Best practices for the execution, analysis, and data storage of plant single-cell/nucleus transcriptomics.

Single-cell and single-nucleus RNA-sequencing technologies capture the expression of plant genes at an unprecedented resolution. Therefore, these technologies are gaining traction in plant molecular and developmental biology for elucidating the transcriptional changes across cell types in a specific tissue or organ, upon treatments, in response to biotic and abiotic stresses, or between genotypes. Despite the rapidly accelerating use of these technologies, collective and standardized experimental and analytical procedures to support the acquisition of high-quality data sets are still missing. In this commentary, we discuss common challenges associated with the use of single-cell transcriptomics in plants and propose general guidelines to improve reproducibility, quality, comparability, and interpretation and to make the data readily available to the community in this fast-developing field of research.

Analysis of Xylem Cells by Nucleus-Based Transcriptomics and Chromatin Profiling.

Nuclei contain essential information for cell states, including chromatin and RNA profiles - features which are nowadays accessible using high-throughput sequencing applications. Here, we describe analytical pipelines including nucleus isolation from differentiated xylem tissues by fluorescence-activated nucleus sorting (FANS), as well as subsequent SMART-seq2-based transcriptome profiling and assay for transposase-accessible chromatin (ATAC)-seq-based chromatin analysis. Combined with tissue-specific expression of nuclear fluorescent reporters, these pipelines allow obtaining tissue-specific data on gene expression and on chromatin structure and are applicable for a large spectrum of cell types, tissues, and organs. Considering, however, the extreme degree of differentiation found in xylem cells with programmed cell death happening during vessel element formation and their role as a long-term depository for atmospheric CO2 in the form of wood, xylem cells represent intriguing and relevant objects for large-scale profilings of their cellular signatures.

OBERON3 and SUPPRESSOR OF MAX2 1-LIKE proteins form a regulatory module driving phloem development.

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.

Has China's Young Thousand Talents program been successful in recruiting and nurturing top-caliber scientists?

In this study, we examined China's Young Thousand Talents (YTT) program and evaluated its effectiveness in recruiting elite expatriate scientists and in nurturing the returnee scientists' productivity. We find that YTT scientists are generally of high caliber in research but, as a group, fall below the top category in pre-return productivity. We further find that YTT scientists are associated with a post-return publication gain across journal-quality tiers. However, this gain mainly takes place in last-authored publications and for high-caliber (albeit not top-caliber) recruits and can be explained by YTT scientists' access to greater funding and larger research teams. This paper has policy implications for the mobility of scientific talent, especially as early-career scientists face growing challenges in accessing research funding in the United States and European Union.

Application of physical training in conjunction with rehabilitation in male soccer injury / Aplicação de treinamento físico em conjunto à reabilitação de lesões no futebol masculino / Aplicación de entrenamiento físico junto con la rehabilitación de lesiones en el fútbol masculino

ABSTRACT Introduction: Soccer is one of the sports with the highest incidence of injuries, generated both by the high performance required on the field and by physical conflicts between players. A fast rehabilitation is essential for the player's performance. It has been empirically observed that an early recovery in patients' rehabilitation is associated with physical training compared to players who received only the traditional rehabilitation. Objective: Verify the physical training influence on rehabilitation sports injuries in male soccer players. Method: 180 cases of male adolescents with sports injuries admitted to a Taiyuan hospital were selected. A division into two groups was randomly computerized to avoid statistical differences in the intensity of the injuries. The control group (14.3±2.45 years old) was treated with the standard protocol, while the experimental group (15.3±2.95 years old) received an intervention with physical training. Interviews and questionnaires were conducted involving analysis of time and severity of the injury, laterality, and location. The control group received treatment based on this information. The recovery rate and intervention satisfaction rate were collected. SPSS22.0 Statistical Software was used for student's t-test and chi-square test. Results: Treatment efficiency was 82 (91.11%) in the control group versus 88 (97.78%) in the experimental group. The degree of dissatisfaction was 1 (2.11%) versus 8 (10%). The perceived overall satisfaction was 80 (89%) versus 87 (96.67%), (p <0.05). Conclusion: Rehabilitation associated with physical training intervention improved satisfaction and treatment efficiency. Evidence Level II; Therapeutic Studies - Investigating the results.

