Hnf1b Is Essential for Nephron Segmentation During Nephrogenesis

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HNF1b Is Essential for Nephron Segmentation during Nephrogenesis

† † Richard W. Naylor,* Aneta Przepiorski,* Qun Ren, Jing Yu, and Alan J. Davidson*

*Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; and †Department of Cell Biology, University of Virginia, Charlottesville, Virginia

ABSTRACT Nephrons comprise a blood filter and an epithelial tubule that is subdivided into proximal and distal segments, but what directs this patterning during kidney organogenesis is not well understood. Using zebrafish, we found that the HNF1b paralogues hnf1ba and hnf1bb, which encode homeodomain transcription factors, are essential for normal segmentation of nephrons. Embryos deficient in hnf1ba and hnf1bb did not express proximal and distal segment markers, yet still developed an epithelial tubule. Initiating hnf1ba/b expression required Pax2a and Pax8, but hnf1ba/b-deficient embryos did not exhibit the expected downregulation of pax2a and pax8 at later stages of development, suggesting complex regulatory loops involving these molecules. Embryos deficient in hnf1ba/b also did not express the irx3b transcription factor, which is responsible for differentiation of the first distal tubule segment. Reciprocally, embryos deficient in irx3b exhibited downregulation of hnf1ba/b transcripts in the distal early segment, suggesting a segment-specific regulatory circuit. Deficiency of hnf1ba/b also led to ectopic expansion of podocytes into the proximal tubule domain. Epistasis experiments showed that the formation of podocytes required wt1a, which encodes the Wilms’ tumor suppressor-1 transcription factor, and rbpj,which encodes a mediator of canonical Notch signaling, downstream or parallel to hnf1ba/b. Taken together, these results suggest that Hnf1b factors are essential for normal segmentation of nephrons during kidney organogenesis.

J Am Soc Nephrol 24: 77–87, 2013. doi: 10.1681/ASN.2012070756

How renal progenitors differentiate into the differ- podocytes is dependent on the activity of the ent nephron segments is poorly understood. The Wilms’ tumor suppressor-1a (Wt1a) transcription zebrafish pronephric kidney has emerged as a useful factor and Notch signaling, similar to observations model to study nephrogenesis due to the high degree in mammals.7–9 The remaining renal progenitors of cellular and genetic conservation with the mam- differentiate into a tubule with distinct proximal malian nephron but without a dependency on prior and distal segments. This process involves a con- renal structures.1 The zebrafish pronephros com- siderable refinement in the expression domains prises a single midline renal corpuscle attached to of early expressed renal genes such as pax2a,and two tubules that are segmented into proximal and the activation of later-acting genes such as irx3b distal segments, including the proximal convoluted tubule (PCT), proximal straight tubule (PST), distal early tubule (DE), and distal late tubule (DL).2 The zebrafish pronephros arises from the in- Received July 31, 2012. Accepted October 9, 2012. termediate mesoderm (IM) during development Published online ahead of print. Publication date available at and initially expresses the transcription factor genes www.jasn.org. 3–5 pax2a, pax8,andlhx1a. The anteriormost cells Correspondence: Dr. Alan J. Davidson, Department of Molec- adopt a podocyte fate and express genes such as ular Medicine and Pathology, University of Auckland, Auckland nephrin and podocin, encoding critical components 1142, New Zealand. Email: [email protected] of the glomerular blood filter.6 The formation of Copyright © 2013 by the American Society of Nephrology

