Sall1 Maintains Nephron Progenitors and Nascent Nephrons by Acting As Both an Activator and a Repressor

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Sall1 Maintains Nephron Progenitors and Nascent Nephrons by Acting as Both an Activator and a Repressor

Shoichiro Kanda,* Shunsuke Tanigawa,* Tomoko Ohmori,* Atsuhiro Taguchi,* Kuniko Kudo,* † ‡ | † Yutaka Suzuki, Yuki Sato, Shinjiro Hino,§ Maike Sander, Alan O. Perantoni,¶ Sumio Sugano, Mitsuyoshi Nakao,§** and Ryuichi Nishinakamura***

Departments of *Kidney Development and §Medical Cell Biology, Institute of Molecular Embryology and Genetics, and ‡Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto, Japan; †Department of Medical Genome Sciences, University of Tokyo, Tokyo, Japan; |Departments of Pediatrics and Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California; ¶Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland; and **CREST, Japan Science and Technology Agency, Saitama, Japan

ABSTRACT The balanced self-renewal and differentiation of nephron progenitors are critical for kidney development and controlled, in part, by the transcription factor Six2, which antagonizes canonical Wnt signaling- mediated differentiation. A nuclear factor, Sall1, is expressed in Six2-positive progenitors as well as differentiating nascent nephrons, and it is essential for kidney formation. However, the molecular functions and targets of Sall1, especially the functions and targets in the nephron progenitors, remain unknown. Here, we report that Sall1 deletion in Six2-positive nephron progenitors results in severe progenitor depletion and apoptosis of the differentiating nephrons in mice. Analysis of mice with an inducible Sall1 deletion revealed that Sall1 activates genes expressed in progenitors while repressing genes expressed in differentiating nephrons. Sall1 and Six2 co-occupied many progenitor-related gene loci, and Sall1 bound to Six2 biochemically. In contrast, Sall1 did not bind to the Wnt4 locus suppressed by Six2. Sall1-mediated repression was also independent of its binding to DNA. Thus, Sall1 maintains nephron progenitors and their derivatives by a unique mechanism, which partly overlaps but is distinct from that of Six2: Sall1 activates progenitor-related genes in Six2-positive nephron progenitors and represses gene expression in Six2-negative differentiating nascent nephrons.

J Am Soc Nephrol 25: 2584–2595, 2014. doi: 10.1681/ASN.2013080896

The nephron is a basic functional unit of the kidney, ureteric bud-derived Wnt9b, thereby maintaining which includes the glomerulus, proximal and distal the self-renewal of nephron progenitors.2–4 How- renal tubules, and the loop of Henle. The mamma- ever, the progenitors gradually lose Six2 expression lian kidney, the metanephros, is formed by recipro- cally inductive interactions between two precursor tissues, namely the metanephric mesenchyme and Received August 27, 2013. Accepted February 26, 2014. the ureteric bud. The mesenchyme contains neph- S.K., S.T., and T.O. contributed equally to this work. ron progenitors that express a transcription factor, Published online ahead of print. Publication date available at Six2. When Six2-positive cells are labeled using www.jasn.org. Six2GFPCre, a mouse strain expressing Cre recom- ﬂ Correspondence: Prof. Ryuichi Nishinakamura, Department of binase fused to green uorescent protein (GFP) un- Kidney Development, Institute of Molecular Embryology and der the control of the Six2 promoter, they give rise to Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860- nephron epithelia in vivo.1 Six2 opposes the canon- 0811, Japan. Email: [email protected] ical Wnt-mediated differentiation evoked by Copyright © 2014 by the American Society of Nephrology

2584 ISSN : 1046-6673/2511-2584 JAmSocNephrol25: 2584–2595, 2014 www.jasn.org BASIC RESEARCH and start to differentiate. These differentiating cells express D). Six2-positive nephron progenitors were undetectable (Fig- Wnt4, which further enhances the differentiation. Six2 binds ure 1, E and F), and development of the nephron components, to the Wnt4 enhancer to ensure that only a subset of progen- including glomerular podocytes, proximal renal tubules, the itors differentiate at each time point. Through this balance loop of Henle, and distal renal tubules, was significantly im- between self-renewal and differentiation, progenitors sequen- paired (Figure 1, G–N). tially transit to pretubular aggregates, renal vesicles, and C- and Sall1 mutant kidneys were already smaller than controls at S-shaped bodies, which eventually develop into nephron epi- E14.5 (Figure 2, A and B). Six2 was still expressed in the Sall1 thelia. mutants, but the number of Six2-positive cells was signifi- spalt (sal)wasfirst isolated from Drosophila as a region- cantly less (Figure 2, C and D). Sall1 was expressed in not specific homeotic gene, and it encodes a nuclear protein char- only the Six2-positive nephron progenitor region but also, acterized by multiple double zinc finger motifs.5 Humans and the differentiating nephrons located deeper inside of the kid- mice each have four known sal-like genes (known as SALL1–4 ney (Figure 2E). Sall1 expression in both populations was sig- in humans and Sall1–4 in mice). Mutations in SALL1 and nificantly less in Sall1 mutants, although expression in the SALL4 have been associated with Townes–Brocks and Okihiro stroma, the outermost layer of the kidney, was not affected syndromes, respectively, both of which are autosomal domi- (Figure 2F, asterisk). This finding reflects the spatially re- nant diseases that involve abnormalities in various organs, in- stricted activity of Cre recombinase in Six2GFPCre mice. cluding ears, limbs, heart, and kidneys.6,7 We have shown that We next isolated kidneys at E12.5 and cultured them for 3 Sall1 is expressed in the metanephric mesenchyme and that days in vitro.BecauseSix2GFPCre mice also express GFP Sall1 knockout mice exhibit kidney agenesis resulting from driven by the Six2 promoter, Six2-positive nephron progeni- failureofuretericbudattractiontothemesenchymeat tors could be monitored by time-lapse confocal microscopy. day 11.5 of gestation (E11.5).8 However, Sall1 should have Sall1 mutants showed a comparable GFP signal at the begin- additional roles, because it continues to be expressed in the ning of the culture (Figure 2, G and H). During the culture, the metanephric mesenchyme after ureteric bud invasion. We pre- nephron progenitors in the Six2GFPCre kidney rapidly viously showed the presence of nephron progenitors in Sall1-positive mesenchyme by establishing a novel colony assay.9 Because Six2 is expressed in Sall1-high mesenchymal cells, the Sall1-high and Six2-positive mesenchyme represents a nephron progenitor population in the embryonic kidney.10 However, the role of Sall1 in the progenitors remains unknown. There- fore, we generated mice lacking Sall1 in Six2- positive progenitors and their derivatives and found that Sall1 is, indeed, essential for maintenance of these populations.

RESULTS

Sall1 Deletion Causes Depletion of Nephron Progenitors Accompanied by Reduction of Nephron Structures To gain insights into the roles of Sall1 in nephron progenitors, we crossed the floxed allele of Sall1 with Six2GFPCre BAC transgenic mice expressing a fusion protein of Sall1 GFP and Cre recombinase in the progeni- Figure 1. deletion depletes nephron progenitors and their derivatives. (A) 1 Kidneys in a newborn control mouse (P0). ad, Adrenal gland; bl, bladder; kid, kidney; tor population. Six2GFPCre;floxed Sall1 flox/flox ov, ovary. (B) Kidney size is reduced in a newborn Six2GFPCre;Sall1 mouse (P0). mice were born at Mendelian frequency, te, Testis. (C and D) Hematoxylin-eosin staining of newborn kidneys. Severe dys- flox/flox but all of them died shortly after birth genesis is observed in the Six2GFPCre;Sall1 mouse kidney. Scale bar, 100 mm. with abnormally small kidneys (Figure 1, (E–N) Immunostaining for Six2 (nephron progenitor), Wt1 (nephron progenitor and A and B). The mutant kidneys contained podocyte), LTL (proximal renal tubule), THP (the loop of Henle), and NCC (distal renal multiple glomerular cysts, dilated renal tu- tubule). Development of the nephron components is significantly impaired in the flox/flox bules, and thin cortexes (Figure 1, C and Six2GFPCre;Sall1 mouse kidney.

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expanded around the branching ureteric bud tips (Figure 2I, Supplemental Video 1). However, in the Sall1 mutants, the signal became almost undetectable within 48 hours of culture (Figure 2J, Supplemental Video 2). We also conﬁrmed the reduction of GFP-positive progenitors by FACS analysis (Figure 2, K and L). Thus, progenitor depletion occurs between E12.5 and E14.5 in the absence of Sall1. Cell cycle analysis of the GFP-positive populations did not show any signiﬁcant differences (Figure 2, M and N), indicating that progenitor depletion is unlikely caused by proliferation defects.

Sall1 Deletion Impairs Self-Renewal of Nephron Progenitors and Induces Apoptosis in the Differentiating Nephrons We then used lineage tracing to examine the fate of Sall1 mutant nephron progenitors. We crossed Six2GFPCre mice with a re- porter strain, in which the CAG promoter, floxed stop sequences, and the tandem dimer Tomato coding sequence have been in- serted into the Rosa26 locus.11 As reported previously,1 Six2-positive progenitors gave rise to most components of the nephron (Figure 3A). In contrast, the differentiation of nephrons was severely impaired in Sall1 mutants at birth (Figure 3B), most pro- foundly in the proximal tubules and the loop of Henle. At E14.5 in the control, a significant portion of progenitor descendants was still located in the Six2-positive region Figure 2. Nephron progenitors are depleted in midgestation embryos. (A and B) where they were originated (arrowheads in m Hematoxylin-eosin staining of E14.5 kidneys. Scale bar, 100 m. (C and D) Im- Figure 3, C and C9), whereas the remaining munostaining for Six2. There are fewer Six2-positive cells relative to controls. (E and F) cells had joined the neural cell adhesion mol- Immunostaining for Sall1. There is much less expression of Sall1 in the mutant nephron components. *Sall1 in the stroma remains expressed. (G–J) Expansion of nephron ecule (NCAM)-positive differentiating pop- 9 progenitors in organ culture. (G and H) Kidneys at the beginning of the culture (E12.5). ulation (arrows in Figure 3, C and C ). In the (I and J) Kidneys after 2 days of the culture. The GFP signal in the mutant kidney becomes Sall1 mutants, the number of labeled cells weaker during the course of the culture. Time-lapse videos are shown in Supplemental located in the Six2-positive region was sig- flox/flox Video 1 (Six2GFPCre) and Supplemental Video 2 (Six2GFPCre;Sall1 ). Scale bar, 100 nificantly smaller (arrowheads in Figure 3, D mm. (K and L) FACS analysis of the GFP-positive cells. Kidneys were isolated at E12.5 and and D9), suggesting that the self-renewal ca- cultured for 2 days. The proportion of GFP-positive progenitors is significantly less in the pacity of Sall1 mutant progenitors is im- mutant. Average (SD) of three samples. A, area; FSC, forward scatter; PI, propidium iodide. paired. There was a small contribution of (M and N) Cell cycle analysis of GFP-positive cells. There is no difference between the labeled cells to the differentiating nephrons, Sall1 mutant and the control. Average (SD) of three samples. (O–T) Dual immunostaining flox/flox flox/flox but the morphology of these structures was of Sall1 and Six2 in Sall1 and Six2GFPCre;Sall1 kidneys at E13.5. Sall1 ex- different from the well organized S-shaped pression (red) is reduced in some of the nephron progenitors (arrowheads) and differentiating nephrons (arrows). The expression of Sall1 is not affected in the stroma (*). There bodies seen in the controls (arrows in Figure 9 9 are fewer Six2-positive cells (green), but most of them are negative for Sall1. ub, Ureteric 3, C and D ). Nevertheless, cell fates were bud. Scale bar, 40 mm. restricted to the NCAM-positive nephron lineages, and no signs of transdifferentiation to other lineages were detected (Figure 3D). At E13.5, we detected apoptotic cells in the

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also undetectable (Figure 3, G and H), which is consistent with Figure 2N. Considering the absence of apparent apoptosis, proliferation defects, or aberrant lineage conversion in Sall1- deiﬁcient nephron progenitors, depletion of these progenitors could result from the skewed balance to differentiation versus self-renewal, which is followed by apoptosis in the differentiating nephrons.

