Axon guidance genes control hepatic artery development.

Earlier data on liver development demonstrated that morphogenesis of the bile duct, portal mesenchyme and hepatic artery is interdependent, yet how this interdependency is orchestrated remains unknown. Here, using 2D and 3D imaging, we first describe how portal mesenchymal cells become organised to form hepatic arteries. Next, we examined intercellular signalling active during portal area development and found that axon guidance genes are dynamically expressed in developing bile ducts and portal mesenchyme. Using tissue-specific gene inactivation in mice, we show that the repulsive guidance molecule BMP co-receptor A (RGMA)/neogenin (NEO1) receptor/ligand pair is dispensable for portal area development, but that deficient roundabout 2 (ROBO2)/SLIT2 signalling in the portal mesenchyme causes reduced maturation of the vascular smooth muscle cells that form the tunica media of the hepatic artery. This arterial anomaly does not impact liver function in homeostatic conditions, but is associated with significant tissular damage following partial hepatectomy. In conclusion, our work identifies new players in development of the liver vasculature in health and liver regeneration.

Upregulation of ROBO3 promotes proliferation, migration and adhesion of AML cells and affects the survival of AML patients.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy, which is the most common and severe acute leukemia in adults. Its occurrence, development and prognosis are affected by many factors, and more research is still needed to further guide its treatment. Here, we found that roundabout3 (ROBO3) was associated with poor prognosis in AML through bioinformatics analysis. We then found that overexpression of ROBO3 promoted AML cell proliferation, adhesion and migration while knockdown of ROBO3 had opposite effects. We subsequently found that ROBO3 regulated CD34 expression in AML cells, and this regulatory effect may be achieved through the Hippo-YAP pathway. The inhibitors of this pathway, K-975 and verteporfin, showed an inhibitory effect on AML cells with high ROBO3 expression. ROBO3 was also found to be significantly increased in bone marrow samples from AML patients. Our research indicates that ROBO3 plays an important role in the development of AML, which suggests that ROBO3 can be a prognostic biomarker and potential therapeutic target for AML.

Molecular and Cellular Insights: A Focus on Glycans and the HNK1 Epitope in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a synaptic disorder with a GABA/glutamate imbalance in the perineuronal nets and structural abnormalities such as increased dendritic spines and decreased long distance connections. Specific pregnancy disorders significantly increase the risk for an ASD phenotype such as preeclampsia, preterm birth, hypoxia phenomena, and spontaneous miscarriages. They are associated with defects in the glycosylation-immune placental processes implicated in neurogenesis. Some glycans epitopes expressed in the placenta, and specifically in the extra-villous trophoblast also have predominant functions in dendritic process and synapse function. Among these, the most important are CD57 or HNK1, CD22, CD24, CD33 and CD45. They modulate the innate immune cells at the maternal-fetal interface and they promote foeto-maternal tolerance. There are many glycan-based pathways of immunosuppression. N-glycosylation pathway dysregulation has been found to be associated with autoimmune-like phenotypes and maternal-autoantibody-related (MAR) autism have been found to be associated with central, systemic and peripheric autoimmune processes. Essential molecular pathways associated with the glycan-epitopes expression have been found to be specifically dysregulated in ASD, notably the Slit/Robo, Wnt, and mTOR/RAGE signaling pathways. These modifications have important effects on major transcriptional pathways with important genetic expression consequences. These modifications lead to defects in neuronal progenitors and in the nervous system's implementation specifically, with further molecular defects in the GABA/glutamate system. Glycosylation placental processes are crucial effectors for proper maternofetal immunity and endocrine/paracrine pathways formation. Glycans/ galectins expression regulate immunity and neurulation processes with a direct link with gene expression. These need to be clearly elucidated in ASD pathophysiology.

Biallelic pathogenic variants in roundabout guidance receptor 1 associate with syndromic congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

A crucial role for Arf6 in the response of commissural axons to Slit.

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

Quantitative Proteomics Reveals Molecular Network Driving Stromal Cell Differentiation: Implications for Corneal Wound Healing.

The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-ß. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGß1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.

Motor neuron migration and positioning mechanisms: New roles for guidance cues.

