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During vertebrate somitogenesis an inherent segmentation clock coordinates the spatiotemporal signaling to generate segmented structures that pattern the body axis. Using our experimental and quantitative approach, we study the cell movements and the genetic oscillations of her1 expression level at single-cell resolution simultaneously and scale up to the entire pre-somitic mesoderm (PSM) tissue. From the experimentally determined phases of PSM cellular oscillators, we deduced an in vivo frequency profile gradient along the anterior-posterior PSM axis and inferred precise mathematical relations between spatial cell-level period and tissue-level somitogenesis period. We also confirmed a gradient in the relative velocities of cellular oscillators along the axis. The phase order parameter within an ensemble of oscillators revealed the degree of synchronization in the tailbud and the posterior PSM being only partial, whereas synchronization can be almost complete in the presumptive somite region but with temporal oscillations. Collectively, the degree of synchronization itself, possibly regulated by cell movement and the synchronized temporal phase of the transiently expressed clock protein Her1, can be an additional control mechanism for making precise somite boundaries.

Microridges are evolutionarily conserved actin-rich protrusions present on the apical surface of the squamous epithelial cells. In zebrafish epidermal cells, microridges form self-evolving patterns due to the underlying actomyosin network dynamics. However, their morphological and dynamic characteristics have remained poorly understood owing to lack of automated segmentation methods. We achieved ~97% pixel-level accuracy with the deep learning microridge segmentation strategy enabling quantitative insights into their bio-physical-mechanical characteristics. From the segmented images, we estimated an effective microridge persistence length as ~0.61μm. We discovered the presence of mechanical fluctuations and found relatively greater stresses stored within patterns of yolk than flank, indicating distinct regulation of their actomyosin networks. Furthermore, spontaneous formations and positional fluctuations of actin clusters within microridge influenced pattern rearrangements over short length/time-scales. Our framework allows large-scale spatiotemporal analysis of microridges during epithelial development and probing of their responses to chemical and genetic perturbations to unravel the underlying patterning mechanisms.

We study spatial correlations between oscillator phases in the steady state of a Kuramoto model, in which phase oscillators that are randomly distributed in space interact with constant strength but within a limited range. Such a model could be relevant, for example, in the synchronization of gene expression oscillations in cells, where only oscillations of neighboring cells are coupled through cell-cell contacts. We analytically infer spatial phase-phase correlation functions from the known steady-state distribution of oscillators for the case of homogenous frequencies and show that these can contain information about the range and strength of interactions, provided the noise in the system can be estimated. We suggest a method for the latter, and also explore when correlations appear to be ergodic in this model, which would enable an experimental measurement of correlation functions to utilize temporal averages. Simulations show that our techniques also give qualitative results for the model with heterogenous frequencies. We illustrate our results by comparison with experimental data on genetic oscillations in the segmentation clock of zebrafish embryos.

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that microridges are formed of F-actin networks and require the function of the Arp2/3 complex for their maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, grow laterally long, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that a microridge is formed of a network of F-actin and requires the function of the Arp2/3 complex for its maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

The I-conotoxin superfamily (I-Ctx) is known to have four disulfide bonds with the cysteine arrangement C-C-CC-CC-C-C, and the members inhibit or modify ion channels of nerve cells. Recently, Olivera and co-workers (FEBS J. 2005; 272: 4178–4188) have suggested that the previously described I-Ctx should now be divided into two different gene superfamilies, namely, I1 and I2, in view of their having two different types of signal peptides and exhibiting distinct functions. We have revisited the 28 entries presently grouped as I-Ctx in UniProt Swiss-Prot knowledgebase, and on the basis of in silico analysis have divided them into I1 and I2 superfamilies. The sequence analysis has provided a framework for in silico annotation enabling us to carry out computer-based functional characterization of the UniProtKB/TrEMBL entry Q59AA4 from Conus miles and to predict it as a member of the I2 superfamily. Furthermore, we have predicted the mature toxin of this entry and have proposed that it may be an inhibitor of voltage-gated potassium channels. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.

