IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport.

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.

Dynamic remodeling model based on chemotaxis of slime molds.

Social infrastructure networks, essential for daily life and economic activities, encompass utilities such as water, electricity, roads, and telecommunications. Dynamic remodeling of these systems is crucial for responding to continuous changes, unexpected events, and increased demand. This study proposes a new dynamic remodeling model inspired by biological mechanisms, focusing on a model based on the chemotaxis of slime molds. Slime molds adapt spontaneously to environmental changes by remodeling through the growth and degeneration of tubes. This capability can be applied to optimizing and dynamic remodeling social infrastructure networks. This study elucidated the chemotactic response characteristics of slime molds using biological experiments. The mold's response was observed by considering changes in the concentration of chemicals as environmental changes, confirming that slime molds adapt to environmental changes by shortening their periodic cycles. Subsequently, based on this dynamic response, we propose a new dynamic model (oscillated Physarum solver, O-PS) that extends the existing Physarum solver (PS). Numerical simulations demonstrated that the O-PS possesses rapid and efficient path-remodeling capabilities. In particular, within a simplified maze network, the O-PS was confirmed to have the same shortest-path searching ability as the PS, while being capable of faster remodeling. This study offers a new approach for optimizing and dynamically remodeling social infrastructure networks by mimicking biological mechanisms, enabling the rapid identification of solutions considering multiple objectives under complex constraints. Furthermore, the variation in convergence speed with oscillation frequency in the O-PS suggests flexibility in responding to environmental changes. Further research is required to develop more effective remodeling strategies.

The scheduling of adolescence with Netrin-1 and UNC5C.

Dopamine axons are the only axons known to grow during adolescence. Here, using rodent models, we examined how two proteins, Netrin-1 and its receptor, UNC5C, guide dopamine axons toward the prefrontal cortex and shape behaviour. We demonstrate in mice (Mus musculus) that dopamine axons reach the cortex through a transient gradient of Netrin-1-expressing cells - disrupting this gradient reroutes axons away from their target. Using a seasonal model (Siberian hamsters; Phodopus sungorus) we find that mesocortical dopamine development can be regulated by a natural environmental cue (daylength) in a sexually dimorphic manner - delayed in males, but advanced in females. The timings of dopamine axon growth and UNC5C expression are always phase-locked. Adolescence is an ill-defined, transitional period; we pinpoint neurodevelopmental markers underlying this period.

40†years of homeodomain transcription factors in the Drosophila nervous system.

Drosophila nervous system development progresses through a series of well-characterized steps in which homeodomain transcription factors (HDTFs) play key roles during most, if not all, phases. Strikingly, although some HDTFs have only one role, many others are involved in multiple steps of the developmental process. Most Drosophila HDTFs engaged in nervous system development are conserved in vertebrates and often play similar roles during vertebrate development. In this Spotlight, we focus on the role of HDTFs during embryogenesis, where they were first characterized.

Improved Double Deep Q-Network Algorithm Applied to Multi-Dimensional Environment Path Planning of Hexapod Robots.

Detecting transportation pipeline leakage points within chemical plants is difficult due to complex pathways, multi-dimensional survey points, and highly dynamic scenarios. However, hexapod robots' maneuverability and adaptability make it an ideal candidate for conducting surveys across different planes. The path-planning problem of hexapod robots in multi-dimensional environments is a significant challenge, especially when identifying suitable transition points and planning shorter paths to reach survey points while traversing multi-level environments. This study proposes a Particle Swarm Optimization (PSO)-guided Double Deep Q-Network (DDQN) approach, namely, the PSO-guided DDQN (PG-DDQN) algorithm, for solving this problem. The proposed algorithm incorporates the PSO algorithm to supplant the traditional random selection strategy, and the data obtained from this guided approach are subsequently employed to train the DDQN neural network. The multi-dimensional random environment is abstracted into localized maps comprising current and next level planes. Comparative experiments were performed with PG-DDQN, standard DQN, and standard DDQN to evaluate the algorithm's performance by using multiple randomly generated localized maps. After testing each iteration, each algorithm obtained the total reward values and completion times. The results demonstrate that PG-DDQN exhibited faster convergence under an equivalent iteration count. Compared with standard DQN and standard DDQN, reductions in path-planning time of at least 33.94% and 42.60%, respectively, were observed, significantly improving the robot's mobility. Finally, the PG-DDQN algorithm was integrated with sensors onto a hexapod robot, and validation was performed through Gazebo simulations and Experiment. The results show that controlling hexapod robots by applying PG-DDQN provides valuable insights for path planning to reach transportation pipeline leakage points within chemical plants.

Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons.

During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. The SMP core-matrisome was characterized and computationally predicted to form a basement membrane-like structure tailored for motor axon guidance, including basement membrane-associated ECM proteins, as collagen XV-B, one of the earliest core-matrisome gene transcribed in differentiating SMPs and the glycoprotein Tenascin C. To investigate how contact-mediated guidance cues are organized along the motor path to exert their function in vivo, we used microscopy-based methods to analyze and quantify motor axon navigation in tnc and col15a1b knock-out fish. We show that motor axon shape and growth rely on the timely expression of the attractive cue Collagen XV-B that locally provides axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. Our study offers evidence that not only the composition of ECM cues but their topology critically influences motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury as regenerating nerves follow their original path.

The geometry of photopolymerized topography influences neurite pathfinding by directing growth cone morphology and migration.

Objective. Cochlear implants provide auditory perception to those with severe to profound sensorineural hearing loss: however, the quality of sound perceived by users does not approximate natural hearing. This limitation is due in part to the large physical gap between the stimulating electrodes and their target neurons. Therefore, directing the controlled outgrowth of processes from spiral ganglion neurons (SGNs) into close proximity to the electrode array could provide significantly increased hearing function.Approach.For this objective to be properly designed and implemented, the ability and limits of SGN neurites to be guided must first be determined. In this work, we engineer precise topographical microfeatures with angle turn challenges of various geometries to study SGN pathfinding and use live imaging to better understand how neurite growth is guided by these cues.Main Results.We find that the geometry of the angled microfeatures determines the ability of neurites to navigate the angled microfeature turns. SGN neurite pathfinding fidelity is increased by 20%-70% through minor increases in microfeature amplitude (depth) and by 25% if the angle of the patterned turn is made obtuse. Further, we see that dorsal root ganglion neuron growth cones change their morphology and migration to become more elongated within microfeatures. Our observations also indicate complexities in studying neurite turning. First, as the growth cone pathfinds in response to the various cues, the associated neurite often reorients across the angle topographical microfeatures. Additionally, neurite branching is observed in response to topographical guidance cues, most frequently when turning decisions are most uncertain.Significance.Overall, the multi-angle channel micropatterned substrate is a versatile and efficient system to assess neurite turning and pathfinding in response to topographical cues. These findings represent fundamental principles of neurite pathfinding that will be essential to consider for the design of 3D systems aiming to guide neurite growthin vivo.

olf413 an octopamine biogenesis pathway gene is required for axon growth and pathfinding during embryonic nervous system development in Drosophila melanogaster.

OBJECTIVE: Neurotransmitters have been extensively studied as neural communication molecules. Genetic associations discovered, and indirect intervention studies in Humans and mammals have led to a general proposition that neurotransmitters have a role in structuring of neuronal network during development. olf413 is a Drosophila gene annotated as coding for dopamine beta-monooxygenase enzyme with a predicted function in octopaminergic pathway. The biological function of this gene is very little worked out. In this study we investigate the requirement of olf413 gene function for octopamine biogenesis and developmental patterning of embryonic nervous system. RESULT: In our study we have used the newly characterized neuronal specific allele olf413SG1.1, and the gene disruption strain olf413MI02014 to dissect out the function of olf413. olf413 has an enhancer activity as depicted by reporter GFP expression, in the embryonic ventral nerve cord, peripheral nervous system and the somatic muscle bundles. Homozygous loss of function mutants show reduced levels of octopamine, and this finding supports the proposed function of the gene in octopamine biogenesis. Further, loss of function of olf413 causes embryonic lethality. FasII staining of these embryos reveal a range of phenotypes in the central and peripheral motor nerves, featuring axonal growth, pathfinding, branching and misrouting defects. Our findings are important as they implicate a key functional requirement of this gene in precise axonal patterning events, a novel developmental role imparted for an octopamine biosynthesis pathway gene in structuring of embryonic nervous system.

