Axon fasciculation in the developing olfactory nerve
© Miller et al; licensee BioMed Central Ltd. 2010
Received: 21 May 2010
Accepted: 19 August 2010
Published: 19 August 2010
Olfactory sensory neuron (OSN) axons exit the olfactory epithelium (OE) and extend toward the olfactory bulb (OB) where they coalesce into glomeruli. Each OSN expresses only 1 of approximately 1,200 odor receptors (ORs). OSNs expressing the same OR are distributed in restricted zones of the OE. However, within a zone, the OSNs expressing a specific OR are not contiguous - distribution appears stochastic. Upon reaching the OB the OSN axons expressing the same OR reproducibly coalesce into two to three glomeruli. While ORs appear necessary for appropriate convergence of axons, a variety of adhesion associated molecules and activity-dependent mechanisms are also implicated. Recent data suggest pre-target OSN axon sorting may influence glomerular convergence. Here, using regional and OR-specific markers, we addressed the spatio-temporal properties associated with the onset of homotypic fasciculation in embryonic mice and assessed the degree to which subpopulations of axons remain segregated as they extend toward the nascent OB. We show that immediately upon crossing the basal lamina, axons uniformly turn sharply, usually at an approximately 90° angle toward the OB. Molecularly defined subpopulations of axons show evidence of spatial segregation within the nascent nerve by embryonic day 12, within 48 hours of the first OSN axons crossing the basal lamina, but at least 72 hours before synapse formation in the developing OB. Homotypic fasciculation of OSN axons expressing the same OR appears to be a hierarchical process. While regional segregation occurs in the mesenchyme, the final convergence of OR-specific subpopulations does not occur until the axons reach the inner nerve layer of the OB.
In the adult mouse olfactory system, there is a precise topographic organization between the olfactory epithelium (OE) and the olfactory bulb (OB). Regionally defined markers, such as olfactory cell adhesion molecule (OCAM), discriminate between olfactory sensory neuron (OSN) axons innervating the dorsal and ventral domains in the OB, while the final convergence of OSN axons into glomeruli reflects odor receptor (OR) expression [1–4]. However, the spatio-temporal correlates related to the segregation of subpopulations of OSN axons within the developing olfactory nerve remain unknown.
The initial development of the primary olfactory pathway, from the OE to the OB, begins at embryonic day (E)9 with the differentiation of neurons within the olfactory placode (OP). The first OSN axons that cross the basal lamina of the developing OE are seen at E10 to E10.5 ; at later ages these coalesce into fascicles surrounded by presumptive olfactory ensheathing cells. The OSN axons appear to follow a scaffold of migrating neurons that emerge from the OP beginning at E10. In what is collectively termed the migratory mass (MM), the OSN axons intermingle with these migrating neurons as they extend towards the presumptive OB. OSN axons first contact and penetrate the telencephalic vesicle on approximately E11 [5–7]. However, most OSN axons remain restricted to the presumptive olfactory nerve layer (pONL) until E15 when glomerulogenesis begins. This phase, the 'waiting period', is similar to that seen for thalamocortical axons in the subcortical plate and may contribute to the segregation of molecularly distinct subpopulations of OSN axons .
Each mouse OSN expresses only 1 of approximately 1,200 OR genes, and axons from OSNs expressing the same OR coalesce in two to three glomeruli per OB, typically one lateral and one medial glomerulus [9–12]. Consistent with a possible role in axon-axon interactions, ORs are expressed in OSN growth cones where they may contribute to ligand-induced Ca2+ transients [13, 14]. In the absence of an OR, axons fail to converge to the appropriate glomerulus [11, 12, 15] while substitution of a different OR partially redirects axon coalescence [15, 16]. Similarly, changes in functional activity and the expression of adhesion molecules can influence axon coalescence, independent of OR expression [17–19]. Thus, a consensus is emerging that OSN axon coalescence and/or targeting may be a hierarchical process in which multiple mechanisms determine regional positioning in the OB, while OR expression underlies the final convergence of axons within the inner nerve layer and the formation of glomeruli .
Recent work introduced the concept of pre-target axon sorting - a gross order and organization within an axon bundle prior to contacting the target - as a potential mechanism in OSN axon coalescence [1, 2], but there is continuing controversy about the application of this notion to the olfactory nerve, or the spatio-temporal correlates of axon sorting that would be necessary during early embryonic development.
