Falling Stereotypes and New Cell Models in Neurobiology

A major goal in neurobiology is to study the mechanisms by which neurites navigate towards their targets to establish functional synapses. While a large amount of information is available on the extra cellular signals driving neuritogenesis and synaptogenesis, major gaps exist in the comprehension of the intracellular events. <br><br>Rho family GTPases regulate the dynamic of the actin cytoskeleton and plays a crucial role in regulating neuronal development. In particular, Rac has been implicated in axon elongation and guidance, in dendritic growth and in the regulation of dendritic spines formation and plasticity. There are three Rac genes in mammals: Rac1, Rac2 and Rac3, sharing about 90% protein identity, all participating in several functions, including actin dynamics and adhesion (Hall, 1998). Rac1 is ubiquitous, while Rac2 is specific to the hematopoietic lineage and Rac3 is developmentally regulated in the vertebrate brain (Corbetta et al., 2005), with the highest expression during neurite branching and synaptogenesis (Bolis et al., 2003). The co-expression of Rac1 and Rac3 in the vertebrate nervous system suggests that they are needed to solve individual developmental and maintenance issues in the nervous system. <br><br>The aim of my work is to analyse the function of Rac1 and Rac3 during nervous system development. I took advantage of both <i>in vitro</i> and <i>in vivo</i> approaches to answer this question. Comparison between wild-type and Rac3-/- hippocampal neurons showed no differences in neuritogenesis and synaptogenesis processes. On the other hand, Rac1 depletion by RNAi in both wild-type and Rac3-/- hippocampal neurons during early stage of neuronal development showed specific alteration of the dendritic development without affecting axonal outgrowth. Reduced Rac1 levels impaired growth cone dynamics and F-actin levels in both neurites and growth cones. These results show that endogenous Rac1 rather than Rac3 activity is essential for early dendritic development, but not required for the initial establishment of neuronal polarity and axonal development. Depletion of either Rac1 or Rac3 during later stages of <i>in vitro</i> neuronal development did not severe dendritic spines morphogenesis, while double depletion strongly impaired the formation and maturation of dendritic spines. All together these <i>in vitro</i> results reinforce the hypothesis that Rac1 and Rac3 are both important for neuronal development underlying a specific role for Rac3 in later events of neuronal development. <br><br>We next evaluated the effects of the depletion of Rac1 and Rac3 <i>in vivo</i>. I generated mice with conditional deletion of Rac1 in neurons (Rac1N), by crossing Rac1Flox/Flox mice (Walmsley et al., 2003) with transgenic Synapsin-Cre mice (Zhu et al., 2001) and double RaclN/Rac3-/- mice. <br><br>As Rac3-/-, also Rac1N developed normally and was fertile. Both Rac3-/- and Rac1N did not reveal major abnormalities in the brain cytoarchitecture, cell layering or alteration in the general organization of dendrites and synapses. Double RaclN/Rac3-/- mice died around 13 days after birth (P13) and showed strong neurological defects. Morphological analysis revealed evident alterations in the hippocampus. The abnormalities in the organization of the hippocampus may be correlated with the frequent spontaneous epileptic seizures developing after P7 in these mice. We found that double depletion of Rac1 and Rac3 induced an increase of WAVE1 (Ser310) and PAK1 (Thr423) phosphorylation, and reduced the total levels of RhoA. All together the data obtained in this thesis show for the first time that both Rac1 and Rac3 are important for neuronal development, while mutation of each gene leads to viable animals with no evident anatomical defects due to partial compensatory effects.

Students in elementary statistics traditionally see experiments and data as words and numbers in a text. They receive little exposure to the important statistical activities of sample selection, data collection, experimental design, development of statistical models, the need for randomization, selection of factors, etc. They often leave the first course without a firm understanding of the role of applied statistics or of the statistician in scientific investigations. In an attempt to improve elementary statistics education, we have developed a statistics laboratory similar to those of other elementary science courses. We will discuss our experiences in teaching a laboratory component with the traditional elementary statistics course. In each lab session, students, working in teams, discuss the design of an experiment, carry out the experiment, and analyze their data using Minitab on a Macintosh or MS-DOS based computer. The students then individually either answer a series of short answer questions or write a formal scientific report. The labs are designed to be relatively inexpensive and portable. They do not require a prior background in science, statistics or computing.

