrogenic differentiation. At stage 19 and 20, this behavior was only observed in cultures of the distal most 50-100 microns of the limb bud mesenchyme. Between stages 25 and 27, distal zone cultures failed to become entirely chondrogenic. At all stages, subdistal zone cultures always contained substantial areas of nonchondrogenic cells. The different behavior observed between distal zone and corresponding subdistal zone cultures appears to be a consequence of the presence of somite-derived presumptive muscle cells in the latter, since no such difference was observed in analagous cultures prepared from muscle-free wing buds. The high capacity of the distal zone for cartilage differentiation supports a view of pattern formation in which inhibition of cartilage is an important component. However, its consistent behavior in vitro indicates that micromass cultures do not reflect the in vivo differences between the distal zones at different stages. The subdistal region retains a high capacity of cartilage differentiation and the observed behavior in micromass reflects interactions with a different cell population.     

Onset of troponin synthesis in the chick wing bud.

Indirect antibody labeling techniques were used to determine when cells in the chick embryo wing bud begin to synthesize troponin. Frozen sections of stage 22 through stage 27 wing buds were treated with antibodies to the troponin complex and fluorescein-labeled antiimmunoglobulin. Cells producing detectable quantities of troponin were found first in late stage 24 or early stage 25 wing buds; all wing buds stage 25 and older contained labeled cells. Cells synthesizing troponin were initially localized in the muscle-forming areas of the wing bud nearest to the body wall. As the wing bud developed, cells located in more distal areas of the wing bud became labeled with fluorescent antibody, and the number of cells engaged in troponin synthesis increased in all areas. At all stages in which labeling occurred, some cells contained fluorescent cross-striations. When placed in the context of recent studies on the appearance of myofibrillar proteins, these results indicate that myogenic cells in the chick limb bud begin to synthesize large quantities of troponin at approximately the same time as the other muscle contractile proteins.     

Further studies of hormone-sensitive sodium and potassium transport in red cells from developing chick embryos

Sodium and potassium transport in the definitive series of chick embryo red cells changes significantly, both qualitatively and quantitatively, during maturation. Sodium efflux and potassium influx consist of three parts: a ouabain-sensitive, a furosemide-sensitive, and a ouabain-furosemide-insensitive component. In chick red cells of most ages, the ouabain-sensitive and furosemide-sensitive parts of the cation fluxes do not overlap. Cation transport in the more mature red cells is increased significantly by epinephrine, whereas cation transport in red cells from younger embryos is stimulated much less. This is a beta-adrenergic effect of epinephrine and is mediated by cyclic AMP. The relative lack of response in younger embryos is not due to the absence of beta-adrenergic receptor or the lack of production of cyclic AMP. Ouabain has no effect on the hormone-sensitive sodium or potassium transport. On the other hand, furosemide nearly completely abolishes the effect of epinephrine. In addition, there is a good correlation between furosemide-sensitive components of both sodium and potassium transport and the epinephrine-sensitive component. Furosemide has no effect on cyclic AMP levels in the presence or absence of epinephrine. This suggests that furosemide may act directly on the cation transport system. In the red cells from younger embryos, furosemide-sensitive units are present but cannot be fully activated by epinephrine. Therefore, the lack of the hormone effect on cation movements in these cells is consistent with the view that the appropriate units are present, but do not respond fully to intracellular cyclic AMP levels.     

Dissociated limb bud cells of chick embryos can express lens specificity when reaggregated and cultured in vitro

Limb bud cells of chick embryos (stages 23–24) were dissociated into single cells, reaggregated, and cultured in vitro for about a week. δ-Crystallin, generally thought to be a lens-specific protein in the chick, was detected in the aggregates by indirect immunofluorescent staining, double immunodiffusion test, and immunoelectrophoresis with specific antiserum against δ-crystallin. Cells containing δ-crystallin were distributed in epidermal cell clusters and also in mesenchymal tissues surrounding cartilage nodules in the aggregates. Those cells in mesenchymal tissues were shown to have originated from the mesoderm of the limb bud, and those in epidermal cell clusters probably originated from the ectoderm. The possible cellular origin of this appearance of δ-crystallin was discussed.     

