© 2003 by The Society for Integrative and Comparative Biology
Viewing Cell Movements in the Developing Neuroendocrine Brain1
1 Colorado State University, Department of Biomedical Sciences, Fort Collins, Colorado 80523
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Many studies suggest that migratory guidance cues within the developing brain are diverse across many regions. To better understand the early development and differentiation of select brain regions, an in vitro method was developed using selected inbred and transgenic strains of embryonic mice. In particular, organotypic slices are used to test factors that influence the movements of neurons during brain development. Thick 250 µm slices cut on a vibrating microtome are prepared and maintained in vitro for 0–3 days. Nissl stain analyses often show a uniform distribution of cells in the regions of interest on the day of plating (embryonic days 12–15). After 3 days in vitro, cellular aggregation suggesting nuclear formation or the changing position of cells with a defined phenotype show that reasonably normal cell movements occur in several regions. Movements in vitro that mimic changes in vivo suggest that key factors reside locally within the plane of the slices. Video microscopy studies are used to follow the migration of fluorescently labeled cells in brain slices from mice maintained in serum-free media for 1 to 3 days. Transgenic mice with selective promoter driven expression of fluorescent proteins allow us to view specific cell types (e.g., neurons expressing gonadotropin-releasing hormone). The accessibility of an in vitro system that provides for relatively normal brain development over key brief windows of time allows for the testing of important mechanisms.
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INTRODUCTION |
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There is a growing recognition of disorders of neuronal migration with striking clinical consequences, and an appreciation for the molecular mechanisms that may underlie them (Gleeson and Walsh, 2000
). Many studies suggest that migratory guidance cues within the developing brain are diverse across many regions and multiple methods are needed to dissect key molecular mechanisms. In vitro slice preparations from mammalian embryos have provided powerful tools for studying the migratory behavior of cells in many regions of the developing nervous system. Experiments have shown behaviors that were not easy to predict a priori, particularly novel orientations and patterns of cell migration (e.g., Andersen et al., 1997a, b; Nadarajah et al., 2002). The ability to introduce specific reagents in vitro has provided for a determination of the role of calcium channels (Komuro and Rakic, 1992) and NMDA receptors (Komuro and Rakic, 1993) on cell migration in the cerebellum. Organotypic slices have long been used to study the developing hypothalamus from the perspective of neurite outgrowth (Toran-Allerand, 1976, 1980) and gene expression (Wray et al., 1989; Thomas et al., 1998), but not for examining cell migration. We adapted in vitro organotypic slice procedures to directly examine the movements of cells within parts of the brain associated with neuroendocrine functions; particularly the preoptic area (Tobet et al., 1994; Hendersen et al., 1999) and hypothalamus (Tobet et al., 1999; Dellovade et al., 2001; Davis et al., 2002a), and to study the migration of GnRH neurons across the developing mouse nasal septum and into and through the rostral forebrain (Tobet et al., 1996; Bless et al., 2000). The combination of in vitro studies with in vivo studies allows hypotheses to be tested in a way that ensures cross-validation of results.
