J Am Soc Nephrol 11: S116–S119, 2000 Secreted Molecules in Metanephric Induction

THOMAS J. CARROLL and ANDREW P. McMAHON Department of Molecular and Cellular Biology, Biological Laboratories, Harvard University, Cambridge, Massachusetts.

Abstract. Nearly 50 yr ago, Clifford Grobstein made the ob- the classic model of metanephric induction. The studies of the servation that the ureteric bud induced the nephrogenic mes- classic ureteric inducer performed to date have most likely enchyme to undergo tubulogenesis. Since that discovery, sci- been characterizations of a mesenchyme-specific inducer, entists have attempted to characterize the molecular nature of Wnt-4, and its role in tubulogenesis. Ureteric induction most the inducer. To date, no single molecule that is both necessary likely involves a series of distinct events that provide prolif- and sufficient for nephric induction has been identified. Be- erative, survival, and condensation signals to the mesenchyme, cause of recent insights regarding the role of several secreted integrating the growth of the ureteric system with tubulogen- molecules in tubulogenesis, it has become necessary to revise esis.

The developmental biologic processes of the kidney have been logenesis. The conclusion drawn from this discovery was that the subject of intense study for more than 100 yr (for review, the ureteric bud induces tubulogenesis within the surrounding see reference (1). All three vertebrate kidney types (pro- mesenchyme. During further investigation, it was discovered nephros, mesonephros, and metanephros) are derivatives of a that a number of tissues, including, most notably, a dorsal region of the embryo known as the intermediate mesoderm. In portion of the embryonic spinal cord, are able to substitute for mice, a portion of the mesonephric duct, known as the meta- the ureter in this inductive interaction. Although the spinal cord nephric bud, branches dorsally approximately 10.5 d after certainly plays no role in the normal induction of the mesen- coitus and invades the caudal-most portion of the nephrogenic chyme, it was assumed that this functional mimicry is attrib- mesenchyme. The bud branches several times while growing utable to the expression of the endogenous inducer within the peripherally and eventually forms the collecting duct system of spinal cord tissue. In fact, the spinal cord seems to be a far the mature kidney. Shortly after invasion by the bud, the more effective inducer than the ureter itself and, since this mesenchyme juxtaposed to the bud condenses, undergoes an discovery, it has been used in place of the ureter in the majority epithelial transformation, and fuses with the duct, forming the of studies on metanephric induction. For example, observations metanephric tubule. The tubule elongates and proceeds through that the inductive event could take place across a filter (sug- a series of intermediate forms, known as the comma- and gesting that the inducer was a secreted molecule) but required S-shaped bodies, before ultimately forming the mature renal a large enough pore size to allow cell/cell contact (suggesting tubule. The proximal-most region of the tubule forms a spe- that the secreted molecule was closely associated with the cell cialized structure, i.e., the glomerulus, which is vascularized membrane) were made using spinal cord as the inductive and is the primary filtration apparatus of the organ. Mature source. murine kidneys consist of approximately 0.5 million precisely In recent years, developmental biologists have sought the arranged functional units, or nephrons, connected to the col- molecules responsible for a number of inductive signals that lecting duct system. Because of the importance of the kidney in were first identified embryologically, including the ureteric maintaining proper blood chemistry values, the correct ar- inducer. On the basis of the initial characterizations made by rangement and number of nephrons are essential. Grobstein, candidate molecules for the kidney inducer should It has been nearly 50 yr since Clifford Grobstein discovered satisfy a number of criteria (7). First, and most important, a that interactions between the ureteric bud and the adjacent candidate molecule must be expressed in the ureteric bud at the mesenchyme affect the normal development of the metaneph- time of induction, i.e., 10.5 d after coitus in mice. Second, a ric kidney (2–6). Grobstein revealed that, if the ureteric bud is candidate molecule must be a secreted factor that is closely separated from the metanephric mesenchyme at an early stage associated with the cell membrane. Finally, if the molecule is of development, then the mesenchyme fails to undergo tubu- truly involved in tubule induction, then it must be both neces- sary and sufficient for normal induction. This model makes the assumption that there exists a single Correspondence to Dr. Andrew P. McMahon, Department of Molecular and inducing molecule, which is the simplest hypothesis in the Cellular Biology, Biological Laboratories, Harvard University, 16 Divinity absence of contradicting data. However, to date, no single Avenue, Cambridge, MA 02138. Phone: 617-496-3757; Fax: 617-496-3763; E-mail: [email protected] molecule that satisfies all of these criteria has been identified. 