Korotkova DD , Lyubetsky VA , Ivanova AS , Rubanov LI , Seliverstov AV , Zverkov OA , Martynova NY , Nesterenko AM , Tereshina MB , Peshkin L , Zaraisky AG .

R01 HD073104 NICHD NIH HHS

	Graphical Abstract
	Figure 1. The 2- and 3-Species Selection Modes for Genes with No Orthologs in the Upper Species (A and B) The schemes demonstrate the case when gene X is considered to be the lost gene for the 2- and 3-species modes. Gene X only has homolog X∗∗ but no ortholog in the upper species because the neighbors of X in the basic (for 2-species mode, A) or lower (for 3-species mode, B) species (Z, S, Y or Q∗, R∗, Y, respectively) have no homologs in the upper species, where the neighbors of gene X∗∗ are O and P. The red ticks indicate the borders of the “window” in which local synteny has been checked. The size of the window, l, was chosen in this particular work to be 2 Mbp for all genes: l = l1 = l2. Thin and bold arrows indicate homologous and orthologous genes in the upper and lower species, respectively. (С and D) The schemes demonstrate the case in which gene X is considered to be preserved for the 2- and 3-species modes, respectively. Gene X has an ortholog X∗∗ in the upper species because at least one of the neighbors of X in the basic (for 2-species mode, C) or lower (for 3-species mode, D) species (Z, S, Y or Q∗, R∗, Y, respectively) has a homolog in the upper species, where the neighbors of gene X∗∗ are Y∗ and S∗.
	Figure 2. Investigation of the Expression and Function of Genes Identified As a Result of Bioinformatic Screening during X. laevis Tadpole Tail Regeneration and Analysis of the Structure of c-Answer Protein (A) qRT-PCR analysis of c-answer expression in the blastema during tail regeneration at the indicated day post-amputation. All of the results were normalized to the geometric mean expression of two housekeeping genes (ef1alfa and odc) as previously described (Ivanova et al., 2013). (B) Injection of anti-sense morpholino oligonucleotides to c-answer mRNA inhibits tadpole tail regeneration. (B’) Fluorescent image of the same embryos shown in (B) demonstrating the distribution of the co-injected tracer fluorescein lysine dextran (FLD). (C) Alignment of c-Answer with FGFR4. (D) Localization of the secreted hybrid of EGFP and c-Answer on the membrane of the animal cap cell as revealed by confocal microscopy. (E) Schematics of the c-Answer deletion mutants used in the co-immunoprecipitation and functional analysis experiments. (F) Western blotting with a FLAG-antibody after coIP of FLAG-c-Answer with different Myc-tagged c-Answer deletion mutants, as shown in (C). (G) The c-Answer homodimer model according to the coIP results shown in (D). Scale bars: 1 mm in (B) and 200 nm in (D). See also Figures S1 and S2.
	Figure 3. Analysis of c-answer Expression during the Early Embryonic Development and in the Processes of Tail and Hindlimb Bud Regeneration of X. laevis (A) Temporal expression of c-Answer as revealed by RNA sequencing (RNA-seq) analysis (Yanai et al., 2011). (B and C) In situ hybridization with a c-Answer probe in the whole-mounted middle neurula (B) and the frozen sagittal section of the embryo at the middle neurula stage (C). Anr, anterior neural ridge; npl, neural plate; tm, trunk mesoderm. (D and E) Whole-mount in situ hybridization with a sense probe to c-answer (control) of the amputated tail (D) and hindlimb bud (E) at day 1 post-amputation. (F and G) Whole-mount in situ hybridization with an anti-sense probe to c-answer of the amputated tail (F) and hindlimb bud (G) on day 1 post-amputation. Increased c-Answer expression is observed in the wound epithelium (we) and blastema (bl). (H and I) Frozen histological sections of the regenerating tail (H) and hindlimb bud (I) hybridized whole-mount with an anti-sense probe to c-answer. Scale bars: 500 μm in (B) and (C) and 50 μm in (D)–(I). See also Figure S3.
	Figure 4. Effects of c-answer Downregulation by Knockdown with Anti-Sense Morpholino Oligonucleotides and the CRISPR/Cas9 Knockout on Tadpole Brain Development and Tail Regeneration (A) c-answer knockdown with c-answer MO1 injections into the dorsal right blastomere at the 4-cell stage results in diminishing of the overall tadpole size, especially of the forebrain and eye (n = 180; 85% morphants), compared to that of the left side (control). Overlay with the fluorescent image demonstrates the distribution of the co-injected tracer FLD. (B) Tadpole in which c-Answer was knocked out in 2nd exon with CRISPR/Cas9 technology has a smaller size then the wild-type tadpole at the same stage. (C, C’, D, and D’) Tail regeneration in tadpoles with c-answer knockdown (C) or knockout (D) is inhibited compared to that of the wild-type control (see E for quantification). (E) Diagram showing the distribution of tail regeneration phenotypes in tadpoles injected with the indicated MO or components of the CRISPR/Cas9 system. (F) Analysis of the indicated marker genes expression by qRT-PCR in tips of the amputated tails of Xenopus laevis tadpoles at stage 41 (see for the scheme of the experiments and for specific primers structures; Ivanova et. al. 2018). The results were normalized by geometric median of odc and ef1alfa housekeeping genes expression described in Ivanova et al. (2013). The expression level of each gene at 0 dpa in the control piece of tissue was taken as one arbitrary unit. Scale bars: 1 mm in (A) and (B) and 250 μm in (C)–(D’). Figures S1, S4, S5, and S6.
