Development Of Dorsal Root Ganglia Research Articles

Cleaved Delta like 1 intracellular domain regulates neural development via Notch signal-dependent and -independent pathways

ABSTRACTNotch-Delta signaling regulates many developmental processes, including tissue homeostasis and maintenance of stem cells. Upon interaction of juxtaposed cells via Notch and Delta proteins, intracellular domains of both transmembrane proteins are cleaved and translocate to the nucleus. Notch intracellular domain activates target gene expression; however, the role of the Delta intracellular domain remains elusive. Here, we show the biological function of Delta like 1 intracellular domain (D1ICD) by modulating its production. We find that the sustained production of D1ICD abrogates cell proliferation but enhances neurogenesis in the developing dorsal root ganglia (DRG), whereas inhibition of D1ICD production promotes cell proliferation and gliogenesis. D1ICD acts as an integral component of lateral inhibition mechanism by inhibiting Notch activity. In addition, D1ICD promotes neurogenesis in a Notch signaling-independent manner. We show that D1ICD binds to Erk1/2 in neural crest stem cells and inhibits the phosphorylation of Erk1/2. In summary, our results indicate that D1ICD regulates DRG development by modulating not only Notch signaling but also the MAP kinase pathway.

The expression of prosaposin and its receptors, GRP37 and GPR37L1, are increased in the developing dorsal root ganglion.

Prosaposin (PSAP), a highly conserved glycoprotein, is a precursor of saposins A–D. Accumulating evidence suggests that PSAP is a neurotrophic factor, as well as a regulator of lysosomal enzymes. Recently, the orphan G-protein-coupled receptors GPR37 and GPR37L1 were recognized as PSAP receptors, but their functions have not yet been clarified. In this study, we examined the distribution of PSAP and its receptors in the dorsal root ganglion (DRG) during development using specific antibodies, and showed that PSAP accumulates primarily in lysosomes and is dispersed throughout the cytoplasm of satellite cells. Later, PSAP colocalized with two receptors in satellite cells, and formed a characteristic ring shape approximately 8 weeks after birth, during a period of rapid DRG development. This ring shape, which was only observed around larger neurons, is evidence that several satellite cells are synchronously activated. We found that sortilin, a transporter of a wide variety of intracellular proteins containing PSAP, is strongly localized to the inner side of satellite cells, which contact the neuronal surface. These findings suggest that PSAP and GPR37/GPR37L1 play a role in activating both satellite and nerve cells.

Nociceptor subtypes are born continuously over DRG development

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.

Integrin-Linked Kinase (ILK) Plays an Important Role in the Laminin-Dependent Development of Dorsal Root Ganglia during Chicken Embryogenesis

Integrin-linked kinase (ILK) is mainly localized in focal adhesions where it interacts and modulates the downstream signaling of integrins affecting cell migration, adhesion, and survival. The interaction of dorsal root ganglia (DRG) cells, being part of the peripheral nervous system (PNS), with the extracellular matrix (ECM) via integrins is crucial for proper PNS development. A few studies have focused on ILK’s role in PNS development, but none of these have focused on chicken. Therefore, we decided to investigate ILK’s role in the development of Gallus gallus domesticus’s DRG. First, using RT-PCR, Western blotting, and in situ hybridization, we show that ILK is expressed in DRG. Next, by immunocytochemistry, we show ILK’s localization both intracellularly and on the cell membrane of DRG neurons and Schwann cell precursors (SCPs). Finally, we describe ILK’s involvement in multiple aspects of DRG development by performing functional experiments in vitro. IgG-mediated interruption of ILK’s action improved DRG neurite outgrowth, modulated their directionality, stimulated SCPs migration, and impacted growth cone morphology in the presence of laminin-1 or laminin-1 mimicking peptide IKVAV. Taken together, our results show that ILK is important for chicken PNS development, probably via its exposure to the ECM.

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons.

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors.

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these components form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signaling, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unexpected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic phenotype. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the balance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernumerary DRG sensory neurons in a wild-type background, and can rescue the sensory neuron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10(baz1) protein’s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lateral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates.

Ontogenic expression profiles and oxaliplatin regulation of leptin expression in mice dorsal root ganglion.