1046-6673/1111-0116 Several secreted molecules, including members of the bone Journal of the American Society of Nephrology morphogenetic protein, fibroblast growth factor (FGF), insulin- Copyright © 2000 by the American Society of Nephrology like growth factor, and Wnt families of growth factors, are J Am Soc Nephrol 11: S116–S119, 2000 Secreted Molecules in Metanephric Induction S117 expressed in the invading ureteric bud; however, none of them Wnt-7b are capable of inducing tubulogenesis and thus cannot be the A second family member that is expressed in the developing classic inducer (for review, see reference 8). Although it was metanephros is Wnt-7b (10). Unlike Wnt-4, Wnt-7b is ex- recently demonstrated that members of the wingless/int family pressed within the epithelium of the ureteric bud. However, the of secreted glycoproteins are capable of inducing tubulogenesis time at which Wnt-7b mRNA can first be detected within the in vivo (9), to date only one Wnt (Wnt-11) that is expressed in ureteric epithelium (13.5 d after coitus) is not consistent with the tip of the ureteric bud during the inductive phase has been this molecule playing a role in induction. Furthermore, Wnt-7b identified (10). Unfortunately, as discussed below, this mole- mRNA is excluded from the tips of the ureter, the region cule is not sufficient to induce tubulogenesis in vitro. If a single thought to possess inductive properties, further diminishing the inductive factor exists, it cannot be one of the already charac- possibility of a role for this molecule in the inductive process. terized Wnt proteins. Furthermore, current data suggest that the Preliminary genetic analyses suggest that Wnt-7b plays a role Wnt signal originates not from the ureter but rather from the in duct maintenance or growth (Carroll, Pepicelli, & McMa- metanephric mesenchyme (11). In light of these data, it seems hon, unpublished observations), rather than induction. appropriate to reevaluate the nature of the metanephric inducer and the role of secreted proteins in the process of induction. Wnt-11 A search for Wnt genes expressed in the developing kidney revealed the previously uncharacterized Wnt-11 (10). The ex- Role of Wnt Proteins in Kidney Development pression pattern of this gene was particularly intriguing to The Wnt proteins have been demonstrated to play essential nephrologists. Wnt-11 is expressed within the mesonephric roles in a number of developmental processes in a wide range duct just before metanephric induction. As the ureteric bud of organisms (for review, see reference (12) or www. invades the metanephric blastema, Wnt-11 expression intensi- stanford.edu/ϳrnusse/pathways/allcomp.html). The discovery fies within the leading edge of the ureteric bud. As the ureter that cell lines expressing Wnt-1 are capable of inducing met- branches, Wnt-11 expression is maintained at the tips of each anephric mesenchyme to undergo tubulogenesis (9) led to the branch but is lost in the intervening epithelium. Wnt-11 con- hypothesis that Wnt proteins may function as the classic met- tinues to be expressed at the tips of the ureter as they move anephric inducer. In fact, the spinal cord, which is capable of toward the cortex of the developing kidney, which is a region substituting for the endogenous inducer, expresses several Wnt of continuing induction. proteins, providing further circumstantial evidence that this Wnt-11 expression closely matches that expected for a mol- important family of molecules may be involved in metanephric ecule involved in tubule induction. However, when cell lines induction. Because Wnt-1 is not expressed in developing kid- expressing Wnt-11 were co-cultured with metanephric mesen- neys, it has been hypothesized that this molecule mimics chyme, they failed to induce tubulogenesis (11). Wnt-11 there- another Wnt in in vitro experiments. To date, four Wnt proteins fore fails to satisfy at least one of the criteria (sufficiency) for that are expressed in the developing metanephros have been a metanephric inducer. It seems unlikely that this molecule identified, i.e., Wnt-4, -6, -7b, and -11 (10). Because Wnt-6 is plays a role in induction. Quite surprisingly, in this same assay expressed at extremely low levels and is thought not to play a it was found that cell lines expressing Wnt-4 were capable of major role in kidney development, it is not further discussed. inducing tubulogenesis. This result is surprising in light of the We concentrate on the prospective roles of Wnt-4, -7b, and -11 fact that Wnt-4 was previously shown to act downstream of the in induction and later nephric development. ureteric signal. Furthermore, it has been shown that metaneph- ric mesenchyme itself is not capable of inducing tubulogenesis (so-called homeogenetic induction) (14). The fact that a gene Wnt-4 that is expressed within the metanephric condensations and The first Wnt shown to be involved in kidney development acts downstream of the ureteric signal is capable of inducing was Wnt-4 (13). Wnt-4 is expressed in the aggregating meta- tubulogenesis is quite unexpected. nephric mesenchyme at 11.5 d after coitus, shortly after ure- teric bud invasion. Targeted ablation of this gene results in Signal Relay in Tubulogenesis embryos with severely hypoplastic kidneys. In the absence of As mentioned above, nearly all of the characterizations of Wnt-4, the metanephric mesenchyme condenses normally but metanephric induction have been performed using spinal cord fails to aggregate into pretubule clusters and undergo tubulo- as the inductive tissue, under the assumption that this tissue genesis. Morphologic and molecular marker analyses suggest mimics the endogenous inducer. Because the Wnt molecules that Wnt-4 is involved in the transition of tubular mesenchyme behave identically to the spinal cord in this in vitro assay, it has to epithelium. Interestingly, the ureteric epithelium of mutant also been assumed that they mimic the ureteric signal. How- kidneys undergoes several rounds of branching, suggesting that ever, the ability of Wnt-4 to induce tubulogenesis necessitates tubulogenesis is not required for this process. The expression a reevaluation of these findings. patterns and phenotypes of Wnt-4 mutants suggest that Wnt-4 If the Wnt molecules truly mimic the ureteric inducer, then is a mesenchymal factor that acts downstream of the initial co-culture of a Wnt-expressing cell line with Wnt-4 mutant inductive events. Therefore, Wnt-4 cannot be the ureteric in- mesenchyme should not induce tubulogenesis (because Wnt-4 ducer. has been shown to produce its effect downstream of the ure- S118 Journal of the American Society of Nephrology J Am Soc Nephrol 11: S116–S119, 2000 teric signal). Quite surprisingly, this was not the case. Several combination with ␤-FGF and transforming growth factor-␣ Wnt-expressing cell lines could induce tubulogenesis in Wnt-4 (TGF-␣), supports full differentiation of cultured metanephric mutant mesenchyme, suggesting that the Wnt proteins produce mesenchyme (19,20). Recently, Barasch et al. (21) demon- their effects at the level of or downstream of Wnt-4 (11). strated that leukemia inhibitory factor (LIF), which by itself is Therefore, it seems that the Wnt proteins do not mimic the unable to induce tubulogenesis, can induce full differentiation classic inductive signal at all. The characteristics of the classic when supplemented with ␤-FGF and TGF-␣, suggesting that it inducer were defined using spinal cord as the inductive tissue. may be the unidentified ureteric factor. Interestingly, although The spinal cord, like the Wnt proteins, was assumed to mimic LIF is expressed in the ureter, it seems to act downstream of the endogenous ureteric signal. The ability of the spinal cord to the primary inductive signal, inasmuch as markers of the initial induce tubulogenesis might be attributable to the fact that it inductive response (Pax-2 and Wnt-4) are expressed in cultured expresses several Wnt proteins (including Wnt-4), rather than mesenchyme in its absence (21). This observation correlates the possibility that it expresses the endogenous inducer. To well with the fact that ␤-FGF by itself can cause condensation investigate this possibility, the inductive properties of the spi- but not tubulogenesis of metanephric mesenchyme (18). How- nal cord were reexamined (11). If the spinal cord mimics a ever, it is surprising that the expression of Wnt-4 in mesen- ureteric signal, its inductive abilities should be blocked by chyme cultured with ␤-FGF and TGF-␣ does not lead to Wnt-4 mutant mesenchyme. However, if the spinal cord is tubulogenesis, as would be expected from the in vitro experi- simply mimicking the Wnt signal, then it should be able to ments. There are clearly regulatory events that we do not now induce tubulogenesis in Wnt-4 