J Am Soc Nephrol 24: 77–87, 2013 ISSN : 1046-6673/2401-77 77 BASIC RESEARCH www.jasn.org that is essential for the expression of DE segment-specific compromised the activity of the cdh17 promoter (n=5 of 68 genes.10 injected embryos were weakly eGFP+) compared with the in- In this study, we identified paralogues of hepatocyte nuclear tact promoter (n=36 of 64 injected embryos were eGFP+;Fig- factor-1 beta (hnf1ba and hnf1bb), encoding homeodomain ure 1A) consistent with Hnf1b factors acting as direct inducers transcription factors, as essential regulators of nephron for- of cdh17. mation. Zebrafish embryos deficient in hnf1ba/hnf1bb ectopically express podocyte markers in the region where the Nephron Expression of cdh17 is Lost in proximal tubule normally forms. Although an epithelial tu- hnf1ba/b-Deficient Embryos bule arises from the remaining IM, it fails to express segment- We next investigated the effect of hnf1ba/b deficiency on cdh17 specific solute transporter genes. Epistasis experiments expression in vivo. Embryos homozygous for hi1843, a reces- revealed that hnf1ba/b acts upstream or in parallel to wt1a sive retrovirus-induced mutation in hnf1ba,17 develop pericar- and Notch signaling to restrict podocyte fate. Taken together, dial edema and die around 5 dpf (Supplemental Figure 3A). these results implicate Hnf1b factors as key determinants of Incrosses of hnf1bahi1843/+ fish resulted in a quarter of the off- nephron segmentation that act to restrict podocyte formation spring with reduced cdh17 expression in the DL segment and activate segment-specific gene expression programs. (Figure 1B). We hypothesized that this restricted loss of cdh17 transcripts was due to redundant activities of hnf1bb in the other segments. Consistent with this, injection of an hnf1bb RESULTS morpholino into hnf1bahi1843 homozygous embryos induced a loss of cdh17 expression from all segments (Figure 1B). Geno- Hnf1b Factors Directly Regulate the cdh17 Promoter typing confirmed these embryos were homozygous for the To better understand nephrogenesis, we developed a trans- hnf1bahi1843 allele (data not shown). No effect on cdh17 expres- genic line in which enhanced green fluorescence protein (eGFP) sion was seen with hnf1bb knockdown alone (Supplemental was expressed in the pronephros. The cadherin-17 (cdh17) gene Figure 3B), suggesting that the activity of hnf1bb in the pro- was chosen because it is highly expressed in all tubule segments.11 nephros is redundant with hnf1ba. Taken together, these results Although a 4.6 kb cdh17 promoter was initially identified,12 confirm that Hnf1b factors regulate cdh17 expression in vivo. we subsequently found that 1.2 kb of genomic sequence upstream of the start codon is equally sufficient (Figure 1A). The Nephron Segmentation, but not Epithelialization, Fails spatiotemporal expression of this shorter promoter recapitu- to Occur in hnf1ba/b-Deficient Embryos lates the endogenous cdh17 gene expression pattern with eGFP To assess the effect of hnf1ba/b deficiency on nephron segmen- fluorescence/transcripts being detected in all tubule segments tation, we examined a range of segment-restricted markers. from the 8-somite stage to 3 days postfertilization (dpf) (Supple- We found that hnf1ba/b-deficient animals lack expression of mental Figure 1, A–C). all segment markers at 24 hours postfertilization (hpf) includ- We performed a deletion mapping analysis to identify crit- ing atp1a1a.4 (all segments), slc20a1a and megalin (PCT seg- ical transcription factor binding sites in the cdh17 promoter ment), etv5a and trpm7 (PSTsegment), slc12a1 and kcnj1 (DE (Supplemental Figure 2A). By transiently expressing these de- segment), slc12a3 (DL segment), and clcnk (DE and DL seg- letion constructs in embryos, a 300 bp region (2900 to 2600 ments; Figure 2). In addition, transcripts for odf3b and rfx2, nucleotides) was identified as being essential for promoter activ- markers of multi-cilliated cells, are also absent in hnf1ba/b- ity (n=17 of 48 injected embryos were eGFP+; Figure 1A). This deficient embryos (Figure 2). By contrast, relatively normal element contains sequence motifs similar to the consensus bind- expression was found for the cloacal markers gata3 and aqua- ing site for HNF1b, a homeodomain-containing transcription porin3a (Figure 2). factor implicated in kidney development13 (Supplemental Fig- To investigate if the pronephric tubules were apoptosing in ure 2B). hnf1ba/b-deficient embryos, we performed acridine orange The zebrafish genome contains two Hnf1b paralogues, staining but did not detect any significant cell death in the hnf1ba and hnf1bb.14 An analysis of their expression patterns kidney (data not shown). Sectioning of hnf1ba/b-deficient showed that they are expressed in the developing pronephros and unaffected siblings stained for atp1a1a.4 transcripts from the 8-somite stage onward (Supplemental Figure 1, D showed the presence of a tubule in the mutants, suggesting and E), similar to previous reports.14–17 Whereas hnf1ba is that epithelialization of the IM was not blocked (insets in Fig- expressed in all tubule segments, transcripts for hnf1bb are ure 2A). In support of this, we found expression of the epi- restricted to the proximal tubule segments and DE tubule seg- thelial markers laminin5, cadherin1 (cdh1), and epcam,inthe ment with barely detectable levels in the DL segment (Supple- tubule of hnf1ba/b-deficient embryos (Figure 2B). A closer mental Figure 1E). On the basis of these results, Hnf1ba and examination of the tubules by electron microscopy revealed Hnf1bb were considered excellent candidates for regulating small cuboidal epithelial cells with apical junctional com- cdh17 expression. We examined the effect of deleting one of plexes and a basement membrane. However, the brush border the putative Hnf1b binding sites (position 2750) in the cdh17 and tall morphology of the epithelial cells that characterize the promoter (Supplemental Figure 2B). This mutation severely PCT segment was absent and the tubule was collapsed with a

