Xenopus: A Prince Among Models for Pronephric Kidney Development
Elizabeth A. Jones
Molecular Physiology, Department of Biological Sciences, Warwick University, Coventry, United Kingdom
Address correspondence to: Dr. Elizabeth A. Jones, Molecular Physiology, Department of Biological Sciences, Warwick University, Coventry CV4 7AL, UK. Phone: 00-44-2476-523061; Fax: 00-44-2476-523701; E-mail: elizabeth.jones{at}warwick.ac.uk
Recent advances in techniques that are available to study themolecular development of the frog Xenopus make developmentalstudies using this amphibian amenable to experimentation. Thisreview outlines some of the attractive features of this modelorganism and describes how these techniques can be and are beingused in studies on the organogenesis of the larval amphibiankidney, the pronephros. The roles of micromanipulation, grafting,and in vitro culturing of animal caps are discussed as toolsin the analysis of kidney development and as a source of tissuefor subtractive hybridization strategies. The importance ofexpression cloning and functional analysis of newly identifiedpronephros-specific genes are also described. Finally, transgenesisand electroporation are discussed as potentially new methodsof gene delivery to the pronephros. These techniques can beused to help identify the gene networks that control organogenesisof this larval kidney form, which will undoubtedly have applicabilityto higher vertebrate kidney development.
Since the early 1990s, there has been a resurgence of interestin studies aimed at the molecular dissection of the amphibianpronephros, the larval kidney of the frog. In general, the pseudotetraploidXenopus laevis has been the experimental amphibian of choice;however, with the genetic advantages afforded by its diploidcousin Xenopus tropicalis, this animal will surely soon enterthe frame. Xenopus tropicalis, although somewhat smaller insize, has much faster development than Xenopus laevis, reachingmaturity in 4 to 6 mo. This coupled with its diploid genomemakes the possibility of successful genetic screens a likelihoodrather than pure fantasy. For those whose major interest isthe pathology of the human kidney, it might seem an obscurechoice of developmental model in which to study the vertebratekidney. However, during the course of this, review I hope toconvince the skeptical reader that Xenopus actually is a princeamong models for vertebrate kidney organogenesis, which canprovide new and exciting functional information to all researcherswho have an interest in nephrology.
The kidneys of higher vertebrates form from three successivestructures: the pronephros, the mesonephros, and finally themetanephros. Each of these forms is progressively more complexin the number and organization of the nephrons, and in mammals,only the last two forms are functional. However, the formationof all three forms of kidney is essential, with each subsequentorgan being dependent on the presence of the former structurefor its origin (1). The successive derivation of progressivekidney forms occurs throughout vertebrate evolution. As someof the molecular steps involved in early induction, patterning,and differentiation events are established, it has become clearthat recapitulation of genetic programs that control these developmentprograms occurs (reviewed in 2–5). Because the pronephrosis the first and simplest structure to form, this perhaps makesit an appropriate place to start to unravel the mysteries ofkidney organogenesis. However, in higher vertebrates, the pronephrosforms only as a rudimentary organ that does not function. Furthermore,the embryonic stage at which pronephros formation occurs isinaccessible in utero; thus, an alternative system in whichto study these earliest events is essential.
The frog can provide such a system. Xenopus can be hormonallystimulated to produce eggs throughout the year. These eggs canbe fertilized in vitro, and then all subsequent developmentoccurs in either water or simple salt solution. No feeding isnecessary until approximately 6 d of development, by which timeall of the major organ systems have formed and are functional.The large size and external development make the frog embryoeasy to manipulate. Thus, grafting; explanting small piecesof tissue; and microinjection of DNA, mRNA, and protein allare possible, making the frog embryo one of the systems of choiceto identify the function of molecules during development.
Considerable progress has been made on both the developmentaland the molecular events that take place during pronephros formation(2,4,6–8). The pronephros is a simple nonintegrated nephronthat forms from the intermediate mesoderm on either side ofthe embryo. This simple paired organ has three major compartments:the glomus, tubules, and duct. The glomus forms from the splanchnicintermediate mesoderm and is the vascularized filtration unit.It projects into the coelomic space, where blood filtrate isreleased (8). The tubules form opposite the glomus from thesomatic intermediate mesoderm and form a convoluted mass. Threenephrostomes, ciliated funnels, open into the coelom and functionto drive waste from the coelom into the tubules (6). The nephrostomesopen into the collecting tubules, which leads to the commontubule. The common tubule eventually joins the pronephric duct,which opens to the exterior via the cloaca (7). The study ofeach of these components has been aided by the discovery ofmolecular or immunohistochemical markers that decorate eachof these structures in a specific manner (9). Figure 1 showseach of these component structures and their positional relationshipto one another.