mutant mesenchyme. Rather understand, and further investigation is needed. surprisingly, the latter hypothesis seems to be correct. Spinal cord is capable of inducing tubulogenesis just as well in Wnt-4 Conclusion mutant mesenchyme as in wild-type mesenchyme. This finding Currently available data support a multifactorial process for strongly suggests that the spinal cord signal is acting at the metanephric induction. Tubulogenesis most likely requires level of or downstream of Wnt-4 within the metanephric mes- multiple sequential inductive signals from the ureter, in com- enchyme. This further suggests that most studies of the nature bination with feedback from the mesenchyme to the ureter of the metanephric inducer that have been performed to date (Figure 1). After the initial inductive event (possibly involving have actually characterized a mesenchymal inducer, Wnt-4. Met- ␤-FGF), the mesenchyme is signaled to condense. Further anephric induction seems to involve a series of events that orig- differentiation (tubulogenesis) requires additional signals, inates in the ureter but includes mesenchyme-specific signals. which, at least in vitro, can be provided by the LIF molecule. Placing Wnt-4 within this system is more difficult. The exper- Ureteric Inducer 2000 iments by Barasch et al. (21) suggest that Wnt-4 is downstream The data reviewed here suggest that much of what we know of the FGF-like signal but upstream of LIF. However, at least (or thought we knew) regarding the nature of the ureteric in vitro, Wnt-4 alone is sufficient to induce tubulogenesis in inducer is probably incorrect. The studies performed to date the absence of additional ureteric factors, seemingly in contra- have most likely characterized Wnt-4 and its role in tubulo- diction to these results. Further investigation is certainly genesis. These recent discoveries present nephrologists with a needed to establish the organizational relationships of these number of unanswered questions. What do we know regarding molecules during kidney development. It would be interesting the molecular nature of the ureteric signal? Because we must to test the ability of Wnt-4 to induce tubulogenesis in LIF ignore much of the previous data (because it was obtained mutant mesenchyme. Unfortunately, neither LIF nor its recep- using spinal cord), the only fact we know definitely is that the tors manifest a severe kidney phenotype when mutated (per- ureter is necessary for tubulogenesis. It remains unclear haps because of molecular redundancy), precluding these ex- whether the ureter itself actually induces tubulogenesis. There periments. However, assessments of the ability of LIF to are several alternative explanations for the lack of tubulogen- induce tubulogenesis in Wnt-4 mutant mesenchyme can be esis after removal of the ureter. For example, the ureter could performed and should help us to better understand this series of provide survival and/or proliferative signals to the metanephric events. mesenchyme (dead cells do not form tubules). This is sup- Regardless of how these signals relate to each other, it seems ported by the observations that mesenchyme cultured in the quite clear that multiple signals from the ureteric bud and the absence of an inducer undergoes apoptosis (15) and some metanephric mesenchyme cooperate to integrate the survival, signals produced by the ureter, including bone morphogenetic proliferation, and condensation of the metanephric blastema protein-7, promote survival and growth of the mesenchyme but with ureteric branching. This sequence leads to a tightly reg- do not induce tubulogenesis (16,17). However, other factors ulated and precisely controlled process of nephric morphogen- produced by the ureter, including ␤-FGF (18), cause cultured esis. With increased understanding, we should be able to dif- metanephric mesenchyme to condense, suggesting that this ferentiate each of these events at both the molecular and structure provides more than just survival or proliferative sig- morphologic levels. The next several years should provide us nals. These data suggest that metanephric induction is not a with evidence that will further clarify these processes. What- single event. Further support for this notion is derived from ever the future brings, it seems certain that the classic one- several recent studies. It has been demonstrated that an uniden- signal model for metanephric induction is greatly oversimpli- tified factor (or factors) from a ureteric bud cell line, in fied. The future holds great promise for the discovery of unique J Am Soc Nephrol 11: S116–S119, 2000 Secreted Molecules in Metanephric Induction S119

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