Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans

C. elegans develops through four larval stages (L1 to L4) separated by molts. The identity of larval stages is mostly determined by stage-specific expression of heterochronic genes, which constitute an intrinsic genetic timer. However, extrinsic cues such as food availability or population density also modulate the developmental timing of C. elegans by mechanisms that remain largely unknown. To investigate a potential role of the nervous system in the temporal regulation of C. elegans development, we pharmacologically manipulated nicotinic neurotransmission, which represents a prominent signaling component in C. elegans nervous system. Exposure to the nicotinic agonist DMPP during post-embryonic development is lethal at the L2/L3 molt. Specifically, it delays cell divisions and differentiation during the L2 stage but does not affect the timing of the molt cycle, hence causing exposure of a defective L3 cuticle to the environment after the L2/L3 molt. Forcing development through a previously uncharacterized L2 diapause resynchronizes these events and suppresses DMPP-induced lethality. Nicotinic acetylcholine receptors (nAChRs) containing the UNC-63 subunit are required, probably in neurons, to trigger the action of DMPP. Using a forward genetic screen, we further demonstrated that the nuclear hormone receptor (NHR) DAF-12 is necessary to implement the developmental effects of DMPP. Therefore, a novel neuroendocrine pathway involving nAChRs and the NHR DAF-12 can control the speed of stage-specific developmental events in C. elegans. Activation of DMPP-sensitive nAChRs during the second larval stage uncouples a molting timer and a developmental timer, thus causing a heterochronic phenotype that is lethal at the subsequent molt.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

Recovery, distribution, and development of Acanthocheilonema viteae third- and early fourth-stage larvae in adult jirds

Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.     

Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies

Cell culture is an invaluable tool for investigation of basic biological processes. However, technical hurdles including low cell yield, poor cell differentiation and poor attachment to the growth substrate have limited the use of this tool for studies of the genetic model organism Caenorhabditis elegans. This protocol describes a method for the large-scale culture of C. elegans embryo cells. We also describe methods for in vitro RNA interference, fluorescence-activated cell sorting of embryo cells and imaging of cultured cells for patch-clamp electrophysiology studies. Developing embryos are isolated from gravid adult worms. After eggshell removal by enzymatic digestion, embryo cells are dissociated and plated onto glass substrates. Isolated cells terminally differentiate within 24 h. Analysis of gene expression patterns and cell-type frequency suggests that in vitro embryo cell cultures recapitulate the developmental characteristics of L1 larvae. Cultured embryo cells are well suited for physiological analysis as well as molecular and cell biological studies. The embryo cell isolation protocol can be completed in 5-6 h.     

A stepwise method for the isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries

Methods for the stepwise isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries are described. Both cell types can be isolated in pure culture with high yields. Dogs are a common species used in the study of atherosclerosis and coronary artery disease. Capacity to isolate endothelial cells and smooth muscle cells from individual canine coronary arteries should prove useful in the study of coronary artery disease.     

Multiple phenotypes resulting from a mutagenesis screen for pharynx muscle mutations in Caenorhabditis elegans

We describe a novel screen to isolate pharyngeal cell morphology mutants in Caenorhabditis elegans using myo-2::GFP to rapidly identify abnormally shaped pharynxes in EMS (Ethyl Methanesulfonate) mutagenized worms. We observed over 83 C. elegans lines with distinctive pharyngeal phenotypes in worms surviving to the L1 larval stage, with phenotypes ranging from short pharynx, unattached pharynx, missing cells, asymmetric morphology, and non-adherent pharynx cells. Thirteen of these mutations have been chromosomally mapped using Single Nucleotide Polymorphisms (SNPs) and deficiency strain complementation. Our studies have focused on genetically mapping and functionally testing two phenotypes, the short pharynx and the loss of muscle cohesion phenotypes. We have also identified new alleles of sma-1, and our screen suggests many genes directing pharynx assembly and structure may be either pharynx specific or less critical in other tissues.     

