Characterization of in vitro transcriptional responses of dorsal root ganglia cultured in the presence and absence of blastema cells from regenerating salamander limbs.
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ABSTRACT: During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema-dorsal root ganglia (DRG) co-culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for five days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1,541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function in injury and neurodevelopmental responses observed for mammalian DRG. We then cultured 5-day DRG explants for an additional five days with or without co-cultured blastema cells. On Day 10, we identified 27 genes whose expression in cultured DRG was significantly affected by the presence or absence of blastema cells. Overall, our study established a DRG-blastema in vitro culture system and identified candidate genes for future investigations of axon regrowth, nerve-blastema signaling, and neural regulation of limb regeneration.
Project description:BackgroundFollowing amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs.ResultsWe identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation.ConclusionOur data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.
Project description:Sensory neurons with cell bodies situated in dorsal root ganglia convey information from external or internal sites of the body such as actual or potential harm, temperature or muscle length to the central nervous system. In recent years, large investigative efforts have worked toward an understanding of different types of DRG neurons at transcriptional, translational, and functional levels. These studies most commonly rely on data obtained from laboratory animals. Human DRG, however, have received far less investigative focus over the last 30 years. Nevertheless, knowledge about human sensory neurons is critical for a translational research approach and future therapeutic development. This review aims to summarize both historical and emerging information about the size and location of human DRG, and highlight advances in the understanding of the neurochemical characteristics of human DRG neurons, in particular nociceptive neurons.
Project description:Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration.
Project description:Dorsal root ganglion (DRG) neurons detect sensory inputs and are crucial for pain processing. They are often studied in vitro as dissociated cell cultures with the assumption that this reasonably represents in vivo conditions. However, to the best of our knowledge, no study has directly compared genome-wide transcriptomes of DRG tissue in vivo versus in vitro or between laboratories and culturing protocols. Comparing RNA sequencing-based transcriptomes of native to cultured (4 days in vitro) human or mouse DRG, we found that the overall expression levels of many ion channels and G-protein-coupled receptors specifically expressed in neurons are markedly lower although still expressed in culture. This suggests that most pharmacological targets expressed in vivo are present under the condition of dissociated cell culture, but with changes in expression levels. The reduced relative expression for neuronal genes in human DRG cultures is likely accounted for by increased expression of genes in fibroblast-like and other proliferating cells, consistent with their mitotic status in these cultures. We found that the expression of a subset of genes typically expressed in neurons increased in human and mouse DRG cultures relative to the intact ganglion, including genes associated with nerve injury or inflammation in preclinical models such as BDNF, MMP9, GAL, and ATF3. We also found a striking upregulation of a number of inflammation-associated genes in DRG cultures, although many were different between mouse and human. Our findings suggest an injury-like phenotype in DRG cultures that has important implications for the use of this model system for pain drug discovery.
Project description:A peripheral nerve injury results in disruption of the fiber that usually protects axons from the surrounding environment. Severed axons from the proximal nerve stump are capable of regenerating, but axons are exposed to a completely new environment. Regeneration recruits cells that produce and deposit key molecules, including growth factor proteins and fibrils in the extracellular matrix (ECM), thus changing the chemical and geometrical environment. The regenerating axons thus surf on a newly remodeled micro-landscape. Strategies to enhance and control axonal regeneration and growth after injury often involve mimicking the extrinsic cues that are found in the natural nerve environment. Indeed, nano- and micropatterned substrates have been generated as tools to guide axons along a defined path. The mechanical cues of the substrate are used as guides to orient growth or change the direction of growth in response to impediments or cell surface topography. However, exactly how axons respond to biophysical information and the dynamics of axonal movement are still poorly understood. Here we use anisotropic, groove-patterned substrate topography to direct and enhance sensory axonal growth of whole mouse dorsal root ganglia (DRG) transplanted ex vivo. Our results show significantly enhanced and directed growth of the DRG sensory fibers on the hemi-3D topographic substrates compared to a 0 nm pitch, flat control surface. By assessing the dynamics of axonal movement in time-lapse microscopy, we found that the enhancement was not due to increases in the speed of axonal growth, but to the efficiency of growth direction, ensuring axons minimize movement in undesired directions. Finally, the directionality of growth was reproduced on topographic patterns fabricated as fully 3D substrates, potentially opening new translational avenues of development incorporating these specific topographic feature sizes in implantable conduits in vivo.
