Developmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease

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Developmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease

Arianna Fiorentino, Armelle Christophorou, Filippo Massa, Serge Garbay, Magali Chiral, Mette Ramsing, Maria Rasmussen, Marie-Claire Gubler,

Bettina Bessieres, Laurence Heidet, et al.

To cite this version:

Arianna Fiorentino, Armelle Christophorou, Filippo Massa, Serge Garbay, Magali Chiral, et al.. De-

velopmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease. Cell Reports, Elsevier

Inc, 2020, 33 (4), pp.108304. �10.1016/j.celrep.2020.108304�. �hal-03050838�

Developmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease

Graphical Abstract

Highlights

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Renal glomerulogenesis implies the separation of vascular and urinary poles (VP/UP)

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Pole separation is due to a protuberance whose emergence is controlled by HNF1B

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The lack of the protuberance leads to UP trapping/

constriction inside the VP

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UP constriction prevents primary urine outflow and gives rise to glomerular cysts

Authors

Arianna Fiorentino,

Armelle Christophorou, Filippo Massa, ..., Laurence Heidet, Evelyne Fischer, Marco Pontoglio

Correspondence

evelyne.fischer@inserm.fr (E.F.), marco.pontoglio@inserm.fr (M.P.)

In Brief

Renal glomerulogenesis, a complex morphogenetic process, is still not fully understood. Fiorentino et al. discover a genetically programmed process whose developmental dysfunction leads to self- strangled glomeruli that generate cysts upon filtration. This process tackles the need to separate the urinary and vascular poles in early nephron precursors.

Fiorentino et al., 2020, Cell Reports 33 , 108304 October 27, 2020 ª 2020 The Authors.

https://doi.org/10.1016/j.celrep.2020.108304 ll

Report

Developmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease

Arianna Fiorentino,

Armelle Christophorou,

Filippo Massa,

Serge Garbay,

Magali Chiral,

Mette Ramsing,

Maria Rasmussen,

Marie-Claire Gubler,

Bettina Bessieres,

Laurence Heidet,

Evelyne Fischer,

*

and Marco Pontoglio

*

1

Universite´ de Paris, Institut Necker-Enfants Malades (INEM), Epigenetics and Development Team, INSERM U1151, CNRS UMR 8253, 75015 Paris, France

2

Department of Pathology, Randers Regional Hospital, 8930 Randers, Denmark

3

Department of Genetics, Vejle Hospital, Lillebælt Hospital, University of Southern Denmark, 7100 Vejle, Denmark

4

INSERM U1163/Centre de Re´fe´rence MARHEA, H^ opital Necker-Enfants Malades, 75015 Paris, France

5

Service d’Histologie-Embryologie-Cytoge´ne´tique, H^ opital Necker-Enfants Malades, 75015 Paris, France

6

Present address: Inovarion Paris, France

7

Present address: Institut de Biologie de l’Ecole Normale Supe´rieure, Ecole Normale Supe´rieure, CNRS, INSERM, Universite´ Paris Sciences et Lettres, 75005 Paris, France

8

These authors contributed equally

9

Senior author

10

Lead Contact

*Correspondence: evelyne.fischer@inserm.fr (E.F.), marco.pontoglio@inserm.fr (M.P.) https://doi.org/10.1016/j.celrep.2020.108304

SUMMARY

The architecture of renal glomeruli is acquired through intricate and still poorly understood developmental steps. In our study we identify a crucial glomerular morphogenetic event in nephrogenesis that drives the re- modeling/separation of the prospective vascular pole (the future entrance of the glomerular arterioles) and the urinary pole (the tubular outflow). We demonstrate that this remodeling is genetically programmed. In fact, in mouse and human, the absence of HNF1B impairs the remodeling/separation of the two poles, leading to trapping and constriction of the tubular outflow inside the glomerulus. This aberration gives rise to obstruc- tive glomerular dilations upon the initiation of primary urine production. In this context, we show that phar- macological decrease of glomerular filtration significantly contains cystic expansion. From a developmental point of view, our study discloses a crucial event on glomerular patterning affecting the ‘‘inside-outside’’ fate of the epithelia in the renal glomerulus.

INTRODUCTION

Human kidneys filter and produce approximately 180 L of pri- mary urine every day. The handling of such a remarkably large volume of fluid is possible thanks to the highly specialized struc- ture of nephrons. In particular, the glomerulus, the blood filtration unit of the nephron, is normally characterized by two opposite connections. On one side is the vascular pole (VP), where the afferent and efferent arterioles convey and recover blood, respectively. On the other side is the urinary pole (UP), the outflow connection where primary urine produced by the glomerular ultrafiltration of blood can freely flow from the glomer- ular urinary space to the connected tubular lumen.

Glomerular cysts are characterized by the dilation of glomer- ular capsule. The mechanisms that lead to this pathology are not understood. This type of lesions is frequently observed in renal developmental disorders (congenital abnormalities of the kidney and urogenital tract [CAKUT]). A considerable proportion of these renal developmental abnormalities are linked to gene mutations. One of the most prevalent genetic defects respon-

sible for CAKUT is represented by mutations in hepatocyte nu- clear factor 1B (HNF1B) (Ulinski et al., 2006; Decramer et al., 2007; Heidet et al., 2010), a gene that encodes for a homeo- POU transcription factor (De Simone et al., 1991; Rey-Campos et al., 1991). HNF1B mutations may lead to kidney agenesis, renal multicystic hypodysplasia, and, notably, glomerular cysts (Bingham et al., 2001; Massa et al., 2013; Heliot et al., 2013).

Despite the remarkable penetrance of this phenotype, the morphogenetic steps controlled by HNF1B during glomerulo- genesis are still poorly understood.

