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. 2021 Mar 11;10:e59540. doi: 10.7554/eLife.59540

Figure 2. Wnt signalling antagonises Notch activity.

(a–d’) Schematic representation of the Piggybac, Tol2, and RCAS constructs used for β-catenin gain- (GOF) and loss-of-function (LOF) experiments. The PB-βcat-GOF and T2-βcat-GOF contain the full-length β-catenin including the α-catenin binding domain (αCat-BD), 12 Armadillo domains, the transactivator (TS) motif, and the S33Y mutation preventing phosphorylation and degradation. The RCAS-βcat-LOF and T2-βcat-LOF constructs drive expression of a truncated form of β-catenin comprising the Armadillo repeats only. (b–d’) Activity of the Wnt reporter in E3 otocysts co-electroporated with either T2-EGFP (control; b–b’), PB-βcat-GOF (c–c’), or RCAS-βcat-LOF (d–d’). Note the ventral expansion of the Wnt reporter fluorescence in (c–c’) and its restriction to the most dorsal part of the otocyst in (d–d’). (e–g’) Activity of the Notch reporters T2-Hes5::nd2EGFP or Hes5::d2FP635 in E3 otocysts co-electroporated with either T2-mCherry (control, e–e’), T2-βcat-LOF (f–f’), or PB-βcat-GOF (g–g’). The Notch reporter is normally activated in the anterior (arrowhead) and to a lesser extent posterior (asterisk) prosensory domains of the otocyst (e–e’). It is strongly upregulated in dorsal regions transfected with the T2-βcat-LOF construct (brackets in f–f’), but barely detectable in otocysts co-electroporated with PB-βcat-GOF (g–g’). On the other hand, manipulation of Notch activity had no discernible effect on the activity of the Wnt reporter (Figure 2—figure supplement 1).

Figure 2.

Figure 2—figure supplement 1. Manipulating Notch activity does not affect Wnt signalling.

Figure 2—figure supplement 1.

(a–c’) Whole mounts of E3 chicken otocysts co-electroporated with Wnt reporter 5TCF::H2B-RFP and a control plasmid T2-EGFP or constructs activating (pNICD1-EGFP) and blocking (pDN-MAML1-EGFP) Notch signalling. There are no major changes in the dorso-ventral pattern of activation of the Wnt reporter in response to gain- and loss-of-Notch function (a’, b’, c’).