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Axon projection is a spatial- and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections in Drosophila’s small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compelling in vivo model to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions.

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, –3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and –3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.

Direct binding of netrin receptorswith dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains amajor unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic ß-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a keyMT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction. [ABSTRACT FROM AUTHOR]

Abstract Targeted radionuclide therapy is a revolutionary tool for the treatment of highly spread metastatic cancers. Most current approaches rely on the use of vectors to deliver radionuclides to tumor cells, targeting membrane‐bound cancer‐specific moieties. Here, we report the embryonic navigation cue netrin‐1 as an unanticipated target for vectorized radiotherapy. While netrin‐1, known to be re‐expressed in tumoral cells to promote cancer progression, is usually characterized as a diffusible ligand, we demonstrate here that netrin‐1 is actually poorly diffusible and bound to the extracellular matrix. A therapeutic anti‐netrin‐1 monoclonal antibody (NP137) has been preclinically developed and was tested in various clinical trials showing an excellent safety profile. In order to provide a companion test detecting netrin‐1 in solid tumors and allowing the selection of therapy‐eligible patients, we used the clinical‐grade NP137 agent and developed an indium‐111‐NODAGA‐NP137 single photon emission computed tomography (SPECT) contrast agent. NP137‐111In provided specific detection of netrin‐1‐positive tumors with an excellent signal‐to‐noise ratio using SPECT/CT imaging in different mouse models. The high specificity and strong affinity of NP137 paved the way for the generation of lutetium‐177‐DOTA‐NP137, a novel vectorized radiotherapy, which specifically accumulated in netrin‐1‐positive tumors. We demonstrate here, using tumor cell‐engrafted mouse models and a genetically engineered mouse model, that a single systemic injection of NP137‐177Lu provides important antitumor effects and prolonged mouse survival. Together, these data support the view that NP137‐111In and NP137‐177Lu may represent original and unexplored imaging and therapeutic tools against advanced solid cancers.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronalspecific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies. [ABSTRACT FROM AUTHOR]

Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well‐characterized role as a tissue‐level axon guidance cue, in mediating oncogenic stress‐induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras‐induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress‐induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl‐CoA generation and systemic metabolism. Supplementation of carnitine or acetyl‐CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism. Synopsis: NetrinB is a secreted protein that acts as a regulator of cell migration and an axon guidance cue. The current study identifies a role for NetB as a humoral signaling molecule that mediates systemic effects of local oncogenic stress on organismal metabolism in Drosophila. Ras‐induced dysplastic cells upregulate and secrete NetrinB.Inhibition of either NetrinB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress‐induced organismal death.NetrinB from the dysplastic tissue suppresses carnitine biosynthesis in the fat body.Supplementation of carnitine or acetyl‐CoA improves fly survival under oncogenic stress. [ABSTRACT FROM AUTHOR]

Simple Summary: The current outcomes for early diagnosis and late treatment of clear cell renal cell carcinoma (ccRCC) are unsatisfactory. This study aimed to determine the diagnostic and prognostic value of Netrin family genes for ccRCC and to explore new therapeutic targets. We systematically analyzed the expression profile of Netrin family genes in ccRCC using a bioinformatics approach and identified three genes, including NTN4, as potential biomarkers for ccRCC prognosis. In vitro and in vivo experiments confirmed that NTN4 regulates the growth and invasion of ccRCC cells by inhibiting the Wnt/β-catenin signaling pathway. Targeting this pathway through NTN4 may be a promising strategy for ccRCC treatment. The results of this study may have important implications for developing personalized cancer treatment strategies and identifying new biomarkers for ccRCC. Clear cell renal cell carcinoma (ccRCC, or KIRC) is the most common type of kidney cancer, originating within the renal cortex. The current outcomes for early diagnosis and late treatment of ccRCC are unsatisfactory. Therefore, it is important to explore tumor biomarkers and therapeutic opportunities for ccRCC. In this study, we used bioinformatics methods to systematically evaluate the expression and prognostic value of Netrin family genes in ccRCC. Through our analysis, three potential biomarkers for ccRCC were identified, namely NTNG1, NTNG2, and NTN4. Moreover, we performed in vitro and in vivo experiments to explore the possible biological roles of NTN4 and found that NTN4 could regulate ccRCC development through Wnt/β-catenin signaling. We elucidate the molecular mechanism by which NTN4 modulates β-catenin expression and nuclear translocation to inhibit ccRCC progression, providing a new theoretical basis for developing therapeutic targets for ccRCC. Thus, we suggest that Netrin-related studies may offer new directions for the diagnosis, treatment, and prognosis of ccRCC patients. [ABSTRACT FROM AUTHOR]

