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Tissue clearing and expansion microscopy have significantly advanced neuroscience by enabling the visualization of spatial information about biological components, from tissue-level architecture to nanoscale structures. Currently, their application is limited to tissues without prolonged fixation, which excludes long-term preserved specimens, such as human brains. To address this issue, we present a new multiplex labeling and expansion-applicable method for centimeter-sized archived tissue (MOCAT). We successfully applied MOCAT to formalin-fixed paraffin-embedded (FFPE) mouse, pig, and human brain blocks up to 15 years old. The reliability of MOCAT was demonstrated by the indistinguishable spatial and quantitative information of biomarkers obtained from FFPE intact mouse brains compared to that from freshly prepared SDS-delipidated intact mouse brains. Additionally, MOCAT-processed FFPE samples exhibited superior long-term antigenicity preservation and suitability for advanced expansion microscopy in human brain compared to those kept in 10% formalin. In summary, MOCAT combined with FFPE fulfills the demand for long-term tissue preservation and organ-level multiplex imaging in super-resolution.

Current tissue clearing and labeling approaches require freshly prepared samples to avoid problems related to over-fixation, such as loss of antigenicity or difficulty of tissue clearing. Thus, the predicament of specimens with long-term preservation limits the application of state-of-art technology to archived human brain tissues. Here, we present the development and validation of multiplex labeling of centimeter-sized archived tissue (MOCAT)—a formalin-fixed paraffin-embedding (FFPE)–based sample preservation and delipidation technique for centimeter-level tissue clearing and immunolabeling. We demonstrate that MOCAT sufficiently delipidates whole mouse brain and human brain blocks and is superior to 10% formalin for long-term antigenicity preservation; moreover, the spatial information of biomarkers offered by MOCAT-processed intact mouse brains is identical to that offered by freshly prepared SDS-delipidated intact mouse brains. MOCAT not only satisfies the need for long-term tissue storage before performing volumetric multiplex imaging for research purposes but also has the potential to be used in human clinical examination.

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In

Mouse hippocampus retains the capacity for neurogenesis throughout lifetime, but such plasticity decreases with age. Adult hippocampal neurogenesis (AHN) involves the birth, maturation, and synaptic integration of newborn granule cells (GCs) into preexisting hippocampal circuitry. While functional integration onto adult-born GCs has been extensively studied, maturation of efferent projections onto CA3 pyramidal cells is less understood, particularly in aged brain. Here, using combined light and reconstructive electron microscopy (EM), we describe the maturation of mossy fiber bouton (MFB) connectivity with CA3 pyramidal cells in young adult and aged mouse brain. We found mature synaptic contacts of newborn GCs were formed in both young and aged brains. However, the dynamics of their spatiotemporal development and the cellular process by which these cells functionally integrated over time were different. In young brain newborn GCs either formed independent nascent MFB synaptic contacts or replaced preexisting MFBs, but these contacts were pruned over time to a mature state. In aged brain only replacement of preexisting MFBs was observed and new contacts were without evidence of pruning. These data illustrate that functional synaptic integration of AHN occurs in young adult and aged brain, but with distinct dynamics. They suggest elimination of preexisting connectivity is required for the integration of adult-born GCs in aged brain.

Significance During neural development, growing axons navigate over long distances to reach their targets. A critical step in this process is the regulation of its surface receptors on the axon’s growth cone in response to environmental cues. We focus on how the UNC-5 receptor in Caenorhabditis elegans motor axons is regulated during axon repulsion. By combining C. elegans genetics, biochemistry, and imaging, we found that MAX-1 SUMOylation and AP-3 complex have significant roles in UNC-5–mediated axon repulsion. Our findings reveal how SUMOylation and AP-3–mediated trafficking and degradation interact to help the growing axon find its final target., During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5–mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5–mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex β subunit gene, apb-3. Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3. Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3–mediated trafficking and degradation of UNC-5 receptors during axon guidance.

