Le Bouffant R et al. (2014), Differential expression of arid5b isoforms in X...

XB-ART-49639

Int J Dev Biol 2014 Jan 01;585:363-8. doi: 10.1387/ijdb.140029mu.

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Differential expression of arid5b isoforms in Xenopus laevis pronephros.

Le Bouffant R , Cunin AC , Buisson I , Cartry J , Riou JF , Umbhauer M .

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Arid5b belongs to the ARID family of transcription factors characterised by a helix-turn-helix motif- based DNA-binding domain called ARID (A-T Rich Interaction Domain). In human, alternative splicing leads to long and short isoforms (isoform1 and 2, respectively) which differ in their N-terminal part. In this study, we report the cloning and expression pattern of Xenopus laevis arid5b. We have isolated a full length cDNA that shows homology with the human arid5b isoform1. Furthermore, 5'RACE experiments revealed the presence of a shorter isoform equivalent to the human isoform2. Temporal expression analysis by RT-qPCR indicated that X. laevis arid5b isoform1 and isoform2 are differentially expressed during development. Isoform1 is strongly expressed maternally, while isoform2 expression is essentially restricted to tailbud stages. Spatial expression analysis by whole mount in situ showed that arid5b is predominantly expressed in the developing pronephros. Arid5b mRNAs are detected in the antero-dorsal part of the pronephros anlage at the early tailbud stage and later on, in the proximal part of the pronephric tubule. RT-qPCR analyses with primers that allow to discriminate isoform1 from isoform2 showed that the latter is enriched in the pronephros anlage. In agreement with a specific pronephric signature of the isoform2, we also observed that isoform2 but not isoform1 is upregulated in animal caps induced to form pronephric tissue in response to activin A and retinoic acid. These results indicate that the two arid5b isoforms are differentially expressed and likely play different roles during early Xenopus development.

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Species referenced: Xenopus laevis
Genes referenced: arid5b dll1 odc1 pax8 slc12a1

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	Fig. 1. Characterization of a X. laevis ortholog of arid5b. (A) Predicted ARID domain amino acid sequence comparison: G. gallus (ac Q5ZJ69); H. sapiens (ac Q14865); X. tropicalis (F6QQ73); D. rerio (E7F888). (B) Synteny blocks containing arid5b genes in H. sapiens; M. musculus and X. tropicalis genomes. Genes organization in the human arid5b gene region was used as basis for comparison. Chromosomal localization is indicated. The scaffold number is given for X. tropicalis. Relative spacing between the genes is not shown. (C) Phylogenetic tree of arid5b genes from various vertebrate species constructed using the neighbour-joining method. Accession numbers used are shown.
	Fig. 2 (Left). Temporal expression of arid5b during Xenopus embryonic development. Expression of arid5b analysed by RT-qPCR at cleavage (stages 5 and 9), gastrula (stage 11), neurula (stages 14) and tailbud stages (stages 22 and 28). Primers were designed in order to amplified either both isoforms (total arid5b) (A), isoform1 (B,D) or isoform2 (C,E). The relative quantities of mRNA were determined with ODC (A,B,C) or total arid5b (D,E) mRNA as the endogenous reporter. Isoform1 is strongly maternally expressed; isoform2 is mainly expressed during tailbud stages. Average values from three independent experiments.
	Fig. 3 (Right). Expression of arid5b at the early gastrula stage. RT-qPCR analyses for total arid5b, isoform1 and isoform2 were performed on dissected explants from early gastrula stage embryo (stage10.5). Embryos were dissected either into ventral and dorsal halves or into dorsal marginal zone (DMZ), ventral marginal zone (VMZ), lateral marginal zone (LMZ) ectoderm and endoderm. Both isoforms are ubiquitously expressed at the early gastrula stage. Average values from three independent experiments.
	Fig. 4 (Left). Spatial expression of arid5b during Xenopus development. In situ hybridization of whole (A-D, G-L) or transverse fractured embryos (E,F) at the indicated stages of development with antisense probe for arid5b (nucleotide 775 to 1612 of isoform1) (A,B,D,E,H,J,L,I), arid5b arid5b arid5b sens arid5b-slc12a1 arid5b slc12a1 arid5b-slc12a1 arid5b arid5b delta1 delta1 arid5b-slc12a1 st 22 st 25 st 25 st 31 st 25 st 25 st 35/36 st 35/36 st 35/36 st 35/36 st 35/36 st 35/36 G B C D E F H I J K L A delta1 (C,F), slc12a1 (I,K,L) and with control sense probe for arid5b (G). Lateral views with anterior to the right (A-D,G,H,J-L). Transverse section at the level of the proximal pronephric tubule. In I and L, arid5b is revealed in light blue and slc12a1 in purple. Arid5b mRNAs are detected in the anterior pronephric anlage during tailbud stages in a broader domain than delta1. At tadpole stage 35/36, arid5b expression is restricted to the proximal part of the tubule and does not overlap with slc12a1 expression which is specific for the intermediate and the first segment of the distal tubule. Scale bars are 0.3 mm.
	Fig. 5 (Right). Arid5b isoform2 is specifically expressed in the pronephros. Expression of isoform1, isoform2 and pax8 analysed by RT-qPCR in different embryonic regions (see materials and methods) dissected at tailbud stage 25. Isoform2 as well as pax8 are strongly expressed in pronephric anlage in comparison to other embryonic tissues (statistically significant for all). Isoform1 is slightly more expressed in the head in comparison to other tissues. Average values from three independent experiments. * P<0.05.
	Fig. 6. Arid5b isoform2 is specifically induced in animal caps treated with activin A and RA. RT-qPCR analysis of arid5b isoform1 and isoform2, as well as pax8 expression in induced blastula animal caps. Animal caps were dissected at blastula stage 9, incubated for 3 hours in presence of RA and activin A, or mock solution for the control group. Animal caps were further cultured in 1X MBS for 48h hours and processed for RT-qPCR. A significant increase of isoform2 expression is observed in response to RA and activin A. Average values from three independent experiments. * P<0.05 ** P < 0.005.
	arid5b (AT rich interactive domain 5B (MRF1-like)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
	arid5b (AT rich interactive domain 5B (MRF1-like)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior left, dorsal up.