Inducible Sall1 Deletion Phenocopies the Conditional Sall1 Mutant Cre activity in the Six2GFPCre mice is mosaic, and it takes several days to completely delete Sall1 in the entire progenitor- derived populations.1 Dual immunostaining for Sall1 and Six2 at E13.5 showed that Six2 was retained in the Sall1-null cells (Figure 2, O–T), suggesting that Sall1 reduction does not lead to an immediate loss of Six2. We intermittently observed re- sidual nephron formation in mutant mice, but these structures were always positive for Sall1 (Supplemental Figure 1A), indicating that only escapers from Six2GFPCre-mediated deletion can form nephrons. To identify the direct molecular events downstream of Sall1, we analyzed inducible Sall1 mutants fl (Sall1CreER/ ox). On tamoxifen treatment at E12.5, this mutant strain had smaller kidneys at birth, which we reported previously.12 We found that Six2-positive nephron progenitors were lost in the newborn mutant (Figure 4, A and B), and the development of most nephron components was significantly impaired (Figure 4, C–H). The differentiating nephrons ex- hibited apoptosis at E14.5 (Figure 4, I–K). Thus, the inducible Sall1 mutant strain phenocopied the Six2GFPCre-dependent Sall1 deletion. Sall1 expression was already reduced at E13.5, 1 day after tamoxifen treatment, whereas Six2 was retained in the Sall1-null cells (Figure 4, L–Q), which is consistent with the results shown in Figure 2, O–T. At E14.5, Six2 expression still remained (Figure 4, R–U). Although FGF9 and FGF20 maintain nephron progenitors,13 Etv4, with expression in the mesenchyme that correlates with FGF activity,14,15 showed normal Figure 3. Sall1 deletion impairs self-renewal of nephron progeni- expression (Supplemental Figure 1, B and C). Furthermore, ad- flox/flox tors and induces apoptosis in differentiating nephrons. Lineage dition of FGF2 to the Six2GFPCre;Sall1 kidney in organ trace analysis of the nephron progenitors. Scale bar , 100 mm. (A culture did not rescue the phenotypes (Supplemental Figure 1, D and B) Tandem dimer Tomato (tdTomato) is stained by the anti–red and E). Lef1 is an indicator of Wnt activity, and it is normally fluorescent protein antibody (blue). The development of nephrons, expressed in differentiating nephrons but not progenitors.15 especially proximal tubules (*) and the loop of Henle (**), is severely There was no upregulation of Lef1 in the progenitor regions 2 Sall1 impaired in the mutant at P0. (C and D) Immunostaining days after tamoxifen treatment (Supplemental Figure 1, F and at E14.5 shows the reduction of tdTomato-positive (red) nephron G). In addition, expression patterns of Wnt9b (in the ureteric progenitors (arrowheads) and the NCAM/tdTomato-positive 9 9 bud stalk) and Wnt11 (in the ureteric bud tip) were not signif- (yellow) differentiating nephrons (arrows). C and D show higher – magnification. (E and F) Immunostaining for cleaved caspase 3 icantly altered (Supplemental Figure 1, H K). In contrast, Six2- fi (brown) at E13.5. Apoptotic cells are detected in the differentiating positive progenitors were signi cantly reduced at E15.5, and nephrons of the Sall1 mutants. (G and H) Immunostaining for many nascent nephrons were formed simultaneously (Figure phosphohistone-H3 (PHH3 [brown]). Proliferation defects are not 4, V, V9,W,andW9). These renal vesicles were aberrantly large observed in the Sall1 mutant. ub, ureteric bud. considering the reduced progenitor population. Therefore, Sall1 deletion is likely to accelerate premature differentiation that leads to the progenitor depletion. Concomitant apoptosis in the dif- Sall1-deficient differentiating nephrons (arrows in Figure 3, ferentiating nephrons could cause the nephron loss at birth. E and F). Apoptosis in the progenitors was less prominent (ar- However, ectopic Lef1 expression was not observed in the pro- rowheads in Figure 3, E and F), and proliferation defects were genitors (arrowheads in Figure 4, X and Y), suggesting that the

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Figure 4. Inducible Sall1 deletion phenocopies the conditional Sall1 mutant. Tamoxifen was injected at E12.5 and analyzed later. (A–H) Immunostaining for Six2 (nephron progenitor), WT1 (nephron progenitor and podocyte), LTL (proximal renal tubule), and THP (the loop CreER/flox of Henle) at P0. Development of the nephron components is significantly impaired in the Sall1 mouse kidney. Scale bar, 100 mm. (I–K) Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (red) and NCAM (green) staining at E14.5. (J and K) Apoptotic cells are detected in differentiating nephrons in the mutant. (K) Apoptosis is more prominent in the differentiating nephrons located deeper inside of the kidney. Arrows, differentiating nephrons; arrowheads, nephron progenitors. Scale bar, 40 mm. (L–Q) Dual immunostaining for Sall1 and Six2 at E13.5. Sall1 expression (red) is reduced in some of the nephron progenitors (arrowheads) and differentiating nephrons (arrows). Most of the Six2-positive cells (green) are negative for Sall1 in the mutant. *Stroma. Scale bar, 40 mm. (R–U) Immunostaining for Sall1 and Six2 at E14.5. Scale bar, 100 mm. (V and W) Immunostaining for Six2 at E15.5. Note that the differentiating nephrons (renal vesicles; arrows) were aberrantly large considering the reduced progenitors (arrowheads) in the Sall1 mutant. Six2 was slightly overstained, and therefore, the nascent nephrons expressing Six2 weakly were detectable. Scale bar, 40 mm. V9 and W9 show higher magnification. (X and Y) Immunostaining for Lef1 (Wnt activity) at E15.5. Lef1 is +/flox expressed in the differentiating nephrons (arrows) and excluded from the progenitors (arrowheads; dotted regions) in both Sall1 CreER/flox and Sall1 kidneys. Scale bar, 40 mm. ub, ureteric bud.

premature differentiation is not caused by the Six2/Wnt-medi- contrast to the gradual Six2GFPCre-mediated deletion, al- ated mechanism. though it is formally possible that Sall1 in the stroma could It is noteworthy that Sall1 expression in the inducible strain also play a role. was reduced in not only nephron progenitor-derived lineages but also, the cortical stroma (compare Figure 4Q with Figure Sall1 Is an Activator in Nephron Progenitors and a 2T). However, the phenotype similarities of the two mutant Repressor in Differentiating Nephrons strains indicate that Sall1 expressed in the nephron lineage To identify downstream targets of Sall1, we performed micro- has a major role in kidney development, at least until birth. array analysis using the kidneys of inducible Sall1 mutant mice, The large renal vesicles in the inducible strain are likely to along with controls, harvested 24 and 48 hours after tamoxifen result from the simultaneous Sall1 deletion, which is in treatment at E12.5. We also performed microarray analysis

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fl fl using kidneys from Six2GFPCre;Sall1 ox/ ox mice and controls confirmed the Sall1 binding by ChIP-quantitative PCR (Sup- at E14.5. We then compared gene expression among the four plemental Figure 3C). genotypes and picked up genes that overlapped in all these Co-occupancy of Sall1 and Six2 was mainly limited to comparisons; 31 genes (41 probes) were downregulated im- progenitor-related genes and not detectable at the gene loci mediately on Sall1 deletion (Figure 5A, Supplemental Table 1). related to differentiation, extracellular matrix, or ureteric bud We further performed microarray analysis using sorted attraction (Supplemental Table 3). Sall1 binding sites signifi- Six2GFP-positive progenitors from embryonic kidneys and cantly overlapped within 500 bp from the Six2 binding sites found that 25 of 31 genes (31 of 41 probes) were more abun- (Figure 6B). The Six2-bound regions were similar to those dantly expressed in the Six2-positive progenitors, indicating regions reported by Park et al.,3 including Six2 and Wnt4 en- that Sall1 positively regulates these progenitor-related genes. hancers (Figure 6, Supplemental Tables 1 and 3). Extracted This list includes Cited1, Osr1,andRobo2, which are expressed Six2 binding consensus sequences were also identical in the Six2-positive domains and have important roles in kid- (GNAACNNNANNC). In contrast, Sall1 binding consensus ney development,16–19 and also, heretofore unappreciated sequences were enriched with A and T (Figure 6C), which is genes, such as Megf9. Quantitative RT-PCR and histologic consistent with previous reports, including our own work.20,21 analysis confirmed marked reduction of Cited1 and a slight Furthermore, we confirmed the binding of Sall1 to the loci decrease of Osr1 (Figure 5, B–E, Supplemental Figure 1L). mentioned above by an electrophoretic mobility shift assay The microarray analysis also identified 56 genes (72 probes) (EMSA) (Figure 7A). In addition, the immunoprecipitation that were upregulated immediately on Sall1 deletion (Figure assay using Sall1Flag embryonic kidneys as well as overex- 5A, Supplemental Table 2). Interestingly, 47 of 56 genes (56 of pression analysis showed that Sall1 bound to Six2 (Figure 7, B 72 probes) were more abundantly expressed in the Six2- and C). This interaction was still observed in the presence of negative population. Because Sall1 is expressed in not only deoxyribonuclease and ribonuclease and confirmed by the re- Six2-positive progenitors but also, the Six2-negative differenti- combinant proteins generated in vitro (Figure 7, C and D). ating nephrons, Sall1 may negatively regulate a subset of genes in Thus, these two proteins bind to each other directly. the latter population. This gene list included Nkx6.1, with func- It is reported that Six2 binds to differentiation-related gene tion in the kidney that has not been identified. The significant loci, such as Wnt4, Fgf8,andBmp7, and suppresses these genes increase of this gene in the Sall1 mutants was confirmed by in the nephron progenitors.3 However, we did not detect any quantitative RT-PCR (Supplemental Figure 1L). Immunostain- Sall1 binding throughout a few hundred kilobases of these loci ing showed that Nkx6.1 was, indeed, increased in the differen- (Figure 6D, Supplemental Figure 2C), indicating that Sall1 tiating nephrons of the inducible Sall1 mutants, whereas it was functions independently of the inhibitory roles of Six2. expressed only weakly in the control (Figure 5, F and G). This Thus, the cooperation between Sall1 and Six2 could be limited increase was also confirmed in the Six2GFPCre-dependent Sall1 to the progenitors. deletion (Figure 5, H and I); therefore, Sall1 could function as a Sall1 binding was also undetectable in most of the dere- negative regulator in the differentiating nephrons, while func- pressed loci, including the Nkx6.1 locus (Figure 6E, Supple- tioning as a positive regulator in the progenitors. mental Table 2), suggesting that Sall1-mediated repression is independent of direct binding to DNA. We detected binding Progenitor-Related Loci but Not Differentiation- of Sall1 with endogenous HDAC2 and Mi2b, which are com- Related Loci Are Co-Occupied by Sall1 and Six2 ponents of the histone deacetylase (HDAC)-containing Mi2/ We next performed chromatin immunoprecipitation (ChIP) nucleosome remodeling deacetylase (NuRD) complex, in followed by sequencing (ChIP-Seq) analysis using embryonic Sall1Flag embryonic kidneys and the overexpression analysis kidneys. We found that Sall1 and Six2 co-occupy the loci (Figure 7, B and C). These observations are consistent with encoding the genes that are downregulated in the absence of the hypothesis that Sall1 could function as a repressor in the Sall1,suchasOsr1, Robo2,andMegf9. This result indicates differentiating nephrons where Six2 expression has disap- that these genes are the direct Sall1 targets (Figure 6A, Sup- peared. There could exist another DNA binding molecule plemental Table 1), although the Sall1 peaks in the Cited1 that bridges the Sall1 and Mi2/NuRD complex with the locus were equivocal. In addition, many loci containing genes Nkx6.1 locus. essential for kidney development, such as Eya1, Pax2, Wt1,and Gdnf, as well as Sall1 and Six2 themselves are co-occupied by both Sall1 and Six2 (Figure 6A, Supplemental Figure 2A). One DISCUSSION of the major exceptions is the Hox cluster, which is occupied only by Sall1 (Supplemental Figure 2B). To rule out nonspe- Sall1 is expressed in Six2-positive progenitors as well as cificbindingoftheSall1antibody,wegeneratedanother differentiating nascent nephrons, and Sall1 deletion results mouse strain that contains Flag-tagged Sall1 (Sall1Flag)by in depletion of both populations. Sall1 maintains nephron homologous recombination (Supplemental Figure 3A). The progenitors and their derivatives by a mechanism that partly expression pattern of Sall1Flag was identical to that of endog- overlaps but is distinct from that of Six2. Sall1 activates enous Sall1 proteins (Supplemental Figure 3B), and we progenitor-related genes in Six2-positive nephron progenitors

