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Developmental Biology 257 (2003) 356Ð370 www.elsevier.com/locate/ydbio

Regulation of metanephric kidney development by growth/differentiation factor 11

Aurora F. Esquela* and Se-Jin Lee

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Received for publication 2 January 2003, revised 31 January 2003, accepted 31 January 2003

Abstract Growth/differentiation factor 11 (Gdf11) is a transforming growth factor ␤ family member previously shown to control anterior/posterior patterning of the axial skeleton. We now report that Gdf11 also regulates kidney organogenesis. Mice carrying a targeted deletion of Gdf11 possess a spectrum of renal abnormalities with the majority of mutant animals lacking both kidneys. Histological analysis revealed a failure in ureteric bud formation at the initial stage of metanephric development in most Gdf11 mutant embryos examined. The metanephric mesenchyme of mutant embryos lacking a ureteric bud was found to be defective in the expression of glial cell line-derived neurotrophic factor (Gdnf), a gene known to direct ureteric bud outgrowth. The addition of Gdnf protein to urogenital tracts taken from Gdf11 null embryos induced ectopic ureteric bud formation along the Wolffian duct. Our studies suggest that Gdf11 may be important in directing the initial outgrowth of the ureteric bud from the Wolffian duct by controlling the expression of Gdnf in the metanephric mesenchyme. © 2003 Elsevier Science (USA). All rights reserved.

Keywords: Gdf11; Gdnf; Kidney organogenesis; Metanephric development; Ureteric bud

Introduction inductive interactions between the ductal epithelium and the nephrogenic mesenchyme result in the specification of dis- Kidney organogenesis is a highly organized and complex tinct nephric cell types and the spatial organization of the process (Grobstein, 1955; Saxen, 1987). In mammals, kid- kidney. Signals from the ureteric bud promote both the ney development occurs in three stages. At embryonic stage survival of the metanephric mesenchyme and the induction day 8 (E8) in mice, the pronephros, comprised of a pro- of the surrounding metanephric mesenchyme to condense nephric duct and tubules, differentiates from intermediate and differentiate into tubular structures, which will later mesoderm in the anterior region of the embryo. The meso- form the nephrons of the kidney. The metanephric mesen- nephros appears at E9.5 when the pronephric duct (Wolffian chyme, in turn, sends signals out to the ureteric bud, which duct) extends caudally to the cloaca and induces the adja- will induce the ureteric epithelium to grow and branch into cent mesonephric mesenchyme to condense and form a the metanephric mesenchyme. The ureteric branches will linear array of nephric tubules. Both the pro- and mesone- ultimately form the collecting duct system of the kidney. phros regress shortly after their formation. The third stage of A number of secreted molecules have been shown to kidney development begins with the formation of the de- signal between the ureteric bud and the metanephric mes- finitive kidney, the metanephros (E11). The initiation of enchyme in order to control the coordinated development of metanephric development occurs when an epithelial bud the definitive kidney (Kuure et al., 2000; Schedl and Hastie, grows out from the posterior region of the Wolffian duct and 2000; Davies, 2001). Glial cell line-derived neurotrophic invades the loose metanephric mesenchyme. Bidirectional factor (Gdnf) directs the first stage of metanephric develop- ment, the outgrowth of a ureteric bud from the Wolffian

* Corresponding author. Fax: ϩ1-410-614-7079 duct. Gdnf is secreted from the metanephric mesenchyme E-mail addresses: [email protected] (A.F. Esquela), [email protected] (S.-J. and binds to its receptor, Ret receptor tyrosine kinase Lee). (c-Ret), and the coreceptor, glycosylphosphatidyl-inositol-

0012-1606/03/$ Ð see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0012-1606(03)00100-3 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356–370 357 linked Gdnf family receptor ␣1 (Gfr␣1), which are highly Materials and methods expressed in the posterior portion of the Wolffian duct, to induce ureteric bud formation (Pachnis, 1993; Durbec et al., Generation of Gdf11 mutant mice 1996; Jing et al., 1996; Treanor et al., 1996). The majority of mice carrying a targeted deletion for Gdnf, c-Ret,or Gdf11 mutant mice were previously described (McPher- Gfr␣1 exhibit a complete failure in ureteric bud formation ron et al., 1999), and the genotypes of the mice or embryos (Schuchardt et al., 1994, 1996; Moore et al., 1996; Pichel et were determined on tail or yolk sac extracts by Southern al., 1996; Sanchez et al., 1996; Enomoto et al., 1998). In blot analysis (McPherron et al., 1999). Mice were housed in addition to these data, Gdnf has been shown in vitro to be the Johns Hopkins University School of Medicine Animal capable of inducing ectopic ureteric bud formation along Facility under the guidelines for animal experimentation various portions of the Wolffian duct and directing the held by the School of Medicine. growth of these buds toward a localized source (Pepicelli et al., 1997; Sainio et al., 1997), further supporting the impor- Histological and in situ analysis tance of Gdnf in controlling ureteric bud outgrowth. The Gdnf/c-Ret/Gfr␣1 signaling pathway is also essen- Mice heterozygous for Gdf11 were crossed, and the re- tial in subsequent stages of kidney development. Gdnf di- sulting embryos were collected in phosphate-buffered saline rects the continued growth and branching of the ureteric bud (PBS) and fixed in 4% paraformaldehyde overnight. The into the surrounding metanephric mesenchyme by binding fixed embryos were then soaked in 0.5 M sucrose/PBS, to its receptors on the tips of the ureteric branches. Gdnf embedded in O.C.T. compound (Tissue-Tek), and frozen. protein has been shown to increase the number of ureteric Samples were sectioned at 12 ␮m thickness with a mic- branches and the amount of cell proliferation in the ureteric rotome cryostat. Sections for histological analysis were branching tips when added to kidney rudiments in culture stained with hematoxylin and eosin. (Vega et al., 1996; Pepicelli et al., 1997; Sainio et al., 1997). In situ hybridization analysis was performed by using Exogenously added Gdnf protein has also been shown to digoxigenin-labeled riboprobes (UTP/DIG, Roche) on fro- increase the transcription of its own receptor, c-Ret, and zen sections as described (Wilkinson, 1992), with blocking Wnt11 at the tips of the ureteric branches (Pepicelli et al., and antibody incubation steps as described in Heller et al. 1997). Thus, the Gdnf/c-Ret/Gfr␣1 signaling pathway is (1998). Slides were mounted in a water-based medium important in the formation of the ureteric bud at the initial (Aquamount) and photographed. Both sense and antisense stage of metanephric development but is also needed at later riboprobes for each gene were used in each set of experi- stages to control the development of the ureteric tree. ments. The riboprobes used for the Gdf11 transcript corre- In this paper, we describe the role of a secreted protein, sponded only to the pro-region and the 3Ј UTR in order to growth/differentiation factor 11 (Gdf11), that appears to avoid cross-hybridization to myostatin. Probes correspond- work upstream of the Gdnf/c-Ret/Gfr␣1 signaling pathway ing to c-Ret, Eya1, Emx2, Gdnf, Lim1, and Six2 were gen- in order to regulate mammalian kidney organogenesis. erated by PCR using primers based on the published se- Gdf11 is a member of the transforming growth factor ␤ quences from GenBank. The probe for WT1 was obtained (TGF␤) family of growth and differentiation factors and is from the American Type Culture Collection (ATCC). The closely related to myostatin (McPherron et al., 1997). At Pax2 and ActRIIB probes were kindly provided by G. early stages of embryonic development, Gdf11 is expressed Dressler (University of Michigan, Ann Arbor) and J. Mas- at highest levels in the primitive streak region and in the tail sague (Memorial Sloan-Kettering Cancer Center, New bud. At later stages, Gdf11 is widely expressed in a variety York), respectively. of tissues, including the neural tube, the dorsal root gan- glion, the retina, and the limb bud (Gamer et al., 1999; TUNEL analysis McPherron et al., 1999; Nakashima et al., 1999). Previ- ously, we described the generation of mice carrying a tar- DNA nick-end labeling of tissue sections was carried out geted deletion of Gdf11 (McPherron et al., 1999). We according to published procedures (Gavrieli et al., 1992) using showed that homozygous null mice for Gdf11 have dramatic digoxigenin-labeled 11-2Ј,3Ј ddUTP (Roche) on frozen sec- homeotic transformations of the axial skeleton, suggesting tions as described (Wilkinson, 1992), with blocking and anti- that Gdf11 is essential for the proper specification of posi- body incubation steps as described in Heller et al. (1998). tional identity. Here, we report that Gdf11 also plays an important role during kidney development. Our findings Explant cultures indicate that Gdf11 may direct the initiation of metanephric kidney development by controlling the expression of Gdnf Metanephric kidney rudiments were dissected from in the metanephric mesenchyme and thereby inducing ure- E11.5 mouse embryos in Leibovitz L-15 medium (Gibco, teric bud outgrowth. Gdf11 is the earliest acting secreted Invitrogen Corporation) supplemented with 1% fetal bovine factor identified to date that specifically directs the forma- serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and tion of the definitive kidney, the metanephros. 100 U/ml streptomycin. Cultures were placed on nucleopore 358 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356–370

