Pioneer Axons Utilize a Dcc Signaling-Mediated Invasion Brake to Precisely Complete Their Pathfinding Odyssey.

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.

Isolation and Culture of Dorsal Root Ganglia (DRG) from Rodents.

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.

Neuronally Enriched RUFY3 Is Required for Caspase-Mediated 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.

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system.

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.

Nociceptor-like rat dorsal root ganglion neurons express the angiotensin-II AT2 receptor throughout development.

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.

Local synthesis of dynein cofactors matches retrograde transport to acutely changing demands.

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 p150Glued. 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.

An ensemble of regulatory elements controls Runx3 spatiotemporal expression in subsets of dorsal root ganglia proprioceptive neurons.

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.

Optogenetic activation of axon guidance receptors controls direction of neurite outgrowth.

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.

Application of atomic force microscopy for investigation of Na(+),K(+)-ATPase signal-transducing function.

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 10⁻¹° 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.

Nf2-Yap signaling controls the expansion of DRG progenitors and glia during DRG development.

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.

Whole-mount imaging of mouse embryo sensory axon projections.

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.

MLLT11/AF1q is differentially expressed in maturing neurons during development.

Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11 or ALL1 fused from chromosome 1q (MLLT11/AF1q) is a highly conserved 90 amino acid protein that functions in hematopoietic differentiation. Its translocation to the Trithorax locus has been implicated in malignancies of the hematopoietic system. However, the spatio-temporal profile of MLLT11 expression during embryonic development has not been characterized. Here we show that MLLT11 has a remarkably specific expression pattern in the developing central and peripheral nervous system. We find high levels of MLLT11 transcript and protein expression in the developing marginal zone of the cortex and spinal cord. MLLT11 co-localized with Tbr2 in the developing subplate region of the cortex and expanded to encompass the cortical plate at late fetal stages. Expression in the peripheral nervous system initiated at E9.5 in the facio-acoustic cranial ganglia and elaborated to identify all the cranio-facial and dorsal root ganglia by E10.5. We also observed expression in the eye and gastrointestinal tract, where MLLT11 transcripts localized to Tuj1-positive inner retinal layer and autonomic neurons, respectively. Altogether these results show that MLLT11 is a pan-neuronal marker, suggesting a role in neural differentiation in the central nervous system and neural crest-cell derived peripheral ganglia.

The negative influence of high-glucose ambience on neurogenesis in developing quail embryos.

Gestational diabetes is defined as glucose intolerance during pregnancy and it is presented as high blood glucose levels during the onset pregnancy. This condition has an adverse impact on fetal development but the mechanism involved is still not fully understood. In this study, we investigated the effects of high glucose on the developing quail embryo, especially its impact on the development of the nervous system. We established that high glucose altered the central nervous system mophologically, such that neural tube defects (NTDs) developed. In addition, we found that high glucose impaired nerve differentiation at dorsal root ganglia and in the developing limb buds, as revealed by neurofilament (NF) immunofluorescent staining. The dorsal root ganglia are normally derived from neural crest cells (NCCs), so we examine the delamination of NCCs from dorsal side of the neural tube. We established that high glucose was detrimental to the NCCs, in vivo and in vitro. High glucose also negatively affected neural differentiation by reducing the number and length of neurites emanating from neurons in culture. We established that high glucose exposure caused an increase in reactive oxidative species (ROS) generation by primary cultured neurons. We hypothesized that excess ROS was the factor responsible for impairing neuron development and differentiation. We provided evidence for our hypothesis by showing that the addition of vitamin C (a powerful antioxidant) could rescue the damaging effects of high glucose on cultured neurons.

Neuroprotective effects of resveratrol on embryonic dorsal root ganglion neurons with neurotoxicity induced by ethanol.

