Figure 1.
Generation of Aldoc-Venus knock-in mice and Venus expression in their brain.
A, Schematic representations of Aldoc genome, targeting vector and targeted genome. Black boxes indicate exons. Black boxes indicate the probes for Southern blot analysis. Semicircles indicate FRT sequences. Met, initial methionine; DT, diphtheria toxin gene; Venus, Venus gene; pgk-gb2neo, neomycin-resistant gene expression cassette; Sc, ScaI; EV, EcoRV. B, PCR genotyping of mice tail DNA detects 383 bp band from the wile-type allele and 1169 bp band from the targeted allele. C-H, Bright field illumination (C–E) and epifluorescence (F–H) photomicrographs of the whole brain of adult littermates (75-day old) bred from heterozygous parents (C and F, wild type; D and G, heterozygote; E and H, homozygote). I–K. Epifluorescence photomicrographs of sagittal sections of the brain of a heterozygote. Scale bar in K applies to I–K. Abbreviations for anatomical terms in this and following figures: 3V, third ventricle; 4V, forth ventricle; I–VI, layer I–VI; I–X, lobules I–X; a–c, sublobules a–c (as in VIa); ac, anterior commissure; AIN, anterior interposed nucleus; Amy, amygdala; APT, anterior pretectal nucleus; beta, subnucleus beta; C, caudal; cc, corpus callosum; Ce, cerebellum; Cop, copula pyramidis; CPu, caudate and putamen; Cr I, crus I of the ansiform lobule; CF, climbing fiber; Cr II, crus II of the ansiform lobule; Cx, cerebral cortex; D, dorsal; DAO, dorsal accessory olive; DC, dorsal cap of Kooy; DCo, dorsal cochlea nucleus; DH, dorsal horn; DLH, dorsolateral hump (of the AIN); DLP, dorsolateral protuberance (of the MN); EPl, external plexiform layer (olfactory bulb); fi, fimbria of hippocampus; Fl, flocculus; GCL, ganglion cell layer; gl, granule cell layer (cerebellum); GP, globus pallidus; GrO, granular cell layer of olfactory bulb; Hip, hippocampus; Hy, hypothalamus; ic, internal capsule; IC, inferior colliculus; INL, inner nuclear layer (retina); IO, inferior olive; IPl, internal plexiform layer (olfactory bulb); L, lateral; LMol, lacunosum moleculare layer; LN, lateral nucleus; Lt, left; LV, lateral ventricle; M, medial; MAO, medial accessory olive; ml, molecular layer (cerebellum); MN, medial nucleus; Olf, olfactory bulb; ONL, outer nuclear layer (retina); Or, oriens layer of hippocampus; PAG, periaqueductal gray; Par, paramedian lobule; pcl, PC layer; pf, primary fissure; PFl, paraflocculus; PIN, posterior interposed nucleus; PN, pontine nucleus; Py, pyramidal cell layer of hippocampus; R, rostral; Rad, stratum radiatum of hippocampus; Rt, right; SC, superior colliculus; Sim, simple lobule; SN, substantia nigra; Th, thalamus; V, ventral; VH, ventral horn; ZI, zona incerta.
Figure 2.
Venus expression in the nervous system other than the retina in Aldoc-Venus mice.
A, Ampullas of the vertical semicircular canals. Whole mount preparation. B and C, Dorsal cochlear nucleus in a parasagittal section. High magnification (C) shows a labeled cartwheel cell. D–E, Spinal cord. F–K, coronal sections of the brain at different levels from the hindbrain to olfactory bulb. Squares (in B, E, H–K) indicate the areas that are magnified in separate panels. L–P, Double labeling with DAPI in dorsal root ganglion, CA1 of the hippocampus, olfactory bulb, corpus callosum and cerebral cortex, respectively, in medium magnification. Q, Confocal photomicrograph of double labeling of Venus and DAPI in an Aldoc-positive area of the cerebellar cortex. All PCs expressed Venus strongly at their soma, dendrites and axon terminals. Paraflocculus in a coronal section. Filled arrowheads indicate somata of Bergmann glias, while an open arrowheads indicate an astrocytes in the granular layer. Both of them expressed Venus moderately. R, Confocal photomicrograph of Venus expression in Bergmann glias in a mostly Aldoc-negative area. Lobule V in a parasagittal section. Asterisks indicate PCs. Arrowheads indicate vertical processes of Bergmann glias. Scale bar in K applies to D–K. Scale bar in P applies to L–P. Scale bar in R applies to Q and R. See the legends for Figure 1 for abbreviations.
