A Switch From Canonical to Noncanonical Wnt Signaling Mediates Early Differentiation of Human Neural Stem Cells

Abstract

Wnt/b-catenin signaling is essential for neurogenesis but less is known about b-cateninindependent Wnt signals. We show here that Wnt/activator protein-1 (AP-1) signaling drives differentiation of human embryonic stem cell and induced pluripotent stem cell-derived neural progenitor cells. Neuronal differentiation was accompanied by a reduction in b-catenin/Tcfdependent transcription and target gene expression, increased levels and/or phosphorylation of activating transcription factor 2 (ATF2), cyclic AMP response element-binding protein, and c-Jun, and increased AP-1-dependent transcription. Inhibition of Wnt secretion using the porcupine inhibitors IWP-2 and Wnt-C59 blocked neuronal differentiation, while activation or inhibition of Wnt/b-catenin signaling had no effect. Neuronal differentiation increased expression of several Wnt genes, including WNT3A, silencing of which reduced differentiation. Addition of recombinant Wnt-3a to cells treated with IWP-2 or Wnt-C59 increased AP-1 levels and restored neuronal differentiation. The effects of Wnt-3a could not be blocked by addition of Dkk-1 or IWR-1, suggesting the involvement of noncanonical signaling. Consistent with this, restoration of neuronal differentiation by Wnt-3a was reduced by inhibition of Jun N-terminal kinase (JNK) and by gene silencing of ATF2. Together, these observations suggest that b-catenin-independent Wnt signals promote neural stem/progenitor cell differentiation in a signaling pathway involving Wnt-3a, JNK, and ATF2. STEM CELLS 2014;32:3196-3208

Canonical and noncanonical Wnt signaling in neural stem/progenitor cells

“…This could be partially More recently, mES cell differentiation was found to be accompanied by activation of noncanonical signaling via increased expression of Tcf3 [171], which is known to signal independently of -catenin in several contexts [172]. This is consistent with our findings in human NSCs, where Wnt-3a promotes differentiation via JNK/ATF2 independently of β-catenin [163], and with a recent study in human iPS cells, which showed that Wnt-3 and Wnt-9B cooperate to promote dopaminergic differentiation, with canonical signaling maintaining proliferation and noncanonical signaling, involving JNK, driving differentiation [173]. These studies indicate that noncanonical signaling can play an important role during the differentiation of hES cells and warrant further studies of noncanonical Wnt [174].…”

“…In human embryonal carcinoma cells, Wnt-11 maintains neural progenitor cell proliferation but prevents further differentiation, which instead is driven by another noncanonical Wnt, Wnt-4 [68]. We recently found that noncanonical Wnt -1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3a signaling stimulates differentiation of hES cells and iPS cells, something that could account for some of the controversy found in the literature regarding the role of Wnt-3a [163]. The nature of the response to Wnt-3a, canonical versus noncanonical, are likely to be influenced by the relative levels of Wnt receptors, secreted Wnt antagonists and intracellular effectors expressed by target cells.…”

Comprehensive profiling reveals mechanisms of SOX2-mediated cell fate specification in human ESCs and NPCs

“…These results indicate that enhanced canonical Wnt signaling may be partially responsible for neuronal differentiation defects induced by SOX2 KD and that canonical Wnt signaling needs to be silenced when neuronal differentiation begins in hNPCs. The inhibitory role of canonical Wnt signaling in neuronal differentiation in hNPCs is in agreement with the finding by Bengoa et al [17], but is in contrast to the inductive role of Wnt signaling in mouse adult neurogenesis [10], possibly reflecting a species-specific function of the pathway in neuronal differentiation. However, the reduced cell number due to massive cell death, as indicated by increased levels of active caspase3 ( Figure 5A), was only seen after SOX2 KD but not after ChIR99021 treatment, suggesting the presence of additional mechanism underlying SOX2's role in supporting cell survival during neuronal differentiation.…”

“…The role of canonical Wnt signaling in hNPCs is less studied. Recently, Wnt signaling was reported to be dispensable for hNPC generation and proliferation; instead, it contributes to the specification of the anterior and posterior identity [16], and hinders neuronal differentiation from hNPCs [17]. These findings suggest a difference in the role of Wnt signaling between human and mouse cells.…”

Multiple roles of Ulk4 in neurogenesis and brain function

“…13 canonical Wnt signaling is crucial for driving NSC self-renewal and expansion. For example, Fzd1 regulates cell fate commitment, and Fzd1 knockdown markedly decreases newborn neurons and increases the generation of astrocytes in adult hippocampus.…”