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The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires., Competing Interests: Declaration of competing interest I declare I do not have any competing interests., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Neurons have a unique polarized nature that must adapt to environmental changes throughout their lifespan. During embryonic development, axon elongation is led by the growth cone, 1 culminating in the formation of a presynaptic terminal. After synapses are formed, axons elongate in a growth cone-independent manner to accompany body growth while maintaining their ultrastructure and function. 2 , 3 , 4 , 5 , 6 To further understand mechanical strains on the axon shaft, we developed a computer-controlled stretchable microfluidic platform compatible with multi-omics and live imaging. Our data show that sensory embryonic dorsal root ganglia (DRGs) neurons have high plasticity, with axon shaft microtubules decreasing polymerization rates, aligning with the direction of tension, and undergoing stabilization. Moreover, in embryonic DRGs, stretch triggers yes-associated protein (YAP) nuclear translocation, supporting its participation in the regulatory network that enables tension-driven axon growth. Other than cytoskeleton remodeling, stretch prompted MARCKS-dependent formation of plasmalemmal precursor vesicles (PPVs), resulting in new membrane incorporation throughout the axon shaft. In contrast, adolescent DRGs showed a less robust adaptation, with axonal microtubules being less responsive to stretch. Also, while adolescent DRGs were still amenable to strain-induced PPV formation at higher stretch rates, new membrane incorporation in the axon shaft failed to occur. In summary, we developed a new resource to study the biology of axon stretch growth. By unraveling cytoskeleton adaptation and membrane remodeling in the axon shaft of stretched neurons, we are moving forward in understanding axon growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The dorsal funiculus in the spinal cord relays somatosensory information to the brain. It is made of T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons. Our previous study has shown that Slit signaling is required for proper guidance during bifurcation, but loss of Slit does not affect all DRG axons. Here, we examined the role of the extracellular molecule Netrin-1 (Ntn1). Using wholemount staining with tissue clearing, we showed that mice lacking Ntn1 had axons escaping from the dorsal funiculus at the time of bifurcation. Genetic labeling confirmed that these misprojecting axons come from DRG neurons. Single axon analysis showed that loss of Ntn1 did not affect bifurcation but rather altered turning angles. To distinguish their guidance functions, we examined mice with triple deletion of Ntn1, Slit1, and Slit2 and found a completely disorganized dorsal funiculus. Comparing mice with different genotypes using immunolabeling and single axon tracing revealed additive guidance errors, demonstrating the independent roles of Ntn1 and Slit. Moreover, the same defects were observed in embryos lacking their cognate receptors. These in vivo studies thus demonstrate the presence of multi-factorial guidance mechanisms that ensure proper formation of a common branched axonal structure during spinal cord development., Competing Interests: BC, KN, AY, LG, AJ, MT, LM No competing interests declared, (© 2024, Curran et al.)

The cell-adhesion molecule NEPH1 is required for maintaining the structural integrity and function of the glomerulus in the kidneys. In the nervous system of Drosophila and C. elegans, it is involved in synaptogenesis and axon branching, which are essential for establishing functional circuits. In the mammalian nervous system, the expression regulation and function of Neph1 has barely been explored. In this study, we provide a spatiotemporal characterization of Neph1 expression in mouse dorsal root ganglia (DRGs) and spinal cord. After the neurogenic phase, Neph1 is broadly expressed in the DRGs and in their putative targets at the dorsal horn of the spinal cord, comprising both GABAergic and glutamatergic neurons. Interestingly, we found that PRRXL1, a homeodomain transcription factor that is required for proper establishment of the DRG-spinal cord circuit, prevents a premature expression of Neph1 in the superficial laminae of the dorsal spinal cord at E14.5, but has no regulatory effect on the DRGs or on either structure at E16.5. By chromatin immunoprecipitation analysis of the dorsal spinal cord, we identified four PRRXL1-bound regions within the Neph1 introns, suggesting that PRRXL1 directly regulates Neph1 transcription. We also showed that Neph1 is required for branching, especially at distal neurites. Together, our work showed that Prrxl1 prevents the early expression of Neph1 in the superficial dorsal horn, suggesting that Neph1 might function as a downstream effector gene for proper assembly of the DRG-spinal nociceptive circuit., (© 2024. The Author(s).)

