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Sensory neurons in the DRG express Slit1-3 and Robo1-2 Double staining of Slit1-3 and Robo1-2 with NeuN in the DRG shows all sensory neurons in the DRG express Slit1-3 and Robo1-2. The neuronal marker NeuN labels both large and small diameter sensory neurons. Merged images show that Slit1 (A), Slit2 (B), Slit3 (C), Robo1 (D) and Robo2 (E) are all expressed by both large and small diameter sensory neurons. The yellow colour in merged images shows the co-localization of Slit1-3 and Robo1-2 signal with NeuN staining. Double staining of NeuN with Slit1-3 and Robo1-2 antibodies thus confirms that all the sensory neurons in the DRG express Slit1-3 and Robo1-2. Staining with Hoechst dye (Ho) is also shown (blue) to identify cell nuclei within the tissue.
Source publication
The secreted glycoproteins, Slit1-3, are classic axon guidance molecules that act as repulsive cues through their well characterised receptors Robo1-2 to allow precise axon pathfinding and neuronal migration. The expression patterns of Slit1-3 and Robo1-2 have been most characterized in the rodent developing nervous system and the adult brain, but...
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Citations
... Expression analysis showed that SCs express Robo 1,2 and 4 ( Figure 4-figure supplement 1f), 261 consistent with previous findings that Robo1 is expressed by SCs (Carr et al., 2017). To determine 262 whether Slit acts via the Robo receptors, we performed knockdown experiments for Robo1 and ...
... Here, we show that Slit2/Slit3 signalling is responsible for the homotypic CIL 419 signal between SCs and that this signal is required for the efficient directional collective SC 420 migration that is required for nerve repair. This is consistent with reports that Slit2, Slit3 and 421 Robo1 are expressed by myelinating and non-myelinating SCs (Carr et al., 2017;Wang et al., 422 2013) and that a Slit2 signal can repulse a SC in culture (Wang et al., 2013). The ability of a 423 secreted Slit signal to act as a repellent over both longer and short distances, likely requires cell-424 specific mechanisms that regulate whether the Slit molecules remain tethered to the cell surface 425 Simpson, Kidd, et al., 2000). ...
Collective cell migration is fundamental for the development of organisms and in the adult, for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during rat Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell-surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective Schwann cell migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased Schwann cell collective migration and increased clustering of Schwann cells within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.
... During the process of carcinogenesis, there is an inactivation of the SLIT2, ROBO1 and Robo2 genes, being therefore considered as tumor suppressor genes [42,43] . Both genes are extremely important during the process of nerve formation and repair. ...
Studies have shown that Schwann cells participate in the tumor microenvironment, producing several factors that benefit cancer cells. During this process, Schwann cells are dedifferentiated and help the process of cancer cellular proliferation. These cells then migrate to the region close to the tumor tissue and assist the development of the neoplastic cell. In this context, the present study aimed to evaluate the influence of Schwann cells on prostate cancers. We investigated the association between Schwann cells and prostate cancer often associated with perineural invasion. Initially, we used the GEO Datasets platform from the GEO repository to identify a database reporting gene expression in Schwann cells in a neoplastic context. Briefly, the database contains the expression results from experiments in which two factors produced by tumor cells were added to cell cultures. Comparisons were made between samples from the first and third passages. We then used these data to perform differential gene expression analysis and crossed data from upregulated genes with differential expression data from negative and positive perineural invasion prostate cancers. We observed that the “axon guidance” pathway was upregulated in negative perineural invasion prostate cancers. Meanwhile, upregulated mRNAs activate the “axon guidance” and, together with ROBO1 and MPZ upregulation, inhibit perineural invasion pathways. Both genes are also associated with Schwann cell migration inhibition. PER3 , NR3C1 , PPARGC1A , TIMP3 , ID2 , PDE6B , and CAVIN1 were upregulated in negative perineural tumors, while SLC25A10 was upregulated. We also observed upregulated genes in positive perineural invasion: PPARGC1A , TIMP3, S100A8 , ID2 , DEFB1 , AQP3 , ASS1 , PDE6B , NEFH , and CAVIN1 . AQP3 and NEFH were upregulated only in positive perineural invasion tumors and PER3 and NR3C1 were upregulated only in negative perineural invasion samples. We believe that Circadian rhythm and/or melatonin disruption could be associated with Schwann cells dedifferentiation; consequently, Schwann cells produce different factors that will participate in various processes of tumor progression. These processes may also be involved in tumor invasion into the perineural tissue in prostate cancer.
