Neuropilin2 regulates the guidance of post-crossing spinal commissural axons in a subtype-specific manner
© Tran et al.; licensee BioMed Central Ltd. 2013
Received: 19 April 2013
Accepted: 19 July 2013
Published: 31 July 2013
Spinal commissural axons represent a model system for deciphering the molecular logic that regulates the guidance of midline-crossing axons in the developing central nervous system (CNS). Whether the same or specific sets of guidance signals control the navigation of molecularly distinct subtypes of these axons remains an open and largely unexplored question. Although it is well established that post-crossing commissural axons alter their responsiveness to midline-associated guidance cues, our understanding of the repulsive mechanisms that drive the post-crossing segments of these axons away from the midline and whether the underlying guidance systems operate in a commissural axon subtype-specific manner, remains fragmentary at best.
Here, we utilize axonally targeted transgenic reporter mice to visualize genetically distinct dorsal interneuron (dI)1 and dI4 commissural axons and show that the repulsive class 3 semaphorin (Sema3) guidance receptor Neuropilin 2 (Npn2), is selectively expressed on the dI1 population and is required for the guidance of post-crossing dI1, but not dI4, axons. Consistent with these observations, the midline-associated Npn2 ligands, Sema3F and Sema3B, promote the collapse of dI1, but not dI4, axon-associated growth cones in vitro. We also identify, for the first time, a discrete GABAergic population of ventral commissural neurons/axons in the embryonic mouse spinal cord that expresses Npn2, and show that Npn2 is required for the proper guidance of their post-crossing axons.
Together, our findings indicate that Npn2 is selectively expressed in distinct populations of commissural neurons in both the dorsal and ventral spinal cord, and suggest that Sema3-Npn2 signaling regulates the guidance of post-crossing commissural axons in a population-specific manner.
KeywordsSemaphorins Atoh1 Neurog2 Development Spinal cord
Central nervous system
Class 3 semaphorin
Distinct populations of commissural neurons are widely distributed along the dorsoventral (D-V) and mediolateral (M-L) axes of the developing vertebrate spinal cord, and can be distinguished by their morphology, cell body position, gene expression patterns and axonal trajectories [1–6]. Although all commissural neurons extend axons across the floor plate (FP), an intermediate target at the ventral midline of the spinal cord, whether the same guidance signals control the pathfinding of each subtype towards (pre-crossing) and away (post-crossing) from the floor plate remains a largely unexplored issue.
The basic-helix-loop-helix (bHLH) transcription factors Atoh1, Neurog1 and Neurog2 define specific neuronal progenitor populations that give rise to genetically distinct dI1, dI2 and dI4 dorsal commissural neurons in the embryonic mouse spinal cord [7–9]. Enhancer elements derived from these bHLH factors direct reporter expression to distinct populations of commissural axons as they project toward, across and beyond the FP [10–16]. An antibody specific for GAD65, a rate-limiting enzyme required for GABA synthesis, labels ventral commissural neurons in the embryonic rat spinal cord [17, 18]. Together, these markers provide tools for investigating molecular mechanisms that control the guidance of dorsal and ventral commissural axon subtypes.
Post-crossing commissural axons must lose and gain responsiveness to midline attractants and repellents, respectively, in order to successfully project away from the FP . Repulsive signaling resulting from interactions between Robo receptors on post-crossing commissural axons and their Slit ligands on FP cells prevents multiple populations of commissural axons from re-crossing, and lingering at, the FP [16, 19–22]. Inhibitory interactions between the Npn2 receptor and the ventral midline-associated Sema3s, Sema3B and Sema3F [23–25], facilitate the switch in responsiveness exhibited by post-crossing commissural axons [26–29]. However, it remains to be determined whether Sema3-Npn2 interactions regulate the pathfinding of all or only specific subsets of commissural axons.
