Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin

Summary Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, -actinin and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryospecific isoform with a different molecular mass was detected.     

Tropomyosin isoforms in developing chicken gizzard smooth muscle

In the embryonic smooth muscle of chicken gizzards we found 4 high-Mr-type and 5 low-Mr-type tropomyosin isoforms in addition to alpha- and beta-isoforms reported already. The criteria by which they were identified as tropomyosin isoforms were as follows: 1) anomalous reduction of electrophoretic mobility in the presence of urea, 2) cross reactivity with antisera against tropomyosins, 3) inclusion in a tropomyosin preparation obtained by the usual method for tropomyosin purification, and 4) binding ability to skeletal muscle actin. At the early stages of development, the amounts of these isoforms were larger than those of alpha- and beta-isoforms, but they gradually decreased at later stages and finally disappeared completely after hatching. Our previous study of gizzard smooth muscle showed that the amount ratio of accumulated tropomyosin to gamma-actin was reasonably constant in the development after hatching, while, at the earlier embryonic stages (7-14 d of incubation), it was lower than expected. The isoforms found in this study were present in amounts large enough to bring the ratio at the earlier stages up to the constant amount ratio observed after hatching. Therefore, the coordinate accumulation of actin and tropomyosin was suggested to occur even at the embryonic stages.     

Differentiation of smooth muscle cells in chicken gizzard

During development of the chicken gizzard, a thick layer of undifferentiated cells (mesenchymal cells) is constructed, and the cells differentiate into smooth muscle cells or connective tissues. We found that the differentiation of smooth muscle cells occurred first near the outer surface of the gizzard and the differentiated area spread to the inside of the gizzard. Therefore, we assumed that the differentiation of most of the smooth muscle cells in the gizzard is induced by differentiated smooth muscle itself. When undifferentiated cells from gizzard of 7-day-old embryo (Hamburger and Hamilton's stages 26-27) were cultured on a coverglass coated with extract of gizzard that contained differentiated smooth muscle cells, the cells attached to the coverglass and differentiated into smooth muscle cells. On the other hand, extract of gizzard from 7-day-old embryo did not induce the differentiation of smooth muscle cells, though it induced the attachment of cells. We found that activity for the differentiation of smooth muscle cells appeared when differentiated smooth muscle cells appeared in developing gizzard. Gizzard contained higher activity for the differentiation of smooth muscle cells than the other tissues. Transforming growth factor-beta (TGF-beta), which induces the differentiation of vascular smooth muscle cells, did not induce the differentiation of smooth muscle cells in gizzard, though extract of aorta induced the differentiation of smooth muscle cells in gizzard. The results obtained here support evidence that the differentiation of most of the smooth muscle cells in gizzard is induced by a self-catalytic mechanism in which differentiated smooth muscle itself induces the differentiation of smooth muscle cells.     

Accumulation of tropomyosin in developing chicken gizzard smooth muscle

Smooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to alpha and beta isoforms (Hosoya et al. (1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of alpha and beta isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.     

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.     

Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle

An actin polymerization-inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization-inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schr?er, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-520]. In this paper, a 21-kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21-kDa peptide was identified as a polymerization-inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21-kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization-inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization-inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization-inhibiting protein contains mainly polymerization-inhibiting peptides and only some contaminants, and that all the polymerization-inhibiting peptides are proteolytic fragments stemming from a common precursor.     

Studies on the soluble phosphodiesterases of chicken gizzard smooth muscle.

In this study we describe the identification of four soluble forms of cyclic nucleotide phosphodiesterase from chicken gizzard smooth muscle. These isoenzymes were separated from one another by ion-exchange chromatography on DEAE-cellulose and by calmodulin-Sepharose affinity chromatography. Each form migrates as a single discrete band when it is electrophoresed on non-denaturing polyacrylamide gels and stained for phosphodiesterase activity. Each form is also eluted as a single peak on gel-permeation chromatography, giving apparent Mr values of 114 000, 116 000, 122 000 and 59 000. All four enzymes have apparent Km values in the 0-20 microM range, although their relative specificities for cyclic AMP and cyclic GMP differ. Two of the forms bind to calmodulin in a Ca2+-dependent manner; however, only one is activated by calmodulin. The interaction of the second calmodulin-binding form with calmodulin is disrupted by the papaverine derivative verapamil without significantly altering the hydrolytic activity of the enzyme.     

Expression of immunoreactive myosin and myoglobin in the developing chicken gizzard

Summary Antibodies to adult-type myosin and myoglobin from chicken gizzard were used to study the expression of these proteins during chicken embryogenesis. Using the indirect immunofluorescent technique, myosin was detected as discrete fluorescent foci in the central part of the presumptive chicken gizzard as early as day 5 of development. During the following days, immunoreactive myosin extends both craniocaudally as well as laterally and reaches the serosal and luminal borders by day 13/14. On day 16, the adult fascicular pattern is achieved. As judged by enzymelinked immunoassay and spectroscopic methods, myoglobin did not appear until day 18.Dedicated to Mrs. C.F. Schoenberg, Department of Anatomy, Cambridge, Great Britain     