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Figure 5. Sall1 is an activator in nephron progenitors and a repressor in differentiating nephrons. (A) Venn diagrams of the left show the flox/flox CreER/flox overlap of decreased (upper panels) or increased (lower panels) probes in Six2GFPCre;Sall1 kidneys and Sall1 kidneys 24 and 48 hours after tamoxifen treatment. The circle graphs on the right show the distributions of the decreased or increased genes in Six2- GFP–positive or -negative cells. Gene numbers are smaller than probe numbers because of the overlaps of the probes. KO, knockout. (B and C) Immunostaining for Cited1. Tamoxifen was injected at E12.5 and analyzed at E14.5. The expression of Cited1 is significantly CreER/flox decreased in the Sall1 kidney. Scale bar, 100 mm. (D and E) In situ hybridization of Osr1. The expression of Osr1 is mildly CreER/flox reduced in the Sall1 kidney at E14.5. (F–I) Immunostaining for Nkx6.1. The expression of Nkx6.1 in the differentiating nephrons CreER/flox flox/flox (arrows) is significantly increased in both the Sall1 kidney treated with tamoxifen and the Six2Cre;Sall1 kidney at E14.5.

but is not involved in Six2-mediated suppression of the Wnt4/ could still impair the self-renewal of nephron progenitors. Fgf8 pathway. This network view of stem cells may explain why Sall1 deletion The expression changes of the Sall1 target genes were does not cause an immediate loss of Six2, despite Sall1 binding to unexpectedly mild, considering the severe phenotypes. We the Six2 enhancer. previously showed that Sall4 is essential for the maintenance of In differentiating nephrons where Six2 expression has embryonic stem (ES) cells.22 Sall4 forms a network with other disappeared, Sall1 is likely to function as a repressor. It is nuclear factors by both protein–protein interaction and mu- proposed that Sall family members function as transcriptional tual transcriptional activation, thereby maintaining pluripo- repressors by interacting with the Mi2/NuRD complex.24–26 tency.21,23 In this type of network, deletion of Sall4 alone leads However, genes endogenously repressed by Sall1 in the kidney to mild changes in a subset of the components but still results in remain elusive. We show that Sall1 suppresses multiple genes, stem cell depletion. Likewise, Sall1 binds to many progenitor- including Nkx6.1. Nkx6.1 is essential for neuron and pancreas related gene loci, but in terms of transcription, a subset of them development,27,28 and Nkx6.1 misexpression in uncommitted is affected moderately. The additive effects of these changes pancreas progenitors using the Nkx6.1OE mouse speciﬁes an

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Figure 6. Progenitor-related loci but not differentiation-related loci are co-occupied by Sall1 and Six2. (A) ChIP-Seq analysis of Sall1 and Six2 within the progenitor-related loci (mm9 coordinates). Asterisks show peaks co-occupied by Sall1 and Six2, and diamonds show peaks reported in a study by Park et al.3 Red asterisks correspond to the regions used for the EMSA in Figure 7. The co-occupied peak in the Osr1 locus was not detected in the stringent peak calls, but Sall1 binding was veriﬁed by an EMSA. (B) Venn diagram showing the

J Am Soc Nephrol 25: 2584–2595, 2014 Sall1 Maintains Nephron Progenitors 2591 BASIC RESEARCH www.jasn.org endocrine fate.29 We overexpressed Nkx6.1 in vivo in nephron the offspring was performed by PCR using a forward primer, progenitor-derived lineages by crossing the Six2GFPCre with 59-CTGGGAACGTGGAAAAACTG-39, and two reverse primers, the Nkx6.1OE mouse (Supplemental Figure 4). Although the 59-CACTCTGGCAGCTTTAGCTTG-39 and 59-GTCATCGTCCTTG- expression was mosaic, two of five Six2GFPCre;Nkx6.1OE mice TAGTC-39, producing products of 153 bp for the control allele showed hypoplastic kidneys with scattered Six2-positive pro- and 178 bp for the mutant allele. Homozygous mice showed no genitors. Therefore, the increase of Nkx6.1 could affect kidney apparent abnormalities. All animal experiments were performed in development. In ES cells, Sall4 binds to the Mi2/NuRD accordance with institutional guidelines and ethical review commit- complex and represses aberrant expression of Cdx2, a critical tees. transcription factor that stimulates differentiation to trophectoderm.25 Therefore, dual functions of Sall family pro- In Situ Hybridization and Immunostaining teins in stem/progenitor cells are well conserved. To address Histologic examinations were performed as described previously.31 the role of Sall1 in differentiating nephrons more precisely, we Mice were fixed in 10% formalin, embedded in paraffin, and cut into performed Wnt4Cre-mediated Sall1 deletion.However,the 6-mm sections. In situ hybridization was performed using an auto- phenotype was again lost in progenitors, although it was not mated Discovery System (Roche) according to the manufacturer’s as complete as that in Six2GFPCre-mediated deletion (Sup- protocols. Templates for the probes were generated by RT-PCR and plemental Figure 5A). This observation was caused by leaky sequenced. Immunostaining was carried out automatically using a excision in nephron progenitors (Supplemental Figure 5B). BlueMap or DABmap Kit and the automated Discovery System or Thus, the relative importance of Sall1 as an activator versus manually for immunofluorescence staining. The following primary repressor remains to be clarified. antibodies were used: anti-Six2 (Proteintech); anti-Sall122,32 (Perseus Wepropose that Sall1 positively regulates targets in nephron Proteomics); anti-Sall1 (AB31526; Abcam, Inc.), anti-Cited1 progenitors and suppresses aberrant gene expression in the (Thermo Fisher Scientific); anti-Wt1 (Santa Cruz Biotechnology); differentiating nephrons, thereby maintaining these popula- LTL (Vector Laboratories); anti-THP (Santa Cruz Biotechnology); tions. Regulation of different targets as a positive and negative anti-NCC (EMD Millipore); anticytokeratin (Sigma-Aldrich); regulator could depend on the interacting proteins that are anti-NCAM (Developmental Studies Hybridoma Bank; EMD available in progenitors (Six2) or differentiating nephrons Millipore); anti-Nkx6.1 (R&D Systems); anticleaved caspase 3 (Mi2/NuRD), although identification of molecules recruiting (Cell Signaling Technology); anti–phosphohistone-H3 (EMD Sall1 to the repressed loci will be necessary. Millipore); anti-red fluorescent protein (Rockland); anti-Lef1 (Cell Signaling Technology); and anti-DDDDK tag (Abcam, Inc.). The monoclonal (Perseus Proteomics) and polyclonal (Abcam, CONCISE METHODS Inc.) anti-Sall1 antibodies gave no background signals in Sall1 mutant kidneys. Terminal deoxynucleotidyl transferase-mediated Generation of the Mutant Mice digoxigenin-deoxyuridine nick-end labeling assays were performed fl Sall1 ox, Sall1CreER,andNkx6.1OE mice were described previ- using an ApopTag Plus fluorescein in situ apoptosis detection kit ously.12,25,29 Six2GFPCre BAC transgenic and Wnt4GFPCre mice was (EMD Millipore), and the signal was enhanced by Alexa 594-conjugated provided by Andrew McMahon.1,3 The R26RtandemdimerTomato streptavidin (Invitrogen). mouse was obtained from The Jackson Laboratory.11 Tamoxifen treatment was described previously.12 To generate the Sall1Flag mice, the Organ Culture of the Embryonic Kidney 59 EcoRI–HindIII Sall1 genomic fragment containing exon 3 of Sall1 E12.5 kidneys were cultured on Millicell Cell Culture Inserts (EMD fused with a Flag tag (5.5 kb) as well as the 39 HindIII–ClaI fragment Millipore) placed in glass-bottomed dishes containing DMEM media (2.8 kb) were incorporated into a vector containing Neo flanked by with 10% serum. Time-lapse confocal images were taken using loxP sites and diphtheria toxin A-subunit in tandem. The targeting a CellVoyager CV1000 (Yokogawa) and processed using Imaris vector was electroporated into E14.1 ES cells, and 8 of 280 G418- (Bitplane). The APC BrdU flow kit (BD Pharmingen) was used for resistant clones were correctly targeted as determined by Southern cell cycle analysis as described.25 blotting analyses using 59 or 39 probes after EcoRV or HindIII diges- tion, respectively. The three correctly targeted ES clones were used to Tamoxifen Treatment generate germ-line chimeras that were bred with C57BL/6J female Tamoxifen (70 mg/kg body wt) was administered intraperitoneally mice at the Center for Animal Resources and Development at into pregnant female mice as described.12 Because tamoxifen treat- Kumamoto University. The Neo cassette was deleted by crossing the ment hindered the ability of mice to give birth, we euthanized the mutant mice with mice expressing Cre ubiquitously.30 Genotyping of pregnant mother at the expected birth date.

number of peaks bound by Sall1 or Six2 across the whole genome. Sall1 binding peaks within 500 bp from Six2 binding peaks are classiﬁed as co-occupied. (C) The enriched de novo Sall1 binding motif recovered from ChIP-Seq peak regions. (D and E) No Sall1 binding peaks in the Wnt4 or Nkx6.1 loci. Diamond shows the peak reported in a study by Park et al.3

2592 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2584–2595, 2014 www.jasn.org BASIC RESEARCH

Microarray and Quantitative RT-PCR fl fl fl fl Two sets of Six2GFPCre;Sall1 ox/ ox versus Sall1 ox/ ox fl at E14.5 and three pairs of Sall1CreER/ ox versus fl Sall1+/ ox (two pairs for 24 hours and one pair for 48 hours after tamoxifen treatment at E12.5) were analyzed. Mircoarray analysis was performed by using Agilent whole-mouse genome (4344,000; v2) or SurePrint G3 mouse gene expression (8360,000). The data were nor- malized by GeneSpring GX software (Agilent Technologies). Microarray platforms were com- bined using Entrez Gene ID, and differentially expressed genes (.1.5-fold) were extracted. The array data have been deposited with the Na- tional Center for Biotechnology Information Gene Expression Omnibus (accession no. GSE45845). RNA was isolated from dissected kidneys using an RNeasy Plus Micro Kit (Qiagen) and then reverse-transcribed with random primers using the Superscript VILO cDNA Synthesis Kit (Invitrogen). Quantitative PCR was carried out using the Dice Real Time System Thermal Cycler (Takara Bio) and Thunderbird SYBR qPCR Mix (Toyobo). All the samples were nor- malized by the b-actin expression.

ChIP-Seq and Immunoprecipitation ChIP analysis was performed as described.3,33 Briefly, 12 kidneys at E16.5 per sample were fixed for 30 minutes, sonicated, and mixed with anti-Six2 antibody (Proteintech), anti- Sall1 antibody (Abcam, Inc.), anti-Flag M2 antibody (Sigma-Aldrich), or rabbit or mouse IgG (Santa Cruz Biotechnology) at 4°C overnight followed by precipitation using Dynabeads M-280 conjugated with anti-rabbit or -mouse IgG (Invitrogen). Templates for ChIP-Seq analysis were prepared using the ChIP-Seq sample ’ Figure 7. Sall1 binds to the progenitor-related loci and biochemically associates with prep kit (Illumina) following the manufacturer s Six2 and Mi2/NuRD. (A) EMSA of Sall1 showing Sall1 binding to the progenitor-related instructions. Sequencing was carried out on loci. Competitor MT, mutated competitor oligonucleotides; competitor WT, wild-type Illumina HiSeq2000 platform, and at least 20 competitor oligonucleotides; IgG, negative control for the supershift (*) by the anti- million 36-nucleotide single end sequences Sall1 antibody (Sall1 ab); Sall1, in vitro-translated Sall1 proteins; vector, in vitro- were generated for each sample. The sequence translated lysates from the empty vector. (B) Binding of Sall1 with Six2 (upper panel) or data were submitted to Genbank/DNA Data the Mi2/NuRD complex (lower panel) in the kidney. Control and Sall1Flag E15.5 Bank of Japan (accession no. DRA000957). Se- kidneys were immunoprecipitated using the anti-Flag antibody and then blotted with quences were mapped to the mouse genome the indicated antibodies. IP, immunoprecipitation. (C) Binding of Sall1 with Six2 in- (mm9) allowing two base mismatches. Peak dependent of DNase and RNase treatment. Flag-Sall1 and myc-Six2 were overex- call was carried out using MACS (http://liulab. pressed in human embryonic kidney 293 cells followed by IP using the anti-Flag dfci.harvard.edu/MACS/). Six2 peaks giving antibody. Endogenous HDAC2 also bound to overexpressed Sall1. (D) Direct binding , fi of the recombinant Sall1 and Six2 proteins generated in vitro. Recombinant proteins P 0.001 were ltered with the following param- 2 3 $ $ prepared by the rabbit reticulocyte lysate system were mixed, immunoprecipitated, eters: 10 LOG10 (P value) 150, tags 39, and blotted with the indicated antibodies. DNase, deoxyribonuclease; RNase, ribo- fold$8, and false discovery rate#1.4. Sall1 peaks nuclease; Six2, in vitro-translated Six2 proteins. giving P,0.001 were filtered with the following parameters: 2103LOG10 (P value) $30, tags$35, fold$7, and false discovery rate#2.5.

J Am Soc Nephrol 25: 2584–2595, 2014 Sall1 Maintains Nephron Progenitors 2593 BASIC RESEARCH www.jasn.org

Multiple EM for Motif Elicitation-ChIP was used to identify the Sall1 3. Park J-S, Ma W, O’Brien LL, Chung E, Guo J-J, Cheng J-G, Valerius MT, and Six2 binding motif. Transfections were performed using Lipo- McMahon JA, Wong WH, McMahon AP: Six2 and Wnt regulate self- fectamine 2000 reagent (Invitrogen). Human embryonic kidney renewal and commitment of nephron progenitors through shared gene regulatory networks. Dev Cell 23: 637–651, 2012 293T cells or kidneys at E15.5 were lysed with lysis buffer (Cell Sig- 4. Karner CM, Das A, Ma Z, Self M, Chen C, Lum L, Oliver G, Carroll TJ: fi naling Technology) and sonicated on ice. Lysates were clari ed by Canonical Wnt9b signaling balances progenitor cell expansion and centrifugation and incubated with beads conjugated with the anti- differentiation during kidney development. Development 138: 1247– Flag M2 antibody for 1 hour at 4°C. Beads were washed three times 1257, 2011 with Tris-buffered saline-1% NP40, and bound proteins were eluted 5. Kühnlein RP, Frommer G, Friedrich M, Gonzalez-Gaitan M, Weber A, Wagner-Bernholz JF, Gehring WJ, Jäckle H, Schuh R: spalt encodes an with 100 mM glycine-HCl (pH 2.6) and analyzed by Western blotting evolutionarily conserved zinc finger protein of novel structure which using the anti-Mi2b (Santa Cruz Biotechnology) or anti-HDAC2 provides homeotic gene function in the head and tail region of the (Santa Cruz Biotechnology) antibodies. Drosophila embryo. EMBO J 13: 168–179, 1994 6. Kohlhase J, Wischermann A, Reichenbach H, Froster U, Engel W: Mu- tations in the SALL1 putative transcription factor gene cause Townes- EMSA Nat Genet – Recombinant mouse Sall1 protein was prepared by the rabbit re- Brocks syndrome. 18: 81 83, 1998 ’ 7. Kohlhase J, Heinrich M, Schubert L, Liebers M, Kispert A, Laccone F, ticulocyte lysate system (Promega) according to the manufacturer s Turnpenny P, Winter RM, Reardon W: Okihiro syndrome is caused by 34 protocol. EMSAs were performed as described elsewhere. In vitro- SALL4 mutations. Hum Mol Genet 11: 2979–2987, 2002 translated Sall1 proteins (10 ng in 3 ml) were incubated at 4°C for 30 8. Nishinakamura R, Matsumoto Y, Nakao K, Nakamura K, Sato A, minutes with a [g-32P]ATP-labeled double-stranded oligonucleotide Copeland NG, Gilbert DJ, Jenkins NA, Scully S, Lacey DL, Katsuki M, in the presence or absence of unlabeled double-stranded competitors Asashima M, Yokota T: Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development. Development 128: 3105– (Supplemental Table 4). Supershift assays were performed by addi- 3115, 2001 tional incubation with an anti-Sall1 monoclonal antibody or mouse 9. Osafune K, Takasato M, Kispert A, Asashima M, Nishinakamura R: IgG for 20 minutes before electrophoresis. The bound products were Identification of multipotent progenitors in the embryonic mouse resolved in a 4% nondenaturing polyacrylamide gel in 0.53Tris- kidney by a novel colony-forming assay. Development 133: 151–161, Borate/EDTA buffer, and then, they were exposed to a radioactive 2006 Kidney Int imaging plate and detected by an FLA-3000 laser scanner (Fuji Photo 10. Nishinakamura R: Stem cells in the embryonic kidney. 73: 913–917, 2008 Film). 11. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H: A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13: 133–140, 2010 ACKNOWLEDGMENTS 12. Inoue S, Inoue M, Fujimura S, Nishinakamura R: A mouse line expressing Sall1-driven inducible Cre recombinase in the kidney mes- We thank R. Matoba for microarray analysis, T. Horiuchi, K. enchyme. Genesis 48: 207–212, 2010 Imamura, and M. Tosaka for chromatin immunoprecipitation and 13. Barak H, Huh SH, Chen S, Jeanpierre C, Martinovic J, Parisot M, Bole- Feysot C, Nitschké P, Salomon R, Antignac C, Ornitz DM, Kopan R: sequencing, M. Aoki for FACS analysis, and Y. Kaku, S. Inoue, and FGF9 and FGF20 maintain the stemness of nephron progenitors in S. Fujimura for histological analysis. We also thank K. Shimamura, mice and man. Dev Cell 22: 1191–1207, 2012 T. Miyata, Y. Xi, J. Kreidberg, L. O’Brien, and A.P. McMahon for 14. Brown AC, Adams D, de Caestecker M, Yang X, Friesel R, Oxburgh L: helpful advice. FGF/EGF signaling regulates the renewal of early nephron progenitors Development – Thisstudywas supportedby KAKENHIGrants 23390228, 25111725, during embryonic development. 138: 5099 5112, 2011 15. Mugford JW, Yu J, Kobayashi A, McMahon AP: High-resolution gene and 221S0002 from Ministry of Education, Culture, Sports, Science and expression analysis of the developing mouse kidney defines novel Technology, Japan. Research in the laboratory of M.S. was supported by cellular compartments within the nephron progenitor population. Dev National Institutes of Health Grant R01-DK68471. Biol 333: 312–323, 2009 16. Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM, Mortlock DP, Baldwin HS, de Caestecker M: Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme con- DISCLOSURES tains self-renewing progenitor cells and gives rise exclusively to Dev Biol – None. nephronic epithelia. 313: 234 245, 2008 17. James RG, Kamei CN, Wang Q, Jiang R, Schultheiss TM: Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells. De- REFERENCES velopment 133: 2995–3004, 2006 18. Mugford JW, Sipilä P, McMahon JA, McMahon AP: Osr1 expression 1. Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, demarcates a multi-potent population of intermediate mesoderm that McMahon AP: Six2 defines and regulates a multipotent self-renewing undergoes progressive restriction to an Osr1-dependent nephron nephron progenitor population throughout mammalian kidney de- progenitor compartment within the mammalian kidney. Dev Biol 324: velopment. Cell Stem Cell 3: 169–181, 2008 88–98, 2008 2. Self M, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G: 19. Grieshammer U, Le Ma, Plump AS, Wang F, Tessier-Lavigne M, Martin Six2 is required for suppression of nephrogenesis and progenitor re- GR: SLIT2-mediated ROBO2 signaling restricts kidney induction to a newal in the developing kidney. EMBO J 25: 5214–5228, 2006 single site. Dev Cell 6: 709–717, 2004

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20. Yamashita K, Sato A, Asashima M, Wang P-C, Nishinakamura R: Mouse 28. Sander M, Sussel L, Conners J, Scheel D, Kalamaras J, Dela Cruz F, homolog of SALL1, a causative gene for Townes-Brocks syndrome, Schwitzgebel V, Hayes-Jordan A, German M: Homeobox gene Nkx6.1 binds to A/T-rich sequences in pericentric heterochromatin via its C- lies downstream of Nkx2.2 in the major pathway of beta-cell formation terminal zinc ﬁnger domains. Genes Cells 12: 171–182, 2007 in the pancreas. Development 127: 5533–5540, 2000 21. Lim CY, Tam W-L, Zhang J, Ang HS, Jia H, Lipovich L, Ng H-H, Wei C-L, 29. Schaffer AE, Freude KK, Nelson SB, Sander M: Nkx6 transcription fac- Sung WK, Robson P, Yang H, Lim B: Sall4 regulates distinct transcrip- tors and Ptf1a function as antagonistic lineage determinants in multi- tion circuitries in different blastocyst-derived stem cell lineages. Cell potent pancreatic progenitors. Dev Cell 18: 1022–1029, 2010 Stem Cell 3: 543–554, 2008 30. Sakai K, Miyazaki J: A transgenic mouse line that retains Cre re- 22. Sakaki-Yumoto M, Kobayashi C, Sato A, Fujimura S, Matsumoto Y, combinase activity in mature oocytes irrespective of the cre transgene Takasato M, Kodama T, Aburatani H, Asashima M, Yoshida N, transmission. Biochem Biophys Res Commun 237: 318–324, 1997 Nishinakamura R: The murine homolog of SALL4, a causative gene in 31. Fujimura S, Jiang Q, Kobayashi C, Nishinakamura R: Notch2 activation Okihiro syndrome, is essential for embryonic stem cell proliferation, in the embryonic kidney depletes nephron progenitors. JAmSoc and cooperates with Sall1 in anorectal, heart, brain and kidney de- Nephrol 21: 803–810, 2010 velopment. Development 133: 3005–3013, 2006 32. Sato A, Kishida S, Tanaka T, Kikuchi A, Kodama T, Asashima M, 23. Kim J, Chu J, Shen X, Wang J, Orkin SH: An extended transcriptional Nishinakamura R: Sall1, a causative gene for Townes-Brocks syndrome, network for pluripotency of embryonic stem cells. Cell 132: 1049–1061, enhances the canonical Wnt signaling by localizing to heterochromatin. 2008 Biochem Biophys Res Commun 319: 103–113, 2004 24. Lauberth SM, Rauchman M: A conserved 12-amino acid motif in Sall1 33. Tanaka S, Miyagi S, Sashida G, Chiba T, Yuan J, Mochizuki-Kashio M, recruits the nucleosome remodeling and deacetylase corepressor Suzuki Y, Sugano S, Nakaseko C, Yokote K, Koseki H, Iwama A: Ezh2 complex. JBiolChem281: 23922–23931, 2006 augments leukemogenicity by reinforcing differentiation blockage in 25. Yuri S, Fujimura S, Nimura K, Takeda N, Toyooka Y, Fujimura YI, acute myeloid leukemia. Blood 120: 1107–1117, 2012 Aburatani H, Ura K, Koseki H, Niwa H, Nishinakamura R: Sall4 is es- 34. Tanigawa S, Lee CH, Lin CS, Ku CC, Hasegawa H, Qin S, Kawahara A, sential for stabilization, but not for pluripotency, of embryonic stem Korenori Y, Miyamori K, Noguchi M, Lee LH, Lin YC, Steve Lin CL, cells by repressing aberrant trophectoderm gene expression. Stem Nakamura Y, Jin C, Yamaguchi N, Eckner R, Hou DX, Yokoyama KK: Jun Cells 27: 796–805, 2009 dimerization protein 2 is a critical component of the Nrf2/MafK com- 26. Denner DR, Rauchman M: Mi-2/NuRD is required in renal progenitor plex regulating the response to ROS homeostasis. Cell Death Dis 4: cells during embryonic kidney development. Dev Biol 375: 105–116, e921, 2013 2013 27. Sander M, Paydar S, Ericson J, Briscoe J, Berber E, German M, Jessell TM, Rubenstein JL: Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates. This article contains supplemental material online at http://jasn.asnjournals. Genes Dev 14: 2134–2139, 2000 org/lookup/suppl/doi:10.1681/ASN.2013080896/-/DCSupplemental.

J Am Soc Nephrol 25: 2584–2595, 2014 Sall1 Maintains Nephron Progenitors 2595 Supplemental data

Supplementary Figure 1. Analysis of the Sall1 mutant mice (A) (A’: higher magnification) Nephrons formed in the Six2GFPCre;Sall1flox/flox mice at E14.5 retain Sall1 expression. Sall1 is stained red and Six2 is green. Scale bar=40 m. (B)(C) In situ hybridization of Etv4 (FGF activity) in Sall1+/flox and Sall1CreER/flox kidneys at E14.5 (48hr after tamoxifen treatment), showing the intact expression in the progenitors (arrowheads). Expression in ureteric buds reflects Gdnf activity. Scale bar=100 m. (D)(E) Immunostaining of Six2 (green) and cytokeratin 8 (red) in Sall1flox/flox (D) and Six2Cre;Sall1flox/flox (E) kidneys in culture. E12.5 kidneys were treated with FGF2 (50ng/ml) and heparin (1ug/ml) and cultured for 2 days, but the phenotypes of the Sall1 mutants were not rescued. Scale bar=100 m. (F)(G) Immunostaining of Lef1 (Wnt activity) Sall1+/flox and Sall1CreER/flox kidneys at E14.5 (48hr after tamoxifen treatment). Lef1 is expressed in the differentiating nephrons (arrows) and excluded from the progenitors (arrowheads) in both samples. Scale bar=100 m. (H)(I) Whole mount in situ hybridization of Wnt9b in Sall1+/flox (H) and Sall1CreER/flox (I) kidneys 48hr after tamoxifen treatment. Scale bar=100 m. (J)(K) Whole mount in situ hybridization of Wnt11 in Sall1+/flox (J) and Sall1CreER/flox (K) kidneys 48hr after tamoxifen treatment. Scale bar=100 m. (L) Quantitative RT-PCR of E14.5 kidneys. Sall1+/flox mice and Sall1CreER/flox mice were treated with tamoxifen at E12.5. The columns represent means ± SD (n=5). Primers are listed in Supplementary Table 4.

Supplementary Figure 2. Sall1 binding to gene loci that are essential for kidney development (A) Sall1 and Six2 co-occupy Pax2, Wt1, Gdnf, and Sall1 loci. Asterisk: peaks co-occupied by Sall1 and Six2. Diamond: peaks reported in a study by Park et al. 3 (B) Sall1 but not Six2 binds to the loci of Hox gene clusters. (C) Six2 but not Sall1 binds to the Fgf8 and Bmp7 loci. The peak marked by # is an artifact, which is also detected in the IgG control sample.

Supplementary Figure 3. Generation and analysis of Sall1Flag mouse (A) Targeting strategy for Sall1Flag knock-in mouse. The stop codon of Sall1 was replaced by the Flag tag. H: HindIII; E: EcoRV. The genomic DNA was digested with

1 EcoRV or HindIII and hybridized with 5’ or 3’ probes, respectively. (B) Flag and Sall1 immunostaining of Sall1Flag mouse kidneys at E15.5. Scale bar=100 m. (C) ChIP-qPCR analysis of the Six2 locus using Sall1Flag mouse kidneys and the anti-Flag antibody. A binding peak is observed at region 2, which corresponds to the marked peak in Figure 6A. Primers are listed in Supplementary Table 4.

Supplementary Figure 4. Overexpression of Nkx6.1 in vivo affects kidney development (A) Successful overexpression of Nkx6.1 in Six2Cre: Nkx6.1 OE kidneys at E14.5, shown by immunostaining using the anti-Nkx6.1 antibody. (B) A mild size reduction of the Six2Cre: Nkx6.1 OE kidneys at P0. Hematoxylin-eosin staining. (C) Scattered Six2-positive nephron progenitors in the Six2Cre: Nkx6.1 OE kidneys at P0. Scale bar=100 m.

Supplementary Figure 5. Wnt4Cre-mediated deletion is not specific to differentiating nascent nephrons (A) Wnt4Cre-mediated Sall1 deletion at E14.5. (B) Lineage trace analysis of Wnt4Cre analyzed at E14.5. tdTomato is stained by the anti-RFP antibody (blue). Arrowheads: nephron progenitors; arrows: differentiating nephrons; ub: ureteric bud. Scale bar=50 m.

Supplementary Video 1. Time-lapse analysis of nephron progenitor development in the control kidney Six2CreGFP kidneys were cultured at E12.5 on Millicell Cell Culture Inserts placed in dishes containing DMEM with 10% serum. The confocal time-lapse images were taken using a CellVoyager CV1000. GFP-positive nephron progenitors expand as the ureteric bud branches.

Supplementary Video 2. Time-lapse analysis of nephron progenitor depletion in Sall1 deficiency Six2CreGFP; Sall1flox/flox kidneys were cultured at E12.5 on Millicell Cell Culture Inserts placed in glass-bottomed dishes containing DMEM with 10% serum. Confocal time-lapse images were taken using a CellVoyager CV1000. GFP signal decreased rapidly in the absence of Sall1.

Supplementary Table 1. Down-regulated genes in the absence of Sall1 Decreased expression of a series of genes in Sall1 mutant kidneys compared with that in their respective controls. Most of the down-regulated genes in the absence of Sall1 are expressed more abundantly in Six2(+) cells than in Six2(-) cells. Sall1- and Six2-binding peaks detected in our ChIP-Seq analysis were compared with Six2-binding peaks in the study by Park et al.3 (mm9 coordinates). Asterisks and diamonds correspond to those in Figure 6 and Supplementary Figure 2.

Supplementary Table 2. Up-regulated genes in the absence of Sall1 Increased gene expression in a series of Sall1 mutant kidneys compared with their respective controls. Most of the up-regulated genes are expressed more abundantly in Six2(-) cells than in Six2(+) cells. Sall1- and Six2-binding peaks detected in our ChIP-Seq analysis were compared with Six2-binding peaks in the study by Park et al.3 (mm9 coordinates).

Supplementary Table 3. Expression and ChIP-seq analysis of kidney developmental genes Sall1- and Six2-binding peaks detected in our ChIP-Seq analysis were compared with Six2-binding peaks in the study by Park et al.3 (mm9 coordinates). Asterisks and diamonds correspond to those in Figure 6 and Supplementary Figure 2.

Supplementary Table 4. Primers for ChIP-qPCR and quantitative RT-PCR, and oligonucleotides for EMSA.

3 Supplementary Figure 1 A A’ Sall1 F G Lef1 ub ub

B C Etv4 H I Wnt9b ub ub

D E J K Wnt11

100 p<0.001 p<0.001 10 p<0.001 p=0.014 p<0.001 p<0.001

0.1

0.01 WT KO WT KO WT KO WT KO WT KO WT KO Sall1 Six2 Cited1 Osr1 Odz2 Nkx6.1 Supplementary Figure 2 A Pax2 Wt1 (chr19:44,660,000-44,960,000) (chr2:104,800,000-105,,040,000)

IgG IgG * Sall1 Sall1 *

IgG IgG

Six2 * Six2 *

Sall1 Gdnf (chr8:91,450,000-92,326,000) (chr15:74,000,000-7,800,000)

IgG IgG

Sall1 * * ** Sall1 * IgG IgG

  Six2 *  *  * * * Six2 *

B Hoxa1, a2,a3, a4, a5, a6, a7, a9, a10, a11, a13 (chr6 : 52,000,001-52,250,000)

IgG

Sall1

IgG

Six2

C Fgf8 Bmp7 (chr19:45,670,001-45,820,000) (chr2:172,600,001-173,872,000)

# IgG IgG

# Sall1 Sall1

IgG IgG   Six2 Six2 Supplementary Figure 3 A B A Probes H Wild type H E H E

Flag E

DTA Neo

Flag E Sall1-Flag Neo(+) H E H E

Neo 6.0 kb 1.0kb WT 15.5 kb Sall1-Flag 12 kb WT 8 kb Sall1-Flag

Cre

Flag Sall1-Flag Neo(-) H E H E

Sall1+/+ Sall1Flag/+ Sall1Flag/+ B Flag Flag Sall1

Flag Sall1

C 0.03 IgG 0.02 a-Flag

%Input 0.01

1 2 3 Supplementary Figure 4 Nkx6.1OE Six2GFPCre;Nkx6.1OE A B Nkx6.1

C D

E F Six2 Supplementary Figure 5 Wnt4GFPCre: A Sall1flox/flox Sall1flox/flox

Six2

Sall1

Wnt4GFPCre; B tdTomato tdTomato

ub ub ub Supplementary Table 1

Six2 Log2 Log2 Log2 Log2 Sall1 Six2 GeneSymbol GeneName binding (Sall1CreER/flox 48hr) (Sall1CreER/flox 24hr) (Six2Cre; Sall1flox/flox) (Six2 GFP+ vs GFP-) binding binding (Park et al.; ref3)

Ocm oncomodulin -3.78 -2.59 -1.78 7.05 absent chr5:144,793,500-144,793,600 chr5:144,793,471-144793622 Cbp/p300-interacting transactivator with Cited1 -2.85 -2.20 -4.16 4.80 absent chrX:99,515,160-99,515,460 absent Glu/Asp-rich carboxy-terminal domain 1 Sall1 sal-like 1 (Drosophila) -2.78 -0.74 -1.25 1.64 chr8:91,559,382-91,564,748 chr8:91,569,591-91,571,276 chr8:91,910,504-91,914,563 chr8:91,912,350-91,913,805 chr8:91,935,147-91,937,179* chr8:91,935,304-91,937,293* chr8:91,936,103-91,936,284 chr8:91,962,070-91,964,145 chr8:91,938,409-91,940,376 chr8:91,939,275-91,939,431 chr8:91,982,978-91,984,393 chr8:92,085,399-92,088,663 chr8:92,085,787-92,088,204 chr8:92,092,399-92,094,667* chr8:92,129,157-92,130,804* chr8:92,094,825-92,096,530 chr8:92,182,190-92,186,278* chr8:92,182,364-92,184,152* chr8:92,130,054-92,130,238 chr8:92,184,846-92,186,022 chr8:92,197,793-92,199,765* chr8:92,198,196-92,199,608* chr8:92,239,090-92,242,534 chr8:92,230,587-92,232,637 Dync1i1 dynein cytoplasmic 1 intermediate chain 1 -2.30 -1.12 -1.65 3.30 absent chr6:5,683,998-5,685,382 chr6:5,684,379-5,684,728 Dkk1 dickkopf homolog 1 (Xenopus laevis) -2.15 -1.00 -1.69 -5.32 absent absent absent Dkk1 dickkopf homolog 1 (Xenopus laevis) -2.03 -1.00 -1.73 -5.57 shown in the other column UDP-N-acetyl-alpha-D-galactosamine: Galntl2 polypeptide N-acetylgalactosaminyltransferase- -1.98 -2.46 -2.66 4.14 absent absent absent like 2 Pdyn prodynorphin -1.93 -1.30 -3.93 5.35 absent chr2:129,523,851-129,526,042 absent Fgf1 fibroblast growth factor 1 -1.83 -2.48 -1.68 5.40 absent chr18:39,034,094-39,035,438 chr18:39,034,516-39,034,806 chr18:39,040,772-39,042,700 chr18:39,041,961-39,042,307 Moxd1 monooxygenase, DBH-like 1 -1.42 -1.11 -1.20 1.57 absent absent absent Fgf1 fibroblast growth factor 1 -1.17 -0.61 -1.62 4.35 shown in the other column Gm6213 predicted gene 6213 -1.16 -0.63 -1.65 -5.01 absent absent absent Capn6 calpain 6 -1.10 -0.67 -0.85 2.99 absent absent absent Lbx2 ladybird homeobox homolog 2 (Drosophila) -1.07 -0.78 -1.29 2.79 absent absent absent Tcfap2c transcription factor AP-2, gamma -1.07 -0.69 -0.80 -4.06 absent absent absent Wnt6 wingless-related MMTV integration site 6 -1.04 -1.06 -1.45 -3.01 absent absent absent H2-Eb1 histocompatibility 2, class II antigen E beta -1.02 -0.65 -1.13 0.49 absent absent absent Osr1 odd-skipped related 1 (Drosophila) -1.01 -0.85 -0.88 5.15 chr12:9,265,136-9,266,406 chr12: 9,583,063-98,855,408 chr12:9,663,412-9,665,066 chr12:9,9904852-9,906,011* chr12:10,134,583-10,136,241 chr12:10,141,956-10,143,714 chr12:10,237,565-10,239,324 chr12:10,238,413-10,238,627 Robo2 roundabout homolog 2 (Drosophila) -1.00 -0.70 -0.66 3.56 chr16:73,529,347-73,531,168 chr16:73,530,336-73,530,636 chr16:73,533,609-73,533,909 chr16:74,457,123-74,459,457* chr16:74,457,244-74,459,474* chr16:74,458,499-74,458,753 chr16:74,464,261-74,466,231 chr16:74,467,369-74,469,755 chr16:74,513,516-74,516,223* chr16:74,514,010-74,516,020* chr16:74,514,689-74,515,212 C2cd4b C2 calcium-dependent domain containing 4B -0.99 -1.27 -1.41 5.18 absent absent absent Nefl neurofilament, light polypeptide -0.95 -0.79 -0.92 3.70 absent absent absent Stk32a serine/threonine kinase 32A -0.95 -1.14 -2.31 3.50 absent chr18:43,442,666-43,444,253 chr18:43,443,559-43,443,715 Osr1 odd-skipped related 1 (Drosophila) -0.94 -0.85 -0.82 4.72 shown in the other column Snap91 synaptosomal-associated protein 91 -0.93 -1.10 -3.60 3.30 absent chr9:86,878,689-86,882,629 absent chr9:86,891,164-86,893,355 Pcnxl2 pecanex-like 2 (Drosophila) -0.92 -1.09 -1.29 2.75 absent chr8:128,395,717-128,397,635 chr8:128,396,405-128,396,560 Has2 hyaluronan synthase 2 -0.92 -0.76 -1.16 4.18 absent chr15:56,349,183-56,350,888 chr15:56,350,004-56,350,207 Nefl neurofilament, light polypeptide -0.87 -0.79 -0.89 4.50 shown in the other column sparc/osteonectin, cwcv and kazal-like Spock2 -0.86 -1.08 -1.89 3.74 absent chr10:59,559,940-59,561,286 chr10:59,568,493-59,569,043 domains proteoglycan 2 chr10:59,567,851-59,570,032 Has2 hyaluronan synthase 2 -0.84 -0.76 -1.30 4.81 shown in the other column growth arrest and DNA-damage-inducible, Gadd45gip1 -0.83 -0.88 -1.01 0.37 absent absent absent gamma interacting protein 1 1700024P16Rik RIKEN cDNA 1700024P16 gene -0.83 -0.57 -0.70 -1.92 absent absent absent Stk32a serine/threonine kinase 32A -0.82 -2.95 -1.87 2.20 shown in the other column Robo2 roundabout homolog 2 (Drosophila) -0.80 -0.49 -0.75 3.93 shown in the other column Wnt6 wingless-related MMTV integration site 6 -0.79 -1.06 -1.16 -1.59 shown in the other column Pnmt phenylethanolamine-N-methyltransferase -0.79 -0.70 -1.78 5.00 absent absent absent Ppm1e protein phosphatase 1E (PP2C domain containing) -0.75 -0.83 -1.47 3.13 absent absent absent Robo2 roundabout homolog 2 (Drosophila) -0.72 -0.70 -1.25 3.43 shown in the other column 2610028E06Rik RIKEN cDNA 2610028E06 gene -0.72 -0.63 -3.77 3.74 absent absent absent sparc/osteonectin, cwcv and kazal-like Spock2 -0.71 -1.08 -1.53 4.30 shown in the other column domains proteoglycan 2 Krt23 keratin 23 -0.66 -1.02 -0.62 -6.09 absent absent absent Megf9 multiple EGF-like-domains 9 -0.63 -0.69 -0.94 1.51 chr4:70,149,257-70,152,337* chr4:70,149,909-70,151,851* absent chr4:70,157,125-70,159,298* chr4:70,157,444-70,159,250* chr4:70,160,472-70,162,492 chr4:70,195,594-70,199,117 chr4:70,163,076-70,164,382 chr4:70,195,701-70,198,887 UDP-Gal:betaGlcNAc beta B3galt5 -0.62 -1.01 -1.70 1.04 absent chr5:150,477,007-150,478,593 chr5:150,477,654-150,477,982 1,3-galactosyltransferase, polypeptide 5 Supplementary Table 2