In this study, we utilize transgenic reporter mouse lines that selectively label dI1 or dI4 dorsal commissural axons and anti-mouse GAD65 as a marker for ventral commissural axons to assess the role(s) of Sema3-Npn2 signaling in the pathfinding of these distinct axon populations. Whereas the Atoh1-tauGFP reporter has previously been shown to specifically label dI1 neurons [11, 15], here, we demonstrate that a novel transgenic mouse line, Neurog2-tauGFP, targets GFP to a subset of the Neurog2 expressing progenitors in the dorsal neural tube that give rise to dI4 commissural neurons. We find that dI1, but not dI4, commissural axons express Npn2, and require this receptor for navigating on the contralateral side of the ventral midline and for Sema3-mediated collapse of their growth cones in vitro. We also show, for the first time, that a GABAergic population of ventral commissural axons is present in the embryonic mouse spinal cord and that Npn2 regulates the guidance of their contralateral projections. Together, these findings indicate that Npn2 regulates the pathfinding of contralateral commissural projections in a subtype-specific manner.
Atoh1 and Neurog2 enhancers drive GFP expression in spinal dI1 and dI4 commissural neurons, respectively, and dI1, but not dI4, neurons express Npn2
Pre-crossing and post-crossing segments of dI1 and dI4 commissural axons can be separately visualized by confocal microscopy
The guidance of post-crossing, but not pre-crossing, dI1 axons is perturbed in Neuropilin 2 (Npn2) mutant spinal cords
Our observation that Npn2 is expressed on both pre- and post-crossing segments of dI1 axons (see Figure 2A) raised the possibility that this Sema3 receptor is required for these axons to navigate to, across, and/or beyond the ventral midline in the embryonic mouse spinal cord. Further, our finding that anti-Npn2 does not label most dI4 axons (see Figure 2B) suggests that Npn2 is selectively required for the guidance of dI1 axons. In order to test these possibilities, we used the visualization strategy described above to assess the consequences of inactivating Npn2 or its ligands, Sema3F/Sema3B, on the pathfinding of pre- and post-crossing dI1 and dI4 axons. Specifically, we separately crossed the Atoh1-tauGFP and Neurog2-tauGFP reporter lines with Npn2, Sema3F or Sema3B knockout mice and utilized confocal microscopy to selectively visualize pre- and post-crossing dI1 or dI4 axons in these mutant mice.
Post-crossing dI1, but not dI4, axon-associated growth cones collapse in the presence of Sema3B, Sema3F or Slit2, in vitro
Ventral spinal commissural neurons and their axons express GAD65
Neuropilin 2 is required for the guidance of post-, but not pre-, crossing GABAergic ventral commissural axons
Labeling strategies for visualizing discrete populations of commissural neurons in the developing mouse spinal cord
By exploiting unilateral in ovo electroporation in the embryonic chick spinal cord, we previously showed that Atoh1 and Neurog1 enhancer elements direct reporter expression to distinct classes of dorsal commissural and characterized the trajectories of their pre- and post-crossing axons [16, 31]. Here, we used enhancer elements to generate Atoh1-tauGFP and Neurog2-tauGFP reporter mice and to visualize the trajectories adopted by dI1 and dI4 commissural axons, respectively, in the embryonic mouse spinal cord. Although pre-crossing dI4 axons projected to the FP along a more lateral route than their dI1 counterparts, confocal microscopy facilitated the optical separation of planes containing pre- and post-crossing axon segments, revealing that a large number of both dI1 and dI4 post-crossing axons extend away from the FP along diagonal trajectories. Supporting the validity of these observations, the post-crossing projections of mouse dI1 axonal subtypes closely resembled those displayed by their chick counterparts . We also used an anti-mouse GAD65 antibody to identify, for the first time, a ventral population of commissural neurons that extends post-crossing axons into the both the ventral and lateral funiculus of the mouse spinal cord. These particular findings are consistent with our previous observations in the embryonic rat spinal cord [17, 18, 36, 37].
