Concave-to-convex curve conversion of fiber cells correlates with Y-shaped suture formation at the poles of the rodent lens.

The eye lens contains convexly curved fiber cells that align in concentric layers around the lens anterior-posterior pole axis. For lens fiber differentiation at the equator, cells elongate with their apical and basal tips migrating towards the anterior and posterior poles, respectively. At each pole, the fiber tips meet opposing tips of other fiber cells, to form a suture. Although umbilical or point sutures are observed in fish and birds, line, Y- or star-shaped sutures are detected in other vertebrate lenses. Sutures that do not converge at the point are thought to result from intricate movements of the fiber tips, rather than a straightforward migration along a meridional path. The triggers that give rise to these variations are currently not understood. Our findings revealed that in the mouse embryo, the early-stage lens contains only concave curved fibers, and later, a zone of concave-to-convex curve conversion develops. At this point, a nascent suture in a linear shape appears at the posterior pole and subsequently progresses into a V-shape. This V-shape appears to further develop into a Y-shape as a branch extends from the apex of the V-shape. In lens of zebrafish and Xenopus larvae that form point sutures, this curve-conversion zone is not observed. In lens of adult birds (e.g. zebra finch) that form a point suture, these too also lack a curve-conversion zone. In our previous studies, we demonstrated that murine lens fibers undergoing curve conversion extend membrane protrusions, or lamellipodia, at their basal membranes. In line with this, we did not observe protrusions at the basal tips of fibers in the non-mammalian lenses of zebrafish, Xenopus, and zebra finch in which curve conversion does not occur. We propose that the concave-to-convex conversion in rodent lenses introduces defined paths for fiber cell tips, leading to a more elaborate and complex suture formation, compared to the simple point suture of lower vertebrates.

Strategic targeting of Cas9 nickase induces large segmental duplications.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

Mentalis nerve branches supplying the lower lip revisited: a study of human fetuses and donated elderly cadavers.

PURPOSE: Little information is known about the mentalis nerve course from the lower lip approximation margin (free margin) to the upper lip. Likewise, no difference in nerve distribution has been observed between the cutaneous and mucosal parts of the lip. Therefore, this study reexamined mentalis nerve morphology. METHODS: For macroscopic observations, three fresh cadavers were dissected (one male and two females; aged 78-93). We also evaluated histological sections obtained from five donated elderly cadavers (two males and three females, aged 82-96 years) and 15 human fetuses (11-40 weeks or crown-rump length 80-372 mm). Immunohistochemical analysis for S100 protein and tyrosine hydroxylase was performed. RESULTS: In both fetuses and adult cadavers, one to three nerve branches ran upward in the submucosal tissue from the mental foramen. Near the free margin of the lip, some branches passed through the orbicularis oris muscle layer toward the lip skin, whereas others followed a reversed J-shaped course along the free margin. Nerve twigs ran in parallel beneath the mucosa, whereas wavy nerve twigs attached to the basal lamina of the lip epidermis. The difference in nerve endings abruptly occurred at the skin-mucosal junction. Tyrosine hydroxylase-positive sympathetic nerve twigs surrounded arteries and formed a branch composed of S100-negative unmyelinated fibers. CONCLUSION: The lower lip skin was innervated by a perforating branch passing through the orbicularis oris muscle, that was different from the lip mucosa. A sudden change in the nerve ending configuration at the mucocutaneous junction seemed to develop postnatally.

Exploratory Study of Growth of Circumference of Mandibular Fossa Adjacent to Petrous Portion of Temporal Bone Using Dried Skulls.

The morphogenetic process of development of the circumference of the mandibular fossa during tooth eruption, which involves the replacement of deciduous teeth with permanent teeth, is strongly affected by occlusion. To the best of our knowledge, no studies have investigated the effect of occlusion on this process. This study investigated the morphogenetic process of development during tooth eruption using dried skulls harvested from Indian donors. The average distance between the ala-major-squamosa suture and the foramen ovale according to age group was as follows: 3.24 mm in the 8-month-old group and 8.92 mm in the adult group. The average distance between the ala-major-squamosa suture and the apex of the articular tubercle according to age groups was as follows: 10.38 mm in the 8-month-old group and 19.34 mm in the adult group. The average distance between the point of intersection of the petrosquamous fissure and petrotympanic fissure located on the perpendicular line drawn posteriorly from the shortest distance of the medio-lateral axis between the ala-major-squamosa suture and the apex of the articular tubercle according to age group was as follows: 9.68 mm in the 8-month-old group and 14.3 mm in the adult group. These results suggest that the mandibular fossa is strongly affected by load due to occlusion, unlike the growth of the neurocranium. This indicates that the effect of occlusion is a secondary element in the morphogenetic process of development of the circumference of the mandibular fossa.