Sensory neurogenesis in the dorsal root ganglion (DRG) occurs in two waves of differentiation with larger, myelinated proprioceptive and low-threshold mechanoreceptor (LTMR) neurons differentiating before smaller, unmyelinated (C) nociceptive neurons. This temporal difference was established from early birthdating studies based on DRG soma cell size. However, distinctions in birthdates between molecular subtypes of sensory neurons, particularly nociceptors, is unknown. Here, we assess the birthdate of lumbar DRG neurons in mice using a thymidine analog, EdU, to label developing neurons exiting mitosis combined with co-labeling of known sensory neuron markers. We find that different nociceptor subtypes are born on similar timescales, with continuous births between E9.5 to E13.5, and peak births from E10.5 to E11.5. Notably, we find that thinly myelinated Aδ-fiber nociceptors and peptidergic C-fibers are born more broadly between E10.5 and E11.5 than previously thought and that non-peptidergic C-fibers and C-LTMRs are born with a peak birth date of E11.5. Moreover, we find that the percentages of nociceptor subtypes born at a particular timepoint are the same for any given nociceptor cell type marker, indicating that intrinsic or extrinsic influences on cell type diversity are occurring similarly across developmental time. Overall, the patterns of birth still fit within the classical "two wave" description, as touch and proprioceptive fibers are born primarily at E10.5, but suggest that nociceptors have a slightly broader wave of birthdates with different nociceptor subtypes continually differentiating throughout sensory neurogenesis irrespective of myelination., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Axons navigate through the embryo to construct a functional nervous system. A missing part of the axon navigation puzzle is how a single axon traverses distinct anatomic choice points through its navigation. The dorsal root ganglia (DRG) neurons experience such choice points. First, they navigate to the dorsal root entry zone (DREZ), then halt navigation in the peripheral nervous system to invade the spinal cord, and then reinitiate navigation inside the CNS. Here, we used time-lapse super-resolution imaging in zebrafish DRG pioneer neurons to investigate how embryonic axons control their cytoskeleton to navigate to and invade at the correct anatomic position. We found that invadopodia components form in the growth cone even during filopodia-based navigation, but only stabilize when the axon is at the spinal cord entry location. Further, we show that intermediate levels of DCC and cAMP, as well as Rac1 activation, subsequently engage an axon invasion brake. Our results indicate that actin-based invadopodia components form in the growth cone and disruption of the invasion brake causes axon entry defects and results in failed behavioral responses, thereby demonstrating the importance of regulating distinct actin populations during navigational challenges. SIGNIFICANCE STATEMENT Correct spatiotemporal navigation of neuronal growth cones is dependent on extracellular navigational cues and growth cone dynamics. Here, we link dcc -mediated signaling to actin-based invadopodia and filopodia dynamics during pathfinding and entry into the spinal cord using an in vivo model of dorsal root ganglia (DRG) sensory axons. We reveal a molecularly-controlled brake on invadopodia stabilization until the sensory neuron growth cone is present at the dorsal root entry zone (DREZ), which is ultimately essential for growth cone entry into the spinal cord and behavioral response., (Copyright © 2021 the authors.)

Transplantation of embryonic motor neurons has been shown to improve motor neuron survival and innervation of neuromuscular junctions in peripheral nerves. However, there have been no reports regarding transplantation of sensory neurons and innervation of sensory receptors. Therefore, we hypothesized that the transplantation of embryonic sensory neurons may improve sensory neurons in the skin and innervate Merkel cells and Meissner's corpuscles. We obtained sensory neurons from dorsal root ganglia of 14-day rat embryos. We generated a rat model of Wallerian-degeneration by performing sciatic nerve transection and waiting for one week after. Six months after cell transplantation, we performed histological and electrophysiological examinations in naïve control, surgical control, and cell transplantation groups. The number of nerve fibers in the papillary dermis and epidermal-dermal interface was significantly greater in the cell transplantation than in the surgical control group. The percent of Merkel cells with nerve terminals, as well as the average number of Meissner corpuscles with nerve terminals, were higher in the cell transplantation than in the surgical control group, but differences were not significant between the two groups. Moreover, the amplitude and latency of sensory conduction velocity were evoked in rats of the cell transplantation group. We demonstrated that the transplantation of embryonic dorsal root ganglion cells improved sensory nerve fiber number and innervation of Merkel cells and Meissner's corpuscles in peripheral nerves., (© 2021 John Wiley & Sons Ltd.)

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Preparations of peripheral sensory neurons from rodents are essential for studying the molecular mechanism of neuronal survival and physiology. Although, isolating and culturing these neurons proves difficult, often these preparations are contaminated with nonneuronal proliferating cells. Here, we describe an isolation method using a Percoll gradient and an antimitotic reagent to significantly reduce the nonneuronal cell contamination while maintaining the integrity of the rodent sensory dorsal root ganglia (DRG) neurons.

Direct intercellular communication via gap junctions has an important role in the development of the nervous system, ranging from cell migration and neuronal differentiation to the formation of neuronal activity patterns. This study characterized and compared the specific spatio-temporal expression patterns of connexins (Cxs) 37, 43 and 45 during early human developmental stages (since the 5th until the 10th developmental week) in the spinal cord (SC) and dorsal root ganglia (DRG) using double immunofluorescence and transmission electron microscopy. We found the expression of all three investigated Cxs during early human development in all the areas of interest, in the SC, DRG, developing paravertebral ganglia of the sympathetic trunk, notochord and all three meningeal layers, with predominant expression of Cx37. Comparing the expression of different Cxs between distinct developmental periods, we did not find significant differences. Specific spatio-temporal pattern of Cxs expression might reflect their relevance in the development of all areas of interest via cellular interconnectivity and synchronization during the late embryonic and early fetal period of human development.

