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FGF signals are involved in the differentiation of notochord cells and mesenchyme cells of the ascidian Halocynthia roretzi

Yoshie Shimauchi^,, Seiko D. Murakami and Nori Satoh^*

Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Present address: Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan
The first two authors contributed equally to this work

View larger version (72K): [in a new window]   Fig. 1. (A) Nucleotide and predicted amino acid sequences of HrFGFR cDNA. The insert of the cDNA encompasses 3,199 bp including 18 adenylyl residues located at the 3' end. The ATG at positions 118-120 represents the putative start codon for the HrFGFR protein. An asterisk indicates the termination codon. The transmembrane domain is indicated by dotted-underline, and the intracellular tyrosine kinase domains are underlined. Bold, italic capitals indicate cysteine residues forming disulfide bridges characteristic of immunogloblin (Ig)-like domains. Bold capitals indicate amino acid residues characteristic of the tyrosine kinase domain. Arrows indicate two regions of amino acid residues that were used in the molecular phylogenetic analysis shown in Fig. 2. (B) Schematic representation of the structural and functional domains of HrFGFR and the mutant dnHrFGFR. The FGF receptor contains two Ig-like domains bounded by cysteine residues that form disulfide bridges (Ig2 and Ig3), a transmembrane domain (TM), and an intracellular tyrosine kinase domain split by 14 amino acids. dnHrFGFR represents a mutant construct in which the entire tyrosine kinase domain starting from amino acid 369 (marked by an arrow on HrFGFR) was deleted.

View larger version (19K): [in a new window]   Fig. 2. HrFGFR is a routeness of the chordate FGFR family members. A molecular phylogenetic tree was constructed by means of a neighbor-joining method by comparison of amino acid sequences of the tyrosine kinase domains shown in Fig. 1. The mouse VEGFR2 and PDGFR were used as the outgroup. The numbers indicate the relative robustness of each node as assessed by bootstrap analysis (100 replications).

View larger version (45K): [in a new window]   Fig. 3. Genomic Southern analysis of the HrFGFR gene. The genomic DNA prepared from a single H. roretzi was digested with EcoRI (E), HindIII (H) or PstI (P), resolved by electrophoresis, and transferred to a nylon membrane. Blots were hybridized with two different 32P-labeled DNA probes (common C-terminal probe and specific Ig3 domain probe) and washed under high stringency conditions. Bands that show the same mobility at both probes are marked by arrowheads. Each lane was loaded with 10 µg digested DNA.

View larger version (26K): [in a new window]   Fig. 4. Temporal expression of HrFGFR. Northern blots of poly(A)+ RNA prepared from the fertilized eggs (F), 64-cell stage embryos (64), neurulae (N), and tailbud-stage embryos (TB), hybridized with a 32P-labeled probe prepared from the C-terminal domain (upper) and Ig3 domain (lower). Each lane was loaded with 5 µg poly(A)+ RNA. Exposure time was 1.5 days.

View larger version (101K): [in a new window]   Fig. 5. Spatial expression of HrFGFR as revealed by whole-mount in situ hybridization with DIG-labeled antisense probes. (A) An unfertilized egg. (B) A fertilized egg. (C) An 8-cell stage embryo, lateral view. (D,D') A 16-cell stage embryo viewed from (D) the animal pole (future ventral side) and (D') vegetal pole (future dorsal side). (E,E') A 32-cell stage embryo; (E) animal view and (E') vegetal view. (F) A 64-cell stage embryo, animal view. (G) A 110-cell stage embryo, animal view. (H) A gastrula, vegetal view. (I,I') A neurula, (I) dorsal view and (I') lateral view. Signals for zygotic expression are evident in cells of the epidermis in the posterior region. (J,J') An early tailbud embryo viewed from the lateral (J) and dorsal (J') side. (K) A mid-tailbud embryo, lateral view. Signal is seen in epidermal cells of the tail. The scale bar is 100 µm for all panels.

View larger version (50K): [in a new window]   Fig. 6. Effects of injection of mRNAs for HrFGFR or dnHrFGFR on expression of endoderm-specific AP (A-A''), muscle-specific AChE (B-B"), epidermis-specific HrEpiC gene (C-C''), and notochord-specific HrBra (D-D'',E-E'') and Not1 antigen (F-F''). Development of the first three markers and the Not1 antigen was examined at the mid-tailbud stage (A-C,F), while that of HrBra was examined at the 110-cell (D-D'') and initial tailbud stages (E-E''). (A-E) control, uninjected embryos, (A'-E') embryos injected with HrFGFR mRNA; (A''-E'') embryos injected with dnHrFGFR mRNA.

View larger version (28K): [in a new window]   Fig. 7. Semi-quantitative RT-PCR analysis of the amount of HrBra mRNA in normal embryos and dnHrFGFR mRNA-injected embryos. Negative control (RT-) was without reverse transcriptase in cDNA synthesis.

View larger version (52K): [in a new window]   Fig. 8. Effects of injection of dnHrFGFR mRNA on expression of HrCA1, assessed by whole-mount in situ hybridization. (A) Expression of HrCA1 in a control tailbud embryo. Strong signals are evident in mesenchyme cells (mch) and in several neuronal cells (nu) in the anterior and dorsal trunk, while weak signal is evident in notochord cells (N, dotted line) and muscle cells (mu). (B) Embryo injected with HrFGFR mRNA. (C-E) Three examples of embryos injected with dnHrFGFR mRNA. The expression of HrCA1 was downregulated or suppressed. Scale bar is 100 µm for all panels.

View larger version (84K): [in a new window]   Fig. 9. Effects of injection of dnHrFGFR mRNA on expression of HrTBB2, assessed by whole-mount in situ hybridization. (A) Expression of HrTBB2 in a control tailbud embryo. Signals are evident in neuronal cells (nu) in the anterior and dorsal trunk and dorsal tail regions. Embryo injected with (B) HrFGFR mRNA and (C) dnHrFGFR mRNA. The expression pattern of HrTBB2 is disturbed. Scale bar, 100 µm for all panels.