Role of the Polarity Protein Scribble for Podocyte Differentiation and Maintenance

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribbleflox/flox mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.


Introduction
The glomerular filtration barrier is a unique structure characterized by a precise three dimensional framework of podocytes that elaborate long, regularly spaced, interdigitating foot processes, enveloping the glomerular capillaries. Neighbouring podocytes are connected by the slit diaphragm, a specialized cell junction and the only cell-cell contact of mature podocytes, that bridges the filtration slit between podocyte foot processes [1]. We recently demonstrated that the evolutionarily conserved apical Par3-Par6-aPKC complex (Par complex), a fundamental regulator of apicobasal cell polarity, interacts with the slit diaphragm proteins Nephrin and Neph1 [2]. During podocyte differentiation the Par complex and the cell-cell contacts of immature podocytes migrate from apical towards basal aspects of the podocyte cell membrane, where primary processes and foot processes subsequently develop.
In epithelial cells apicobasal cell polarity is established by the asymmetric distribution of three core polarity complexes, the apical Crumbs complex, consisting of Crumbs, PALS1 and PATJ, the apical Par complex localizing at the tight junctions and the basolateral Scribble complex, comprising the proteins Scribble, Dlg and Lgl [3]. In Zebrafish, morpholino knockdown of the apical polarity protein Crumbs2b causes disorganization of podocyte foot process architecture and loss of slit diaphragms [4]. In addition, podocyte-specific deletion of aPKCiota in mice results in foot process effacement, nephrotic syndrome, progressive glomerulosclerosis and death at 3-4 weeks after birth [5,6] underlining the critical importance of apical polarity complexes for podocyte differentiation and maintenance. However, the relevance of the basolateral Scribble complex for podocyte function is yet completely unclear.
Scribble is a large cytoplasmic scaffold protein of the leucinerich repeat (LRR) and PDZ domain (LAP) family with 16 Nterminal LRRs and 4 C-terminal PDZ domains [7]. Targeting of LAP family members to the lateral membrane depends on their LRR domains [8]. In polarized renal epithelial cells Scribble localizes to the adherens junctions and the lateral membrane in an E-Cadherin dependant manner [9], and knockdown of Scribble results in delayed tight junction assembly, increased cell motility and reduced adhesion similar to the phenotype of E-cadherin knockdown [10]. In wound healing assays Scribble is essential for polarization of migrating cells, recruitment of CDC42 and Rac1 to the leading edge and directed migration [11].
In mice, point mutations of Scribble (circletail and rumpelstilzchen mutations) cause severe impairment of neural tube development with craniorachischisis and neonatal death [12,13]. A similar phenotype is described for the loop-tail mouse [14], which displays a mutation in the gene encoding for the planar cell polarity (PCP) protein Vangl2 [15,16]. Both proteins interact genetically and physically [17,18,19].
Scribble is targeted to proteasomal degradation by the high risk papilloma virus protein E6-E6AP ubiquitin-protein ligase complex [20], and cervical neoplasms are associated with reduced Scribble protein levels [21]. Further, downregulation and mislocalization of Scribble promotes cell transformation and mammary tumorigenesis [22], suggesting Scribble as a tumor suppressor. In recently Figure 1. Migration of apical and basolateral polarity proteins during podocyte differentiation. Frozen kidney sections of newborn Wistar rat (P0) were stained using antibodies against the apical membrane protein Podocalyxin, the apical polarity protein Par3 and the basolateral polarity protein Scribble and were subjected to confocal laser microscopy. Since glomerular development is asynchronous, kidneys of newborn rats display various glomerular developmental stages. Each panel displays the expression pattern of the accordant proteins during glomerular development (from left to right): Developmental stages ranging from comma-shaped body (I), s-shaped body (II), capillary loop stage (III to IV), to a maturing glomerulus (V). published work Scribble heterozygosity causes prostate hyperplasia, while prostate-specific knockout of Scribble results in loss of cellular polarity, elevated proliferation and progression to intraepithelial neoplasia [23]. These data underline the fundamental role of Scribble for the establishment and maintenance of epithelial cell polarity, cell migration and tissue architecture.
Here we analysed the spatiotemporal expression of Scribble during glomerular development and generated podocyte-specific and constitutive Scribble knockout mice to investigate the role of Scribble in podocyte differentiation and maintenance.

