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The Journal of Neuroscience, June 1, 2000, 20(11):4129-4137
Granule Cells and Cerebellar Boundaries: Analysis of Unc5h3 Mutant Chimeras
Dan Goldowitz1, Kristin M. Hamre1, Stefan A. Przyborski2, and Susan L. Ackerman2 1 Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee 38163, and 2 The Jackson Laboratory, Bar Harbor, Maine 04609
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Mutations in the Unc5h3 gene, a receptor for the netrin 1 ligand, result in abnormal migrations of both Purkinje and granule cells to regions outside the cerebellum and of granule cells to regions within the cerebellum. Because both Purkinje and granule cells express this molecule, we sought to determine whether one or both of these cell types are the primary target of the mutation.
Chimeric mice were made between wild-type ROSA26 transgenic mouse embryos (whose cells express
-galactosidase) and Unc5h3 mutant embryos. The resulting chimeric brains exhibited a range of phenotypes. Chimeras that had a limited expression of the extracerebellar phenotype (movement of cerebellar cells into the colliculus and midbrain tegmentum) and the intracerebellar phenotype (migration of granule cells into white matter) had a normal-appearing cerebellum, whereas chimeras that had more ectopic cells had attenuated anterior cerebellar lobules. Furthermore, the colonization of colliculus and midbrain tegmentum by cerebellar cells was not equivalent in all chimeras, suggesting different origins for extracerebellar ectopias in these regions.
The granule cells of the extracerebellar ectopias were almost entirely derived from Unc5h3/Unc5h3 mutant embryos, whereas the ectopic Purkinje cells were a mixture of both mutant and wild-type cells. Intracerebellar ectopias in the chimera were composed exclusively of mutant granule cells. These findings demonstrate that both inside and outside the cerebellum, the granule cell is the key cell type to demarcate the boundaries of the cerebellum.
Key words: mouse; cerebellum; Purkinje cells; rostral cerebellar malformation; rcm; neuronal migration; neurological mutant mice
INTRODUCTION
The molecular dissection of compartments in the CNS has made impressive strides of late (Rubenstein et al., 1994
; Lee and Jessell, 1999
Recent studies of the mouse mutation rostral cerebellar malformation [rcm (Lane et al., 1992
Both granule cell neuroblasts and Purkinje cells express Unc5h3 during their egress from the primary germinal epithelium and subsequent development (Ackerman et al., 1997
We have used experimental murine chimeras to determine the cellular target of the Unc5h3 mutation as well as to explore how Unc5h3 regulates cerebellar development. By examining the genotype of cells in phenotypically mutant locations, we find that Purkinje cells cross cerebellar boundaries regardless of their genotype, whereas ectopic granule cells are almost completely of the mutant genotype. Thus, the granule cell neuroblast is the pioneer cell in establishing cerebellar boundaries.
MATERIALS AND METHODS
Mice and generation of experimental murine chimeras. Unc5h3 and ROSA26 mice were used from the colonies maintained at the Jackson Laboratory (Bar Harbor, ME). The Unc5h3 mutation (termed Unc5h3rcmTgN(Ucp)1.23Kz) arose at the Jackson Laboratory as an insertional event from a transgenic experiment (Ackerman et al., 1997
The Gtrosa26 (ROSA26) mouse was the wild-type component of the chimera, and the strain was used to mark cell genotype in chimeras. The ROSA26 mouse has a
Chimeric mice were generated in a manner that has been described previously (Goldowitz and Mullen, 1982a
Mice (>60 d of age) were intracardially perfused over a 20 min time period with room temperature saline and 4% paraformaldehyde fixative in 0.1 M phosphate buffer, pH 7.4. The brains were dissected out of the skull and placed in 0.1 M phosphate buffer, pH 7.4. Before sectioning, the brains were infiltrated with 30% sucrose in the same buffer to act as a cryoprotectant. The brains were sectioned in the sagittal plane on a cryostat at a thickness of 10-15 µm and mounted on Superfrost Plus slides.
