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PubMed2001

Cytoskeletal reorganization during the formation of oligodendrocyte processes and branches.

During oligodendrocyte development, signals relevant to process formation must be transduced into appropriate changes in cytoskeletal organization. We have explored how microtubules and microfilaments interact during the outgrowth and branching of oligodendrocyte processes in culture. We observed that microfilaments are enriched in the peripheral region beneath the plasma membrane and constitute the major cytoskeletal element at the leading edge of the process, which is also enriched in the B-isoform of the non-muscle myosin II heavy chain. Microtubules form a dense bundle within the process and splay before extending into the leading edge and branches, following tracks laid by microfilaments. Pharmacologic disruption of microfilaments and microtubules compromised normal process outgrowth and branching. However, microtubules rapidly reinvaded most processes after removal of both antimicrotubule and antimicrofilament drugs, but the reinvasion was severely compromised if the antimicrofilament drug was retained. These results are consistent with the hypothesis that microfilaments guide the local reorganization of microtubules for the elongation of oligodendrocyte processes and the formation of new branches.

Actin Cytoskeleton↗

PubMed2006

Branching stochastic processes with immigration in analysis of renewing cell populations.

This paper considers the utility of a new class of stochastic branching processes with non-homogeneous immigration in modeling complex renewing cell systems. Such systems typically include the population of stem cells that provides an inexhaustible supply of cells necessary for maintaining the cellular composition of a tissue. A stem cell may be induced to transform (differentiate) into a progenitor cell. Progenitor cells retain the ability to proliferate and their function is believed to provide a quick proliferative response to an increased demand for cells in the population. There may be several sub-types of progenitor cells. Terminally differentiated cells do not divide under normal conditions; they are responsible for maintaining tissue-specific functions. Recent advancements in experimental techniques offer considerable scope for quantitative studies of in vivo cell kinetics based on stochastic modeling of renewing cell populations. However, no ready-made theory is currently available to take full advantage of these advancements. This paper introduces such a theory with a special focus on its feasibility in biological applications.

Cell Death↗

PubMed1982

Scanning electron microscopic observations of rat tibia using the HCl-collagenase method.

The metaphysis of rat tibia was examined by scanning electron microscopy (SEM) using the HCl-collagenase method (EVAN et al., 1976) and the three-dimensional structure of osteocytes and the relationship between osteocytes and osteoblasts were demonstrated. After the HCl-collagenase treatment, the bone matrix near the exposed surface was completely removed but the cellular components retained their structural integrity. Young osteocytes located close to osteoblasts showed flattened, spindly shapes. They were connected with adjacent osteoblasts by short, wide and slightly branched processes. On the other hand, the osteocytes located in the deeper portion of trabecular bone were smaller in size, being round or oval and flattened in shape. They were connected with both osteoblasts and neighboring osteocytes by long and more extensively branched processes forming a complicated network. From these results, it is suggested that osteoblasts extend their processes perpendicularly to the bone surface, with these processes branching off and connecting to each other at the osteoid layer.

Animals↗

PubMed1996

Glial cells in transected optic nerves of immature rats. I. An analysis of individual cells by intracellular dye-injection.