Figure 1. Morphologic components of the pronephros of Xenopus laevis embryos. (A) The pronephros is made up of three major structural components: The glomus, which is the filtration device; the tubules, which connect to the coelom via three nephrostomes; and the duct, which exits to the cloaca. (B and C) Immunohistochemical staining with the monoclonal antibodies 3G8 and 4A6 allows detection of the tubules and duct, respectively (Photograph courtesy of R. Collins) (9). (D) In situ analysis with the WT-1 marker identifies glomus tissue (photograph courtesy of C. Haldin). (E) Cryostat section of a WT-1 in situ–stained embryo shows the glomus projecting into the coelom. (F and G) In situ analysis with a 4a-tubulin probe decorates the ciliated nephrostomes (Chen and Jones, unpublished observations, 2004).
Vertebrates that live in a freshwater aquatic environment haveto have active excretory systems to excrete the excess fluidthat enters the body through the skin. Larval amphibia are noexception to this and as a consequence need to actively reabsorbsalts to prevent ion depletion. The pronephros functions bythe same filtration and resorption processes as any other vertebratenephron, and the process has to be highly efficient (10–12;reviewed in 6). The urine in amphibia is delivered into thecoelomic space after filtration in the glomus. It then entersthe larval nephron via ciliated funnels, the nephrostomes, whichdrive the urine into the pronephric tubules. Aquatic amphibia,such as Xenopus, excrete ammonia that readily diffuses awayin an aquatic environment rather than urea. Urea is producedby amphibia only after metamorphosis and the adoption of a terrestrialhabitat. Once in the tubules, a variety of transporters andco-transporters ensure the setting up of an electrochemicalgradient and the efficient absorption of salts and other macromoleculesin an analogous manner to that achieved in higher vertebratekidneys.
Which Molecular Signals Direct Pronephric Induction and Patterning?
The pronephros forms between the paraxial mesoderm (presumptivesomites) and the lateral plate just ventral to somites 3 to5. All of the components of the pronephros arise from mesodermaltissue, and little is known about the signals received by presumptivepronephric mesoderm that instruct it to become the pronephros.It is known, however, that a number of different signals thatinduce and pattern the structure are given. The anterior somitesare essential for pronephros development and provide an essentialfirst signal. If the body plan of Xenopus is perturbed and somitesdisrupted, as occurs in ultraviolet-treated embryos, then thepronephroi do not form (13). Furthermore unspecified intermediatemesoderm from the pronephric region can be instructed to formpronephric tubules in tissue recombination experiments withdissected anterior somites. The exact timing and nature of thissignal is as yet unidentified, although we know that it is relativelylocalized to the anterior somites. It is thought that perhapsthis signal is responsible for setting up the pronephric field.
The downstream targets of this signal are the transcriptionfactor Pax8 and the LIM/homeodomain protein Xlim-1. It is likelythat Xlim-1 is essential for pronephros development becauseit is essential for all aspects of higher vertebrate kidneydevelopment. Furthermore, when dominant negative Xlim-1 constructsare overexpressed in Xenopus embryos, development of the pronephrictubules is inhibited at early tailbud stages. This suggeststhat Xlim-1 is not essential for the initiation of the pronephrosbut is essential for the subsequent growth and elongation ofthe pronephric tubules (14). Initially, Pax8 and Xlim-1 havedifferent expression domains, suggesting that they are regulatedby different signals. It has been suggested that Pax8 acts asa maintenance factor for Xlim-1, although it is unclear whatactivates Xlim-1 in its initial domain of expression. The regionof the embryo, which expresses both Xlim-1 and Pax8, includesall three domains of the pronephros: glomus, tubules, and duct.
Subsequent patterning events in the pronephric anlagen showthat mediolateral patterning has occurred. The Wilms tumor gene-1orthologue xWT1 is expressed only in the medial portion of thepronephric anlagen, consistent with its later distribution inthe glomus (15). This distribution may result from the inhibitoryinfluence of the epidermis on xWT1 expression perhaps via BMPsignaling. xWT1 also inhibits the expression of Pax8 and Xlim-1,thereby confining their expression to the lateral portion ofthe pronephric anlagen, which will give rise to the tubulesand duct.