Background Activity in neurons drives afferent competition that is critical for the refinement of nascent neural circuits. In ferrets, when an eye is lost in early development, surviving retinogeniculate afferents from the spared eye spread across the thalamus in a manner that is dependent on spontaneous retinal activity. However, how this spontaneous activity, also known as retinal waves, might dynamically regulate afferent terminal targeting remains unknown. Methods We recorded retinal waves from retinae ex vivo using multi-electrode arrays. Retinae came from ferrets who were binocular or who had one eye surgically removed at birth. Linear mixed effects models were used to investigate the effects of early monocular enucleation on retinal wave activity. Results When an eye is removed at birth, spontaneous bursts of action potentials by retinal ganglion cells (RGCs) in the surviving eye are shorter in duration. The shortening of RGC burst duration results in decreased pairwise RGC correlations across the retina and is associated with the retinal wave-dependent spread of retinogeniculate afferents previously reported in enucleates. Conclusion Our findings show that removal of the competing eye modulates retinal waves and could underlie the dynamic regulation of competition-based refinement during retinogeniculate development.

Defects in neuronal connectivity of the brain are well documented among schizophrenia patients. Although the schizophrenia susceptibility gene Disrupted-in-Schizophrenia 1 (DISC1) has been implicated in various neurodevelopmental processes, its role in regulating axonal connections remains elusive. Here, a heterologous DISC1 transgenic system in the relatively simple and well-characterized Caenorhabditis elegans motor neurons has been established to investigate whether DISC1 regulates axon guidance during development. Transgenic DISC1 in C. elegans motor neurons is enriched in the migrating growth cones and causes guidance defects of their growing axons. The abnormal axonal phenotypes induced by DISC1 are similar to those by gain-of-function rac genes. In vivo genetic interaction studies revealed that the UNC-73/TRIO-RAC-PAK signaling pathway is activated by ectopic DISC1 in C. elegans motor axons. Using in vitro GST pull-down and coimmunoprecipitation assays, we found that DISC1 binds specifically to the amino half of spectrin repeats of TRIO, thereby preventing TRIO's amino half of spectrin repeats from interacting with its first guanine nucleotide exchange factor (GEF) domain, GEF1, and facilitating the recruitment of RAC1 to TRIO. In cultured mammalian cells, RAC1 is activated by increased TRIO's GEF activity when DISC1 is present. These results together indicate that the TRIO-RAC-PAK signaling pathway can be exploited and modulated by DISC1 to regulate axonal connectivity in the developing brain.

Throughout the nervous system, neurons restrict their connections to specific depths or “layers” of their targets to constrain the type and number of synapses they make. Despite the importance of lamina-specific synaptic connectivity, the mechanisms that give rise to this feature in mammals remain poorly understood. Here we examined the cellular events underlying the formation of lamina-specific retinal ganglion cell (RGC) axonal projections to the superior colliculus (SC) of the mouse. By combining a genetically encoded marker of a defined RGC subtype (OFF-αRGCs) with serial immunoelectron microscopy, we resolved the ultrastructure of axon terminals fated for laminar stabilization versus those fated for removal. We found that OFF-αRGCs form synapses across the full depth of the retinorecipient SC before undergoing lamina-specific arbor retraction and synapse elimination to arrive at their mature, restricted pattern of connectivity. Interestingly, we did not observe evidence of axon degeneration or glia-induced synapse engulfment during this process. These findings indicate that lamina-specific visual connections are generated through the selective stabilization of correctly targeted axon arbors and suggest that the decision to maintain or eliminate an axonal projection reflects the molecular compatibility of presynaptic and postsynaptic neurons at a given laminar depth.

Axon guidance and synapse formation are important developmental events for establishing a functional neuronal circuitry. These two related cellular processes occur in a coordinated fashion but previous studies from multiple model organisms seemed to suggest that axon guidance and synapse formation are mediated by distinct molecular cues. Thus, axon guidance molecules are responsible for guiding the navigating axon toward its target area, while other adhesion or ligand-receptor molecules specify the synapse formation within the target area. However, accumulative evidence has shown that axon guidance molecules can regulate the localization and formation of pre-synaptic and post-synaptic components during synapse formation. These results demonstrate a role for axon guidance molecules in synapse formation and provide insight into how axon guidance and synapse formation are coordinated at the molecular level.