Fig. 1. Renal defects in mice lacking Gdf11.(AÐC) Urogenital tracts of Gdf11ϩ/ϩ, Gdf11ϩ/Ϫ and Gdf11Ϫ/Ϫ newborn male (A) and female (B, C) mice. Note the absence of kidneys and ureters bilaterally (A, B) or unilaterally (C). (DÐQ) Hematoxylin and eosin (H&E)-stained transverse sections of wild type (D, F, H, J, L, N, and P) and Gdf11 mutant (E, G, I, K, M, O, and Q) mice. (D, E) Normal development of mesonephric tubules (arrows) in both Gdf11ϩ/ϩ and Gdf11Ϫ/Ϫ E10.5 embryos. The formation of the metanephric mesenchyme and the posterior extension of the Wolffian duct (arrowhead) for a Gdf11Ϫ/Ϫ E10.5 embryo in (G) resembled that of a wild type E10.5 embryo (F). Absence of ureteric bud formation in an E11.5 Gdf11Ϫ/Ϫ embryo is shown in (I). (K) Complete absence of kidney tissue (arrowhead) in an E12.5 Gdf11 mutant embryo. (M, O) An E14.5 Gdf11Ϫ/Ϫ embryo with unilateral kidney development. (N, O) Higher magnifications of (L) and (M) respectively. The newborn kidney shown in (Q) was taken from a Gdf11Ϫ/Ϫ animal that exhibited unilateral kidney formation and was found to be underdeveloped when compared with a kidney taken from a wild type littermate (P). Abbreviations: a, adrenal gland; b, bladder; g, gonad; k, kidney; m, metanephric mesenchyme; mt, mesonephric tubules; o, ovary; t, testis; u, ureter; ub, ureteric bud; ut, uterus; wd, Wolffian duct. The scale bars represent 200 ␮m. (DÐG) Taken at the same magnification. membranes (Whatman) of 0.1-␮m pore size and cultivated each day, and were evaluated under a phase-contrast micro- in a transwell system containing F12/DMEM medium scope. Photographs were taken every 24 h in culture. Gdf11 (Gibco) with 10% FBS, 4 mM glutamine, 100 U/ml peni- mutant embryos could be readily distinguished from their cillin, and 100 U/ml streptomycin. Cultures were grown at wild type or heterozygous littermates based on their axial

37¡Cin5%CO2 for up to 96 h, with a change of medium skeletal abnormalities at the time of dissection, but in all A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 359

Fig. 2. Expression of Gdf11 and ActRIIB during normal kidney organogenesis. Transverse sections from wild type embryos at E10.5 (A, B, F, G, K, and L), E11.5 (C, H, and M), E12.5 (D, I, and N), and E14.5 (E, J, and O) were analyzed by in situ hybridization for the expression patterns of Gdf11 (AÐE), ActRIIB (FÐJ), and the kidney marker gene, Pax2 (KÐO). (A, B) Gdf11 was expressed at E10.5 in the mesonephric tubules, the Wolffian duct, and at lower levels in the metanephric mesenchyme. Gdf11 mRNA was detected in both the metanephric mesenchyme and the ureteric bud at E11.5 (C). At E12.5, Gdf11 was expressed in the developing ureter, the ureteric branches, as well as in a population of uninduced metanephric mesenchyme (m) in the periphery of the developing kidney (D). Lower levels of Gdf11 expression could be detected in the condensed metanephric mesenchyme at this stage. Gdf11 continued to be expressed in the ureter, ureteric branches, and condensed metanephric mesenchyme at E14.5 (E). Expression for ActRIIB, a potential receptor for Gdf11, was also detected in the mesonephric kidney, the Wolffian duct, and at low levels in the metanephric mesenchyme at E10.5 (F, G). ActRIIB expression was later detected in both the metanephric mesenchyme and the ureteric bud at E11.5 (H), and continued to be expressed in these mesenchymal and epithelial derivatives at E12.5 (I) and E14.5 (J). The kidney marker gene, Pax2, was also expressed in the mesonephros, the Wolffian duct, the ureteric bud, the metanephric mesenchyme, and their derivatives within the developing kidney (KÐO). Abbreviations: cm, condensed metanephric mesenchyme; m, metanephric mesenchyme; mt, mesonephric tubules; u, ureter; ub, ureteric bud; wd, Wolffian duct. The scale bars represent 200 ␮m. All panels were taken at the same magnification shown in (A), with the exception of (E) (L), and (O). cases the genotypes were later confirmed by using Southern explants were prepared for whole-mount immunostaining by blot analysis. washing the cultures in phosphate-buffered saline, 0.1% For the Gdnf experiments, entire urogenital tracts, con- Tween (PBT), and 10% sheep serum for several hours. The sisting of the Wolffian duct and both the mesonephros and explants were then incubated overnight at 4¡C in PBT, 1% metanephros regions, were dissected from E11.5 wild type sheep serum, and a polyclonal rabbit anti-EHS laminin (Sig- or Gdf11 mutant embryos and grown in the presence or ma) that recognizes the epithelialized nephrons of the kidney absence of Gdnf protein (50 ng/ml; recombinant rat Gdnf; (Ekblom et al., 1980). The explants were then washed in PBT R&D Systems). and incubated for4hatroom temperature in PBT, 1% sheep For the recombination experiments, epithelial and mes- serum, and rhodamine-conjugated secondary antibodies (Santa enchymal components of the E11.5 kidney rudiments were Cruz Laboratories). Explants were washed with PBT and isolated by incubating the rudiments on ice for 6 min with mounted on slides with a water-based medium (Aquamount). 0.25% trypsin/EDTA (Gibco) and then separated by me- Similar procedures were followed for explants stained with chanical manipulation using 27-gauge needles. The tubulo- fluorescein-conjugated Dolichos biflorus-agglutinin (DBA) genesis assays used wild type or Gdf11 mutant spinal cord (Vector Laboratories) to visualize the Wolffian duct system cultured next to wild type or Gdf11 mutant separated met- and the ureteric tree (Laitinen et al., 1987). anephric mesenchyme. Spinal cord taken from E11.5 em- bryos was obtained from the cervical regions and dissected free from surrounding tissues and ganglia. Results

