Peripheral nerve development in zebrafish requires muscle patterning by tcf15/paraxis.

The vertebrate peripheral nervous system (PNS) is an intricate network that conveys sensory and motor information throughout the body. During development, extracellular cues direct the migration of axons and glia through peripheral tissues. Currently, the suite of molecules that govern PNS axon-glial patterning is incompletely understood. To elucidate factors that are critical for peripheral nerve development, we characterized the novel zebrafish mutant, stl159, that exhibits abnormalities in PNS patterning. In these mutants, motor and sensory nerves that develop adjacent to axial muscle fail to extend normally, and neuromasts in the posterior lateral line system, as well as neural crest-derived melanocytes, are incorrectly positioned. The stl159 genetic lesion lies in the basic helix-loop-helix (bHLH) transcription factor tcf15, which has been previously implicated in proper development of axial muscles. We find that targeted loss of tcf15 via CRISPR-Cas9 genome editing results in the PNS patterning abnormalities observed in stl159 mutants. Because tcf15 is expressed in developing muscle prior to nerve extension, rather than in neurons or glia, we predict that tcf15 non-cell-autonomously promotes peripheral nerve patterning in zebrafish through regulation of extracellular patterning cues. Our work underscores the importance of muscle-derived factors in PNS development.

Pretraining of basic skills on a virtual reality vitreoretinal simulator: A waste of time.

PURPOSE: To investigate whether pretraining of basic skills in virtual vitreoretinal surgery affected the performance curve when proceeding to procedure-specific modules. METHODS: This study was a prospective, randomized, controlled, two-centre study. Medical students were randomized into two groups: Group 1 pretrained basic psycho-motor skills (Navigation Training level 2 and Bimanual Training level 3) until they reached their performance curve plateau. Hereafter, both groups trained on the procedure-specific modules (Posterior Hyaloid level 3 and ILM Peeling level 3) until they reached their performance curve plateau. Plateau was defined as three consecutive sessions with the same score with an acceptable variation. The primary outcome was time used to reach performance curve plateau in the procedure-specific modules. RESULTS: A total of 68 medical students were included, and equally randomized into two groups. The participants in Group 1 used a median time of 88 minutes to reach plateau in the basic skills modules but did not differ from Group 2 in time to reach plateau on the procedure-specific modules (183 min versus 210 min, p = 0.40) or in the amplitude of plateau. Group 1 and 2 differed significantly in the starting score of ILM peeling level 3 (0 (0-0) versus 3.5 (0-75), p = 0.03). CONCLUSION: We were not able to show positive skill transfer from basic skills training to the procedure-specific modules in time, starting score or amplitude of plateau. Thus, we recommend that aspiring vitreoretinal surgeons proceed directly to simulation-based training of procedures instead of spending valuable training time on basic skills training.

Open-Ended Inquiry into Zebrafish Nerve Development Using Image Analysis.

Open-ended laboratory projects increase student success and retention in the sciences. However, developing organismal-based research projects is a challenge for students with restricted laboratory access, such as those attending courses remotely. Here I describe the use of image analysis of zebrafish neural development for authentic research projects in an introductory biology laboratory course. Zebrafish are a vertebrate model that produce large numbers of externally and rapidly developing embryos. Because zebrafish larvae are transparent, fluorescent reporters marking nervous system structures can be imaged over time and analyzed by undergraduate scientists. In the pilot of this project, remote first-year college students independently developed biological questions based on an image collection comparing zebrafish mutants and wild-type siblings. Students created and mastered techniques to analyze position, organization, and other morphological features of developing neurons and glia in the images to directly test their biological questions. At the end of the course, students communicated their project results in journal article format and oral presentations. Students were able to hone skills in organismal observation and data collection while studying remotely, and they reported excitement at applying lecture-based knowledge to their own independent questions. This module can be adapted by other instructors for both students on- and off-campus to teach principles of neural development, data collection, data analysis, and scientific communication.

Schwann cell development: From neural crest to myelin sheath.