In vertebrates, class 3 semaphorins (SEMA3) control axon behaviour by binding to neuronal cell surface receptors composed of a ligand binding subunit termed neuropilin (NRP) and a signal transduction subunit of the A-type plexin family (PLXNA). We have determined the requirement for SEMA3/NRP/PLXN signalling in the development of the facial nerve, which contains axons from two motor neuron populations, branchiomotor and visceromotor neurons. Loss of either SEMA3A/NRP1 or SEMA3F/NRP2 caused defasciculation and ectopic projection of facial branchiomotor axons. In contrast, facial visceromotor axons selectively required SEMA3A/NRP1. Thus, the greater superficial petrosal nerve was defasciculated, formed ectopic projections and failed to branch in its target area when either SEMA3A or NRP1 were lost. To examine which A-type plexin conveyed SEMA3/neuropilin signals during facial nerve development, we combined an expression analysis with loss of function studies. Even though all four A-type plexins were expressed in embryonic motor neurons, PLXNA1 and PLXNA2 were not essential for facial nerve development. In contrast, loss of PLXNA4 phenocopied the defects of SEMA3A and NRP1 mutants, and loss of PLXNA3 phenocopied the defects of SEMA3F and NRP2 mutants. The combined loss of PLXNA3 and PLXNA4 impaired facial branchiomotor axon guidance more severely than loss of either plexin alone, suggesting that both pathways normally cooperate; in contrast, loss of both plexins did not impair facial visceromotor defects any worse than loss of PLXNA4. We conclude that PLXNA3 and PLXNA4 synergise to pattern the facial nerve, whereby both are required in branchiomotor neurons, but only PLXNA4 is essential for visceromotor neurons.

SummaryAdult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.

Semaphorin signaling plays integral roles in multiple developmental processes. Branching morphogenesis is one such role that has not been thoroughly explored. Here, we show in mice that functional blockage of neuropilin 1 (Npn1) inhibits cleft formation in the developing submandibular gland (SMG) cultured ex vivo. This Npn1-dependent morphogenesis is mediated by Sema3A and Sema3C in an additive manner, and can be abolished by decreasing the expression of plexin A2 or plexin D1. VEGF, another known Npn1 ligand, has no apparent effects on SMG development. FGF signaling, which also mediates SMG branching morphogenesis, acts in parallel with semaphorin signaling. Finally,in contrast to the effect of FGF signaling, we find that semaphorins do not stimulate the proliferation of SMG epithelial cells. Instead, the semaphorin signals act locally on the epithelial cells to facilitate SMG cleft formation.

Current models of retinogeniculate development have proposed that connectivity between the retina and the dorsal lateral geniculate nucleus (dLGN) is established by gradients of axon guidance molecules, to allow initial coarse connections, and by competitive Hebbian-like processes, to drive eye-specific segregation and refine retinotopy. Here we show that when intereye competition is eliminated by monocular enucleation, blocking cholinergic stage II retinal waves disrupts the intraeye competition-mediated expansion of the retinogeniculate projection and results in the permanent disorganization of its laminae. This disruption of stage II retinal waves also causes long-term impacts on receptive field size and fine-scale retinotopy in the dLGN. Our results reveal a novel role for stage II retinal waves in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, allowing for correct laminar patterning and the even allocation of synaptic territory. These findings should contribute to answering questions regarding the role of neural activity in guiding the establishment of neural circuits.

During early development of the central nervous system (CNS), there is an exuberant outgrowth of projections which later need to be refined to achieve precise connectivity. One widely used strategy for this refinement is axon pruning. Axon pruning has also been suggested to be involved in creating more diverse connection patterns between different species. An understanding of the mechanism of pruning, however, has been elusive in the CNS. Recent studies have focused on a stereotyped pruning event that occurs within the mossy fibers of the developing vertebrate hippocampus. In the following discussion, we will review the cellular and molecular factors that are known to regulate pruning in the hippocampus and highlight some advantages this system presents for future studies on pruning in the developing CNS.