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Figure 1. Hnf1b regulates endogenous cdh17 expression. (A) 1.2 kb of genomic sequence upstream from the first exon of the cdh17 locus is able to drive eGFP expression in the pronephros of transiently transgenic zebrafish embryos (arrowheads). A 300 bp fragment of this cdh17 promoter sequence can recapitulate eGFP fluorescence observed in the full 1.2 kb promoter. Deletion of an Hnf1 binding motif at position 2750 of the full 1.2 kb cdh17 promoter severely reduces transgenesis (embryos are photographed at 5 dpf). (B) At 24 hpf, cdh17 expression in hnf1bahi1843/hi1843 embryos is reduced in the distal segments of the pronephros compared with wild-type embryos. hnf1bbmo knockdown in hnf1bahi1843/hi1843 embryos (hnf1ba/b-deficient) completely inhibits cdh17 expression in the pronephros. small lumen (Figure 3A). On the basis of these findings, we embryos, transcripts for pax2a, pax8, lhx1a,andmecom all fail conclude that hnf1ba and hnf1bb are required to activate a to downregulate and instead persist in proximal regions of the nephron segmentation program in kidney progenitors inde- pronephros at 24 hpf (Figure 3B). These findings indicate that pendently of signals that induce their epithelialization. the Hnf1b transcription factors regulate the spatiotemporal expression changes of pax2a, pax8, mecom,andlhx1a that occur Early Acting Renal Regulators Are Not Downregulated before the appearance of mature nephron segments. in hnf1ba/b-Deficient Embryos Tobetter understand the basis of the nephron defects in hnf1ba/ Ectopic Podocyte Formation Occurs in b-deficient embryos, we examined the expression of renal reg- hnf1ba/b-Deficient Embryos ulators pax2a, pax8, lhx1a,andmecom.4,18–24 Expression of In zebrafish, podocyte progenitors arise around the 12-somite pax2a, pax8, and mecom is lost from proximal portions of the stage and express wt1a and wt1b,orthologsofWt1.9,25,26 These pronephros after the 15-somite stage (excluding podocyte cells migrate to the midline and express podocyte genes such progenitors in the case of pax2a) but is retained in the DL nephrin and podocin.6 In hnf1ba/b-deficient embryos at 24 hpf, segment.4,10,19 Expression of lhx1a is lost in all segments but the expression domain of wt1a and wt1b was ectopically ex- retained in podocyte progenitors.9,20 In hnf1ba/b-deficient panded into the region where the PCT segment normally

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progenitor formation (Figure 4C, arrowhead). At this stage, expression of hnf1ba and wt1b is mutually exclusive in wild- type embryos, as revealed by double in situ hybridization staining (Figure 4D). Taken together, these results suggest that Hnf1b factors act in presumptive PCT progenitors to prevent these cells adopting a podocyte progenitor fate.

