Abstract
Gene duplication is a fundamental source of a new gene in the process of evolution. A duplicated gene is able to accept many kinds of mutations that could lead to loss of function or novel phenotypic diversity. Alternatively, the duplicated genes complementarily lose part of their functions to play original roles as a set of genes, a process called subfunctionalization. Pseudotetraploid frog Xenopus laevis has four sets of genes, and it is generally thought that the alloalleles in X. laevis have mutually indistinguishable functions. In this paper, we report differences and similarities between Xhairy2a and Xhairy2b in the neural crest, floor plate, and prechordal plate. Knockdown studies showed that Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate. Temporal expression pattern analysis revealed that Xhairy2a is a maternal factor having lower zygotic expression than Xhairy2b, while Xhairy2b is not loaded in the egg but has high zygotic expression. Spatial expression pattern analysis demonstrated that future floor plate expression is shared by both alloalleles, but Xhairy2b expression in the neural crest is much higher than Xhairy2a expression, consistent with the results of individual knockdown experiments. Therefore, our data suggest that subfunctionalization occurs in Xhairy2.
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References
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Acknowledgment
We thank Dr. S. Nishimatsu (Kawasaki Medical School) for technical advice on whole-mount in situ hybridization.
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Communicated By T. Hollemann
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Fig. S1
Two alleles of the Xhairy2 gene show functional differences. a Morpholino antisense oligos (mo’s) to target Xhairy2a and Xhairy2b individually. Numbers on bases are of Xhairy2a gene. b Western blot analysis to confirm effects and specificity of morpholinos designed to inhibit translation of Xhairy2a and Xhairy2b genes. Embryos at the four-cell stage were ventrally injected with 800 pg of mRNA of myc-tagged (denoted by MT) Xhairy2a or Xhairy2b construct and/or morpholino (denoted by mo; h2A mo, 6.8 ng; h2B mo, 6.9 ng; 5mis-h2A mo, 6.8 ng; 5mis-h2B mo, 6.9 ng). In -mo constructs (lanes 5, 6, 13, and 14), the target sequences of corresponding morpholino were deleted to test sequence specificity of each morpholino. The sequence of 5 mis-h2A mo was 5′-GCATTTTCATTTGCTTGTAAAGTAA-3′. Xhairy2b-MT/pCS2, -mo Xhairy2b-MT/pCS2, and the sequence of 5 mis-h2B mo were described elsewhere (Yamaguti et al. 2005). Xhairy2a-MT/pCS2 and -mo Xhairy2a-MT/pCS2AT+ were constructed in similar ways. Plus sign indicates presence of corresponding myc-tagged mRNA or mo, and minus sign indicates absence. Coomassie brilliant blue staining of the gel served as loading control. IB Immunoblot, CBB Coomassie brilliant blue. (GIF 59.8 kb)
Fig. S2
Xhairy2a and Xhairy2b have equal level of transcriptional repression. Four-cell-stage embryos were ventrally injected with 240 pg of the luciferase reporter vector and 800 pg of the indicated mRNA, and harvested at stage 10 for measurement of luciferase activity. lacZ mRNA served as negative control. The luciferase reporter vector (denoted by N6: Luc) carries six repeats of N-box sequences where Xhairy2 binds, followed by constitutively active β-actin promoter and luciferase coding sequences (Murato et al. 2006) (GIF 9.87 kb)
Fig. S3
Examination of Xhairy2a-, Xhairy2b-, and Xhairy2-specific probes to verify that each of the probes is specific enough to be used in spatial expression pattern analysis of Xhairy2a and Xhairy2b by means of WISH. In vitro examination was performed by means of RNA dot blot analysis (a and b), and this was followed by in vivo examination with WISH (c and d). a Nucleotide sequence similarity between Xhairy2a and Xhairy2b cDNA is schematically shown. 5′-UTR shows 61% sequence similarity, suggesting that specific probes can be made from this region albeit the short nucleotide (around 120 bp). In the coding region (around 900 bp), Xhairy2a and Xhairy2b are 93% identical, suggesting that a probe recognizing both Xhairy2a and Xhairy2b can be made from this region to view Xhairy2 expression as a whole. b RNA dot blot analysis to confirm the specificity of Xhairy2a- and Xhairy2b-specific probes. As shown in a, we synthesized DIG-labeled Xhairy2a- or Xhairy2b-specific probe for chemiluminescence detection and synthesized fluorescein-labeled Xhairy2b probe for direct membrane staining to show the presence of spotted sense RNA (both Xhairy2a and Xhairy2b). b i, b iii X-ray film images. The specific probes showed strong sensitivity depending on the amount of loaded sense RNA. No cross-hybridization was observed. Spotted sense RNA is indicated at the left side of the panels. b ii, b iv The hybridized membranes were stained with NBT/BCIP to check equal loading of Xhairy2a and Xhairy2b sense RNAs (see “Materials and methods” for details). The probe is indicated at the lower right corner of each panel. c Schematic summary of WISH-based examination to verify the specificity of specific probes shown in d. At the eight-cell stage, 200 pg of Xhairy2a mRNA with FLD was injected into one future ventral blastomere, and the same amount of Xhairy2b mRNA with FLD was injected into Xhairy2a’s neighbor blastomere on the right (viewed from the animal pole with dorsal side up, Xhairy2a mRNA is on the right side and Xhairy2b in on the left side). The injected regions were checked with FLD fluorescein at stage 9 and photographed. The embryos were then fixed and subjected to WISH with the indicated probes. d The results of WISH-based examination. d i–viii Injected regions are indicated by FLD fluorescein (white arrows). Images were taken before fixation. d i′–viii′ Corresponding samples were fixed and stained with the probe indicated at the lower right corner of each panel (Xhairy2a/b indicates simultaneous staining with Xhairy2a and Xhairy2b). In d i′, iii′, v′, and vii′, in which only FLD was injected as control, no signal was detected. In d ii′, iv′, vi′, and viii′, signals (white arrowheads) were detected, as expected. Note that no cross-hybridization was observed although the specific probes were used against the overexpressed mRNAs, demonstrating accuracy of their recognition. In addition, as shown in d ii′, full-length Xhairy2b probe as the Xhairy2 probe indeed detected both Xhairy2a and Xhairy2b (GIF 160 kb)
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Murato, Y., Nagatomo, K., Yamaguti, M. et al. Two alloalleles of Xenopus laevis hairy2 gene—evolution of duplicated gene function from a developmental perspective. Dev Genes Evol 217, 665–673 (2007). https://doi.org/10.1007/s00427-007-0176-x
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DOI: https://doi.org/10.1007/s00427-007-0176-x