The developmental sequence of events that shape nephrons was disclosed decades ago (Saxe´n and Sariola, 1987; Potter, 1965). These steps are sequentially represented by epithelial structures that progressively evolve into S-shaped bodies (SSBs) (reviewed by Schedl, 2007). At the SSB stage, nephron precursors are schematically represented by a folded tubule composed of three different portions: (1) a distal segment con- nected to the ureteric bud (UB), which will give rise to the distal tubule; (2) a medial segment that will generate the proximal tu- bule and Henle’s loop; and (3) a proximal segment (PS) that

Cell Reports 33, 108304, October 27, 2020 ª 2020 The Authors. 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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will give rise to the future glomerulus. At the end of their matura- tion, SSBs activate a bipartite genetic program: on one hand, an intense proliferative phase (Fischer et al., 2006; Saburi et al., 2008) expands the tubular compartment; on the other hand, a complex remodeling process in the most PS leads to the pro- gressive formation of mature glomeruli. In this context, the events that remodel the relative position of the UP and the VP are still very poorly characterized.

Here we show that the remodeling of the VP and UP is genet- ically programmed and that its impairment may lead to glomer- ular cysts in mouse and human renal development. In addition, we discover that a pharmacologically induced decrease of glomerular filtration prevents glomerular cysts expansion.

RESULTS AND DISCUSSION

The Topology of the VP and UP Is Remodeled during Glomerulogenesis

The structure of glomeruli is normally schematically represented with the VP and UP diametrically opposite each other. Our results experimentally demonstrated that this peculiar setting is a robust and rather invariant parameter. In fact, by adopting a three- dimensional (3D) reconstruction approach on transparent mouse kidneys, we showed that the vast majority (90%) of mature glomeruli have VP-UP angles between 150

and 180

(Figure 1A).

However, remarkably, nephron precursors are not generated in this conformation. In SSBs (Figure 1B), the PS forms a cup-like structure (Figure 1B, lower scheme). In this context, the rim of the glomerular cup corresponds to the prospective VP. Remark- ably, at this stage, the prospective UP is immediately adjacent to and contiguous with the VP (Figure 1B, lower scheme). We noticed that the further maturation of SSBs is characterized by a dramatic remodeling of the VP-UP junction. By inspecting the 3D structure of nephron precursors, we realized that during the progressive invagination of the glomerular cup that occurs at this stage, the VP is separated from the UP by the appearance and emergence of a ‘‘protuberance’’ (Figures 1C and 1D). An important topological consequence of this remodeling is that the UP is finally connected with the glomerulus by abutting the outer surface of the glomerulus (parietal layer). With time, the UP and VP progressively acquire their final diametrically opposite positions (Figures 1C and 1D). It is worth noting that during this transition, podocyte precursors maintain their immature epithelial configuration. Nephron precursors in this conformation can be described as in a ‘‘precapillary loop’’ (PCL) stage (Figure 1D).

We then verified if the peculiar process of VP and UP separation occurs in a similar way during glomerular maturation in humans.

Our results showed that this process followed the same pattern observed in mouse embryos (Figure S1A).

Hnf1b Deficiency Impairs the Separation of VP from UP Glomerulogenesis requires the coordination of a complex set of events. The coordination of these events very likely relies on the implementation of genetic programs controlled by specific tran- scription factors. In fact, the loss of function of crucial transcrip- tion factors may lead to dramatic malformations that in turn may give rise to severe developmental pathologies. Given the impor- tant role of HNF1B in nephrogenesis, we sought to investigate if

glomerular cysts in HNF1B deficiency may be linked to a possible developmental distortion of glomerulogenesis. For this aim, we decided to monitor the glomerular maturation in (3D) reconstructions of murine Hnf1b-deficient (Massa et al., 2013) early nephron precursors (Six2-Cre;Hnf1b

), before the occurrence of cysts. Indeed, our results showed that Hnf1b-deficient embryonic nephron precursors were character- ized by a drastically abnormal spatial organization. During SSB maturation, the UP and its connected tubule are aberrantly in- serted via the VP. In other words, instead of being connected to the external parietal layer, the UP and its connected tubule are trapped inside the glomerulus (Figures 2A–2D and 3D; recon- structions shown in Video S1, upper panels). The expression pattern of Peanut lectin (tubular cells) and Wt1 (podocyte) further confirmed that the topology of the developing glomerulus in the mutant is highly aberrant (Figures 2E and 2F). This malformation was highly penetrant, as it was observed in virtually all early mutant nephron precursors (compare animations of optical serial sections in Video S2). Next, we wanted to verify if the topological aberration that we observed in nephron precursors of mouse embryos was also present in humans. Our results showed that most of early nephron precursors in fetuses with HNF1B muta- tions (18 nephron precursors out of 34 analyzed in two fetuses carrying HNF1B mutations) were significantly distorted in a way that is similar to what was observed in mouse embryonic kidneys lacking Hnf1b (Massa et al., 2013; Heliot et al., 2013).

In addition, almost half of the more advanced glomerular precur- sors were aberrantly shaped with tubules inside the glomerular cup (26 nephron precursors out of 52 analyzed in two HNF1B- deficient fetuses; compare Figure S1D with Figure S1E and z stack animations shown in Video S3 upper panels as an example). In addition, 3D reconstructions showed that nephron precursors had an aberrant insertion of the UP in the inner layer of the glomerular cup instead of being connected on the outer layer (Figures 2G and 2H and the corresponding animations in Video S1, lower panels). These observations suggest that the same topological distortion is present in Hnf1b deficiency in mouse and human.

As HNF1B deficiency led to a drastic distortion of glomerulo- genesis, it was important to characterize the expression pattern of Hnf1b in relation to this developmental process. To this end, we took advantage of a LacZ knockin allele (Hnf1b

) (Coffinier et al., 1999) in which the expression of the beta-galactosidase cassette (LacZ) fully recapitulates the expression pattern of Hnf1b. These results showed that Hnf1b is expressed in all tubular cells and in the parietal precursor cells. On the other hand, Hnf1b is not expressed in podocytes or in their precursors (Figures 2I and 2J). It is also known that podocyte precursors are characterized by a strong expression of Wt1 (Figures 2K and 2L).