... Slit guidance ligand proteins known as SLITs, including SLIT1, SLIT2, and SLIT3, were discovered to be highly conserved in several species (20). In the early stages, SLITs were identified as chemorepellents that regulated axon crossing in the middle of the brain (21). ...
Objective(s)
Slit guidance ligand 3 (SLIT3) has been identified as a potential therapeutic regulator against fibroblast activity and fibrillary collagen production in an autocrine manner. However, this research aims to investigate the potential role of SLIT3 in cardiac fibrosis and fibroblast differentiation and its underlying mechanism.
Materials and Methods
C57BL/6 mice (male, 8-10 weeks, n=47) were subcutaneously infused with Ang II (2.0 mg/kg/day) for 4 weeks. One to two-day-old Sprague-Dawley (SD) rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg) and ketamine (50 mg/kg) and the cardiac fibroblast was isolated aseptically. The mRNA and protein expression were analyzed using RT-qPCR and Western blotting.
Results
The SLIT3 expression level was increased in Ang II-induced mice models and cardiac fibroblasts. SLIT3 significantly increased migrated cells and α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts. Ang II-induced increases in mRNA expression of collagen I (COL1A1), and collagen III (COL3A1) was attenuated by SLIT3 inhibition. SLIT3 knockdown attenuated the Ang II-induced increase in mRNA expression of ACTA2 (α-SMA), Fibronectin, and CTGF. SLIT3 suppression potentially reduced DHE expression and decreased malondialdehyde (MDA) content, and the superoxide dismutase (SOD) and catalase (CAT) levels were significantly increased in cardiac fibroblasts. Additionally, SLIT3 inhibition markedly decreased RhoA and ROCK1 protein expression, whereas ROCK inhibitor Y-27632 (10 μM) markedly attenuated the migration of cardiac fibroblasts stimulated by Ang II and SLIT3.
Conclusion
The results speculate that SLIT3 could significantly regulate cardiac fibrosis and fibroblast differentiation via the RhoA/ROCK1 signaling pathway.
... A search for the SHN3-regulated angiogenic effector mediating these effects found that many canonical angiogenic factors, most notably including Vegfa, were not regulated by Shn3. Instead, a search for secreted candidate angiogenic factors found that the axon guidance cue Slit3 was both transcribed and secreted at higher levels by SHN3-deficient osteoblasts [33][34][35]. This regulation of SLIT3 production was mediated by the SHN3-ERK MAPK signaling axis, as the increase in SLIT3 levels was also observed in mice with the knock-in mutation of the Shn3 ERK-binding D-domain. ...
Often, disorders of impaired bone formation involve not only a cell intrinsic defect in the ability of osteoblasts to form bone, but moreover a broader dysfunction of the skeletal microenvironment that limits osteoblast activity. Developing approaches to osteoanabolic therapy that not only augment osteoblast activity but moreover correct this microenvironmental dysfunction may enable both more effective osteoanabolic therapies and also addressing a broader set of indications where vasculopathy or other forms microenvironment dysfunction feature prominently. We here review evidence that SHN3 acts as a suppressor of not only the cell intrinsic bone formation activity of osteoblasts, but moreover of the creation of a local osteoanabolic microenvironment. Mice lacking Schnurri3 (SHN3, HIVEP3) display a very robust increase in bone formation, that is due to de-repression of ERK pathway signaling in osteoblasts. In addition to loss of SHN3 augmenting the differentiation and bone formation activity of osteoblasts, loss of SHN3 increases secretion of SLIT3 by osteoblasts, which in a skeletal context acts as an angiogenic factor. Through this angiogenic activity, SLIT3 creates an osteoanabolic microenvironment, and accordingly treatment with SLIT3 can increase bone formation and enhance fracture healing. These features both validate vascular endothelial cells as a therapeutic target for disorders of low bone mass alongside the traditionally targeted osteoblasts and osteoclasts and indicate that targeting the SHN3/SLIT3 pathway provides a new mechanism to induce therapeutic osteoanabolic responses.