It is often critical to improve the limited regenerative capacity of the peripheral nerves and direct neural growth towards specific targets, such as surgically implanted bioengineered constructs. One approach to accomplish this goal is to use extrinsic neurotrophic factors. The candidate factors first need to be identified and characterized in in vitro tests for their ability to direct the neurite growth. Here, we present a simple guidance assay that allows to assess the chemotactic effect of signaling molecules on the growth of neuronal processes from dorsal root ganglia (DRG) using only standard tissue culture materials. We used this technique to quantitatively determine the combined and individual effects of the ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth. We demonstrated that these two neurotrophic factors, when applied in a 1:1 combination, but not individually, induced directed growth of neuronal processes towards the source of the gradient. This chemotactic effect persists without significant changes over a wide (10-fold) concentration range. Moreover, we demonstrated that other, more general growth parameters that do not evaluate growth in a specific direction (such as, neurite length and trajectory) were differentially affected by the concentration of the CNTF/GNDF mixture. Furthermore, GDNF, when applied individually, did not have any chemotactic effect, but caused significant neurite elongation and an increase in the number of neurites per ganglion., Competing Interests: The authors have declared that no competing interests exist.

During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13-16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

The molecular players regulating the axon degeneration pathway have been identified using in vitro experimental models. Here, we describe an in vitro assay to assess the axonal fragmentation induced by mechanical injury to axons in cultured mouse embryonic dorsal root ganglion (DRG) neurons. DRG neurons are pseudounipolar and therefore suitable for an assay of axonal degeneration after injury. In addition, the time course of the axonal fragmentation is stereotyped, enabling the identification of reagents that either expedite or impede the degeneration process. With an image-based quantification method, the in vitro degeneration assay can be utilized as a platform supporting high-throughput screens for pharmacological or genetic reagents delaying axon degeneration.

Peripheral neuropathies can have various origins, from genetic to acquired causes, and affect altogether a large group of people in the world. Current available therapies aim at helping the disease symptoms but not to correct or stop the development of the disease. Primary neuronal cultures represent an essential tool in the study of events related to peripheral neuropathies as they allow to isolate the affected cell types, often originating in complex tissues in which they account for only a few percentage of cells. They provide a powerful system to identifying or testing compounds with potential therapeutic effect in the treatment of those diseases. Friedreich's ataxia is an autosomal recessive neurodegenerative disorder, which is characterized by a progressive spinocerebellar and sensory ataxia. Proprioceptive neurons of the dorsal root ganglia (DRG) are the primary affected cells. The disease is triggered by a mutation in the gene FXN which leads to a reduction of the frataxin protein. In order to study the neurophysiopathology of the disease at the cellular and molecular levels, we have established a model of primary cultures of DRG sensory neurons in which we induce the loss of the frataxin protein. With such a model we can alleviate the issues related to the complexity of DRG tissues and low amount of sensory neuron material in adult mouse. Hereby, we provide a protocol of detailed and optimized methods to obtain high yield of healthy mouse DRG sensory neuron in culture.

Autonomous mechanisms of axon degeneration are frequently studied in vitro by mechanical axon injury of isolated sensory neurons. This has led to major advances in understanding the molecular pathways governing axon degeneration. However, this approach does not pay attention to potential glial mechanisms for the regulation of axon death. Here, I describe a straightforward protocol to seed purified rat Schwann cells on neuronal cultures in order to study the interaction between axons and these glia during axon degeneration.

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro . We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca 2+ , and here, we address the mechanism of Ca 2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca 2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca 2+ influx in sensory axon degeneration.

Objective: The cost and low success rates of the neurological drug development pipeline have diverted the pharmaceutical industry to 'nerve-on-a-chip' systems as preclinical models to streamline drug development. We present a novel micro-engineered 3D hydrogel platform for the culture of myelinated embryonic peripheral neural tissue to serve as an effective in vitro model for electrophysiological and histological analysis that could be adopted for preclinical testing., Approach: Dorsal root ganglions (DRG) from 15 d old embryonic rats were cultured in 3D hydrogel platforms. The interaction between Schwann cells (SC) and neurons during axonal development and regeneration affects the direction of growth and the synthesis of myelin sheaths. Induction of myelination was performed with two approaches: the addition of exogenous SC and promoting migration of endogenous SC., Main Results: Histological analysis of the preparation utilizing exogenous SC showed aligned, highly fasciculated axonal growth with noticeable myelin sheaths around axons. Separately, electrophysiological testing of the preparation utilizing endogenous SC showed increased amplitude of the compound action potential and nerve conduction velocity in the presence of ascorbic acid (AA)., Significance: This platform has immense potential to be a useful and translatable in vitro testing tool for drug discovery and myelination studies.

The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target-derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret-expressing neurons in the DRG complete developmental cell death prior to TrkA-expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701-717, 2018., (© 2018 Wiley Periodicals, Inc.)

Cobalt is a trace element that localizes in the human body as cobalamin, also known as vitamin B12. Excessive cobalt exposure induces a peripheral neuropathy, the mechanisms of which are yet to be elucidated. We investigated how cobalt may affect mitochondrial motility in primary cultures of rat dorsal root ganglion (DRG). We observed mitochondrial motility by time-lapse imaging after DsRed2 tagging via lentivirus, mitochondrial structure using transmission electron microscopy (TEM), and axonal swelling using immunocytochemical staining. The concentration of cobaltous ion (Co 2+ ) required to significantly suppress mitochondrial motility is lower than that required to induce axonal swelling following a 24-h treatment. Exposure to relatively low concentrations of Co 2+ for 48 h suppressed mitochondrial motility without leading to axonal swelling. TEM images indicated that Co 2+ induces mitochondrial destruction. Our results show that destruction of the axonal mitochondria precedes the axonal degeneration induced by Co 2+ exposure.

