Journal: bioRxiv

Article Title: The level of HAND1 controls the specification of multipotent cardiac and extraembryonic progenitors

doi: 10.1101/2024.08.15.607916

Figure Lengend Snippet: A) Transcription factor motif enrichment analysis of ATAC-seq data from sorted cells at day 7.5. The results are from an input of all regions of open chromatin uniquely identified in each of the 4 population classes as indicated by the colored boxes and based only on SOX17-Tom and NKX2-5-GFP status. For each motif, the average background value is indicated by the dotted line. Significance above background for any given population *p<1E-6. B) Typical EBs at day 10 under control or SB-treated (day 2–3) conditions, from wild-type, YAP1-null, WT1-null, HOXB1–3-null, and HAND1-null hESCs. The brightfield image is overlayed by the NXK2-5-GFP signal, with quantification by flow cytometry shown in C). Each point displays the result of an independent biological experiment. Data are represented as mean ± SD (n=3–7). Significance between any population was assessed by One-Way ANOVA with Tukey’s multiple comparison test *p<0.05. D) Typical flow cytometric analysis of GFP fluorescence in wild-type and HAND1-null EBs at day 10 with and without SB treatment. E) Immunostaining of whole mount day 12 EBs stained for α-actinin (red) and WT1 (green). Scale bars represent 300 μm. Con, control/vehicle; EB, embryoid bodies. See also Figure S2 and Figure S3 .

Article Snippet: The lentiviral packaging vectors (pMDLgp.pRRE [Addgene ID 12251]; pMD.G [Addgene ID 12259] and pRSV-Rev [Addgene ID 12253]), together with the transgene-containing destination vector (TO-HAND1 as described above or TO-MYC [Addgene ID 19775]), were transfected into Lenti-X 293T cells (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Control, Flow Cytometry, Comparison, Fluorescence, Immunostaining, Staining

Journal: bioRxiv

Article Title: The level of HAND1 controls the specification of multipotent cardiac and extraembryonic progenitors

doi: 10.1101/2024.08.15.607916

Figure Lengend Snippet: MNN-UMAP plots of A) wild-type and B) HAND1-null cells from day 3–10 of differentiation colored by cell type. The dashed red lasso highlights the loss of epicardial and fibroblast-like cells in the HAND1-null. A’) The frequency of each cell type by day in wild-type and B’) HAND1-null. C) The levels of prominent markers identifying each population in wild-type cells.

Article Snippet: The lentiviral packaging vectors (pMDLgp.pRRE [Addgene ID 12251]; pMD.G [Addgene ID 12259] and pRSV-Rev [Addgene ID 12253]), together with the transgene-containing destination vector (TO-HAND1 as described above or TO-MYC [Addgene ID 19775]), were transfected into Lenti-X 293T cells (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: The level of HAND1 controls the specification of multipotent cardiac and extraembryonic progenitors

doi: 10.1101/2024.08.15.607916

Figure Lengend Snippet: A-D) UMAP plots from Monocle3: A) colored by cell type in the wild-type and C) in the HAND1-null line; A’) colored by pseudotime with predicted trajectories overlayed, in the wild-type and C’) in the HAND1-null line. Endpoint cell types and FHF/JCF- and SHF-like populations have been annotated and their marker frequencies shown. B) Gene expression of lineage and cell type markers for the wild-type and D) HAND1-null lines. E) SCENIC GRN analyses of cardiac progenitors extracted from the two lineage trajectories of the wild-type differentiation and the single lineage of the HAND1-null trajectory. F) Activity of selected regulons enriched in cardiac progenitor lineages mapped to UMAP plots of wild-type and HAND1-null cells. White indicates the regulon activity is below threshold. The number of gene targets in each regulon is indicated following the name of the TF. G) Heatmap of relative regulon activity by cell line, cell type, and subpopulation, grouped by lineage and stage of enrichment. The cardiac progenitor data represent cells extracted from each lineage. *Identified in HAND1-null population. # Identified in CPC lineage analysis. DEG, differentially expressed genes; FHF, first heart field; JCF, juxta-cardiac field; SHF, second heart field; GRN, gene regulatory network; SCENIC, single-cell regulatory network interference and clustering. See also Figures S4 and S5 .

