| dc.description.abstracteng | The ventricular system of the mammalian brain consists of the two lateral, a third, and a fourth ventricle that interconnect and are filled with circulating cerebrospinal fluid (CSF) (1). CSF streams along the ventricular walls propelled by bundles of motile cilia that protrude from the apical surface of wall-forming ependymal cells (14). Planar cell polarity (PCP), which is created by proteins that are asymmetrically expressed in ependymocytes, controls cilia beating direction (113,166–169). PCP has two components referred to as translational and rotational polarity. Translational polarity is quantified by a vector ((CC) ⃗) extending from the cell center to the center of the cilia bundle. Each cilium docks to a cylindrical, cytoplasmic basal body that extends a basal foot. The angular direction of the foot relative to the axis of the cylindrical basal body is referred to as rotational polarity (19,120).
Explants consisting of flat-mounts of the ventral part of the third ventricle (v3V) show in ex vivo cultures very intricate flow patterns that are organized into eight modules. Within each of the modules, fluid is transported uniformly but flow directions differ between modules (18).
Aim 1 of this thesis was to investigate whether the flow pattern observed ex vivo reflects the in vivo flows. This was achieved by determining, throughout the v3V, translational and rotational polarities and hence cilia beating directions. In addition, we analyzed the cellular expression pattern of the PCP protein Vangl1 in ependymocytes. By means of automated confocal microscopy and custom-made segmentation analysis, PCP and Vangl1 expression sites of >20.000 ependymocytes of the v3V wall were determined in multiple freshly sacrificed mice. Both, the localization of Vangl1 within ependymocytes and translational and rotational polarities presage modular an in vivo flow pattern in the v3V that strongly resembles that seen in ex vivo v3V explants.
Aim 2 was to search for complex features in the architecture of the v3V beyond the PCP. We computed the apical surface area of ependymocytes, the area and shape of cilia bundles, and the length of the (CC) ⃗ vector. We discovered that ependymocytes of the cell morphology in the v3V are heterogeneous in (large and small cells, elongated and round cilia bundles etc.). However, cells of a given characteristic are found in the same location of the v3V of all mice analyzed. For example, there are elongated arrays of large surface area ependymocytes in the rostral part of the v3V that extend from the site of CSF influx to the ventral edge of the v3V.
Aim 3 was to search for temporal changes in the flow directions in the v3V. We discovered that over the span of a mouse circular arrays of cilia driving a whirl-like flow emerge at particular sites. In addition, and specific for the left-side of the v3V wall, the flow pattern changes in several of the eight modules. A particular striking example was seen in the ventral part of flow 6. Prior to the switching, cilia beat towards the posterior end of the v3V, while after the switch, they beat in the opposite direction. Interestingly, this switch would alter the CSF flow from one targeting the tanycytes to one leading away from the tanycytes.
Aim 4 was to identify genes that are involved in the temporal changes of CSF flow in the v3V. We made the important observation that the above-mentioned switches in flow directions do not occur in Per1-/- Per2-/- double mutants. While wild type individuals develop a left-side asymmetry in the fifth week, in Per1-/- Per2-/- double mutants, the flow pattern remains symmetrical. Hence, on both walls flow persistently direct CSF towards the tanycyte region. Period genes are key regulators of circadian timing and influence a vast variety of physiological and pathophysiological processes in postnatal animals.
In conclusion, this thesis shows that the v3V architecture and the polarization of its cells is more complex than that of other tissues with multiciliated cells. This complexity is undoubtedly established during v3V development. We propose that the complex flow pattern suggests the existence of a novel way communication in the brain in which solutes are transported to particular regions by means of directionally beating cilia bundles. The timed switch of the orientation of cilia beating in flow 6 supports this hypothesis and shows that in each area planar polarity can be regulated independently from the rest. Furthermore, it exemplifies how ependymal development occurs in juvenile mice and may be controlled, at least in part, by circadian clock genes. | de |