Immunofluorescence staining Kidney defects in mice lacking Gdf11

Kidney explants were removed from culture at desired time Mice homozygous for a targeted deletion in the Gdf11 points and then fixed and stored in methanol at Ϫ20¡C. Kidney gene died within 1 day after birth (McPherron et al., 1999). 360 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370

In the course of investigating the cause of death in these mutant kidneys were abnormally small in size and pos- animals, we noted that nearly all mutant mice had kidney sessed a reduced number of nephrons, histological exami- defects that were evident on gross examination of the inter- nation revealed that these kidneys were normal in their nal organs. Gdf11 mutant mice showed a wide spectrum of overall architecture (Fig. 1Q). Hence, in the absence of kidney defects, with the majority of mutants (28 out of 47 Gdf11, initiation of metanephric development was severely examined) showing complete bilateral renal agenesis. Most impaired because a ureteric bud failed to form in the pos- of the remaining mutants exhibited milder kidney defects terior region of the Wolffian duct. However, in a subset of ranging from unilateral agenesis (12 out of 47 examined) to mutant embryos in which kidney development did occur, it smaller sized kidneys on one or both sides. Unilateral renal appeared to progress in a normal fashion. agenesis was also observed in a small percentage of het- erozygous mice (5 out of 93 examined). Dissection of the Gdf11 expression in wild type embryos throughout kidney urogenital tracts showed that all animals lacking a kidney on organogenesis one or both sides also exhibited loss of the corresponding ureter. However, regardless of the presence or absence of To assess if the normal mRNA expression patterns for kidneys in these mice, the adrenal glands, male and female Gdf11 correlate with the timing of the kidney defects ob- reproductive tracts, and bladder appeared normal in both served in mice null for this gene, in situ analysis using their placement and structure (Fig. 1AÐC). digoxigenin-labeled riboprobes corresponding to Gdf11 was In order to determine the stage at which abnormal kidney done at several time points throughout kidney organogene- development first becomes apparent in Gdf11 mutant em- sis in wild type embryos. Gdf11 was expressed in the meso- bryos, we analyzed the kidneys at various times during nephric tubule system, which is well established at E10.5 embryonic development. Histological examination revealed (Fig. 2A). Gdf11 mRNA was also present in the Wolffian that the formation of the intermediate kidney, the mesone- duct and at low levels in the metanephric mesenchyme that phros, appeared grossly normal at E10.5 in Gdf11 mutant had not yet been invaded by a ureteric bud in E10.5 wild mice (Fig. 1D and E). We also noted that the Wolffian duct type embryos (Fig. 2B). During the initial stage of meta- had extended posteriorly and the metanephric mesenchyme nephric development at E11.5, Gdf11 was found to be had formed in these E10.5 mutant embryos (5/5) in a similar expressed both in the metanephric mesenchyme and in the fashion as their wild type littermates (Fig. 1F and G). invading ureteric bud epithelium that had grown off the Defects were first observed at E11.5, when metanephric posterior region of the Wolffian duct (Fig. 2C). At E12.5, kidney development is initiated. At this stage, the wild type Gdf11 was expressed in the ureter and the ureteric branches ureteric bud has grown out from the Wolffian duct and of the developing kidney and in a population of uninduced invaded the metanephric mesenchyme (Fig. 1H). In the metanephric mesenchyme within the renal cortex (Fig. 2D). majority of Gdf11 mutant mice observed (17/23), the ure- Gdf11 mRNA could also be detected at low levels in the teric bud failed to form and branch into the metanephric condensed metanephric mesenchyme at this stage. By mesenchyme, although the Wolffian duct was clearly E14.5, when metanephric development is well established, present (Fig. 1I). A small number of mutant animals showed Gdf11 continued to be expressed in the ureter and ureteric normal uni- or bilateral ureteric bud formation at this stage branches, in the uninduced metanephric mesenchyme (6/23) (data not shown). By E12.5 in wild type embryos, the within the periphery of the kidney, and in the condensed ureteric bud has branched extensively into the metanephric metanephric mesenchyme differentiating into nephrons mesenchyme, and the metanephric mesenchyme has begun (Fig. 2E). A comparison of the expression patterns for to condense around the ureteric tips and differentiate into Gdf11 and Pax2, a kidney marker which is known to be epithelial nephron-like structures (Fig. 1J). In most E12.5 expressed in both the epithelial and mesenchymal renal Gdf11 mutant embryos analyzed (13/14), these structures lineages (Figs. 2K-2O; Dressler et al., 1990), showed that, were absent in the area where the kidney should have although Gdf11 and Pax2 mRNA were both expressed in developed (Fig. 1K). the ureteric bud, the metanephric mesenchyme, and their In wild type mice, metanephric development is well derivatives, Gdf11 appeared to be expressed at lower levels established by E14.5, as pretubular structures have begun to in the condensing metanephric mesenchyme and developing differentiate into nephrons, and a collecting duct system has nephrons. In addition, Gdf11 mRNA was detected in the started to form (Fig. 1L and N). Analysis of embryos null uninduced metanephric mesenchyme within the kidney pe- for Gdf11 at E14.5 revealed complete absence of kidney riphery at E12.5 and E14.5, which was a region devoid of structures either bilaterally (4/7) (data not shown) or unilat- Pax2 expression. In situ analysis of sections from newborn erally (3/7) (Fig. 1M and O). Kidney tissue that was present kidneys showed decreased Gdf11 expression at this stage, in mutant embryos at this stage sometimes appeared under- but low levels of expression could be detected in the glo- developed but was histologically normal. Kidneys isolated meruli, distal tubules, and collecting ducts (data not shown). from newborn Gdf11 mutant mice that showed unilateral In summary, wild type Gdf11 mRNA expression was de- renal development were also analyzed and compared with tected in both the ureteric and mesenchymal derivatives their wild type littermates (Fig. 1P and Q). Although the throughout metanephric development. These expression A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 361 patterns correlated with the timing of the renal defects development is to induce expression of Gdnf in the meta- observed in the Gdf11 mutant embryos. nephric mesenchyme. Notably, those E11.5 Gdf11 defective embryos (6 out of 6 examined) that possessed uni- or bilat- Expression of kidney marker genes in E11.5 Gdf11 eral invasion of the ureteric bud into the metanephric mes- homozygous mutant embryos enchyme did show appropriate expression of Gdnf in the invaded nephric mesenchyme (data not shown). Thus, there Histological studies had shown that the majority of E11.5 may be another factor or cell signaling pathway redundant Gdf11 homozygous mutant embryos showed a complete with Gdf11 that allows for the proper expression of Gdnf in absence of ureteric bud invasion into the metanephric mes- the mutant metanephric mesenchyme and therefore rescue enchyme (Fig. 3A and B). In order to determine whether the of ureteric bud formation in a minority of Gdf11 mutants. renal tissue observed in these Gdf11 null embryos had formed and differentiated properly, the expression patterns TUNEL analysis during early stages of kidney of several kidney marker genes previously reported to be development in Gdf11 mutant embryos expressed at the earliest stages of metanephric development were analyzed by using in situ hybridization. We examined Our expression studies confirmed that, at E11.5, the the expression of mRNAs encoding the receptor tyrosine metanephric mesenchyme was present in Gdf11 null em- kinase, c-Ret, and the transcription factors Emx2, Lim1, and bryos regardless of the presence or absence of ureteric bud Pax2, which are normally expressed in the Wolffian duct formation (Fig. 3). However, our histological analysis and in the ureteric bud of wild type embryos at E11.5 (Fig. showed that, by E12.5, the majority of Gdf11 mutant em- 3C, E, G, and I; Dressler et al., 1990; Pachnis et al., 1993; bryos completely lacked renal tissue in the area where Avantaggiato et al., 1994; Shawlot and Behringer, 1995; kidney development should have occurred (Fig. 1K). In Miyamoto et al., 1997). As shown in Fig. 3D, F, H, and J, order to determine the fate of the uninvaded metanephric all of these genes were expressed in the Wolffian duct in mesenchyme noted in the majority of mutant embryos at mice null for Gdf11 at E11.5, confirming that the Wolffian E11.5, transverse sections were analyzed by in situ hybrid- duct had extended posteriorly in the mutant embryos. How- ization at the level of the developing gonads in E12.5 ever, we were unable to detect expression of these genes in mutant embryos in the area where kidney organogenesis the region in or near the metenephric mesenchyme, consis- was clearly underway in wild type littermates using kidney tent with the absence of ureteric bud invasion in the major- markers expressed in the nephric mesenchyme (Eya1, Gdnf, ity of E11.5 mutant embryos examined. We also examined and WT1; Pritchard-Jones et al., 1990; Pelletier et al., 1991; the expression of the transcription factors Pax2, Eya1, Six2, Armstrong et al., 1992; Pichel et al., 1996; Sanchez et al., and WT1, which are normally expressed in the metanephric 1996; Towers et al., 1998; Donovan et al., 1999; Xu et al., mesenchyme before the initial invasion of the ureteric bud 1999), the ureteric branches (c-Ret; Pachnis et al., 1993; and continue to be expressed in the induced metanephric Avantaggiato et al., 1994), or both derivatives (Pax2; mesenchyme (Fig. 3I, K, M, and O; Dressler et al., 1990; Dressler et al., 1990). None of the genes known to be Pritchard-Jones et al., 1990; Pelletier et al., 1991; Arm- expressed in the developing kidney (c-Ret, Eya1, Gdnf, strong et al., 1992; Oliver et al., 1995; Donovan et al., 1999; Pax2, and WT1) were detected in E12.5 Gdf11 mutant Xu et al., 1999; Brophy et al., 2001). As shown in Fig. 3J, embryos in the region where the kidney would normally L, N, and P, Pax2, Eya1, Six2, and WT1 mRNA were reside at the level of the developing gonads (data not appropriately expressed in the uninvaded metanephric mes- shown). Therefore, the metanephric mesenchyme that was enchyme of Gdf11 homozygous null embryos at E11.5, observed in Gdf11 mutant embryos lacking ureteric bud suggesting that the metanephric mesenchyme had formed formation at E11.5 was not detected a day later at E12.5 in and differentiated toward a nephric lineage. most Gdf11 mutants in the area where kidney development Although these results confirm that both the Wolffian should have taken place. duct and the metanephric mesenchyme had formed at E11.5 In order to determine whether loss of kidney tissue oc- in Gdf11 homozygous mutant embryos, expression of met- curred through an apoptotic mechanism during these early anephric mesenchyme markers was not completely normal. stages in Gdf11 null embryos, we carried out TUNEL anal- Specifically, expression of Gdnf, which is normally present ysis on transverse sections taken from wild type and mutant in the metanephric mesenchyme (Fig. 3Q; Pichel et al., embryos. There was no apoptosis detected in the metaneph- 1996; Sanchez et al., 1996; Towers et al., 1998), was not ric mesenchyme of E11.5 Gdf11 mutant embryos that failed detected in any mutant Gdf11 embryo (9 out of 9 examined) to form a ureteric bud when compared with wild type that failed to form a ureteric bud (Fig. 3R). Given that Gdnf embryos (Fig. 4AÐD). Similarly, we did not detect an in- is known to be essential for the initiation of ureteric bud crease in TUNEL staining in E12.5 mutant embryos at the outgrowth from the Wolffian duct, our expression studies level of the developing gonads in the region where the suggest that the failure of ureteric bud formation in Gdf11 kidneys are clearly present in wild type embryos (Fig. mutant embryos may be a direct result of the lack of Gdnf 4EÐH). However, examination of sections taken from re- expression and that a primary role for Gdf11 in kidney gions of E12.5 mutant embryos just posterior to the devel- 362 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370