P21 activated kinases (PAKs) are major downstream effectors of rac-related small GTPases that regulate various cellular processes. We have identified the new PAK gene max-2 in a screen for mutants disrupted in UNC-6/netrin-mediated commissural axon guidance. There are three Caenorhabditis elegans PAKs. We find that each C. elegansPAK represents a distinct group previously identified in other species. Here we examine their roles in the postembryonic migration of the P cell neuroblasts and the axon guidance of the ventral cord commissural motoneurons(VCCMNs). We find that the two PAKs, max-2 and pak-1, are redundantly required for P cell migration and function with UNC-73/Trio and the rac GTPases (CED-10 and MIG-2). During axon guidance of the VCCMNs, PAK-1 also acts with the rac GTPases, CED-10 and MIG-2, and is completely redundant with MAX-2. Interestingly, we find that unlike MAX-2 activity during P cell migration, for motoneuron axon guidance max-2 is also required in parallel to this PAK-1 pathway, independent of rac GTPase signaling. Finally,we provide evidence that MAX-2 functions downstream of the UNC-6/netrin receptor UNC-5 during axon repulsion and is an integral part of its signaling.

During the development of the nervous system, neurons must first migrate to their appropriate locations and then send out axons to make connections. Various environmental cues guide these migrating neurons and growing axons. After axons reach their target regions, neuronal contacts are created through the formation of synapses. Because excess axonal branches and synaptic contacts are often formed during early development, they are pruned or eliminated at later stages to create specific neuronal connections. Several groups of ligand-receptor pairs have been identified to regulate each of these cellular events. Evidence also indicates that these same molecules may be used in multiple developmental processes. Here, we discuss semaphorins and plexins, the largest family of axon guidance ligand-receptor pairs. Because the roles of semaphorins in neuronal migration and axonal repulsion have been extensively reviewed, we will focus on plexin receptors. We will discuss how semaphorin signals are specifically passed through these receptors into cells and how plexins mediate their newly identified roles in axon pruning and synaptic development.

Background Spontaneous retinal activity (SRA) is important during eye-specific segregation within the dorsal lateral geniculate nucleus (dLGN), but the feature(s) of activity critical for retinogeniculate refinement are controversial. Pharmacologically or genetically manipulating cholinergic signaling during SRA perturbs correlated retinal ganglion cell (RGC) spiking and disrupts eye-specific retinofugal refinement in vivo, consistent with an instructive role for SRA during visual system development. Paradoxically, ablating the starburst amacrine cells (SACs) that generate cholinergic spontaneous activity disrupts correlated RGC firing without impacting retinal activity levels or eye-specific segregation in the dLGN. Such experiments suggest that patterned SRA during retinal waves is not critical for eye-specific refinement and instead, normal activity levels are permissive for retinogeniculate development. Here we revisit the effects of ablating the cholinergic network during eye-specific segregation and show that SAC ablation disrupts, but does not eliminate, retinal waves with no concomitant impact on normal eye-specific segregation in the dLGN. Results We induced SAC ablation in postnatal ferret pups beginning at birth by intraocular injection of a novel immunotoxin selective for the ferret vesicular acetylcholine transporter (Ferret VAChT-Sap). Through dual-patch whole-cell and multi-electrode array recording we found that SAC ablation altered SRA patterns and led to significantly smaller retinal waves compared with controls. Despite these defects, eye-specific segregation was normal. Further, interocular competition for target territory in the dLGN proceeded in cases where SAC ablation was asymmetric in the two eyes. Conclusions Our data demonstrate normal eye-specific retinogeniculate development despite significant abnormalities in patterned SRA. Comparing our current results with earlier studies suggests that defects in retinal wave size, absolute levels of SRA, correlations between RGC pairs, RGC burst frequency, high frequency RGC firing during bursts, and the number of spikes per RGC burst are each uncorrelated with abnormalities in eye-specific segregation in the dLGN. An increase in the fraction of asynchronous spikes occurring outside of bursts and waves correlates with eye-specific segregation defects in studies reported to date. These findings highlight the relative importance of different features of SRA while providing additional constraints for computational models of Hebbian plasticity mechanisms in the developing visual system. Electronic supplementary material The online version of this article (doi:10.1186/1749-8104-9-25) contains supplementary material, which is available to authorized users.

