The mechanism of cell elongation during lens fiber cell differentiation

Lens fiber formation is characterized by extensive cell elongation. Earlier studies have shown that lens cell elongation in vitro can occur in the absence of microtubules and is associated with a proportional increase in cell volume. We have previously suggested that lens fiber cell elongation is directly caused by an increase in cell volume. In this report, lenses from 3- and 6-day-old chicken embryos were three-dimensionally reconstructed from serial sections to provide a measure of cell volume and length during various stages of primary and secondary lens fiber formation. In both cases, cell volume was highly correlated with cell length during lens cell elongation. In addition, during primary lens fiber formation, large intercellular spaces between lens vesicle cells disappeared as these cells began to elongate to form lens fibers. Loss of intercellular spaces would be expected if increasing cell volume were responsible for cell elongation. Finally, results of experiments in which the lens capsule was cut with a fine tungsten needle suggested that the capsule was elastic and normally under tension. These findings were used to formulate a model which accounts for the major events in lens morphogenesis based on (1) the regulation of cell volume, (2) the junctions present between lens cells, and (3) the constraint provided by the elasticity of the lens capsule.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

The biochemical and ultrastructural demonstration of collagen during early heart development

A correlative ultrastructural and biochemical study was made of cardiac collagen in the chick embryo, spanning stages 9- to 11 (6 to 13 somites). Analysis (carboxymethyl cellulose chromatography, SDS acrylamide gel electrophoresis and levels of proline hydroxylation) of collagen synthesized in situ permitted classification of this collagen as type I-like (α1:α2 = 2:1). Correlative electron microscopy of hearts fixed in situ showed the appearance of striated collagen fibrils in the cardiac jelly, thus complementing the biochemical findings. Although the electron microscope showed the presence of developing basal laminae and laminalike material, synthesis of the type of collagen reported as unique to basal laminae was not detected, and we propose that these basal laminae may lack type IV collagen. Embryonic stages 9- to 11 are the earliest stages in which collagen synthesis has been demonstrated, and this is the first report of the occurrence of collagen in cardiac jelly of early hearts.     

Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians

Fertilization of frog eggs by frog sperm is inhibited if the egg's membrane potential is positive (N. L. Cross and R. P. Elinson, 1980, Dev. Biol.75, 187–198); however, fertilization of salamander eggs by salamander sperm does not depend on membrane potential (M. Charbonneau, M. Moreau, B. Picheral, J. P. Vilain, and P. Guerrier, 1983, Dev. Biol.98, 304–318). Since salamander sperm can fertilize frog eggs, we have investigated whether this cross-fertilization is voltage dependent. If, during insemination with Notophthalmus sperm, Xenopus eggs were voltage clamped between +7 and +20 mV, fertilization proceeded in $\text{7}\text{10}$ (70%) of the clamped eggs, compared to $\text{38}\text{48}$ (79%) of the neighboring eggs. In control experiments in which voltage-clamped Xenopus eggs were inseminated with Xenopus sperm, fertilization proceeded in only $\text{1}\text{10}$ (10%) of the clamped eggs, compared to $\text{59}\text{60}$ (98%) of the neighbors. Similar results were obtained with cross-fertilization experiments between Notophthalmus sperm and Rana eggs. These experiments indicate that the voltage dependence of fertilization depends on the species of sperm.     

The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development

The cuticle of the free-living nematode Caenorhabditis elegans is a proteinaceous extracellular structure that is replaced at each of four postembryonic molts by the underlying hypodermis. The cuticles of the adult and three juvenile stages (L1, Dauer larva, L4) have been compared ultrastructurally and biochemically. Each cuticle has an annulated surface and comprises two main layers, an inner basal layer and an outer cortical layer. The adult cuticle has an additional clear layer which separates the basal and cortical layers and is traversed by regularly arranged columns of electron-dense material. The fine structure of the cortical layer is similar in cuticles from different stages while that of the basal layer is stage specific. Purified cuticles were obtained by sonication and treatment with sodium dodecyl sulfate (SDS) and their component proteins solubilized with a sulfhydryl reducing agent. The degree of cuticle solubility is stage specific and the insoluble structures for each cuticle were localized by electron microscopy. Analysis of ³⁵S-labeled soluble cuticle proteins by SDS-polyacrylamide gel electrophoresis yields unique banding patterns for each stage. Most proteins are of high molecular weight (100–200 K) and are restricted to particular stages. Sixteen of the nineteen major proteins characterized are specifically degraded by bacterial collagenase. The results indicate that the different molts are not reiterative, but require the integration of both unique and shared gene functions. The potential use of stage-specific cuticle differences to identify and characterize regulatory genes controlling cuticle-type switching during development is discussed.     