In principle, the labeling strategies that we describe here should make it possible to assess and compare the consequences of inactivating any receptor-ligand system or other factor(s) on the guidance of dorsal and ventral commissural axons in the embryonic mouse spinal cord. The roles of a given guidance molecule in directing the growth of dorsal and ventral commissural axons could be investigated in the same embryos by labeling spinal cord preparations derived from dI1 or dI4 (dorsal populations) reporter mice, which have been labeled with anti GAD65 (ventral population) and crossed with mice deficient in the candidate gene. Alternatively, dI1 or dI4 reporter mice could be mated with GAD65 reporter animals (see ) - with a dorsal reporter line harboring a GFP reporter and the GAD65 line carrying a mCherry reporter, or vice versa - and the dual-labeled mice crossed with a knockout line of interest. In addition to evaluating the consequences of inactivating putative guidance factors on the pathfinding of dorsal and ventral commissural neurons within the spinal cord proper, it should also be possible to assess the effects of these perturbations on the long-distance projections of these axons to their brain targets utilizing the labeling strategies described herein. Alternatively, these particular analyses could be carried out by delivering dI1, dI4 and GAD65 reporter constructs into the embryonic mouse spinal cord via unilateral in utero electroporation (see [31, 39]). Based on our previous findings in chick  and rat  embryos, we expect that at least a subset of mouse dI1 and dI4 commissural axons will project within spinocerebellar tracts, whereas a significant number of GAD65-positive mouse GABAergic commissural axons will assemble into spinomesencephalic tracts. Given that the GAD65 neurons we describe in this study are born slightly before the neurons that compose most spinal ascending axon tracts (see ), we further suggest that the GAD65-positive neurons, in particular, likely represent pioneer neurons, which contribute axons to some of these tracts.
Compensatory roles of Sema3B and Sema3F in guiding post-crossing Atoh1 (dI1) commissural axons
Using Atoh1-tauGFP reporter mice, we show that dI1 post-crossing commissural axons require Npn2 for their contralateral trajectory away from the floor plate. In addition, we show that both Sema3B and Sema3F promote robust collapse of post-crossing dI1 axon-associated growth cones. However, neither the Sema3B nor the Sema3F single knockouts phenocopy the Npn2 mutant mice. Thus, our results raise the possibility that Sema3B and Sema3F have compensatory or redundant roles in mediating post-crossing Npn2-positive commissural axon guidance. Interestingly, Slit ligands have been shown to operate in a collaborative manner to regulate midline crossing of commissural axons in both the spinal cord and retina [20, 22, 41]. To determine whether Sema3B and Sema3F have redundant roles in driving post-crossing dI1 projections away from the FP we would need to assess axon pathfinding in Sema3B and Sema3F double knockout spinal cords. However, the close association between the Sema3B and Sema3F loci, which are located less than 0.072 mega base pairs away from each other on the same chromosome, precludes the execution of these experiments. Thus, additional experiments, beyond the scope of this study, are required to definitively determine whether Sema3B and Sema3F operate in concert to guide post-crossing Npn2-positive axons away from the FP.
Neuropilin 2 selectively regulates the guidance of a subset of post-crossing commissural axons
Utilizing dI1 and dI4 reporter mice and anti-GAD65 immunohistochemistry we assess here, for the first time, the consequences of inactivating Npn2 on the pathfinding of distinct subsets of dorsal and ventral commissural axons. Consistent with our observation that Npn2 is expressed on dI1 and GAD65-positive, but not dI4, commissural axons, we show that Npn2 is selectively required for the contralateral pathfinding of dI1 and GABAergic ventral commissural axons. Notably, the inactivation of Npn2 more profoundly disrupts the guidance of dorsal as opposed to ventral commissural axons; in Npn2 null embryos most post-crossing dI1 axons fail to project away from the FP along diagonal trajectories, whereas GAD65-postiive post-crossing exhibit rather minor de-fasciculation defects within the ventral funiculus. It is interesting to note in this regard that the midline attractant, Netrin-1, has been shown to preferentially guide dorsal as opposed to ventral spinal commissural axons . Despite the fact that post-crossing dI4 axons also project away from the FP along diagonal trajectories, which are similar in shape to post-crossing dI1 projections, these axons pathfind normally in mice lacking Npn2. As indicated above, this is consistent with our finding that dI4 neurons/axons do not express Npn2. Interestingly, Robo2 is required for driving most dorsal post-crossing axons away from the ventral midline . Accordingly, it is possible that Robo2 has a major role in directing Npn2-negative dI4 commissural axons away from the FP along diagonal trajectories. Together, our observations support the view that Npn2 regulates commissural axon guidance in a population-specific manner and raise the possibility that Atoh1 or another transcription factor expressed by post-mitotic dI1 neurons directly regulates the expression of Npn2. However, our preliminary studies indicate that Npn2 expression is unaltered in mice lacking Atoh1 (EC and ZK, unpublished observation) and Npn2 was not identified in a systematic screen for direct lineage-specific in vivo targets of Atoh1.