The most widely used method (Brockes' method) for preparing primary Schwann cell culture uses neonatal rat sciatic nerves as the primary source of Schwann cells. The procedure is relatively simple and yields a highly purified population of Schwann cells in a short period of time. The method has also been used to prepare Schwann cells from mice, however, with limitation. For example, Brockes' method is not applicable when the genotypes of mouse neonates are unknown or if the mouse mutants do not develop to term. We described a method ideal for preparing Schwann cells in a transgenic/knockout mouse study. The method uses embryonic dorsal root ganglia as the primary source of Schwann cells and allows preparing separate, highly purified Schwann cell cultures from individual mouse embryos in less than 2 weeks.

The ability to understand in great details, at the molecular level, the process of myelination in the peripheral nervous system (PNS) is, in no minor part, due to the availability of an in vitro culture model of PNS myelination. This culture system is based on the ability to prepare large population of highly purified Schwann cells and dorsal root ganglia neurons that, once co-cultured, can be driven to form in vitro well-defined myelinated axon units. In this chapter, we present our detailed protocols to establish these cell cultures that are derived from modifications of procedures developed 35-40 years ago.

It was recently described that Galectin-1 (Gal-1) promotes axonal growth after spinal cord injury. This effect depends on protein dimerization, since monomeric Gal-1 fails to stimulate axonal re-growth. Gal-1 is expressed in vivo at concentrations that favor the monomeric species. The aim of the present study is to investigate whether endogenous Gal-1 is required for spinal axon development and normal locomotor behavior in mice. In order to characterize axonal development, we used a novel combination of 3-DISCO technique with 1-photon microscopy and epifluorescence microscopy under high power LED illumination, followed by serial image section deconvolution and 3-D reconstruction. Cleared whole lgals-1 -/- embryos were used to analyze the 3-D cytoarchitecture of motor, commissural, and sensory axons. This approach allowed us to evaluate axonal development, including the number of fibers, fluorescence density of the fiber tracts, fiber length as well as the morphology of axonal sprouting, deep within the tissue. Gal-1 deficient embryos did not show morphological/anatomical alterations in any of the axonal populations and parameters analyzed. In addition, specific guidance receptor PlexinA4 did not change its axonal localization in the absence of Gal-1. Finally, Gal-1 deficiency did not change normal locomotor activity in post-natal animals. Taken together, our results show that development of spinal axons as well as the locomotor abilities observed in adult mice are independent of Gal-1. Supporting our previous observations, the present study further validates the use of lgals-1 -/- mice to develop spinal cord- or traumatic brain injury models for the evaluation of the regenerative action of Gal-1., (© 2017 Wiley Periodicals, Inc.)

Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor. Monitoring the dynamics of Septins, which play key roles in yeast polarity, indicates that Septin 7 tags process sites for re-initiation of process growth following mitosis. Interfering with Septins blocks this mechanism. Thus, Septins store polarity features during mitotic rounding so that daughters can reconstitute the initial progenitor polarity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Current research in prosthetic device design aims to mimic natural movements using a feedback system that connects to the patient's own nerves to control the device. The first step in using neurons to control motion is to make and maintain contact between neurons and the feedback sensors. Therefore, the goal of this project was to determine if changes in electrode resistance could be detected when a neuron extended a neurite to contact a sensor. Dorsal root ganglia (DRG) were harvested from chick embryos and cultured on a collagen-coated carbon nanotube microelectrode array for two days. The DRG were seeded along one side of the array so the processes extended across the array, contacting about half of the electrodes. Electrode resistance was measured both prior to culture and after the two day culture period. Phase contrast images of the microelectrode array were taken after two days to visually determine which electrodes were in contact with one or more DRG neurite or tissue. Electrodes in contact with DRG neurites had an average change in resistance of 0.15 MΩ compared with the electrodes without DRG neurites. Using this method, we determined that resistance values can be used as a criterion for identifying electrodes in contact with a DRG neurite. These data are the foundation for future development of an autonomous feedback resistance measurement system to continuously monitor DRG neurite outgrowth at specific spatial locations.

