<i>apol1</i> interacts with <i>myh9</i> in an anemic context.

<p>To test for epistatic effects of <i>apol1</i> and <i>myh9</i> in zebrafish, we first co-injected both <i>apol1-</i>MO (1.0ng/nl dose) and <i>myh9-</i>MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between <i>apol1</i> and <i>myh9</i> in the context of <i>atpif1a</i> suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of <i>apol1</i> and <i>myh9</i>. (D, F) These data are reproduced using butafenacil induced anemia (0.195 μM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (<i>n =</i> 19); <i>atpif1a</i> MO injected (<i>n =</i> 14); <i>apol1-</i>MO+<i>myh9</i>-MO (<i>n =</i> 12); <i>apol1-</i>MO+<i>myh9</i>-MO+<i>atpif1a</i>-MO (n = 11); Butafenacil (n = 48); But+<i>myh9-</i>MO+<i>apol1-</i>MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.</p

Comparison of APOL1 human and zebrafish protein sequences and relevance to the zebrafish kidney.

<p>Protein domain schematic of (A) zebrafish APOL1 and (B) human APOL1 is shown, with zebrafish domains (NP_001025309) aligned to the human protein (NP_001130012) and coded based on summarized consensus scores (Gonnet PAM 250 matrix, Clustal Omega, Cambridge, UK; <i>S</i>, secretory domain, <i>PFD</i>, pore-forming domain, <i>B</i>, BH3 domain, <i>MAD</i>, membrane-addressing domain, <i>SRA</i>, serum resistance-associated binding domain). Prominent regions of the human and zebrafish alignments are expanded, including the (C) BH3 domain and (D) SRA binding domain, and consensus symbols are displayed (* (asterisk), fully conserved;: (colon), >0.5 in the Gonnet PAM 250 matrix;. (period), = <0.5 in the Gonnet PAM 250 matrix). The leucine zipper domain (codons 365–392 in <i>APOL1</i>, underline), and the location of the G1 and G2 risk alleles in CKD in African Americans (S342G/I384M and ΔN388Y389) are highlighted in red. (E) Podocytes from adult glomeruli of <i>pod</i>::NTR-mCherry zebrafish were flow-sorted and evaluated for <i>apol1</i> RNA expression through RT-PCR. <i>apol1</i> is expressed in fluorescence-activated cell sorted (FACS) podocytes and the adult liver. FACS podocytes also express zebrafish <i>podocin</i> (<i>nphs2)</i> but a purkinje-cell marker, <i>wdr81</i>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005349#pgen.1005349.ref029" target="_blank">29</a>], was undetectable. NT = non-template reverse transcription control; L = dissected adult liver cells from <i>pod</i>::NTR-mCherry zebrafish; P = fluorescence-activated cell sorted podocytes from dissected glomeruli of <i>pod</i>::NTR-mCherry zebrafish; Em = 5 dpf whole-zebrafish embryo cDNA.</p

<i>apol1</i> morphant zebrafish embryos display generalized edema and glomerular filtration defects indicative of nephropathy.

