A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development.

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate.

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).

Functional maturation of drug transporters in the developing, neonatal, and postnatal kidney.

Because renal function in newborns is immature, the pharmacokinetics of drugs administered to neonates vary significantly from adult patients. The establishment of drug transport systems is a key process in the functional maturation of the nephron. However, a thorough examination of the expression of the main drug transporters in the kidney throughout all stages of development (embryonic, postnatal, and mature) has yet to be carried out, and the functional (physiological) impact is not well understood. Using time-series microarray data, we analyzed the temporal behavior of mRNA levels for a wide range of SLC and ABC transporters in the rodent kidney throughout a developmental time series. We find dynamic increases between the postnatal and mature stages of development for a number of transporters, including the proximal tubule-specific drug and organic anion transporters (OATs) OAT1 (SLC22a6) and OAT3 (SLC22a8). The OATs are the major multispecific basolateral drug, toxin, and metabolite transporters in the proximal tubule responsible for handling of many drugs, as well as the prototypical OAT substrate para-aminohippurate (PAH). We therefore performed specific in vivo pharmacokinetic analysis of the transport of PAH in postnatal and maturing rodent kidney. We show that there is a 4-fold increase in PAH clearance during this period. Clearance studies in Oat1 and Oat3 knockouts confirm that, as in the adult, Oat1 is the principle transporter of PAH in the postnatal kidney. The substantial differences observed supports the need for better understanding of pharmacokinetics in the newborn and juvenile kidney compared with the adult kidney at the basic and clinical level.

Hs2st mediated kidney mesenchyme induction regulates early ureteric bud branching.

Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFbeta superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st(-)(/)(-) kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st(-/-) UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development.

Protein kinase A regulates GDNF/RET-dependent but not GDNF/Ret-independent ureteric bud outgrowth from the Wolffian duct.

Embryonic kidney development begins with the outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) into the adjacent metanephric mesenchyme (MM). Both a GDNF-dependent and GDNF-independent (Maeshima et al., 2007) pathway have been identified. In vivo and in vitro, the GDNF-dependent pathway is inhibited by BMPs, one of the factors invoked to explain the limitation of UB formation in the unbudded regions of the WD surrounding the UB. However, the exact mechanism remains unknown. Here a previously described in vitro system that models UB budding from the WD was utilized to study this process. Because Protein kinase A (PKA) activation has been shown to prevent migration, morphogenesis and tubulogenesis of epithelial cells (Santos et al., 1993), its activity in budded and non-budded portions of the GDNF-induced WD was analyzed. The level of PKA activity was 15-fold higher in the unbudded portions of the WD compared to budded portions, suggesting that PKA activity plays a key role in controlling the site of UB emergence. Using well-characterized PKA agonists and antagonists, we demonstrated that at various levels of the PKA-signaling hierarchy, PKA regulates UB outgrowth from the WD by suppressing budding events. This process appeared to be PKA-2 isoform specific, and mediated by changes in the duct rather than the surrounding mesenchyme. In addition, it was not due to changes in either the sorting of junctional proteins, cell death, or cell proliferation. Furthermore, the suppressive effect of cAMP on budding did not appear to be mediated by spread to adjacent cells via gap junctions. Conversely, antagonism of PKA activity stimulated UB outgrowth from the WD and resulted in both an increase in the number of buds per unit length of WD as well as a larger surface area per bud. Using microarrays, analysis of gene expression in GDNF-treated WDs in which the PKA pathway had been activated revealed a nearly 14-fold decrease in Ret, a receptor for GDNF. A smaller decrease in GFRα1. a co-receptor for GDNF, was also observed. Using Ret-null WDs, we were able to demonstrate that PKA regulated GDNF-dependent budding but not GDNF-independent pathway for WD budding. We also found that BMP2 was higher in unbudded regions of the GDNF-stimulated WD. Treatment of isolated WDs with BMP2 suppressed budding and resulted in a 3-fold increase in PKA activity. The data suggests that the suppression of budding by BMPs and possibly other factors in non-budded zones of the WD may be regulated in part by increased PKA activity, probably partially through downregulation of Ret/GFRα1 coreceptor expression.

The instructive role of metanephric mesenchyme in ureteric bud patterning, sculpting, and maturation and its potential ability to buffer ureteric bud branching defects.

