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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00611

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Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Department of Biology, Duke University, Durham, NC 27708, USA

View larger version (24K): [in a new window]   Fig. 1. Schematic representation of the SpEndo16 promoter. (A) Relative position of 56 binding sites within the 2.2 kb region that has been shown to drive SpEndo16 expression (Yuh et al., 1994). Twelve unique factors (brown ovals) each interact with only one binding site, six `common factors' (colored rectangles) interact with a few identical (or nearly identical) binding sites, and the structural protein GCF1 (blue ovals) interacts with 23 sites in the SpEndo16 promoter. [Figure adapted from Yuh and Davidson (Yuh and Davidson, 1996)]. (B) Binding sites within the SpEndo16 promoter are clustered into six functionally distinct modules that serve to activate (+) or repress (-) transcription. (C) Logic circuit diagram showing interactions between binding sites within modules A and B of the SpEndo16 promoter based on transient expression assays [Figure adapted from Yuh et al. (Yuh et al., 2001a). Note that binding sites in modules A and B interact extensively.

View larger version (51K): [in a new window]   Fig. 2. Whole-mount in situ hybridization showing LvEndo16 transcription. At the hatched blastula (A) and mesenchyme blastula (B) stages (lateral views), LvEndo16 is expressed throughout the vegetal plate. Vegetal views (a,b) reveal that the PMCs (black arrow), which are derived from the center of the vegetal plate, do not express LvEndo16. As gastrulation proceeds, LvEndo16 is expressed throughout the invaginating archenteron, as seen in lateral (C) and vegetal (c) views. Near the end of gastrulation, LvEndo16 expression still extends throughout the archenteron (D). Expression is downregulated in SMCs (white arrow) as they ingress and migrate away from the tip of the archenteron. A lateral view (E) reveals that LvEndo16 expression also is downregulated in the anterior third of the archenteron (prospective foregut, asterisk) as it bends to make contact with the oral ectoderm. LvEndo16 continues to be expressed in the middle third (prospective midgut) and posterior third (prospective hindgut) of the archenteron. Lateral (F) and aboral (G) views show that LvEndo16 expression is completely extinguished in the prospective foregut, but is maintained in the prospective midgut (black arrowhead) and hindgut (white arrowhead) at the prism stage. LvEndo16 expression persists in both the midgut and hindgut of the pluteus larva until at least the four-arm stage (H-J). Scale bars: 50 µm for A-G; 100 µm for H-J.

View larger version (21K): [in a new window]   Fig. 3. Schematic comparison of Endo16 transcription in S. purpuratus and L. variegatus. The pattern of Endo16 expression (shown in blue) is relatively conserved between S. purpuratus (A) and L. variegatus (B). (Asterisks indicate prospective foregut.) However, Endo16 expression is downregulated in the posterior third of the archenteron (prospective hindgut) only in S. purpuratus. (Arrows indicate hindgut.) SpEndo16 expression persists in the midgut, while LvEndo16 expression persists in both the midgut and hindgut of the pluteus larva.

View larger version (98K): [in a new window]   Fig. 4. LvEndo16 promoter sequence. Shown here is the sequence from -2373 to +83 relative to the transcriptional start site. This sequence includes the promoter, the 5' UTR, and the first exon; +83 is the position of the first intron. A microsatellite consisting of TAC repeats from -1632 to -1850 is underlined. The ATG start codon is boxed.

View larger version (181K): [in a new window]   Fig. 5. Transient expression assays of the 2.2 kb upstream sequence injected into L. variegatus eggs. (A) Microinjection of a LvEndo16-GFP reporter construct resulted in fluorescence in the vegetal plate at the mesenchyme blastula stage. (B) During gastrulation, fluorescence is detected in the archenteron. (C) A ventral view showing fluorescence in the midgut of the pluteus larva. (D) A lateral view showing fluorescence in both the midgut and hindgut of the pluteus larva.

View larger version (57K): [in a new window]   Fig. 6. Alignment of module A of the Endo16 promoter from S. purpuratus and L. variegatus. Sequences extend upstream 335 bp and 345 bp relative to the transcriptional start site for L. vareigatus and S. purpuratus, respectively. (Asterisks indicate a nucleotide match.) Transcription factor binding sites identified in module A of the SpEndo16 promoter are outlined by a red box. The Otx and Z binding sites occur only once within the SpEndo16 promoter, although there are multiple binding sites for the proteins CG, CP and GCF1.

View larger version (20K): [in a new window]   Fig. 7. Schematic representation of the Endo16 promoter in S. purpuratus (A) and L. variegatus (B). The LvEndo16 promoter sequence indicates only those binding sites identified in module A of S. purpuratus. Results from transient expression assays indicate that additional binding sites required for LvEndo16 expression are likely to occur in the 2.2 kb region, but have not yet been identified. (An asterisk indicates that a nucleotide substitution or indel occurs within a binding site compared to the Endo16 promoter sequence in S. purpuratus.) A dot plot (C) and feature maps (D-F) were generated by FamilyRelations based on a seqcomp analysis of the Endo16 promoter (Brown et al., 2002). Alignment of the SpEndo16 and LvEndo16 promoter sequences is noted in the upper right corner of a dot plot (C), corresponding to module A. This is also evident at the right of a feature map (D). In neither case is there convincing evidence for sequence similarity upstream of module A. This result is supported by pairwise comparisons of the Endo16 promoter sequence with BAC sequence from the opposite species. Only one region of conservation corresponding to module A is detected in a pairwise comparison of the SpEndo16 promoter sequence and a BAC sequence from L. variegatus that contains the LvEndo16 locus (E). The reciprocal analysis revealed two regions of conservation, corresponding to module A as well as a microsatellite consisting of TAC repeats (F).

View larger version (139K): [in a new window]   Fig. 8. Reciprocal cross-species transient expression assays using the Endo16 promoter. GFP reporter constructs were microinjected in a reciprocal cross-species experimental design. Images were captured at three stages of development: mesenchyme blastula (A,D,G,J), gastrula (B,E,H,K), and pluteus larva (C,F,I,L). Microinjection of SpEndo16-GFP into S. purpuratus eggs results in a pattern of GFP expression that recapitulates the results of in situ hybridization of the endogenous gene (Nocente-McGrath et al., 1989; Ransick et al., 1993), and as observed by Yuh et al. (Yuh et al., 1994) in transient expression assays (A-C). Microinjection of LvEndo16-GFP into S. purpuratus eggs results in the same pattern of GFP expression (D-F). Note that it does not drive GFP expression in the hindgut of the pluteus larva (F). Microinjection of SpEndo16-GFP into L. variegatus eggs produces ectopic fluorescence in the SMCs as well their pigment cell derivatives (G-I). As in the reciprocal experiment, no fluorescence is detected in the hindgut of the pluteus larva (I). Microinjection of LvEndo16-GFP into L. variegatus eggs results in a pattern of GFP expression (J-L) that recapitulates the results of in situ hybridization of the endogenous gene as shown in Fig. 2. Fluorescence persists in both the midgut and hindgut of the pluteus larva (L).

View larger version (23K): [in a new window]   Fig. 9. Summary of reciprocal cross-species transient expression assays using the Endo16 promoter. Microinjection of the Endo16 promoter into eggs of the same species results in a pattern of GFP expression (green) that recapitulates the results of in situ hybridization (A,D). Microinjection of LvEndo16-GFP into S. purpuratus eggs results in a host-specific pattern of GFP expression (B), while microinjection of SpEndo16-GFP into L. variegatus eggs results in a donor-specific pattern of GFP expression with ectopic fluorescence in the SMCs and their pigment cell derivatives (C). These data indicate that evolutionary changes have arisen both cis and trans to the Endo16 gene. (Arrows indicate hindgut. Arrowheads indicate ectopic fluorescence in the SMCs and pigment cells.)

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