A self-generated Toddler gradient guides mesodermal cell migration

The sculpting of germ layers during gastrulation relies on coordinated migration of progenitor cells, yet the cues controlling these long-range directed movements remain largely unknown. While directional migration often relies on a chemokine gradient generated from a localized source, we find that zebrafish ventrolateral mesoderm is guided by the uniformly expressed and secreted protein Toddler/ELABELA/Apela, acting as a self-generated gradient. We show that the Apelin receptor, which is specifically expressed in mesodermal cells, has a dual role during gastrulation, acting as a scavenger receptor to generate a Toddler gradient, and as a chemokine receptor to sense this guidance cue. Thus, we uncover a single receptor-based self-generated gradient as the enigmatic guidance cue that can robustly steer the directional migration of mesoderm through the complex and continuously changing environment of the gastrulating embryo.

CRMP4-mediated fornix development involves semaphorin-3E signaling pathway

Neurodevelopmental axonal pathfinding plays a central role in correct brain wiring and subsequent cognitive abilities. Within the growth cone, various intracellular effectors transduce axonal guidance signals by remodeling the cytoskeleton. Semaphorin-3E (Sema3E) is a guidance cue implicated in development of the fornix, a neuronal tract connecting the hippocampus to the hypothalamus. Microtubule-Associated Protein 6 (MAP6) has been shown to be involved in the Sema3E growth-promoting signaling pathway. In this study, we identified the Collapsin Response Mediator Protein 4 (CRMP4) as a MAP6 partner and a crucial effector in Sema3E growth-promoting activity. CRMP4-KO mice displayed abnormal fornix development reminiscent of that observed in Sema3E-KO mice. CRMP4 was shown to interact with the Sema3E tripartite receptor complex within Detergent-Resistant Membrane (DRM) domains, and DRM domain integrity was required to transduce Sema3E signaling through the Akt/GSK3 pathway. Finally, we showed that the cytoskeleton-binding domain of CRMP4 is required for Sema3E's growth-promoting activity, suggesting that CRMP4 plays a role at the interface between Sema3E receptors, located in DRM domains, and the cytoskeleton network. As the fornix is affected in many psychiatric diseases, such as schizophrenia, our results provide new insights to better understand the neurodevelopmental components of these diseases.

Multiple Functions of Draxin/Netrin-1 Signaling in the Development of Neural Circuits in the Spinal Cord and the Brain

Axon guidance proteins play key roles in the formation of neural circuits during development. We previously identified an axon guidance cue, named draxin, that has no homology with other axon guidance proteins. Draxin is essential for the development of various neural circuits including the spinal cord commissure, corpus callosum, and thalamocortical projections. Draxin has been shown to not only control axon guidance through netrin-1 receptors, deleted in colorectal cancer (Dcc), and neogenin (Neo1) but also modulate netrin-1-mediated axon guidance and fasciculation. In this review, we summarize the multifaceted functions of draxin and netrin-1 signaling in neural circuit formation in the central nervous system. Furthermore, because recent studies suggest that the distributions and functions of axon guidance cues are highly regulated by glycoproteins such as Dystroglycan and Heparan sulfate proteoglycans, we discuss a possible function of glycoproteins in draxin/netrin-1-mediated axon guidance.

Cbln1 regulates axon growth and guidance in multiple neural regions

The accurate construction of neural circuits requires the precise control of axon growth and guidance, which is regulated by multiple growth and guidance cues during early nervous system development. It is generally thought that the growth and guidance cues that control the major steps of axon guidance have been defined. Here, we describe cerebellin-1 (Cbln1) as a novel cue that controls diverse aspects of axon growth and guidance throughout the central nervous system (CNS). Cbln1 has previously been shown to function in late neural development to influence synapse organization. Here we find that Cbln1 has an essential role in early neural development. Cbln1 is expressed on the axons and growth cones of developing commissural neurons and functions in an autocrine manner to promote axon growth. Cbln1 is also expressed in intermediate target tissues and functions as an attractive guidance cue. We find that these functions of Cbln1 are mediated by neurexin-2 (Nrxn2), which functions as the Cbln1 receptor for axon growth and guidance. In addition to the developing spinal cord, we further show that Cbln1 functions in diverse parts of the CNS with major roles in cerebellar parallel fiber growth and retinal ganglion cell axon guidance. Despite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.

The PH/MyTH4/FERMmolecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of C. elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally-expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild-type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

Analysis of the ENU-3 protein family in nervous system development in C. elegans.

