Whole-mount Immunohistochemistry: A High-throughput Screen for Patterning Defects in the Mouse Cerebellum

Abstract

S U M M A R Y Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II ( ϭ aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila , chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction. (Hawkes et al. 1985Hawkes and Leclerc 1987; Brochu et al. 1990;Hawkes and Gravel 1991; Ahn et al. 1994;Hawkes and Herrup 1996;Hawkes 1997;Hawkes and Eisenman 1997;Walther et al. 1998).Isolation of zebrin II cDNA clones from cerebellar expression libraries has revealed a 98% identity to the metabolic isoenzyme aldolase C (Ahn et al. 1994). Zebrin II is a 36-kD polypeptide expressed strongly by a subset of Purkinje cells arranged in parasagittal stripes that are reproducible in detail between individuals and conserved across species [e.g., rat (Leclerc et al. 1990), opossum (Doré et al. 1990, and mouse (Eisenman and Hawkes 1993)]. Purkinje cells that are immunoreactive for zebrin II express the antigen in their dendrites, somata, and axons but not in the nucleus (Brochu et al. 1990). A detailed map of zebrin II expression in the mouse has been published (Eisenman and Hawkes 1993). Almost every feature of cerebellar structure and function is influenced by the positional information encoded in the Purkinje cells. For example, both climbing and mossy fibers terminate in discrete parasagittal stripes that align with the Purkinje cell stripes (reviewed in Hawkes 1997) and, as a result, the cerebellar functional map also consists of stripes and patches (e.g., Chockkan and Hawkes 1994;Hallem et al. 1999).

Age-Dependent Sexually-Dimorphic Asymmetric Development of the Ferret Cerebellar Cortex

“…The midline of each side was defined by the position of the cerebral longitudinal fissure. We further segmented four transverse domains of the cerebellar cortex, primarily based on the expression pattern of zebrin II/aldolase C [29,30]: left and right sides of the anterior zone (AZ) (vermal lobules I-V), central zone (CZ) (vermal lobules VI-VII, lobules simplex, and crura I and II of ansiform lobules), posterior zone (PZ) (vermal lobules VIII-IXa, and paramedian lobule), and the nodular zone (NZ) (vermal lobules IXb-X, paraflocculus and flocculus). Boundaries of those domains were defined by major fissures/sulci (Supplemental Figure S1) primarily according to our previous study [15].…”

“…Four transverse domains of the cerebellar cortex were delineated primarily based on the zebrin II/aldolase C expression pattern [29,30]. Their volumes were examined to clarify when regional volume laterality of the cerebellum appeared during postnatal development.…”

“…Thus, AQ analysis revealed that the anticlockwise torque asymmetry (rostrally rightward biased, and caudally leftward biased or symmetrical) of the cerebellar cortex was acquired in both sexes during PDs 42 to 90, although the leftward laterality was more enhanced in males than in females. [29,30]. Their boundaries were defined by major [29,30].…”

“…[29,30]. Their boundaries were defined by major [29,30]. Their boundaries were defined by major fissures/sulci (Supplemental Figure S1) primarily according to our previous study [3].…”

“…Volumetric analysis was conducted to measure the cerebellar volume and to evaluate the cerebellar torque asymmetry, based on ex vivo MRI with a high spatial resolution. The cerebellar torque asymmetry was assessed by the difference in asymmetry quotient (AQ) of volumes of four cerebellar transverse domains, which were segmented primarily based on the zebrin II/aldolase C expression pattern [29,30].…”