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doi: 10.1242/10.1242/dev.00521

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Suppression of macho-1-directed muscle fate by FGF and BMP is required for formation of posterior endoderm in ascidian embryos

Keisuke Kondoh?, Kenji Kobayashi* and Hiroki Nishida{dagger}

? Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Yokohama 226-8501, Japan
* Present address: Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan



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  		Fig. 1. Cell lineage of endoderm in the ascidian. (A) Positions of the anterior (orange) and posterior (yellow) endoderm and their precursor blastomeres. Eight-cell embryo is lateral view. Anterior is towards the left, animal pole is upwards. Sixty-four-cell embryo is vegetal view. Anterior is upwards. (B) Lineage tree starting from B4.1 posterior-vegetal blastomeres of an eight-cell embryo. Endoderm lineage is yellow. Minor fates are indicated in parentheses.

 


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  		Fig. 2. Dissociation of the descendant cells of the isolated B4.1 blastomeres at the 16-, 32- and 64-cell stages. After dissociation, cells were cultured as partial embryos until hatching stage. Then endoderm formation was detected by expression of alkaline phosphatase (ALP) (A), and muscle formation was monitored by expression of myosin (B). Endoderm formation was suppressed, and every partial embryo developed into muscle.

 


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  		Fig. 4. Effects of MEK and FGFR inhibitor on formation of the posterior endoderm. (A,B) B4.1 blastomeres were isolated at the eight-cell stage and cultured as partial embryos until hatching stage. They expressed both ALP and myosin according to expectation from the cell lineage. Scale bar: 100 µm. (C) B4.1 partial embryos were fixed at the 64-cell stage. Muscle actin gene is expressed in the nuclei of two blastomeres. (D-F) Treatment with a MEK inhibitor, U0126, resulted in loss of endoderm differentiation. Every constituent cell of the partial embryos expressed myosin protein. Actin mRNA is detectable in six blastomeres. (G) MEK inhibitor did not affect endoderm formation in A4.1 isolates. (H-J) Treatment with FGFR inhibitor, SU5402, also resulted in loss of endoderm differentiation. Every constituent cell expressed myosin protein. Actin expression is detectable in six blastomeres. (K) FGFR inhibitor did not affect endoderm formation in A4.1 isolates. (L) Period of sensitivity to MEK inhibitor. B4.1 blastomeres were isolated at the eight-cell stage, then treatment with MEK inhibitor was initiated at various stages. The period of sensitivity ends at the 64-cell stage. A4.1 partial embryos were not sensitive to MEK inhibitor. n, number of partial embryos examined.

 


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  		Fig. 3. Treatment of dissociated cells with bFGF and BMP4. Descendant cells of the isolated B4.1 blastomeres were dissociated at the 16- and 32-cell stages. Endoderm formation was detected by expression of ALP, and muscle formation was monitored by expression of myosin. (A,B) During dissociation, blastomeres were treated with bFGF protein in sea water. In A, ALP-negative partial embryos consist of very small cells that look like mesenchyme cells. (C,D) Blastomeres were treated with BMP4 protein.

 


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  		Fig. 5. Inhibition of macho-1 function is enough to allow endoderm formation in the absence of cell interaction. (A-D) B4.1 blastomeres were isolated from eight-cell embryos and cultured as partial embryos. Embryos were treated with DMSO as controls. Expression of ALP and myosin was detected to monitor the formation of endoderm and muscle cells. (E-H) The partial embryos were treated with a MEK inhibitor, U0126, to inhibit MEK-MAPK signaling. In F, every constituent cell expressed myosin. (A,B,E,F) Control morpholino antisense oligonucleotide (MO) was injected into fertilized eggs. (C,D,G,H) Antisense MO complementary to macho-1 mRNA was injected to inhibit macho-1 translation. Slight staining in D and H is background caused by mitochondria. Muscle formation was totally suppressed. In G, ALP expression is visible even when embryos were treated with MEK inhibitor. Scale bar: 100 µm.

 


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  		Fig. 6. Redundant BMP signaling induces posterior endoderm formation. (A-D) Cell divisions were permanently arrested at the 110-cell stage by cytochalasin B, and whole embryos were cultured until hatching stage. Then expression of ALP (A,B) and myosin (C,D) was detected. Vegetal views. Arrows indicate the posterior endoderm blastomeres. Treatment with MEK inhibitor did not affect ALP expression in the posterior endoderm blastomeres. Arrowheads indicate presumptive mesenchyme blastomeres that ectopically express muscle myosin. (E,F) The anterior-vegetal A4.1 blastomeres were removed from embryos ({Delta} A4.1) with a fine glass needle at the eight-cell stage. The embryos were cultured without cleavage-arrest until hatching stage, and ALP expression was detected. Without A4.1 blastomeres, embryos became sensitive to MEK inhibitor (F). (G-I) B4.1 partial embryos treated with DMSO (G), MEK inhibitor (H), and both MEK inhibitor and BMP4 protein (I). In I, BMP4 restored ALP expression. Scale bars: 100 µm.

 


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  		Fig. 7. Suppression of muscle fate in non-muscle lineages by cell interactions is required for proper patterning of the posterovegetal quarter of early ascidian embryos. When cell interactions were inhibited by cell dissociation or treatment with MEK inhibitor, every B-line cell assumed muscle fate as directed by macho-1. macho-1 protein would distribute into all B-line cells (pink area). Mes, mesenchyme; TVC, trunk ventral cell.

 

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© The Company of Biologists Ltd 2003