Fig. 3. Kidney marker expression in Gdf11 homozygous mutant embryos at E11.5. Sections for histological analysis in (A) and (B) were stained with hematoxylin and eosin (H&E). Transverse sections were taken from E11.5 wild type (A, C, E, G, I, K, M, O, Q, and S) and Gdf11 mutant embryos lacking a ureteric bud (B, D, F, H, J, L, N, P, R, and T) and analyzed for the expression of c-Ret (C, D), Emx2 (E, F), and Lim1 (G, H) in the Wolffian duct and the ureteric bud; Pax2 (I, J) in both the epithelial and mesenchymal derivatives; and Eya1 (K, L), Six2 (M, N), WT1 (O, P), and Gdnf (Q, R) in the metanephric mesenchyme by in situ hybridization. Note that Gdnf expression was not detected in the metanephric mesenchyme of any Gdf11Ϫ/Ϫ embryo that failed to form a ureteric bud (R). ActRIIB, a potential receptor for Gdf11, was expressed in the metanephric mesenchyme and the ureteric bud in wild type embryos (S) and in the metanephric mesenchyme and the Wolffian duct in mutant embryos at this stage (T). The arrows point to the metanephric mesenchyme, and the arrowheads point to either the ureteric bud in Gdf11ϩ/ϩ or the Wolffian duct in Gdf11Ϫ/Ϫ embryos. Note that in (C) and (K), the sections were cut at a slightly skewed angle and include only one of two ureteric buds invading the metanephric mesenchyme. Abbreviations: m, metanephric mesenchyme; ub, ureteric bud; wd, Wolffian duct. The scale bar represents 150 ␮m. oping gonads revealed extensive apoptosis in the metaneph- These explants were grown for 120 h on nucleopore filter ric mesenchyme that had not been invaded by a ureteric bud rafts and then stained with fluorescein-conjugated Dolichos (Fig. 4I and J). The expression of metanephric mesenchy- biflorusÐagglutinin (DBA) to visualize the ureteric tree by mal-specific genes (Eya1, Pax2, WT1) in this area confirmed immunofluorescence. Explant cultures of kidney rudiments the identity of this tissue (Fig. 4KÐM). These results suggest taken from Gdf11 null embryos in which the ureteric bud that metanephric mesenchyme that is not invaded by a ureteric had clearly invaded the metanephric mesenchyme showed bud at the beginning of kidney development in mutant em- extensive ureteric bud branching and nephric tubule forma- bryos undergoes programmed cell death around E12.5. tion comparable to rudiments taken from heterozygote lit- termates (Fig. 5A and B). However, metanephric rudiments In vitro explant cultures of E11.5 Gdf11 null metanephric taken from Gdf11 null embryos that were devoid of ureteric rudiments bud formation did not differentiate in culture (Fig. 5C). Instead, the mutant metanephric mesenchyme in these cul- In order to examine renal development more closely in tures degenerated within 48 h presumably because the ure- Gdf11 null embryos undergoing uni- or bilateral kidney teric bud normally provides survival factors to the meta- formation, we observed the growth and differentiation of nephric mesenchyme at this stage (Saxen, 1987; Koseki et whole E11.5 metanephric rudiments using explant cultures. al., 1992). A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 363

