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Fgf/MAPK signalling is a crucial positional cue in somite boundary formation

Atsushi Sawada^1,2, Minori Shinya^1,2, Yun-Jin Jiang³, Atsushi Kawakami¹, Atsushi Kuroiwa² and Hiroyuki Takeda^1,*,

¹ Division of Early Embryogenesis, National Institute of Genetics, Mishima, 411-8540 Japan
² Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
³ Vertebrate Development Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
^* Present address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo. Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

View larger version (71K): [in a new window]   Fig. 1. Fgf/MAPK signalling in the presomitic mesoderm (PSM). Lateral views with anterior towards the top (A-F,M,N) and dorsal views with anterior towards the top (G-L) are shown. Black arrows indicate ERK activation domain in the PSM. (A) Immunostaining of a two-somite embryo with an antibody against an activated (di-phosphorylated) form of ERK (MAPK). The activation is detected in the anterior telencephalon, midbrain/hindbrain boundary and the PSM (arrow). (B) A sagittal section, taken from a three-somite embryos, shows that the anterior PSM, approximately four- to five-somite wide, is devoid of staining. Black arrowhead in B indicates the last formed somite boundary; red arrowheads in A,B roughly indicate the anterior border of the positive staining in PSM. (C) The immunostaining (at the three-somite stage) is greatly reduced after 8 minutes exposure to SU5402. (D-I) The activation pattern of ERK at the 10-somite stage in wild-type (D,G), fused somites (fss; E,H) and after eight/deltaD (aei; F,I) embryos. Black arrowheads indicate the activation in newly formed somites. Because fss embryos show no segmentation, they were fixed when their siblings reached the 10-somite stage. (J-M) fgf8 expression at the 10-somite stage in wild-type (J,M), fss (K) and aei (L) embryos. Black arrowheads indicate the expression in newly formed somites. (N) FGFR1 expression at the 10-somite stage in wild type. Broad expression in the PSM and anterior restricted expression in segmented somites are observed. The black arrowhead indicates the expression in newly formed somite. (O) In a western blot, the antibody recognizes a major band about 50 kDa, whose level is sensitive to SU5402. The intensity of each band was normalized by use of a SU5402-insensitive minor band (about 25 kDa, shown in the lower panel). The relative intensity is shown at the bottom of each lane. Scale bars: 100 µm (bar in A is applicable to A,C-N).

View larger version (127K): [in a new window]   Fig. 2. Manipulation of an Fgf signal alters somite size. (A-F) The effect of SU5402 treatment on somitogenesis. Lateral views of live embryos at the seven-somite (A,B) and at the 10-somite (C,D) are shown. The somite number is indicated by numbers near the somites. The embryos were incubated in control (A,C) or SU5402-containing medium (B,D) for 8 minutes at the two-somite stage, followed by intense washing. Larger somites (arrowheads in B,D) are observed at the level of seventh and eighth somites, as compaired with those of a control embryo (arrowheads in A,C). (E,F) Sagittal sections from control (E) and treated (F) embryos confirm the formation of large somites. Note that a large somite (asterisk in F) contains more somitic cells that exhibit no cellular abnormality. (G-J) Transplantation of Fgf-soaked beads. Dorsal views are shown with anterior towards the top. The beads soaked in the medium containing Fgf8b or BSA (control) were transplanted on the left side of the tailbud region at the two-somite stage and fixed after various time periods. Red arrowheads indicate the location of the beads in the paraxial mesoderm. (G,H) Ectopic ERK activation is induced by Fgf-soaked bead (H) but not by a control bead (G). (I,J) The control bead does not affect somitogenesis (I). By contrast (J), the segment borders are anteriorly displaced and a small somite (black arrowhead) is formed anterior to the Fgf bead. Red circles along both sides of the notochord indicate the positions of the segment border, and the circles corresponding to each segment are connected with a broken line. (K,L) Sagittal sections (anterior to the left) of the segmented mesoderm from the embryos transplanted with BSA- (K) or Fgf8b-soaked (L) beads (red asterisks). The segment borders are indicated by broken lines. Regular spaced somites are seen anterior to the BSA beads, while the boundary formation is disturbed and smaller somites are formed anterior to the Fgf8 bead. Scale bars: 100 µm in A,G,I; 50 µm in E,K. Bar in A applies to A-D; bar in E applies to E,F; bar in G applies to G,H; bar in I applies to I,J; bar in K applies to K,L.

