Adult pericardial nephrocyte function is dependent on <i>dKlf15</i>.

<p>(<b>A & B</b>) <i>dKlf15</i> was conditionally silenced in adult nephrocytes using Hand-Target (<i>Target</i>) to drive <i>UAS</i>-<i>dKlf15</i>^<i>RNAi</i>. Flies were reared at 18°C until eclosion then either maintained at 18°C (<b>A’, B’;</b> no knock-down) or placed at 29°C (<b>A”</b>, <b>B”</b>; RNAi permitted) for 4 days. Adult hearts were then stained with wheat germ agglutinin (red) and antibodies to the nephrocyte marker Amnionless (green). Scale bars = 100 μm. (<b>C</b>) Quantification of Amnionless fluorescence signal. ***P<0.001; <i>n</i> = 8 hearts per genotype. (<b>D</b>) Pericardial nephrocytes in semi-intact heart preparations after incubation with fluorescently tagged 10 kDa dextran (green) for 5 or 30 minutes. Nephrocytes of all genotypes associated with dextran when adults were kept at the non-permissive temperature of 18°C (<b>D’</b>), whereas at 29°C, the permissive temperature for silencing, the <i>dKlf15</i> RNAi line could no longer associate with dextran (<b>D”</b>). (<b>E</b>) Quantification of fluorescence after incubation for 0, 5 or 30 minutes with fluorescently-tagged. *P<0.05, ***P<0.001compared to control (<i>Target</i> genotype), <i>ns</i> = not significantly different from control; <i>n</i> = 12–16 nephrocytes from 4 individual flies per genotype, per time-point. (<b>F</b>) Control (<i>Target</i> flies outcrossed to <i>w</i>^<i>1118</i>, (F’)) and <i>Target</i> > <i>dKlf15</i>^<i>RNAi</i> (F”) flies reared at 18°C then transferred to 29°C for 4 days and fed silver nitrate for one week. Upper and middle panels show the pericardial nephrocytes in control flies containing silver (brown pigment, arrows) but not when <i>dKlf15</i> had been silenced (arrowheads). Scale bar = 100μm and 50μm. Lower panels show that pericardial nephrocytes of both genotypes could still be identified with wheat-germ agglutinin (red). Scale bar = 20μm. (<b>G &H</b>) Quantification of nephrocytes and the percentage of nephrocytes containing silver nitrate. <i>ns</i> = not significantly different from the control, ***P<0.001; <i>n</i> = 8 flies per genotype.</p

<i>dKlf15</i> regulates the post-embryonic differentiation of pericardial nephrocytes.

<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>^<i>+</i>) and <i>dKlf15</i>^<i>NN</i> 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>^<i>+/+</i>) and mutant <i>dKlf15</i>^<i>NN</i> 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>^<i>NN</i> 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

<i>dKlf15</i> is critical for nephrocyte development.

<p>(A) Schematic of the <i>dKlf15</i> gene. An allele of <i>dKlf15</i>, originally called <i>Bteb2</i>^{<i>f06447</i>}, and now renamed <i>dKlf15</i>^<i>NN</i>, contains a piggyBac insertion (white box) within the coding region. The red bars define the primer sequence used to detect <i>dKlf15</i> expression. (B) <i>dKlf15</i> expression in adult heart from control (<i>CaS</i>) and mutant (<i>dKlf15</i>^<i>NN</i>) flies. (C) Phase contrast micrographs of the heart from wild type (C’) and <i>dKlf15</i>^<i>NN</i> mutant females (C”) overlaid with fluorescence images of a Hoechst-stained adult heart; nuclei are re-coloured black against a white background. Arrows indicate pericardial nephrocytes, HT = heart tube; scale bar = 50 μm. (D) Micrographs of Hoechst-stained larval oesophagus (OE) at the point it meets the proventriculus (PV) in wild type and <i>dKlf15</i>^<i>NN</i> mutants (D’ % D”, respectively); GC = garland cells (arrow); scale bar = 25 μm. (E) Adult heart from wild type (E’), <i>dKlf15</i>^<i>NN</i> mutants (E”) or flies where <i>dKlf15</i> had been silenced using either <i>dot-Gal4</i> or <i>Hand-Gal4</i> (E”‘ & E”“, respectively), stained with wheat-germ agglutinin (red) and phalloidin^FITC (green); arrow indicates pericardial nephrocytes; scale bar = 100 μm. (F) Adult heart stained with antibodies to Amnionless (green) and phalloidin (red); hearts were from female flies heterozygous for the mutant <i>dKlf15</i>^<i>NN</i> allele and a wild type copy of the allele (F’) or heterozygous for the <i>dKlf15</i>^<i>NN</i> mutant allele and a deletion spanning the <i>dKlf15</i> locus (F”; (<i>Df(1)ED6727</i>)); HT = heart tube.</p

Survival of <i>dKlf15</i> mutants.