The introduction of microtechniques in elementary science courses: I. Blood glucose determination

The introduction of microtechniques in elementary science courses: II. Serum cholesterol and cholesterol ester determination

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Efficient neurotransmission is essential for organism survival and is enhanced by myelination. However, the genes that regulate myelin and myelinating glial cell development have not been fully characterized. Data from our lab and others demonstrates that cd59, which encodes for a small GPI-anchored glycoprotein, is highly expressed in developing zebrafish, rodent, and human oligodendrocytes (OLs) and Schwann cells (SCs), and that patients with CD59 dysfunction develop neurological dysfunction during early childhood. Yet, the function of Cd59 in the developing nervous system is currently undefined. In this study, we demonstrate that cd59 is expressed in a subset of developing SCs. Using cd59 mutant zebrafish, we show that developing SCs proliferate excessively and nerves may have reduced myelin volume, altered myelin ultrastructure, and perturbed node of Ranvier assembly. Finally, we demonstrate that complement activity is elevated in cd59 mutants and that inhibiting inflammation restores SC proliferation, myelin volume, and nodes of Ranvier to wildtype levels. Together, this work identifies Cd59 and developmental inflammation as key players in myelinating glial cell development, highlighting the collaboration between glia and the innate immune system to ensure normal neural development. Editor's evaluation This work investigates the function of the small GPI-anchored protein Cd59, a protein known to suppress complement-mediated inflammation, in Schwann cell development and myelination, using zebrafish as a model system. This article will be of interest to developmental biologists, glial biologists, and immunologists as it suggests the interesting and novel findings that Cd59 regulates Schwann cell development, mainly by modulating Schwann cell proliferation. https://doi.org/10.7554/eLife.76640.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest The nervous system of vertebrates is made of up of complex networks of nerve cells that send signals to one another. In addition to these cells, there are a number of supporting cells that help nerves carry out their role. Schwann cells, for example, help nerve cells to transmit information faster by wrapping their long extensions in a fatty membrane called myelin. When myelin is not produced properly, this can disturb the signals between nerve cells, leading to neurological defects. Schwann cells mature simultaneously with nerve cells in the embryo. However, it was not fully understood how Schwann cells generate myelin during development. To investigate, Wiltbank et al. studied the embryos of zebrafish, which, unlike other vertebrates, are transparent and develop outside of the womb. These qualities make it easier to observe how cells in the nervous system grow in real-time using a microscope. First, the team analyzed genetic data collected from the embryo of zebrafish and mice to search for genes that are highly abundant in Schwann cells during development. This led to the discovery of a gene called cd59, which codes for a protein that also interacts with the immune system. To find out whether Schwann cells rely on cd59, Wiltbank et al. deleted the cd59 gene in zebrafish embryos. Without cd59, the Schwann cells produced too many copies of themselves and this, in turn, impaired the appropriate production of myelin. Since the protein encoded by cd59 normally balances inflammation levels, it was possible that losing this gene overactivated the immune system in the zebrafish embryos. In support of this hypothesis, when the cd59 mutant embryos were treated with an anti-inflammatory drug, this corrected Schwann cell overproduction and restored myelin formation. Taken together, these findings reveal how inflammation helps determine the number of Schwann cells produced during development, and that cd59 prevents this process from getting carried away. This suggests that the nervous system and immune system may work together to build the nervous system. In the future, it will be interesting to investigate whether cd59 acts in a similar way during the development of the human nervous system. Introduction Myelin is a highly specialized, lipid-rich membrane that insulates axons in the nervous system to enhance neurotransmission (Ritchie, 1982) while preserving axon health (Stadelmann et al., 2019). Myelin is produced and maintained by myelinating glial cells such as motor exit point glia (Fontenas and Kucenas, 2021; Fontenas and Kucenas, 2018; Smith et al., 2014) and myelinating Schwann cells (SCs; Figure 1A; Ackerman and Monk, 2016; BEN GEREN, 1954; Jessen and Mirsky, 2005) in the peripheral nervous system (PNS) as well as oligodendrocytes (OLs) in the central nervous system (CNS) (Figure 1B; Ackerman and Monk, 2016; Maturana, 1960; Nave and Werner, 2014; Peters, 1960a; Peters, 1960b). Normal myelin and myelinating glial cell development facilitate an efficient and effective nervous system, ensuring precise motor, sensory, and cognitive function (Almeida and Lyons, 2017; Berger et al., 2006; Stadelmann et al., 2019; Wei et al., 2019). When this process is impaired, as seen with inherited disorders that cause abnormal myelination, patients present with neurological dysfunction that often leads to severe physical and intellectual disabilities (Berger et al., 2006; Stadelmann et al., 2019; van der Knaap and Bugiani, 2017; Wei et al., 2019). Figure 1 with 1 supplement see all Download asset Open asset cd59 is expressed in myelinating glial cells during nervous system development. (A) Timeline of Schwann cell (SC) (orange) development (top panel). SC developmental stages for zebrafish (hours post fertilization [hpf]) and mice (embryonic day [E]) are indicated in the bottom panel. (B) Timeline of oligodendrocyte (OL) (blue) development. OL developmental stages for zebrafish (hpf) and mice (E and postnatal day) are indicated in the bottom panel. (C) Scatter plot of cd59 expression (TPM) in oligodendrocyte lineage cells (OLCs), SCs, and neurons (N) at 72 hpf (left; mean ± SEM: OLC: 2145.1 ± 1215.1; SC: 40.1 ± 16.3; N: 240.5 ± 173.3; dot = replicate) as well as SCs at 36 and 72 hpf (right; mean ± SEM: 36 hpf: 0.0 ± 0.0, 72 hpf: 40.2 ± 16.3; dot = replicate). (D) Schematic of the relative cd59/Cd59a expression in developing SCs (orange) and OLs (blue) determined from RNAseq analysis in Figure 1—figure supplement 1. Developmental stage numbers correspond with stages indicated in (A) and (B). Artwork created by Ashtyn T. Wiltbank with Illustrator (Adobe) based on previous schematics and electron micrographs published in Ackerman and Monk, 2016; Cunningham and Monk, 2018; Jessen and Mirsky, 2005. Figure 1—source data 1 Source data for cd59 bulk, RNAseq expression depicted in Figure 1C. Data contributed to scatter plot of cd59 expression (TPM) in oligodendrocyte lineage cells (OLCs), Schwann cells (SCs), and neurons (N) at 72 hours post fertilization (hpf) (left; mean ± SEM: OLC: 2145.1 ± 1215.1, SC: 40.1 ± 16.3; N: 240.5 ± 173.3; data point = replicate) as well as SCs at 36 and 72 hpf (right; mean ± SEM: 36 hpf: 0.0 ± 0.0, 72 hpf: 40.2 ± 16.3; data point = replicate). https://cdn.elifesciences.org/articles/76640/elife-76640-fig1-data1-v1.xlsx Download elife-76640-fig1-data1-v1.xlsx Despite the importance of myelin, we still lack a complete understanding of myelin and myelinating glial cell development. With the recent boon of transcriptomic and proteomic analyses, many genes and proteins have been highlighted as differentially expressed during myelinating glial cell development, yet their precise functions remain unknown. For example, cd59, a gene that encodes for a small, glycosylphosphotidylinositol (GPI)-anchored glycoprotein, is a particularly interesting candidate for exploration. This gene features in several RNA sequencing (RNAseq) and proteomic analyses of zebrafish and rodent myelinating glial cells (Gerber et al., 2021; Howard et al., 2021; Marisca et al., 2020; Marques et al., 2018; Marques et al., 2016; Piller et al., 2021; Saunders et al., 2019; Siems et al., 2021; Wolbert et al., 2020; Zhu et al., 2019), all of which demonstrate high expression of cd59 in developing oligodendrocyte lineage cells (OLCs) and SCs. cd59 continues to be expressed in juvenile and adult zebrafish (Saunders et al., 2019; Siems et al., 2021; Sun et al., 2013) and becomes the fourth most abundant CNS myelin protein in adult zebrafish (Siems et al., 2021). Phylogenetic analysis demonstrates that Cd59 is conserved within vertebrate evolution (Siems et al., 2021), and accordingly, CD59 protein is also found in developing and adult human myelin (Erne et al., 2002; Koski et al., 2002; Scolding et al., 1998; Zajicek et al., 1995). Collectively, these data indicate that cd59 is a key component of the genetic program that orchestrates myelinating glial cell development. However, despite this knowledge of Cd59 expression, little is known about the function of Cd59 in the developing nervous system. Outside of the nervous system, Cd59 is best known as a complement inhibitory protein. Complement is a molecular cascade within the innate immune system that aids in cell lysis of invading pathogens or aberrant cells in the body (Merle et al., 2015). This process lacks specificity and requires inhibitory proteins, such as Cd59, to protect healthy cells from lytic death (Davies et al., 1989). Cd59, specifically, acts at the end of the complement cascade where it binds to complement proteins 8 and 9 (C8 and C9, respectively) to prevent the polymerization of C9 and the subsequent formation of lytic pores (also known as membrane attack complexes [MACs] or terminal complement complexes [TCCs]) in healthy cell membranes (Meri et al., 1990; Ninomiya and Sims, 1992; Rollins et al., 1991). This interaction is important throughout the adult body, including the nervous system, where CD59a is neuroprotective in models of multiple sclerosis (Mead et al., 2004) and neuromyelitis optica (Yao and Verkman, 2017). Beyond complement-inhibitory functions, Cd59 can also facilitate vesicle signaling in insulin-producing pancreatic cells (Golec et al., 2019; Krus et al., 2014), suppress cell proliferation in T cells responding to a viral infection (Longhi et al., 2005) or smooth muscle cells in models of atherosclerosis (Li et al., 2013), and orchestrate proximal-distal cell identity during limb regeneration (Echeverri and Tanaka, 2005). Like complement inhibition, these processes are critical to normal nervous system function (Almeida et al., 2021; Baron and Hoekstra, 2010; Reiter and Bongarzone, 2020; White and Krämer-Albers, 2014). However, it is unclear exactly what role Cd59 is playing in myelinating glial cell development. Though the precise function is unknown, it is clear that CD59 does impact human nervous system development. Patients with CD59 dysfunction, such as those with congenital CD59 deficiency (Haliloglu et al., 2015; Höchsmann and Schrezenmeier, 2015; Karbian et al., 2018; Solmaz et al., 2020) or germline paroxysmal nocturnal hemoglobinuria (Johnston et al., 2012), present with polyneuropathies during infancy and continue to have nervous system dysfunction throughout their lives. Intriguingly, these neurological symptoms persist even with complement inhibition, the most common treatment for congenital CD59 deficiency (Höchsmann and Schrezenmeier, 2015). Together, these observations further indicate that CD59 has an additional role in the developing nervous system and requires further investigation. Here, utilizing the zebrafish model, we examined the role of Cd59 in the developing nervous system. In this study, we found that cd59 is highly expressed in a subset of developing SCs in addition to mature OLs and myelinating and nonmyelinating SCs. We chose to focus on SCs and found that cd59 mutant zebrafish have excessive SC proliferation. These mutants also have impaired myelin and node of Ranvier development. Finally, we demonstrate that complement activity increases in cd59 mutants and that unregulated inflammation contributes to SC overproliferation and mutant larvae may also have aberrant myelin and node of Ranvier formation. Overall, this data demonstrates that developmental inflammation stimulates SC proliferation and that this process is balanced by Cd59 to ensure normal SC and myelinated nerve development. Results Cd59 is expressed in myelinating glial cells during nervous system development Myelinating glial cell development is a complex process (Figure 1A and B) orchestrated by a genetic program that is not yet fully elucidated. To identify unexplored genes that may impact myelinating glial cell or myelin development, we evaluated