Cis-regulatory organization of the Pax6 gene in the ascidian Ciona intestinalis - PubMed (original) (raw)

Comparative Study

Cis-regulatory organization of the Pax6 gene in the ascidian Ciona intestinalis

Steven Q Irvine et al. Dev Biol. 2008.

Abstract

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information was extended here using in-situ hybridization, and shows that CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 we tested 8 kb of sequence using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An "eye enhancer" was localized to the first intron, which controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord.

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Figures

Fig. 1

Fig. 1. CiPax6 transcript expression pattern visualized by whole-mount in-situ hybridization

Anterior is to the left in all panels. (A) Gastrula stage dorsal view. Bilateral pair of A9.30 cells staining (arrowheads). (B) Neurula stage dorsal view with CiPax6 expression in bilateral strips of neuroectoderm. (C, D) Early tailbud embryo in dorsal and lateral views, respectively. Expression is in entire anterior-posterior length of the nascent CNS. (E, F) Mid-tailbud stage embryo in dorsal and lateral views, respectively. Expression is detected in the sensory vesicle (arrow) and nerve cord, but has become down-regulated in the forming visceral ganglion (brackets). (G) Optical section through a mid-tailbud embryo showing transcripts detected in the two lateral cell ranks of the nerve cord (arrows), which consists of only 4 ependymal cells in cross-section, one dorsal, one ventral and two lateral. (H, I) Dorsal and lateral views, respectively of late tailbud embryos, showing persistent staining in the anterior sensory vesicle and nerve cord. In the higher power view (H), staining is visible in the visceral ganglion, but absent from the neck (n, brackets) and most posterior sensory vesicle. n, neck; nc; nerve cord; sv, sensory vesicle; vg, visceral ganglion.

Fig. 2

Fig. 2. Ciona sequence alignment, transgene diagrams and scoring

(A) mVista sequence alignment plot between C. intestinalis and C. savignyi, with CiPax6 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. Major conserved non-coding sequences (CNSs) are identified by letters in ovals. (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. Dotted horizontal lines indicate sequences not in the transgene. (In these cases the cloned sequences are connected to each other with short linkers.) (C) Scoring chart for expression driven by each transgene in C. intestinalis embryos at mid-late tailbud stages. Number of “+” symbols denotes relative intensity and penetrance of lacZ expression. “-” indicates lack of expression. “+/-” indicates that expression is present in some embryos at a low level. * refers to limited expression in central sensory vesicle only.

Fig. 3

Fig. 3. Major CNS enhancers are located upstream of CiPax6 coding sequence

Photomicrographs of β-galactosidase (β-gal) histochemical assays of lacZ expression driven by various reporter transgenes. Mid-tail (upper panels, A, C, E, G, I, K) and late tail (lower panels, B, D, F, H, J, L) embryos in lateral view with anterior to the left, except as noted. Transgene name and diagram are located below each embryo pair. (A, B) Expression from upstream, intron 1, and intron 4 fragments together. β-gal staining is seen in the entire CNS (arrowheads). Expression is also seen in 2 muscle cells on each side of the trunk, derived from the A8.16 lineage (arrows, see text). These cells also stain with the CiP6-2.5U, -2.0U, and -1.8U constructs. (C, D) 2.5 kb of upstream sequence alone drives expression in the entire CNS, as well as ectopic expression in epidermis (arrows) (D inset) dorsal detailed view of trunk of late tail CiP6-2.5U embryo. (E, F) Specific expression in the sensory vesicle (sv) is driven by a construct with 1986 bp. of upstream sequence. At mid tailbud stage sporadic expression is also seen in the caudal nerve cord (arrowheads) (F inset) Right lateral view of late tail embryo with CiP6-2.0U transgene expression detected with an anti-lacZ antibody. Expression can be seen in the sensory vesicle surrounding the pigmented otolith and ocellus. A cell in the caudal visceral ganglion (arrowhead) also reacts with the lacZ antibody (white arrowhead). (G, H) Deletion of 211 bp. containing the UA CNE results in loss of most CNS expression except for slight expression in the caudal nerve cord at mid tailbud stage (arrowheads). Ectopic expression in mesenchyme appears in this truncated construct (arrows). (I, J) Addition of the distal sequence containing the UB CNE to the -1.8U construct results in gain of some sensory vesicle expression (white arrowheads) in addition to the low level expression in the nerve cord (arrowhead). This construct exhibits some ectopic expression in tail muscle cells at late stages (arrows). (K, L) A construct with only 0.6 kb. of upstream sequence shows only ectopic β-gal expression in trunk mesenchyme at late stages (arrows).

Fig. 4

Fig. 4. Intron 1 contains elements driving expression in photoreceptors

β-gal histochemistry (A-E, H, K) and anti-β-gal immunofluorescent detection (F, G, I, J, L, M) of lacZ reporter transgenes. Constructs are identified below and to the left of corresponding images. (A, C) are mid-tailbud stage in lateral view. Others are late tailbud stages in lateral views, except B, D and K, which are dorsal views, and L which is frontal. (A, B) Construct with 2.5 kb. upstream region and entire intron 1. Expression is in entire CNS, as well as ectopic expression in lineage A8.16 muscle cells (arrows) as seen in Fig. 3. Note expression in bilateral ranks of caudal nerve cord cells (arrowheads) (C-G) Intron 1 fragment connected with 200 bp. of basal promoter sequence. Expression is in the central sensory vesicle (arrowheads). Ectopic expression is also in trunk mesenchyme (arrows). In (F) note expression in photoreceptor array associated with the ocellus (white arrowhead).(H-J) Expression driven from the proximal 1 kb. of intron 1 sequence containing the I1A and I1B CNEs. Variable β-gal signal is seen in ventral and caudal portions of the central sensory vesicle. (K-L) Expression driven from the distal 300 bp. of intron 1 containing the I1C CNE. Variable β-gal signal is in similar regions to that driven by the proximal intron 1 fragment.

Fig. 5

Fig. 5. Intron 4 sequence downregulates expression in the neck and visceral ganglion

Whole mount in-situ hybridization using a lacZ antisense riboprobe, except A and B, which are β-gal histochemical staining. Transgene name and diagram are located below each embryo pair. Top row (A, C, E, G) are mid-tailbud stages, and second row are lateral (B, D, H), or dorsolateral (F) views of late tailbud stages. (A, B) Intron 4 alone drives only ectopic expression in trunk mesenchyme at late stages (arrow). (C-H) LacZ transcript expression is shown for reporter transgenes with upstream and Intron 1 sequence (C, D), upstream and Intron 4 sequence (E, F), and upstream and Intron 1 and Intron 4 sequence (G, H). Note that expression becomes downregulated in the neck and anterior visceral ganglion in the transgenes incorporating intron 4 sequence (brackets). Expression in the caudal nerve cord is reduced in E, F compared with constructs incorporating Intron 1 (C, D & G, H) (arrowheads). However at late stages (H) Intron 4 sequence represses nerve cord expression even in the presence of Intron 1 (arrows). Refer to Table 1 for scoring of multiple embryos for each transgene.

Fig. 6

Fig. 6. Comparisons of _Pax6 cis_-regulation between mouse, Ciona and fly

For each species a VISTA plot of the Pax6 genomic region is shown above a diagram of the exon-intron organization and experimentally verified regulatory regions. Mouse Pax6 (A) is aligned with both human and Fugu rubripes Pax6, C. intestinalis Pax6 (B) is aligned with C. savignyi Pax6, and Drosophila melanogaster eyeless (C) is aligned with the corresponding region in D. pseudoobscura. Mouse data from Kammandel et al. (1999) and Xu et al. (1999). Drosophila data from Hauck et al. (1999) and Adachi et al. (2003).

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