RESUMO Introdução: O futebol é um dos esportes com maior incidência de lesões, geradas tanto pelo alto desempenho exigido em campo quanto por conflitos físicos entre jogadores. Uma breve reabilitação é essencial para o rendimento do jogador. Percebe-se, de forma empírica, uma alta precoce em pacientes submetidos ao treinamento físico associado, quando comparados aos jogadores destinados à reabilitação tradicional. Objetivo: Verificar a influência do treinamento físico na reabilitação de lesões esportivas nos jogadores de futebol masculino. Método: Foram selecionados 180 casos de adolescentes masculinos com lesões esportivas internados neste hospital. A divisão em dois grupos foi efetuada aleatoriamente de forma computadorizada afim de evitar diferenças estatísticas na intensidade das lesões. O grupo controle (idade média 14,3±2,45 anos) foi tratado com o protocolo padrão enquanto ao experimental (idade média 15,3±2,95 anos) foi adicionada uma intervenção com treinamento físico. Foram efetuadas entrevistas e questionários envolvendo análise de tempo e gravidade da lesão, lateralidade e localização. O grupo controle recebeu o tratamento norteado nessas informações. Foram coletados a taxa de recuperação e a taxa de satisfação de intervenção. Foi utilizado o Software Estatístico SPSS22.0 para os testes t de student e qui-quadrado. Resultados: A eficiência do tratamento foi de 82 (91,11%) no grupo controle contra 88 (97,78%) no grupo experimental. O grau de insatisfação foi de 1 (2,11%) contra 8 (10%). A percepção de satisfação total foi de 80 (89%) contra 87 (96,67%), (p <0,05). Conclusão: A reabilitação associada à intervenção de treinamento físico demonstrou melhorar a taxa de satisfação e a eficiência do tratamento. Nível de evidência II; Estudos Terapêuticos - Investigação de Resultados.

RESUMEN Introducción: El fútbol es uno de los deportes con mayor incidencia de lesiones, generadas tanto por el alto rendimiento requerido en el campo como por los conflictos físicos entre los jugadores. Una pronta rehabilitación es esencial para el rendimiento del jugador. Se ha observado empíricamente que una recuperación temprana en la rehabilitación de los pacientes se asocia con la adición del entrenamiento físico en comparación con los jugadores que sólo recibieron la rehabilitación tradicional. Objetivo: Verificar la influencia del entrenamiento físico en la rehabilitación de lesiones deportivas en jugadores de fútbol masculinos. Métodos: Se seleccionaron 180 casos de adolescentes masculinos con lesiones deportivas ingresados en un hospital de Taiyuan. Se realizó una división en dos grupos de forma aleatoria para evitar diferencias estadísticas en la intensidad de las lesiones. El grupo de control (14,3±2,45 años) fue tratado con el protocolo estándar, mientras que el grupo experimental (15,3±2,95 años) recibió una intervención con entrenamiento físico. Se realizaron entrevistas y cuestionarios que incluían el análisis del tiempo y la gravedad de la lesión, la lateralidad y la localización. El grupo de control recibió un tratamiento basado en esta información. Se recogió el índice de recuperación y el índice de satisfacción de la intervención. Se utilizó el software estadístico SPSS22.0 para la prueba t de student y la prueba chi-cuadrado. Resultados: La eficacia del tratamiento fue de 82 (91,11%) en el grupo de control frente a 88 (97,78%) en el grupo experimental. El grado de insatisfacción fue de 1 (2,11%) frente a 8 (10%). La satisfacción global percibida fue de 80 (89%) frente a 87 (96,67%), (p <0,05). Conclusión: La rehabilitación asociada a la intervención de entrenamiento físico mejoró la satisfacción y la eficacia del tratamiento. Nivel de evidencia II; Estudios terapéuticos - Investigación de resultados.

