Contribution of vasculature to stem integrity in Arabidopsis thaliana.

In plants, coordinated growth is important for organ mechanical integrity because cells remain contiguous through their walls. So far, defects in inflorescence stem integrity in Arabidopsis thaliana have mainly been related to epidermal defects. Although these observations suggest a growth-limiting function at the stem cortex, deeper layers of the stem could also contribute to stem integrity. The nac secondary cell wall thickening promoting factor1 (nst1) nst3 double-mutant background is characterized by weaker vascular bundles without cracks. By screening for the cracking phenotype in this background, we identified a regulator of stem cracking, the transcription factor INDETERMINATE DOMAIN9 (IDD9). Stem cracking was not caused by vascular bundle breakage in plants that expressed a dominant repressor version of IDD9. Instead, cracking emerged from increased cell expansion in non-lignified interfascicular fiber cells that stretched the epidermis. This phenotype could be enhanced through CLAVATA3-dependent cell proliferation. Collectively, our results demonstrate that stem integrity relies on three additive mechanical components: the epidermis, which resists inner cell growth; cell proliferation in inner tissues; and growth heterogeneity associated with vascular bundle distribution in deep tissues.

Modulation of NAC transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis.

In Arabidopsis, secondary cell walls (SCW) are formed in fiber cells and vessel cells in vascular tissue for providing plants with mechanical strength and channels for the long distance transportation of water and nutrients. NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) acts as a key gene for the initiation of SCW formation through a hierarchical transcription network. In this study, we report that NST activity is modulated by the NAC domain transcription factor XYLEM NAC DOMAIN1 (XND1) during plant growth. Using yeast two-hybrid screening and in vivo protein interaction analysis, XND1 was identified as an NST-interacting protein that modulates NST1 activity. XND1 and NST1 were co-localized in the nucleus and the interaction of XND1 with NST1 resulted in inhibition of NST1 transactivation activity. In the process of inflorescence growth, XND1 was expressed with a similar pattern to NST1. Up-regulation of XND1 in fiber cells repressed SCW formation. The study demonstrates that NST1 activity is modulated by XND1 in the regulation of secondary cell walls formation.

Visualization of adult fish lens fiber cells.

The crystalline lens of a vertebrate eye is a gradient-index lens and grows throughout life by addition of new lens fiber cells in the periphery. In fish, the growing ball-shaped lens maintains sophisticated optical properties throughout life by maintaining the distribution of refractive index relative to the increasing radius of the lens. During this process, the central fibers must increase refractive index by increasing the cytosolic concentration of crystallin proteins. However, only the youngest, most peripheral lens fiber cells have the ability to synthesize proteins. Unfortunately, the hardness of fish lenses makes investigation of the cellular anatomy impossible with traditional histological methods. We have developed a method for visualizing lens fiber cells across the diameter of the lens in adult fish. The method relies on sectioning embedded lenses with a high-speed power saw and observing the cut surface with a scanning electron microscope (SEM). The combination of SEM and image analysis allowed for precise tracking of the positions of individual cell fiber cells. As an application of the method, we present a cell thickness profile, i.e. the distribution of cells thicknesses and their relative positions along the lens's radius. Combined with detailed optical studies, which by mathematical reasons only are possible on ball-shaped lenses, our method can lead to new insights into the mechanism governing the functional and cellular development of vertebrate lenses.

BNIP3L/NIX is required for elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during eye lens organelle-free zone formation.