Vertebrate nervous system function requires glial cells, including myelinating glia that insulate axons and provide trophic support that allows for efficient signal propagation by neurons. In vertebrate peripheral nervous systems, neural crest-derived glial cells known as Schwann cells (SCs) generate myelin by encompassing and iteratively wrapping membrane around single axon segments. SC gliogenesis and neurogenesis are intimately linked and governed by a complex molecular environment that shapes their developmental trajectory. Changes in this external milieu drive developing SCs through a series of distinct morphological and transcriptional stages from the neural crest to a variety of glial derivatives, including the myelinating sublineage. Cues originate from the extracellular matrix, adjacent axons, and the developing SC basal lamina to trigger intracellular signaling cascades and gene expression changes that specify stages and transitions in SC development. Here, we integrate the findings from in vitro neuron-glia co-culture experiments with in vivo studies investigating SC development, particularly in zebrafish and mouse, to highlight critical factors that specify SC fate. Ultimately, we connect classic biochemical and mutant studies with modern genetic and visualization tools that have elucidated the dynamics of SC development. This article is categorized under: Signaling Pathways > Cell Fate Signaling Nervous System Development > Vertebrates: Regional Development.

Mini-Review - Teaching Writing in the Undergraduate Neuroscience Curriculum: Its Importance and Best Practices.

In neuroscience and other scientific disciplines, instructors increasingly appreciate the value of writing. Teaching students to write well helps them succeed in school, not only because they perform better on assessments but also because well-structured writing assignments improve learning. Moreover, the ability to write well is an essential professional skill, because good clear writing in conjunction with good clear thinking results in increased success in fellowship applications, grant proposals, and publications. However, teaching writing in neuroscience classrooms is challenging for several reasons. Students may not initially recognize the importance of writing, teachers may lack training in the pedagogy of writing instruction, and both teachers and students must commit substantial time and effort to writing if progress is to be made. Here, we detail effective strategies for teaching writing to undergraduates, including scaffolding of teaching assignments, both within a class and across a curriculum; use of different types of writing assignments; early integration of writing into courses; peer review and revision of assignments; mentoring by student tutors; and use of defined rubrics. We also discuss how these strategies can be utilized effectively in the context of multicultural classrooms and labs.

[Simulation-based training in ophthalmologic surgery].

Over the past few decades, simulation-based training has become an increasingly widespread learning tool for training in medical procedures. This type of training provides the opportunity to learn in a safe simulation-based environment, where both technical and diagnostic skills can be developed. In Denmark, there has been developed evidence for simulation-based training in ophthalmology in cataract and vitreoretinal surgery. This form of evidence-based training is meaningful for real clinical setting, and in this review, we argue, that is important to apply for all medical fields.

Prenatal Predictors of Media Use During Infancy.

Parental attitudes and behavior can impact infant media use, though all existing research examines this after the baby is born. However, many studies suggest that prenatal attitudes and behavior can influence parenting practices around many different types of parenting decisions. This study examines whether this extends to parenting practices surrounding media use during infancy. Participants consisted of 170 mothers who completed a number of questionnaires at two time points (prenatally and when infant was ∼16 months old). Results revealed that parents had many concerns about media before their child was born. More supportive prenatal attitudes regarding using media as a coping strategy was associated with higher levels of infant media use and technoference (i.e., when media interfere with the parent-child relationship). Additionally, prenatal media use by mothers was associated with higher levels of infant media use, but lower levels of technoference. Finally, prenatal depression was associated with higher technoference, while parental efficacy was associated with higher infant video chat. Implications of the study include discussion regarding media use at medical office prenatal visits and creating a family media plan before the birth of the child.

In vivo identification of small molecules mediating Gpr126/Adgrg6 signaling during Schwann cell development.