Hnf1b Factors Act Downstream of Retinoic Acid Signaling during Podocyte Formation Retinoic acid (RA) signaling is needed for both podocyte and proximal tubule segment formation.2,10 We therefore asked whether the effects of hnf1ba/b deficiency on podocyte formation were dependent on RA. We blocked RA signaling in hnf1ba/b- deficient embryos from the early gastrula stage to the 15-somite stage using diethy- laminobenzaldehyde (DEAB).27 As previously reported, this results in a loss of podocytes and tubules composed only of distal segments (Figure 4E and Wingert et al.2). We found an absence of wt1b+ podocytes at 24 hpf in DEAB-treated hnf1ba/b- deficient embryos (Figure 4E), suggesting that Hnf1b factors act downstream of RA. We also found that expression of the DL marker, slc12a3, was abrogated in DEAB- treated hnf1ba/b-deficient embryos consistent with a requirement for Hnf1b factors to promote tubule differentiation downstream of RA (Figure 4E). Figure 2. Nephron segmentation fails to occur in hnf1ba/b-deficient embryos. (A) atp1a1a.4 is expressed in all tubule segments of wild-type pronephroi at 24 hpf, but is Hnf1b Factors Act Upstream or not expressed in hnf1ba/b-deficient embryos. Sectioning of hnf1ba/b-deficient em- Parallel to wt1a and Notch Signaling bryos in situ hybridized for atp1a1a.4 reveals the presence of a tubule (inset, arrow- during Podocyte Formation heads). (B) laminin5, cdh1, and epcam expression is normal in hnf1ba/b-deficient We previously demonstrated that the forma- embryos, and laminin5 is clearly present in tubules of sectioned embryos (inset). (C) tion of podocyte progenitors is dependent on fi Hnf1ba/b-de cient animals lack expression of slc20a1a and megalin (PCT segment), wt1a and rbpj, encoding a transcriptional etv5a and trpm7 (PST segment), slc12a1 and kcnj1 (DE segment), slc12a3 (DL seg- mediator of the Notch pathway.9 To deter- ment), clcnk (DE and DL segments), and odf3b and rfx2 (markers of multi-cilliated cells). aquaporin3 (marker of the cloaca) expression is unaffected by hnf1ba/b- mine the epistatic relationship between deficiency. gata3 expression in corpuscle of stannius cells is lost in hnf1ba/b-deficient hnf1ba/b and wt1a and rbpj,weusedmor- embryos, but is maintained in the duct segment. (All photos are lateral views except pholinos to knockdown wt1a and rbpj in for slc20a1a and megalin, which are dorsal views.) hnf1ba/b-deficient embryos. Embryos deficient in all of these factors failed to develop podocytes, indicating that the development forms (Figure 4A). At 48 hpf, a similar expansion in nephrin+ of ectopic podocytes in hnf1ba/b-deficient animals is dependent and podocin+ cells was observed in hnf1ba/b-deficient embryos on wt1a and Notch signaling (Figure 4F). We also found that and these cells failed to fuse at the midline (Figure 4, A and B). the expression of jagged2a (jag2a), encoding a Notch ligand Tobetter determine the timing of this fate change, we examined implicated in podocyte formation,9 also failed to downregulate hnf1ba/b embryos at the 15-somite stage and observed an in- in the proximal portion of the pronephros in hnf1ba/b-deficient creased number of wt1b+ cells, indicative of increased podocyte animals (Figure 4G).

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bodies (Figure 5A). Similarly, in S-shaped bodies, Hnf1b and Wt1 showed nonoverlapping expression domains (Figure 5A). These ﬁndings are consistent with a conserved role for Hnf1b factors in regulating the boundary between podocyte and tubule progenitors.

