Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.
Anatomical congruence of peripheral nerves and blood vessels is well recognized in a variety of tissues. Their physical proximity and similar branching patterns suggest that the development of these networks might be a coordinated process. Here we show that large diameter coronary veins serve as an intermediate template for distal sympathetic axon extension in the subepicardial layer of the dorsal ventricular wall of the developing mouse heart. Vascular smooth muscle cells (VSMCs) associate with large diameter veins during angiogenesis. In vivo and in vitro experiments demonstrate that these cells mediate extension of sympathetic axons via nerve growth factor (NGF). This association enables topological targeting of axons to final targets such as large diameter coronary arteries in the deeper myocardial layer. As axons extend along veins, arterial VSMCs begin to secrete NGF, which allows axons to reach target cells. We propose a sequential mechanism in which initial axon extension in the subepicardium is governed by transient NGF expression by VSMCs as they are recruited to coronary veins; subsequently, VSMCs in the myocardium begin to express NGF as they are recruited by remodeling arteries, attracting axons toward their final targets. The proposed mechanism underlies a distinct, stereotypical pattern of autonomic innervation that is adapted to the complex tissue structure and physiology of the heart.
In developing limb skin, peripheral nerves provide a spatial template that controls the branching pattern and differentiation of arteries. Our previous studies indicate that nerve-derived VEGF-A is required for arterial differentiation but not for nerve-vessel alignment. In this study, we demonstrate that nerve-vessel alignment depends on the activity of Cxcl12-Cxcr4 chemokine signaling. Genetic inactivation of Cxcl12-Cxcr4 signaling perturbs nerve-vessel alignment and abolishes arteriogenesis. Further in vitro assays allow us to uncouple nerve-vessel alignment and arteriogenesis, revealing that nerve-derived Cxcl12 stimulates endothelial cell migration, whereas nerve-derived VEGF-A is responsible for arterial differentiation. These findings suggest a coordinated sequential action in which nerve Cxcl12 functions over a distance to recruit vessels to align with nerves, and subsequent arterial differentiation presumably requires a local action of nerve VEGF-A in the nerve-associated vessels.
Cerebral cavernous malformations (CCM) are irregularly shaped and enlarged capillaries in the brain that are prone to hemorrhage, resulting in headaches, seizures, strokes and even death in patients. The disease affects up to 0.5% of the population and the inherited form has been linked to mutations in one of three genetic loci, CCM1, CCM2 and CCM3. To understand the pathophysiology underlying the vascular lesions in CCM, it is critical to develop a reproducible mouse genetic model of this disease. Here, we report that limited conditional ablation of Ccm2 in young adult mice induces observable neurological dysfunction and reproducibly results in brain hemorrhages whose appearance is highly reminiscent of the lesions observed in human CCM patients. We first demonstrate that conventional or endothelial-specific deletion of Ccm2 leads to fatal cardiovascular defects during embryogenesis, including insufficient vascular lumen formation as well as defective arteriogenesis and heart malformation. These findings confirm and extend prior studies. We then demonstrate that the inducible deletion of Ccm2 in adult mice recapitulates the CCM-like brain lesions in humans; the lesions display disrupted vascular lumens, enlarged capillary cavities, loss of proper neuro-vascular associations and an inflammatory reaction. The CCM lesions also exhibit damaged neuronal architecture, the likely cause of neurologic defects, such as ataxia and seizure. These mice represent the first CCM2 animal model for CCM and should provide the means to elucidate disease mechanisms and evaluate therapeutic strategies for human CCM.
Gli3, one of three vertebrate Gli transcription factors in Hedgehog (Hh) pathway, is processed into a repressor form (Gli3R) in the absence of Hh signal and acts as the major negative transducer of the pathway. Although the role of Gli3 in embryonic patterning has been extensively studied, its role in cortical neurogenesis, especially in the regulation of neural progenitors in proliferation and cell fate specification, is largely unknown. To bypass the patterning defects caused by loss of Gli3, we conditionally deleted Gli3 after patterning was complete in mouse. Our results from birthdating and in utero electroporation experiments demonstrate that the Gli3, specifically Gli3R, is critical for specifying the fate of cortical neurons that are generated following a stereotypical temporal order. Moreover, Gli3 is required for maintaining the cortical progenitors in active cell cycle, suggesting that cells may acquire differentiated status as they turn off Gli3 expression during neurogenesis.
A woman had anosognosia for hemiplegia as a manifestation of brainstem infarction. She had no mental or neuropsychological disturbances, and had involvement of the brainstem in the frontal/parietal-subcortical circuits to the right cerebral hemisphere. Brainstem lesions that disrupt frontal/parietal-subcortical areas may affect anosognosia for hemiplegia.
Semaphorin-3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin-1 and plexin-A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as "proximal bifurcation." In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal-like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin-dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development.
Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.
We introduce a whole-mount immunohistochemistry method for analyzing intricate vascular network formation in mouse embryonic tissues. Laser scanning confocal microscopy with multiple labeling allows for robust imaging of blood and lymphatic vessel branching morphogenesis with excellent resolution.
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