<p>Representative live images of (A) sham-injected control larvae, and (B) <i>apol1</i> morpholino (MO) injected larvae at 5 dpf. <i>apol1</i> morphants display pericardial and yolk sac edema. (C) Injection of increasing doses of <i>apol1</i>-MO demonstrate dose-dependent effects when scored for generalized edema (<i>n</i> = 35–65 embryos/injection; repeated three times) compared to control larvae at 5 dpf. <i>apol1</i> morpholino injected embryos were complemented with the respective human mRNA to <i>APOL1</i> (100pg/nl) and scored for generalized edema at 5 dpf. (D) Ectopic expression of <i>APOL1</i> rescues significantly the edema phenotype observed in <i>apol1</i> morphants (1.0 ng/nl dose). We observed no significant phenotypes when <i>APOL1</i> human mRNA is injected alone. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hpi. (E) Representative eye image series of zebrafish larvae for each injection group show a relatively stable or a decrease in fluorescence intensity over time compared to sham-injected controls. (F) Bar graphs summarize the fluorescence changes observed for each injection group for <i>apol1</i> morphant larvae. Reduction in fluorescence intensity over the pupil was calculated relative to the 24 hpi time point; <i>apol1</i> morphants display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, indicative of compromised glomerular filtration and proteinuria. These defects were rescued significantly when MO was co-injected with orthologous human mRNA. (G-I) Compared to (G) sham-injected controls, the glomerular ultrastructure of (H) <i>apol1</i> morphant zebrafish display partial effacement of podocyte foot process (* asterisks), although the glomerular basement membrane (filled arrowheads) appears normal. Microvillus protrusions (open arrowheads) are also apparent in the urinary space. (I) Ultrastructure defects are rescued upon co-injection of human wild-type mRNA (100pg). Scale bar, 500nm. White bars, normal; black bars, edema. MO concentrations are in μg/μl, with 1nl injected into each embryo. C, sham-injected control; NI, non-injected control. Dextran values are in relative fluorescent intensity, mean ± SE. Control, sham-injected control (<i>n =</i> 29); MO, <i>apol1</i> morpholino injected (<i>n =</i> 26); <i>apol1-</i>MO+mRNA (<i>n =</i> 28). h.p.f., hours post-fertilization; h.p.i., hours post-injection. *p<0.001.</p

<i>In vivo</i> modeling of human <i>APOL1</i> variants associated with disease.

<p><i>apol1</i> MO injected larvae were complemented with the respective human mRNA corresponding to <i>APOL1</i> G1 (S342G/I384M) (100pg/nl) and G2 (100pg/nl) risk variants and scored for edema formation at 5 dpf (n = 26–65 embryos/injection; repeated three times). (A, B) Neither risk variant of <i>APOL1</i> rescues significantly the edema phenotype observed in <i>apol1</i> morphants. However, when human <i>APOL1</i> G2 mRNA was injected alone (B), a significant number of embryos develop edema compared to sham-injected controls, suggesting a possible dominant-negative effect of the G2 altered protein. (C, D) <i>apol1</i> morpholino injected larvae were complemented with human mRNA corresponding to either (C) <i>APOL1</i> G1 I384M or (D) <i>APOL1</i> G1 S342G and scored for edema formation at 5 dpf (n = 48–93 embryos/injection; repeated two times). Each individual variant comprising <i>APOL1</i> G1 risk rescues significantly edema formation in <i>apol1</i> morphant embryos, suggesting that both G1 variants must be present to confer loss of APOL1 function. (E-F) <i>apol1</i> morphants co-injected with human <i>APOL1</i> G1 or G2 mRNA fail to rescue filtration defects as indicated by dextran clearance, while larvae injected with G2 mRNA alone display increased clearance over time. (G) Titration of G2 injected embryos with increasing concentrations of human WT <i>APOL1</i> mRNA show a significant reduction in edema formation of developing embryos at 5 dpf. (H) Zebrafish embryos injected with <i>APOL1</i> G2 mRNA (100pg/nl) alone display glomerular aberrations similar to that of <i>myh9</i> suppressed larvae, with microvillus protrusions present (open arrowheads), although the glomerular basement membrane appears normal (filled arrowheads). Podocyte foot processes (* asterisk) are apparent, although sparsely present. (I) Embryos injected with <i>APOL1</i> G1 mRNA (100pg/nl) alone display normal glomerular ultrastructure. Scale bar, 500nm. White bars, normal; black bars, edema. C, sham-injected control; NI, non-injected control. *p<0.05.</p

<i>myh9</i> expression in the context of <i>apol1</i>/<i>APOL1</i> modulation.

