Espiritu EB, Crunk AE, Bais A, Hochbaum D, Cervino AS, Phua YL, Butterworth MB, Goto T, Ho J, Hukriede NA, Cirio MC.

P30 DK079307 NIDDK NIH HHS , R01 DK069403 NIDDK NIH HHS , R00 DK087922 NIDDK NIH HHS , T32 DK061296 NIDDK NIH HHS , R01 DK103776 NIDDK NIH HHS , R01 HD053287 NICHD NIH HHS , R01 DK102843 NIDDK NIH HHS

             miRNA binding

	Figure 1. The functional domains of Fry directly interact with constitutive active Ldb1-Lhx1. (a) TAP-LL-CA protein contains a calmodulin binding peptide c, a streptavidin binding peptide s, dimerization domain (DD) of Ldb1 protein, Ldb1-Chip conservative domain (LCCD), nuclear localization signal (dark gray bar), Lhx1 homeodomain (HD), and Lhx1 C-terminal conserved regions (1–5). (b) Full-length Xenopus Fryl and Fry proteins. Furry domain (FD, purple bar), two leucine-zipper motifs (gray bars) and a coiled-coil structure (red bar). The percentage of sequence similarity between different regions of these proteins is indicated. The FD + LZ domain fusion version of Fry contains the N-terminal FD domain and C-terminal domains (LZ). The aminoacid numbers of the original protein are indicated. (c) Western blot analysis of immunoprecipitated complexes of transfected HEK-293T cells. Cells were co-transfected with myc-LL-CA and HA-FD + LZ, immunoprecipitated (IP) and blotted (IB) as indicated. IP of untransfected cells and without cell lysate were used as controls for the assay. The red asterisks indicate the bands of interest.
	Figure 2. Fry is co-expressed with lhx1 in the intermediate mesoderm and pronephric kidney of Xenopus embryos. (a–m) Fry and lhx1 expression in Xenopus embryos. (a) Crossed section of a S10 embryo stained for lhx1. (b) Magnification of the region marked in a. (c,d) Transverse section of a S15 embryo with lhx1. (d) Magnification of the area marked in c. (e) Expression of lhx1 in a S18 embryo. (f) Crossed section of a S10 embryo stained for fry. (g) Magnification of the area marked in f. (h,i) Expression of fry in S15 embryos. (h) Transverse section. (i) Magnification of the marked area in h. (j) Expression of fry in a S18 embryo. (k–m) Expression of fry in tadpole stages. Involuting marginal zone (imz), lateral mesoderm (lm), pronephric anlage (pa), intermediate mesoderm (im), notochord (nc), somitic mesoderm (sm), pronephros (pr). Representative embryos are shown.
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, cross section, NF stage 10.
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, transverse section, NF stage 15, dorsal up.
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, transverse section, NF stage 18, dorsal up.
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35-36, lateral view, anterior left, dorsal up.
	Figure 3. The size of the kidney field is reduced in Fry-depleted embryos. (a–d) Pax8 expression in S21 (stage 21) embryos, lateral views. Arrow points to the otic vesicle. (a) Uninjected embryo. (b) Random-MO injected embryo. (c) Fry-MO injected embryo. (d) Fry-MO + FD + LZ mRNA (400 pg) coinjected embryo. (e) Percentage of embryos with reduced (RE), absent (AE) or not affected (NA) pax8 expression. Uninjected (N = 2, 35), Random-MO (N = 2, 23), Fry-MO 5 ng (N = 2, 18), 15 ng (N = 3, 53), 20 ng (N = 4, 70), Fry-MO 20 ng + FD + LZ mRNA 200 pg (N = 2, 39), Fry-MO 20 ng + FD + LZ mRNA 400 pg (N = 2, 51), FD + LZ mRNA 400 pg (N = 2, 46). Data on graph is presented as mean. (f–h) wt1 expression in S24 (stage 24) embryos. (f) Uninjected (n = 20), (g) control and (h) injected (n = 20) sides of the same embryo are shown. (i–k) 3G8 immunostaining. (i) Uninjected (n = 25), (j) control and (k) injected (n = 28) sides of the same embryo. Magnifications of the boxed areas are shown in each panel. (l–n) β1-NaK-ATPase expression in S39 (stage 39) embryos. (l) Uninjected (n = 22). (m) Control and (n) injected (n = 23) sides of the same embryo, arrow points to pronephros positive staining. Representative embryos are shown. Embryos at 8-cell were injected 1x V2 with 15 or 20 ng of the morpholino or as indicated.
