Cell interactions in the developing epidermis of the leech Helobdella triserialis

In embryonic development of the leech Helobdella triserialis, each of the four paired ectodermal teloblasts contributes some progeny to a characteristic dorsal or ventral territory of the epidermis. To ascertain the relative roles of cell lineage and cell interactions in generating the highly regular epidermal distribution pattern of the various ectodermal cell lines, a series of experiments was carried out in which the ablation of particular teloblasts was combined with the intracellular injection of cell lineage tracers. The results showed that, after the ablation of an OP proteloblast, or of an O, P, or Q teloblast, the epidermal progeny of the remaining ipsilateral and contralateral teloblasts spread into the territory normally occupied by the epidermal progeny of the ablated teloblast. In this spreading process, cells may cross the ventral midline but not the dorsal midline. The spread of epidermal progeny of one teloblast in response to ablation of another teloblast is contrasted with the failure of the neuronal progeny of one teloblast to replace any missing neural tissue. It appears, therefore, that all epidermal cell lines are of equal developmental potential, regardless of their teloblast of origin, with the eventual location of any epidermal cell in the body wall being governed by interactions between cells within the developing epidermis.     

Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle

Three myosin heavy chain isoforms with unique peptide maps appear sequentially in the development of the chicken pectoralis major muscle. An embryonic isoform is expressed early and throughout development in the embryo. A second isoform appears just after hatching and predominates by 10 days ex ovo. A third isoform, indistinguishable from adult myosin heavy chain, predominates by 8 weeks after hatching. This sequence of myosin isoform change does not, however, appear during myogenesis in vitro. In cultures prepared from embryonic myoblasts only embryonic myosin heavy chain is expressed. This is true even in cultures maintained for 30 days. Myosin light chain expression also changes in vivo with a progressive increase in fast light chain 3 accumulation. In vitro, however, this shift to increasing fast light chain 3 accumulation does not occur. The results indicate that the myosin heavy chain and light chain pattern observed in vitro is identical to that of the embryonic muscle and that the conditions necessary for the shift in expression to a more mature myosin phenotype are not present in myogenic cultures. These cultures are therefore potentially of great value in probing further the neural and humoral determinants of muscle fiber maturation and growth.     

Ectodermal interactions during neurogenesis in the glossiphoniid leech Helobdella triserialis

Morphogenetic cell interactions during development were studied by combining cell ablation and cell lineage tracing techniques in embryos of the leech Helobdella triserialis. Ablation of an identified ectodermal teloblast, or teloblast precursor blastomere, on one side of an early embryo was often found to result in the later abnormal migration of the progeny cells of the corresponding contralateral, nonablated teloblast to the ablated side of the embryo; such abnormal migration was termed “midline violation.” Two different kinds of midline violation were observed. Crossover: after ablation of an N teloblast individual stem cell progeny of the contralateral N teloblast sometimes cross the ventral midline of the germinal plate of the embryo. Switching: after ablation of an OPQ teloblast precursor bandlets of stem cells produced by the contralateral O, P, or Q teloblasts sometimes switch to the germinal band of the ablated side at the site of origin of the germinal bands. The occurrence of crossover and switching shows that the eventual site occupied by a progeny cell of a particular teloblast is not automatically determined by its lineage, but also depends on interactions with other cells. Midline violation in the leech embryo CNS does not constitute true regulation, however, since the restoration of neurons to the ablated side is accompanied by a neuron deficit on the nonablated side. The occurrence of the two distinct kinds of midline violation, crossover and switching, may be explained by the relative position of the stem cell bandlets within the germinal bands, and by the geometrical features of the formation of the germinal plate from the germinal bands.     

Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell

A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20–200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as a supercoiled molecule, and imprecise when pOT is introduced in linear form.     

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied inRana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca²⁺ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca²⁺-sensitive CSF, 1CSF, and the Ca²⁺-insensitive CSF, 2CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.     

Effects of environmental stress or ACTH treatment during pregnancy on maternal and fetal plasma androstenedione in the rat

Prenatal stress applied during the last trimester of pregnancy has been shown to alter fetal development and influence adult sexual behavior. Since androstenedione (Δ⁴) has the potential to participate in differentiation processes, this study was designed to assess the effect of prenatal stress on maternal and fetal Δ⁴ titers. Restraint/illumination/heat (environmental stress) or ACTH injections were used to stress pregnant rat dams beginning on Day 14 of pregnancy. Blood samples and organ weights were obtained from nonpregnant animals, pregnant rats on Days 5, 10, 15, 18, and 20 of pregnancy, and fetuses on Days 18 and 20 of gestation. Maternal and male and female fetal Δ⁴ titers were determined by radioimmunoassay. ACTH and environmental stress significantly reduced fetal body weight and male anogenital distance. Environmental stress also significantly reduced the size of 20-day fetal adrenals and testes. Each treatment caused significant short-term (1 hr after treatment) and long-term (16 hr after treatment) elevation of maternal plasma Δ⁴ on Days 15 and 18 of gestation, but only short-term elevation of Δ⁴ titers on Day 20. ACTH treatment did not cause long-term elevation of fetal Δ⁴ although both ACTH treatment and environmental stress generated a significant short-term increase in fetal Δ⁴ titers. Environmental stress produced long-term elevation of fetal Δ⁴ in 18-day fetuses of both sexes and in 20-day female fetuses. It is concluded that maternal stress and exogenous ACTH significantly elevate maternal and fetal Δ⁴ titers during the prenatal period postulated to be critical in sexual differentiation of the rat brain.     

