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First published online December 21, 2007
doi: 10.1242/10.1242/dev.011494

Development 135, 281-290 (2008)
Published by The Company of Biologists 2008
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Maternal-zygotic medaka mutants for fgfr1 reveal its essential role in the migration of the axial mesoderm but not the lateral mesoderm

Atsuko Shimada1,*,{dagger}, Mina Yabusaki1,*, Hitomi Niwa1,*, Hayato Yokoi1,{ddagger}, Kohei Hatta2, Daisuke Kobayashi1 and Hiroyuki Takeda1,{dagger}

1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
2 Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan.


Figure 1
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  		Fig. 1. Schematic representation of the production of an MZ hdf (fgfr1) medaka mutant by germline replacement.

 

Figure 2
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  		Fig. 2. Development and transfer of a donor germline into a hybrid host. (A,B) Parental strains used for the production of interspecific medaka hybrids. (A) A Kaga female. (B) A Hainan male. (C,D) A chimeric host embryo at 1 day (four-somite stage) (C) and 3 days (D) post-fertilization showing a successful transfer of donor PGCs (arrows). The somatic contribution of the donor cells to the anterior neuroectoderm lineage is also shown (* in C). (E) A representative matured host female with a cluster of fertilized eggs (arrow). (F-H) Confirmation of a successful germline transfer by detection of the pigmentation marker gene, b. (F) F1-hybrid; B/B. (G) Donor; b/b. (H) Progeny of a transplanted hybrid host female; b/b. The embryos shown are at 2 days post-fertilization. (I-K) Cross sections of ovaries from a 4-week-old Kaga strain (H), non-transplanted hybrid (J) and transplanted F1-hybrid host (K). The transplanted hybrid ovary contains growing oocytes (arrows in K). (L) Confirmation of germline transfer by detection of strain-specific genetic markers. Two M-markers (Kimura et al., 2004Go) were amplified from genomic DNAs of Kaga, Hainan, F1-hybrid, donor and the progeny of the F1-hybrid transplanted with donor PGCs. Note that the patterns of marker amplification for the progeny are identical to those of the donor. Scale bars: 200 µm.

 

Figure 3
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  		Fig. 3. Gross morphology of the hdf (fgfr1) mutants at segmentation stages. (A-H) Dorsal views of the head (left) and trunk (right) regions at the six-somite stage (st. 21) (A-D) and nine-somite stage (st.22) (E-H). Asterisks in D and H indicate posterior limits of the trunk. (I-T) Cross sections at the levels of forebrain (I-L), midbrain (M-P) and hindbrain (Q-T) indicated by the dashed lines, a-c in E-H, respectively. (U) A dorsal view of the anterior head of the MZ embryo at the 30-somite stage. The anterior head structures are relatively normal in the zygotic (B,F,J,N,R) and maternal (C,G,K,O,S) mutants, although the head in the zygotic mutant is slightly larger than wild type (A,E,I,M,Q). By contrast, in the MZ mutant the brains display abnormal morphologies with a cyclops appearance (D,H,U and arrow in L), large and flattend midbrain (D,H, and arrow in P) and extra hindbrain (arrow in T). Note that mesenchymal cells are abnormally accumulated at the level of the midbrain to hindbrain (asterisks in P and T). Scale bars: 100 µm. A, anterior; LE, lens; M, maternal; MHB, midbrain-hindbrain boundary; MZ, maternal-zygotic mutants; P, posterior; RE, retina; WT, wild type; Z, zygotic.

 

Figure 4
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  		Fig. 4. Mesoderm induction in the MZ hdf (fgfr1) medaka mutant. (A-P) Expression of the early mesoderm markers, no tail (ntl) (A-D) and fgf8 (E-H), dorsal mesoderm marker, chordin (I-L), and the Fgf-downstream target, sprouty4 (M-P), in each of the indicated types of hdf (fgfr1) mutant at the early gastrula stage (50% epiboly, st. 15). Dorsal (upper) and lateral (lower) views of the same embryo are shown in A-D, E-H and M-P. Arrows indicate the organizer region; arrowheads in N indicate weak expression of sprouty4. The embryos of each genotype express ntl (A-D), fgf8 (E-H) and chordin (I-L) in the blastoderm margin. (Q-V) ntl and sprouty4 expression in SU5402-treated embryos at the early gastrula stage (50% epiboly, st. 15). Embryos were treated with SU5402 at concentrations of 0 (2% DMSO) (Q,T), 0.05 (R,U), and 0.1 mg/ml (S,V) from morula to 50% epiboly. The level of ntl expression persisted at the 0.1 mg/ml concentration, whereas sprouty4 expression was undetectable. M; maternal; MZ; maternal zygotic fgfr1 mutants; WT, wild type; Z, zygotic.

