Tereshina MB et al. (2019), Agr2-interacting Prod1-like protein Tfp4 from X...

XB-ART-55806

Genesis 2019 May 01;575:e23293. doi: 10.1002/dvg.23293.

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Agr2-interacting Prod1-like protein Tfp4 from Xenopus laevis is necessary for early forebrain and eye development as well as for the tadpole appendage regeneration.

Tereshina MB , Ivanova AS , Eroshkin FM , Korotkova DD , Nesterenko AM , Bayramov AV , Solovieva EA , Parshina EA , Orlov EE , Martynova NY , Zaraisky AG .

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The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.

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Species referenced: Xenopus laevis
Genes referenced: ag1 agr2 agr3

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	Figure 1. Multiple alignment and phylogenetic analysis of Xenopus Tfp4 with other Tfp‐like proteins and spatio‐temporal expression patterns of Tfp4 during the early development in comparison to Agr2. (a) The multiple alignment of Xenopus laevis and Xenopus tropicalis Tfp4 with Lypd6, CD59 and salamander Prod1 primary structures demonstrates the presence of all highly conserved amino acid residues of the Tfp‐domain in Xenopus Tfp4. (b) Phylogenetic analysis (UPGMA) clusters XtTfp4 and XlTfp4 to Prod1 rather than to CD59 or Lypd6. (c) The results of in situ hybridization in X. laevis embryos at stages14 and 25 (dark purple staining) show overlapping expression patterns of Tfp4 and Agr2 in the anterior ectoderm (st.14, dorsal view) and in the cement and hatching glands (st.25, frontal view). The dotted line points the neural plate area. cg, Cement gland; hg, Hatching gland
	Figure 2. Spatio‐temporal expression pattern of Tfp4 during regeneration. (a) The qRT‐PCR data demonstrate activation of Tfp4 expression during regeneration at 1–2 days post amputation (dpa) of tail or hindlimb (st.52). Data are represented as mean ± SD, statistical significance was determined by t‐test, p < .05 (asterisk). (b) Results of in situ hybridization show high Tfp4 expression level in wound epithelium (we) of hindlimb at 1 day after amputation (dpa). By 2 dpa expression in wound epithelium becomes weaker. (c) Results of in situ hybridization demonstrate high Tfp4 expression level in wound epithelium (we) and some regions of tail fin (yellow arrows) at 1 day after amputation (dpa) of tadpole's tail. expression of Tfp4 at 2 dpa is less strong and is present in wound epithelium and blastema (bl). By 5 dpa we could not detect Tfp4 expression in almost regenerated tail. In situ hybridization with control sense‐digTfp4 shows no staining in the hindlimb at 2 dpa or tail at 1dpa. Dashed red line points the amputation level
	Figure 3. Spatio‐temporal expression pattern of Tfp4 during regeneration. (a) The qRT‐PCR data demonstrate activation of Tfp4 expression during regeneration at 1–2 days post amputation (dpa) of tail or hindlimb (st.52). Data are represented as mean ± SD, statistical significance was determined by t‐test, p < .05 (asterisk). (b) Results of in situ hybridization show high Tfp4 expression level in wound epithelium (we) of hindlimb at 1 day after amputation (dpa). By 2 dpa expression in wound epithelium becomes weaker. (c) Results of in situ hybridization demonstrate high Tfp4 expression level in wound epithelium (we) and some regions of tail fin (yellow arrows) at 1 day after amputation (dpa) of tadpole's tail. expression of Tfp4 at 2 dpa is less strong and is present in wound epithelium and blastema (bl). By 5 dpa we could not detect Tfp4 expression in almost regenerated tail. In situ hybridization with control sense‐digTfp4 shows no staining in the hindlimb at 2 dpa or tail at 1dpa. Dashed red line points the amputation level.
	Figure 4. Eye development is affected by synergistic action of Tfp4 and Agr2 if they are co‐overexpressed in Xenopus embryos. (a) The transmitted light and fluorescent images of tadpoles st. 38 injected by single Tfp4 or Agr2 mRNA or both Agr2 + Tfp4 mRNAs demonstrate eye enlargement on the injected side. The fluorescent images show the injection distribution in the tadpole (b) Statistics of tadpoles' percent with enlarged eye from tadpoles groups with Tfp4, Agr2, or Tfp4 + Agr2 overexpression. The results are statistically significant at p < .05 (asterisk), t‐test. red dashes indicate the diameter of the eye, which was used for the eye size changes calculations. (c) The transmitted light and fluorescent images of tadpoles st. 38 overexpressing Tfp4 or Agr2 against the background of downregulation by MO of Agr2 or Tfp4, respectively demonstrate no eye enlargement