Fig. 4. In situ detection of apoptosis in E11.5 and E12.5 Gdf11 homozygous mutant embryos defective in ureteric bud formation. Wild type (A, B, E, and F) and Gdf11 mutant (C, D, and GÐN) embryos at E11.5 (AÐD) and E12.5 (EÐN) were analyzed for apoptosis by using the TUNEL assay. Transverse sections shown in (A, C, E, G and I) were stained with hematoxylin and eosin (H&E) for histological analysis. No increased apoptosis was detected at E11.5 (D) or E12.5 (H) for Gdf11 mutant embryos in the area where kidney development should have taken place when compared with their wild type littermates (B, F). However, sections taken from E12.5 Gdf11 mutant embryos just below the level of the developing gonads showed extensive apoptosis in the uninvaded metanephric mesenchyme (J). The histological section in (I) shows the presence of metanephric mesenchyme that was not invaded by a ureteric bud. The expression of metanephric mesenchyme-specific markers, Eya1 (K), Pax2 (L), and WT1 (M), confirmed the identity of this renal tissue. ActRIIB, a potential receptor for Gdf11, was also expressed in the mutant metanephric mesenchyme undergoing apoptosis (N). The arrows in (IÐN) point to the metanephric mesenchyme in the E12.5 mutant embryos. Abbreviations: g, gonad; k, kidney; m, metanephric mesenchyme; ub, ureteric bud; wd, Wolffian duct. The scale bar represents 200 ␮m.

Because our in situ hybridization studies had shown that DBA in order to visualize the Wolffian duct system and the Gdnf expression was absent in the uninvaded metanephric ureteric tree. In the absence of Gdnf protein, E11.5 Gdf11 mesenchyme of Gdf11 mutant embryos (Fig 3R), we tested mutant explants that lacked a ureteric bud at the time of whether the addition of Gdnf protein to explants taken from dissection, failed to form a ureteric bud after 96 h in culture E11.5 Gdf11 null embryos could induce ureteric bud out- (Fig. 6C). As shown in Fig. 6D, multiple ureteric bud growth from the mutant Wolffian ducts. The entire urogen- structures were observed growing out along the Wolffian ital system was dissected out of E11.5 Gdf11 mutant em- duct of Gdf11 mutant embryos in the presence of Gdnf (6 bryos that clearly lacked ureteric bud formation and grown out of 7 experiments). The frequency of ectopic ureteric bud in culture for 96 h on filter rafts in the presence or absence formation along the length of the Wolffian duct in Gdf11 of Gdnf protein (50 ng/ml). Explants were then stained with mutant embryos was noted to be much more extensive when 364 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 compared with wild type cultures in the presence of Gdnf cellular signaling during kidney development. We therefore protein (Fig. 6B and D), although the reason for this differ- examined the expression patterns for ActRIIB during meta- ence is unclear. These experiments demonstrate that the nephric organogenesis and compared these expression pat- Gdf11 mutant Wolffian ducts were competent to undergo terns with those for Gdf11. In E10.5 wild type embryos, ureteric bud formation and the addition of Gdnf protein to ActRIIB mRNA was detected in the mesonephric tubules, Gdf11 mutant kidney rudiments could rescue ureteric bud the Wolffian duct, and at low levels in the metanephric outgrowth from the Wolffian duct. These results taken to- mesenchyme that had not yet been invaded by a ureteric bud gether with our in situ hybridization studies suggest that the (Fig. 2F and G). At E11.5, ActRIIB was expressed in both primary defect in Gdf11 mutant embryos is a lack of Gdnf the metanephric mesenchyme and the ureteric bud of the expression in the metanephric mesenchyme, which leads to developing kidney (Fig. 2H). By E12.5, ActRIIB expression a failure of ureteric bud outgrowth from the Wolffian duct. was detected both in the metanephric mesenchyme condens- We also investigated whether the mutant metanephric ing around the tips of the ureteric branches and in the mesenchyme from Gdf11 null embryos that failed to form a ureteric branches themselves (Fig. 2I; Feijen et al., 1994). ureteric bud by E11.5 was competent to undergo later stages Robust expression of ActRIIB mRNA continued at E14.5 in of kidney development, specifically nephron formation. the developing ureter, the ureteric branches, the condensed Normally, the ureteric bud provides signals to the meta- metanephric mesenchyme, and differentiating nephrons nephric mesenchyme that induce the mesenchyme to con- (Fig. 2J). Therefore, ActRIIB mRNA patterns resembled dense and differentiate into nephron structures (Saxen, that of Gdf11 at early stages of kidney organogenesis with 1987). However, when E11.5 spinal cord is cultured to- both genes being expressed in the mesonephric tubules, the gether with separated E11.5 metanephric mesenchyme, the Wolffian duct, the ureteric bud, and the metanephric mes- spinal cord can substitute for the ureteric bud and induce the enchyme (Fig. 2AÐC and FÐH). However, unlike ActRIIB, renal tissue to undergo a similar tubulogenic program (Fig. Gdf11 was expressed in a population of uninduced meta- 7A; Grobstein, 1955, 1956). To test whether mutant meta- nephric mesenchyme in the renal cortex and was expressed nephric mesenchyme isolated from E11.5 Gdf11 null em- at lower levels in the condensed metanephric mesenchyme bryos defective in ureteric bud formation was competent to at E12.5 and E14.5 (Fig. 2D, E, I, and J). The findings that undergo tubulogenesis, mutant Gdf11 mesenchyme was cul- Gdf11 and ActRIIB are essential for proper renal develop- tured together with Gdf11 null spinal cord for 96 h on ment and are both expressed in the developing kidney are nucleopore filter rafts. E11.5 Gdf11 null spinal cord was consistent with the hypothesis that Gdf11 may use ActRIIB used in these experiments to eliminate the contribution of for cellular signaling during kidney organogenesis. secreted Gdf11 protein to the cultures, since we had noted We also investigated ActRIIB expression during meta- during our original in situ hybridization analysis in wild nephric development in embryos lacking Gdf11. At E11.5, type embryos that Gdf11 was robustly expressed in the ActRIIB expression was detected both in the Wolffian duct dorsal half of the developing spinal cord (data not shown). and the metanephric mesenchyme of Gdf11 null embryos As shown in Fig. 7C, mutant metanephric mesenchyme was that failed to form a ureteric bud (Fig. 3T). In a subset of capable of responding to the inductive signals from the E11.5 mutant embryos exhibiting bilateral kidney develop- Gdf11 null spinal cord and forming tubules (5 out of 5 ment, ActRIIB expression resembled that seen in wild type experiments) in a similar fashion as the control cocultures embryos (data not shown). At E12.5 in Gdf11 null embryos, (10 out of 11 experiments) (Fig. 7A). The metanephric ActRIIB expression was not detected in the area where the tubule structures formed in these experiments were visual- kidney normally resides (data not shown), but transverse ized by staining the cultures with an anti-laminin antibody, sections taken posterior to the developing gonads in these which recognizes epithelialized tissue. These data confirm mutants showed expression of ActRIIB in the metanephric that metanephric mesenchyme from homozygous null mesenchyme undergoing extensive apoptosis (Fig. 4N). Gdf11 embryos that failed to form a ureteric bud was com- These data show that, in Gdf11 mutant embryos, ActRIIB petent to undergo later stages of kidney development in- continues to be expressed in the metanephric mesenchyme volving nephron formation. and the Wolffian duct, although ureteric bud formation has failed to occur. Activin type IIB receptor kidney expression patterns

