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- ZII Expression
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The advantages of the embryonic chick as a model for studying neural development range from the relatively low cost of fertilized eggs to the rapid rate of development. We investigated in ovo cerebellar development in the chick, which has a nearly identical embryonic period as the mouse (19–22 days). We focused on three antigens: Calbindin (CB), Zebrin II (ZII), and Calretinin (CR), and our results demonstrate asynchronous expression patterns during cerebellar development. Presumptive CB+ Purkinje cells are first observed at embryonic day (E)10 in clusters in posterior cerebellum. At E12, corresponding with global expression of CB across the cerebellum, Purkinje cells began to express ZII. By E14–E16, Purkinje cells disperse into a monolayer and develop a pattern of alternating immunopositive and immunonegative ZII stripes. CR is initially expressed by clusters of presumptive Purkinje cells in the nodular zone at E8. However, this expression is transient and at later stages, CR is largely confined to the granule and molecular layers. Before hatch (E18–E20), Purkinje cells adopt a morphologically mature phenotype with complex dendritic arborizations. Comparing this data to that seen in mice, we found that the sequence of Purkinje cell formation, protein expression, and development is similar in both species, but these events consistently begin ∼5–7 days earlier in the precocial chick cerebellum, suggesting an important role for heterochrony in neurodevelopment. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.
The cerebellum is responsible for coordinating precision and timing of complex motor functions, cognitive and perceptual analysis, and memory and emotional processing (Middleton and Strick, 1998; Larouche and Hawkes, 2006; Sultan and Glickstein, 2007; Baumann and Mattingley, 2012). Underlying this diverse functionality is a characteristic histological organization and pattern of cellular compartmentalization. It is understood that the adult cerebellum of various mammals and birds demonstrates a conserved pattern of antigen expression by major cell types of the cerebellum, including Purkinje cells and granule cells. Although these immunoreactive compartments correspond to physiologically defined zones (Gravel et al., 1987; Ji and Hawkes, 1994; Voogd et al., 2003; Sugihara and Shionda, 2004; Voogd and Ruigrok, 2004; Pakan and Wylie, 2008; Pakan et al., 2010, 2011), little is known about their development outside of mammals. Here, we provide new information on embryonic cerebellar development in one of the most common developmental models, the domestic chicken (Gallus gallus domesticus). Domestic chickens (hereafter, chicks) have a lengthy history of use in the study of neurodevelopment (Waddington, 1930; Le Douarin, 1973; Keynes and Stern, 1984; Lumsden and Kiecker, 2004, Ma et al., 2012), offering the advantages of accessibility throughout embryogenesis (Hamburger and Hamilton, 1951; Schmutz and Grimwood, 2004) and rapid development to a fully precocial state (characterized by high levels of motor coordination and evidence of olfactory, visual, and auditory abilities; Stark, 1993). In addition, embryonic chicks are amenable to various in ovo manipulations including microsurgical tissue grafting and lineage tracing methods that form the foundation of our understanding of the commitment and determination of different neural cell types as well as their interactions during development (reviewed by Sauka-Spengler and Barembaum, 2008).
The cerebellum of birds, similar to that of mammals, has a complex and hierarchical morphology, beginning with subdivision of the cortex into three major lobes (anterior, posterior, and flocculonodular) separated by prominent fissures. Each lobe has a number of folds known as folia (= lobules in mammals) (Larsell, 1972). On the basis of molecular markers and afferent projections, the avian cerebellum is further compartmentalized into a series of transverse zones. The pattern of zonation includes the lingular zone (LZ; folium I), anterior zone (AZ; folia II–V), central zone (CZ; folia VI–VII), posterior zone (PZ; folia VIII–IX), and nodular zone (NZ; posterior folium IX and folium X) (pigeons: Pakan et al., 2007; hummingbirds: Iwaniuk et al., 2009; adult chickens: Marzban et al., 2010). These transverse zones are divided mediolaterally by species-specific and antigen-specific parasagittal striping patterns, shown as alternating subsets of immunopositive and immunonegative Purkinje cells (Wylie et al., 2011; see Apps and Hawkes, 2009 for review). In coronal and sagittal sections, the mature cerebellum is organized into three distinct layers each with a unique combination of cell types. The molecular layer includes stellate and basket cell interneurons, Purkinje cell dendrites, and parallel fibers. Deep to this, the Purkinje cell layer is a monolayer of Purkinje cell somata. Next, the granule cell layer contains primarily granule cells, Golgi cells, unipolar brush cells, and Lugaro cells (see Ambrosi et al., 2007) as well as afferent mossy fiber terminals.
