Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs.

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Species-specific models in toxicology: in vitro epithelial barriers.

Species-specific in vitro epithelial barriers represent interesting predictive tools for risk assessment evaluation in toxicological studies. Moreover, these models could be applied either as stand-alone methods for the study of absorption, bioavailability, excretion, transport, effects of xenobiotics, or through an Integrated Testing Strategy. The aim of this review is to give a comprehensive overview of in vitro species-specific epithelial barrier models from bovine, dog and swine. Bovine mammary epithelial barrier as a fundamental instrument for the evaluation of the toxicant excretion, the blood brain barrier as a useful first approach in toxicological and pharmacological studies, the porcine intestinal barrier, the canine skin barrier, and finally the pulmonary barrier from bovine and swine species are described in this review.

Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage.

Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point.

Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner.

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer's disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces.

Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1µM) concentration of MeHgCl. ß-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to µm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.

Neural stem cells from human cord blood on bioengineered surfaces--novel approach to multiparameter bio-tests.

Stem cell technology combined with emerging surface nano/micro-technologies provides a new tool for better understanding of the mechanisms involved in cell fate decisions and compound-induced adverse reactions. This article provides state-of-the-art on the development of modern multiparameter bio-tests based on interactions between neural stem cells derived from human cord blood and bioengineered surfaces. Cell growth platforms with controlled content, geometry and spatial distribution of bioactive and stem cell attractive areas were fabricated either by micro-contact printing or piezoelectric spotting of polycationic biomolecules or extracellular matrix proteins (ECM) on cell-repellent surfaces. HUCB-NSCs were shown to adhere, differentiate and respond to neurotoxic MeHgCl on functional domains in a manner dependent on protein type and concentration, cell density and serum conditions. While receptor-mediated interactions with ECM proteins under absence of serum promote neuronal differentiation, non-specific adhesion to polycationic molecules maintain cells attached to the surface in non-differentiated stage. Functional domains were further engineered to create "smart" microenvironment by immobilizing to the surface signaling molecules together with ECM proteins. Stimulation of selected intracellular pathways by molecules of Wnt, Shh, CNTF or Notch type resulted in differentiation of HUCB-NSC to either neuronal or astroglial lineage. Sensor techniques applied to HUCB-NSC included measurements of electrical activity using multielectrode array chips. Spontaneous electrical field potentials of HUCB-NSCs were dependent upon developmental stage of tested cells. Bioengineered surfaces, on protein microarrays and micro-electrode array chips provide a novel approach to the multiparameter bio-tests by adding an important information on the sensitivity of certain molecular pathways and functional cellular responses to selected neurotoxins.

Stem-cell culture on patterned bio-functional surfaces.

Bio-functional surfaces have been created by printing proteins on antifouling surfaces in a customised geometry. Human umbilical cord neural stem cells incubated on the samples readily attach to the protein defined domains, where they have been monitored during 21 days of culture. The stability of the pattern varies with the density of cells anchored to the microstamped proteins. Highly packed cell patterned domains favoured non-differentiated mode, while low-density areas allowed the spreading out of the cells and differentiation. Tailoring the geometry (pattern size and distances) enables improving the monitoring of the stem cells' developmental processes. The biocompatible surfaces can serve as a model to study processes accompanying stem cell neural lineage commitment.

Function of ID1 protein in human cord blood-derived neural stem-like cells.

The effect of dominant-negative regulator of basic helix-loop-helix (bHLH) transcription factors, an ID1 protein, on growth and differentiation of neural stem-like cell line derived from human umbilical cord blood (HUCB-NSC) was investigated. This nontransformed, mesodermal germ layer-originated line contains high levels of ID1 protein, whose intercellular distribution reflects HUCB-NSC differentiation status. Whereas cells remained undifferentiated and self-renewing in serum-free (SF) cultures, ID1 protein, although highly expressed, did not attain cell nuclei and was localized mainly in cytoplasm. In long-term-expanded cultures of partially committed (primed) HUCB-NSC grown in a low serum concentration (LS cultures) ID1 protein became translocated toward cell nuclei. Further neuronal differentiation of the cells, either spontaneous in the presence of serum or induced by neuromorphogens (dBcAMP, RA), resulted in almost complete depletion of ID1 mRNA and protein. Accordingly, HUCB-NSC transfectants overexpressing the ID1 gene were significantly inhibited in their differentiation. Notably, only neuronal and not glial development was affected after ID1 overexpression. A similar gain-of-function effect of ID1 transfection was observed in human NSC-like line (DEV) of medullobastoma origin, which is constitutively devoid of ID1 expression. Thus, our results on HUCB-NSC confirm further its neural-specific behavior and the crucial role of ID1 protein as a potent negative regulator of neural stem cell differentiation, pointing out that this protein distribution between cytoplasmic and nuclear cell compartments can be one of the most important steps in differentiation signal transduction.