Leptin is widely distributed in many tissues, including the nervous system. However, the ontogeny of leptin expression in the dorsal root ganglion (DRG) is unclear. Recent studies have shown that leptin is involved in the regulation of neuropathic pain induced by nerve injury. Our previous results showed that exogenous leptin administration alleviated the pain behaviors induced by chronic constriction sciatic nerve injury. In the present study, the ontogenic expression of leptin was detected in the DRG of the mouse embryo at days 15.5 (E15.5), E17.5, and E19.5 of gestation and in the postnatal mouse at days 5 (P5), P15, and P25, and in the adult mouse. Leptin immunoreactivity and mRNA were not found in DRG at E15.5. The percentage of leptin immunopositive (leptin) neurons was about 27% at E17.5. It continued to increase to about 70% at P5. From P5 to P15, there was no significant change. The proportion of DRG neurons positive for leptin decreased after P15 and there were about 41% leptin neurons in adults. The expression profile of leptin mRNA is similar to leptin immunoreactivity. Oxaliplatin (OXA) is an effective platinum-based drug used as first-line chemotherapy for advanced colorectal cancer. However, it may induce neuropathic pain. In the current study, we found that the expression of leptin was increased in the lumbar 4-6 DRG of OXA-treated mice. These results indicate that leptin is involved in the regulation of DRG development and OXA-induced neuropathic pain.

Runx3-regulated expression of two Ntrk3 transcript variants in dorsal root ganglion neurons.

Somatosensation is divided into proprioception and cutaneous sensation. Dorsal root ganglion (DRG) neurons project their fibers toward peripheral targets including muscles and skin, and centrally to the spinal cord. Proprioceptive DRG neurons transmit information from muscle spindles and Golgi tendon organs to the spinal cord. We previously showed that Runt-related transcription factor 3 (Runx3) is expressed in these neurons and their projections to the ventral spinal cord and muscle spindles are lost in Runx3-deficient (Runx3(-/-) ) mouse embryos. Although Runx3 is likely to contribute to the fate decision and projection of proprioceptive DRG neurons, the precise roles for Runx3 in these phenomena are unknown. To identify genes regulated by Runx3 in embryonic DRGs, we performed microarray analyses using cDNAs isolated from wild-type and Runx3(-/-) DRGs of embryonic day (E) 12.5 and selected two transcript variants of the tyrosine kinase receptor C (TrkC) gene. These variants, Ntrk3 variant 1 (Ntrk3-v1) and variant 2 (Ntrk3-v2), encode full-length and truncated receptors of neurotrophin-3, respectively. Using double in situ hybridization, we found that most of Ntrk3-v1 mRNA expression in E14.5 DRGs depended on Runx3 but that more than half of Ntrk3-v2 mRNA one were expressed in a Runx3-independent manner. Furthermore, our data revealed that the rate of Ntrk3-v1 and Ntrk3-v2 colocalization in DRGs changed from E14.5 to E18.5. Together, our data suggest that Runx3 may play a crucial role in the development of DRGs by regulating the expression of Ntrk3 variants and that DRG neurons expressing Ntrk3-v1 but not Ntrk3-v2 may differentiate into proprioceptive ones.

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.

Role of myosin Va in neuritogenesis of chick dorsal root ganglia nociceptive neurons

Myosin-Va, widely distributed throughout the developing nervous system, is involved in the transport of vesicles and other intracellular components with its globular tail domain (GTD) implicated in cargo recognition/interaction. Inactivation of myosin-Va in dorsal root ganglia (DRG) neurons of chick embryos, in vitro, decreases the rate of filopodial extension. MYO5A mutant mice have severe neurological defects. We have found that the overexpression of GTD in DRG cultures reduces the number of neurons with long neurites (above fourfold cell body length) and increased the number of neurons with short or no neurites. However, if transfection occurred after the onset of neuritogenesis, this was not seen. In embryo, we characterized the expression pattern of myosin-Va during neuritogenesis of TrkA-positive cells at different stages of chick DRG development. Myosin-Va expression was detected starting from HH25. At this stage, it was present in cells both with and without neurites. The presence of myosin-Va in DRG neurites persisted throughout the last stage analysed (HH34). The data suggest that Myosin Va can participate in embryonic DRG neuritogenesis.

Low-Density Lipoprotein Receptor Related protein-1 (LRP1)-Dependent Cell Signaling Promotes Neurotrophic Activity in Embryonic Sensory Neurons

Developing sensory neurons require neurotrophic support for survival, neurite outgrowth and myelination. The low-density lipoprotein receptor-related protein-1 (LRP1) transactivates Trk receptors and thereby functions as a putative neurotrophin. Herein, we show that LRP1 is abundantly expressed in developing dorsal root ganglia (DRG) and that LRP1-dependent cell signaling supports survival, neurite extension and receptivity to Schwann cells even in the absence of neurotrophins. Cultured embryonic DRG neurons (E15) were treated with previously characterized LRP1 ligands, LRP1-receptor binding domain of α2-macroglobulin (RBD), hemopexin domain of MMP-9 (PEX) or controls (GST) for two weeks. These structurally diverse LRP1 ligands significantly activated and sustained extracellular signal-regulated kinases (ERK1/2) 5-fold (p<0.05), increased expression of growth-associated protein-43(GAP43) 15-fold (P<0.01), and increased neurite outgrowth 20-fold (P<0.01). Primary sensory neurons treated with LRP1 ligands survived > 2 weeks in vitro, to an extent equaling NGF, a finding associated with canonical signaling mechanisms and blockade of caspase-3 cleavage. LRP1 ligand-induced survival and sprouting were blocked by co-incubation with the LRP1 antagonist, receptor associated protein (RAP), whereas RAP had no effect on NGF-induced activity. Site directed mutagenesis of the LRP1 ligand, RBD, in which Lys1370 and Lys1374 are converted to alanine to preclude LRP1 binding, were ineffective in promoting cell signaling, survival or inducing neurite extension in primary sensory neurons, confirming LRP1 specificity. Furthermore, LRP1-induced neurite sprouting was mediated by Src-family kinase (SFK) activation, suggesting transactivation of Trk receptors. Co-cultures of primary embryonic neurons and Schwann cells showed that LRP1 agonists promoted axonal receptivity to myelination to Schwann cells. Collectively, these findings identify LRP1 as a novel and perhaps essential trophic molecule for sensory neuronal survival and development.

Modulation of dorsal root ganglion development by ErbB signaling and the scaffold protein Sorbs3

The multipotent cells of the vertebrate neural crest (NC) arise at the dorsal aspect of the neural tube, then migrate throughout the developing embryo and differentiate into diverse cell types, including the sensory neurons and glia of the dorsal root ganglia (DRG). As multiple cell types are derived from this lineage, it is ideal for examining mechanisms of fate restriction during development. We have isolated a mutant, ouchless, that specifically fails to develop DRG neurons, although other NC derivatives develop normally. This mutation affects the expression of Sorbs3, a scaffold protein known to interact with proteins involved in focal adhesions and several signaling pathways. ouchless mutants share some phenotypic similarities with mutants in ErbB receptors, EGFR homologs that are implicated in diverse developmental processes and associated with several cancers; and ouchless interacts genetically with an allele of erbb3 in DRG neurogenesis. However, the defect in ouchless DRG neurogenesis is distinct from ErbB loss of function in that it is not associated with a loss of glia. Both ouchless and neurogenin1 heterozygous fish are sensitized to the effects of ErbB chemical inhibitors, which block the development of DRG in a dose-dependent manner. Inhibitors of MEK show similar effects on DRG neurogenesis. We propose a model in which Sorbs3 helps to integrate ErbB signals to promote DRG neurogenesis through the activation of MAPK and upregulation of neurogenin1.

Dok5 is involved in the signaling pathway of neurotrophin-3 against TrkC-induced apoptosis

TrkC is a dependence receptor and many reports have shown that neurotrophin-3 promotes cell survival by inhibiting TrkC-induced apoptosis in many cell lines. However, the identity of the adaptor protein involved in the NT-3/TrkC signaling pathway regulating cell death and survival remains unclear. The downstream of tyrosine kinase/docking protein (Dok) adaptor protein 5 is one substrate of the TrkC receptor. Because NT-3 and its receptor, TrkC, are strongly expressed by sensory neurons, we measured the expression of Dok5 and TrkC in the developing mouse spinal cord and dorsal root ganglia (DRG). We found that the number of cells positive for both Dok5 and TrkC decreases with DRG development. Immunoprecipitation and immunofluorescence staining showed that Dok5 interacted with TrkC and partially colocalized with TrkC in DRG neurons. In HEK293T cells, TrkC triggered apoptosis, but NT-3 prevented TrkC-induced apoptosis. Interestingly, siRNA knockdown of Dok5 expression partially prevented the protection of NT-3 against TrkC-induced apoptosis by regulating the activity of caspase-3. Taken together, we concluded that Dok5 is necessary for NT-3 signaling to block TrkC-induced apoptosis.

