Satellite glial cell manipulation prior to axotomy enhances developing dorsal root ganglion central branch regrowth into the spinal cord.

The central and peripheral nervous systems (CNS and PNS, respectively) exhibit remarkable diversity in the capacity to regenerate following neuronal injury with PNS injuries being much more likely to regenerate than those that occur in the CNS. Glial responses to damage greatly influence the likelihood of regeneration by either promoting or inhibiting axonal regrowth over time. However, despite our understanding of how some glial lineages participate in nerve degeneration and regeneration, less is known about the contributions of peripheral satellite glial cells (SGC) to regeneration failure following central axon branch injury of dorsal root ganglia (DRG) sensory neurons. Here, using in vivo, time-lapse imaging in larval zebrafish coupled with laser axotomy, we investigate the role of SGCs in axonal regeneration. In our studies we show that SGCs respond to injury by relocating their nuclei to the injury site during the same period that DRG neurons produce new central branch neurites. Laser ablation of SGCs prior to axon injury results in more neurite growth attempts and ultimately a higher rate of successful central axon regrowth, implicating SGCs as inhibitors of regeneration. We also demonstrate that this SGC response is mediated in part by ErbB signaling, as chemical inhibition of this receptor results in reduced SGC motility and enhanced central axon regrowth. These findings provide new insights into SGC-neuron interactions under injury conditions and how these interactions influence nervous system repair.

Rohon-beard neurons do not succumb to programmed cell death during zebrafish development.

During neural development, sculpting of early formed circuits by cell death and synaptic pruning is necessary to generate a functional and efficient nervous system. This allows for the establishment of rudimentary circuits which necessitate early organism survival to later undergo subsequent refinement. These changes facilitate additional specificity to stimuli which can lead to increased behavioral complexity. In multiple species, Rohon-Beard neurons (RBs) are the earliest mechanosensory neurons specified and are critical in establishing a rudimentary motor response circuit. Sensory input from RBs gradually becomes redundant as dorsal root ganglion (DRG) neurons develop and integrate into motor circuits. Previous studies demonstrate that RBs undergo a dramatic wave of cell death concurrent with development of the DRG. However, contrary to these studies, we show that neurogenin1+ (ngn1) RBs do not undergo a widespread wave of programmed cell death during early zebrafish development and instead persist until at least 15 days post fertilization (dpf). Starting at 2 dpf, we also observed a dramatic medialization and shrinkage of ngn1+ RB somas along with a gradual downregulation of ngn1 in RBs. This alters a fundamental premise of early zebrafish neural development and opens new avenues to explore mechanisms of RB function, persistence, and circuit refinement.

Glia at Transition Zones.

Neural cells are segregated into their distinct central nervous system (CNS) and peripheral nervous system (PNS) domains. However, at specialized regions of the nervous system known as transition zones (TZs), glial cells from both the CNS and PNS are uniquely present with other specialized TZ cells. Herein we review the current understanding of vertebrate TZ cells. The article discusses the distinct cells at vertebrate TZs with a focus on cells that are located on the peripheral side of the spinal cord TZs. In addition to the developmental origin and differentiation of these TZ cells, the functional importance and the role of TZ cells in disease are highlighted. This article also reviews the common and unique features of vertebrate TZs from zebrafish to mice. We propose challenges and open questions in the field that could lead to exciting insights in the field of glial biology.

Chimeric efferocytic receptors improve apoptotic cell clearance and alleviate inflammation.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

Cd59 and inflammation regulate Schwann cell development.

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.

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.

Transforming growth factor-beta signaling modulates perineurial glial bridging following peripheral spinal motor nerve injury in zebrafish.

