The mechanisms governing the navigation of commissural axons during embryonic development have been extensively investigated in the past years, often using the drosophila ventral nerve cord and the ...spinal cord as model systems. Similarities but also specificities in the general strategies, the molecular signals as well as in the regulatory pathways controlling the response of commissural axons to the guidance cues have been found between species. Whether the semaphorin signaling contributes to midline crossing in the fly nervous system remains unknown, while in contrast, it does play a prominent contribution in vertebrates. In this review we discuss the functions of the semaphorins during commissural axon guidance in the developing spinal cord, focusing on the family member semaphorin 3B (Sema3B) in the context of midline crossing in the spinal cord.
Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the ...early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4⁻/⁻ NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Metastatic melanoma patients carrying a BRAFV600 mutation can be treated with a combination of BRAF and MEK inhibitors (BRAFi/MEKi), but innate and acquired resistance invariably occurs. Predicting ...patient response to targeted therapies is crucial to guide clinical decision. We describe here the development of a highly efficient patient‐derived xenograft model adapted to patient melanoma biopsies, using the avian embryo as a host (AVI‐PDXTM). In this in vivo paradigm, we depict a fast and reproducible tumor engraftment of patient samples within the embryonic skin, preserving key molecular and phenotypic features. We show that sensitivity and resistance to BRAFi/MEKi can be reliably modeled in these AVI‐PDXTM, as well as synergies with other drugs. We further provide proof‐of‐concept that the AVI‐PDXTM models the diversity of responses of melanoma patients to BRAFi/MEKi, within days, hence positioning it as a valuable tool for the design of personalized medicine assays and for the evaluation of novel combination strategies.
Synopsis
We conceived an efficient patient‐derived xenograft model for melanoma samples, using the avian embryo as a host (AVI‐PDXTM), preserving key phenotypic traits. Response to targeted therapies is reliably modeled in the AVI‐PDXTM, making it a valuable preclinical tool to assess combination treatments.
The AVI‐PDXTM allows a highly efficient tumor take of patient melanoma biopsies, which is not conditioned by the tumor stage.
Proliferative and invasive phenotypes of melanoma cells are preserved in the AVI‐ PDXTM.
Sensitivity and resistance to BRAFi/MEKi can be modeled in the AVI‐PDXTM as well as synergies with other drugs.
Preclinical assessment of the efficacy of targeted therapies in the AVI‐PDXTM is predictive of melanoma patient clinical responses.
We conceived an efficient patient‐derived xenograft model for melanoma samples, using the avian embryo as a host (AVI‐PDXTM), preserving key phenotypic traits. Response to targeted therapies is reliably modeled in the AVI‐PDXTM, making it a valuable preclinical tool to assess combination treatments.
The wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. To overcome the limited number of guidance cues, modulation of axon ...responsiveness is crucial for specifying accurate trajectories. We report here a novel mechanism by which ligand/receptor co-expression in neurons modulates the integration of other guidance cues by the growth cone. Class 3 semaphorins (Sema3 semaphorins) are chemotropic guidance cues for various neuronal projections, among which are spinal motor axons navigating towards their peripheral target muscles. Intriguingly, Sema3 proteins are dynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed in most of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selective manipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilin receptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenous Nrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where this modulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. We show first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration of motoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limb-derived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.
