Islet1 Deletion Causes Kidney Agenesis and Hydroureter Resembling CAKUT

BASIC RESEARCH www.jasn.org

† ‡ Yusuke Kaku,* Tomoko Ohmori,* Kuniko Kudo,* Sayoko Fujimura, Kentaro Suzuki, | Sylvia M. Evans,§ Yasuhiko Kawakami, and Ryuichi Nishinakamura*

*Department of Kidney Development and †Liaison Laboratory Promotion Facility, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; ‡Department of Developmental Genetics Research, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan; §Skaggs School of Pharmacy and Department of Medicine, University of California, San Diego, La Jolla, California; and |Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota

ABSTRACT Islet1 (Isl1) is a transcription factor transiently expressed in a subset of heart and limb progenitors. During studies of limb development, conditional Isl1 deletion produced unexpected kidney abnormalities. Here, we studied the renal expression of Isl1 and whether it has a role in kidney development. In situ hybridization revealed Isl1 expression in the mesenchymal cells surrounding the base of the ureteric bud in mice. Conditional deletion of Isl1 caused kidney agenesis or hypoplasia and hydroureter, a phenotype resembling human congenital anomalies of the kidney and urinary tract (CAKUT). The absence of Isl1 led to ectopic branching of the ureteric bud out from the nephric duct or to the formation of accessory buds, both of which could lead to obstruction of the ureter-bladder junction and consequent hydroureter. The abnormal elongation and poor branching of the ureteric buds were the likely causes of the kidney agenesis or hypoplasia. Furthermore, the lack of Isl1 reduced the expression of Bmp4, a gene implicated in the CAKUT-like phenotype, in the metanephric region before ureteric budding. In conclusion, Isl1 is essential for proper development of the kidney and ureter by repressing the aberrant formation of the ureteric bud. These observations call for further studies to investigate whether Isl1 may be a causative gene for human CAKUT.

J Am Soc Nephrol 24: 1242–1249, 2013. doi: 10.1681/ASN.2012050528

The mammalian kidney, the metanephros, is mechanical obstruction of the ureter or malforma- formed by reciprocally inductive interactions be- tion of the smooth muscle layer causes dilatation of tween two precursor tissues: the metanephric mes- the ureter (hydroureter) and, in more severe cases, enchyme and the ureteric bud. The metanephric of the renal pelvis (hydronephrosis). The combi- mesenchyme attracts the ureteric bud tips toward nation of kidney and ureter defects caused by the mesenchyme and subsequently induces branch- gene mutations is termed congenital anomalies ing of the ureteric buds. The attracted ureteric bud of the kidney and urinary tract (CAKUT) and tips in turn induce the mesenchymal cells to differentiate into the epithelia of the glomeruli and renal tubules. Impairment of these processes Received May 28, 2012. Accepted March 24, 2013. can lead to a variety of abnormal developmental Y.K. and T.O. contributed equally to this work. disorders of the kidney. Meanwhile, the stalks of the Published online ahead of print. Publication date available at ureteric buds elongate and differentiate into col- www.jasn.org. lecting ducts and the ureter. The ureteric epithelium Correspondence: Prof. Ryuichi Nishinakamura, Department of also interacts with the surrounding ureteral mes- Kidney Development, Institute of Molecular Embryology and enchyme, which differentiates into the smooth Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860- muscle layer of the ureter that pushes the urine 0811, Japan. Email: [email protected] downward by peristaltic movements. Therefore, Copyright © 2013 by the American Society of Nephrology