bulk and single-cell (sc) RNAseq datasets that assessed myelinating glial cells during nervous system development (Gerber et al., 2021; Howard et al., 2021; Marisca et al., 2020; Marques et al., 2018; Marques et al., 2016; Piller et al., 2021; Saunders et al., 2019; Wolbert et al., 2020; Zhu et al., 2019). Across multiple datasets, cd59 (zebrafish) or Cd59a (mouse), which encodes for a small, GPI-anchored glycoprotein that has no known function in the developing nervous system, is expressed in SCs and OLCs (Figure 1C, Figure 1—figure supplement 1). When looking at SCs, data from developing zebrafish and mice indicated that cd59/Cd59a, respectively, is minimally expressed in neural crest cells (NCCs; the multipotent progenitors that give rise to SCs; Figure 1—figure supplement 1A; Howard et al., 2021; Zhu et al., 2019) but increases in expression in SC precursors (SCPs; Figure 1—figure supplement 1B.1,B.2; Gerber et al., 2021), immature SCs (iSCs; Figure 1—figure supplement 1B.1,B.2; Gerber et al., 2021), pro-myelinating SCs (pmSCs; Figure 1—figure supplement 1B.1,B.2; Gerber et al., 2021), and mature myelinating SCs (MSCs) and non-myelinating SCs (MSCs and NMSCs; Figure 1C, Figure 1—figure supplement 1B.3,C.1,C.2; Gerber et al., 2021; Piller et al., 2021; Saunders et al., 2019; Wolbert et al., 2020). This data indicates that Cd59 may be important during early stages of SC development as well as in mature MSCs and NMSCs (Figure 1D). In contrast, bulk RNAseq of zebrafish oligodendrocyte progenitor cells (OPCs) and OLs indicated that cd59 is expressed near the onset of myelination (72 hours post fertilization [hpf]; Figure 1C; Piller et al., 2021). scRNAseq (Marisca et al., 2020; Marques et al., 2018; Marques et al., 2016) and in situ hybridization (ISH; Siems et al., 2021) of zebrafish and mouse OLCs showed that cd59/CD59a is mostly expressed in mature OLs and not OPCs (Figure 1—figure supplement 1D). CD59 protein is also present within newly formed human myelin sheaths as is evident in immunostaining of third trimester fetal brains (Zajicek et al., 1995). Based on these findings, it is likely that Cd59 does not play a role in early stages of OLC development but could influence mature, myelinating OL function (Figure 1D). Intrigued by this elevated expression of cd59 in developing myelinating glial cells, we verified that cd59 RNA was expressed in OLCs and SCs with chromogenic ISH (CISH) and fluorescent ISH (FISH) at 3 days post fertilization (dpf) (Figure 2A–C). Morphology, location, and co-expression with sox10:megfp, which is a marker for both OLCs and SCs, showed that cd59 RNA is expressed in both cell types at 3 dpf (Figure 2A–C). Beyond expression in the heart and pancreas, cd59 expression is largely confined to developing myelinating glial cells at this stage. Figure 2 with 2 supplements see all Download asset Open asset cd59 is expressed in Schwann cells (SCs) and oligodendrocytes (OLs). (A) Bright-field images of whole-mount chromogenic in situ hybridization (CISH) showing cd59 expression (purple) in the heart, pancreas, dorsal and ventral oligodendrocyte lineage cells (OLCs) (dOLCs and vOLCs, respectively; filled arrows), and posterior lateral line nerve (pLLN) SCs (empty arrows) at 3 days post fertilization (dpf). Schematic (right panel) indicates location of spinal cord, notochord, and pLLN in transverse section. (B) Fluorescent in situ hybridization (FISH) (RNAscope; ACD) showing cd59 expression (cyan) in sox10:megfp-positive, pLLN SCs (cd59-positive orange cells indicated by empty arrows), and spinal cord OLs (cd59-positive orange cells indicated by filled arrows) at 3 dpf in transverse sections (top row). Representative image (top row) displays one SC on each pLLN (left and right empty arrows) as well as multiple OLs in the spinal cord (filled arrows). Images were acquired with confocal imaging. (C) Imaris renderings of the confocal images shown in (B), including the full image (left panel). From the full image (left panel, C), a single SC (indicated with the open magenta arrow and enlarged in the middle panel, C’) and a single OLC (indicated with the filled magenta arrow and enlarged in the right panel, C’’) illustrate the cd59 puncta localized within the myelinating cells. (D) Mosaic labeling showing a cd59:tagrfp-positive OL in the spinal cord (left) and two SCs on the pLLN (right) at 3 dpf. (E) Immunofluorescence (IF) showing cd59:tagrfp expression (cyan) in Sox10-positive SCs (orange) along the pLLN at 3 and 7 dpf (left and right panels, respectively). Double-positive cells are indicated with white arrows. White dashed lines outline the pLLN. Sox10-positive pigment cells outside of the dashed lines were not included in the analysis. (F) Scatter plot of percent cd59:tagrfp-positive SCs on the pLLN from 3 to 7 dpf (mean ± SEM: 3 dpf: 9.4 ± 1.5; 4 dpf: 10.0 ± 1.0; 5 dpf: 7.8 ± 1.2; 6 dpf: 4.4 ± 0.6; 7 dpf: 5.8± 0 .6; p-values: 3 vs. 6 dpf: p=0.0126, 4 vs. 6 dpf: p=0.0095, 4 vs. 7 dpf: Pp0.0477; dot = 1 fish). Data collected from somites 11–13 (~320 µm) and normalized to units per 100 µm. These data were compared with a one-way ANOVA with Tukey’s post-hoc test using GraphPad Prism. All fluorescent images were acquired with confocal imaging. Scale bars: (A) lateral view, 100 µm; transverse section, 25 µm; (B, D) 10 µm; (E) 25 µm. Artwork created by Ashtyn T. Wiltbank with Illustrator (Adobe). Figure 2—source data 1 Source data for the quantification of percent cd59:tagrfp-positive cells per 100 µm depicted in Figure 2E. Data contributed to scatter plot of percent cd59:tagrfp-positive Schwann cells (SCs) on the posterior lateral line nerve (pLLN) from 3 to 7 days post fertilization (dpf) (mean ± SEM: 3 dpf: 9.4 ± 1.5; 4 dpf: 10.0 ± 1.0; 5 dpf: 7.8 ± 1.2; 6 dpf: 4.4 ± 0.6; 7 dpf: 5.8 ± 0.6; p-values: 3 vs. 6 dpf: p=0.0126, 4 vs. 6 dpf: p=0.0095, 4 vs. 7 dpf: p=0.0477; data point = 1 fish). These data were compared with a one-way ANOVA with Tukey’s post-hoc test using GraphPad Prism. Data collected from somites 11–13 (~320 μm) and normalized to units per 100 μm. https://cdn.elifesciences.org/articles/76640/elife-76640-fig2-data1-v1.xlsx Download elife-76640-fig2-data1-v1.xlsx To study these cells in vivo, we created a transcriptional reporter zebrafish line for cd59 (herein referred to as cd59:tagrfp). This line utilizes 5 kb of the cd59 promoter as well as the first exon and intron, the latter containing an enhancer that is important for cd59 