Targeted radionuclide therapy is a revolutionary tool for the treatment of highly spread metastatic cancers. Most current approaches rely on the use of vectors to deliver radionuclides to tumor cells, targeting membrane‐bound cancer‐specific moieties. Here, we report the embryonic navigation cue netrin‐1 as an unanticipated target for vectorized radiotherapy. While netrin‐1, known to be re‐expressed in tumoral cells to promote cancer progression, is usually characterized as a diffusible ligand, we demonstrate here that netrin‐1 is actually poorly diffusible and bound to the extracellular matrix. A therapeutic anti‐netrin‐1 monoclonal antibody (NP137) has been preclinically developed and was tested in various clinical trials showing an excellent safety profile. In order to provide a companion test detecting netrin‐1 in solid tumors and allowing the selection of therapy‐eligible patients, we used the clinical‐grade NP137 agent and developed an indium‐111‐NODAGA‐NP137 single photon emission computed tomography (SPECT) contrast agent. NP137‐111In provided specific detection of netrin‐1‐positive tumors with an excellent signal‐to‐noise ratio using SPECT/CT imaging in different mouse models. The high specificity and strong affinity of NP137 paved the way for the generation of lutetium‐177‐DOTA‐NP137, a novel vectorized radiotherapy, which specifically accumulated in netrin‐1‐positive tumors. We demonstrate here, using tumor cell‐engrafted mouse models and a genetically engineered mouse model, that a single systemic injection of NP137‐177Lu provides important antitumor effects and prolonged mouse survival. Together, these data support the view that NP137‐111In and NP137‐177Lu may represent original and unexplored imaging and therapeutic tools against advanced solid cancers. Synopsis: During development, netrin‐1 is released and diffusible in the microenvironment. We showed that in cancer, netrin‐1 accumulates in the extracellular matrix. We used this property to follow its in vivo accumulation by radiolabeling and to target netrin‐1‐expressing tumor cells with radioimmunotherapy. In cancer tissues, netrin‐1 is secreted but not diffusible.Netrin‐1 interacts with many components of the extracellular matrix and accumulates in the tumor intercellular space.Anti‐netrin‐1 monoclonal antibody can be radiolabeled to detect in vivo the presence of netrin‐1 as a diagnostic test.Anti‐netrin‐1 monoclonal antibody can be conjugated with Lutetium 177 to cargo the radioelement and destroy tumor cells in vivo. [ABSTRACT FROM AUTHOR]

The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.

Background: Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane‐bound receptors such as Uncoordianted‐5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. Results: Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated‐5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. Conclusion: Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages. [ABSTRACT FROM AUTHOR]

Laminins (LMs) are essential components of all basement membranes where they regulate an extensive array of tissue functions. Alternative splicing from the laminin α3 gene produces a non‐laminin but netrin‐like protein, Laminin N terminus α31 (LaNt α31). LaNt α31 is widely expressed in intact tissue and is upregulated in epithelial cancers and during wound healing. In vitro functional studies have shown that LaNt α31 can influence numerous aspects of epithelial cell behavior via modifying matrix organization, suggesting a new model of laminin auto‐regulation. However, the function of this protein has not been established in vivo. Here, a mouse transgenic line was generated using the ubiquitin C promoter to drive inducible expression of LaNt α31. When expression was induced at embryonic day 15.5, LaNt α31 transgenic animals were not viable at birth, exhibiting localized regions of erythema. Histologically, the most striking defect was widespread evidence of extravascular bleeding across multiple tissues. Additionally, LaNt α31 transgene expressing animals exhibited kidney epithelial detachment, tubular dilation, disruption of the epidermal basal cell layer and of the hair follicle outer root sheath, and ~50% reduction of cell numbers in the liver, associated with depletion of hematopoietic erythrocytic foci. These findings provide the first in vivo evidence that LaNt α31 can influence tissue morphogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Projection neurons of the spinal cord dorsal horn which transmit pain, itch, and temperature information to the brain comprise the anterolateral system (AS). A recent molecular and genetic study showed that many developing AS neurons express the transcription factor Phox2a and provided insights into the mechanisms of their ontogeny and wiring of nociceptive neuronal circuits. Here, we show that the loss of the axonal guidance and neuronal migration signal netrin1 results in impaired migration of mouse Phox2a+ AS neurons into the spinal lamina I. Furthermore, we show that in the absence of Dab1, an intracellular transducer of the neuronal migration signal reelin, the migration of spinal lamina V and lateral spinal nucleus Phox2a+ AS neurons is impaired, in line with deficits in nociception seen in mice with a loss of reelin signaling. Together, these results provide evidence that netrin1 and reelin control the development of spinal nociceptive projection neurons, suggesting a mechanistic explanation for studies that link sequence variations in human genes encoding these neurodevelopmental signals and abnormal pain sensation. [ABSTRACT FROM AUTHOR]