Log2 Log2 Log2 Log2 Sall1 Six2 Six2 binding GeneSymbol GeneName (Sall1CreER/flox 48hr) (Sall1CreER/flox 24hr) (Six2Cre; Sall1flox/flox) (Six2 GFP+ vs GFP-) binding binding (Park et al.; ref3)

Odz2 odd Oz/ten-m homolog 2 (Drosophila) 3.70 2.48 2.13 -1.53 absent chr11:36,685,840-36,687,519 chr11:36,686,562-36,686,793 Odz2 odd Oz/ten-m homolog 2 (Drosophila) 3.62 2.48 2.10 -1.42 absent chr11:36,810,638-36,812,020 Odz2 odd Oz/ten-m homolog 2 (Drosophila) 3.47 2.48 1.47 0.18 absent Nkx6-1 NK6 homeobox 1 3.10 2.14 1.53 -1.47 absent absent absent 3110082D06Rik RIKEN cDNA 3110082D06 gene 2.68 1.54 2.40 -2.74 absent absent absent Trhr thyrotropin releasing hormone receptor 2.64 1.07 1.86 0.12 chr15:44,020,317-44,022,935 absent absent Krt36 keratin 36 2.32 2.08 2.65 2.07 absent chr11:99,964,150-99,964,350 absent 3110082D06Rik RIKEN cDNA 3110082D06 gene 2.31 1.54 2.32 -1.56 absent absent absent Krt36 keratin 36 2.13 1.99 2.67 2.00 shown in the other column shown in the other column shown in the other column Gm6792 predicted gene 6792 2.04 0.61 0.63 -3.21 absent absent absent Rnf152 ring finger protein 152 1.81 1.33 1.68 -1.26 absent absent absent Kera keratocan 1.78 1.97 2.05 -1.90 absent chr10:97,053,826-97,055,372 chr10:97,054,393-97,054,612 Pcdh8 protocadherin 8 1.66 0.78 1.02 4.37 absent absent absent Rnf152 ring finger protein 152 1.47 1.33 1.76 -0.86 shown in the other column shown in the other column shown in the other column Ccl21a chemokine (C-C motif) ligand 21A 1.45 1.30 1.48 -1.39 absent absent absent Lrrc4c leucine rich repeat containing 4C 1.42 0.60 1.68 -0.86 absent absent absent Slitrk6 SLIT and NTRK-like family, member 6 1.40 1.40 0.69 -5.30 absent absent absent Saa2 serum amyloid A 2 1.36 0.98 0.75 5.13 absent absent absent Ebf2 early B-cell factor 2 1.36 0.66 0.69 -1.03 absent absent absent Fap fibroblast activation protein 1.34 0.76 0.89 -0.37 absent absent absent Fap fibroblast activation protein 1.34 0.76 1.56 -5.15 shown in the other column shown in the other column shown in the other column Krt15 keratin 15 1.30 0.87 2.08 2.63 absent absent absent Rnf152 ring finger protein 152 1.28 1.33 1.93 -0.65 shown in the other column shown in the other column shown in the other column Snph syntaphilin 1.24 1.01 1.02 1.46 absent absent absent Vegfc vascular endothelial growth factor C 1.24 0.62 1.17 -1.20 absent absent absent Gdpd2 glycerophosphodiester phosphodiesterase domain containing 2 1.24 0.89 0.74 -0.03 absent absent absent Angptl1 angiopoietin-like 1 1.21 1.45 0.88 -2.97 absent absent absent Gabrb3 gamma-aminobutyric acid (GABA) A receptor, subunit beta 3 1.20 0.63 0.72 -1.77 absent absent absent Oit3 oncoprotein induced transcript 3 1.18 0.84 1.09 -3.46 absent absent absent Foxp2 forkhead box P2 1.18 1.09 1.36 -3.50 absent absent absent Lypd1 Ly6/Plaur domain containing 1 1.16 0.87 0.82 -0.05 absent absent absent Egfl6 EGF-like-domain, multiple 6 1.12 0.78 0.97 -1.57 chrX:162,947,271-162,948,745 absent absent Aspn asporin 1.09 2.14 1.52 -5.27 absent absent absent 5430435G22Rik RIKEN cDNA 5430435G22 gene 1.05 0.67 0.68 -1.45 absent absent absent Dtna dystrobrevin alpha 1.05 0.83 0.80 2.06 absent absent absent Napsa napsin A aspartic peptidase 1.05 0.47 0.98 -2.72 absent absent absent 1700028P14Rik RIKEN cDNA 1700028P14 gene 1.04 0.61 0.73 -0.46 absent absent absent 0.99 1.37 1.32 -0.59 chr5:84,813,557-84,815,455 absent absent Epha5 Eph receptor A5 chr5:85,097,634-85,099,671 Rspo2 R-spondin 2 homolog (Xenopus laevis) 0.97 1.90 1.51 -0.42 absent absent absent Ebf3 early B-cell factor 3 0.96 0.62 1.77 -3.46 absent absent absent Foxp2 forkhead box P2 0.95 0.78 1.36 -2.18 shown in the other column shown in the other column shown in the other column Dtna dystrobrevin alpha 0.95 0.83 0.70 1.90 shown in the other column shown in the other column shown in the other column Shisa9 shisa homolog 9 (Xenopus laevis) 0.95 0.85 0.74 -2.70 absent absent absent Ebf3 early B-cell factor 3 0.94 0.62 1.38 0.00 shown in the other column shown in the other column shown in the other column Sfrp4 secreted frizzled-related protein 4 0.92 1.26 1.95 -4.32 absent absent absent Htr2b 5-hydroxytryptamine (serotonin) receptor 2B 0.90 0.80 0.62 1.60 absent chr1:88,015,600-88,017,322 absent Fibin fin bud initiation factor homolog (zebrafish) 0.87 0.75 1.18 -4.77 absent absent absent Ryr3 ryanodine receptor 3 0.86 0.60 0.75 -2.07 absent absent absent Nov nephroblastoma overexpressed gene 0.84 1.41 1.30 -2.10 chr15:54,477,140-54,478,129 absent absent Dtna dystrobrevin alpha 0.82 0.83 0.73 0.71 shown in the other column shown in the other column shown in the other column Col5a3 collagen, type V, alpha 3 0.76 0.64 1.17 -3.92 chr9:20,557,700-20,558,700 absent absent Ddx4 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 0.76 1.01 0.64 -0.20 absent absent absent 0.75 0.93 1.16 -4.17 chr5:13,325,149-13,327,960 absent absent sema domain, immunoglobulin domain (Ig), chr5:13,397,505-13,402,459 Sema3a short basic domain, secreted, (semaphorin) 3A chr5:13,402,643-13,406,956 chr5:13,426,453-13,427,898 Bmp5 bone morphogenetic protein 5 0.75 0.72 0.86 -2.33 absent absent absent Oit3 oncoprotein induced transcript 3 0.75 0.84 0.77 -0.83 shown in the other column shown in the other column shown in the other column Cldn1 claudin 1 0.75 1.30 1.55 -4.40 absent absent absent Tbx1 T-box 1 0.75 0.72 1.03 -3.54 absent absent absent triggering receptor expressed on Trem2 0.72 0.61 0.68 0.06 absent absent absent myeloid cells 2 membrane-spanning 4-domains, subfamily A, Ms4a7 0.72 0.66 1.38 0.71 absent absent absent member 7 Fxyd7 FXYD domain-containing ion transport regulator 7 0.70 0.68 1.22 -0.78 absent absent absent Cldn1 claudin 1 0.69 1.30 1.28 -5.01 shown in the other column shown in the other column shown in the other column membrane-spanning 4-domains, subfamily A, Ms4a7 0.69 0.66 1.43 0.75 shown in the other column shown in the other column shown in the other column member 7 Col12a1 collagen, type XII, alpha 1 0.68 0.74 0.81 -4.30 absent absent absent Trem2 triggering receptor expressed on myeloid cells 2 0.65 0.61 0.77 -0.00 shown in the other column shown in the other column shown in the other column sema domain, immunoglobulin domain (Ig), Sema3a 0.64 0.83 1.26 -5.00 shown in the other column shown in the other column shown in the other column short basic domain, secreted, (semaphorin) 3A Pcdh20 protocadherin 20 0.64 0.88 1.37 -3.22 absent absent absent March11 membrane-associated ring finger (C3HC4) 11 0.62 0.72 0.66 -4.09 absent absent absent Parm1 prostate androgen-regulated mucin-like protein 1 0.61 0.64 1.13 -1.91 absent absent absent Bmp5 bone morphogenetic protein 5 0.61 0.72 0.75 0.44 shown in the other column shown in the other column shown in the other column Figf c-fos induced growth factor 0.60 0.82 1.18 -4.93 absent absent absent Ccdc113 coiled-coil domain containing 113 0.60 0.91 1.13 -4.03 absent absent absent Slurp1 secreted Ly6/Plaur domain containing 1 0.59 0.49 1.25 -0.70 absent absent absent Supplementary Table 3

Six2 Log2 Log2 Log2 Log2 Sall1 Six2 Gene Symbol binding (Sall1CreER/flox 48hr) (Sall1CreER/flox 24hr) (Six2Cre; Sall1flox/flox) (Six2 GFP+ vs GFP-) binding binding (Park et al.; ref3)

Progenitor-related -2.8 -0.72 -1.27 1.65 chr8:91,559,382-91,564,748 chr8:91,569,591-91,571,276 chr8:91,910,504-91,914,563 chr8:91,912,350-91,913,805 chr8:91,935,147-91,937,179* chr8:91,935,304-91,937,293* chr8:91,936,103-91,936,284 chr8:91,962,070-91,964,145 chr8:91,938,409-91,940,376 chr8:91,939,275-91,939,431 chr8:91,982,978-91,984,393 Sall1 chr8:92,085,399-92,088,663 chr8:92,085,787-92,088,204 chr8:92,092,399-92,094,667* chr8:92,129,157-92,130,804* chr8:92,094,825-92,096,530 chr8:92,182,190-92,186,278* chr8:92,182,364-92,184,152* chr8:92,130,054-92,130,238 chr8:92,184,846-92,186,022 chr8:92,197,793-92,199,765* chr8:92,198,196-92,199,608* chr8:92,239,090-92,242,534 chr8:92,230,587-92,232,637 -0.52 -0.5 -1.62 5.87 chr17:86,087,020-86,088,664 chr17:86,087,682-86,087,809 Six2 chr17:86,145,975-86,149,207* chr17:86,146,706-86,149,340* chr17:86,147,979-86,148,273 chr17:86,196,249-86,200,295 chr17:86,198,467-86,198,745 0.29 0.17 -0.57 1.22 chr19:44,706,202-44,707,935* chr19:44,705,424-44,708,047* chr19:44,743,902-44,746,867 chr19:44,718,402-44,720,395 Pax2 chr19:44,743,623-44,745,539 chr19:44,827,480-44,829,598 chr19:44,978,154-44,978,284 -0.35 0.09 -1.09 0.04 chr1:14,234,375-14,235,693 chr1:14,235,103-14,235,289 chr1:14,451,499-14,453,661 chr1:14,558,658-14,561,609 chr1:14,560,514-14,560,929 Eya1 chr1:14,577,583-14,579,048 chr1:14,616,816-14,618,109 chr1:14,624,565-14,627,013* chr1:14,624,969-14,626,797* chr1:14,625,742-14,625,927 chr1:14,662,024-14,663,490 chr1:14,706,626-14,706,934 0.15 0.09 -0.49 1.15 chr2:104,863,154-104,864,824* chr2:104,862,758-104,865,023* absent chr2:104,882,731-104,886,149 chr2:104,867,037-104,868,414 Wt1 chr2:104,902,608-104,904,049 chr2:104,928,528-104,931,383 chr2:104,957,226-104,958,678

