Stromal Cell-SLIT3/Cardiomyocyte-ROBO1 Axis Regulates Pressure Overload-Induced Cardiac Hypertrophy.

BACKGROUND: Recently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress. METHODS: We performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling. RESULTS: We first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction. CONCLUSIONS: Collectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Control of T-shaped Bifurcation by Multiple Guidance Mechanisms during Dorsal Funiculus Development in the Spinal Cord.

The dorsal funiculus in the spinal cord relays somatosensory information to the brain. It is made of T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons. Our previous study has shown that Slit signaling is required for proper guidance during bifurcation, but loss of Slit does not affect all DRG axons. Here, we examined the role of the extracellular molecule Netrin-1 (Ntn1). Using wholemount staining with tissue clearing, we showed that mice lacking Ntn1 have axons escaping from the dorsal funiculus at the time of bifurcation. Genetic labeling confirmed that these misprojecting axons come from DRG neurons. Single axon analysis showed that the defect does not affect bifurcation but rather alters turning angles. To distinguish their guidance functions, we examined mice with triple deletion of Ntn1, Slit2, and Slit2 and found a completely disorganized dorsal funiculus. Comparing mice with different genotypes using immunolabeling and single axon tracing revealed additive guidance defects, demonstrating the independent roles of Ntn1 and Slit. Moreover, the same defects were observed in embryos lacking their cognate receptors. These in vivo studies thus demonstrate the presence of multi-factorial guidance mechanisms that ensure proper formation of a common branched axonal structure during spinal cord development.

WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors.

Axon pathfinding is controlled by attractive and repulsive molecular cues that activate receptors on the axonal growth cone, but the full repertoire of axon guidance molecules remains unknown. The vertebrate DCC receptor family contains the two closely related members DCC and Neogenin with prominent roles in axon guidance and three additional, divergent members - Punc, Nope, and Protogenin - for which functions in neural circuit formation have remained elusive. We identified a secreted Punc/Nope/Protogenin ligand, WFIKKN2, which guides mouse peripheral sensory axons through Nope-mediated repulsion. In contrast, WFIKKN2 attracts motor axons, but not via Nope. These findings identify WFIKKN2 as a bifunctional axon guidance cue that acts through divergent DCC family members, revealing a remarkable diversity of ligand interactions for this receptor family in nervous system wiring. One-Sentence Summary: WFIKKN2 is a ligand for the DCC family receptors Punc, Nope, and Prtg that repels sensory axons and attracts motor axons.

Up-regulation of cholesterol synthesis pathways and limited neurodegeneration in a knock-in Sod1 mutant mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene ( SOD1 ) are associated with ∼20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. To generate a model that more closely represents patient gene expression, we created a knock-in point mutation (G85R, a human ALS-causing mutation) in the endogenous mouse Sod1 gene, leading to mutant SOD1 G85R protein expression. Heterozygous Sod1 G85R mutant mice resemble wild type, whereas homozygous mutants have reduced body weight and lifespan, a mild neurodegenerative phenotype, and express very low mutant SOD1 protein levels with no detectable SOD1 activity. Homozygous mutants exhibit partial neuromuscular junction denervation at 3-4 months of age. Spinal cord motor neuron transcriptome analyses of homozygous Sod1 G85R mice revealed up-regulation of cholesterol synthesis pathway genes compared to wild type. Transcriptome and phenotypic features of these mice are similar to Sod1 knock-out mice, suggesting the Sod1 G85R phenotype is largely driven by loss of SOD1 function. By contrast, cholesterol synthesis genes are down-regulated in severely affected human TgSOD1 G93A transgenic mice at 4 months. Our analyses implicate dysregulation of cholesterol or related lipid pathway genes in ALS pathogenesis. The Sod1 G85R knock-in mouse is a useful ALS model to examine the importance of SOD1 activity in control of cholesterol homeostasis and motor neuron survival. SIGNIFICANCE STATEMENT: Amyotrophic lateral sclerosis is a devastating disease involving the progressive loss of motor neurons and motor function for which there is currently no cure. Understanding biological mechanisms leading to motor neuron death is critical for developing new treatments. Using a new knock-in mutant mouse model carrying a Sod1 mutation that causes ALS in patients, and in the mouse, causes a limited neurodegenerative phenotype similar to Sod1 loss-of-function, we show that cholesterol synthesis pathway genes are up-regulated in mutant motor neurons, whereas the same genes are down-regulated in transgenic SOD1 mice with a severe phenotype. Our data implicate dysregulation of cholesterol or other related lipid genes in ALS pathogenesis and provide new insights that could contribute to strategies for disease intervention.