The formation and life-long growth of the ocular lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To achieve their mature structure and transparent function, newly formed lens fiber cells undergo a series of cellular remodeling events including the complete elimination of cellular organelles to form the lens organelle-free zone (OFZ). To date, the mechanisms and requirements for organelle elimination by lens fiber cells remain to be fully elucidated. In previous studies, we detected the presence of mitochondria contained within autophagolysosomes throughout human and chick lenses suggesting that proteins targeting mitochondria for degradation by mitophagy could be required for the elimination of mitochondria during OFZ formation. Consistently, high-throughput RNA sequencing of microdissected embryonic chick lenses revealed that expression of a protein that targets mitochondria for elimination during erythrocyte formation, called BCL2 interacting protein 3-like (BNIP3L/NIX), peaks in the region of lens where organelle elimination occurs. To examine the potential role for BNIP3L in the elimination of mitochondria during lens fiber cell remodeling, we analyzed the expression pattern of BNIP3L in newborn mouse lenses, the effect of its deletion on organelle elimination and its co-localization with lens organelles. We demonstrate that the expression pattern of BNIP3L in the mouse lens is consistent with it playing an important role in the elimination of mitochondria during lens fiber cell organelle elimination. Importantly, we demonstrate that deletion of BNIP3L results in retention of mitochondria during lens fiber cell remodeling, and, surprisingly, that deletion of BNIP3L also results in the retention of endoplasmic reticulum and Golgi apparatus but not nuclei. Finally, we show that BNIP3L localizes to the endoplasmic reticulum and Golgi apparatus of wild-type newborn mouse lenses and is contained within mitochondria, endoplasmic reticulum and Golgi apparatus isolated from adult mouse liver. These data identify BNIP3L as a novel requirement for the elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell remodeling and they suggest a novel function for BNIP3L in the regulation of endoplasmic reticulum and Golgi apparatus populations in the lens and non-lens tissues.

Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

MAIN CONCLUSION: Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.

Intrinsic and extrinsic regulatory mechanisms are required to form and maintain a lens of the correct size and shape.

Understanding how tissues and organs acquire and maintain an appropriate size and shape remains one of the most challenging areas in developmental biology. The eye lens represents an excellent system to provide insights into regulatory mechanisms because in addition to its relative simplicity in cellular composition (being made up of only two forms of cells, epithelial and fiber cells), these cells must become organized to generate the precise spheroidal arrangement that delivers normal lens function. Epithelial and fiber cells also represent spatially distinct proliferation and differentiation compartments, respectively, and an ongoing balance between these domains must be tightly regulated so that the lens achieves and maintains appropriate dimensions during growth and ageing. Recent research indicates that reciprocal inductive interactions mediated by Wnt-Frizzled and Notch-Jagged signaling pathways are important for maintaining and organizing these compartments. The Hippo-Yap pathway has also been implicated in maintaining the epithelial progenitor compartment and regulating growth processes. Thus, whilst some molecules and mechanisms have been identified, further work in this important area is needed to provide a clearer understanding of how lens size and shape is regulated.

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism.

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.

The water permeability of lens aquaporin-0 depends on its lipid bilayer environment.

Aquaporin-0 (AQP0), the primary water channel in lens fiber cells, is critical to lens development, organization, and function. In the avascular lens there is thought to be an internal microcirculation associated with fluid movement. Although AQP0 is known to be important in fluid fluxes across membranes, the water permeability of this channel has only been measured in Xenopus oocytes and in outer lens cortical membranes, but not in inner nuclear membranes, which have an increased cholesterol/phospholipid ratio. Here we measure the unit water permeability of AQP0 in different proteoliposomes with cholesterol/phospholipid ratios and external pHs similar to those found in the cortex and nucleus of the lens. Osmotic stress measurements were performed with proteoliposomes containing AQP0 and three different lipids mixtures: (1) phosphatidylcholine (PC) and phosphatidylglycerol (PG), (2) PC, PG, with 40 mol% cholesterol, and (3) sphingomyelin (SM), PG, with 40 mol% cholesterol. At pH 7.5 the unit permeabilities of AQP0 were 3.5 ± 0.5 × 10(-14) cm(3)/s (mean ± SEM), 1.1 ± 0.1 × 10(-14) cm(3)/s, and 0.50 ± 0.04 × 10(-14) cm(3)/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. For lipid mixtures at pH 6.5, corresponding to conditions found in the lens nucleus, the AQP0 permeabilities were 1.5 ± 0.4 × 10(-14) cm(3)/s and 0.76 ± 0.03 × 10(-14) cm(3)/s in PC:PG:cholesterol and SM:PG:cholesterol, respectively. Thus, although AQP0 unit permeability can be modified by changes in pH, it is also sensitive to changes in bilayer lipid composition, and decreases with increasing cholesterol and SM content. These data imply that AQP0 water permeability is regulated by bilayer lipid composition, so that AQP0 permeability would be significantly less in the lens nucleus than in the lens cortex.

