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Gene Expr Patterns
2018 Dec 01;30:55-63. doi: 10.1016/j.gep.2018.08.001.
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Molecular characterization of wdr68 gene in embryonic development of Xenopus laevis.
Bonano M
,
Martín E
,
Moreno Ruiz Holgado MM
,
Silenzi Usandivaras GM
,
Ruiz De Bigliardo G
.
???displayArticle.abstract??? WDR68, also known as DCAF7, is a WD40 repeated domain protein highly conserved in eukaryotic organisms in both plants and animals. This protein participates in numerous cellular processes and exerts its function through interaction with other proteins. In the present work, we isolated, sequenced and characterized cDNA corresponding to the wdr68 gene in embryos of the amphibian Xenopus laevis. Syntenic analysis revealed high conservation of the genomic region containing the WDR68 locus in amniotes. Nevertheless, in fishes and amphibians, we observed that the tandem genes surrounding wdr68 undergoes certain rearrangements with respect to the organization found in amniotes. We also defined the temporal and spatial expression pattern of the wdr68 gene in the development of Xenopus laevis through whole mount in situ hybridization and RT-PCR techniques. We observed that wdr68 is ubiquitously expressed during early embryonic development but, during the neurula stage, it undergoes a strong expression in the neural tube and in the migratory cephalic streams of the neural crest. At the tailbud stages, it is strongly expressed in the cephalic region, particularly in otic and optic vesicles, in addition to branchial arches. In contrast, wdr68 transcripts are localized in the somitic mesoderm in the trunk. The expression area that includes the migratory neural crest of the head and the branchial arches suggest that this gene would be involved in jaws formation, probably through a hierarchical relationship with the component genes of the endothelin-1/endothelin receptor type A cell signaling pathway.
Fig. 1. Unrooted phylogenetic tree showing the molecular relationship between WDR68 homologous proteins from different animals and plants, drawn by the TreeDyn algorithm from the data generated by CLUSTAL OMEGA.
Fig. 2. Alignment of WDR68 protein sequences. The sequence characterized in the present work, corresponding to Xenopus laevis (KX910100.1), was aligned with the sequences from Xenopus tropicalis (NP_989097), zebrafish (NP_956363), chicken (NP_001072972), mouse (AAH48722) and fruit fly (AAF50953). The comparison showed a high degree of conservation of the WD40 domain, indicated by a violet box. Asterisks indicate identical amino acids between the aligned sequences; colons indicate conserved substitutions; dot indicates semi conserved-substitutions. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Analysis of the conserved syntenic regions containing the WDR68 locus in amphibian genome: X. laevis (subgenome L and subgenome S) and X. tropicalis; in fishes: zebrafish and medaka; in birds: chicken; in three species of mammals: human, cat and elephant. Genes are represented by colored boxes while arrow indicates the orientation of the transcription unit. Boxes with the same color represent orthologous genes. Genomic regions were not drawn to scale in order to reduce complexity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Expression pattern for the wdr68 gene during Xenopus early development. (A, B) Dorsal view of embryo. (C–F) Side view. (G, H) Transverse sections. (A) wdr68 expression is ubiquitous during early stages of development. (B, C) Since the late neurula stage, there is an enrichment in its expression in the head region (black arrowhead in B), in the migratory streams of the cranial neural crest (black arrowheads in C) and in the developing somites (white arrow). (D) At stage 25, wdr68 transcripts are evident in the otic vesicle. In the somitic region (black arrow), they acquire a segmental pattern. (E, F) Since the tailbud stage, wdr68 is strongly marked in the branchial arches (brackets). (G) Front section at the level of the branchial arches (blue broken line in F). Notice that the ectodermal cell population corresponding to the neural crest expresses wdr68. (H) Transverse section at the trunk level (red broken line in F). wdr68 expression is evident on the floor of the neural tube and in somitic mesoderm. References: BA, branchial arches; CNC, cranial neural crest; Ect, ectoderm; OpV, optic vesicle; OtV, otic vesicle; n, notochord; NC, neural crest; NT, neural tube. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. RT-PCR analysis of wdr68 gene expression in early embryonic development of Xenopus laevis. A maternal source of wdr68 transcripts can be observed. This molecular approach shows that the level of wdr68 expression is quite similar throughout developmental stages: when zygotic expression starts (st. 9), along the neurulation (st 12,5–25), at the tailbud stage (st. 30–35) and at the tadpole stage (st. 45). ef1 alpha was used as loading control. A mix without DNA template was used as negative control.
Fig. 6. RT-PCR analysis showing the ubiquitous expression of wdr68â¯at mid neurula stage. (A) Scheme representing a transverse section of a stage 16 embryo. Explants were dissected from ectodermal tissue, mesoderm, neural plate and neural crest in order to carry out total RNA purification for the subsequent RT-PCR. (B) Semiquantitative PCR showing a similar wdr68 expression level in the three ectodermal domains of neural crest, neural plate and lateral ectoderm. A lower expression was detected in the paraxial mesoderm. A mix without DNA template was used as negative control. Expression of ef1 alpha was used as loading control.
Fig. 7. Comparison between embryonic expressions of wdr68, ppet-1, ece-1 and ednra genes. (A) Antero dorsal view of mid-neurula stage. ppet-1 transcripts are detected in the mesoderm underlying the neural crest (asterisks) (Bonano et al., 2008). (B) Dorsal view of an embryo at neurula stage showing ece-1 expression in the lateral neural folds area (asterisks), in the ectoderm and mesoderm surrounding neural crest territory (Bonano et al., 2008). (C) Dorsal view of an embryo at neurula stage showing wdr68 expression at the cephalic region and in somitic mesoderm. (D) Lateral view of tailbud stage embryos. ednra is expressed in the branchial arches (arrow heads), in otic and optic vesicles and also in the migratory truncal neural crest streams. (E) At tailbud stages, the head shows a strong expression of wdr68, mainly at the otic and optic vesicles and in the branchial arches. At the trunk level, wdr68 is expressed in the somitic mesoderm. References: Op V, optic vesicle; Ot V, otic vesicle; n, notochord; np, neural plate; s, somites.
dcaf7 ( DDB1 and CUL4 associated factor 7 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
