Nkx1-2 is a transcriptional repressor and is essential for the activation of Brachyury in P19 mouse embryonal carcinoma cell

Abstract

Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm.

Early molecular events during retinoic acid induced differentiation of neuromesodermal progenitors

“…forebrain/midbrain). NMPs are unique in that they co-express Sox2 and T (Brachyury) that drive the neuroectodermal lineage and the mesodermal lineage, respectively (Martin and Kimelman, 2012; Olivera-Martinez et al, 2012; Tsakiridis et al, 2014; Wymeersch et al, 2016), along with Nkx1-2 which is a marker of all caudal progenitors (Delfino-Machin et al, 2005; Tamashiro et al, 2012; Sasai et al, 2014). A positive-feedback FGF and WNT signaling loop maintains an undifferentiated state in caudal progenitors that promote body axis extension (Ciruna and Rossant, 2001; Aulehla et al, 2003; Dunty et al, 2008; Naiche et al, 2011; Martin and Kimelman, 2012; Olivera-Martinez et al, 2012; Jurberg et al, 2014; Cunningham et al, 2015b).…”

CDX4 regulates the progression of neural maturation in the spinal cord

“…4C (mSax1,Nkx1.2), which has been shown to act as a repressor in mouse cell lines (Tamashiro et al, 2012) and chicken embryos (Sasai et al, 2014). As previously suggested (Sasai et al, 2014), overexpression of mouse Sax1 represses Pax6 (Fig.…”

“…4, 6). It is likely, however, that Cdx4 regulation of Sax1 is mediated by Wnt and FGF, as these pathways are under Cdx control (Chawengsaksophak et al, 2004;Savory et al, 2009;van Rooijen et al, 2012), and they are required for Sax1 transcription (Bertrand et al, 2000;Tamashiro et al, 2012). In this scenario, dominant-negative Cdx4 would downregulate Sax1 by blocking Wnt and FGF signaling (Fig.…”

“…Cdx4, at the core of the transcription factor network, provides an integration point for the inputs to regulate effector genes, as Cdx4 transcription is directly regulated by FGF, Wnt and RA (Chang et al, 2016;Lee and Skromne, 2014). FGF and Wnt promote pluripotency by directly activating pluripotency marker Sax1 (Diez del Corral et al, 2002;Tamashiro et al, 2012), but also initiate the loss of pluripotency and primes cells towards differentiation by activating Cdx4, an indirect repressor of Sax1 (Fig 4B). A similar phenomenon is observed in the regulation of Pax6, with FGF repressing (Bertrand et al, 2000) and Cdx4 activating in a RA-dependent manner the transcription of Pax6 (Fig.…”

“…These vectors use the chicken Actin promoter to drive high gene expression levels, and carry a GFP gene as a reporter for transcription. Genes of interest were either cloned into vectors in the laboratory (Cdx4, VP16Cdx4, EnRCdx4, mNkx1.2; for details see supplementary material), or obtained already in the appropriate vector from other laboratories (Pax6-pCIG and EnRPax6-pCIG were kindly provided by Dr. Francois Medevielle (Bel-Vialar et al, 2007); and mNkx1.2-pEF2 was kindly provided by Dr. Yusuke Marikawa (Tamashiro et al, 2012)). …”