Motor neurons differentiate from progenitor cells and cluster as motor nuclei, settling next to the floor plate in the brain stem and spinal cord. Although precise positioning of motor neurons is critical for their functional input and output, the molecular mechanisms that guide motor neurons to their proper positions remain poorly understood. Here, we review recent evidence of motor neuron positioning mechanisms, highlighting situations in which motor neuron cell bodies can migrate, and experiments that show that their migration is regulated by axon guidance cues. The view that emerges is that motor neurons are actively trapped or restricted in static positions, as the cells balance a push in the dorsal direction by repulsive Slit/Robo cues and a pull in the ventral direction by attractive Netrin-1/DCC cues. These new functions of guidance cues are necessary fine-tuning to set up patterns of motor neurons at their proper positions in the neural tube during embryogenesis.

Slit/Robo signals prevent spinal motor neuron emigration by organizing the spinal cord basement membrane.

The developing spinal cord builds a boundary between the CNS and the periphery, in the form of a basement membrane. The spinal cord basement membrane is a barrier that retains CNS neuron cell bodies, while being selectively permeable to specific axon types. Spinal motor neuron cell bodies are located in the ventral neural tube next to the floor plate and project their axons out through the basement membrane to peripheral targets. However, little is known about how spinal motor neuron cell bodies are retained inside the ventral neural tube, while their axons can exit. In previous work, we found that disruption of Slit/Robo signals caused motor neuron emigration outside the spinal cord. In the current study, we investigate how Slit/Robo signals are necessary to keep spinal motor neurons within the neural tube. Our findings show that when Slit/Robo signals were removed from motor neurons, they migrated outside the spinal cord. Furthermore, this emigration was associated with abnormal basement membrane protein expression in the ventral spinal cord. Using Robo2 and Slit2 conditional mutants, we found that motor neuron-derived Slit/Robo signals were required to set up a normal basement membrane in the spinal cord. Together, our results suggest that motor neurons produce Slit signals that are required for the basement membrane assembly to retain motor neuron cell bodies within the spinal cord.

Mind the (sr)GAP - roles of Slit-Robo GAPs in neurons, brains and beyond.

The Slit-Robo GTPase-activating proteins (srGAPs) were first identified as potential Slit-Robo effectors that influence growth cone guidance. Given their N-terminal F-BAR, central GAP and C-terminal SH3 domains, srGAPs have the potential to affect membrane dynamics, Rho family GTPase activity and other binding partners. Recent research has clarified how srGAP family members act in distinct ways at the cell membrane, and has expanded our understanding of the roles of srGAPs in neuronal and non-neuronal cells. Gene duplication of the human-specific paralog of srGAP2 has resulted in srGAP2 family proteins that may have increased the density of dendritic spines and promoted neoteny of the human brain during crucial periods of human evolution, underscoring the importance of srGAPs in the unique sculpting of the human brain. Importantly, srGAPs also play roles outside of the nervous system, including during contact inhibition of cell movement and in establishing and maintaining cell adhesions in epithelia. Changes in srGAP expression may contribute to neurodevelopmental disorders, cancer metastasis and inflammation. As discussed in this Review, much remains to be discovered about how this interesting family of proteins functions in a diverse set of processes in metazoans and the functional roles srGAPs play in human disease.

Motor axons are guided to exit points in the spinal cord by Slit and Netrin signals.

In the spinal cord, motor axons project out the neural tube at specific exit points, then bundle together to project toward target muscles. The molecular signals that guide motor axons to and out of their exit points remain undefined. Since motor axons and their exit points are located near the floor plate, guidance signals produced by the floor plate and adjacent ventral tissues could influence motor axons as they project toward and out of exit points. The secreted Slit proteins are major floor plate repellents, and motor neurons express two Slit receptors, Robo1 and Robo2. Using mutant mouse embryos at early stages of motor axon exit, we found that motor exit points shifted ventrally in Robo1/2 or Slit1/2 double mutants. Along with the ventral shift, mutant axons had abnormal trajectories both within the neural tube toward the exit point, and after exit into the periphery. In contrast, the absence of the major ventral attractant, Netrin-1, or its receptor, DCC caused motor exit points to shift dorsally. Netrin-1 attraction on spinal motor axons was demonstrated by in vitro explant assays, showing that Netrin-1 increased outgrowth and attracted cultured spinal motor axons. The opposing effects of Slit/Robo and Netrin-1/DCC signals were tested genetically by combining Netrin-1 and Robo1/2 mutations. The location of exit points in the combined mutants was significantly recovered to their normal position compared to Netrin-1 or Robo1/2 mutants. Together, these results suggest that the proper position of motor exit points is determined by a "push-pull" mechanism, pulled ventrally by Netrin-1/DCC attraction and pushed dorsally by Slit/Robo repulsion.

