Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.     

On the role of the connective tissue in the patterning of the chick limb musculature

Summary By modifying the temporal relationship between connective tissue and myogenic cell invasion during early limb bud development new evidence of the organizing role of the connective tissue was obtained.Muscle cell-deprived wing buds were allowed to grow up to stages 22 to 27 of Hamburger and Hamilton, when they received a transplant of quail myogenic cells (somitic mesoderm or wing premuscular mass) into the dorsal face of their presumptive upper arm. Muscular arrangement in forearm and hand was analyzed 4 days later. In 8 out of 14 of those cases which had received a graft of premuscular mass before stage 25 of Hamburger and Hamilton, muscle development took place distally to the graft-site in accordance with the wing segment.     

Cytodifferentiation of the paraventricular nucleus in the chick embryo

The developmental changes in the cytoarchitecture of the hypothalamic paraventricular nucleus of the chick embryo were studied with particular emphasis on the differentiation of the magnocellular neurons. These cells can be distinguished from the parvocellular elements starting from stages 34--35 (Hamburger and Hamilton 1951) in Golgi-impregnated specimens. At the same stages, electron microscopy reveals dense-core granules, resembling the characteristic elementary granules of the neurosecretory material in the cytoplasm of the larger neurons. In addition, a few immature synapses were observed on these magnocellular perikarya. The present observations suggest that the early onset of neurosecretion in this area may be neurally regulated during early phases of development.     

Effect of spatial position of uterine quail blastoderms cultured in vitro on bilateral symmetry formation

Summary A method of in vitro culture for uterine quail blastoderms has been developed, which allows them to develop from cleavage throughout gastrulation and further: stages 4–10 of Hamburger and Hamilton (1951). The method consists of cultivating the blastoderms on egg albumen in a vertical position; this permits about 50% of the blastoderms explanted before area pellucida formation to develop bilateral symmetry and to form normal primitive streak, somites and head structures. Development of the blastoderms explanted after their area pellucida was already formed, occurred normally and was not influenced by their spatial position in the culture.This work was performed as part of project no. 09.7.1.5.2 of the Polish Academy of Sciences     

Relative growth rates of maxillary mesenchyme in the chick embryo

Chick embryos were injected with [3H]-thymidine at days 3-7 of incubation and were fixed and embedded in plastic. The embryos were divided into three stage groupings of development [Hamburger and Hamilton: J Morphol 88:49-92, 1951], and labeling indices were determined for each of the following delineated regions within the maxillary process at each stage: region 1, subepithelial mesenchyme located at the medial side of the maxillary process adjacent to the roof of the stomodeum; region 2, subepithelial mesenchyme at the ventral tip of the maxillary process (as seen on cross section); region 3, subepithelial mesenchyme at the lateral portion of the maxillary process below the eye; and region 4, interior mesenchyme defined as the central portion of the maxillary process and separated from the epithelium by the three other regions. Results indicated that differences exist among the regions examined and that these differences were stage specific. At stages 19-21 and stages 24-25 1/2, growth rates were higher in subepithelial regions than interiorly. At stages 28-29, however, a statistically significant difference among the regions was not found. These results suggested that there is an association between growth rates in the maxillary process mesenchyme and its proximity to the overlying epithelium and that these effects are related to the stage of development.     

Differentiation of troponin in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy

The differentiation of troponin (TN) in cardiac and skeletal muscles of chicken embryos was studied by indirect immunofluorescence microscopy. Serial sections of embryos were stained with antibodies specific to TN components (TN-T, -I, and -C) from adult chicken cardiac and skeletal muscles. Cardiac muscle began to be stained with antibodies raised against cardiac TN components in embryos after stage 10 (Hamburger and Hamilton numbering, 1951, J. Morphol. 88:49-92). It reacted also with antiskeletal TN-I from stage 10 to hatching. Skeletal muscle was stained with antibodies raised against skeletal TN components after stage 14. It also reacted with anticardiac TN-T and C from stage C from stage 14 to hatching. It is concluded that, during embryonic development, cardiac muscle synthesizes TN-T and C that possess cardiac- type antigenicity and TN-I that has antigenic determinants similar to those present in cardiac as well as in skeletal muscles. Embryonic skeletal muscle synthesizes TN-I that possesses antigenicity for skeletal muscle and TN-T and C which share the antigenicities for both cardiac and skeletal muscles. Thus, in the development of cardiac and skeletal muscles, a process occurs in which the fiber changes its genomic programming: it ceases synthesis of the TN components that are immunologically indistinguishable from one another and synthesizes only tissue-type specific proteins after hatching.     

