Figure 1.
Organisation of Hha and Hhb loci in Lampetra (Lf) and Petromyzon (Pm).
A, Sequence information available after library screening (Lf cosmids), in silico searches (Pm contigs and trace archives) and PCR amplification is schematically depicted. Drawing is not to scale. The top row shows a “generic” Hh gene, with 3 exons (black boxes) and 2 introns containing previously described regulatory elements (coloured ovals, ar-A, B, C, and D; see text). Below, Hha genes are drawn in blue/purple, Hhb genes are drawn in green/kaki. The sizes of introns and the overall length from ATG to Stop codons are indicated. Restriction sites for enzymes EcoRI (E), HindIII (H) and XhoI (X) used for Southern analysis of the cosmids are indicated (restriction sites located on the Lawrist7 vector are not indicated). The trace archives for putative PmHhb exonic sequences have the following IDs (plus and minus are directions for assembly). For exon1: gnl|ti|1427444168+, gnl|ti|1179676842+, gnl|ti|1483498011+, and gnl|ti|1482777522-. For exon2: gnl|ti|1484276315+ and gnl|ti|1482717043+. For exon3: gnl|ti|1470810056+, gnl|ti|1213886743+, gnl|ti|1193744006-, and gnl|ti|1192802884-. B, Southern blot of Lf cosmids HH2, 3, and 4, using the partial previously isolated Lf cDNA encompassing most of exon1 (174 nt) and part of exon2 (100 nt) [23] as a probe (see Figure 2C for probe localisation on cDNA). Restriction enzymes and band sizes (in bp, obtained from in silico restriction digest of the HH cosmid sequences) are indicated. C, Similarity between each coding region and entire locus (in parenthesis) of the lamprey Hh genes. Nucleotide sequences were aligned with the CHAOS/DIALIGN program and the percent identity was calculated using the ClustalX program (ver2.11; [27]). The values show high similarities between the orthologs even when including intronic regions (see the value in the parentheses), while it is much lower between paralogs despite genes of the same species. D, An example of alignment showing the high sequence identity between orthologs and the lower sequence identity between paralogs of lamprey Hh genes. The sequence shown is at the level of the intron2-exon3 junction. The splice acceptor site (AG) is indicated.
Figure 2.
Phylogenetic analysis of lamprey's Hhs.
Inset: a minimal representation of the Hedgehog family in vertebrates, to highlight the classically-described relationships between Sonic, Indian, and Desert groups (see text, Introduction). A and B are Neighbour-Joining (NJ) and Bayesian phylogenetic trees (aligned aminoacids) of 28 (NJ) and 27 (Bayes) Hedgehog family members including the presently found lamprey members (black arrowhead), with the fly and amphioxus Hhs used as out-group. Bootstrap values are given and the 3 orthology groups are indicated on the right (Sonic, Shh; Indian, Ihh; and Desert, Dhh). C is an amino-acid alignment of lamprey Hh proteins with gnathostome family members. The functional domains (HH signal and Hint domain) are indicated, as well as the exon (E) junction positions (e., g., E1/E2). The regions corresponding to Hha- and Hhb-specific in situ hybridization probes used in Figure 3 are also indicated, as well as the previously isolated Lf probe used in [23].
Figure 3.
Compared expression patterns for LfHha and LfHhb in Lampetra embryos.
A-D show in toto in situ hybridisation photographs for LfHha (A and B; left) and LfHhb (C and D; right). Stages are indicated. Anterior is to the left and dorsal is to the top. Dotted lines delineate the brain in A, and the telencephalon and pineal gland in A to D. E-L show in situ hybridisation photographs for LfHha (E-H; left) and LfHhb (I-L; right) on transverse sections (except L, saggital section) through the head of stage 27 Lampetra embryos. Sections are organised from the most anterior to the more posterior parts of the embryos. M-N show schematic summary drawings of the expression patterns for LfHha (M; left) and LfHhb (N; right), where Hh expression is red and the intensity of red is related to the expression density observed reproducibly on many sectioned embryos in independent experiments. In all panels, arrowheads indicate expression in a placodal structure identified as the nasohypophyseal placode (nhp) underlying the diencephalon in the continuity of the prechordal plate (pcp), arrows point to lower lip expression, and asterisks indicate background trapping in the branchial arches cavities. Abbreviations are: di, diencephalon; fp, floor plate; h, hypothalamus; p, pineal gland; pcp, prechordal plate; mb, midbrain; mhb, mid-hindbrain boundary; n, unidentified neuronal populations; nhp, nasohypophyseal placode; no, notochord; t, telencephalon; tg, tegmentum; zli, zona limitans intrathalamica.
Figure 4.
Conserved non coding elements (CNEs) in lamprey's Hha and Hhb.
A is a search of CNEs in PmHha and LfHhb gene sequences, confined with the Latimeria Shh enhancers ar-A, ar-B, and ar-C. Both genes show conserved peaks in the putative enhancer regions. A' is a close-up on ar-C. B is a search of CNEs in intron2 using LfHhb as baseline, showing high conservation between lamprey Hhs, and dispersion of ar-C sub-elements throughout the Latimeria sequence. C is a direct visualization of the underlying nucleotide alignments between coelacanth, lampreys and rat Shh/Hh at the level of C1. Note that the motif conservation extends on the 5′ side in lamprey genes, and is particularly shifted in the case of Hha. Analyses were performed with the global alignment algorithm MLAGAN and visualized with VISTA plot, with Calc Window and Min Cons Width of 20 bp respectively, and 70% Cons Identity. In the baselines, purple boxes indicate the exon positions, while the blue, red, and yellow ovals are the positions of ar-A, ar-B and ar-C, respectively. Conservation peaks representing putative CNEs appear in pink.
Figure 5.
Functional analysis of lamprey CNEs in zebrafish embryos.
A, LfHhb intron2 enhancer activity visualized in representative zebrafish embryos at 26hpf, at low (left) and high magnification (right) focusing on the notochord and floor plate. B, LfHhb ar-C1 enhancer activity visualized in two representative embryo at 26hpf, one at low (left) and one at high magnification (right) focusing on the notochord and floor plate. A schematic representation of the reporter constructs is shown in each case. It contains a zebrafish shha -0.8 kb minimal promoter driving GFP expression under control of the 3.1 kb intron2 (shown in A) or the 30 bp LfHhb ar-C1 (shown in B).
Table 1.
Quantification of GFP immuno-positive zebrafish embryos after reporter constructs injection at one-cell stage.