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Mice that lack astrotactin have slowed neuronal migration

¹ Laboratory of Developmental Neurobiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA
² Medizinische Fakultät, Neurologische Universitätsklinik, Hoppe-Seyler-Straße 3, D-72076 Tübingen, Germany

View larger version (49K): [in a new window]   Fig. 1. Targeting strategy of astrotactin locus. (A) Wild-type astrotactin locus (WTL): black rectangles represent exons. The black lines on either side represent the 5' and 3' external probes used to detect homologous recombination by Southern blot. Astrotactin targeting vector (TV): a 168 bp exon containing the translation initiation site (ATG) was designed to be replaced with a pgk1-neo cassette. pgk1-neo and HSV1-tk were used as positive and negative selection markers, respectively. (B) Targeted locus after homologous recombination (ML): BglII digests liberate 8.0 kb and 6.5 kb fragments from the wild-type and targeted alleles, respectively. PstI digests liberate 9.5 kb and 7.8 kb fragments from the wild-type and targeted alleles. SpeI digests give 10.0 kb for wild-type allele and 8.4 kb for the mutant allele. (C) Western blot analysis indicates that this mutant produces no astrotactin protein in astrotactin null animals.

View larger version (82K): [in a new window]   Fig. 2. In vitro and in vivo migration assays show that granule cells migrate more slowly in mice lacking astrotactin. (A) In vitro migration assay. The majority of wild-type granule cells migrate faster than 10 µm/h (gray) whereas the majority of granule cells from astrotactin null mice migrate more slowly than 10 µm/h. (B-G) Midline sagittal sections of cerebella from mice after varying survival times after intraperitoneal injection of BrdU. Brown peroxidase product shows cells that have taken up BrdU, sections were counterstained with Hematoxylin. In all figures, the EGL is at the top and is marked yellow, the IGL is marked by a blue bar. Six hours after BrdU injection there are similar numbers of heavily labeled cells in wild type (B) and astrotactin null (C) EGL. After 24 hours survival there are more labeled cells in the molecular layer and IGL of wild-type (D) cerebella than in astrotactin null mice (E). Forty-eight hours after injection, there are many labeled cells in the IGL and heavily labeled cells in the EGL of wild-type mice (F), whereas there are still a substantial number in the EGL and fewer in the IGL of the mutant mice (G). Scale bars: 30 µm.

View larger version (127K): [in a new window]   Fig. 3. Morphology of granule cells in vitro (A-D), in vivo (E-H) and glia (I,J). (A) Brightfield micrograph of wild type granule cells in a migration culture. Note the elongated cell profiles as granule cells migrate along glial fibers. The same culture following Tuj-1 immunohistochemistry is seen in B. (C) An equivalent migration culture with astrotactin null granule cells. Cell profiles are more rounded and are not as closely associated with the glial process. Tuj-1 stain of the same culture reveals that the rounded profiles are differentiated neurons. (E-J) Sagittal sections through cerebellum with EGL at top of figure. (E) Wild-type P6 Nissl stained section showing normal morphology of migrating granule cells in the molecular layer. Note that cells are ‘tear drop’ in shape (arrows). (F) Section showing the more rounded granule cells (arrows) in the molecular layer of P6 astrotactin null mice. (G) Nissl section of a P8 wild-type mouse with elongated migrating profiles in the molecular layer above the much larger Purkinje cells. (H) Section from an astrotactin null P8 mouse showing rounded granule cell profiles in the EGL (arrow). (I,J) Bergmann glia labeled with GFAP in normal and mutant mice. Scale bars: 30 µm.

View larger version (21K): [in a new window]   Fig. 4. (A) Assay to assess binding of granule cells to glial substrates. Asterisks indicate significant differences (P<0.01) in the ability of wild-type and mutant cells to adhere to glia. (B) Comparisons of the relative area occupied by granule cells in the cerebella of P6, P15 and adult normal and mutant mice (*P<0.01). (C) Comparison of the relative number (corrected for areas observed) of TUNEL-positive cells in the EGL and IGL of normal and mutant P6 mice (*P<0.05).

View larger version (103K): [in a new window]   Fig. 5. Morphological changes to the cerebellum of astrotactin null mice. Nissl stained sections of normal (A) and mutant P15 mice (B) reveal that the dispersion of the EGL is delayed in astrotactin null mice. Note also the presence of pyknotic nuclei in B (arrowhead). (C) Nissl stained parasagittal section from a P15 astrotactin null mouse showing an ectopic clump of granule cells in the molecular layer (arrow). (D) Nissl stained parasagittal section from a P15 astrotactin null mouse showing Purkinje cells with abnormal morphology (arrowhead) adjacent to Purkinje cells that appear normal. Note how the abnormal cells are oriented out of the plane of the section. (E-H) Confocal images of double immunohistochemistry showing glia (GFAP staining in green) and Purkinje cells (calbindin staining in red) on parasagittal sections. (E) Wild-type adult, showing normal Purkinje cell morphology. In F,G, the dendrites of Purkinje cells from astrotactin null mice are seen to spread across into territories normally occupied by neighboring cells. Note that the columnar organization (revealed by the GFAP staining) of the cerebellum is otherwise normal. (H) Ectopic Purkinje cells in a section cut from a P19 astrotactin null mouse. Scale bars: 30 µm (A,B,D,E-H); 60 µm (C).

View larger version (28K): [in a new window]   Fig. 6. Histogram showing the performance of wild type and astrotactin null mice in a Rota-rod behavioral assay. Note the consistent significantly (P<0.01) lower performances of astrotactin null mice in all exercises, except on the final day of the acceleration test (#) when compared with age- and size-matched wild-type mice. Batch 1 refers to the first set tests for each mouse and Batch 2 refers to the second test for each mouse.