Fibroblast growth factor-induced lens fiber cell elongation is driven by the stepwise activity of Rho and Rac.

The spheroidal shape of the eye lens is crucial for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that, in the mouse lens, membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating that lamellipodium formation is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controlling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin and actin reduced the height of both early and later fibers, indicating that elongation of these fibers relies on actomyosin contractility. Consistent with this, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose that it does so through regulation of Rho activity.

Fibroblast Growth Factor-induced lens fiber cell elongation is driven by the stepwise activity of Rho and Rac.

The spheroidal shape of the eye lens is critical for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating lamellipodium is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin, and actin reduced the height of both early and later fibers, indicating elongation of these fibers relies on actomyosin contractility. Consistently, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose it to do so through regulation of Rho activity.

Confined-microtubule assembly shapes three-dimensional cell wall structures in xylem vessels.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

The Japanese Epidemiologic Study for Perioperative Anaphylaxis, a prospective nationwide study: clinical signs, severity, and therapeutic agents.

BACKGROUND: Diagnosis of perioperative anaphylaxis is difficult because of its non-specific and variable signs and symptoms. Therapeutic agents used to treat anaphylaxis and anaesthesiologist responses also vary depending on the case, which might affect outcomes; however, only a few studies have focused on these factors. METHODS: This prospective study of perioperative anaphylaxis, a part of the Japanese Epidemiologic Study for Perioperative Anaphylaxis, investigated the clinical signs, its severity, therapeutic drugs, epinephrine administration, and anaesthesiologist responses in cases of perioperative anaphylaxis to assess trends and variability. Shock index was used to assess severity of cardiovascular collapse. RESULTS: In 43 patients analysed in this study, cardiovascular signs (88.4%) were the most frequent, followed by skin (81.4%) and respiratory signs (60.5%). The presence of signs increased during the clinical course. The median time from the first signs to diagnosis of anaphylaxis was 10 (5.0-17.8) min. The rates of epinephrine use were 30.2% (unused), 48.8% (i.v.), and 20.9% (i.m.). The median time from diagnosis of anaphylaxis to epinephrine administration was 7 (inter-quartile range: 1.5-8.0) min. Antihistamines and corticosteroids were each used in 69.8% of cases. The worst shock index was higher in patients who received i.v. epinephrine (2.77 [0.90] mean [standard deviation]) than in both no epinephrine use cases (1.35 [0.41]) and i.m. epinephrine cases (1.89 [0.77] (P<0.001]). CONCLUSIONS: The clinical signs and treatments of perioperative anaphylaxis are variable, and the choice regarding epinephrine administration is based on symptom severity. CLINICAL TRIAL REGISTRATION: UMIN000035350.

The Japanese Epidemiologic Study for Perioperative Anaphylaxis, a prospective nationwide study: allergen exposure, epidemiology, and diagnosis of anaphylaxis during general anaesthesia.

BACKGROUND: Diagnosis of perioperative anaphylaxis is often challenging. This study describes the utility of a newly developed tool for identifying patients with a high possibility of anaphylaxis, and aimed to investigate the frequency of anaphylaxis with each drug during the perioperative period in Japan. METHODS: This study included patients with anaphylaxis of Grade 2 or higher severity during general anaesthesia at 42 facilities across Japan in 2019 and 2020. We developed and adopted a unique objective evaluation tool yielding a composite score for diagnosing anaphylaxis, which includes the results of skin tests and basophil activation tests, and clinical scores for perioperative anaphylaxis. The number of cases using each drug and the total number of anaphylaxis cases were investigated to calculate the frequency of anaphylaxis. RESULTS: General anaesthesia was performed in 218 936 cases, which included 55 patients with suspected perioperative anaphylaxis. The developed composite score diagnosed 43 of them with a high probability of anaphylaxis. The causative agent was identified in 32 cases. Plasma histamine levels showed high diagnostic accuracy for anaphylaxis. The top causative agents were rocuronium (10 cases in 210 852 patients, 0.005%), sugammadex (7 cases in 150 629 patients, 0.005%), and cefazolin (7 cases in 106 005 patients, 0.007%). CONCLUSIONS: We developed a composite tool to diagnose anaphylaxis, and found that the combination of tryptase levels, skin testing, and basophil activation testing results and clinical score improved the certainty of anaphylaxis diagnosis. The incidence of perioperative anaphylaxis in our study was 1 in about 5000 general anaesthesia cases. CLINICAL TRIAL REGISTRATION: UMIN000035350.