While building the nervous system, regions of some developing axons are eliminated; this can also happen as a result of axonal injury. During development, many axon branches that are formed in excess of an organism's needs are fated for removal in a process called axon pruning. By contrast, when axons are injured the axon segment distal to the injury site is compartmentalized and eliminated. In both cases, the end result is similar -- a region of an axon is selected for removal. Recent evidence suggests that there are some similarities in the cellular and molecular mechanisms that regulate axon elimination in development and during axonal injury.

The netrin UNC-6 repels motor axons by activating the UNC-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the UNC-5 receptor signaling pathway.

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Here, we show that plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, we provide evidence that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo.

Summary: Programmed cell death is a pivotal process that regulates neuronal number during development. Key regulators of this process are members of the BCL-2 family. Using mRNA differential display, we identified a Bcl-2 family gene, Blm-s (Bcl-2-like molecule, short form), enriched in postmitotic neurons of the developing cerebral cortex. BLM-s functions as a BH3-only apoptosis sensitizer/derepressor and causes BAX-dependent mitochondria-mediated apoptosis by selectively binding to prosurvival BCL-2 or MCL-1. When challenged with γ-irradiation that produces DNA double-strand breaks (DSBs), Blm-s is transcriptionally upregulated in postmitotic immature neurons with concurrently increased apoptosis. RNAi-mediated depletion of Blm-s protects immature neurons from irradiation-induced apoptosis. Furthermore, Blm-s is a direct target gene of p53 and AP1 via the ataxia telangiectasia mutated (ATM)- and c-Jun N-terminal kinase (JNK)-signaling pathways activated by DSBs. Thus, BLM-s is likely an apoptosis sensor activated by DSBs accumulating in postmitotic immature neurons. : Liu et al. describe a Bcl-2 family gene, Blm-s, that is specifically expressed in postmitotic immature neurons during embryonic development. When the developing brain is challenged with γ-irradiation that produces DNA double-strand breaks (DBSs), Blm-s is upregulated in the neurons that undergo cell death. Depletion of Blm-s prevents immature neurons from irradiation-induced death. The authors also show that Blm-s is a direct target of p53 and AP1 via the signaling pathways activated by DSBs.

Topographic maps with a defined spatial ordering of neuronal connections are a key feature of brain organization. Such maps are believed to develop in response to complementary position-specific labels in presynaptic and postsynaptic fields. However, the complementary labeling molecules are not known. In the well-studied visual map of retinal axons projecting to the tectum, the labels are hypothesized to be in gradients, without needing large numbers of cell-specific molecules. We recently cloned ELF-1 as a ligand for Eph family receptors. Here, RNA hybridization shows matching expression gradients for ELF-1 in the tectum and its receptor Mek4 in the retina. Binding activity detected with alkaline phosphatase fusions of ELF-1 and Mek4 also reveals gradients and provides direct evidence for molecular complementarity of gradients in reciprocal fields. ELF-1 and Mek4 may therefore play roles in retinotectal development and have properties predicted of topographic mapping labels.

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration.

Stretch reflex circuits are a prime example of wiring specificity in the vertebrate spinal cord. Homonymous sensory afferents and motoneurons typically form monosynaptic connections, while neurons innervating antagonistic or unrelated muscles do not. Pecho-Vrieseling et al. now show that the semaphorin Sema3E and its receptor Plexin-D1 prevent monosynaptic connectivity in the cutaneous maximus muscle stretch reflex circuit.

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.