AT2 receptor (AT2R) plays a functional role in foetal development. Its expression declines in most tissues soon after birth but stays high in sensory areas of the adult nervous system. In the dorsal root ganglia (DRG) the expression pattern of AT2R during development and the identity of the subpopulation expressing it remain unknown. Using a combination of semi-quantitative PCR, western blotting and immunohistochemistry we examined the expression of AT2R at mRNA and protein levels in rat DRGs from embryonic day 15 (E15) until postnatal day 30 (PN30). We found that both AT2R mRNA and protein levels exhibited only minor (statistically non-significant) fluctuations from E15 to PN30. Detailed quantitative analysis of ABC/DAB AT2R staining showed a) that the receptor was present in most neurons at E15 and E18 and b) that postnatally it was predominantly expressed by small DRG neurons. Given that small neurons are putative C-nociceptors and the proposed role of AT2R in neuropathic pain, we next examined whether these AT2R-positive neurons co-localized with Ret and trkA embryonically and with IB4-binding postnatally. Most AT2R-positive neurons expressed trkA embryonically and bound IB4 postnatally. We found strong positive statistically highly significant correlations between AT2R cytoplasmic%intensities and trkA at E15/E18 and with Ret only at E18. Cytoplasmic AT2R also strongly and positively correlated with IB4-binding at PN3, 15 and 30. Our demonstration that a subpopulation of C-nociceptor-like neurons expresses AT2R during development supports a role for this receptor in neuropathic pain., (Copyright © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.)

Cytoplasmic dynein mediates retrograde transport in axons, but it is unknown how its transport characteristics are regulated to meet acutely changing demands. We find that stimulus-induced retrograde transport of different cargos requires the local synthesis of different dynein cofactors. Nerve growth factor (NGF)-induced transport of large vesicles requires local synthesis of Lis1, while smaller signalling endosomes require both Lis1 and p150 Glued . Lis1 synthesis is also triggered by NGF withdrawal and required for the transport of a death signal. Association of Lis1 transcripts with the microtubule plus-end tracking protein APC is required for their translation in response to NGF stimulation but not for their axonal recruitment and translation upon NGF withdrawal. These studies reveal a critical role for local synthesis of dynein cofactors for the transport of specific cargos and identify association with RNA-binding proteins as a mechanism to establish functionally distinct pools of a single transcript species in axons.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs-dubbed R1, R2, and R3-that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs' ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs., (© 2016 Appel et al.; Published by Cold Spring Harbor Laboratory Press.)

A cyclic GMP (cGMP) signaling pathway, comprising C-type natriuretic peptide (CNP), its guanylate cyclase receptor Npr2, and cGMP-dependent protein kinase I, is critical for the bifurcation of dorsal root ganglion (DRG) and cranial sensory ganglion axons when entering the mouse spinal cord and the hindbrain respectively. However, the identity and functional relevance of phosphodiesterases (PDEs) that degrade cGMP in DRG neurons are not completely understood. Here, we asked whether regulation of the intracellular cGMP concentration by PDEs modulates the branching of sensory axons. Real-time imaging of cGMP with a genetically encoded fluorescent cGMP sensor, RT-PCR screens, in situ hybridization, and immunohistology combined with the analysis of mutant mice identified PDE2A as the major enzyme for the degradation of CNP-induced cGMP in embryonic DRG neurons. Tracking of PDE2A-deficient DRG sensory axons in conjunction with cGMP measurements indicated that axon bifurcation tolerates increased cGMP concentrations. As we found that the natriuretic peptide scavenger receptor Npr3 is expressed by cells associated with dorsal roots but not in DRG neurons itself at early developmental stages, we analyzed axonal branching in the absence of Npr3. In Npr3-deficient mice, the majority of sensory axons showed normal bifurcation, but a small population of axons (13%) was unable to form T-like branches and generated turns in rostral or caudal directions only. Taken together, this study shows that sensory axon bifurcation is insensitive to increases of CNP-induced cGMP levels and Npr3 does not have an important scavenging function in this axonal system., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this study, we analyzed the role of drebrin, through shRNA-mediated depletion and overexpression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 min treatment with the branch-inducing signal nerve growth factor increases the levels of axonal drebrin. This study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1092-1110, 2016., (© 2016 Wiley Periodicals, Inc.)

Peripheral nerve myelination is a complex event resulting from spatially and temporally regulated reciprocal interactions between the neuron and myelin-forming Schwann cells. The dynamic process and the protein functional modules and networks that operate throughout the myelination process are poorly understood because of a lack of methodologies suitable for observing specific changes in the Schwann cell/neuron-unit. The identification of the precise roles for the proteins participating in the functional modules and networks that participate in the myelination process is hindered by the cellular and molecular complexity of the nervous tissue itself. We have developed an approach based on a myelinating dorsal root ganglion explant model that allows distinguishing clear, reproducible and predictable differences between the biochemical properties and the genomic and proteomic expression profiles of both cellular components of the Schwann cell/neuron unit at different stages of the myelination process. This model, derived from E13.5 C57BL/6J mouse embryos, is sufficiently robust for use in identifying the protein functional networks and modules related to peripheral nerve myelin formation. The genomic expression profiles of the selected neuronal, Schwann cell and myelin-specific proteins in the cultures reflect in vivo profiles reported in the literature, and the structural and ultrastructural properties of the myelin, as well as the myelination schedule of the cultures, closely resemble those observed in peripheral nerves in situ. The RNA expression data set is available through NCBI gene expression omnibus accession GSE60345. We have developed a reproducible and robust cell culture-based approach, accompanied by a genome-wide expression data set, which allows studying myelination in the peripheral nervous system at the proteomic and transcriptomic levels in Schwann cells and neurons. Myelinating dorsal root explant cultures, prepared from C57BL/6J mouse embryos, present distinct developmental stages comparable to those observed in a peripheral nerve in situ. This model can be used for identifying the protein functional networks and modules related to peripheral nerve myelin formation., (© 2016 International Society for Neurochemistry.)