Pax2a and Pax8 Are Required for hnf1ba/b Expression in the Nephron The pax2a and pax8 genes are expressed before the initiation of hnf1ba/b in the IM, making them excellent candidates as upstream inducers.4 To explore this, we examined embryos deficient in pax2a and pax8 by injecting pax2a morpholinos into embryos from a pax8rfp/+ incross (where the pax8 gene has been disrupted by the DSRed gene30). In approximately a quarter of the injected embryos, representing presumptive pax2a/pax8 doubly deficient animals, we observed a severe reduction of transcripts for both hnf1ba (25.5%; n=51) and hnf1bb (21.9%; n=41) at 24 hpf (Figure 5B). No effect on hnf1ba/b expression was seen in embryos singly deficient in either pax2a or pax8 (data not shown). These observations indicate that pax2a and pax8 are required for expression of hnf1ba/b in the pronephros. If pax2a/8 acts upstream of hnf1ba/b, then we would expect nephrogenesis to be disrupted in pax2a/8-deficient animals to a similar extent as in hnf1ba/b-deficient animals. Toconfirm this, we examined pax2a/pax8 deficiency on the expression of Figure 3. Epithelial characteristics are retained, but early acting mature segment markers and found a severe downregulation renal regulators are not downregulated in hnf1ba/b-deficient of cdh17, slc20a1a (PCT segment), slc12a1 (DE segment), and embryos. (A) Electron microscopy images of 3 dpf wild-type (top slc12a3 (DL segment) in presumptive pax2a/8-deficient em- panels) and hnf1ba/b-deficient (bottom panels) embryos sec- bryos (Figure 5B). tioned through the proximal tubule region. Black box indicates the area magnified in the right-hand panels. (B) Expression of Irx3b-Deficient Embryos Display Downregulated early acting renal regulators pax2a, pax8, lhx1a,andmecom are Expression of hnf1ba in the DE Segment maintained in all segments of the pronephros of hnf1ba/b- We previously showed that knockdown of irx3b results in de- deficient embryos. bb, brush border; ejc, epithelial junctional fective DE segment differentiation with a loss of slc12a1 and complex; m, mitochondria; n, nucleus. Scale bars, 0.5 mm. kcnj1 expression.10 Given that the DE segment also fails to form in hnf1ba/b-deficient embryos, we investigated the epistatic relationships between hnf1ba/b and irx3b.In To explore whether Hnf1b factors can suppress podocyte hnf1ba/b-deficient embryos at 24 hpf, irx3b was not expressed, formation when overexpressed, we injected wild-type embryos consistent with the loss of the DE segment in these animals with synthetic hnf1ba mRNA. Although a dose of hnf1ba mRNA (Figure 6A). We next investigated whether irx3b was required was chosen that rescued nephron formation in hnf1ba/b- to maintain hnf1ba expression in the DE segment. Wild-type deficient embryos (data not shown), we failed to observe embryos were injected with irx3b morpholinos and expression a reduction in wt1a+ or wt1b+ podocytes in the injected embryos of hnf1ba was examined at 24 hpf. We found that transcripts for at 24 hpf (n=35 of 35 injected embryos with wt1a+ podocytes hnf1ba were reduced in the DE segment of the majority of irx3b- and n=31 of 31 injected embryos with wt1b+ podocytes; Fig- deficient embryos (82.6%; n=46), together with cdh17 (78%; ure 4H). This suggests that forced expression of Hnf1b factors ‘ ’ fi n=41), atp1a1a.4 (67%; n=39) and clcnk (100%; n=48; Figure in the normal podocyte domain is not suf cient to block 6B). These data suggest that Hnf1b factors are initially required podocyte specification. to induce irx3b but irx3b is then needed at later stages to maintain Mammalian nephrons develop from pretubular aggregates hnf1ba expression in the DE segment. that develop into renal vesicles and then comma- and S-shaped bodies with a proximal-distal axis.28 Proximal cells in these structures express Wt1 and are believed to contribute to DISCUSSION podocytes, whereas the other portions likely give rise to tubule segments.29 In E15.5 mouse kidneys, we found transcripts for Hnf1b, and the closely related factor Hnf1a, have emerged as Hnf1b in the distal, but not proximal, domain of comma-shaped important regulators of epithelial differentiation in multiple