Final patterning events involve the dorso/ventral patterningof the anlagen exemplified by the Wnt-4 expression pattern.At stage 26, this is restricted to the dorso/lateral componentof the pronephros (16) and members of the Notch pathway (Serrateand Delta), which also become localized in the dorsal compartment(17). Activation of Notch signaling specifies "not duct," thusdefining a compartment of the pronephros that gives rise tothe tubules (17).
These molecular signals are accompanied by morphologic changes.The first sign of pronephric segregation is a change in cellshape in the somatic intermediate mesoderm at early tailbudstages. The glomus also starts to form at this stage, the glomusprimordium being split off from that of the tubule and ductas the intermediate mesoderm is split into splanchnic and somaticmesoderm by the formation of the coelom (8). Shortly after thetubule primordium has formed, it starts to change shape, indicatingits final pattern. The dorsal edge becomes molded to indicatethe position of the three nephrostomes, and ventrally, the mostdistal element of the tubule is thrown anteriorly. By stage25, the main body of the pronephros is obvious as a solid massof cells below somites 3 and 4 and the duct has started to extendposteriorly to the cloaca. The nephrostomes are first evidentat stage 28. From stage 30, the tubules start to develop a lumen.The pronephros becomes fully functional at stage 38, but thetubules continue to coil, forming a convoluted mass by feeding(6). Having set out some of the molecular players and morphologicevents that occur in pronephros development, we now considersome of the advantages afforded by this amphibian experimentalsystem.
Micromanipulation and Grafting in Amphibian Pronephric Development
One of the major advantages of amphibian embryos is that developmentoccurs in simple saline solution throughout the stages whennormal organogenesis occurs. Small explants of material canbe dissected from early embryos and cultured in the presenceor absence of growth factors. Such tissue is conveniently dissectedfrom the blastula-stage embryo, when the embryo consists oftwo basic types of cells: those in the animal pole, which willgive rise to epidermis and neural tissue, and those in the vegetalregion, which will give rise to the endoderm or gut. The mesodermarises at the equator, after an inductive event between thesetwo cell types (18). The animal pole cells can be dissectedwith an eyebrow hair knife to give an animal cap (Figure 2A).This material if cultured in saline alone will differentiateinto atypical epidermis but if treated with the growth factoractivin at 10 ng/ml and retinoic acid at 10–5 M will differentiateto pronephros at high frequency (19). These induced pronephrictubules express genes characteristic of pronephric differentiationin the correct sequence and approximately the same time at whichthey would be expressed during normal development, suggestingthat the material induced in vitro parallels normal development(20). These pronephroi, induced in vitro, have been shown tobe functional when transplanted into nephrectomized host embryos(14). Initially, it was thought that only pronephric tubuleswere induced in this way, but now it is clear that conditionscan be identified in which duct is also able to differentiate,using 10–4 M RA and 10 ng/ml activin (21). Reverse transcriptase–PCRanalysis for the glomus molecular marker xWT1 also defines conditionsfor glomus tissue development (22). Thus, all of the componentsof the pronephros can be induced in vitro. This ability to generatepronephros tissue in vitro has been invaluable in the developmentof the subtractive hybridization strategies used in the identificationof new genes expressed in the pronephros (20,23,24). In fact,the animal caps can act as a source of undifferentiated cellsthat are capable of differentiating under the appropriate treatmentinto a wide variety of cell types (reviewed in 25).
Figure 2. Manipulative techniques in Xenopus embryos. (A) Animal caps can be dissected from blastula-stage embryos with eyebrow hair knives. The addition of activin and retinoic acid at appropriate concentrations instructs the formation of pronephric tissues at high frequency. (B) Animal caps can be used as inert chambers to culture dissected pronephric primordia in specification assays (19,23). (C) The two-cell embryo can be injected with mRNA transcribed in vitro from pools of cDNA clones to identify genes with a functional role in pronephros development (24). The injected side is co-labeled with a lineage label (GFP or -galactosidase), which allows the identification of the experimental side of the embryo versus the contralateral control. The embryos then are analyzed by in situ hybridization for genes that mark early pronephric territories, e.g., Xlim-1 (blue). Differences in the experimental versus the control side of the embryo indicate a role for the injected mRNA in pronephric development.