Circular Approaches in Small-Scale Food Production.

Globally, food production is one of the main water and energy consumers. Having in view the growing population on global scale, a higher efficiency of food production is needed. Circular approaches offer a large potential to enhance the efficiency of food production and have a long tradition in the food production process of mankind. However, industrial farming has interdicted traditional cycle-closed farming approaches leading to a variety of environmental challenges. The contribution illustrates the basics of traditional gardening and farming approaches and describes how their characteristics are adapted in innovative modern farming systems like aquaponic, permaculture, urban farming, as well as recovered traditional farming systems. The approach to combine traditional farming methods with modern ones will provide multiple benefits in the future to ensure food security. There is to be underlined that such a strategy holds a substantial potential of circular flux management in small scale food production. This potential could be transposed to a larger scale also, particularly in terms of agroforestry and integrated plant and animal husbandry or integrated agriculture and aquaculture. In this way, small-scale food production holds a large potential for the future implementation of the water-energy-food security nexus.

Tissue-specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3-ROBO1.

Although mesenchymal stem/stromal cells (MSCs) are being explored in numerous clinical trials as proangiogenic and proregenerative agents, the influence of tissue origin on the therapeutic qualities of these cells is poorly understood. Complicating the functional comparison of different types of MSCs are the confounding effects of donor age, genetic background, and health status of the donor. Leveraging a clinical setting where MSCs can be simultaneously isolated from discarded but healthy bone and thymus tissues from the same neonatal patients, thereby controlling for these confounding factors, we performed an in vitro and in vivo paired comparison of these cells. We found that both neonatal thymus (nt)MSCs and neonatal bone (nb)MSCs expressed different pericytic surface marker profiles. Further, ntMSCs were more potent in promoting angiogenesis in vitro and in vivo and they were also more motile and efficient at invading ECM in vitro. These functional differences were in part mediated by an increased ntMSC expression of SLIT3, a factor known to activate endothelial cells. Further, we discovered that SLIT3 stimulated MSC motility and fibrin gel invasion via ROBO1 in an autocrine fashion. Consistent with our findings in human MSCs, we found that SLIT3 and ROBO1 were expressed in the perivascular cells of the neonatal murine thymus gland and that global SLIT3 or ROBO1 deficiency resulted in decreased neonatal murine thymus gland vascular density. In conclusion, ntMSCs possess increased proangiogenic and invasive behaviors, which are in part mediated by the paracrine and autocrine effects of SLIT3.

NELL2-Robo3 complex structure reveals mechanisms of receptor activation for axon guidance.

Axon pathfinding is critical for nervous system development, and it is orchestrated by molecular cues that activate receptors on the axonal growth cone. Robo family receptors bind Slit guidance cues to mediate axon repulsion. In mammals, the divergent family member Robo3 does not bind Slits, but instead signals axon repulsion from its own ligand, NELL2. Conversely, canonical Robos do not mediate NELL2 signaling. Here, we present the structures of NELL-Robo3 complexes, identifying a mode of ligand engagement for Robos that is orthogonal to Slit binding. We elucidate the structural basis for differential binding between NELL and Robo family members and show that NELL2 repulsive activity is a function of its Robo3 affinity and is enhanced by ligand trimerization. Our results reveal a mechanism of oligomerization-induced Robo activation for axon guidance and shed light on Robo family member ligand binding specificity, conformational variability, divergent modes of signaling, and evolution.

TAG-1 Multifunctionality Coordinates Neuronal Migration, Axon Guidance, and Fasciculation.

Neuronal migration, axon fasciculation, and axon guidance need to be closely coordinated for neural circuit assembly. Spinal motor neurons (MNs) face unique challenges during development because their cell bodies reside within the central nervous system (CNS) and their axons project to various targets in the body periphery. The molecular mechanisms that contain MN somata within the spinal cord while allowing their axons to exit the CNS and navigate to their final destinations remain incompletely understood. We find that the MN cell surface protein TAG-1 anchors MN cell bodies in the spinal cord to prevent their emigration, mediates motor axon fasciculation during CNS exit, and guides motor axons past dorsal root ganglia. TAG-1 executes these varied functions in MN development independently of one another. Our results identify TAG-1 as a key multifunctional regulator of MN wiring that coordinates neuronal migration, axon fasciculation, and axon guidance.

Cell migration and axon guidance at the border between central and peripheral nervous system.