Application of Pathfinding Algorithms in Partial Discharge Localization in Power Transformers.

The introduction of artificial intelligence (AI) to ultra-high-frequency (UHF) partial discharge (PD) monitoring systems in power transformers for the localization of PD sources can help create a robust and reliable system with high usability and precision. However, training the AI with experimental data or data from electromagnetic simulation is costly and time-consuming. Furthermore, electromagnetic simulations often calculate more data than needed, whereas, for localization, the signal time-of-flight information is the most important. A tailored pathfinding algorithm can bypass the time-consuming and computationally expensive process of simulating or collecting data from experiments and be used to create the necessary training data for an AI-based monitoring system of partial discharges in power transformers. In this contribution, Dijkstra's algorithm is used with additional line-of-sight propagation algorithms to determine the paths of the electromagnetic waves generated by PD sources in a three-dimensional (3D) computer-aided design (CAD) model of a 300 MVA power transformer. The time-of-flight information is compared with results from experiments and electromagnetic simulations, and it is found that the algorithm maintains accuracy similar to that of the electromagnetic simulation software, with some under/overestimations in specific scenarios, while being much faster at calculations.

Orienteering with One Endomorphism.

In supersingular isogeny-based cryptography, the path-finding problem reduces to the endomorphism ring problem. Can path-finding be reduced to knowing just one endomorphism? It is known that a small degree endomorphism enables polynomial-time path-finding and endomorphism ring computation (in: Love and Boneh, ANTS XIV-Proceedings of the Fourteenth Algorithmic Number Theory Symposium, volume 4 of Open Book Ser. Math. Sci. Publ., Berkeley, 2020). An endomorphism gives an explicit orientation of a supersingular elliptic curve. In this paper, we use the volcano structure of the oriented supersingular isogeny graph to take ascending/descending/horizontal steps on the graph and deduce path-finding algorithms to an initial curve. Each altitude of the volcano corresponds to a unique quadratic order, called the primitive order. We introduce a new hard problem of computing the primitive order given an arbitrary endomorphism on the curve, and we also provide a sub-exponential quantum algorithm for solving it. In concurrent work (in: Wesolowski, Advances in cryptology-EUROCRYPT 2022, volume 13277 of Lecture Notes in Computer Science. Springer, Cham, 2022), it was shown that the endomorphism ring problem in the presence of one endomorphism with known primitive order reduces to a vectorization problem, implying path-finding algorithms. Our path-finding algorithms are more general in the sense that we don't assume the knowledge of the primitive order associated with the endomorphism.

The geometry of photopolymerized topography influences neurite pathfinding by directing growth cone morphology and migration.

Cochlear implants (CIs) provide auditory perception to those with profound sensorineural hearing loss: however, the quality of sound perceived by a CI user does not approximate natural hearing. This limitation is due in part to the large physical gap between the stimulating electrodes and their target neurons. Therefore, directing the controlled outgrowth of processes from spiral ganglion neurons (SGNs) into close proximity to the electrode array could provide significantly increased hearing function. For this objective to be properly designed and implemented, the ability and limits of SGN neurites to be guided must first be determined. In this work, we engineered precise topographical microfeatures with angle turn challenges of various geometries to study SGN pathfinding. Additionally, we analyze sensory neurite growth in response to topographically patterned substrates and use live imaging to better understand how neurite growth is guided by these cues. In assessing the ability of neurites to sense and turn in response to topographical cues, we find that the geometry of the angled microfeatures determines the ability of neurites to navigate the angled microfeature turns. SGN neurite pathfinding fidelity can be increased by 20-70% through minor increases in microfeature amplitude (depth) and by 25% if the angle of the patterned turn is made more obtuse. Further, by using engineered topographies and live imaging of dorsal root ganglion neurons (DRGNs), we see that DRGN growth cones change their morphology and migration to become more elongated within microfeatures. However, our observations also indicate complexities in studying neurite turning. First, as the growth cone pathfinds in response to the various cues, the associated neurite often reorients across the angle topographical microfeatures. This reorientation is likely related to the tension the neurite shaft experiences when the growth cone elongates in the microfeature around a turn. Additionally, neurite branching is observed in response to topographical guidance cues, most frequently when turning decisions are most uncertain. Overall, the multi-angle channel micropatterned substrate is a versatile and efficient system to assess SGN neurite turning and pathfinding in response to topographical cues. These findings represent fundamental principles of neurite pathfinding that will be essential to consider for the design of 3D systems aiming to guide neurite growth in vivo.