TRPV1 is involved in abdominal hyperalgesia in a mouse model of lipopolysaccharide-induced peritonitis and influences the immune response via peripheral noradrenergic neurons.

AIMS: The transient receptor potential vanilloid subfamily 1 (TRPV1) not only plays a role as a nociceptor but also has some regulatory effects on the immune system. We investigated the effects of TRPV1 on abdominal pain and the immune system in lipopolysaccharide (LPS)-induced peritonitis and the association between TRPV1 and peripheral noradrenergic neurons. MAIN METHODS: Experiments were performed in 8- to 14-week-old male wild-type (WT) and TRPV1 knockout (KO) mice. The mice were intraperitoneally injected with a non-lethal dose of LPS. Pain assessment and investigation of changes in the immune system were performed. Denervation of sympathetic nerves and the noradrenergic splenic nerve was induced by intraperitoneal administration of 6-hydroxydopamine. KEY FINDINGS: The levels of serum cytokines were not significantly different in WT mice and TRPV1 KO mice. Abdominal mechanical hyperalgesia was greater in WT mice than in TRPV1 KO mice from 6 h to 3 days. The numbers of macrophages, neutrophils, dendritic cells, and CD4 T cells in the spleens of TRPV1 KO mice were significantly increased compared to those in WT mice 4 days after LPS administration. By noradrenergic denervation, the numbers of those cells in WT mice increased to levels comparable to those in TRPV1 KO mice. SIGNIFICANCE: In LPS-induced peritonitis, abdominal inflammatory pain was transmitted via TRPV1. In addition, TRPV1 had an anti-inflammatory effect on the spleen in the late phase of peritonitis. This anti-inflammatory effect was thought to be mediated by activation of the sympathetic nervous system and/or noradrenergic splenic nerve induced by TRPV1 activation.

Types of anomalies in two-dimensional video-based gait analysis in uncontrolled environments.

Two-dimensional video-based pose estimation is a technique that can be used to estimate human skeletal coordinates from video data alone. It is also being applied to gait analysis and in particularly, due to its simplicity of measurement, it has the potential to be applied to gait analysis of large populations. However, it is considered difficult to completely homogenize the environment and settings during the measurement of large populations. Therefore, it is necessary to appropriately deal with technical errors that are not related to the biological factors of interest. In this study, by analyzing a large cohort database, we have identified four major types of anomalies that occur during gait analysis using OpenPose in uncontrolled environments: anatomical, biomechanical, and physical anomalies and errors due to estimation. We have also developed a workflow for identifying and correcting these anomalies and confirmed that this workflow is reproducible through simulation experiments. Our results will help obtain a comprehensive understanding of the anomalies to be addressed during pre-processing for 2D video-based gait analysis of large populations.

7,8-Dihydroxy-3-(4'-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells.

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

Intrathecal morphine exacerbates paresis with increasing muscle tone of hindlimbs in rats with mild thoracic spinal cord injury but without damage of lumbar α-motoneurons.

Adverse effects of morphine on locomotor function after moderate to severe spinal cord injury (SCI) have been reported; however, the effects after mild SCI without damage of lumbar α-motoneurons have not been investigated. We investigated the effects of lumbar intrathecal morphine on locomotor function after mild thoracic SCI and the involvement of classic opioid receptor activation. A mild thoracic contusive SCI was induced in adult rats at the T9-T10 spine level under sevoflurane anesthesia. We evaluated the effects of single doses of intrathecal morphine and selective µ-, δ-, and κ-opioid receptor agonists, continuous infusion of intrathecal morphine for 72 hours, and administration of physiological saline on locomotor function and muscle tone in the hindlimbs. The numbers of damaged and total α-motoneurons in the lumbar spinal cord were also investigated. Single doses of morphine aggravated residual locomotor function after SCI but did not affect functional recovery. Single doses of morphine and µ- and δ-opioid receptor agonists significantly aggravated residual locomotor function with increases in muscle tone after SCI, and the effects of the drugs were reversed by naloxone. In contrast, continuous infusion of morphine led to persistent decline in locomotor function with increased muscle tone, which was not reversed by naloxone, but did not increase the number of damaged lumbar α-motoneurons. These results indicate that a single dose of morphine at an analgesic dose transiently increases muscle tone of the hindlimbs via activation of spinal µ- and δ- opioid receptors, resulting in further deterioration of locomotor function in the acute phase of mild SCI. Our results also suggest that an increased dose of morphine with prolonged administration leads to persistent decline in locomotor function with increased muscle tone via mechanisms other than direct activation of classical opioid receptors. Morphine should be used cautiously even after mild SCI.

