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First published online 3 September 2003
doi: 10.1242/dev.00732

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Maternal macho-1 is an intrinsic factor that makes cell response to the same FGF signal differ between mesenchyme and notochord induction in ascidian embryos

Kenji Kobayashi^1,*,, Kaichiro Sawada¹, Hiroki Yamamoto¹, Shuichi Wada^2,*, Hidetoshi Saiga² and Hiroki Nishida¹

¹ Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
² Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minamiohsawa, Hachiohji, Tokyo 192-0397, Japan

View larger version (45K): [in a new window]   Fig. 1. Fate specification in the vegetal hemisphere of ascidian embryos. (A-C) Endoderm (En)-lineage cells are yellow. Mesenchyme (Mes)-lineage cells are shown in green and muscle (Mus)-lineage cells in red. Notochord (Not)- and nerve cord (NC)lineage cells are pink and purple, respectively. (A) Tailbud embryos. Lateral view. Anterior is towards the left. Upper and lower diagrams illustrate midsagittal and parasagittal sections, respectively. (B) 32-cell stage embryo. Vegetal view. Light-blue arrows indicate direction of induction of FGF signaling. Red hatching indicates the location of the posterior-vegetal cytoplasm (PVC). (C) 64-cell stage embryo. Blastomeres connected with blue bars are sister blastomeres. (D) A directed signaling and asymmetric division model of the 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. Light-blue arrows indicate direction of induction of FGF signaling. (a) Schematic drawing representing embryo at the 32-cell stage. Endoderm precursors (En) emanate the inductive FGF signal (light-blue arrows) to neighboring anterior and posterior blastomeres and polarize them. The PVC (red hatching) brings about different responsiveness of posterior marginal cells. (b) Asymmetric divisions occur at the 64-cell stage in both the anterior and posterior marginal zones. For precise positions of blastomeres, see (C). (c) Without inductive signal, both daughter blastomeres in the anterior region assume the default nerve cord fate (NC), and those in the posterior region assume the default muscle fate (Mus). (d) When isolated blastomeres receive inducing FGF signal all over the surface, both daughter cells develop into notochord (Not) or mesenchyme (Mes), depending on absence or presence of PVC, respectively. (E,F) The results of PVC removal and PVC transplantation, respectively. Light-blue arrows indicate direction of induction of FGF signaling. (E) When the PVC is removed, ectopic notochord is induced in the position of presumptive mesenchyme in the posterior region. Mesenchyme formation is suppressed. (F) PVC transplantation to the anterior region of intact eggs results in ectopic mesenchyme and muscle formation in the anterior region. On the other hand, notochord formation is suppressed. NC, nerve cord; Not, notochord; En, endoderm; Mes, mesenchyme; Mus, muscle.

View larger version (59K): [in a new window]   Fig. 2. Effect of suppression of macho-1 function by MO on formation of muscle and notochord. (A-C) Expression of the muscle myosin protein. (A) Control larva in which 300 pg of control MO was injected into fertilized egg. Anterior is towards the left. (B) 100 pg and (C) 300 pg of macho-1 MO was injected. (D-F) Expression of notochord-specific Not1 antigen. (D) Control tailbud embryo that was injected with 300 pg of control MO. (E) 100 pg and (F) 300 pg of macho-1 MO was injected. (B',C',E',F') Morphologies of embryos are shown in B,C,E,F, respectively. Scale bars: 100 µm.

View larger version (57K): [in a new window]   Fig. 3. Formation of mesenchyme detected with Mch-3 antibody in macho-1-deficient and -overexpressing embryos. (A,B) Expression of mesenchyme-specific Mch-3 antigen. (A) Control larva in which control MO was injected. Anterior is towards the left. (B) 100 pg of macho-1 MO was injected. (C) Diagram illustrating the vegetal hemisphere of the 110-cell stage embryo. Presumptive mesenchyme blastomeres are indicated in green. (D-G) Expression of mesenchyme-specific Mch-3 antigen. Embryos whose cleavages were arrested at the 110-cell stage. (D) Control embryo injected with control MO. Anterior is upwards. (E) 100 pg of macho-1 MO was injected. (F) PVC-removed embryo. (G) 50 pg of macho-1 mRNA was injected. White arrowheads indicate ectopic expression of Mch-3 antigen in the anterior half. Anterior is upwards. (H) Lateral view of the eight-cell stage embryo and fate map. Nerve cord (NC), notochord (Not), endoderm (En), mesenchyme (Mes) and muscle (Mus)-forming areas are indicated. Color of each area is the same as in Fig. 1. (I) Expression of mesenchyme-specific Mch-3 antigen in the partial embryos derived from isolated blastomere of the eight-cell embryos. Embryos were injected with 50 pg of control macho-1 mRNA that lacks the zinc-finger domain or 50 pg of macho-1 mRNA. After isolation, blastomeres were treated with FGF or BSA only. Scale bars: 100 µm.