Figure 3.
Identification of Aldoc expressing neurons and glia in the retina with confocal photomicroscopy.
A, Transverse section of the retina under low magnification. Only this photo was taken with an epifluorescence microscope. This retina section was obtained from a perfused mouse. B, Intermediate magnification photos of a cross section labeled with immunostaining of Venus with anti-GFP antibody (left subpanel), DAPI staining (center subpanel) and double labeling (right subpanel). C–H, High magnification photos of cross sections labeled with immunostaining of Venus (most left subpanel), immunostaining of calbindin (C), recoverin (D), Pax6 (E), cone arrestin (F), glutamine synthetase (G) or protein kinase C (H) (next most left subpanel), DAPI staining (next most right subpanel), and triple labeling (most right subpanel). Arrowheads indicate a population of ganglion cell (B), horizontal cell (C), rod photoreceptor cells (D), amacrine cells (E), cone photoreceptor cells (F), Müller glia cells (G) and bipolar cell (H). See the legends for Figure 1 for abbreviations.
Figure 4.
Comparison of the striped Aldoc or Venus expression pattern among wild type (Aldoc+/+), heterozygote (Aldoc+/Venus) and homozygote (AldocVenus/Venus) mice.
A–F, Photomicrographs of Aldoc immunostaining in a wild type (the most left column), Aldoc immunostaining, double labeling and Venus in a heterozygote (the three center columns), and Venus in a homozygote (the most right column) in horizontal sections of the cerebella at the similar levels (A, rostral cerebellum with three narrow zones; B, Paravermal lobule VIa; C, Lobule VIII; D, Junction between lobules IXc and X; E, Flocculus, dorsal part; F, Flocculus, ventral part). Arrowheads in D indicate areas with higher Aldoc/Venus expression in lobule X. White and black arrowheads in E and F indicate rostral and caudal parts of the flocculus that had different Aldoc/Venus expression levels. Sections from the wild type and heterozygote were processed with Aldoc immunostaining with Texas red-conjugated secondary antibody in the same session with the same antibody solutions. Photos of Aldoc immunostaining and Venus were, respectively, processed with the same exposure and adjustment settings for all mice in A–F. Scale bar under F applies to A–F. G, Higher magnification photomicrographs of Aldoc immunostaining (left panel), double labeling (center panel) and Venus (right panel) of a coronal section of the cerebellum (lobule VIII) in a heterozygote, showing complete match at the cell population level.
Figure 5.
Development of Venus expression pattern in the cerebellum.
A–L, Photomicrographs of dorsocaudal aspect of the cerebellum in whole-brain preparations of heterozygotes at P5, every postnatal day between P9 and P17, P35 and P77. All photomicrographs were taken and adjusted with the same protocol. Scale bar in L applies to A–L.
Figure 6.
Detailed Aldoc expression pattern in the cerebellar vermis and hemisphere. examined in Aldoc-Venus mice.
A–E, Aspects of vermal surface. F, Reconstruction of the unfolded entire vermal cortex (molecular layer) by SSAA from serial sections. The rostral and caudal parts (lobules I–V and lobules VII–X) were from serial coronal sections in a mouse and the central part (lobule V–VII) were from serial horizontal sections in another mouse. G–I, Aspects of hemispheral surface. J, Reconstruction of the unfolded entire hemispheral cortex (molecular layer) by SSAA from serial sections. Since folial organization is complicated, SSAAs in individual folia are combined to show the entire cortex as a mosaic of the SSAAs. K, The revised scheme of the Aldoc expression pattern drawn on the unfolded cerebellar cortex based on the SSAAs shown in F and J. Note that darker colors mean more intense expression of Venus in K, opposite to photomicrographs. The mosaic displays of SSAAs were shrunk by 45% in the rostrocaudal (vertical) direction to fit to the space in F and J. Arrowheads indicate the apex of lobules in F and J. Scale bar in E applies to A–E. Scale bar in I applies to G–I. See the legends for Figure 1 for abbreviations.
Figure 7.
Inter-individual variation in Venus expression pattern in the cerebellum.
