Involvement of SIP1 in positioning of somite boundaries in the mouse embryo



Periodical production of somites provides an excellent model system for understanding genesis of metameric structures underlying embryonic development. This study reports production of somites with roughly half rostro-caudal length in homozygous Sip1 (Smad-interacting protein 1) knockout mouse embryos. This altered periodicity of somitogenesis is caused by the rostral expansion of the expression domain of genes involved in the maintenance of unsegmented state of paraxial mesoderm, e.g., Fgf8, Wnt3a, Dll3, and Tbx6. This is accompanied by the rostral extension of oscillatory gene expression such as L-fng, Hes7, and Dll1, and the rostrally shifted termination of Raldh2 expression that continues from the anterior embryonic side. The phenotype of Sip1−/− embryo introduces a new molecular component SIP1 in positioning of somite boundaries, and provides support for the current “clock and wavefront” model. Developmental Dynamics 234:332–338, 2005. © 2005 Wiley-Liss, Inc.


In early mouse embryos, the Sip1 (Smad- interacting protein 1) gene, encoding a member of the ZFHX1 family of two-handed zinc finger transcriptional factors (Remacle et al.,1999), is expressed mainly in neuroectoderm, paraxial mesoderm, and neural crest cells. To investigate the function of the Sip1 gene during embryo development, we generated Sip1-null mutant mice by gene targeting (Higashi et al.,2002). Using mutant embryos, we have previously shown that Sip1 is essential for the development of vagal neural crest and migration of cranial neural crest cells (Van de Putte et al.,2003). Homozygous mutant (Sip1−/−) embryos die around E9.5 accompanied by cardio-vascular dysfunction (Van de Putte et al.,2003).

We describe here another important phenotype, namely development of defective somites displaying shortened spatial periodicity. Somite segmentation occurs periodically in embryonic development and produces regularly arranged metameric structures. A generally accepted model of somite segmentation assumes interaction between the two systems, i.e., intracellular oscillation of expression of a set of genes in the paraxial mesoderm that provides the basis for periodical somite segmentation, and a caudally-regressing front of gene activities that are involved in maintaining the unsegmented state of paraxial mesoderm. Evidence indicates that Notch signaling, involving Lunatic Fringe (L-fng) and HES7, steers the former oscillation mechanism, which operates in 2-h cycles in mouse embryos. The rostrally shallower and caudally regressing FGF/WNT signal gradient in the paraxial mesoderm underlies the latter mechanism (Dubrulle et al.,2001; Sawada et al.,2001; Aulehla et al.,2003). Here we show that the rostro-caudal truncation of somites in Sip1−/− embryos is presumably caused by persistent and slow-regressing FGF/WNT signals, which we interpret as slower caudal displacement of the “wavefront” in the current “clock and wavefront” model (Dubrulle et al.,2001; Aulehla et al.,2003).


Sip1 Expression in the Neural Plate and The Paraxial Mesoderm

In gastrulating mouse embryos, Sip1 is expressed in a pair of paraxial zones in the dorsal view (Fig. 1A). This expression pattern is a composite of signals in the neural plate and paraxial mesoderm. The signal in the neural plate is strong rostral to the node, and that in the paraxial mesoderm occurs with spatial periodicity in the rostral boundaries of each of already formed somites and is strongest in the next-forming somite (Fig. 1B, C, note that Fig. 1C shows the same embryo as B after removal of neural plate anterior to the broken line).

Figure 1.

Sip1 expression in a wild-type mouse embryo at E8.5 detected by in situ hybridization. A: Dorsal view. Strong Sip1 expression in the neural plate is shown. The asterisk indicates the position of the node. B: Enlargement of the caudal end of an embryo showing the superimposement of Sip1 in signals of the neural plate and paraxial mesoderm. C: The same specimen as B, but the neural plate was peeled-off in the part rostral to the broken line, and the signals in the paraxial mesoderm are shown. The last-formed somite boundary is indicated by the black arrowhead, and the boundary to be formed by the white arrowhead.