Translocation of apical and basolateral polarity proteins during podocyte differentiation
Previously, we identified that the aPKC complex translocates from the apical to basal membranes during podocyte differentiation, preceding the development of primary and foot processes [5]. To study the protein localisation of Scribble during glomerular development, we co-stained the apical membrane marker Podocalyxin and Par3 with Scribble in newborn rat kidney sections. Whereas Par3 is already expressed during comma-shaped body stage and localizes to the apical sited cell-cell contacts, expression of Podocalyxin starts during s-shaped body stage, when Par3 and the cell-cell contacts migrate along the lateral side of

Development of podocyte foot processes in cultured embryonic Scribble knockout kidneys and in circletail mutant mice
Since Cre expression under control of the NPHS2-promoter starts late during glomerular development [24,25], a developmental phenotype of Scribble knockout in podocytes might be masked in Scribble flox/flox ; NPHS2.Cre mice. To study the effect of Scribble knockout on early glomerular development and podocyte differentiation, we analyzed kidneys of constitutive Scribble knockout mice. Scribble flox/flox mice were crossed with Cre deleter mice to create constitutive Scribble knockout mice (Figure 6 A). Most constitutive Scribble knockout animals die during embryonic development between E12.5 and E14.5, before podocyte maturation is finished. To monitor the glomerular development we harvested the kidneys at E12.5 for kidney culture experiments.  (Figure 6 E). We next analysed homozygous circletail mutant mice, which express a shortened Scribble protein lacking the third and fourth c-terminal PDZ domains leading to craniorachischisis, gastroschisis and late embryonic death around E18 [12]. No obvious morphological alteration of podocyte foot During podocyte differentiation the cell-cell contacts translocate from the apical site along the lateral membrane to basal, where primary processes and foot processes develop. Par3 localizes to the cell-cell contacts, moves with them to basal and localizes to the cell-cell contact of mature podocytes, the slit diaphragm, which bridges the filtration slit between foot processes. Scribble localizes basal of Par3 at the cell-cell junctions and the basolateral membrane of immature podocytes. In mature podocytes Scribble localizes to the basolateral membrane, which is defined as the area beneath the slit-diaphragm. Differentiation of podocytes is accompanied with an enlargement of the apical membrane area, marked by Podocalyxin, whose expression starts parallel with the translocation of the cell-cell contacts to basal in s-shaped body stage. Relative to this apical membrane enlargement the basolateral membrane, marked by Scribble, shrinks to the area beneath the slitdiaphragm in mature podocytes. doi:10.1371/journal.pone.0036705.g003 processes could be detected in these mice (Figure 6 F), indicating that Scribble is dispensable for regular foot process development.

Discussion
In epithelial cells Par3 is located at the tight junctions marking the border between apical and basolateral membrane compartments [3]. Previously we demonstrated that Par3 co-localizes with the tight junction protein ZO-1 at the apical cell-cell contacts of immature podocytes as well as at the slit diaphragm of mature podocytes [2,5]. Here we could demonstrate that another polarity protein, Scribble, is enriched in podocytes. During differentiation the immature cell-cell contacts and Par3 as well as Scribble translocate towards the basal aspects of the podocyte, followed by primary and foot process development ( Figure 1). Interestingly, Scribble appears to localize basally of Par3 during podocyte differentiation as well as in mature podocytes. In agreement, in polarized MDCK cells it has been described that Scribble localizes basal of the tight junctions at the adherens junctions [9]. Strikingly, mature podocytes do not feature classical tight or adherens junctions but a unique cell-cell contact, the slit diaphragm, which bridges foot processes of neighbouring podocytes. The slit diaphragm displays characteristics of both, tight junctions with associated proteins such as ZO-1 [26], Jam4 [27] and Par3 [2] as well as adherens junctions with proteins like P-cadherin [28]. This unique cell-cell contact makes it difficult to predict the function of single junctional components. In MDCK epithelial cells knockdown of Scribble causes delayed tight junction assembly and disrupts cell adhesion [10]. However, it remained completely unclear whether Scribble also significantly contributes to the development and maintenance of podocyte slit diaphragms. Therefore, we generated podocyte-specific Scribble knockout mice. Unexpectedly, no morphological or clinical abnormalities could be detected in these mice (Figure 4 and 5). Since the NPHS2promoter activates Cre expression relatively late during glomerular development [24,25], a developmental phenotype of podocytespecific deletion of Scribble might be masked. For this reason we analyzed constitutive Scribble knockout mice and circletail mice, in which Scribble is truncated after the second PDZ domain, which results in late embryonic death [12]. Constitutive Scribble knockout mice die intraembryonally between E12.5 and E14.5. To study glomerular development we performed kidney culture experiments. Podocytes in constitutive Scribble knockout kidney culture as well as podocytes of circletail mice develop foot processes being connected by the slit diaphragm showing no morphological abnormalities compared to control podocytes indicating that Scribble is dispensable for podocyte foot process development ( Figure 6). However, if other basal polarity proteins are involved in podocyte shape formation, has to be addressed in future studies.
In summary, while Scribble seems to be important for general epithelial cell- [9] and pronephros [29] development, it appears to be dispensable for podocyte function and development. This unexpected result underlines the unique features of polarity programs shaping the complex three-dimensional podocyte architecture. Striking characteristics of developing podocytes are the expansion of apical membranes going along with a shrinking of basal membrane compartments (as illustrated in Figure 1 and 3). This appears to be congruent with the observation that podocyte differentiation is rather being driven by apical polarity complex signaling while Scribble signaling is not required for the specialized podocyte architecture.