The frontier between the podocyte precursors and tubular cells

represents a boundary at which these two transcription factors

are mutually exclusively expressed. In maturing SSBs, this

boundary is characterized by the presence of a few cells (transi-

tional cells) that are programmed to express both Wt1 and Hnf1b

at lower levels (Figures 2I, 2K, and 2O, arrowhead). As previously

mentioned, the first step in VP-UP remodeling is characterized

by the appearance of a ‘‘protuberance.’’ Our results showed

that this structure buds exactly at this boundary. This

Figure 1. The Remodeling of the Vascular and Urinary Poles during Glomerulogenesis

(A) Left: representation of the angle between the vascular pole (VP) and the urinary pole (UP) in a maturing mouse embryonic glomerulus; Laminin (green),

(red), and DAPI (blue). Right: distribution of the measured angles in mature glomeruli from 15 day postnatal mice (n = 40).

(B) Schematic representation of an S-shaped body (SSB) connected to a ureteric bud (UB). The SSB is composed of three portions: the proximal segment (PS), medial segment (MS), and distal segment (DS). In this configuration, the prospective VP, represented by the rim of the glomerular cup, and the UP are adjacent each other.

(C and D) Schematic representations of glomerular precursors showing the critical sequential morphogenetic steps in the proximal segment during glomer- ulogenesis (C). The corresponding immunofluorescence images are shown below in (D);

in red, Claudin-1 (Cldn1) in green, and DAPI in blue. The UP is separated from the VP by the emergence of a protuberance (see arrows in C and D). At the end of the separation process, VP and UP progressively reach their typical diametrically opposite position. Dashed yellow arrows indicate the distance between the VP and the UP at different stages of glomerular maturation.

See also Figure S1.

Cell Reports 33, 108304, October 27, 2020 3

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protuberance is formed by podocyte precursors (Wt1

/no LacZ) in the inner side and by transitional cells (Wt1

/LacZ

) on the outer side (see the more advanced developmental step of SSBs in Figures S1F, S1H, and S1J and Video S4).

Remarkably, the absence of Hnf1b prevents the budding of the ‘‘protuberance’’ (Figures S1G, S1I, and S1K; Video S4).

The defective emergence of this structure leads to the acquisi- tion of an aberrant configuration in which the tubule connecting Figure 2. Abnormal Glomerular Precursor Remodeling in Hnf1b Deficiency

(A–F) Schematic representations (A and B) and immunofluorescence (C–E) of control and mutant ‘‘precapillary’’ loop stage nephron precursors: Texas red X- phalloidin (PHAL) in red and Laminin in green (C and D),

in red and Peanut lectin in green (E and F), and DAPI in blue (C–E). During maturation, the urinary pole (UP) is separated from podocyte precursors by a protuberance (arrow in C). In mutant, the UP is trapped inside the glomerular cup (D).

(G and H) Tridimensional reconstruction of human nephron precursors. In (H) is represented the abnormal tubular and UP localization due to

deficiency compared with control (G). Scale bars: 50

(I–P) Immunofluorescence of mouse embryonic maturing SSBs. Beta-galactosidase expression (LacZ knockin under the control of

promoter) in green,

in red, and Synaptopodin (Synpo) in white. (I and J) Beta-galactosidase/LacZ detection illustrates the expression pattern of

expressed in tubular cells and not in podocyte precursors. In controls, cells expressing

/LacZ

(arrowheads in I and K) separate the tubular cells from the podocyte layer. In mutants, the inner layer of the glomerular cup is composed of

/LacZ

cells (arrowheads in J and L) at the tubular junction;

cells not expressing

is seen in developing podocytes in mutant (arrow in N) and control (arrow in M).

(O and P) Merge of (I), (K), and (M) (O) and (P) merge of (J), (L), and (N). Scale bars: 10

m.

See also Figures S1 and S2.

to the UP remains trapped inside the glomerulus. As the protu- berance normally outgrowths at the frontier of the expression of Hnf1b and Wt1, we assumed that the absence of Hnf1b might have perturbed the genetic programs of the cells at this frontier.

In this context, the use of the LacZ reporter (knocked in into the Hnf1b locus) allowed us to analyze the destiny of cells that were programmed to express Hnf1b in the absence of its HNF1beta protein expression. Our results showed that the mutually exclu- sive pattern of Hnf1b and Wt1 was not perturbed by the absence of Hnf1b (Figures 2J, 2L, 2P, S1G, S1I, and S1K; Video S4).

Despite the maintenance of this boundary, the organization of the glomerular structure was heavily disrupted. In particular, gene expression pattern of glomerular markers further clarified some aspects of the structurally aberrant glomerular precursors (Figure S2). Our results showed that, as expected, the inner epithelial layer of control glomerular precursors was composed exclusively by podocyte precursors, as they expressed homoge- neously high levels of Wt1 and Synaptopodin (Wt1

, Synpo), two markers of podocytes (Figures 2K, 2M, and 2O). In Hnf1b deficiency instead, the trapping of the tubule inside the glomer- ulus led also to the inclusion of parietal cells in the inner layer (Figures 2J and 2P). In this respect, it is worth noting that despite this aberration, mutant glomerular precursors developed normal podocytes that were perfectly comparable with those of wild- type precursors with Wt1

and Synpo expression (Figures 2L, 2N, and 2P).

The malformation of Hnf1b-deficient nephron precursors high- lights important mechanistic aspects of glomerulogenesis.

