Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours.

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.

Prion Function and Pathophysiology in Non-Mammalian Models.

More than thirty years have passed since the discovery of the prion protein (PrP) and its causative role in transmissible spongiform encephalopathy. Since a combination of both gain- and loss-of-function mechanisms may underlay prion pathogenesis, understanding the physiological role of PrP may give important clues about disease mechanisms. Historically, the primary strategy for prion research has involved the use of human tissue, cell cultures and mammalian animal models. Nevertheless, experimental difficulties of in vivo studies and controversial observations obtained in these systems have stimulated the search for alternative animal models. PrPC is highly conserved in mammals, and PrPC-related orthologs are expressed in zebrafish, a vertebrate model organism suitable to study the mechanisms associated with human diseases. Invertebrate models, as they do not express PrPC have served to investigate the neurotoxic mechanisms of mammalian PrP. Here we overview most recent advances in the study of PrP function in normal and pathogenic conditions based on non-mammalian studies, highlighting the contribution of zebrafish, fly and worms to our current understanding of PrP biology.

Computational methods for analysis of dynamic events in cell migration.

Cell migration is a complex biological process that involves changes in shape and organization at the sub-cellular, cellular, and supra-cellular levels. Individual and collective cell migration can be assessed in vitro and in vivo starting from the flagellar driven movement of single sperm cells or bacteria, bacterial gliding and swarming, and amoeboid movement to the orchestrated movement of collective cell migration. One key technology to access migration phenomena is the combination of optical microscopy with image processing algorithms. This approach resolves simple motion estimation (e.g. preferred direction of migrating cells or path characteristics), but can also reveal more complex descriptors (e.g. protrusions or cellular deformations). In order to ensure an accurate quantification, the phenomena under study, their complexity, and the required level of description need to be addressed by an adequate experimental setup and processing pipeline. Here, we review typical workflows for processing starting with image acquisition, restoration (noise and artifact removal, signal enhancement), registration, analysis (object detection, segmentation and characterization) and interpretation (high level understanding). Image processing approaches for quantitative description of cell migration in 2- and 3-dimensional image series, including registration, segmentation, shape and topology description, tracking and motion fields are presented. We discuss advantages, limitations and suitability for different approaches and levels of description.

Células Troncales en el Desarrollo y las Perspectivas de Reprogramación Celular para la Regeneración / Stem Cells in Development and the Perspectives of Cellular Reprogramming for Regeneration

Los organismos multicelulares se desarrollan a partir de una sola célula: el cigoto. A lo largo de su ontogenia, las células que derivan del cigoto despliegan distintos programas celulares, los cuales son estabilizados por mecanismos epigenéticos. Los programas de las células troncales son más inclusivos, siendo mayor el silenciamiento que la activación de genes durante el proceso de diferenciación celular. Experimentalmente, se ha logrado que células en estado de diferenciación terminal reactiven el programa de células troncales y recuperen su pluripotencialidad, proceso llamado reprogramación. Esto despierta esperanzas en el avance de una medicina regenerativa con nuevas capacidades para el tratamiento de enfermedades crónicas, sin las restricciones éticas del uso de células embrionarias.

Multicellular organisms develop from one cell: the zygote. During ontogeny, cells derived from the zygote display different cellular programs that are stabilized through epigenetic mechanisms. The programs of stem cells seem more inclusive, and during the process of differentiation a larger number of genes are silenced than activated. The reactivation of pluripotency recovers of the stem cell program in terminally differentiated cells has been achieved experimentally. This process, called reprogramming, brings new hope for the development of a regenerative medicine with new capabilities for the treatment of chronic diseases, without the ethic restrains imposed by the use of embryonic cells.

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

Hedgehog and Fgf signaling pathways regulate the development of tphR-expressing serotonergic raphe neurons in zebrafish embryos.

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Asymmetry in the epithalamus of vertebrates.

The epithalamus is a major subdivision of the diencephalon constituted by the habenular nuclei and pineal complex. Structural asymmetries in this region are widespread amongst vertebrates and involve differences in size. neuronal organisation, neurochemistry and connectivity. In species that possess a photoreceptive parapineal organ, this structure projects asymmetrically to the left habenula, and in teleosts it is also situated on the left side of the brain. Asymmetries in size between the left and right sides of the habenula are often associated with asymmetries in neuronal organisation, although these two types of asymmetry follow different evolutionary courses. While the former is more conspicuous in fishes (with the exception of teleosts), asymmetries in neuronal organisation are more robust in amphibia and reptiles. Connectivity of the parapineal organ with the left habenula is not always coupled with asymmetries in habenular size and/or neuronal organisation suggesting that, at least in some species, assignment of parapineal and habenular asymmetries may be independent events. The evolutionary origins of epithalamic structures are uncertain but asymmetry in this region is likely to have existed at the origin of the vertebrate, perhaps even the chordate, lineage. In at least some extant vertebrate species, epithalamic asymmetries are established early in development, suggesting a genetic regulation of asymmetry. In some cases, epigenetic factors such as hormones also influence the development of sexually dimorphic habenular asymmetries. Although the genetic and developmental mechanisms by which neuroanatomical asymmetries are established remain obscure, some clues regarding the mechanisms underlying laterality decisions have recently come from studies in zebrafish. The Nodal signalling pathway regulates laterality by biasing an otherwise stochastic laterality decision to the left side of the epithalamus. This genetic mechanism ensures a consistency of epithalamic laterality within the population. Between species, the laterality of asymmetry is variable and a clear evolutionary picture is missing. We propose that epithalamic structural asymmetries per se and not the laterality of these asymmetries are important for the behaviour of individuals within a species. A consistency of the laterality within a population may play a role in social behaviours between individuals of the species.