Here, we show that segregation of subpopulations of axons within the developing olfactory nerve is evident by E12. Using both regional and OR-specific markers, we demonstrate that molecularly homotypic axons follow parallel courses through the mesenchyme, prior to the formation of the cribriform plate, and maintain a spatial congruency as they begin to establish a primitive nerve layer in the developing OB. Homotypic axon fasciculation appears to be a hierarchical process. While regional segregation is prominent within the olfactory nerve, convergence of OR-specific subpopulations of OSN axons, and the initiation of glomerulogenesis, do not occur until the axons cross into the inner nerve layer of the OB.
Materials and methods
Mice were used as follows: pregnant, time-mated CD-1 (Charles River, Wilmington, MA, USA), AC3 knockout heterozygous [19–21]; M72-GFP and P2-LacZ mice (gift from Dr Peter Mombaerts). The M72-GFP mice and the P2-lacZ mice were crossed to create a new heterozygous line containing constructs for both P2 and M72. For the genetically engineered lines, embryonic mice were derived from timed-pregnant females (day of the vaginal plug = E0), which were euthanized using CO2, followed by cervical dislocation. Embryonic and postnatal pups were swiftly decapitated and placed in 4% paraformaldehyde in phosphate-buffered saline (PBS; 0.1 M phosphate buffer and 0.9% NaCl, pH 7.4) at 4°C overnight. After fixation, tissue was washed in PBS overnight. Neonatal and embryonic tissue was cryoprotected by immersion in 30% sucrose in PBS at 4°C until the tissue sank, embedded in OCT compound (Tissue-Tek; Miles Laboratories Inc., Elkhart, IN, USA), and frozen in a slurry of dry ice and ethanol. Embryonic specimens were sagittally or coronally sectioned at 20 μm using a cryostat (Reichert-Jung 2800 Frigocut E). Sections were thaw mounted onto SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA), air-dried, and stored at -20°C until needed. All procedures undertaken in this study were approved by the Animal Care and Use Committees of Yale University and conformed to NIH guidelines.
Immunohistochemistry and confocal microscopy
DBA (biotin conjugated)
Gift from Y Yoshihara
WFA (fluorescein conjugated)
Gift from J Cloutier
Dk α Rb 555/488
Dk α Gt 488/555
Dk α Rt Cy3
Dk α Ch Cy3
Images were acquired with a Leica confocal microscope, using 20×, 40× or 63× objectives, the latter two oil-immersion. Digital images were collected from a single optical plane, approximately 1 μm thick. Digital images were color balanced using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA). The composition of the images was not altered in any way. Plates were constructed using Corel Draw 10.0 for the Macintosh (Corel, Ottawa, Ontario, Canada).
Serial 20-μm sections were imaged using an Olympus BX51 Epifluorescent Microscope. The olfactory nerve pathway was reconstructed from the serial sections using IMARIS software (Bitplane AG, St. Paul, MN, USA). Due to the thickness of the sections, the z-parameter needed to be artificially set so that the reconstruction appears continuous. Hence, the x- and y-axes are proportional, while the z-axis is set to maximize viewing of the images. When surfaces were rendered, the colocalization function was used to illustrate the expression patterns of neuropilin 1 (NRP-1) and neural cell adhesion molecule (NCAM).
Quantitative analyses were performed on timed-pregnant M72/P2 embryos at E16. Images were rotated so that axon fascicles were parallel to the longitudinal axis of the image. The distance across the entire fascicle was defined on the basis of NCAM staining. The width of each M72 or P2 fascicle was defined based on lacZ or GFP staining. The distance of the M72/P2 axons was defined as the outer point on the leftmost fascicle to the outer point of the rightmost fascicle. The distance was measured using the Measure and Label function in Image J.