<p>Zebrafish embryos were injected with either <i>apol1</i>-MO (1.0ng/nl dose), <i>APOL1</i> G1 (S342G:I384M) mRNA (100pg), or <i>APOL1</i> G2 (100pg) mRNA alone, in the absence (white bars) or presence (black bars) of <i>atpif1</i>α-MO. Total RNA at 5 dpf or 3 dpf (<i>APOL1</i> G2/<i>atpif1α</i>-MO embryos did not survive to 5 dpf) was extracted and reverse-transcribed with random primers to obtain whole-embryo cDNA. <i>myh9</i> expression was determined by quantitative real-time PCR and relative expression was calculated against <i>actb1</i>. (A) <i>apol1</i>-MO injected embryos do not display any significant changes in <i>myh9</i> expression compared to sham-injected control embryos. Additionally, <i>APOL1</i> G1 expression does not alter <i>myh9</i> expression alone, however, under <i>atpif1</i>α-induced anemia, we observe an increase in <i>myh9</i> expression. (B) <i>APOL1</i> G2 expression results in a significant decrease in <i>myh9</i> expression compared to sham-injected control embryos, suggesting that the altered APOL1 protein may regulate <i>myh9 in vivo</i>. (C) Co-injection of <i>APOL1</i> G2 (100pg) and human WT <i>MYH9</i> (n = 31–60; repeated two times), does not rescue edema formation caused by <i>APOL1</i> G2 expression in 5 dpf larvae, suggesting that the interaction between <i>APOL1</i> G2 and <i>MYH9</i> may be indirect. Relative expression values are mean ± SE in triplicate with two biological replicates. * = p<0.05; ** = p<0.01.</p

<i>apol1</i>-CRISPR F0 zebrafish embryos reproduce phenotypes observed in <i>apol1</i> morphants.

<p>(A) Schematic of the zebrafish <i>apol1</i> locus and location of the guide RNA (gRNA) target used for <i>apol1</i>-CRISPR experiments; the primers used to PCR-amplify the target region are shown (arrowheads). (B) At 1 dpf, a representative sampling of 8 founders and 8 non-injected controls were selected and subjected to T7 endonuclease 1 (T7E1) assay. The appearance of T7E1 fragments at ~180bp indicate positive gRNA targeting of exon 3 in the <i>apol1</i> locus. No T7E1 fragments were detected in non-injected control embryos. In total, 25 out of 41 founders subjected to T7E1 assay showed the presence of T7E1 fragments, indicating that ~61% of founders have insertion/deletions (indels) in the exon 3 region of <i>apol1</i>. (C) Multiple sequence alignment of <i>apol1</i> reference sequence (ENSDARG00000007425) to <i>apol1</i>-CRISPR variants generated from PCR amplification and subsequent TA cloning and sequencing of two representative <i>apol1</i>-gRNA/CAS9 injected founders. 13 PCR-cloned sequences are shown, representing four wild-type variants (c1-4) and all indel types detected among 50 PCR-clones (c5-13). Of 50 total PCR-clones sequenced, 31 showed detectable indels, representing an estimated 62% mosaicism in <i>apol1</i>-CRISPR/CAS9 injected founders. Lines mark the specific sequence targeted by the <i>apol1</i>-gRNA (exon3) and the location of the PAM recognition motif (i.e. TGG). (D) <i>apol1</i>-gRNA and CAS9 co-injected embryos were scored for edema formation at 5 dpf (n = 26–31 embryos/injection, repeated three times; *p<0.001). (E) <i>apol1-</i>gRNA and CAS9 co-injected embryos display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, similar to that of <i>apol1-</i>MO injected embryos (*p<0.001). Bar graphs summarize the changes for each injection group. Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19–21); <i>apol1</i>-gRNA+CAS9 (n = 11–17); <i>apol1</i>-gRNA alone (n = 13–14), repeated 2 times. (F) <i>apol1</i>-CRISPR/CAS9 injected embryos display podocyte foot process effacement at 5 dpf, similar to that of <i>apol1</i> morphant larvae. Ultrastructural defects appear less severe when compared to <i>apol1-</i>MO injected embryos, however, including less foot process effacement and the absence of microvilli in the urinary space. Filled arrowheads, glomerular basement membrane. Scale bar, 500nm.</p