Different Concentrations of FGF Ligands, FGF2 or FGF8 Determine Distinct States of WNT-Induced Presomitic Mesoderm

Abstract

Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the na€ ıve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm. STEM CELLS 2016;34:1790-1800 SIGNIFICANCE STATEMENTOur study adds a new dimension to the current understanding of the Wnt-mediated PSM induction of EpiSCs by deciphering the importance of FGF concentration in the lineage commitment toward different presomitic mesoderm (PSM) states. We believe that this study thereby provides valuable information for directed differentiation of ESCs. Furthermore, this method of differentiation toward PSM cell states can be a valuable method for studying the dynamics of gene regulation during PSM formation and musculoskeletal disorders that arise during early development.

Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells

“…Consistent with previous reports (Loh et al, 2016; Xi et al, 2017; Chal et al, 2015; Umeda et al, 2012; Sudheer et al, 2016), CHIR99021 efficiently induced DLL1 + /PAX3-GFP − cells (56.3±3.1%) (C in Fig. 1D and …”

Making muscle: skeletal myogenesisin vivoandin vitro

“…The generation of cells with a posterior presomitic mesoderm fate from PSCs in vitro was analyzed using mESC reporter lines for Msgn1 expression (Chal et al, 2015;Sudheer et al, 2016) (Box 1). Optimal induction of posterior presomitic mesoderm fate was observed when mESCs were pretreated with BMP4 to differentiate them into epiblast-like cells, followed by treatment combining a Wnt activator (R-spondin 3 or the GSK3β inhibitor CHIR99021) and a BMP inhibitor [LDN-193189 (Cuny et al, 2008)], which prevented the induced presomitic mesoderm cells from drifting to a lateral plate mesoderm fate (Chal et al, 2015).…”

“…One of the first examples of this was the brachyury-GFP mESC reporter line, which was used to analyze mesoderm induction in vitro (Fehling et al, 2003;Gadue et al, 2006). More recently, ESC lines that harbor fluorescent reporters for the expression of key paraxial mesoderm/myogenic lineage markers, namely Msgn1, Pax3, myogenin (Myog) and Pax7, have been used to track the sequential differentiation of PSCs toward paraxial mesoderm and skeletal muscle (Chal et al, 2015;Sudheer et al, 2016). mesoderm cells induced in vitro also expressed cyclic genes such as Hes7, suggesting that the segmentation machinery is activated in vitro (Chal et al, 2015).…”

Why the short face? Developmental disintegration of the neurocranium drives convergent evolution in neotropical electric fishes

“…All the signaling molecules discussed above are known to perform multiple functions during development (Dworkin, Boglev, Owens, & Goldie, 2016; Harada, Sato, & Nakamura, 2016; McCarthy et al., 2016; Sudheer et al., 2016; Wada et al., 2005). It is therefore unlikely that modulation of these signaling molecules is regulated by a single gene.…”

“…This species was also found to be highly integrated in development despite being brachycephalic. & Goldie, 2016;Harada, Sato, & Nakamura, 2016;McCarthy et al, 2016;Sudheer et al, 2016;Wada et al, 2005). It is therefore unlikely that modulation of these signaling molecules is regulated by a single gene.…”