Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

Real-world practice patterns and outcomes in Veterans with relapsed/refractory diffuse large B-cell lymphoma.

Aim: To describe practices and outcomes in veterans with relapsed/refractory diffuse large B-cell lymphoma. Patients & methods: Using Veteran Affairs Cancer Registry System and electronic health record data, we identified relapsed/refractory diffuse large B-cell lymphoma patients completing second-line treatment (2L) in 2000-2016. Treatments were classified as aggressive/nonaggressive. Analyses included descriptive statistics and the Kaplan-Meier estimation of progression-free survival and overall survival. Results: Two hundred and seventy patients received 2L. During median 9.7-month follow-up starting from 2L, 470 regimens were observed, averaging 2.7 regimens/patient: 219 aggressive, 251 nonaggressive. One hundred and twenty-one patients proceeded to third-line, 50 to fourth-line and 18 to fifth-line treatment. Median progression-free survival in 2L was 5.2 months. Median overall survival was 9.5 months. Forty-four patients (16.3%) proceeded to bone marrow transplant. Conclusion: More effective, less toxic treatments are needed and should be initiated earlier in treatment trajectory.

Cadherin 6 promotes neural crest cell detachment via F-actin regulation and influences active Rho distribution during epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is a complex change in cell phenotype that is important for cell migration, morphogenesis and carcinoma metastasis. Loss of epithelial cell adhesion and tight regulation of cadherin adhesion proteins are crucial for EMT. Cells undergoing EMT often display cadherin switching, where they downregulate one cadherin and induce expression of another. However, the functions of the upregulated cadherins and their effects on cell motility are poorly understood. Neural crest cells (NCCs), which undergo EMT during development, lose N-cadherin and upregulate Cadherin 6 (Cdh6) prior to EMT. Cdh6 has been suggested to suppress EMT via cell adhesion, but also to promote EMT by mediating pro-EMT signals. Here, we determine novel roles for Cdh6 in generating cell motility during EMT. We use live imaging of NCC behavior in vivo to show that Cdh6 promotes detachment of apical NCC tails, an important early step of EMT. Furthermore, we show that Cdh6 affects spatiotemporal dynamics of F-actin and active Rho GTPase, and that Cdh6 is required for accumulation of F-actin in apical NCC tails during detachment. Moreover, Cdh6 knockdown alters the subcellular distribution of active Rho, which is known to promote localized actomyosin contraction that is crucial for apical NCC detachment. Together, these data suggest that Cdh6 is an important determinant of where subcellular actomyosin forces are generated during EMT. Our results also identify mechanisms by which an upregulated cadherin can generate cell motility during EMT.

Rho activation is apically restricted by Arhgap1 in neural crest cells and drives epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transitions (EMTs) are crucial for morphogenesis and carcinoma metastasis, yet mechanisms controlling the underlying cell behaviors are poorly understood. RhoGTPase signaling has been implicated in EMT; however, previous studies have yielded conflicting results regarding Rho function, and its role in EMT remains poorly understood. Elucidation of precise Rho functions has been challenging because Rho signaling is highly context dependent and its activity is tightly regulated spatiotemporally within the cell. To date, few studies have examined how Rho affects cell motility in intact organisms, and the pattern of Rho activity during motile cell behaviors of EMT has not been determined in any system. Here, we image endogenous active Rho during EMT in vivo, and analyze effects of Rho and Rho-kinase (ROCK) manipulation on cell motility in vivo. We show that Rho is activated in a discrete apical region of premigratory neural crest cells during EMT, and Rho-ROCK signaling is essential for apical detachment and generation of motility within the neuroepithelium, a process that has been poorly understood. Furthermore, we find that Arhgap1 restricts Rho activation to apical areas, and this restriction is necessary for detachment. Our results provide new insight into mechanisms controlling local Rho activation and how it affects dynamic cell behaviors and actomyosin contraction during key steps of EMT in an intact living organism.

Understanding the working alliance with clients diagnosed with anorexia nervosa.

The therapeutic working alliance is a vital ingredient of psychotherapy, specifically for clients diagnosed with anorexia nervosa, as progress is often slow and treatment difficult. This qualitative phenomenological study investigated the experiences of eight clients with anorexia nervosa and seven therapists who work with this population, regarding which therapist factors aided in and challenged the working alliance formation in individual psychotherapy. Data was gathered through semi-structured interviews. Some helpful therapist factors included collaboration, appropriate self-disclosure, providing a warm and safe environment, and willingness to be contacted outside of a session. Unhelpful factors included lack of attunement and objectivity and failure to individualize treatment.

Calsyntenin-1 regulates axon branching and endosomal trafficking during sensory neuron development in vivo.