During the development of the nervous system, neurons are guided to their final targets by several well-known guidance cues. In Caenorhabditis elegans the expression of the UNC-6/Netrin guidance cue along the ventral cord attracts axons that express UNC-40, while repulsing axons that express both the UNC-5 and UNC-40 receptors. Lack of both UNC-40 and the novel protein ENU-3 enhanced the ventral guidance defects of the AVM and PVM (Yee et al., 2014). This suggests that ENU-3 functions in an UNC-6 dependent pathway parallel to UNC-40 in controlling migrations towards the ventral nerve cord. Mutations in all proteins of the ENU-3 family also enhance the motor neuron axon outgrowth defects of strains lacking UNC-6 or the UNC-5 receptor, thus they function in a parallel unknown pathway (Yee et al., 2011). Expression analyses in HeLa cells have determined that ENU-3 and one of its paralogs, C38D4.1 localize to the nuclear membrane/ER while another of its paralogs, K01G5.3 is an intracellular membrane-associated protein.

Analysis of the ENU-3 protein family in nervous system development in C. elegans.

During the development of the nervous system, neurons are guided to their final targets by several well-known guidance cues. In Caenorhabditis elegans the expression of the UNC-6/Netrin guidance cue along the ventral cord attracts axons that express UNC-40, while repulsing axons that express both the UNC-5 and UNC-40 receptors. Lack of both UNC-40 and the novel protein ENU-3 enhanced the ventral guidance defects of the AVM and PVM (Yee et al., 2014). This suggests that ENU-3 functions in an UNC-6 dependent pathway parallel to UNC-40 in controlling migrations towards the ventral nerve cord. Mutations in all proteins of the ENU-3 family also enhance the motor neuron axon outgrowth defects of strains lacking UNC-6 or the UNC-5 receptor, thus they function in a parallel unknown pathway (Yee et al., 2011). Expression analyses in HeLa cells have determined that ENU-3 and one of its paralogs, C38D4.1 localize to the nuclear membrane/ER while another of its paralogs, K01G5.3 is an intracellular membrane-associated protein.

Growth Factors as Axon Guidance Molecules: Lessons From in vitro Studies

Growth cones at the tips of extending axons navigate through developing organisms by probing extracellular cues, which guide them through intermediate steps and onto final synaptic target sites. Widespread focus on a few guidance cue families has historically overshadowed potentially crucial roles of less well-studied growth factors in axon guidance. In fact, recent evidence suggests that a variety of growth factors have the ability to guide axons, affecting the targeting and morphogenesis of growth cones in vitro. This review summarizes in vitro experiments identifying responses and signaling mechanisms underlying axon morphogenesis caused by underappreciated growth factors.

Blood vessels regulate primary motor neuronal pathfinding in zebrafish via exosome contained microRNA-22

A precise neuro-vascular communication is crucial to orchestrate directional migration and patterning of the complex vascular network and neural system. However, how blood vessels are involved in shaping the proper neuronal formation has not been fully understood. 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 ECs resulted in the patterning defects of both ISVs and PMNs. However, neuronal block of miR-22a mainly led to the axonal defects of PMN. Then we demonstrated that endothelial miR-22a regulates PMNs axonal navigation via exosome pathway. Sema4c was identified as a potential target of miR-22a through transcriptomic analysis and in silico analysis. Furthermore, luciferase assay and EGFP sensor assay in vivo confirmed the binding of miR-22a with 3’-UTR of sema4c. In addition, Down-regulation of sema4c in the miR-22a morphants significantly neutralized the aberrant patterning of vascular and neural networks. Our study revealed that miR-22a acted as a dual guidance cue coordinating vascular and neuronal patterning and expanded the repertoire of guidance molecules, which might be of use therapeutically to guide vessels and nerves in the relevant diseases that affect both systems.

Anti-Inflammatory Role of Netrin-4 in Diabetic Retinopathy

Diabetic retinopathy is characterized by dysfunction of the retinal vascular network, combined with a persistent low-grade inflammation that leads to vision-threatening complications. Netrin-4 (NTN4) is a laminin-related secreted protein and guidance cue molecule present in the vascular basal membrane and highly expressed in the retina. A number of studies inferred that the angiogenic abilities of NTN4 could contribute to stabilize vascular networks and modulate inflammation. Analyzing human specimens, we show that NTN4 and netrin receptors are upregulated in the diabetic retina. We further evaluated a knock-out model for NTN4 undergoing experimental diabetes induced by streptozotocin. We investigated retina function and immune cells in vivo and demonstrated that NTN4 provides a protective milieu against inflammation in the diabetic retina and prevents cytokine production.