We have previously shown that as a consequence of disabling Slit-Robo signaling in chick or mouse embryos post-crossing dI1 and dI2 axons fail to project diagonally away from the FP [16, 31]. These phenotypes closely resemble the contralateral pathfinding defects displayed by dI1 axons in mice lacking Npn2, raising the possibility that Robo and Npn receptors functionally and/or physically interact to regulate commissural axon pathfinding. Notably, Robo-Npn2 interactions facilitate cortical interneuron migration within the embryonic mouse forebrain . Alternatively, repulsive Robo receptors and Npn2 may separately regulate the guidance of heretofore-unidentified subsets of dI1 axons. We do not favor this possibility since the dI1 axons disrupted as a result of disabling Robo signaling in electroporated chick embryos, and those perturbed following inactivation of Npn2 in transgenic reporter mouse embryos, were visualized utilizing the same Atoh1 enhancer elements. Given that dI1 axons likely contribute to the spinocerebellar tract and GABAergic ventral commissural axons presumably assemble into spinomesencephalic tracts (see above), it would be interesting to determine, in future studies, whether inactivation of Npn2 disrupts the formation of these longitudinally projecting ascending axonal tracts. If the loss of Npn2 perturbs dI1 axons from forming a spinocerebellar tract in mouse embryos, this would be consistent with the targeting phenotype we observed in chick embryos following the disruption of Slit-Robo signaling , and represent another functional parallel between the roles of Robo and Npn receptors in commissural axon pathfinding.
Consistent with commissural axons gaining responsiveness to midline repellents only after they cross the FP, it was previously shown that the growth of cultured post-, but not pre-, crossing commissural axons are responsive to midline-associated Sema3s . Complementing and extending this observation by identifying a subset of dorsal commissural axons that gain responsiveness to midline inhibitory cues, we show here that Sema3B/3F selectively promotes the collapse of post-crossing dI1 (but not dI4) commissural axon-associated growth cones. Given that we find Npn2 expressed on both pre- and post-crossing segments of dI1 axons it is not clear why growth cones associated with post-crossing axons are selectively responsive to midline Semas, but the underlying mechanism could involve axon segment-specific receptor processing [27, 29] or silencing . It is well established that Npns form holoreceptor complexes with class A Plexins in order to mediate repulsion [25, 46, 47]. Accordingly, commissural axon-associated PlexinA1  is a good candidate for facilitating the response of post-crossing dI1 growth cones to Sema3B/3F and this possibility can be addressed by analyzing the consequences of inactivating Plexin A1 in Atoh1-tauGFP reporter mice.