Expanding of Life Strategies in Placozoa: Insights From Long-Term Culturing of Trichoplax and Hoilungia.

Placozoans are essential reference species for understanding the origins and evolution of animal organization. However, little is known about their life strategies in natural habitats. Here, by maintaining long-term culturing for four species of Trichoplax and Hoilungia, we extend our knowledge about feeding and reproductive adaptations relevant to the diversity of life forms and immune mechanisms. Three modes of population dynamics depended upon feeding sources, including induction of social behaviors, morphogenesis, and reproductive strategies. In addition to fission, representatives of all species produced "swarmers" (a separate vegetative reproduction stage), which could also be formed from the lower epithelium with greater cell-type diversity. We monitored the formation of specialized spheroid structures from the upper cell layer in aging culture. These "spheres" could be transformed into juvenile animals under favorable conditions. We hypothesize that spheroid structures represent a component of the innate immune defense response with the involvement of fiber cells. Finally, we showed that regeneration could be a part of the adaptive reproductive strategies in placozoans and a unique experimental model for regenerative biology.

The role of FYCO1-dependent autophagy in lens fiber cell differentiation.

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1-/-) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1-/- mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1-/- mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type.

Advances about the Roles of Membranes in Cotton Fiber Development.

Cotton fiber is an extremely elongated single cell derived from the ovule epidermis and is an ideal model for studying cell development. The plasma membrane is tremendously expanded and accompanied by the coordination of various physiological and biochemical activities on the membrane, one of the three major systems of a eukaryotic cell. This review compiles the recent progress and advances for the roles of the membrane in cotton fiber development: the functions of membrane lipids, especially the fatty acids, sphingolipids, and phytosterols; membrane channels, including aquaporins, the ATP-binding cassette (ABC) transporters, vacuolar invertase, and plasmodesmata; and the regulation mechanism of membrane proteins, such as membrane binding enzymes, annexins, and receptor-like kinases.

Constant lens fiber cell thickness in fish suggests crystallin transport to denucleated cells.

The crystalline lens of the vertebrate eye grows throughout life. This growth may be enormous in fish, while the lens must be functional from larva to adult. During growth, the fiber cells of the lens must increase the concentration of specific proteins (crystallins) in the cytoplasm to increase refractive index. However, the bulk of the fiber cells in a vertebrate lens are denucleated and have no organelles to synthesize proteins. To study how this problem is solved, we first measured lens fiber cell thickness in the Nile tilapia, a teleost fish. In the lenses from 25 fish, in two size groups, fibers were considerably thinner than in other vertebrates. Fiber thickness was about constant along the radius of the lens and the same between the size groups. Since our results provided no evidence for shrinkage of lens fiber cells with growth (expected if protein concentration is increased by expelling water) we included eight additional teleost species to elucidate the mechanism by which the cells increase crystallin concentration. In all species, fiber cell thickness was about constant throughout the lens, with species-specific values. The changes in fiber cell thickness expected from an increase in crystallin concentration by removal of water were modeled. Shrinkage in cell thickness by up to 66% would have been necessary to reach the required crystallin concentration. We conclude that crystallin concentration in denucleated lens fiber cells is increased by transport of proteins from synthetically competent cells in the periphery of the lens.

Identification of active signaling pathways by integrating gene expression and protein interaction data.

BACKGROUND: Signaling pathways are the key biological mechanisms that transduce extracellular signals to affect transcription factor mediated gene regulation within cells. A number of computational methods have been developed to identify the topological structure of a specific signaling pathway using protein-protein interaction data, but they are not designed for identifying active signaling pathways in an unbiased manner. On the other hand, there are statistical methods based on gene sets or pathway data that can prioritize likely active signaling pathways, but they do not make full use of active pathway structure that link receptor, kinases and downstream transcription factors. RESULTS: Here, we present a method to simultaneously predict the set of active signaling pathways, together with their pathway structure, by integrating protein-protein interaction network and gene expression data. We evaluated the capacity for our method to predict active signaling pathways for dental epithelial cells, ocular lens epithelial cells, human pluripotent stem cell-derived lens epithelial cells, and lens fiber cells. This analysis showed our approach could identify all the known active pathways that are associated with tooth formation and lens development. CONCLUSIONS: The results suggest that SPAGI can be a useful approach to identify the potential active signaling pathways given a gene expression profile. Our method is implemented as an open source R package, available via https://github.com/VCCRI/SPAGI/ .