Epithelial remodelling plays a crucial role during development. The ability of epithelial sheets to temporarily lose their integrity as they fuse with other epithelial sheets underpins events such as the closure of the neural tube and palate. During fusion, epithelial cells undergo some degree of epithelial-mesenchymal transition (EMT), whereby cells from opposing sheets dissolve existing cell-cell junctions, degrade the basement membrane, extend motile processes to contact each other, and then re-establish cell-cell junctions as they fuse. Similar events occur when an epithelium is wounded. Cells at the edge of the wound undergo a partial EMT and migrate towards each other to close the gap. In this review, we highlight the emerging role of Netrins in these processes, and provide insights into the possible signalling pathways involved. Netrins are secreted, laminin-like proteins that are evolutionarily conserved throughout the animal kingdom. Although best known as axonal chemotropic guidance molecules, Netrins also regulate epithelial cells. For example, Netrins regulate branching morphogenesis of the lung and mammary gland, and promote EMT during Drosophila wing eversion. Netrins also control epithelial fusion during optic fissure closure and inner ear formation, and are strongly implicated in neural tube closure and secondary palate closure. Netrins are also upregulated in response to organ damage and epithelial wounding, and can protect against ischemia-reperfusion injury and speed wound healing in cornea and skin. Since Netrins also have immunomodulatory properties, and can promote angiogenesis and re-innervation, they hold great promise as potential factors in future wound healing therapies. [ABSTRACT FROM AUTHOR]

Chronic pain is often present at more than one anatomical location, leading to chronic overlapping pain conditions. Whether chronic overlapping pain conditions represent a distinct pathophysiology from the occurrence of pain at only one site is unknown. Using genome-wide approaches, we compared genetic determinants of chronic single-site versus multisite pain in the UK Biobank. We found that different genetic signals underlie chronic single-site and multisite pain with much stronger genetic contributions for the latter. Among 23 loci associated with multisite pain, nine loci replicated in the HUNT cohort, with the DCC netrin 1 receptor (DCC) as the top gene. Functional genomics identified axonogenesis in brain tissues as the major contributing pathway to chronic multisite pain. Finally, multimodal structural brain imaging analysis showed that DCC is most strongly expressed in subcortical limbic regions and is associated with alterations in the uncinate fasciculus microstructure, suggesting that DCC-dependent axonogenesis may contribute to chronic overlapping pain conditions via corticolimbic circuits. [ABSTRACT FROM AUTHOR]

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through midoogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis. [ABSTRACT FROM AUTHOR]

Axon projection is a spatial- and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections in Drosophila 's small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compelling in vivo model to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions., Competing Interests: JL, YW, XL, JH, YT No competing interests declared, (© 2024, Liu et al.)

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis., Competing Interests: PP, JM, KZ, DP, SW, YR, MH, RR, BG, RC, TS, FD No competing interests declared, PM Founder of Netris Pharma, (© 2023, Perampalam et al.)