The glial response to Wallerian degeneration was studied in optic nerves following unilateral enucleation in immature rats, aged 21 days old (P21). The three-dimensional morphology of dye-filled glia was determined in intact nerves, at post-enucleation day 21 in normal nerves from untreated P21 rats, by correlating laser scanning confocal microscopy and camera lucida drawings of single cells. In normal and transected nerves, the majority of dye-filled cells comprized astrocytes (54% and 65%, respectively). In normal P21 nerves, the predominant astrocyte form had a complex stellate morphology and had a centrally-located cell body from which branching processes extended randomly. Two other distinct forms were transverse and longitudinal astrocytes, which had a polarized process extension in a plane perpendicular or parallel to the long axis of the nerve, respectively. These forms were recognized in transected nerves also, but astrocytes in transected nerves had a simple morphology on the whole, and extended few, dense processes which branched infrequently. Quantitative analysis of astrocyte morphology confirmed that individual astrocytes underwent considerable remodelling in response to Wallerian degeneration. A prominent reaction was that astrocytes had withdrawn radial processes and extended a greater proportion of processes longitudinally, parallel to the long axis of the nerve and along the course of degenerated axons. A further, notable feature of transected nerves was the development of novel longitudinal forms and of hypertrophic astroglia. These results indicated that all astrocytes became reactive following enucleation and that glial scar formation was not the function of a single astrocyte subtype. Oligodendrocytes in transected nerves had lost their myelin sheaths and appeared as small cells with numerous bifurcating processes which extended radially, but a small number of oligodendrocytes were recognized which apparently supported myelin sheaths (9%, compared to 40% in normal nerves). In addition, there was a significant population of indeterminate cells in transected nerves (26%, compared to 6% in normal nerves) and, although some of these were identified as microglia/macrophages, it was concluded that many were likely to be dedifferentiated oligodendrocytes.

Age Factors↗

PubMed2001

Regulation of human cytotrophoblast morphogenesis by hepatocyte growth factor/scatter factor.

In vitro morphogenesis of epithelial cells to form tube-like structures is regulated by hepatocyte growth factor-scatter factor (HGF/SF). The placenta is a rich source of HGF/SF, and its absence in mice has been shown to lead to impaired placental growth and embryonic death. There is no information in the literature regarding in vitro morphogenesis of human cytotrophoblasts or the effect of HGF/SF on this process. In this study, cytotrophoblasts were isolated from human placentae obtained from all three trimesters of gestation and cultured on the recombinant basement membrane matrix (Matrigel). Under these conditions, cytotrophoblasts participated in morphogenetic events including formation of spheroid-like structures, radial linear processes with branching, and invaded Matrigel and formed large, tube-like structures. The presence of a developing lumen was documented in the linear projections arising from spheroids and in the tube-like structures by both confocal and transmission electron microscopy. Immunohistochemistry was used to characterize the phenotype of the cells, and staining with anti-cytokeratin and anti-E-cadherin antibodies confirmed the presence of cytotrophoblasts in both the spheroids and tube-like structures. Recombinant HGF (rHGF) significantly increased the invasive activity of cytotrophoblasts isolated from the first and second (P < 0.001) and third trimesters (P < 0.01). In addition, rHGF significantly increased the percentage of spheroids with branching processes in the first and second trimesters (P < 0.05). Anti-HGF antibody inhibited both these effects in a dose-dependent manner, indicating the specificity of the above findings. This study provides new evidence indicating that HGF/SF regulates invasion and branching morphogenesis of cytotrophoblasts throughout gestation, with maximum effects in the first and second trimester. These findings may help to elucidate the importance of the reduced expression of HGF/SF identified in placentae from women with preeclampsia or intrauterine growth restriction and suggest that HGF/SF may serve as an important candidate in therapeutic intervention strategies.

Cadherins↗

PubMedDate not supplied

Msx-2 expression and glucocorticoid-induced overexpression in embryonic mouse submandibular glands.

