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First published online 2 October 2008
doi: 10.1242/dev.025817

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Functional resolution of duplicated hoxb5 genes in teleosts

¹ Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
² Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
³ Centre de recherche en cancérologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, L'Hôtel-Dieu de Québec, Québec G1R 2J6, Canada.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Constructs used to generate transgenic animals. (A-D) The reporter transgene was inserted into the first exon of the zebrafish hoxb5a (A) or hoxb5b (C) gene. (B) The J3 element was deleted from the hoxb5a construct. (D) J3 was inserted into the hoxb5b construct. (E-G) Individual conserved non-coding elements (CNEs) from mouse and zebrafish Hoxb5 loci were coupled to a β-globin minimal promoter and the EGFP or lacZ reporter genes (black rectangles). a and b represent the hoxba and hoxbb zebrafish complexes, respectively. In A-D, hatched rectangles indicate exon sequences; black triangles, loxP sites; amp, ampicillin resistance gene; neo, neomycin resistance gene. Mm, Mus musculus; Dr, Danio rerio.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Conserved non-coding elements in the Hoxb5 genes of vertebrates. Sequences from mouse, human, zebrafish and Takifugu were compared using Pipmaker. The mouse Hoxb5 locus is used as a reference and is shown on the horizontal axis. Sequences from human (Hs), zebrafish (Dr) and Takifugu rubripes (Tr) are compared and the similarity is shown on the vertical axis. CNE positions are indicated by shading: red, J1; blue, J2; green, J3. A sequence similarity lower than 50% was observed between the J2 elements from Takifugu hoxb5a and mouse Hoxb5 using a different algorithm. Identical results to those shown were obtained when teleost sequences were used as a reference. The annotations at the top refer to the mouse reference sequence: exons (black boxes), simple repeats (vertical rectangles) and CpG islands (white and gray rectangles).

View larger version (88K): [in this window] [in a new window]   Fig. 3. Embryonic expression of mouse and zebrafish Hoxb5 genes. (A-D) Sagittal sections of E12.5 mouse embryos. The antisense Hoxb5 probe consisted of a 430 bp fragment from the Hoxb5 cDNA that includes the N-terminal part of the Hoxb5 protein (Krumlauf et al., 1987). (E-J) Whole-mount in situ hybridization on (E,F) 24 hpf, (G,I) 48 hpf and (H,J) 72 hpf zebrafish embryos using antisense riboprobes for hoxb5a (b5a, 550 bp probe) and hoxb5b (b5b, 600 bp probe). Zebrafish embryos are shown as lateral views. Arrows point to the embryonic neural tube. ba, branchial arches; g, gut; gn, trigeminal ganglion; l, lung; pd, pronephric duct; s, somites. Scale bars: 500 µm in A-D; 250 µm in E-J.

View larger version (80K): [in this window] [in a new window]   Fig. 4. Expression of lacZ reporter transgenes in mouse embryos. E10.5 (A-H) and E12.5 (I-P) transgenic mouse embryos, as indicated. A,C,E,G,I,K,M,O are lateral views; B,D,F,H,J,L,N,P are dorsal views. Arrows indicate the neural tube and asterisks indicate somites. fg, facioacoustic ganglion; ng, nodose ganglion. Scale bars: 500 µm.

View larger version (131K): [in this window] [in a new window]   Fig. 5. Analysis of reporter transgene expression on sagittal sections and in the internal organs of mouse embryos. (A-H) Sagittal sections from E13.5 transgenic mouse embryos, as indicated. Arrows indicate the neural tube. (I-L) Internal organs from E13.5 transgenic embryos, as indicated. cp, cartilage primordium; DRG, dorsal root ganglia; g, gut; k, kidney; st, stomach; ag, adrenal glands; X, trigeminal ganglia. Scale bars: 330 µm in A-D,F,G; 1000 µm in E,H; 125 µm in I,J,L; 250 µm in K.

View larger version (72K): [in this window] [in a new window]   Fig. 6. Expression of EGFP reporter transgenes in zebrafish embryos. (A-I) Lateral views (anterior to the left) of primary transgenic zebrafish embryos injected with (A-C) hoxb5aEGFP, (D,E) hoxb5bEGFP, (F,G) hoxb5aJ3EGFP or (H,I) hoxb5binsJ3EGFP. Developmental stages are indicated. Arrows indicate cells of the developing nervous system; arrowheads mark somites and muscle cells. Approximately 60% of the embryos injected with hoxb5bEGFP and 50% of embryos injected with hoxb5binsJ3EGFP showed EGFP expression, as compared with only 30% of hoxb5bEGFP and 37.5% of hoxb5aJ3EGFP embryos. Scale bars: 250 µm. (J,K) Relative copy numbers of EGFP transcripts detected by RT-PCR in primary transgenic embryos injected with hoxb5aEGFP (b5a), hoxb5bEGFP (b5b), hoxb5aJ3EGFP (b5aJ3) or hoxb5binsJ3EGFP (b5binsJ3) constructs. Column heights represent relative copy numbers of EGFP transcripts after normalization to endogenous hoxb5a expression. Each bar represents the average of three groups of transgenic embryos and error bars indicate s.e.m.

View larger version (100K): [in this window] [in a new window]   Fig. 7. Enhancer activity of individual Hoxb5 non-coding elements in transgenic mouse embryos. (A-H) Reporter transgene expression was analyzed in primary transgenic embryos whose age varied from E12.5 to E14.5. The name and origin of the tested CNE are shown at the bottom right-hand corner of each panel. Arrowheads mark somite derivatives; arrows point to the neural tube. Mm, Mus musculus; Dr, Danio rerio; a and b refer to the hoxba and hoxbb zebrafish complexes, respectively. Scale bar: 1000 µm.

View larger version (124K): [in this window] [in a new window]   Fig. 8. Enhancer activity of individual Hoxb5 non-coding elements in transgenic zebrafish. (A-G) Lateral views, anterior to the left. Arrows in B and E indicate cells of the developing CNS. Arrowheads indicate somites in A and muscle cells in D-G. The name and origin of each regulatory element are indicated as in Fig. 7. Age, in hpf, is indicated. Scale bars: 500 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Phylogenetic analysis of Hoxb5 loci from human, mouse, zebrafish and Takifugu. (A) Phylogenetic tree predicted by the DDC model. Its topology is based on the assumption that hoxba and hoxbb teleost complexes resulted from a duplication event that happened after the separation of the fish and tetrapod lineages and before the divergence of Takifugu and zebrafish lineages. (B-F) Phylogenetic trees built with MEGA3 based on the following sequences: (B) Hoxb5 coding sequences (850 bp); (C) 5' upstream region of Hoxb5 genes (130 bp); (D) J1 CNE (255 bp); (E) J2 CNE (150 bp); (F) CNEs present in all Hoxb5 loci (535 bp), for which the 5' upstream region, J1 and J2 were joined in a continuous single sequence for each Hoxb5 locus. As the J3 CNE was only found in the hoxb5a complexes of zebrafish and Takifugu, it was not included in this analysis. Hs, human; Mm, mouse; Dr, zebrafish; Tr, Takifugu. The scale bar indicates an estimated evolutionary distance of 0.05 nucleotide substitutions per site. Numbers at nodes indicate the bootstrap values.

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