The laminins (LM) are a family of basement membranes glycoproteins with essential structural roles in supporting epithelia, endothelia, nerves and muscle adhesion, and signaling roles in regulating cell migration, proliferation, stem cell maintenance and differentiation. Laminins are obligate heterotrimers comprised of α, β and γ chains that assemble intracellularly. However, extracellularly these heterotrimers then assemble into higher-order networks via interaction between their laminin N-terminal (LN) domains. In vitro protein studies have identified assembly kinetics and the structural motifs involved in binding of adjacent LN domains. The physiological importance of these interactions has been identified through the study of pathogenic point mutations in LN domains that lead to syndromic disorders presenting with phenotypes dependent on which laminin gene is mutated. Genotype-phenotype comparison between knockout and LN domain missense mutations of the same laminin allows inferences to be drawn about the roles of laminin network assembly in terms of tissue function. In this review, we will discuss these comparisons in terms of laminin disorders, and the therapeutic options that understanding these processes have allowed. We will also discuss recent findings of non-laminin mediators of laminin network assembly and their implications in terms of basement membrane structure and function.

The laminins (LM) are a family of basement membranes glycoproteins with essential structural roles in supporting epithelia, endothelia, nerves and muscle adhesion, and signaling roles in regulating cell migration, proliferation, stem cell maintenance and differentiation. Laminins are obligate heterotrimers comprised of α, β and γ chains that assemble intracellularly. However, extracellularly these heterotrimers then assemble into higher-order networks via interaction between their laminin N-terminal (LN) domains. In vitro protein studies have identified assembly kinetics and the structural motifs involved in binding of adjacent LN domains. The physiological importance of these interactions has been identified through the study of pathogenic point mutations in LN domains that lead to syndromic disorders presenting with phenotypes dependent on which laminin gene is mutated. Genotype-phenotype comparison between knockout and LN domain missense mutations of the same laminin allows inferences to be drawn about the roles of laminin network assembly in terms of tissue function. In this review, we will discuss these comparisons in terms of laminin disorders, and the therapeutic options that understanding these processes have allowed. We will also discuss recent findings of non-laminin mediators of laminin network assembly and their implications in terms of basement membrane structure and function. [ABSTRACT FROM AUTHOR]

In mammals, excitatory cortical neurons develop from the proliferative epithelium and progenitor cells in the ventricular zone and subventricular zone, and migrate radially to the cortical plate, whereas inhibitory GABAergic interneurons are born in the ganglionic eminence and migrate tangentially. The migration of newly born cortical neurons is tightly regulated by both extracellular and intracellular signaling to ensure proper positioning and projections. Non-cell-autonomous extracellular molecules, such as growth factors, axon guidance molecules, extracellular matrix, and other ligands, play a role in cortical migration, either by acting as attractants or repellents. In this article, we review the guidance molecules that act as cell–cell recognition molecules for the regulation of neuronal migration, with a focus on netrin family proteins, their receptors, and related molecules, including neogenin, repulsive guidance molecules (RGMs), Down syndrome cell adhesion molecule (DSCAM), fibronectin leucine-rich repeat transmembrane proteins (FLRTs), and draxin. Netrin proteins induce attractive and repulsive signals depending on their receptors. For example, binding of netrin-1 to deleted in colorectal cancer (DCC), possibly together with Unc5, repels migrating GABAergic neurons from the ventricular zone of the ganglionic eminence, whereas binding to α3β1 integrin promotes cortical interneuron migration. Human genetic disorders associated with these and related guidance molecules, such as congenital mirror movements, schizophrenia, and bipolar disorder, are also discussed.

In the adult mouse brain, neurogenesis occurs mainly in the ventricular-subventricular zone (V-SVZ) and the subgranular zone of the hippocampal dentate gyrus. Neuroblasts generated in the V-SVZ migrate to the olfactory bulb via the rostral migratory stream (RMS) in response to guidance molecules, such as netrin-1. We previously showed that the related netrin-5 (NTN5) is expressed in Mash1-positive transit-amplifying cells and doublecortin-positive neuroblasts in the granule cell layer of the olfactory bulb, the RMS, and the subgranular zone of the adult mouse brain. However, the precise role of NTN5 in adult neurogenesis has not been investigated. In this study, we show that proliferation in the neurogenic niche is impaired in NTN5 knockout mice. The number of proliferating (EdU-labeled) cells in NTN5 KO mice was significantly lower in the V-SVZ, whereas the number of Ki67-positive proliferating cells was unchanged, suggesting a longer cell cycle and decreased cell division in NTN5 KO mice. The number of EdU-labeled cells in the RMS and olfactory bulb was unchanged. By contrast, the numbers of EdU-labeled cells in the cortex, basal ganglia/lateral septal nucleus, and corpus callosum/anterior commissure were increased, which largely represented oligodendrocyte lineage cells. Lastly, we found that chain migration in the RMS of NTN5 KO mice was disorganized. These findings suggest that NTN5 may play important roles in promoting proliferation in the V-SVZ niche, organizing proper chain migration in the RMS, and suppressing oligodendrogenesis in the brain.