The abnormalities in kidney development and in ante- Discussion rior/posterior patterning of the axial skeleton seen in Gdf11 mutant embryos are strikingly similar to the abnormalities In the present study, we have shown that Gdf11 is im- reported in mice lacking the activin type IIB receptor portant for kidney organogenesis. Although a subset of (ActRIIB) (Oh and Li, 1997; McPherron et al., 1999). The mutant animals exhibited uni- or bilateral kidney develop- similar phenotypes of these mutant mice, as well as recent ment, the majority of mice lacking Gdf11 had complete evidence showing that Gdf11 can bind to ActRIIB (Oh et renal agenesis. Analysis of the urogenital tracts of newborn al., 2002), raise the possibility that Gdf11 uses ActRIIB for animals lacking Gdf11 revealed that the adrenal glands, A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 365 male and female reproductive tracts, and bladder appeared between the metanephric mesenchyme and the posterior normal regardless of their kidney defects. Therefore, the portion of the Wolffian duct. initial specification of the intermediate mesoderm during Metanephric mesenchyme from E12.5 Gdf11 mutant em- embryogenesis appeared unperturbed, and rather abnormal- bryos detected in regions posterior to the developing gonads ities appeared to be restricted to kidney development. More- was shown by TUNEL analysis to undergo extensive apo- over, because the formation of the mesonephros proceeded ptosis. This nephric mesenchyme appropriately expressed normally in mice lacking Gdf11, we conclude that this gene the kidney markers Eya1, Pax2, and WT1, confirming the specifically controls the formation of the definitive kidney, identity of this tissue. The marked increase of apoptosis in the metanephros. Histological studies showed that the pri- this region was not surprising since it is believed that the mary defect in most Gdf11 homozygous null animals ap- ureteric bud provides signals that promote the survival of peared to be a failure of ureteric bud outgrowth from the the metanephric mesenchyme as well as directs the conden- Wolffian duct and the subsequent invasion of the ureteric sation and epithelization of the mesenchyme (Koseki et al., bud into the metanephric mesenchyme. 1992; Barasch et al., 1996). Transfilter organ culture exper- The expression of various kidney markers in the most iments in vitro have shown that E11.5 metanephric mesen- severely affected Gdf11 mutant embryos at E11.5 confirmed chyme separated from the invaded ureteric bud will undergo that the Wolffian duct and the metanephric mesenchyme apoptosis and degenerate within 48 h in culture (Fig. 7B; had developed but that the mesenchyme was not competent Saxen, 1987; Koseki et al., 1992). In addition, gene disrup- to induce ureteric bud formation and invasion into the neph- tion studies for c-Ret, Eya1, Gdnf, and WT1 all report ric mesenchyme. We noted that the Wolffian duct properly increased apoptosis in the mutant metanephric mesenchyme expressed c-Ret, Emx2, Lim1, and Pax2, although the ure- that was not invaded by the ureteric bud (Kreidberg et al., teric bud failed to form in the majority of null embryos 1993; Sanchez et al., 1996; Schuchardt et al., 1996; Xu et analyzed. Furthermore, Eya1, Pax2, Six2, and WT1 were al., 1999), similar to what we observed in Gdf11 mutant expressed in the uninvaded metanephric mesenchyme of embryos. We therefore conclude that the absence of ureteric E11.5 Gdf11 null embryos, suggesting that the metanephric bud formation in most Gdf11 null embryos directly resulted mesenchyme had formed and begun to differentiate toward in the degeneration of the metanephric mesenchyme a nephric lineage. However, Gdnf failed to be expressed in through an apoptotic mechanism around E12.5. Gdf11 mutant metanephric mesenchyme that was not in- Explant culture studies further suggest that the failure of vaded by a ureteric bud. Gdnf has been shown to be a the metanephric mesenchyme to differentiate may result not metanephric mesenchyme-derived signal that binds to its from the lack of Gdf11 signaling directly on this tissue but receptors, c-Ret and GFR␣1, in the posterior region of the rather as an indirect effect of the failure to form a ureteric Wolffian duct and directs the formation of the ureteric bud bud. When the metanephric mesenchyme was isolated from and its invasion into the mesenchyme (Schuchardt et al., E11.5 Gdf11 null embryos defective for ureteric bud for- 1994, 1996; Enomoto et al., 1998). The majority of mice mation and grown together with dorsal spinal cord, a strong homozygous null for Gdnf have a failure of ureteric bud inducer of tubulogenesis, the mutant tissue was capable of outgrowth (Moore et al., 1996; Pichel et al., 1996; Sanchez forming condensates that went on to differentiate into et al., 1996). Gdnf is also important at later stages of kidney nephron-like structures. In this respect, the phenotype of development and induces further ureteric growth and Gdf11 mutant embryos appears to be distinct from the branching by binding to its receptors located at the tips of phenotypes of WT1 or Pax2 mutant embryos, which have the ureteric branches. Our expression studies suggest that also been shown to have defects in ureteric bud outgrowth failure of the ureteric bud to form in the majority of Gdf11 (Kreidberg et al., 1993; Torres et al., 1995). Unlike Gdf11 null embryos may result from a lack of Gdnf signaling mutant mesenchyme, WT1 or Pax2 mutant mesenchyme is

Fig. 5. Differentiation of whole E11.5 Gdf11 null kidney rudiments in explant culture. Urogenital tracts of E11.5 Gdf11ϩ/Ϫ and Gdf11Ϫ/Ϫ embryos were isolated and grown in culture for 120 h. The heterozygote kidney shown in (A) exhibited extensive growth and differentiation in culture, which was evident by the well-formed ureteric tree stained with fluorescein-conjugated Dolichos biflorusÐagglutinin (DBA). A kidney rudiment from a Gdf11 null littermate, which clearly possessed a ureteric bud at the time of dissection, exhibited normal development in culture (B). However, the Gdf11Ϫ/Ϫ kidney shown in (B) appeared smaller in size when compared with the Gdf11ϩ/Ϫ kidney (A). Kidney rudiments isolated from Gdf11 null embryos that were devoid of ureteric bud formation did not differentiate in culture (C), and a ureteric tree was never detected when the explants were stained with DBA after 120 h (data not shown). The arrows in (A) and (B) indicate the differentiating kidney, and the black arrowhead in (C) indicates the position of the Wolffian duct. The scale bars represent 250 ␮m. Fig. 6. Effect of Gdnf protein on kidney explants taken from E11.5 Gdf11 mutant embryos lacking a ureteric bud. E11.5 wild type (A, B) and Gdf11 mutant (C, D) explants consisting of the Wolffian duct and the mesonephric and metanephric regions were cultured in the presence (B, D) or absence (A, C) of Gdnf protein (50 ng/ml) for 96 h. Ectopic ureteric bud formation along the Wolffian duct was visualized by immunofluorescence staining using fluorescein- conjugated Dolichos biflorusÐagglutinin (DBA). Wild type urogenital explants cultured in the presence of Gdnf protein showed additional ectopic epithelial buds growing out from the Wolffian duct as indicated by the arrowheads (B). Gdf11 mutant explants that clearly lacked a ureteric bud at the time of dissection failed to differentiate in the absence of Gdnf protein (C). Gdf11 mutant explants grown in the presence of Gdnf protein showed extensive ectopic ureteric bud formation along the Wolffian duct (D) when compared with the wild type in (B). Abbreviations: ub, ureteric bud; wd, Wolffian duct. The scale bar represents 200 ␮m. 366 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 367

