Expression of gamma-adducin is associated with regions of morphogenetic cell movement in the chick embryo

The adducin family of cytoskeletal proteins are known to mediate actin driven cell movements. Here we describe the cloning and expression pattern of a gene encoding gamma-adducin in the chick embryo. Expression of this gene is strikingly restricted to the epithelial cell layer (with a few exceptions including emerging notochord and lateral mesoderm). Gamma-adducin is detected at particularly high levels in cell populations undergoing important morphogenetic movements, such as epiblast approaching the primitive streak, regressing spinal cord primordia and closing neural tube.     

Expression of chick Coactosin in cells in morphogenetic movement

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed‐end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti‐Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.     

Tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.     

Functional and structural characteristics of polyribosomes associated with chick embryo cell nuclei

Polyribosomes bound to the outer nuclear membrane was isolated from purified preparations of chicken embryo cell nuclei. These polyribosomes were shown to consist fractions forming unstable complexes with the nuclear membrane which can be separated from the latter by treatment with high ionic strength buffer solutions. Using sedimentation and gradient density analyses, the nuclei-bound RNP complexes were shown to be predominantly composed of 80S monosomes which take an active part in collagen polypeptide synthesis in cell-free protein-synthesizing systems. A comparison of sedimentation properties and collagen-synthesizing activity of nuclei-bound polyribosomes and cytoplasmic polyribosomes forming unstable complexes with endoplasmic membranes, it was concluded that the nuclei-bound 80S monosomes are an early step in the formation of cytoplasmic polyribosomes.     

Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle

This study provides evidence that cells destined to segment together into somites have a degree of cell division synchrony. We have measured the duration of the cell division cycle in somite and segmental plate cells of the chick embryo as 9.5 h using [3H]thymidine pulse- and-chase. Treatment of embryos with any of a variety of inhibitors known to affect the cell division cycle causes discrete periodic segmental anomalies: these anomalies appear about 6-7 somites after treatment and, in some cases, a second anomaly is observed 6 to 7 somites after the first. Since somites take 1.5 h to form, the 6- to 7- somite interval corresponds to about 9-10 h, which is the duration of the cell cycle as determined in these experiments. The anomalies are similar to those seen after heat shock of 2-day chick embryos. Heat shock and some of the other treatments induce the expression of heat-shock proteins (hsp); however, since neither the expression nor the distribution of these proteins relate to the presence or distribution of anomalies seen, we conclude that hsps are not responsible for the pattern of segmental anomalies observed. The production of periodic segmental anomalies appears to be linked to the cell cycle. A simple model is proposed, in which we suggest that the cell division cycle is involved directly in gating cells that will segment together.     

Acceleration of early chick embryo morphogenesis by insulin is associated with altered expression of embryonic genes

In the present study, we show that insulin accelerates early morphogenesis in gastrulating chick embryo explants cultured in vitro, whereas antiserum to insulin adversely affects this process. Comparison between length of body axis of control and treated embryos clearly brings out the significant acceleration of development by excess insulin (0.175 to 17.5 nM). In embryos treated with 87.5 and 175 nM insulin, a high occurrence of abnormalities is observed. Treatment of embryos with antiserum to porcine insulin results in a high percentage of abnormalities, particularly in the forming neural tube. In situ hybridization of whole embryos using digoxigenin-labeled riboprobes showed that insulin modifies the expression of crucial developmental genes within 2 hours. While Brachyury, a pan-mesodermal marker gene, ERNI, the earliest known marker for neural induction in chick, and noggin, important in neural tube patterning, are upregulated, expression of goosecoid, necessary for gastrulation movements, does not appear to be significantly altered. During the same time, insulin does not exert any mitogenic effect on chick embryonic cells as assessed by nuclear counts. These findings demonstrate that insulin plays an important role in the early morphogenesis of the chick embryo. The function of insulin appears to be mediated by specific genes which orchestrate pattern formation during early development.     

Kinetics of chick embryo cell types in culture

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.     

Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo

$\text{T}\text{T}$ embryos have been first distinguishable at 8 days post coitum by their gross morphological abnormalities. By quantitative morphometry of histological sections, anomalies in the homozygotes were expressed numerically. At 8 days p.c., morphologically identifiable T-homozygotes had an increased number of ectodermal and a reduced number of mesodermal cells compared to the wild type. At 7 days p.c., embryos with a low mesoderm/ectoderm ratio were found only in litters of $\text{T}\text{+}\text{×}\text{T}\text{+}$ matings at the expected frequency. At 6 days p.c., one-fourth of the embryos in $\text{T}\text{+}\text{×}\text{T}\text{+}$ litters showed a delay in the transition from cuboidal to squamous endoderm. No such embryos were found in the +/+ × +/+ matings. In 6-, 7-, and 8-day mutant embryos, cells proliferated at statistically normal rates. Therefore, it may be said that advanced morphological irregularities of 8-day homozygotes cannot be accounted for by anomalies in cell proliferation. When the total cell number was 5 × 10⁴/embryo (8 days), a sudden change was observed in the regional distribution of mesodermal and ectodermal cells along the anteroposterior axis of $\text{T}\text{T}$ embryos. Since no regional difference in the cell cycle time was observed, these abnormalities may best be explained by anomalies in cell migration. These results strongly suggest abnormal morphology of $\text{T}\text{T}$ mutants resulting from defects in morphogenetic movement.     

Altered synthesis of myosin light chains is associated with contractility in cultures of differentiating chick embryo breast muscle

Cultured chick embryo skeletal muscle cells normally synthesize only the embryonic isoform of mysoin. We have found that aneural muscle cultures that become or are provoked into an extremely contractile state will begin to synthesize a pattern of myosin light chains typical of maturing muscle. Immunoblots with neonatal and adult specific monoclonal antibodies did not reveal a corresponding isozyme transition in myosin heavy chain. These results demonstrate a correlation between contractility and the regulation of myosin light chain maturation, and also suggest that the transitions of heavy and light chain synthesis during development do not appear to be under close coordinate regulation.     

Expression of alphaVbeta3 integrin in the chick embryo aortic endothelium

The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.     

N-cadherin is crucial for heart formation in the chick embryo

The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured at stages 5–12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N-cadherin activity.     

Peridermal cell patterning in the feather-forming skin of the chick embryo

The shape, distribution, and orientation of peridermal cells were examined in the dorsolumbar skin of $\text{7}\text{1}\text{2}\text{-}\text{day}$ chick embryos. Most feather rudiments of middorsal and lateral rows showed a marked cephalocaudal polarity. A similar polarity was found in the prospective rudiments of skin areas lateral to the last-formed row. On the cranial slope and apex of rudiments, cells are convex and predominantly elongated at right angles with respect to the cephalocaudal axis, whereas on the caudal slope, most cells are flat, polygonal, surrounded by a border-line ridge, and oriented predominantly with their long axis parallel to the cephalocaudal axis. The significance of this pattern is discussed in view of the fact that the epidermis is the determinant tissue in feather orientation.     

Extracellular matrix fibrils and cell contacts in the chick embryo

Summary The migration of neural crest and sclerotome cells and the extension of ventral root axons in chick embryos at stages 16–20 were studied by light microscopy as well as scanning and transmission electron microscopy at the leg bud level of fixed specimens. Extensive cellular movements take place in association with an extracellular matrix consisting of microfibrils. The neural crest and sclerotome cells migrate into the large matrix-filled extracellular space surrounding the neural tube and notochord, apparently using microfibril bundles as substratum. The cells exhibit pseudopodia which are closely associated with the matrix fibrils. The fibrils around the notochord show a spatial arrangement indicating that the sclerotome cells are contact-guided to their subsequent positions. Mutual cell contacts, including those established by cell processes, frequently show cytoplasmic electron dense plaques at adjacent membranes. These small plaque contacts might be correlated to contact inhibition of locomotion between the cells and participate in the guidance of cells. The growth cones of extending axons exhibit filopodia contacting both surrounding mesenchyme cells and extracellular fibrils. The orientation of the axons might thus be affected by contacts with cell surfaces as well as with extracellular material.Technical assistance was given by Mrs. Kerstin Ahlfors, Mrs. Charlotte Fällström, Mrs. Annika Kylberg and Mrs. Stine SöderströmSupported by grants from The Swedish Natural Science Research Council