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Figure 4. Ectopic podocyte formation occurs in hnf1ba/b-deficient embryos. (A) wt1a and wt1b are ectopically expressed in the proximal tubule domain of hnf1ba/b-deficient embryos at 24 hpf. Similarly, nephrin+ and podocin+ cells are expanded in hnf1ba/b - deficient embryos at 48 hpf. (B) Normal midline fusion of the wt1b+ podocytes at 24 hpf does not occur in hnf1ba/b-deficient embryos. (C) At the 15-somite stage of development, we observe an expansion of wt1b+ cells in hnf1ba/b -deficient embryos (arrowhead). (D) At the 15-somite stage, wt1b and hnf1ba expression are not overlapping and mark distinct regions of the renal epithelium (wt1b+ podocytes and hnf1ba+ tubules). (E) hnf1ba/b deficiency does not rescue loss of wt1b+ podocytes in DEAB-treated embryos. Ex- pansion of DL tubule marker slc12a3 into proximal tubule segments of DEAB-treated embryos is observed, but in hnf1ba/b-deficient embryos treated with DEAB, slc12a3 expression is completely prevented. (F) Morpholino knockdown of wt1a and rbpj1 prevents formation of ectopic wt1b+ cells in hnf1ba/b-deficient embryos. (G) jagged2a (jag2a) expression is maintained in the proximal pronephros of hnf1ba/b deficient embryos. (H) hnf1ba mRNA overexpression does not suppress wt1a+ and wt1b+ cell formation.

organs.16,17,31–37 In the developing mouse kidney, Hnf1b is ex- genes involved in cystic kidney disease.14,32,41,42 Although pressed in the ureteric bud, which branches into the collecting HNF1b mutations in humans support a link to cyst formation, duct system, as well as the tubules.38,39 The role of Hnf1b in the they are also associated with severe congenital abnormalities of kidney has been challenging to study because its constitutive the kidney, suggestive of an early role of HNF1b in nephrogen- inactivation leads to early lethality.38 A number of studies using esis.40,43 Experiments in frogs have further implicated Hnf1b different approaches to overcome this have implicated Hnf1b in factors in nephron formation but determining their precise ureteric bud growth/branching16,32,40 and the regulation of function has been elusive.36,44,45 Here, using the zebraﬁsh

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Figure 6. Irx3b regulates hnf1ba expression in the DE segment. (A) hnf1ba/b-deﬁcient embryos do not express irx3b in the cen- tral region of the pronephric tubules. (B) irx3b-deﬁcient embryos are created by morpholino knockdown. In these embryos, we observe reduced hnf1ba, cdh17, atp1a1a.4, and clcnk expression in the DE segment (arrowheads).