Neurogenic potential of human umbilical cord blood: neural-like stem cells depend on previous long-term culture conditions.

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB-NPs). Due to further expansion of these cells we have established the first human umbilical cord blood-derived neural-like stem cell line (HUCB-NSC) growing in serum-free (SF) or low-serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB-NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB-NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co-cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co-cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial-specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB-NSC on the neurogenic signals provided by surrounding environment in vitro.

Human cord blood-derived neural stem cell line--possible implementation in studying neurotoxicity.

Neural stem cell line developed from human umbilical cord blood (HUCB-NSC) [Buzanska et al., 2003. Journal of Neurochemistry 85, 33] is an ethically uncontroversial source of stem cells, able to differentiate into neuronal, astrocytic and oligodendroglial lineages. Developmental fate decisions of HUCB-NSC can be experimentally manipulated in vitro by the presence of trophic factors, mitogenes and neuromorphogenes, but can also be influenced by neurotoxins. In this report two-dimensional (2-D) and three-dimensional (3-D) HUCB-NSC cultures are introduced as useful models for testing developmental neurotoxicity. For 2-D culture models we established a standardized method for the assessment of the growth rate and cell differentiation in 96-well plates. The proliferative capacity of the HUCB-NSC was monitored by the MTT test while their ability to differentiate into neural-like cells by immunocytochemistry of beta-tubulin III and MAP-2 for neurons, GFAP and S-100beta for astrocytes and GalC for oligodendrocytes. The 3-D culture of HUCB-NSC is represented by neurospheres. Proliferation and migration of the intermediate precursors from attached neurospheres are shown to be controlled and altered by various growth factors and further modulated by the extracellular matrix component-fibronectin. Thus, neurospheres derived from the HUCB-NSC line can represent a suitable model of the activation of dormant stem cells residing in their niche, and can be used for neurotoxic studies.

Human cord blood-derived cells attain neuronal and glial features in vitro.

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons, astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes - 11% of the total population of differentiating cells.

Glutamine transport in C6 glioma cells: substrate specificity and modulation in a glutamine deprived culture medium.

A previous study has shown that glutamine (Gln) uptake in C6 cells grown in a standard medium containing 2 mM Gln, is predominantly mediated by a sodium-dependent system that is inhibited by ASC system substrates alanine (Ala), serine (Ser), cysteine (Cys) and threonine (Thr), shows pH sensitivity and partial tolerance to substitution of Na+ by Li+, features compatible with system ASCT2 that is strongly expressed in cultured astrocytes. The uptake was not inhibited by the model system A substrate alpha-(methylamino)isobutyric acid (MeAiB), and glycine (Gly) or proline (Pro), indicating that the substrate-regulated system A as defined by routine criteria is relatively inactive in these cells (Dolinska et al., 2000). In this study we compared the uptake of radiolabeled Gln and a model ASC substrate -Thr in cells grown to the same density in Gln-containing and Gln-deprived media. Cells grown in the absence of Gln showed a reduced activity of system ASC-mediated Gln uptake, and the system lost tolerance for Li+ and became somewhat more resistant to lowering pH of the medium. In contrast to cultured astrocytes deprived of Gln, the overall Gln uptake activity in C6 cells adapted to grow in a medium without Gln was lower than in cells grown in a Gln containing medium, and the uptake by system A remained inactive. C6 cells cultured both in the presence and absence of Gln expressed ASCT2 mRNA, indicating that system ASCT2-mediated Gln uptake is modulated at a posttranscriptional level. In contrast to Gln uptake, Thr uptake was more active in cells cultured in the absence of Gln and showed neither pH dependence nor lithium tolerance in either medium, which is typical of an uptake mediated by the widespread ASCT1 isoform of system ASC. In C6 cells grown in the presence or absence of Gln alike, approximately 20% of the sodium-dependent Gln uptake was resistant to MeAiB+Thr, indicating contribution of system N. The N system-mediated uptake in C6 cells grown in the absence, but not in the presence of Gln was not inhibited by glutamate (Glu) that conforms to the characteristics of the glial N system variant, SN1.

