comparative transcriptomic analysis identifies ... · comparative transcriptomic analysis...

Sugano et al. Evolutionarily conserved DCT gene products 1

Comparative transcriptomic analysis identifies

evolutionarily conserved gene products in the vertebrate

renal distal convoluted tubule

Yuya Sugano1,2, Chiara Cianciolo Cosentino1,2, Dominique Loffing-Cueni1,

Stephan C. F. Neuhauss2# and Johannes Loffing1,3#

1Institute of Anatomy, 2Institute of Molecular Life Sciences, University of Zurich,

Zurich, Switzerland, 3Swiss National Center of Competence in Research “Kidney.CH”,

Zurich, Switzerland

#Corresponding authors

Corresponding authors:

Johannes Loffing Stephan Neuhauss

Institute of Anatomy, Institute of Molecular Life Sciences,

University of Zurich University of Zurich

Winterthurerstrasse 190, Winterthurerstrasse 190,

CH-8057 Zurich, Switzerland CH-8057 Zurich, Switzerland

Phone: +41 (0) 44 635 53 20 Phone: +41 (0)44 635 60 40

Fax: + 41 (0) 44 635 57 02 Fax: +41 (0)44 635 68 97

Email: [email protected] Email: [email protected]

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Abstract

Understanding the molecular basis of the complex regulatory networks controlling

renal ion transports is of major physiological and clinical importance. In this study,

we aimed to identify evolutionarily conserved critical players in the function of the

renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We

generated a transgenic zebrafish line with expression of the red fluorescent mCherry

protein under the control of the zebrafish DCT-specific promoter of the thiazide-

sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate

from zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of

the mammalian DCT, for subsequent RNA-seq analysis. We next compared this

zebrafish DL transcriptome to the previously established mouse DCT transcriptome

and identified a subset of gene products significantly enriched in both the teleost DL

and the mammalian DCT, including SLCs and nuclear transcription factors.

Surprisingly, several of the previously described regulators of NCC (e.g. SPAK, KLHL3,

ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the

zebrafish DL expressed enriched levels of related homologues. Functional

knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in

the zebrafish pronephros, similar to what was seen previously in knockout mice for

its homologue, Ppp1r1a. The present work is the first report on global gene

expression profiling in a specific nephron portion of the zebrafish kidney, an

increasingly used model system for kidney research. Our study suggests that

comparative analysis of gene expression between phylogenetically distant species

may be an effective approach to identify novel regulators of renal function.

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Introduction

The mammalian renal distal convoluted tubule (DCT) plays a critical role in the

regulation of the whole body NaCl balance and hence, the control of blood pressure

[22]. Moreover, the DCT participates in the regulation of renal K+, Ca2+ and Mg2+

excretion [28,34,35]. The thiazide-sensitive sodium chloride cotransporter (NCC) is

the major apical Na+ and Cl- transport pathway in the DCT [23]. The activity of NCC is

regulated by several kinases including WNKs, SPAK, OSR1 and SGK1. Moreover,

several proteins involved in ubiquitylation (e.g. Nedd4-2, Kelch-like 3 and Cullin 3)

were found to regulate NCC either via direct ubiquitylation of the transporter or via

ubiquitylation of signaling molecules involved in the control of NCC activity [13,31].

The relevance of the DCT, NCC and its regulators is evidenced by two hereditary

diseases with mirroring symptoms [3]. In Gitelman syndrome (GS), patients suffer

from renal salt wasting with hypokalemia, hypocalciuria and hypomagnesemia due

to loss-of-function mutations in NCC [12]. By contrast, in Gordon syndrome,

mutations in WNK1, WNK4, Kelch-like 3 (KLHL3) or Cullin 3 increase NCC activity

and lead to familial hyperkalemic hypertension (FHHt) with hypercalciuria and

hypermagnesemia [1,20].

Our previous work on the transcriptome of the mouse DCT revealed that the

expression of many of these NCC regulators is enriched in the DCT [26]. This allowed

us to identify protein phosphatase 1 inhibitor 1 (I1) as a new regulator of NCC,

confirming that transcriptomic analysis is a powerful method to identify relevant

genes for specific functions along the nephron [2,4,27]. However, a challenge of

transcriptomic analysis resides in the large amount of dataset produced by this type

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of comprehensive analysis [5]. This renders selection of candidate genes for further

functional analyses usually difficult. Moreover, testing the in vivo relevance of

candidate genes is complicated by the limited availability of genetically modified

mouse models and the long generation time of mice. To circumvent these limitations,

we reasoned that the zebrafish Danio rerio might serve as an advantageous tool. The

zebrafish is an established vertebrate model because of its small adult size, its high

fecundity, the high degree of genomic conservation to humans and the availability of

genetic tools for rapid and sophisticated genetic manipulations [31]. For kidney

research, the zebrafish proofed already to be an excellent system to study

mechanisms of renal development and pathologies including acute kidney injury,

glomerular disease and polycystic kidney degeneration [6,8,25]. What makes the

zebrafish also attractive for studies on the regulation of renal ion transporters is the

fact that almost all of the major renal ion transporters and channels are conserved in

zebrafish and that the orthologues exhibit the same segmental distribution along the

nephron of mammals and the zebrafish [38]. Consistently, the zebrafish possess the

orthologue of the mammalian NCC that is specifically expressed in the distal late (DL)

segment, the corresponding segment of the mammalian DCT.