Sfrp-1 and sfrp-2 are expressed in overlapping and distinct domains during chick development

Secreted frizzled related proteins (Sfrps) are thought to bind and regulate Wnt activity through a cysteine rich domain that is highly similar to that of Frizzled receptors. To investigate possible roles for Sfrps in chick development, we have isolated partial cDNAs encoding Sfrp-1 and Sfrp-2 and have thoroughly characterized the expression patterns of both genes. Both sfrp-1 and sfrp-2 are expressed at all stages of development analyzed, ranging from Hamburger and Hamilton stage 4 through stage 32. Expression of both sfrp-1 and sfrp-2 is observed in mesodermal and ectodermal derivatives, while sfrp-1 is also found in endodermal lineages.     

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.     

Immunohistological study of the expression of glucagon in dorsal pancreatic endoderm in the early chick embryo

An immunohistological study demonstrated that glucagon first appears in the dorsal pancreatic endoderm of the chick embryo at stage 16 of Hamburger and Hamilton during normal development. It was also shown that the self-differentiation potency of the isolated dorsal endoderm to express glucagon in vitro in the absence of adjoining tissues appears at stage 11.     

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.     

Compatibility of chick embryo eye anlagen with the ectoderm of the early amphibian gastrula in vitro

Eye vesicles were isolated from the early chick embryos (stage 9+ after Hamburger and Hamilton, 1951) and combined with the Rana temporaria early gastrula ectoderm (EGE) in vitro. The tissues were jointly incubated in medium 199 diluted twice with deionized water at 22 +/- 1 degree for 7-8 days or the eye vesicles were removed from the EGE ectoderm within 16-18 h. At the joint long-term incubation of these tissues, a toxic effect of the chick embryonic tissues on the EGE cells was noted. In none of the experiments, the inducing effect of the eye vesicle on the EGE was found. Similar data were obtained when the EGE was jointly cultivated with the brain (stage 9-10) and retina (stage 15) of chick embryos. The brain of the chick embryos at stage 15 exerted a weak neuralizing effect on the EGE. In the control experiments, the eye vesicles explanted with the chick embryonic ectoderm remained viable till the end of cultivation but no lentoids formed in the ectoderm. The absence of lens-inducing effect at the joint cultivation of the chick embryonic eye vesicles with the EGE is considered as a result of disturbance of the synthesis or secretion of the corresponding agents rather than a sequence of the species "incompatibility" of the inductor and reacting tissue. Hence, the use of "xenogenic" tissue recombinants is not justified when analyzing the lens-inducing activity of the eye vesicles.     

The spatial pattern and temporal sequence in which feather germs arise in the white Leghorn chick embryo

Feather germs arise in a specific sequence and spatio-temporal pattern within each of 10 feather areas on the White Leghorn chick embryo. The time of feather germ initiation was determined by histological and gross macroscopic analyses. Protruding feather germs are sequentially visualized in the dorsal, thigh, breast, head, humoral, ventral, wing, eye, and external auditory meatus feather areas, respectively, from stage 31- to stage 39+ [V. Hamburger and H.L. Hamilton (1951) J. Morphol. 88, 49-92]. The rate at which successive feather tracts appear was found to differ for different feather areas and was not simply due to the size of a feather area. Feather germ histogenesis was examined in the dorsal, thigh, breast, ventral, wing, and tail feather areas. The stages of feather germ histogenesis, examined on the wing feather area, are similar to those previously described for the dorsal surface. Gross and histological analyses gave different times and temporal sequences of feather germ visualization. Some feather areas were readily visualized at the time of feather germ initiation, while others showed a lag between the histological appearance of feather germs and their macroscopic visualization. Thus, macroscopic observations do not accurately reflect the pattern of histogenesis.     