The transcription factor Casz1 is required for proper assembly of vertebrate vasculature and heart morphogenesis as well as for temporal control of Drosophila neuroblasts and mouse retina progenitors in the generation of different cell types. Although Casz1 function in the mammalian nervous system remains largely unexplored, Casz1 is expressed in several regions of this system. Here we provide a detailed spatiotemporal characterization of Casz1 expression along mouse dorsal root ganglion (DRG) and dorsal spinal cord development by immunochemistry. In the DRG, Casz1 is broadly expressed in sensory neurons since they are born until perinatal age. In the dorsal spinal cord, Casz1 displays a more dynamic pattern being first expressed in dorsal interneuron 1 (dI1) progenitors and their derived neurons and then in a large subset of embryonic dorsal late-born excitatory (dILB) neurons that narrows gradually to become restricted perinatally to the inner portion. Strikingly, expression analyses using Prrxl1-knockout mice revealed that Prrxl1, a key transcription factor in the differentiation of dILB neurons, is a positive regulator of Casz1 expression in the embryonic dorsal spinal cord but not in the DRG. By performing chromatin immunoprecipitation in the dorsal spinal cord, we identified two Prrxl1-bound regions within Casz1 introns, suggesting that Prrxl1 directly regulates Casz1 transcription. Our work reveals that Casz1 lies downstream of Prrxl1 in the differentiation pathway of a large subset of dILB neurons and provides a framework for further studies of Casz1 in assembly of the DRG-spinal circuit., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

During amniote embryogenesis the nervous and vascular systems interact in a process that significantly affects the respective morphogenesis of each network by forming a "neurovascular" link. The importance of neurovascular cross-talk in the central nervous system has recently come into focus with the growing awareness that these two systems interact extensively both during development, in the stem-cell niche, and in neurodegenerative conditions such as Alzheimer's Disease and Amyotrophic Lateral Sclerosis. With respect to the peripheral nervous system, however, there have been no live, real-time investigations of the potential relationship between these two developing systems. To address this deficit, we used multispectral 4D time-lapse imaging in a transgenic quail model in which endothelial cells (ECs) express a yellow fluorescent marker, while neural crest cells (NCCs) express an electroporated red fluorescent marker. We monitored EC and NCC migration in real-time during formation of the peripheral nervous system. Our time-lapse recordings indicate that NCCs and ECs are physically juxtaposed and dynamically interact at multiple locations along their trajectories. These interactions are stereotypical and occur at precise anatomical locations along the NCC migratory pathway. NCCs migrate alongside the posterior surface of developing intersomitic vessels, but fail to cross these continuous streams of motile ECs. NCCs change their morphology and migration trajectory when they encounter gaps in the developing vasculature. Within the nascent dorsal root ganglion, proximity to ECs causes filopodial retraction which curtails forward persistence of NCC motility. Overall, our time-lapse recordings support the conclusion that primary vascular networks substantially influence the distribution and migratory behavior of NCCs and the patterned formation of dorsal root and sympathetic ganglia., (Published by Elsevier Inc.)

Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue.

SIX1 homeodomain protein is one of the essential key regulators of sensory organ development. Six1-deficient mice lack the olfactory epithelium, vomeronasal organs, cochlea, vestibule and vestibuloacoustic ganglion, and also show poor neural differentiation in the distal part of the cranial ganglia. Simultaneous loss of both Six1 and Six4 leads to additional abnormalities such as small trigeminal ganglion and abnormal dorsal root ganglia (DRG). The aim of this study was to understand the molecular mechanism that controls Six1 expression in sensory organs, particularly in the trigeminal ganglion and DRG. To this end, we focused on the sensory ganglia-specific Six1 enhancer (Six1-8) conserved between chick and mouse. In vivo reporter assays using both animals identified an important core region comprising binding consensus sequences for several transcription factors including nuclear hormone receptors, TCF/LEF, SMAD, POU homeodomain and basic-helix-loop-helix proteins. The results provided information on upstream factors and signals potentially relevant to Six1 regulation in sensory neurons. We also report the establishment of a new transgenic mouse line (mSix1-8-NLSCre) that expresses Cre recombinase under the control of mouse Six1-8. Cre-mediated recombination was detected specifically in ISL1/2-positive sensory neurons of Six1-positive cranial sensory ganglia and DRG. The unique features of the mSix1-8-NLSCre line are the absence of Cre-mediated recombination in SOX10-positive glial cells and central nervous system and ability to induce recombination in a subset of neurons derived from the olfactory placode/epithelium. This mouse model can be potentially used to advance research on sensory development.