	Figure 5. Effects of the Overexpression of c-Answer and Its Deletion Mutants on Tadpole Brain Development and Tail Regeneration (A and B) Inhibition of foxg1 (A) and rax (B) expression on the right side of middle neurula embryos by injection into the right dorsal blastomere at the 4-cell stage. (C) Overexpression of wild-type c-answer results in the development of an ectopic telencephalic hemisphere (3) in addition to normal hemispheres (1 and 2). (D and E) Ectopic eye differentiation in tadpoles overexpressing wild-type c-answer in the form of one normal-sized eye (D) or three small eyes (E). (F and G) Ectopic expression of foxg1 (F) and rax (G) on the right side of middle neurula embryos following the injection of c-answer mRNA into the right dorsal blastomere at the 4-cell stage. (H and H’) Overexpression of wild-type c-answer rescues tail regeneration during the “refractory” period (see I for quantification). (I) Diagram showing the distribution of the regenerating tail phenotypes in the control tadpoles and those overexpressing wild-type c-answer. Tails were amputated during the refractory period. (J) Overexpression of the deltaC-c-Answer mutant leads to a telencephalon size increase and ectopic RPE differentiation. (K) Overexpression of the extracellular domain of c-Answer results in a slight increase in the telencephalic and eye size on the injected (right) side. (L) Overexpression of the deltaN-c-Answer mutant inhibits the development of the telencephalon and eye on the injected side. (M and N) Ectopic foxg1 (M) and rax (N) expression on the left side of middle neurula embryos injected with deltaC-c-Answer mRNA into the left dorsal blastomere at the 4-cell stage. (O) Inhibition of foxg1 expression in the lateral part of the endogenous right expression domain of foxg1. Scale bars: 500 μm in (A), (B), (D)–(G), and (M)–(O); 1 mm in (C) and (L); and 250 μm in (H) and (H’). See also Figure S7.
	Figure 6. Analysis of the Binding Capacity of c-Answer and Its Deletion Mutants to the FGFR1–4 and P2ry1 Receptors (A) Schematic of the experiments. FLAG- and Myc-tagged proteins were separately expressed from synthetic mRNAs in the embryos, and the embryonic extracts were mixed for coIP and analyzed by western blotting. (B–D) Western blotting analysis with anti-Myc or anti-FLAG antibodies after coIP of Myc-c-Answer with FLAG-Piezo1, FGFR1–4, P2Y1 (B), of FLAG-FGFR4 with different c-Answer deletion mutants of Myc-c-Answer (C), or FLAG-P2ry1 with different deletion mutants of Myc-c-Answer (D). Cont. means the control western blotting with indicated antibody (Myc or FLAG) after application on anti-Myc or anti-FLAG resin of only Myc-c-Answer, FLAG-FGFR4, or FLAG-P2Y1, without preliminary coIP.
	Figure 7. c-Answer Promotes Signaling through the FGFR4 and P2ry1 Receptors, Related to Video S1 and Video S2 (A) Schematic of the experiment performed to analyze the effects of c-Answer on the expression of the MAP/ERK pathway luciferase reporter pSPE-Luc pGL4.33. The stress-activated reporter AP-1-Luc pGL4 was used as a control. (B) Diagram showing the Luc signal analysis results for the two reporters in the animal caps of embryos expressing the indicated proteins. (C) Schematic of the experiment performed to analyze the effects of c-Answer on the Ca2+ flux in response to the addition of the P2ry1 agonist ADP to animal cap cells expressing the Ca2+ sensor Case12 and purinergic receptor P2ry1. (D) Fluorescent images of cells expressing the indicated proteins before and after ADP addition. (E) Diagram showing the results of Case12 signal analysis in animal cap cells expressing the indicated proteins. Scale bars: 200 μm in (D).