When the glomerular cup starts its invagination, some cells will be destined to remain inside and form the inner layer of the glomerular cup, whereas others (parietal cells) become the outer layer of this cup. When Hnf1b is not expressed, the topological boundary of inner-fated cells is extended and includes the UP.

A remarkable observation is that this topological boundary coin- cides with the frontier of the expression pattern of Hnf1b itself.

Hnf1b Deficiency Leads to the Obstruction of the Glomerular Tubular Outflow

Our results showed that Hnf1b-deficient nephron precursors were characterized by the fact that the lumen of the trapped tu- bule was collapsed (see arrow in Figure 3B or Video S5 and its quantification in Figure S3). It is known that lumen formation in renal nephron precursor is a complex and highly coordinated process. In this context, it has been reported that the inactivation of Afadin leads to defective tubular lumen formation that, howev- er, does not lead to the appearance of glomerular cysts (Gao et al., 2017). However, it should be noted that in this context, em- bryos die before the onset of glomerular filtration. It is interesting to note that Hnf1b has been shown to control lumen formation in the gut of zebrafish larvae (Bagnat et al., 2007). In this study, the authors showed that zebrafish larvae carrying mutations in Hnf1b failed to specify a single lumen and instead developed multiple ones. We therefore wondered if the collapsed tubular lumen we observed was a consequence of a defective lumen for- mation. Our results clearly indicated that in the context of nephron precursors, the absence of Hnf1b did not lead to any major dysfunction in lumen formation. In fact, phalloidin staining of early nephron precursors (vesicles, comma-shaped bodies,

and SSBs) showed that their lumen was normally formed (Fig- ures 3D–3F, as an example). In addition, no major defects in api- cobasal polarity were observed in mutant nephron precursors.

Claudin 1 (Cldn1) was correctly detected at the subapical tight junctions (Figures S4A–S4D), whereas aPKCzeta (Prkcz) was normally localized at the apical compartment of the tubular cells (Figures S4E and S4F). Remarkably, these two proteins were also detected in correspondence of the collapsed lumen section in more advanced mutant nephron precursors (Figures S4B and S4F, arrows). In addition, in human mutant nephron precursors, we could clearly see that the lumen was normally formed, as it was detected with apical-specific Peanut lectin staining (Fig- ure S4G). We therefore suggest that the focal pattern of the re- striction of the lumen that we observed is instead due to a local mechanical circular strain around the tubule. In support of this scenario is the fact that the collapsed luminal segment system- atically localized in correspondence of the maximal constriction of the external diameter of the tubule.

We then tried to understand how the tubular outflow might have been strangled. It is known that during nephrogenesis, SSBs are characterized by a vascular cleft that is typically invaded by mesangial and endothelial cells. In Hnf1b deficiency, this cleft is very close to the second invagination that normally forms on the opposite side (Figures 3G and 3H). This aberrant configuration probably favors the formation of a unique cavity that surrounds 360

the tubular outflow. When mesangial (ex- pressing Pdgfrb) and endothelial cells (stained by GSA) colo- nized this cavity (Figures 3I–3N; Video S3, lower panels) they finally encircle the trapped tubule. Remarkably, phalloidin stain- ing showed that the constriction of the tubule was surrounded by F-actin filament rings in mesangial cells (see the confocal images in Figures 3O–3Q). Therefore, this spatial configuration leads to the constriction of the glomerular outflow tubule just above its connection with the UP.

Glomerular Filtration Leads to Glomerular Cystic Expansion

Despite the aberrant topology of Hnf1b-deficient glomerular precursors, the glomerular inner layer developed normally differentiated podocytes (Figures 2L and 2N), suggesting that the filtration of blood could eventually take place. However, in this context, the restriction of the lumen of the outflow tubule might prevent the filtered primary urine from freely flowing out of the glomerulus (Figures 4A–4C; Video S5, as an example of early and late dilation steps, respectively). Our working hy- pothesis postulates that the start of glomerular filtration in a context in which glomerular precursors have an obstructed tubular outflow may lead to the generation and expansion of glomerular cysts. This hypothesis predicts that any maneuver that might lead to a decrease in glomerular filtration should contain the expansion of the volume of cysts. To verify this pre- diction, in order to decrease the glomerular filtration rate (GFR), we treated pregnant mice with losartan, a renin-angiotensin system inhibitor known to be able to decrease GFR also in em- bryos (Stevenson et al., 1996). Pregnant female mice were treated with this drug every day during their last week of gesta- tion, just before the morphogenesis of the first glomerular structures starts to take place. Renin-angiotensin blockade

Cell Reports 33, 108304, October 27, 2020 5

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during kidney development is known to lead to severe defects in nephrogenesis. In fact, in control embryos, losartan treat- ment tended to generate defects in tubular expansion of neph- rons (data not shown). On the other hand, remarkably, we showed that losartan led to a drastic reduction of the average volume of glomerular cysts in Hnf1 deficiency (Figures 4D and 4E). In particular, a prominent fraction of glomeruli of mutant embryos from treated mothers were normally sized without any cystic expansion. This indicates that decreasing GFR drastically prevented the expansion or the generation of glomerular cysts in mutant embryos.

The hypothesis of an obstructive mechanism in glomerular cyst formation has been object of an important and controversial debate tracing back to the first theories of Virchow in the late 1880s (Virchow, 1869; Potter, 1972; Woolf et al., 2002). Indeed, glomerular cysts can be promptly generated in vivo with experi- mental models of urinary tract obstruction during embryogenesis

(Kitagawa et al., 1999, 2004). On the other hand, using serial sec- tions, several groups have shown the absence of overt urinary outflow obstruction in glomerulocystic patients. For this reason, the scientific community tended to abandon this hypothesis in the context of human pathology (Liu et al., 2000). In a way, our study may reconcile this controversy. Indeed, we demonstrated that defective VP-UP remodeling leads to the trapping and constriction of the tubular outflow inside the glomerulus. Upon the start of filtration, primary urine builds up in the urinary space of the strangled glomerulus. This leads to the expansion of the urinary space and, eventually, to the extrusion of the trapped tu- bule from the VP. Therefore, the aberrant glomerular/tubular or- ganization is expected to lead to a transient obstruction that is resolved by the glomerular expansion mediated by the primary urine pressure. This kind of topological defect could be at the origin of other types of glomerulocystic disease that arise during development.

