Notch3 and DeltaB maintain Müller glia quiescence and act as negative regulators of regeneration in the light-damaged zebrafish retina.

Damage to the zebrafish retina stimulates resident Müller glia to reprogram, reenter the cell cycle, divide asymmetrically, and produce neuronal progenitor cells that amplify and differentiate into the lost neurons. The transition from quiescent to proliferative Müller glia involves both positive and negative regulators. We previously demonstrated that the Notch signaling pathway represses retinal regeneration by maintaining Müller glia quiescence in zebrafish. Here we examine which Notch receptor is necessary to maintain quiescence. Quantitative RT-PCR and RNA-Seq analyses reveal that notch3 is expressed in the undamaged retina and is downregulated in response to light damage. Additionally, Notch3 protein is expressed in quiescent Müller glia of the undamaged retina, is downregulated as Müller glia proliferate, and is reestablished in the Müller glia. Knockdown of Notch3 is sufficient to induce Müller glia proliferation in undamaged retinas and enhances proliferation during light damage. Alternatively, knockdown of Notch1a, Notch1b, or Notch2 decreases the number of proliferating cells during light damage, suggesting that Notch signaling is also required for proliferation during retinal regeneration. We also knockdown the zebrafish Delta and Delta-like proteins, ligands for the Notch receptors, and find that the deltaB morphant possesses an increased number of proliferating cells in the light-damaged retina. As with Notch3, knockdown of DeltaB is sufficient to induce Müller glia proliferation in the absence of light damage. Taken together, the negative regulation of Müller glia proliferation in zebrafish retinal regeneration is mediated by Notch3 and DeltaB.

Climate anomalies and childhood growth in Peru.

Climate change has been linked to poor childhood growth and development through maternal stress, nutritional insults related to lean harvests, and exposure to infectious diseases. Vulnerable populations are often most susceptible to these stressors. This study tested whether susceptibility to linear growth faltering is higher among Peruvian children from indigenous, rural, low-education, and low-income households. High-resolution weather and household survey data from Demographic and Health Survey 1996-2012 were used to explore height-for-age z-scores (HAZ) at each year of life from 0 to 5. Rural, indigenous children at age 0-1 experience a HAZ reduction of 0.35 units associated with prenatal excess rainfall which is also observed at age 4-5. Urban, non-indigenous children at age 4-5 experience a HAZ increase of 0.07 units associated with postnatal excess rainfall, but this advantage is not seen among rural, indigenous children. These findings highlight the need to consider developmental stage and social predictors as key components in public health interventions targeting increased climate change resilience.

Depression and Marijuana Use among a Sample of Urban Females: Is Stage of Development Important?

BACKGROUND: Increasing rates of marijuana use and high rates of depression among females are major public health concerns and ambiguous study results suggest a complex association between the two. Inconsistent findings may be due to differences in sample characteristics (e.g., females vs. males) and result from differences in the relationship over time (i.e., across developmental stages). OBJECTIVES: Presently, the extent to which the relationship between depression and marijuana use differed for females in emerging adulthood, compared to young adult females, was examined. METHODS: Two hundred forty females (mean age = 23.9 ± 3.4 years) from low-income urban neighborhoods completed a semi-structured HIV-risk interview, Beck's Depression Inventory II, and provided a urine sample that was tested for the presence of psychoactive drugs. RESULTS: Approximately 35% tested positive for marijuana and 21% were experiencing depression. Results from unadjusted logistic regression analyses indicated a relationship between depression and marijuana use (OR = 2.1; 95% CI = 1.08-3.93). African Americans had higher odds of testing positive for marijuana, compared to Whites (OR = 4.6; 95% CI = 2.52-8.68). In the full model, the relationship between depression and marijuana use did not differ by developmental stage. CONCLUSIONS: Interventions aimed at reducing depression may prove beneficial for decreasing marijuana use among females between 18 and 29 years of age residing in disadvantaged communities. However, more research is needed to identify risk factors for marijuana use and to explicate the relationship between marijuana use and depression. Special consideration should be given to African American females.

Coordination in theory, coordination in practice: the case of the Clusters.