	Figure S1. Testing of MO specificity and efficiency, Related to Figure 2 and Figure S4. A-D. Scheme of the morpholino target sites location on mRNA of the X. laevis orthologs of X. tropicalis genes: prothymosin homolog, sfrpx, c-answer, and paramyosin-like. For testing specificity of these MOs, mRNA encoding this protein tagged fron C-terminus with Myc-epitop was injected into each blastomere of 2-cell X. laevis embryos (100pg/blastomere) either alone or in mixture with indicated MOs (8nl of 0.2 mM water solution). The injected embryos were collected at the middle gastrula stage and analyzed for presence of Myc-tagged proteins by Western blotting with anti-Myc antibody (see Materials and Methods for details). E. Testing of specificity of c-answer MO2. F-H. Different types of tail phenotypes in tadpoles injected with c-answer vivoMO (0,9 mM) directly into the tail directly before the amputation. I. Diagram showing the distribution of tail regeneration phenotypes in tadpoles injected with the control vivoMO and c-answer vivoMO in two different concentrations. Scale bars on F-H 250 µm.
	Figure S2. Alignment of c-Answer from different species and its localization in cell membrane, Related to Figure 2. A. Alignment of c-Answer from the indicated could-blooded animals. B. Co-localization of EGFP-c-Answer with cell membrane marker mKate2-mem on cell membrane of the ectodermal cell of Xenopus laevis embryo at the middle gastrula stage. Scale bars on B 200 nm.
	Figure S3. Distribution of c-answer, the forebrain primordium markers foxg1 and rax and the cement gland marker ag1 expression according to single cell sequencing data in embryos at stages 16, 18, 20 and 22 (Briggs et al., 2018), Related to Figure 3. A. Stage 16, all embryonic cells arranged by their expression profile similarity (Briggs et al., 2018), clustered and colored according to the tissue subtype, where they belong. The area corresponding to cells of anterior neural plate including eye primordia is circled by white line. For legend see https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?cgibin/client_datasets/xenopus_embryo_timecourse_v2/S16 B. Stage 16, cells expressing c-answer are colored green. The intensity of green color depends on the number of c-answer mRNA present in cell, more intense color relates to greater amount of c-answer mRNA molecules per cell. The area corresponding to cells of anterior neural plate including eye primordia is circled by white line. C. Stage 16, cells expressing marker of the telencephalic and dorsal eye primordia, foxg1, are colored green. The area corresponding to cells of anterior neural plate including eye primordia is circled by white line. D. Stage 16, cells expressing marker of the eye and forebrain primordia, rax, are colored green. The area corresponding to cells of anterior neural plate including eye primordia is circled by white line. E. Stage 18, all embryonic cells are clustered and colored according to the tissue subtype, where they belong. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. For legend see https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?cgibin/client_datasets/xenopus_embryo_timecourse_v2/S18 F. Stage 18, cells expressing c-answer are colored green. The area corresponding to cells of anterior neural plate including eye primordia is circled by white line. G. Stage 18, cells expressing marker of the eye and forebrain primordia, foxg1, are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. H. Stage 18, cells expressing marker of the eye and forebrain primordia, rax, are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. I. Stage 20, all embryonic cells are clustered and colored according to the tissue subtype, where they belong. . The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. For legend see https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?cgibin/client_datasets/xenopus_embryo_timecourse_v2/S20 J. Stage 20, cells expressing c-answer are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. K. Stage 20, cells expressing marker of the eye and forebrain primordia, foxg1, are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. L. Stage 20, cells expressing marker of the eye and forebrain primordia, rax, are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. M. Stage 22, all embryonic cells are clustered and colored according to the tissue subtype, where they belong. For legend see https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?cgibin/client_datasets/xenopus_embryo_timecourse_v2/S22 N. Stage 22, cells expressing c-answer are colored green. The area corresponding to cells of anterior neural plate including eye primordia is circled by white line. O. Stage 22, cells expressing marker of the eye and forebrain primordia, foxg1, are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line. P. Stage 22, cells expressing marker of the eye and forebrain primordia, rax, are colored green. The area corresponding to cells of anterior neural tube including eye primordia is circled by white line.
	Figure S4. The results of embryo genotyping after c-answer CRISPR/Cas9 knockout on two target sites, Related to Figure 4. A. Diagram showing percentage of mutations after CRISPR/Cas9 procedure in ten randomly picked embryos at late gastrula stage injected with sgRNA for target site in 2nd c-answer exon of c-answer and Cas9 protein (see Figure S4 for the target site location). B. Diagram showing percentage of mutations after CRISPR/Cas9 procedure in ten randomly picked embryos at late gastrula stage injected with sgRNA for target site in 6th c-answer exon and Cas9 protein (see Figure S4 for the target site location). C. Examples of deletions and insertions in 2nd c-answer exon after CRISPR/Cas9 procedure. D. Examples of deletions and insertions in 6th c-answer exon after CRISPR/Cas9 procedure.
