Differentiation of Extracutaneous Melanocytes in Embryos of the Turtle,Trionyx sinensis japonicus

The present study investigates the mode of differentiation of neural crest-derived melanocytes in the embryos of the soft-shell turtle, Trionyx sinensis japonicus. DOPA reaction-positive melanoblasts were first detected in 10-day-old embryos. Melanocyte differentiation in terms of pigmentation takes place from the day 16 of development. Melanin pigments were found in the dorsal integument as well as in various extracutaneous tissues such as skeletal muscle, dorsal aorta, peritoneum, blood vessels, choroid, lung, bone marrow, fat tissues and in the connective tissue of the nose. These results suggest the presence of a specific environmental regulation of the melanoblast differentiation in the soft-shell turtle.     

Differentiation of Lens and Pigment Cells in Cultures of Brain Cells of Chick Embryos

Dissociated cells of brains (tel- and diencephalons) of 3.5-day-old chick embryos were cultivated in vitro under the cell culture conditions which are known to be permissive for neural retinal cells (NR cells) to transdifferentiate into lens and/or pigmented epithelial cells (PE cells). The differentiation of lentoid bodies (LBs) with lens-specific (δ-crystallin and PE cells with melanin granules was observed in such brain cultures.
LBs appeared in two different phases, i. e., 2–3 days and 16–30 days of cultivation, and after 40 days of culture these structures were formed in all 60 culture dishes. Sometimes, LBs were observed in foci of PE cells formed during earlier stages of brain cultures. When similar brain cultures were prepared with older embryos of 5-, 8.5-, 14-, and 16-days of incubation, no differentiation of lens and PE cells was observed.     

Mammary Gland Specific Knockdown of the Physiological Surge in Cx26 during Lactation Retains Normal Mammary Gland Development and Function

Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26^fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function.     

The spike component of the fertilization potential in the toad, Bufo japonicus: changes during meiotic maturation and absence during cross-fertilization

Immature oocytes of the toad, Bufo japonicus, inseminated between first- and second-meiotic metaphase, exhibited polyspermy. Monospermy occurred when the oocytes had reached second-meiotic metaphase. Electrical recording during insemination of the immature oocyte showed fast-rising and slow-rising spikes followed by a gradual shift to a positive membrane potential. The number of fast spikes in each oocyte corresponded well with the number of sperm observed in cytological sections. Mature oocytes elicited one fast spike followed by a rapid rise to a positive plateau. Ion-substitution experiments indicated that, like the plateau, the initial fast spike is mediated mainly by increased permeability of the oocyte plasma membrane to halides such as Cl- or I-. When inseminated with sperm of the newt, Cynops pyrrhogaster, mature Bufo oocytes exhibited polyspermy accompanied by a gradual hyperpolarization and a slowly developing positive plateau, without the fast spike that occurs in self-species fertilization. These results indicated that the spike component of the fertilization potential can be dissociated from the plateau component, and may be elicited by different mechanisms.     

Transient and Specific Expression of a Cysteine Endopeptidase Associated with Autolysis during Differentiation of Zinnia Mesophyll Cells into Tracheary Elements

Endopeptidase activities during the differentiation of Zinniacells into tracheary elements (TEs) were examined with severalpeptidyl 4-methyl-7-coumarylamido (MCA) as substrates. The activitythat hydrolysed carbobenzoxy-Phe-Arg-MCA (Z-Phe-Arg-MCA) atpH 5 increased in a differentiation-related manner: this activity,which was not observed in freshly isolated mesophyll cells wasinduced by the combination of -naphthaleneacetic acid (NAA)and 6-benzyladenine (BA) that is necessary for differentiationof TEs, but not by NAA or BA alone. The activity in cells culturedin TE-inductive medium that contained both NAA and BA increasedvery rapidly between the 48th and 60th hours of culture, whenthe number of TE increased rapidly. A protease responsible forthis activity with a molecular mass of 30 kDa was partiallypurified from cells which had been cultured in the TE-inductivemedium and included many immature TEs. Strong inhibition by[L-3-trans-carboxyoxiran-2-carbonyl]-L-Leu-agmatin (E-64), andactivation by dithiothreitol (DTT) indicated that this proteasebelongs to a family of cysteine endopeptidases (EC 3.4.22). ¹Present address: Department of Material and Biological Engineering,Tsuruoka National College of Technology, Inooka-Sawada 104,Tsuruoka, Yamagata, 997 Japan     

Enzymatic Activities modified during Multiplication and Differentiation of Neuroblastoma Cells

THE neuroblastoma cell system of Augusti-Tocco and Sato¹ is useful for studying neurone differentiation and function. The cells multiply rapidly in vitro and have low oxygen consumption². When serum is withdrawn from the culture medium, cellular expansion³, increase of acetylcholinesterase activity⁴ and increase of oxygen consumption² occur. The differences in respiratory metabolism of neuroblastoma cells, depending on whether they are proliferating or differentiating, may suggest major modifications of intermediary metabolism.     

