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Regulation of the muscle-specific expression and function of an ascidian T-box gene, As-T2

Yasuo Mitani1,*,§, Hiroki Takahashi1,2,{ddagger},§ and Nori Satoh1,¶

1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
2 PRESTO, Japan Science and Technology Corporation, Japan
* Present address: National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
{ddagger} Present address: Department of Developmental Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan
§ These authors contributed equally to this work



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  		Fig. 1. Effects of microinjection of As-T2/EnR mRNA on the expression of muscle-specific actin gene (HrMA4) and myosin heavy chain gene (HrMHC), assessed by whole-mount in situ hybridization. (A-F) Expression of HrMA4 and (G-Q) HrMHC. (A-C,G-I,O-Q) Embryos around the 110-cell stage, (D-F,J-L) at the early tailbud stage and (M,N) at the 64-cell stage. (A,D,G,J,M) Control embryos; (B,E,H,K,N) embryos developed from eggs injected with 0.2 µg/µl As-T2/EnR mRNA and (C,F,I,L) those injected with 0.01 µg/µl As-T2/EnR mRNA. (O-Q) Co-injection of 0.2 µg/µl As-T2/EnR mRNA with (O) 0.05 µg/µl As-T2, (P) 0.2 µg/µl As-T2 or (Q) 0.1 µg/µl HrBra mRNA. Scale bar: 100 µm.

 


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  		Fig. 2. Minimal promoter for specific expression of As-T2 in two embryonic regions (muscle and the tip of the tail) of Halocynthia embryos. (A) Various deletion constructs examined to determine essential flanking sequences. (B) Frequency of embryos with the reporter gene expression and embryonic regions of expression. Numbers in graphs indicate the number of positive embryos. The deletion constructs indicated at the bottom are the same as those in A.

 


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  		Fig. 3. Expression of lacZ in Halocynthia tailbud-stage embryos that developed from eggs injected with various deletion constructs of pAs-T2/lacZ. (A) Injection of p(-2164)As-T2/lacZ resulted in the expression of the reporter gene in muscle cells (Mu; arrow) and TT cells (arrowhead). En, endoderm; N, notochord. Scale bar: 100 µm. (B) Injection of p(-2164 ~ -555)As-T2/lacZ resulted in the expression of lacZ in TT (tip of the tail) cells. (C) Injection of p(-230)As-T2/lacZ resulted in the expression of lacZ in muscle cells (Mu).

 


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  		Fig. 4. (A) Nucleotide sequence of the 5'-flanking region of As-T2, including the regions for its muscle-specific (shown with red letters) and TT-specific (shown with blue letters) expression. A putative Gli protein-binding motif is shown with a yellow underline. The transcription start site is shown as +1, and eleven deduced amino acids are shown in the lower right-hand corner. The purple box indicates the Tp (proximal T-binding motif) and the green box indicates the Td (distal T-binding motif). (B) Suggested motifs and sequences responsible for the specific expression of As-T2. (C) Sequences of T protein-binding motifs shared by various T-related genes. Dots indicate conserved nucleotides.

 


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  		Fig. 5. Two T-binding motifs and autoregulation of As-T2. (A-D) Expression of lacZ in the 110-cell stage embryos when p(-230)As-T2/lacZ was injected into fertilized eggs without (A) or with As-T2 mRNA (B,C) or HrBra mRNA (D). (B) As-T2 mRNA was tagged with a sequence encoding GFP, and the expression of GFP in embryos showed the proper translation of As-T2 mRNA. (E-H) Expression of lacZ in the 110-cell stage embryos when p(-351)As-T2/lacZ was injected into fertilized eggs alone (E) or with As-T2 mRNA (F,G) or HrBra mRNA (H). (F) GFP expression confirming proper translation of injected As-T2 mRNA. The red box indicates the putative minimal promoter of As-T2; and Td indicates the distal and Tp the proximal T-binding motif. Injected eggs were allowed to develop to the 110-cell stage, and then cleavage was arrested for about 12 hours before detection of the reporter gene expression. Scale bar: 100 µm.

 


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  		Fig. 6. Requirement of the distal and proximal T-binding motifs of As-T2 for its upregulation assessed by the reporter gene expression. (A,B) p(-351)({Delta}Td)As-T2/lacZ was injected solely (A) or with As-T2 mRNA (B). (C,D) p(-351)({Delta}Tp)As-T2/lacZ injected solely (C) or with As-T2 mRNA (D). Injected eggs were allowed to develop to the 110-cell stage, and then cleavage was arrested for about 12 hours before detection of the reporter gene expression.

 


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  		Fig. 7. Nucleotide sequences of the 5' flanking region of (A) HrMA4 and (B) HrMHC. There is a T-binding motif in HrMA4 (shown by a red stippled box) and in HrMHC (a pink box). Sequences of T-binding motifs are compared (A) between mouse T consensus, HrMA4 and As-T2 (Tp), and (B) between mouse T consensus, HrMHC and As-T2 (Tp). Black dots represent nucleotides shared by the T consensus, and white dots those shared by As-T2 (Tp).

 


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  		Fig. 8. Efficiency of T-binding motifs in expression of HrMA4 (A-D) and HrMHC (E-H). (A-D) The reporter gene expression in embryos at the 110-cell stage injected with p(-216)HrMA4/lacZ (A), p(-216)HrMA4/lacZ with As-T2 mRNA (B), p(-216)({Delta}T)HrMA4/lacZ (C) and p(-216)({Delta}T)HrMA4/lacZ with As-T2 mRNA (D). (E-H) The reporter gene expression in embryos at the 110-cell stage injected with p(-132)HrMHC/lacZ (E), p(-132)HrMHC/lacZ with As-T2 mRNA (F), p(-132)({Delta}T)HrMHC/lacZ (G) and p(-132)({Delta}T)HrMHC/lacZ with As-T2 mRNA (H). Injected eggs were allowed to develop to the 110-cell stage, and then cleavage was arrested for about 12 hours before detection of the reporter gene expression.

 


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  		Fig. 9. Possible functional circuitry of As-T2 associated with muscle differentiation in Halocynthia embryos (see text for details).

 

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