Studies have established that ethanol (EtOH) consumption results in damage to the peripheral nervous systems. Although the pathobiological mechanism is still unclear, oxidative stress is known to play an important role in EtOH-induced neurotoxicity. Because resveratrol (Res) is attracting increased attention due to its antioxidative properties, we investigated the neuroprotective efficacy of Res in ethanol-treated embryonic dorsal root ganglion (DRG) neurons in vitro. Organotypic DRG explants and a dispersed cell culture model were used to evaluate the effects of Res on EtOH-induced neurotoxicity. Res increased the number of extended nerve fibers and neurons that migrated from the DRG explants. Hoechst 33342 staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling analysis showed that the EtOH-induced apoptosis was inhibited by Res. The effects of Res were blocked by the 5'-adenosine monophosphate-activated protein kinase inhibitor Compound C and the sirtuin 1 inhibitor nicotinamide. The elevation of oxidative/nitrosative stress, as measured by the amount of reactive oxygen species, malondialdehyde, nitrite, glutathione and superoxide dismutase activity, was also attenuated by Res. The data from the present study indicate that Res protects DRG neurons from EtOH-induced neurotoxicity. Res and its derivative may be effective for the treatment of diseases characterized by axonopathy and neuron loss induced by EtOH.

Positional differences of axon growth rates between sensory neurons encoded by Runx3.

The formation of functional connectivity in the nervous system is governed by axon guidance that instructs nerve growth and branching during development, implying a similarity between neuronal subtypes in terms of nerve extension. We demonstrate the molecular mechanism of another layer of complexity in vertebrates by defining a transcriptional program underlying growth differences between positionally different neurons. The rate of axon extension of the early subset of embryonic dorsal root ganglion sensory neurons is encoded in neurons at different axial levels. This code is determined by a segmental pattern of axial levels of Runx family transcription factor Runx3. Runx3 in turn determines transcription levels of genes encoding cytoskeletal proteins involved in axon extension, including Rock1 and Rock2 which have ongoing activities determining axon growth in early sensory neurons and blocking Rock activity reverses axon extension deficits of Runx3(-/-) neurons. Thus, Runx3 acts to regulate positional differences in axon extension properties apparently without affecting nerve guidance and branching, a principle that could be relevant to other parts of the nervous system.

Tlx3 and Runx1 act in combination to coordinate the development of a cohort of nociceptors, thermoceptors, and pruriceptors.

Neurons in the mouse dorsal root ganglia (DRGs) are composed of a variety of sensory modalities, such as pain-related nociceptors, itch-related pruriceptors, and thermoceptors. All these neurons are derived from late-born neurons that are initially marked by the expression of the nerve growth factor receptor TrkA. During perinatal and postnatal development, these TrkA lineage neurons are globally segregated into Ret-expressing and TrkA-expressing subtypes, and start to express a variety of sensory receptors and ion channels. The runt domain transcription factor Runx1 plays a pivotal role in controlling these developmental processes, but it remains unclear how it works. Here we showed that the homeodomain transcription factor Tlx3, expressed broadly in DRG neurons, is required to establish most Runx1-dependent phenotypes, including the segregation of TrkA-expressing versus Ret-expressing neurons and the expression of a dozen of sensory channels and receptors implicated in sensing pain, itch and temperature. Expression of Runx1 and Tlx3 is independent of each other at prenatal stages when they first establish the expression of these channels and receptors. Moreover, overexpression of Runx1 plus Tlx3 was able to induce ectopic expression of sensory channels and receptors. Collectively, these studies suggest that genetically Tlx3 acts in combination with Runx1 to control the development of a cohort of nociceptors, thermoceptors, and pruriceptors in mice.

Establishment of immortalized Schwann cells derived from rat embryo dorsal root ganglia.

Schwann cells (SCs) play an important role in the development, function and regeneration of peripheral nerves. They can enhance both peripheral and central nerve regeneration by providing a supportive environment for neurite outgrowth through the release of neurotrophic factors. However, use of primary SCs for in vitro models is limited because these cells are difficult to prepare and maintain in high yield and purity under common cell culture conditions. Human telomerase reverse transcriptase (hTERT) expression induces immortalization of various cell types without substantial alterations of their phenotypes. Therefore, in this study we transfected SCs with hTERT to establish a reliable cell source and observed the effect of hTERT on SCs. In order to accomplish this, SCs were isolated from rat embryo dorsal root ganglions, transfected with hTERT at early passage (passage 3). SCs passage 4, 8, 12 and 30 after transfection (hTERT-SCs) were used for immunocytochemistry, RT-PCR and western blotting. Results showed that all the early (passage 4) and late (passage 30) passage hTERT-SCs expressed hTERT mRNA and gained full telomerase activity. The transfection did not alter the mRNA expression of senescence-associated genes, such as p53 and p16. The expression of BDNF (brain-derived neurotrophic factor) was significantly decreased as cell passage increased, compared to the untransfected control. On the other hand, the expression of NGF (nerve growth factor ) was elevated at early passages (passages 4 and 8) and decreased at late passages (12 and 30). These data indicate that the use of specific immortalization techniques can establish SC lines that retain characteristics of typical primary SCs, and different mechanisms responsible for regulating NGF and BDNF expression. This is the first report regarding the immortalization of SCs derived from rat embryo dorsal root ganglions. These cells are useful in studies investigating the cellular mechanisms and regenerative processes of SCs.

