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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-DeltaSTU
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, p21Cip1, demonstrating specificity for cdki
function (Vernon et al., 2003). Notch-ICD downregulates Xic1 (Figure 2A, B) and
prevents neuron differentiation (Figure 3C). Conversely, X-DeltaSTU upregulates Xic1
(Figure 2D) and promotes formation of ectopic primary neurons (Chitnis et al., 1995).
Therefore, co-injection of Xic1 Mos with X-DeltaSTU mRNA was used to investigate
whether Xic1 is required for the formation of primary neurons even when Notch
signalling is blocked. X-DeltaSTU 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-DeltaSTU 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.
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
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-DeltaSTU, (B) X-DeltaSTU + Con Mo or (C) X-DeltaSTU
+ 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, NotchICD (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, 1191-1202.
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,
659-671.
Jean, D., Ewan, K. and Gruss, P. (1998) Molecular regulators involved in vertebrate eye
development. Mech Dev, 76, 3-18.
Ma, Q., Kintner, C. and Anderson, D.J. (1996) Identification of neurogenin, a vertebrate
neuronal determination gene. Cell, 87, 43-52.
Vernon, A.E. and Philpott, A. (2003) The developmental expression of cell cycle
regulators in Xenopus laevis. Gene Expr Patterns, 3, 179-192.
Figure S1
A
∆STU
B
D
D'
C
∆STU +
Con Mo
ICD + E
NGN
D''
p27Xic1
∆STU +
p27Xic1Mo
ICD +
NGN +
XMyT-1
p27Xic1
E'
E''
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