	Figure S5. Effects of c-answer downregulation by knockdown with anti-sense morpholino oligonucleotide 2 (MO2) and the CRISPR/Cas9 knockout with sgRNA to the target site in 6th exon on tadpole brain development and tail regeneration, Related to Figure 4. A. c-answer knockdown with c-answer MO2 injections into the dorsal right blastomere at the 4- cell stage results in diminishing of the overall tadpole size, especially of the forebrain and eye, compared to that of the left side (control). Overlay with the fluorescent image demonstrates the distribution of the co-injected tracer FLD. B. Tadpoles in which c-Answer was knocked out in 6th exon with CRISPR/Cas9 technology have smaller size, especially eyes, then the wild-type tadpoles at the same stage. B and D. Tail regeneration in tadpoles with c-answer MO2 knockdown or knockout in 6th exon, respectively, is inhibited compared to that of the wild-type control (see Figure 4). E. Tadpole in which c-Answer was knocked out in 6th exon with CRISPR/Cas9 technology have smaller size then the wild-type tadpoles at the same stage. F. Diagram showing the distribution of tail regeneration phenotypes in tadpoles injected with canswer MO2 or indicated components of the CRISPR/Cas9 system. Scale bars: A, B, D, E 500 µm; C 1mm
	Figure S6. Whole-mount in situ hybridization with probes to the transcripts of the indicated genes of embryos injected with c-answer MO1 or c-answer mRNA variants, Related to Figure 5. A. Inhibition of the pax6 expression on the right side of the middle neurula embryos injected with c-answer MO1 into the right dorsal blastomere at 4-cell stage. Left side is not injected control. A'. The fluorescent image for A demonstrating the distribution of green fluorescent tracer, FLD, co-injected with c-answer MO1. B. Ectopic pax6 expression in the middle neurula embryos injected with c-answer MO1 into 2 blastomeres at 2-cell stage. C. pax6 expression in the control (not injected) middle neurula embryos. D. Ectopic pax6 expression on the right side of the middle neurula embryos injected with deltaCc-answer mRNA into the right dorsal blastomere at 4-cell stage. Left side is not injected control. D'. The fluorescent image for D demonstrating the distribution of green fluorescent tracer, FLD, co-injected with deltaC-c-answer mRNA. E. No effect on xanf1 expression in the middle neurula embryos injected with c-answer MO1 into the left dorsal blastomere at 4-cell stage. E'. The fluorescent image for E demonstrating the distribution of green fluorescent tracer, FLD, co-injected with c-answer MO1. F. No effect on xanf1 expression in the middle neurula embryos injected with the control canswer misMO into the left dorsal blastomere at 4-cell stage. F'. The fluorescent image for F demonstrating the distribution of green fluorescent tracer, FLD, co-injected with c-answer misMO. G. No effect on six3 expression in the middle neurula embryos injected with c-answer MO1 into the right dorsal blastomere at 4-cell stage. G'. The fluorescent image for G demonstrating the distribution of green fluorescent tracer, FLD, co-injected with c-answer MO1. H. No effect on six3 expression in the middle neurula embryos injected with c-answer mRNA into 2 blastomeres at 2-cell stage. I. six3 expression in the control (not injected) middle neurula embryo. J. No effect on en1 expression in the middle neurula embryos injected with c-answer MO1 into 2 blastomeres at 2-cell stage. J can be referred to as not injected control. J'. The fluorescent image for H demonstrating the distribution of green fluorescent tracer, FLD, co-injected with c-answer MO1. K. Inhibition of the en1 expression in the middle neurula embryos injected with c-answer mRNA into 2 blastomeres at 2-cell stage. L. En1 expression in the control (not injected) middle neurula embryo. Scale bars 500 µm for all photos is shown on A.
	Figure S7. Phenotypes of tadpoles after c-answer knockout and overexpression of c-answer variants, Related to Figure 4 and Figure 5. A. Tadpoles with c-answer knockout in 2nd exon (right) have smaller eyes than the control ones (left). B and B'. Local injection of c-Answer MO mixed with FLD tracer in one blastomere, which give rise to the middle part of the cement gland, at 16-cell stage resulted in the inhibition of the cement gland differentiation in this part. The embryo at stage 26 is shown from the ventral side, anterior to the top. С. Tadpole overexpressing wild-type c-Answer has ectopic cement gland on the head process (Zerbino et al.). С'. Whole-mount in situ hybridization with the probe to foxg1 of the same tadpole as on C reveals additional telencephalon within the head process (arrow head). D. QRT-PCR analysis of c-answer mRNA in the tail tips of stage 46 tadpoles developed from the wild-type embryos and from embryos injected with this mRNA. E-G. Ectopic RPE differentiation and enlarged telencephalon on the injected side in tadpoles overexpressing wild-type c-Answer (arrows). Yellow bars indicate the telencephalon anterior and posterior borders. H. Ectopic eye in tadpole overexpressing wild-type c-Answer. Scale bars: A 250 µm; B-C', E-H 500 µm.

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