Background The development of the corticospinal tract (CST) in higher vertebrates relies on a series of axon guidance decisions along its long projection pathway. Several guidance molecules are known to be involved at various decision points to regulate the projection of CST axons. However, previous analyses of the CST guidance defects in mutant mice lacking these molecules have suggested that there are other molecules involved in CST axon guidance that are yet to be identified. In this study, we investigate the role of plexin signaling in the guidance of motor CST axons in vivo. Results Expression pattern studies show that plexin-A3, plexin-A4, and neuropilin-1 are expressed in the developing cerebral cortex when the motor CST axons originating from layer V cortical neurons are guided down to the spinal cord. By analyzing mutant mice, we show that motor CST axons that turn dorsally to cross the midline at the pyramidal decussation require plexin-A3 and plexin-A4 signaling. Although other CST guidance defects are found in neuropilin-1 mutants, this dorsal turning defect is not observed in either neuropilin-1 or neuropilin-2 mutants, suggesting that the local cues that activate plexin signaling at the dorsal turning point are membrane-bound semaphorins. Further expression pattern study and mutant analysis indicate that Sema6A is one of the local cues for motor CST axon turning at the pyramidal decussation. Conclusion Dorsal turning and midline crossing at the pyramidal decussation is a crucial step to properly direct CST axons into the dorsal spinal cord. We show that the signaling of plexin-A3, plexin-A4, and Sema6A is at least partially required for dorsal turning of the CST axons, while neuropilin-1 is required for proper fasciculation of the tract at midline crossing. Together with previous reports, these results demonstrate that several guidance cues are specifically utilized to regulate the dorsal turning and midline crossing of developing CST axons.

Neurons in the developing CNS tend to send out long axon collaterals to multiple target areas. For these neurons to attain specific connections, some of their axon collaterals are subsequently pruned—a process called stereotyped axon pruning. One of the most striking examples of stereotyped pruning in the CNS is the pruning of corticospinal tract (CST) axons. The long CST collaterals from layer V neurons of the visual and motor cortices are differentially pruned during development. Here we demonstrate that select plexins and neuropilins, which serve as coreceptors for semaphorins, are expressed in visual cortical neurons at the time when CST axon collaterals are stereotypically pruned. By analyzing mutant mice, we find that the pruning of visual, but not motor, CST axon collaterals depends on plexin-A3, plexin-A4, and neuropilin-2. Expression pattern study suggests that Sema3F is a candidate local cue for the pruning of visual CST axons. Using electron microscopic analysis, we also show that visual CST axon collaterals form synaptic contacts in the spinal cord before pruning and that the unpruned collaterals in adult mutant mice are unmyelinated and maintain their synaptic contacts. Our results indicate that the stereotyped pruning of the visual and motor CST axon collaterals is differentially regulated and that this specificity arises from the differential expression of plexin receptors in the cortex.

During development, the semaphorin family of guidance molecules is required for proper formation of the sympathetic nervous system. Plexins are receptors that mediate semaphorin signaling, but how plexins function during sympathetic development is not fully understood. Using phenotypic analyses of mutant mice in vivo, expression pattern studies, and in vitro assays, we show that plexin-A3 and plexin-A4 are essential for normal sympathetic development. This study confirms our previous in vitro findings that the two plexins differentially regulate the guidance of sympathetic axons. In addition, we find that semaphorin signaling through plexin-A3 and plexin-A4 restricts the migration of sympathetic neurons, but these two plexins function redundantly since migration defects are only observed in plexin-A3/-A4 double mutants. Surprisingly, our analysis also indicates that plexin-A3 and plexin-A4 are not required for guiding neural crest precursors prior to reaching the sympathetic anlagen. Immunoprecipitation studies suggest that these two plexins independently mediate secreted semaphorin signaling. Thus, plexin-A3 and plexin-A4 are expressed in newly-differentiated sympathetic neurons, but not their neural crest precursors. They function cooperatively to regulate the migration of sympathetic neurons and then differentially to guide the sympathetic axons.