The atomised nature of the humanitarian system has led to frequent and repeated attempts to coordinate humanitarian activity. Since 2005, some of the best resourced, and arguably most successful, coordination mechanisms have been the humanitarian Clusters, groups of UN (United Nations) and non-UN actors that engage in sectoral coordination of humanitarian response (such as the provision of healthcare and water) at the global and country level. Nevertheless, it is not clear exactly what 'coordination' means in the context of a Cluster. Formal guidance suggests that they should be aiming to create a single, joint strategy to guide the activities of members. Actual experience of the Clusters, however, indicates that looser forms of coordination are more effective. This finding resonates with organisational theory, and with the experience of emergency management professionals beyond the international humanitarian sector. To capitalise fully on the success of the Clusters, policymakers may need to rethink their attitudes to, and expectations of, coordination.

Text Message Delivered Peer Network Counseling for Adolescent Smokers: A Randomized Controlled Trial.

Although adolescent tobacco use has declined in the last 10 years, African American high school seniors' past 30-day use has increased by 12 %, and as they age they are more likely to report lifetime use of tobacco. Very few urban youth are enrolled in evidenced-based smoking prevention and cessation programming. Therefore, we tested a text messaging smoking cessation intervention designed to engage urban youth through an automated texting program utilizing motivational interviewing-based peer network counseling. We recruited 200 adolescents (90.5 % African American) into a randomized controlled trial that delivered either the experimental intervention of 30 personalized motivational interviewing-based peer network counseling messages, or the attention control intervention, consisting of text messages covering general (non-smoking related) health habits. All adolescents were provided smart phones for the study and were assessed at baseline, and at 1, 3, and 6 months post intervention. Utilizing repeated measures general linear models we examined the effects of the intervention while controlling for race, gender, age, presence of a smoker in the home, and mental health counseling. At 6 months, participants in the experimental condition significantly decreased the number of days they smoked cigarettes and the number of cigarettes they smoked per day; they significantly increased their intentions not to smoke in the future; and significantly increased peer social support among girls. For boys, participants in the experimental condition significantly reduced the number of close friends in their networks who smoke daily compared to those in the control condition. Effect sizes ranged from small to large. These results provide encouraging evidence of the efficacy of text messaging interventions to reduce smoking among adolescents and our intervention holds promise as a large-scale public health preventive intervention platform.

Development and Outcomes of a Text Messaging Tobacco Cessation Intervention With Urban Adolescents.

BACKGROUND: This paper describes the development of an urban adolescent text messaging tobacco cessation intervention and preliminary findings from a randomized, controlled trial. The authors successfully adapted a face-to-face intervention into a personalized, automated, and interactive 5-day texting protocol. METHODS: Respondent-driven sampling was used beginning at a community substance abuse facility. Seventy-two tobacco-dependent adolescents were randomized into an automated computer texting program that delivered either the experimental condition of 30 motivational interviewing- and social network counseling-based personalized messages or the attention control condition consisting of a texting program covering general (non-smoking-related) health habits. All teens were provided smartphones for the study and were assessed at baseline and at 1, 3, and 6 months post intervention. Analyses examined condition×time interactions. RESULTS: At 6 months, the experimental condition decreased the number of cigarettes smoked in the past 30 days, increased intentions not to smoke in the future, and increased peer social support compared with controls. Effect sizes were moderate to large. CONCLUSIONS: These findings are unique, as they target urban adolescents with a mobile health format and add to the growing literature on the efficacy of text-delivered interventions.

Clcf1/Crlf1a-mediated signaling is neuroprotective and required for Müller glia proliferation in the light-damaged zebrafish retina.