Development of the olfactory nerve
The OE arises from the OP, a specialized epithelial thickening in the rostro-lateral aspect of the head. The OP is separated from the telencephalon by the frontonasal mesenchyme. The interactions between the OP and the mesenchyme are necessary for the proper development of the molecular and cellular diversity in the OE as well as the establishment of the axon trajectories that comprise the olfactory nerve . The adult olfactory nerve includes over approximately 1,200 subpopulations of axons, each representative of 1 from a candidate genome of 1,200 different ORs. Additionally, there are larger subpopulations of axons that express regional markers, including, but not limited to, NAD(P)H:quinone oxidoreductase 1 (NQO1), OCAM, NRP-1, Roundabout2 (Robo2), and various cell surface carbohydrates. While NRP-1, Robo2, and some cell surface carbohydrates have been implicated in axon targeting and the proper formation of the olfactory nerve, other molecules such as NQO1 and OCAM have not yet been shown to play a functional role related to axon targeting in the OB. Nonetheless, these markers are effective as tools for testing hypotheses regarding the molecularly homotypic convergence of subpopulations of OSN axons during the formation of the olfactory nerve.
Before assessing the behavior of subpopulations of axons as the olfactory nerve forms, it is critical to understand the initial developmental events that give rise to the primary afferent pathway. Cells first differentiate and express neuronal markers, such as β-tubulin III, within the OP at E9 (AM Miller et al., submitted). Subsequently, by E10, two different populations of neurons differentiate within the OP, the OSNs that will remain within the OE, and those neurons that will exit the OE and join the MM. The MM cells migrate out by E10, prior to the outgrowth of OSN axons, and coalesce to form a scaffold that extends toward the as yet undifferentiated basal telencephalon (AM Miller et al., submitted). Beginning at E10 to E10.5 in mice, the OSN axons extend across the basal lamina  and join the scaffold of MM cells, turning towards the telencephalon [23, 24]. Initially, the trajectories of MM cells and the OSN axons rely on a combination of guidance cues within the mesenchyme, axon-axon interactions, and telencephalic-derived chemotropic factors [25, 26]. By E11.5 the OSN axons contact the rostral-most tip of the telencephalon and innervate the presumptive OB  (AM Miller et al., submitted). The first synapses are detected in the OB at E14, but are not found in high numbers until E15 at the onset of glomerulogenesis [8, 27, 28].
Homotypic regional axon segregation begins in the mesenchyme
NQO1+ axons are topographically restricted to the rostral and ventral aspects of the nerve, whereas the OCAM+ axons track caudally and dorsally (Figure 2A, B). The expression pattern of NQO1 and OCAM did not vary significantly along the medial-lateral axis. Interestingly, a population of cells expressing NQO1 also appears in the nerve, primarily in the caudal aspect. These cells are a subset of MM cells (AM Miller et al., submitted). While the cells comprising the MM emerge from the OP as early as E10, there is no evidence of the NQO1+ population until E12.
Next, we assessed the topography of axons expressing NRP-1, the ligand-binding subunit for Semaphorin 3A [31–34]. NRP-1 is expressed by a subset of OSNs whose axons terminate in medial and lateral glomeruli . Unlike NQO1 and OCAM, which are expressed in distinct zones within the OE, NRP-1 does not conform to zonal patterning in the OE .
Axons expressing NRP-1 occupy a spatially distinct region in the nerve. Laterally, NRP-1 is most strongly expressed rostrally (Figure 2C, open arrow; see Additional file 1 for single channel localization), but medially, expression shifts caudally (Figure 2D; Additional file 1). A rostral-to-caudal transition zone can be recognized at the lateral-medial midline. To better visualize this segregation, we created a three-dimensional reconstruction of the olfactory nerve. In a lateral view, NRP-1 expression is visible in the rostral nerve and axon bundles (Figure 2E; Additional file 2). The axon fascicles targeting the central and caudal aspects of the nerve are primarily NRP-1-/NCAM+. Similarly, there is a region that is consistently NRP-1- (NCAM+; red) in the central-most aspect of the nerve (Figure 2E, asterisk; Additional file 2). NRP-1 also stains blood vessels, which follow a trajectory parallel to the axons extending up from the OE.