Determination of Unc5h3 genotype in chimeric mice. Experimental murine chimeras were composed of either genetically +/Unc5h3 or Unc5h3/Unc5h3 cells. To determine the Unc5h3 genotype in each chimera, both phenotypic and expression analyses were performed. First, the brain phenotype was examined to determine whether ectopic cerebellar cells were present in the midbrain or brainstem. It was concluded that a genetically Unc5h3/Unc5h3 embryo contributed to the chimera if there were obvious populations of ectopic cells in these regions. Second, the expression of the Unc5h3 transcript was examined in Purkinje cells by in situ hybridization/autoradiography as described previously (Ackerman et al., 1997
Identification of granule, Purkinje, and radial glia cells in chimeras. Three major cerebellar cell types were examined in the present analysis. The cerebellar granule cell was identified by its characteristic pattern of condensed chromatin on the inside of the nuclear envelope within a small round nucleus (Goldowitz and Mullen, 1982b
Sections for immunohistochemistry were rinsed with PBS with 0.2% Triton X-100 (PBS/T), blocked with 2% nonfat dry milk, and incubated in the primary antibody overnight at room temperature. The following day, sections were rinsed, lightly fixed in 1% paraformaldehyde for 2 min, and processed for
Demonstration of cell genotype in chimeras using
To ensure that expression of the
Determination of percentage chimerism. The allocation of wild-type and mutant Purkinje and granule cells was analyzed by determining the percentage chimerism within normally and abnormally positioned locations. Percentage chimerism was defined as the percentage of cells within an individual population that are genotypically Unc5h3. Purkinje and granule cells were quantified within the cerebellum proper and in ectopic populations in the colliculus. The cerebellum proper was subdivided into the anterior and posterior lobes using the primary fissure as the demarcation point, whereas the ectopic collicular populations were subdivided into regions that were adjacent or distant to the cerebellum. To estimate the percentage chimerism in the Purkinje cell population, all calbindin-immunopositive Purkinje cells within a given subdivision were counted. Purkinje cells were determined as genotypically +/+ if they possessed several blue puncta of
The degree of ectopia within the midbrain (extracerebellar) and in the white matter (intracerebellar) was assessed and given a score between 1 and 5, where 1 was the phenotypically normal cerebellum and 5 was the phenotypically Unc5h3/Unc5h3 cerebellum. The scores were qualitatively estimated and based on the size of the ectopia (extracerebellar) and the number and size of ectopic clusters (intracerebellar).
Reconstruction of normal, mutant, and chimeric cerebella and determination of normal and ectopic cerebellar area. Quantitative measures of the normal and abnormal cerebellum were obtained by measuring the area occupied by normally positioned granule cells (in the internal granule layer), intracerebellar ectopic granule cells (granule cells in the white matter), and extracerebellar granule cells (in the midbrain tegmentum and inferior colliculus) in Unc5h3/Unc5h3 mutant chimeras, wild-type (+/Unc5h3
ROSA26) chimeras, and Unc5h3/Unc5h3 mutant mice. Sections from chimeric mutant, chimeric control, and mutant cerebella were assigned to bins (from 1 to 10) that were evenly spaced along the medial-lateral extent of the hemicerebellum. A representative section in each bin from each cerebellum was measured using a Neurolucida Imaging system attached to a Nikon microscope. Traces of whole cerebella were made using a 4× objective, and the granule cell territories were outlined using a 25× objective. The program calculated the area encompassed by the IGL, intracerebellar, and extracerebellar ectopic granule cells for each section.
RESULTS
Identification of Unc5h3/Unc5h3 chimeras
Mice homozygous for the Unc5h3 mutation are ataxic and have brain abnormalities, including ectopic granule and Purkinje cells in the midbrain and brainstem, and disruptions of the trilaminar structure of the cerebellar cortex (Lane et al., 1992
As detailed in Materials and Methods, the first criterion used to ascertain that chimeras were composed of Unc5h3/Unc5h3 cells was the presence of a mutant phenotype, similar to that found in Unc5h3 mutant mice. The principal mutant phenotype is the anterior invasion of the inferior colliculus and midbrain tegmentum by cerebellar cells (Figs. 1B, 2A). In addition to these extracerebellar ectopias, Unc5h3 mice have abnormalities in cell position within the cerebellum (Fig. 1B). Boundaries between the white matter and the IGL are often blurred by the presence of granule cells in the white matter. Additionally, small areas of the IGL are devoid of granule cells and overlying Purkinje cells. These granule cell "divots" are typically found in proximity to granule cell ectopias in the white matter (Figs. 1B, 4A). The other obvious abnormality in Unc5h3 mutant mice is a reduction in the size of the cerebellum, primarily attributable to a loss of anterior cerebellar structures (Fig. 1A,B; Table 1). Thus, a chimeric brain that exhibited any or all of the above abnormalities met the first criterion for assigning the non-wild-type genotype as Unc5h3/Unc5h3.