Generation of Short Somites in Sip1−/− Embryos

We examined somitogenesis in Sip1−/− mutant embryos during gastrulation at stages earlier than E9, when mutant embryos were still healthy. In Sip1−/− embryos of up to the 7-somite stage, the number of somites was the same as in wild-type embryos. However, the somites were shorter in the rostro-caudal dimension and irregular in the shape of their boundary. This is illustrated by in situ hybridization of Uncx4.1, marking the caudal half of already segmented and the next-forming somites (Fig. 2A, B), and was confirmed in parasagittal sections (Fig. 2C–F). In contrast, the size of somites and unsegmented paraxial mesoderm in the lateral dimension was normal (Fig. 2G–J). Analysis of cell proliferation by BrdU labeling and of apoptosis using TUNEL technique indicated no significant difference with wild-type embryos (data not shown). The length of trunk paraxial mesoderm, measured between the rostral margin of the second somite and the caudal tip of the embryo, was not significantly different among embryos regardless of the Sip1 genotype and of the developmental stages (Fig. 2K), indicating that the caudal extension of the unsegmented paraxial mesoderm was unaffected even after the 7-somite stage. Thus, in Sip1−/− mutant embryos, only the length of the area occupied by the segmented somites was reduced, and this was compensated for by the extension of the unsegmented, more caudal region (Fig. 2L).

Figure 2.

Short somites generated in Sip1−/− embryos. A,B: Uncx4.1 in situ hybridization to mark the posterior half of somite in the wild-type (+/+) (A) and Sip1−/− (B) embryos. C–F: Parasagittal paraffin sections of wild-type (C) and Sip1−/− embryo (E) stained with Hematoxilin-Eosin showing the paraxial mesoderm. D and F are an enlargement of C and E, respectively. G–J: Transverse sections of analogous wild-type (G, H) and Sip1−/− (I, J) embryos showing both segmented (G, I) and unsegmented (H, J) regions, at the levels indicated in C and E. K: The length of trunk paraxial mesoderm at various developmental stages in wild-type and Sip1−/− embryos, as measured from the second somite to the posterior end of embryos. L: The proportion of the length of the segmented region to that of trunk paraxial mesoderm in wild-type, Sip1+/− and Sip1−/− embryos. M–O: Embryos at E9.0 when a normal embryo (M) has 18 somites, and a Sip1−/− embryo (N) has been arrested for somite segmentation at the seven pair of somites. O: Enlargement of the boxed area in N showing seven regularly arranged, short somites. P: Schematic presentation of the aberrant somite morphology and overall somitogenesis in wild-type and Sip1−/− embryos. Scale bars = 100 μm (C–F), 50 μm (G–J).

Although somite segmentation proceeded up to the 7-somite stage in Sip1−/− embryos, no somite cleavage occurred beyond this stage (Fig. 2M–O, and the schematic presentation in Fig. 2P).

Rostral Expansion of the Region of Gene Oscillation

Lunatic fringe (L-fng) and Hes7 are representative genes that display oscillatory expression in the unsegmented paraxial mesoderm, the most rostral stripe halting at the next-forming somite boundary in mouse embryos (Fig. 3Aa and Ac), and have been documented to participate in somite segmentation (Bessho et al.,2001a; Cole et al.,2002; Morales et al.,2002; Dale et al.,2003).

Figure 3.

A: Whole-mount in situ hybridization to detect expression of the oscillatory genes in embryo at 6-somite stage (E8.5). a,c,e,g: Normal embryos. b,d,f,h: Sip1−/− embryos. L-fng (a, b), Hes7 (c, d), Dll1 (e, f), Axin2 (g, h). B: A set of embryos at the 7-somite stage (E8.5) demonstrating the oscillatory expression of mouse Dll1 gene in the unsegmented paraxial mesoderm. a–c: Embryos hybridized with the Fgf8 probe showing three progressive stages of oscillating L-fng gene expression (Bessho et al.,2001b; Serth et al.,2003). d–f: The same embryos developed for Dll1 signal after stripping the L-fng signal. The most caudal peak of Dll1 expression is marked by the asterisk. Black arrowheads indicate the position of the latest somite cleavage.

Dll1 expression has not been considered to oscillate (Hrabe de Angelis et al.,1997), presumably after over-development of the hybridization signals. However, we found that its expression oscillates synchronously with L-fng, as shown by hybridization of the same embryo specimens using L-fng and Dll1 probes (Fig. 3B). This observation is consistent with the oscillatory expression of deltaC, a zebrafish homologue of mouse Dll1 (Jiang et al.,2000).

It is also known that Axin2 is strongly expressed in the caudal end of the embryo and its expression continues to the anterior paraxial mesoderm, rostrally ending with one or two stripes of Axin2 expression in the rostral margins of presumptive somites (Fig. 3Ag) (Aulehla et al.,2003).

