REVIEW

Gene regulatory network of renal primordium development

Michael Marcotte & Richa Sharma & Maxime Bouchard

Received: 1 August 2013 /Revised: 6 September 2013 /Accepted: 6 September 2013 /Published online: 9 October 2013# IPNA 2013

Abstract Animal development progresses through the step-wise deployment of gene regulatory networks (GRN) encodedin the genome. Comparative analyses in different species andorgan systems have revealed that GRN blueprints are com-posed of subcircuits with stereotypical architectures that areoften reused as modular units. In this review, we report theevidence for the GRN underlying renal primordium develop-ment. In vertebrates, renal development is initiated by theinduction of a field of intermediate mesoderm cells competentto undergo lineage specification and nephric (Wolffian) ductformation. Definition of the renal field leads to the activationof a core regulatory subcircuit composed of the transcriptionfactors Pax2/8, Gata3 and Lim1. These transcription factorsturn on a second layer of transcriptional regulators while alsoactivating effectors of tissue morphogenesis and cellular spe-cialization. Elongation and connection of the nephric duct tothe cloaca (bladder/urethra primordium) is followed by meta-nephric kidney induction through signals emanating from themetanephric mesenchyme. Central to this process is the acti-vation and positioning of the glial cell line-derived neuro-trophic factor (Gdnf)–Ret signaling pathway by networksubcircuits located in the mesenchyme and epithelial tissuesof the caudal trunk. Evidence shows that each step of the renalprimordium developmental program is regulated by structuredGRN subunits organized in a hierarchical manner. Under-standing the structure and dynamics of the renal GRN willhelp us understand the intrinsic phenotypical variability ofcongenital anomalies of the kidney and urinary tract and guideour approaches to regenerative medicine.

Keywords Pax2/Pax8 .Gata3 .Lxh1/Lim1 .Gene regulatorynetwork .Mouse pronephros/mesonephros . Kidney

Gene regulatory networks in development

Organogenesis requires a systematic diversification and dif-ferentiation of cells into organized structures. This is achievedthrough the gradual elaboration of genetic networks that sus-tain developmental programs. It is the hierarchy and thestructured nature of these networks that allow the great plas-ticity and fidelity of embryo development.

Comparative studies of gene regulatory networks (GRNs)from a spectrum of species have revealed the presence ofconserved subcircuit architectures organized in hierarchical se-quences [1, 2]. In vertebrates GRNs regulate key processes suchas hematopoietic stem cell homeostasis, mesoderm developmentand heart organogenesis [3]. In these networks, each subcircuitplays a specific role, either as a regulator or effector of cellfunction. An important regulatory subcircuit, referred to as thekernel, is of crucial importance at the onset of structure or organformation [4]. Kernel subcircuits are typically composed oftranscription factors setting up organogenetic programs. Due totheir importance, kernel subcircuits show a high degree ofevolutionary conservation [4]. Such conservation is also seenin signaling pathways that represent modular subcircuits used indifferent developmental contexts. Effector units, on the otherhand, are involved in receiving the information from regulatorysubcircuits and translating them into cellular activities, such aschanges in cell shape, proliferation or migration. The finaloutput of these regulatory networks is performed by cell differ-entiation subcircuits that maintain tissue homeostasis and assignspecific physiological roles to the different units of the organ [4].

The renal primordium (or pro/mesonephros) represents aunique system to study gene network organization due to itsrelative simplicity as well as the increasing number of geneticregulators identified as necessary for its development.

