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    • The WNT signaling pathway is a

    2018-11-08

    The WNT signaling pathway is a primary determinant in assigning an A/P positional identity to NPCs. This instructional cue is imparted early during NPC generation and once this identity is established, it is stable and cannot be altered through exogenous manipulation of the WNT pathway. Using a WNT reporter line, we show that endogenous WNT signaling is highly variable among individual c-myc inhibitor as they acquire a NPC phenotype, with cells of posterior identity expressing WNT reporter activity. In addition to expressing markers of posteriorly fated NPCs, most notably genes of the HOX gene cluster, these cells also express multiple WNT ligands. In contrast, NPCs with anterior identity, as detected by a lack of WNT reporter activity, express multiple WNT antagonists. These differences in expression of WNT agonist and antagonist resemble those observed in the developing neural tube in vivo, with posterior tissues expressing WNT proteins and anterior tissues expressing WNT antagonists such as DKK1 and FRZB (Hashimoto et al., 2000; Leyns et al., 1997). These opposing WNT signals generate an endogenous gradient of WNT activity, which divides the embryonic neural tube along the A/P axis into distinct progenitor domains, each of which gives rise to specific regionalized neurons (Ciani and Salinas, 2005; Kiecker and Niehrs, 2001; Nordström et al., 2002). These progenitor domains have regionally specific gene-expression profiles and differentiation predispositions despite similar levels of expression of the pan-neural markers SOX1 and SOX2 (Pevny et al., 1998; Wood and Episkopou, 1999; Zappone et al., 2000). Here, we showed that NPCs exhibited a broad range of endogenous WNT activity that conferred specific regionalized fates despite comparable expression levels of SOX1 and SOX2, perhaps mimicking the same developmental events that are seen during early in vivo neural tube development. Therefore, the local WNT microenvironment tightly regulates the WNT activity status and hence the positional identity of NPCs. A somewhat unexpected implication of these gene-expression patterns is that WNT signaling appears to be acting cell autonomously, with WNT signaling activity restricted to those cells expressing WNT genes. Although WNT signaling activity is present in a graded fashion in these NPC cultures, WNT proteins are acting in an autocrine rather than paracrine manner. Furthermore, expression of WNT antagonists may mute the response in cells near or adjacent to WNT secreting cells. A more careful analysis of this cell-based system will likely yield important mechanistic insights into the dynamic nature of WNT signaling during development. This restricted WNT signaling activity observed in NPC cultures is consistent with the notion that WNT proteins act locally (Habib et al., 2013) and exhibit minimal, if any, extracellular diffusion. A recent study demonstrated that flies expressing an engineered membrane-tethered Wingless (a fly WNT protein) are viable and normally patterned, suggesting that the spread of Wingless is dispensable for patterning and growth (Alexandre et al., 2014). Similarly, in our cell-based system, WNT proteins act locally and do not signal to distant cells. In addition, expression of WNT antagonists in the WNT− populations may act to block paracrine WNT signaling activity. This local WNT activity is not the result of the physical separation of distinct WNT expressing domains, since this localized activity is retained in a mixed and seemingly homogeneous cell culture system. While endogenous WNT signaling activity is a major source of heterogeneity among individual NPCs, exogenous manipulation of this signaling pathway can be exploited to impart specific positional identities to NPCs during their generation from hPSCs, thereby reducing cellular heterogeneity. Activation of WNT signaling with purified WNT3a protein or a GSK-3β inhibitor (CHIR98014) led to the generation of NPCs with a hindbrain/spinal cord identity, whereas inhibition of WNT signaling with a PORCN inhibitor (IWP2) to block endogenous WNT protein processing led to the generation of NPCs with a forebrain phenotype. As shown in this and other studies (Li et al., 2009; Pankratz et al., 2007), in the absence of any WNT pathway manipulations, NPCs generated form hPSCs are generally biased toward an anterior fate, suggesting that endogenous WNT signaling in these culture systems is relatively low and insufficient to promote posterior fates. Consequently, ectopic activation of WNT signaling produces a prominent shift from an anterior to a posterior fate. In contrast, in the absence of WNT signaling (through IWP2 addition), the relative increase of anterior-related markers, though statistically significant, is less pronounced.