1242 ISSN : 1046-6673/2408-1242 JAmSocNephrol24: 1242–1249, 2013 www.jasn.org BASIC RESEARCH constitutesa majorcause ofrenalfailure in the perinatalperiod in humans.1 Bmp4 is expressed in the mesenchyme surrounding the ureteric stalk, and Bmp4 heterozygous mutant mice display abnormalities that mimic human CAKUT, including hypo- plastic kidneys and hydroureter.2 These mice exhibit ectopic or accessory ureteric bud formation, which results in misconnection of the ureter to the urinary bladder or duplicated ureters. It has been proposed that Bmp4 has dual functions, namely inhibition of ectopic budding of the ureteric tips and promotion of ureteric stalk elongation,2 although the pre- cise mechanisms remain unknown. Shh is expressed in the ureteric epithelium, and its deletion results in reduced expression of Bmp4 in the surrounding mesenchyme and eventually hydroureter.3 The transcription factors Tbx18 and Sox9 are expressed in the ureteric mesenchyme, and their deletion also leads to hydroureter resulting from impaired development of the smooth muscle layer of the ureter.4,5 In this study, we identify Islet1 (Isl1; Mouse Genome In- formatics), which encodes an LIM-homeodomain protein, as a key regulator of kidney and ureter development. Mice deficient for Isl1 die by embryonic day (E) 11.5 and exhibit developmental arrest of motor neurons.6 Isl1 is transiently expressed in a subset of heart and limb progenitors and is re- quired for the normal development of these tissues.7,8 In the course of conditional Isl1 deletion in the lateral mesoderm using Hoxb6Cre for analysis of limb development,9 kidney abnormalities were observed. This finding was unexpected because no previous literature has reported Isl1 expression in the developing kidney to the best of our knowledge. There- fore, we examined the expression and roles of Isl1 in kidney development in detail. Figure 1. Isl1 is transiently expressed in the mesenchyme surrounding the kidney primordia. (A–D) In situ hybridization of Isl1 RESULTS during kidney development. Note that Isl1 is expressed in the mesenchyme surrounding the ureteric buds until E11.5 but rap- Isl1 Is Transiently Expressed in the Mesenchyme idly decreases thereafter. The signal at E14.5 is absent except for Surrounding the Ureteric Bud Stalk the preaortic sympathetic ganglia (*). (E) Whole-mount in situ First, we examined the expression patterns of Isl1 during kid- hybridization of Isl1 in the E14.5 kidney. (F–H) Immunostaining for Hoxb6Cre ney development by in situ hybridization. At E10.5, Isl1 was Isl1 using an anti-Isl1 antibody. (I and J) Cre activity in Hoxb6Cre;tdTomato expressed in tissues surrounding the cloaca but was excluded mice. Sections of reporter mice were stained with an anti-RFP antibody. Note the significant overlap of from the kidney primordia, comprising the metanephric mes- Isl1 and Hobx6Cre expression. Black arrowheads, ureteric bud enchyme and nephric duct (Figure 1A). At E11.5, a similar stalk; white arrowheads, ureter; cl, cloaca; im, intermediate me- Isl1 expression pattern was observed, in that expression was soderm; kid, kidney; lpm, lateral plate mesoderm; mm, meta- detected in the ureteric mesenchyme surrounding the ureteric nephric mesenchyme; nd, nephric duct. Scale bars: 100 mm. bud stalk and cloaca but was not expressed in the metanephric mesenchyme or ureteric bud epithelia (Figure 1B). Subse- quently, Isl1 expression was rapidly downregulated at E12.5 weakly in the lateral and intermediate mesoderm at E9.5 (Figure 1C), and no Isl1 expression was observed in the kid- and robustly in the ureteric mesenchyme at E11.5. (Figure 1, ney proper or the ureter at E14.5 (Figure 1D). These obser- G and H). vations were confirmed by whole-mount in situ hybridiza- Next, the Cre recombinase activity of Hoxb6Cre mice was tion, showing no Isl1 signals in the kidney or ureter at E14.5 examined. This mouse strain is often used for deletion of genes (Figure 1E). Staining with an anti-Isl1 antibody confirmed in lateral mesoderm-derived tissues, such as the limb buds.10 these observations, including the negative expression in the To examine the Cre activity in the developing kidney, ureter at E14.5 (Figure 1F). Nuclear Isl1 staining was observed we crossed Hoxb6Cre mice with a reporter strain, in which

J Am Soc Nephrol 24: 1242–1249, 2013 Isl1 Knockout Causes CAKUT 1243 BASIC RESEARCH www.jasn.org the CAG promoter, stop sequences ﬂanked by loxP sites, and the tandem dimer Tomato (tdTomato) are inserted into the Rosa26 locus.11 Owing to the presence of the potent CAG promoter, this mouse strain serves as a very sensitive reporter for Cre recombinase activity. When these mice were crossed with Hoxb6Cre mice, the tdTomato signals were detected mainly in the lateral mesoderm and weakly in the intermediate mesoderm at E9.5 (Figure 1I), which is consistent with the reported Cre activity.10 At E11.5, Cre activity was broadly detected, except for the ureteric bud epithelia (Figure 1J). Although the activity in the metanephric mesenchyme was mosaic, the regions with Cre activity cover all the Isl1-positive populations described above. Thus Hoxb6Cre mice are useful for efﬁcient deletion of Isl1 during kidney development. The non-nuclear signal in the ureteric epithelium in Figure 1H, which is not consistent with the data obtained by in situ hybridization, could result from background staining of the antibody. In addition, the cryptic Isl1 expression in the ureteric epithelium does not affect our analysis because Hobx6Cre does not delete genes in this population.