The use of GFP to selectively label cell populations provides an additional strong technique for developmental studies of cell migration. Experiments using slice preparations have utilized fluorescent dyes such as DiI (e.g., O'Rourke et al., 1992), Oregon green (Nadarajah et al., 2001; 2002), and others (Alifragis et al., 2002) to label cells in vitro. The migration of cells in selected locations can be readily followed, but the chemical identity of the labeled cells is not as easily determined. The discovery of GFP in the jellyfish, Aequorea victoria, and its adaptation for transgenic and viral use, allows the targeted expression of a fluorescent marker that can be visualized in live cells (Chalfie et al., 1994; Marshall et al., 1995; Okada et al., 1999). GFP is a 238 amino acid protein that absorbs short wavelength light and emits green light (Chalfie et al., 1994). New mutated forms of GFP that are more intensely fluorescent provide more avenues for its use in vertebrate systems (Moriyoshi et al., 1996; Zernicke-Goetz et al., 1996). By combining GFP with promoters that drive expression in selected cell populations, movements and migration of identified cell populations can be visualized. We are currently utilizing 3 lines of transgenic mice in which specific neuronal promoters are used to drive either GFP (Suter et al., 2000; Stallings et al., 2002) or yellow fluorescent protein expression (YFP; Feng et al., 2000). Cells in the ventromedial nucleus of the hypothalamus (VMH) have been shown to selectively express steroidogenic factor-1 (SF-1; Ikeda et al., 1994; Shinoda et al., 1995; Roselli et al., 1997; Dellovade et al., 2000; Ikeda et al., 2001). The linkage of GFP downstream from a promoter that drives selective expression of SF-1 (Stallings et al., 2002) allows us to follow the migration of cells destined for the VMH (Davis et al., 2004). The mouse gonadotropin-releasing hormone (GnRH) gene promoter has been utilized by two groups to drive GFP expression in GnRH neurons (Spergel et al., 1999; Suter et al., 2000), and we have used mice from one of these lines (Suter et al., 2000) to examine GnRH neuron migration directly (Bless et al., 2002). Thy-1 is a cell surface glycoprotein (immunoglobulin superfamily) whose expression in the brain is restricted to neurons, and is spatially and temporally regulated during development leading to expression in the majority of neurons at maturity (Barlow and Huntley, 2000). The Thy-1 promoter has been used to drive YFP expression in a wide variety of neurons in adults (Feng et al., 2000), and we have used the same line of mice at embryonic ages to examine the movement of cells in the preoptic area and hypothalamus.
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MATERIALS AND METHODS |
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Animals
Timed pregnant mice of various strains were obtained from the animal facility at the Shriver Center of the University of Massachusetts Medical School. All mice were maintained in plastic cages with bedding (Sani-Chips, P.J. Murphy Inc. and Carefresh Total Care Bedding, Absorption Corp.) in a 14hr:10hr light: dark cycle (lights on 07:00) with Agway Prolab rat, mouse, hamster 2000 formula and tap water provided ad lib. Pregnant mice (day sperm plugs found = Day 0) were fed Lab Diet Mouse Diet (11% protein; PMI Feeds, Inc.). For some experiments, the mitotic marker bromodeoxyuridine (BrdU) is injected on specific days during gestation to determine the positions of neurons born on specific days at subsequent points in development (Miller and Nowakowski, 1988
). Pregnant mice are injected intraperitoneally with 25 mg/kg BrdU in 0.05 M PBS (pH 7.4). To obtain embryos, pregnant mice were anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg) and pups removed one at a time for slice preparations. Neonates on postnatal days 8–21 (P8, P12) were anesthetized using ketamine (80 mg/kg) and xylazine (8 mg/kg). Animal care was in full accordance with institutional guidelines.
In vitro studies: coverslip model
Heads or brains are dissected free from mouse embryos in cold Krebs buffer. After embedding in 8% low melting temperature agarose (Sigma Type VII-A), slices (250 µm thick) were cut using a vibrating microtome (Leica VT1000S) and placed into cold sterile-filtered Krebs buffer containing 0.01 M HEPES, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.1 mg/ml gentamicin. The plane of section is based on the model system studied and the expected orientation of migration. Thus, sagittal sections are cut to examine the migration of GnRH neurons from the nasal compartment into the brain (Tobet et al., 1996) and coronal sections are cut to examine the differentiation of nuclear groups in the forebrain (Henderson et al., 