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Figure 1. Chimeric cerebella exhibit a range of phenotypes. Hematoxylin and eosin-stained parasagittal sections through the superior cerebellar peduncle are shown from (A) an Unc5h3/+ control, (B) an Unc5h3/Unc5h3 animal, and (C-E) three Unc5h3/Unc5h3
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Table 1. Area (in µm2) occupied by granule cells in the cerebellum proper and ectopically in the midbrain tegmentum and colliculus (extracerebellar) and within the cerebellar white matter (intracerebellar) in mutant control (Unc5h3/Unc5h3;ROSA26), chimera control (+/+ The second criterion used to confirm the presence of Unc5h3/Unc5h3 cells was the presence of Purkinje cells that lacked Unc5h3 expression as detected by autoradiographic in situ hybridization. The number of silver grains over Purkinje cells was scored by an investigator (S.A.P.) naive to the results of the first criterion. In all cases in which the Unc5h3 component was genotyped as Unc5h3/Unc5h3 according to criterion one, the Purkinje cell grain counts supported this assignment, i.e., there were numerous unlabeled Purkinje cells (data not shown).
Using these criteria, we identified 5 Unc5h3/Unc5h3
Chimeric Unc5h3/Unc5h3 cerebella exhibited a range of phenotypes
Although the Unc5h3/Unc5h3 cerebellum lacked the anterior lobules of the mediolateral cerebellum (Fig. 1B), formation of these lobules in the five Unc5h3/Unc5h3 chimeras was variable (Fig. 1C-E). For example, chimera 1 had a normal-appearing pattern of lobulation (Fig. 1C), whereas chimeras 4 and 5 had attenuated anterior cerebellar lobules (Fig. 1D,E).
The size of the extracerebellar ectopia was also variable in chimeric mice. In larger ectopias, cerebellar cells were more organized and recapitulated typical cerebellar morphology with the appearance of granule cell, Purkinje cell, and molecular layers (Fig. 1E). In lateral regions of the brain, large ectopias formed folia-like structures (Fig. 2B,C). These ectopias were composed of numerous streams and pockets of cells that extended anteriorly as far as the border between the inferior and superior colliculi in the dorsal mesencephalon and to the interpeduncular fossa in the ventral mesencephalon. The middle cerebellar peduncle appeared to be a caudal boundary to these ventrally located, ectopic cerebellar neurons. In the larger ectopias, the ventralmost ectopic neurons formed a cell-dense cap over the interpeduncular fossa, with sparse streams of cells that connected to the other larger mass of ectopic cells that colonize the inferior colliculus (IC) (Fig. 2D, arrowheads). In other chimeric brains, the ectopic cells did not extend as far or colonize as much territory as these larger ectopias (Fig. 2C).
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Figure 2. The inferior colliculus and midbrain tegmentum are differentially colonized by Unc5h3 mutant cells in the chimeric brain. Parasagittal sections through the lateral cerebellum of (A) an Unc5h3/Unc5h3 mouse and (B-D) three Unc5h3/Unc5h3
The two masses of ectopic cerebellar neurons, one that formed in the colliculus and the other that formed in the midbrain tegmentum, appeared to have different origins. Two chimeras (4 and 3) exhibited reciprocal patterns of cerebellar ectopias. In chimera 4 there were large numbers of ectopic cells in the inferior colliculus, whereas a more limited set of ectopic cells existed in the midbrain tegmentum (Fig. 2C). Conversely, chimera 3 had fewer ectopic cells in the inferior colliculus but a large ectopia in the tegmentum (Fig. 2D). A third, chimera 1, had only a small dorsal ectopia in the inferior colliculus, with no ectopic cells in the ventral midbrain tegmentum (Fig. 1C).