Isl1 Deletion Causes Kidney Agenesis or Hydroureter: A CAKUT-Like Phenotype flox/flox All the Hoxb6Cre;Isl1 mice died shortly after birth flox/flox flox/+ flox/+ Figure 2. Isl1 deletion causes kidney agenesis or hydroureter: (n=11), while Isl1 , Isl1 ,andHoxb6Cre;Isl1 flox/flox a CAKUT-like phenotype. (A) Kidneys in a newborn Isl1 mice were apparently normal (n=9, n=13, and n=21, respec- mouse (P0). ad, adrenal gland; bl, bladder; kid, kidney; ov, ovary. flox/flox tively). Regarding the 22 presumptive urogenital systems in (B) Bilateral kidney agenesis in a Hoxb6Cre;Isl1 mouse (P0). Hoxb6Cre;Isl1flox/flox the 11 mutant mice, 11 (50%) showed (C) Unilateral kidney agenesis accompanied by hydroureter (*) flox/flox complete kidney agenesis (Figure 2, A and B), 10 (45.5%) and hydronephrosis (**) in a Hoxb6Cre;Isl1 mouse (P0). (D exhibited hydronephrosis and hydroureter (Figure 2C), and and E) Hematoxylin-eosin staining of E16.5 kidneys. Severe ure- flox/flox one (4.5%) had normal-sized kidneys. At E16.5, 50% of the ter dilatation (*) is observed in the Hoxb6Cre;Isl1 mouse. kidneys examined (three of six) were absent in Hoxb6Cre; The mutant kidney (**) is squashed. (F and G) Immunostaining for flox/flox Isl1 mice, and dilatation of the pelvis and ureter all along smooth muscle actin (SMA) in the ureter. Note that the mutant the tract was observed in the remaining three kidneys (Fig- ureteric wall is thinner but expresses SMA (white arrowheads). (H a ure 2, D and E). The mutant ureter had a thinner wall but and I) Immunostaining for Sm22 in the ureter at E16.5. (J and K) a retained the smooth muscle layer, which was confirmed by Immunostaining for Sm22 in the ureteral mesenchyme at E14.5. (L and M) In situ hybridization of Myocardin in the ureteral mes- immunostaining for smooth muscle actin (Figure 2, F and enchyme at E14.5. Scale bars: 100 mm. G). The mutant smooth muscle layer was also positive for Sm22a, a maturation marker for this ureteric mesenchyme- derived lineage (Figure 2, H–K). Myocardin,anotherdif- expression in the kidney proper was significantly reduced ferentiation marker expressed earlier than Sm22a,was (Figure 3B). At E14.5, Isl1 was still expressed, albeit weakly, also detected in the Isl1 mutants (Figure 2, L and M), in- at the ureterovesical junction (Figure 3C), as well as in the dicating that maturation of the ureteral mesenchyme was region along the urethra (Figure 3D). unaffected in the absence of Isl1. Therefore, the hydroureter Ink injected into the pelvis filled up the dilated pelvis and is unlikely to result from developmental impairment of the ureters, but did not reach the urinary bladder in any of the ureteric mesenchyme and could be caused by other mecha- newborn mutants (n=4), suggesting the existence of urinary nisms, such as mechanical obstruction in the lower urinary tract obstruction (Figure 3, E–G). We also observed duplicated tract. ureters in one mutant, as shown by negative ink flow in the second ureter (Figure 3G). Serial sectioning of the ure- The Ureter Is Not Properly Connected to the Urinary ter-bladder (ureterovesical) junctions of these samples, as well Bladder in the Absence of Isl1 as those at E14.5, revealed that the ureters failed to reach the We then focused on Isl1 expression in the lower urinary tract. urinary bladder and ended blindly in nine of 11 Isl1 mutant Isl1 was robustly expressed along the nephric duct, at the base mice examined (Figure 3, H and I). In addition, we found of the ureteric stalk, and in the genital tubercle at E11.5 (Figure impaired formation of the urethra in a subset of the mutants 3A). This expression was retained at E12.5 when the (three of nine), all of which had the abnormal ureterovesical

