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First published online 28 April 2004
doi: 10.1242/dev.01145

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The ascidian Mesp gene specifies heart precursor cells

Yutaka Satou^1,*,, Kaoru S. Imai^1,* and Nori Satoh^1,2

¹ Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan
² CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 333-0012, Japan

View larger version (40K): [in a new window]   Fig. 1. (A-D) Schematic representation of ascidian heart development. (A) A pair of heart lineage cells (B7.5) in the bilaterally symmetrical 110-cell embryo. (B) Heart progenitors called trunk ventral cells (TVCs) in the tailbud embryo, which differentiate first on each lateral side of the embryo and (C) then fuse ventrally in the swimming larva. In (B,C), upper panels show lateral views and lower panels show ventral views. A pair of anterior muscle cells is also derived from B7.5 (yellow). (D) The heart in the juvenile. (E-H) The developmental fate of the B7.5 blastomere. (E) One of the B7.5 blastomeres is labeled with DiI (arrow). (F) A tailbud embryo and (G) a larva in which DiI was injected into B7.5 at the 110-cell stage. The TVCs are labeled with DiI (arrows), as well as two anterior muscle cells (arrowheads). (H) A juvenile in which DiI was injected into B6.3 at the 32-cell stage. The heart (white arrow), germline cells (black arrow) and degenerated larval tail muscle cells (white arrowhead) are labeled.

View larger version (17K): [in a new window]   Fig. 2. Ciona Mesp gene. A molecular phylogenetic tree generated by the neighbor-joining method based on the alignment of the bHLH domain indicating orthology between Mesp proteins of two Ciona species (enclosed by boxes) and other animals. 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 (cut-off value=50%). E proteins of Drosophila (daughterless), C. intestinalis (Ci-E12/E47) and humans (TFE2) are used as outgroups. Proteins of animals other than ascidians are shown by their accession number, species abbreviation and protein name. Species abbreviations are as follows: HS, Homo sapiens; MM, Mus musculus; DR, Danio rerio; DM, Drosophila melanogaster; Ci, Ciona intestinalis; Cs, Ciona savignyi.

View larger version (151K): [in a new window]   Fig. 3. Cs-Mesp is expressed transiently and specifically in (A) B7.5 blastomeres at the 110-cell stage, (B) their daughter cells (B8.9 and B8.10) at the early to mid-gastrula stage, and (C) their granddaughter cells (B9.17, B9.18, B9.19 and B9.20) at the late gastrula stage. (D) The expression is not observed at the neurula stage. The signals are shown by arrows. Scale bar represents 100 µm.

View larger version (47K): [in a new window]   Fig. 4. Cs-Mesp is essential for specification of TVCs. (A-E) Expression of genes in TVCs of control and (A'-E') Cs-Mesp-MO1-injected embryos. (A,A') Cs-Nkx; (B,B') Cs-HAND; (C,C') NoTrlc; (D,D') a gene orthologous to the C. intestinalis ID00152 gene; and (E,E') ATP2A1/2/3. Inserts in (A,A',B,B') are ventral views of the trunk region showing Cs-Nkx and Cs-HAND expression. Arrows in (A-E) indicate TVCs. Red arrows in A'-E' indicate suppressed expression of downstream genes in TVCs. (F,F') Tailbud embryos in which the B7.5-lineage cells are labeled with DiI. (F) In a control embryo, TVCs (white arrow) and two anterior muscle cells (arrowheads) are labeled. (F') In a Cs-Mesp-MO1-injected embryo, labeled cells are not observed in the region in which TVCs should be located (a red arrow). Instead, four labeled cells are observed in the tail (arrowheads).

View larger version (97K): [in a new window]   Fig. 5. Differentiation of tissues other than TVC in Cs-Mesp knockdown embryos. (A) Suppression of Cs-Mesp with specific morpholino did not affect larval morphology or the expression of (B) endoderm-specific histochemical activity of alkaline phosphatase (arrow), (C) epidermis-specific gene Cs-Epi1, (D) pan-neural marker gene Cs-ETR, (E) mesenchyme-specific gene Cs-Mech1, (F) notochord-specific gene Cs-fibrn, and (G) a muscle-specific actin gene Cs-MA1. (H,I) Expression of Cs-MA1 in (H) control and (I) Cs-Mesp knockdown embryos, which are arrested at the 110-cell stage. As indicated by arrows, a pair of B7.5 blastomeres expresses Cs-MA1 both in control and Cs-Mesp knockdown embryos.

View larger version (96K): [in a new window]   Fig. 6. Cs-Mesp is essential for juvenile heart development. (A) A juvenile with two gill-slits developed from a control egg and (A') that from a Cs-Mesp-MO1-injected egg. Black and red arrows indicate the heart and the region in which the heart should be found.

View larger version (84K): [in a new window]   Fig. 7. Expression of Cs-Mesp is under the control of ß-catenin and Cs-macho1. (A-C) Expression of Cs-Mesp at the late gastrula stage. (A) Control embryos and embryos injected with (B) ß-catenin and (C) Cs-macho1 morpholino. (D) Quantification of the amount of Cs-Mesp mRNA in control embryos and experimental embryos developed from eggs injected with ß-catenin and Cs-macho1 morpholino. Quantification was performed by real-time RT-PCR. The relative amounts of mRNA compared with those in control embryos are shown.