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First published online 2 March 2005
doi: 10.1242/dev.01707

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Making very similar embryos with divergent genomes: conservation of regulatory mechanisms of Otx between the ascidians Halocynthia roretzi and Ciona intestinalis

¹ Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachiohji, Tokyo 192-0397, Japan
² Head Organizer Project, Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuou-Ku, Kobe, Hyougo 650-0047, Japan
³ LGPD, IBDM, Case 907, Campus de Luminy, F-13288 Marseille Cedex 09, France

View larger version (47K): [in a new window]   Fig. 1. The upstream (5402-1473 bp) and the first intron (812-24 bp) regions are capable of directing Hr-Otx transcription during cleavage stages. (A) The structure of 5' region of Hr-Otx, including the putative endogenous promoter (TATAA) and intron 1. Boxes on the gray line indicate exons; ATG indicates the translation start site, which is located in the second exon. The light gray lines are the genomic regions examined for their transcription-driving activity. (B,B',D,D') Expression of Hr-Otx detected by whole-mount in situ hybridization. (C,C',E-F') Transcription of lacZ in embryos injected with the reporter construct harboring the upstream region 5402-1473 (C,C',E,E'), or the intron region 812-24 (F,F'), as visualized by in situ hybridization. In injected embryos, not all Hr-Otx-expressing cells are stained because of the mosaic inheritance of injected DNA. Specimens at the 32-cell stage (B-C') and 64-cell stage (D-F') are shown. (B-F) Animal view; (B'-F') vegetal view. A schematic representation of in situ hybridization is shown on the right side of each specimen (C,C',E-F'). Cells expressing Hr-Otx are colored, according to their derivation from the 8-cell embryo (H), as follows: a-line, dark green; b-line, light green; A-line, orange; and B-line, yellow. Blue dots indicate the cells in which lacZ transcripts were detected. Below the specimens of whole-mount in situ hybridization, the identity of the lacZ-transcribing cell line is indicated. (G) Frequency of lacZ -positive cells for a given cell line in embryos injected with the construct harboring the region 5402-1473bp or 812-23bp, examined at the 32-cell stage (top) and 64-cell stage (bottom). Color codes are as in H. Gray indicates ectopic expression. Number in parenthesis indicates number of embryos examined.

View larger version (20K): [in a new window]   Fig. 2. The 3.5-kb upstream region of Ciona Otx is capable of reproducing endogenous Otx expression pattern in Halocynthia embryos. (A) The structure of the 5' region of Ci-Otx. Boxes on the gray line indicate exons. ATG indicates the translation start site, which is located in the second exon. The light gray line represents the upstream region included in the construct –3541 (Bertrand et al., 2003). (B,B') Transcription of lacZ in 64-cell stage embryos injected with the reporter construct –3541, as visualized by whole-mount in situ hybridization. Animal (B) and vegetal (B') views are shown. Schematic representation of the in situ hybridization specimen is shown right (B,B'). Cells with endogenous Hr-Otx expression are colored as in Fig. 1. Blue dots indicate the cells in which lacZ transcripts were detected. Below the in situ hybridization specimens, the lacZ -positive cell line is indicated. (C) Frequency of lacZ-transcript-positive cells for the given cell line in embryos injected with the –3541 construct, examined at the 64-cell stage.

View larger version (40K): [in a new window]   Fig. 3. Line-specific regulatory modules responsible for regulating Hr-Otx transcription during cleavage stage. (A) Schematic representation of the regions, 5402-1473 bp and 812-24 bp. For analysis of their transcriptional activity, regions lacking the putative endogenous Hr-Otx promoter were placed upstream of the HrMA4a basal promoter. Dark gray lines indicate the region that exhibited lacZ transcription in Hr-Otx-expressing cells. The positions of the line-specific transcription regulatory modules (#1-#6) are indicated at the bottom. The regions marked by light gray lines directed lacZ transcription only weakly during cleavage stage. (B-G') Transcription of lacZ in 64-cell stage embryos injected with reporter constructs harboring regulatory modules #1 (B,B'), #2 (C,C'), #3 (D,D'), #4 (E,E'), #5 (F,F') and #6 (G,G'), as visualized by whole-mount in situ hybridization. Animal (B-G) and vegetal (B'-G') views are shown. The identity of the lacZ-transcribing cell line is indicated below the in situ hybridization specimens. Asterisk indicates that module #6 also drives lacZ transcription in all a-line cells from the 110-cell stage onward. (H) Frequency of lacZ-positive cells for a given cell line in 64-cell embryos injected with constructs harboring modules #1-#6. For color codes see Fig. 1

View larger version (27K): [in a new window]   Fig. 4. Introduction of mutations into the BSs for Lhx, Fox and T-box proteins resulted in a loss of lacZ transcription in the vegetal hemisphere. (A-D) Frequency of lacZ-positive cells for a given cell in 64-cell embryos injected with reporter constructs harboring the regulatory modules indicated on the left side. Red, yellow and blue ovals represent the binding sites for Lhx, Fox and T-box proteins, respectively. (E) Frequency of lacZ-transcribing cells for a given cell lineage in 64-cell embryos injected with reporter constructs harboring the DNA fragments shown on the left side. DNA fragment Lhx/Fox-BS contains the single Fox-binding site and the two Lhx-binding sites from regulatory module #6. DNA fragments Lhx-BS and Fox-BS include four binding sites for Lhx and Fox, respectively, which are from regulatory module #6. The DNA fragment T-box-BS includes four copies of the T-box-binding site present in the regulatory module #3. The nucleotides marked yellow, red and blue represent the putative binding sites for Fox, Lhx and T-box proteins, respectively. For color codes in histograms, see Fig. 1.

View larger version (37K): [in a new window]   Fig. 5. The regulatory modules of Hr-Otx are capable of directing line-specific transcription in Ciona intestinalis embryos. The reporter constructs containing regulatory modules #2 (A), #3 (B), #4 (C,D), #5 (E,E') and #6 (F) were electroporated into Ciona fertilized eggs, and the resulting embryos were examined for lacZ transcripts at the 64-cell (A-C,E,E',F) or 76-cell (D) stage. Animal (A,E) and vegetal (B-D,E',F) views are shown. Shown below the images are the identity of the lacZ-expressing cell line in the Ciona embryo and a schematic representation of the in situ hybridization specimen. (G,H) Frequency of lacZ-positive cells for a given cell line in 64-cell (G) or 76-cell (H) embryos injected with reporter constructs harboring the regulatory modules indicated on the left side. For color codes, see Fig. 1.

View larger version (14K): [in a new window]   Fig. 6. Comparison of the Otx upstream region in Ciona intestinalis and Halocynthia roretzi. Gray lines indicate the 5' genomic region of Otx genes. Boxes indicate exons; light and dark gray indicate the untranslated and translated regions, respectively. Blue lines indicate the transcription regulatory modules identified in each ascidian species; below the lines, the distribution of putative transcription factor-binding sites (colored small ovals) is indicated for each regulatory module. The name of the cell lineage in which lacZ transcripts were detected is indicated for each regulatory module. Color codes for transcription factors and the nucleotide sequences of their binding sites are indicated at the bottom. In Ciona, asterisks indicate sites conserved between C. intestinalis and C. savignyi.