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Spatially differentiated expression of quadruplicated green-sensitive RH2 opsin genes in zebrafish is determined by proximal regulatory regions and gene order to the locus control region.

Tsujimura T, Masuda R, Ashino R, Kawamura S - BMC Genet. (2015)

Bottom Line: The zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns.Importantly, competition for the RH2-LCR activity among the replicates is critical in this collective regulation, facilitating differentiation of expression among them.This combination of specificity and generality enables seemingly complicated spatial differentiation of duplicated opsin genes characteristic in fish.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, 277-8562, Chiba, Japan. t-tsujimura@umin.ac.jp.

ABSTRACT

Background: Fish are remarkably diverse in repertoires of visual opsins by gene duplications. Differentiation of their spatiotemporal expression patterns and absorption spectra enables fine-tuning of feature detection in spectrally distinct regions of the visual field during ontogeny. Zebrafish have quadruplicated green-sensitive (RH2) opsin genes in tandem (RH2-1, -2, -3, -4), which are expressed in the short member of the double cones (SDC). The shortest wavelength RH2 subtype (RH2-1) is expressed in the central to dorsal area of the adult retina. The second shortest wave subtype (RH2-2) is expressed overlapping with RH2-1 but extending outside of it. The second longest wave subtype (RH2-3) is expressed surrounding the RH2-2 area, and the longest wave subtype (RH2-4) is expressed outside of the RH2-3 area broadly occupying the ventral area. Expression of the four RH2 genes in SDC requires a single enhancer (RH2-LCR), but the mechanism of their spatial differentiation remains elusive.

Results: Functional comparison of the RH2-LCR with its counterpart in medaka revealed that the regulatory role of the RH2-LCR in SDC-specific expression is evolutionarily conserved. By combining the RH2-LCR and the proximal upstream region of each RH2 gene with fluorescent protein reporters, we show that the RH2-LCR and the RH2-3 proximal regulatory region confer no spatial selectivity of expression in the retina. But those of RH2-1, -2 and -4 are capable of inducing spatial differentiation of expression. Furthermore, by analyzing transgenic fish with a series of arrays consisting of the RH2-LCR and multiple upstream regions of the RH2 genes in different orders, we show that a gene expression pattern related to an upstream region is greatly influenced by another flanking upstream region in a relative position-dependent manner.

Conclusions: The zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns. The input from these proximal elements collectively dictates the actual gene expression pattern of the locus, context-dependently. Importantly, competition for the RH2-LCR activity among the replicates is critical in this collective regulation, facilitating differentiation of expression among them. This combination of specificity and generality enables seemingly complicated spatial differentiation of duplicated opsin genes characteristic in fish.

No MeSH data available.


Translocation of the RH2-LCR revealed gene-order dependent competition for the RH2 regulation. a Translocation of the RH2-LCR. The RH2-LCR was removed from the original position and then re-inserted into the immediate downstream of RH2-3 in the RH2-PAC clones [18]. b Transverse sections of the adult transgenic zebrafish retinas of the PAC constructs where RH2-2 (left), RH2-3 (middle) and RH2-4 (right) are replaced with the GFP reporter respectively. The GFP signals appear as green. Note that in the left panel the green signal is saturated and only the autofluorescence from the retina is captured. The dorsal side is at the top, and the ventral side is at the bottom. Scale bars = 100 μm
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Fig4: Translocation of the RH2-LCR revealed gene-order dependent competition for the RH2 regulation. a Translocation of the RH2-LCR. The RH2-LCR was removed from the original position and then re-inserted into the immediate downstream of RH2-3 in the RH2-PAC clones [18]. b Transverse sections of the adult transgenic zebrafish retinas of the PAC constructs where RH2-2 (left), RH2-3 (middle) and RH2-4 (right) are replaced with the GFP reporter respectively. The GFP signals appear as green. Note that in the left panel the green signal is saturated and only the autofluorescence from the retina is captured. The dorsal side is at the top, and the ventral side is at the bottom. Scale bars = 100 μm

Mentions: To further test the effect of relative distance with each other in such context-dependent regulation of the RH2 locus, we established transgenic zebrafish lines of modified RH2-PAC clones, in which the RH2-LCR was translocated from the 15-kb upstream of the gene array to the region immediately downstream of RH2-3 in the RH2-PAC (Fig. 4a). We previously showed that this configuration increased the expression level of RH2-3 and decreased those of RH2-1 and RH2-2 in larvae by transient transgenic assays with GFP reporters [18]. Consistently, in the adult transgenic fish carrying the RH2-PAC clone with GFP replacing RH2-3, the expression of RH2-3/GFP was clearly extended towards the dorsal area of the retina (Fig. 4bmiddle). By contrast, the expression of RH2-2/GFP was abolished (Fig. 4bleft) and that of RH2-4/GFP was maintained in the ventral area and the dorsal tip of the retina in this configuration (Fig. 4bright). Thus, upon the translocation, the closer RH2-3 became the target of the RH2-LCR in the central to dorsal area of the retina. The LCR activity promoting the more distant RH2-1/RH2-2 was reduced. In the ventral retina, RH2-4 was still predominantly activated, excluding the activation of RH2-3.Fig. 4


Spatially differentiated expression of quadruplicated green-sensitive RH2 opsin genes in zebrafish is determined by proximal regulatory regions and gene order to the locus control region.