Figure 3. Obstruction of Urinary Pole in Hnf1b Deficiency

(A–H) Immunofluorescence of murine S-shaped bodies (A and B), renal vesicles (C and D), and comma-shaped bodies (E and F). F-actin detected with Texas red X-Phalloidin (PHAL) in red, Laminin in green, and DAPI in blue. Subapical F-actin is detected both in control and mutant developing nephron precursors. The arrow in (B) indicates a noticeable constriction of the tubular lumen. (G and H) In early control nephron precursors, the second cleft (arrow in G) is relatively distant from the vascular cavity (arrowhead in G). In mutant precursors the two clefts are much closer to each other. This might contribute to the formation of a unique cavity (H, arrowhead and arrow) around the trapped tubule.

(I–N) Immunostaining with

lectin (GSA) in red,

in white, and DAPI in blue. In later stage of development, endothelial (GSA) and mesangial cells (expressingPdgfrb) (I and K, respectively) colonize the cavity of the vascular cleft. In mutant precursors instead, capillaries (J) and mesangial cells (L) colonize the unique cavity that surrounds 360

the tubule. (M) Merge of (I) and (K); (N) merge of (J) and (L). T, tubule.

(O–Q) Projections of confocal images of Texas red X-Phalloidin (PHAL) staining show that the constriction of the tubule (arrows in O and Q) is associated with the organization of F-actin filaments. In (P), the tubular subapical actin is visible (arrowhead) also in correspondence of the constriction. Scale bars: 10

See also Figures S3 and S4.

Figure 4. Losartan Constrains Glomerular Cystic Volume

(A–C) Immunofluorescence of control and mutant capillary loop stage nephron precursors (A and B) and their corresponding schematic representations in (C).

Texas red X-Phalloidin (PHAL) in red, Laminin in green, and DAPI in blue. In control glomeruli, the urinary pole (UP) is positioned opposite to the vascular pole (VP).

The maturation of the filtration barrier correlates with the expansion of the obstructed mutant glomeruli. T, tubule. Scale bars: 10

m.

(legend continued on next page) Cell Reports 33, 108304, October 27, 2020 7

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Our study highlights important mechanisms that govern neph- rogenesis. We have identified a key morphogenetic event that is represented by the emergence of the glomerular protuberance.

We suppose that HNF1B might control the transcriptional activa- tion of crucial effectors that could be directly involved in this event. The emergence of the protuberance accounts for the tran- sition of SSBs to the PCL stage, when the prospective VP and UP are separated and progressively acquire their eventual opposite stereotypical conformation (Figure 1C). In summary, we demon- strated that aberrations of the transition between SSBs and PCLs may lead to transient obstructive glomerular cysts. Finally, we discovered an unappreciated function of Hnf1b orchestrating the inside-outside patterning of glomeruli.

STAR + METHODS

Detailed methods are provided in the online version of this paper and include the following:

d

KEY RESOURCES TABLE

d

RESOURCE AVAILABILITY

Lead Contact

B

Materials Availability

Data and Code Availability

d

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mouse strains

B

Human samples

d

METHOD DETAILS

Losartan treatment

B

Cyst measurements after Losartan treatment

Immunofluorescence in mouse kidney samples

Immunofluorescence in human kidney samples

3D reconstructions in human kidney samples

Tubular lumen opening measurements

Vascular-urinary poles angles measurements

d

QUANTIFICATION AND STATISTICAL ANALYSIS

Angles between urinary and vascular poles in wild-type

mouse

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

celrep.2020.108304.

ACKNOWLEDGMENTS

We thank all the members of Marco Pontoglio’s lab, Luisa Dandolo, Jerome Rossert, and Fabiola Terzi for helpful discussions and critical reading of the manuscript. We are very grateful to the platforms of Imagerie Cellulaire, Mor- phologie et Histologie, and the animal house facility of the Cochin and INEM Institutes. We thank Thomas Blanc and Mohamad Zaidan for postnatal mouse transparent kidneys. This work was supported by Fondation pour la Re- cherche Me´dicale (DEQ23726), Fondation Bettencourt-Schueller, the Euro-

pean Community’s Seventh Framework Programme (FP7)/2009 (agreement 241955, SYSCILIA) and FP7/2012 Marie Curie ITN (agreement 317246, TRAN- CYST), WHOAMI?, Agence Nationale de la Recherche (ANR) (ANR-11-LABX- 0071, Idex ANR-11-IDEX-0005-02).

AUTHOR CONTRIBUTIONS

E.F. and M.P. conceived the project, supervised the work, and wrote the manuscript. M. Ramsing, M. Rasmussen, M.-C.G., and L.H. provided human kidney specimens. A.F., A.C., F.M., M.C., and S.G. performed the experi- ments. A.F., A.C., L.H., F.M., and M.C. analyzed and interpreted the data.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: May 11, 2020 Revised: July 28, 2020 Accepted: October 2, 2020 Published: October 27, 2020

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Development

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(D) Embryonic day (E) 18.5 kidney sections stained with green fluorescent lectins (Peanut and

lectins and wheat germ agglutinin) and with

lectin in red. These sections illustrate the typical effect of losartan treatment on the degree of the expansion of glomerular cysts. Scale bar:

325

(E) Glomerular volume measured in E18.5 embryos from mothers daily treated with losartan or with saline during the last week of gestation (mean

SD). *p

0.05

and ****p

0.0001 (n = 42, 84, 102, 55 for controls non-treated, mutants non-treated, mutants treated, and controls treated, respectively). Statistical significance

was evaluated with Kruskal-Wallis test with Dunn’s multiple-comparisons test.