The renal primordium

Renal development begins at embryonic day (E) 8.5 of mousedevelopment with specification of the renal identity from

M. Marcotte :R. Sharma :M. Bouchard (*)Goodman Cancer Research Centre and Department of Biochemistry,McGill University, Montreal, 1160 Pine Avenue W.,Montreal, QC, Canada H3A 1A3e-mail: maxime.bouchard@mcgill.ca

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urogenital progenitor cells located in the intermediate meso-derm (IM), at the level of the sixth to eighth somites. Thisprocess is followed by nephric duct formation through mesen-chymal–epithelial transitions of newly committed cells [5]. Thenephric duct grows caudally down the embryo trunk to fusewith the cloaca (bladder/urethra primordium). As it elongatescaudally, the nephric duct initiates mesonephric tubule forma-tion in the adjacent nephric cord to form the mesonephros [6].In amniotes, the caudal part of the mesenchymal nephric cordspecializes into metanephric mesenchyme, which drives meta-nephros (definitive kidney) development by inducing uretericbud (UB) formation and subsequent branching. In turn, uretertips induce nephron formation from nephrogenic progenitorslocated in the metanephric mesenchyme [7]. In lower verte-brates, such as zebrafish and frog, the mesonephros constitutesthe functional form of the kidney [7].

Inducing renal fate

Extensive studies in the chick and frog systems have describedthe roles of neighboring tissues, namely the paraxial mesoderm,the lateral plate mesoderm and the overlying ectoderm, in renalfield specification within the IM [8–10]. Detailed informationon the mechanism and dynamics of renal lineage induction is,however, still elusive. It was shown that the ectoderm overlyingthe intermediate and lateral plate mesoderm is required for theexpression of the renal markers Pax2 and Lim1 (also calledLhx1) and for nephric duct extension [11]. Interestingly, thisactivity can be partially substituted by exogenous bone mor-phogenetic protein-4 (Bmp4) activity [11](Fig. 1a). As Bmp4expression in the lateral plate mesoderm is lost by ectodermremoval [11], Bmp may act through self-maintenance in thelateral plate mesoderm where it regulates gene expression inboth the IM and lateral plate mesoderm in a dose-dependentmanner [9]. In explant cultures, Bmp activation of the IMmarker Odd-skipped related 1 (Osr1), is blocked by cyclohex-imide, indicating that the activation of Osr1 by Bmp signalingis translation-dependent and therefore indirect [9]. In Xenopusand Danio rerio , Osr1 promotes the expression of Pax2 andLim1 , suggesting that it plays a role in establishing kidneyprecursor cells. In the mouse, the role of Osr1 in lineagespecification is less clear as the pro/mesonephros form normallyin Osr1-deficient embryos [9]. However, Osr1 is cruciallyinvolved in metanephric mesenchyme formation [12].

In addition to ectodermal and lateral plate inductive activ-ities, signals from the trunk paraxial mesoderm (somitic field)are required for the induction of the chick pronephros [9, 10].Physical separation of the paraxial mesoderm and IM resultsin a loss of renal marker expression in the IM [10]. Thus,along the medial–lateral axis, Bmp signaling from the lateralplate mesoderm seems to act in conjunction with unidentifiedparaxial mesoderm signals to induce renal fate in the IM.

Other important regulators of IM definition along the medial-lateral axis have been identified. Among them, Foxc1 and Foxc2play an important role in preventing IM expansion into theparaxial mesoderm region [13]. Conversely, Nodal-like signal-ing is necessary for maintaining Pax2 expression and IM fateand for preventing paraxial mesoderm expansion [14]. Note-worthy, Bmp signaling is required for the activation of Pax2 byNodal. In this system,Nodal andFoxC genes therefore establisha bistable exclusion motif in which mutually repressive signalsdefine different tissue/cellular states. Here, the Nodal–FoxCmotif defines the paraxial to IM boundary and sets up thecompetence of IM cells to initiate renal gene expression inresponse to Bmp and other inductive signals (Fig. 1a).