 

Figure 5
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  		Fig. 5. The expression of anterior neural markers and sprouty4 in the MZ hdf (fgfr1) medaka mutant. (A-D) Bud-stage (st. 18) embryos were hybridized with pax2, krox20 and ntl probes. (E-H) Twelve-somite-stage (st. 23) embryos were hybridized with bf1, pax2, krox20 and ntl probes. (I-P) Bud-stage (st. 18, I-L) and 12-somite-stage (st. 23, M-P) embryos were hybridized with a sprouty4 probe. The anterior is at the top. The genotypes of the embryos are indicated on the top of each column of panels. Note that the expression levels of bf1 at anterior telencephalon (black arrow in H), pax2 at MHB (red arrow in H) and sprouty4 at MHB (red arrow in P) are reduced in the MZ embryos at the 12-somite stage. AT, anterior telencephalon; M; maternal; MHB, midbrain-hindbrain boundary; MZ; maternal zygotic mutants; r3, rhombomere 3; r5, rhombomere 5; WT; wild type; Z, zygotic.

 

Figure 6
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  		Fig. 6. Impaired development of the prechordal plate in the MZ hdf (fgfr1) medaka mutant. (A-D) Sagittal sections of the bud-stage (st. 18) embryos hybridized with a gooescoid (gsc) probe that stains the prechordal plate. Anterior head region (anterior to the left) is shown. Arrows indicate the expression domain of gsc (i.e. the prechordal plate) underlying the neural epithelium. Note that in the MZ embryo (D), the expression domain of gsc does not cover the entire anterior head region but is prematurely terminated. (E,F) Early migration of the prechordal plate at 50% epiboly (st 15). Dorsal views of maternal (E) and MZ (F) embryos stained with a gsc probe. (E',F') Sagittal sections through the dorsal midline of the embryos shown in E and F. Dashed lines in E',F' represent the border between the epiblast (EP, the future neural epithelium) and the migrating axial mesoderm (arrows). Scale bars: 100 µm in A-D; 50 µm in E',F'. M; maternal; MZ; maternal zygotic fgfr1 mutants; NE, neural epithelium; WT; wild type; Z, zygotic.

 

Figure 7
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  		Fig. 7. Gastrulation movement in the MZ hdf (fgfr1) medaka mutant embryo. (A-J) Epiboly movement in the M (A-E) and MZ (F-J) mutants; the same embryo is shown at all stages in both cases. Dorsal views (animal pole is up) (A,B,F,G). Dorsal-vegetal views (C,D,H,I). Vegetal views (E,J). Dashed lines indicate the border between the blastoderm and the yolk sac. The arrow in J shows a narrow gap between the lateral marginal cells. Note that completion of epiboly in the MZ mutant occurs at the same time as in the M mutant except in the midline region (E,J). (K-R) Movement of the axial and lateral mesoderm cells of the MZ hdf (fgfr1) mutant. Axial (K) or lateral (N) mesoderm cells were labeled via the UV-mediated uncaging of DMNB-caged fluorescein-dextran at the shield stage (st. 14), and traced for anterior migration (L,M) and dorsal migration (O,P), respectively. Arrows in K and N indicate the embryonic shield. The white arrows in L,M,O,P indicate locations of the labeled mesoderm cells at 6 hours after uncaging. The yellow arrows in O,P highlight the enveloping layer. Initial labeling positions are represented by pink arrows in L,M,O,P. (Q,R) Graphs comparing the migration of labeled cells in M (blue) and MZ (red) mutant embryos. Anterior migration (Q) was quantified by the ratio of the length of labeled cells relative to the yolk sac diameter. Dorsal migration (R) was quantified by the angle between the central position of the labeled cells relative to their initial position at the shield stage.

 

Figure 8
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  		Fig. 8. Cell-autonomous role of Fgfr1 during anterior migration of axial mesoderm cells in the medaka. (A) Schematic illustration of the shield transplantation experiment. Donor cells were labeled by both rhodamine-dextran and biotin-dextran. The migrating ability of the transplanted cells was then assayed at the bud stage (st. 18) by assessing the position of the anterior limits of the labeled cells (indicated by the arrow). (B-G) Lateral views of the representative transplanted embryos. Anterior is to the left. Rhodamine-labeled cells in the live embryos (B-D) or biotin-labeled cells in the fixed embryos (E-G) contribute to the axial mesoderm in the host embryos. (B,E) Donor, wild type; host, wild type. Transplanted cells reach the most anterior mesoderm region. (C,F) Donor, MZ mutant; host, wild type. The anterior limit of the migrating cells shifts posteriorly compared with the control transplant in B,E. (D,G) Donor, wild type; host, MZ mutant. The anterior limit of the transplanted cells is located at the most anterior region. Arrowheads indicate the positions of the anterior limits of the migratory cells. (H-S) Cross sections of the anterior neural regions. (H-M) Cross sections at the level of the eye vesicle region (indicated by the dashed lines, a, in E-G). (N-S) Cross sections at the level of the hindbrain region (indicated by the dashed lines, b, in E-G). (K-M,Q-S) Higher magnification of the dashed boxes shown in H-J and N-P, respectively. Biotin-labeled cells contribute to the prechordal mesoderm in the host embryos (arrowheads in K-M and Q-S). Scale bars: 100 µm in B,E; 50 µm in H,N; 10 µm in K,Q.

 

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