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Fig. 4. Localization and function of Kir4.1 in the frog embryo. Using antibodies to Kir4.1 that reveal clean bands of the predicted size (corresponding to single subunits and dimers) (A), immunohistochemistry revealed that the unfertilized egg localized Kir4.1 protein throughout the vegetal half, excluding a sickle-shaped (bowl shaped, in three dimensions) area under the animal pole (B). In contrast, after fertilization, the protein is located throughout the animal hemisphere (C). At the 2-cell stage, Kir4.1 protein localization exhibits a range of patterns consistent with finger-like projections which can be observed at different stages of a movement from an equatorial pool towards the animal pole (D–F). The patterns are suggestive of a movement that bends to follow the contour of the animal pole edge and then returns to the equatorial pool, forming triangle-like areas including at the cell membrane (F–H). At the 2-cell stage, localization is symmetrical along the LR axis and results in Kir4.1 presence at the cell cortex in sections taken at the animal pole (I). Sections taken in the vegetal hemisphere exhibit a more complex localization, being present throughout the cytoplasm of the ventral cells and in rod-like patterns in the dorsal cells (blue arrows, J). The normal sequence of animal pole-directed movement of Kir4.1 protein (B–H) was disrupted by manipulation of the cytoplasmic transport machinery. Inhibition of myosin by Blebbistatin often introduced an asymmetry along the LR axis and abolished the appearance of well-defined fingers (L). Depolarization of microtubules somewhat disrupted the well-formed pattern (M). Inhibition of dynein motor protein activity and disruption of the actin cytoskeleton resulted in much more drastic changes in the normal pattern, resulting in most of the Kir4.1 protein localizing to the animal pole in bulk (N and O). Red arrows indicate positive signal. Yellow ovals in each panel indicate plane of section, as indicated in (K); orientation of sections taken perpendicular to the AV axis is shown in (K′). Functional roles for Kir4.1 in left–right patterning were tested by misexpression of a dominant negative construct designed to trap endogenous Kir4.1 protein subunits in the ER at the 1-cell stage, and scoring laterality of the heart, gut, and gall-bladder at st. 43. The raw data are shown in Table 2. In contrast to control (uninjected) embryos and embryos injected with Kir2.1-ER mRNA, all of which exhibited >97% normal laterality (P), embryos injected with Kir4.1-ER mRNA exhibited 25% incidence of independent randomization of the three organs assayed (heterotaxia), including reversals of the heart (Q), and gut and gall-bladder (R). Red arrowheads indicate apex of the heart; yellow arrowheads indicate direction of gut coil vertex; green arrowhead indicates position of the gall bladder. |