Gpr126/Adgrg6, an adhesion family G protein-coupled receptor (aGPCR), is required for the development of myelinating Schwann cells in the peripheral nervous system. Myelin supports and insulates vertebrate axons to permit rapid signal propagation throughout the nervous system. In mammals and zebrafish, mutations in Gpr126 arrest Schwann cells at early developmental stages. We exploited the optical and pharmacological tractability of larval zebrafish to uncover drugs that mediate myelination by activating Gpr126 or functioning in parallel. Using a fluorescent marker of mature myelinating glia (Tg[mbp:EGFP-CAAX]), we screened hypomorphic gpr126 mutant larvae for restoration of myelin basic protein (mbp) expression along peripheral nerves following small molecule treatment. Our screens identified five compounds sufficient to promote mbp expression in gpr126 hypomorphs. Using an allelic series of gpr126 mutants, we parsed the ability of small molecules to restore mbp, suggesting differences in drug efficacy dependent on Schwann cell developmental state. Finally, we identify apomorphine hydrochloride as a direct small molecule activator of Gpr126 using combined in vivo/in vitro assays and show that aporphine class compounds promote Schwann cell development in vivo. Our results demonstrate the utility of in vivo screening for aGPCR modulators and identify small molecules that interact with the gpr126-mediated myelination program.

2017 Midwest Zebrafish Meeting Report.

The 2017 Midwest Zebrafish meeting was held from June 16 to 18 at the University of Cincinnati, sponsored by the Cincinnati Children's Hospital Divisions of Developmental Biology, Molecular Cardiovascular Biology, and Gastroenterology, Hepatology, and Nutrition. The meeting, organized by Saulius Sumanas, Joshua Waxman, and Chunyue Yin, hosted >130 attendees from 16 different states. Scientific sessions were focused on morphogenesis, neural development, novel technologies, and disease models, with Steve Ekker, Stephen Potter, and Lila Solnica-Krezel presenting keynote talks. In this article, we highlight the results and emerging themes from the meeting.

The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6.

Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options.

The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons.

Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons.

Neurobiology: Myelin Goes Where the Action Is.

There is increasing evidence that neuronal activity modulates how axons are wrapped in myelin. Two recent studies demonstrate that activity-dependent vesicle release from neurons regulates myelination in vivo.

The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with laminin-211.

Myelin ensheathes axons to allow rapid propagation of action potentials and proper nervous system function. In the peripheral nervous system, Schwann cells (SCs) radially sort axons into a 1:1 relationship before wrapping an axonal segment to form myelin. SC myelination requires the adhesion G protein-coupled receptor GPR126, which undergoes autoproteolytic cleavage into an N-terminal fragment (NTF) and a seven-transmembrane-containing C-terminal fragment (CTF). Here we show that GPR126 has domain-specific functions in SC development whereby the NTF is necessary and sufficient for axon sorting, whereas the CTF promotes wrapping through cAMP elevation. These biphasic roles of GPR126 are governed by interactions with Laminin-211, which we define as a novel ligand for GPR126 that modulates receptor signaling via a tethered agonist. Our work suggests a model in which Laminin-211 mediates GPR126-induced cAMP levels to control early and late stages of SC development.

A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.

Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes and immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs (GPR126 and GPR133) functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the seven-transmembrane helix domain, which triggers G protein activation. Our studies show high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a mode of aGPCR activation and may prompt the development of specific ligands for this currently untargeted GPCR family.

A transcriptional program promotes remodeling of GABAergic synapses in Caenorhabditis elegans.

Although transcription factors are known to regulate synaptic plasticity, downstream genes that contribute to neural circuit remodeling are largely undefined. In Caenorhabditis elegans, GABAergic Dorsal D (DD) motor neuron synapses are relocated to new sites during larval development. This remodeling program is blocked in Ventral D (VD) GABAergic motor neurons by the COUP-TF (chicken ovalbumin upstream promoter transcription factor) homolog, UNC-55. We exploited this UNC-55 function to identify downstream synaptic remodeling genes that encode a diverse array of protein types including ion channels, cytoskeletal components, and transcription factors. We show that one of these targets, the Iroquois-like homeodomain protein, IRX-1, functions as a key regulator of remodeling in DD neurons. Our discovery of irx-1 as an unc-55-regulated target defines a transcriptional pathway that orchestrates an intricate synaptic remodeling program. Moreover, the well established roles of these conserved transcription factors in mammalian neural development suggest that a similar cascade may also control synaptic plasticity in more complex nervous systems.

