Expression of the Distalless-B gene in Ciona is regulated by a pan-ectodermal enhancer module

Abstract

The Ci-Dll-B gene is an early regulator of ectodermal development in the ascidian Ciona intestinalis (Imai et al., 2006). Ci-Dll-B is located in a convergently transcribed bigene cluster with a tandem duplicate, Ci-Dll-A. This clustered genomic arrangement is the same as those of the homologous vertebrate Dlx genes, which are also arranged in convergently transcribed bigene clusters. Sequence analysis of the C. intestinalis Dll-A-B cluster reveals a 378bp region upstream of Ci-Dll-B, termed B1, which is highly conserved with the corresponding region from the congener Ciona savignyi. The B1 element is necessary and sufficient to drive expression of a lacZ reporter gene in a pattern mimicking the endogenous expression of Ci-Dll-B at gastrula stages. This expression pattern which is specific to the entire animal hemisphere is activated preferentially in posterior, or b-lineage, cells by a central portion of B1. Expression in anterior, or a-lineage cells, can be activated by this central portion in combination with the distal part of B1. Anterior expression can also be activated by the central part of B1 plus both the proximal part of B1 and non-conserved sequence upstream of B1. Thus, cis-regulation of early Ci-Dll-B expression is activated by a required submodule in the center of B1, driving posterior expression, which works in combination with redundant submodules that respond to differentially localized anterior factors to produce the total animal hemisphere expression pattern. Interestingly, the intergenic region of the cluster, which is important for expression of the Dlx genes in vertebrates, does not have a specific activating function in the reporter genes tested, but acts as an attenuator in combination with upstream sequences.

Fig. 1. Ciona sequence alignment, diagrams of larger reporter transgenes and scoring

(A) mVista sequence alignment plot between C. intestinalis and C. savignyi Ci-Dll-B-A clusters. Exons shown as blue-green boxes. Curve represents levels of sequence identity in a 50 bp window. Blue-shaded peaks are in exons while pink-shaded peaks are in non-coding sequence. Peaks of conserved non-coding sequences (CNSs) tested here are denoted as B1, B2, and ig. Distances from transcription start sites of Ci-Dll-B and Ci-Dll-A shown in kilobases (kb). (B) Diagrams of transgene constructs. Non-coding sequence is represented by red bars aligned with corresponding sequence in Vista plot above. LacZ reporter gene insertion is represented by a blue bar. Extent of expression of each transgene in reporter experiments is shown at right as percentages of aggregate anterior and posterior animal quadrants with staining for ß-galactosidase. * significantly different (p<0.05) in Kruskal-Wallis test; + posterior expression significantly different (p<0.05). Refer to Supplementary Tables 1 and 2 for numbers of embryos scored and statistics.

Fig. 2. Reporter transgene expression – whole cluster and upstream 1 kb

(A-E) CiDB-A whole cluster construct β-galactosidase staining pattern. (B, C) Animal and vegetal views, respectively, at early gastrula stage. (D, E) Animal and vegetal views, respectively, at late gastrula stage. (F-H) Ci-DB-1.0 transgenic embryos. (G) Animal and (H) vegetal pole views at early gastrula stage. Cells in H which appear on the vegetal side (arrowheads) are b8.17 and b8.19 cells derived from the animal hemisphere (Nishida, 1986).

Fig. 3. Reporter transgene expression

(A-F) Representative animal pole views of transgenic embryos. Name and a diagram of the reporter transgenes electroporated in each case are below the corresponding photos.

Fig. 4. Diagrams of reporter transgenes associated with the B1 conserved sequence element

Representation of the position of the B1 CNS within the CiDB-1.0 construct (top). Predicted transcription factor binding sites are shown by colored ovals. Below are diagrams of reporter transgene constructs. The Ci-Dll-B promoter is incorporated, except where noted “fkh” which indicates the non-coding sequence linked to a silent basal Ci-FoxA-a promoter (Harafuji et al., 2002) (Constructs CiDB-0.2-0.44, CiDB-0.35-0.62 and CiDB-0.35-0.62mG. CiFk5'A is the empty Ci-FoxA-a promoter vector.) Chart at right shows the extent of expression of each transgene as percentages of aggregate number of anterior and posterior animal quadrants with staining for ß-galactosidase. Refer to Supplementary Tables 1 and 2 for numbers of embryos scored and statistics.

Fig. 5. Reporter transgene expression – B1 region analysis

(A-H) Representative animal pole views of transgenic embryos. Name and a diagram of the reporter transgenes electroporated in each case are below the corresponding photos. (A) Deletion of the distal 170 bp of B1 has little effect on expression compared with CiDB-1.0., while deletion of the proximal 140 bp (B) causes some loss of expression. (C) 440 bp upstream of Ci-Dll-B drives expression only in the posterior animal hemisphere, similar to that seen when the proximal 211 bp of B1 is tested upstream of a heterologous Ci-FoxA-a promoter (D). On the other hand the more distal 276 bp of B1 is able to drive expression in all quadrants of the animal hemisphere, upstream of both the native Ci-Dll-B promoter (G), and the stronger Ci-FoxA-a promoter (E) Mutation of a canonical GATA site in this fragment (F) reduces, but does not eliminate expression in the same domain. Truncation of the same fragment to 163 bp near the middle of B1 still drive expression, but mostly in the posterior quadrants.

Fig. 6. Model for Ci-Dll-B regulation

Diagram of working model of Ci-Dll-B B1 element organization. In this scheme, consistent with our results, a central part of B1 (labeled “a”) is required for any expression, which is confined to the posterior (b-line) cells of the animal hemisphere. Anterior expression is gained when either the distal part of B1, “b” is added, or the combination of proximal B1, “d” and the region upstream of B1, “c”. Elements “b”,“c”, or “d” on their own or together, without “a”, do not drive expression.