Implication of SLIT3-ROBO1/ROBO2 in granulosa cell proliferation, differentiation and follicle selection in the prehierarchical follicles of hen ovary.

The SLIT/ROBO pathway has been implicated in prehierarchical follicular development of hen ovary by an intrafollicular autocrine and/or paracrine fashion. SLIT3, one of the key components of the SLIT/ROBO family, serves as a ligand that potentially interacts with the four receptors, ROBO1, ROBO2, ROBO3 and ROBO4. But the exact roles and regulatory mechanism of SLIT3 in chicken ovarian follicle development remain largely unclear. The present study was conducted to investigate the potential roles and molecular regulation of SLIT3 in granulosa cell (GC) proliferation, differentiation and follicle selection within the prehierarchical follicles of hen ovary. We found that SLIT3 interacts physically with the four ROBO receptors, but the expression of the ROBO1 and ROBO2 genes are more susceptible to the regulation of SLIT3 ligand than that of the ROBO3 and ROBO4 genes. Moreover, the siRNA-mediated knockdown of SLIT3 in the follicular GCs leads to a significant increase in cell proliferation. Conversely, overexpression of SLIT3 results in a remarkable reduction in GC proliferation. Furthermore, the overexpressed SLIT3 has notably decreased the mRNA and protein expression levels of follicle-stimulating hormone (FSHR), growth and differentiation factor 9 (GDF9), steroidogenic acute regulatory protein (STAR) and cytochrome P450 11A1 (CYP11A1) in the GCs. These results indicated that SLIT3 may play an inhibitory effect on GC proliferation, differentiation and follicle selection, and these suppressive actions of SLIT3 in the GC proliferation can be prohibited by the siRNA-mediated knockdown of ROBO1 and ROBO2 receptors. The current data provide a basis for further investigation of molecular mechanisms of SLIT3-ROBO1/2 pathway in controlling the prehierarchical follicle development of the hen ovary.

Robo signaling regulates the production of cranial neural crest cells.

Slit/Robo signaling plays an important role in the guidance of developing neurons in developing embryos. However, it remains obscure whether and how Slit/Robo signaling is involved in the production of cranial neural crest cells. In this study, we examined Robo1 deficient mice to reveal developmental defects of mouse cranial frontal and parietal bones, which are derivatives of cranial neural crest cells. Therefore, we determined the production of HNK1+ cranial neural crest cells in early chick embryo development after knock-down (KD) of Robo1 expression. Detection of markers for pre-migratory and migratory neural crest cells, PAX7 and AP-2α, showed that production of both was affected by Robo1 KD. In addition, we found that the transcription factor slug is responsible for the aberrant delamination/EMT of cranial neural crest cells induced by Robo1 KD, which also led to elevated expression of E- and N-Cadherin. N-Cadherin expression was enhanced when blocking FGF signaling with dominant-negative FGFR1 in half of the neural tube. Taken together, we show that Slit/Robo signaling influences the delamination/EMT of cranial neural crest cells, which is required for cranial bone development.

Conserved signaling mechanisms in Drosophila heart development.

Signal transduction through multiple distinct pathways regulates and orchestrates the numerous biological processes comprising heart development. This review outlines the roles of the FGFR, EGFR, Wnt, BMP, Notch, Hedgehog, Slit/Robo, and other signaling pathways during four sequential phases of Drosophila cardiogenesis-mesoderm migration, cardiac mesoderm establishment, differentiation of the cardiac mesoderm into distinct cardiac cell types, and morphogenesis of the heart and its lumen based on the proper positioning and cell shape changes of these differentiated cardiac cells-and illustrates how these same cardiogenic roles are conserved in vertebrates. Mechanisms bringing about the regulation and combinatorial integration of these diverse signaling pathways in Drosophila are also described. This synopsis of our present state of knowledge of conserved signaling pathways in Drosophila cardiogenesis and the means by which it was acquired should facilitate our understanding of and investigations into related processes in vertebrates. Developmental Dynamics 246:641-656, 2017. © 2017 Wiley Periodicals, Inc.