Cell Fate Decisions Within the Vascular Cambium-Initiating Wood and Bast Formation.

Precise coordination of cell fate decisions is a hallmark of multicellular organisms. Especially in tissues with non-stereotypic anatomies, dynamic communication between developing cells is vital for ensuring functional tissue organization. Radial plant growth is driven by a plant stem cell niche known as vascular cambium, usually strictly producing secondary xylem (wood) inward and secondary phloem (bast) outward, two important structures serving as much-needed CO2 depositories and building materials. Because of its bidirectional nature and its developmental plasticity, the vascular cambium serves as an instructive paradigm for investigating principles of tissue patterning. Although genes and hormones involved in xylem and phloem formation have been identified, we have a yet incomplete picture of the initial steps of cell fate transitions of stem cell daughters into xylem and phloem progenitors. In this mini-review perspective, we describe two possible scenarios of cell fate decisions based on the current knowledge about gene regulatory networks and how cellular environments are established. In addition, we point out further possible research directions.

Control of cambium initiation and activity in Arabidopsis by the transcriptional regulator AHL15.

Plant secondary growth, which is the basis of wood formation, includes the production of secondary xylem, which is derived from meristematic cambium cells embedded in vascular tissue. Here, we identified an important role for the Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) transcriptional regulator in controlling vascular cambium activity. The limited secondary xylem development in inflorescence stems of herbaceous Arabidopsis plants was significantly reduced in ahl15 loss-of-function mutants, whereas constitutive or vascular meristem-specific AHL15 overexpression produced woody inflorescence stems. AHL15 was required for enhanced secondary xylem formation in the woody suppressor of overexpression of constans 1 (soc1) fruitfull (ful) double loss-of-function mutant. Moreover, we found that AHL15 induces vascular cambium activity downstream of the repressing SOC1 and FUL transcription factors, most likely similar to how it enhances lateral branching by promoting biosynthesis of the hormone cytokinin. Our results uncover a novel pathway driving cambium development, in which AHL15 expression levels act in parallel to and are dependent on the well-established TDIF-PXY-WOX pathway to differentiate between herbaceous and woody stem growth.

Strigo-D2-a bio-sensor for monitoring spatio-temporal strigolactone signaling patterns in intact plants.

Strigolactones (SLs) are a class of plant hormones that mediate biotic interactions and modulate developmental programs in response to endogenous and exogenous stimuli. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established, and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor that enables the examination of SL signaling distribution at cellular resolution and is capable of rapid response to altered SL levels in intact Arabidopsis (Arabidopsis thaliana) plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged, indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. The specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts with highly instructive analytical depth.

Tissue-specific transcriptome profiling of the Arabidopsis inflorescence stem reveals local cellular signatures.

Genome-wide gene expression maps with a high spatial resolution have substantially accelerated plant molecular science. However, the number of characterized tissues and growth stages is still small due to the limited accessibility of most tissues for protoplast isolation. Here, we provide gene expression profiles of the mature inflorescence stem of Arabidopsis thaliana covering a comprehensive set of distinct tissues. By combining fluorescence-activated nucleus sorting and laser-capture microdissection with next-generation RNA sequencing, we characterized the transcriptomes of xylem vessels, fibers, the proximal and distal cambium, phloem, phloem cap, pith, starch sheath, and epidermis cells. Our analyses classified more than 15,000 genes as being differentially expressed among different stem tissues and revealed known and novel tissue-specific cellular signatures. By determining overrepresented transcription factor binding regions in the promoters of differentially expressed genes, we identified candidate tissue-specific transcriptional regulators. Our datasets predict the expression profiles of an exceptional number of genes and allow hypotheses to be generated about the spatial organization of physiological processes. Moreover, we demonstrate that information about gene expression in a broad range of mature plant tissues can be established at high spatial resolution by nuclear mRNA profiling. Tissue-specific gene expression values can be accessed online at https://arabidopsis-stem.cos.uni-heidelberg.de/.