expression (Tone et al., 1999), to drive cytoplasmic expression of TagRFP in cd59-expressing cells. With in vivo, confocal imaging, mosaic labeling with our cd59:tagrfp construct revealed labeling of OLs and SCs with myelin-like processes (Figure 2D), indicating that cd59 is expressed in mature myelinating glial cells. Bulk and scRNAseq data indicated that cd59 is expressed during early stages of SC development whereas cd59 expression was restricted to mature OLs. To verify this expression pattern, we looked at cd59 RNA expression at earlier stages in the SC lineage. At 24 hpf, using CISH and FISH, we not observe cd59 expression in we see it in the and of the spinal cord at this stage (Figure supplement 1A and At 36 and hpf, when to develop and we expression of cd59 with in cells along the posterior lateral line nerve (pLLN) in the (Figure supplement and 72 hpf, we cd59 expression within SCs along the pLLN (Figure supplement and These data demonstrate that cd59 is expressed in developing SCs, the expression seen in the RNAseq datasets we analyzed (Figure 1—figure supplement 1). From our ISH we that cd59 was not expressed in SCs with spinal motor nerves not cd59 (Figure 2A–C). cd59 expression was largely confined to SCs along the pLLN (Figure 2A–C). To investigate this expression we our cd59:tagrfp This line in labeling of cd59-expressing pLLN SCs, which we using with a cd59 in cd59:tagrfp-positive SCs (Figure supplement To verify that cd59:tagrfp-positive SCs expressed SC including 10 and myelin protein a we examined pLLN SCs in and gene larvae these with in confocal imaging. cd59:tagrfp expression was in and SCs at 72 and 7 dpf (Figure supplement that cd59:tagrfp-positive SCs other known SC To determine whether cd59 was expressed in all pLLN SCs, we (IF) to cd59:tagrfp-positive SCs with an which all SCs along the pLLN. when looking at SCs from 3 to 7 cd59:tagrfp was expressed in a subset of pLLN SCs of 4.4 ± to 9.4 ± of pLLN SCs; Figure and We also that cd59 expression was expressed in a subset of OLs (Figure and was in Taken together, these data demonstrate that cd59 expression is in developing myelinating glial cells. of cd59 mutant zebrafish with findings demonstrate that cd59 is expressed in a subset of developing SCs. However, it is unclear how Cd59 SC development. In other cells, Cd59 has many functions, including cell lysis (Davies et al., signaling (Golec et al., 2019; Krus et al., 2014), cell proliferation et al., et al., et al., et al., and proximal-distal cell identity (Echeverri and Tanaka, 2005). is little known about Cd59 in the developing nervous system, To the function of Cd59 in SC development, we cd59 mutant zebrafish with the end of the first exon Figure we an with a that the exon 2 a (herein referred to as mutant Figure and sequencing analysis of mutant revealed that multiple were produced compared to wildtype cd59, which produced a single (Figure supplement 1A and of the mutant indicated that an early was in the a protein compared to the wildtype Figure supplement and the proteins the and (Figure supplement and the mutant Cd59 protein was to be We also an additional cd59 mutant which was produced with the RNA This mutant a 6 near the end of exon 2 that not the (Figure supplement and D) and produced a single mutant (Figure supplement of the mutant the of two in the which a protein compared to wildtype Cd59 Figure supplement and Though the protein was not we that with the prevent protein to the and protein Collectively, mutant larvae have perturbed RNA that in formation and protein and mutant larvae have a these both mutants were to have impaired Cd59 Figure 3 with 1 supplement see all Download asset Open asset of cd59 mutant zebrafish with (A) Schematic of zebrafish cd59 The cd59 encodes the protein with the indicated in The is indicated in The dashed lines indicate the and the (B) for wildtype cd59 and mutant showing a at the between exon 2 (blue) and 2 of the cd59 (C) Schematic of Cd59 protein made in wildtype panel) and mutant bottom panel). (D) Bright-field images of wildtype and mutant larvae at 72 hours post fertilization (hpf) showing no as a of cd59 (E) in situ hybridization (CISH) showing oligodendrocytes (OLs) in the spinal cord (filled arrows) and Schwann cells (SCs) on the posterior lateral line nerve (pLLN) (empty arrows) at 72 hpf in transverse (F) CISH showing OLs in the spinal cord (filled arrows) and SCs on the pLLN (empty arrows) at 72 hpf in transverse CISH showing SCs on the pLLN (empty arrows) and neurons in the (filled arrows) at 72 hpf in transverse Fluorescent in situ hybridization (FISH) (RNAscope; ACD) showing cd59 expression (cyan) in SCs (orange) along the pLLN at 72 Representative images each a transverse of µm) of single SC on the pLLN. Imaris renderings show cd59 puncta that are localized within each Scatter plot of the number of cd59 RNA puncta in pLLN SCs (mean ± SEM: ± ± dot = 1 = 7 per These data were compared with using GraphPad Prism. CISH and images were acquired with confocal imaging. Scale bars: (A) 25 µm; µm. Artwork created by Ashtyn T. Wiltbank with Illustrator (Adobe). Figure data 1 Source data for the quantification of the number of cd59 puncta in Schwann cells (SCs) at 72 hours post fertilization (hpf) depicted in Figure Data contributed to scatter plot of the number of cd59 RNA puncta in posterior lateral line nerve (pLLN) SCs (mean ± SEM: ± ± data point = 1 = 7 per These data were compared with using GraphPad Prism. Download To our evaluation of these we to developmental between cd59 mutant larvae and their wildtype and were and produced embryos with no or (Figure Figure supplement we to SCs were present in mutant which were in the of our Using we wildtype and mutant larvae with RNA (also known as early response or and at 72 wildtype and mutant larvae and SCs along the pLLN (Figure indicating that Cd59 was not for SC or expression of genes for SC and myelination. that SCs were we were how cd59 expression was in mutant SCs. to our mutant embryos produced RNA with (Figure supplement RNA with can a process by which normal and mutant gene expression is RNA et al., et al., and et al., 2014). To determine mutant RNA we to wildtype and mutant larvae with a cd59 (Figure and of cd59 RNA puncta showed that cd59 expression was reduced in mutant SCs compared to wildtype SCs (Figure indicating that mutant RNA was and could in a of protein. to the lack of a for Cd59, we were to determine these in RNA