Rostro–caudal morphogenetic field definition

The initial induction of renal fate in the IM occurs only at thelevel of the sixth to eighth somite along the rostro–caudal axis,suggesting that these cells possess an intrinsic competence torespond to medial–lateral inducing signals. Recent studies haveidentified signaling molecules that define the renal morphoge-netic field along the rostro–caudal axis. One such morphogen isretinoic acid (RA) [15–19]. RA cooperates with roof-plate-derived activin in inducing Lim1 expression in the IM [20, 21](Fig. 1a). The fact that RA induces Lim1 expression in thepresence of a translation inhibitor suggests a direct regulatoryinteraction [15]. Recent data further show that RA acts throughthe regulation of the patterning transcription factor HoxB4 [21].HoxB4 is indeed required to set the competence of IM cells torespond to renal specification signals along the rostro–caudal axis[8]. In chick embryos, misexpression of HoxB4 in anterior non-renal IM, either by RA administration or by plasmid-mediatedoverexpression, results in ectopic renal gene expression [21].Notably, Hox gene induction by RA precedes that of Lim1 ,while the endogenous expression domain of Hoxb4 overlapswith that of Lim1 in the pronephros [18]. Thus, RA may definethe renal morphogenetic field through HoxB4 gene expressionand subsequently regulate nephric duct formation by direct Lim1gene activation in a feedforward subcircuit pattern. Interestingly,anotherHox gene,HoxA6 , is associated with the specification ofthe caudal limit of the renal morphogenetic field. Misexpressionof HoxA6 in prospective duct-forming regions of the IM resultsin repression of duct formation [22]. It would therefore appearthat HoxA6 has the potential to inhibit the RA–HoxB4–Lim1regulatory subcircuit (Fig. 1a). Whether or not this subcircuit isconserved in mammals remains to be determined.

Pax2/8, a bottleneck of the renal gene regulatory network

Evidence so far suggests that the renal field acquires its com-petence by genetic programs acting along the mediolateral and

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rostro–caudal axes. Renal inducing factors, such as Bmps, arethen able to act on competent intermediate progenitor cells toinduce the renal cell fate. The earliest known markers of therenal fate are the transcription factors Pax2, Pax8 and Lim1 [10,23–29]. Pax2 and Lim1 are activated by Bmp signaling [9] andare accepted readouts of renal lineage specification. According-ly, Pax2 and Pax8 were identified as both necessary and suffi-cient for specification of the nephric lineage [30]. In Pax2/8double mutant embryos, there are no morphological signs ofnephric duct formation, concomitant with the absence of ex-pression of the nephric duct markers Ret and Lim1 [30]. As aresult, the pro/mesonephros fails to form and the IM undergoesapoptosis at E9.5 [30].

To date, only Pax2/8 -deficient embryos show a completefailure to induce the renal lineage. However, other transcrip-tion factors, such as Gata3 and Lim1, also play an importantrole in early renal primordium morphogenesis. Lim1 is ini-tially expressed in the IM and lateral plate mesoderm at E7.5and E8.5 but becomes restricted to the nephric duct primordi-um by E9.5 [31–34]. Conventional as well as conditional genetargeting approaches have shown that Lim1 is required fornephric duct extension down the trunk, possibly bymaintaining nephric duct survival [33]. Consequently, Lim1 -deficient embryos fail to form a metanephros [33, 35, 36].However, Lim1 deficiency does not prevent renal lineagespecification as pro/mesonephros formation still occurs inLim1 -deficient embryos [33, 35, 37]. On the other hand,

targeted inactivation of Gata3 causes wayward growth ofthe nephric duct down the trunk and a loss of Ret expression[38]. This defect leads to an absence of the metanephros due toa failure of the nephric duct to complete its extension to thecloaca [38]. These findings implicate Gata3 as a key regulatorof guidance molecules that control nephric duct extension. Asis the case for Lim1 deficiency, Gata3 mutant embryos stillform the renal cell fate lineage [38].

In an effort to establish the early gene regulatory network ofpro/mesonephros development, Boualia et al. recentlyperformed a systematic analysis of Pax2/8, Lim1 and Gata3molecular function [37]. This analysis demonstrated thatGata3and Lim1 both depend on Pax2/8 function, while Pax2 andPax8 regulation is independent of Gata3 and Lim1 [33, 35, 37,38] (Fig. 1b). In addition, Gata3 and Lim1 were shown to berequired for each other’s expression, thereby establishing abistable double positive feedback loop [37]. A direct transcrip-tional regulation was shown for Pax2 on Lim1 andGata3 [37],and for Gata3 on Lim1 . Hence, together, Pax2/8, Gata3 andLim1 form a kernel subcircuit that stably establishes the renalprimordium transcriptional program (Fig. 1b).