It is well known that the process of branching morphogenesis requires epithelial-mesenchymal interactions. One outstanding model for the study of tissue interactions during branching morphogenesis is the embryonic mouse submandibular gland (SMG). Although it has been clearly demonstrated that the branching pattern is dependent on interactions between the epithelium and the surrounding mesenchyme, little is known about the molecular mechanism underlying the branching process. One group of transcription factors that likely participates in the control of epithelial-mesenchymal inductive interactions are the Msx-class of homeodomain-containing proteins. In this paper, we focus on Msx-2 because its developmental expression is correlated with inductive interactions, suggesting that Msx-2 may play a functional role during cell-cell interactions. We demonstrate the expression of Msx-2 mRNA and protein to be primarily in the branching epithelia with progressive embryonic (E13 to E15) SMG development and, to a lesser extent, in the mesenchyme. We also show that Msx-2 is expressed by embryonic SMG primordia cultured under defined conditions. In addition, to begin to delineate a functional role for Msx-2, we employed an experimental strategy by using exogenous glucocorticoid (CORT) treatment of embryonic SMGs in vitro and in vivo to significantly enhance branching morphogenesis and evaluate the effect of CORT treatment on embryonic SMG Msx-2 expression. A marked increase in Msx-2 transcripts and protein is detected with in vitro and in vivo CORT treatment. Our studies indicate that one mechanism of CORT regulation of salivary gland morphogenesis is likely through the modulation of Msx-2 gene expression.

Animals↗

PubMed1989

A three-dimensional reconstruction of the ruffled border of osteoclasts.

Osteoclasts of rat parietal bones were cultured and observed under the transmission electron microscope. The processes forming the ruffled border were studied three-dimensionally by a reconstruction method. After the observation of one plane (A-plane), serial ultrathin sections were made at right angles to the A-plane (B-plane). The processes of the ruffled border in the B-plane were reconstructed, based on the A-plane features, with Nikon Cosmozone 2S. The three-dimensional structure of the ruffled border of the osteoclasts was composed of two kinds of processes: one finger-like and the other plate-like. Both finger-like and plate-like processes branched from the stem processes projecting directly from the cell body. The long plate-like processes branched into secondary plates, extending perpendicularly to the bone surface.

Animals↗

PubMed2004

A branching model for the spread of infectious animal diseases in varying environments.

This paper is concerned with a stochastic model, describing outbreaks of infectious diseases that have potentially great animal or human health consequences, and which can result in such severe economic losses that immediate sets of measures need to be taken to curb the spread. During an outbreak of such a disease, the environment that the infectious agent experiences is therefore changing due to the subsequent control measures taken. In our model, we introduce a general branching process in a changing (but not random) environment. With this branching process, we estimate the probability of extinction and the expected number of infected individuals for different control measures. We also use this branching process to calculate the generating function of the number of infected individuals at any given moment. The model and methods are designed using important infections of farmed animals, such as classical swine fever, foot-and-mouth disease and avian influenza as motivating examples, but have a wider application, for example to emerging human infections that lead to strict quarantine of cases and suspected cases (e.g. SARS) and contact and movement restrictions.

Animals↗

PubMed2005

Crystal structure and functional implications of Pyrococcus furiosus hef helicase domain involved in branched DNA processing.

DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

Adenosine Triphosphatases↗

PubMed2000

Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis.

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 microm) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 microm PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF- and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dnBMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.

Cell Line, Transformed↗

PubMed1995

Mathematical modeling of dendritic growth in vitro.

The dendritic branching pattern of cultured hippocampal neurons was analyzed to obtain mathematical parameters that fit the time-dependent growth of dendrites under limited extrinsic influence. Cultured neurons were stained with a non-toxic carbocyanine dye (diO) and pyramidal-shaped neurons that were physically separated from one another were analyzed at post-plating days 1, 2, 3, 4, 6 and 7. The geometric branching pattern of the dendrites was analyzed using a mathematical model that incorporates random effects in the form of a Galton-Watson branching process where splitting of one branch is statistically independent of the splitting of all other branches, and deterministic effects in the form of a parameter that measures the extent to which dense patterns (clusters) or sparse patterns (elongated trees) are formed. The geometric branching pattern of the dendrites was analyzed using a mathematical model that incorporates random and deterministic effects. The model parameters were estimated via the method of maximum likelihood. The data suggest that in vitro basal dendrites grow according to a purely random branching process without pronounced dense or sparse patterns, while apical dendrites tend to form elongated trees with fewer secondary bifurcations. This trend is quantified, and it depends on the culture conditions in which the neurons are grown. The quantitative assessment of various influences on dendritic growth patterns are discussed.