Zebrafish possess the innate ability to fully regenerate any neurons lost following a retinal injury. This response is mediated by Müller glia that reprogram and divide asymmetrically to produce neuronal precursor cells that differentiate into the lost neurons. However, little is understood about the early signals that induce this response. Ciliary neurotrophic factor (CNTF) was previously shown to be both neuroprotective and pro-proliferative within the zebrafish retina, however CNTF is not expressed following injury. Here we demonstrate that alternative ligands of the Ciliary neurotrophic factor receptor (CNTFR), such as Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine receptor-like factor 1a (Crlf1a), are expressed within Müller glia of the light-damaged retina. We found that CNTFR, Clcf1, and Crlf1a are required for Müller glia proliferation in the light-damaged retina. Furthermore, intravitreal injection of CLCF1/CRLF1 protected against rod photoreceptor cell death in the light-damaged retina and induced proliferation of rod precursor cells in the undamaged retina, but not Müller glia. While rod precursor cell proliferation was previously shown to be Insulin-like growth factor 1 receptor (IGF-1R)-dependent, co-injection of IGF-1 with CLCF1/CRLF1 failed to induce further proliferation of either Müller glia or rod precursor cells. Together, these findings demonstrate that CNTFR ligands have a neuroprotective effect and are required for induction of Müller glia proliferation in the light-damaged zebrafish retina.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes through Müller glia (MG) reprogramming and asymmetric cell division that produces a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, do MG reprogram to a developmental retinal progenitor cell (RPC) state? Second, to what extent does regeneration recapitulate retinal development? And finally, does loss of different retinal cell subtypes induce unique MG regeneration responses? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. Here we show that injury induces MG to reprogram to a state similar to late-stage RPCs. However, there are major transcriptional differences between MGPCs and RPCs, as well as major transcriptional differences between activated MG and MGPCs when different retinal cell subtypes are damaged. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes.

Gene expression profile of the regeneration epithelium during axolotl limb regeneration.

Urodele amphibians are unique among adult vertebrates in their ability to regenerate missing limbs. The process of limb regeneration requires several key tissues including a regeneration-competent wound epidermis called the regeneration epithelium (RE). We used microarray analysis to profile gene expression of the RE in the axolotl, a Mexican salamander. A list of 125 genes and expressed sequence tags (ESTs) showed a ≥1.5-fold expression in the RE than in a wound epidermis covering a lateral cuff wound. A subset of the RE ESTs and genes were further characterized for expression level changes over the time-course of regeneration. This study provides the first large scale identification of specific gene expression in the RE.

Retinal regeneration requires dynamic Notch signaling.

Retinal damage in the adult zebrafish induces Müller glia reprogramming to produce neuronal progenitor cells that proliferate and differentiate into retinal neurons. Notch signaling, which is a fundamental mechanism known to drive cell-cell communication, is required to maintain Müller glia in a quiescent state in the undamaged retina, and repression of Notch signaling is necessary for Müller glia to reenter the cell cycle. The dynamic regulation of Notch signaling following retinal damage also directs proliferation and neurogenesis of the Müller glia-derived progenitor cells in a robust regeneration response. In contrast, mammalian Müller glia respond to retinal damage by entering a prolonged gliotic state that leads to additional neuronal death and permanent vision loss. Understanding the dynamic regulation of Notch signaling in the zebrafish retina may aid efforts to stimulate Müller glia reprogramming for regeneration of the diseased human retina. Recent findings identified DeltaB and Notch3 as the ligand-receptor pair that serves as the principal regulators of zebrafish Müller glia quiescence. In addition, multi-omics datasets and functional studies indicate that additional Notch receptors, ligands, and target genes regulate cell proliferation and neurogenesis during the regeneration time course. Still, our understanding of Notch signaling during retinal regeneration is limited. To fully appreciate the complex regulation of Notch signaling that is required for successful retinal regeneration, investigation of additional aspects of the pathway, such as post-translational modification of the receptors, ligand endocytosis, and interactions with other fundamental pathways is needed. Here we review various modes of Notch signaling regulation in the context of the vertebrate retina to put recent research in perspective and to identify open areas of inquiry.

Prophylactic Activation of Shh Signaling Attenuates TBI-Induced Seizures in Zebrafish by Modulating Glutamate Excitotoxicity through Eaat2a.