Atherosclerotic lesions are characterized by lipid infiltration in regions with high rates of endothelial cell turnover. The present investigation was designed to elucidate the route of macromolecular transport across vascular endothelium. The aorta and vena cava of male Sprague-Dawley rats were perfusion-fixed after the intravenous injection of Evans-blue albumin (EBA) or horseradish peroxidase (HRP). Fluorescence microscopic examination of en face preparation of the aorta stained with hematoxylin allowed the identification of endothelial cells that underwent mitosis, together with the localization and quantification of fluorescent spots for EBA leakage. The HRP specimens were subjected to histochemical treatment, and HRP leakage was seen as brown spots under the light microscope. Silver nitrate stain was added in both EBA and HRP studies to outline cell boundaries and to visualize stigmata, stomata, and dead cells. In the aorta, almost every dividing cell showed junctional leakage to albumin and HRP, with clustering of leaky spots around the branch orifices. Time-dependent studies showed gradual increases in the diameter and number of these heterogeneously sized leaky spots, which finally fused to sizes corresponding to the "blue areas" for EBA or "brown areas" for HRP. Compared with arteries, veins had fewer mitotic cells, but more dead cells and diffuse dye-staining areas, indicating a more rapid transport of macromolecules. The leaky spots in the artery were associated mainly with mitotic cells, dead cells, and stigmata, whereas those in the vein occurred primarily at regions with dead cells. These results suggest that the preferential association of the enhanced transport of macromolecules with mitosis in the arterial as compared to venous endothelium and the differential behavior in transmural transport between arteries and veins may form the basis for the predilection of atherosclerosis in arteries.

The segregation and maintenance of eye-specific inputs in the dorsal lateral geniculate nucleus (dLGN) during early postnatal development requires the patterned spontaneous activity of retinal waves. In contrast to the development of the mouse, ferret eye-specific segregation is not complete at the start of stage III glutamatergic retinal waves, and the remaining overlap is limited to the C/C1 lamina of the dLGN. To investigate the role of patterned spontaneous activity in this late segregation, we disrupted retinal waves pharmacologically for 5 day windows from postnatal day (P) 10 to P25. Multi-electrode array recordings of the retina in vitro reveal that the cholinergic agonist epibatidine disrupts correlated retinal activity during stage III waves. Epibatidine also prevents the segregation of eye-specific inputs in vivo during that period. Our results reveal a novel role for cholinergic influence on stage III retinal waves as an instructive signal for the continued segregation of eye-specific inputs in the ferret dLGN.

Regressive events play a key role in modifying neural connectivity in early development. An important regressive event is the pruning of neuronal processes. Pruning is a strategy often used to selectively remove exuberant neuronal branches and connections in the immature nervous system to ensure the proper formation of functional circuitry. In the following review, we discuss our present understanding of the cellular and molecular mechanisms that regulate the pruning of axons during neuronal development as well as in neurological diseases. The evidence suggests that there are several similarities between the mechanisms that are involved in developmental axon pruning and axon elimination in disease. In summary, these findings provide researchers with a unique perspective on how developmental plasticity is achieved and how to develop strategies to treat complex neurological diseases.