Magnetic Separation of Phagosomes of Defined Age from Tetrahymena thermophila

ABSTRACT. We describe a new mass isolation procedure for both pure and stage-specific phagosomes from Tetrahymena thermophila . We prepared magnetic iron dextran particles about 1 μm in diameter to label the phagosomes. The oral apparatus of the cells concentrated these particles so readily that after 1 min the majority of the cells had formed a single phagosome. A short wash removed non-ingested particles, enabling us to follow the age-dependent changes of a single labeled phagosome through the cell. Phagosomes of different ages, including very young and nascent phagosomes, were removed easily from the non-magnetic cell debris of mechanically homogenized cells by means of a permanent magnet. The isolated phagosomes are pure as tested by enzymatic assays and light and electron microscopy. Since the yield of pure phagosomes of all ages is high (∼ 90%), this method could be generally applied for phagosome isolation from ciliates.     

Developmental regulation of a novel outwardly rectifying mechanosensitive anion channel in Caenorhabditis elegans

The nematode Caenorhabditis elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. However, the relative inaccessibility of somatic cells in adult animals greatly limits direct electrophysiological studies of channel activity. We developed methods to routinely isolate and patch clamp C. elegans embryo cells and oocytes and to culture and patch clamp neurons and muscle cells. Dissociated embryonic cells express a robust outwardly rectifying anion current that is activated by membrane stretch and depolarization. This current, termed I(Cl,mec), is inhibited by anion and mechanosensitive channel inhibitors. I(Cl,mec) has broad anion selectivity and the channel has a unitary conductance of 5-7 picosiemens. I(Cl,mec) is not detectable in whole-cell or isolated patch recordings from oocytes, cultured muscle cells, and cultured neurons but is expressed in single cell and later embryos. Channel density is high, and the current is observed in >80% of membrane patches. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I(Cl,mec) channels. The apparent activation of I(Cl,mec) shortly after fertilization and its down-regulation in terminally differentiated cells suggests that the channel may play important roles in embryogenesis and/or cytokinesis.     

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.     

A primary culture system for functional analysis of C. elegans neurons and muscle cells

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.     

Real-time analysis of Drosophila post-embryonic haemocyte behaviour

Background

The larval stage of the model organism Drosophila is frequently used to study host-pathogen interactions. During embryogenesis the cellular arm of the immune response, consisting of macrophage-like cells known as plasmatocytes, is extremely motile and functions to phagocytise pathogens and apoptotic bodies, as well as produce extracellular matrix. The cellular branch of the larval (post-embryonic) innate immune system consists of three cell types—plasmatocytes, crystal cells and lamellocytes—which are involved in the phagocytosis, encapsulation and melanisation of invading pathogens. Post-embryonic haemocyte motility is poorly understood thus further characterisation is required, for the purpose of standardisation.

Methodology

In order to examine post-embryonic haemocyte cytoskeletal dynamics or migration, the most commonly used system is in vitro cell lines. The current study employs an ex vivo system (an adaptation of in vitro cell incubation using primary cells), in which primary larval or pre-pupal haemocytes are isolated for short term analysis, in order to discover various aspects of their behaviour during events requiring cytoskeleton dynamics.

Significance

The ex vivo method allows for real-time analysis and manipulation of primary post-embryonic haemocytes. This technique was used to characterise, and potentially standardised, larval and pre-pupal haemocyte cytoskeleton dynamics, assayed on different extracellular matrices. Using this method it was determined that, while larval haemocytes are unable to migrate, haemocytes recovered from pre-pupae are capable of migration.     

Isolation, stages of differentiation, and long term maintenance of adult rat myocytes

Although a variety of techniques have been developed to isolate myocytes from adult hearts, the long term viability of such cells has only recently been investigated. In addition, relatively little is known about the stages of differentiation such cells proceed through following isolation. In the present study myocytes were isolated using two techniques, one involving retrograde perfusion via the aorta, and the other involving mechanical "shearing." In addition, several modifications were made to minimize the trauma normal associated with isolating myocytes from adult hearts. Both techniques yielded a high percentage of rod-shaped, quiescent myocytes, although myocytes isolated using the "shearing" method were less likely to remain viable for more than 24 hours. With both techniques those cells which remained viable for more than 24 hours proceeded through an identical pattern of differentiation leading to stable, attached cells which remained viable for up to four weeks. These results demonstrate that with the appropriate isolation techniques it is possible to maintain adult myocardial cells in culture for lengthy periods of time.     

Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function

Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 x 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 x 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.     

lin-28 controls the succession of cell fate choices via two distinct activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

Preparation of single cells from aggregated Taxus suspension cultures for population analysis

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.