Structural characterization and statistical-mechanical model of epidermal patterns

In proliferating epithelia of mammalian skin, cells of irregular polygonal-like shapes pack into complex nearly flat two-dimensional structures that are pliable to deformations. In this work, we employ various sensitive correlation functions to quantitatively characterize structural features of evolving packings of epithelial cells across length scales in mouse skin. We find that the pair statistics in direct and Fourier spaces of the cell centroids in the early stages of embryonic development show structural directional dependence, while in the late stages the patterns tend towards statistically isotropic states. We construct a minimalist four-component statistical-mechanical model involving effective isotropic pair interactions consisting of hard-core repulsion and extra short-ranged soft-core repulsion beyond the hard core, whose length scale is roughly the same as the hard core. The model parameters are optimized to match the sample pair statistics in both direct and Fourier spaces. By doing this, the parameters are biologically constrained. Our model predicts essentially the same polygonal shape distribution and size disparity of cells found in experiments as measured by Voronoi statistics. Moreover, our simulated equilibrium liquid-like configurations are able to match other nontrivial unconstrained statistics, which is a testament to the power and novelty of the model. We discuss ways in which our model might be extended so as to better understand morphogenesis (in particular the emergence of planar cell polarity), wound-healing, and disease progression processes in skin, and how it could be applied to the design of synthetic tissues

Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains

Multi-omic profiling reveals the ataxia protein sacsin is required for integrin trafficking and synaptic organization

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia caused by mutations in SACS, which encodes the protein sacsin. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament disorganization, and progressive death of cerebellar Purkinje neurons. It is unclear why the loss of sacsin causes these deficits or why they manifest as cerebellar ataxia. Here, we perform multi-omic profiling in sacsin knockout (KO) cells and identify alterations in microtubule dynamics and mislocalization of focal adhesion (FA) proteins, including multiple integrins. Deficits in FA structure, signaling, and function can be rescued by targeting PTEN, a negative regulator of FA signaling. ARSACS mice possess mislocalization of ITGA1 in Purkinje neurons and synaptic disorganization in the deep cerebellar nucleus (DCN). The sacsin interactome reveals that sacsin regulates interactions between cytoskeletal and synaptic adhesion proteins. Our findings suggest that disrupted trafficking of synaptic adhesion proteins is a causal molecular deficit in ARSACS

Deterministic diffusion fiber tracking improved by quantitative anisotropy

Diffusion MRI tractography has emerged as a useful and popular tool for mapping connections between brain regions. In this study, we examined the performance of quantitative anisotropy (QA) in facilitating deterministic fiber tracking. Two phantom studies were conducted. The first phantom study examined the susceptibility of fractional anisotropy (FA), generalized factional anisotropy (GFA), and QA to various partial volume effects. The second phantom study examined the spatial resolution of the FA-aided, GFA-aided, and QA-aided tractographies. An in vivo study was conducted to track the arcuate fasciculus, and two neurosurgeons blind to the acquisition and analysis settings were invited to identify false tracks. The performance of QA in assisting fiber tracking was compared with FA, GFA, and anatomical information from T 1-weighted images. Our first phantom study showed that QA is less sensitive to the partial volume effects of crossing fibers and free water, suggesting that it is a robust index. The second phantom study showed that the QA-aided tractography has better resolution than the FA-aided and GFA-aided tractography. Our in vivo study further showed that the QA-aided tractography outperforms the FA-aided, GFA-aided, and anatomy-aided tractographies. In the shell scheme (HARDI), the FA-aided, GFA-aided, and anatomy-aided tractographies have 30.7%, 32.6%, and 24.45% of the false tracks, respectively, while the QA-aided tractography has 16.2%. In the grid scheme (DSI), the FA-aided, GFA-aided, and anatomy-aided tractographies have 12.3%, 9.0%, and 10.93% of the false tracks, respectively, while the QA-aided tractography has 4.43%. The QA-aided deterministic fiber tracking may assist fiber tracking studies and facilitate the advancement of human connectomics. © 2013 Yeh et al