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization.

BACKGROUND: Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. METHODS AND RESULTS: Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well. CONCLUSIONS: These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis.

Motor neuron cell bodies are actively positioned by Slit/Robo repulsion and Netrin/DCC attraction.

Motor neurons differentiate from a ventral column of progenitors and settle in static clusters, the motor nuclei, next to the floor plate. Within these cell clusters, motor neurons receive afferent input and project their axons out to muscle targets. The molecular mechanisms that position motor neurons in the neural tube remain poorly understood. The floor plate produces several types of guidance cues with well-known roles in attracting and repelling axons, including the Slit family of chemorepellents via their Robo receptors, and Netrin1 via its DCC attractive receptor. In the present study we found that Islet1(+) motor neuron cell bodies invaded the floor plate of Robo1/2 double mutant mouse embryos or Slit1/2/3 triple mutants. Misplaced neurons were born in their normal progenitor column, but then migrated tangentially into the ventral midline. Robo1 and 2 receptor expression in motor neurons was confirmed by reporter gene staining and anti-Robo antibody labeling. Mis-positioned motor neurons projected their axons longitudinally within the floor plate, and failed to reach their normal exit points. To test for potential counteracting ventral attractive signals, we examined Netrin-1 and DCC mutants, and found that motor neurons shifted dorsally in the hindbrain and spinal cord, suggesting that Netrin-1/DCC signaling normally attracts motor neurons closer to the floor plate. Our results show that motor neurons are actively migrating cells, and are normally trapped in a static position by Slit/Robo repulsion and Netrin-1/DCC attraction.

New insights into implication of the SLIT/ROBO pathway in the prehierarchical follicle development of hen ovary.

The SLIT/Roundabout (ROBO) pathway is involved in follicle development of mammalian ovary, and 2 secreted hormones activin A and inhibin A have potential roles in modulation of the SLIT/ROBO system, but the related actions remain poorly understood in bird. The aims of the present study were to examine the spatial and temporal expression of the SLIT ligand genes (SLIT1, SLIT2, and SLIT3) and their receptor ROBO1, ROBO2, ROBO3, and ROBO4 genes in various-sized prehierarchical follicles during hen ovary development and the effects of activin A and inhibin A on the expression of these genes in the cultured hen follicles. Our result demonstrated that the transcripts of the 3 SLIT genes were highly expressed in the developing follicles and expression patterns of the SLIT transcripts were different from those of ROBO genes detected by real-time quantitative reverse transcriptase PCR. Both SLIT and ROBO transcripts were predominantly expressed in oocytes and granulosa cells from the prehierarchichal follicles examined by in situ hybridization. The localization for SLIT and ROBO proteins was revealed by immunohistochemistry similar to the spatial distribution of their transcript. In cultured follicles (4 to 8 mm in diameter), the expression levels of SLIT and ROBO members are hormonally regulated by activin A (10 ng/mL) and/or inhibin A (20 ng/mL) after treatment for 24 h. However, the expression of only SLIT2, SLIT3, and ROBO3 mRNA presented a directly opposite response to activin A and inhibin A hormones. These results indicate that SLIT/ROBO pathway is implicated in the prehierarchical follicular development of the hen ovary by an intrafollicular autocrine and/or paracrine action, and is influenced by activin A and inhibin A hormones.

Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-ß, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

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 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 SC 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 SC collective migration and increased clustering of SCs 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.

SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo.

Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called "pericytic" MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, "non-pericytic" MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications.

Role of the SLIT-ROBO signaling pathway in renal pathophysiology and various renal diseases.

SLIT ligand and its receptor ROBO were initially recognized for their role in axon guidance in central nervous system development. In recent years, as research has advanced, the role of the SLIT-ROBO signaling pathway has gradually expanded from axonal repulsion to cell migration, tumor development, angiogenesis, and bone metabolism. As a secreted protein, SLIT regulates various pathophysiological processes in the kidney, such as proinflammatory responses and fibrosis progression. Many studies have shown that SLIT-ROBO is extensively involved in various aspects of kidney development and maintenance of structure and function. The SLIT-ROBO signaling pathway also plays an important role in different types of kidney disease. This article reviews the advances in the study of the SLIT-ROBO pathway in various renal pathophysiological and kidney disorders and proposes new directions for further research in this field.