Modeling a variant of unknown significance in the Drosophila ortholog of the human cardiogenic gene NKX2.5

Figures

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  Fig. 1.. Functional analysis of TinR321N in vitro. (A) Comparison of the homeodomain sequences of human (Hs) NKX2.5 and Drosophila melanogaster (Dm) Tinman (Tin). The location of the point mutation being analyzed is indicated. (B) Purified native proteins used for DNA-binding assays. Equivalent amounts of wild-type (WT) MBP-Tin (lane 3) and mutated MBP-Tin (lane 4) were purified. The purified MEF2 was used for a different project. (C) Electrophoretic mobility shift assay demonstrating a specific interaction between WT Tin and the Multiplexin (Mp) Tin-binding site. In the absence of added protein, the free probe has high mobility (lane 1), but a portion of this probe has reduced mobility in the presence of WT Tin protein (lane 2). This interaction is specific, because the intensity of the bound complex is reduced in the presence of a 300-fold excess of non-radioactive Tin sequence (lane 3) but not reduced in the presence of excess non-radioactive mutant (mt) sequence (lane 4). Representative gel from duplicate experiments. (D) Electrophoretic mobility shift assay testing the ability of WT MBP-Tin homeodomain (MBP-Tin HD) and MBP-TinR321N HD to bind to a consensus Tin-binding site in the Mp…

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  Fig. 2.. The Tin homeodomain interacts directly with Doc1. GST alone or fused to Doc1 (GST-Doc1) was immobilized as solid-phase bait on glutathione agarose, washed extensively, and subsequently incubated without or with 10 µM MBP alone or MBP-Tin (WT or R321N mutant) homeodomain (HD) prey proteins as indicated. Following incubation, reactions were extensively washed and resolved by SDS-PAGE. Owing to the overlapping molecular masses of GST-Doc1 and MBP-Tin, bound MBP-Tin was visualized in western blots using an anti-MBP antibody (top panel, arrowhead). Ponceau Red staining of the blot was done following western blot development to ensure equal GST and GST-Doc1 loading and transfer (bottom panel). Binding to GST was minimal for all MBP proteins. GST-Doc1 did not show a significant interaction with MBP alone but did bind both WT MBP-Tin and MBP-TinR321N at this single concentration. Binding to the MBP-TinR321N appeared reduced compared to that to WT MBP-Tin. Data for interactions with WT MBP-Tin are representative of five independent experiments. Data for interaction with MBP-TinR321N are from a single experiment, but were further investigated in Fig. 3.

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  Fig. 3.. The TinR321N isoform interacts with Doc1, but with a significantly reduced affinity. (A) WT and R321 Tin isoforms were purified to near homogeneity (arrow); 10 µg of each protein preparation is shown. (B) Equal amounts of GST-Doc1 were immobilized on glutathione agarose, washed extensively, and subsequently incubated in the presence of increasing concentrations (0-500 nM) of WT MBP-Tin or MBP-TinR321N. Following incubation, reactions were extensively washed and resolved by SDS-PAGE. Bound MBP-Tin proteins were visualized by western blot analysis using an anti-MBP antibody (left panels); GST-Doc1 was visualized using Ponceau Red staining of blots (right panels). WT MBP-Tin (top-left panel) bound robustly across the concentration range tested, whereas MBP-TinR321N (bottom-left panel) was significantly impaired in GST-Doc1 binding. (C) Saturation binding curves of the mean±s.d. from five independent experiments demonstrate the effects of R321N mutation on Doc1 binding. Bound MBP-Tin proteins were quantified in ImageJ and normalized to the total GST-Doc1 for respective samples. AU, arbitrary units.

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  Fig. 4.. Generation and analysis of a tinR321N allele in vivo. (A) Sequence reads of WT tin (top) and the tinR321N allele (bottom), from PCR products generated from homozygous adults. The Arg codon (5′-CGA) is altered to Asn (5′-AAC), and no other changes to the tin coding sequence were observed. (B) There were no observable changes in heart cell patterning of tinR321N homozygotes compared to vasa-Cas9 controls. Cardiac cells are labeled with anti-H15 (blue) and anti-Svp (green). A subset of pericardial cells is labeled with anti-Odd (red). n≥10 embryos for each genotype. (C) Larval hearts from the A6 abdominal segment of vas-Cas9 (control) and tinR321N homozygous larvae, labeled with anti-Alpha-actinin (also known as Actn; red) to visualize cardiac myofibrils. No consistent differences in heart structure were observed between control and mutant (representative images shown, see text for quantification). Scale bars: 50 µm.

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  Fig. 5.. tinR321N adults have defects in heart cell patterning. (A-B″) Whole-mount preparations of w1118 (control; A) and tinR321N homozygous (B) adult hearts, stained for accumulation of F-actin (also known as Act5C; green) and β-Integrin (red). Although the overall structure of the heart did not differ between genotypes based upon the F-actin accumulation, accumulation of Integrin was irregular in the mutants (B′) compared to controls (A′). In controls, a single prominent line of Integrin stain was observed along the ventral midline of the heart, traced by a blue line in A″. This line of stain was irregular in the tinR321N homozygotes (B″). To quantify the irregular nature of the line, the length of the blue line was calculated relative to that of a single straight line along the ventral midline (magenta in A″ and B″), to give a ratio of curve/straight. (C) The calculated ratio for w1118 (n=15) was significantly less than that for tinR321N (n=17), indicating that the line was more irregular in the mutants than in the controls. This phenotype was stronger in tinEC40/+ heterozygotes (n=7). Error bars represent s.e.m. (D) Combining tinR321N with tinEC40 to create tinR321N/tinEC40…