Now, we hypothesized that also the most relevant genes involved in regulation of the

DCT function are evolutionarily conserved and hence expressed in both mouse and

zebrafish distal tubules. This should allow us to filter down the vast number of DCT-

enriched genes to the most relevant genes and that could be then tested later for their

significance in the zebrafish. To validate this idea, we (i) developed a transgenic

zebrafish with red fluorescent mCherry protein expression in the DL driven by the

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promoter of zebrafish slc12a3 encoding NCC, (ii) isolated the DL tubules from the

adult transgenic zebrafish for RNA-seq, (iii) compared the established transcriptome

of the zebrafish DL with the previously obtained transcriptome of the mouse DCT and

(vi) tested the functional significance of one candidate gene by morpholino-based

knockdown in the zebrafish larval pronephros using custom-made antibodies for

zebrafish NCC. We present a proof-of-concept for the use of the cross-species

transcriptomic analysis in isolating candidate genes for novel regulators of renal

function.

Materials and methods

Zebrafish lines and husbandry

Zebrafish were maintained under a 14h/10h light/dark cycle. Fish were bred as

previously described and the embryos were raised at 28 ⁰C in E3 medium [37]. For

microdissection of DL segments, transgenic zebrafish with mCherry expression in the

DL segments were generated through the Tol2 gateway system [18]. The 1kb

fragment of the promoter of slc12a3 was amplified from WIK genomic DNA with

Phusion High-fidelity DNA polymerase (Thermo Scientific). The primers contained

attB4 and attB1 sequences at the 5’ ends of the forward and reverse primers,

respectively. The PCR product was first subcloned into the pDONR-P4P1R vector and

then recombined into the pDestTol2CG2 vector with mCherry middle element and the

3’-polyA element using the Gateway Vector Conversion kit (Invitrogen).

Plasmid DNAs for microinjection were purified using the plasmid purification kit

(Macherey-Nagel, Oensingen, Switzerland). Tol2 transposase mRNA was in vitro

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transcribed using mMessage mMachine Sp6 kit (Life Technologies). The expression

construct was co-injected into fertilized embryos at the one-cell stage with

transposase mRNAs. The injected embryos were screened for cmlc2:eGFP marker and

positive embryos were raised to adulthood. F0 founders were outcrossed to wild-type

zebrafish and resulting F1 offsprings were then screened for mCherry expression in

the DLs. Positive F1 adults were subsequently outcrossed to wild-types to obtain the

stable F2 generation.

Whole-mount in situ hybridization

A digoxigenin-labeled RNA probe was prepared by in vitro transcription of the

zebrafish slc12a3 cDNA fragment using the Roche DIG-RNA Labeling Kit (Roche

Diagnostics, Rotkreuz, Switzerland). Zebrafish embryos were fixed in 4 % PFA in PBS

at 4 ⁰C overnight and whole-mount in situ hybridization was performed as previously

described in [33].

DL tubule microdissection

Tubule isolation from the mesonephros was performed according to [9] with some

modifications to the zebrafish adult mesonephros. Approximately, 20 adult

transgenic zebrafish Tg(slc12a3:mCherry) were anaesthetized and the kidneys were

dissected. Isolated kidneys were incubated in 10 mM DTT for 1 h at room

temperature. Kidneys were then washed in 1 X PBS and digested in 5 mg/ml

collagenase (Roche Diagnostics, Rotkreuz, Switzerland) in Hank’s saline at 28.5 ⁰C for

1 h. DL segments were identified by mCherry transgene expression and

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microdissected from the rest of the kidneys under the stereomicroscope. mCherry+

DL segments and mCherry- structures were transferred into 1.5 ml Eppendorf tubes

by pippeting for RNA extractions. Three biological replicates were prepared for each

sample group.

RT-PCR

cDNAs were reverse-transcribed from RNAs of mCherry+ and mCherry- samples. PCR

was performed with a primer pair for slc12a3 (5'- TGGCTTGGCTAGAGATTG -3' and

5'- ATTCATGTTTTTGCCTGC - 3') under the following conditions: 5 min at 94 ⁰C and

then 42 cycles of 30 s at 94 ⁰C, 30 s at 56 ⁰C, and 1 min 72 ⁰C, prior to the final

extension of 5 min at 72 ⁰C.