c-Maf is required for the development of dorsal horn laminae III/IV neurons and mechanoreceptive DRG axon projections.

Establishment of proper connectivity between peripheral sensory neurons and their central targets is required for an animal to sense and respond to various external stimuli. Dorsal root ganglion (DRG) neurons convey sensory signals of different modalities via their axon projections to distinct laminae in the dorsal horn of the spinal cord. In this study, we found that c-Maf was expressed predominantly in the interneurons of laminae III/IV, which primarily receive inputs from mechanoreceptive DRG neurons. In the DRG, c-Maf⁺ neurons also coexpressed neurofilament-200, a marker for the medium- and large-diameter myelinated afferents that transmit non-noxious information. Furthermore, mouse embryos deficient in c-Maf displayed abnormal development of dorsal horn laminae III/IV neurons, as revealed by the marked reduction in the expression of several marker genes for these neurons, including those for transcription factors MafA and Rora, GABA(A) receptor subunit α5, and neuropeptide cholecystokinin. In addition, among the four major subpopulations of DRG neurons marked by expression of TrkA, TrkB, TrkC, and MafA/GFRα2/Ret, c-Maf was required selectively for the proper differentiation of MafA⁺/Ret⁺/GFRα2⁺ low-threshold mechanoreceptors (LTMs). Last, we found that the central and peripheral projections of mechanoreceptive DRG neurons were compromised in c-Maf deletion mice. Together, our results indicate that c-Maf is required for the proper development of MafA⁺/Ret⁺/GFRα2⁺ LTMs in the DRG, their afferent projections in the dorsal horn and Pacinian corpuscles, as well as neurons in laminae III/IV of the spinal cord.

The transcription factor c-Maf controls touch receptor development and function.

The sense of touch relies on detection of mechanical stimuli by specialized mechanosensory neurons. The scarcity of molecular data has made it difficult to analyze development of mechanoreceptors and to define the basis of their diversity and function. We show that the transcription factor c-Maf/c-MAF is crucial for mechanosensory function in mice and humans. The development and function of several rapidly adapting mechanoreceptor types are disrupted in c-Maf mutant mice. In particular, Pacinian corpuscles, a type of mechanoreceptor specialized to detect high-frequency vibrations, are severely atrophied. In line with this, sensitivity to high-frequency vibration is reduced in humans carrying a dominant mutation in the c-MAF gene. Thus, our work identifies a key transcription factor specifying development and function of mechanoreceptors and their end organs.

The metalloproteinase inhibitor Reck is essential for zebrafish DRG development.

The neural crest is a migratory, multipotent cell lineage that contributes to myriad tissues, including sensory neurons and glia of the dorsal root ganglia (DRG). To identify genes affecting cell fate specification in neural crest, we performed a forward genetic screen for mutations causing DRG deficiencies in zebrafish. This screen yielded a mutant lacking all DRG, which we named sensory deprived (sdp). We identified a total of four alleles of sdp, all of which possess lesions in the gene coding for reversion-inducing cysteine-rich protein containing Kazal motifs (Reck). Reck is an inhibitor of metalloproteinases previously shown to regulate cell motility. We found reck function to be both necessary for DRG formation and sufficient to rescue the sdp phenotype. reck is expressed in neural crest cells and is required in a cell-autonomous fashion for appropriate sensory neuron formation. In the absence of reck function, sensory neuron precursors fail to migrate to the position of the DRG, suggesting that this molecule is crucial for proper migration and differentiation.