Mice
LoxP sites were introduced into the introns 1 and 8 flanking Scribble exons 2-8 in 129S1/SvlmJ mouse embryonic stem cells, using a neo cassette as a selectable marker flanked by frt sites. Targeted stem cells were injected into blastocysts of C57BL/6J mice to obtain chimeric floxed mice. After germline transmission, the mice were crossed to C57BL/6J mice expressing flp recombinase to remove the neo cassette. Progeny containing a floxed Scribble allele lacking the neo cassette (Scribble flox/+ ) were further backcrossed to C57BL/6J mice. Cre-mediated recombination causes a frame shift and early stop of translation. In addition floxed mice were crossed to C57BL/6J Cre deleter mice to excise the loxP flanked genomic region (exons 2-8) and to generate heterozygous mice carrying the constitutive knockout allele (Scribble 2/+ ) [30,31]. Construction of the targeting vector, generation and injection of targeted stem cells and subsequent generation of chimeric mice, Scribble flox/+ and Scribble 2/+ mice were performed by genOway (Lyon, France).
NPHS2.Cre mice were kindly provided by Lawrence Holzman (Renal, Electrolyte and Hypertension Division, University of Pennsylvania School of Medicine Philadelphia, PA, USA) [24]. For genotyping or detection of deletion of Scribble exons 2-8 DNA was isolated from tail clip or isolated glomeruli, respectively. For detection of WT or the loxP site, the primers were tccagttagcactcaggcgtcagg (forward) and cagctccgagaggttctcacagtcc (reverse). For detection of the deletion, primers were accccagtgctctctggtgtttttattg (forward) and cagctccgagaggttctcacagtcc (reverse). For detecting the circletail mutation, primers were ctagccctcccccccc (forward; locked nucleic acid primer) and cctgggactgagaaggacat (reverse). All animal studies were approved by the Committee on Research Animal Care, Regierungsprä sidium Freiburg and by the Washington University Animal Studies Committee.

Protein Overload and Subsequent Analysis
Scribble Dpodocyte mice and control littermates (n = 5 each) received endotoxin-free BSA (Sigma A9430) (250 mg/ml, dissolved in PBS) intraperitoneally for 4 consecutive days (10 mg/g body weight) [32,33]. Urinary albumin excretion rates were analyzed before injections and at days 1 to 6 after the first injection.

Urine and serum analyses
Urinary albumin and urinary creatinine were measured using a fluorimetric albumin test kit (Progen) and an enzymatic creatinine kit (Labor+Technik) following the manufacturer's instructions. Proteinuria was expressed as mg albumin/mg creatinine.

Metanephric kidney culture
Timed matings were set up with constitutive Scribble heterozygous knockout mice; the date of the vaginal plug was designated as day 0. Metanephric kidneys were microdissected from the embryos at embryonic day 12.5 and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% Penicillin and Streptomycin at 37uC and 5% CO 2 on 0.4 mm transwell inserts [35]. The medium was replaced every 48 h. The kidney cultures were harvested after up to 6 days in culture. For immunofluorescence staining, the cultures were fixed in 4% paraformaldehyde, dehydrated in 15% and 30% sucrose, frozen in OCT and sectioned into 6 mm thick sections.

Morphological analysis
Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin or Epon and further processed for PAS staining or transmission electron microscopy, respectively.

Immunofluorescence staining of kidney sections
Kidneys were frozen in OCT compound and sectioned at 6 mm (Leica Kryostat). The sections were fixed with 4% paraformaldehyde, blocked in PBS containing 5% BSA and incubated for 1 hour with primary antibodies as indicated. After PBS rinse for several times, fluorophore-conjugated secondary antibodies (Invitrogen) were applied for 30 minutes. Images were taken using a Zeiss laser scan microscope equipped with a 636 water immersion objective or a Zeiss fluorescence microscope equipped with a 56, a 206 and a 406 oil immersion objective. To determine the number of podocytes per glomerular section, kidney sections were stained against the podocyte nuclear marker WT1. WT1-positive cells were counted in 30 glomeruli per mouse per condition (n = 3 for each condition).

Immunogold electron microscopy
Fixed samples of rat kidney were embedded in Lowicryl K4M resin (Electron Microscopy Sciences), and ultrathin sections were labeled by an indirect immunogold protocol, as described [36].

Statistical Analyses
Data were expressed as the mean 6 SEM. Statistical comparisons were performed using two-tailed Student's t-test if not stated otherwise. Differences with P,0.05 were considered significant.