Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct

When meiotic maturation of primary oocytes of the starfish Asterias forbesi is induced by 1-methyladenine, rapid and striking changes in the pattern of protein synthesis detectable by electrophoresis occur after germinal vesicle breakdown. These include a decline in relative labeling with [³⁵S]methionine of several polypeptides synthesized in the oocyte, and increased labeling and new appearance of several polypeptides. Fertilization does not result in other detectable changes. The population of total mRNA translatable in a rabbit reticulocyte lysate cell-free system does not change, but the distribution of mRNAs between polysomes and the postribosomal supernatant reflects the changes observed in vivo. Thus these changes are regulated at the translational level. A review of the literature indicates that translationally mediated changes in patterns of protein synthesis during maturation of oocytes may be a widespread phenomenon.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

Enzyme-induced aziridine formation by isolated hepatocytes

The metabolism of 2-bromoethylaminonaphthoquinone in hepatocytes isolated from rats was studied. This compound was chemically inert in the reaction system used. However, in buffer solution containing isolated hepatocytes, it was gradually converted into aziridinylnaphthoquinone. Under the same reaction conditions, 4-chlorobutylaminonaphthoquinone also gave the cyclization products, pyrrolidinylnaphthoquinone. Cellular GSH decreased in both reactions.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.     

Pattern formation during early amphibian development: Embryogenesis in inverted anuran and urodele eggs

Early embryogenesis was monitored in Xenopus, Rana (anurans), and Ambystoma (urodele) eggs which were inverted at various times between fertilization and first cleavage. The pattern of cleavage furrow formation, site of involution, and extent of organogenesis were observed. In several instances, pattern formation was dramatically altered. The small/large blastomere pattern was, for example, reversed in some inverted embryos. Developmental arrest at early organogenesis usually followed pattern reversal. By employing a series of tissue transplantations, it was possible to establish that the activity of the primary embryonic organizer of inverted embryos was diminished drastically. The developmental competence of the prospective ectoderm of inverted embryos was, however, reversed. Incomplete organogenesis in inverted embryos is therefore probably due to either abnormal mesoderm formation or defective tissue interactions.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.     

Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.     

Differences in iodinated peptides and thyroid hormone formation after chemical and thyroid peroxidase-catalyzed iodination of human thyroglobulin

The distribution of iodine among the polypeptides of human goiter thyroglobulin (Tg) was examined. Tg was iodinated in vitro with ¹³¹I to levels of 2 to 84 gram atoms (g.a.)/mol using thyroid peroxidase (TPO) or a chemical iodination system. The samples were reduced, alkylated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two low-molecular-weight peptides appeared preferentially in radioautograms of the sodium dodecyl sulfate (SDS) gels of TPO-iodinated samples. Iodination of these peptides increased sharply in the TPO-treated Tg as the level of total iodine/ molecule rose. Radioiodine was incorporated into these same gel regions in the chemically treated Tg, but only after much higher levels of total iodination were reached. Differences in iodoamino acid distribution were also noted between the chemically and enzymatically iodinated thyroglobulins. In the chemically iodinated samples, little thyroxine (T₄) was synthesized, even at high iodine levels. In the TPO-treated samples only small amounts of T₄ were seen below 14 g.a. total I/mol, while at or above that level of iodination T₄ formation increased sharply. To examine the coupling process, Tg was chemically iodinated, excess I^? removed, and the samples treated with TPO and a H₂O₂-generating system in the absence of iodide. Radioautograms obtained from SDS-polyacrylamide gels of reduced and alkylated protein from such coupling assays showed an increase in the level of iodine in the low-molecular-weight peptides after TPO treatment. Thyroxine production also increased with TPO treatment. The addition of free DIT (a known coupling enhancer) to the [¹³¹I]Tg/TPO incubation increased both the production of T₄ and the amount of iodine in the smaller polypeptides. Two-dimensional maps prepared from CNBr-digested TG showed differences between the coupled and uncoupled samples. Our observations confirm the importance of the lowmolecular-weight peptides derived from Tg in thyroid hormone synthesis. At total iodine levels above 14 g.a./mol Tg in enzymatically treated samples there is selective incorporation of iodine into both the low-molecular-weight polypeptides and into thyroid hormone.     

Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates

It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.     

Oxidative metabolism of hydralazine. Evidence for nitrogen centered radicals formation

The oxidative metabolism of hydralazine, a hydrazine-containing hypotensive drug, has been studied using a spin-trapping technique. In the presence of Cu²⁺, Fe²⁺ and Fe³⁺, hydralazine rapidly forms a nitrogen-centered-DMPO adduct with a^N = 15.0G, a_β^H = 16.7G and a_β^N = 2.55G. While catalase has a very small inhibitory effect, superoxide dismutase completely inhibits the formation of the DMPO adduct. Mass spectral analysis of the adduct indicates that the hydralazyl radical is trapped with DMPO. Human red blood cells also catalyze the formation of a nitrogen-centered-DMPO adduct, a^N = 15.9G, a_β^H = 19.4G and a_β^N = 1.7G, which is different than that obtained with metal ions. DMPO-H adduct is also formed in the red cells from hydralazine.     

Lack of correlation between agglutinability,the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.     