Self-formation of concentric zones of telencephalic and ocular tissues and directional retinal ganglion cell axons.

The telencephalon and eye in mammals are originated from adjacent fields at the anterior neural plate. Morphogenesis of these fields generates telencephalon, optic-stalk, optic-disc, and neuroretina along a spatial axis. How these telencephalic and ocular tissues are specified coordinately to ensure directional retinal ganglion cell (RGC) axon growth is unclear. Here, we report self-formation of human telencephalon-eye organoids comprising concentric zones of telencephalic, optic-stalk, optic-disc, and neuroretinal tissues along the center-periphery axis. Initially-differentiated RGCs grew axons towards and then along a path defined by adjacent PAX2+ VSX2+ optic-disc cells. Single-cell RNA sequencing of these organoids not only confirmed telencephalic and ocular identities but also identified expression signatures of early optic-disc, optic-stalk, and RGCs. These signatures were similar to those in human fetal retinas. Optic-disc cells in these organoids differentially expressed FGF8 and FGF9; FGFR inhibitions drastically decreased early RGC differentiation and directional axon growth. Through the RGC-specific cell-surface marker CNTN2 identified here, electrophysiologically excitable RGCs were isolated under a native condition. Our findings provide insight into the coordinated specification of early telencephalic and ocular tissues in humans and establish resources for studying RGC-related diseases such as glaucoma.

Structure-function dynamics of engineered, modular neuronal networks with controllable afferent-efferent connectivity.

Objective.Microfluidic devices interfaced with microelectrode arrays have in recent years emerged as powerful platforms for studying and manipulatingin vitroneuronal networks at the micro- and mesoscale. By segregating neuronal populations using microchannels only permissible to axons, neuronal networks can be designed to mimic the highly organized, modular topology of neuronal assemblies in the brain. However, little is known about how the underlying topological features of such engineered neuronal networks contribute to their functional profile. To start addressing this question, a key parameter is control of afferent or efferent connectivity within the network.Approach.In this study, we show that a microfluidic device featuring axon guiding channels with geometrical constraints inspired by a Tesla valve effectively promotes unidirectional axonal outgrowth between neuronal nodes, thereby enabling us to control afferent connectivity.Main results.Our results moreover indicate that these networks exhibit a more efficient network organization with higher modularity compared to single nodal controls. We verified this by applying designer viral tools to fluorescently label the neurons to visualize the structure of the networks, combined with extracellular electrophysiological recordings using embedded nanoporous microelectrodes to study the functional dynamics of these networks during maturation. We furthermore show that electrical stimulations of the networks induce signals selectively transmitted in a feedforward fashion between the neuronal populations.Significance.A key advantage with our microdevice is the ability to longitudinally study and manipulate both the structure and function of neuronal networks with high accuracy. This model system has the potential to provide novel insights into the development, topological organization, and neuroplasticity mechanisms of neuronal assemblies at the micro- and mesoscale in healthy and perturbed conditions.

Learning the Biochemical Basis of Axonal Guidance: Using Caenorhabditis elegans as a Model.