This study reports on the measured change in the attitude of students toward teaching science as a result of their experiences in an elementary activity-based science course for preservice elementary teachers. Overall, the students in this study showed a positive shift in attitude as measured by the Revised Science Attitude Scale instrument that was administered at the beginning and the end of the semester. When the researchers looked at specific subscales within the instrument, one of the four subscales did not show a statistically significant change in attitude. The course is required for all elementary preservice teachers.

Wnt inhibitor factor-1 (WIF-1) is an extracellular antagonist of Wnts secreted proteins, first characterized as an expressed sequence tag in the human retina. WIF-1 belongs to the secreted Frizzled-related protein (sFRP) class, which can directly bind to Wnt proteins, prevent Wnts from binding to their receptors in vertebrates. Wif1 is expressed in the nervous system of mouse, Xenopus, zebrafish and human. It has been shown that WIF-1 affects the formation of somites in Xenopus embryos and inhibits rod production in retinal histogenesis by binding to Wnt4 in mice. Histological information of Wif1 expression during the development of the central nervous system has been reported in mouse, Xenopus and zebrafish and the strong embryonic expression suggests Wif1 may play an essential role in the spatial and temporal regulation of Wnt signals in development of central nervous system. The Wnt pathway plays a key role in the patterning of the nervous system. However, insights into the function of Wif1 in the development of the central nervous system are rather limited. Selecting suitable stage and target according to the expression pattern may contribute to understanding the function of Wif1.