Intercellular and intracellular cilia orientation is coordinated by CELSR1 and CAMSAP3 in oviduct multi-ciliated cells.

The molecular mechanisms by which cilia orientation is coordinated within and between multi-ciliated cells (MCCs) are not fully understood. In the mouse oviduct, MCCs exhibit a characteristic basal body (BB) orientation and microtubule gradient along the tissue axis. The intracellular polarities were moderately maintained in cells lacking CELSR1 (cadherin EGF LAG seven-pass G-type receptor 1), a planar cell polarity (PCP) factor involved in tissue polarity regulation, although the intercellular coordination of the polarities was disrupted. However, CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a microtubule minus-end regulator, was found to be critical for determining the intracellular BB orientation. CAMSAP3 localized to the base of cilia in a polarized manner, and its mutation led to the disruption of intracellular coordination of BB orientation, as well as the assembly of microtubules interconnecting BBs, without affecting PCP factor localization. Thus, both CELSR1 and CAMSAP3 are responsible for BB orientation but in distinct ways; their cooperation should therefore be critical for generating functional multi-ciliated tissues.

SUPPRESSOR OF MAX2 1-LIKE 5 promotes secondary phloem formation during radial stem growth.

As a pre-requisite for constant growth, plants produce vascular tissues at different sites within their post-embryonic body. Interestingly, the formation of vascular tissues during longitudinal and radial expansion of shoot and root axes differs fundamentally with respect to its anatomical configuration. This raises the question to which level regulatory mechanisms of vascular tissue formation are shared throughout plant development. Here, we show that, similar to primary phloem formation during longitudinal growth, the cambium-based formation of secondary phloem depends on the function of SUPPRESSOR OF MAX2 1-LIKE (SMXL) genes. In particular, local SMXL5 deficiency results in the absence of secondary phloem. Moreover, the additional disruption of SMXL4 activity increases tissue production in the cambium region without secondary phloem being formed. Using promoter-reporter lines, we observed that SMXL4 and SMXL5 activities are associated with different stages of secondary phloem formation in the Arabidopsis stem. Based on genome-wide transcriptional profiling and expression analyses of phloem-related markers, we concluded that early steps of phloem formation are impaired in smxl4;smxl5 double mutants and that the additional cambium-derived cells fail to establish phloem-related features. Our results showed that molecular mechanisms determining primary and secondary phloem formation share important properties, but differ slightly with SMXL5 playing a more dominant role in the formation of secondary phloem.

Protection of DAergic neurons mediates treadmill running attenuated olfactory deficits and olfactory neurogenesis promotion in depression model.

In this study, we aimed to test the effects of treadmill running on depression induced olfactory functions and OB neurogenesis in depression model. Depression model was created with chronic unpredictable mild stress (CUMS) and treadmill running was performed as the antidepressant treatment. Behavioral results showed that treadmill running not only attenuated the depression mood but also improved the olfactory discrimination and sensitivity in CUMS depression model. Immune-staining further indicates treadmill running promoted neurogenesis in hippocampal OB region. Moreover, treadmill running prevented the loss of DAergic neurons in glomerular layer of OB region, indicating the critical role of DAergic neuronal functions in regulating treadmill running mediated olfactory functions. In depression model, inhibiting DAergic neurons by intra-OB injection of 6-OHDA resulted in the compromised improving effects of treadmill running olfactory discrimination. In conclusion, treadmill running could attenuate depression associated olfactory deficits by promoting olfactory neurogenesis and improve DAergic neural functions.

Biophysics in oviduct: Planar cell polarity, cilia, epithelial fold and tube morphogenesis, egg dynamics.

