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ALPK2 Promotes Cardiogenesis in Zebrafish and Human Pluripotent Stem Cells.

ABSTRACT: Cardiac development requires coordinated biphasic regulation of the WNT/β-catenin signaling pathway. By intersecting gene expression and loss-of-function siRNA screens we identified Alpha Protein Kinase 2 (ALPK2) as a candidate negative regulator of WNT/β-catenin signaling in cardiogenesis. In differentiating human embryonic stem cells (hESCs), ALPK2 is highly induced as hESCs transition from mesoderm to cardiac progenitors. Using antisense knockdown and CRISPR/Cas9 mutagenesis in hESCs and zebrafish, we demonstrate that ALPK2 promotes cardiac function and cardiomyocyte differentiation. Quantitative phosphoproteomics, protein expression profiling, and β-catenin reporter assays demonstrate that loss of ALPK2 led to stabilization of β-catenin and increased WNT signaling. Furthermore, cardiac defects attributed to ALPK2 depletion can be rescued in a dose-dependent manner by direct inhibition of WNT signaling through the small molecule XAV939. Together, these results demonstrate that ALPK2 regulates β-catenin-dependent signaling during developmental commitment of cardiomyocytes.

SUBMITTER: Hofsteen P

PROVIDER: S-EPMC5993047 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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ALPK2 Promotes Cardiogenesis in Zebrafish and Human Pluripotent Stem Cells.

Hofsteen Peter P Robitaille Aaron Mark AM Strash Nicholas N Palpant Nathan N Moon Randall T RT Pabon Lil L Murry Charles E CE

iScience 20180401

Cardiac development requires coordinated biphasic regulation of the WNT/β-catenin signaling pathway. By intersecting gene expression and loss-of-function siRNA screens we identified Alpha Protein Kinase 2 (ALPK2) as a candidate negative regulator of WNT/β-catenin signaling in cardiogenesis. In differentiating human embryonic stem cells (hESCs), ALPK2 is highly induced as hESCs transition from mesoderm to cardiac progenitors. Using antisense knockdown and CRISPR/Cas9 mutagenesis in hESCs and zebr ...[more]

PMID: 29888752

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Project description:BackgroundStudying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction.MethodsWe examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.ResultsThe combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway.ConclusionsVPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

Dataset Information

ALPK2 Promotes Cardiogenesis in Zebrafish and Human Pluripotent Stem Cells.

Publications

ALPK2 Promotes Cardiogenesis in Zebrafish and Human Pluripotent Stem Cells.

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