Human medulloblastoma cell line DEV is a potent tool to screen for factors influencing differentiation of neural stem cells.

The aim of our study was to investigate whether a human neural cell line could be used as a reliable screening tool to examine the functional conservation, in humans, of transcription factors involved in neuronal or glial specification in other species. Gain-of-function experiments were performed on DEV cells, a cell line derived from a human medulloblastoma. Genes encoding nine different transcription factors were tested for their influence on the process of specification of human DEV cells towards a neuronal or glial fate. In a first series of experiments, DEV cells were transfected with murine genes encoding transcription factors known to be involved in the neuronal differentiation cascade. Neurogenins-1, -2, and -3; Mash-1; and NeuroD increased the differentiation of DEV cells towards a neuronal phenotype by a factor of 2-3.5. In a second series of experiments, we tested transcription factors involved in invertebrate glial specification. In the embryonic Drosophila CNS, the development of most glial cells depends on the master regulatory gene glial cell missing (gcm). Expression of gcm in DEV cells induced a twofold increase of astrocytic and a sixfold increase of oligodendroglial cell types. Interestingly, expression of tramtrack69, which is required in all Drosophila glial cells, resulted in a fourfold increase of only the oligodendrocyte phenotype. Expression of the related tramtrack88 protein, which is not expressed in the fly glia, or the C. elegans lin26 protein showed no effect. These results show that the Drosophila transcription factor genes tested can conserve their function upon transfection into the human DEV cells, qualifying this cell line as a screening tool to analyze the mechanisms of neuronal and glial specification.

Extracellular signal-regulated kinase suppression is not involved in apoptosis of neuroblastoma N2a cells induced by protein kinase C inhibition.

A model study for the mechanism of cell death in vitro was established on the neuroblastoma N2a cell line. By differential staining of living cells with Hoechst 33258 and propidium iodide, or by Terminal-dUTP-Transferase-Nick-End Labelling (TUNEL), cJun/AP1 immunoreactivity, and DNA laddering, we show that the N2a cell line responded with apoptosis to protein kinase C (PKC) inhibition. Two different classes of PKC inhibitors (staurosporine and Gö6976) at concentrations generally regarded as PKC-selective (10 nM and 50 nM, respectively), significantly increased the number of apoptotic cells in N2a cultures. The cells started to die 2-3 hours after the treatment; then, at 6 and/or 24 hours, approximately 30-40% of the cells acquired the apoptotic feature. This response was dependent on neither cell differentiation nor PKC and bcl2 gene and protein expression, but exclusively on the concomitant withdrawal of serum from growth medium. Furthermore, both of the experimental manipulations (serum deprivation and PKC inhibition) synergically, but to different extents, suppressed the extracellular signal regulated kinase (ERK) pathway. Their pro-apoptotic effect, however, was neither mimicked nor modified by an additional inhibition of MEK/ERK kinase by 50 microM PD 98059, resulting in an 80% inhibition of its initial activity. We conclude, therefore, that apoptosis of N2a cells triggered by PKC-inhibition, as well as its abrogation by serum, is totally independent of concomitant modulations of ERK activity also evoked by the above treatments.

Delayed induction of apoptosis by ammonia in C6 glioma cells.