Depending on its nano-spacing, ALCAM promotes cell attachment and axon growth.

ALCAM is a member of the cell adhesion molecule (CAM) family which plays an important role during nervous system formation. We here show that the two neuron populations of developing dorsal root ganglia (DRG) display ALCAM transiently on centrally and peripherally projecting axons during the two phases of axon outgrowth. To analyze the impact of ALCAM on cell adhesion and axon growth, DRG single cells were cultured on ALCAM-coated coverslips or on nanopatterns where ALCAM is presented in physiological amino-carboxyl terminal orientation at highly defined distances (29, 54, 70, 86, and 137 nm) and where the interspaces are passivated to prevent unspecific protein deposition. Some axonal features (branching, lateral deviation) showed density dependence whereas others (number of axons per neuron, various axon growth parameters) turned out to be an all-or-nothing reaction. Time-lapse analyses revealed that ALCAM density has an impact on axon velocity and advance efficiency. The behavior of the sensory axon tip, the growth cone, partially depended on ALCAM density in a dose-response fashion (shape, dynamics, detachment) while other features did not (size, complexity). Whereas axon growth was equally promoted whether ALCAM was presented at high (29 nm) or low densities (86 nm), the attachment of non-neuronal cells depended on high ALCAM densities. The attachment of non-neuronal cells to the rather unspecific standard proteins presented by conventional implants designed to enhance axonal regeneration is a severe problem. Our findings point to ALCAM, presented as 86 nm pattern, for a promising candidate for the improvement of such implants since this pattern drives axon growth to its full extent while at the same time non-neuronal cell attachment is clearly reduced.

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.

The role and mechanism of activin A in neurite outgrowth of embryonic dorsal root ganglia of chicken

Activin A, a member of the transforming growth factor β (TGFβ) superfamily, plays an essential role in neuron survival as a neurotrophic and neuroprotective factor in the central nervous system. However, the effects and mechanisms of action of activin A on the neurite outgrowth of dorsal root ganglia (DRG) remain unclear. In the present study, we found that activin A is expressed in DRG collected from chicken embryos on embryonic day 8 (E8). Moreover, activin A induced neurite outgrowth of the primary cultured DRG and maintained the survival of monolayer-cultured DRG neurons throughout the observation period of ten days. Follistatin (FS), an activin-binding protein, significantly inhibited activin A-induced neurite outgrowth of DRG, but failed to influence the effect of nerve growth factor (NGF) on DRG neurite outgrowth. Furthermore, the results showed that activin A significantly upregulated mRNA expression of activin receptor type IIA (ActRIIA) and calcitonin gene-related peptide (CGRP) in DRG, and stimulated serotonin (5-HT) production from DRG, indicating that activin A might induce DRG neurite outgrowth by promoting CGRP expression and stimulating 5-HT release. These data suggest that activin A plays an important role in the development of DRG in an autocrine or paracrine manner.

Age‐related changes in two‐pore domain acid‐sensitive K+ channel expression in rat dorsal root ganglion neurons