View larger version (58K): [in a new window]   Fig. 5. Muscle and nerve cord formation in macho-1-deficient and -overexpressing embryos. (A-D) Expression of muscle myosin protein in embryos whose cleavage was arrested at the 110-cell stage. (E-I,E'-G') Expression of HrETR-1 in (E I) cleavage-arrested 110-cell embryos and (E'-G') 118-cell stage embryos without cleavage arrest. (A,E,E') Embryos injected with control MO. Anterior is upwards. (B,F,F') PVC-removed embryos. (C,G,G') Embryos injected with (C,G) 100 pg and (G') 300 pg of macho-1 MO. White arrowheads indicate ectopic expression of HrETR-1. Black arrowheads indicate the posterior blastomeres that show no signal. Anterior is upwards. (D,H) 100 pg of macho-1 mRNA was injected. (I) PVC-transplanted embryo. (J) The vegetal hemisphere of the 110-cell stage embryo. Color of blastomeres is the same as in Fig. 1. Scale bars: 100 µm.

View larger version (94K): [in a new window]   Fig. 4. Expression of notochord markers in macho-1-deficient and - overexpressing embryos. (A-D) Expression of HrBra gene at the 110-cell stage. (E-H,G',H') Expression of notochord-specific Not1 antigen in embryos whose cleavage was arrested at the 110-cell stage. (A,E) Embryos injected with control MO. Anterior is upwards. (B,F) PVC-removed embryos. (C,G,G') Embryos injected with (C,G) 100 pg and (G') 300 pg of macho-1 MO. White arrowheads indicate ectopic expression in mesenchyme precursors. Anterior is upwards. (D,H,H') Embryos injected with (D,H') 100 pg and (H) 50 pg of macho-1 mRNA. (I) The vegetal hemisphere of the 110-cell stage embryo. Color of blastomeres is the same as in Fig. 1. (J M) Expression of Not1 antigen in the partial embryos derived from isolated mesenchyme precursor blastomeres. (J,K) B8.5 partial embryos. (L,M) B7.7 partial embryos. (J,L) Isolates from control embryos. (K,L) Isolates from embryos injected with 100 pg of macho-1 MO. Scale bars: 100 µm.

View larger version (63K): [in a new window]   Fig. 6. Hrsna is downstream of macho-1 and inhibits notochord formation. (A) Expression of Hrsna at the 64-cell stage in embryos injected with mutant macho-1 mRNA (100 pg, control), macho-1 mRNA (100 pg) and macho-1 MO (100 pg). Anterior is upwards. (B) Morphology of the embryo injected with 100 pg of Hrsna mRNA. (C) Expression of notochord-specific Not1 antigen in cleavage-arrested 110-cell embryos injected with lacZ mRNA (100 pg, control) or Hrsna mRNA (100 pg). Anterior is upwards. (D) Expression of HrBra in the 110-cell stage embryos injected with lacZ mRNA (100 pg, control) or Hrsna mRNA (100 pg). Anterior is upwards. Scale bars: 100 µm.

View larger version (29K): [in a new window]   Fig. 7. Two-step model of binary fate specification in the marginal cells of the vegetal hemisphere in ascidian embryos. (A) There are two distinct steps to specify the four cell types. The first step is inheritance (or not) of macho-1. The second step is receipt (or not) of the FGF signal. (B) Presence or absence of macho-1 is responsible for making cell responses different in mesenchyme and notochord induction. The molecular identity of the PVC factor shown in Fig. 1D is the macho-1 product.