... As a divergent member of its family, ROBO3 only shows a weak capacity to interact with SLITs [19]. ROBO3 is most highly expressed during development in the central nervous system, and its expression is maintained in the adult nervous and sensory organ systems [22,23]. Notably, ROBO3 has a major role in commissural axon midline crossing, where it controls the switch between chemo-attraction and chemo-repulsion [24]. ...
Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development. Cell Death and Disease (2022) 13:762 ; https://doi.
... The absence of full-length dystrophin in mdx muscle partially compromises this interaction, which may be protected by SPL inhibition. The effects of SPL inhibition on genes associated with axon guidance could potentially be related to changes in the SC population, as SCs are known to produce and export axon guidance molecules in response to muscle injury [35][36][37][38]. This finding seems to be unique to our study and is particularly interesting in consideration of the peripheral neuropathy and axonopathy associated with SGPL1 mutations and SPL insufficiency syndrome [39]. ...
Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.
... In the peripheral nervous system of the adult mouse, all three Slits, as well as Robo1 and 2, are expressed in sensory and motor neurons. Robo1 is also expressed in blood vessels associated with peripheral nerves (Carr, Parkinson, & Dun, 2017). ...
Proper innervation of peripheral organs helps to maintain physiological homeostasis and elicit responses to external stimuli. Disruptions to normal function can result in pathophysiological consequences. The establishment of connections and communication between the central nervous system and the peripheral organs is accomplished through the peripheral nervous system. Neuronal connections with target tissues arise from ganglia partitioned throughout the body. Organ innervation is initiated during development with stimuli being conducted through several types of neurons including sympathetic, parasympathetic, and sensory. While the physiological modulation of mature organs by these nerves is largely understood, their role in mammalian development is only beginning to be uncovered. Interactions with cells in target tissues can affect the development and eventual function of several organs, highlighting their significance. This chapter will cover the origin of peripheral neurons, factors mediating organ innervation, and the composition and function of organ-specific nerves during development. This emerging field aims to identify the functional contribution of innervation to development which will inform future investigations of normal and abnormal mammalian organogenesis, as well as contribute to regenerative and organ replacement efforts where nerve-derived signals may have significant implications for the advancement of such studies.
... We also found SLIT2 and its receptor ROBO1, previously shown to be expressed by both axons and Schwann cells, and ROBO2 that is mainly expressed by axons in mice [85] in the lesional samples of our dataset. Schwann cell-expressed NTN1, which participates in axon regeneration following nerve injury [86], is also affected in psoriatic lesions. ...
An increasing amount of evidence indicates the critical role of the cutaneous nervous system in the initiation and maintenance of psoriatic skin lesions by neurogenic inflammation. However, molecular mechanisms affecting cutaneous neurons are largely uncharacterized. Therefore, we reanalyzed a psoriatic RNA sequencing dataset from published transcriptome experiments of nearly 300 individuals. Using the Ingenuity Pathway Analysis software, we associated several hundreds of differentially expressed transcripts (DETs) to nervous system development and functions. Since neuronal projections were previously reported to be affected in psoriasis, we performed an in-depth analysis of neurite formation-related process. Our in silico analysis suggests that SEMA-PLXN and ROBO-DCC-UNC5 regulating axonal growth and repulsion are differentially affected in non-lesional and lesional skin samples. We identified opposing expressional alterations in secreted ligands for axonal guidance signaling (RTN4/NOGOA, NTNs, SEMAs, SLITs) and non-conventional axon guidance regulating ligands, including WNT5A and their receptors, modulating axon formation. These differences in neuritogenesis may explain the abnormal cutaneous nerve filament formation described in psoriatic skin. The processes also influence T-cell activation and infiltration, thus highlighting an additional angle of the crosstalk between the cutaneous nervous system and the immune responses in psoriasis pathogenesis, in addition to the known neurogenic pro-inflammatory mediators.