Preoperative and postoperative complications of cerebrospinal fluid drainage in descending thoracic and thoraco-abdominal aortic aneurysm surgery: a single-center retrospective study.

PURPOSE: Cerebrospinal-fluid drainage (CSFD) has been performed to prevent paraplegia in descending thoracic or thoraco-abdominal aortic aneurysm (DTA/TAAA) surgery; however, CSFD itself has a risk of severe complications. We retrospectively investigated the incidence rates of CSFD-related preoperative and postoperative complications. METHODS: Patients who underwent DTA/TAAA surgery with a CSFD catheter that was inserted on the day before surgery were enrolled. The incidence rates of complications from spinal puncture until DTA/TAAA surgery were investigated as preoperative CSFD complications, and the incidence rates from DTA/TAAA surgery to postoperative day 7 were investigated as CSFD-related postoperative complications. RESULTS: Preoperative CSFD complications were analyzed in 123 cases. DTA/TAAA surgery was postponed due to bloody cerebrospinal fluid (2.5%) and due to meningitis (1.7%). The incidence rate of mild preoperative complications was 32.4%. Postoperative CSFD complications were analyzed in 108 cases. Intracranial hemorrhage occurred in 3.9% of cases in open surgery and other postoperative severe CSFD complications did not occur. The incidence rates of moderate/mild complications in open surgery were 2.6%/14.3% and those in TEVAR were 3.2%/19.4%. CONCLUSION: Bloody cerebrospinal fluid and meningitis, which are severe complications associated with spinal puncture, occurred within 1 day after spinal puncture. The incidence rates of moderate/mild complications were high in both the preoperative and postoperative periods. These results showed that CSFD catheter insertion and management should be performed carefully with consideration given to the risks and benefits of CSFD.

Subfascial infiltration of 0.5% ropivacaine, but not 0.25% ropivacaine, exacerbates damage and inflammation in surgically incised abdominal muscles of rats.

Ropivacaine-induced myotoxicity in surgically incised muscles has not been fully investigated. We evaluated the effects of infiltration anesthesia with ropivacaine on damage, inflammation and regeneration in the incised muscles of rats undergoing laparotomy. Ropivacaine or saline was infiltrated below the muscle fascia over the incised muscles. Pain-related behaviors and histological muscle damage were assessed. Macrophage infiltration at days 2 and 5 and proliferation of satellite cells at day 5 were detected by CD68 and MyoD immunostaining, respectively. Pain-related behaviors were inhibited by 0.25% and 0.5% of ropivacaine for 2 h after surgery. Single infiltration of 0.5% ropivacaine did not induce injury in intact muscles without incision, but single and repeated infiltration of 0.5% ropivacaine significantly augmented laparotomy-induced muscle injury and increased the numbers of CD68-positve macrophages and MyoD-positive cells compared to those in rats with infiltration of saline or 0.25% ropivacaine. In contrast, there were no significant differences in them between rats with saline infusion and rats with 0.25% ropivacaine infiltration. In conclusion, single or repeated subfascial infiltration of 0.25% ropivacaine can be used without exacerbating the damage and inflammation in surgically incised muscles, but the use of 0.5% ropivacaine may be a concern because of potentially increased muscle damage.

Secondary cell wall patterning-connecting the dots, pits and helices.

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.

Development and Regeneration of Muscle, Tendon, and Myotendinous Junctions in Striated Skeletal Muscle.

Owing to a rapid increase in aging population in recent years, the deterioration of motor function in older adults has become an important social problem, and several studies have aimed to investigate the mechanisms underlying muscle function decline. Furthermore, structural maintenance of the muscle-tendon-bone complexes in the muscle attachment sites is important for motor function, particularly for joints; however, the development and regeneration of these complexes have not been studied thoroughly and require further elucidation. Recent studies have provided insights into the roles of mesenchymal progenitors in the development and regeneration of muscles and myotendinous junctions. In particular, studies on muscles and myotendinous junctions have-through the use of the recently developed scRNA-seq-reported the presence of syncytia, thereby suggesting that fibroblasts may be transformed into myoblasts in a BMP-dependent manner. In addition, the high mobility group box 1-a DNA-binding protein found in nuclei-is reportedly involved in muscle regeneration. Furthermore, studies have identified several factors required for the formation of locomotor apparatuses, e.g., tenomodulin (Tnmd) and mohawk (Mkx), which are essential for tendon maturation.

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.