Chicken GRIFIN: A homodimeric member of the galectin network with canonical properties and a unique expression profile.

Occurrence of the adhesion/growth-regulatory galectins as family sets the challenge to achieve a complete network analysis. Along this route taken for a well-suited model organism (chicken), we fill the remaining gap to characterize its seventh member known from rat as galectin-related inter-fiber protein (GRIFIN) in the lens. Its single-copy gene is common to vertebrates, with one or more deviations from the so-called signature sequence for ligand (lactose) contact. The chicken protein is a homodimeric agglutinin with capacity to bind ß-galactosides, especially the histo-blood group B tetrasaccharide, shown by solid-phase/cell assays and a glycan microarray. Mass spectrometric identification of two lactose-binding peptides after tryptic on-bead fragmentation suggests an interaction at the canonical region despite a sequence change from Arg to Val at the site, which impairs reactivity of human galectin-1. RT-PCR and Western blot analyses of specimen from adult chicken organs reveal restriction of expression to the lens, here immunohistochemically throughout its main body. This report sets the stage for detailed structure-activity studies to define factors relevant for affinity beyond the signature sequence and to perform the first complete network analysis of the galectin family in developing and adult organs of a vertebrate.

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation.

In humans, loss of TBC1D20 (TBC1 domain family, member 20) protein function causes Warburg Micro syndrome 4 (WARBM4), an autosomal recessive disorder characterized by congenital eye, brain, and genital abnormalities. TBC1D20-deficient mice exhibit ocular abnormalities and male infertility. TBC1D20 is a ubiquitously expressed member of the family of GTPase-activating proteins (GAPs) that increase the intrinsically slow GTP-hydrolysis rate of small RAB-GTPases when bound to GTP. Biochemical studies have established TBC1D20 as a GAP for RAB1B and RAB2A. However, the cellular role of TBC1D20 still remains elusive, and there is little information about how the functional loss of TBC1D20 causes clinical manifestations in WARBM4-affected children. Here we evaluate the role of TBC1D20 in cells carrying a null mutant allele, as well as TBC1D20-deficient mice, which display eye and testicular abnormalities. We demonstrate that TBC1D20, via its RAB1B GAP function, is a key regulator of autophagosome maturation, a process required for maintenance of autophagic flux and degradation of autophagic cargo. Our results provide evidence that TBC1D20-mediated autophagosome maturation maintains lens transparency by mediating the removal of damaged proteins and organelles from lens fiber cells. Additionally, our results show that in the testes TBC1D20-mediated maturation of autophagosomes is required for autophagic flux, but is also required for the formation of acrosomes. Furthermore TBC1D20-deficient mice, while not mimicking severe developmental brain abnormalities identified in WARBM4 affected children, display disrupted neuronal autophagic flux resulting in adult-onset motor dysfunction. In summary, we show that TBC1D20 has an essential role in the maturation of autophagosomes and a defect in TBC1D20 function results in eye, testicular, and neuronal abnormalities in mice implicating disrupted autophagy as a mechanism that contributes to WARBM4 pathogenesis.

Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells.

ß1-Integrin is a heterodimeric transmembrane protein that has roles in both cell-extra-cellular matrix and cell-cell interactions. Conditional deletion of ß1-integrin from all lens cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional approach was used to delete ß1-integrin solely from the lens fiber cells. This deletion resulted in two distinct phenotypes with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of "clear" conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with the loss of the F-actin network. Physiological measurements found that the lens fiber cells had a twofold increase in gap junctional coupling, perhaps due to differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In summary, ß1-integrin plays a role in maintaining the cellular morphology and homeostasis of the lens fiber cells.