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Binary specification of nerve cord and notochord cell fates in ascidian embryos

Takuya Minokawa^1,*, Kasumi Yagi², Kazuhiro W. Makabe² and Hiroki Nishida^1,

¹ Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
² Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
^* Present address: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (38K): [in a new window]   Fig. 1. Cell lineages of posterior nerve cord and notochord, and expression pattern of HrETR-1. (A-E) Schematic diagrams illustrating ascidian embryos. (A) Tailbud embryo showing nerve cord and notochord. (B) Eight-cell embryo (lateral view; anterior is upwards and vegetal pole is towards the left). (C-E) 32-, 64- and 110-cell embryos, respectively (vegetal views; anterior is upwards). Presumptive nerve cord blastomeres are colored purple. Presumptive notochord blastomeres are colored pink. Short red bars between blastomeres indicate daughter cells generated by the previous cleavages. (F) Lineage tree showing nerve cord and notochord lineages. Only lineage of bilateral half is shown. (G-I) Spatial expression of HrETR-1, as revealed by whole-mount in situ hybridization. (G) Neural plate stage embryo (dorsal view; anterior is towards the left). Expression is seen in most cells of the neural plate, including both the presumptive nerve cord region derived from A4.1 blastomeres and the presumptive brain/palp regions derived from a4.2 blastomeres. White dots indicate the boundary between a4.2 derivatives and A4.1 derivatives. (H,I) Early tailbud embryo: (H) lateral view; (I) dorsal view. Expression is evident in cells of the larval nervous system, including brain, posterior nerve cord and palps.

View larger version (38K): [in a new window]   Fig. 2. (A-D) Expression of HrETR-1 in partial embryos derived from isolated blastomeres at the eight-cell stage. Isolated blastomere is shown in each panel. (A) Partial embryo derived from A4.1 blastomere, which has nerve cord fate. Several cells in the partial embryo express HrETR-1. (B) Partial embryo derived from a4.2 blastomere, which has brain and palp fates. No expression of HrETR-1 is observed. (C) Partial embryo derived from b4.2 blastomere. (D) Partial embryo derived from B4.1 blastomere. (E-H) Partial embryos derived from presumptive nerve cord blastomeres at the 16- (E), 24- (F), 44- (G) and 76-cell (H) stages. All express HrETR-1. In F, two partial embryos are shown. Scale bar: 100 µm.

View larger version (154K): [in a new window]   Fig. 3. Expression of HrETR-1 in fully dissociated cells originating from blastomeres in animal hemisphere (A) and in vegetal hemisphere (B). Cells expressing HrETR-1 (arrowheads) are present only in B. Scale bar: 100 µm.

View larger version (49K): [in a new window]   Fig. 4. Expression of notochord and nerve cord markers in partial embryos derived from A7.3 presumptive notochord blastomeres isolated at 44-cell stage (A,C,E) and at 64-cell stage (B,D,F). (A,B) Morphological observation. Arrowheads indicate cells with typical morphology of notochord cells in partial embryos. (C,D) Expression of Not-1 antigen. (E,F) Expression of HrETR-1. Arrows indicate expression of HrETR-1. Scale bar: 50 µm.

View larger version (41K): [in a new window]   Fig. 5. Expression of notochord and nerve cord markers in two-celled partial embryos. (A) Diagram of the experimental procedure to make two-celled partial embryos. See text for details. (B,D) Two-celled partial embryos isolated before notochord induction. (B) Expression of Not-1 antigen. No expression is observed in either blastomere. (D) Expression of HrETR-1. Both blastomeres show HrETR-1 expression. (C,E) Two-celled partial embryos isolated after notochord induction. (C) Expression of Not-1 antigen. The larger blastomere expresses Not-1. The smaller shows no trace of Not-1 expression. (E) Expression of HrETR-1. The smaller blastomere expresses HrETR-1. The larger one shows no HrETR-1 expression. Scale bar: 100 µm.

View larger version (48K): [in a new window]   Fig. 6. Expression of notochord and nerve cord markers in two-celled partial embryos treated with bFGF. (A) Diagram to show the experimental procedure to make two-celled partial embryos treated with bFGF. (B,D,F) Expression of tissue-specific markers in two-celled partial embryos treated with bFGF. (B) Expression of Not-1 antigen. Both presumptive notochord blastomere (larger blastomere) and presumptive nerve cord blastomere (smaller one) show strong Not-1 expression. (D,F) Expression of HrETR-1 (D) and HrTBB2 (F) is not seen in either blastomere. (C,E,G) Expression of tissue-specific markers in two-celled partial embryos without FGF treatment. (C) Not-1 antigen is not expressed in either blastomere. (E,G) Both blastomeres show HrETR-1 (E) and HrTBB2 (G) expression. Scale bar: 100 µm.

View larger version (57K): [in a new window]   Fig. 7. Expression of notochord and nerve cord markers in two-celled partial embryos treated with MEK inhibitor and FGFR inhibitor. (A) Diagram of the experimental procedure to make two-celled partial embryos treated with inhibitors (inh.) of FGF signaling. (B,E,H) Treatment with MEK inhibitor. (B) Not-1 is not expressed in either blastomere. (E,H) Both blastomeres in two-celled partial embryo show HrETR-1 (E) and HrTBB2 (H) expression. (C,F,I) Treatment with FGFR inhibitor. (C) Not-1 is not expressed in either blastomere. (F,I) Both blastomeres show HrETR-1 (F) and HrTBB2 (I) expression. (D,G,J) Treatment with DMSO. (D) The larger blastomere (notochord blastomere) expresses Not-1. The smaller one (nerve cord blastomere) does not. (G) Only smaller blastomere expresses HrETR-1. (J) Only smaller blastomere expresses HrTBB2. Scale bar: 100 µm.

View larger version (28K): [in a new window]   Fig. 8. A directed signal and asymmetric division model of tissue specification mechanism in the vegetal hemisphere of the ascidian embryo. The model is applicable to both the anterior and posterior margins of the vegetal hemisphere. (A) Schematic drawing representing embryo at 32-cell stage. Endoderm precursors (En) emanate inductive FGF-like signal (green arrows) to neighboring anterior and posterior blastomeres and polarize them. Posterior-vegetal cytoplasm (PVC; red oblique lines) causes different responsiveness in posterior marginal cells. (B) Asymmetric divisions occur at the 64-cell stage. In the anterior region, one daughter cell that faces the inducer and does not have the PVC assumes notochord fate (Not). In the posterior region, one daughter cell that faces the inducer and contains the PVC adopts mesenchyme fate (Mes). (C) Without inductive signal, both daughter blastomeres in the anterior region assume default nerve cord fate (NC), and those in the posterior region assume default muscle fate (Mus). (D) When isolated blastomeres receive FGF signal all over the surface, both daughter cells develop into notochord or mesenchyme, depending on absence or presence of PVC.