1999; Tobet et al., 1999). Alternate angles for cutting are used to confirm the predominant orientation of migration. After the final brain from a litter is cut (no longer than 2hr total time), the slices are left in the cold for an additional 15 min and then incubated for 35 min in MEM containing 10% fetal calf serum with 134 units/ ml penicillin, 0.13 mg/ml streptomycin, 1.34 mM glutamine, and 0.5% glucose for 35 min at 36°C in an incubator with 5% CO2. More recent experiments have shown that 10% fetal calf serum is not necessary in this step, and we currently use a medium that is 2% B-27 supplement with other factors kept constant. Per 100 ml, the medium is comprised of 94.3 ml DMEM F12 Phenol Red Free/ 2 ml B-27 supplement/ 1 ml glutamate/ 1.33 ml Pen/Strep/ 248 µl L-glutamine/ 1.1 ml D-glucose). Slices are then washed in the same media and placed on round coverslips that have been precoated (Table 1, adapted from Roberts et al., 1993) with poly-L-lysine and Vitrogen (Cohesion Technology, Inc.). We also have had recent success using 35 mm plastic dishes with preinserted glass bottoms (MatTek Corporation) that we also coat with Vitrogen. Excess media is removed and the sections are put back in the incubator. It is critical that the incubator be highly humidified (i.e., close as possible to 100%). After 1hr, the slices are covered with approximately 50 µl of a Vitrogen solution comprised of 1 ml Vitrogen, 125 µl 10x MEM, 23 µl pen-strep (10,000 units penicillin and 10 mg streptomycin per ml), and 33 µl of 1 M sodium carbonate. After another 90 min, serum-free medium is added to the slices (Neurobasal Medium with B27 supplement (GIBCO BRL Laboratories) and supplemented with 134 units/ml penicillin, 0.13 mg/ml streptomycin, 1.34 mM glutamine, and 0.5% glucose. Slices are kept at 36°C in an incubator with 5% CO2. After specific culture periods, some slices are fixed in 2% acrolein (from Sigma-Aldrich Co.; 90% stock) or 4% methanol-free formaldehdye (from Polysciences Inc.; 10% stock) for 15–30 min at room temperature and stored in 30% sucrose in 0.1 M PB for frozen sectioning on a sliding microtome at 50 µm for Nissl stain analyses and immunocytochemistry. Other slices were stored following fixation in 0.1 M PB for processing as whole slices in immunocytochemical procedures.
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Video microscopy and analysis
Slices are removed from the incubator and washed three times with sterile filtered 1X Krebs. The coverslip with the slice to be viewed is taken out of the 35 mm dish and placed in the center of a coverslip holder and locked in to insure viewing directly through the bottom of the coverslip. Krebs buffer or media is then pipetted over the slice and the holder is placed into a heated stage with 5% CO2, 95% balance air lightly passed over the top, and fresh media provided at the rate of 4 ml/h. Images are collected using a 20X plan achromat phase objective (0.75 n.a.) on a Nikon TE200 inverted microscope with a Dage RC300 video camera. A 4-D script was created for IPLab Spectrum software (Scanalytics, Inc.) that provided for automated shutter control and the collection of 3-D stacks of three images per time point (every 5 min) that provided a clearer image of each field. After video collection, phase images were taken from each slice to record the location of each field. For analysis, each image stack is analyzed as single frame by selecting the brightest pixel value from the stack for each x–y coordinate of the stack. These frames are then compiled into a video sequence. Video sequences are then adjusted for slice movement by matching non-drifting background objects that appear in each frame. Video sequences are analyzed for absolute distance and net distance traveled by each moving cell, the speed and top speed of each moving cell, and the percentage of migratory time of each moving cell. A moving cell is defined as an object that travels at least 1 cell diameter (about 12 µm) over the course of a video sequence. Absolute distance is calculated as the sum of distances traveled from frame to frame. Net distance is calculated as the distance between a cell's starting position (location in 1st frame) and ending position (location in last frame). Speed is calculated by dividing the duration of time that a cell was visible in a video sequence by the absolute distance traveled by that cell.
In vitro studies: Transwell model
Slices are treated identically as for coverslips through cutting, incubation at 4°C, and then at 36°C in media for 35 min. Slices then are gently laid on transwell membranes in 6 well dishes (Fisher, Catalog #07200169) with 2 ml of incubation media. After 2 or 3 days in vitro, media is removed and sections fixed as described above.