Later during pro/mesonephros development, Pax2 formsanother positive feedback loop circuit with the transcriptionfactor Emx2 [39]. Subsequent to its induction in the IM, Pax2activates Emx2 transcription [39]. At this stage, Pax2 expres-sion is independent of Emx2. At E11.5, however, Pax2 ex-pression is lost in the nephric duct and ureter of Emx2 mutant

Fig. 1 Gene regulatory subcircuits of pro/mesonephros development. aRenal lineage specification subcircuit. b Core regulatory subcircuit com-posed of Pax2/8, Gata3 and Lim1 and their regulatory outputs. c Retregulatory subcircuit whereby Wnt-β-catenin AND Pax2/8 are requiredfor Gata3 expression, while Gata3 AND retinoic acid signaling are bothnecessary for full activation of the Ret receptor tyrosine kinase. d Gene

regulatory subcircuit leading to glial cell line-derived neurotrophic factor(Gdnf) expression in the metanephric mesenchyme. See text for details.Gene regulatory network (GRN) subcircuits were generated with theBioTapestry software. Arrows and blunted lines Positive and negativeregulatory events, respectively. These interactions are not necessarilydirect. Dotted lines refer to long-term maintenance

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embryos, suggesting that Emx2 feeds back to maintain Pax2expression, forming what appears to be an indirect positivefeedback loop. This regulatory system presumably sustainsthe expression of Pax2 and Emx2 over time. The Pax2;Emx2network unit is important for proper ureter budding asPax2;Emx2 double heterozygous embryos show congenitalanomalies of the kidney and urinary tract (CAKUT)-like renalmalformations [39].

An analysis of the downstream gene regulatory networkfurther revealed that the Pax2/8–Gata3–Lim1 core circuit acti-vates a deeper layer of gene regulatory molecules, as well aseffector molecules. Regulators such as Emx2 [39], Evi1, Id4and Plac8 [37] indeed require Pax gene function. Interestingly,the Pax2–Plac8 regulatory interaction has also been observed inthe zebrafish pronephros [40]. In this system, Plac8 was iden-tified as a negative regulator of Pax2 and was postulated to bean addition to the renal gene network that allows elaborateglomerular development, as mutant embryos resembleaglomerular renal systems [40]. The Pax2–Gata3–Lim1 kerneladditionally regulates effector molecules, such as the key reg-ulator of metanephros induction Nephronectin [41] as well asWfdc2 , Pcdh19 and Ret [37, 42]. Direct regulatory events havebeen shown for Gata3 on the Ret promoter [37, 38, 42] and forLim1 on the Nephronectin locus [37]. Lim1 is additionallyrequired for the maintenance of E-cadherin and Wnt9b in thepro/mesonephros [35]. Of interest is the reported role of epi-thelial nephronectin to activate Gdnf in the metanephric mes-enchyme through binding to alpha8beta1 integrin [41]. Thispoints to a regulation of Gdnf expression in the metanephricmesenchyme by nephric duct regulators, through a relativelyshallow transcriptional cascade involving Pax2–Lim1–nephronectin and alpha8beta1integrin (Fig. 1b, d). Hence, thePax2–Gata3–Lim1 kernel is a major driver of pro/mesonephrosmorphogenesis (Fig. 1b).