Organs and tissues in multi-cellular organisms exhibit various morphologies. Tubular organs have multi-scale morphological features which are closely related to their functions. Here we discuss morphogenesis and the mechanical functions of the vertebrate oviduct in the female reproductive tract, also known as the fallopian tube. The oviduct functions to convey eggs from the ovary to the uterus. In the luminal side of the oviduct, the epithelium forms multiple folds (or ridges) well-aligned along the longitudinal direction of the tube. In the epithelial cells, cilia are formed orienting toward the downstream of the oviduct. The cilia and the folds are supposed to be involved in egg transportation. Planar cell polarity (PCP) is developed in the epithelium, and the disruption of the Celsr1 gene, a PCP related-gene, causes randomization of both cilia and fold orientations, discontinuity of the tube, inefficient egg transportation, and infertility. In this review article, we briefly introduce various biophysical and biomechanical issues in the oviduct, including physical mechanisms of formation of PCP and organized cilia orientation, epithelial cell shape regulation, fold pattern formation generated by mechanical buckling, tubulogenesis, and egg transportation regulated by fluid flow. We also mention about possible roles of the oviducts in egg shape formation and embryogenesis, sinuous patterns of tubes, and fold and tube patterns observed in other tubular organs such as the gut, airways, etc.

Bifacial cambium stem cells generate xylem and phloem during radial plant growth.

A reduced rate of stem cell division is considered a widespread feature which ensures the integrity of genetic information during somatic development of plants and animals. Radial growth of plant shoots and roots is a stem cell-driven process that is fundamental for the mechanical and physiological support of enlarging plant bodies. In most dicotyledonous species, the underlying stem cell niche, the cambium, generates xylem inwards and phloem outwards. Despite the importance and intriguing dynamics of the cambium, the functional characterization of its stem cells is hampered by the lack of experimental tools for accessing distinct cambium sub-domains. Here, we use the hypocotyl of Arabidopsis thaliana to map stem cell activity in the proliferating cambium. Through pulse labeling and genetically encoded lineage tracing, we find that a single bifacial stem cell generates both xylem and phloem cell lineages. This cell is characterized by a specific combination of PXY (TDR), SMXL5 and WOX4 gene activity and a high division rate in comparison with tissue-specific progenitors. Our analysis provides a cellular fate map of radial plant growth, and suggests that stem cell quiescence is not a general prerequisite for life-long tissue production.This article has an associated 'The people behind the papers' interview.

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling.

The Wnt signaling pathway can be grouped into two classes, the ß-catenin-dependent and ß-catenin-independent pathways. Wnt5a signaling through a ß-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.

Double-edged sword of interdisciplinary knowledge flow from hard sciences to humanities and social sciences: Evidence from China.

Humanities and Social Sciences (HSS) increasingly absorb knowledge from Hard Sciences, i.e., Science, Technology, Agriculture and Medicine (STAM), as testified by a growing number of citations. However, whether citing more Hard Sciences brings more citations to HSS remains to be investigated. Based on China's HSS articles indexed by the Web of Science during 1998-2014, this paper estimated two-way fixed effects negative binomial models, with journal effects and year effects. Findings include: (1) An inverse U-shaped curve was observed between the percentage of STAM references to the HSS articles and the number of citations they received; (2) STAM contributed increasing knowledge to China's HSS, while Science and Technology knowledge contributed more citations to HSS articles. It is recommended that research policy should be adjusted to encourage HSS researchers to adequately integrate STAM knowledge when conducting interdisciplinary research, as over-cited STAM knowledge may jeopardize the readability of HSS articles.

Bifacial stem cell niches in fish and plants.

Embryonic development is key for determining the architecture and shape of multicellular bodies. However, most cells are produced postembryonically in, at least partly, differentiated organs. In this regard, organismal growth faces common challenges in coordinating expansion and function of body structures. Here we compare two examples for postembryonic growth processes from two different kingdoms of life to reveal common regulatory principles: lateral growth of plants and the enlargement of the fish retina. In both cases, growth is based on stem cell systems mediating radial growth by a bifacial mode of tissue production. Surprisingly, although being evolutionary distinct, we find similar patterns in regulatory circuits suggesting the existence of preferable solutions to a common developmental problem.

A Wnt5 Activity Asymmetry and Intercellular Signaling via PCP Proteins Polarize Node Cells for Left-Right Symmetry Breaking.