AIM: Experimental models are a powerful aid in visualizing molecular phenomena. This work reports how the worm Caenorhabditis elegans (C. elegans) can be effectively explored for students to learn how molecular cues dramatically condition axonal guidance and define nervous system structure and behavior at the organism level. Summary of work: A loosely oriented observational activity preceded detailed discussions on molecules implied in axonal migration. C. elegans mutants were used to introduce second-year medical students to the deleterious effects of gene malfunctioning in neuron response to extracellular biochemical cues and to establish links between molecular function, nervous system structure, and animal behavior. Students observed C. elegans cultures and associated animal behavior alterations with the lack of function of specific axon guidance molecules (the soluble cue netrin/UNC-6 or two receptors, DCC/UNC-40 and UNC-5H). Microscopical observations of these strains, in combination with pan-neuronal GFP expression, allowed optimal visualization of severely affected neurons. Once the list of mutated genes in each strain was displayed, students could also relate abnormal patterns in axon migration/ventral and dorsal nerve cord neuron formation in C. elegans with mutated molecular components homologous to those in humans. SUMMARY OF RESULTS: Students rated the importance and effectiveness of the activity very highly. Ninety-three percent found it helpful to grasp human axonal migration, and all students were surprised with the power of the model in helping to visualize the phenomenon.

Neuronal filopodia: From stochastic dynamics to robustness of brain morphogenesis.

Brain development relies on dynamic morphogenesis and interactions of neurons. Filopodia are thin and highly dynamic membrane protrusions that are critically required for neuronal development and neuronal interactions with the environment. Filopodial interactions are typically characterized by non-deterministic dynamics, yet their involvement in developmental processes leads to stereotypic and robust outcomes. Here, we discuss recent advances in our understanding of how filopodial dynamics contribute to neuronal differentiation, migration, axonal and dendritic growth and synapse formation. Many of these advances are brought about by improved methods of live observation in intact developing brains. Recent findings integrate known and novel roles ranging from exploratory sensors and decision-making agents to pools for selection and mechanical functions. Different types of filopodial dynamics thereby reveal non-deterministic subcellular decision-making processes as part of genetically encoded brain development.

Buckling Susceptibility of a K-File during the Initial Negotiations of Narrow and Curved Canals Using Different Manual Techniques.

(1) Background: One possible way to investigate the potential impact or susceptibility of buckling on different manual techniques is to measure compressive loads during canal negotiation. The higher their values, the easier and quicker the critical load level to buckling is reached, leading to possible instrument lateral deformation. The objective of the present study was to investigate the impacts of compressive loads on a small K-file manipulated with different techniques for canal negotiation in simulated narrow and curved canals. (2) Methods: The tooth model selected was a plastic double-curved premolar 23 mm long (DRSK Group AB, Kasernvagen 2, SE-281 35, Hassleholm, Sweden) with an extremely narrow canal lumen to mimic a very difficult anatomical scenario. An experienced endodontist performed the negotiation of 90 of these artificial teeth randomly assigned to 3 different groups of 30 blocks each, respectively, using 3 different techniques: Group A: watch winding/pull (WW) motion; Group B: balanced forces (BF) technique; Group C: envelope of motion (EOM). The measurement system was based on the use of a dynamometer, Instron, Ltd. (model 2525-818 2kN f.s.), linked to a data acquisition unit HBM MGC+ to test all the compression and tensile loads, including all the peaks. (3) Results: All data acquired were processed by the CATMAN AP HBM software. Multiple comparisons for the highest compressive loads estimated the mean difference between WW vs. BF techniques of 3.60 [95% confidence interval (CI): 2.85 to 4.35, p < 0.001], WW vs. EOM of −1.76 (95% CI: −2.11 to 1.40, p < 0.001), and BF vs. EOM −5.36 (95% CI: −6.04 to −4.67, p < 0.001). (4) Conclusions: In conclusion, among the tested manual motions, the BF technique (Group B) was the most susceptible to buckling with the highest compressive load. WW motion (Group A) and EOM (Group C) were less susceptible to buckling than the BF technique. Therefore, a pressure-free manipulation of manual files, such as WW motion or EOM, can help reduce the susceptibility to buckling during the negotiation of narrow-curved canals.