Alterations in the cell cycle of Drosophila imaginal disc cells precede metamorphosis

Flow cytometric analyses of imaginal disc and brain nuclei of Drosophila melanogaster have been made throughout the third larval instar. In wing, haltere, and leg discs the proportion of cells in the G₂M phase of the cell cycle (tetraploid cells) increases with larval age. In contrast, in the eye disc and in brain the proportion of tetraploid cells, already low at the outset of the instar, declines further. Measurement of growth rates for disc and brain tissue during the same developmental period was carried out by the cell counting procedure of Martin (1982). Our results are consistent with the conclusion that imaginal discs grow exponentially with an apparent doubling time of 5–10 hr from the resumption of cell division (in the first or second larval instar) until about 95 hr, when the apparent doubling time increases. Cell numbers increase until at least 5 hr after formation of white prepupae (122 hr), but during the preceding 10 hr the rate of increase is low. Thus, for wing and leg discs, but not for the eye disc and brain, the declining growth rate is associated with an increase in the proportions of tetraploid cells. In conjunction with cell counts and flow cytometry, fluorometric determination of disc DNA content at 112 hr indicated that the diploid DNA content of imaginal disc nuclei is 0.45 pg.     

The interaction of chemically modified human plasma alpha1-antitrypsin with porcine pancreatic elastase.

The elastase inhibitory capacity of human plasma α₁-antitrypsin was determined following chemical modification of lysyl and arginyl residues. Modification of the guanidino group had no effect upon the inhibitory activity, while acetylation, citraconylation, and trinitrophenylation of the lysyl ?-amino group brought about a loss of elastase inhibitory capacity.     

Three-dimensional hair follicular differentiation of a trichilemmoma cell line in vitro

A cell line was established in vitro from a benign hair follicular tumor of human trichilemmoma. Individual and organized cellular differentiation of this cell line was studied. When these cells were cultured for a long time (more than 3 weeks) without subculture, they started to pile up spontaneously. A part of the pile became indented and simultaneously the opposite side of the indentation budded out. The bud slowly elongated 2 to 3 mm in length in 8 to 12 weeks in culture. Light and electron microscopy revealed the internal structure of piles and elongated buds to be a three-dimensional hair follicular structure. The cells in the outermost layer were least mature. These were cuboid in shape and contained glycogen. The cells in the middle layer were more differentiated with a decreased amount of glycogen and an increased number of tonofilaments and desmosomes. The cells in the innermost layer were most differentiated. Cells were flat in shape and highly convoluted. The cell membrane was thickened as observed in cornified cells in vivo. These organized differentiations were also confirmed by histochemical and immunocytochemical studies; using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide method, free sulfhydryl groups were detected but disulfide bonds were absent in the early cell culture. Disulfide bonds increased slowly and accumulated in the innermost layer of piles. Accumulation of keratin substances, detected by indirect immunofluorescence method using anti-human keratin antibody, was also observed specifically in the piles. These results suggest that an established cell line of human trichilemmoma spontaneously produced, without stromal influence, hair follicular structures as well as individual cell differentiations in vitro as do trichilemmal (hair follicular) cells in vivo.     

Modulation of fertilization by the vitellus after interaction with peptides released by hamster sperm-zona pellucida contact

Two to three minutes after hamster sperm make contact with and adhere to the surface of homologous zonae pellucidae in vitro, the first of several sets of peptides (S1 peptides) is released into the supernatant. This release occurs whether the zonae have or have not been mechanically separated from the vitellus (cellular part of the egg). Presence of the S1 peptides is detected by means of a sperm-egg assay, the premature binding assay. This assay is based on the ability of an aliquot of the medium, in which sperm are interacting with the zona surface, to induce early binding, upon addition of the aliquot to a second drop of interacting gametes. To determine if the vitellus affected the 2-min S1 peptides the ultrafiltrates of the supernatants containing them, released through sperm-egg and sperm-zona interactions, were fractionated on Biogel P-6 and their elution profiles were compared using the premature binding assay. The sperm-egg ultrafiltrates were resolved into two main domains of activity, while those of the sperm-zona formed three. The ultrafiltrates collected 2 min after the interaction of sperm with eggs or with isolated zonae were compared for their abilities to inhibit the penetration of the zona pellucida, a previously demonstrated capacity of the 2-min sperm-egg S1 peptides. The ultrafiltrate containing the sperm-zona peptides, except at a very low level, failed to inhibit penetration significantly. However, when the sperm-zona ultrafiltrate was preincubated with eggs then the resulting supernatant inhibited penetration in a dose-related manner, and the three-domain elution profile, characteristic of the sperm-zona ultrafiltrate, was converted to the egg-like two-domain profile. Taken together these data suggest that the 2-min S1 peptides consist of several subpopulations, at least one of which interacts with the vitellus. The resulting solution then acquires the ability to inhibit penetration of the egg by the sperm in a dose-related manner. Taken together these data indicate that by interacting with at least one of the components of the 2-min peptides, the vitellus is involved in regulating sperm-zona interactions.     

Temporal changes in embryonic nerve cell recognition: correlate with cholinergic development in aggregate cultures

Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in vivo development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development.     

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.