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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00652

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A Twist-like bHLH gene is a downstream factor of an endogenous FGF and determines mesenchymal fate in the ascidian embryos

Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

View larger version (101K): [in a new window]   Fig. 2. Expression profiles of genes characterized in the present study, as revealed by whole-mount in situ hybridization. (A) Schematic representation of ascidian embryos from the 32-cell stage to the tailbud stage. A7.6-line mesenchyme cells (TLCs) are shown in red. At the 32-cell stage, progenitor cells (A6.3) of A7.6 are shown in pink, because the developmental fate of A6.3 is not restricted to mesenchyme. B8.5-line and B7.7-line mesenchymal cells are shown in blue and yellow. Prior to the 110-cell stage and the 64-cell stage, the progenitors of B8.5 and B7.7 are not restricted to mesenchyme and, therefore, they are shown in light blue and light yellow, respectively. Up to the 110-cell stage, the colors identify individual blastomeres and the names for the colored blastomeres are shown. After the 110-cell stage, the colors identify cell populations sharing their origins. (B-F) Expression of Cs-Twist-like1 (B), Cs-Twist-like2 (C), Cs-NoTrlc (D), Cs-Mist (E) and Cs-Hex (F). (G,H) Expression of Cs-Mist (G) and Cs-Hex (H) in embryos arrested at the 110-cell stage. Embryos from the 32-cell stage to late gastrula stage are shown in a vegetal view and tailbud embryos are shown in a lateral view. Scale bar represents 100 µm.

View larger version (60K): [in a new window]   Fig. 1. (A) Alignment of amino acid residues of the bHLH domain of Twist-like1 and Twist-like2 of two Ciona species, and Twist proteins, atonal proteins and N-twist proteins of other animals. (B) A molecular phylogenetic tree generated by the neighbor-joining method based on the alignment of the bHLH domain. The number on each node indicates the percentage of times that a node was supported in 1000 bootstrap pseudoreplications. The tree is shown as a bootstrap consensus tree (cutoff value=50%). Mouse neurogenin1 is used as an outgroup protein. Proteins of animals other than ascidians are shown by their accession number, species abbreviation and protein name. Species abbreviations are as follows: MM, mouse; HS, human; RN, rat; XL, Xenopus laevis; DR, zebrafish; BB, Branchiostoma belcheri; DM, Drosophila melanogaster; CE, Caenorhabditis elegans; EC, Enchytraeus coronatus; HA, Helix aspersa; PV, Patella vulgata; TT, Transennella tantilla; IO, Ilyanassa obsoleta.

View larger version (86K): [in a new window]   Fig. 7. Effects of suppression by the Cs-NoTrlc morpholino on the mesenchyme-specific gene Cs-Mech1 (A,A') and Cs-Twist-like2 (B,B'). (A,B) Control embryos. (A',B') Embryos developed from eggs injected with the Cs-NoTrlc morpholino. (B,B') Embryos arrested at the 110-cell stage and (B and B' insets) tailbud embryos. White arrowheads in A' and B' indicate loss of expression of Cs-Mech1 and Cs-Twist-like2, respectively, in TLCs (compare with expression shown by black arrowheads in A and B). (C,C') Expression of Cs-Twist-like2 in control (C) and Cs-NoTrlc-mRNA overexpressing (C') embryos.

View larger version (91K): [in a new window]   Fig. 9. Effect of functional suppression of Cs-FoxD on the differentiation of TLCs. Expression of Cs-Twist-like2 (A,A') and Cs-Twist-like1 (B,B',C,C') in control embryos (A-C) and embryos developed from eggs injected with the Cs-FoxD morpholino (A'-C'). The loss of expression of the genes in TLCs is shown by white arrowheads. (A,A',C,C') Embryos arrested at the 110-cell stage, in which ectopic expression of Cs-Twist-like1 and Cs-Twist-like2 was evident in B-line notochord cells (black arrows).

View larger version (58K): [in a new window]   Fig. 3. Effects of functional suppression of Cs-Fgf9/16/20 on the expression of Cs-Twist-like1 in the late gastrula (A,A'), Cs-Twist-like2 in the tailbud embryo (B,B'), Cs-NoTrlc in the 64-cell embryo (C,C'), Cs-Mist in the tailbud embryo (D,D') and Cs-Hex in the tailbud embryo (E,E'), as assessed by in situ hybridization. (A-E) Control embryos and (A'-E') embryos developed from eggs injected with the Cs-Fgf9/16/20 morpholino.