Cleavage of A-CAM by endogenous proteinases in cultured lens cells and in developing chick embryos

We describe two truncated forms of A-CAM (N-cadherin) and present evidence suggesting that both forms are proteolytically derived from the intact A-CAM molecule. The first is a membrane-bound fragment of A-CAM displaying an apparent molecular weight of 78 kDa. This polypeptide, containing the C-terminal portion of the protein, may be generated in cultured chicken lens cells, either by a short treatment with trypsin-EGTA, or by endogenous proteinase(s) during incubation in low Ca2+ medium. Immunofluorescent labeling of normal and EGTA-treated cells indicated that the 78-kDa fragment is uniformly distributed over the cell surface. Moreover, staining of developing chick embryos with pairs of antibodies which distinguish the 78-kDa fragment from intact A-CAM indicated that, at early stages of sclerotome dissociation in developing somites, a truncated derivative of the molecule is generated. The second truncated form of A-CAM is a 97-kDa polypeptide which is constitutively released by cultured lens cells into the culture medium in the presence of normal medium. We present evidence that the 97-kDa molecule is proteolytically derived from A-CAM by the action of an endogenous proteinase. We discuss possible mechanisms leading to the formation of these two truncated derivatives and their possible involvement in the physiological modulation of A-CAM-mediated interactions.     

The anatomy, ultrastructure and fluid dynamics of the developing vasculature of the embryonic chick wing bud

The spatiotemporal sequence of vascular pattern development in the embryonic chick wing bud and surrounding shoulder, flank and belly regions is detailed for Hamburger-Hamilton (1951) stages 20-25. Vasculature was microinjected with an unreactive aqueous tracer (aniline blue), and major traffic patterns were visualized. Formation of extensive avascular regions and the emergence of chondrogenic phenotypes are correlated with the retreat of the vasculature from the wing core. Ultrastructural studies of vascular cells show that vessels remain monolayered and undifferentiated until stage 25, after the adult vascular pattern has been laid down. Vascular cytodifferentiation occurs only in the cells of the brachial artery until stage 35, with the veins and capillaries retaining an 'early' morphology. This vascular pattern may be an important component reflecting or directing limb pattern development.     

High-affinity binding of testosterone in serum from normal developing chick embryos and during the graft-versus-host reaction

The sera of developing chicken embryos contain high-affinity, low-capacity protein binding sites for testosterone. The affinities remain constant throughout development, with mean values for the association constants of approx. 3.6 X 10(8) M-1 at 25 degrees C, whereas the concentration of sites varies markedly as a function of age: from approx. 2 nmol/g serum proteins in 11-day embryos, it rises to a peak of approx. 5-8 nmol at 14-16 days, then drops to approx. 2.6 nmol at 18 days and only 0.8-1 nmol in adults. Testosterone binding is inhibited by corticosterone, progesterone and dihydrotestosterone, and is little affected by estradiol. The testosterone and corticosterone binding properties of chicken sera show close similarities: parallel ontogenic patterns; constant ratios, throughout development, of the equilibrium binding parameters of the two steroids; mutual binding inhibition. The evidence strongly suggests that the two activities are associated, at least in part, with a common protein carrier(s). In growing embryos which undergo a graft-versus-host reaction, elicited by the graft of adult spleen tissue at 9 days of age, the testosterone and corticosterone binding activities are significantly decreased. This decrease is due to a fall in the number of sites, whereas association constants are not affected. This is the first high-affinity, saturable, testosterone-binding property to be described in an embryonic serum.     

Nerve-dependent and -independent tenascin expression in the developing chick limb bud.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.     

Cell fusion and differentiation in cultured chick muscle cells

A method for the quantitative estimation of myoblast fusion and the effects of variables in the culture conditions on the extent of fusion are described. The onset of fusion is delayed both by feeding and by lowering the initial cell density, but effects of alterations in sera and embryo extracts are evident only at later stages of differentiation. Using a sensitive fluorimetric assay for creatine phosphokinase, enzyme activity was determined throughout the culture period and related to the extent of cell fusion. The increase in activity during a 7-day culture period is biphasic, the first small increase (2–5 ×), which begins after 40–50 h in culture, being apparently closely related to cell fusion. There is a 48 h delay before the second, much larger (up to 25 ×) increase begins.     

Collagen synthesis in the muscle of developing chick embryos.

Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443--453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.     

Apoptosis in the chick wing bud and the permanence of FGF-2 rescue

Summary Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The opaque patch (OP) and posterior necrotic zone (PNZ) were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by inhibition of protein synthesis with cycloheximide. Both regions showed rescue with cycloheximide as determined by the chromium release assay and examination of electron micrographs. Also, the permanence of basic fibroblast growth factor (FGF-2) rescue in the OP and PNZ was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. This research shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation and is prevented by inhibition of protein synthesis with cycloheximide, biochemically characterizing this death as apoptosis. It also suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ.