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

	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.
	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 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.
	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.

Agrawal, The miR-30 miRNA family regulates Xenopus pronephros development and targets the transcription factor Xlim1/Lhx1. 2009, Pubmed

Agrawal, The miR-30 miRNA family regulates Xenopus pronephros development and targets the transcription factor Xlim1/Lhx1. 2009, Pubmed
Agulnick, Interactions of the LIM-domain-binding factor Ldb1 with LIM homeodomain proteins. 1996, Pubmed
Barnes, Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis. 1994, Pubmed
Bouchard, Nephric lineage specification by Pax2 and Pax8. 2002, Pubmed
Breving, The complexities of microRNA regulation: mirandering around the rules. 2010, Pubmed
Byun, Fryl deficiency is associated with defective kidney development and function in mice. 2018, Pubmed
Carroll, Wilms' tumor suppressor gene is involved in the development of disparate kidney forms: evidence from expression in the Xenopus pronephros. 1996, Pubmed
Carroll, Synergism between Pax-8 and lim-1 in embryonic kidney development. 1999, Pubmed
Cartry, Retinoic acid signalling is required for specification of pronephric cell fate. 2006, Pubmed
Cebrian, The number of fetal nephron progenitor cells limits ureteric branching and adult nephron endowment. 2014, Pubmed
Chen, Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos. 2001, Pubmed
Chen, MiR-23b controls TGF-β1 induced airway smooth muscle cell proliferation via direct targeting of Smad3. 2017, Pubmed
Chen, Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development. 2002, Pubmed
Chiba, MST2- and Furry-mediated activation of NDR1 kinase is critical for precise alignment of mitotic chromosomes. 2009, Pubmed
Chu, Dicer function is required in the metanephric mesenchyme for early kidney development. 2014, Pubmed
Cirio, Lhx1 is required for specification of the renal progenitor cell field. 2011, Pubmed
Cong, The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis. 2001, Pubmed
Costello, Lhx1 functions together with Otx2, Foxa2, and Ldb1 to govern anterior mesendoderm, node, and midline development. 2015, Pubmed
Dagle, Oligonucleotide-based strategies to reduce gene expression. 2001, Pubmed
Dawid, LIM domains: multiple roles as adapters and functional modifiers in protein interactions. 1998, Pubmed
DeLay, Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9. 2018, Pubmed
Denby, MicroRNA-214 antagonism protects against renal fibrosis. 2014, Pubmed
Desvignes, Evolution of the miR199-214 cluster and vertebrate skeletal development. 2014, Pubmed
Diep, Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. 2011, Pubmed
Emoto, Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons. 2004, Pubmed
Enkhmandakh, The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer. 2006, Pubmed
Flynt, Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. 2007, Pubmed
Gawantka, Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning. 1998, Pubmed
Gentsch, Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus. 2018, Pubmed
Gomez, MicroRNAs as novel therapeutic targets to treat kidney injury and fibrosis. 2016, Pubmed
Goto, Xenopus furry contributes to release of microRNA gene silencing. 2010, Pubmed
Griffiths-Jones, miRBase: tools for microRNA genomics. 2008, Pubmed
Hayette, AF4p12, a human homologue to the furry gene of Drosophila, as a novel MLL fusion partner. 2005, Pubmed
Hiratani, Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos. 2001, Pubmed
Ho, The pro-apoptotic protein Bim is a microRNA target in kidney progenitors. 2011, Pubmed
Hopwood, MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. 1989, Pubmed
Hukriede, Conserved requirement of Lim1 function for cell movements during gastrulation. 2003, Pubmed
Huttlin, Architecture of the human interactome defines protein communities and disease networks. 2017, Pubmed
Ikeda, Furry protein promotes aurora A-mediated Polo-like kinase 1 activation. 2012, Pubmed
Juan, Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. 2009, Pubmed
Kitamoto, Temporal regulation of global gene expression and cellular morphology in Xenopus kidney cells in response to clinorotation. 2005, Pubmed
Klockenbusch, Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1. 2010, Pubmed
Kodjabachian, A study of Xlim1 function in the Spemann-Mangold organizer. 2001, Pubmed
Li, MicroRNA filters Hox temporal transcription noise to confer boundary formation in the spinal cord. 2017, Pubmed
Marrone, MicroRNA-17~92 is required for nephrogenesis and renal function. 2014, Pubmed
Martello, MicroRNA control of Nodal signalling. 2007, Pubmed
Mathelier, Large scale chromosomal mapping of human microRNA structural clusters. 2013, Pubmed
Matthews, LIM-domain-binding protein 1: a multifunctional cofactor that interacts with diverse proteins. 2003, Pubmed
Mauch, Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm. 2000, Pubmed
Mitchell, Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm. 2007, Pubmed
Nagai, Furry promotes acetylation of microtubules in the mitotic spindle by inhibition of SIRT2 tubulin deacetylase. 2013, Pubmed
Nagai, Multifaceted roles of Furry proteins in invertebrates and vertebrates. 2014, Pubmed
Nagalakshmi, Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney. 2011, Pubmed
Nakagawa, Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis. 2015, Pubmed
Perkins, Transport properties of toad kidney epithelia in culture. 1981, Pubmed
Quinlan, A common variant of the PAX2 gene is associated with reduced newborn kidney size. 2007, Pubmed
Rogler, Knockdown of miR-23, miR-27, and miR-24 Alters Fetal Liver Development and Blocks Fibrosis in Mice. 2017, Pubmed
Romaker, MicroRNAs are critical regulators of tuberous sclerosis complex and mTORC1 activity in the size control of the Xenopus kidney. 2014, Pubmed
Schuchardt, Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development. 1996, Pubmed
Seufert, Developmental basis of pronephric defects in Xenopus body plan phenotypes. 1999, Pubmed
Shawlot, Requirement for Lim1 in head-organizer function. 1995, Pubmed
Sudou, Dynamic in vivo binding of transcription factors to cis-regulatory modules of cer and gsc in the stepwise formation of the Spemann-Mangold organizer. 2012, Pubmed
Swanhart, Characterization of an lhx1a transgenic reporter in zebrafish. 2010, Pubmed
Taira, The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos. 1992, Pubmed
Taira, Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus. 1994, Pubmed
Tang, Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation. 2008, Pubmed
Thaler, LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. 2002, Pubmed
Thiagarajan, Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling. 2011, Pubmed
Tsang, Lim1 activity is required for intermediate mesoderm differentiation in the mouse embryo. 2000, Pubmed
Weber, SIX2 and BMP4 mutations associate with anomalous kidney development. 2008, Pubmed
Wessely, MicroRNAs in kidney development: lessons from the frog. 2010, Pubmed
Yasuoka, Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification. 2014, Pubmed
Yatim, NOTCH1 nuclear interactome reveals key regulators of its transcriptional activity and oncogenic function. 2012, Pubmed