1244 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 1242–1249, 2013 www.jasn.org BASIC RESEARCH

mesenchyme and the ureteric bud epithelia (Figure 4, C and D). Isl1 was expressed in the ureteric mesenchyme surrounding the base of the ureteric bud stalk and the nephricductinthewild-typekidneys (Figure 4E; see also Figures 1 and 3A), which was abolished in the mutant kidneys (Figure 4F). However, some mutant kidneys showed residual Isl1 expression, as evaluated by immunostaining, probably owing to the mosaic deletion of Hoxb6Cre as reported previously,9 which could partly explain the variable kidney sizes at E14.5. If the kidney is formed to some extent, hydronephrosis and/or hydroureter could be a prominent phenotype owing to the obstruction at the ureterovesical junction. Tbx18 and Sox9 were expressed in the ureteric mesenchyme at this stage, and Figure 3. The ureter is not properly connected to the urinary bladder in the absence of their expression levels were unaffected Isl1. (A and B) Whole-mount in situ hybridization of Isl1 at E11.5 (A) and 12.5 (B). (C and D) Section in situ hybridization of Isl1 at the E14.5 ureterovesical junction (C) and in the mutant kidneys, although the ex- along the urethra (D). (E–G) Antegrade ink injection into the pelvis. Note that the ink in pression domains were slightly narrower the mutant does not reach the bladder. Duplicated ureters are shown in G. *Second (Figure 5, A–D). We also found that ureter is not filled with ink. (H and I) Hematoxylin-eosin (HE) staining of the ureter- these two genes were expressed in the ovesical junctions at E14.5. The mutant ureter (white arrowhead) fails to connect to the ureteric mesenchyme close (or proximal) bladder. (J and K) HE staining of male urethral regions at E14.5. (L and M) HE staining to the metanephric mesenchyme, while of the E14.5 kidneys. The mutant kidney is reduced in size. Black arrowheads, ure- Isl1 was distributed in the distant (or dis- terovesical junctions; white arrowheads, blunt-ended ureter; bl, bladder; gt, genital tal) domains at the base of the ureteric tubercle; kid, kidney; mm, metanephric mesenchyme; nd, nephric duct. Scale bars: stalksandaroundthenephricduct,al- m 100 m. though these two domains overlapped to some extent. We confirmed these obser- junctions (Figure 3, J and K). Therefore, the hydroureter in the vations by whole-mount in situ hybridization (Figure 5, Isl1 mutants is mainly caused by impaired connection of the E–H). ureter to the bladder. Serial sectioning of the mutant ureteric buds suggested that However, at E14.5, when no hydronephrosis became the direction of the buds might be altered (see Figures 4B and apparent because of the negligible urine production, the 5D). Therefore, we visualized the ureter branching by crossing kidney size ranged from normal to complete agenesis among the Isl1 mutants with Hoxb7GFP mice. At E12.5, while the 10 kidneys examined (two agenesis, four hypoplasia, four control ureteric stalks were connected close to the urogenital normal size; see Figure 3, L and M, for hypoplasia), suggesting sinus, the mutant ureteric bud branched poorly and was still that the development of the kidney proper was also impaired connected to the common nephric duct (Figure 4, G and H). independently of the urinary tract obstruction. Because we did The mutant ureter on the other side failed to bud. At E11.5, the not detect any defects in metanephric mesenchymal genes, ureteric bud elongated from the nephric duct and branched such as Six2 or Cited1, in the mutant kidneys, we searched into the metanephric mesenchyme in the control mice (Figure for additional abnormalities. 4I). In the absence of Isl1, however, the ureteric budding oc- curred ectopically, as shown by a longer common nephric Ectopic or Accessory Ureteric Buds Are Formed in the duct, in three kidneys examined (Figure 4J). We also found Absence of Isl1 an accessory ureteric bud in one mutant kidney (Figure 4K). At E11.5, the ureteric buds became elongated and invaded into Whole-mount in situ hybridization of Pax2 using additional the metanephric mesenchyme in the wild-type kidneys (Figure samples confirmed these observations (Figure 4, L–N). Seven 4A). In contrast, the mutant kidneys showed variable pheno- of nine kidneys examined at E11.5 showed cranially posi- types at this step. Overall, 50% of the kidneys examined (6 of tioned and poorly branched ureteric buds (Figure 4M), 12) showed impaired elongation of the ureteric buds (Figure while two exhibited accessory buds (Figure 4N). These defects 4B), while the remaining buds reached the metanephric mes- (i.e., ectopically positioned or accessory ureteric buds) could enchyme. In the former, apoptosis was observed in the ureteric lead to misconnection of the ureterovesical junction or

J Am Soc Nephrol 24: 1242–1249, 2013 Isl1 Knockout Causes CAKUT 1245 BASIC RESEARCH www.jasn.org

