Differential distribution of tau proteins in developing cat cerebellum.

The differential distribution and phosphorylation of tau proteins in cat cerebellum was studied with two well characterized antibodies, TAU-1 and TAU-2. TAU-1 detects tau proteins in axons, and the epitope in perikarya and dendrites is masked by phosphorylation. TAU-2 detects a phosphorylation-independent epitope on tau proteins. The molecular composition of tau proteins in the range of 45 kD to 64 kD at birth changed after the first postnatal month to a set of several adult variants of higher molecular weights in the range of 59 kD to 95 kD. The appearance of tau proteins in subsets of axons corresponds to the axonal maturation of cerebellar local-circuit neurons in granular and molecular layers and confirms previous studies. Tau proteins were also identified in synapses by immunofluorescent double-staining with synapsin I, located in the pinceau around the Purkinje cells, and in glomeruli. Dephosphorylation of juvenile cerebellar tissue by alkaline phosphatase indicated indirectly the presence of differentially phosphorylated tau forms mainly in juvenile ages. Additional TAU-1 immunoreactivity was unmasked in numerous perikarya and dendrites of stellate cells, and in cell bodies of granule cells. Purkinje cell bodies were stained transiently at juvenile ages. During postnatal development, the intensity of the phosphate-dependent staining decreased, suggesting that phosphorylation of tau proteins in perikarya and dendrites may be essential for early steps in neuronal morphogenesis during cat cerebellum development.

Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Evolutionary and convergence stability for continuous phenotypes in finite populations derived from two-allele models.

The evolution of a quantitative phenotype is often envisioned as a trait substitution sequence where mutant alleles repeatedly replace resident ones. In infinite populations, the invasion fitness of a mutant in this two-allele representation of the evolutionary process is used to characterize features about long-term phenotypic evolution, such as singular points, convergence stability (established from first-order effects of selection), branching points, and evolutionary stability (established from second-order effects of selection). Here, we try to characterize long-term phenotypic evolution in finite populations from this two-allele representation of the evolutionary process. We construct a stochastic model describing evolutionary dynamics at non-rare mutant allele frequency. We then derive stability conditions based on stationary average mutant frequencies in the presence of vanishing mutation rates. We find that the second-order stability condition obtained from second-order effects of selection is identical to convergence stability. Thus, in two-allele systems in finite populations, convergence stability is enough to characterize long-term evolution under the trait substitution sequence assumption. We perform individual-based simulations to confirm our analytic results.

Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10.

Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.

Fission yeast: in shape to divide.

How are cell morphogenesis and cell cycle coordinated? The fission yeast is a rod-shaped unicellular organism widely used to study how a cell self-organizes in space and time. Here, we discuss recent advances in understanding how the cell acquires and maintains its regular rod shape and uses it to control cell division. The cellular body plan is established by microtubules, which mark antipodal growth zones and medial division. In turn, cellular dimensions are defined by the small GTPase Cdc42 and downstream regulators of vesicle trafficking. Yeast cells then repetitively use their simple rod shape to orchestrate the position and timing of cell division.

Characterization of larval moulting cycles in Maja brachydactyla (Brachyura, Majidae) reared in the laboratory

The moulting cycles of all larval instars (zoea I, zoea II, and megalopa) of the spider crab Maja brachydactyla Balss 1922 were studied in laboratory rearing experiments. Morphological changes in the epidermis and cuticle were photographically documented in daily intervals and assigned to successive stages of the moulting cycle (based on Drach's classification system). Our moult-stage characterizations are based on microscopical examination of integumental modifications mainly in the telson, using epidermal condensation, the degree of epidermal retraction (apolysis), and morphogenesis (mainly setagenesis) as criteria. In the zoea II and megalopa, the formation of new setae was also observed in larval appendages including the antenna, maxillule, maxilla, second maxilliped, pleopods, and uropods. As principal stages within the zoea I moulting cycle, we describe postmoult (Drach's stages A–B combined), intermoult (C), and premoult (D), the latter with three substages (D0, D1, and D2). In the zoea II and megalopa, D0 and D1 had to be combined, because morphogenesis (the main characteristic of D1) was unclear in the telson and did not occur synchronically in different appendices. The knowledge of the course and time scale of successive moult-cycle events can be used as a tool for the evaluation of the developmental state within individual larval instars, providing a morphological reference system for physiological and biochemical studies related to crab aquaculture.

Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe beta-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the beta-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased beta-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/beta-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Roles of mGluR5 in synaptic function and plasticity of the mouse thalamocortical pathway.

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of cortical sensory maps. However, its precise roles in the synaptic function and plasticity of thalamocortical (TC) connections remain unknown. Here we first show that in mGluR5 knockout (KO) mice bred onto a C57BL6 background cytoarchitectonic differentiation into barrels is missing, but the representations for large whiskers are identifiable as clusters of TC afferents. The altered dendritic morphology of cortical layer IV spiny stellate neurons in mGluR5 KO mice implicates a role for mGluR5 in the dendritic morphogenesis of excitatory neurons. Next, in vivo single-unit recordings of whisker-evoked activity in mGluR5 KO adults demonstrated a preserved topographical organization of the whisker representation, but a significantly diminished temporal discrimination of center to surround whiskers in the responses of individual neurons. To evaluate synaptic function at TC synapses in mGluR5 KO mice, whole-cell voltage-clamp recording was conducted in acute TC brain slices prepared from postnatal day 4-11 mice. At mGluR5 KO TC synapses, N-methyl-D-aspartate (NMDA) currents decayed faster and synaptic strength was more easily reduced, but more difficult to strengthen by Hebbian-type pairing protocols, despite a normal developmental increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents and presynaptic function. We have therefore demonstrated that mGluR5 is required for synaptic function/plasticity at TC synapses as barrels are forming, and we propose that these functional alterations at the TC synapse are the basis of the abnormal anatomical and functional development of the somatosensory cortex in the mGluR5 KO mouse.

Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation.

Lymphatic valves are essential for efficient lymphatic transport, but the mechanisms of early lymphatic-valve morphogenesis and the role of biomechanical forces are not well understood. We found that the transcription factors PROX1 and FOXC2, highly expressed from the onset of valve formation, mediate segregation of lymphatic-valve-forming cells and cell mechanosensory responses to shear stress in vitro. Mechanistically, PROX1, FOXC2, and flow coordinately control expression of the gap junction protein connexin37 and activation of calcineurin/NFAT signaling. Connexin37 and calcineurin are required for the assembly and delimitation of lymphatic valve territory during development and for its postnatal maintenance. We propose a model in which regionally increased levels/activation states of transcription factors cooperate with mechanotransduction to induce a discrete cell-signaling pattern and morphogenetic event, such as formation of lymphatic valves. Our results also provide molecular insights into the role of endothelial cell identity in the regulation of vascular mechanotransduction.

Oxygen uptake during early cardiogenesis of the chick.

Oxidative metabolism of the isolated embryonic heart of the chick has been determined using a spectrophotometric technique allowing global as well as localized micromeasurements of the O2 uptake. Entire hearts, excised from embryos of 10 somites (primordia fused, stage 10 HH) and 40 somites (S shaped, stage 20 HH) were placed in a special chamber under controlled metabolic conditions where they continued to beat spontaneously and regularly. During the 32 h of development, the O2 consumption of the whole heart increased from 0.9 +/- 0.1 to 5.3 +/- 0.8 nmol O2/h. These values corrected for protein content were, however, comparable (0.45 nmol O2.h-1.micrograms-1). At stage 10-12, the O2 uptake varied along the cardiac tube (from 0.74 to 1.0 nmol O2.h-1.mm-2). From stage 10 to 20, the O2 uptake per unit area of ventricle wall increased from 0.7 +/- 0.2 to 1.8 +/- 0.2 nmol O2.h-1.mm-2, and the O2 uptake per myocardial volume during one cardiac cycle varied from 7 to 2.5 nmol O2/cm3. These results indicate that, despite an intense morphogenesis, the cardiac tissue has a rather low and stable oxidative metabolism, although the O2 requirement of the whole heart increases significantly. Moreover, the normalized suprabasal aerobic energy expenditure decreases throughout early cardiogenesis. The functional integrity of the isolated embryonic heart combined with the experimental possibilities of the microtechnique make the preparation appropriate for studying the changes in cardiac metabolism during development.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

Cytotaxonomy of Simulium cauchense Floch & Abonnenc and Simulium quadrifidum Lutz (Diptera: Simuliidae) in Brazilian Amazonia

Simulium cauchense Floch & Abonnenc and Simulium quadrifidum Lutz are widely distributed in the Amazon region and are morphologically similar at the larval and pupal stages. Chromosomally, these species are readily distinguished by the position of the nucleolar organizer, which is in the short arm of chromosome I in S. cauchense and in the long arm of chromosomes III in S. quadrifidum. They also differ by three fixed inversions. Sex chromosomes are undifferentiated in both species. Chromosomal resolution of the two species allowed us to evaluate four structural features previously used as diagnostic aids at the larval stage. Characters that distinguish larvae of the two species are the number of branches and branching patterns of the dorsal abdominal setae and the dark band on each primary fan. Branching patterns of the gill histoblasts were often diagnostic, with S. quadrifidum exhibiting more proximal branching and S. cauchense more distal branching. Sites where both species occurred sometimes had larvae with one petiole branching proximally and the other distally; in these cases examination of the chromosomes permitted assignment of the specimen to species. Pigmentation patterns of larvae, on the other hand, are highly variable. Color typically is sex linked in both species.

Effects of brood manipulation costs on optimal sex allocation in social hymenoptera.

In eusocial Hymenoptera, queens and workers are in conflict over optimal sex allocation. Sex ratio theory, while generating predictions on the extent of this conflict under a wide range of conditions, has largely neglected the fact that worker control of investment almost certainly requires the manipulation of brood sex ratio. This manipulation is likely to incur costs, for example, if workers eliminate male larvae or rear more females as sexuals rather than workers. In this article, we present a model of sex ratio evolution under worker control that incorporates costs of brood manipulation. We assume cost to be a continuous, increasing function of the magnitude of sex ratio manipulation. We demonstrate that costs counterselect sex ratio biasing, which leads to less female-biased population sex ratios than expected on the basis of relatedness asymmetry. Furthermore, differently shaped cost functions lead to different equilibria of manipulation at the colony level. While linear and accelerating cost functions generate monomorphic equilibria, decelerating costs lead to a process of evolutionary branching and hence split sex ratios.