Purkinje cell–specific expression of human Dyrk1A in zebrafish.A, schematic presentation of the bidirectional construct for the generation of a stable transgenic line driving HA-tagged hDyrk1A expression in Purkinje cells. Two ca8 promoter-derived PC-specific enhancer element (cpce) dimers in opposite orientation (yellow triangles) are linked to E1B basal promoters on both sides directing the expression of mClover (right cistron) and HA-hDyrk1A (left cistron) to Purkinje cells. The construct is not drawn to scale. B–M, dorsal views (anterior to the left) onto the left and right cerebellar PC hemispheres (B) recorded by confocal microscopy. Transient transgenic 4 dpf larvae show expression of hDyrk1A, visualized by αHA antibody staining, and (C) FynmClover fluorescence in Purkinje cells. D, the merged image reveals coexpression of both proteins. Colocalization of the PC-specific αParvalbum antibody staining (E) and FynmClover (F) in the merged image (G) indicate that the hDyrk1A expression is confined to cerebellar PCs. H–M, whole-mount immunostaining on F2 larvae of a stable Tg(ca8-hDryk1A-mClover) line. H, maximal brightness projections of confocal microscopy image stack recordings after αHA antibody staining visualize hDyrk1A expression in the cerebellum. I, the corresponding mClover fluorescence labels PCs, whereas the merged image (J) demonstrates reliable coexpression of both proteins. Images at higher magnification display membrane-tagged mClover (K and K’) and nuclear/cytoplasmic hDyrk1A (L and L’) colocalized within the same cells (M and M’). White arrowheads indicate Purkinje cells. The scale bar represents 50 μm (B–M) and 20 μm (K’–M’). cpce, ca8 promoter–derived PC-specific enhancer element; dpf, days post fertilization; Dyrk1A, dual-specificity tyrosine phosphorylation–regulated kinase 1A; HB, hindbrain; PCs, Purkinje cells; TeO, tectum opticum.
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