Fig. 7. Analysis of tubule formation using Gdf11 mutant kidney rudiments. E11.5 metanephric mesenchyme was obtained from Gdf11ϩ/ϩ (A, B) or Gdf11Ϫ/Ϫ embryos defective in ureteric bud formation (C) and cultured next to E11.5 wild type (A) or Gdf11 mutant spinal cord (C) for 96 h. Gdf11 mutant metanephric mesenchyme cultured next to mutant spinal cord showed similar epithelization of the mesenchyme observed in (A) confirming that the mutant metanephric mesenchyme can respond to inductive signals (C). The presence of nephric tubules in (A) and (C) was confirmed by staining the explants with an anti-laminin antibody (␣-lam) and then visualized by immunofluorescence. Wild type metanephric mesenchyme cultured alone for 96 h quickly degenerated (B) and was negative for anti-laminin staining (data not shown). The arrows in (AÐC) point to the metanephric mesenchyme. Abbreviation: sc, spinal cord. The scale bars represent 200 ␮m. unable to form tubule structures when grown together with gene expression in the presumptive metanephric mesen- dorsal spinal cord in culture (Kreidberg et al., 1993; Brophy chyme and/or Wolffian duct resulting in defective ureteric et al., 2001). Therefore, in contrast to both WT1 and Pax2, bud formation. which perform two independent roles during kidney forma- In this respect, a recent report describing the disruption tion, directing ureteric bud formation as well as inducing of the Hox11 paralogous group in mice suggests that the tubulogenesis, Gdf11 appears to control ureteric bud out- Hox genes are important for the proper patterning of the growth from the posterior portion of the Wolffian duct but kidney along the anterior/posterior axis (Wellik et al., is not essential in later developmental events, such as tubule 2002). Specifically, the triple disruption of the Hox11 formation. paralogous group resulted in ureteric bud defects and lack of Two possible mechanisms could explain the kidney de- Gdnf expression in the mutant metanephric mesenchyme fects observed in the Gdf11 homozygous null mice. One similar to renal abnormalities observed in the Gdf11 mutant possibility is that these are secondary effects resulting from mice. It has also been proposed that the Hox11 paralogues the anterior/posterior patterning abnormalities present in work together with the Pax-Eya-Six network to pattern the these embryos. We previously reported that all Gdf11 null kidney since Hox11 triple mutants also do not express Six2 mice possessed anterior homeotic transformations of their in the mutant metanephric mesenchyme, and therefore, vertebral column, had posterior displacement of their hind Hox11 defects resemble the renal abnormalities reported for limbs, and showed alterations in Hox gene expression the Eya1 disruption (Xu et al., 1999). within the prevertebrae (McPherron et al., 1999). Gdf11 has For several reasons, we do not believe that the kidney also been shown to induce Hox gene expression in the chick defects seen in the Gdf11 mutant embryos result from global limb and in neural explants, which supports the idea that alterations in anterior/posterior patterning. First, our histo- Gdf11 works upstream of the Hox genes to control “posi- logical analysis and in situ hybridization studies of the tional identity” along the anterior/posterior axis (Gamer et Gdf11 mutant embryos argue that both the Wolffian duct al., 1999; Liu et al., 2001). It is possible that the kidney and metanephric mesenchyme formed normally and ex- defects observed in the Gdf11 mutant embryos occurred due pressed all kidney-specific markers appropriately with the to a misalignment of crucial tissues involved in kidney exception of Gdnf, suggesting that the original patterning of formation along the body axis or a shift or a mistiming of the renal tissue was unperturbed. If the defects in kidney 368 A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 development did result from misalignment of crucial tis- During our analysis of Gdf11 null embryos, we observed sues, for example, one might have expected that expression that a subset of mutant animals exhibited normal uni- or of more than a single kidney marker gene might have been bilateral kidney development. Histological studies revealed altered. Second, if Gdf11 worked upstream of the Hox11 that, if a ureteric bud formed in Gdf11 mutant mice, kidney genes during kidney organogenesis, we would predict that development appeared to progress in a normal fashion. In Gdf11 homozygous mutants would also fail to express Six2 situ analysis further showed that E11.5 Gdf11 mutant ani- in their mutant metanephric mesenchyme, which is not the mals possessing a ureteric bud appropriately expressed all case. Thus, Gdf11 may have an independent role in control- of the kidney markers tested, including Gdnf. Kidney de- ling ureteric bud formation that is unrelated from its role in velopment in this subset of Gdf11 mutant animals could axial patterning. have been rescued either by a parallel pathway that controls We favor the possibility that Gdf11 plays a direct role ureteric bud formation or by a redundant member of the during the initial stage of metanephric kidney development TGF␤ superfamily. A number of genes have recently been by inducing the expression of Gdnf in the metanephric identified that are essential for ureteric bud outgrowth and mesenchyme and thereby controlling ureteric bud out- function upstream of Gdnf. These include the tumor sup- growth from the posterior region of the Wolffian duct. This pressor gene WTI (Kreidberg et al., 1993), the transcription hypothesis is supported by in vitro studies in which we factors Eyal (Xu et al., 1999) and Pax2 (Torres et al., 1995), added Gdnf protein to explant cultures that consisted of the and the Hox11 paralogous genes (Wellik et al., 2002). entire Wolffian duct system and the surrounding tissues Therefore, it is possible that there are several parallel path- taken from both wild type and Gdf11 E11.5 null embryos. ways involved in directing the proper formation of the We found that Gdf11 null cultures, which were devoid of ureteric bud that could rescue the Gdf11 phenotype. Another ureteric bud outgrowth at the time of dissection, were in- possibility is that kidney development was rescued in a duced by Gdnf protein to form numerous ectopic ureteric subset of the Gdf11 mutant embryos by myostatin, which is buds along the Wolffian duct. The analysis of the wild type 92% identical to Gdf11 within its C-terminal domain expression patterns for Gdf11 during various stages of kid- (McPherron et al., 1999) and has been shown to bind to ney organogenesis further supports our hypothesis that ActRIIB, the presumptive receptor for Gdf11 (see below) Gdf11 may regulate expression of Gdnf. We observed (Lee and McPherron, 2001; Oh et al., 2002). Analysis of Gdf11 expression in the mesonephros, the Wolffian duct, mice homozygous null for both Gdf11 and myostatin would and the metanephric mesenchyme at E10.5, prior to ureteric address the possibility of redundant function. bud formation. Gdnf is also expressed in the mesonephros The abnormalities in axial skeleton patterning and kid- and the metanephric mesenchyme at this stage (Pichel et al., ney development in Gdf11 mutant embryos are similar, 1996; Sanchez et al., 1996; Towers et al., 1998). Gdf11 although more severe than the defects observed in embryos mRNA was later detected in the metanephric mesenchyme mutant for a TGF␤ type II serine/threonine kinase receptor, and the invading ureteric bud at E11.5, a time when Gdnf activin receptor type IIB (ActRIIB) (Oh and Li, 1997; mRNA is specifically localized to the metanephric mesen- McPherron et al., 1999). Specifically, ActRIIB-deficient chyme (Pichel et al., 1996; Sanchez et al., 1996; Towers et mice have anterior transformations of their spinal column, al., 1998). As kidney development progresses, Gdf11 including three extra thoracic vertebrae, and a spectrum of mRNA becomes localized to the ureteric branches, to a kidney abnormalities, such as