(n=4) (Figure 6F). Similarly, we observed Bmp4 reduction in all of the Isl1 mutants examined at E11.5 (n=8), while Bmp4 was expressed along the nephric duct and around the ureteric bud stalk in the control mice (Figure 1, G and H). This reduction is consistent with the similar CAKUT-like phenotypes in both Isl1 and Bmp4 mutant mice. However, BMP4 supplementation to the organ cultures starting at E10.5 did not signiﬁcantly rescue the Isl1 mutant phenotypes, which are variable in nature (Sup- plementary Figure 1). BMP4 addition inhibited ureteric budding and metanephros development even in control kidneys as reported previously,2 while lower concentrations had minimal effects on the Isl1 mutants. It is possible that strict spatio- temporal regulation of BMP activity is necessary for proper ureteric budding, and this cannot be mimicked faithfully Figure 4. Ectopic or accessory ureteric buds are formed in the absence of Isl1. (A and B) Hematoxylin-eosin staining of E11.5 kidneys. The ureteric bud tips invade into the by exogenous BMP4 addition. Indeed, metanephric mesenchyme in the wild-type kidney, but not in the mutant kidney. (C there are no reports of successful rescue and D) Immunostaining for cleaved caspase 3. Positive signals are detected in the of the kidney phenotypes even in Bmp4 ureteric mesenchyme and also in the ureteric bud stalk (black arrowhead). (E and F) heterozygotes. Although Bmp4-indepen- Immunostaining for Isl1. Isl1 expression around the ureteric stalk and the nephric duct dent mechanisms could also be involved, is absent in the mutant kidney. (G and H) Visualization of ureteric branching at E12.5 the phenotypic similarities between the using Hoxb7GFP. The mutant ureter (*) is still connected to the nephric duct, and not Isl1 and Bmp4 mutants, and Bmp4 reduc- to the urogenital sinus (***). No ureteric bud exists on the other side. (I–K) Ureteric tion in the Isl1 mutants, suggest that Isl1 is buds at E11.5. Note the poor branching of the ureteric bud and longer common essential for urogenital development, at nephric duct in the mutant (bidirectional arrows). An accessory ureteric bud (**) is least partly by regulating Bmp4. observed in K. (L–N) Whole-mount in situ hybridization of Pax2, showing staining of both the ureteric buds and the metanephric mesenchyme. Ectopic and accessory (**) ureteric buds are observed in M and K, respectively. Black arrowhead, ureteric bud stalk; white arrowhead, ureteric bud tip; white arrow, nephric duct; kid, kidney; mm, DISCUSSION metanephric mesenchyme. Scale bars: 100 mm. We have shown that Isl1 deletion results in kidney agenesis/hypoplasia and hydro- duplicated ureters and eventually hydroureter. The subse- ureter that resembles CAKUT. Isl1 is expressed in the ureteric quent poor branching of the ureteric buds is likely to cause mesenchyme at the base of the ureteric buds and is subse- the kidney agenesis/hypoplasia observed in the Isl1 mutants. quently maintained in the lower urinary tract. In the absence of Isl1, the ureteric budding occurs ectopically along the neph- Bmp4 Expression Is Reduced in the Absence of Isl1 ric duct or duplicated ureters are formed, both of which could At E10.5, a stage before the ureteric bud is formed, a bulge was lead to the impaired connection at the ureterovesical junction observed in the nephric duct in the presumptive metanephric and subsequent hydroureter. Because Isl1 is not expressed in region of the control mice (Figure 6A), which was not apparent the ureteric bud epithelia or the metanephric mesenchyme, its in the Isl1 mutants (Figure 6B). The size of the Pax2-positive action should be non–cell-autonomous. It is well established metanephric mesenchyme in the Isl1 mutants was similar to that Bmp4 reduction causes CAKUT-like phenotypes. Bmp4 that in the control mice (Figure 6, C and D). heterozygous mice show a variety of kidney abnormalities, Mice heterozygous for Bmp4 showed variable kidney sizes including kidney hypoplasia, and hydroureter.2 Bmp4 inhibits and hydroureter resulting from ectopic or accessory ureteric ureteric budding, and its deletion leads to ectopic or accessory buds,2 similar to the phenotypes of the Isl1 mutant mice. At ureteric bud formation. Our data indicate that Isl1 deletion E10.5, Bmp4 was expressed along the nephric duct and around results in reduced Bmp4 expression, thereby mimicking the the metanephric mesenchyme (Figure 6E). Bmp4 expression, Bmp4 heterozygous mutant phenotypes. especially in the presumptive metanephric region, was signif- Although Isl1 is weakly expressed in the intermediate icantly reduced in all of the Isl1 mutant kidneys examined mesoderm at E9.5, we did not detect apparent abnormalities in