In Sip1−/− embryos, the domain of paraxial mesoderm expressing these genes is rostrally expanded. One or two additional stripes of L-fng and Hes7 expression occurred in the unsegmented paraxial mesoderm, with the most rostral stripe reaching nearly to the last-formed segmental boundary (Fig. 3Ab and Ad). In addition to L-fng and Hes7, the striped expression domain of Dll1 also showed anterior extension (Fig. 3Af). Axin2 expression was also rostrally expanded, similar to other oscillatory genes, and a stripe of Axin2 expression occurred just caudal to the most recently formed segmentation boundary (Fig. 3Ah).

Rostral Expansion of Fgf8 Expression in the Sip1−/− Embryos

The above observations suggested that the unsegmented region of paraxial mesoderm was expanded rostrally. Several genes, including Fgf8, are known to be involved in the maintenance of unsegmented state. At various stages of somitogenesis, Fgf8 mRNA was detected in the unsegmented region more rostrally extended in Sip1−/− embryos than normal, as shown using an exon-specific probe (Fig. 4A–D). Interestingly, when an intron-specific probe was used to detect nascent transcripts, its expression domain was identical between Sip1−/− and wild-type embryos (Fig. 4E,F). This suggests that rostral expansion of Fgf8 expression is not caused by expansion of the cell population actively transcribing Fgf8 but rather results from extended stabilization of Fgf8 transcripts in the paraxial mesoderm of Sip1−/− embryos. In addition, the expression of Wnt3a, Dll3, and Tbx6 was also extended rostrally (Fig. 4G–L). Therefore, Sip1 deficiency caused general rostral expansion of the expression domain of the genes involved in the maintenance of the unsegmented state of paraxial mesoderm.

Figure 4.

In situ hybridization to detect expression of genes involved in the maintenance of unsegmented state of paraxial mesoderm. A,C,E,G,I,K: Wild-type embryos. B,D,F,H,J,L: Sip1−/− embryos. Fgf8 mRNA at the 3-somite stage (A, B). Other embryos are shown at the 6-somite stage. Fgf8 mRNA expression (C, D). Fgf8 intron sequence expression (E, F). Wnt3a expression (G, H). Dll3 expression (I, J). Tbx6 expression (K, L). Black arrowheads indicate the position of the latest somite cleavage.

Raldh2 Expression in Sip1−/− Embryos

It is known that Fgf8 and retinoic acid synthesis mediated by Raldh2 (retinaldehyde dehydrogenase 2) gene are mutually repressive, and retinoic acid affects Fgf8 mRNA distribution in the paraxial mesoderm (Diez del Corral et al.,2003). We thus examined whether the Raldh2 expression in the paraxial mesoderm is altered in Sip1−/− embryos, using in situ hybridization of Raldh2 transcripts. In wild-type embryos, the domain of strong Raldh2 expression in paraxial mesoderm starts from the rostral region and terminates at the position one somite length more caudal to the last somite cleavage, namely in the position rostral to the block of the strongest Sip1 expression (Figs. 5A and 6A). In the Sip1−/− embryos, the boundary of strong Raldh2 expression was shifted rostrally, in a way complementary to rostral extension of Fgf8 mRNA distribution (Figs. 5B and 6A). This raises the possibility that Sip1 activity is involved in modulating the mutually repressive Raldh2-Fgf8 interaction.

Figure 5.

Raldh2 expression in wild-type (A) and Sip1−/− embryos (B) at the 6-somite stage. Black arrowheads indicate the position of the latest somite cleavage. The domain of strong Raldh2 expression is bracketed.

Figure 6.

The summary of observations and possible models of SIP1 function in the regulation of somitogenesis. A: Comparison of Sip1, Fgf8, and Raldh2 expression in the paraxial mesoderm of wild-type and Sip1−/− embryo employing the 3-somite stage as model. B: Possible models of regulation of Fgf8 mRNA distribution by SIP1 in wild-type embryos. Black and white arrowheads indicate the positions of the latest somite cleavage and next-forming somite boundaries, respectively. Model 1: SIP1 activity leads to promotion of Fgf8 mRNA degradation in the same Sip1-expressing cells. Model 2: SIP1 primarily augments Raldh2 expression, and secondarily promotes Fgf8 mRNA degradation. RA indicates retinoic acid.


Based on our observations in this study, we find it likely that the rostral expression of Fgf8 regresses more slowly in a caudal direction in Sip1−/− mutants than in normal embryos. According to the “clock and wavefront” model (Cooke and Zeeman,1976), a slower caudal regression of the “wavefront” indeed results in shorter inter-segmental distance. We find that (1) the rostral expansion of expression of Fgf8, which is involved in the maintenance of unsegmented state of paraxial mesoderm (Fig. 4), (2) the rostral extension of the domain of oscillatory gene expression (Fig. 3A), and (3) the shorter somites produced in Sip1−/− embryo (Fig. 2) are consistent with this model.