In the adult mouse brain, neurogenesis occurs mainly in the ventricular-subventricular zone (V-SVZ) and the subgranular zone of the hippocampal dentate gyrus. Neuroblasts generated in the V-SVZ migrate to the olfactory bulb via the rostral migratory stream (RMS) in response to guidance molecules, such as netrin-1. We previously showed that the related netrin-5 (NTN5) is expressed in Mash1-positive transit-amplifying cells and doublecortin-positive neuroblasts in the granule cell layer of the olfactory bulb, the RMS, and the subgranular zone of the adult mouse brain. However, the precise role of NTN5 in adult neurogenesis has not been investigated. In this study, we show that proliferation in the neurogenic niche is impaired in NTN5 knockout mice. The number of proliferating (EdU-labeled) cells in NTN5 KO mice was significantly lower in the V-SVZ, whereas the number of Ki67-positive proliferating cells was unchanged, suggesting a longer cell cycle and decreased cell division in NTN5 KO mice. The number of EdU-labeled cells in the RMS and olfactory bulb was unchanged. By contrast, the numbers of EdU-labeled cells in the cortex, basal ganglia/lateral septal nucleus, and corpus callosum/anterior commissure were increased, which largely represented oligodendrocyte lineage cells. Lastly, we found that chain migration in the RMS of NTN5 KO mice was disorganized. These findings suggest that NTN5 may play important roles in promoting proliferation in the V-SVZ niche, organizing proper chain migration in the RMS, and suppressing oligodendrogenesis in the brain. [ABSTRACT FROM AUTHOR]

During embryonic development in bilaterally symmetric organisms, correct midline crossing is important for the proper formation of functional neural circuits. The aberrant development of neural circuits can result in multiple neurodevelopmental disorders, including horizontal gaze palsy, congenital mirror movement disorder, and autism spectrum disorder. Thus, understanding the molecular mechanisms that regulate proper axon guidance at the midline can provide insights into the pathology of neurological disorders. The signaling mechanisms that regulate midline crossing have been extensively studied in the Drosophila ventral nerve cord and the mouse embryonic spinal cord. In this review, we discuss these axon guidance mechanisms, highlighting the most recent advances in the understanding of how commissural axons switch their responsiveness from attractants to repellents during midline crossing. • Conserved mechanisms of axon guidance at the midline are explored. • Advances in understanding how axons change their responsiveness are highlighted. • Diverse strategies for regulation of axon guidance receptors are discussed. [ABSTRACT FROM AUTHOR]

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages but has never been found to participate in gastrulation – the earliest cell migration in metazoan embryogenesis. Here, we found that the netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguingly, Hau-netrin-1 was expressed in the N lineage, which gives rise in part to the ventral midline of ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. Given that the leech netrin is expressed in the precursor cells of the ventral midline during gastrulation, we propose that a heterochronic change from the midline netrin expression had taken place in the evolution of a novel mode of gastrulation in the directly developing leech embryos. Image 1 • Netrin and netrin receptors are expressed during leech gastrulation. • Leech netrins are expressed in cell lineages contributing to future midline. • Netrin guides mesoderm migration during leech gastrulation. • Netrin may contribute to the evolution of a new mode of gastrulation. [ABSTRACT FROM AUTHOR]