Quantum Dot Labeling of Stem Cells during Proliferation and Differentiation

This article has no abstract.     

Increase of Specific Activity, Electrophoretic Type-Transition and Gene Expression of Alkaline Phosphatase during Endodermal Differentiation of F9 Mouse Embryonal Carcinoma Cells

In F9 mouse embryonal carcinoma cells, the specific activity of alkaline phosphatase (ALPase) increases markedly during endodermal differentiation induced by retinoic acid (RA) treatment, but the specific 5'-nucleotidase activity of a similar ecto-phosphatase increases only temporally. Polyacrylamide disc gel electrophoresis showed that F9 cells express only type I ALPase, whereas RA-treated F9 cells express both type I and type II ALPases. Type II ALPase is a minor form on day 1 of RA treatment and becomes the major form on day 4. RA-treated F9 cells also expressed mRNAs for endoderm cell-specific molecules, such as α-fetoprotein, type IV collagen and laminin B1 chain, but their expression of M₂-type pyruvate kinase mRNA of an essential non-ectoenzyme remains constant throughout endodermal differentiation. Northern blot analyses showed that type I ALPase was encoded by a liver (L)/bone (B)/kidney (K)/placenta (P)-type mRNA. The expression of L/B/K/P-type ALPase mRNA was induced in RA-treated F9 cells, but its increase preceded that of ALPase specific activity. These results suggest that the expression of L/B/K-type ALPase is regulated at the translational and/or post-translational level. The differential inhibition of ALPases by L-phenylalanine/L-homoarginine and the thermal inactivation (56°C for 60 min) inferred that type II ALPase was also an L/B/K-type isozyme.     

Steroid Producing Cells during Ovarian Differentiation of the Tilapia, Sarotherodon niloticus

In order to examine the initial appearance and development of the steroid producing cells (SPCs) during the process of ovarian differentiation, histology and ultrastructure of tilapia ( Sarotherodon niloticus ) ovaries were investigated from 10 to 50 days after hatching. In gonads of fry at 23–26 days after hatching, initial ovarian differentiation was confirmed by the differentiation of stromal aggregations in the proximal and distal region of the gonad on the side facing the lateral wall. This represents the initial formation of the ovarian cavity. At the same time as ovarian differentiation, a few large cells appeared initially in the vicinity of blood vessels. They have some of the ultrastructural features characteristic of SPCs such as a moderate number of mitochondria with tubular cristae, a large amount of smooth endoplasmic reticulum and many free ribosomes. Based on these ultrastructural criteria, together with the present finding that these cells further differentiated into the typical SPCs at older stages, these cells were identified as SPCs. Thereafter, by 30–50 days, SPCs increased gradually in number in the area enclosing the blood vessels of ovaries. The increase in SPCs coincided with the development of germ cells, including the multiplication of oogonia and the transformation from oogonia to oocytes.     

Phenotypic Adaptations Help Rhodococcus erythropolis Cells during the Degradation of Paraffin Wax

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax that contains 20–40 carbon chain hydrocarbons. The paraffin wax may accumulate inside production tubes, pipelines, and processing facilities, and also in tankers during petroleum transportation. There are few bacterial strains that are able to degrade solid substrates. In the present study, the biodegradation of paraffin is evaluated using Rhodococcus erythropolis cells. This bacterium is able to grow using paraffin wax from an oil refinery plant as the sole carbon source. The cells grow as a thick biofilm over the solid substrate, make scale‐like structures that increase the area of the initially smooth surface of paraffin, produce biosurfactants, and become more negatively charged than ethanol‐ or glucose‐grown cells. When paraffin wax is supplied as microparticles, to increase the cell–substrate contact area and to simulate paraffin precipitation, the cells also adjust the composition of the fatty acids of the phospholipids of the cellular membrane to decrease its fluidity and paraffin biodegradation increases considerably. The study suggests that the phenotypic adaptation of R. erythropolis cells may be used to degrade paraffin wax under real conditions.     