	Figure 5. Synergistic interaction of Fry and Lhx1 to pattern the kidney field. (a–d) Pax8 expression in S21 (stage 21) embryos, lateral views. (e) Percentage of S21 embryos with abnormal pax8 expression under different treatments. Uninjected (N = 3, 41), Fry-MO (N = 3, 56), Lhx1-AS (N = 3, 65), Fry-MO + Lhx1-AS (N = 3, 75) (N = number of experiments, number of embryos). (f–i) 3G8 immunostaining of S32 (stage 32) embryos. (j) Percentage of S32 embryos with reduced or absent 3G8 staining. Uninjected (N = 3, 122), Fry-MO (N = 3, 88), Lhx1-AS (N = 3, 79), Fry-MO + Lhx1-AS (N = 3, 81). (k–n) β1-NaK-ATPase expression in S39 (stage 39) embryos. (o) Percentage of embryos with abnormal β1-NaK-ATPase expression. Uninjected (N = 6, 168), Fry-MO (N = 6, 165), Lhx1-AS (N = 6, 193), Fry-MO + Lhx1-AS (N = 6, 183). Reduced (RE), absent (AE) or not affected (NA) expression/staining. 8-cell embryos were injected 1x V2 with 2.5 ng of Fry-MO and/or 50 pg of Lhx1-AS. Representative embryos are shown. Data in the graphs is presented as means. Statistical significance was evaluated using Fisher’s exact test ****p < 0.0001.
	Figure 6. Synergistic interaction of Fry and Lhx1 in the paraxial mesoderm. (a–d) In situ hybridization of S21 (stage 21) embryos for myoD. Asterisk indicates the injected side of the embryo. (e–h) In situ hybridization of S15 embryos for aldh1a2. (a,e) Uninjected embryos. (i) Percentage of embryo with reduced (RE) or not affected (NA) myoD expression. Uninjected (N = 3, 85), Fry-MO (N = 3, 84), Lhx1-AS (N = 3, 78), Fry-MO + Lhx1-AS (N = 3, 73). (j) Percentage of embryo with reduced (RE) or not affected (NA) aldh1a2 expression. Uninjected (N = 3, 82), Fry-MO (N = 3, 86), Lhx1-AS (N = 3, 88), Fry-MO + Lhx1-AS (N = 3, 84) (N = number of experiments, number of embryos). Embryos at 8-cell were injected 1x V2 with 2.5 ng of Fry-MO and/or 50 pg of Lhx1-AS as indicated on each panel. Representative embryos are shown. Data in graphs is presented as means. Statistical significance was evaluated using Fisher’s exact test ****p < 0.0001.
	Figure 7. miRNA deep sequencing of Fry- and Lhx1-depleted embryos. (a) Experimental procedure followed to generate the samples for miRNA deep sequencing. Both dorsal blastomeres of 4-cell embryos were injected. Dorsal halves were isolated and processed for miRNA deep sequencing. (b) Selected nine miRNA clusters with increased levels of their respective miRs upon either lhx1 or fry depletion. The values are the normalized number of counts for each miR.
	Figure 8. Validation of miRNA deep sequencing data by real-time qRT-PCR. (a) Schematic of the miR-199a/214 and miR-23b/27b/24a clusters in the X. tropicalis genome. The Xenopus tropicalis chromosome (Xtr) where each cluster is located is indicated. The miR-199a/214 cluster is located within intron 14 of the dynamin3 gene and has a length of ~6 kb while the miR-23b/27b/24a cluster is intergenic and has a length of ~1 kb. The grey colored rectangles indicate the position of the mature miRNAs within the pre-miRNA structures. Arrows indicate transcription direction. (b) Expression levels of miRs within the miR-199a/214 and miR-23b/27b/24a clusters were determined in Lhx1- and/or Fry-depleted embryos. 8-cell embryos were injected 2x V2 with 2.5 ng of Fry-MO and/or 50 pg of Lhx1-AS. Error bars indicate standard deviation derived from three repeats of the PCR reactions with different biological samples. Statistical significant differences were determined by a one-tailed paired t-test. n.s., non-significant; *p < 0.05; **p < 0.01.
	Figure 9. Overexpression of miR-199a/214 and miR-23b/27b reduce the kidney field size. (a–i) Pax8 expression in S21 (stage 21) embryos injected with LNA mimics. (a,b,f and g) Injected with LNA control. (a,f) Uninjected side (ctrl). (b,g) Injected side with LNA control. (c,d) Embryo injected with LNA miR-199a + miR214. Uninjected (c) and injected (d) sides of the same embryo. (h,i) Embryos injected with LNA miR-27b + miR23b. Uninjected (h) and injected (i) sides of the same embryo. (e) Percentage of embryos with abnormal pax8 expression field. Uninjected (N = 3, 63); LNA ctrl. (N = 3, 64); LNA miR-199a + miR-214 (N = 3, 76, 49% reduced or absent expression) (N = number of experiments, number of embryos, % of affected embryos). (j) Percentage of embryos with abnormal pax8 expression field. Uninjected (N = 3, 66); LNA ctrl. (N = 3, 65); LNA miR-27b + miR-23b (N = 3, 59, 64% reduced or absent expression). Reduced (RE), absent (AE) or not affected (NA) field of expression. Data in graphs is presented as means. ****p < 0.0001, **p < 0.01 Fisher’s exact test
	Supplementary Figure S1. Fry-like interacts with the constitutive active Ldb1-Lhx1 complex. Western blot analysis of total nuclear extracts or immunoprecipitated complexes of transfected HEK-293T cells. Cells were transfected either with myc-V or myc-LL-CA, immunoprecipitated (IP) with anti-myc antibody and blotted (IB) either with anti-myc or FRYL antibody. * This band might be a product of degradation of myc-LL- CA.