Immunocytochemistry
To detect specific immunoreactive antigens, sections or slices (at 4°C) are pretreated with 0.1 M glycine in 0.05 M PBS (pH 7.5; sections from perfusion fixed tissue only) followed by 0.5% sodium borohydride in 0.05 M PBS and 5% normal goat serum (NGS) with 0.3% Triton-X 100 (Tx)/PBS and 1% hydrogen peroxide. Washes with PBS separate each step and times are extended for 250 µm thick slices versus 50 µm thick sections. The tissue is then incubated with the designated antisera for 2–3 nights (in vivo sections) or 6 nights (in vitro slices). For secondary antibody processing, tissue was washed with PBS/1% NGS with 0.02% Triton-X100 prior to incubation with the appropriate biotinylated secondary antibody (Vector Laboratories) for 2 hr at room temperature (in vivo sections) or overnight at 4°C (in vitro slices). After washes in PBS/0.02% Triton-X100 (all at room temperature) tissue is then incubated with Vectastain ABC reagent (Vector Laboratories). Black reaction product is produced in 50 µm tissue sections using 0.25% 3,3'-diaminobenzidine (DAB, freshly dissolved in tris buffered saline and filtered) with 0.2% nickel ammonium sulfate and 0.02% hydrogen peroxide during a 5 min reaction period. Brown reaction product was produced in 250 µm slices using DAB in PBS without nickel. DAB was prepared without hydrogen peroxide for thick slices and hydrogen peroxide was added after a 15 min pre incubation, and reactions were allowed to proceed for 20 min.
All sections or slices are mounted onto gelatin-coated slides. The tissue sections from experiments where DAB is used for visualization are dehydrated and coverslipped using Permount. Fluorescent tissue sections or slices are coverslipped using Vectashield reagent (Vector Laboratories) or using an aqueous mounting media that dries following application (Accurate Chemical and Scientific Corp.).
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RESULTS AND DISCUSSION |
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Using our slice model, after 2 or 3 days in vitro there is significant evidence of cell rearrangements simply based on Nissl stain analyses. For example, in hypothalamic slices, cell groups that are not evident at the time of embryo harvesting and in vitro placement become evident after 2 to 3 days in vitro (e.g., Tobet et al., 1999
; Henderson et al., 1999; Fig. 1A–E). In each case, the rearrangements result in nuclear formation similar to that found over the comparable period in vivo. Formation of apparently normal cell groups in vitro in coronal sections from either the preoptic area (Henderson et al., 1999) or the medial basal hypothalamus (Tobet et al., 1999) suggests that key factors reside locally within the plane of the slices. This is notable in light of the fact that there are cell movements that are known to occur in the rostral caudal dimension (e.g., GnRH neurons, see below). Thus for the formation of at least one cell group in the preoptic area and for the VMH there may be minimal contributions from factors located rostral or caudal to the location of the nascent nuclei. This does not imply that there is no contribution from factors that influence tangential migration as there is significant evidence for migration in both regions that is not along the orientation of radial glial fibers, but rather more dorsal-ventral in orientation (Henderson et al., 1999; Dellovade et al., 2001).
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An interesting twist on movement relative to the orientation of radial glia fibers arises in the preoptic area where there are dueling orientations of radial glial fibers ventral to the anterior commissure (Tobet et al., 1995
; Henderson et al., 1999). One way to visualize the alternate migration of cells along these orthogonal planes is to view the changing positions of cells labeled with the mitotic indicator BrdU. In one experiment, pregnant mice were injected on E15 and slices were placed in vitro for 2–3 days. When these slices were processed for BrdU immunocytochemistry, labeled nuclei were noted in progressive lateral and ventral positions in the region of the preoptic area, consistent with migration of cells in both directions along the orientation of radial glial fibers (Fig. 2). Similarly, video microscopy studies showed migration in this same region along both dorsal-ventral and medial-lateral orientations (Henderson et al., 1999). Experiments using retroviral methods in developing chick brain in vivo to label clones of cells with a lacZ reporter also suggested that there is significant dorsal ventral migration in the hypothalamus (Arnold-Aldea and Cepko 1996; Golden et al., 1997). While each method has potential methodological caveats, the concordance of the results using the different methods lends promise to the conclusions reached.