Precise regulation of axon branching is crucial for neuronal circuit formation, yet the mechanisms that control branch formation are not well understood. Moreover, the highly complex morphology of neurons makes them critically dependent on protein/membrane trafficking and transport systems, although the functions for membrane trafficking in neuronal morphogenesis are largely undefined. Here we identify a kinesin adaptor, Calsyntenin-1 (Clstn-1), as an essential regulator of axon branching and neuronal compartmentalization in vivo. We use morpholino knockdown and a Clstn-1 mutant to show that Clstn-1 is required for formation of peripheral but not central sensory axons, and for peripheral axon branching in zebrafish. We used live imaging of endosomal trafficking in vivo to show that Clstn-1 regulates transport of Rab5-containing endosomes from the cell body to specific locations of developing axons. Our results suggest a model in which Clstn-1 patterns separate axonal compartments and define their ability to branch by directing trafficking of specific endosomes.

Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.

Neurons must develop complex structure to form proper connections in the nervous system. The initiation of axons in defined locations on the cell body and their extension to synaptic targets are critical steps in neuronal morphogenesis, yet the mechanisms controlling axon formation in vivo are poorly understood. The centrosome has been implicated in multiple aspects of neuronal morphogenesis; however, its function in axon development is under debate. Conflicting results from studies of centrosome function in axonogenesis suggest that its role is context dependent and underscore the importance of studying centrosome function as neurons develop in their natural environment. Using live imaging of zebrafish Rohon-Beard (RB) sensory neurons in vivo, we discovered a spatiotemporal relationship between centrosome position and the formation of RB peripheral, but not central, axons. We tested centrosome function by laser ablation and found that centrosome disruption inhibited peripheral axon outgrowth. In addition, we show that centrosome position and motility are regulated by LIM homeodomain transcription factor activity, which is specifically required for the development of RB peripheral axons. Furthermore, we show a correlation between centrosome mislocalization and ectopic axon formation in bashful (laminin alpha 1) mutants. Thus, both intrinsic transcription factor activity and extracellular cues can influence centrosome position and axon formation in vivo. This study presents the first positive association between the centrosome and axon formation in vivo and suggests that the centrosome is important for differential neurite formation in neurons with complex axonal morphologies.

High-sensitivity real-time analysis of nanoparticle toxicity in green fluorescent protein-expressing zebrafish.

Gold nanoparticles (AuNP) show great potential for diagnostic and therapeutic application in humans. A great number of studies have tested the cytotoxicity of AuNP using cell culture. There is, however, an urgent need to test AuNP in vertebrate animal models that interrogate biodistribution and complex biological traits like organ development, whole body metabolism, and cognitive function. The sheer number of different compounds precludes the use of small rodent model for initial screening. The extended fish embryo test (FET) is used here to bridge the gap between cell culture and small animal models. A study on the toxicity of ultrasmall AuNP in wild type and transgenic zebrafish is presented. FET faithfully reproduce all important findings of a previous study in HeLa cells and add new important information on teratogenicity and hepatotoxicity that could not be gained from studying cultured cells.

Repulsion or adhesion: receptors make the call.

Repulsive signaling plays a prominent role in regulating cell-cell interactions and is fundamental to multiple developmental processes. A proper balance between repulsion from and adhesion to other cells or the extracellular matrix is also important. Semaphorin-Plexin and ephrin-Eph ligand-receptor pairs compose two major repulsive signaling systems. Recent advances have elucidated mechanisms by which Semaphorin-Plexin and ephrin-Eph signaling control repulsion versus adhesion. Semaphorins act through a complex signaling pathway to inhibit integrin-mediated adhesion, allowing cell repulsion. Ephrin-Eph interactions can directly mediate cell adhesion and several mechanisms control whether ephrin-Eph binding and signaling induces repulsion or adhesion.

Mechanistic insights from animal models of neurofibromatosis type 1 cognitive impairment.

Neurofibromatosis type 1 (NF1) is an autosomal-dominant neurogenetic disorder caused by mutations in the gene neurofibromin 1 (NF1). NF1 predisposes individuals to a variety of symptoms, including peripheral nerve tumors, brain tumors and cognitive dysfunction. Cognitive deficits can negatively impact patient quality of life, especially the social and academic development of children. The neurofibromin protein influences neural circuits via diverse cellular signaling pathways, including through RAS, cAMP and dopamine signaling. Although animal models have been useful in identifying cellular and molecular mechanisms that regulate NF1-dependent behaviors, translating these discoveries into effective treatments has proven difficult. Clinical trials measuring cognitive outcomes in patients with NF1 have mainly targeted RAS signaling but, unfortunately, resulted in limited success. In this Review, we provide an overview of the structure and function of neurofibromin, and evaluate several cellular and molecular mechanisms underlying neurofibromin-dependent cognitive function, which have recently been delineated in animal models. A better understanding of neurofibromin roles in the development and function of the nervous system will be crucial for identifying new therapeutic targets for the various cognitive domains affected by NF1.