Numerous molecularly distinct subsets of commissural neurons are distributed throughout the vertebrate spinal cord (see Background). Despite this well-established diversity, whether the same set of guidance receptors-ligands controls the pathfinding of all midline-crossing commissural axon populations or whether the directed growth of each population is regulated by a particular subset of these guidance systems, as has elegantly been shown for spinal motor axons [48, 49], remains a key open question in the field. In large part, the lack of robust and reproducible labeling systems that can be used to reliably visualize, and assess the consequences of a given perturbation on, distinct classes of commissural axons has precluded population-specific analyses of commissural axon pathfinding. Here, we utilize novel genetic labeling strategies and immunohistochemistry to elucidate the distinct axonal trajectories of genetically specific populations of commissural neurons along the dorsoventral spinal cord. In addition, we show that the dI1, not the dI4, population of commissural neurons derived from Atoh1 progenitors, and a subset of the GABAergic ventral commissural neurons express the Npn2 receptor on all segments of their axons. However, only the contralateral/post-crossing segments of dI1, Atoh1-GFP labeled axons respond to Sema3-mediated repulsion. Taken together our findings show that Sema3-Npn2 signaling is required for the pathfinding of distinct subtypes of contralateral commissural axons in the developing mouse spinal cord.
All mice were maintained on a C57BL/6 background and at least three backcrosses were performed for each line. For timed pregnancies, embryonic day 0.5 (E0.5) was considered to be noon on the day the vaginal plug was observed. The Neurog2-tauGFP transgenic mice were generated using the TgN2-7tauGFP transgene according to standard procedures by the University of Texas Southwestern Medical Center Transgenic Facility. The transgene contains a 1 kb enhancer (chr3:127337663 to 127338713 from mouse build mm9) from the 3’ end of the Neurog2 gene that has been mutated such that, in combination with the β-globin basal promoter, it directs reporter expression to a subset of Neurog2-expressing progenitors within the dorsal neural tube in transgenic mice . The tauGFP cassette is as published for Atoh1tauGFP. The Npn2 and Sema3F mutant mice were maintained as previously described . The Sema3B mutant mouse line was purchased from the Jackson Laboratory (strain #006705) and maintained according to the instructions on their website. In all cases, genotyping was performed using the PCR and DNA samples generated from mouse ear or tail tissue biopsies. Pregnant dams were exposed to compressed carbon dioxide and sacrificed by cervical dislocation and the embryos were removed by cesarean section. The Institutional Animal Care and Use Committees of the Albert Einstein College of Medicine and Rutgers University collectively approved the animal-use protocols.
Timed-pregnant mice were sacrificed at a given embryonic day and the embryos were harvested in cold PBS. Embryos were then immersion-fixed in cold 4% Paraformaldehyde for 2 to 4 h and equilibrated in a 30% sucrose solution overnight, before being embedded in Tissue-Tek OCT compound (Miles Scientific, Elkhart, IN, USA) and frozen at −80°C. Embryos were cryosectioned at 16 or 20 μm using a Leica Cryostat and the sections were mounted on Superfrost Plus microscope slides (Fisher Scientific International) and allowed to air dry for 16 h at room temperature.
Immunohistochemical labeling of the sections was performed essentially as described [51, 52]. A goat polyclonal antibody specific for Npn2 (cat. no. AF567; R&D Systems, Minneapolis, MN, USA) was used at 15 μg/ml, and a rabbit monoclonal anti-GAD2/GAD65 was used at 1:1000 (cat. no. 5843, Cell Signaling, Danvers, MA, USA) was used at 1:1000, with each antibody diluted in 10% donkey serum and 0.1% Triton X-100 and applied to the sections after blocking in the same buffer. Appropriate secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA).
Analyses of open-book spinal cord preparations
Open-book preparations of embryonic spinal cords were generated from the various reporter mice in a given genetic background as described . To visualize the projections of the resident GFP-labeled axons, the open-book preparations were flat-mounted and imaged using an Olympus Fluoview 500 confocal microscope. Image stacks/Z series were analyzed using Metamorph Imaging Software (Universal Imaging, Inc.). To analyze the relative density of GFP-labeled axons contained within a particular portion of these preparations, the images were set to threshold using the auto-threshold function and the number of GFP-positive pixels was counted. Comparable levels of the rostral-caudal axis of the spinal cord were analyzed in each set of experiments, and the thresholding of the images was kept consistent between all image sets. Data was represented as an average of the area containing fluorescence over the threshold value. Data sets between mutant and control groups were then compared using a Student’s-T test.