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.

Mechanical Regulation of Three-Dimensional Epithelial Fold Pattern Formation in the Mouse Oviduct.

Epithelia exhibit various three-dimensional morphologies linked to organ function in animals. However, the mechanisms of three-dimensional morphogenesis remain elusive. The luminal epithelium of the mouse oviduct forms well-aligned straight folds along the longitudinal direction of the tubes. Disruption of the Celsr1 gene, a planar cell polarity-related gene, causes ectopically branched folds. Here, we evaluated the mechanical contributions of the epithelium to the fold pattern formation. In the mutant oviduct, the epithelium was more intricate along the longitudinal direction than in the wild-type, suggesting a higher ratio of the longitudinal length of the epithelial layer to that of the surrounding smooth muscle (SM) layer (L-Epi/SM ratio). Our mathematical modeling and computational simulations suggested that the L-Epi/SM ratio could explain the differences in fold branching between the two genotypes. Longitudinal epithelial tensions were increased in well-aligned folds compared with those in disorganized folds both in the simulations and in experimental estimations. Artificially increasing the epithelial tensions suppressed the branching in simulations, suggesting that the epithelial tensions can regulate fold patterning. The epithelial tensions could be explained by the combination of line tensions along the epithelial cell-cell boundaries with the polarized cell arrays observed in vivo. These results suggest that the fold pattern is associated with the polarized cell array through the longitudinal epithelial tension. Further simulations indicated that the L-Epi/SM ratio could contribute to fold pattern diversity, suggesting that the L-Epi/SM ratio is a critical parameter in the fold patterning in tubular organs.

Dynamics of planar cell polarity protein Vangl2 in the mouse oviduct epithelium.

The planar cell polarity (PCP) pathway regulates morphogenesis in various organs. The polarized localization is a key feature of core PCP factors for orchestrating cell polarity in an epithelial sheet. Several studies using Drosophila melanogaster have investigated the mechanism of the polarized localization. However, to what extent these mechanisms are conserved and how the polarization of core PCP factors is maintained in mature vertebrates are still open questions. Here, we addressed these questions by analyzing the dynamics of Vangl2, a member of core PCP factors, in the mouse oviduct epithelium. Multiple core PCP factors including Vangl2 were expressed in the mouse oviduct in postnatal stages. Vangl1, Vangl2 and Frizzled6 had polarized localization in the oviduct epithelium. Exogenously introduced expression of green fluorescent protein (GFP)-tagged core PCP factors by electroporation revealed that Vangl1, Vangl2 and Prickle2 are localized on the ovarian side of the cell periphery in the oviduct. To visualize the Vangl2 dynamics, we generated the R26-Vangl2-EGFP transgenic mice. In these mice, Vangl2-EGFP was ubiquitously expressed and showed polarized localization in multiple organs including the oviduct, the trachea, the lateral ventricle and the uterus. Fluorescence recovery after photobleaching (FRAP) analysis in the mature oviduct revealed that Vangl2 in the enriched subdomain of cell periphery (cellular edge) was more stable than Vangl2 in the less-enriched cellular edge. Furthermore, when a subregion of a Vangl2-enriched cellular edge was bleached, the Vangl2-enriched subregion neighboring the bleached region in the same cellular edge tended to decrease more intensities than the neighboring sub-region in the next Vangl2-enriched cellular edge. Finally, the polarization of Vangl2 was observed in nocodazole treated mouse viduct, suggesting the maintenance of Vangl2 asymmetry is independent of microtubule formation. Taken together, we revealed the characteristics of Vangl2 dynamics in the oviduct epithelium, and found that Vangl2 forms stable complex at the enriched cellular edge and forms compartments. Our data collectively suggest that the mechanism for maintenance of Vangl2 asymmetry in mature mouse oviduct is different from the microtubule dependent polarized transport model, which has been proposed for the reinforcement of the asymmetry of two core PCP proteins, Flamingo and Dishevelled, in the developing fly.