1246 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 1242–1249, 2013 www.jasn.org BASIC RESEARCH

Figure 6. Bmp4 expression is reduced in the absence of Isl1. Figure 5. Tbx18 and Sox9 expression in the ureteric mesen- Whole-mount in situ hybridization is shown. (A and B) Ret ex- chyme is unaffected in the absence of Isl1.(AandB)In situ hy- pression at E10.5 shows the absence of the bulge in the nephric bridization of Tbx18. Tbx18 is expressed in the ureteric mesen- duct in the Isl1 mutant. (C and D) Pax2 is expressed not only in chyme close to the metanephric mesenchyme, but not around the nephric duct but also in the metanephric mesenchyme at the nephric duct. (C and D) Immunostaining for Sox9. Sox9 is E10.5. (E and F) Bmp4 expression is reduced, especially in the expressed in the ureteric mesenchyme close to the metanephric metanephric region, in the Isl1 mutant at E10.5. (G and H) Bmp4 mesenchyme. Sox9 is also expressed in the ureteric bud tips at expression is also reduced at E11.5. White arrows, nephric duct; this stage. (E–H) Whole-mount in situ hybridization of Ret, Tbx18, *metanephric mesenchyme. Scale bars: 100 mm. Isl1,andBmp4 in the wild-type kidneys at E11.5. Ret is expressed along the nephric duct and in the ureteric bud, and is most Tbx18 abundant in the ureteric bud tips. is broadly expressed in and Bmp4 is conserved, yet distinct, in the development of the ureteric mesenchyme close to the ureteric bud tip. In con- Isl1 Bmp4 different organs. trast, and are expressed around the base of the ure- Isl1 teric bud stalk (yellow arrowheads). White arrowhead, ureteric However, there should also be -independent regulation Bmp4 Bmp4 bud tip; white arrow, nephric duct; mm, metanephric mesen- of because expression is not completely abolished chyme. Scale bars: 100 mm. in the absence of Isl1. In addition, the expression of these two genes does not completely overlap. For example, Isl1 is not expressed in the ureteric mesenchyme at E14.5, while Bmp4 the metanephric mesenchyme before ureteric budding. There- is expressed and plays a major role in the development of this fore, kidney agenesis or hypoplasia caused by the absence of Isl1 tissue.3 It was recently reported that Bmp4 deletion in mice could mainly be explained by impaired elongation and poor using Isl1Cre causes lethality during mid-gestation with more branching of the ureteric buds. Indeed, ureteric bud-speciﬁc severe phenotypes than the Isl1 mutants, including abnormal deletion of the BMP receptor ALK3 (Bmpr1a)alsoleadsto development of the lower limbs and pelvis, as well as kidney kidney hypoplasia.12 and bladder agenesis.14 Isl1Cre-mediated gene deletion oc- AlinkbetweenIsl1 and Bmp4 has also been reported in the curs in all cells that express Isl1 at least once. Indeed, when development of other organs. In tooth development, Isl1 and Isl1Cre was bred into the indicator strain, Isl1Cre activity at Bmp4 are coexpressed in the incisor epithelium, and their E14.5 was found in most of the urogenital tissues except for expression is interdependent.13 Inhibition of Isl1 expression the ureteric epithelia, including the metanephric and ure- causes a reduction in Bmp4 and a corresponding loss of Msx1 teral mesenchyme, bladder, and urethra (Supplementary in the underlying mesenchyme. Isl1 also marks a subset of Figure 2). Therefore, Isl1-independent Bmp4 expression in cardiac progenitors, and Bmp4 and Bmp7 are expressed in these tissues should also play important roles in urogenital regions that overlap with or are adjacent to Isl1-expressing development. cells.8 Isl1Cre-dependent deletion of Bmpr1a leads to abnor- Isl1 has not been considered as a candidate gene for the mal heart and limb development. Thus, the interaction of Isl1 CAKUT phenotype, partly because its expression is excluded

J Am Soc Nephrol 24: 1242–1249, 2013 Isl1 Knockout Causes CAKUT 1247 BASIC RESEARCH www.jasn.org from the major part of the kidney primordia, such as the This study was supported in part by Grants-in-Aid from the metanephric mesenchyme and ureteric buds. To the best of Ministry of Education, Culture, Sports, Science and Technology our knowledge, mutations in human ISL1 have been reported (MEXT) and by the Global COE Program (Cell Fate Regulation Re- in a family with type 2 diabetes,15 but not in CAKUT. Because search and Education Unit), MEXT, Japan. the present data clearly indicate that mutations in Isl1 can cause a CAKUT-like phenotype, human ISL1 mutations should be examined in a subset of patients who have CAKUT, DISCLOSURES with or without diabetes. None.