Tsujimura T, Masuda R, Ashino R, Kawamura S - BMC Genet. (2015)

Translocation of the RH2-LCR revealed gene-order dependent competition for the RH2 regulation. a Translocation of the RH2-LCR. The RH2-LCR was removed from the original position and then re-inserted into the immediate downstream of RH2-3 in the RH2-PAC clones [18]. b Transverse sections of the adult transgenic zebrafish retinas of the PAC constructs where RH2-2 (left), RH2-3 (middle) and RH2-4 (right) are replaced with the GFP reporter respectively. The GFP signals appear as green. Note that in the left panel the green signal is saturated and only the autofluorescence from the retina is captured. The dorsal side is at the top, and the ventral side is at the bottom. Scale bars = 100 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4634787&req=5

Fig4: Translocation of the RH2-LCR revealed gene-order dependent competition for the RH2 regulation. a Translocation of the RH2-LCR. The RH2-LCR was removed from the original position and then re-inserted into the immediate downstream of RH2-3 in the RH2-PAC clones [18]. b Transverse sections of the adult transgenic zebrafish retinas of the PAC constructs where RH2-2 (left), RH2-3 (middle) and RH2-4 (right) are replaced with the GFP reporter respectively. The GFP signals appear as green. Note that in the left panel the green signal is saturated and only the autofluorescence from the retina is captured. The dorsal side is at the top, and the ventral side is at the bottom. Scale bars = 100 μm
Mentions: To further test the effect of relative distance with each other in such context-dependent regulation of the RH2 locus, we established transgenic zebrafish lines of modified RH2-PAC clones, in which the RH2-LCR was translocated from the 15-kb upstream of the gene array to the region immediately downstream of RH2-3 in the RH2-PAC (Fig. 4a). We previously showed that this configuration increased the expression level of RH2-3 and decreased those of RH2-1 and RH2-2 in larvae by transient transgenic assays with GFP reporters [18]. Consistently, in the adult transgenic fish carrying the RH2-PAC clone with GFP replacing RH2-3, the expression of RH2-3/GFP was clearly extended towards the dorsal area of the retina (Fig. 4bmiddle). By contrast, the expression of RH2-2/GFP was abolished (Fig. 4bleft) and that of RH2-4/GFP was maintained in the ventral area and the dorsal tip of the retina in this configuration (Fig. 4bright). Thus, upon the translocation, the closer RH2-3 became the target of the RH2-LCR in the central to dorsal area of the retina. The LCR activity promoting the more distant RH2-1/RH2-2 was reduced. In the ventral retina, RH2-4 was still predominantly activated, excluding the activation of RH2-3.Fig. 4

Bottom Line: The zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns.Importantly, competition for the RH2-LCR activity among the replicates is critical in this collective regulation, facilitating differentiation of expression among them.This combination of specificity and generality enables seemingly complicated spatial differentiation of duplicated opsin genes characteristic in fish.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, 277-8562, Chiba, Japan. t-tsujimura@umin.ac.jp.

ABSTRACT

Background: Fish are remarkably diverse in repertoires of visual opsins by gene duplications. Differentiation of their spatiotemporal expression patterns and absorption spectra enables fine-tuning of feature detection in spectrally distinct regions of the visual field during ontogeny. Zebrafish have quadruplicated green-sensitive (RH2) opsin genes in tandem (RH2-1, -2, -3, -4), which are expressed in the short member of the double cones (SDC). The shortest wavelength RH2 subtype (RH2-1) is expressed in the central to dorsal area of the adult retina. The second shortest wave subtype (RH2-2) is expressed overlapping with RH2-1 but extending outside of it. The second longest wave subtype (RH2-3) is expressed surrounding the RH2-2 area, and the longest wave subtype (RH2-4) is expressed outside of the RH2-3 area broadly occupying the ventral area. Expression of the four RH2 genes in SDC requires a single enhancer (RH2-LCR), but the mechanism of their spatial differentiation remains elusive.

Results: Functional comparison of the RH2-LCR with its counterpart in medaka revealed that the regulatory role of the RH2-LCR in SDC-specific expression is evolutionarily conserved. By combining the RH2-LCR and the proximal upstream region of each RH2 gene with fluorescent protein reporters, we show that the RH2-LCR and the RH2-3 proximal regulatory region confer no spatial selectivity of expression in the retina. But those of RH2-1, -2 and -4 are capable of inducing spatial differentiation of expression. Furthermore, by analyzing transgenic fish with a series of arrays consisting of the RH2-LCR and multiple upstream regions of the RH2 genes in different orders, we show that a gene expression pattern related to an upstream region is greatly influenced by another flanking upstream region in a relative position-dependent manner.

Conclusions: The zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns. The input from these proximal elements collectively dictates the actual gene expression pattern of the locus, context-dependently. Importantly, competition for the RH2-LCR activity among the replicates is critical in this collective regulation, facilitating differentiation of expression among them. This combination of specificity and generality enables seemingly complicated spatial differentiation of duplicated opsin genes characteristic in fish.

No MeSH data available.