Next, we rendered surfaces onto the same three-dimensional reconstruction using the colocalization feature on IMARIS to assess the segregation between the NRP-1+/NCAM+ regions (yellow) and the NRP-1-/NCAM+ regions (red; Additional file 2). For clarity, the NRP-1+/NCAM- area, which stains the mesenchyme and blood vessels only, was not included in the reconstruction. In a medial view (Additional file 3A), axons expressing NRP-1 are seen traversing the nerve rostral-to-caudal to join the caudal aspect of the nerve where NRP-1+ axons are located. Laterally, as discussed above, there is a central/ventral portion of the nerve that remains NRP-1-. Medially, strong NRP-1 expression is seen rostrally. Laterally (Additional file 3B), NRP-1 expression is segregated to the rostral region of the nerve, and the associated fascicles. Generally, in the caudal region, there is an absence of NRP-1 expression. These segregation patterns are consistent with the confocal images displayed above, and reinforce the notion that NRP-1+ axons sort in a consistent, spatially defined manner.
Two plant lectins implicated in axon guidance, Dolichos biflorus agglutinin (DBA) and Wisteria floribunda agglutinin (WFA), bind to subpopulations of cell surface carbohydrates expressed by subsets of OSNs. In the adult mouse OB, DBA primarily labels dorsal glomeruli [36, 37]. WFA is expressed by E12.5, and at E15.5 is present in a subset of axons that terminate in the dorsal main OB .
In the nascent nerve, DBA labels a subset of axons in a dorsal patch midway across the olfactory nerve pathway (Figure 4B; open arrow). There is also diffuse DBA staining in the rostral region of the nerve, as well as a small patch that is strongly DBA+ in the rostral/dorsal part of the nerve (Figure 4B). The caudal aspect of the nerve has the lowest level of staining (Figure 4B). WFA expression was also strongest in a patch in the dorsal part of the nerve, midway across (Figure 4C; open arrow). Its pattern of expression appears similar to that of DBA. This was expected because early postnatal expression of DBA and WFA is overlapping (AMM and CAG, unpublished data). In the fascicles entering the nerve rostrally, colocalization or overlap of WFA+/NCAM+ axons is found. In the nerve itself, however, staining is strongest in the described dorsal patch, with lower levels caudally (Figure 4C).
Robo2, a well-established axon guidance molecule, has a graded pattern of expression in the OE, high dorsomedial and low ventrolateral . This regional pattern is also found in the OB, where Robo2 is most strongly expressed in the OSN axons that project to the dorsomedial aspect. Similarly, Robo2 is expressed in a gradient-like pattern in the olfactory nerve (Figure 4D). The gradient-like pattern is apparent going from the rostral-to-caudal aspect of the nerve. The caudal end is devoid of Robo2 staining (Figure 4D). Axon fascicles coming from the basal lamina to target the middle-to-caudal end of the nerve do not show distinct segregation, appearing orange (Figure 4D, open arrow). Robo2 staining also appears in the mitral cell and external plexiform layers of the OB (Figure 4D, asterisk). While these regional markers demonstrate unique segregation patterns, each also illustrates that axons coursing towards different regions in the OB are segregating within the mesenchyme. Because some of the markers we have used to assess axon sorting, such as NRP-1, do not shown evidence of zonal segregation within the OE, these data suggest that developmental pre-target OSN axon sorting within the mesenchyme is important in establishing the topography between the OE and OB.
Segregation of regional markers in the olfactory nerve pathway is altered in the absence of adenylyl cyclase 3
The expression patterns of NRP-1 in the AC3 KO mouse are significantly different. In the nerve layer of the OB, NRP-1 expression is very weak in the ventral axon fascicles contacting the OB (Figure 5B, B'; Additional file 4). However, the deeper layers of the OB, including the external plexiform layer, have strong lateral NRP-1 expression that is more prominent than in the heterozygotes (Figure 5A versus 5B; Additional file 4). Instead of exhibiting distinct segregation as the axons enter the nerve layer, NRP-1 expression diminishes, and only NCAM staining can be observed (Figure 5B'; Additional file 4). In the axon bundles, while low NRP-1 expression is evident by light orange bundles, it is notably less than that of the heterozygote (Figure 5B, B'; Additional file 4).
The expression patterns of DBA in the AC3 heterozygotes versus the homozygous KO mice provides a clearer picture of the altered spatial segregation of axons. As described earlier, DBA expression is typically restricted in a subset of axons in the most dorsal aspect of the OB [36, 37] (Figure 5C; Additional file 4). However, in the homozygous AC3 KO mice, DBA staining covered a broader area extending fully across the dorsal aspect of the developing OB, from midline medial to midline lateral (Figure 5D; Additional file 4). In summary, by examining both DBA and NRP-1 levels and expression patterns, we can conclude that the aberrant glomerular targeting in the AC3 mice is likely a manifestation of improper regional segregation within the developing olfactory nerve pathway that can be seen as early as E15, at the onset of glomerulogenesis.