CONCISE METHODS REFERENCES

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Yu J, Carroll TJ, McMahon AP: Sonic hedgehog regulates proliferation cordance with our institutional guidelines and ethical review com- and differentiation of mesenchymal cells in the mouse metanephric kidney. Development 129: 5301–5312, 2002 mittees. Mice carrying the Cre allele were genotyped with forward 4. Airik R, Bussen M, Singh MK, Petry M, Kispert A: Tbx18 regulates the 9 9 primer Cre 1 (5 -AGG TTC GTT CAC TCA TGG A-3 ) and reverse development of the ureteral mesenchyme. JClinInvest116: 663–674, primer Cre 2 (59-TCG ACC AGT TTA GTT ACC C-39). PCR ampli- 2006 fications were performed under identical conditions using GoTaq 5. Airik R, Trowe M-O, Foik A, Farin HF, Petry M, Schuster-Gossler K, DNA polymerase (Promega), with denaturation at 95°C for 5 mi- Schweizer M, Scherer G, Kist R, Kispert A: Hydroureternephrosis due to loss of Sox9-regulated smooth muscle cell differentiation of the ureteric nutes, 35 cycles of 95°C for 30 seconds, 58°C for 60 seconds, and mesenchyme. Hum Mol Genet 19: 4918–4929, 2010 fi 72°C for 30 seconds, and a nal extension at 72°C for 7 minutes. The 6. Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM: Requirement PCR products were analyzed by electrophoresis in a 1.2% agarose gel for LIM homeobox gene Isl1 in motor neuron generation reveals a and visualized by ethidium bromide staining. motor neuron-dependent step in interneuron differentiation. Cell 84: 309–320, 1996 fi In situ 7. Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J, Evans S: Isl1 identi es a Hybridization and Immunostaining cardiac progenitor population that proliferates prior to differentiation Histologic examinations were performed as described pre and contributes a majority of cells to the heart. Dev Cell 5: 877–889, viously.19,20 Mice were fixed in 10% formalin, embedded in paraffin, 2003 and cut into 6-mm sections. In situ hybridization was performed using 8. Yang L, Cai C-L, Lin L, Qyang Y, Chung C, Monteiro RM, Mummery CL, an automated Discovery System (Roche) according to the manufac- Fishman GI, Cogen A, Evans S: Isl1Cre reveals a common Bmp pathway in heart and limb development. Development 133: 1575–1585, 2006 turer’s protocols. The probe for Bmp4 was a kind gift from Dr. N. Ueno 9. Itou J, Kawakami H, Quach T, Osterwalder M, Evans SM, Zeller R, (National Institute for Basic Biology, Japan). Templates for other Kawakami Y: Islet1 regulates establishment of the posterior hindlimb probes were generated by RT-PCR and sequenced. Immunostaining field upstream of the Hand2-Shh morphoregulatory gene network in was carried out automatically using a BlueMap Kit (Roche) and the mouse embryos. Development 139: 1620–1629, 2012 Discovery System. The following primary antibodies were used: 10. Lowe LA, Yamada S, Kuehn MR: HoxB6-Cre transgenic mice express Cre recombinase in extra-embryonic mesoderm, in lateral plate and anti-Isl1 (Abcam); anti–smooth muscle actin (Abcam); anti- limb mesoderm and at the midbrain/hindbrain junction. Genesis 26: a Sm22 (Abcam); anti-pSmad1/5/8 (Cell Signaling Technology); 118–120, 2000 anti-Sox9 (Millipore); anti–cleaved caspase 3 (Cell Signaling Tech- 11. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng nology); anti-RFP (Rockland); and anti–cytokeratin 8 (Develop- LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H: A robust and mental Studies Hybridoma Bank). For whole-mount in situ hybrid- high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13: 133–140, 2010 ization, samples were fixed with 4% paraformaldehyde and 12. Di Giovanni V, Alday A, Chi L, Mishina Y, Rosenblum ND: Alk3 controls processed using an automated InsituPro VS (Intavis AG) according nephron number and androgen production via lineage-specificeffects to the manufacturer’sprotocol. in intermediate mesoderm. Development 138: 2717–2727, 2011 13. Mitsiadis TA, Angeli I, James C, Lendahl U, Sharpe PT: Role of Islet1 in the patterning of murine dentition. Development 130: 4451–4460, 2003 ACKNOWLEDGMENTS 14. Suzuki K, Adachi Y, Numata T, Nakada S, Yanagita M, Nakagata N, Evans SM, Graf D, Economides A, Haraguchi R, Moon AM, Yamada G: Reduced BMP signaling results in hindlimb fusion with lethal pelvic/ We thank G. Yamada for helpful discussions and S. Usui for technical urogenital organ aplasia: a new mouse model of sirenomelia. PLoS assistance. ONE 7: e43453, 2012