Ammonia is a neurotoxin whose administration in large doses causes coma and death of the exposed animals, but whether and in what degree these whole body effects are related to the death of CNS cells is not known. Since the downstream effects of ammonia in cultured CNS cells appear to be partly mediated by overactivation of several putative signalling mechanisms characteristic for the apoptotic program, we speculated that ammonia neurotoxicity may be apoptogenic. In this study, C6 glioma cells grown in 2% serum were exposed to 5 mM or 10 mM NH(4)Cl (ammonia) for 96 h and tested for the appearance of apoptosis by (a) Hoechst staining, (b) TUNEL reaction and (c) DNA ladder, at different times of exposure. In cultures exposed to either 5 mM or 10 mM ammonia, about 10% of the cells were found to enter apoptosis at 48 h of exposure, and the number of apoptotic cells rose to 30% at 72 h, and to 50% at 96 h of exposure, respectively. The first transduction signal purportedly involved in apoptosis, activation of PKCalphabeta, was transient and appeared already after 3-6 h of treatment. Coincident with pronounced manifestation of apoptosis (at 72 h and even more at 96 h of exposure) was an increased transfer of the transcription factor NFkappaB from cytoplasmto nucleus as revealed by EMSA assay. The number of cells affected by ammonia-induced apoptosis was markedly reduced by incubation with a NOS inhibitor, L-NAME at 100 microM concentration. The results indicate that ammonia-induced apoptosis is a result of a complex interplay of at least three signalling molecules: NO, PKC and the transcription factor NFkappaB, with NFkappaB being possibly involved in the induction of iNOS and generation of toxic levels of NO in the cells.

Molecular subdivision of the cortex of dividing Tetrahymena is coupled with the formation of the fission zone.

In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis.

Okadaic acid promotes cell division in synchronized Tetrahymena pyriformis and in the cell division-arrested (cdaA1) temperature-sensitive mutant of T. thermophila.

1. Okadaic acid (OA) at 0.5 to 1 microM accelerated the onset and completion of division in heat-synchronized Tetrahymena pyriformis, especially where cells had been transiently delayed in the presence of dimethyl sulfoxide (DMSO). 2. The cell division-arrested mutant, cdaA1, of Tetrahymena thermophila ceased dividing after being shifted from the permissive temperature of 22 degrees C to the restrictive temperature of 37 degrees C, but continued to grow without forming fission furrows, resulting in deformed "monsters". In the presence of 1 microM OA, monster formation was completely inhibited, and over 20% of the mutant cells at 37 degrees C proceeded through a further apparently normal division. Evidence is presented for the first time that the potent and relatively selective PP2A inhibitor, okadaic acid (OA) can promote the entry and completion of Tetrahymena cell division as opposed to simply aiding the premature appearance of M-phase events seen in other cell systems. In this regard, the differential response to the combined action of OA and the kinase inhibitor 6-dimethyl-aminopurine (6-DMAP) at chosen stages of the cell cycle is shown. At early division, inhibitory effects of 6-DMAP were enhanced by the presence of OA, whereas in advanced stages of division, OA treatment by-passed 6-DMAP-induced inhibition and accelerated cells through division. The results are discussed in terms of the actions of these drugs on phosphorylation/dephosphorylation events responsible for driving division.

Relationship between spatial pattern of basal bodies and membrane skeleton (epiplasm) during the cell cycle of Tetrahymena: cdaA mutant and anti-membrane skeleton immunostaining.

Microtubular basal bodies and epiplasm (membrane skeleton) are the main components of the cortical skeleton of Tetrahymena. The aim of this report was to study functional interactions of basal bodies and epiplasm during the cell cycle. The cortex of Tetrahymena cells was stained with anti-epiplasm antibody. This staining produced a bright epiplasmic layer with a dark pattern of unstained microtubular structures. The fluorescence of the anti-epiplasm antibody disappeared at sites of newly formed microtubular structures, so the new basal body domains and epiplasmic layer could be followed throughout the cell cycle. Different patterns of deployment of new basal bodies were observed in early and advanced dividers. In advanced dividers the fluorescence of the epiplasmic layer diminished locally within the forming fission line where the polymerization of new basal bodies largely extincted. In wild type Tetrahymena, the completion of the micronuclear metaphase/anaphase transition was associated with a transition from the pattern of new basal body deployment and epiplasm staining of the early divider to the pattern of the advanced dividers. The signal for the fission line formation in Tetrahymena (absent in cdaA1 Tetrahymena mutationally arrested in cytokinesis) brings about 1) transition of patterns of deployment of basal bodies and epiplasmic layer on both sides of the fission line; and 2) coordination of cortical divisional morphogenesis with the micronuclear mitotic cycle.

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila.

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35 degrees C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity.