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First published online 19 November 2003
doi: 10.1242/dev.00847

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Morpholino-based gene knockdown screen of novel genes with developmental function in Ciona intestinalis

Lixy Yamada^1,*, Eiichi Shoguchi^2,*, Shuichi Wada^2,*, Kenji Kobayashi^1,*, Yasuaki Mochizuki^1,*, Yutaka Satou¹ and Nori Satoh^1,2,

¹ Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
² CREST, Japan Science and Technology Agency, Tokyo, Japan

View larger version (54K): [in a new window]   Fig. 1. Control experiments. (A) Morphology of an uninjected control larva. Trunk and tail are indicated by a bracket and a broken line, respectively. (B) Morphology of a larva developed from an egg injected with Ci-Bra morpholino. (C) Expression of notochord marker Ci-talin (arrowhead) in a tailbud embryo visualized by whole-mount in situ hybridization. (D) The absence of Ci-talin expression (white arrowhead) in a tailbud embryo injected with Ci-Bra morpholino. (E) Recovery of Ci-talin expression (arrowhead) in an embryo at the tailbud stage co-injected with Ci-Bra morpholino and its mRNA. Scale bars: 100 µm for A,B; 50 µm for C-E.

View larger version (110K): [in a new window]   Fig. 2. Morphology of larvae showing the `disorganized body plan' phenotype. Examples of embryos at the stage corresponding to normal tadpole larvae (A,F-I,J,L,M) or tailbud embryos (B-E,K,N), showing the `disorganized body plan' phenotype upon injection with morpholinos. The name of the genes examined is shown in the bottom left-hand corner of each panel. See text for details. Scale bar: 50 µm.

View larger version (112K): [in a new window]   Fig. 3. Morphology of larvae showing the `abnormal tail' phenotype. The arrowhead in A-C,E,F shows palps, and the arrow in D,F shows the sensory pigment cells. The name of the gene examined is shown in the bottom left-hand corner of each panel. See text for details. Scale bar: 50 µm.

View larger version (68K): [in a new window]   Fig. 4. Effects of suppression of cieg003h01 function with specific morpholino. (A,C,E,G,I,K) Control embryos. (B,D,F,H,J,L) Embryos injected with specific morpholino against the gene only. (M,N) Embryos co-injected with morpholino and synthetic mRNA. Expression of epidermis marker, Ci-Epi1 (A,B); muscle marker, AchE (C,D); mesenchyme marker, ciad005j06 (E,F); nervous system marker, Ci-ETR (G,H); notochord marker, Ci-talin (I,J); and endoderm marker, ALP (K,L,N). In M, the arrow indicates the tip of the tail and bars show the border between the trunk and tail. All embryos were fixed at the stage corresponding to tailbud stage. See text for details. Scale bar: 50 µm.

View larger version (148K): [in a new window]   Fig. 5. Effects of suppression of citb018a09 function with specific morpholino. (A-C) Morphology of larvae injected with control lacZ morpholino (A), or the first (B) or second (C) forms of citb018a09-specific morpholino. (D-I) Whole-mount in situ hybridization showing the expression of a marker gene for the whole nervous system, Ci-ETR (D,E), and two genes involved in the function of the photosensory organ, Ci-opsin3 (F,G) and Ci-arr (H,I). (D,F,H) Control embryos. (E,G,I) Embryos injected with the first form of citb018a09-specific morpholino. See text for details. Scale bar: 50 µm.