Heliot, C., Desgrange, A., Buisson, I., Prunskaite-Hyyryla¨inen, R., Shan, J., Vainio, S., Umbhauer, M., and Cereghini, S. (2013). HNF1B controls prox- imal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2. Development

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(2000). Digital glomerular reconstruction in a patient with a sporadic adult form of glomerulocystic kidney disease. Am. J. Kidney Dis.

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controls nephron tubular development. Development

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241–255.

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A description of a fetal syndrome associated with HNF1B mutation and a wide intrafamilial disease variability. Am. J. Med. Genet. A.

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Cell Reports 33, 108304, October 27, 2020 9

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STAR + METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Polyclonal rabbit anti-Jagged1 (H-114) antibody

Santa Cruz Biotechnology Cat#sc-8303; RRID:AB_649685

Polyclonal rabbit anti-Synaptopodin (H- 140)

Santa Cruz Biotechnology Cat#sc-50459; RRID:AB_2201163

Monoclonal anti-Anticorps PKC z (H-1) Santa Cruz Biotechnology Cat#sc-17781; RRID:AB_628148 Polyclonal rabbit anti-CADHERIN 6 Gift from Dr Gregory Dressler (Mah et al.,

Developmental Biology 223, 38–53 (2000)

N/A

Texas Red-X Phalloidin Invitrogen Cat#T7471

Rabbit Anti-Mouse Laminin Antibody, Unconjugated,

Sigma-Aldrich Cat#L9393; RRID:AB_477163

Rabbit anti-Pax-2 Polyclonal Antibody Invitrogen Cat#71-6000; RRID:AB_2533990

Peanut lectin (PNA)-Fluorescein labeled Vector laboratories Cat#RL1072 Chicken Anti-beta Galactosidase

Polyclonal Antibody

Abcam Cat#ab9361; RRID:AB_307210

Anti-Wilms’ Tumor 1 (WT1) Protein monoclonal mouse antibody

Dako Cat#M3561; RRID:AB_2304486

PDGF Receptor beta (28E1) Rabbit mAb antibody

Cell Signaling Cat#3169; RRID:AB_2162497

Polyclonal rabbit anti-CLAUDIN-1 antibody Abcam Cat#ab15098; RRID:AB_301644

Lotus Tetragonolobus Lectin (LTL), Fluorescein

Vector Laboratories Cat#FL-1321

Wheat Germ Agglutinin (WGA), Fluorescein Vector Laboratories Cat#FL-1021

Anti-rabbit Alexa Fluor 488 Thermofisher Cat#A32790; RRID:AB_2762833

Anti-chick Alexa Fluor 488 Thermofisher Cat#A32931; RRID:AB_2762843

Anit-rabbit Alexa Fluor 647 Thermofisher Cat#A32795; RRID:AB_2762835

Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I), Rhodamine

Vector Laboratories Cat#RL-1102-2

Goat anti-mouse Alexa Fluor 568 Thermofisher Cat # A-11004; RRID:AB_2534072

Biological Samples

Human control fetal renal tissue This paper N/A

Human HNF1B-deficient fetal renal tissue with still active nephrogenic zone

(Rasmussen et al., 2013) N/A

Human HNF1B-deficient fetal renal tissue without nephrogenic zone

This Paper (HNF1B and mechanisms of cystic renal hypodysplasia; Agence de BioMedecine Project PFS13-013)

N/A

Chemicals, Peptides, and Recombinant Proteins BABB (Benzyl-alcohol; Benzyl-benzoate;

1:2)

Sigma Aldrich Cat#B_6630; Cat# B-1042

Sodium Azide Sigma Aldrich Cat# No. 08591-1ML-F

Losartan Sigma Aldrich Cat# 114798-26-4

Critical Commercial Assays

M.O.M detection Kit Vector laboratories Cat# BMK-2202

Experimental Models: Organisms/Strains

Mouse: Tg(Six2-EGFP/cre)1Amc/J Jackson Laboratory JAX 009606

Mouse: mixed background: Hnf1b

Coffinier et al., 1999 N/A

Mouse: mixed background: Hnf1b

Coffinier et al., 2002 N/A

(Continued on next page)

RESOURCE AVAILABILITY Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Marco Pontoglio (marco.pontoglio@inserm.fr).

Materials Availability

All mouse strains in this study are available from the Lead Contact (marco.pontoglio@inserm.fr) with a completed Materials Transfer Agreement.

Data and Code Availability

This study did not generate/analyze [datasets/code].

EXPERIMENTAL MODEL AND SUBJECT DETAILS Mouse strains

Six2-Cre mice were described in Kobayashi et al. (2008). Hnf1b

and Hnf1b

mice were described in Coffinier et al. (1999) and Coffinier et al. (2002), respectively. Six2-Cre; Hnf1b

or Six2-Cre; Hnf1b

male or female were crossed with Hnf1b

mice to obtain Hnf1b-deficient embryos. These male and female embryos were referred as mutants (described in Massa et al. [2013]) and were carrying either a Six2-Cre; Hnf1b

or Six2-Cre; Hnf1b

genotype, whereas Six2-Cre; Hnf1b

or Six2-Cre; Hnf1b

male and female embryos from the same litter were used as controls. Kidneys were dissected from male and female embryos at E17.5 and E18.5. Kidneys from 3 wild-type mice were dissected at postnatal P15 to evaluate the mature glomerular configuration.

All mice were kept in individually ventilated cages together with littermates under a 12h light/dark cycle and were provided with water and food ad libitum. Animals were handled according to national and European community guidelines. Animal facility is licensed by the French Ministry of Agriculture (Agreement A 75-14-02), and protocols were approved by the ethics committee at Cochin Institute.