Synthesis of tubulin and actin during the preimplantation development of the mouse

The relative rates of synthesis of actin and tubulin during mouse preimplantation development have been investigated utilizing O'Farrell's two-dimensional polyacrylamide gel system and internal protein markers. During mouse preimplantation development, rates of protein synthesis remain low and are little changed until the 8-cell stage when a rapid increase is evident. From the 8-cell stage on, a much higher rate of synthesis is maintained. The rate of synthesis of actin remains also at a steady low level in the unfertilized and fertilized ovum. However, by the 8-cell stage actin synthesis has increased 10-fold. Our measurements include the blastocyst, at that point in development actin synthetic rates are almost 90-fold higher than in the unfertilized ovum. While this precipitous increase is proceeding, incorporation of [³H]leucine into total protein increases only 7-fold. Synthesis of actin in the blastocyst represents 5.7% of total protein synthesis. The rate of tubulin synthesis, unlike actin, more closely parallels the increments in total protein synthetic rates. At the blastocyst stage it has increased 14-fold and its synthesis represents almost 2% of total protein synthesis. These results are discussed with reference to some of the physiological changes taking place during preimplantation development.     

Effect of procaine on development of ‘ridges’ and ‘domes’ in primary cell cultures from mammary glands of untreated virgin mice: The role of cell density in the occurrence of ‘domes’

Primary cell cultures from mammary glands of virgin mice that were not pretreated with hormones were subjected to: (1) procaine; (2) insulin+ prolactin +hydrocortisone; (3) a combination of (1) and (2). Procaine caused a ‘ridge’ effect similar to that of the hormones. The combination of procaine with the hormones caused a still stronger ‘ridge’ effect as well as the formation of ‘domes’. The formation of ‘domes’ is suggested to be dependent on cell density.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

The multicomponent nature of teratoma cell adhesion factor

The multicomponent nature of teratoma cell adhesion factor has been demonstrated. Fractionation of crude ascites fluid on a DEAE cellulose ion exchange column shows that two or more components are involved in teratoma adhesion factor (TAF) activity. Glycoproteins (or proteoglycans) in fractionated ascites fluid were localized in polyacrylamide gels. The possible role of these sugar-containing molecules in teratoma cell adhesion and current hypotheses on the mechanism of carbohydrate involvement in intercellular adhesion are discussed.     

Hyperproduction of dihydrofolate reductase in Diplococcus pneumoniae by mutation in the structural gene: The absence of an effect on other enzymes of folate coenzyme biosynthesis

A unique group of mutations (ame^r) in the dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase, EC 1.5.1.3.) structural gene of Diplococcus pneumoniae determine a marked overproduction of the corresponding enzyme protein. Since findings with these mutations relate to a key metabolic function and may be important to the regulation of folate coenzyme synthesis in general, the same group of multations were also examined for their effects on a number of related enzymic activities. Mutant and wild-type cell-free extracts, in addition to dihydrofolate reductase activity, exhibited both dihydropteroate and dihydrofolate synthetic activities under the conditions employed. Four folate coenzyme-related enzyme activities could also be demonstrated with the same preparations. These are mediated by the following enzymes, serine hydroxymethyl transferase (l-serine: tetrahydrofolate 10-hydroxymethyl tranferase, EC 2.1.2.1), 5, 10-methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.5), 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) and glutamate formiminotransferase (N-formimino-l-glutamate: tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5). The ame^r mutations examined in the current study determined 3–80-fold increases in dihydrofolate reductase in comparison to the wild type. However, none of the other folate-related enzyme activities were altered. The possible significance of these findings in light of previous results is discussed.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Temperature-shift analysis of conidial development in Aspergillus nidulans

Temperature-shift experiments have been performed on spore-originated colonies of 11 thermosensitive aconidial mutants of Aspergillus nidulans in order to determine the latest time of shift to the restrictive temperature that prevents the initiation of conidiation. This time defines the beginning of the thermosensitive period (TSP) of the mutant. Eight of the mutants have TSPs that begin in the 7-hour period (32–39 hr) just prior to the first appearance of conidia-bearing structures, while 3 of the mutants have TSPs that begin later and very close to the time of onset of conidiation (45 hr). Thus no mutant of the set has a TSP that begins during the first 32 hr of vegetative growth of spore-originated colonies. For all mutants, an upshift performed after the beginning of the TSP allows initiation of conidiation at close to the normal time and at the normal rate, but results in an abrupt cessation of conidiation at some fixed time after upshift, characteristic of the mutant. The mutant whose TSP begins the earliest (aco-49) is exceptional in that, if conidiation is suppressed by growth of colonies in submerged culture, this mutant becomes thermoinsensitive during vegetative submerged growth; in contrast, the remaining 10 mutants become thermoinsensitive only after the suppressive condition has been relieved. We discuss the possibility that this exceptional mutant is defective in a function required for initiation of the process that ultimately results in the formation of conidia.     

