A High-Content Screen Reveals New Small-Molecule Enhancers of Ras/Mapk Signaling as Probes for Zebrafish Heart Development

Zebrafish is the preferred vertebrate model for high throughput chemical screens to discover modulators of complex biological pathways. We adapted a transgenic zebrafish line, Tg(dusp6:EGFP), which reports on fibroblast growth factor (Fgf)/Ras/Mapk activity, into a quantitative, high-content chemical screen to identify novel Fgf hyperactivators as chemical probes for zebrafish heart development and regeneration. We screened 10,000 compounds from the TimTec ActiProbe library, and identified several structurally distinct classes of molecules that enhanced Fgf/Ras/Mapk signaling. We chose three agents—ST020101, ST011282, and ST006994—for confirmatory and functional studies based on potency, repeatability with repurchased material, favorable whole organism toxicity, and evidence of structure–activity relationships. Functional follow-up assays confirmed that all three compounds induced the expression of Fgf target genes during zebrafish embryonic development. Moreover, these compounds increased cardiac progenitor populations by effecting a fate change from endothelial to cardiac progenitors that translated into increased numbers of cardiomyocytes. Interestingly, ST006994 augmented Fgf/Ras/Mapk signaling without increasing Erk phosphorylation, suggesting a molecular mechanism of action downstream of Erk. We posit that the ST006994 pharmacophore could become a unique chemical probe to uncover novel mechanisms of Fgf signaling during heart development and regeneration downstream of the Mapk signaling node

A Quantitative Systems Pharmacology Platform Reveals NAFLD Pathophysiological States and Targeting Strategies

Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence with a heterogeneous and complex pathophysiology that presents barriers to traditional targeted therapeutic approaches. We describe an integrated quantitative systems pharmacology (QSP) platform that comprehensively and unbiasedly defines disease states, in contrast to just individual genes or pathways, that promote NAFLD progression. The QSP platform can be used to predict drugs that normalize these disease states and experimentally test predictions in a human liver acinus microphysiology system (LAMPS) that recapitulates key aspects of NAFLD. Analysis of a 182 patient-derived hepatic RNA-sequencing dataset generated 12 gene signatures mirroring these states. Screening against the LINCS L1000 database led to the identification of drugs predicted to revert these signatures and corresponding disease states. A proof-of-concept study in LAMPS demonstrated mitigation of steatosis, inflammation, and fibrosis, especially with drug combinations. Mechanistically, several structurally diverse drugs were predicted to interact with a subnetwork of nuclear receptors, including pregnane X receptor (PXR; NR1I2), that has evolved to respond to both xenobiotic and endogenous ligands and is intrinsic to NAFLD-associated transcription dysregulation. In conjunction with iPSC-derived cells, this platform has the potential for developing personalized NAFLD therapeutic strategies, informing disease mechanisms, and defining optimal cohorts of patients for clinical trials

Heterotaxy patient cohorts analyzed by targeted and whole exome sequencing analyses.

<p><b>(A)</b> 25 heterotaxy (HTX) patients from Children’s National Medical Center were sequence analyzed with targeted ciliome sequencing (yellow) to identify cilia-related mutations. This includes 13 HTX patients with airway ciliary dysfunction (CD) and 12 patients with normal airway cilia function (without CD). This analysis identified <i>DNAH6</i> as the only candidate gene with mutations found exclusively in patients with CD (n = 2). <b>(B)</b> Whole-exome sequencing (pink) was conducted in 23 HTX patients from Cincinnati Children’s Hospital, with 1 patient identified with a novel homozygous <i>DNAH6</i> mutation. <b>(C)</b> <i>DNAH6</i> amplicon resequencing (blue) was conducted on 72 HTX patients from Tokyo Women’s Medical University and 54 patients from Children’s Hospital of Philadelphia. Of these 126 patients, 6 were found to have heterozygous <i>DNAH6</i> mutation. The latter 6 patients were further analyzed by whole exome sequencing (pink). In 5 of these patients, additional heterozygous mutations were found in other PCD genes including, 4 mutations in <i>DNAH5</i>.</p

<i>DNAH6</i> and PCD gene mutations identified in heterotaxy patients.

<p><i>DNAH6</i> and PCD gene mutations identified in heterotaxy patients.</p

<i>Dnah6</i> genetically interacts with <i>Dnai1</i> and <i>Dnah5</i> to cause heterotaxy and PCD.

<p><b>(A,B)</b> Embryos injected with subthreshold dose of <i>dnah6</i> and <i>dnai1</i> MO show increased heart looping defects compared with Ctrl MO injections (n = 177, p-value = 3.8x10^-8), or single injection of either <i>dnai1</i> (p = 9.29x10^-9) or <i>dnah6</i> (p = 2.04x10^-8) MO at the same MO dose (A). Similar results were observed with subthreshold <i>dnah5/dnah6</i> double MO knockdown (n = 82; p = 1.74x10^-5, Bonferroni corrected)<b>. (C,D)</b> Reciliating mouse airway epithelia from wildtype (+/+) and heterozygous (+/-) <i>Dnai1</i> knockout (C) or <i>Dnah5</i> mutant (D) mice show robust ciliation and ciliary motion (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s017" target="_blank">S5 Movie</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s018" target="_blank">S6 Movie</a>). 30nM <i>Dnah6</i> siRNA had no effect on ciliation or cilia motility in wildtype airway epithelia, but in heterozygous <i>Dnai1 or Dnah5</i> mutant airway, ciliation was reduced and ciliary motion was dyskinetic (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s017" target="_blank">S5 Movie</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s018" target="_blank">S6 Movie</a>). With 50nM siRNA, little or no cilia was seen in wildtype and heterozygous <i>Dnai1</i> or <i>Dnah5</i> mutant mouse airway.</p

<i>dnah6</i> morpholino knockdown in zebrafish embryo cause laterality defects.

<p><b>(A,B)</b><i>dnah6</i> MO injected embryos exhibited curly tail and cardiac edema phenotype at 48 hours post fertilization (hpf). <b>(C,D)</b> <i>dnah6</i> MO injected embryos at 48hpf exhibited heart (C) and gut (D) looping defects, including failure to loop (middle panels) and reversal looping (right panels). <b>(E)</b> In <i>situ</i> hybridization analysis revealed abnormal right sided (R), bilateral (B), and absent (Ab) <i>spaw</i> expression after <i>dnah6</i> MO knockdown. <b>(F,G)</b> Heart/gut looping defects were observed in <i>dnah6 MO</i>-injected embryos, including normal (N), right sided (R), and straight (St) heart/gut looping phenotypes. <b>(H).</b> KV cilia were shorter (middle) in <i>dnah6</i> AUG-MO but not <i>dnah6</i> spl-MO injected embryos (p-value = 0.0017). <b>(I)</b>. High-speed videomicroscopy showed reduction in KV motile cilia of <i>dnah6</i> MO injected embryos (p = 1.6x10^-4 for 7.5 ng and p = 1.1x10^-5 for 10 ng <i>dnah6</i> MO; two-tailed Student’s t-test). <b>(J)</b> Little bead movement (color tracing) was observed in <i>dnah6</i> MO injected embryos. <b>(K)</b> KV flow was absent or reduced with <i>dnah6</i> knockdown (Chi-square test, p-value = 0.0192).</p

The complex genetics of hypoplastic left heart syndrome.

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD. Nat Genet 2017 Jul; 49(7):1152-59