As Sip1 is expressed both in the neural and mesodermal tissue (Fig. 1), it is formally possible that Sip1 expressed in the neural plate determines the rostral limit of Fgf8 mRNA distribution in the paraxial mesoderm. However, since the somite segmentation proceeds normally in the absence of the neural plate, as demonstrated using chicken embryos (Chapman et al.,1996), it is more likely that Sip1 expressed in the paraxial mesoderm determines the rostral limit of Fgf8 mRNA distribution, which is indicative of “wavefront” activity in the paraxial mesoderm (Fig. 6B).

It is known that expression of Raldh2, leading to retinoic acid synthesis, and distribution of Fgf8 mRNA are mutually exclusive (Diez del Corral et al.,2003). In Sip1−/− mutant embryos the boundary of strong Raldh2 expression and Fgf8 mRNA was rostrally shifted (Fig. 5), in support of this interaction. Thus, two alternative models for involvement of SIP1 function in regulation of somite segmentation can be considered, either SIP1 regulating Fgf8 mRNA or SIP1 primarily regulating Raldh2 expression (Fig. 6B). In either model, Fgf8 mRNA is regulated for its turnover, as Sip1−/− genotype does not affect Fgf8 transcription per se (Fig. 4E, F), consistent with the previous notion that steepness of Fgf8 mRNA gradient in the paraxial mesoderm is determined by regulation of mRNA stability (Dubrulle and Pourquie,2004). It is possible that SIP1 augments the activity of machinery for Fgf8 mRNA degradation through its transcriptional regulation (Model 1, Fig. 6B). Alternatively, SIP1 may augment Raldh2 expression at the Raldh2/Fgf8 boundary, leading to promotion of Fgf8 mRNA degradation (Model 2, Fig. 6B). Overall, our observations on the defects of somitogenesis in Sip1−/− embryos suggest that SIP1 activity is involved in the positioning of somite boundaries by modulating the Raldh2/Fgf8 interaction boundary in the paraxial mesoderm.



Sip1 +/− heterozygous mice (Higashi et al.,2002) were maintained in ICR background and mated to obtain homozygous (−/−) embryos. The mice and embryos were handled according to the guidelines of the Committee for Animal Experiments, Osaka University, in the research project approved by the committee (approval number 05-025).

Plasmid and In Situ Hybridization

Whole-mount in situ hybridization was done using digoxigenin-labeled probes and color development using NBT (Nitro blue tetrazolium chloride)/BCIP (5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt) as described by Uchikawa et al. (1999), except that 1% SDS was used instead of CHAPS. A full-length Sip1 cDNA sequence was used for preparing the antisense probe. Other probes were originally gifts from A. Mansouri (Uncx4.1; Mansouri et al.,1997), R. Johnston (L-fng; Johnston et al.,1997), R. Kageyama (Hes7; Bessho et al.,2001b), A. Gossler (Dll1; Bettenhausen et al.,1995), P. H. Crossley (Fgf8; Crossley and Martin,1995), S. Takada (Wnt3a; Takada et al.,1994), R. Beddington (Dll3; Dunwoodie et al.,1997), and D. Chapman (Tbx6; Chapman et al.,1996). Fgf8 intron probe was prepared according to Dubrulle and Pourquie (2004).

For detection of Dll1 mRNA, color development with solution of NBT/BCIP was performed at 4°C overnight, rather than at room temperature, in order to avoid over-development of the hybridization signal. For sequential detection of L-fng and Dll1 in the same embryo specimens, the probes were labeled by fluorescein and digoxigenin, respectively, and color development using alkaline phosphatase-conjugated anti-fulorescein and INT (2-[4-lodophenyl]-3- [4-nitrophenyl]-5-phenyl-tetrazolium chloride)/BCIP was done first, followed by photographic recording and decolorization in methanol. Then Dll1 mRNA was detected as described above.

Measurement of the Length of Paraxial Mesoderm

The length from the rostral end of the second somites to the caudal end of embryo, as well as that of segmented domain, was determined as a trajectory through the dorso-ventral midpoints of paraxial mesoderm, using printout of lateral embryonic images. More than eight embryos were examined for each somite stage.


We appreciate stimulating discussions with Drs. O. Pourquie and Y. Takahashi and members of the Kondoh and Huylebroeck laboratories. We thank Drs. H. Hamada and Y. Saga for provision of in situ hybridization probes. M.M. was a Research Fellow of the Japan Society for the Promotion of Sciences.