KLC4 shapes axon arbors during development and mediates adult behavior.

Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches, proper microtubule (MT) dynamics, and endosomal transport. Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that klc4 mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating klc4 as a new gene involved in stress response circuits.

Pregnancy-associated plasma protein-aa regulates endoplasmic reticulum-mitochondria associations.

Endoplasmic reticulum (ER) and mitochondria form close physical associations to facilitate calcium transfer, thereby regulating mitochondrial function. Neurons with high metabolic demands, such as sensory hair cells, are especially dependent on precisely regulated ER-mitochondria associations. We previously showed that the secreted metalloprotease pregnancy-associated plasma protein-aa (Pappaa) regulates mitochondrial function in zebrafish lateral line hair cells (Alassaf et al., 2019). Here, we show that pappaa mutant hair cells exhibit excessive and abnormally close ER-mitochondria associations, suggesting increased ER-mitochondria calcium transfer. pappaa mutant hair cells are more vulnerable to pharmacological induction of ER-calcium transfer. Additionally, pappaa mutant hair cells display ER stress and dysfunctional downstream processes of the ER-mitochondria axis including altered mitochondrial morphology and reduced autophagy. We further show that Pappaa influences ER-calcium transfer and autophagy via its ability to stimulate insulin-like growth factor-1 bioavailability. Together our results identify Pappaa as a novel regulator of the ER-mitochondria axis.

Muscle contractions guide rohon-beard peripheral sensory axons.

Multiple molecular cues guide neuronal axons to their targets during development. Previous studies in vitro have shown that mechanical stimulation also can affect axon growth; however, whether mechanical force contributes to axon guidance in vivo is unknown. We investigated the role of muscle contractions in the guidance of zebrafish peripheral Rohon-Beard (RB) sensory axons in vivo. We analyzed several mutants that affect muscle contraction through different molecular pathways, including a new mutant allele of the titin a (pik) gene, mutants that affect the hedgehog signaling pathway, and a nicotinic acetylcholine receptor mutant. We found RB axon defects in these mutants, the severity of which appeared to correlate with the extent of muscle contraction loss. These axons extend between the muscle and skin and normally have ventral trajectories and repel each other on contact. RB peripheral axons in muscle mutants extend longitudinally instead of ventrally, and the axons fail to repel one another on contact. In addition, we showed that limiting muscle movements by embedding embryos in agarose caused similar defects in peripheral RB axon guidance. This work suggests that the mechanical forces generated by muscle contractions are necessary for proper sensory axon pathfinding in vivo.

The cell adhesion molecule Tag1, transmembrane protein Stbm/Vangl2, and Lamininalpha1 exhibit genetic interactions during migration of facial branchiomotor neurons in zebrafish.

Interactions between a neuron and its environment play a major role in neuronal migration. We show here that the cell adhesion molecule Transient Axonal Glycoprotein (Tag1) is necessary for the migration of the facial branchiomotor neurons (FBMNs) in the zebrafish hindbrain. In tag1 morphant embryos, FBMN migration is specifically blocked, with no effect on organization or patterning of other hindbrain neurons. Furthermore, using suboptimal morpholino doses and genetic mutants, we found that tag1, lamininalpha1 (lama1) and stbm, which encodes a transmembrane protein Vangl2, exhibit pairwise genetic interactions for FBMN migration. Using time-lapse analyses, we found that FBMNs are affected similarly in all three single morphant embryos, with an inability to extend protrusions in a specific direction, and resulting in the failure of caudal migration. These data suggest that tag1, lama1 and vangl2 participate in a common mechanism that integrates signaling between the FBMN and its environment to regulate migration.

Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.

The induction and migration of neural crest cells (NCCs) are essential to the development of craniofacial structures and the peripheral nervous system. A critical step in the development of NCCs is the epithelial to mesenchymal transition (EMT) that they undergo in order to initiate migration. Several transcription factors are important for the NCC EMT. However, less is known about the effectors regulating changes in cell adhesion, the cytoskeleton, and cell motility associated with the EMT or about specific changes in the behavior of cells undergoing EMT in vivo. We used time-lapse imaging of NCCs in the zebrafish hindbrain to show that NCCs undergo a stereotypical series of behaviors during EMT. We find that loss of cell adhesion and membrane blebbing precede filopodial extension and the onset of migration. Live imaging of actin dynamics shows that actin localizes differently in blebs and filopodia. Moreover, we find that disruption of myosin II or Rho-kinase (ROCK) activity inhibits NCC blebbing and causes reduced NCC EMT. These data reveal roles for myosin II and ROCK in NCC EMT in vivo, and provide a detailed characterization of NCC behavior during EMT that will form a basis for further mechanistic studies.