A–F, Photomicrographs of vermal lobules VII–VIII in five adult mice (A–E) and a schematic representation of the striped expression pattern of Venus in this area (F). G–L, Photomicrographs of the right side of lobules VI and its hemispheral extension, simple lobule and crus I in five adult mice (G–K) and a schematic representation of the striped expression pattern of Venus in this area (L). For abbreviations of anatomical terms, see the legends for Figure 1. Arrowhead in J indicates a trace of the blood vessel. Scale bar in E applies to A–F. Scale bar in K applies to G–L.
Figure 8.
Venus expression pattern in the flocculus and paraflocculus.
A, Photomicrograph of the flocculus in the ventral outer surface of the whole-mount preparation of the right cerebellum. Venus expression in the rostral part was weaker than in the caudal part. B–D, Photomicrograph of double labeling of PLCB4 in a parasagittal section of the flocculus. Superimposing (D) of immunostaining of PLCB4 (B) and Venus fluorescence (C) in the same section indicate that PLCB4 and Venus are expressed in a complementary pattern; the rostral part that was weak in Venus expression expressed PLCB4. Arrowheads indicate the boundary between the Venus-positive and -negative areas. Inset in the right bottom indicates the position of the photomicrograph. E, Schematic illustrating lobular organization and unfolding of the flocculus. F, The Aldoc expression pattern in the paraflocculus and flocculus depicted in the unfolded scheme, based on the SSAA (G). G, SSAA from serial parasagittal sections of the paraflocculus and flocculus. The displays of SSAA were shrunk by 50% in the dorsoventral (vertical) direction. Red curve indicate the boundary between stripes 8a+ and 8b+. Arrowheads indicate the apex of lobules. Scale bar in D applies to B–D. See the legends for Figure 1 for abbreviations.
Figure 9.
Relationship of the 9+/9− boundary in the flocculus with the known floccular compartmentalization.
A, Comparison of the Venus expression pattern (left), the immunostained HSP25 expression pattern (center) and double labeling (right) in photomicrographs of a horizontal section (top), SSAA from serial horizontal sections (middle), and mapping in the unfolded scheme (bottom), of the paraflocculus and flocculus. Arrowheads in the top panels indicate the boundary between Aldoc-positive and -negative areas, which coincided with the boundary between HSP25-positive and -negative areas, in the flocculus. Arrowheads in the center panels indicate the apex of the lobule. The displays of SSAA were shrunk by 50% in the dorsoventral (vertical) direction. The mapping was based on SSAA. B, Three dimensional reconstruction of the HSP25 and Venus labeling patterns in the paraflocculus and flocculus. Caudal and rostral views of the paraflocculus and a lateral view of the flocculus are shown. Red and pink areas indicate strong and moderate HSP25 expression. Purple areas indicate Aldoc-negative area. C, Schematic showing correspondence between the Aldoc stripes and functional zones in the flocculus. D, Anterograde labeling of olivocerebellar climbing fibers by a tracer injection into the dorsal cap subnucleus of the inferior olive. Comparison of labeled climbing fibers (center), Venus expression (left) and double labeling (right) indicates that labeled climbing fibers were located in the Aldoc-negative area. E, Injection site of tracer (Alexa Fluor 594-conjugated dextran amine) in the dorsal cap subnucleus of the inferior olive. Contour of the inferior olive subnuclei (blue) was drawn by referring to another photo of intrinsic and Venus fluorescence of the same section. See the legends for Figure 1 for abbreviations.
Figure 10.
Venus expression pattern in the mouse cerebellar nuclei.
A–F, Parasagittal sections showing the cerebellar nuclei at different mediolateral levels, separated by about 0.24 mm. After taking photos of Venus fluorescence (green), the sections were stained with thionine (magenta) and photographed again. Two photos were superimposed to produce double-stained images (see Methods). Thus, the photos can be regarded as double labeling of Aldoc and thionine. Dotted curves indicate contours of the cerebellar nuclei and boundaries of Aldoc-positive and -negative divisions of the cerebellar nuclei. Asterisk in C indicates the caudoventral crust-like aldolase C-positive area in the posterior interposed nucleus. G, Confocal photomicrograph of the Aldoc-positive part. Asterisks in G indicate large neurons in the cerebellar nucleus. H, Three dimensional reconstruction of the right cerebellar nuclei of mouse. While the contour of the entire cerebellar nuclei is shown with wire frames, the Aldoc positive part is shown with gray solid. Scale bar in F applies to A–F. See the legends for Figure 1 for abbreviations.