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the α- and β- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using ³⁵SO₄ and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t_½ = 50 versus 150 minutes, respectively) and during the deinduction period (t_½ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.     

Connection between the Synthesis of Differentiation Specific Proteins and the Capacity of Cells to Respond to Cytokinin in the Moss Funaria

The differentiation stage of the caulonema in Funaria hygrometrica protonema is distinguished from the chloronema stage by three additional protein bands (CSP) in the soluble protein fraction. During the change of caulonema to chloronema, which is induced by isolation of single filaments (regeneration), the CSP disappear. This is not the consequence of an accelerated degradation or turnover but of a gradual termination in the de novo synthesis of CSP during regeneration as demonstrated by pulse-chase experiments with l -[4,5–³H] leucine. Cytokinin inhibits the termination of the synthesis of CSP. The decrease in synthesis parallels the decrease in ability of the isolated caulonema cells to respond to cytokinin via bud formation. It is therefore concluded that the CSP are involved in the competence of caulonema cells to respond to cytokinins.     

Live Imaging of Whole Mouse Embryos during Gastrulation: Migration Analyses of Epiblast and Mesodermal Cells

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.     

Specific Entrainment of Mitral Cells during Gamma Oscillation in the Rat Olfactory Bulb

Local field potential (LFP) oscillations are often accompanied by synchronization of activity within a widespread cerebral area. Thus, the LFP and neuronal coherence appear to be the result of a common mechanism that underlies neuronal assembly formation. We used the olfactory bulb as a model to investigate: (1) the extent to which unitary dynamics and LFP oscillations can be correlated and (2) the precision with which a model of the hypothesized underlying mechanisms can accurately explain the experimental data. For this purpose, we analyzed simultaneous recordings of mitral cell (MC) activity and LFPs in anesthetized and freely breathing rats in response to odorant stimulation. Spike trains were found to be phase-locked to the gamma oscillation at specific firing rates and to form odor-specific temporal patterns. The use of a conductance-based MC model driven by an approximately balanced excitatory-inhibitory input conductance and a relatively small inhibitory conductance that oscillated at the gamma frequency allowed us to provide one explanation of the experimental data via a mode-locking mechanism. This work sheds light on the way network and intrinsic MC properties participate in the locking of MCs to the gamma oscillation in a realistic physiological context and may result in a particular time-locked assembly. Finally, we discuss how a self-synchronization process with such entrainment properties can explain, under experimental conditions: (1) why the gamma bursts emerge transiently with a maximal amplitude position relative to the stimulus time course; (2) why the oscillations are prominent at a specific gamma frequency; and (3) why the oscillation amplitude depends on specific stimulus properties. We also discuss information processing and functional consequences derived from this mechanism.     

Characterization of Mesonephric Cells That Migrate into the XY Gonad during Testis Differentiation

In mouse fetal gonads, sex differentiation begins at 10.5-11.5 days postcoitum (dpc). With XY gonads of 12.5 dpc, cord-like structures are visible and stromal cells migrate from adjacent mesonephros, unlike in XX gonads. However, the migrated mesonephric cells, except for the endothelial cells, have not been specifically identified because they have not expressed differentiation markers over the course of organ coculture in previous experiments. In this study, we have for the first time succeeded in isolating only the mesonephric cells that migrate into the XY gonad from the mesonephros with alive and then cultured these cells in vitro through the use of an organ coculture system using EGFP-transgenic mice and a FACS Vantage. The migrated and isolated cells were used for morphological and molecular characterization. The migrated mesonephric cells contained three cell forms; a sharp cell form, a round cell form, and a cluster-forming cell. The sharp cells have the characters of peritubular myoid cells. The round cells and cluster-forming cells have the potential to differentiate into Leydig cells, as some of them are 3beta-HSD-positive. In in vitro culture of migrated mesonephric cells, the cluster-forming cells proliferated well and then differentiated into round cells, suggesting that the cluster-forming cells may be stem or precursor cells for the round cells. Thus, our findings provide important information related to the migration and differentiation of migrated mesonephric cells in the XY gonad.     

Characterization of PHB1 and Its Role in Mitochondrial Maturation and Yolk Platelet Degradation during Development of Artemia Embryos

Background

To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts) with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown.

Principal Finding

Our study investigated the role of prohibitin 1 (PHB1) in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein.

Conclusions/Significance

PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.