Clustered fine compartmentalization of the mouse embryonic cerebellar cortex and its rearrangement into the postnatal striped configuration - PubMed (original) (raw)

Clustered fine compartmentalization of the mouse embryonic cerebellar cortex and its rearrangement into the postnatal striped configuration

Hirofumi Fujita et al. J Neurosci. 2012.

Abstract

Compartmentalization is essential for a brain area to be involved in different functions through topographic afferent and efferent connections that reflect this organization. The adult cerebellar cortex is compartmentalized into longitudinal stripes, in which Purkinje cells (PCs) have compartment-specific molecular expression profiles. How these compartments form during development is generally not understood. To investigate this process, we focused on the late developmental stages of the cerebellar compartmentalization that occur from embryonic day 17.5 (E17.5), when embryonic compartmentalization is evidently observed, to postnatal day 6 (P6), when adult-type compartmentalization begins to be established. The transformation between these compartmentalization patterns was analyzed by mapping expression patterns of several key molecular markers in serial cerebellar sections in the mouse. A complete set of 54 clustered PC subsets, which had different expression profiles of FoxP2, PLCβ4, EphA4, Pcdh10, and a reporter molecule of the 1NM13 transgenic mouse strain, were distinguished in three-dimensional space in the E17.5 cerebellum. Following individual PC subsets during development indicated that these subsets were rearranged from a clustered and multilayered configuration to a flattened, single-layered and striped configuration by means of transverse slide, longitudinal split, or transverse twist spatial transformations during development. The Purkinje cell-free spaces that exist between clusters at E17.5 become granule cell raphes that separate striped compartments at P6. The results indicate that the ∼50 PC clusters of the embryonic cerebellum will ultimately become the longitudinal compartments of the adult cerebellum after undergoing various peri- and postnatal transformations that alter their relative spatial relationships.

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Figures

Model 1.

3D representation of E17.5 PC clusters. All PC clusters and immature fissures in the E17.5 hemicerebellum (left-hand side) are represented in the 3D space. The contents are the same as shown in Figures 5_F–J_ and 8_B_. This file contains a list of 3D objects to show or hide individual clusters and fissures. They can also be viewed from any directions. “Left,” “Top,” “Front,” “Right,” “Bottom,” “Back” views (in the view list of the file) actually shows the left (lateral), caudal, ventral, right (medial), rostral, dorsal aspects of the representation. Cluster vt1 and its midline cross section (“midline”) are formed with multiple objects for technical reason in this 3D representation. Scale bars, 500 μm.

View 3D

Figure 1.

Development of the β-gal expression pattern in the 1NM13 transgenic mouse strain. A–E, Whole-mount β-gal visualization in the 1NM13 cerebellum at E17.5 (A), P1 (B), P3 (C), P6 (D), and P12 (E). Dorsal view. Filled and open arrowheads indicate paramedian and hemispheral areas that begin to express β-gal earlier than other areas. These two areas can be clearly followed from E17.5 to P12, although many other β-gal-positive areas emerged between P0 and P12. Lobulation of the cerebellum also developed during this period. F, Adult-type longitudinal compartments represented by the aldolase C expression pattern mapped on the unfolded scheme of the entire cerebellar cortex (Sugihara and Quy, 2007). The β-gal expression pattern in the 1NM13 cerebellum at P12 is closely related to the aldolase C expression pattern (Furutama et al., 2010). Scale bar in E applies to A–E.

Figure 2.

Compartmentalization into clusters in the E17.5 PC layer. A, Lateral view of whole-mount β-gal1NM13 visualization in the E17.5 brain. Lines indicate the level and direction of sections shown in B–M. B–E, Photomicrographs of immunostaining for PC-marker molecules, FoxP2 (B) and RORα (C). These photos and DAPI counterstaining were merged into artificial color channels in a coronal section of the cerebellum at E17.5 (D). Square in D indicates the area of the magnified photo in E. Arrowheads in E indicate PC-sparse space between PC clusters, which were filled with cells that were labeled with DAPI. F–M′, Double-staining for β-gal1NM13 (blue) and FoxP2 (brown) in coronal (F–I) and horizontal (J–M) sections. Note that FoxP2 expression is variable in intensity among PC clusters. Accompanying illustrations (F′–M′) depict outlines of the PC clusters in the section with temporary names (see Results). Bluish colors in some depicted clusters in F′–M′ indicate the general expression intensity of β-gal1NM13 in the clusters. Areas outside of the cerebellum have been trimmed. The relative position of individual sections within the entire caudorostral or dorsoventral range of the cerebellum is indicated as percentage in F–M′. Scale bar in D applies to B–D; scale bar in I′ applies to F–M′. Abbreviations in this and subsequent figures are as follows: I–X, Lobules I–X; a-c, sublobules a–c (as in VIa); C, caudal; CN, cerebellar nuclei; Cop, copula pyramidis; cp, choroid plexus; Cr I, crus I of the ansiform lobule; Cr II, crus II of the ansiform lobule; D, dorsal; DC, dorsal cochlear nucleus; Fl, flocculus; f. ic, intercrural fissure; f. pc, preculminate fissure; f. pl, posterolateral fissure; f. pr, primary fissure; f. sec, secondary fissure; GT, germinal trigone; L, lateral; M, medial; Par, paramedian lobule; PFl, paraflocculus; R, rostral; Sim, simple lobule; V, ventral.