Animal cap and explant studies have also been used to definethe time at which signals are given to intermediate mesodermto instruct the formation of the pronephros (22,25). In theseexperiments, explants were taken from progressively earlierpronephros or presumptive pronephros and cultured between twoanimal caps in a Holtfreter sandwich until elements of the pronephroscould be identified immunohistochemically or assayed by reversetranscriptase–PCR (Figure 2B). These experiments showthat tubule and glomus are specified by stage 12.5 just aftergastrulation and that duct is specified at stage 14. Also, ithas been shown, by grafting experiments, that anterior somitescan provide a signal that can convert unspecified intermediatemesoderm to pronephric tissue (13). These experiments have informedthe search for the initial inducers of the pronephros, whichis now being carried out by expression cloning.
Identification of New Genes with Functional Roles in Pronephric Development
Because Xenopus embryos can be microinjected with ease, theyoffer an ideal system to assay expression libraries to identifygene function in the development of tissues and/or organs. Thepronephros, because it develops close to the surface of theembryo and can be easily visualized by the expression of specificmarkers such as Xlim-1 and Pax8, represents an ideal organ inwhich to identify genes that affect organogenesis. Such large-scalescreens have been carried out, the most notable success beingthat of Grammer et al. (27). A total of 50,000 clones from atadpole stage cDNA library were screened in pools of 96 individualclones. The pools were transcribed in vitro, and the mRNA wasintroduced into one cell of the two-cell embryo to target oneside of the embryo with the exogenous mRNA. Embryos then werecultured and assayed using in situ markers to define particulardevelopmental structures. Pax8 was used to define the developingpronephros at stage 28 (Figure 2C). Pools with consistent developmentaleffects were subpooled, and 66 clones were identified with adefinable biologic activity representing 57 discrete cDNA. Althoughpools were identified with pronephric disrupting activity, nospecific genes were isolated with such activity. Grammer etal. (27) suggested that this might be due to the broad approachtaken with both the initial library and the screening. A morespecific screen using cDNA from more defined regions of theembryo might yield results in pronephric organogenesis. Becausewe now can identify tissues with biologic activities in thedevelopment of the pronephros, expression screening of thissort is clearly an attractive and a focused approach to identifying,functionally, the genes involved in the earliest pronephricevents. The availability of full-length expression clones inX. tropicalis from four developmental stages will also increasethe chance of identifying developmentally relevant genes (28).
The ability to obtain differentiated pronephros from animalcaps that have been incubated in retinoic acid and activin (seeprevious section) has also led to the successful use of differentialhybridization or subtractive hybridization strategies to identifynew genes with a functional role in pronephric development (20,23,24,29).These approaches have used the high level of pronephric tissuethat differentiates in treated caps as a source of mRNA enrichedin transcripts from pronephric genes. Two highly specific pronephricgenes have been cloned after subtractive hybridization, whichare not expressed significantly in other tissues: Annexin IVand XSMP-30. Two others, the zinc finger protein XCH3–3band XCIRP the cold-inducible RNA binding protein have somewhatwider distributions. XCIRP-1, Annexin IV, and XCH3–3ball have been shown to have a functional role in pronephricdevelopment by either overexpression analysis or by morpholinooligonucleotide knockdown (Table 1) (24,29,31).
The subtractive hybridization approaches just described arethe most powerful ways by which to identify molecules that areactive in pronephric kidney development, using the embryo itselfas the experimental readout. New genes that have been identifiedthrough a functional screen have, by definition, a functionalrole in kidney development and patterning. However, for newgenes that have been identified as being expressed in the pronephros,the next challenge is to identify whether they potentially functionin developmental processes themselves. For higher vertebrates,the major techniques for analysis of function involve the productionof either conditional or nonconditional transgenic animals.This is an expensive and time-consuming process that does notalways lead to an analyzable phenotype, because of either functionalredundancy or embryonic lethality. Xenopus, however, offersa system whereby overexpression by injection of normal or mutantmRNA or reduction of expression after injection of morpholinooligonucleotides can be achieved quickly and inexpensively (29).This approach has been used to great advantage in a varietyof systems in Xenopus, including the pronephros (Figure 3).
