BASIC RESEARCH www.jasn.org Islet1 Deletion Causes Kidney Agenesis and Hydroureter Resembling CAKUT † ‡ 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 hybridiza- tion 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 resem- bling 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 ab- normal 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 miscon- nection of the ureter to the urinary bladder or duplicated ure- ters. 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 expres- sion 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 develop- ment 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 ex- pressed 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 sur- rounding 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 pro- moter, 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 meso- derm 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).