1. Two-pore domain K⁺ (K(2P) ) channel expression influences brain development. The K(2P) channels, including two-pore domain acid-sensitive K⁺ (TASK) channels, contribute to the setting of the resting membrane potential of neurons. In addition to neurons in the brain, dorsal root ganglion (DRG) neurons also express K(2P) channels. The aim of the present study was to identify postnatal changes in the expression of TASK channels in DRG neurons. 2. Expression of TASK channels (TASK-1, TASK-2 and TASK-3) was compared between neonatal (postnatal Day (P) 1 or P2) and adult (P120) rat DRG using semiquantitative polymerase chain reaction, western blot analysis, immunostaining and the patch-clamp technique. 3. In adult (P120) rat DRG, expression of TASK-2 mRNA and protein was downregulated, whereas TASK-3 mRNA and protein expression was upregulated. There were no consistent changes in TASK-1 mRNA and protein expression. Single-channel recordings showed very low TASK-2- and TASK-3-like channel expression in P1-P2 DRG neurons (∼10% in TASK-2 and ∼3% in TASK-3). In P120 DRG, there was a reduction in the detection of TASK-2-like channels, whereas the detection of TASK-3-like channels increased. 4. These results show that TASK-2 and TASK-3 mRNA and protein expression undergoes age-related changes in DRG neurons, indicating that TASK-2 and TASK-3 channels are likely to contribute to the setting of the resting membrane potential of DRG neurons in neonates and adults, separately or together, during DRG development.

The role of the transcription factor Rbpj in the development of dorsal root ganglia

BackgroundThe dorsal root ganglion (DRG) is composed of well-characterized populations of sensory neurons and glia derived from a common pool of neural crest stem cells (NCCs), and is a good system to study the mechanisms of neurogenesis and gliogenesis. Notch signaling is known to play important roles in DRG development, but the full scope of Notch functions in mammalian DRG development remains poorly understood.ResultsIn the present study, we used Wnt1-Cre to conditionally inactivate the transcription factor Rbpj, a critical integrator of activation signals from all Notch receptors, in NCCs and their derived cells. Deletion of Rbpj caused the up-regulation of NeuroD1 and precocious neurogenesis in DRG early development but led to an eventual deficit of sensory neurons at later stages, due to reduced cell proliferation and abnormal cell death. In addition, gliogenesis was delayed initially, but a near-complete loss of glia was observed finally in Rbpj-deficient DRG. Furthermore, we found P75 and Sox10, which are normally expressed exclusively in neuronal and glial progenitors of the DRG after the NCCs have completed their migration, were co-expressed in many cells of the DRG of Rbpj conditional knock-out mice.ConclusionsOur data indicate that Rbpj-mediated canonical Notch signaling inhibits DRG neuronal differentiation, possibly by regulating NeuroD1 expression, and is required for DRG gliogenesis in vivo.

Glial precursors clear sensory neuron corpses during development via Jedi-1, an engulfment receptor

During the development of peripheral ganglia 50 % of the neurons generated undergo apoptosis. How the massive numbers of corpses are removed is unknown. We report that satellite glial cell precursors are the primary phagocytic cells for apoptotic corpse removal in developing mouse dorsal root ganglia (DRG). Confocal and electron microscopic analysis revealed that glial precursors, not macrophages, are responsible for clearing most of the dead DRG neurons. Moreover, we identified Jedi-1, a novel engulfment receptor, and MEGF10, a purported engulfment receptor, as homologs of the invertebrate engulfment receptors Draper and CED-1 expressed in the glial precursor cells. Expression of Jedi-1 or MEGF10 in fibroblasts facilitated binding to dead neurons and knocking down either protein in glial cells, or over expressing truncated forms lacking the intracellular domain, inhibited engulfment of apoptotic neurons. Together, these results reveal the cellular and molecular mechanism by which neuronal corpses are culled during DRG development.

Protein tyrosine phosphatase receptor type O regulates development and function of the sensory nervous system

The roles of protein tyrosine phosphatases (PTPs) in differentiation and axon targeting by dorsal root ganglion (DRG) neurons are essentially unknown. The type III transmembrane PTP, PTPRO, is expressed in DRG neurons, and is implicated in the guidance of motor and retinal axons. We examined the role of PTPRO in DRG development and function using PTPRO −/− mice. The number of peptidergic nociceptive neurons in the DRG of PTPRO −/− mice was significantly decreased, while the total number of sensory neurons appeared unchanged. In addition, spinal pathfinding by both peptidergic and proprioceptive neurons was abnormal in PTPRO −/− mice. Lastly, PTPRO −/− mice performed abnormally on tests of thermal pain and sensorimotor coordination, suggesting that both nociception and proprioception were perturbed. Our data indicate that PTPRO is required for peptidergic differentiation and process outgrowth of sensory neurons, as well as mature sensory function, and provide the first evidence that RPTPs regulate DRG development.