Axon trajectory in OR-specific subpopulations of axons
Next, we turned our attention to OR-specific subpopulations of axons (M72 and P2) to determine how the smallest subsets of homotypic OSN axons behave during the development of the olfactory nerve. To study these OR-specific axon subpopulations, we used genetically engineered homozygous mice, P2-IRES-taulacZ and M72-IRES-tauGFP (kind gift from Dr P Mombaerts) and heterozygous P2-lacZ/M72-GFP mice that we bred to visualize the behavior of the M72+ and the P2+ axon subpopulations at E16, while the olfactory nerve is forming. While M72+ and P2+ OSNs can been seen earlier in these mice, we chose to look at E16 because this was the first age at which there were sufficient numbers of OSNs expressing each marker to assess the interaction and behavior between homotypic (M72:M72; P2:P2) and heterotypic (M72:P2) axons and fascicles.
Next, we analyzed heterozygous P2-lacZ/M72-GFP mice to assess the spatial segregation of specific OR-subpopulations expressed in different zones of the OE (P2 is in zone 2 and M72 in zone 1) and projecting to different regions of the OB (P2 axons to the caudal-ventral region of the medial and lateral OB; M72 axons to the caudal-dorsal region of the medial and lateral OB). Generally, the two OR-specific subpopulations remained segregated (Figure 8B, C), though there were instances when single P2+ and M72+ axons appeared closely apposed as they crossed paths (Figure 8A, A'). Isolated axons were also found occasionally in a regional domain defined primarily by the other OR subpopulation (Figure 8C, C').
Here, we: (1) demonstrate that pre-target axon sorting begins as early as embryonic day 12, less than 48 hours after the first OSNs axons have crossed the basal lamina; (2) show that regional sorting occurs as the axons travel through the lamina propria into the mesenchyme, and is clearly evident within the olfactory nerve pathway; (3) illustrate that while regional segregation occurs in the mesenchyme, ultimate coalescence of OR-specific subpopulations does not occur until the OSN axons cross into the inner nerve layer of the bulb; (4) suggest that in the absence of functional activity, pre-target axon sorting is perturbed, which could be the underlying mechanism for the glomerular mistargeting seen in the AC3 KO mice. Based on these data we propose that beginning at the earliest stages of olfactory development, a hierarchical model for homotypic fasciculation can be observed. Regional segregation occurs in the mesenchyme, but OR homotypic axons do not completely fasciculate until they reach the inner nerve layer of the OB.
Regional homotypic axon segregation occurs in the mesenchyme
At E10, two distinct populations of neurons differentiate within the olfactory placode; the OSNs and the MM cells. Following the migration of the MM cells across the basal lamina, the OSN axons begin to extend across the basal lamina where they come in contact with the MM cells and the ensheathing cells to form clusters that ultimately coalesce as the MM. When the OSN axons move through the lamina propria, into the mesenchyme, they sort according to their molecular phenotype. Clear regional segregation can be visualized with an assortment of markers, including NQO1/OCAM, NRP-1, Robo2, and plant lectins. While our understanding of the functional role of these molecules in the olfactory system remains unclear, several, including NRP-1, Robo2, and the lectins, have been implicated in axon targeting. Given our data, it seems plausible that these and other molecules influence the sorting of OSN axons as they cross the basal lamina. This suggests that the regional topographic organization of the OB is established in the mesenchyme.
Pre-target axon sorting is perturbed in the absence of AC3
Previous work suggested that altering cAMP expression severely affects axon convergence and glomerular formation [17, 19, 20]. Axons from OSNs expressing OR I7 exhibit targeting errors and aberrant glomerular formation when cAMP production is decreased. Of further relevance to the data reported here, the OR I7 axons typically label for NRP-1, but in AC3 KO mice, NRP-1 expression is greatly reduced in parallel with the perturbation of I7 axon targeting and glomerular formation . This suggests that the level of NRP-1 expression may be modulated by cAMP and is a determinant of OSN axon coalescence/fasciculation. To explore this hypothesis further, we examined axon fasciculation and the expression of regional markers during embryogenesis in the absence of AC3. Our data showed a clear loss of the distinct regional segregation that normally occurs within the mesenchyme, prior to the axons innervating the OB. While levels of NRP-1 were reduced, minimal expression was seen in the OB nerve layer and axon bundles, unlike the postnatal day 20 mouse . Our data therefore suggest strongly that the aberrant glomerular formation seen in older mice following down-regulation of cAMP is a function of perturbed regional sorting of axons within the embryonic mesenchyme, prior to axons reaching the nascent OB. Moreover, our data are consistent with the plausible hypothesis that one of the molecular mechanisms downstream of cAMP may be NRP-1 expression [2, 41].