The ability of the mammalian brain to maintain spatial representations of external or internal information for short periods of time has been associated with sustained neuronal spiking and reverberatory neural network activity in the medial entorhinal cortex. Here, we show that conditional genetic deletion of netrin-1 or the netrin receptor deleted-in-colorectal cancer (DCC) from forebrain excitatory neurons leads to deficits in short-term spatial memory. We then demonstrate that conditional deletion of either netrin-1 or DCC inhibits cholinergic persistent firing and show that cholinergic activation of muscarinic receptors expressed by entorhinal cortical neurons promotes persistent firing by recruiting DCC to the plasma membrane. Together, these findings indicate that normal short-term spatial memory function requires the synergistic actions of acetylcholine and netrin-1. [Display omitted] • Deletion of netrin-1 or its receptor DCC perturbs spatial working memory • Netrin-1 loss impairs cholinergic persistent firing in layer V entorhinal cortex • Cholinergic activation increases surface DCC to generate plateau potentials • Netrin-1 and cholinergic activation synergize to generate persistent firing Glasgow et al. show that loss of netrin-1 or DCC disrupts short-term spatial memory and is required for cholinergic persistent firing, a cellular substrate for working memory. Cholinergic receptor activation recruits plasma membrane DCC, which works synergistically with netrin-1 to promote cationic-channel-mediated plateau potentials in layer V entorhinal neurons. [ABSTRACT FROM AUTHOR]

Modulating gene expression, either by transfection or by RNA-interference (RNAi), is a powerful means for studying the functions of essential genes during nervous system development and maintenance. Both approaches depend on the successful delivery of the corresponding nucleic acids into the targeted cells. Here we demonstrate the fast, accurate and highly localized biolistic delivery of plasmid-DNA and dsRNA into central neurons and muscle cells in internal layers of living leech embryos using carrier particles launched with a recently developed pneumatic capillary gun. This delivery method is used to perform RNAi to silence the expression of the axon guidance factor netrin in muscle cells and central neurons of leech embryos, and to induce ectopic expression of actin tagged with a fluorescent protein in both types of cells. We also show the biolistic transfection and staining in monolayer cultures of mammalian cells and carry out independent injection of two different lipophilic tracking dyes with a single capillary gun.

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. We show that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring.

Neurons have evolved specialized growth structures to reach and innervate their target cells. These growth cones express specific receptor molecules that sense environmental cues and transform them into steering decisions. Historically, various concepts of axon guidance have been developed to better understand how axons reach and identify their targets. The essence of these efforts seems to be that growth cones require solid substrates and that major guidance decisions are initiated by extracellular cues. These sometimes highly conserved ligands and receptors have been extensively characterized and mediate four major guidance forces: chemoattraction, chemorepulsion, contact attraction and contact repulsion. However, during development, cells, too, do migrate in order to reach molecularly-defined niches at target locations. In fact, axonal growth could be regarded as a special case of cellular migration, where only a highly polarized portion of the cell is elongating. Here, I combine several examples from genetically tractable model organisms, such as Drosophila or zebrafish, in which cells and axons are guided by attractive cues. Regardless, if these cues are secreted into the extracellular space or exposed on cellular surfaces, migrating cells and axons seem to keep close contact with these attractants and seem to detect them right at their source. Migration towards and along such substrate-derived attractants seem to be particularly robust, as genetic deletion induces obvious searching behaviors and permanent guidance errors. In addition, forced expression of these factors in ectopic tissues is highly distractive too, regardless of the pattern of other endogenous cues. Thus, guidance and migration towards and along attractive tissues is a powerful steering mechanism that exploits affinity differences to the surroundings and, in some instances, determines growth trajectories from source to target region.

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.

Direct binding of netrin receptors with dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains a major unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic β-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a key MT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

A neuron's contribution to the information flow within a neural circuit is governed by the structure of its dendritic arbor. The geometry of the dendritic arbor directly determines synaptic density and the size of the receptive field, both of which influence the firing pattern of the neuron. Importantly, the position of individual dendritic branches determines the identity of the neuron's presynaptic partner and thus the nature of the incoming sensory information. To generate the unique stereotypic architecture of a given neuronal subtype, nascent branches must emerge from the dendritic shaft at preprogramed branch points. Subsequently, a complex array of extrinsic factors regulates the degree and orientation of branch expansion to ensure maximum coverage of the receptive field whilst constraining growth within predetermined territories. In this review we focus on studies that best illustrate how environmental cues such as the Wnts and Netrins and their receptors sculpt the dendritic arbor. We emphasize the pivotal role played by the actin cytoskeleton and its upstream regulators in branch initiation, outgrowth and navigation. Finally, we discuss how protocadherin and DSCAM contact-mediated repulsion prevents inappropriate synapse formation between sister dendrites or dendrites and the axon from the same neuron. Together these studies highlight the clever ways evolution has solved the problem of constructing complex branch geometries. • The architecture of the dendritic tree is unique to each neuronal subtype. • Development of functional neural circuitry is determined by dendrite shape. • Dendrite branch geometry is tightly regulated by adhesion and guidance receptors. • Branch initiation and growth is driven by dynamic actin remodelling. [ABSTRACT FROM AUTHOR]