Background: GABAA receptor-mediated neurotransmission is greatly influenced by cation-chloride cotransporter activity during developmental stages. In embryonic neurons Na-K-2Cl (NKCC1) cotransporters mediate active chloride uptake, thus increasing the intracellular chloride concentration associated with GABA-induced depolarization. At fetal stages near term, oxytocin-induced NKCC1 downregulation has been implicated in the developmental shift from depolarizing to hyperpolarizing GABA action. Mature dorsal root ganglion neurons (DRGN), however, express high NKCC1 levels and maintain high intracellular chloride levels with consequent GABA-induced depolarization., Results: Gramicidin-perforated patch-clamp recordings were used to assess the developmental change in chloride homeostasis in rat cultured small DRGN at the embryonic day 16 (E16) and 19 (E19). The results were compared to data previously obtained in fetal DRGN at E14 and in mature cells. A significant NKCC1 downregulation, leading to reduction in excitatory GABAergic transmission, was observed at E16 and E19., Conclusion: These results indicate that NKCC1 activity transiently decreases in DRGN at fetal stages near term. This developmental shift in GABAergic transmission may contribute to fetal analgesia and neuroprotection at birth.

The Young's modulus of 10-12-day-old chick embryos' sensory neurons cultivated in dissociated cell culture was measured using a PeakForce Quantitative Nanomechanical Mapping atomic force microscopy. The native cells were tested in control experiments and after application of ouabain. At low "endogenous" concentration of 10⁻¹⁰ M, ouabain tended to increase the rigidity of sensory neurons. We hypothesize that this trend resulted from activation of Na⁺,K⁺-ATPase signal-transducing function.

Dorsal root ganglia (DRG) sensory neurons arise from heterogeneous precursors that differentiate in two neurogenic waves, respectively controlled by Neurog2 and Neurog1. We show here that transgenic mice expressing a Zeb1/2 dominant-negative form (DBZEB) exhibit reduced numbers of nociceptors and altered pain sensitivity. This reflects an early impairment of Neurog1-dependent neurogenesis due to the depletion of specific sensory precursor pools, which is slightly later partially compensated by the contribution of boundary cap cells (BCCs). Indeed, combined DBZEB expression and genetic BCCs ablation entirely deplete second wave precursors and, in turn, nociceptors, thus recapitulating the Neurog1(-/-) neuronal phenotype. Altogether, our results uncover roles for Zeb family members in the developing DRGs; they show that the Neurog1-dependent sensory neurogenesis can be functionally partitioned in two successive phases; and finally, they illustrate plasticity in the developing peripheral somatosensory system supported by the BCCs, thereby providing a rationale for sensory precursor diversity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) control synaptic specificity through hetero- or homophilic interactions in different regions of the nervous system. In the developing spinal cord, monosynaptic connections of exquisite specificity form between proprioceptive sensory neurons and motor neurons, however, it is not known whether IgSF molecules participate in regulating this process. To determine whether IgSF molecules influence the establishment of synaptic specificity in sensory-motor circuits, we examined the expression of 157 IgSF genes in the developing dorsal root ganglion (DRG) and spinal cord by in situ hybridization assays. We find that many IgSF genes are expressed by sensory and motor neurons in the mouse developing DRG and spinal cord. For instance, Alcam, Mcam, and Ocam are expressed by a subset of motor neurons in the ventral spinal cord. Further analyses show that Ocam is expressed by obturator but not quadriceps motor neurons, suggesting that Ocam may regulate sensory-motor specificity in these sensory-motor reflex arcs. Electrophysiological analysis shows no obvious defects in synaptic specificity of monosynaptic sensory-motor connections involving obturator and quadriceps motor neurons in Ocam mutant mice. Since a subset of Ocam+ motor neurons also express Alcam, Alcam or other functionally redundant IgSF molecules may compensate for Ocam in controlling sensory-motor specificity. Taken together, these results reveal that IgSF molecules are broadly expressed by sensory and motor neurons during development, and that Ocam and other IgSF molecules may have redundant functions in controlling the specificity of sensory-motor circuits.

Our understanding of the role of somatosensory feedback in regulating motility during chicken embryogenesis and fetal development in general has been hampered by the lack of an approach to selectively alter specific sensory modalities. In adult mammals, pyridoxine overdose has been shown to cause a peripheral sensory neuropathy characterized by a loss of both muscle and cutaneous afferents, but predominated by a loss of proprioception. We have begun to explore the sensitivity of the nervous system in chicken embryos to the application of pyridoxine on embryonic days 7 and 8, after sensory neurons in the lumbosacral region become post-mitotic. Upon examination of the spinal cord, dorsal root ganglion and peripheral nerves, we find that pyridoxine causes a loss of neurotrophic tyrosine kinase receptor type 3-positive neurons, a decrease in the diameter of the muscle innervating nerve tibialis, and a reduction in the number of large diameter axons in this nerve. However, we found no change in the number of Substance P or calcitonin gene-related peptide-positive neurons, the number of motor neurons or the diameter or axonal composition of the femoral cutaneous nerve. Therefore, pyridoxine causes a peripheral sensory neuropathy in embryonic chickens largely consistent with its effects in adult mammals. However, the lesion may be more restricted to proprioception in the chicken embryo. Therefore, pyridoxine lesion induced during embryogenesis in the chicken embryo can be used to assess how the loss of sensation, largely proprioception, alters spontaneous embryonic motility and subsequent motor development., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