The central and peripheral nervous system (CNS and PNS, respectively) are composed of distinct neuronal and glial cell types with specialized functional properties. However, a small number of select cells traverse the CNS-PNS boundary and connect these two major subdivisions of the nervous system. This pattern of segregation and selective connectivity is established during embryonic development, when neurons and glia migrate to their destinations and axons project to their targets. Here, we provide an overview of the cellular and molecular mechanisms that control cell migration and axon guidance at the vertebrate CNS-PNS border. We highlight recent advances on how cell bodies and axons are instructed to either cross or respect this boundary, and present open questions concerning the development and plasticity of the CNS-PNS interface.

Diverse spinal commissural neuron populations revealed by fate mapping and molecular profiling using a novel Robo3Cre mouse.

The two sides of the nervous system coordinate and integrate information via commissural neurons, which project axons across the midline. Commissural neurons in the spinal cord are a highly heterogeneous population of cells with respect to their birthplace, final cell body position, axonal trajectory, and neurotransmitter phenotype. Although commissural axon guidance during development has been studied in great detail, neither the developmental origins nor the mature phenotypes of commissural neurons have been characterized comprehensively, largely due to lack of selective genetic access to these neurons. Here, we generated mice expressing Cre recombinase from the Robo3 locus specifically in commissural neurons. We used Robo3 Cre mice to characterize the transcriptome and various origins of developing commissural neurons, revealing new details about their extensive heterogeneity in molecular makeup and developmental lineage. Further, we followed the fate of commissural neurons into adulthood, thereby elucidating their settling positions and molecular diversity and providing evidence for possible functions in various spinal cord circuits. Our studies establish an important genetic entry point for further analyses of commissural neuron development, connectivity, and function.

Corrigendum: GnRH-1 Neural Migration From the Nose to the Brain Is Independent From Slit2, Robo3 and NELL2 Signaling.

[This corrects the article DOI: 10.3389/fncel.2019.00070.].

GnRH-1 Neural Migration From the Nose to the Brain Is Independent From Slit2, Robo3 and NELL2 Signaling.

Gonadotropin releasing hormone-1 (GnRH-1) neurons play a pivotal role in controlling pubertal onset and fertility once they reach their hypothalamic location. During embryonic development, GnRH-1 neurons migrate from the nasal area to the hypothalamus where they modulate gonadotropin release from the pituitary gland. Defective migration of the GnRH-1 neurons to the brain, lack of GnRH-1 secretion or signaling cause hypogonadotropic hypogonadism (HH), a pathology characterized by delayed or absence of puberty. Binding of the guidance cue Slit2 to the receptor roundabout 3 (Robo3) has been proposed to modulate GnRH-1 cell motility and basal forebrain (bFB) access during migration. However, evidence suggests that Neural EGFL Like 2 (NELL2), not Slit2, binds to Robo3. To resolve this discrepancy, we analyzed GnRH-1 neuronal migration in NELL2, Robo3, and Slit2 knock-out mouse lines. Our data do not confirm a negative effect for monogenic Robo3 and Slit2 mutations on GnRH-1 neuronal migration from the nasal area to the brain. Moreover, we found no changes in GnRH-1 neuronal migration in the brain after NELL2 loss-of-function. However, we found that Slit2 loss-of-function alters the patterning of GnRH-1 cells in the brain, suggesting that Slit2 loss-of-function affects GnRH-1 cell positioning in the brain in a Robo3 independent fashion. Our results challenge previous theories on GnRH-1 neuronal migration mechanisms and provide a new impetus to identify and understand the complex genetic mechanisms causing disorders like Kallmann syndrome (KS) and HH.

Meninges-derived cues control axon guidance.

The axons of developing neurons travel long distances along stereotyped pathways under the direction of extracellular cues sensed by the axonal growth cone. Guidance cues are either secreted proteins that diffuse freely or bind the extracellular matrix, or membrane-anchored proteins. Different populations of axons express distinct sets of receptors for guidance cues, which results in differential responses to specific ligands. The full repertoire of axon guidance cues and receptors and the identity of the tissues producing these cues remain to be elucidated. The meninges are connective tissue layers enveloping the vertebrate brain and spinal cord that serve to protect the central nervous system (CNS). The meninges also instruct nervous system development by regulating the generation and migration of neural progenitors, but it has not been determined whether they help guide axons to their targets. Here, we investigate a possible role for the meninges in neuronal wiring. Using mouse neural tissue explants, we show that developing spinal cord meninges produce secreted attractive and repulsive cues that can guide multiple types of axons in vitro. We find that motor and sensory neurons, which project axons across the CNS-peripheral nervous system (PNS) boundary, are attracted by meninges. Conversely, axons of both ipsi- and contralaterally projecting dorsal spinal cord interneurons are repelled by meninges. The responses of these axonal populations to the meninges are consistent with their trajectories relative to meninges in vivo, suggesting that meningeal guidance factors contribute to nervous system wiring and control which axons are able to traverse the CNS-PNS boundary.

Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2.

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2-mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)-mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

Genetic analysis reveals that amyloid precursor protein and death receptor 6 function in the same pathway to control axonal pruning independent of ß-secretase.

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that ß-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that ß-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning.

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.

Autocrine/juxtaparacrine regulation of axon fasciculation by Slit-Robo signaling.

Axons travel to their targets in bundles or fascicles, but the molecules regulating fasciculation remain incompletely characterized. We found that Slit2 and its Robo receptors are expressed by motor axons, and that inactivation of Slit2 or Robo1 and Robo2 in mice caused axons to defasciculate prematurely at muscle targets. In vitro, Slit2 secreted by motoneurons regulated fasciculation through Robo1 and Robo2. These results support the idea that Slit2 promotes axon fasciculation via an autocrine and/or juxtaparacrine mechanism.

Collaborative and specialized functions of Robo1 and Robo2 in spinal commissural axon guidance.

Commissural neurons project axons across the floor plate at the spinal cord ventral midline. After crossing, commissural axons turn rostrally, sort into distinct positions within the ventrolateral funiculus, and never reenter the floor plate. Robo1 and Robo2 are receptors for the midline repellents Slit1-Slit3, and upregulation of Robos in post-crossing axons allows expulsion from the floor plate and prevents recrossing. Before crossing, Robo-mediated repulsion is attenuated by the divergent family member Robo3/Rig-1. To define the relative contributions of Robo family members to commissural axon guidance in mice, we studied commissural axon trajectories in combination mutants between Robo1, Robo2, and Robo3. Our results suggest the existence of another receptor contributing to Slit repulsion because the failure of midline crossing in Robo3 mutants is rescued largely but not entirely by loss of both Robo1 and Robo2 and because axon guidance defects in mice lacking both Robo1 and Robo2 are less severe than in mice lacking all Slits. Analysis of post-crossing axon trajectories indicates that Robo1 and Robo2 collaborate to prevent axons from reentering the gray matter and projecting dorsally alongside contralateral pre-crossing axons. We also discovered a previously unappreciated division of labor between Robo1 and Robo2 in post-crossing axons. Robo2 is required for axons to project away from the floor plate into the lateral funiculus. In contrast, Robo1 prevents axonal stalling after crossing. Our results reveal specialized and complementary actions of Robo1 and Robo2 in commissural axon guidance and suggest the existence of an as yet unidentified Slit receptor.

GA-binding protein is dispensable for neuromuscular synapse formation and synapse-specific gene expression.

The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and other "synaptic" genes is required for synapse-specific transcription. The Ets domain transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression. Inactivation of GABPalpha, the DNA-binding subunit of GABP, leads to early embryonic lethality, preventing analysis of synapse formation in gabpalpha mutant mice. To study the role of GABP at neuromuscular synapses, we conditionally inactivated gabpalpha in skeletal muscle and studied synaptic differentiation and muscle gene expression. Although expression of rb, a target of GABP, is elevated in muscle tissue deficient in GABPalpha, clustering of synaptic AChRs at synapses and synapse-specific gene expression are normal in these mice. These data indicate that GABP is dispensable for synapse-specific transcription and maintenance of normal AChR expression at synapses.

CD24 is expressed by myofiber synaptic nuclei and regulates synaptic transmission.

The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further. We show that CD24, which encodes a small, variably and highly glycosylated, glycosylphosphatidylinositol (GPI)-linked protein, is expressed preferentially by myofiber synaptic nuclei in embryonic and adult muscle, and that CD24 expression is restricted to the central region of muscle independent of innervation. Moreover, we show that CD24 has a role in presynaptic differentiation, because synaptic transmission is depressed and fails entirely, in a cyclical manner, after repetitive stimulation of motor axons in CD24 mutant mice. These deficits in synaptic transmission, which are accompanied by aberrant stimulus-dependent uptake of AM1-43 from axons, indicate that CD24 is required for normal presynaptic maturation and function. Because CD24 is also expressed in some neurons, additional experiments will be required to determine whether pre- or postsynaptic CD24 mediates these effects on presynaptic development and function.