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First published online 14 March 2007
doi: 10.1242/dev.02825

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Cell fate polarization in ascidian mesenchyme/muscle precursors by directed FGF signaling and role for an additional ectodermal FGF antagonizing signal in notochord/nerve cord precursors

¹ Faculty of Marine Bioscience and Technology, Kangnung National University, Gangneung Daehangno, Gangneung 210-702, Republic of Korea.
² Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.

View larger version (62K): [in this window] [in a new window]   Fig. 1. Diagrams showing the fates of cells in the vegetal hemisphere of ascidian embryos. Endoderm-lineage cells are colored yellow. Those for notochord, nerve cord, the brain, mesenchyme and muscle are shown in pink, purple, blue, green and red, respectively. (A,B) Lateral views; anterior is to the left. (A) Tailbud embryos. Upper and lower diagrams illustrate midsagittal and parasagittal sections, respectively. (B) An eight-cell-stage embryo. Fate map of major larval tissues and the name of each blastomere are indicated. (C,D) Vegetal views. Anterior is up. The cells shown by ellipses at the top of each diagram are blastomeres in the animal hemisphere, which are behind the vegetal hemisphere. (C) A 32-cell-stage embryo. Cells expressing FGF9/16/20 are highlighted by black dots. Light blue arrows indicate direction of FGF signaling. Red hatching indicates the proposed area where macho-1 functions. (D) A 64-cell-stage embryo. The pairs of blastomeres connected with bars are sister blastomeres. Br, brain; En, endoderm; Epi, epidermis; Mes, mesenchyme; Mus, muscle; NC, nerve cord; Not, notochord.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Effect of endoderm transplantation. (A,B) Diagrams of the experimental design for endoderm transplantation. Ectoderm cells were removed from early 32-cell host embryos (red twisted arrows). Donor endoderm cells were isolated from early 32-cell embryos. The isolated endoderm cells were transplanted to the ectoderm-depleted region (red arrowheads) so that the presumptive mesenchyme (A) and notochord (B) cells are sandwiched with two groups of endoderm cells. (C-N) Expression of tissue-specific markers in un-manipulated control (left column), endoderm-transplanted (middle column) and ectoderm-transplanted control (right column) embryos. (C-K) Vegetal views. (L-N) Animal views. Anterior is up. (C-E) Expression of the mesenchymespecific Mch-3 antigen in embryos in which cleavage was arrested at the 110-cell stage with cytochalasin B. Arrowhead in D indicates a muscle blastomere that ectopically expressed the Mch-3 antigen. (F-H) Expression of the muscle actin gene at the 110-cell stage. (I-K) Expression of notochord-specific Hr-Bra at the 110-cell stage. Arrowheads in J indicate cells of nerve cord lineage that ectopically expressed Hr-Bra. (L-N) Expression of neural plate-specific Hr-ETR1 in cleavage-arrested 110-cell embryos. Two rows of expression were observed in brain-lineage (arrowheads b) and nerve cord-lineage (arrowheads n) cells. The percentages at the bottom of each photo represent the proportion of embryos that ectopically expressed the Mch-3 antigen and Hr-Bra, and that showed reduced expression of actin and ETR1. Scale bar: 100 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 3. Reversal of FGF signal direction causes reversal of mesenchyme/muscle asymmetric division. Expression of mesenchyme marker (Mch-3) in cleavage-arrested 110-cell embryos (A-G), and of muscle actin in 110-cell-stage embryos (A'-G'). All photos are vegetal pole views; anterior is up. Only the left side of each embryo was manipulated. Diagrams of the experimental designs are shown at the top of the panels. (A,A') The A4.1 and B4.1 blastomeres on the left side were injected with Hr-FGF9/16/20 MO at the eight-cell stage. (B,B') A b4.2 blastomere was injected with FGF mRNA at the eight-cell stage. (C,C') Endodermtransplanted embryos. Donor endoderm cells were injected with FGF MO (MOEn-T) at the eight-cell stage. (D-G,D'-G') b-line ectoderm-transplanted embryos. b-line donor cells were injected with either the FGF mRNA (D,E, mRNA-b4.2-T) or venus YFP mRNA (G, YFP-b4.2-T), and were transplanted. The host embryos had FGF MO (D,E, MO-A4.1-B4.1) or control MO (G, cMO-A4.1-B4.1) injected. (E,E') A subset of the b4.2 descendant cells injected with FGF mRNA (arrowhead in E') were transplanted. In E', the midline of the embryo is indicated by a broken line. (E'') Schematic representation of the results in E and E'. Normal positions of mesenchyme (green) and muscle (red) fates (right half) and altered positions of the fates on the manipulated side (left half) are shown. (F,F') b-line cells were transplanted without injection as a control. The percentage in E represents the proportion of embryos in which muscle-lineage but not mesenchymelineage cells expressed the Mch-3 antigen. The percentage in E' indicates the proportion of embryos in which mesenchyme-lineage but not musclelineage cells expressed actin. Scale bar: 100 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Robust fate specification in nerve cord/notochord precursors. Expression of a notochord marker (Hr-Bra) in 