Evolutionary Origins of the Neural Crest and Neural Crest Cells

I evaluate the lines of evidence—cell types, genes, gene pathways, fossils—in putative chordate ancestors—cephalochordates and ascidians—pertaining to the evolutionary origin of the vertebrate neural crest. Given the intimate relationship between the neural crest and the dorsal nervous system during development, I discuss the dorsal nervous system in living (extant) members of the two groups, especially the nature, and genes, and gene regulatory networks of the brain to determine whether any cellular and/or molecular precursors (latent homologues) of the neural may have been present in ancestral cephalochordates or urochordates. I then examine those fossils that have been interpreted as basal chordates or cephalochordates to determine whether they shed any light on the origins of neural crest cell (NCC) derivatives. Do they have, for example, elements of a head skeleton or pharyngeal arches, two fundamental vertebrate characters (synapomorphies)? The third topic recognizes that the origin of the neural crest in the first vertebrates accompanied the evolution of a brain, a muscular pharynx, and paired sensory organs. In a paradigm-breaking hypothesis—often known as the ‘new head hypothesis’—Carl Gans and Glen Northcutt linked these evolutionary innovations to the evolution of the neural crest and ectodermal placodes (Gans and Northcutt Science 220:268-274, 1983. doi:10.1126/science.220.4594.268; Northcutt and Gans The Quarterly Review of Biology 58:1–28, 1983. doi:10.1086/413055). I outline the rationale behind the new head hypothesis before turning to an examination of the pivotal role played by NCCs in the evolution of pharyngeal arches, in the context of the craniofacial skeleton. Integrations between the evolving vertebrate brain, muscular pharynx and paired sensory organs may have necessitated that the pharyngeal arch skeletal system—and subsequently, the skeleton of the jaws and much of the skull (the first vertebrates being jawless)—evolved from NCCs whose developmental connections were to neural ectoderm and neurons rather than to mesoderm and connective tissue; mesoderm produces much of the vertebrate skeleton, including virtually all the skeleton outside the head. The origination of the pharyngeal arch skeleton raises the issue of the group of organisms in which and how cartilage arose as a skeletal tissue. Did cartilage arise in the basal proto-vertebrate from a single germ layer, cell layer or tissue, or were cells and/or genes co-opted from several layers or tissues? Two recent studies utilizing comparative genomics, bioinformatics, molecular fingerprinting, genetic labeling/cell selection, and GeneChip Microarray technologies are introduced as powerful ways to approach the questions that are central to this review.     

Rapid myosin phosphorylation transients in phasic contractions in chicken gizzard smooth muscle

In intact smooth muscle strips from chicken gizzard, electrical stimulation and carbachol elicited brief, phasic contractions which were associated with a very rapid, transient phosphorylation of the 20 kDa myosin light chains. The phosphorylation transients reached their peak after 3 s and 6 s and preceded that of force. Phosphorylation was not significantly different from basal levels after 10 s and 30 s while force still amounted to 50% of the peak value. The rate of tension decline could be increased by cessation of stimulation or by addition of atropine, even at apparently basal phosphorylation levels suggesting a phosphorylation independent regulation.     

Striated myofibrils in anti-myosin stained,isolated chicken gizzard smooth muscle cells

Summary Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM.Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 m. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5±0.2 m. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.     

Purification and characterization of a neurite outgrowth factor from chicken gizzard smooth muscle

Chicken gizzard extract contains a macromolecular glycoprotein that promotes neurite outgrowth of dissociated neurons from the ciliary ganglia of chick embryos. Using conventional purification procedures, the factor responsible for the neurite outgrowth (neurite outgrowth factor (NOF)) was purified about 2000-fold to an apparent single protein band (as judged by agarose-polyacrylamide gel electrophoresis). Twenty fmol/cm2 of the purified NOF bound to the culture well was sufficient to exert maximal neuritic response of cultured ciliary ganglia neurons from 8-day-old chick embryos. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that NOF migrated as a single polypeptide of 700 and 210 kDa under nonreducing and reducing conditions, respectively. NOF stained with periodic acid-Schiff reagent and had a sedimentation coefficient of 12 s, a Stokes radius of 114 A, and an isoelectric point of about 5.1. Gizzard NOF was trypsin-sensitive, but resistant to treatment with heparinase, beta-galactosidase, and neuraminidase. Antibody prepared against the purified NOF blocked NOF activity in a dose-dependent manner. The antibody did not inhibit the biological activity of mouse laminin, although it cross-reacted weakly with laminin. Immunohistochemical analysis showed that the antibody against NOF strongly stained the extracellular matrix of cells in thin sections of gizzard, skeletal muscle, heart, liver, and ciliary ganglion, and also the membrane and the cytoplasm of cultured gizzard muscle cells. The present data suggest that gizzard NOF is a novel extracellular matrix glycoprotein which has a role in neurite outgrowth promotion from peripheral neurons in vivo. Although unlikely, the possibility that the NOF is a chick laminin could not be excluded.     

Striated myofibrils in anti-myosin stained, isolated chicken gizzard smooth muscle cells.

Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM. Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 micrometer. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5 +/- 0.2 micrometer. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gr?schel-Stewart.