Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification

Abstract

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

Using human pluripotent stem cell differentiated endothelial cells to study the differential regulation in PAH

“…1d). Mesoderm cells treated with ROCK inhibitor is efficient to differentiate into skeletal myocytes [30], so we predicted that Y27632 in the process of EC differentiation plays the same role as their protocol. In order to further explore the inhibitor(Y27632)'s function, we designed a serial concentration gradient experiment (Fig.…”

Bmi1 inhibitor PTC-209 promotes Chemically-induced Direct Cardiac Reprogramming of cardiac fibroblasts into cardiomyocytes

“…3C), some of which playing pivotal roles in heart development. As the case of Iroquois homeobox gene 1 (Irx1), encoding a cardiac transcription factor important for the development of ventricular conduction system 20 ; Krüppel-like factor 5 (Klf5), a zinc finger-containing transcription factor involved in many different cellular processes, ranging from the governance of pluripotency of embryonic stem cell to regulation of cardiovascular pathophysiology [21][22][23] ; the transcriptional modulator Cited2, involved in Brachyury, Mesp1, Isl1, Gata4 and Tbx5 expression during cardiac differentiation of embryonic stem cells 24 ; Nuclear receptor subfamily 4, group A, member 2 (NR4A2), also known as Nurr1, a member of the NR4A orphan nucleus receptor family involved in the immediate early response to different stress-stimuli, with some roles in cardiac remodelling 25 ; Tbx6, a member of the evolutionarily conserved T-box family of transcription factors that are essential regulators of normal embryonic development, critical for mesoderm induction and subsequent lineage diversification by regulation of Nkx2-5 expression 26 ; JMJD2A/KDM4A, a member of the JmjC domain-containing family JMJD2 of histone demethylases that catalyse the demethylation of trimethylated H3K9 (H3K9me3) and H3K36 (H3K36me3), involved in promotion of cardiac hypertrophy 27 , as well as the c-Jun dimerization protein 2, JDP2, member of the basic leucine zipper (bZIP) superfamily which typically suppresses transcription through binding to CRE and TRE DNA promoter elements and recruiting histone deacetylases 28 ; Rp58 (also known Znf238, Zfp238, Zbtb18), a sequence-specific transcriptional repressor which inhibits Id genes (Id1-4) playing a central and evolutionarily conserved role during muscle formation 29 ; the ubiquitous protein PHF10, a subunit of the PBAF chromatin-remodelling complexes which plays crucial role in antagonizing Polycomb action during development 30 ; the forkhead box transcription factors Foxd1, which has been recently described as a promoter of iPSC generation 31 ; the myocyte-specific enhancer factor MEF2b 32 .…”

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries

“…Several methodologies have been employed for the molecular biological analysis of direct reprogramming. Comprehensive transcriptome analyses using RNA-seq have been performed frequently, and differential expression gene analysis between Continued on next page: 199) Transcription factor studies indicated that almost all of the reprogramming-related transcription factors played vital roles during the reprogramming process, e.g., by remodeling histone and DNA modifications, initiating the target transcription network, and erasing the transcriptional signature of the source cells. Recently, the activities of specific transcription factors, termed pioneer factors, seem pivotal for remodeling the chromatin states of cell conversion.…”

“…Recently, single-cell analyses, such as single-cell RNA sequencing (scRNA-seq), have been performed in many direct reprogramming studies. 149),181),187),192), 199) These studies have clarified many features of direct reprogramming processes. For example, a limiting step and an obstruction factor, 187) a stem cell-like transition state, 181) and multiple trajectories 192) of the direct cell fate conversions were discovered using scRNA-seq data.…”