The first author’s name appears incorrectly in the published article. The correct name is: Rajasekaran Bhavna. The correct citation is: Bhavna R, Uriu K, Valentin G, Tinevez J-Y, Oates AC (2016) Object Segmentation and Ground Truth in 3D Embryonic Imaging. PLoS ONE 11(6): e0150853. doi:10.1371/journal.pone.0150853

During vertebrate embryo development, the body axis is subdivided into repeated structures, called somites. Somites bud off from an un-segmented tissue called the pre-somitic mesoderm (PSM) in a sequential and periodic manner, tightly controlled by an in built molecular clock, called the "segmentation clock". According to current understanding, the clock is comprised of: (i) an autonomous cellular oscillator consisting of an intracellular negative feedback loop of Her genes within the PSM cells, (ii) Delta-ligand and Notch-receptor coupling that facilitates synchronization of oscillators among the PSM cells, (iii) Tissue level waves of gene expression that emerge in the posterior PSM and move coherently towards anterior, leading to global arrest of oscillations in the form of somites. However, the movement of cellular oscillators within the PSM before the formation of somitic furrows, a prominent feature of the tissue as observed through this work has not been experimentally considered as a constituent of the segmentation clock so far. Our work aims to incorporate movement of cellular oscillators in the framework of the segmentation clock. It is well known from theoretical studies that the characteristics of relative motion of oscillators affect their synchronization properties and the patterns of oscillations they form. Particularly, theoretical studies by Uriu et al., PNAS (2010) suggest that cell movements promotes synchronization of genetic oscillations. Here, we established experimental techniques and image analysis tools to attain quantitative insight on (i) diffusion co-efficient of cellular oscillators, (ii) dynamics of a population of oscillators, within the PSM aiming towards concomitant understanding of the relationship between movement and synchronization of cellular oscillators. In order to quantitatively relate cellular oscillators and their motion within the PSM, I established imaging techniques that enabled visualization of fluorescenctly labeled nuclei as readouts of cell positions in live embryo, and developed dedicated segmentation algorithm and implemented tracking protocol to obtain nuclei positions over time in 3D space. Furthermore, I provide benchmarking techniques in the form of artificial data that validate segmentation algorithm efficacy and, for the first time proposed the use of transgenic embryo chimeras to validate segmentation algorithm performance within the context of in vivo live imaging of embryonic tissues. Preliminary analysis of our data suggests that there is relatively high cell mixing in the posterior PSM, within the same spatial zone where synchronous oscillations emerge at maximum speed. Also, there are indications of gradient of cell mixing along the anterior-posterior axis of the embryo. By sampling single cell tracks with the help of nuclei markers, we have also been able to follow in vivo protein oscillations at single cell resolution that would allow quantitative characterization of coherence among a population of cellular oscillators over time. Our image analysis work flow allows testing of mutant embryos and perturbation of synchrony dynamics to understand the cause-effect relationship between movement and synchronization properties at cellular resolution. Essentially, through this work, we hope to bridge the time scales of events and cellular level dynamics that leads to highly coordinated tissue level patterns and thereby further our understanding of the segmentation clock mechanism.

A novel conotoxin pl14a containing 25 amino acid residues with an amidated C-terminus from vermivorous cone snail, Conus planorbis belongs to J-conotoxin superfamily and this is the first conotoxin, which inhibits both nicotinic acetylcholine receptor subtypes and Kv1.6 channel. We have attempted through bioinformatics approaches to elucidate the extent of specificity of pl14a towards Kv1 channel subtypes (Kv1.1-Kv1.6). Our work provides rationale for the relatively high specificity and binding mode of pl14a to Kv1.6 channel. The pl14a peptide contains two types of structural elements, namely the putative dyad (Lys18 and Tyr19) and basic residue ring constituted of arginine residues. We have carried out in silico docking studies so as to assess the contribution of one or combination of both structural elements of pl14a in blocking of Kv1.6 channel. For this purpose, we have built by homology modelling, the theoretical 3D structure of Kv1.6 channel based on the available crystal structure of mammalian shaker Kv1.2 channel. Docking studies suggest that positively charged residues ring may be involved in the blocking mechanism of Kv1.6 channel. The models suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kappaM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. The newly detected pharmacophore for pl14a interacting with Kv1.6 channel provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively-charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profiles against subtypes of the potassium channels for these conopeptides.