110-cell embryos (A-E), and of a nerve cord marker (Hr-ETR1) in cleavage-arrested 110-cell embryos (A'-E'). Anterior is up. (A-E,A') Vegetal views. (B'-E') Animal views. Manipulation was carried out on the left side of embryos with the exception of those in E and E', where both sides were manipulated. (A,A') 110-cell embryos developed from eight-cell embryos where A4.1 was injected with Hr-FGF9/16/20 MO. Black arrowheads indicate notochord blastomeres that did not express Bra but expressed ETR1. Yellow arrowheads represent notochord blastomeres that weakly expressed Bra. (B,B') a4.2 was injected with FGF mRNA. Red arrowheads represent nerve cord blastomeres that weakly expressed Bra. (C,C') A4.1 and a4.2 blastomeres were injected with FGF MO and mRNA, respectively. ETR1 is expressed broadly in the anterior-animal hemisphere in B' and C'. (D,D') Control embryos to C and C' in which cMO (A4.1) and venus YFP mRNA (a4.2) were injected. (E,E') Endoderm-transplanted embryos. Donor endoderm cells were injected with FGF MO at the eight-cell stage. Black arrowheads indicate nerve cord blastomeres that ectopically expressed Bra. Scale bar: 100 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Suppression of notochord fate by a signal from ectoderm. (A-B') Expression of Hr-Bra in 110-cell embryos (A,B; vegetal views) and of ETR-1 in cleavage-arrested 110-cell embryos (A',B'; animal views). Anterior is up. (A,A') Embryos with a-line cells removed at the early 32-cell stage. Arrowheads indicate cells in the nerve cord lineage that ectopically expressed Bra. (B,B') Embryos with a-line cells removed at the early 64-cell stage. (C-E') Expression of Hr-Bra and ETR-1 in partial embryos. (C,C') Every a-line cell (Ecto) and nerve cord/notochord precursor was co-isolated from early 32-cell embryos and cultured until the 110-cell stage. Arrowheads in C' indicate cells of notochord lineage. They did not express ETR1. (D,D',E,E') The same co-isolate as in C was recombined at the notochord side with a-line cells (D,D') or mesenchyme precursors (E,E'). In D', four rows of ETR1-expressing cells were observed, consisting of two rows of notochord and nerve cord precursors flanked by two rows of brain cells. Br, brain; Epi, epidermis; Mes, mesenchyme; NC, nerve cord; Not, notochord precursors. Scale bar: 100 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 6. Suppression of MAPK activation and FoxA expression by an ectoderm signal. (A,B) Activation of MAPK (ERK) in an unmanipulated control 44-cell-stage embryo (A) and a 44-cell-stage embryo with a-line cells removed at the early 32-cell stage (B). Anterior views. Arrowheads indicate nuclei of the four nerve cord lineage cells to be compared. Yellow arrowheads represent ectopic activation of MAPK in the medial nerve cord precursors. a, animal pole; v, vegetal pole. (A',B') Nuclear staining with DAPI to show the position of blastomeres and their nuclei. Midlines are indicated by broken lines. Pink and white letters below nuclei show blastomeres with and without activated MAPK in their nuclei, respectively. (C-F) Expression of Hr-FoxA (C,D) and Hr-Bra (E,F) in 110-cell embryos. Vegetal views. Anterior is up. (C) An unmanipulated 110-cell-stage embryo. (D) An embryo with a-line cells removed at the early 32-cell stage. Note that the uppermost layer of nerve cord cells is ectopically stained. Dots in the diagrams (C',D') signify the blastomeres that expressed FoxA. (E) A a4.2 blastomere on the left side was injected with Hr-FGF9/16/20 mRNA at the eight-cell stage. Red arrowheads indicate weak ectopic expression of Bra. (F) A4.1 and a4.2 blastomeres on the left side were injected with FoxA and FGF mRNAs at the eight-cell stage, respectively. Black arrowheads indicate nerve cord lineage cells that ectopically expressed Bra. The percentages shown in C-F represent the proportion of embryos that showed ectopic expression of FoxA (C,D) and Bra (E,F) in cells of nerve cord lineage. Scale bar: 100 µm. Ecto, ectoderm; En, endoderm; N, notochord; NC, nerve cord.

View larger version (45K): [in this window] [in a new window]   Fig. 7. A model for patterning of the vegetal hemisphere in ascidian embryos. (A) Patterning in normal embryos. Cell types are highlighted by the same color code as in Fig. 1. At the division to the 64-cell stage, two kinds of asymmetric division take place in the anterior (NC vs. Not) and posterior (Mes vs. Mus) marginal zones. macho-1 is a maternal and intrinsic competence factor for mesenchyme induction. Light blue arrows indicate FGF signal. (B) Direction of asymmetry for segregation of muscle and mesenchyme fates is determined by the direction from which the FGF signal comes. (C) Polarity of asymmetric division that produces nerve cord and notochord precursors is determined by the direction from which the inhibitory signal to suppress notochord fate comes. The signal inhibits activation of MAPK by the FGF signal and expression of FoxA on the nerve cord side. Presence of the ectodermal signal seems to be more crucial for generation of the medial nerve cord precursors (A7.4 cells in Fig. 1) than that of the lateral precursors (A7.8 cells). See text for details. Ecto, ectoderm; En, endoderm; Mes, mesenchyme; Mus, muscle; NC, nerve cord; Not, notochord.