Vertebrate gastrulation: calcium waves orchestrate cell movements.

A recent study reveals that the propagation of intercellular calcium signals is closely associated with the generation of convergent extension movements during Xenopus gastrulation. Such signals provide a mechanism whereby large populations of cells can communicate to generate orchestrated cell movements.

A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon.

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl(-/-) embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl(-/-) embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3beta can restore eye and telencephalic fates to mbl(-/-) embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.

Expression of nk2.1a during early development of the thyroid gland in zebrafish.

We show here that a zebrafish orthologue of the Thyroid Transcription Factor-1 (TTF-1), nk2.1a, is expressed in the developing thyroid gland. Using a fate mapping approach we found that an early nk2.1a expression domain in the endoderm adjacent to the heart follows morphogenetic movements of the lower jaw, ending up in the region in which the mature thyroid gland is located. We therefore suggest that nk2.1a labels the thyroid precursor cells from somitogenesis stages onwards.

Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation.

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.

A nodal signaling pathway regulates the laterality of neuroanatomical asymmetries in the zebrafish forebrain.

Animals show behavioral asymmetries that are mediated by differences between the left and right sides of the brain. We report that the laterality of asymmetric development of the diencephalic habenular nuclei and the photoreceptive pineal complex is regulated by the Nodal signaling pathway and by midline tissue. Analysis of zebrafish embryos with compromised Nodal signaling reveals an early role for this pathway in the repression of asymmetrically expressed genes in the diencephalon. Later signaling mediated by the EGF-CFC protein One-eyed pinhead and the forkhead transcription factor Schmalspur is required to overcome this repression. When expression of Nodal pathway genes is either absent or symmetrical, neuroanatomical asymmetries are still established but are randomized. This indicates that Nodal signaling is not required for asymmetric development per se but is essential to determine the laterality of the asymmetry.

Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis.

We have taken advantage of the optical transparency of zebrafish embryos to investigate the patterns of cell division, movement and shape during early stages of development of the central nervous system. The surface-most epiblast cells of gastrula and neurula stage embryos were imaged and analysed using a computer-based, time-lapse acquisition system attached to a differential interference contrast (DIC) microscope. We find that the onset of gastrulation is accompanied by major changes in cell behaviour. Cells collect into a cohesive sheet, apparently losing independent motility and integrating their behaviour to move coherently over the yolk in a direction that is the result of two influences: towards the vegetal pole in the movements of epiboly and towards the dorsal midline in convergent movements that strengthen throughout gastrulation. Coincidentally, the plane of cell division becomes aligned to the surface plane of the embryo and oriented in the anterior-posterior (AP) direction. These behaviours begin at the blastoderm margin and propagate in a gradient towards the animal pole. Later in gastrulation, cells undergo increasingly mediolateral-directed elongation and autonomous convergence movements towards the dorsal midline leading to an enormous extension of the neural axis. Around the equator and along the dorsal midline of the gastrula, persistent AP orientation of divisions suggests that a common mechanism may be involved but that neither oriented cell movements nor shape can account for this alignment. When the neural plate begins to differentiate, there is a gradual transition in the direction of cell division from AP to the mediolateral circumference (ML). ML divisions occur in both the ventral epidermis and dorsal neural plate. In the neural plate, ML becomes the predominant orientation of division during neural keel and nerve rod stages and, from late neural keel stage, divisions are concentrated at the dorsal midline and generate bilateral progeny (C. Papan and J. A. Campos-Ortega (1994) Roux's Arch. Dev. Biol. 203, 178-186). Coincidentally, cells on the ventral surface also orient their divisions in the ML direction, cleaving perpendicular to the direction in which they are elongated. The ML alignment of epidermal divisions is well correlated with cell shape but ML divisions within the neuroepithelium appear to be better correlated with changes in tissue morphology associated with neurulation.

Initial pH as a determining factor of glucose consumption and lactic and acetic acid production in oral streptococci.

Lactic and acetic acid production was evaluated from six strains of oral streptococci, viz Streptococcus gordonii, Streptococcus mutans, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguis and Streptococcus sobrinus cultured in the presence of 1, 5, 10 mM glucose and without glucose, at initial pH values of 5, 5.5, 6 and 7. S. sobrinus and S. salivarius caused the greatest decreases in pH. At pH values of 5 and 5.5, lactic acid and acetic acid production in the species tested was discordant with residual glucose levels. Acid production from protein was especially great in S. mutans and S. salivarius.

Conduction velocity groups in the retino-tectal and retino-thalamic visual pathways of the pigeon (Columbia livia).

The anatomical characteristics of the avian visual system are well known. However, there are wide gaps in our knowledge with respect to the physiological characteristics of their visual system. For example, we lack both an operational identification of the different ganglion cell types present in the retinae of birds, and a description of their presumptive differential central projections. The results presented here address this latter point by classifying the conduction velocity groups of fibers present in the optic tract of the pigeon. We report the existence of at least 5 groups of axons in the optic tract of the pigeon, with conduction velocities of 22-18 m/s, 12-10 m/s, 8 m/s, 6 m/s and less than 2.5 m/s. All five groups project to the tectum but only the four fastest groups project to the dorsal thalamic complex. The homologies with the populations of retinal axons found in cats are discussed.