Plexin signaling is required for stereotyped pruning of long axon collaterals in the vertebrate CNS; however, a cellular basis for plexins on stereotyped pruning has not been determined. Using quantitative electron microscopy and immunocytochemistry, we found that infrapyramidal mossy fiber axon collaterals form transient synaptic complexes with basal dendrites of CA3 pyramidal cells in the early postnatal mouse hippocampus. At later postnatal ages, these synaptic complexes stop maturing and are removed before stereotyped pruning by a mechanism that does not involve axon degeneration and glial cell engulfment. In knock-out mice that lack plexin-A3 signaling, the synaptic complexes continue to mature, and, as a result, the collaterals are not pruned. Thus, our results suggest that intact plexin-A3 signaling contributes to synaptic complex elimination, which is associated with stereotyped axon pruning.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that participate in both exocytic and endocytic vesicular transport pathways via mechanisms that are only partially understood. Although several ARF-like proteins (ARLs) are known, their biological functions remain unclear. To characterize its molecular properties, we cloned mouse and human ARL4 (mARL4 and hARL4) cDNA. The appearance of mouse ARL4 mRNA during embryonic development coincided temporally with the sequential formation of somites and the establishment of brain compartmentation. Using ARL4-specific antibody for immunofluorescence microscopy, we observed that endogenous mARL4 in cultured Sertoli and neuroblastoma cells was mainly concentrated in nuclei. When expressed in COS7 cells, ARL4-T34N mutant, predicted to exist with GDP bound, was concentrated in nucleoli. Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL4 interacted with importin-alpha through its C-terminal NLS region and that the interaction was not nucleotide-dependent. Like ARL2 and -3, recombinant hARL4 did not enhance cholera toxin-catalyzed auto-ADP-ribosylation. Its binding of guanosine 5'-O-(thiotriphosphate) was modified by phospholipid and detergent, and the N terminus of hARL4, like that of ARF, was myristoylated. Our findings suggest that ARL4, with its distinctive nuclear/nucleolar localization and pattern of developmental expression, may play a unique role(s) in neurogenesis and somitogenesis during embryonic development and in the early stages of spermatogenesis in adults.

Publisher Summary A large number of so-called orphan receptors have been identified that appear by their structure to be cell surface receptors for polypeptide ligands, yet have no known ligand. This has implied an opportunity to identify corresponding ligands that would be novel cell-cell signaling molecules. One strategy to identify such molecules has been to use the extracellular domain of the orphan receptor, fused to an alkaline phosphatase (AP) or immunoglobulin Fc tag that can serve as an affinity probe to identify the corresponding ligand(s). This approach is widely applicable, and has been used to identify a large number of novel ligands. An analogous strategy, using ligands fused to AP or Fc tags, has similarly been used to identify novel receptors. The chapter describes the use of such fusion proteins to identify and clone novel ligands or receptors. It also describes methods for the molecular characterization of ligands, receptors, and their interactions. An efficient procedure to identify a good source for cloning is to test embryonic or adult tissues by in situ staining, using a receptor or ligand-AP fusion protein as a probe to identify locations of high expression.

Mek4 and Sek are tyrosine kinases with expression patterns in the mouse embryo that suggest important functions in early development. However, like all Eph family kinases, both were identified as orphan receptors without known ligands. We show that Mek4 and Sek soluble receptor-alkaline phosphatase fusion proteins can be used in a procedure termed RAP in situ to identify regions of ligand expression in the mouse embryo. Based on this spatial information, a cDNA expression library was prepared, and was screened with the fusion proteins to identify Eph ligand family-1 (ELF-1). In cell lines and embryos, ELF-1 is membrane bound by a phosphatidylinositol tail, a feature that may account for unique biological functions. Its sequence is homologous with B61, a ligand for the Eck kinase, defining a family of related ligands. The expression domains of ELF-1, Mek4 and Sek indicate potential roles in embryonic patterning.

The kit ligand (KL) is one of several growth factors that are active as transmembrane molecules and can also be proteolytically cleaved to yield soluble forms. We have investigated the signals and structural determinants that control the cleavage of KL. Presentation at the membrane appears to be critical, because no cleavage occurs in variants that lack a transmembrane domain. Signals in the cytoplasmic domain do not seem to be required, because cleavage was not blocked by removal of the C-terminal valine residue, deletion of the whole cytoplasmic tail, or the replacement of the cytoplasmic tail that occurs in the Sl17H mutation. KL thus appears to differ from transforming growth factor-alpha, which apparently requires a C-terminal valine as a signal for cleavage. Although proteolysis must be tightly restricted to the correct cell surface proteins and sites within each protein, cleavage of KL does not seem to be determined entirely by a requirement for a specific substrate sequence. However, the effects of deletion or insertion variants of KL suggest that cleavage may be limited to sites within a specific range of distances from the cell membrane.