Figure 3.

Distinction of clusters according to molecular expression profiles in the E17.5 PC layer. A–L, Double- or triple-staining for markers of PC clusters and FoxP2 accompanied by drawings of individual PC clusters (B, E, H, K) and measurements of expression intensity of the marker (C, F, I, L) in coronal sections at a similar rostrocaudal level. β-Gal1NM13 labeling was photographed under bright field (see Materials and Methods). Scale bar in J applies to A, D, G, J.

Figure 4.

Expression of early cerebellar markers in E17.5 cerebellum at various levels of coronal and horizontal sections. A–R′, Double-immunostaining for each of the early markers (red or magenta, A–F; Pcdh10; G–L, PLCβ4; M–R, EphA4) and FoxP2 (green) accompanied by a drawing of individual PC clusters in the section (A′–R′). β-gal1NM13 is also visualized in D–F. The drawings can be regarded as an atlas of the PC clusters (with our temporary names) at a particular level of the coronal or horizontal section. The magenta-like colors in drawings of some clusters indicate the intensity of expression of the marker in the section. Areas outside of the cerebellum are trimmed. The relative position (%) of individual sections from dorsal to ventral or from caudal to rostral is indicated. The sectioning plane for M–O was slightly tilted toward the rostroventral-caudodorsal direction compared with other horizontal sections. Scale bar in R′ applies to A–R′.

Figure 5.

3D reconstruction of all E17.5 PC clusters. A–E, Methods for the 3D reconstruction of PC clusters. Contours of individual clusters depicted in consecutive coronal sections of the cerebellum (A–C) were aligned in 3D space (D). PC clusters were then reconstructed by using the “loft” command in Rhinoceros software (E). F–J, Reconstruction of all PC clusters in the E17.5 hemicerebellum (right-hand side in F, H–J; left-hand side in G) viewed from various directions. The clusters are shown with the temporary name, where the color indicates the β-gal1NM13 expression level. Yellow bars indicate cerebellar fissures that were recognized at E17.5. Opaque whitish areas circumscribed by an orange line indicate the inner part of the cerebellum (or clusters viewed through it). Scale bar in C applies to A–C.

Figure 6.

Compartmentalization into stripes in the P6 PC layer. A, Lateral view of whole-mount β-gal1NM13 visualization in the P6 brain. Lines indicate the level and direction of sections shown in B–I, M, N. B–E, Coronal sections double-labeled for β-gal1NM13 (blue) and calbindin (brown) (B, C) and for β-gal1NM13 (blue) and PLCβ4 (brown) (D, E) at nearly the same level in different mice. Magnified photos in C and E show the same β-gal1NM13-positive stripes bordered by GC raphes (filled and open arrowheads). F, G, Horizontal sections labeled for EphA4 alone, and EphA4 and β-gal1NM13, respectively in different mice. H, Coronal section double-labeled for β-gal1NM13 (green, bright field) and Pcdh10 (magenta, fluorescence). I, Horizontal section double-labeled for β-gal1NM13 and PLCβ4. J–L, Serial section alignment analysis for striped compartments in the PC layer. The PC layer in the rostral wall of lobule VIa was clipped from 25 consecutive sections including that in I and aligned in order (J). Striped compartments (indicated by temporary names), which were recognized by distinct molecular expression profiles and by separation by GC raphes, were mapped with expression profiles of β-gal1NM13 (K) and PLCβ4 (L). GC raphes are indicated by thick lines. M, N, Horizontal sections in other levels showing striped compartmentalization of the PC layer distinguished by the β-gal1NM13 and PLCβ4 expression profiles. Straight lines in I, M, N indicate the continuity of PC gaps. Filled and open arrowheads in I–M indicate the same GC raphes in lobule VIa as in C, E. Red arrowheads in F–H, M, N indicate the same compartment (it2). Scale bar in H applies to B, D, F–H; scale bar in E applies to C, E; scale bar in N applies to I, M, N.