Cited1 -2.87 -2.18 -4.18 4.8 absent chrX:99514,619-99,515,712 absent -0.99 -0.82 -0.87 5.11 chr12:9,265,136-9,266,406 chr12: 9,583,063-98,855,408 chr12:9,663,412-9,665,066 Osr1 chr12:9,9904852-9,906,011* chr12:10,134,583-10,136,241 chr12:10,141,956-10,143,714 chr12:10,237,565-10,239,324 chr12:10,238,413-10,238,627 -1.52 -1.52 -1.67 5.52 FGF1 absent chr18:39,034,094-39,035,438 chr18:39,034,516-39,034,806 chr18:39,040,772-39,042,700 chr18:39,041,961-39,042,307 FGF2 0.07 0.14 -0.15 -0.47 absent absent absent 0.04 -0.1 -0.68 1.7 FGF9 absent chr14:59,107,122-59,108,879 chr14:59,107,730-59,108,069 chr14:59,134,560-59,136,448 chr14:59,135,528-59,135,796 FGF20 -0.34 -0.4 -0.32 6.85 absent chr8:41,324,122-41,326,059 chr8:41,325,166-41,325,419 -1.08 -0.27 -0.74 1.15 absent chr2:172,667,102-172,668,820 chr2:172,667,933-172,668,149 BMP7 chr2:172,758,882-172,762,510 chr2:172,760,469-172,760,754 Hoxa2 chr6:52,112,980-52,116,365 absent absent Hoxa5 chr6:52,159,318-52,165,129 Hoxa9 chr6:52,175,369-52,182,159 Diffrentiation Wnt9b -0.01 -0.06 0.5 -3.52 absent absent absent Wnt4 0.37 -0.02 -0.71 -1.59 absent chr4:136,777,571-136,774,226 chr4:136,773,187-136,773,353 LEF1 0.19 -0.1 0.29 -4.26 absent absent chr3:130440576-130440757 Dkk1 -2.1 -0.98 -1.73 -5.71 absent absent absent FGF8 0.14 -0.13 -1.05 1.3 absent chr19:45,695,222-45,697,760 chr19:45695446-45695814 Jag1 -0.07 0.06 -0.83 -3.87 absent chr2:136,990,923-136,992,487 absent Notch1 0.16 0.29 -0.75 -0.41 absent chr2:26,396,629-26,398,109 absent Notch2 0.07 0.22 -0.14 0.24 absent chr3:97,740,586-97,742,523 absent Hnf1b -0.14 0.37 -0.82 -3.62 chr11:83,629,065-83,631,720 absent absent Adhesion/repulsion Integrin a3 0.18 0.13 0.05 0.84 absent absent absent Integrin a8 -0.12 -0.31 -0.82 2.28 absent chr2:11,940,289-11,942,124 chr2:11,941,243-11,941,615 -0.26 -0.03 0.05 -0.67 absent chr8:130,948,221-130,949,370 chr8:130,948,920-130,949,219 Integrin b1 chr8:131,011,518-131,013,343 -0.75 -0.57 -1.01 3.42 chr16:73,529,347-73,531,168 chr16:73,530,336-73,530,636 chr16:73,533,609-73,533,909 chr16:74,457,123-74,459,457* chr16:74,457,244-74,459,474* chr16:74,458,499-74,458,753 Robo2 chr16:74,464,261-74,466,231 chr16:74,467,369-74,469,755 chr16:74,513,516-74,516,223* chr16:74,514,010-74,516,020* chr16:74,514,689-74,515,212 Slit2 0.01 -0.05 -0.01 -0.4 chr5:47,947,543-47,949,658 chr5:47,947,543-47,949,658 chr5:47,948,014-47,948,303 Extracellular matrix Nephronectin 0.16 -0.1 -0.84 -2.71 absent absent absent Laminin alpha1 -0.28 0 -1.17 -2.95 absent absent chr17:67982218-67982477 laminin alpha5 0.05 0.07 -0.31 0.03 absent absent absent laminin beta1 0.12 0.14 -0.06 -1.56 absent absent absent laminin beta2 0.22 0.17 0.2 0.04 absent absent absent laminin gamma1 0.08 0.18 -0.22 -1.1 absent absent absent COL4A3 0.94 0.25 0.38 absent absent absent COL4A4 0.45 0.07 0.58 -1.56 absent absent absent COL4A5 0.05 -0.01 0.58 -0.2 absent absent absent Ureteric bud attraction -0.76 -0.66 -0.06 1.11 chr15:7,411,984-7,413,234 chr15:7,412,321-7,412,817 chr15:7,463,342-7,465,268 chr15:7,472,514-7,474,370 Gdnf chr15:7,551,156-7,552,891 chr15:7,562,479-7,563,621 chr15:7,573,709-7,576,260* chr15:7,574,199-7,575,862* chr15:7,647,427-7,648,858 Ret -0.5 -0.25 -0.24 -6.67 absent absent absent Etv4 -0.06 -0.27 -1.22 1.37 absent chr11:101,632,947-101,634,979 chr11:101,633,863-101,634,122 Etv5 0.04 -0.1 -1.3 0.49 absent absent absent Sprouty1 -0.12 -0.15 -0.6 -0.46 absent absent chr3:37,538,702-37,538,895 FGF10 0.04 -0.07 -0.3 2.19 absent absent absent -0.15 0.23 -0.21 -1.06 absent chr14:47,195,029-47,195,717 chr14:47,195,735-47,195,901 BMP4 absent chr14:47,213,357-47,215,049 chr14:47,213,638-47,214,076 absent chr14:47,337,540-47,338,880 chr14:47,338,153-47,338,279 Gremlin -0.43 0.05 -1.38 -0.83 absent absent absent -0.29 -0.32 -1.67 2.85 Kif26b chr1:180,457,002-180,459,602 chr1:180,613,106-180,614,972 chr1:180,613,939-180,614,184 Supplementary Table 4

Primers for ChIP-qPCR Six2-1F 5’-CAG CAG CTG TAT CCA GGT CA-3 Six2-1R 5’-CAA TGG GCA CAT GAA CAA AG-3’ Six2-2F 5’-GGG GTT TAT GGT TCA CAA CG-3’ Six2-2R 5’-GGG AAG AAA AAC CAT GTC CA-3’ Six2-3F 5’-CCA TGA GAG ATT CCC TTC CA-3’ Six2-3R 5’-CCG GAG TAC TGA AGC CAG AG-3’

Primers for qRT-PCR b-actin F 5’-CATCCGTAAAGACCTCTATGCCAAC-3’ b-actin R 5’-ATGGAGCCACCGATCCACA-3’ Sall1 F 5’-TGTCAAGTTCCCAGAAATGTTCCA-3’ Sall1 R 5’-ATGCCGCCGTTCTGAATGA-3’ Six2 F 5’-GCAACTTCCGCGAGCTCTAC-3’ Six2 R 5’-GCCTTGAGCCACAACTGCTG-3’ Osr1 F 5’-ACTGATGAGCGACCTTACACCTG-3’ Osr1 R 5’-ACTTGTGAGTGTAGCGTCTTGTGGA-3’ Cited1 F 5’-GTCGAGGCCTGCACTTGATG-3’ Cited1 R 5’-CCAAGGTTGGAGTAGGCCAGAG-3’ Odz2 F 5’-GAGGTCCCAGATTCACATCAGTCA-3’ Odz2 R 5’-TTTCCATCCAGGCGTTCCA-3’ Nkx6.1 F 5’-AGCACGCTTGGCCTATTCTC-3’ Nkx6.1 R 5’-CACTTGGTCCTGCGGTTCTG-3’

Oligonucleotides for EMSA Six2 consensus Name Sequence Position of Sall1 probe Position of Six2 binding motif GNAACNNNANNC

1 Osr1-Wt-Fw 5’-GTATATATTATCCTGCAAAAGAAAAGCATAAC-3’ 394bp up stream of Sall1 chr12:9,905,826-9,905,856 chr12:9,905,421-9,905,432 GGAACCTGACAC 2 Osr1-Mt-Fw 5’-GTATATATTgTCCTGCAAAAGAAAAGCAcAAC-3’ binding motif

3 Six2-Wt-Fw 5’-CTGGAGAAATAAAACTGAGTTATTGTTTCC-3’ 366bp down stream of chr17:86,147,858-86,147,887 chr17:86,148,253-86,148,264 GAAACCCGACCC 4 Six2-Mt-Fw 5’-CTGGAGAAAcAAAACTGAGTTgTTGTTTCC-3’ Sall1 binding motif

5 Eya1-Wt-Fw 5’-CATAAATAGAGTAAATACATTTAATTTTCATTTCTATATTATC-3’ 257bp down stream of chr1:14,625,520-14625562 chr1:14,625,819-14,625,830 GTAACTACTCCC 6 Eya1-Mt-Fw 5’-CAccAAcAGAGTAggTACATTTggccTTCATccCTATATggTC-3’ Sall1 binding motif

7 Robo2-Wt-Fw 5’-CAGATAGTTTCATAATGAAATTTTCTTTTATGTTGAATCC-3’ GTTTGAGTTAC 435bp down stream of chr16:74,514,526-74,514,565 chr16:74,515,053-74,515,063 8 Robo2-Mt-Fw 5’-CAGATAGccTCAcAATGGGATTcTCTTctgTGTTGggTCC-3’ (complementary) Sall1 binding motif

9 Megf9-Wt-Fw 5’-GGCACTAAATAAAACGGCAAAGTCATAAAAATTTAATTTG-3’ GTTTTCAGTTCC 349bp down stream of chr4:70,150,276-70,150,315 chr4:70,150,664-70,150,675 10 Megf9-Mt-Fw 5’-GGCACTAgAcAggACGGCAAAGTCAcAccAATcTccTTTG-3’ (complementary) Sall1 binding motif

11 Cited1-Wt-Fw 5’-TTACAAGCAATTAAAATACACTAAAATAGATTTATACAGG -3’ 42bp up stream of Sall1 chrX:99,515,395-99,515,434 chrX:99,515,343-99,515,353 GGAAATGACTC 12 Cited1-Mt-Fw 5’-TTACAAGCAAccAggATACACTAggATAGAggTcTACAGG -3’ binding motif