Trajectory and organization of OR-specific subpopulations of axons in the developing nerve
We previously demonstrated with high resolution confocal and electron microscopy that the stable homotypic fasciculation of OSN axons expressing the same OR did not necessarily occur prior to the axons entering the inner nerve layer, proximal to the target glomerulus . In many instances axons 'followed tortuous or isolated trajectories before entering the appropriate glomerulus.' However, there was a stark discrepancy in the behavior of OR-specific OSN axons in the outer versus the inner olfactory nerve layer. In the former, most OR-specific axons coursed individually or in small fascicles. As axons entered the inner olfactory nerve layer, they formed larger homotypic fascicles immediately prior to coalescing into a glomerulus .
To extend these studies, we investigated the embryonic fasciculation of OR-specific subpopulations of OSN axons. Specifically, we asked if there was evidence of an OR-specific glomerular map within the embryonic mesenchyme. Genetically engineered P2-IRES-taulacZ and M72-IRES-tauGFP and heterozygous P2-lacZ/M72-GFP mice were processed for high-resolution confocal microscopy to examine the organization and fasciculation of OR-specific OSN axons while traversing the mesenchyme.
We found that as OSN axons exit the basal lamina they make a stereotypic approximately 90° turn towards the OB. As the OR-specific subpopulations course through the mesenchyme they are heterotypically fasciculated; M72-GFP axons do not necessarily appose each other and P2-tauLacZ axons do not necessarily appose each other. While in many instances two axons of the same OR subpopulation are in very close proximity, or in some cases apposed, the positioning is not stable. Overall, the rule is that multiple isolated axons, or small fascicles, traverse the mesenchyme following parallel paths within the same larger fascicle, but there is no precedent for them to be adjacent or apposed. This argues against the 'contextual model of self-sorting' proposed by Mombaerts  in which the OR, or an OR-containing complex, is responsible for homophilic and heterophilic interactions that promote the sorting of OSN axons into glomeruli. Were this the case, the adhesive forces/interactions between the ORs expressed in the growth cones and along the shaft of the axons would seem likely to promote homotypic fasciculation whenever the axons were in close proximity or in apposition. However, through the mesenchyme our data show that such interactions are transient, without evidence of stable homotypic fasciculation. It may be that the role of the OR is topographically defined and that the nature of the binding partners changes at the time of glomerular coalescence, much as the properties of guidance molecules can change from repulsive to attractive during development (for review see [44, 45]).
We have shown that within 48 hours of the first OSN axons crossing the basal lamina of the OE, there is regional, pre-target axon sorting occurring within the mesenchyme. Using a panel of well-established regional markers, we demonstrated that regional segregation occurs in a topographically restricted pattern and is activity-dependent. Interestingly, while the OR-specific subpopulations do show a clear pattern of regional segregation within the nerve, there is no evidence of homotypic OR fasciculation until the OSN axons reach the inner nerve layer of the OB.
Dolichos biflorus agglutinin
green fluorescent protein
neural cell adhesion molecule
olfactory cell adhesion molecule
olfactory sensory neuron
presumptive olfactory nerve layer
Wisteria floribunda agglutinin.
The authors express their appreciation to Ms Dolores Montoya and Ms Christine Kaliszewski for excellent technical support. We also thank the members of the Greer lab for their encouragement and many constructive discussions. We thank Dr Jean-Francois Cloutier and Jin Hyung Cho for providing us with the Robo2 antibody and for their technical assistance. This work has been supported in part by NIDCD DC00210, DC007880, and AG028054 to CAG; AMM was supported by the Yale MSTP Program (GM0720) and F30 DC010324.
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