Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression

Abstract

The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step toward unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH) is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4), NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first 3-weeks of life.

Keywords: circuit; development; patterning; purkinje cells; topography.

Figure 1

NFH is expressed in the cerebellar nuclei of newborn mice. (A) An unstained brain at P0 illustrating the cutting planes used to acquire the tissue sections shown in panel (B). The sagittal plane is shown in red and the coronal plane in blue. (B) NFH labels the CN and not Purkinje cells. Purkinje cells were identified with Calbindin, a specific marker for Purkinje cells. Background staining was detected in the choroid plexus. Abbreviations: CN, cerebellar nuclei; CP, choroid plexus; NFH, neurofilament heavy chain; P0, postnatal day 0; PCs, Purkinje cells; and the cardinal lobes are ABL, anterobasal lobe; ADL, anterodorsal lobe; CEL, central lobe; POS, posterior lobe; INL, inferior lobe. The cardinal lobes were named according to the nomenclature of Altman and Bayer (1997). Scale bar = 200 μm for the sagittal sections and = 500 μm for the coronal sections.

Figure 2

NFH is expressed in parasagittal zones at postnatal day 2. (A) At P2, NFH is heavily expressed in all three sets of cerebellar nuclei [fastigial nucleus (Fn), interpositus nucleus (In), dentate nucleus (Dn)], in the commissural axons from the Fn (arrow), and weakly in broad zones of Purkinje cells in the vermis and hemispheres. (B) High power image of the midline (m) showing Purkinje cell zones (asterisks). Scale bar in (A) = 500 μm and (B) = 100 μm.

Figure 3

NFH is expressed in zones throughout postnatal development. Wholemount immunohistochemistry demonstrates that NFH is expressed in zones of Purkinje cells at P7 (A–C), P12 (D–F), and P20 (G–I). Note that all regions of the vermis and hemispheres are compartmentalized into zones. The vermis lobules are numbered with Roman numerals. Scale bars = 1 mm.

Figure 4

Zone formation in the vermis revealed by NFH expression. (A) Schematic indicating the region of the vermis that is shown in panels (B–D). (B) At the end of the first postnatal week, NFH expression reveals clear zones in the posterior lobules. (C) By P12, the immunopositive zones in lobule VII have expanded laterally, and immunonegative zones decrease in width accordingly. This expansion is obvious at the midline (brackets). (D) By P20, strong expression at the lateral margins of each zone emerge (asterisks), while weaker expression persists within the center of each zone (double arrows). The negative zones in lobule IX have resolved by P20 (compare to narrow zones marked by arrows in panels B and C).

Figure 5

NFH zone boundaries are sharpened in the developing hemispheres. (A) Schematic indicating the region of the hemisphere that is shown in panels (B–D). (B) NFH expression reveals a raphe-like pattern in Crus II at P7. (C) By P12, the NFH pattern consists of clear-cut zones (asterisks in C and D). (D) The overall pattern at P20 is identical to P12, although each zone is now sharply delineated. Abbreviation: LS, lobulus simplex; PML, paramedian lobule; COP, copula pyramidis. Scale bar = 500 μm.

Figure 6

NFH and neurogranin are expressed in complementary zones. (A) NFH is expressed in broad zones at P2. (B) Neurogranin is expressed in a series of early postnatal Purkinje cell zones. (C) The pattern of NFH is complementary to the pattern of neurogranin at P2. The arrows point to zonal boundaries and the vertical dotted lines highlight complementary domains at the midline. Scale bar = 250 μm.

Figure 7

NFH and PLCß4 have a complex zonal relationship. NFH (green) and PLCß4 (magenta) have corresponding patterns in the anterior lobules at P12 (A–C) and P20 (G–I). (D–F) In the posterior cerebellum, the widespread expression of NFH overlaps with PLCß4 positive and PLCß4 negative zones (arrows). (J–L) By P20, all NFH positive and negative zones (asterisks) overlap with PLCβ4 zones. Scale bar in (C) = 500 μm [applies to (A,B,G–I)] and the scale bar in (L) = 500 μm (applies to D–F, J,K).