<p>(A) Stage 16 embryos stained with antibodies to Odd-skipped (Odd) or Even-skipped (Eve) and Tinman (Tin) and β3 tubulin. (B) Wild type (<i>dKlf15</i><sup><i>+</i></sup>) and <i>dKlf15</i><sup><i>NN</i></sup> on a <i>Hand-GFP</i> background to mark cardiomyocytes and pericardial nephrocytes (arrows). (C) Number of Even-skipped, Odd-skipped and Tinman positive cells in stage 16 embryos; <i>n</i> = 8–12 embryos per genotype. (D) Number and size of nephrocytes in larvae at different stages. ***P<0.001; <i>n</i> = 8–14 larvae per genotype; (note, nephrocyte were too infrequent to quantify at L3 stage). (E) Ultrastructure of nephrocytes from L3 stage wild type (<i>dKlf15</i><sup><i>+/+</i></sup>) and mutant <i>dKlf15</i><sup><i>NN</i></sup> larvae. Arrows indicate slit diaphragms. Scale bars = 5 μm (upper panels); = 1 μm (lower panels). (F) <i>Hand-GFP</i> wild type and <i>dKlf15</i><sup><i>NN</i></sup> lines expressing a <i>sticks and stones</i> reporter (red). Nephrocytes in L1 (arrows); nephrocytes in L3 (asterisks); grey line defines the heart. (G) Nuclear morphology of <i>Hand-GFP</i>-positive cells co-stained with wheat germ agglutinin (red), scale bar = 25 μm. (H) Micrographs show the Hand-GFP fluorescence signal of the ‘wing hearts’ (arrows) seen through the cuticle of the scutellum of pupa.</p
