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. 2016 Mar 1;30(5):535-52.
doi: 10.1101/gad.274142.115.

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

Nathan P Young  1 , Anwesh Kamireddy  1 , Jeanine L Van Nostrand  1 , Lillian J Eichner  1 , Maxim Nikolaievich Shokhirev  2 , Yelena Dayn  3 , Reuben J Shaw  1
Affiliations

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

Nathan P Young et al. Genes Dev. .

Abstract

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation.

Keywords: AMPK; Tfeb; Wnt; embryonic stem cells; endoderm; germ layer specification; lysosomes.

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Figures

Figure 1.
Figure 1.
Dynamic AMPK signaling during EB differentiation and generation and characterization of AMPK double-knockout ESCs. (A) RT-qPCR analysis of AMPK subunits during EB differentiation. γ3 was not detected at any time point. Data are from two independent experiments. Bar graphs depict mean ± SEM. (B) ESCs and differentiating EBs were lysed on the indicated days 1 h after a medium change and subjected to Western blotting with the antibodies listed. (C) Western blot analysis of AMPKα1 and AMPKα2 in wild-type (WT) parental and two independent AMPK double-knockout (DKO) CRISPR clones. (D) Immunoblot on lysates from wild-type and AMPK double-knockout ESCs following vehicle or 1 h of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) treatment (0.5 mM). (E) Levels of pluripotency markers Oct4 and Nanog in wild-type and AMPK double-knockout ESCs as determined by Western blot. (F) Bright-field images of wild-type and two AMPK double-knockout ESC lines grown on feeders indicating normal ESC-like morphology (panel i) and equivalent amounts of alkaline phosphatase activity between the different genotypes (panel ii). Bar, 100 µm. (G) Proliferation curves of wild-type and AMPK double-knockout ESCs grown in the absence of feeders in both high glucose (HG; 25 mM) and low glucose (LG; 2.5 mM). n = 2 samples per condition.
Figure 2.
Figure 2.
Differentiation defects of AMPK double-knockout ESCs. (A) Bright-field images of representative wild-type (WT) and AMPK double-knockout (DKO) EBs differentiated for 12 d in high-glucose conditions. Bar, 100 µm. (B) Hematoxylin and eosin (H&E)-stained sections of wild-type and AMPK double-knockout day 12 EBs in high glucose. Images at the right correspond to boxed sections from the left. Bar, 50 µm. (C) Gene set enrichment analysis (GSEA) plots from high-throughput whole-transcriptome sequencing (mRNA-seq) profiles of wild-type and AMPK double-knockout EBs differentiated for 12 d in high glucose. (Panel i). The top two plots correspond to general differentiation defects of AMPK double-knockout cells. (Panel ii). The bottom plots highlight endoderm (liver) versus ectoderm (neuronal) germ layer skewing in wild-type versus double-knockout EBs, respectively. (NES) Normalized enrichment score; (FDR) false discovery rate. (D) Elevated levels of Oct4 mRNA in day 12 AMPK double-knockout EBs compared with wild type, as determined by RT-qPCR. (***) P < 0.005; (*) P < 0.05, compared with wild type. Relative abundance of Oct4 was normalized to wild-type ESC levels. (E) Heat map depicting relative mRNA expression of several endoderm and ectoderm markers in wild-type and double-knockout EBs at day 8 (d8) and day 12 (d12) of differentiation in high and low glucose. Values were calculated from mRNA-seq data. (F) RT-qPCR analysis of selected endoderm (top) and ectoderm (bottom) markers in high-glucose day 12 EBs derived from either wild-type or AMPK double-knockout cells confirming germ layer skewing between different genotypes. Data are from three independent experiments. Average ± SEM is plotted. (*) P < 0.05; (#) P < 0.005, compared with wild type. (G) Immunofluorescence (IF) on fixed sections of wild-type and AMPK double-knockout samples after 12 d of EB differentiation in high glucose. GATA4 and Nestin served as endoderm and ectoderm markers, respectively. Bar, 50 µm. (H) Representative whole-mount fluorescence images of embryonic day 8.5 (E8.5) embryos that were injected with tdTomato-positive wild-type or AMPK double-knockout ESCs ex vivo at E2.5 prior to blastocyst implantation into recipient mothers. DAPI staining was performed to outline the embryo. Bar, 200 µm. Statistical significance was determined by Student's t-test.
Figure 3.
Figure 3.