Neurons have evolved specialized growth structures to reach and innervate their target cells. These growth cones express specific receptor molecules that sense environmental cues and transform them into steering decisions. Historically, various concepts of axon guidance have been developed to better understand how axons reach and identify their targets. The essence of these efforts seems to be that growth cones require solid substrates and that major guidance decisions are initiated by extracellular cues. These sometimes highly conserved ligands and receptors have been extensively characterized and mediate four major guidance forces: chemoattraction, chemorepulsion, contact attraction and contact repulsion. However, during development, cells, too, do migrate in order to reach molecularly-defined niches at target locations. In fact, axonal growth could be regarded as a special case of cellular migration, where only a highly polarized portion of the cell is elongating. Here, I combine several examples from genetically tractable model organisms, such as Drosophila or zebrafish, in which cells and axons are guided by attractive cues. Regardless, if these cues are secreted into the extracellular space or exposed on cellular surfaces, migrating cells and axons seem to keep close contact with these attractants and seem to detect them right at their source. Migration towards and along such substrate-derived attractants seem to be particularly robust, as genetic deletion induces obvious searching behaviors and permanent guidance errors. In addition, forced expression of these factors in ectopic tissues is highly distractive too, regardless of the pattern of other endogenous cues. Thus, guidance and migration towards and along attractive tissues is a powerful steering mechanism that exploits affinity differences to the surroundings and, in some instances, determines growth trajectories from source to target region. [ABSTRACT FROM AUTHOR]

Abstract Studies in the fruit fly Drosophila melanogaster have provided many fundamental insights into the genetic regulation of neural development, including the identification and characterization of evolutionarily conserved axon guidance pathways and their roles in important guidance decisions. Due to its highly organized and fast-developing embryonic nervous system, relatively small number of neurons, and molecular and genetic tools for identifying, labeling, and manipulating individual neurons or small neuronal subsets, studies of axon guidance in the Drosophila embryonic CNS have allowed researchers to dissect these genetic mechanisms with a high degree of precision. In this review, we discuss the major axon guidance pathways that regulate midline crossing of axons and the formation and guidance of longitudinal axon tracts, two processes that contribute to the development of the precise three-dimensional structure of the insect nerve cord. We focus particularly on recent insights into the roles and regulation of canonical midline axon guidance pathways, and on additional factors and pathways that have recently been shown to contribute to axon guidance decisions at and near the midline. [ABSTRACT FROM AUTHOR]

Abstract The Drosophila motor system starts to assemble during embryonic development. It is composed of 30 muscles per abdominal hemisegment and 36 motor neurons assembling into nerve branches to exit the CNS, navigate within the muscle field and finally establish specific connections with their target muscles. Several families of guidance molecules that play a role controlling this process as well as transcriptional regulators that program the behavior of specific motor neuron have been identified. In this review we summarize the role of both groups of molecules in the motor system as well as their relationship where known. It is apparent that partially redundant guidance protein families and membrane molecules with different functional output direct guidance decisions cooperatively. Some distinct transcriptional regulators seem to control guidance of specific nerve branches globally directing the expression of groups of pathfinding molecules in all motor neurons within the same motor branch. [ABSTRACT FROM AUTHOR]

Astrocytes have critical functions throughout the central nervous system (CNS) and have emerged as regulators of synaptic development and function. With their highly complex morphologies, they are able to interact with thousands of synapses via peripheral astrocytic processes (PAPs), ensheathing neuronal axons and dendrites to form the tripartite synapse. In this way, astrocytes engage in crosstalk with neurons to mediate a variety of CNS processes including the regulation of extracellular matrix protein signaling, formation and maintenance of the blood-brain barrier (BBB), axon growth and guidance, homeostasis of the synaptic microenvironment, synaptogenesis, and the promotion of synaptic diversity. In this review, we discuss several key astrocyte signaling factors (thrombospondins, netrins, apolipoproteins, neuregulins, bone morphogenetic proteins, and neuroligins) in the maintenance and regulation of synapse formation. We also explore how these astrocyte signaling factors are impacted by and contribute to substance abuse, particularly alcohol and cocaine use.

