EMBO REPORT SUPPLEMENTARY SECTION. Quantitation of mitotic cells after perturbation of Notch signalling.

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1 EMBO REPORT SUPPLEMENTARY SECTION Quantitation of mitotic cells after perturbation of Notch signalling. Notch activation suppresses the cell cycle indistinguishably both within and outside the neural plate at neural plate stage (Figure 1A-C) and retains this activity at tailbud stages in the ectoderm. The extent of cell cycle inhibition was quantified by counting the number of ph3-expressing cells in a given area on the injected versus the uninjected side of the embryo at stage 22. Notch-ICD, XSu(H)-ANK and X-Delta STU reduced the number of ph3 positive cells by 69%, 51% and 43% respectively (p<0.001). Neurons do not form in the absence of Xic1 even when Notch signalling is blocked. We demonstrated previously that injection of Xic1 antisense morpholino oligonucleotides (Xic1 Mo) inhibits expression of Xic1 protein at least to late neurula stages, as verified by western blotting (Vernon et al., 2003). This Mo-induced loss of Xic1 protein blocks XNGNR1-mediated primary neurogenesis and neuron loss is fully rescued by co-injection of the mammalian cdki, p21 Cip1, demonstrating specificity for cdki function (Vernon et al., 2003). Notch-ICD downregulates Xic1 (Figure 2A, B) and prevents neuron differentiation (Figure 3C). Conversely, X-Delta STU upregulates Xic1 (Figure 2D) and promotes formation of ectopic primary neurons (Chitnis et al., 1995). Therefore, co-injection of Xic1 Mos with X-Delta STU mrna was used to investigate whether Xic1 is required for the formation of primary neurons even when Notch signalling is blocked. X-Delta STU mrna was injected either alone, in combination with a control morpholino (Con Mo) or with Xic1 Mo. Both alone and in combination with Con Mo, X-Delta STU overexpression produced a substantial increase in the density of primary neurons (69% of embryos, n=32 and 80% of embryos, n=12 respectively) (Figure S1A, B). However, primary neuron formation was completely inhibited in the presence of Xic1 Mo (100%, n=27) (Figure S1C). Therefore, blocking lateral inhibition only induces formation of primary neurons in the presence of Xic1. Notch signalling regulates cyclin A2 and cdk2 transcripts in the nervous system.

2 How does Notch pathway activation simultaneously downregulate Xic1 and arrest the cell cycle in the neural plate? Although there are many potential targets, one possible mechanism for the observed inhibition of cell cycle progression by Notch signalling is through the inhibition of positive cell cycle regulators, such as cdks and cyclins. By midneural plate stages, cyclin A2 and cdk2 messages become detectable and are most strongly expressed in prospective dorso-anterior neural tissue (Vernon and Philpott, 2003) and Figure S2A, B), although expression is much weaker in the posterior neural plate. The effect of Notch signalling on cyclin A2 and cdk2 messages in prospective neural tissue was determined by in situ hybridization at mid-neurula stages. Overexpression of Notch-ICD and XSu(H)-ANK reduces the amount of cyclin A2 and cdk2 expression in neural tissue (Figure S2A, B, D, E arrows). We also overexpressed Xsu(H)-DBM, a dominant negative construct that has been reported to inhibit Notch signalling. In our hands, we did not see any affect of Su(H)-DBM on primary neurons. However, its overexpression did increase the number of phospho-histone H3-expressing cells in the neural plate (39% p<0.001) and it does expand the region of cyclin A2 and cdk2 expression, (Figure S2, G and H) Notch signalling can disrupt the formation of anterior neural structures, sometimes resulting in their reduction (Coffman et al., 1993). Therefore, the expression of Pax6, a marker of the presumptive eye field (Jean et al., 1998), was examined to determine whether non-specific disruption of anterior structures is responsible for the reduction in cyclin A2 and cdk2 observed upon Notch activation. While Notch-ICD and XSu(H)-ANK disrupted Pax6 expression in a minority of embryos (35%, n=52 and 15%, n=41 respectively) (Figure S2C, F), cyclin A2 (61%, n=64 and 45%, n=50 respectively) (Figure S2A, B) and cdk2 (63%, n=54 and 37%, n=47 respectively) (Figure S2D, E) expression was more severely affected. This difference indicates that the reduction of cyclin A2 and cdk2 transcripts is unlikely to be entirely the result of disruption of the anterior structures in which they are most highly expressed. Indeed, the disruption of anterior structures seen later in development may result from inappropriate cell cycle cessation due to the downregulation of cyclin A2 and cdk2 by Notch signalling. Due to weak staining, we were unable to determine the effect of perturbation of Notch signalling

3 on cyclin A2 and cdk2 expression in the posterior neural plate. While these molecules may be targets for Notch signalling in this region, it is quite possible that other cell cycle regulators, for instance members of the Rb pathway, are targeted to regulate cell proliferation in this area. Figure S1 Xic1 is required for primary neuron formation when Notch signalling is blocked Embryos were injected with (A) X-Delta STU, (B) X-Delta STU + Con Mo or (C) X-Delta STU + Xic1 Mo with βgal as a tracer (light blue) and assayed for Nβtub expression (purple) by in situ hybridization (injected side to left). Figure S2 Notch activation suppresses cyclin A2 and cdk2 expression Embryos were injected with (A-C) Notch-ICD, (D-F) XSu(H)-ANK, or (G-I) XSu(H)- DBM, along with βgal (light blue, injected side to left), and assayed for cyclin A2, cdk2, and Pax6 expression at stage 16 by in situ hybridization as labelled. Anterior views show that Notch-ICD (A, B) and XSu(H)-ANK (D, E) downregulate both cyclin A2 and cdk2 (arrows) while XSu(H)-DBM (G, H) upregulates them (arrowheads). In contrast, Notch- ICD (C), XSu(H)-ANK (F), and XSu(H)-DBM (I) have little effect on Pax6. Bellefroid, E.J., Bourguignon, C., Hollemann, T., Ma, Q., Anderson, D.J., Kintner, C. and Pieler, T. (1996) X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation. Cell, 87, Coffman, C.R., Skoglund, P., Harris, W.A. and Kintner, C.R. (1993) Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell, 73, Jean, D., Ewan, K. and Gruss, P. (1998) Molecular regulators involved in vertebrate eye development. Mech Dev, 76, Ma, Q., Kintner, C. and Anderson, D.J. (1996) Identification of neurogenin, a vertebrate neuronal determination gene. Cell, 87,

4 Vernon, A.E. and Philpott, A. (2003) The developmental expression of cell cycle regulators in Xenopus laevis. Gene Expr Patterns, 3,

5 Figure S1 A B C STU STU + Con Mo STU + p27 Xic1 Mo D ICD + E ICD + NGN NGN + XMyT-1 p27 Xic1 D' D'' E' E'' p27 Xic1

6 Figure S2 A ICD B ICD C ICD D A2 ANK E K2 ANK F Pax6 ANK G A2 DBM H K2 DBM I Pax6 DBM A2 K2 Pax6