Figure 3. Early cleavage divisions in Xenopus divide the embryo into regions that are fated to become specific structures. At the eight-cell stage, the ventral vegetal cells are fated to contribute to the pronephros. At the 32-cell stages from the ventral C tier, C2 and C3 contribute to this tissue. Microinjection of mRNA into individual blastomeres therefore can be used to deliver specific gene products to the developing pronephros.
Initial studies focused on genes that were cloned in Xenopusand already had been shown to have a role in kidney developmentin higher vertebrates such as xWT1, Xlim-1, Pax8, Notch, Wnt-4,and Xlmx-1b (14–17,32,33). All of these genes have beenshown to have effects on pronephric development (summarizedin Table 1). These studies, however, add to our knowledge, becauseno mouse knockout has been analyzed at the pronephric stageof development, so the exact timing of the effect in highervertebrates is unknown because transgenic embryos are analyzedonly at later stages of development. Perhaps the power of theamphibian system is best exemplified as a means of analyzinggenes that are expressed in the pronephros and for which noprevious functional data are available. Thus Annexin IV, XCIRP1,and XCH3b all are shown to have newly identified roles in kidneydevelopment (Table 1) (24,29,31).
One of the most significant advances in recent years has beenthe development of transgenesis in the frog (33–38). Thistechnique involves the restriction enzyme mediated incorporationof DNA into the genome before the first cell division, thusensuring that all of the cells of the subsequent embryo carrythe DNA and that germline transmission from reared adult transgenicfrogs is possible (Figure 4A). Both X. laevis and X. tropicalishave been used successfully in this procedure, which withina few hours of microinjection can generate hundreds of individualtransgenic embryos. Many of the transgenic animals have beenmade with constructs that incorporate GFP as a reporter systemto monitor expression (39,40). The external development of thetadpole allows the transgenic animals to be monitored at multipletimes over the course of early development to establish theexpression pattern of the transgene. Multiple transgenes havealso been used both carried on the same plasmid and on differentplasmids, which allow easy identification of transgenic animalsas well as identification of overlapping regions of gene expressionindicating potential interactions (39,40).
Figure 4. Transgenic techniques in Xenopus embryos. (A) Restriction enzyme mediated incorporation transgenesis has been used successfully in X. laevis and X. tropicalis to generate transgenic embryos and frog lines (31–35). (B) The binary Gal4-UAS system has been adapted for use in the frog to allow tissue-specific expression of the transgene (39). (C) Cre recombinase transgenics also allow tissue-specific transgene expression (42). Colored areas in tadpoles indicate regions of tissue-specific gene expression, in this case illustrating fluorescence reporter gene expression in the somites (muscle blocks). ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein.
Transgenic animals can either be studied in a transient manneror established as lines. It is at this point that the diploidgenome and a generation time of 4.5 to 6 mo of X. tropicalisbecome significant advantages compared with the pseudotetraploidgenome of X. laevis and a generation time of 12 mo or more (39).Transgenic animals can be used in identical ways to wild-typeanimals. Microinjection, grafting, and dissection all can becarried out, and genetic screening strategies are also beingdeveloped (40,41). Transgenic techniques are now becoming morerefined, allowing more targeted gene expression. Four differentapproaches have been taken: The use of the binary Gal4-UAS system,the development of modified progesterone RU-486 and Tetracycline-oninducible systems, the use of heat shock-inducible transgenes,and most recently the development of a Cre-Lox recombinase system(42–45).
The binary Gal4-UAS system was developed in Drosophila for targetedmis-expression (46,47). This approach depends on the generationof transgenic activator lines, which express the yeast transcriptionfactor Gal4, and effector lines in which the gene of interestis flanked by multiple copies of the upstream activator sequences,UAS, capable of binding Gal4. Crossing two such lines will resultin the expression of the effector transgene in the spatial andtemporal pattern of the promoter driving the Gal4 activator(Figure 4B). Depending on the promoter used, tissue-specificexpression can be achieved (42). This system can be made hormone-inducibleif, in the activator line, the Gal4 activator is fused to themutated human progesterone receptor and the VP16 transcriptionalactivation domain. Addition of the synthetic progesterone hormoneanalogue RU-486 will prevent sequestration of the fusion proteinin the cytoplasm and, after nuclear entry, will result in effectiveligand-inducible transactivation of the gene of interest (48).A similar strategy used a Gal4–retinoid X receptor fusionas the activator transgene to identify naturally occurring retinoidX receptor ligand hotspots after UAS-GFP expression in liveembryos (49).