Human samples

Kidney sections from eight human male and female fetuses carrying a HNF1B mutation between 21 and 33 weeks of gestation (GW) were analyzed (patient 1: 21GW+3days, patient 2: 22GW; patient 3: 26GW, patient 4: 22GW, patient 5: 30GW, patient 6: 33GW, pa- tient 7: 23GW, patient 8: 33GW). In addition, one normal human fetus (25 GW) was analyzed. Among the samples, only two siblings (patient 1 and patient 2 (Rasmussen et al., 2013)) presented with a still active nephrogenic zone (Figures S1B and S1C). A generalized premature exhaustion of the renal progenitor pool in fetuses is a condition commonly found in HNF1B-deficiency and more in general in hypodysplastic CAKUT patients (Bingham et al., 2001). Nephron precursors and glomerular counting were performed on equiva- lent surface of three histological sections from the two human fetuses harboring a nephrogenic zone (Rasmussen et al., 2013). This study was conducted with the approval of the ‘‘Comite´ de Protection des Personnes pour la recherche biome´dicale Ile de France 2.’’

Tissue sample from the fetus were received with written informed consents from the parents.

METHOD DETAILS Losartan treatment

Losartan (Sigma Aldrich) was given to pregnant females fostering embryos that were segregating for Hnf1b-deficiency (mothers were either Six2-Cre; Hnf1b

crossed with males Hnf1b f/f or Hnf1b

crossed with males Six2-Cre; Hnf1b

). Two groups of three mouse pregnant female each were treated starting on E12 after conception by oral gavage with a dose of 150 mg/Kg administered twice a day. Two groups of three pregnant female were treated by oral gavage with saline and used as control. Male and female embryos were collected at E18.5, genotyped and kidneys were dissected and processed for whole mount staining (Figure 4D).

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Software and Algorithms

Imaris Oxford Instruments https://imaris.oxinst.com/

Fiji/ImageJ ImageJ Fiji https://fiji.sc RRID: SCR_002285

Imod University of Colorado https://bio3d.colorado.edu/imod/

download.html#Development

Graphpad Prism 8 https://www.graphpad.com:443/

RRID:SCR_002798

Cell Reports 33, 108304, October 27, 2020 e2

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Embryonic kidneys were fixed overnight at 4

C in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) diluted in PBS, rinsed three times 30 minutes in PBS, and incubated with a combination of fluorescein-coupled lectins (LTL, PNA (peanut) and WGA) and Rhodamine-GSA for 5 days at 4

C in PBST (PBS with 0.5% Tween 20) with the addition of 0.1% Na Azide (Sigma Aldrich). Kidneys were then rinsed in PBST (several washes during 24hours) and progressively dehydrated in increasing concentrations of Methanol (Carbo Elma) (30%, 70%, 90% and 100% two hours in each bath) and kept overnight in 100% Methanol at 4

C. Transparency was obtained by immerging kidneys in a BABB (Benzyl-alcohol; Benzyl-benzoate; 1:2) (Sigma-Aldrich) solution as described in Kolesova´

et al. (2016).

Cyst measurements after Losartan treatment

Acquisition of stacks were obtained from kidneys kept in BABB at 10X magnification with a spinning disk confocal microscopy (Nikon Ti Eclipse) equipped with a Yokogawa CSU-X1 confocal head. It is known that the maturation of renal glomeruli is an asynchronous process since several different glomerular precursors, at different developmental stages, may coexist in the same embryonic kidney.

The deepest structures, next to the cortico-medullary junction, correspond to the oldest and more differentiated ones. We measure glomerular volumes specifically in the deepest (oldest and fully differentiated) layers of glomeruli in embryonic kidneys. Only that old- est 2 layers of developing glomeruli were considered. Measurements were done with Imaris software (https://imaris.oxinst.com/).

Immunofluorescence in mouse kidney samples

Male and female embryonic mouse kidneys (E17.5-E18.5) were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) in PBS overnight at 4

C and washed in PBS three times for 30 min at room temperature.

For paraffin sections, thin sections (5 m m) of paraffin-embedded kidneys were performed with a microtome. Paraffin sections were then dewaxed in 100% Toluene and progressively rehydrated using progressive baths of decreasing Ethanol (Carlo Erba) concen- trations (Ethanol 100%, Ethanol 90%, Ethanol 70% and Ethanol 30%, one minute each) and rinsed in PBS. These sections were then subjected to antigen retrieval in boiling citrate-based buffer (Dako, S2369) for 15 minutes, and let cool down at room temperature during approximately one hour in a Becker. After rinsing in PBS, sections were processed with a MOM Kit (Vector Labs, BMK- 2202) according to the manufacturer’s instructions with slight modifications to the original procedure. Briefly, after the blocking steps, sections were incubated in a combination of primary antibodies and/or lectins diluted in MOM working solution for one hour. The combination of primary antibodies are the following ones: anti-WT1 and anti-Laminin antibodies (Figure 1A), anti-WT1 and anti- CLAUDIN-1 antibodies (Figure 1D); anti-WT1 antibody and Peanut Lectin (Figures 2C and 2D); anti-WT1, anti-betagalactosidase and anti-Synaptopodin antibodies (Figures 2I–2P); anti-WT1 and anti-betagalactosidase antibodies (Figures S1F–S1K); anti-betaga- lactosidase and anti-Pax2 antibodies (Figures S2A–S2E); anti-WT1, anti-betagalactosidase and anti-Jag1 antibodies (Figures S2G–