Figure 7.

Local rearrangement of PC subsets from E17.5 to P6. A–C′, Coronal sections of the cerebellum at the central level immunostained for FoxP2 and PLCβ4 at E17.5 (A), P0 (B), and P1 (C). Individual clusters (indicated by temporary names) were mapped in A′–C′. D–F′, Serial section alignment analysis performed in serial horizontal sections of the posterior hemisphere of the E17.5 (D), P0 (E), and P6 (F) cerebella, which were double-labeled for β-gal1NM13 and FoxP2 (D, E) or for β-gal1NM13 and calbindin-D28k (F). Compartmental organization recognized in the analysis is mapped on the schematic drawings (D′–F′) along with the expression profiles of β-gal1NM13. GC raphes are indicated by thick lines. The density of PCs was low at the bottom of the secondary fissure in E (f. sec).

Figure 8.

Modes of transformation of PC subsets during peri- and postnatal cerebellar development. A, Schematics of modes of transformation; longitudinal split (a), transverse slide (b), and transverse twist (c). B–E, “Longitudinal split”-type transformation in EphA4-positive cluster it3 in crus I. Cluster it3 in the E17.5 cerebellum was shown in dorsal view of whole-mount EphA4 immunostaining (Ba) and in three-dimensional reconstruction with (Bb) and without (Bc) other clusters. Coronal sections immunostained for FoxP2 and EphA4 at E17.5 (C) and P1 (D) shows split of cluster it3 at P1. Serial section alignment analysis for horizontal sections labeled for β-gal1NM13 and EphA4 at P6 (E) shows separation of it3. Cluster/compartment it3 is colored consistently in mappings (Cb, Db, Eb). Red and cyan arrowheads indicate the rostral and caudal parts of cluster/compartment it3, respectively. F–H, “Transverse slide”-type transformation in four clusters/compartments in crus II (it3, hp1, hp2, and hp3). Horizontal sections of the central cerebellum (lobules IV–V, crus I, and crus II-paramedian lobule-copula pyramidis) immunostained for FoxP2 and PLCβ4 at E17.5 (Fa) and P1 (Ga) shows change of cluster disposition as depicted in the accompanying drawings (Fb, Gb). Serial section alignment analysis for horizontal sections labeled for β-gal1NM13 and PLCβ4 at P6 (H) shows striped alignment of the four compartments in crus II. These compartments are colored consistently in mappings (Fb, Gb, Hb). Red and cyan arrowheads indicate compartments hp1 and hp3, respectively. I–K, “Transverse twist”-type transformation in the paraflocculus. Serial section alignment analysis shows two compartments (it2 and hp2) were continuous across the secondary fissure at E17.5 (Ia) as depicted in the accompanying drawing (Ib). Since these subsets consistently express β-gal1NM13, they were followed in the caudal view of whole-mount β-gal1NM13 visualization at E17.5 (J) and P3 (K). The ventral part in the paraflocculus was twisted rostrolaterally in these clusters. Red and cyan arrowheads indicate clusters/compartments it2 and hp2, respectively.

Figure 9.

Correspondence of molecular expression patterns in compartments throughout the entire cerebellar cortex between E17.5 and P6. A–H, Expression patterns of β-gal1NM13, PLCβ4, EphA4 and Pcdh10 in PC clusters/compartments mapped on the unfolded scheme of the cerebellar cortex at E17.5 and P6. The labeling intensity of molecules is indicated by the darkness of colors. Compartment boundaries in the deep PC layer are shown with dashed curves in the E17.5 schemes. Thick lines indicate PC gaps and GC raphes. I, J, Clear PC gaps at E17.5 and GC raphes at P6. Each color indicates correspondence. K, Intensity of the molecular expression in individual PC subsets represented by light, medium, or dark colors in the E17.5 (top) and P6 (bottom) cerebellum. Darker colors indicate higher expression intensity. Slashed cells in P6 β-gal1NM13 expression mean that the subset may be separated into multiple small subsets that have different β-gal1NM13 expression profiles. Cells with “?” in P6 β-gal1NM13 expression means that the observation was not clear.

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Clustered fine compartmentalization of the mouse embryonic cerebellar cortex and its rearrangement into the postnatal striped configuration - PubMed (original) (raw)