Animals↗

PubMed2005

Critical branching captures activity in living neural networks and maximizes the number of metastable States.

Recent experimental work has shown that activity in living neural networks can propagate as a critical branching process that revisits many metastable states. Neural network theory suggests that attracting states could store information, but little is known about how a branching process could form such states. Here we use a branching process to model actual data and to explore metastable states in the network. When we tune the branching parameter to the critical point, we find that metastable states are most numerous and that network dynamics are not attracting, but neutral.

Animals↗

PubMed2005

Vere-Jones' self-similar branching model.

Motivated by its potential application to earthquake statistics as well as for its intrinsic interest in the theory of branching processes, we study the exactly self-similar branching process introduced recently by Vere-Jones. This model extends the ETAS class of conditional self-excited branching point-processes of triggered seismicity by removing the problematic need for a minimum (as well as maximum) earthquake size. To make the theory convergent without the need for the usual ultraviolet and infrared cutoffs, the distribution of magnitudes m' of daughters of first-generation of a mother of magnitude m has two branches m < m' with exponent beta - d and m' > m with exponent beta + d, where beta and d are two positive parameters. We investigate the condition and nature of the subcritical, critical, and supercritical regime in this and in an extended version interpolating smoothly between several models. We predict that the distribution of magnitudes of events triggered by a mother of magnitude m over all generations has also two branches m' < m with exponent and with exponent beta - h, with h=d squareroot of (1-s), where s is the fraction of triggered events. This corresponds to a renormalization of the exponent d into h by the hierarchy of successive generations of triggered events. For a significant part of the parameter space, the distribution of magnitudes over a full catalog summed over an average steady flow of spontaneous sources (immigrants) reproduces the distribution of the spontaneous sources with a single branch and is blind to the exponents beta, d of the distribution of triggered events. Since the distribution of earthquake magnitudes is usually obtained with catalogs including many sequences, we conclude that the two branches of the distribution of aftershocks are not directly observable and the model is compatible with real seismic catalogs. In summary, the exactly self-similar Vere-Jones model provides an attractive new approach to model triggered seismicity, which alleviates delicate questions on the role of magnitude cutoffs in other non-self-similar models. The new prediction concerning two branches in the distribution of magnitudes of aftershocks could be tested with recently introduced stochastic reconstruction methods, tailored to disentangle the different triggered sequences.

Journal Article↗

PubMed1993

Modulation of HGF-induced tubulogenesis and branching by multiple phosphorylation mechanisms.

MDCK cells cultured in Type I collagen gels can be induced to develop branching tubular structures with demonstrable lumens in the presence of hepatocyte growth factor (HGF). As we have now shown by immunofluorescent localization of specific marker proteins, these tubules retain apical-basolateral polarity. However, the secondary signaling events which lead to these characteristic morphogenetic changes induced by HGF remain largely unelucidated. In order to examine these signaling events, particularly the role of protein phosphorylation in the formation of branching tubular structures, Madin-Darby canine kidney (MDCK) cells in collagen gels were treated with HGF plus well-characterized agents that affect protein phosphorylation. We quantified the formation of branching processes, an early step in the development of tubular structures in this model. Protein kinase C (PKC) inhibition resulted in more complex branching processes in the presence of HGF, when compared with HGF alone. In contrast, treatment with activators of protein kinase A (PKA), as well as calmodulin antagonists, caused a marked decline in process formation. Consistent with an important role for protein phosphorylation in HGF-induced morphogenesis, protein phosphatase inhibition by okadaic acid or calyculin A was found to markedly inhibit process formation. Tyrosine kinase (TK) inhibition also decreased the percentage of processes. This is consistent with data indicating that one of the HGF receptors is identical to the c-met protooncogene product, which is known to possess TK activity. Our results suggest that the HGF-mediated induction of branching processes in MDCK cells, an early step in the development of branching tubular structures, can be modulated by multiple phosphorylation mechanisms including those mediated by PKC, PKA, and Ca2+/calmodulin-dependent kinase(s). We discuss how these phosphorylation events may play crucial roles in determining the degree of tubule formation and their length, as well as the extent of their arborization during the early development of epithelial tissues.