Based on differential patterns of protein expression, Purkinje cells and granule cells constitute a heterogeneous neuronal population (Hawkes and Leclerc, 1987; Hawkes and Turner, 1994; Armstrong et al., 2000; reviewed in Apps and Hawkes, 2009). Previous studies in mice have demonstrated that while the pattern of protein expression by Purkinje cells and granule cells is static among adults, it is dynamic during development (Leclerc et al., 1988; Hawkes and Eisenman, 1997; Armstrong et al., 2001). In this study, we used western blot analysis and immunohistochemistry to document the onset and expression pattern of three proteins of interest at embryonic (E) days 8, 10, 12, 14, 16, 18, and 20 during chick development, focusing specifically on the antigens Calbindin (CB), Zebrin II (ZII), and Calretinin (CR).
CB is a 28-kDa calcium-binding protein expressed by cerebellar Purkinje cells (e.g., Wassef et al., 1985; Celio, 1990; Bastianelli and Pochet, 1993). In embryonic mice, CB is expressed in a heterogeneous pattern of immunopositive and immunonegative Purkinje cell clusters arranged on either side of the midline starting at E16 (Wassef et al., 1985). Beginning perinatally and continuing throughout postnatal development and adulthood, CB is globally expressed by all Purkinje cells in rodents and birds (rat: Celio, 1990; mouse: Ozol et al., 1999; pigeon: Pakan et al., 2007; adult chicken: Marzban et al., 2010). CB acts primarily as an intracellular Ca2+ buffer, and has been shown to play a role in Purkinje cell-dependent motor learning in CB−/− mice (reviewed in Schwaller et al., 2002).
ZII (aldolase C), is a 36-kDa glycolytic isozyme (Ahn et al., 1994) that, similar to CB, is selectively expressed by Purkinje cells within the cerebellum. Unlike CB, ZII expression in adults is discontinuous, leading to the recognition of transverse zones (Ozol et al., 1999) and parasagittal stripes (Hawkes and Leclerc, 1987; reviewed by Larouche and Hawkes, 2006) in the adult cerebellum. In mice, ZII is first expressed around postnatal day 5 (P5) in Purkinje cells of the posterior lobe (PZ and NZ: lobules VIII–X). From these posterior positions, ZII expression spreads dynamically to increasingly more anterior and lateral cells until global expression in all Purkinje cells is seen at ∼P12. As postnatal development continues, approximately half of the ZII immunopositive (ZII+) Purkinje cells stop expressing this protein (Leclerc et al., 1988; Eisenman and Hawkes, 1993; Hawkes and Herrup, 1996; reviewed in Armstrong and Hawkes, 2000). The result is a conserved striping pattern of ZII+ and ZII− Purkinje cells. In addition to mice, the pattern of ZII stripes has been mapped in numerous species including rat (Brochu et al., 1990), opossum (Doré et al., 1990), guinea pig (Larouche et al., 2003), hedgehog (Sillitoe et al., 2003), fish (Brochu et al., 1990), pigeon (Pakan et al., 2007), hummingbird (Iwaniuk et al., 2009), and adult chicken (Marzban et al., 2010). In adult avian species, ZII+ Purkinje cells are organized into a pattern of parasagittal stripes within the AZ and PZ, and Purkinje cells of the LZ, CZ, and NZ are almost uniformly ZII+ (Pakan et al., 2007; Iwaniuk et al., 2009; Marzban et al., 2010).
CR is a 29-kDa calcium-binding protein that is expressed by a variety of cells in the cerebellar cortex. In adult rodents, these include granule cells, unipolar brush cells, Golgi cells, stellate and basket cells, and Lugaro cells as well as mossy fibers and climbing fibers (Rogers, 1989; Résibois and Rogers, 1992; Floris et al., 1994; Diño et al., 1999; Nunzi et al., 2002; reviewed in Baimbridge et al., 1992).
In posthatch chicks, CR expression was seen in stellate and basket cells, mossy fibers and a subset of climbing fibers (Rogers, 1989; De Castro et al., 1998; reviewed in Bastianelli, 2003), while granule cells were CR-immunonegative (Rogers, 1989; Bastianelli and Pochet, 1993). During development, the transient expression of CR in the granule cell layer of the chick cerebellum has previously been reported (Bastianelli and Pochet, 1993). Although the physiological function of CR in the central nervous system remains poorly understood, Bastianelli and Pochet (1993) suggest that the induction and/or expression of CR, and other calcium-binding proteins, may be linked to neurodegeneration. CR-deficient mice display impaired motor coordination suggesting a role in cerebellar function (Schiffmann et al., 1999; Dove et al., 2000; Gall et al., 2003).
Our results demonstrate a unique sequence and pattern of expression for each of CB, ZII, and CR during cerebellar development in the chick, similar to that seen in mammals. However, the developmental expression of each antigen was consistently earlier in chick despite the fact that both species have a similar embryonic period of 19–22 days.