Intracerebellar ectopias, composed of granule cells within the white matter of the cerebellum proper, were also found in the chimeric cerebella although, they were less frequent than those found in nonchimeric Unc5h3/Unc5h3 mice (Fig. 1, compare black arrows in B with those in D and E; Tables 1, 2). The brain of chimera 1, which is composed of mainly wild-type cells, had a small but distinct extracerebellar ectopia, whereas it was difficult to distinguish any clear intracerebellar ectopias (Fig. 1C). This finding suggests that a much larger percentage of genotypically mutant cells is needed for the formation of these ectopias. Alternatively, the Unc5h3 gene may play more of a role in controlling the rostral-caudal boundaries of the cerebellum and be less important in the establishment of the deep boundary of the IGL.
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Table 2. Phenotype and percentage chimerisma in Unc5h3/Unc5h3 A second type of intracerebellar abnormality in the Unc5h3/Unc5h3 mouse was the presence of acellular regions or divots in the IGL of the lateral cerebellum (Fig. 1B, asterisks). These divots occurred much less frequently than white matter ectopias but were always associated with the abnormal presence of granule cells in the white matter. In chimeras, these divots were also found associated with white matter ectopias. The occurrence of this sort of abnormality was proportional to the extent of the other mutant phenotypes found in each chimera (data not shown).
To quantify these differences, we analyzed areas of normally and ectopically placed cerebellar granule cells (Table 1). The medial cerebellum provided the most marked contrast in normal cerebellar area when comparing the Unc5h3/Unc5h3 mutant and normal cerebellum. On the other hand, most of the cerebellar ectopias in the Unc5h3/Unc5h3 mutant mouse were in the lateral cerebellum. Thus, measures were obtained from the appropriate regions to examine these phenotypes in three chimeric cerebella (1, 3, and 5). As shown in Table 1, chimeric cerebella had a range of mutant phenotypes that were intermediate between the Unc5h3/Unc5h3 mutant and wild-type controls.
The ROSA26 marking system and chimeric cerebella
The validity of the ROSA26 marker for distinguishing genotypically mutant and nonmutant cells in chimeric brains was established in control tissue. Previous reports have indicated that cells from the ROSA26 lineage can be identified by the presence of
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Figure 3. The Unc5h3 mutation acts in granule cells and not Purkinje cells. Parasagittal sections of the cerebellum and adjoining inferior colliculus are shown from chimera 2 (A-C) and chimera 5 (D-H). The boundary between normal cerebellum (to the right) and the extracerebellar ectopia (to the left) is denoted by longer black arrows in A, B, D, E, and I. Wild-type (ROSA26) cells are labeled with the blue
To establish a baseline for the analysis of mutant chimera brains, we examined the general distribution of cells from the Unc5h3 and ROSA26 lineages in +/Unc5h3
Ectopic Purkinje cells were a mixture of mutant and wild-type genotypes
Like Unc5h3/Unc5h3 mice, Purkinje cells, as marked by calbindin immunopositivity, were present in the inferior colliculus and midbrain tegmentum of chimeric mice. In the inferior colliculus these cells were always present with neighboring granule cells. However, at the farthest extent of the ectopic expansion, ectopic granule cells were found without accompanying Purkinje cells. In fact there was the impression that a threshold number of granule cells needed to be present in the collicular ectopia before Purkinje cells colonized that area. In contrast, rare isolated sets of Purkinje cells could be found without accompanying granule cells in the midbrain tegmentum of chimeras. These Purkinje cells were never at the leading edge of the ectopia, suggesting that these cells did not pioneer the formation of the ectopia.