uni- or bilateral agenesis or population of uninduced metanephric mesenchyme in the hypoplasia of both kidneys. Recent studies have also shown renal cortex, and, to a lesser extent, to the condensed met- that Gdf11 can bind to ActRIIB (Oh et al., 2002), which anephric mesenchyme and differentiating nephrons. Like- raises the possibility that Gdf11 may use ActRIIB for cel- wise, Gdnf is expressed at later stages of kidney organo- lular signaling in the kidney. Therefore, we determined genesis, throughout the cortical region of the kidney and whether ActRIIB is expressed at the appropriate time and in most prominently in the condensed metanephric mesen- the appropriate tissues during metanephric kidney develop- chyme, but is downregulated in the differentiating nephric ment in order to act as the receptor for Gdf11. We showed tubules (Pichel et al., 1996; Sanchez et al., 1996; Towers et that ActRIIB mRNA could be detected in the mesonephros, al., 1998). Our in situ hybridization analysis shows that the Wolffian duct, and the metanephric mesenchyme prior Gdf11 is expressed early enough during kidney develop- to ureteric bud formation and in the metanephric mesen- ment to direct ureteric bud outgrowth from the Wolffian chyme and the invading ureteric bud at the initiation of duct, possibly by regulating the expression of Gdnf in the metanephric development in wild type embryos. ActRIIB metanephric mesenchyme. However, based on our results, it expression continued in these derivatives throughout kidney is not clear whether Gdf11 signaling is required in the organogenesis. Based on the expression patterns for metanephric mesenchyme or the Wolffian duct in order to ActRIIB and Gdf11 within the developing kidney and the regulate Gdnf expression and promote ureteric bud out- similar disruption phenotypes for these genes, it is possible growth. Furthermore, later expression patterns for Gdf11 in that Gdf11 uses ActRIIB for cellular signaling during renal the kidney suggest that Gdf11 may play multiple roles organogenesis. However, the examination of the expression during renal development. patterns for Gdf11 and ActRIIB did not reveal which tissue, A.F. Esquela, S.-J. Lee / Developmental Biology 257 (2003) 356Ð370 369 the metanephric mesenchyme or the Wolffian duct, was Avantaggiato, V., Dathan, N.A., Grieco, M., Fabien, N., Lazzaro, D., responsible for the initial signal that induced the formation Fusco, A., Simeone, A., Santoro, M., 1994. Developmental expression of the ureteric bud. of the RET proto-oncogene. Cell Growth Differ. 5, 305Ð311. Barasch, J., Qiao, J., McWilliams, G., Chen, D., Oliver, J.A., Herzlinger, Recent studies suggest that Gdf11 may, in addition to Ac- D., 1997. Ureteric bud cells secrete multiple factors, including bFGF, tRIIB, use a related TGF␤ type II serine/threonine kinase which rescue renal progenitors from apoptosis. Am. J. Physiol. 273, receptor, activin receptor type IIA (ActRIIA), for cellular sig- 757Ð767. naling in the kidney. Gdf11 has been shown to bind to Ac- Brophy, P.D., Ostrom, L., Lang, K.M., Dressler, G.R., 2001. Regulation of tRIIA (Oh et al., 2002), and expression of this receptor has ureteric bud outgrowth by Pax2-dependent activation of the glial de- rived neurotrophic factor gene. Development 128, 4747Ð4756. been observed in the developing kidney (Feijen et al., 1994). Davies, J., 2001. Intracellular and extracellular regulation of ureteric bud ϩ/Ϫ Ϫ/Ϫ ActRIIA /ActRIIB mice possess severe anterior transfor- morphogenesis. J. Anat. 198, 257Ð264. mations of the vertebral column that closely resemble the axial Donovan, M.J., Natoli, T.A., Sainio, K., Amstutz, A., Jaenisch, R., Sariola, abnormalities observed in the Gdf11 null mice (McPherron et H., Kreidberg, J.A., 1999. Initial differentiation of metanephric mes- al., 1999; Oh et al., 2002). Interestingly, 98% of ActRIIAϩ/Ϫ/ enchyme is independent of WT1 and the ureteric bud. Dev. Dyn. 24, Ϫ/Ϫ 252Ð262. ActRIIB animals showed kidney abnormalities compared Dressler, G.R., Deutsch, U., Chowdhury, K., Nornes, H.O., Gruss, P., with only 25% observed in the ActRIIB null animals and 96% 1990. Pax-2, a new murine paired-box containing gene and its expres- observed for the Gdf11 mutant mice (McPherron et al., 1999; sion in the developing excretory system. Development 109, 787Ð795. Oh et al., 2002). These results imply that ActRIIA and Ac- Durbec, P., Marcos-Gutierrez, C.V., Kilkenny, C., Grigoriou, M., Wartio- tRIIB share a redundant function in directing proper kidney waara, K., Suvanto, P., Smith, D., Ponder, B., Costantini, F., Saarma, M., 1996. GDNF signaling through the Ret receptor tyrosine kinase. organogenesis, possibly by using Gdf11 as their ligand. Nature 381, 789Ð793. In summary, we have shown that Gdf11 plays an impor- Ekblom, P., Alitalo, K., Vaheri, A., Timpl, R., Saxen, L., 1980. Induction tant role in the initiation of metanephric development. of a basement membrane glycoprotein in embryonic kidney: possible Based on the renal abnormalities observed in Gdf11 mutant role of laminin in morphogenesis. Proc. Natl. Acad. Sci. USA 77, mice, we conclude that Gdf11 directs ureteric bud out- 485Ð489. growth from the Wolffian duct. Our in situ hybridization Enomoto, H., Araki, A., Jackman, A., Heuckeroth, R.O., Snider, W.D., Johnson Jr., E.M., Milbrandt, J., 1998. GFR␣1-deficient mice have studies and explant culture experiments suggest that Gdf11 deficits in the enteric nervous system and kidneys. Neuron 21, 317Ð signals through ActRIIB to control the expression of Gdnf 324. in the metanephric mesenchyme in order to induce ureteric Feijen, A., Goumans, M.J., van den Eijnden-van Raaij, A.J.M., 1994. bud formation. To our knowledge, Gdf11 is the earliest Expression of activin subunits, activin receptors and follistatin in acting secreted factor identified to date in the molecular postimplantation mouse embryos suggest specific developmental func- tions for different activins. Development 120, 3621Ð3637. pathway that controls metanephric organogenesis. Gamer, L.W., Wolfman, N.M., Celeste, A.J., Hattersley, G., Hewick, R., Rosen, V., 1999. A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos. Dev. Biol. 208, 222Ð232. Acknowledgments Gavrieli, Y., Sherman, Y., Ben-Sasson, S.A., 1992. Identification of pro- grammed cell death in situ via specific labeling of nuclear DNA We thank Gregory Dressler and Joan Massague for provid- fragmentation. J. Cell Biol. 119, 493Ð501. ing the Pax2 and ActRIIB probes and Andrew Dudley for Grobstein, C., 1955. Inductive interactions in the development of the technical advice with the explant cultures. We also thank mouse metanephros. J. Exp. Zool. 130, 319Ð340. Grobstein, C., 1956. Trans-filter induction of tubules in mouse metaneph- Alexandra McPherron for critical reading of the manuscript. rogenic mesenchyme. Exp. Cell Res. 10, 424Ð440. Gdf11 was licensed by Johns Hopkins University to MetaMor- Heller, S., Sheane, C.A., Javed, Z., Hudspeth, A.J., 1998. Molecular mark- phix Inc. (MMI) and sublicensed to Wyeth. S.-J.L. is entitled ers for cell types of the inner ear and candidate genes for hearing to a share of sales royalty received by the University from sales disorders. Proc. Natl. Acad. Sci. USA 95, 11400Ð11405. of this factor. The University, S.-J.L., and A.F.E. also own Jing, S., Wen, D., Yu, Y., Holst, P.L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J.C., Hu, S., Altrock, B.W., MMI stock, which is subject to certain restrictions under Uni- Fox, G.M., 1996. GDNF-induced activation of the ret protein tyrosine versity policy. S.-J.L. is a paid consultant to MMI. The Uni- kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell versity in accordance with its conflict of interest policies is 85, 1113Ð1124. managing the terms of these arrangements. This work was Koseki, C., Herzlinger, D., Al-Awqati, Q., 1992. Apoptosis in metanephric supported by NIH Grant ROIHD35887 (to S.-J.L), and A.F.E. development. J. Cell Biol. 119, 1327Ð1333. 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