pax2a/8-deficient embryos show decreased levels of hnf1ba and hnf1bb. Thus, we propose that Pax2a and Pax8 initiate nephron formation by performing two key functions: (1)in- Figure 5. Mouse Hnf1b is expressed in distal portions of S-shaped ducing the MET of the IM and (2)inducinghnf1ba and bodies and pax2a/pax8-doubly deficient embryos have reduced expression of nephron segment markers. (A) In E15.5 mice, Hnf1b hnf1bb expression. fi is distally restricted in comma-shaped bodies (left panel) and Whereas the absence of the DE segment in hnf1ba/b-de cient Hnf1b and Wt1 show nonoverlapping expression domains in S- animals can be explained by a failure to induce irx3b, the mo- shaped bodies (right panels). (B) pax8rfp/rfp/pax2amo embryos lecular basis for the absence of the other segments is unclear. show reduced expression of hnf1ba, hnf1bb, cdh17, slc20a1a, One possibility is that Hnf1b drives expression of all segment- slc12a1,andslc12a3 at 24 hpf. specific genes but only specific targets are activated due to the presence of unique combinations of transcriptional cofac- tors, repressors, or epigenetic modifications. In support of model, we demonstrate that Hnf1b factors act downstream of Hnf1b factors being “master tubule regulators,” other studies pax2a and pax8 to restrict podocyte fate and to establish the have identified target genes in both proximal and distal segments proximo-distal segmentation pattern of the nephron (Figure including FXYD2 (encoding the g-subunit of Na+K+ATPase),46 7). In addition, evidence for transcriptional regulatory loops OAT1/slc22a6,47 OAT4/slc22a11,48 and URAT1/slc22a12.49 In was identified between hnf1ba/b, pax2a, pax8,andirx3b. addition, a recent bioinformatics approach identified potential Our finding that a tubular epithelium still forms in hnf1ba/b- Hnf1 binding sites in a large number of tubule genes, with a deficient animals suggests that the mesenchymal-to-epithelial predominance in proximal tubule-restricted genes.50 transition (MET) of the IM is an independent process from RA is required for IM cells to adopt a podocyte fate and the activation of segment genes, such as solute transporters. important targets include wt1a51 and the Notch ligand jag2a.2 Although we cannot rule out the possibility that defective mat- These genes are not only expressed by podocyte progenitors, uration of the tubular epithelium contributes to the segmenta- which arise from cells adjacent to somite 3, but also by a subset tion failure, we favor a model whereby Hnf1b factors act as key of PCT progenitors.2,9 These observations suggest that a inducers of nephron differentiation in parallel to MET-inducing mechanism exists to prevent Wt1a and Notch signaling factors. Likely candidates for regulating MET are pax2a and from inducing a podocyte program in PCT progenitors. We pax8,astheIMinPax2/8 double-mutant mouse embryos propose that the Hnf1b factors confer this suppressive activity fails to undergo MET and is lost by apoptosis.22 In zebrafish, giventhattheyactdownstreamofRAbutupstream,orin pax2a and pax8 areexpressedbeforethehnf1b factors and parallel, to Wt1a and the Notch pathway to restrict podocyte

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All animal husbandry adhered to the accredited Code of Animal Conduct and was approved by the University of Auckland Animal Ethics Committee.

Promoter Analyses of the cdh17 Transgene Design A cdh17:GFP transgenic line that drove GFP expression in the pronephros, yolk, and gut11 was initially made using a 4.6 kb genomic fragment upstream of the first exon of the cdh17 locus. We further refined this promoter to a 1.2 kb fragment of 59 upstream sequence and used this construct to undertake a deletion analysis by PCR and subcloning. A consensus HNF1 site at position 2750 was deleted and replaced with a FseI restriction enzyme site by PCR and subcloning. Promoter constructs in the pTol2 transposase vector (50 ng/nl) were injected into one-cell stage embryos together with Tol2 transposase mRNA (120 ng/nl) and imaged at later stages using a Nikon 80i compound microscope and a Hamamatsu ORCA camera. Some of these embryos were raised to adulthood and Figure 7. A model for the spatiotemporal regulation of nephron segmentation by stable transgenic lines expressing GFP were gen- Hnf1b factors in zebrafish. erated to confirm the results from the transient injections. formation. This function is in agreement with the expression Genotyping pattern of hnf1ba, which is non-overlapping with wt1b+ Hnf1bahi1843 fish were isolated from a retroviral insertional mutagenesis podocyte progenitors at the 15-somite stage. In developing mouse screen and are considered to represent a null allele due to insertion nephrons, mutually exclusive expression domains for Wt1 and of the vector into exon 2 of the hnf1ba gene.15,17 To identify embryos Hnf1b are also found, raising the possibility that the regulation carrying the retroviral insert (homozygous or heterozygous), we ex- of podocyte specification by Hnf1b is conserved in mammals. tracted genomic DNA from individual embryos using a lysis buffer (10 Simple overexpression of hnf1ba was not sufficient to inhibit mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.3% Tween20, podocyte specification and further work is needed to understand 0.3% NP40) and proteinase K (10 mg/ml). Embryos were incubated how Hnf1b factors regulate the podocyte/tubule boundary. overnight in the lysis buffer at 55°C, and then incubated at 98°C for 10 In summary, we have demonstrated new roles for the Hnf1b minutes to kill the proteinase K. Primers specific to the LacZ gene factors as major regulators of nephron segmentation and po- present in the vector were used to amplify a 364 bp product from this docyte formation. These functions appear to act indepen- embryo genomic DNA (forward: 59-ATCCTCTAGACTGCCATGG-39; dently to pathways that promote epithelial tubulogenesis, reverse: 59-ATCGTAACCGTGCATCTG-39). To distinguish homozy- strongly suggesting that Hnf1b factors function to induce a gotes from heterozygotes, primers flanking the vector insertion site renal-specific program within a “generic” epithelium. These were used that generated a 271 bp product when the wild-type allele findings provide novel insights into the functions of Hnf1b was present (forward: 59-AATTCAACCAGGCCACAC-39;reverse: during nephrogenesis and will help guide future studies into 59-CGGTTGCACTCCTCCACC-39). We used the following cycling the causes of kidney cysts and renal birth defects in patients conditions: 10 seconds at 98°C denaturation step, 15 seconds at 72°C with HNF1b mutations. extension step for 25 cycles, and a final 10 minutes 72°C extension using the Finnzymes Phusion hot start DNA polymerase.