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Following the changing position of immunocytochemically-identified cells indicates movements within slices maintained in vitro over several days. This is particularly true when the total number of cells can be counted and shown to be the same over time. Cells that contain the peptide GnRH are found in stereotyped positions in the nasal compartment and basal forebrain of embryonic mice. If slices are created on either E12 or E13, then after 1 to 3 days in vitro GnRH neurons are found in positions within sagittal slices that show the normal pattern of movement over the course of development in vivo (Tobet et al., 1996
; Bless et al., 2000). Importantly, the number of GnRH neurons detected after 1–3 days in vitro remains the same at approximately 400–600 neurons per slice. Interestingly, when this same type of experiment was carried out in rats, only 25% of the GnRH neuronal population could be accounted for in vitro after 1, 2 or 3 days (S.A.T., unpublished data). In explants from primate olfactory placodes, GnRH neurons become more detectable after one to three weeks in vitro (Terasawa et al., 1993). In explants from E11 mouse olfactory placode, typically only 25% or fewer of the GnRH neuronal population has been accounted for (Fueshko et al., 1998). For GnRH neurons, a problem is that the neurons can only be detected if they make sufficient GnRH to be detected; there are no independent markers. Thus when the cell number changes it is not determined whether the cells die or simply stop synthesizing peptide. This presents an additional problem in that the missing cells may comprise a subset that may have special properties that are not known.
We use video microscopy to follow the migration of fluorescently labeled cells in brain slices from mice maintained in serum-free media for 1 to 3 days. Our early studies utilized DiI to label cells randomly (Tobet et al., 1994; Henderson et al., 1999; Dellovade et al., 2001; Davis et al., 2002) and showed that cell movements in the hypothalamus were of approximately the same rates as those in other brain regions; between 15 and 30 microns/hr (O'Rourke et al., 1992; Komura and Rakic, 1992, 1993; Nadarajah et al., 2001). Our results provide a picture of the different orientations of movement that occur in different portions of the developing hypothalamus and preoptic area. It was a key finding in cortex that cell migration occurs not only radially from the ventricular zone toward the pial surface, but also tangentially (O'Rourke et al., 1992) from the ganglionic eminence to the cerebral cortex (Anderson et al., 1997b; Jimenez et al., 2002). Video microscopy is now revealing potential changes in the behavior or migrating cells such that neurons may switch from tangential migration to ventricle directed migration prior to a radial migration back out to the cortical plate (Nadarajah et al., 2002). In the hypothalamus and preoptic area there is significant tangential migration (Tobet et al., 1994; Henderson et al., 1999; Dellovade et al., 2001; Davis et al., 2002) in addition to the expected radial migration (Altman and Bayer, 1986). As noted above, tangential migration in hypothalamus has been also suggested by retroviral studies in chick brain (Arnold-Aldea and Cepko, 1996; Golden et al., 1997). We are currently taking advantage of animals that are transgenic for GFP under the control of selective promoters (e.g., the promoters for GnRH (Suter et al.,2000), SF-1 (Stallings et al., 2002), and Thy-1 (Feng et al., 2000). These mice offer the ability to better identify cells that are viewed compared to dye labeling. Furthermore, by labeling significantly larger populations of cells within each slice, we can view the movements of more cells per field than was previously possible. Our studies in the region of the VMH using DiI showed movements that were predominantly perpendicular to the orientation of glial guides close to the third ventricle, but parallel to the orientation of glial processes in the center of the presumptive VMH (Fig. 3). Due to the low level of labeling with DiI per field of view, this picture of movements took many slices over a substantial timeframe to accumulate the data. Using slices from Thy-1/YFP transgenic mice, a comprehensive image of all of the types of movement have been visible even within a single slices (Fig. 4).