Effect of cyclic nucleotides on the morphogenetic and ultrastructural characteristics of the primitive knot in the chick embryo in organotypic cultures

The isolated Hensen's nodes of chick embryos (stage 4 after Hamburger and Hamilton) were cultivated in organotypic cultures after the treatment with 0.5 mM solutions of cAMP and cGMP. Both the control and treated explants were characterized by a wide variability of differentiation of various tissue rudiments. cGMP inhibited reliably the differentiation of notochord. At the ultrastructural level the control and cAMP-treated cultures were characterized by an increased autophagia whereas the treatment with cGMP inhibited the yolk utilization and resulted in the appearance of vast regions of partial necrosis.     

Transition from non-muscle to muscle myosin light chains in chick embryo development

In chick embryo blastoderm the electrophoretic pattern of myosin light chains changes between the 4-somite and the 19-somite stages (stages 8-13 of Hamburger and Hamilton) from that of non-muscle to muscle myosin. This transition seems to follow the differentiation of the myotomes and to be developmentally regulated.     

Scanning electron microscopical observations on the differentiating mesonephros of the chick embryo

Chick embryos were staged according to the method of Hamburger and Hamilton [1951] and fixed. Cross sections through the cephalic fourth of the mesonephric ridges were examined by scanning electron microscopy. The steps in glomerular differentiation could be observed with ease. The first foot processes to appear in podocytes arose directly from the basal surface of the cell body. In a second step, lateral branches appeared and gave off secondary or even tertiary branches that interdigitated with those from neighbouring podocytes, following a pattern that was very similar to the one previously described by other authors in metanephric nephrons. Endothelial pores appeared in the glomerular capillaries at very early stages of the glomerular differentiation. The differentiation of the epithelium of proximal tubules was characterized by the growth of apical microvilli and of finger-like evaginations from the lateral membranes. At stages 20 and 21, the most differentiated glomeruli had only basal foot processes; only after stage 25 did the first generation nephrons reach full maturity. Because during this period the mesonephros is known to produce urine, our results indicate that nephrons start to function before they have completed their differentiation.     

Fate mapping and cell lineage analysis of Hensen's node in the chick embryo.

Fate maps of chick Hensen's node were generated using DiI and the lineage of individual cells studied by intracellular injection of lysine-rhodamine-dextran (LRD). The cell types contained within the node are organized both spatially and temporally. At the definitive primitive streak stage (Hamburger and Hamilton stage 4), Hensen's node contains presumptive notochord cells mainly in its anterior midline and presumptive somite cells in more lateral regions. Early in development it also contains presumptive endoderm cells. At all stages studied (stages 3-9), some individual cells contribute progeny to more than one of these tissues. The somitic precursors in Hensen's node only contribute to the medial halves of the somites. The lateral halves of the somites are derived from a separate region in the primitive streak, caudal to Hensen's node.     

Experimental study of the origin of the parathyroid glands.

The origin of the parathyroid glands was investigated in chick embryos (Gallus domesticus). Pieces of the third branchial arch were grafted, and its ectodermal layer formed a new structure (parathyroid III), which became separated from the placodial ectoderm. This structure continued to develop until, together with neural crest cells which gave rise to the mesenchyme, it formed a distinct parathyroid III gland by stage 28 of Hamburger and Hamilton.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.     

Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts

By immunizing mice with cells from embryonic chick motoneuron cultures, an antiserum was produced which recognizes an antigen(s) restricted to cell surfaces of most, or all, neurons. With the use of this antiserum, the appearance of neuron-specific antigenicity in cells of the embryonic spinal cord was examined by indirect immunofluorescence microscopy. The antigen or set of antigens reacting with this antiserum was first detectable in the neural tube of chick embryos at stage 15–16 (V. Hamburger and H. L. Hamilton, 1951, J. Morphol.88, 49–92). In addition to the neuroblasts located in the mantle layer, some mitotic cells as well as some spindle-shaped cells in the germinal layer were antigen positive. Immunofluorescence microscopy combined with autoradiography revealed that none of the antigen-positive cells could be labeled with [³H]thymidine; thus they do not synthesize DNA, and none of the cells in the DNA synthetic phase expressed the antigen(s). As the neuroblasts do not synthesize DNA after they have differentiated from the germinal cells, we believe that the antigen-positive cells are differentiated elements and that the differentiation of membranes specific for neurons begins already before or during the terminal mitosis of cells which will be defined as neuroblasts.