... We also found SLIT2 and its receptor ROBO1, previously shown to be expressed by both axons and Schwann cells, and ROBO2 that is mainly expressed by axons in mice 74 in the lesional samples of our dataset. Schwann cell-expressed NTN1, which participates in axon regeneration following nerve injury 75 , is also affected in psoriatic lesions. ...
An increasing amount of evidence indicates the critical role of the cutaneous nervous system in the initiation and maintenance of psoriatic skin lesions by neurogenic inflammation. However, molecular mechanisms affecting cutaneous neurons are largely uncharacterized. Therefore, we reanalyzed a psoriatic RNA sequencing dataset from published transcriptome experiments of nearly 300 individuals. Using the Ingenuity Pathway Analysis software, we associated several hundreds of differentially expressed transcripts (DETs) to nervous system development and functions. Since neuronal projections were previously reported to be affected in psoriasis, we performed an in-depth analysis of neurite formation-related processed. Our in silico analysis suggests that SEMA-PLXN and ROBO-DCC-UNC5 regulating axonal growth and repulsion are differentially affected in non-lesional and lesional skin samples. We identified opposing expressional alterations in secreted ligands for axonal guidance signaling (RTN4/NOGOA, NTNs, SEMAs, SLITs) and non-conventional axon guidance regulating ligands, including WNT5A and their receptors, modulating axon formation. These differences in neuritogenesis may explain the abnormal cutaneous nerve filament formation described in psoriatic skin. The processes also influence T cell activation and infiltration, thus highlighting an additional angle of the crosstalk between the cutaneous nervous system and the immune responses in psoriasis pathogenesis, in addition to the known neurogenic pro-inflammatory mediators.
... However, cDNA microarray and bulk mRNA sequencing studies are unable to map upregulated genes to different cell types in the distal nerve stump. Therefore, immunohistochemistry or in situ hybridisation techniques are often used to identify the cell types of an upregulated gene (Carr et al., 2017;Dun et al., 2019;Chen et al., 2020). Recent advances in single-cell RNA sequencing (scRNA-seq) technologies and the development of bioinformatic pipelines have enabled us to rapidly obtain cell type specific gene expression profiles in large numbers of individual cells (Chen et al., 2019). ...
Following peripheral nerve injury, transcription factors upregulated in the distal nerve play essential roles in Schwann cell reprogramming, fibroblast activation and immune cell function to create a permissive distal nerve environment for axonal regrowth. In this report, we first analysed four microarray data sets to identify transcription factors that have at least twofold upregulation in the mouse distal nerve stump at day 3 and day 7 post-injury. Next, we compared their relative mRNA levels through the analysis of an available bulk mRNA sequencing data set at day 5 post-injury. We then investigated the expression of identified TFs in analysed single-cell RNA sequencing data sets for the distal nerve at day 3 and day 9 post-injury. These analyses identified 55 transcription factors that have at least twofold upregulation in the distal nerve following mouse sciatic nerve injury. Expression profile for the identified 55 transcription factors in cells of the distal nerve stump was further analysed on the scRNA-seq data. Transcription factor network and functional analysis were performed in Schwann cells. We also validated the expression pattern of Jun, Junb, Runx1, Runx2, and Sox2 in the mouse distal nerve stump by immunostaining. The findings from our study not only could be used to understand the function of key transcription factors in peripheral nerve regeneration but also could be used to facilitate experimental design for future studies to investigate the function of individual TFs in peripheral nerve regeneration.