Article Snippet: The lentiviral packaging vectors (pMDLgp.pRRE [Addgene ID 12251]; pMD.G [Addgene ID 12259] and pRSV-Rev [Addgene ID 12253]), together with the transgene-containing destination vector (TO-HAND1 as described above or TO-MYC [Addgene ID 19775]), were transfected into Lenti-X 293T cells (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Marker, Expressing, Activity Assay

Journal: bioRxiv

Article Title: The level of HAND1 controls the specification of multipotent cardiac and extraembryonic progenitors

doi: 10.1101/2024.08.15.607916

Figure Lengend Snippet: A) Western blot of HAND1-AM-Tag (Active Motif) at day 3 and 4 of differentiation in different conditions compared to undifferentiated hESCs. B) Motif enrichment analysis of HAND1 ChIP-seq peaks with control (low HAND1) and SB (high HAND1) sets merged. Significance is relative to the background control. C) Doxycycline-inducible HAND1-BFP transgene expressed in HAND1-null cells for cell sorting by HAND1 expression (12-hour induction) and molecular analysis by ATAC- and RNA-seq (both performed on 3 biological replicates). C’) Euler plots for the ATAC-seq analysis representing the number of differentially accessible chromatin regions in HAND1+ vs HAND1-negative cells and the subset with detected HAND1 binding. D) Schematic illustration of the motifs enriched in chromatin with increased or decreased accessibility as a result of HAND1 expression. E) Identification of HAND1[+] (activating) and HAND1[-] (repressing) regulons. F) Chord plot showing significantly enriched gene ontology terms for HAND1 target genes. The red and blue text coloring indicates terms enriched in activated targets and repressed targets respectively. G) UMAP plots showing the activity of constant and HAND1-sensitive regulons in endoderm and mesoderm of wild-type and HAND1-null cells. The activity of the HAND1[+] and HAND1[-] regulons are shown with the domains of strong activity highlighted. H–J) ATAC-seq in HAND1-null and HAND1+ (12 h induction) populations, and HAND1 binding at low-HAND1 (CTRL) and high-HAND1 (SB) levels at the H) FOXF1 locus, I) HOXB cluster and J) CDX2 locus. K) Gene regulatory network model of HAND1. * Direct early target of HAND1. The dashed line indicates a link at high HAND1 levels. EB, embryoid bodies; CTRL, control / vehicle-only; DEG, differentially expressed genes; EMT, epithelial-mesenchymal transition; ExEm, extraembryonic mesoderm; GO, gene ontology; GRN, gene regulatory network; JCF, juxta-cardiac field; WT, wild-type. See also Figure S6 .

Article Snippet: The lentiviral packaging vectors (pMDLgp.pRRE [Addgene ID 12251]; pMD.G [Addgene ID 12259] and pRSV-Rev [Addgene ID 12253]), together with the transgene-containing destination vector (TO-HAND1 as described above or TO-MYC [Addgene ID 19775]), were transfected into Lenti-X 293T cells (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Western Blot, ChIP-sequencing, Control, FACS, Expressing, RNA Sequencing Assay, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: The level of HAND1 controls the specification of multipotent cardiac and extraembryonic progenitors

doi: 10.1101/2024.08.15.607916

Figure Lengend Snippet: A) Schematic illustration of HAND1 level with primitive streak-like patterning of hESCs into cardiac and extraembryonic mesoderm. B) Live images (brightfield and fluorescence) showing HAND1-Tomato in EBs at day 5 of differentiation. C) UMAP plots showing the target genes AUC score (regulon activity) for MYC and N-MYC with differentiation in wild-type cells. White indicates the score is below threshold. D) Target genes AUC score for MYC, N-MYC, PGC-1α (PPARGC1A) and SRF through pseudotime of mesoderm differentiation. E) Scaled target gene AUC scores in bulk populations by differentiation day and impact of induction of a dox-inducible MYC transgene at day 5–6. F) Expansion of HAND1-high, HAND1-low, and HAND1-neg progenitors from SB, control and DMH1-treated conditions respectively. HAND1-high progenitors were maintained in FGF8, BMP4, and CHIR; HAND1-low progenitors were maintained in FGF8, BMP4, and WNT3A, and HAND1-negative progenitors were maintained in FGF8-only (see Methods). Flow cytometric analysis of HAND1-Tom and NKX2-5-GFP after 6 days of expansion. G) Differentiation of progenitors to epicardial and endothelial cells assessed by immunostaining for WT1 and CD31, respectively and additionally by the flow cytometric analysis of CD31. H) Differentiation of progenitors to cardiomyocytes assessed by immunostaining for cTroponin T. I) Quantification of differentiated cell types by population. Data in I represent mean ± SD, n = 3 independent biological experiments. Scale bars represent 150 μm. AUC, area under the curve. EB, embryoid body; Epi, epicardial cells; Endo, endothelial cells; CM, cardiomyocyte. See also Figure S7 .

Article Snippet: The lentiviral packaging vectors (pMDLgp.pRRE [Addgene ID 12251]; pMD.G [Addgene ID 12259] and pRSV-Rev [Addgene ID 12253]), together with the transgene-containing destination vector (TO-HAND1 as described above or TO-MYC [Addgene ID 19775]), were transfected into Lenti-X 293T cells (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Fluorescence, Activity Assay, Control, Immunostaining