Spindle-F-actin interactions in mitotic spindles in an intact vertebrate epithelium.

Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, whether actin filaments (F-actin) are required for or are even present in mitotic spindles has long been controversial. Here we have developed improved methods for simultaneously preserving F-actin and microtubules in fixed samples and exploited them to demonstrate that F-actin is indeed associated with mitotic spindles in intact Xenopus laevis embryonic epithelia. We also find that there is an "F-actin cycle," in which the distribution and organization of spindle F-actin changes over the course of the cell cycle. Live imaging using a probe for F-actin reveals that at least two pools of F-actin are associated with mitotic spindles: a relatively stable internal network of cables that moves in concert with and appears to be linked to spindles, and F-actin "fingers" that rapidly extend from the cell cortex toward the spindle and make transient contact with the spindle poles. We conclude that there is a robust endoplasmic F-actin network in normal vertebrate epithelial cells and that this network is also a component of mitotic spindles. More broadly, we conclude that there is far more internal F-actin in epithelial cells than is commonly believed.

Semaphorin3D regulates axon axon interactions by modulating levels of L1 cell adhesion molecule.

The decision of a growing axon to selectively fasciculate with and defasciculate from other axons is critical for axon pathfinding and target innervation. Fasciculation can be regulated by cell adhesion molecules that modulate interaxonal adhesion and repulsive molecules, expressed by surrounding tissues that channel axons together. Here we describe crosstalk between molecules that mediate these mechanisms. We show that Semaphorin3D (Sema3D), a classic repulsive molecule, promotes fasciculation by regulating L1 CAM levels and axon-axon interactions rather than by creating a repulsive surround. Knockdown experiments show that Sema3D and L1 genetically interact to promote fasciculation. Sema3D overexpression increases and Sema3D knockdown decreases levels of axonal L1 protein. Moreover, excess L1 rescues defasciculation caused by the loss of Sema3D. In vivo time-lapse imaging reveals that Sema3D or L1 knockdown cause identical defects in growth cone behaviors during axon-axon interactions, consistent with a loss of adhesion. These results reveal a novel mechanism by which a semaphorin promotes fasciculation and modulates axon-axon interactions by regulating an adhesion molecule.

Developing a business-practice model for pharmacy services in ambulatory settings.

A business-practice model is a guide, or toolkit, to assist managers and clinical pharmacy practitioners in the exploration, proposal, development and implementation of new clinical pharmacy services and/or the enhancement of existing services. This document was developed by the American College of Clinical Pharmacy Task Force on Ambulatory Practice to assist clinical pharmacy practitioners and administrators in the development of business-practice models for new and existing clinical pharmacy services in ambulatory settings. This document provides detailed instructions, examples, and resources on conducting a market assessment and a needs assessment, types of clinical services, operations, legal and regulatory issues, marketing and promotion, service development and exit plan, evaluation of service outcomes, and financial considerations in the development of a clinical pharmacy service in the ambulatory environment. Available literature is summarized, and an appendix provides valuable citations and resources. As ambulatory care practices continue to evolve, there will be increased knowledge of how to initiate and expand the services. This document is intended to serve as an essential resource to assist in the growth and development of clinical pharmacy services in the ambulatory environment.

Distinct roles for the cell adhesion molecule Contactin2 in the development and function of neural circuits in zebrafish.

Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.

The Kinesin Adaptor Calsyntenin-1 Organizes Microtubule Polarity and Regulates Dynamics during Sensory Axon Arbor Development.

Axon growth and branching, and development of neuronal polarity are critically dependent on proper organization and dynamics of the microtubule (MT) cytoskeleton. MTs must organize with correct polarity for delivery of diverse cargos to appropriate subcellular locations, yet the molecular mechanisms regulating MT polarity remain poorly understood. Moreover, how an actively branching axon reorganizes MTs to direct their plus ends distally at branch points is unknown. We used high-speed, in vivo imaging of polymerizing MT plus ends to characterize MT dynamics in developing sensory axon arbors in zebrafish embryos. We find that axonal MTs are highly dynamic throughout development, and that the peripheral and central axons of sensory neurons show differences in MT behaviors. Furthermore, we show that Calsyntenin-1 (Clstn-1), a kinesin adaptor required for sensory axon branching, also regulates MT polarity in developing axon arbors. In wild type neurons the vast majority of MTs are directed in the correct plus-end-distal orientation from early stages of development. Loss of Clstn-1 causes an increase in MTs polymerizing in the retrograde direction. These misoriented MTs most often are found near growth cones and branch points, suggesting Clstn-1 is particularly important for organizing MT polarity at these locations. Together, our results suggest that Clstn-1, in addition to regulating kinesin-mediated cargo transport, also organizes the underlying MT highway during axon arbor development.