On the Long-range Coordination of Epidermal Planar Cell Polarity

Planar cell polarity (PCP) refers to the collective alignment of cells or groups of cells within the epithelial plane. The establishment of PCP requires an asymmetric distribution of core PCP complexes within each cell across the tissue plane, and this asymmetry functions to inform downstream polarized cellular behaviors such as directed cell migration, unidirectional cilia beating, and oriented cell divisions. While significant progress has been made towards understanding the mechanisms controlling the asymmetric molecular interactions between core PCP complexes, less is known about the global inputs that direct and align this asymmetric protein localization patterns over long distances, especially in vertebrate systems. In this thesis, we explore the mechanisms governing the asymmetric distribution of a core PCP protein, Celsr1 in mammalian skin. We demonstrate that the onset of Celsr1 asymmetry coincides both temporally and spatially with a gradient of tissue deformation oriented along the medial-lateral axis across the back skin. We present evidence that uniaxial tissue strain can act as a long-range polarizing cue for reorienting Celsr1 polarity. Observations both in vivo and in vitro suggest that the effect of tissue anisotropy on Celsr1 polarity is not a direct consequence of cell shape but rather reflects the restructuring of cell-cell interfaces during oriented cell divisions and cell rearrangements that serve to relax tissue strain. Using a clonal analysis approach, we demonstrate that cell intercalations remodel intercellular junctions predominantly between the mediolateral interfaces of neighboring cells. This restructuring of the cell surface polarizes Celsr1, which is slow to accumulate at nascent junctions yet stably associates with persistent junctions. Together, these results demonstrate that tissue anisotropy globally aligns Celsr1 polarity by creating a directional bias in the formation of new cell interfaces while simultaneously restricting Celsr1 along persistent anterior-posterior interfaces

Planar cell polarity: global inputs establishing cellular asymmetry

Many tissues develop coordinated patterns of cell polarity that align with respect to the tissue axes. This phenomenon refers to planar cell polarity (PCP) and is controlled by multiple conserved PCP modules. A key feature of PCP proteins is their asymmetric localization within the tissue plane, whose orientation is guided by global directional cues. Here, we highlight current models and recent findings on the role of tissue-level gradients, local organizer signals, and mechanical forces in establishing the global patterns of PCP

Natural and unanticipated modifiers of RNAi activity in Caenorhabditis elegans.

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains

Analysis of RDE-4 maternal effects.

<p>RDE-4 maternal effects are fully penetrant and expressive. Progeny animals from crosses depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050191#pone-0050191-g004" target="_blank">Figure 4A</a> were scored in each generation for RNAi activity (<i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050191#s2" target="_blank">Materials and Methods</a>). Results from three different crosses are depicted. Three experiments were conducted at 22°C. The maternal effects were not altered by growth temperature. <i>rde-4/+</i> F1 heterozygotes displayed wild-type RNAi activity, as expected. All of the F2 animals, including <i>rde-4/rde-4</i> homozygotes, displayed wild-type RNAi activity due to RDE-4 maternal effects. Some of the F2 progeny were chosen at random, isolated, allowed to produce F3 progeny, and subsequently genotyped by DNA sequencing. Those F2 progeny that were homozygous for <i>rde-4</i> produced F3 progeny that were RNAi- at 100% penetrance and expressivity; those F2 progeny that were heterozygous or wild type produced F3 progeny that were RNAi+ at 100% penetrance and expressivity, which is in keeping with a strong RDE-4 maternal effect. The ratio of wild-type to homozygous <i>rde-4</i> F2 animals was approximately 3∶1, as predicted, and this is reflected in the RNAi activity of the F3s as well. Similar results were observed for both <i>rde-4(ne299)</i> and <i>rde-4(ne309)</i> alleles.</p

RNAi activity in <i>rde-1(yy11)</i> and <i>haf-6(ne335)</i> mutants.

<p>The <i>yy11</i> allele, present as a background mutation in a <i>haf-6(ne335)</i> transgenic stock, was genetically dissected from the <i>haf-6(ne335)</i> mutation and analyzed separately for RNAi activity. (<b>A</b>) <i>yy11</i> provides resistance to transgene-delivered dsRNAs, as evidenced by lack of gene silencing of the GFP reporter in homozygous animals. By contrast, <i>haf-6(ne335)</i> single mutants display wild-type RNAi activity in this transgene assay. (<b>B</b>). Animals homozygous for the <i>yy11</i> allele are RNAi resistant when dsRNAs are delivered by ingestion of bacteria that express dsRNAs. Thus, the <i>rde-1(yy11)</i> mutants displayed strong RNAi phenotypes irrespective of delivery method (<b>A</b>, <b>B</b>), while, as expected, the <i>haf-6(ne335)</i> single mutant was RNAi defective by ingestion only (<b>A, B</b>). Bacteria expressing dsRNA targeting the <i>pop-1</i> and <i>dpy-11</i> genes were used in feeding experiments. (<b>C</b>) Genetic and molecular analysis revealed the nature of the <i>yy11</i> mutation—a transposon insertion in exon 9 of <i>rde-1.</i> DNA sequences flanking the insertion site are indicated.</p

Analysis of RDE-4 maternal effects.

<p>RDE-4 maternal effects are not affected by temperature. Four separate experiments, performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050191#pone-0050191-t001" target="_blank">Table 1</a>, were conducted at 15°C using <i>rde-4(ne309).</i> Similar results were obtained using <i>rde-4(ne299)</i> alleles.</p