Establishing Ret expression in the nephric duct

One of the key effectors downstream of the Pax2–Gata3–Lim1kernel is the Ret receptor tyrosine kinase. In response to itsligand Gdnf, Ret is essential for ureteric budding andbranching, as well as for nephric duct elongation [43] (seebelow). Genetic and molecular evidence shows that Pax2 acti-vates Gata3 , which in turn regulates Ret expression [37, 42,44]. Interestingly, canonicalWnt signaling seems to branch intothis pathway as conditional inactivation of the β-catenin geneCtnnb1 , which leads to a near complete loss of bothGata3 andRet expression but does not affect Pax2 expression in thenephric duct [42, 45]. This observation points to a Gata3–Rettranscriptional unit that requires both Pax2 and β-catenin. Retexpression is also dependent on mesenchymal RA as embryosdeficient for the RA signaling, either through Raldh2 mutationor expressing a dominant negative RA receptor, completely

lose Ret expression while Gata3 expression is unaffected [46,47]. Given the drastic loss of Ret expression in the Gata3 andCtnnb1 mutants, or when RA signaling is disrupted, the net-work architecture apparently includes two logical “AND” gateswhereby two independent factors are necessary to regulate athird one. In this network, Pax2ANDβ-catenin are required forGata3 expression, and RA signaling AND Gata3 are requiredfor expression of Ret (Fig. 1c). Typically, this type of circuitserves to limit gene expression to a region in which the regu-latory signals overlap. Accordingly, the highest levels of Retexpression are in the caudal nephric duct [48], which corre-sponds to the portion of the duct closest to the highest expres-sion of Raldh2 [44]. Interestingly, it is possible that the tran-scription factor Emx2 acts in complement or relay to Gata3 tomaintain Ret expression, as Emx2 is also regulated by Pax2andCtnnb1 [39, 49] and required to maintain Ret expression inthe UB [50].

The Gdnf–Ret–Wnt11 maintenance loop

One of the crucial network subcircuits at the core of ureterbudding and branching is a positive feedback loop betweenGdnf, Ret and Wnt11. Mesenchymally expressed Gdnf bindsRet and its co-receptor Gfra1 at the site of UB induction tostimulate downstream signaling, which results in the expressionofWnt11 that feeds back to maintain Gdnf expression [51]. Thismaintenance of the Gdnf–Ret circuit at the ureter tips allows forcontinued budding and branch elongation of the metanephrickidney. Based on the Gdnf–Ret–Wnt11 feedback loopMenshykau and Iber [52] produced a mathematical model reca-pitulating the budding and branching behaviors of the kidney[52]. This model was robust to changes, to noise and to inputvariations. It thus supports the notion that the Gdnf–Ret–Wnt11feedback loop is at the heart of the kidney branching morpho-genesis and is an excellent example of the application of thenetwork framework to developmental biology.

The transcription factors Etv4 and Etv5 are among the keyeffectors of the core Gdnf–Ret circuit to achieve appropriatebranching morphogenesis (Fig. 2). Accordingly, Etv4/5 dou-ble knockouts display a complete failure in branching mor-phogenesis, as well as reduced expression of the effectors andregulators of branching including Spry1 , Wnt11 , Cxcr4 , MetandMmp14 [53]. The indirect regulation of Spry1 by Ret is animportant example of a negative regulatory loop, which acts todampen the strength of the Ret signaling response (Fig. 2).Spry1 -deficient embryos show supernumerary UBs as a resultof increased Ret signaling in the nephric duct [54], which canbe limited by the reduction ofGdnf dosage [54]. In addition toSpry1, Sema3a also acts through its receptor Nrp1 to nega-tively regulate ureter budding by dampening Gdnf–Ret sig-naling [55] (Fig. 2). Interestingly, inhibition of the Ret-regulated gene Cxcr4 in explant kidney cultures also leads

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to downregulation of Pax2 ,Wnt11 and Gdnf [56], suggestinga relatively complex modulation of theWnt11–Gdnf feedbackmechanism.