Kidney organogenesis depends on reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) to form the UB-derived collecting system and MM-derived nephron. With the advent of in vitro systems, it is clear that UB branching can occur independently of MM contact; however, little has been done to detail the role of MM cellular contact in this process. Here, a model system in which the cultured isolated UB is recombined with uninduced MM is used to isolate the effects of the MM progenitor tissue on the development and maturation of the collecting system. By morphometrics, we demonstrate that cellular contact with the MM is required for vectorial elongation of stalks and tapering of luminal caliber of UB-derived tubules. Expression analysis of developmentally significant genes indicates the cocultured tissue is most similar to an embryonic day 19 (E19) kidney. The likely major contributor to this is the functional maturation of the collecting duct and proximal nephron segments in the UB-induced MM, as measured by quantitative PCR, of the collecting duct-specific arginine vasopressin receptor and the nephron tubule segment-specific organic anion transporter OAT1, Na-P(i) type 2 cotransporter, and Tamm-Horsfall protein gene expressions. However, expression of aquaporin-2 is upregulated similarly in isolated UB and cocultured tissue, suggesting that some aspects of functional maturation can occur independently of MM cellular contact. In addition to its sculpting effects, the MM normalized a "branchless" UB morphology induced by FGF7 or heregulin in isolated UB culture. The morphological changes induced by the MM were accompanied by a reassignment of GFRalpha1 (a receptor for GDNF) to tips. Such "quality control" by the MM of UB morphology may provide resiliency to the branching program. This may help to explain a number of knockout phenotypes in which branching and/or cystic defects are less impressive than expected. A second hit in the MM may thus be necessary to make these defects fully apparent.

Analysis of metagene portraits reveals distinct transitions during kidney organogenesis.

Organogenesis is a multistage process, but it has been difficult, by conventional analysis, to separate stages and identify points of transition in developmentally complex organs or define genetic pathways that regulate pattern formation. We performed a detailed time-series examination of global gene expression during kidney development and then represented the resulting data as self-organizing maps (SOMs), which reduced more than 30,000 genes to 650 metagenes. Further clustering of these maps identified potential stages of development and suggested points of stability and transition during kidney organogenesis that are not obvious from either standard morphological analyses or conventional microarray clustering algorithms. We also performed entropy calculations of SOMs generated for each day of development and found correlations with morphometric parameters and expression of candidate genes that may help in orchestrating the transitions between stages of kidney development, as well as macro- and micropatterning of the organ.

The lectin Dolichos biflorus agglutinin is a sensitive indicator of branching morphogenetic activity in the developing mouse metanephric collecting duct system.

The urine collecting duct system of the metanephric kidney develops by growth and branching morphogenesis of an unbranched progenitor tubule, the ureteric bud. Bud branching is mainly dichotomous and new branches form from existing branch tips, which are also the main sites of cell proliferation in the system. This behaviour, and the fact that some genes (e.g. Wnt11, Sox9) are expressed only in tips, suggests that tip cells are in a specific state of differentiation. In this report, we show that the lectin Dolichos biflorus agglutinin (DBA), hitherto regarded and used as a general marker of developing renal collecting ducts, binds to most of the duct system but does not bind to the very tips of growing branches. The zone avoided by DBA corresponds to the zone that expresses Wnt11, and the zone that shows enhanced cell proliferation. If branching of the ureteric bud of cultured embryonic kidneys is inhibited in organ culture, by blocking the kidney's endogenous glial cell-derived neurothrophic factor (GDNF)-based branch-promoting signals, the DBA-binding zone extends to the very end of the tip but is lost from there when branching is re-activated. Similarly, if excess GDNF is provided to growing kidneys, the DBA-free zone expands. DBA-staining status therefore appears to be a sensitive indicator of the morphogenetic activity of the collecting duct system.

A high-resolution anatomical ontology of the developing murine genitourinary tract.

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.

Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions.

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.

A role for microfilament-based contraction in branching morphogenesis of the ureteric bud.

BACKGROUND: Branching morphogenesis of the ureteric bud/collecting duct epithelium is an important feature of kidney development. Recent work has identified many transcription factors and paracrine signaling molecules that regulate branching, but the physical mechanisms by which these signals act remain largely unknown. The actin cytoskeleton is a common component of mechanisms of morphogenesis. We have therefore studied the expression of, and requirement for actin filaments in the ureteric bud, a branching epithelium of the mammalian kidney. METHODS: Embryonic kidney rudiments were grown in organ culture. Actin expression in kidneys growing normally and those in which branching was inhibited was examined using labeled phalloidin. The morphogenetic effects of inhibiting actin organization and tension using cytochalasin D, butanedione monoxime, and Rho kinase ROCK inhibitors were assessed using immunofluorescence. RESULTS: F-actin is expressed particularly strongly in the apical domains of cells at the tips of branching ureteric bud, but this expression depends on the bud actively growing and branching. Blocking the polymerization of actin using cytochalasin D inhibits ureteric bud branching reversibly, as does blocking myosin function using butadiene monoxime. Inhibiting the activation of ROCK, a known activator of myosin, with the drugs Y27632 or with H1152 inhibits the expression of strong actin bundles in the ureteric bud tips and inhibits ureteric bud branching without inhibiting other aspects of renal development. CONCLUSION: The formation of tension-bearing actin-myosin complexes is essential for branching morphogenesis in the developing kidney.