View larger version (49K): [in a new window]   Fig. 4. Genetic cascade among the transcription factors identified in the present study. (A-C) Control embryos. (A'-C') Experimental embryos developed from eggs injected with the Cs-Twist-like1 morpholino. Expression of Cs-Twist-like2 in the late gastrula embryo (A,A') and in the tailbud embryo (insets in A and A'). (B,B') Cs-Mist expression in the tailbud embryo. (C,C') Cs-Hex expression in the tailbud embryo. (D,D',E,E') Expression of Cs-Twist-like1. (D,E) In control early gastrulae, Cs-Twist-like1 was evident in A7.6 blastomeres (black arrowheads). (D',E') In early gastrulae developed from eggs injected with the Cs-NoTrlc morpholino, Cs-Twist-like1 expression was lost in A7.6 blastomeres (white arrowheads). Embryos shown in E ,E' were arrested at the 110-cell stage and cultivated until the control untreated embryos developed to the late gastrula stage.

View larger version (79K): [in a new window]   Fig. 5. (A-I,A'-I') Suppression of Cs-Twist-like1 using specific morpholinos, showing the effects on: larval morphology (A,A'); the expression of the epidermis-specific gene Cs-Epi1 (B,B'), the pan-neural marker gene Cs-ETR (C,C'), the notochord-specific gene Csfibrn (D,D'), and the muscle-specific actin gene Cs-MA1 (E,E',H,H'); the endoderm-specific histochemical activity of alkaline phosphatase (F,F',I,I'); and the mesenchyme-specific gene Cs-Mech1 (G,G'). (A-I) Control embryos. (A'-I') Embryos developed from eggs injected with the Twist-like1-morpholino. (H,H',I,I') Embryos arrested at the 110-cell stage. Arrows in E' and H' indicate the appearance of extra cells with Cs-MA1 expression. Arrows in I' indicate the occurrence of extra cells with histochemically detected activity of alkaline phosphatase. Arrowheads in I and I' indicate pigmented cells that are not alkaline-phosphatase positive. (J,K) Effect of overexpression of Cs-Twist-like1 mRNA as assessed by expression of Cs-Mech1 (J) and Cs-Twist-like2 (K). (L,M) Co-injection of the Cs-FGF9/16/20 morpholino and Cs-Twist-like1 mRNA resulted in the recovery of Cs-Mech1 (L) and Cs-Twist-like2 (M) expression. The insets in L and M are control embryos injected with the Cs-FGF9/16/20 morpholino and examined for Cs-Mech1 (L) and Cs-Twist-like2 (M) expression.

View larger version (129K): [in a new window]   Fig. 6. Specificity of Cs-Twist-like1 morpholino as assessed by the expression of Cs-Mech1 (A,A') and Cs-Twist-like2 (B,B'). (A,B) Co-injection of the Cs-Twist-like1 morpholino and Cs-Twist-like1 mRNA with the morpholino recognition sequence resulted in loss of expression of Cs-Mech1 (A) and Cs-Twist-like2 (B). (A',B') Co-injection of the Cs-Twist-like1 morpholino and Cs-Twist-like1 mRNA lacking the morpholino recognition sequence resulted in recovery of the expression of Cs-Mech1 (A') and Cs-Twist-like2 (B').

View larger version (121K): [in a new window]   Fig. 8. Specificity of Cs-NoTrlc morpholino as assessed by the expression of Cs-Twist-like2 in tailbud (A,A') and 110-cell-arrested tailbud (B,B') embryos. (A,B) Co-injection of the Cs-NoTrlc morpholino and Cs-NoTrlc mRNA with morpholino recognition sequence resulted in loss of expression of Cs-Twist-like2 in TLCs. (A',B') Co-injection of the Cs-NoTrlc morpholino and Cs-NoTrlc mRNA without a morpholino recognition sequence resulted in recovery of the expression of Cs-Twist-like2.

View larger version (20K): [in a new window]   Fig. 10. Quantification of the amounts of Cs-NoTrlc and EF2 mRNA in control embryos (-), and in experimental embryos developed from eggs injected with the Cs-FoxD morpholino (inj). Quantification was performed with 64-cell embryos and 110-cell embryos by real-time RT-PCR. The relative amounts of mRNA compared with those in control embryos are shown.

View larger version (22K): [in a new window]   Fig. 11. Representation of the genetic cascades involved in the differentiation of B8.5-line (A), B7.7-line (B) and A7.6-line (C) mesenchyme (TLCs). Arrows indicate positive regulation of genes. Dotted arrows indicate a possible involvement that is suggested on the basis of results from another ascidian species. See details in text.

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