First published online 25 June 2008
doi: 10.1242/dev.019877
Development 135, 2555-2562 (2008)
Published by The Company of Biologists 2008
Mesp2 and Tbx6 cooperatively create periodic patterns coupled with the clock machinery during mouse somitogenesis
Masayuki Oginuma1,
Yasutaka Niwa2,
Deborah L. Chapman3 and
Yumiko Saga1,4,*
1 Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540,
Japan.
2 Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
3 Department of Biological Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania, PA 15260, USA.
4 Division of Mammalian Development, National Institute of Genetics, Yata 1111,
Mishima 411-8540, Japan.
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Fig. 1. The temporal regulation of Mesp2 transcription by Notch
signaling. (A-D) Representative Mesp2 transcription states
revealed by high-resolution in situ hybridization with combined antisense
probes corresponding to an intronic region and exons of mouse Mesp2.
(A) No transcription, (B) primary transcription, (C) active transcription and
cytoplasmic accumulation of transcripts, and (D) transcriptional termination.
Magenta, Mesp2 transcripts; blue, DAPI staining. The subcellular
localization of the Mesp2 transcripts revealed by these images is
depicted schematically below each panel. (E-J) Double staining of
Mesp2 transcripts (in situ hybridization) and Notch activity
(anti-NICD antibody) during one cycle of somitogenesis. Mesp2
transcription was not detected in phase II (E,F, n=5), was initiated
during phase III (G,H, n=6), and was further upregulated in phase I
(I,J, n=7). Arrowheads in G-J indicate anterior limits of
Mesp2 transcription. (K-N) Higher magnification of phase III
(K,L) and phase I (M,N) images. Mesp2 transcripts were detectable in
the posterior half of the active-Notch domain with a clear anterior boundary
(dotted lines). The actual numbers of cells showing different subcellular
localization of Mesp2 transcripts are shown on the right of the
panels for phase III and I.
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Fig. 2. Mesp2 transcription occurs at the anterior end of the Tbx6
expression domain. (A-J) Spatio-temporal changes in the
Mesp2 transcription pattern during somitogenesis. Double staining of
Mesp2 and NICD (see Fig.
1E-N) or of Mesp2 and Tbx6 (A-F) was conducted using a
single mouse embryo for each phase. (A,B) Phase II, (C,D) phase III and (E,F)
phase I. The staining patterns for B,D,F are also shown schematically. (G,H)
Magnified images of C,D. (I,J) Magnified images of E,F. The transcriptional
states in I and J were roughly estimated using the subcellular localization
pattern of the Mesp2 transcripts and are shown in the right-hand
panel.
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Fig. 3. Mesp2 induces the degradation of Tbx6 via a ubiquitin-proteasome
pathway. (A-D) Comparison of the expression patterns for Tbx6
protein (A,B) and mRNA (C,D) between wild-type (+/+) and Mesp2-null
(P2L/P2L) mice. Dorsal views, anterior to the left. n=4 (A),
n=3 (B), n=3 (C), n=4 (D). (E) The stability
of Tbx6 was compared in embryonic tails with or without Mesp2. The time was
estimated by the number of somites formed in the wild-type embryo.
(F,G) Caudal portions of E10 embryos were bisected and the left
halves treated with DMSO (control), while the right halves were treated with
MG132 (F, n=10) or PMSF (G, n=3) and immunostained for Tbx6.
(H-M) Double-immunostaining patterns representative of the
relationships between Mesp2, Tbx6 and Notch during somitogenesis. The stained
sections shown in the vertical rows are derived from a single embryo. A
schematic of the Notch activity pattern used to assign the phase of the embryo
is shown in the top panels; phase III (n=6), phase II (n=9),
phase I (n=5). The proteins being detected are indicated in the left
panels. A-P, anterior-posterior; D-V, dorsal-ventral.
