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Fig. 1 Multiple sequence alignment of esr9, esr10, and related pro- helix1, loop and helix follows (Ferre-D’Amare et al., 1993). The
teins. Comparison of the ESR9 and ESR10 protein sequences with evaluation of percentage conservation was done using the BLOSthat
of other vertebrate homologs belonging to the Hairy/enhancer UM62 similarity score matrix. Background intensities represent
of split related (HER) family. The bHLH domain and the orange conservation (darkΩ100%, mediumΩ80% or more, lightΩ60%
domain are well conserved between these proteins as well as the or more). EMBL accession numbers are: esr9, AJ009282, esr10,
tetrapeptid WRPW at the carboxyl terminus. Designation of basic, AJ009285.
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Fig. 2 Evolutionary relationships of the Gridlock, Hairy, and enhancer
of split families of bHLH proteins indicate that esr9 and
esr10 belong to the vertebrate HES5 sub-family. In this rooted
neighbor-joining tree, only the bHLH domain was used for comparison.
The Gridlock family members were used as an outgroup.
The different constituent families are shaded. Numbers above
branches indicate percent support for the nodes defining the families
in distance bootstrap analyses (1000 replicates). Br Brachydanio
rerio, Ce Caenorhabditis elegans, Cp Cynops pyrrhogaster, Cs
Cupienus salei, Dh Drosophila hydei, Dm Drosophila melanogaster,
Dv Drosophila viridis, Gg Gallus gallus, Hs Homo sapiens, Mm
Mus musculus, Rn Rattus norvegicus, Tc Tribolium castaneum, Xl
Xenopus laevis.
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Fig. 3 Expression of esr9 in Xenopus embryos. Xenopus laevis embryos
at different stages of development were used for wholemount
in situ hybridisation analysis of esr9 mRNA localisation.
(a–c) Gastrulae stage 10.5, lateral view, dorsal to the right in a,
vegetal view, dorsal to the top in b, sagittal section, lateral view,
dorsal to the right in c. Neurula stage 13, anterior view, dorsal to
the top in d, dorsal view, anterior to the left in e. Neurula stage 20,
dorsal view, anterior to the left in f, lateral view, anterior to the
left in g. Tailbud stage 30, lateral view, anterior to the left in h, the
inset is a transverse vibratome section at the trunk level, dorsal to
the top. In i, late neurulae stage 20 embryos are in dorsal view,
anterior to the top. The left embryo was stained only for thy2 (red),
the right one double stained with thy2 and esr9 (blue). Note that
the staining for esr9 in the anterior half of S1 (arrowhead) overlaps
that of thy2. (j) A diagram is shown representing a dorsal view of
the paraxial mesoderm on both sides of the notochord, with the
posterior end on the right. The domains of expression of esr9 relative
to that of thy2 are drawn in parallel to the events of segmentation
in Xenopus. Mz, marginal zone; bl, blastopore lip; tg, trigeminal
placode; m, i, l, medial, intermediate and lateral stripes of
primary neuronal precursors; pr, pronephros; cg, cranial ganglias;
sc, spinal cord; so, somitomeres; ey, eyes; br, brain; tbd, tailbud.
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Fig. 4 Cyclic expression of esr9. Embryos at stage 19 (a, aø, b, bø, Expression in the anterior compartment of somitomere S1 is shown
c, cø), stage 21 (d, dø, e, eø, f, fø), stage 24 (g, gø, h, hø, i, iø) are in white, that of S2 in red, and the tailbud and transition zone
shown on a dorso-posterior view, dorsal to the top. a-i depicts em- domains in blue. Between the phase III and the next phase I, a new
bryos after whole-mount in situ hybridisation using an esr9 probe. somites will form from somitomere S4 (not shown here), and a new
aø–iø are schematic drawings portraying the dynamic and periodic somitomere S1 will form from the anterior portion of cells in the
expression in the presomitic mesoderm. In this diagram, neural transition zone domain, in blue. The nomenclature of phase I, II
expression has been omitted for clarity. and III is taken from (Pourquie and Tam, 2001).
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Fig. 5 Cyclic expression of esr9 is autonomous and does not require
protein synthesis. (a) Four examples are shown of embryos
stage 19 and 21 showing left-right asymmetry of esr9 expression.
Posterior view, dorsal to the top. (b) Stage 20 Xenopus embryos
were untreated, or cultivated in presence of 15 mg/ml CHX for 30,
60, and 120 min. Examples of embryos at different phases are
marked: open circle for phase I , asterisk for phase II and closed
circle for phase III. Note the disappearance of the stripy expression
of esr9 after 120 min of CHX treatment.
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