Based on the reported role of Wnt11 in other systems, theWnt11 feedback loop is expected to act mostly through the non-canonical Wnt signaling pathway. In the mouse, Wnt11 inacti-vation results in Gdnf downregulation and kidney hypoplasia[51]. Although Wnt11 is part of the Gdnf–Ret maintenanceloop, Wnt11 mutant embryos have a much milder phenotypethan that of Gdnf , Ret or Gfra1 mutant kidneys, respectively[57–62], suggesting that Wnt11 plays a supportive role in thisregulatory loop. Different Wnt signals act through Frizzledreceptors (Fzd) [63] of which several are expressed in thedeveloping kidney. Among these, Fzd4 and Fzd8 showoverlapping expression in the developing kidney [64]. Interest-ingly, combinatorial deletion of Fzd4 and Fzd8 results insignificantly smaller kidneys due to a reduction in branching,concomitant with reduced Gdnf expression [64]. As this phe-notype closely resembles that ofWnt11-deficiency, it is possiblethat Wnt11 signaling proceeds through these receptors. In ad-dition to the expected regulation of non-canonical signaling byWnt11, the canonical pathway also plays an important role inureter budding and branching. Conditional inactivation of thecanonical intermediate β-catenin (Ctnnb1) specifically in theUB results in dysplastic kidney rudiments, harboring reducedexpression of Gdnf , Ret and Wnt11 [49].

Ret signaling in the nephric duct is further modulated bymesenchymally derived Bmp activity (Fig. 2), as recombinant

Bmp4 is sufficient to repress Gdnf-induced ureter budding,while heterozygosity for Bmp4 results in ectopic ureter bud-ding [65–67]. The negative effect of Bmp4 is counteracted byat least two independent mechanisms that allow the ureter tobud properly. In the mesenchyme, Grem1 binds Bmp to pre-vent Bmp receptor binding. Accordingly, Grem1 mutant em-bryos display renal agenesis, which can be rescued by the lossof either a single allele of Bmp4 or both alleles of Bmp7[67–69]. In addition, the inhibitory role of Bmp signaling onthe Gdnf–Ret circuit is also modulated in the epithelial com-partment through the action of the neuropeptidemolecule NPY[70]. In nephric duct culture experiments, NPY was shown tofacilitate Gdnf-driven budding and was capable of rescuingBmp4-mediated inhibition of budding [70]. Interestingly, NPYwas upregulated in response to Gdnf stimulation, suggestingthe existence of an additional positive feedback loop in whichGdnf activates NPY, thereby further sensitizing the nephricduct to Gdnf by inhibition of Bmp4 repressive activity (Fig. 2).

Activation and spatial restriction of Gdnf expression

The range and level of Ret activation in the nephric duct alsodepends on the regulation ofGdnf expression in the metaneph-ric mesenchyme. Gdnf expression depends on a GRN initiatedby Osr1 and Hox11 [12, 71]. The transcription factor Osr1 isrequired for the expression of Eya1 and Pax2 in the metaneph-ric mesenchyme [12], which are both necessary for Gdnf

Fig. 2 Integrated view of the gene regulatory network (GRN) underlyingpro/mesonephros development and ureteric bud induction. Regulatoryinteractions are derived from molecular and genetic evidence obtained inthe mouse, chick, frog or zebrafish systems. See text for details. GRNwas

generated with the BioTapestry software. Arrows and blunted linesPositive and negative regulatory events, respectively. These interactionsare not necessarily direct. Dotted lines refer to long-term maintenance

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expression [72] (Fig. 1d). Similarly, the loss of Hoxa11 ,Hoxc11 and Hoxd11 results in Gdnf downregulation and fail-ure of metanephric development [71]. Together, Eya1 and Pax2form a molecular complex with the Hox11 paralogs that direct-ly binds and activates the Gdnf , Six2 [73] and Six1 genes [74](Fig. 1d). The latter, together with its paralog Six4, also takespart in Gdnf regulation [75]. Ultimately this cascade results inthe activation of additional regulators of kidney development,including Lim1 , Sal1 and Grem1 (Fig. 1d). In parallel to this,the Gdf11 signaling molecule is also critically involved inGdnfexpression in the metanephric mesenchyme [76] (Fig. 2).

