Inositol 1,4,5-triphosphate receptor is selectively expressed in cerebellum but not cerebellum-like structures of the elasmobranch fish, Leucoraja erinacea.

The Inositol 1,4,5-trisphosphate receptor type 1 protein (Ip3r1) performs an essential role for the induction of cerebellar long-term depression. Here, I describe the use of RT-PCR, qPCR, western blotting and immunohistochemistry to assay Ip3r1 gene expression and localize Ip3r1 protein in the hindbrain of the elasmobranch fish, Leucoraja erinacea. Elasmobranchs are representatives of the most basal, yet extant lineage of gnathostomes, or jawed vertebrates. The cerebellum is a synapomorphy for gnathostomes and thus elasmobranch cerebellar physiology may serve as a proxy for the ancestral state of other jawed vertebrates. LeIp3r1 is selectively expressed in the cerebellum of the little skate and the resultant protein is localized to Purkinje cells. If Ip3r1 performs the same functions in the skate cerebellum as in the mammalian cerebellum, then parallel fiber-Purkinje cell long-term depression through Ip3r1 mediated intracellular calcium regulation may be a conserved feature of cerebellar physiology. Cerebellum and surrounding hindbrain regions termed cerebellum-like structures share a common developmental genetic toolkit. LeIp3r1 expression was lowly detected in cerebellum-like structures indicating that although generatively homologous, the cerebellum and cerebellum-like structures do not share a complete overlap of common expression. Because of the little skate's important phylogenetic placement, performing molecular methodologies to assay targeted gene expression and determine protein localization in the hindbrain can be valuable for our understanding of cerebellar evolution and comparative neural development.

Morphological development of the dorsal hindbrain in an elasmobranch fish (Leucoraja erinacea).

The developmental anatomy of the dorsal hindbrain in an elasmobranch fish, Leucoraja erinacea, is described. We focus on the cerebellum, which is a synapomorphy for gnathostomes. Cerebellar development in L. erinacea, a representative of the most basal gnathostome lineage, may be a proxy for the ancestral state of cerebellar development. We also focus on sensory processing regions termed 'cerebellum-like' structures due to common anatomical and physiological features with the cerebellum. These structures may be considered generatively homologous if they share common developmental features. To test this hypothesis, the morphological development of the cerebellum and cerebellum-like structures must first be described. Of particular importance is the development of common features, such as the molecular layer, which is the defining characteristic of these structures. The molecular layers of the cerebellum and cerebellum-like structures are supplied with parallel fiber axons from distinct granule cell populations. These are the lateral granule mass, the dorsal granular ridge, the medial granule mass, and the granular eminences of the cerebellum. Cerebellar and cerebellar-like development in L. erinacea is similar to development in other elasmobranchs. The temporal order in which these granule cell populations develop suggests an evolutionary history of duplication or expansion of an existing developmental event.

Evidence for generative homology of cerebellum and cerebellum-like structures in an elasmobranch fish based on Pax6, Cbln1 and Grid2 expression.

The majority of neurons in the mammalian brain reside within the cerebellum (Cb). Yet, the evolutionary origins of the Cb are not well understood. There are several sensory nuclei present across vertebrate phylogeny collectively termed cerebellum-like structures (CbLS) due to a shared anatomy and physiology with the Cb. Despite the similarities, the CbLS are clearly not phylogenetically homologous with the Cb. Common structure and function may arise due to a shared genetic and developmental toolkit. To examine this possibility, we used sequence analysis, western blotting, immunohistochemistry and RT-qPCR to test for the expression of three genes that are critical for mammalian cerebellar development in the Cb and CbLS of an elasmobranch fish, Leucoraja erinacea. In the mammalian Cb, Pax6 is necessary for parallel fiber development, while Cbln1 and Grid2 code for proteins necessary for parallel fiber-principal cell synaptogenesis. Pax6 and Cbln1 are expressed by granule cells in the Cb and CbLS of the adult skate and stage 31 embryo. Grid2 is expressed by principal cells in the Cb and CbLS of the adult and stage 31 embryo. RT-qPCR showed this expression is spatially and temporally restricted to the Cb and CbLS. If Pax6, Cbln1 and Grid2 perform the same functions in the skate Cb and CbLS as they do in the mammalian Cb, then these structures may develop using a shared genetic toolkit and be considered generatively homologous. It is possible that the evolutionary genesis of the Cb was the result of duplication or expansion of the cerebellum-like developmental toolkit.