Osmium tetroxide

For scanning electron microscopy, leech specimens were treated with 16% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) or relaxation solution (4.8 mM NaCl₂, 1.2 mM KCl, 10 mM MgCl₂, 8% EtOH) while feeding or relaxing. After treatment, the head region containing the proboscis was cut and fixed in 4% PFA at room temperature overnight. The tissues were washed three times with PBT (1X PBS + 0.1% Tween-20) for 20 min at room temperature, and then fixed in 1% osmium tetroxide (Ted Pella Inc., Redding, CA, USA) in 1 M PBS for 1 h. Osmium tetroxide was removed by washing three times with PBT. Thereafter, the tissues were gradually dehydrated with ethanol (30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% in 1X PBS) for 20 min per step. Dehydrated tissues were treated with stepwise concentrated isopentyl acetate (Alfa Aesar, Ward Hill, MA, USA) (isopentyl acetate: EtOH = 1:3, 1:1, and 3:1) for 15 min per step, and then transferred to 100% isopentyl acetate. After the solution was removed, the samples were dried for 3 days in a fume hood. Dried samples were coated with gold particles and examined with an UltraPlus field emission scanning electron microscope (Carl Zeiss, Oberkochen, BW, Germany).

Small fragments of kidney tissue were fixed in 2% glutaraldehyde (Sigma), then post-fixed with 1% osmium tetroxide (Ted Pella Inc.), embedded in EMBed-812 resin (EMS, Hatfield), cut, stained, and analyzed under transmission electron microscopy (Philips Tecnai 12 Bio TWIN) at 80 kV, with standard magnifications between ×2,500 and ×16,500.
The electron micrographs of every animal were analyzed by setting the percentage of capillary loops with foot process effacement usually along with microvillous transformation of the podocyte and irregularities of the outer surface of the glomerular basement membrane. Then, a comparison was made between micrographs of the same magnification in the different groups of animals.
Images were analyzed in blind by a specialist pathologist.

For transmission electron microscopy (TEM), samples were fixed quickly for 24 hours in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri, USA), 2.5% glutaraldehyde (Ted Pella, Redding, California, USA), and 0.003% CaCl2 (Sigma-Aldrich) in sodium cacodylate buffer (Sigma-Aldrich), post-fixed in 2% osmium tetroxide (Ted Pella), dehydrated in an acetone dilution series (30%, 50%, 70%, and 90%, followed by 3 × 100%), and embedded with Eponate 12 resin (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Sections for TEM were cut to a thickness of 70 nm with diamond knives and a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany). The sections were stained with either 2% (w/v) aqueous uranyl acetate and 1% (w/v) lead citrate. Specimens were viewed with a TecnaiG2 Spirit 120kV (Thermo Fisher, Waltham, Massachusetts, USA) transmission electron microscope.
Due to size variability between wild-type and eas-1(lf) animals (S1B Fig), the relative position of the AMsh cell body on the animal was used to determine where to section the animals. The relative position of the AMsh cell body was measured to be consistent and similar between both genotypes, at around 0.12 times the total length of the animal from the nose tip (S1J Fig). Sections around this region were used for TEM imaging, and the AMsh cell bodies were identified in reference to micrographs from WormAtlas [76 ].