S2L); anti-Cadh6(Mah et al., 2000) and anti-betagalactosidase antibodies (Figures S2M–S2R); anti-WT1, anti-betagalactosidase and anti-CLAUDIN-1 antibodies (Figures S4A–S4D) and anti-Prkcz antibody (Figures S4E and S4F). After several washes in PBS, samples were incubated with appropriate species-specific Alexa Fluor-conjugated secondary antibodies (anti-rabbit and anti-chick Alexa Fluor488, anti-rabbit Alexa Fluor647 and anti-mouse Alexa fluor568, 1:250; Invitrogen) and DAPI (Sigma Aldrich) in PBS for one hour at room temperature. Sections were then rinsed in PBS three times ten minutes and mounted with Vectashield. Sections were observed with a Leica microscope equipped with a Yokogawa CSU-X1M1 spinning disk. Confocal images were analyzed with Fiji/ImageJ (Fiji https://fiji.sc RRID: SCR_002285)

For 3D reconstructions or z stacks, 100-150 m m kidney sections were performed using a vibratome and kept in PBS at 4

C. Sec- tions were then incubated 48h at 4

C in 0.1% PBS/Triton (Sigma Aldrich) / 0.1% Na Azide (Sigma Aldrich) with a combination of pri- mary antibodies and/or lectins: Texas Red X-Phalloidin and anti-Laminin antibody (Figures 2C, 2D, 3A–3H, 3O–3Q, 4A, and 4B; Fig- ure S3), and Rhodamine-GSA and anti-Pdgfrb antibody (Figures 3I–3N). Sections were then washed four times 3 h and kept overnight in PBS at 4

C, incubated with Alexa Fluor-conjugated secondary antibodies (anti-mouse AlexaFluor 568 and/or anti-rabbit Alexa- Fluor 488, 1/250; Invitrogen) and DAPI (Sigma Aldrich) 48h at 4

C in PBS and washed four times 3 hours and kept overnight in PBS at 4

C before mounting them with Vectashield (Vector Laboratories). Sections were observed with a Leica microscope equipped with a Yokogawa CSU-X1M1 spinning disk. 3D-reconstructions were obtained using IMOD software (https://bio3d.colorado.edu/

imod) (Kremer et al., 1996). Animations were prepared with Bitplane/Imaris software.

Immunofluorescence in human kidney samples

Paraffin sections were incubated with anti-WT1 antibody and Peanut lectin (Figures S1B and S1C) or Peanut lectin (Figure S4G) in PBS/ 0.1% Triton at 4

C overnight. After several washes in PBS, samples in (Figures S1B and S1C) were incubated with Alexa Fluor568-conjugated goat anti-mouse antibody (1/250; Invitrogen) in PBS for one hour at room temperature.

Sections were observed with a Leica microscope equipped with a Yokogawa CSU-X1M1 spinning disk. Confocal images were analyzed with ImageJ (NIH).

3D reconstructions in human kidney samples

For 3D reconstructions (Figures 2C and 2D; Figures S1A, S1D, and S1E), small pieces of human paraffin blocks (1mm3) were

dewaxed in Toluene for 2 days with several washes, then rehydrated in decreasing Ethanol concentrations (Ethanol100%, Ethanol

90%, Ethanol 70% and Ethanol 30%) diluted in PBS, one hour each and finally rinsed in PBS. Thick sections were incubated with a

combination of fluorescein-coupled lectins (LTL and PNA(Peanut)) and anti-WT1 antibody diluted in PBS/Triton 0.1%/NaAzide 0.1%

for three days at 37

C. After several washes in PBS during 24hours, sections were incubated with Alexa Fluor-conjugated secondary anti-mouse antibody (1/250; Invitrogen) in 0.1% Na-Azide in PBS and DAPI (Sigma Aldrich) for 3 days at 37

C. After several washes in PBS during 24hours at room temperature, kidney sections were then progressively dehydrated in increasing concentrations of Meth- anol (Methanol 30%, Methanol 70%, Methanol90% and Methanol100%) two hours in each bath and kept overnight in pure Methanol at 4

C. Transparency was obtained by immerging kidneys in a BABB (Benzyl-alcohol; Benzyl-benzoate; 1:2) solution as described in ([CSL STYLE ERROR: reference with no printed form.]). Sections were observed with a Leica microscope equipped with a Yokogawa CSU-X1M1 spinning disk. 3D-reconstructions were obtained using IMOD software (https://bio3d.colorado.edu/imod) (Kremer et al., 1996). Animations were prepared with Bitplane/Imaris software.

Tubular lumen opening measurements

Z stacks from vibratome sections labeled with anti-laminin antibody and Texas Red X- Phalloidin of control and mutant male and female embryos were used to quantify the tubular lumen opening. In developing precapillary structures, three independent measure- ments were performed at the urinary pole in control and at the tubular constriction level in the mutant. Using Fiji/ImageJ (Fiji https://fiji.

sc RRID: SCR_002285), the opening distance was measured between two opposite subapical actin filaments of two tubular cells facing each other, including the thickness of the array itself (evaluated at 0.18 m m per cell). The mean of the three measures for each of the developing structures (n = 10 in control and n = 11 in mutant) was used for the statistical analysis.

Vascular-urinary poles angles measurements

Using juvenile mouse kidney z stacks (15 days after birth), we measured the angles between the lines joining the center of the glomer- ulus and on one hand the VP (identified by the entrance of the glomerular arterioles) and on the other hand the UP. We used z stacks from three wild-type mice (number of measured glomeruli: n = 40). Angles measurements were obtained with Imaris software.

QUANTIFICATION AND STATISTICAL ANALYSIS

Lumen opening measurements were statistically assessed with a Mann-Whitney test (Graphpad Prism). Definition of n = values are reported in the legend of Figure S3 (Means+/ SD).

Angles between urinary and vascular poles in wild-type mouse

Statistical significance was evaluated with Kruskal–Wallis test with Dunn’s multiple comparisons test (Graphpad Prism). Definition of n = values are reported in legends of Figure 4.

Cell Reports 33, 108304, October 27, 2020 e4

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