View larger version (87K): [in a new window]   Fig. 3. Gene expression analyses. Dorsal views are shown with anterior towards the top. (A-C) her1 expression in control (A) and SU5402-treated (B) embryos. The embryos were treated with SU5402 for 8 minutes at the two-somite stage and fixed at the six-somite stage when the prospective large somites are being specified. For comparison, the left half of the control embryo shown in A and the right half of the treated embryo shown in B are shown as a composite (C). In treated embryos, the anterior her1 stripe is missing. Arrows indicate the level of the anterior her1 stripe in a control embryo. (D-F) her1 expression in fused somite (fss) mutants after treated with control (D) or SU5402 (E) medium. A composite (F), the left half of SU5402-treated wild-type embryo shown in B and the right half of SU5402-treated fss embryo in E, shows that the her1 stripe in the intermediate PSM is not maintained in fss–/– background after SU5402 treatment. The arrows in F indicate the level of the intermediate (anteriormost in this case) her1 stripe in the embryo shown in B. As fss embryos show no segmentation, the embryos were treated with SU5402 for 8 minutes when their siblings reached the two-somite stage, and they were fixed when their siblings reached the six-somite stage. (G,H) mesp-a expression at the six-somite stage in control (G) and SU5402-treated (H) embryos. The embryos were treated with SU5402 for 8 minutes at the two-somite stage, fixed at the six-somite stage and followed by in situ hybridization with mesp-a (blue) probe. To make clear the relationship between the mesp-a stripe and the tailbud (indicated by double-headed arrows), the embryos were also stained with the antibody against No tail (Ntl). The notochord and the tailbud is positive for the antibody (yellow). The anterior mesp-a expression domain, which normally demarcates the somite primordium at the position of –II (G) (Durbin et al., 2000; Sawada et al., 2000), posteriorly shifts to the intermediate PSM (H) after treatment of SU5402. (I) A composite picture of papc expression at the six-somite stage in control (left) and SU5402-treated (right) embryos. papc is expressed in four bilateral pairs of stripes in the paraxial mesoderm during the segmentation period (Yamamoto et al., 1998). The first stripe is located in the anterior border of the newest somite formed (I) and the second in the forming somite (0). The two posterior, stronger and broader stripes seem to be located in successive somite primordia (-I and -II). In treated embryos, the positions of the posterior two stripes are posteriorly displaced when compared with control embryos with a wider interval between them. (J-M) her1 expression in the PSM exposed to exogenous Fgfs. The embryos were transplanted with Fgf beads at the two-somite stage, and fixed at the two-somite (J), five-somite (K) and eight-somite (L,M) stages. Red arrowheads indicate the location of the beads in the PSM. The intermediate her1 stripe is anteriorly expanded around the Fgf bead with the posterior border unchanged (L); the anterior her1 stripe is located in more anterior region than on the control side (arrow in L). In fss embryo, the transplanted Fgf-bead exerts the same effect on the intermediate her1 stripe (M; compare with L). Note that the anterior her1 stripes are always missing in fss embryos on both sides of the PSM. (N-P) her1 expression in after eight (aei) mutant after treatment with control (N) or SU5402 (O) medium. A composite (P) shows that the anterior her1 expression shifts posteriorly to the intermediate PSM after treatment with SU5402. The arrows in P indicate the level of the anteriormost her1 expression in control and treated aei embryos. In aei mutants, the first eight to ten somites are normally formed and, thereafter, defects in boundary formation are evident. Thus, the embryos were treated with SU5402 at the four-somite stage, and fixed at the eight-somite stage (later stages than other experiments). The genotyping of the embryos was carried out using PCR (Holley et al., 2000). Scale bar: 100 µm.

View larger version (21K): [in a new window]   Fig. 4. Models of how Fgf/MAPK signalling is involved in somite boundary formation. (A) An exogenous Fgf signal in the anterior PSM maintains the PSM in an immature status for a longer period. This anteriorized the position at which the domain transition of the her1 cyclic expression, from the cycling (in light blue) to the stabilized (in dark blue), takes place. This results in an anteriorly expanded intermediate her1 stripe. (B) In fss embryos, anterior her1 stripe is missing, owing to maturation defects. The her1 expression pattern in fss embryos is apparently similar to that in wild-type embryos treated with SU5402. However, SU5402 treatment of fss embryos discriminates between them. In wild-type embryos treated with SU5402, the anterior her1 stripe disappears owing to a premature domain transition of her1 expression induced by accelerated maturation of the PSM. (C) An Fgf signal activated in the intermediate and posterior PSM antagonizes maturation of PSM cells. In the maturation wavefront, the anterior PSM, which is devoid of MAPK activation, becomes competent to initiate furrow formation in response to the oscillator. When an Fgf signal is transiently compromised by SU5402, the maturation is accelerated and the wavefront is posteriorly expanded, which leads to a posterior shift in furrow formation. The presumptive somites are indicated by Roman numerals.

View larger version (136K): [in a new window]   Fig. 5. The effects of Fgf manipulation on cell death, proliferation and migration in the PSM. (A,B) Sagittal sections (anterior towards the left) showing apoptotic cells (brown, arrowheads) in the PSM after SU5402 treatment (A) and Fgf8b-bead (asterisk) transplantation control (B). (C,D) Sagittal sections (anterior to the left) showing cells in M phase (brown, arrowheads) in the PSM after SU5402 treatment (C) and in an Fgf8b-bead (asterisk) transplantation control (D). (E,F) Cell lineage analysis in the PSM. Fgf8b-soaked bead (asterisks) was transplanted in the tailbud of two-somite stage embryos injected with cased fluorescein. A group of PSM cells at the same axial level of both transplanted and control sides were then fluorescently labelled by laser-assisted uncaging (E). A white arrowhead indicates the tailbud. The relative position of labelled cells (arrows) is still maintained when examined at the 12-somite stage after 5 hours’ incubation (F), although the points of segment furrow formation are anteriorly displaced on the transplanted side. Each segment border of the same numbered somites is connected by a red line. Scale bars: in A, 50 µm for A-D; in E, 100 µm for E,F.