A spatial and temporal map of C. elegans gene expression.

The C. elegans genome has been completely sequenced, and the developmental anatomy of this model organism is described at single-cell resolution. Here we utilize strategies that exploit this precisely defined architecture to link gene expression to cell type. We obtained RNAs from specific cells and from each developmental stage using tissue-specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA-tagging method. We then generated gene expression profiles of more than 30 different cells and developmental stages using tiling arrays. Machine-learning-based analysis detected transcripts corresponding to established gene models and revealed novel transcriptionally active regions (TARs) in noncoding domains that comprise at least 10% of the total C. elegans genome. Our results show that about 75% of transcripts with detectable expression are differentially expressed among developmental stages and across cell types. Examination of known tissue- and cell-specific transcripts validates these data sets and suggests that newly identified TARs may exercise cell-specific functions. Additionally, we used self-organizing maps to define groups of coregulated transcripts and applied regulatory element analysis to identify known transcription factor- and miRNA-binding sites, as well as novel motifs that likely function to control subsets of these genes. By using cell-specific, whole-genome profiling strategies, we have detected a large number of novel transcripts and produced high-resolution gene expression maps that provide a basis for establishing the roles of individual genes in cellular differentiation.

Validation of an electronic system for recording medical student patient encounters.

The Liaison Committee for Medical Education requires monitoring of the students clinical experiences. Student logs, typically used for this purpose, have a number of limitations. We used an electronic system called Patient Tracker to passively generate student encounter data. The data contained in Patient Tracker was compared to the information reported on student logs and data abstracted from the patients charts. Patient Tracker identified 30% more encounters than the student logs. Compared to the student logs, Patient Tracker contained a higher average number of diagnoses per encounter (2.28 vs. 1.03, p<0.01). The diagnostic data contained in Patient Tracker was also more accurate under 4 different definitions of accuracy. Only 1.3% (9/677) of diagnoses in Patient Tracker vs. 16.9% (102/601) diagnoses in the logs could not be validated in patients charts (p<0.01). Patient Tracker is a more effective and accurate tool for documenting student clinical encounters than the conventional student logs.

A new developmentalist role: connecting youth development, mental health, and education.

The RALLY practitioner implements RALLY's prevention and intervention strategies, working with all of the students in a class to deliver nonstigmatizing, developmentally based services. The practitioner model is based on the philosophy that relationships are key to allowing students to achieve their full health and academic potential. RALLY practitioners work within the classroom individually, in small groups, and in after-school time. In all of these contexts, RALLY practitioners focus on four major functions: (1) building strong relationships with students, (2) providing developmental and academic support, (3) referring students to services based on their needs and interests, and (4) connecting with teachers, families, clinicians, after-school staff, and other service providers in order to bridge these worlds. RALLY practitioners offer an in-novative model to bring mental health and educational support into schools. They have varying educational backgrounds, from high school graduates, to college students, to graduate interns in mental health or education professions. Training and supervision, however, are crucial in helping practitioners of all levels to create integrated systems of support that take all of a student's interests and needs into consideration while moving toward academic success.

Reinventing clinical roles and space at school.

Recognizing both the need for youth-focused acute mental health services and the barriers for low-income families to access outside services, the RALLY Program expanded its services to include direct clinical services for students within the school setting. This article explores the challenges, strategies, and benefits of implementing a fluid range of formal and informal clinical interventions within RALLY's nonstigmatizing, developmental, and inclusive approach. Balancing insurance company demands with students' nonbillable needs requires diverse funding streams and responsive programming. Creative use of space, commitment to relationships, and flexibility of roles form the foundation of this approach. Through case studies, the author examines practical and creative applications of developmental theories adaptable to individual students' unique needs. The author concludes with recommendations to the field to strengthen nonstigmatizing services offered to address the holistic needs of youth at school.