Cell Determination and Early Differentiation

In mammals, Myostatin (MSTN) is a known negative regulator of muscle growth and development, but its role in birds is poorly understood. To investigate the molecular mechanism of MSTN on muscle growth and development in chickens, we knocked out MSTN in chicken fetal myoblasts (CFMs) and sequenced the mRNA transcriptomes. The amplicon sequencing results show that the editing efficiency of the cells was 76%. The transcriptomic results showed that 296 differentially expressed genes were generated after down-regulation of MSTN, including angiotensin I converting enzyme (ACE), extracellular fatty acid-binding protein (EXFABP) and troponin T1, slow skeletal type (TNNT1). These genes are closely associated with myoblast differentiation, muscle growth and energy metabolism. Subsequent enrichment analysis showed that DEGs of CFMs were related to MAPK, P13K/AKT, and STAT3 signaling pathways. The MAPK and P13K/AKT signaling pathways are two of the three known signaling pathways involved in the biological effects of MSTN in mammals, and the STAT3 pathway is also significantly enriched in MSTN knock out chicken leg muscles. The results of this study will help to understand the possible molecular mechanism of MSTN regulating the early differentiation of CFMs and lay a foundation for further research on the molecular mechanism of MSTN involvement in muscle growth and development.

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Abstract 305: Single Cell RNA Sequencing of Adult Cardiac c-kit+ Cells in a Murine Lineage Tracing Model

Circulation Research ◽

10.1161/res.117.suppl_1.305 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Bryan D Maliken ◽

Onur Kanisicak ◽

Jeffery D Molkentin

Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Rna Sequencing ◽

Cardiac Injury ◽

Mesenchymal Cells ◽

Lineage Tracing ◽

Tyrosine Kinase Receptor ◽

Early Differentiation ◽

Gfp Reporter ◽

Single Cell Rna Sequencing

A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to eliminate endothelial and leukocyte progenitor contamination, respectively. Following this isolation, cells were examined to determine GFP reporter status and then submitted for single cell RNA sequencing using the Fluidigm A1 system. Clustering of 654 genes from this data identified 6 distinct subpopulations indicating various stages of early differentiation among CD31- and CD45-negative cardiac interstitial cells. Furthermore, comparison of GFP+ c-kit cells to the total non-GFP mesenchymal cells identified 75 differentially expressed transcripts. These unique gene signatures may help parse the genes that underlie cellular plasticity in the heart and define the best molecular lineages for transdifferentiation into cardiac myocytes.

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Changes in the transferrin requirement of cultured chick embryo mesoderm cells during early differentiation

Development ◽

10.1242/dev.95.1.81 ◽

1986 ◽

Vol 95 (1) ◽

pp. 81-93

Author(s):

E. J. Sanders

Keyword(s):

Chick Embryo ◽

Type I Collagen ◽

Specific Effect ◽

Primitive Streak ◽

Cellular Adhesion ◽

Type I ◽

Surface Binding ◽

Surface Receptors ◽

Early Differentiation ◽

Species Specific

Mesodermal tissue from the chick embryo at various stages of early differentiation was cultured in hydrated gels of type I collagen in the presence and absence of transferrin. Primary mesoderm explants from primitive-streak-stage embryos responded to the presence of avian transferrin by significantly improved outgrowth which appeared to be related to the ability of the cells to attach to, and migrate in, the collagen. No evidence was obtained which suggested that this observation was dependent on increased cell proliferation. This outgrowth enhancement was not duplicated by transferrin of human origin. The avian transferrin did not produce this effect on cells cultured on plastic substrata, suggesting that the species-specific effect involves modulation by the extracellular matrix. Mesoderm explants from somite stages of development showed no increase in outgrowth in the presence of either avian or human transferrin as judged by counting the number of outwandering cells. Ultrastructural immunocytochemistry indicated surface binding of transferrin by cells in the gels, and the presence of endogenous transferrin on the surfaces of mesoderm cells in situ and in their extracellular environment. It is suggested that by binding to cell surface receptors, transferrin may be able to influence the strength of cellular adhesion to collagen and hence the capacity for cell locomotion.

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