AMPK double-knockout EBs exhibit defects in lysosome function and Tfeb regulation. (A) GSEA plot showing enrichment of the gene set associated with the KEGG term “lysosome” in wild-type (WT) versus AMPK double-knockout (DKO) day 12 EBs in high glucose. (B) Relative mRNA levels of selected lysosomal genes in wild-type and AMPK double-knockout day 12 EBs in high glucose as assessed by qRT–PCR. n = 3 independent experiments. (*) P < 0.05; (#) P < 0.005 compared with wild type. (C) Plot of corrected log-transformed P-values of KEGG lysosome pathway enrichment in sets of genes identified to be up-regulated in wild type versus double knockout under high-glucose conditions. At each time point (X-axis), the statistical significance of enrichment of genes associated with the KEGG term “lysosome” in wild-type versus AMPK double-knockout samples was determined as described in the Materials and Methods. The resulting P-values were log-transformed and are plotted on the Y-axis. Numbers in parentheses indicate the number of lysosomal genes significantly up-regulated in wild-type samples at each time point. (D) Direct fluorescence images of day 12 wild-type and AMPK double-knockout EBs after 1 h of incubation with DQ-BSA-Green followed by a 2-h chase prior to fixation. Bar, 100 µm. (E) Relative lysosomal activity, as determined by DQ-BSA assay, in day 12 wild-type and AMPK double-knockout EBs in high glucose. Results are from two independent experiments, with at least 10 EBs analyzed for each sample within an experiment. (****) P < 0.0001 compared with wild type . (F) Box and whisker plot of mRNA expression differences across 81 coordinated lysosomal expression and regulation (CLEAR) network genes in AMPK double-knockout versus wild-type samples at the indicated time points. High-glucose conditions only. See the Materials and Methods for details. (##) P < 10−8, one sample t-test for nonzero mean. (G) RT-qPCR analysis of Tfeb in day 8 wild-type and AMPK double-knockout EBs in high glucose. Three independent experiments were performed. (*) P < 0.05; (***) P < 0.0005 compared with wild type . (H) Immunoblot analysis of Tfeb in wild-type and AMPK double-knockout EBs differentiated in either high (HG) or low (LG) glucose for 8 d. (I) Time-course analysis of selected mTOR signaling components during high-glucose EB differentiation of wild-type and double-knockout ESCs. Lysates were immunoblotted with the indicated antibodies. (J) GSEA plot depicting enrichment of a “mTOR inhibition” gene set (derived from everolimus-treated vs. untreated mouse tissues) in wild-type versus double-knockout EBs at day 12. (K) Western blot analysis of day 6 wild-type and AMPK double-knockout EBs. After overnight incubation in the indicated glucose medium, EBs were treated with either vehicle (DMSO) or 500 nM INK128 for 2 h prior to lysate preparation and blotting with indicated antibodies. For standard bar graphs, average ± SEM is plotted. Statistical significance was determined by Student's t-test unless otherwise noted.
Figure 4.
Figure 4.
Proper lysosomal function is required for endoderm differentiation. (A) Immunoblot analysis of Tfeb in EBs derived from wild-type (WT) parental and two independent TfebMUT CRISPR clones. (B) Wild-type and TfebMUT ESCs underwent EB differentiation for 7 d in high (HG) or low (LG) glucose followed by RT-qPCR of selected CLEAR network genes. (*) P < 0.05; (#) P < 0.005 compared with wild-type LG. (C) Relative lysosomal activity, as determined by DQ-BSA assay, in wild-type and TfebMUT2 EBs. Data are from three independent experiments, with 10 EBs analyzed in each experiment. (****) P < 0.0001 compared with wild type . (D,E) RT-qPCR analysis of endoderm (D) and ectoderm (E) markers in day 7 EBs from wild-type and two TfebMUT clones. (*) P < 0.05; (#) P < 0.005 compared with wild type . (F,G) Wild-type ESCs were differentiated into EBs for 7 d in the presence or absence of 2.5 nM bafilomycin A (BafA). The compound was added daily. mRNA levels of selected endoderm (F) and ectoderm (G) genes were assessed by RT-qPCR. (*) P < 0.05; (#) P < 0.005 compared with DMSO. (H) IF analysis depicting wild-type EB-specific colocalization of GATA4, an endoderm marker, with the highly polarized staining of lysosomal marker Lamp2 in the outer layer of cells. Most EBs derived from AMPK double-knockout (DKO) ESCs lack appreciable staining throughout the structure, as shown in the bottom image. Bar, 50 µm. For B and D–G, data are from three independent experiments. For all bar graphs, average ± SEM is plotted. Statistical significance was determined by Student's t-test.
Figure 5.
Figure 5.