The rate of proliferation of cells depends on the proportion of cycling cells and the frequency of cell division. Here, we describe in detail methods for quantifying the proliferative behavior of specific cell types in situ, and use the method to examine cell cycle dynamics in two neural crest derivatives--dorsal root ganglia (DRG) using frozen sections, and the enteric nervous system (ENS) using wholemount preparations. In DRG, our data reveal a significant increase in cell cycle length and a decrease in the number of cycling Sox10+ progenitor cells at E12.5-E13.5, which coincides with the commencement of glial cell generation. In the ENS, the vast majority of Sox10+ cells remain proliferative during embryonic development, with only relatively minor changes in cell cycle parameters. Previous studies have identified proliferating cells expressing neuronal markers in the developing ENS; our data suggest that most cells undergoing neuronal differentiation in the developing gut commence expression of neuronal markers during G2 phase of their last division. Combined with previous studies, our findings show that different populations of neural crest-derived cells show tissue-specific patterns of proliferation., (© 2014 Wiley Periodicals, Inc.)

Molecular mechanisms governing the maintenance and proliferation of dorsal root ganglia (DRG) progenitors are largely unknown. Here we reveal that the Hippo pathway regulates the expansion of DRG progenitors and glia during mammalian DRG development. The key effectors of this pathway, transcriptional coactivators Yap and Taz, are expressed in DRG progenitors and glia during DRG development but are at least partially inhibited from activating transcription. Aberrant YAP activation leads to overexpansion of DRG progenitor and glial populations. We further show that the Neurofibromatosis 2 (Nf2) tumor suppressor inhibits Yap during DRG development. Loss of Nf2 leads to similar phenotypes as does YAP hyperactivation, and deleting Yap suppresses these phenotypes. Our study demonstrates that Nf2-Yap signaling plays important roles in controlling the expansion of DRG progenitors and glia during DRG development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkA(taulacZ) mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkA(taulacZ) line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkA(taulacZ)/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

How animals adjust the size of their organs is a fundamental, enduring question in biology. Here we manipulate the amount of neural crest (NC) precursors for the dorsal root ganglia (DRG) in axolotl. We produce embryos with an under- or over-supply of pre-migratory NC in order to find out if DRG can regulate their sizes during development. Axolotl embryos are perfectly suitable for this research. Firstly, they are optimal for microsurgical manipulations and tissue repair. Secondly, they possess, unlike most other vertebrates, only one neural crest string located on top of the neural tube. This condition and position enables NC cells to migrate to either side of the embryo and participate in the regulation of NC cell distribution. We show that size compensation of DRG in axolotl occurs in 2 cm juveniles after undersupply of NC (up-regulation) and in 5 cm juveniles after oversupply of NC (down-regulation). The size of DRG is likely to be regulated locally within the DRG and not via adaptations of the pre-migratory NC or during NC cell migration. Ipsi- and contralateral NC cell migration occurs both in embryos with one and two neural folds, and contralateral migration of NC is the only source for contralateral DRG formation in embryos with only one neural fold. Compensatory size increase is accompanied by an increase in cell division of a DRG precursor pool (PCNA+/SOX2-), rather than by DRG neurons or glial cells. During compensatory size decrease, increased apoptosis and reduced proliferation of DRG cells are observed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The direct effect of guidance cues on developing and regenerating axons in vivo is not fully understood, as the process involves a multiplicity of attractive and repulsive signals, presented both as soluble and membrane-bound ligands. A better understanding of axon guidance is critical to functional recovery following injury to the nervous system through improved outgrowth and mapping of damaged nerves. Due to their implications as inhibitors to central nervous system regeneration, we investigated the repulsive properties of semaphorin 6A and ephrin-B3 on E15 rat dorsal root ganglion explants, as well as possible interactions with soluble gradients of chemoattractive nerve growth factor (NGF). We employed a 3D biomimetic in vitro choice point model, which enabled the simple and rapid preparation of patterned gel growth matrices with quantifiable presentation of guidance cues in a specifiable manner that resembles the in vivo presentation of soluble and/or immobilized ligands. Neurites demonstrated an inhibitory response to immobilized Sema6A by lumbosacral dorsal root ganglion explants, while no such repulsion was observed for immobilized ephrin-B3 by explants at any spinal level. Interestingly, Sema6A inhibition could be partially attenuated in a concentration-dependent manner through the simultaneous presentation of soluble NGF gradients. The in vitro model described herein represents a versatile and valuable investigative tool in the quest for understanding developmental processes and improving regeneration following nervous system injury.

The development of dissociated cells from rat embryonic spinal ganglion after transplantation to damaged nerve of adult animals was studied using immunohistochemical differentiation markers of neural and glial cells. The cell suspension obtained after dissociation of rat embryonic spinal ganglia (embryonic day 15) was injected into the proximal segment of crushed sciatic nerve. The nerve was damaged by ligation for 40 sec. Progenitor cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before transplantation. BrdU-immunopositive cells were detected in the nerve trunks of recipients on days 1, 21, and 28 after transplantation. Dissociated cells of rat embryonic spinal ganglion (embryonic day 15) survived for at least 4 weeks after transplantation to the nerve and differentiate into NeuN-immunopositive neurons with morphological properties of sensory neurons and satellite cells containing S100 protein.