Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

Abstract

BackgroundNotch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues.Methodology/Principal FindingsTo analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis.Conclusion/SignificanceWe suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context.

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation

“…To analyze this further, we first determined induction of Sox9 expression by Notch1 during EB differentiation. In line with our previous results, 13 we found that Notch1 activation resulted in immediate upregulation of Sox9 expression in ESCs also in the presence of the protein synthesis inhibitor cycloheximide (CHX) used at a concentration and under conditions previously established to inhibit protein synthesis (Figure 2a), suggesting Sox9 to be directly targeted by Notch signaling. Supporting this idea, we have recently shown the potential binding sites of RBP-J within the Sox9 promoter.…”

“…Among the genes that are most likely directly activated by Notch1 signaling in ESCs, we have recently identified Sox9. 13 This idea of direct regulation of Sox9 by Notch1 was strongly supported by the luciferase experiments in this study using Sox9 promoter reporter constructs with mutated RBP-J-binding sites. Sox9 is an essential regulator of neural crest development and chondrogenesis and marks the progenitor population of chondrogenic cells.…”

“…Recently, we have shown that Notch1 signaling upregulates Sox9, an essential transcription factor involved in chondrogenesis, in ESCs and ectodermal, as well as in mesodemal progenitor cells. 13 Thus, we reasoned that Sox9 may be involved in the induction of chondrogenesis by activated Notch1. To analyze this further, we first determined induction of Sox9 expression by Notch1 during EB differentiation.…”

“…Supporting this idea, we have recently shown the potential binding sites of RBP-J within the Sox9 promoter. 13,28 Sox9 was highly inducible by activated Notch1 during early EB differentiation, but not at late time points of differentiation (day 10; Figure 2b). Furthermore, to show the direct nature of Sox9 regulation by Notch1, we used luciferase reporter constructs that were transiently transfected into ES cells.…”

“…Recently, we have shown that Sox9 is a primary, most likely direct target gene of Notch in ESCs, as well as in ectodermal and mesodermal cells. 13 Sox9 is a transcription factor which is expressed throughout all cartilage-generating regions and in mesodermal chondrocyte progenitors. 14 Sox9 regulates the expression of several cartilage-specific matrix components like collagen II, IX, XI and aggrecan.…”