	Supplementary Figure S2. In vitro binding assay of the indicated GST-fusion proteins with in vitro translated FD+LZ (35S)methionine of Fry. TAP-LL-∆C lacks the C-terminal conserved regions 2-5 of Lhx1. Coomassie brilliant blue (CBB) staining is shown as input control for the GST-fusion proteins.
	Supplementary Figure S3. Fryl expression in Xenopus embryos. (a, c) Expression of fryl in S10 (stage 10) and S15 embryos (stage 15). (b, d) Crossed section of S10 and S15 embryos stained for fryl. (e-g) Expression of fryl in late neurula S25 and tailbud stages S29/30 and S35/36. Notochord (nc) and somites (s).
	Supplementary Figure S4. Induction of axis development defects by depletion of Fry and Lhx1. (a) Representation of the experimental procedure. Both dorsal blastomeres of the 4-cell embryos were injected. (b) Percentage of embryos at S33/34 (stage 33/34) with a shortened axis and/or headless phenotype. Uninjected (N=3, 36), Fry-MO 10 ng (N=4, 110), Fry-MO 20 ng (N=6, 148), Lhx1-AS 100 pg (N=3, 59), Lhx1-AS 200 pg (N=1, 12), Lhx1-AS 400 pg (N=3, 29). (c-e) Representative embryos of the selected doses of Fry-MO and Lhx1-AS are shown.
	Supplementary Figure. S5. Overexpression of miR-199a/214 and miR-23b/27b reduce the kidney field size. (a-d, f-i) β1-NaK-ATPase expression in S21 (stage 21) embryos injected with LNA mimics. (a, b, f and g) Embryos injected with LNA control. (a and f) Uninjected side (ctrl). (b and g) Injected side with LNA control. (c and d) Injected side with LNA miR-199a + miR-214. Uninjected (c) and injected (d) sides of the same embryo. (h and i) Embryos injected with LNA miR-27b + miR-23b. Uninjected (h) and injected (i) sides of the same embryo. (e) Percentage of embryos with abnormal β1- NaK-ATPase expression. Uninjected (N=3, 70); LNA ctrl. (N=3, 74); LNA miR-199a + miR-214 (N=3, 92, 39% reduced or absent expression). (j) Percentage of embryos with abnormal β1-NaK-ATPase expression. Uninjected (N=3, 96); LNA ctrl. (N=3, 66); LNA miR-27b + miR-23b (N=3, 63, 46% reduced or absent expression). Reduced (RE), absent (AE) or not affected (NA) field of expression. Data in graphs is presented as means. **** p<0.0001, * p<0.05 Fisher’s exact test.
	Supplementary Figure S6. Overexpression of miR-199a/214 and miR-23b/27b within a V2 blastomere have no significant effects on myoD and aldh1a2 expression. (a-c) In situ hybridization of S21 (stage 21) embryos for myoD. Asterisk indicates the injected side of the embryo. (d-f) In situ hybridization of S15 (stage 15) embryos for aldh1a2. (g) Percentage of embryo with reduced (RE) or not affected (NA) myoD expression. Uninjected (N=2, 50); LNA ctrl. (N=2, 40); LNA miR-199a + miR-214 (N=2, 52); LNA miR-27b + miR-23b (N=2, 45, 8% reduced expression). (h) Percentage of embryos with reduced (RE) or not affected (NA) aldh1a2 expression. Uninjected (N=2, 48); LNA ctrl. (N=2, 44, 2% reduced expression); LNA miR-199a + miR-214 (N=2, 65, 3% reduced expression); LNA miR-27b + miR-23b (N=2, 52).
	Figure 4. Expression of paraxial mesoderm marker genes is reduced in Fry-depleted embryos. (a–c) In situ hybridization of S15 (stage 15) embryos for myoD. (g) Percentage of embryos with reduced (RE) or not affected (NA) myoD expression field revealed by in situ hybridization. Uninjected (N = 3, 92), Random-MO (N = 2, 36), Fry-MO (N = 4, 85). (d–f) In situ hybridization of S15 embryos for aldh1a2. (h) Percentage of embryos with reduced (RE) or not affected (NA) aldh1a2 expression field revealed by in situ hybridization. Uninjected (N = 3, 75), Random-MO (N = 2, 36), Fry-MO (N = 3, 76) (N = number of experiments, number of embryos). As indicated on each panel, 8-cell embryos were injected 1x V2 with 15 ng of the MOs or left uninjected. Asterisk indicates the injected side of the embryo. Representative embryos are shown. Data in graphs is presented as means. Statistical significance was evaluated using Fisher’s exact test ***p < 0.0001.

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