Tfeb overexpression corrects lysosomal defects and increases endodermal gene expression in AMPK double-knockout cells. (A) Schematic of wild-type (WT) Tfeb and caTfeb cDNA constructs. (TA) Transactivation domain; (bHLH) basic helix–loop–helix domain; (LZ) leucine zipper. S142 and S211 denote phosphorylation sites that control nuclear–cytoplasmic shuttling. (B) Western blots of representative GFP and caTfeb-expressing clones following stable transfection of cDNAs into an AMPK double-knockout (DKO) ESC CRISPR line. (C) RT-qPCR analysis of selected CLEAR target genes in wild-type parental and AMPK double-knockout clones expressing either GFP or caTfeb following 7 d of EB differentiation. (D) Direct fluorescence images of day 12 EBs derived from parental or caTfeb-expressing AMPK double-knockout ESCs after 1 h of incubation with DQ-BSA-Green followed by a 2-h chase prior to fixation. Bar, 100 µm. (E) Relative lysosomal activity, as determined by DQ-BSA assay, in day 12 wild-type and AMPK double-knockout (parental or caTfeb-expressing) EBs in high glucose. See the Materials and Methods for details. Results are from two independent experiments, with at least 10 EBs analyzed for each sample within an experiment. (****) P < 0.0001. (F,G) Wild-type parental and AMPK double-knockout ESCs expressing either GFP or caTfeb were differentiated for 7 d followed by RT-qPCR analysis of selected endoderm (F) and ectoderm (G) markers. C, F, and G show data from one representative experiment in which multiple GFP and caTfeb clones (n = 3 per cDNA) were analyzed and compared with wild-type parental controls. Similar results were observed in four independent experiments with additional independently derived cDNA clones. (*) P < 0.05; (#) P < 0.005; (^) P < 0.0005. For all bar graphs, average ± SEM is plotted. Statistical significance was determined by Student's t-test.
Figure 6.
Figure 6.
Attenuated canonical Wnt signaling resulting from lysosomal deregulation contributes to endodermal differentiation defects in AMPK double-knockout and TfebMUT cells. (A) GSEA plot depicting a β-catenin-associated gene set up-regulated in wild-type (WT) versus AMPK double-knockout (DKO) EBs following 4 d of differentiation. (B) Western blots showing dynamics of phospho-β-catenin during the EB differentiation time course of wild-type, AMPK double-knockout, and TfebMUT ESCs. ESCs, day 4 (d4) EBs, and day 7 (d7) EBs were harvested and immunoblotted with the indicated antibodies. (C) Wild-type ESCs were differentiated into EBs in the presence of vehicle (DMSO) or 2.5 nM BafA for the indicated times before being processed and analyzed as in B. (D) IF of Wnt signaling component GSK3β and lysosomal marker Lamp2 in day 8 EBs of the indicated genotypes. Note the subcellular colocalization of GSK3β/ Lamp2 in several cells on the outer layer of wild-type EBs. Insets in the bottom right of each panel depict zoomed-in images from the boxed-in sections. Bar, 10 µm. (E) Immunoblot analysis of day 7 wild-type , AMPK double-knockout, and TfebMUT EBs treated daily with either vehicle (DMSO) or 3 µM CHIR99021 (CHIR). One hour after the last treatment, lysates were harvested and blotted with the indicated antibodies. (F) RT-qPCR analysis of the endoderm marker Sox17 in EBs from E (DM-DMSO). (*) P < 0.05 compared with wild-type DM; (ns) not significant. n = 4 independent experiments. (G) IF analysis of GATA4 in day 8 wild-type, AMPK double-knockout, or TfebMUT EBs either vehicle- or CHIR-treated throughout the differentiation protocol. Bar, 50 µm. The bar graph represents average ± SEM. Statistical significance was determined by Student's t-test.
Figure 7.
Figure 7.
Model depicting how AMPK regulates cell fate decisions through lysosome-dependent control of Wnt signaling. (A) In wild-type cells, AMPK inhibition of mTOR allows sufficient levels of Tfeb to enter the nucleus and transcriptionally up-regulate endolysosomes. This organelle system is required for optimal signaling through the canonical Wnt pathway through its ability to sequester the GSK3β-containing DC, freeing β-catenin from degradation. Active β-catenin then translocates into the nucleus to induce target genes, many of which are important mediators of endodermal differentiation, including Sox17. (B) In AMPK double-knockout cells, increased mTOR signaling blunts Tfeb activity, leading to a defective endolysosomal compartment. As a result, DC inhibition of β-catenin remains intact even in the presence of Wnt, preventing robust activation of endodermal genes.
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