Sema4C Is Required for Vascular and Primary Motor Neuronal Patterning in Zebrafish.

Endothelial cells (ECs) and neurons share a number of common signaling pathways and molecular mediators to orchestrate directional migration and guide the pattern of the vascular network and nervous system. So far, research concerning the functional coupling between vascular and neuronal pathfinding remains insufficient. Semaphorin4C (sema4C), a member of class 4 semaphorins, is initially described in the nervous system, whose role has been demonstrated in diverse biological developments. The present study focused on the role of sema4C in the vascular and neural development process in zebrafish embryos. It confirmed that sema4C is expressed in both the nervous system and intersegmental vessels (ISVs) in zebrafish embryos by diverse expression analysis. It also showed that the knockdown of sema4C caused a serious pathfinding anomaly both in the ISVs and primary motor neurons (PMNs) of zebrafish embryos. In addition, overexpressing exogenous sema4C mRNA in sema4C morphants remarkably neutralized the defective pattern of the vascular and neural system. Collectively, this report suggests that sema4C acts as a dual guiding factor regulating vascular and neuronal development. These findings elucidate a new molecular mechanism underlying blood vessel and nerve development and might serve as groundwork for future research on functional coupling between both systems.

Axons in the Chick Embryo Follow Soft Pathways Through Developing Somite Segments.

During patterning of the peripheral nervous system, motor axons grow sequentially out of the neural tube in a segmented fashion to ensure functional integration of the motor roots between the surrounding cartilage and bones of the developing vertebrae. This segmented outgrowth is regulated by the intrinsic properties of each segment (somite) adjacent to the neural tube, and in particular by chemical repulsive guidance cues expressed in the posterior half. Yet, knockout models for such repulsive cues still display initial segmentation of outgrowing motor axons, suggesting the existence of additional, yet unknown regulatory mechanisms of axon growth segmentation. As neuronal growth is not only regulated by chemical but also by mechanical signals, we here characterized the mechanical environment of outgrowing motor axons. Using atomic force microscopy-based indentation measurements on chick embryo somite strips, we identified stiffness gradients in each segment, which precedes motor axon growth. Axon growth was restricted to the anterior, softer tissue, which showed lower cell body densities than the repulsive stiffer posterior parts at later stages. As tissue stiffness is known to regulate axon growth during development, our results suggest that motor axons also respond to periodic stiffness gradients imposed by the intrinsic mechanical properties of somites.

To Stick or Not to Stick: The Multiple Roles of Cell Adhesion Molecules in Neural Circuit Assembly.

Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly.

MicroRNA-22 coordinates vascular and motor neuronal pathfinding via sema4 during zebrafish development.

A precise guiding signal is crucial to orchestrate directional migration and patterning of the complex vascular network and neural system. So far, limited studies have reported the discovery and functions of microRNAs (miRNAs) in guiding vascular and neural pathfinding. Currently, we showed that the deficiency of miRNA-22a, an endothelial-enriched miRNA, caused dramatic pathfinding defects both in intersegmental vessels (ISVs) and primary motor neurons (PMNs) in zebrafish embryos. Furthermore, we found the specific inhibition of miR-22a in endothelial cells (ECs) resulted in patterning defects of both ISVs and PMNs. Neuronal block of miR-22a mainly led to axonal defects of PMN. Sema4c was identified as a potential target of miR-22a through transcriptomic analysis and in silico analysis. Additionally, a luciferase assay and EGFP sensor assay confirmed the binding of miR-22a with 3'-UTR of sema4c. In addition, downregulation of sema4c in the miR-22a morphants significantly neutralized the aberrant patterning of vascular and neural networks. Then we demonstrated that endothelial miR-22a regulates PMNs axonal navigation. Our study revealed that miR-22a acted as a dual regulatory cue coordinating vascular and neuronal patterning, and expanded the repertoire of regulatory molecules, which might be of use therapeutically to guide vessels and nerves in the relevant diseases.