Spinal motor nerves are necessary for organismal locomotion and survival. In zebrafish and most vertebrates, these peripheral nervous system structures are composed of bundles of axons that naturally regenerate following injury. However, the cellular and molecular mechanisms that mediate this process are still only partially understood. Perineurial glia, which form a component of the blood-nerve barrier, are necessary for the earliest regenerative steps by establishing a glial bridge across the injury site as well as phagocytosing debris. Without perineurial glial bridging, regeneration is impaired. In addition to perineurial glia, Schwann cells, the cells that ensheath and myelinate axons within the nerve, are essential for debris clearance and axon guidance. In the absence of Schwann cells, perineurial glia exhibit perturbed bridging, demonstrating that these two cell types communicate during the injury response. While the presence and importance of perineurial glial bridging is known, the molecular mechanisms that underlie this process remain a mystery. Understanding the cellular and molecular interactions that drive perineurial glial bridging is crucial to unlocking the mechanisms underlying successful motor nerve regeneration. Using laser axotomy and in vivo imaging in zebrafish, we show that transforming growth factor-beta (TGFß) signaling modulates perineurial glial bridging. Further, we identify connective tissue growth factor-a (ctgfa) as a downstream effector of TGF-ß signaling that works in a positive feedback loop to mediate perineurial glial bridging. Together, these studies present a new signaling pathway involved in the perineurial glial injury response and further characterize the dynamics of the perineurial glial bridge.

zMADM (zebrafish mosaic analysis with double markers) for single-cell gene knockout and dual-lineage tracing.

As a vertebrate model organism, zebrafish has many unique advantages in developmental studies, regenerative biology, and disease modeling. However, tissue-specific gene knockout in zebrafish is challenging due to technical difficulties in making floxed alleles. Even when successful, tissue-level knockout can affect too many cells, making it difficult to distinguish cell autonomous from noncell autonomous gene function. Here, we present a genetic system termed zebrafish mosaic analysis with double markers (zMADM). Through Cre/loxP-mediated interchromosomal mitotic recombination of two reciprocally chimeric fluorescent genes, zMADM generates sporadic (<0.5%), GFP+ mutant cells along with RFP+ sibling wild-type cells, enabling phenotypic analysis at single-cell resolution. Using wild-type zMADM, we traced two sibling cells (GFP+ and RFP+) in real time during a dynamic developmental process. Using nf1 mutant zMADM, we demonstrated an overproliferation phenotype of nf1 mutant cells in comparison to wild-type sibling cells in the same zebrafish. The readiness of zMADM to produce sporadic mutant cells without the need to generate floxed alleles should fundamentally improve the throughput of genetic analysis in zebrafish; the lineage-tracing capability combined with phenotypic analysis at the single-cell level should lead to deep insights into developmental and disease mechanisms. Therefore, we are confident that zMADM will enable groundbreaking discoveries once broadly distributed in the field.

Clearing Your Mind: Mechanisms of Debris Clearance After Cell Death During Neural Development.

Neurodevelopment and efferocytosis have fascinated scientists for decades. How an organism builds a nervous system that is precisely tuned for efficient behaviors and survival and how it simultaneously manages constant somatic cell turnover are complex questions that have resulted in distinct fields of study. Although neurodevelopment requires the overproduction of cells that are subsequently pruned back, very few studies marry these fields to elucidate the cellular and molecular mechanisms that drive nervous system development through the lens of cell clearance. In this review, we discuss these fields to highlight exciting areas of future synergy. We first review neurodevelopment from the perspective of overproduction and subsequent refinement and then discuss who clears this developmental debris and the mechanisms that control these events. We then end with how a more deliberate merger ofneurodevelopment and efferocytosis could reframe our understanding of homeostasis and disease and discuss areas of future study.

A novel gene trap line for visualization and manipulation of erbb3b+ neural crest and glial cells in zebrafish.

Glia are a diverse and essential cell type in the vertebrate nervous system. Transgenic tools and fluorescent reporter lines are critical resources to investigate how glial subtypes develop and function. However, despite the many lines available in zebrafish, the community still lacks the ability to label all unique stages of glial development and specific subpopulations of cells. To address this issue, we screened zebrafish gene and enhancer trap lines to find a novel reporter for peripheral glial subtypes. From these, we generated the gSAIzGFFD37A transgenic line that expresses GFP in neural crest cells and central and peripheral glia. We found that the gene trap construct is located within an intron of erbb3b, a gene essential for glial development. Additionally, we confirmed that GFP+ â€‹cells express erbb3b along with sox10, a known glial marker. From our screen, we have identified the gSAIzGFFD37A line as a novel and powerful tool for studying glia in the developing zebrafish, as well as a new resource to manipulate erbb3b+ â€‹cells.

Diversity and convergence within peripheral glia development.

In a recent study, Tasdemir-Yilmaz and colleagues used single-cell RNA sequencing to reveal how diversity among peripheral glia changes with development and also identified unifying genetic profiles that are shared between mature glia. These data highlight new tools and pathways in which to understand peripheral glial development and function.