Approximately 2 million individuals experience a traumatic brain injury (TBI) every year in the United States. Secondary injury begins within minutes after TBI, with alterations in cellular function and chemical signaling that contribute to excitotoxicity. Post-traumatic seizures (PTS) are experienced in an increasing number of TBI individuals that also display resistance to traditional anti-seizure medications (ASMs). Sonic hedgehog (Shh) is a signaling pathway that is upregulated following central nervous system damage in zebrafish and aids injury-induced regeneration. Using a modified Marmarou weight drop on adult zebrafish, we examined PTS following TBI and Shh modulation. We found that inhibiting Shh signaling by cyclopamine significantly increased PTS in TBI fish, prolonged the timeframe PTS was observed, and decreased survival across all TBI severities. Shh-inhibited TBI fish failed to respond to traditional ASMs, but were attenuated when treated with CNQX, which blocks ionotropic glutamate receptors. We found that the Smoothened agonist, purmorphamine, increased Eaat2a expression in undamaged brains compared to untreated controls, and purmorphamine treatment reduced glutamate excitotoxicity following TBI. Similarly, purmorphamine reduced PTS, edema, and cognitive deficits in TBI fish, while these pathologies were increased and/or prolonged in cyclopamine-treated TBI fish. However, the increased severity of TBI phenotypes with cyclopamine was reduced by cotreating fish with ceftriaxone, which induces Eaat2a expression. Collectively, these data suggest that Shh signaling induces Eaat2a expression and plays a role in regulating TBI-induced glutamate excitotoxicity and TBI sequelae.

Gene regulatory networks controlling vertebrate retinal regeneration.

Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick, and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity, and neurogenesis. In zebrafish and chick, the transition from quiescence to reactivity is essential for retinal regeneration, whereas in mice, a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice after injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal.

The outer segment of the vertebrate rod photoreceptor is a highly modified cilium composed of many discrete membranous discs that are filled with the protein machinery necessary for phototransduction. The unique outer segment structure is renewed daily with growth at the base of the outer segment where new discs are formed and shedding at the distal end where old discs are phagocytized by the retinal pigment epithelium. In order to understand how outer segment renewal is regulated to maintain outer segment length and function, we used a small molecule screening approach with the transgenic (hsp70:HA-mCherryTM) zebrafish, which expresses a genetically-encoded marker of outer segment renewal. We identified compounds with known bioactivity that affect five content areas: outer segment growth, outer segment shedding, clearance of shed outer segment tips, Rhodopsin mislocalization, and differentiation at the ciliary marginal zone. Signaling pathways that are targeted by the identified compounds include cyclooxygenase in outer segment growth, γ-Secretase in outer segment shedding, and mTor in RPE phagocytosis. The data generated by this screen provides a foundation for further investigation of the signaling pathways that regulate photoreceptor outer segment renewal.

How time flies: a study of novice skydivers.

Although time distortion is commonly reported during a traumatic experience, there is little research addressing the phenomenon. This study investigated the role of affect in time perception in a very stressful experience by indexing novice tandem skydivers' (N=76) levels of fear and excitement before the skydive and soon after landing. Estimations of how long skydivers thought their experience lasted were obtained after landing. Whereas increased fear was associated with the perception of time passing slowly, increased excitement was associated with the perception of time passing quickly. These data support models of time perception based on avoidance and approach motivations influencing time distortion.

Opportunities for CRISPR/Cas9 Gene Editing in Retinal Regeneration Research.

While retinal degeneration and disease results in permanent damage and vision loss in humans, the severely damaged zebrafish retina has a high capacity to regenerate lost neurons and restore visual behaviors. Advancements in understanding the molecular and cellular basis of this regeneration response give hope that strategies and therapeutics may be developed to restore sight to blind and visually-impaired individuals. Our current understanding has been facilitated by the amenability of zebrafish to molecular tools, imaging techniques, and forward and reverse genetic approaches. Accordingly, the zebrafish research community has developed a diverse array of research tools for use in developing and adult animals, including toolkits for facilitating the generation of transgenic animals, systems for inducible, cell-specific transgene expression, and the creation of knockout alleles for nearly every protein coding gene. As CRISPR/Cas9 genome editing has begun to revolutionize molecular biology research, the zebrafish community has responded in stride by developing CRISPR/Cas9 techniques for the zebrafish as well as incorporating CRISPR/Cas9 into available toolsets. The application of CRISPR/Cas9 to retinal regeneration research will undoubtedly bring us closer to understanding the mechanisms underlying retinal repair and vision restoration in the zebrafish, as well as developing therapeutic approaches that will restore vision to blind and visually-impaired individuals. This review focuses on how CRISPR/Cas9 has been integrated into zebrafish research toolsets and how this new tool will revolutionize the field of retinal regeneration research.