Project description:The proneural transcription factor neurogenin 1 (neurog1) has been shown to be a key regulator of dorsal root ganglion (DRG) neuron development. Here we use a novel transgenic zebrafish line to demonstrate that the neural crest population that gives rise to DRG neurons becomes fate restricted to a neuronal/glial precursor before the onset of neurog1 function. We generated a stable transgenic zebrafish line that carries a modified bacterial artificial chromosome that expresses green fluorescent protein (GFP) under the control of the neurog1 promoter [Tg(neurog1:EGFP)]. In contrast to previously described neurog1 transgenic lines, Tg(neurog1:EGFP) expresses GFP in DRG neuronal precursors cells as they migrate ventrally and after their initial differentiation as neurons. Using this line, we are able to track the fate of DRG neuronal precursor cells during their specification. When Neurog1 function is blocked, either by neurog1 morpholino antisense oligonucleotide injection or in neurog1 mutants, GFP expression initiates in neural crest cells, although they fail to form DRG neurons. Rather, these cells take on a glial-like morphology, retain proliferative capacity, and express glial markers and become associated with the ventral motor root. These results suggest that, within the zebrafish neural crest, there is a fate-restricted lineage that is limited to form either sensory neurons or glia in the developing DRG. Neurog1 acts as the key factor in this lineage to direct the formation of sensory neurons.
Project description:Oxaliplatin induced peripheral neurotoxicity is characterized by an acute cold-induced syndrome characterized by cramps, paresthesias/dysesthesias in the distal limbs and perioral region, that develops rapidly and lasts up to one week affecting nearly all the patients as well as by long-lasting symptoms. It has been previously shown that pharmacological or genetic ablation of TRPA1 responses reduces oxaliplatin-induced peripheral neurotoxicity in mouse models. In the present report, we show that treatment with concentrations of oxaliplatin similar to those found in plasma of treated patients leads to an acidification of the cytosol of mouse dorsal root ganglia neurons in culture and this in turn is responsible for sensitization of TRPA1 channels, thereby providing a mechanistic explanation to toxicity of oxaliplatin. Reversal of the acidification indeed leads to a significantly reduced activity of TRPA1 channels. Last, acidification occurs also in vivo after a single injection of therapeutically-relevant doses of oxaliplatin.
Project description:To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.
Project description:Varicella-zoster virus (VZV) causes varicella, establishes latency in sensory ganglia, and reactivates as herpes zoster. Human dorsal root ganglia (DRGs) xenografts in immunodeficient mice provide a model for evaluating VZV neuropathogenesis. Our investigation of the role of glycoprotein I (gI), which is dispensable in vitro, examines the functions of a VZV gene product during infection of human neural cells in vivo. Whereas intact recombinant Oka (rOka) initiated a short replicative phase followed by persistence in DRGs, the gI deletion mutant, rOkaDeltagI, showed prolonged replication with no transition to persistence up to 70 days after infection. Only a few varicella-zoster nucleocapsids and cytoplasmic virions were observed in neurons, and the major VZV glycoprotein, gE, was retained in the rough endoplasmic reticulum in the absence of gI. VZV neurotropism was not disrupted when DRG xenografts were infected with rOka mutants lacking gI promoter elements that bind cellular transactivators, specificity factor 1 (Sp1) and upstream stimulatory factor (USF). Because gI is essential and Sp1 and USF contribute to VZV pathogenesis in skin and T cells in vivo, these DRG experiments indicate that the genetic requirements for VZV infection are less stringent in neural cells in vivo. The observations demonstrate that gI is important for VZV neurotropism and suggest that a strategy to reduce neurovirulence by deleting gI could prolong active infection in human DRGs.