Pathogenic variants in
L1CAM
, the gene encoding the L1 cell adhesion molecule, are responsible for a wide clinical spectrum including X-linked hydrocephalus with stenosis of the Sylvius aqueduct, ...MASA syndrome (mental retardation, aphasia, shuffling gait, adducted thumbs), and a form of spastic paraplegia (SPG1). A moderate phenotype with mild intellectual disability (ID) and X-linked partial corpus callosum agenesis (CCA) has only been related to
L1CAM
in one family. We report here a second family, including 5 patients with mild to moderate ID and partial CCA without signs usually associated with
L1CAM
pathogenic variations (such as hydrocephalus, pyramidal syndrome, thumb adductus, aphasia). We identified a previously unreported c.3226A > C transversion leading to a p.Thr1076Pro amino acid substitution in the fifth fibronectin type III domain (FnIII) of the protein which co-segregates with the phenotype within the family. We performed in vitro assays to assess the pathogenic status of this variation. First, the expression of the novel p.Thr1076Pro mutant in COS7 cells resulted in endoplasmic reticulum (ER) retention and reduced L1CAM cell surface expression, which is expected to affect both L1CAM-mediated cell-cell adhesion and neurite growth. Second, immunoblotting techniques showed that the immature form of the L1CAM protein was increased, indicating that this variation led to a lack of maturation of the protein. ID associated with CCA is not a common clinical presentation of
L1CAM
pathogenic variants. Genome-wide analyses will identify such variations and it is important to acknowledge this atypical phenotype.
Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we ...reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.
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•Uncoordinated-5 receptor D (Unc5D) and glypican-3 (GPC3) form an octameric complex•Anti-GPC3 nanobodies (Nanoglue, Nanobreak) enhance or weaken Unc5-GPC3 binding•Unc5/GPC3 signaling causes cell contact-repulsion in vitro•In vivo, Unc5/GPC3 modulate the migration of cortical neurons and neuroblastoma cells
Detailed structural analysis and modulation of binding between uncoordinated-5 receptor D (Unc5D) and morphogen receptor glypican-3 (GPC3) reveal how finely balanced Unc5-GPC3 interactions regulate the migration of neuronal and cancer cells.
Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this ...activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue.
Lack of preclinical patient-derived xenograft cancer models in which to conduct large-scale molecular studies seriously impairs the development of effective personalized therapies. We report here an ...in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large-scale RNA-seq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple-negative breast cancer (TNBC) and validated using TNBC standards of care and an investigational therapeutic agent.
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•Human TNBC cells implanted in the avian embryo survive and efficiently form tumors•Anti-tumoral therapies intravenously injected are assessed by 3D imaging•Post-graft large-scale analyses allow studying the mechanism of action of drugs•The AVI-PDX allows preclinical evaluation of therapies and patient stratification
Biological sciences research methodologies; Cancer; Model organism
Axonal projections in the CNS can be categorized as either crossed or uncrossed. Crossing and uncrossing of axons has been explained by attractive and repulsive molecules like Netrin‐1 and Slits, ...which are secreted by midline structures. However, uncrossed projections can be established even in double knockout mice of slit1 and slit2 or of roundabout1 (robo1) and robo2, two receptors for Slits. Here, we found that a novel mechanism mediated by Neuropilin‐2 (Nrp2) contributes to the formation of uncrossed projections of midbrain dopaminergic neurons (mDANs). Nrp2 transcriptional activities were detected in a subset of mDANs, and its protein was expressed in mDAN axons growing through the ipsilateral diencephalon. In nrp2lacZ/lacZ mice, mDAN axons aberrantly grew toward the ventral midline and even crossed it, suggesting that Nrp2 is necessary for the development of mDAN ipsilateral projections. We investigated the involvement of Semaphorin 3B (Sema3B) and Sema3F, two ligands of Nrp2, by analysing mDAN axon trajectories in single or double knockout mice. In both cases, mDAN axons still projected ipsilaterally, suggesting the involvement mechanisms independent of these Sema3s. Nrp2‐deficient mDAN axons retained their responsiveness to Slit2, demonstrating that aberrant mDAN axons in nrp2lacZ/lacZ mice were not indirectly mediated by alterations in Slit/Robo signaling. Taken together, our results indicate that a novel mechanism mediated by Nrp2 contributes to the establishment of uncrossed projections by mDAN axons.
Axonal projections in the CNS are either crossed or uncrossed. Previous works indicated that Slits/Robos signaling controls the formation of uncrossed projections. Here we show that a mechanism mediated by Neuropilin‐2 also regulates the formation of uncrossed projections of midbrain dopaminergic neurons. Interestingly, we found evidence that this mechanism is independent of Sema3s, major ligands for Neuropilin‐2, and several other known guidance cues.
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