Costa S et al. (2017), An Intronic cis-Regulatory Element Is Crucial f...

ECB-ART-45160

PLoS One 2017 Jan 01;121:e0170969. doi: 10.1371/journal.pone.0170969.

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An Intronic cis-Regulatory Element Is Crucial for the Alpha Tubulin Pl-Tuba1a Gene Activation in the Ciliary Band and Animal Pole Neurogenic Domains during Sea Urchin Development.

Costa S , Nicosia A , Cuttitta A , Gianguzza F , Ragusa MA .

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In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the proper territories of the embryo. Additionally, the mutation of the forkhead box consensus sequence binding site in ICR3 prevented Pl-Tuba1a expression.

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Genes referenced: LOC100887844 LOC115919910 LOC587333 pole tubgcp2

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	Fig 1. Expression of the -1.8KbGFP transgene construct during P. lividus embryo development.At the top is a schematic structure (drawn to scale) of the Pl-Tuba1a -1.8KbGFP reporter construct. The bent arrow indicates the TSS. A grey box represents the first exon (5â€™UTR and ATG start codon). Downstream of the first exon there are the first intron and two codons of the second exon. Coloured boxes indicate the four ICRs. For sake of simplicity, only the section of ICR3 inside the intron is shown. The arrowed green box represents the GFP reporter gene cloned in frame with the alpha tubulin codons. Left: triple-merged images (bright-field, GFP fluorescence and Texas Red fluorescence-a) or merged fluorescence and bright-field images (b, c, d). Right: GFP fluorescence images from microinjected embryos. Ã— 20 magnification. (a) 32-cell stage; (b) Blastula stage; (c) Gastrula stage; (d) Pluteus stage. Lv: lateral view; av: animal view.
	Fig 2. Transgene basal expression and loss of expression by TATA box deletion.Gene transfer assay results performed using the -0.1(Î”ICR1-2)GFP (A) and the +20(Î”TATA)GFP (B) constructs. Structure and conventions are the same as in Fig 1. (a) Gastrula stage; (b) Pluteus stage. (A) Merged fluorescence and bright-field images (left) and GFP fluorescence images (right) from microinjected embryos are shown. (B) Triple-merged bright-field, GFP fluorescence and Texas Red fluorescence images (left) proving embryos are microinjected and GFP fluorescence images (right). Ã— 20 magnification. Lv: lateral view; av: animal view.
	Fig 3. Enhancer functions of ICR1 and ICR2.Upstream deletions: luciferase activity progressively decreases after ICR1 and ICR2 sequential deletions. Left: schematic pictures of luciferase reporter constructs. Structure and conventions are the same as in Fig 1, except that the arrowed yellow box represents the Luc reporter gene. Right: luciferase activity measured at gastrula stage (24 hours post fertilization-hpf). The activity of the -1.8KbLuc construct is defined as 100%. Data are expressed as means Â± standard deviation (SD) of triplet measurements of at least three independent experiments. Asterisks denote statistical significance: ** P value â‰¤ 0.0021, * P value = 0.0122.
	Fig 4. Transgene loss of expression by intron deletion.Embryos were microinjected with the -1.8(Î”Intron)GFP construct. Left: triple-merged images (bright-field, GFP fluorescence and Texas Red fluorescence) proving embryos are microinjected. Right: GFP fluorescence images. Structure and conventions are the same as in Fig 1. (a) Gastrula stage; (b) Pluteus stage. Av: animal view.
	Fig 5. ICR3 and ICR4 function: spatial expression.In these construct depictions, ICR3 was enlarged to show deletion details (not to scale). Left: schematic pictures of GFP reporter constructs. Structure and conventions are the same as in Fig 1. Right: merged fluorescence and bright-field images or triple-merged images and fluorescence images from microinjected pluteus stage embryos are shown to observe GFP localization. Ã— 20 magnification. Av: animal view; ov: oral view.
	Fig 6. ICR3 and ICR4 function: quantitative analysis.In these construct depictions, ICR3 was enlarged to show deletion details (not to scale). Left: schematic pictures of luciferase reporter constructs. Structure and conventions are the same as in Fig 2. Right: luciferase activity measured at gastrula stage (24 hpf). The activity of the -1.8KbLuc construct is defined as 100%. Data are expressed as means Â± SD of triplet measurements of at least three independent experiments. Asterisks denote statistical significance: *** P value < 0.0005.
	Fig 7. Transcription factor binding sites in the sequence necessary for proper Pl-Tuba1a expression.Top: wild-type and mutant construct sequences from +94 to +110 bp. Arrows indicate specific nucleotide changes generated by site-directed mutagenesis (T101 to G and T102 to C). Center and bottom: FoxD3 matrix logo (M00130) and Ncx (M00484) matrix logo aligned to sequences. Logos were generated using WebLogo [30].
	Fig 8. A summary of the transcription regulation of the Pl-Tuba1a gene.Chromatin modifications occurring on the Pl-Tuba1a promoter, according to [27] are shown. Transgenic wild type and mutagenized constructs and their expression during embryo development are also shown. See text for details.

References [+] :

Angerer, The evolution of nervous system patterning: insights from sea urchin development. 2011, Pubmed, Echinobase