As shown in Figure 3, C and F, both genotypically ROSA26 Purkinje cells and genotypically Unc5h3/Unc5h3 Purkinje cells were found within the inferior colliculus. There was no preferential localization of the two genotypes of Purkinje cells along the extent of the ectopia. Similar percentages of Unc5h3/Unc5h3 Purkinje cells in the cerebellum proper and ectopic inferior colliculus were found, indicating that mutant and wild-type Purkinje cells had an equal ability to cross the boundary into extracerebellar territory (Table 2). A similar distribution of wild-type and mutant Purkinje cells was also found in the midbrain tegmentum ectopias of chimeras (data not shown).
The vast majority of ectopic granule cells were of the Unc5h3/Unc5h3 genotype
Granule cells were typically the only cerebellar cell type at the farthest reaches of extracerebellar ectopias in chimeric brains, which indicates, as previously suggested by studies of Unc5h3 mutant embryos (Przyborski et al., 1998
Intracerebellar granule cell ectopias were composed almost completely of genotypically Unc5h3/Unc5h3 cells (Fig. 4C-F), and divots occurred in the midst of folia among predominantly wild-type granule cells (Fig. 5A). It is interesting to note that the wild-type cells do not migrate laterally to fill the divot that is presumably created by the migration of Unc5h3/Unc5h3 granule cells into the white matter.
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Figure 4. The Unc5h3 mutation acts intrinsic to intracerebellar granule cells for IGL boundary formation. Shown are parasagittal sections through lateral cerebella of an Unc5h3/Unc5h3 animal (A, B), chimera 5 (C, D), and chimera 2 (E, F). The posterior cerebellar region is shown. The arrows in A, C, and E denote the region shown at higher magnification in B, D, and F, respectively. The white, double-sided arrows (B, D, and F) mark the boundary between the IGL and the white matter. Sections in A and B are stained with hematoxylin and eosin. In C-F, all cells are stained with neutral red, and wild-type (ROSA26) cells are labeled with the blue
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Figure 5. Both genotypically mutant and wild-type radial glial cells are found in extracerebellar ectopias. Parasagittal sections through chimera 5 (A) and chimera 1 (B) were immunostained with an anti-GFAP antibody to label glial cells and counterstained with neutral red. Wild-type cells have one or more blue dots after the
Ectopic radial glia were a mixture of mutant and wild-type genotypes
An anti-GFAP antibody was used to identify glial cell bodies and their processes. In the normal cerebellum, radial glia processes span the distance between the Purkinje cell layer and the pial surface. In contrast, the subpial glial population of the native inferior colliculus is oriented parallel to the pial surface. The morphology of the glia in ectopic regions of chimeric brains was variable, depending on the degree of colonization by cerebellar cells. In chimeras with less organized and less granule cell-populated collicular ectopias, the glial fibers were thickened and lacked clear radial processes (Fig. 5B). However, in the colliculus of chimeras with greater contributions of ectopic cells, the arrangement and morphology of glial processes were similar to that seen in the normal cerebellum (Fig. 5A). The determination of whether the glial cells are genotypically mutant or wild type relies on the double-staining with both
DISCUSSION
The granule cell is the pioneer neuron in establishing cerebellar boundaries
The molecular mechanisms by which neurons commence and cease their migration are beginning to be understood through the analysis of naturally occurring and induced mutations of the mouse genome. The Unc5h3 gene appears to be a key player in cerebellar neuronal migration, as demonstrated by the phenotype of mice with mutations in this gene.
One of the key insights that the Unc5h3 mutant reveals is the role of cells and molecules in determining the regional boundaries that are specific to the cerebellum. What are the means by which the rostral and ventral boundaries of the cerebellum are established? Both Purkinje cells and granule cells express the Unc5h3 gene, and therefore either one or both cell types could be responsible for demarcating the cerebellar territory. Eisenman and Brothers (1998)
Based on two findings from the analysis of Unc5h3/Unc5h3
/
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Figure 6. Schematic representation of the contribution of genotypically mutant (gray) and genotypically wild-type (black) granule cells (circles) and Purkinje cells (P) in Unc5h3/Unc5h3
We did find a small population of wild-type granule cells that participated in the formation of the extracerebellar ectopias. At least two possible explanations exist for this finding. First, ectopic Purkinje cells of either genotype may attract a small number of EGL cells (some of which are wild type in origin) into the ectopic stream. Second, these ectopic, wild-type EGL cells may be passively displaced within the stream of ectopic mutant cells. Whatever the mechanism underlying the presence of wild-type granule cells in these ectopias, it must be tempered with our results that these cells are only found a short distance past the normal cerebellar-collicular boundary and never extend into the more distal aspects of the ectopia.