Alkaloids↗

PubMed2004

Host processing of branched DNA intermediates is involved in targeted transposition of IS911.

A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends. We show here that these IR-targeted events occur by an unusual mechanism. In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction. IR-targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure. The single-end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction. SET intermediates must be processed by the bacterial host to obtain the final insertion products. Sequence analysis of these IR-targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another. These sequence changes cannot be explained by the classic transposition pathway. A model is presented in which the four-way Holliday-like junction created by SET is processed by host-mediated branch migration, resolution, repair and replication. This pathway resembles those described for processing other branched DNA structures such as stalled replication forks.

DNA Transposable Elements↗

PubMed1982

A light microscopic demonstration of amoeboid microglia and microglial cells in the retina of rats of various ages.

The retinae from rats of various ages were stained using the weak silver carbonate method of del Rio-Hortega. In newborn and early postnatal (3-day-old) rats, silver-impregnated round and amoeboidic cells displaying thick stout pseudopodial processes were distributed in the nerve fiber and ganglion cell layers. In 10-day-old rats, the cell bodies were rod-shaped bearing pseudopodia or fusiform showing slender and branching processes. The majority of the cells were found in the ganglion cell layer with occasional ones in the inner plexiforn layer. By 20 days of age, the cells became smaller and exhibited long branching processes. They were located predominantly in the inner plexiform or bipolar cell layer. In the adult animals, all the silver-impregnated cells appeared small and flattened with long branching processes. Based on their morphological features, it was concluded that they were in fact amoeboid microglia and microglial cells present in the developing retina which probably function as macrophages as described in the brain tissues.

Aging↗

PubMed2000

The effective population size of some age-structured populations.

It was shown in a previous paper that if generations are discrete, then the effective population size of a large population can be derived from the theory of multitype branching processes. It turns out to be proportional to the reciprocal of a term that appears in the denominator of expressions for survival probabilities when there is a supercritical positively regular branching process for which the dominant positive eigenvalue of the first moment matrix is slightly larger than 1. If there is an age-structured population with unchanging proportions among sexes and age groups, then the effective population size is shown to be also obtainable from the theory of multitype branching processes. The expression for this parameter has the same form as in the corresponding model for discrete generations, multiplied by an appropriate measure of the average length of a generation. Results are obtained for dioecious random mating populations, populations reproducing partly by selfing, and populations reproducing partly by full-sib mating.

Age Factors↗

PubMed1984

Stochastic models of tumor growth and the probability of elimination by cytotoxic cells.

The probability of tumor extinction due to the action of cytotoxic cell populations is investigated by several one dimensional stochastic models of the population growth and elimination processes of a tumor. The several models are made necessary by the nonlinearity of the processes and the different parameter ranges explored. The deterministic form of the model is T' = gamma 0T - k'6T/(K1 + T) where gamma 0, k'6 and K1 are positive constants. The parameter of most import is lambda 0 = gamma 0 - k'6/K1 which determines the stability of the T = 0 equilibrium. With an initial tumor size of one, a (linear) branching process is used to estimate the extinction probability. However, in the case lambda = 0 when the linearization of the deterministic model gives no information (T = 0 is actually unstable) the branching model is unsatisfactory. This makes necessary the utilization of a density-dependent branching process to approximate the population. Through scaling a diffusion limit is reached which enables one to again compute the probability of extinction. For populations away from one a sequence of density-dependent jump Markov processes are approximated by a sequence of diffusion processes. In limiting cases, the estimates of extinction correspond to that computed from the original branching process. Table 1 summarizes the results.

Cell Survival↗