In this project, a method that had been developed using a single setup was employed to correctly measure the tilt of the Earth’s axis on 21 June 2015. The method is an easily comprehensible and applicable technique that can be used in elementary science and astronomy courses and understood by students of every age group.

Nervous system development of the sea cucumber Stichopus japonicus

In this study, the game-based development approach is proposed for improving the learning motivation, problem solving skills, and learning achievement of students. An experiment was conducted on a learning activity of an elementary school science course to evaluate the performance of the proposed approach. A total of 59 sixth graders from two classes of the elementary school participated in the experiment. One class of 30 students was selected as the experimental group, and the other class of 29 students was the control group. From the experimental results, it was found that the proposed game development-based learning approach could effectively promote the students' problem-solving skills. However, the students' learning achievement and motivations were quite different from our expectations. A discussion of the experimental group interview data is provided and suggestions made.

In conventional classroom driven instructor training, disabled student achieved low personality & motivation, more mental load due to lack of practical learning and unsuitable usage of learning material through different simulated contexts. Therefore, intent of proposed study is to integrate microworld game-based learning to different learning styles of special needs students which enhance their learning performance and encourage them to discover and solve problems they faced during classroom teaching. A quasi experimental design was implemented in the proposed SmartLearn system in natural science behavioral course to examine the effectiveness of the proposed approach. The experimental results showed that students who adopted microworld gaming-based system was able to reduce their cognitive load, enhance learning motivation skills to gain understanding on novel concepts, improve personality to foster interpersonal skills, self-efficacy and improvements in learning achievements in comparison to the conventional learning technology driven approach. The findings of this study provide good evidence for special needs children where association of practically applied learning scenarios with gaming contexts improve their performance which encourages development of such systems for learners suffered from Autism, Dyslexia, Dysgraphia. Alzheimer etc.