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Fig. 4. Initiation of Notch signal oscillation correlates with the onset of
Mesp2 transcription. (A-I) Sections of early stage mouse
embryos ( E7.0) were analyzed by double immunostaining. (A-C) The embryos
were stained with DAPI (A), and with antibodies against NICD and Tbx6 (B,C). A
higher magnification image of B is shown in C. These embryos showed weak NICD
activity without oscillation, and Tbx6 expression (n=13). (D-I)
Sections were also subjected to double staining for Hes7 mRNA and
NICD (D-F), and Lfng mRNA and NICD (G-I). Hes7 and
Lfng mRNAs were weakly expressed, but did not show clear wave-like
patterns (n=10). (J-N) Sections of late-streak stage embryos
just prior to somite formation (E7.5) were stained with DAPI to show the
embryonic structure (J), and double stained for Tbx6 and Mesp2 (K,L), or Mesp2
and NICD (M,N). Higher magnification images for K and M are shown in L and N,
respectively. An oscillatory pattern of Notch activity was detected and the
spatial patterns of the three factors were similar to those of later stage
embryos (n=12). Ex, extraembryonic region; Emb, embryonic region.
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Fig. 5. Effects of the FGF signaling pathway on the regulation of Mesp2
expression. (A-C) The spatial relationship between Mesp2
(using mRNA probe) and Dusp4 was examined by double in situ
hybridization. The posterior border of the Mesp2 expression domain
(round bracket in B) coincides with the anterior limit of the Dusp4
expression domain (border indicated by arrow in A,B). The stage of this embryo
was assigned as phase I. A higher magnification image of B is shown in C.
(D,E) Dusp4 expression revealed by whole-mount in situ
hybridization in wild-type (D, n=4) and Mesp2-null
(P2L/P2L; E, n=2) embryos. (F) An analysis of
PSM-specific Fgfr1 knockout (cKO) mice. Whole-mount in situ
hybridization revealed a posterior shift of the Mesp2 expression
domain in the Fgfr1-cKO embryo (n=8). (G) Caudal
portions of E10.5 embryos were treated with FGF inhibitor (SU5402) or DMSO
(control) for 6 hours. A posterior shift of the Mesp2 expression
domain was observed in the embryos treated with SU5402 (5 out of 6 embryos).
(H) Caudal portions of E10 embryos were bisected and the left halves
were treated with DMSO (control), while the right halves were treated with
SU5402. A posterior shift of the Mesp2 expression domain was observed
in 10 out of 13 SU5402-treated embryos tested. (I-L) Double
immunostaining of sections was employed to examine Tbx6 and Mesp2 (I,J) or
Mesp2 and NICD (K,L) expression in wild-type control (I,K) and
Fgfr1-cKO (J,L) embryos.
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Fig. 6. A model for periodic somitogenesis in the mouse. Schematic
representation of the temporal and spatial changes in the expression patterns
and relationships among Mesp2 (pink), Tbx6 (green), NICD (blue) and FGF
signaling (orange) during a single cycle of somitogenesis. The FGF signal is
provided from the PSM with a posterior-to-anterior gradient. The expected
threshold in the activity defines the determination front that corresponds to
the posterior limit of the Mesp2 expression domain. In phase III,
Notch activity reaches the anterior PSM, where Mesp2 transcription
has been initiated in the cells with Tbx6 expression and lacking negative
effectors such as FGF and Wnt signaling. In phase I, Mesp2 protein accumulates
and suppresses NICD by activating Lfng, and also suppresses Tbx6 protein by
promoting its rapid degradation via the ubiquitin-proteasome pathway. In phase
II, when the next wave of Notch activity has just reached the anterior PSM
region, the three signals (NICD, Mesp2 and Tbx6) show complete segregation,
thus establishing a boundary between NICD and Mesp2 that demarcates the
segmental border, and a boundary between Mesp2 and Tbx6 that demarcates the
next Mesp2 anterior limit and, thus, the next segmental border.
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© The Company of Biologists Ltd 2008