The effect of carbon dioxide on local cerebral blood flow during surface hypothermia in dogs.

Local cerebral blood flows were measured using the hydrogen clearance technique. This was found to be a satisfactory method.During hypothermia, maintenance of an F_ECO₂ above 5% is accompanied by higher LCBF while the opposite occurs with hyperventilation to 3% F_ECO₂.     

Membrane differentiation and de Nova synthesis of the (Na+ + k+)-activated adenosine triphosphatase during development of Artemia salina nauplii.

The developing brine shrimp, Artemia salina, nauplius is explored as a new model for the study of the biogenesis of the cation transport enzyme, (Na⁺ + K⁺)-activated adenosine triphosphatase [(Na, K)-ATPase]. (Na, K)-ATPase activity develops from undetectable levels in preemergent cysts (embryos prior to 12 hr of development) to very high levels in the nauplius after 40 hr of incubation in sea water [Conte, F. P., Droukas, P. C., and Ewing, R. D. 1977). J. Exp. Zool.202, 339], then declines between 44 and 72 hr. Similar ontogenic patterns of enzyme activity development are observed for Mg-ATPase, 5′-nucleotidase, glucose-6-phosphatase, NADH oxidase (rotenone insensitive), and cytochrome oxidase. However, these enzymes show measurable activity in the early cyst stage, and the points at which the activity increases and then reaches a maximum are usually different from those of the (Na, K)-ATPase. These enzyme ontogeny studies demonstrate that membrane differentiation is extensive during the period in naupliar development when (Na, K)-ATPase activity appears, and that the appearance of specific enzymes is asynchronous during embryogenesis. Pulse-chase experiments with NaH¹⁴CO₃ show an increase in the specific radioactivity of the partially purified (Na, K)-ATPase which is maximum when the label is administered at 12–18 hr after the initiation of development. At this time the specific radioactivity increases with purity of the enzyme, whereas in earlier pulse periods the specific radioactivity is higher in the more crude enzyme fractions, suggesting that preferential synthesis of the (Na, K)-ATPase occurs between 12 and 18 hr. Radioactivity is found in the subunits of the partially purified (Na, K)-ATPase isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is maximum for the 12- to 18-hr pulse experiment. These pulse-chase experiments demonstrate that the large increase in (Na, K)-ATPase activity is due to de novo synthesis and establish that the brine shrimp is a workable new model for the study of the biogenesis of the (Na, K)-ATPase.     

Molecular cloning and selection of genes regulated in Aspergillus development

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.     

Remodeling of sperm chromatin following fertilization: nucleosome repeat length and histone variant transitions in the absence of DNA synthesis

Within the first cell cycle following fertilization the average nucleosomal repeat length of sea urchin male pronuclear chromatin declines by 30-40 base pairs to a value typical of that found in the embryo. This decline occurs after a lag of about 30 min postfertilization, and is accompanied by replication of the male chromatin and accumulation of cleavage-stage (CS) core histone variants. When replication is inhibited by greater than 95% with aphidicolin, the decline in repeat length still occurs, although it is slightly retarded. The decline in repeat length also occurs when protein synthesis is blocked by greater than 98% and DNA synthesis by 60-70% with emetine. The adjustment of nucleosome repeat length therefore can occur in vivo without extensive movement of replication forks across the length of the chromatin, or normal progression of the cell cycle, and appears to require no proteins synthesized postfertilization. Blocking of DNA synthesis or protein synthesis also does not prevent the normal histone variant transitions involved in male pronuclear chromatin remodeling. Although their accumulation is slowed, CS core variants eventually become the predominant male pronuclear histones in their classes when replication is inhibited. Since a shortening of the average nucleosomal repeat length of approximately 10-20% is not sufficient to account for this large acquisition of CS variants, some of the sperm (Sp) core histones are probably displaced from the replication-blocked pronucleus. Therefore, accumulation of CS H2A and CS H2B are temporally correlated with the repeat length transition, whereas replication, normal progression of the cell cycle, and the early histone transitions involving SpH1 and SpH2B are not.