Figure 3. Sns and Duf are required for nephrocyte diaphragm formation and normal morphology.

a,b, sns (a, aⁱ) and duf,rst (b) embryonic garland cells lack diaphragms and lacunae. aⁱ, higher magnification of a, showing electron-dense subcortical material (arrowheads). Small lacunae (asterisk) lacking diaphragms are occasionally found (b, arrowhead). c,d, Wild-type (c) and duf (d) third instar garland cells. c, diaphragms (arrowheads) and lacunae (asterisk) densely populate the nephrocyte surface. d, duf nephrocytes have small lacunae (arrowheads) lacking diaphragms and a substantially thickened basement membrane (bm). e,f, SEMs of wild-type (e) and duf (f) third instar garland nephrocytes stripped of basement membrane by collagenase treatment. duf nephrocytes lack the furrows corresponding to diaphragm rows. g,h, Wild-type (g) and duf (h) Viking-GFP (collagen IV) third instar garland cells, stained with anti-GFP (green) showing greater Viking deposition around duf nephrocytes (arrowheads and inset). Garland cell number is also reduced in duf larvae, suggesting that mutant cells ultimately die. i,j, Diaphragm and foot process morphology are abnormal (arrowheads) in sns (i) and human nephrin (j) embryonic overexpression. Scale bars 200nm (a,c,d), 100nm (aⁱ,b), 50nm (i-j), 5μm (e,f). os, oesophagus, pv, proventriculus, tr, trachea.