on the injected side. The fluorescent images show the injection distribution in the tadpole. (d) The measurements of diameter were made in the ImageJ software and were used for calculation of eye diameter (D) differences between mRNA overexpressing side (right) and control non‐injected side (left). The percent mean was calculated from (Dright‐Dleft)/Dleft. t‐Test, p < .05 (asterisk). (e) The in situ hybridization analysis of the eye marker Rx1 expression in the embryos (st.15) injected by the corresponding mRNAs. The fluorescent images show the injection distribution in the embryo. Black arrows indicate Rx1 expression expansion on the injected sides. (f) The differences in percent of embryos with Rx1 expression expansion over‐expressing Tfp4, Agr2, or Tfp4 + Agr2 mRNAs are statistically significant at p < .001 (asterisk), t‐test.
	Figure 5. The synergetic effect of Tfp4 + Agr2 on cell proliferation. The results of immunohistochemistry staining with rabbit anti‐phosphoHistone H3 (Millipore) and anti‐rabbit CF568 (Sigma) in the midneurula stage embryos, injected into one of two blastomeres by Agr2 (150 ng/mkl), or Tfp4 (150 ng/mkl), or Tfp4 + Agr2 mixed with fluorescent tracer FLD. The mitotic cells count was performed using ImageJ. The fold change of mitotic cells number was calculated by dividing of number of pH3B‐positive cells in the injected side of the embryo on number of them in the non‐injected side (N i/N ni). Statistical significance was determined in t‐test, p < .05 (asterisk).
	Figure S1. Tfp4 expression pattern during Xenopus laevis development. The results of in situ hybridization show Tfp4 expression (dark-purple staining) in cells of Xenopus laevis embryos starting from late gastrula stage (a), increasing in non-neural ectoderm at early (b) and late neurula (c). At stage 25 (d) expression is detected in epidermis and cement gland and by tailbud stage 32 (e - lateral view, e’ - front view) Tfp4 transctipts are concentrated in cement and hatching gland cells and progenitor cells of branchial arches, middle and anterodorsal lateral line, olfactory placode, otic vesicle and eye. A-c dorsal view, anterior to the top, d,e – lateral view, e’ – front view. Abbreviations: a – anterodorsal lateral line, ab – branchial arches, bl – blastopore groove, cg – cement gland, e – eye, hg – hatching gland, m – middle lateral line, np – neural plate, nt – neural tube, op – oflcatory placode, ov – otic vesicle.
	Figure S2. Tfp4 MO efficiency test. a. MO sequence. b. Western blotting with anti-flag and anti-tubuline antibody.
	Figure S3. Knock-down of Tfp4 by splice-MO and the rescue experiment. a. The qRT-PCR check of Tfp4 splice-MO effectiveness. The PCR product of Tfp4 intron region is present in embryos injected by Tfp4 splice-MO. b. Statistics of tadpoles’ percent with reduced eye (dark grey columns, measured at st.38) or reduced telencephalon (light grey columns, measured at st.44) from tadpoles groups injected with Tfp4 splice-MO or with the “rescue mix” Tfp4 splice-MO + Tfp4 mRNA. Data on the bar chart are represented as % mean ±SD, the results are statistically significant at p<0,05 (asterisk), t-test. Red dashes indicate the diameter of the eye, which was used for the eye size changes calculations. n- number of tadpoles used in three independent experiments. c,e. The transmitted light and fluorescent images of the tadpole injected with Tfp4 splice-MO into the progenitors of the anterior ectoderm at the right side of the embryo show severe malformations of the head structures, especially of the eye and telencephalon. d,f. The transmitted light and fluorescent images of the rescued tadpoles, which were injected with Tfp4 splice-MO mixed with Tfp4 mRNA into the progenitors of the anterior ectoderm at the right side of the embryo show normalized development of eye and telencephalon.
	Figure S4. Eye development affected by action of XAg1 overexpressed in Xenopus embryos in comparison to co-expression with Tfp4 or to control (EGFP). a. The transmitted light and fluorescent images of tadpoles st. 38 injected by single EGFP or Ag1 mRNA or Ag1+Tfp4 mRNAs demonstrate eye reduction on the Ag1-injected side. b. Statistics of tadpoles’ percent with reduced eye from tadpoles groups with EGFP, Ag1 or Tfp4+Ag1 overexpression. The results are statistically significant at p<0,05 (asterisk), t-test. The measurements of eyes diameters were made in the ImageJ software and were used for calculation of eye diameter (D) differences between mRNA over-expressing side (right) and control non-injected side (left). The percent mean was calculated from (Dright-Dleft)/Dleft. T-test, p<0,05 (asterisk).