Developmental time,cell lineage,and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [³⁵S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.     

The physico-chemical mechanism of mediated transport. I. Facilitated diffusion

On the basis of the currently accepted model for the cell membrane structure, a physico-chemical model for mediated transport is developed and solved for the case of polar non-electrolyte migration through the cell membrane. The model considers the interstitial space defined by the transport protein subunits to be the migration pathway for polar solutes. A Langmuir-type adsorption equilibrium is assumed at the interfaces and a multicomponent diffusion mechanism of solute and water is postulated within the migration pathway, where the polar residues of the transport protein represent another component of the system. Membrane selectivity is governed by the adsorption constants, which are shown to affect strongly the kinetics of transport. Isosmotic transport and the volume change of the cell are important features incorporated in the model, which is shown to fulfill the peculiar properties of facilitated diffusion systems. It is concluded that the same type of pathway can be used for the transport of other polar solutes through existing or induced hydrophilic channels, for which a similar approach is suggested.     

Direct determination of UDP-glucuronic acid in cell extracts by high-performance liquid chromatography

A simple and sensitive method for the direct determination of UDP-glucuronic acid by high-performance liquid chromatography with simultaneous measurement of UDP-glucose was developed. Optimal resolution and separation of UDP-glucuronic acid was attained under isocratic conditions with the ion-pairing agent n-octylamine. Quantitation was sensitive down to 5 pmol for standards and for liver cell extracts. Because this method directly measures UDP-glucuronic acid, it can be used for quantitation in the presence of drugs that interfere with enzymatic methods.     

Oligo(U) sequences present in sea urchin maternal RNA decrease following fertilization

Oligo(U) tracts were identified and measured in RNA from sea urchin eggs and embryos using a quantitative assay based on the amount of [3H]poly(A) protected from RNase T2 in duplexes with the oligo(U). The oligo(U) amounted to 0.0035% of egg RNA (0.063 X 10(-12) g/egg) and decreased to 0.0015% (0.027 X 10(-12) g/embryo) by 2 hr after fertilization. The oligo(U) tracts had a maximum size of 15-30 nucleotides and were associated with two size classes of RNA. In eggs about half were in 100 to 200 nucleotide RNA and half in mRNA-sized molecules. After fertilization, the oligo(U) in the population of large-mRNA-sized molecules was greatly reduced.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Down-regulation of epidermal growth factor receptors in mouse embryos

Previously we have shown (E. D. Adamson, M. J. Deller, and J. B. Warshaw, 1981, Nature (London) 291, 656–659) that epidermal growth factor (EGF) binds specifically to the cells and stimulates the incorporation of tritiated thymidine into DNA of several tissues of mouse embryos in a dose-dependent fashion when tested in vitro. However, in vivo a different response is obtained; exogenous EGF causes reduced incorporation of radiolabeled thymidine compared to buffer-injected control embryos. Several possible explanations are being explored. Here we present evidence that one of the responses of embryonic tissues in vivo to exogenous EGF is “down-regulation” of its receptors.     

Elemental composition of the perivitelline fluid in early Drosophila embryos

Samples of the perivitelline fluid in the polar pockets of preblastoderm Drosophila embryos were analyzed with an electron microprobe, and the results compared with analyses of adult hemolymph. The concentrations of sodium, magnesium, calcium, chlorine, and phosphorus are about the same in these two fluids; but potassium and sulfur are three to four times higher in perivitelline fluid. Moreover, the concentrations of these elements in the anterior and posterior pockets of the same embryos were compared. The former five elements seem to be about 10% more concentrated in the anterior pocket; but the latter two show no significant difference between pockets.     

Autonomous muscle cell differentiation in partial ascidian embryos according to the newly verified cell lineages

Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol.99, 382–394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.     

Developmental compartments in the Drosophila melanogaster wing disc

Distribution of the enzyme aldehyde oxidase (AO) within the pouch of the mature wing disc is precise and differential. General locations of compartmental boundaries have been identified by fate mapping and studies of AO distribution. The suspected locations of the boundaries were verified by analyzing the distribution of AO-negative cells within an AO-stained background in gynandromorphs and in X-ray-induced clones of AO-negative cells. The anterior/posterior border appeared slightly anterior to the junction of the AO⁺ anterior presumptive wing surfaces and AO^? posterior wing surfaces. A narrow band of AO⁺ cells extending proximodistally on both presumptive wing surfaces belongs to the posterior compartment. Two dorsal/ventral (dor./vent.) restrictions were found. The dor./vent. restriction equivalent to the dor./vent. border found in the adult wing was located at the ventral most edge of the AO-stained presumptive wing margin. A second restriction which was less strictly obeyed was found on the dorsal edge of the wing margin. We conclude that the whole presumptive wing margin is part of the dorsal compartment. Within the anterior wing margin an intensively stained oval was also found to be clonally restrictive. Therefore, territories were found within the prospective wing margin for which no such features have been identified in the adult Drosophila melanogaster wing.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.