Zhu X et al. (2012), Characterization and expressional analysis of D...

XB-ART-45884

Gene 2012 Nov 01;5091:77-84. doi: 10.1016/j.gene.2012.08.024.

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Characterization and expressional analysis of Dleu7 during Xenopus tropicalis embryogenesis.

Zhu X , Li Z , Jiang D , Zhao J , Huang L , Zhang J , Huang X .

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We characterized the genomic structure and developmental expression of the Dleu7 (deleted in lymphocytic leukemia, 7) gene in Xenopus tropicalis and the evolution of the gene across species. Within the protein-coding sequence (CDS) region, X. tropicalis Dleu7 consists of two exons and one intron. However, bioinformatic analysis indicates that this 211-amino-acid protein contains no obvious functional domains or known motifs. Reverse-transcription polymerase chain reaction and whole-mount in situ hybridization results revealed that, in addition to its expression in the blood island region, some regions of the central nervous system, and subdomains of the neural tube, X. tropicalis Dleu7 is zygotically expressed primarily in mesoderm tissues such as notochord and muscles during early embryogenesis. Expression in notochord is consistent with results from genome-wide association studies suggesting that DLEU7 is related to human adult height. Expression in the blood island region, where blood cell precursors (including B cells) are generated, implies a potential conserved role for Dleu7 in B-cell development between amphibians and mammals. Expression of Dleu7 in some regions of the central nervous system and subdomains of the neural tube also suggests other functions in development. Phylogenetic analysis indicated that Dleu7 is a vertebrate-specific gene and undergoes strong selective pressure in lower vertebrates but is functionally constrained in higher mammals. When subcellular localization was examined by overexpression of enhanced green fluorescent protein fusion protein, Dleu7 showed centrosome localization with main distribution in cytoplasm. Treating gastrula embryos with SU5402, a small molecular inhibitor of the fibroblast growth factor (FGF) receptor, confirmed that Dleu7 expression in mesoderm is regulated by FGF signaling. Our data provide important clues for pathogenesis and physiology during development from the perspective of evolutionary conservation.

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Species referenced: Xenopus tropicalis
Genes referenced: arf11l dleu7 fgf8 ints6 rnaseh2b shh spryd7 tbxt trim13 wdfy2

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	Fig. 1. Genomic structure of X. tropicalis Dleu7. (A) The genomic structure of X. tropicalis Dleu7 contains two exons and one intron. The full-length cDNA is 2973 bp. The 5′ untranslated region, CDS, and 3′ untranslated region are 18 bp, 636 bp, and 2319 bp, respectively. (B) Amino acid sequence of X. tropicalis Dleu7, consisting of 211 amino acids. The boxed 178–198 amino acid region is predicted to be a transmembrane helix.
	Fig. 2. Phylogenetic trees for Dleu7 genes. The Dleu7 CDSs were used to construct phylogenetic trees: human (Homo sapiens NM_198989.2), chimpanzee (Pan troglodytes XM_001157783.1), orangutan (Pongo pygmaeus XM_0028242 83), macaque (Macaca mulatta XM_001104889), galago (Otolemur garnettii ENSOGAT00000009131), pika (Ochotona princeps ENSOPRT00000012975), rabbit (Oryctolagus cuniculus ENSOCUT00000003939), rat (Rattus norvegicus NM_001106043.1), mouse (Mus musculus NM_173419.2), hedgehog (Erinaceus europaeus ENSEEUT00000014568), opossum (Monodelphis domestica), chicken (Gallus gallus ENSGALT00000027481), frog (Xenopus tropicalis JQ838068), and zebrafish (Danio rerio JQ838069). NJ trees were analyzed by MEGA5 software. Numbers on branch nodes are bootstrap replication values.
	Fig. 3. Amino acid sequences alignment of Dleu7 genes. Amino acid sequences were collected from human (Homo sapiens), chimpanzee (Pan troglodytes), macaque (Macaca mulatta), galago (Otolemur garnettii), rat (Rattus norvegicus), mouse (Mus musculus), opossum (Monodelphis domestica), chicken (Gallus gallus), frog (Xenopus tropicalis), and zebrafish (Danio rerio). X. tropicalis Dleu7 shares low similarity with Dleu7 from other vertebrates. Identity residues from zebrafish to mammals are shown in black; similarity sites are shown in dark gray and light gray.
	Fig. 4. Syntenic analysis of Dleu7 genes. Syntenic analysis of Dleu7 genes in human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), frog (Xenopus tropicalis), and zebrafish (Danio rerio). An asterisk indicates that the gene's information was found in other scaffolds, because the genomic sequence in X. tropicalis is not yet complete. These genes are depicted by relative location. Dleu7 is located on the same loci, neighbored by Wdfy2, Ints6, Fam24a, Rnaseh2b, Kcnrg1, Trim13, Clld6, and Kpna3, in higher vertebrate genomes. In the zebrafish genome, Clld6, Kpna3, and Arl11 are also 3′ downstream of Dleu7, which is consistent with other vertebrate genomes, indicating that the relative positions of Dleu7, Clld6, Kpna3, and Arl11 are highly conserved in these genomes.
	Fig. 5. Selection pressure test for Dleu7. The nonsynonymous-to-synonymous substitution rate ratio, ω (dN/dS), for selection pressure in each phylogenetic tree branches was tested using the M1 model. The ω values are noted on each branch and are notably higher on the branches from zebrafish to hedgehog than on the branches from mouse to human.
	Fig. 6. Temporal and spatial expression of X. tropicalis Dleu7. (A–E) Spatial expression of Dleu7 by RT-PCR at stage 10.5 (gastrula) (A and B), stage 13 (early neurula) (C), stage 23 (late neurula) (D), and stage 28 (tail bud) (E). (F–L) Transverse paraffin section on stage 28 embryos after WISH. The signal of Dleu7 is observed in forehead (F), midbrain and eyes (G), notochord (I), blood island (H and J), neural tube and lateral muscle (K and L). The black, yellow, green, and red arrowheads indicate the signal of Dleu7 in brain, notochord, eye, and spinal cord respectively; the blue arrowheads show the expression of Dleu7 in the blood island (E, H) and lateral muscle (L). (M) Temporal expression of Dleu7. The expression is not detected from eggs to embryos until stage 9 and increases sharply at gastrula stage. The expression decreased gradually during the neurulation, and very weak signal is detected at stage 28 by RT-PCR.
	Fig. 7. Subcellular localization and expressional regulation of X. tropicalis Dleu7. (A–C) Effect of SU5402 treatment on Dleu7 expression. WISH analysis showed that SU5402 treatment reduced expression of Dleu7 in embryos (B) compared with DMSO treatment (A). RT-PCR results (C) confirm the WISH result: expression levels of Dleu7 and Xbra are clearly decreased in embryos treated with SU5402 (1, 3, and 5) compared with DMSO controls (2, 4, and 6). (D–G) Subcellular localization of X. tropicalis Dleu7 in HeLa cells. Nuclei and centrosomes are stained by DAPI (D) and γ-tubulin (F), respectively. Dleu7 can be detected by the expression of EGFP (E). Overlap view of D–F is shown in G.
	dleu7 (deleted in lymphocytic leukemia, 7) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.
	dleu7 (deleted in lymphocytic leukemia, 7) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	dleu7 (deleted in lymphocytic leukemia, 7) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28 lateral view, anterior left, dorsal up.