In vitro collapse assay
Large open-book preparations were dissected from E11.5 mouse embryos of various genotypes in ice-cold, Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) according to previous published methods [33, 34]. For pre-crossing axon containing explants, tungsten needles were used to isolate floor plate-lacking dorsal spinal cord tissue, which contains cell bodies of commissural neurons. In contrast, we isolated floor-plate-attached commissural neuron containing half spinal cord explants as sources of post-crossing axons. Dorsal spinal cord preparations (pre-crossing axons) were supplemented with 250 ng/ml recombinant Netrin-1 (cat. no. 1109-N1-025; R&D Systems, Minneapolis, MN, USA) to promote axon outgrowth. Both dorsal spinal cord (pre-crossing axons) and FP-attached preparations (post-crossing axons) were sectioned into small, approximately square, pieces and these explants were placed at 37°C with minimal media on nitric acid cleaned, Silanized, and Laminin-coated cover slips (mouse, Invitrogen cat# 23017, Carlsbad, CA, USA) in Hanks Balanced Salt Solution (HBSS, Gibco, cat#14170-088, Carlsbad, CA, USA), as described  for at least 2 h to allow adequate time for the tissues to adhere to the cover slips. The explants were then cultured for either 48 h in DMEM media with 1% penicillin/streptomycin/glutamine (Gibco-BRL, Carlsbad, CA, USA), and 1% Bottenstein's N2 supplement (Gibco-BRL, Carlsbad, CA, USA). To induce collapse, media was collected from HEK-293 cells transfected  with mammalian expression vectors containing the coding regions of Slit-2 (gift from Y Rao, National Institute of Biological Sciences) or Sema3F/Sema3B (gifts from A Kolodkin, The Johns Hopkins University). These conditioned media or control medium (from mock-transfected cells) were added to the explant cultures at a dilution of 1:100, and the explants were incubated for an additional hour at 37°C. The culture media was then removed and the explants were fixed for 10 m in pre-warmed 4% PFA with 10% sucrose. After fixation, the explants were washed with PBS and stained with AlexaFluor 568-phalloidin (cat. no. A12380; Molecular Probes, Eugene, OR, USA) and anti-GFP, AlexaFluor 488 conjugate (cat. No. A-21311; Molecular Probes, Eugene, OR, USA). The tips of axons displaying prominently spread growth cones containing lamellipodia and multiple filopodia (visualized by Phalloidin labeling) were scored as non-collapsed, whereas those lacking lamellipodia and multiple filopodia were scored as collapsed.
Photodocumentation and data analyses
All epifluorescence images were captured using a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) and all confocal images were obtained with either a Fluoview 500 microscope (Olympus, Tokyo, Japan) or a Yukogawa CSU10 confocal system, and processed with ImageJ64 (National Institutes of Health, Bethesda, MD, USA). Brightness and contrast of images were adjusted using Adobe Photoshop CS (Adobe, San Jose, CA, USA). All data analyses were carried out using the statistical tests indicated in the Figure Legends and GraphPad Prism (Version 5.0d).
Tracy S Tran and Edward Carlin are co-authors.
This work was supported by a grant from the New Jersey Commission on Spinal Cord Research (CSCR11IRG011) to TST and ZK, and grants from the National Institutes of Health: NINDS to ZK (R01NS038505), NICHD to JEJ (R01HD037932). We thank the following investigators for mice and reagents: Alex Kolodkin, Marc Tessier-Lavigne, and Yi Rao. We also thank Kostantin Dobrenis for assistance with confocal microscopy, Matthew Gavin for help with quantification, and Angela Jevince for assistance with figure preparation. We are grateful to Carol Mason, Hannes Bülow, Jean Hébert, Alex L. Kolodkin, Patricia E. Phelps and Wilma Friedman for providing critical and insightful comments on the manuscript.
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