The negative regulation of Gdnf expression along therostro–caudal axis is also necessary to limit the range of itsactivity, thus ensuring the formation of a single UB at thecorrect location. A number of genes have been identified thatgradually repress Gdnf activity/expression in the rostral mes-enchyme. Among these, Foxc1 acts to restrict both Eya1 andGdnf (Fig. 1d), as its loss results in ectopic UBs leading toduplex kidneys and hydronephrosis [77]. Similarly, bothSlit2- and Robo2-deficient mice show an expansion of Gdnfexpression, resulting in duplex kidneys and hydronephrosis(Fig. 1d). Interestingly, reduction ofGdnf gene dosage in Slit2mutant embryos rescues this phenotype, in accordance with aprimary role for Slit2 inGdnf regulation. Hence, while Gdnf–Ret signaling has conclusively been identified as the mainsignaling event leading to UB formation: a fine and complexregulation of their respective expression and activity turns outto be crucial for normal metanephric kidney induction.

Fibroblast growth factor signaling: complementingthe Gdnf–Ret signaling pathway

While Gdnf–Ret is undoubtedly central to ureter budding, micenull for Gdnf or Ret occasionally form UBs and hypoplastickidneys [78, 79], suggesting that other factors contribute tobudding. Interestingly, mice null for Spry1 and either Ret orGdnf develop fully branched kidneys, although the branchingpattern is disrupted [79]. However, further deletion of a singleallele of Fgf10 in these mice results in kidney agenesis,suggesting that Spry1 may additionally act to inhibit fibroblastgrowth factor (Fgf) signaling [79] (Fig. 2). Presumably, theremoval of this hidden inhibition then allows for Fgf-drivenbranching through bypassing Ret to directly activate Etv4/5[79] (Fig. 2). Furthermore, Fgf10 has also been shown tocooperate with Gdnf in UB induction and branching morpho-genesis [79]. However, the aforementioned ability of Fgf10 toinduce a fully branched kidney in the absence of Gdnf andSpry1 suggests a secondary reciprocal positive feedback loopbetween the metanephric mesenchyme and UB involving Fgfs.While the identity of all the Fgf ligands involved is unclear,several are expressed during kidney development, and it islikely that they act redundantly in this system. Accordingly,

the compound loss of Fgf9 and Fgf20 results in kidney agen-esis [80], whereas Fgf7-null mice display a similar phenotypeto the Fgf10 knockout embryos [81]. Furthermore, loss of theFgf receptor genes Fgfr2 or Fgfrl1 results in a spectrum ofureter branching dysgenesis phenotypes [82–86]. In addition,FgfrL1 has been shown to act upstream of the Etv4 transcrip-tion factor [86]. Thus, while Fgfs seem to have a moderateeffect on ureter budding, they can act as a secondary driver ofgrowth and branching and thereby contribute significantly tometanephros development and on occasion compensate for lossof Gdnf-driven branching.

Conclusion

In all vertebrate species, pro/mesonephros morphogenesis is theinitial phase of renal development. With the identification ofcrucial regulators of this process, we are now in a position toestablish a blueprint of the GRN driving early renal lineagespecification and morphogenesis (Fig. 2). As research pro-gresses, additional regulators will be identified that can be addedto the initial GRN to provide a more accurate understanding ofrenal development and disease. Although developmental defectscan originate from single-gene deficiencies, a more elaborateunderstanding of the renal GRN will gradually help us interpretdevelopmental diseases of the kidney as regulatory networkdeficiencies and provide a molecular explanation for the greatphenotypical variability of the CAKUT disease group.

Acknowledgments Special thanks to Katherine Stewart for her criticalreview of the manuscript. MB’s laboratory is supported by grants fromthe Canadian Institutes for Health Research (MOP-130431) and by theKidney Foundation of Canada. MB holds a Canada Research Chair inDevelopmental Genetics of the Urogenital System.

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