CONCISE METHODS Morpholinos and mRNA Synthesis Previously validated morpholinos to wt1a (59-CACGAACATCAGAACC- Zebraﬁsh Husbandry CATTTTGAG-3926), rbpja/b (59-CAAACTTCCCTGTCACAACA- Zebraﬁsh were maintained and staged according to established proce- GGCGC-3953), pax2a (5-TATGTGCTTTTTCTTACCTTCCGAG-3954) dures.52 Embryos were collected from paired matings of hnf1baHi1843 hnf1bb (59-CTTGGACACCATGTCAGTAAA-3915), and irx3b heterozygous,17 pax8RFP heterozygous,30 or wild-type Tubingen adults. (59- ACCGGGAGGACTGCGGGGAAACTCG-3910) were purchased

84 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 77–87, 2013 www.jasn.org BASIC RESEARCH from Gene Tools LLC and resuspended in 13 Danieau solution. One- treated with 2 mg/ml RNase A at 37°C for 15 minutes, and incubated cell embryos were injected with 1–5 nl of morpholino at 1.75 ng/nl for with anti-Digoxigenin-AP (1:4000) at 4°C overnight. Sections were in- rbpja/b, 3.5 ng/nl for wt1a, 5 ng/nl for hnf1bb, 2.5 ng/nl for irx3b, and 1 cubated with BM purple and color reaction was terminated by fixation ng/nl for pax2a. Embryos were incubated at 28°C to the desired stage, in 4% PFA in PBS. Slides were mounted with the Glycergel mounting fixed in 4% paraformaldehyde, and stored in methanol. Synthetic hnf1ba media. mRNA was synthesized from a pCS2+-hnf1ba cDNA construct15 using the mMessage mMachine kit (Ambion). Zebrafish embryos were injected with 1–5nlof50ng/ml hnf1ba mRNA at the one-cell stage ACKNOWLEDGMENTS and incubated at 28°C to the desired stage, fixed in 4% paraformalde- fi hyde, and stored in methanol. We thank Renee Vivien for assistance with zebra sh and Hilary Holloway for assistance with electron microscopy. This work was funded by National Institutes of Health R01 grant Embryo Chemical Treatments DEAB (Sigma-Aldrich D86256) was dissolved in 100% DMSO to DK069403-06 and Marsden fund 3626456 to A.J.D. make 1 M stocks and 1 ml aliquots were stored at 220°C. Embryos 2 2 were incubated in 1.6310 5 MDEAB/DMSOor1.6310 5 M DMSO (control) in E3 embryo media from 60% epiboly to the 15- DISCLOSURES somite stage, washed three times in E3 embryo media, and further None. incubated until the desired stage of development for analysis. REFERENCES Whole-Mount In Situ Hybridization Analyses Whole-mount in situ hybridization of zebrafish embryos was per- 1. 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