A Drosophila toolkit for HA-tagged proteins unveils a block in autophagy flux in the last instar larval fat body.

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with a POI that harbors different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we have developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: 'HA Frankenbody'. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Mechanistically, lysosomal activity was downregulated at this stage, and endocytosis, but not autophagy, was indispensable for the swelling of lysosomes. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

A novel preparation for histological analyses of intraventricular macrophages in the embryonic brain.

Microglia colonize the brain starting on embryonic day (E) 9.5 in mice, and their population increases with development. We have previously demonstrated that some microglia are derived from intraventricular macrophages, which frequently infiltrate the pallium at E12.5. To address how the infiltration of intraventricular macrophages is spatiotemporally regulated, histological analyses detecting how these cells associate with the surrounding cells at the site of infiltration into the pallial surface are essential. Using two-photon microscopy-based in vivo imaging, we demonstrated that most intraventricular macrophages adhere to the ventricular surface. This is a useful tool for imaging intraventricular macrophages maintaining their original position, but this method cannot be used for observing deeper brain regions. Meanwhile, we found that conventional cryosection-based and naked pallial slice-based observation resulted in unexpected detachment from the ventricular surface of intraventricular macrophages and their mislocation, suggesting that previous histological analyses might have failed to determine their physiological number and location in the ventricular space. To address this, we sought to establish a methodological preparation that enables us to delineate the structure and cellular interactions when intraventricular macrophages infiltrate the pallium. Here, we report that brain slices pretreated with agarose-embedding maintained adequate density and proper positioning of intraventricular macrophages on the ventricular surface. This method also enabled us to perform the immunostaining. We believe that this is helpful for conducting histological analyses to elucidate the mechanisms underlying intraventricular macrophage infiltration into the pallium and their cellular properties, leading to further understanding of the process of microglial colonization into the developing brain.

CD206+ macrophages transventricularly infiltrate the early embryonic cerebral wall to differentiate into microglia.

The relationships between tissue-resident microglia and early macrophages, especially their lineage segregation outside the yolk sac, have been recently explored, providing a model in which a conversion from macrophages seeds microglia during brain development. However, spatiotemporal evidence to support such microglial seeding in situ and to explain how it occurs has not been obtained. By cell tracking via slice culture, intravital imaging, and Flash tag-mediated or genetic labeling, we find that intraventricular CD206+ macrophages, which are abundantly observed along the inner surface of the mouse cerebral wall, frequently enter the pallium at embryonic day 12. Immunofluorescence of the tracked cells show that postinfiltrative macrophages in the pallium acquire microglial properties while losing the CD206+ macrophage phenotype. We also find that intraventricular macrophages are supplied transepithelially from the roof plate. This study demonstrates that the "roof plate→ventricle→pallium" route is an essential path for microglial colonization into the embryonic mouse brain.