Decision points in the migration of mitotically active EGL cells
Granule cell precursors are generated from a specialized portion of the ventricular epithelium, the germinal trigone. These precursors initially migrate in a dorsal direction and subsequently anteriorly and medially over the surface of the cerebellum to form the EGL (Miale and Sidman, 1961
If the ectopic cells in Unc5h3 chimeras arose from misreading a signal at a single origin, then the degree of the ectopias in the chimeric brainstem and colliculus should be dependent solely on the degree of chimerism. In other words, the number of mutant granule cells present at that decision point would determine the extent of ectopias in both the midbrain tegmentum and the colliculus. However, we find that chimeras can exhibit opposite phenotypes with respect to the ectopic colonization of brainstem and colliculus (Fig. 2). Thus the opposing phenotypes seen in chimeras implicate the presence of two separable decision points in the migratory history of the granule cell.
It seems most likely that the granule cells responsible for reading the stop signals comprise the anterior cerebellar compartment. In the normal cerebellum, the first granule cell neuroblasts that leave the germinal trigone are those destined for the anterior-medial lobules (Goldowitz, 1989
A decision point in the migration of postmitotic granule cells
In restricted locations within the Unc5h3/Unc5h3 mutant cerebellum, the boundary between the white matter and the IGL is indistinct, with many granule cells found in the white matter directly below the IGL. This raises the interesting and as yet unanswered question: how are granule cells constrained within the IGL? The present study provides two interesting observations about these intracerebellar ectopias. First, in the Unc5h3/Unc5h3
The mechanism by which these intracerebellar ectopias arise is unclear. White matter ectopias may be created by an exuberant migration of granule cells past the IGL/white matter border. However, within the disrupted Unc5h3/Unc5h3 cerebellum, a distinct IGL can be identified in many areas, demonstrating that only a small percentage of the granule cells are affected. Thus, although the presence of UNC5H3 is obviously one of the important factors that normally constrains granule cells within the IGL, clearly other factors also mediate this function.
Ligands for UNC5H3
UNC5H3 has been demonstrated to bind the secreted protein netrin 1 (Leonardo et al., 1997
There are several possible explanations for the lack of an Unc5h3-like phenotype in Ntn1
The presence of genotypically Unc5h3/Unc5h3 granule cells in the cerebellar white matter of Unc5h3/Unc5h3
Concluding remarks
Our chimeric analysis of the Unc5h3 mutation indicates that single cerebellar cells (members of the granule cell population) read a stop signal to format the ventral and anterior boundaries of the developing cerebellum. Furthermore, the formation of the internal granular layer also relies on the ability of granule cells to read a molecular stop signal, although this molecule may be different from the one that sets the anterior and ventral cerebellar boundaries. More generally, the Unc5h3 mutant mouse presents a fascinating mutation that highlights the possibility that a host of as yet undiscovered molecules (receptors and their ligands) serve as stop signals for migration throughout the CNS.
FOOTNOTES Received Dec. 10, 1999; revised March 2, 2000; accepted March 10, 2000.
This work was supported by grants from National Institutes of Health (NS35900) to S.L.A. and a CORE grant (CA34196) from the National Cancer Institute and The University of Tennessee College of Medicine and Department of Anatomy and Neurobiology. We thank Richard Cushing for technical assistance, Greg Martin and Justin Boyd for assistance in imaging, and Drs. Tom Gridley and Timothy O'Brien for their comments on this manuscript.
Correspondence should be addressed to Dr. Dan Goldowitz, Department of Anatomy and Neurobiology, University of Tennessee, Memphis, 875 Monroe Avenue, Memphis, TN 38163. E-mail: dgold{at}nb.utmem.edu.
Dr. Przyborski's current address: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, United Kingdom S10 2TN.
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