For studying genes that are involved in relatively late developmentalevents such as metamorphosis, inducible systems are a majoradvantage. These systems allow potentially lethal transgenesto be switched on at a precise developmental time, allowingperiods of effective activity to be assessed. In this way, byexpressing a tetracycline-sensitive dominant negative thyroidhormone receptor, controlled by the addition of doxycycline,the period of time in which thyroid hormone controls innervationof the developing limb from the spinal cord has been established(40). The use of heat shock promoters can also temporally controlthe expression of the transgene, thus allowing the gene actionlater in development to be dissected from early effects of thegene (41). Recently, cryopreservation of sperm from a heatshock-inducibletransgenic line opened the way for easier preservation of transgeniclines (50).
The Cre-Lox recombinase system has also been tested in transgenicfrogs (45,51). Cre recombinase was introduced as a transgenedriven by the muscle actin tissue-specific promoter. This transgenicline was crossed with a line that contains a reporter transgenethat contains the cytomegalovirus promoter driving enhancedcyan fluorescent protein, flanked by lox P recombination sites,and enhanced yellow fluorescent protein (Figure 4C). Activationof the Cre recombinase in the muscle was associated with a colorchange in the reporter as a result of removal of enhanced cyanfluorescent protein and replacement by enhanced yellow fluorescentprotein.
These techniques are now ripe for transfer to the study of pronephricdevelopment. All that is needed is a reliable promoter to drivekidney-specific expression. The obvious candidates are promotersthat drive highly specific expression, such as Annexin IV, fromthe earliest time of specification through to the time whenthe pronephros is functional. However, this has not yet beenisolated. An alternative is to take advantage of the fact thatpromoters from other species can faithfully reproduce tissue-specificexpression patterns in the frog (52,53). Although candidatepromoters have been identified in mammals, they have not beentested extensively in the amphibian system (54–57). Oncesuch promoters become available, they can be used to drive genesspecifically in the kidney, which will further aid analysisof the functional networks of genes that are involved in pronephrosformation.
Targeting by transgenesis, however, is completely dependenton the availability of suitable tissue-specific promoters totarget gene expression both temporally and spatially. It isfor this reason that the technique of electroporation has beenexploited for the delivery of plasmids to particular tissuesand organs (reviewed in 58,59). This technique leads to fast,localized expression that has been particularly well exploitedby chick developmental biologists as an alternative to a replication-competentretrovirus system for gene transfer (60). This technique hasbeen applied successfully to Xenopus, in which electroporationinto the neural tube in neurula and tadpole stages has beenachieved (61–63). We are now applying this technique tothe pronephros to specifically overexpress genes with potentialroles in pronephric development and patterning (Kyuno and Jones,unpublished observations, 2004).
Conclusion
In conclusion, the frog provides a simple model vertebrate systemto analyze the development of the pronephric kidney, the firstvertebrate kidney to be formed. The external development andease with which the developing kidney rudiment can be dissectedand/or grafted provide an experimentally tractable system thatis not available in higher vertebrates. That kidney structurescan be induced in vitro has made the identification of new genes,previously unidentified in kidney structures, a realistic proposition.This knowledge then can be transferred to higher vertebratesystems, where it may shed additional light on metanephric development.
The frog provides an experimental system in which the functionof genes can be investigated. This can be achieved by microinjectionof wild-type or mutant mRNA, the effects being scored by analyzingthe morphology and/or gene expression changes in the developingpronephros. This approach can be used for both frog genes andgenes from higher vertebrates, including human. This has beenexploited particularly in the study of human HNF1 (64,65). Inthis study, a frameshift mutation in HNF1 associated with renalagenesis is shown to have a similar effect on pronephros developmentin the frog. This opens the way for wider functional characterizationin the frog system of such mutations that cause significantrenal disease in humans.
The development of genetic techniques such as transgenesis,whereby gene expression can be controlled both spatially andtemporally, offers the real prospect of being able to characterizeand generate the pattern of interacting genes in the nephrogenicnetwork. Such network analysis will certainly inform the medicalworld and perhaps help in the identification of appropriatecourses of therapy and treatment.
Acknowledgments
Work in the author’s laboratory is funded by the BBSRCand the Wellcome Trust.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
fusion as the activator transgene to identify naturally occurring retinoid X receptor 
(64,65). In this study, a frameshift mutation in HNF1
