Functional regionalization of the differentiating cerebellar Purkinje cell population occurs in an activity-dependent manner.

Introduction: The cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive. Methods and results: We show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer. Discussion: Hence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Neurological Disease Modelling for Spinocerebellar Ataxia Using Zebrafish.

The cerebellum integrates sensory information and motor actions. Increasing experimental evidence has revealed that these functions as well as the cerebellar cytoarchitecture are highly conserved in zebrafish compared with mammals. However, the potential of zebrafish for modelling human cerebellar diseases remains to be addressed. Spinocerebellar ataxias (SCAs) represent a group of genetically inherited cerebellar diseases leading to motor discoordination that is most often caused by affected cerebellar Purkinje cells (PCs). Towards modelling SCAs in zebrafish we identified a small-sized PC-specific regulatory element that was used to develop coexpression vectors with tunable expression strength. These vectors allow for in vivo imaging of SCA-affected PCs by high-resolution fluorescence imaging. Next, zebrafish with SCA type 13 (SCA13) transgene expression were established, revealing that SCA13-induced cell-autonomous PC degeneration results in eye movement deficits. Thus, SCA13 zebrafish mimic the neuropathology of an SCA-affected brain as well as the involved loss of motor control and hence provide a powerful approach to unravel SCA13-induced cell biological pathogenic and cytotoxic mechanisms.

Modeling Neurodegenerative Spinocerebellar Ataxia Type 13 in Zebrafish Using a Purkinje Neuron Specific Tunable Coexpression System.

Purkinje cells (PCs) are primarily affected in neurodegenerative spinocerebellar ataxias (SCAs). For generating animal models for SCAs, genetic regulatory elements specifically targeting PCs are required, thereby linking pathological molecular effects with impaired function and organismic behavior. Because cerebellar anatomy and function are evolutionary conserved, zebrafish represent an excellent model to study SCAs in vivo We have isolated a 258 bp cross-species PC-specific enhancer element that can be used in a bidirectional manner for bioimaging of transgene-expressing PCs in zebrafish (both sexes) with variable copy numbers for tuning expression strength. Emerging ectopic expression at high copy numbers can be further eliminated by repurposing microRNA-mediated posttranslational mRNA regulation.Subsequently, we generated a transgenic SCA type 13 (SCA13) model, using a zebrafish-variant mimicking a human pathological SCA13R420H mutation, resulting in cell-autonomous progressive PC degeneration linked to cerebellum-driven eye-movement deficits as observed in SCA patients. This underscores that investigating PC-specific cerebellar neuropathologies in zebrafish allows for interconnecting bioimaging of disease mechanisms with behavioral analysis suitable for therapeutic compound testing.SIGNIFICANCE STATEMENT SCA13 patients carrying a KCNC3R420H allele have been shown to display mid-onset progressive cerebellar atrophy, but genetic modeling of SCA13 by expressing this pathogenic mutant in different animal models has not resulted in neuronal degeneration so far; likely because the transgene was expressed in heterologous cell types. We developed a genetic system for tunable PC-specific coexpression of several transgenes to manipulate and simultaneously monitor cerebellar PCs. We modeled a SCA13 zebrafish accessible for bioimaging to investigate disease progression, revealing robust PC degeneration, resulting in impaired eye movement. Our transgenic zebrafish mimicking both neuropathological and behavioral changes manifested in SCA-affected patients will be suitable for investigating causes of cerebellar diseases in vivo from the molecular to the behavioral level.

Eppur Si Muove: Evidence for an External Granular Layer and Possibly Transit Amplification in the Teleostean Cerebellum.

The secreted signaling factor Sonic Hedgehog (Shh) acts in the floor plate of the developing vertebrate CNS to promote motoneuron development. In addition, shh has dorsal expression domains in the amniote alar plate (i.e., in isocortex, superior colliculus, and cerebellum). For example, shh expressing Purkinje cells act in transit amplification of external granular layer (EGL) cells of the developing cerebellum. Our previous studies had indicated the presence of an EGL in anamniote zebrafish, but a possible role of shh in the zebrafish cerebellar plate remained elusive. Therefore, we used an existing zebrafish transgenic line Tg(2.4shha-ABC-GFP)sb15; Shkumatava et al., 2004) to show this gene activity and its cellular localization in the larval zebrafish brain. Clearly, GFP expressing cells occur in larval alar zebrafish brain domains, i.e., optic tectum and cerebellum. Analysis of critical cerebellar cell markers on this transgenic background and a PH3 assay for mitotic cells reveals that Purkinje cells and eurydendroid cells are completely non-overlapping postmitotic cell populations. Furthermore, shh-GFP cells never express Zebrin II or parvalbumin, nor calretinin. They are thus neither Purkinje cells nor calretinin positive migrating rhombic lip derived cells. The shh-GFP cells also never correspond to PH3 positive cells of the ventral cerebellar proliferative zone or the upper rhombic lip-derived EGL. From this marker analysis and the location of shh-GFP cells sandwiched between calretinin positive rhombic lip derived cells and parvalbumin positive Purkinje cells, we conclude that shh-GFP expressing cells qualify as previously reported olig2 positive eurydendroid cells, which are homologous to the amniote deep cerebellar nuclei. We confirm this using double transgenic progeny of shh-GFP and olig2-dsRed zebrafish. Thus, these zebrafish eurydendroid cells may have the same role in transit amplification as Purkinje cells do in amniotes.

Zebrafish jam-b2 Gal4-enhancer trap line recapitulates endogenous jam-b2 expression in extraocular muscles.

BACKGROUND: Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS: We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS: The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.