<p>(A) Survival of larvae on yeast containing different amounts of silver nitrate (AgNO3); ***P<0.001; <i>n</i> = 3 independent trials starting with 20 eggs per trial. (B) Survival of adult males on control (circles) or AgNO₃ diet (squares); <i>n</i> = 85–120 flies per genotype (housed as 15 flies per vial). Genotype had no effect on lifespan or survival on diet containing silver nitrate (P>0.05). Silver nitrate was toxic to both genotypes (P<0.001, relative to flies on the control diet).</p

Rescue of the <i>dKlf15</i>^<i>NN</i> mutation.

<p>(A) Schematic showing genomic duplications. (B) Adult hearts stained with anti-Amnionless antibodies (green) and phalloidin (red). Male flies were hemizygous for the mutant <i>dKlf15</i> allele (B’); or hemizygous for the mutant allele and a genomic duplication on the third chromosome corresponding to either side of the <i>dKlf15</i> locus (<i>Dp(120)</i> and <i>Dp(122</i>); B” and B”‘, respectively) or covering the <i>dKlf15</i> locus (<i>Dp(473)</i>; B”“); scale bar = 100 μm.</p

<i>dKlf15</i> expression is restricted to nephrocytes.

<p>(A’ & A”) Schematics showing location of the two nephrocyte populations in larval and adult <i>Drosophila</i>, the garland cells (GCs) and pericardial nephrocytes (PNs). Garland cells are at the interface between the paraventriculus (PV) and oesophagus (oe), whereas pericardial nephrocyte are either side of the heart tube (HT); A>P, anterior-posterior. (B’ & B”) Antisera raised to dKlf15 (but not the pre-immune sera, B”), locate to the nucleus of adult pericardial nephrocytes (arrow). (C) Two magnifications of the adult heart immunostained with anti-dKlf15 (1:10) and anti-Amnionless (1:100) antibodies. Arrows denote pericardial nephrocytes, the arrowhead indicates a cardiomyocyte nucleus. Scale bar = 20 μm. (D) The heart in a dissected L3 larva expressing the RedStinger fluorescent reporter driven by <i>dKlf15-Gal4</i> and stained with wheat germ agglutinin which is taken up preferentially by pericardial nephrocytes (blue); HT = heart tube; scale bar = 40 μm. (E) L2 larva stained with anti-Amnionless and anti-dKlf15 showing localisation of dKlf15 to the nucleus of Amnionless-positive pericardial cells (arrow). (F’-F”“) <i>dKlf15-Gal4</i> driven <i>RedStinger</i> expression in living larvae and a dissected adult. Fluorescence is seen in binucleate garland cells next to the paraventriculus (PV) in L3 larvae (arrows). Fluorescence was also detected in pericardial nephrocytes (asterisks) either side of the heart tube (HT) at L2, L3 and adult (Ad) stages. Scale bars = 50 μm.</p

<i>dKlf15</i> overexpression increases the number of functional nephrocytes in adults and prevents age-dependent nephrocyte dysfunction.

<p>(A) Pericardial nephrocytes from control (progeny of <i>Dot-Gal4</i> outcrossed to <i>w</i>^<i>1118</i>; A’) and <i>dKlf15</i> over-expressing flies (<i>Dot-Gal4; UAS-dKlf15</i>; A”) stained with anti-dKlf15 (1:100) and anti-Amnionless antibodies (1:100). Scale bar = 50 μm. (B’) Pericardial nephrocytes from control (progeny of <i>Dot-Gal4</i> outcrossed to <i>w</i>^<i>1118</i>; B’) and <i>dKlf15</i> over-expressing flies (<i>Dot-Gal4; UAS-dKlf15</i>; B”) incubated with 10 kDa dextran and wheat germ agglutinin (to label cell surfaces). Arrow indicates a group of smaller nephrocytes. Scale bar = 80 μm. (C’-C”“) Nephrocytes from one and seven-week-old flies, incubated with 10 kDa dextran (green) and wheat germ agglutinin (red). Arrows indicate nephrocytes that have accumulated dextran; arrowheads identify nephrocytes that have not accumulated dextran. (D) Quantification of nephrocyte numbers and the proportion which were functional as determined by their ability to bind dextran. *P<0.001 compared to wild type and control; ^<i>a</i> P<0.001 compared to one-week-old wild type and control; ^<i>b</i> P<0.001 compared to seven-week-old wild type and control; <i>n</i> = 8–12 flies per genotype / per time-point.</p