Phosphodiesterase Inhibitors Sildenafil and Vardenafil Reduce Zebrafish Rod Photoreceptor Outer Segment Shedding.

Purpose: The vertebrate rod photoreceptor undergoes daily growth and shedding to renew the rod outer segment (ROS), a modified cilium that contains the phototransduction machinery. It has been demonstrated that ROS shedding is regulated by the light-dark cycle; however, we do not yet have a satisfactory understanding of the molecular mechanisms that underlie this regulation. Given that phototransduction relies on the hydrolysis of cGMP via phosphodiesterase 6 (PDE6), we examined ROS growth and shedding in zebrafish treated with cGMP-specific PDE inhibitors. Methods: We used transgenic zebrafish that express an inducible, transmembrane-bound mCherry protein, which forms a stripe in the ROS following a heat shock pulse and serves as a marker of ROS renewal. Zebrafish were reared in constant darkness or treated with PDE inhibitors following heat shock. Measurements of growth and shedding were analyzed in confocal z-stacks collected from treated retinas. Results: As in dark-reared zebrafish, shedding was reduced in larvae and adults treated with the PDE5/6 inhibitors sildenafil and vardenafil but not with the PDE5 inhibitor tadalafil. In addition, vardenafil noticeably affected rod inner segment morphology. The inhibitory effect of sildenafil on shedding was reversible with drug removal. Finally, cones were more sensitive than rods to the toxic effects of sildenafil and vardenafil. Conclusions: We show that pharmacologic inhibition of PDE6 mimics the inhibition of shedding by prolonged constant darkness. The data show that the influence of the light-dark cycle on ROS renewal is regulated, in part, by initiating the shedding process through activation of the phototransduction machinery.

Association of exposure to neighborhood drug activity, neurobehavioral traits, and marijuana use among at-risk African American females.

INTRODUCTION: Theories of relative deprivation suggest African Americans in disadvantaged communities are at increased risk for drug use. This increased risk may be due, in part, to exposure to drugs and drug subcultures. Given the significance of the prefrontal cortex (PFC) functioning in yielding behavior that is strategically guided rather than reactive to environmental demands, it is important to examine the relationship between PFC functioning, neighborhood drug activity and substance use among African Americans residing in high risk communities. METHODS: A sample of 120 young adult African American females was recruited from high-risk neighborhoods. Each completed the modified version of the neighborhood environment scale, a neurobehavioral assessment designed to measure apathy, behavioral disinhibition and executive dysfunction, and provided a urine sample that was tested for the presence of psychoactive drugs. RESULTS: Logistic regression analyses indicated that females with higher scores on behavioral disinhibition were 2.6 times more likely to test positive for marijuana (95%CI = 1.02, 6.57). Neither apathy nor executive dysfunction was related to marijuana use. No relationship emerged between neighborhood drug activity and marijuana use. CONCLUSIONS: Among the neurobehavioral traits considered only behavioral disinhibition was associated with marijuana use, suggesting that different neurobehavioral domains may be uniquely related to marijuana use. For females living in high risk environments, the extent to which they are able to control impulses may provide some protection against marijuana use. Future studies focused on the moderating effects of behavioral disinhibition on the association of exposure to risk environments and marijuana use may prove beneficial. Further, the study adds to the small base of literature supporting the Frontal Systems Behavior Scale as a brief assessment to evaluate frontally-mediated neurobehavioral traits relevant to substance use. However, future studies aimed at examining the influence of neighborhood drug activity might benefit from more precise measures of exposure to neighborhood drug activity. More research to replicate and expand on the present findings is warranted.