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The accessibility of an in vitro system that provides for relatively normal brain development over key brief windows of time allows for the significant testing of important mechanisms. Early slice studies in the cerebellum tested the role of calcium channels and NMDA receptors (Komura and Rakic, 1992
, 1993). We began our studies by determining whether there might be sex differences in cell migration, and by examining the roles of GABA on cell movements in the developing hypothalamus and preoptic area. In the preoptic area we have shown sex differences in cell migration (Henderson et al., 1999), and preliminary data suggests that estradiol may influence the orientation of cell movement in the region (S.A.T., unpublished data). Our studies on the roles of GABA have concentrated on the migration of GnRH neurons from the nasal compartment into the basal forebrain, and on the formation of the VMH. Others have shown a role for GABA in cortical cell migration (Behar et al., 1996, 1998, 2000) acting through both GABAA and GABAB receptor mechanisms. It is clear that GABAergic signaling alters cell movements for both GnRH neurons (Bless et al., 2000; Heger et al., 2003) and cells in the region of the VMH (Dellovade et al., 2001; Davis et al., 2002). While GABAB receptors may play a significant role in the movement of VMH neurons (inhibiting movement; Davis et al., 2002), it is less likely that they play a role in the movement of GnRH neurons. We found no effects of GABAB agonist treatments (baclofen) on GnRH neuron positions in vivo or in vitro (Tobet et al., 2001).
A common feature of cell movements in all brain regions may be the necessity of neurons to detach from fibers that they use for migratory guidance. In the cerebral cortex, reelin has been hypothesized to help signal when migrating cortical neurons release their radial glial guides (Rice and Curran, 2001). For GnRH neurons, GABAergic signaling may help determine the point in the migration when GnRH neurons release their neuronophilic guide fibers that originate with vomeronasal neuroepithelial cells in the peripheral olfactory system. Treatment with the GABAA receptor antagonist bicuculline caused GnRH neurons to release from peripherin immunoreactive fibers earlier in their migratory path than normal. This caused an abnormal pattern of GnRH neuron system organization in the short term (Bless et al., 2000) and a long-term change in the position of GnRH neuron in the region of the organum vasculosum of the lamina terminalis in adulthood (S.A.T., unpublished data). The functional consequence of this was a significant 3-day delay in puberty as indicated by the day of vaginal opening for animals exposed to bicuculline during gestation. In a complementary experiment, transgenic over-expression of the GABA synthesis enzyme, glutamic acid decarboxylase, in a subset of GnRH neurons led to aberrant GnRH neuronal positions in development, and deficits in reproductive function in adult mice (Heger et al., 2003). In the region of the VMH, GABAA receptor agonist (muscimol) or antagonist (bicuculline) treatments in vitro caused cells to alter their orientation of movement relative to radial glial guides. Thus in 3 very different situations of migratory guidance, neuronophilic (GnRH) or gliophilic for laminar (cerebral cortex) or nuclear organization (VMH), the signals for release from guiding fibers may be critical determinants of final positions.
Much information is needed on the nature of cell movements that could result in the formation of nuclear groups, scattered cells with defined phenotypes, and well-ordered layers. Nuclear groups have irregular boundaries that must be discerned. Layers have regular boundaries. In the hypothalamus, many neuroendocrine cells are scattered across regions without clear boundaries, but with highly stereotyped cell distributions. In each case, the cues that determine final cell positions are critical determinants for producing a nervous system that can then be wired for appropriate connectivity. The use of in vitro slices that allow the rearrangement of cells following relatively normal patterns provides a powerful model system for studies that may help determine the cues that shape the developing neuroendocrine brain.
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ACKNOWLEDGMENTS |
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This work was supported by MH57748 and MH61376 (SAT) and HD33441 (GAS and SAT). Special thanks go to members of the lab that contributed to the development of the techniques described in this manuscript, including I. Hanna, T. Chickering, T. Dellovade, E. Bless, and A. Davis as well as key collaborators in adjacent laboratories including J. Crandall and particularly G. A. Schwarting.
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FOOTNOTES |
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1 From the Symposium Contemporary Approaches to Endocrine Signaling presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2003, at Toronto, Canada.
2 E-mail: stuart.tobet{at}colostate.edu
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