1248 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 1242–1249, 2013 www.jasn.org BASIC RESEARCH

15. Shimomura H, Sanke T, Hanabusa T, Tsunoda K, Furuta H, Nanjo K: 19. Fujimura S, Jiang Q, Kobayashi C, Nishinakamura R: Notch2 activation Nonsense mutation of islet-1 gene (Q310X) found in a type 2 diabetic in the embryonic kidney depletes nephron progenitors. JAmSoc patient with a strong family history. Diabetes 49: 1597–1600, 2000 Nephrol 21: 803–810, 2010 16. Sun Y, Dykes IM, Liang X, Eng SR, Evans SM, Turner EE: A central role 20. Uchiyama Y, Sakaguchi M, Terabayashi T, Inenaga T, Inoue S, for Islet1 in sensory neuron development linking sensory and spinal KobayashiC,OshimaN,KiyonariH,NakagataN,SatoY,SekiguchiK, gene regulatory programs. Nat Neurosci 11: 1283–1293, 2008 Miki H, Araki E, Fujimura S, Tanaka SS, Nishinakamura R: Kif26b, 17. Song MR, Sun Y, Bryson A, Gill GN, Evans SM, Pfaff SL: Islet-to-LMO a kinesin family gene, regulates adhesion of the embryonic kid- stoichiometries control the function of transcription complexes that ney mesenchyme. Proc Natl Acad Sci U S A 107: 9240–9245, specify motor neuron and V2a interneuron identity. Development 136: 2010 2923–2932, 2009 18. Srinivas S, Goldberg MR, Watanabe T, D’Agati V, al-Awqati Q, Costantini F: Expression of green ﬂuorescent protein in the ureteric bud of transgenic mice: A new tool for the analysis of ureteric bud mor- This article contains supplemental material online at http://jasn.asnjournals. phogenesis. Dev Genet 24: 241–251, 1999 org/lookup/suppl/doi:10.1681/ASN.2012050528/-/DCSupplemental.

J Am Soc Nephrol 24: 1242–1249, 2013 Isl1 Knockout Causes CAKUT 1249 Supplemental Figure 1

A Isl1flox/flox Hoxb6Cre;Isl1flox/flox

) -

BMP4 ( BMP4 *

BMP4 (+) BMP4 *

B Isl1flox/flox Hoxb6Cre;Isl1flox/flox

) -

BMP4 ( BMP4 *

* BMP4 (+) BMP4 Supplemental Figure 2 Isl1Cre; indicator A B

C D Supplemental Figure 1. BMP4 addition to Isl1 mutant kidneys in organ culture. Each side of the metanephric region at E10.5 was isolated and cultured on a filter at the air/liquid interface for 24 hours, and stained with an anti-cytokeratin 8 antibody to visualize the ureteric buds. (A) BMP4 (1 ng/ml) was added to the culture medium. Four control kidneys and five Isl1 mutant kidneys were used for each condition. (B) Affi-Gel Blue beads (Bio-Rad) soaked for 1 hour in BMP4 (5 g/ml) or albumin (negative control) were placed close to the presumptive ectopic budding sites of the nephric ducts, as shown by dotted circles (six kidneys in each condition). Ureteric budding was impaired or accessory buds (**) were formed in the Isl1 mutants, irrespective of BMP4 treatment. Beads soaked in BMP4 (0.5 g/ml) showed similar results (four kidneys in each group). While the indicated concentrations of BMP4 had minimal effects on the control kidneys, higher concentrations of BMP4 inhibited ureteric budding in both the controls and the Isl1 mutants. When the BMP4-soaked beads were placed on the metanephric side of the nephric duct, we observed more inhibitory effects. White arrowhead: ureteric bud tip; white arrow: nephric duct; *: urogenital sinus. Scale bars: 100 m.

Supplemental Figure 2. Cre activity in Isl1Cre mice. Sections of Isl1Cre;tdTomato reporter mice at E14.5 were stained with an anti-RFP antibody. The signals are observed in the metanephric and ureteral mesenchyme (A), ureteric mesenchyme at the distal ureter (B), urinary bladder (C), and genital tubercle (D). No Cre activity is detected in the ureteric epithelia. Scale bars: 100 m.