There is a growing demand for software developers who have experience writing parallel programs rather than just "parallelizing" sequential systems as computer hardware gets more and more parallel. In order to develop the skills of future software engineers, it is crucial to teach pupils parallelism in elementary computer science courses. We searched the Scopus database for articles on "teaching parallel and distributed computing" and "parallel programming," published in English between 2008 and 2019. 26 papers were included in the study after quality review. As a result, a lab course using the C++ programming language and MPI library serves as the primary teaching tool for parallel and distributed computing.

Much publicity has been given to announcements that full-time first-year graduate student 'enrollments in chemistry are decreasing. To those familiar with the changes in science education during the past decade, both undergraduate and secondary school, this isn't surprising. Although William F. Kieffer, editor of the Journal of Chemical Education , observed in 1966 that we might expect that one of the consequences of the widespread use of CBA and CHEMS would be to arouse even more interest in chemistry as a career, these courses and other curricular attempts to improve elementary science courses in effect have actually alienated many students from Dr. Paul Saltman notes that It is actually appalling to see the textbooks that are widely used in this country and abroad to communicate to young minds thirsting to know and understand. Far too often, authors of textbooks fail to grasp the meaning of science. Dael Wolfle, executive officer of American Association for the ...

Runt-related (Runx) transcription factors play essential roles during development and adult tissue homeostasis and are responsible for several human diseases. They regulate a variety of biological mechanisms in numerous cell lineages. Recent years have seen significant progress in our understanding of the functions performed by Runx proteins in the developing and postnatal mammalian nervous system. In both central and peripheral nervous systems, Runx1 and Runx3 display remarkably specific expression in mostly non-overlapping groups of postmitotic neurons. In the central nervous system, Runx1 is involved in the development of selected motor neurons controlling neural circuits mediating vital functions such as chewing, swallowing, breathing, and locomotion. In the peripheral nervous system, Runx1 and Runx3 play essential roles during the development of sensory neurons involved in circuits mediating pain, itch, thermal sensation and sense of relative position. Runx1 and Runx3 orchestrate complex gene expression programs controlling neuronal subtype specification and axonal connectivity. Runx1 is also important in the olfactory system, where it regulates the progenitor-to-neuron transition in undifferentiated neural progenitor cells in the olfactory epithelium as well as the proliferation and developmental maturation of specific glial cells termed olfactory ensheathing cells. Moreover, upregulated Runx expression is associated with brain injury and disease. Increasing knowledge of the functions of Runx proteins in the developing and postnatal nervous system is therefore expected to improve our understanding of nervous system development, homeostasis and disease.

The basic helix–loop–helix factors play a central role in neuronal differentiation and nervous system development, which involve the Notch and signal transducer and activator of transcription (STAT)/small mother against decapentaplegic signaling pathways. Neural stem cells differentiate into three nervous system lineages, and the suppressor of cytokine signaling (SOCS) and von Hippel-Lindau (VHL) proteins are involved in this neuronal differentiation. The SOCS and VHL proteins both contain homologous structures comprising the BC-box motif. SOCSs recruit Elongin C, Elongin B, Cullin5(Cul5), and Rbx2, whereas VHL recruits Elongin C, Elongin B, Cul2, and Rbx1. SOCSs form SBC-Cul5/E3 complexes, and VHL forms a VBC-Cul2/E3 complex. These complexes degrade the target protein and suppress its downstream transduction pathway by acting as E3 ligases via the ubiquitin–proteasome system. The Janus kinase (JAK) is the main target protein of the E3 ligase SBC-Cul5, whereas hypoxia-inducible factor is the primary target protein of the E3 ligase VBC-Cul2; nonetheless, VBC-Cul2 also targets the JAK. SOCSs not only act on the ubiquitin–proteasome system but also act directly on JAKs to suppress the Janus kinase–signal transduction and activator of transcription (JAK-STAT) pathway. Both SOCS and VHL are expressed in the nervous system, predominantly in brain neurons in the embryonic stage. Both SOCS and VHL induce neuronal differentiation. SOCS is involved in differentiation into neurons, whereas VHL is involved in differentiation into neurons and oligodendrocytes; both proteins promote neurite outgrowth. It has also been suggested that the inactivation of these proteins may lead to the development of nervous system malignancies and that these proteins may function as tumor suppressors. The mechanism of action of SOCS and VHL involved in neuronal differentiation and nervous system development is thought to be mediated through the inhibition of downstream signaling pathways, JAK-STAT, and hypoxia-inducible factor–vascular endothelial growth factor pathways. In addition, because SOCS and VHL promote nerve regeneration, they are expected to be applied in neuronal regenerative medicine for traumatic brain injury and stroke.