Axon guidance molecules play an important role in regulating proper neuronal networking during neuronal development. They also have non-neuronal properties, which include angiogenesis, inflammation, and tumor development. Semaphorin 3F (SEMA3F), a member of the class 3 semaphorins, was initially identified as an axon guidance factor, that repels axons and collapses growth cones. However, SEMA3F has similar effects on endothelial cells (ECs) and tumor cells. In this review, we discuss the novel molecular mechanisms underlying SEMA3F activity in vascular and tumor biology. Recent evidence suggests that SEMA3F functions as a PI3K-Akt-mTOR inhibitor in mammalian cells, including T cells, ECs, and tumor cells. Therefore, SEMA3F may have broad therapeutic implications. We also discuss the key role of axon guidance molecules as regulators of the tumor microenvironment. Netrin-1, a chemoattractant factor in the neuronal system, promotes tumor progression by enhancing angiogenesis and metastasis. Moreover, our recent studies demonstrate that netrin-1/neogenin interactions augment CD4+ T cell chemokinesis and elicit pro-inflammatory responses, suggesting that netrin-1 plays a key role in modulating the function of a tumor and its surrounding cells in the tumor microenvironment. Overall, this review focuses on SEMA3F and netrin-1 signaling mechanisms to understand the diverse biological functions of axon guidance molecules.

The question of whether mossy fiber sprouting is epileptogenic has not been resolved; both sprouting-induced recurrent excitatory and inhibitory circuit hypotheses have been experimentally (but not fully) supported. Therefore, whether mossy fiber sprouting is a potential therapeutic target for epilepsy remains under debate. Moreover, the axon guidance mechanisms of mossy fiber sprouting have attracted the interest of neuroscientists. Sprouting of mossy fibers exhibits several uncommon axonal growth features in the basically non-plastic adult brain. For example, robust branching of axonal collaterals arises from pre-existing primary mossy fiber axons. Understanding the branching mechanisms in adulthood may contribute to axonal regeneration therapies in neuroregenerative medicine in which robust axonal re-growth is essential. Additionally, because granule cells are produced throughout life in the neurogenic dentate gyrus, it is interesting to examine whether the mossy fibers of newly generated granule cells follow the pre-existing trajectories of sprouted mossy fibers in the epileptic brain. Understanding these axon guidance mechanisms may contribute to neuron transplantation therapies, for which the incorporation of transplanted neurons into pre-existing neural circuits is essential. Thus, clarifying the axon guidance mechanisms of mossy fiber sprouting could lead to an understanding of central nervous system (CNS) network reorganization and plasticity. Here, we review the molecular and cellular mechanisms of axon guidance in mossy fiber sprouting by discussing mainly in vitro studies.

Mesenchymal-epithelial transitions (METs) are important in both development and the growth of secondary tumours. Although the molecular basis for epithelial polarity is well studied, less is known about the cues that induce MET. Here we show that Netrins, well known as chemotropic guidance factors, provide a basal polarising cue during the Drosophila midgut MET. Both netrinA and netrinB are expressed in the visceral mesoderm, the substrate upon which midgut cells migrate, while their receptor frazzled (fra) is expressed in midgut cells. Netrins are required to polarise Fra to the basal surface, and Netrins and Fra undergo mutually-dependent endocytosis, with Fra subsequently trafficking to late endosomes. Mutations to fra and netrins affect both migration and MET but to different degrees. Loss of fra strongly delays migration, midgut cells fail to extend protrusions, and apico-basal polarisation of proteins and epithelium formation is inhibited. In netrin mutants, the migration phenotype is weaker and cells still extend protrusions. However, apico-basal polarisation of proteins, including Fra, and FActin is greatly disrupted and a monolayer fails to form. Delocalised accumulations of FActin are prevalent in netrin mutants but not fra mutants suggesting delocalised Fra may disrupt the MET. βPS localisation is also affected in netrin mutants in that a basal gradient is reduced while localisation to the midgut/VM interface is increased. Since a similar effect is seen when endocytosis is inhibited, Netrin and Fra may regulate Integrin turnover. The results suggest Netrin-dependent basal polarisation of Fra is critical for the formation of an epithelium.