Zebrafish NCC antibodies

Antibodies recognizing total zebrafish NCC and NCC phosphorylated at Thr49 and Thr

62 (corresponding to Thr53 and Thr58 in mice) were raised in rabbits by

immunizations with the following keyhole limpet hemocyanin (KLH)-coupled,

synthetic peptides and phosphopeptides corresponding to amino acid sequences of

zebrafish NCC: total zNCC (aa 290-307: C-ATPQKQARGFFSYRADIF), pT49-zNCC (aa

46-52: C-GYD-phosphoT-LDAP), and pT62 (aa 58-67: C-FYTN-phosphoT-EVFGR).

The N-terminal cysteine was added for coupling of peptides to KLH. The antisera were

affinity-purified against the respective immunogenic peptides. For phospho-site

specific antibodies, the primary purification was followed by additional affinity

purification against the non-phosphorylated peptides. Peptide-synthesis,

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immunizations of rabbits, and antibody purifications were custom-made by Pineda

Antibody Services (Berlin, Germany).

Immunoblotting

The human embryonic kidney cell line HEK293 (ATCC, Manassas, Virginia) was

maintained in MEM (life technologies) with 10% FCS (Amimed) and transfected at

70% confluency with mammalian expression vectors (3 ug DNA) encoding for EGFP

(EX-EGFP-M02, Tebu-bio) and zNCC (dslc12a3_pcDNA3.1-C-(k)DYK, (Genscript),

respectively. Two days after transfection, cells were lysed with RIPA Buffer (Pierce)

and processed for immunoblotting according to standard procedures. 50ug of

proteins were loaded per lane and separated by SDS-Page, blotted on nitrocellulose

membrane (Bio-Rad) and subsequently incubated with affinity–purified antibodies

against total zNCC (1:2000), pT49-zNCC (1:1000), and pT62-zNCC (1:200). Binding

of primary antibodies was revealed by incubation with an IRDye 800 conjugated goat-

anti-rabbit IgG (1:10’000, LICOR, Germany) using a LICOR infrared imager (LICOR).

Equal loading and blotting was controlled by incubating the membrane with a

monoclonal antibody against βactin (1:20’000; Sigma-Aldrich) and a respective

IRDye680 labelled secondary antibody goat-anti-mouse IgG (1:10'000; LICOR,

Germany). In a subset of experiments, phosphorylation of NCC was increased by

incubation for 30 minutes of the transfected HEK293 cells in the presence of the

protein phosphatase 1 inhibitor calyculin A (Cell Signaling) at concentration of 20 nM

in the cell culture medium.

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Whole-mount immunofluorescence

Zebrafish larvae and adult kidneys were fixed in 4 % PFA for 30 min at room

temperature (RT). Samples were serially dehydrated and stored in 100% MeOH at -

20 ⁰C at least overnight. Samples were treated with acetone for 7 min at -20 ⁰C and

after washes in 1 X PBST (1 X PBS, 0.1 % Tween 20), blocked in the blocking buffer

(1% BSA, 1% DMSO, 0.1% Tween 20, 5% normal goat serum in 1 X PBS) for 4 hours

at RT. Samples were then incubated with the respective primary antibodies in the

blocking buffer at 4 ⁰C overnight. 1:500 dilution was used for t-zNCC, both pT49-zNCC

and pT62-zNCC. The alpha-subunit of the Na-K-ATPase was detected with a mouse

anti-Na-K-ATPase antibody (Developmental Studies Hybridoma Bank, University of

Iowa) diluted by 1:100. Next day, samples were washed in 1 X PBST several times and

subsequently blocked in the blocking buffer for 3 hours at RT. As a secondary

antibodies, goat anti-rabbit IgG Alexa 488 and goat anti-mouse IgG Alexa 568 were

applied in 1 X PBST with a dilutions of 1:500 at 4 ⁰C overnight. Samples were washed

in 1 X PBST and 1 X PBS and stored in glycerol at 4 ⁰C.

RNA-seq analysis

RNAs were extracted from microdissected DL tubules using the RNA extraction kit

(Macherey-Nagel). Adaptor-ligated cDNA libraries were constructed using Ovation

Single Cell RNA-Seq System (NuGEN). The TruSeq SR Cluster Kit v4-cBot-HS or

TruSeq PE Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster

generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was

performed on the Illumina HiSeq 2500 paired end at 2 X126 bp or single end 126 bp

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using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA). The data are deposited

in the Gene Expression Omnibus database of the National Institutes of Health

(https://www.ncbi.nlm.nih.gov/geo/) under the following accession number

(GSE96519). Full lists of gene expression data were generated by GeneVia

Technologies (Tampere, Finland) and were ordered according to general expression

levels (suppl. table 1) and enrichment in DL (suppl. table 2).

Morpholino injection

Morpholino antisense oligonucleotides (GeneTools LLC, Philamath, Oregon, USA)

were designed to target the translation start sites of the zebrafish ppp1r1b (ppp1r1b-

ATG-MO: 5’-GGATCCATAATGCGCTTTCGTCCTC-3’). The standard morpholino (5’-

CCTCTTACCTCAGTTACAATTTATA-3’) was used as a control. The morpholinos (6

ng/nl) were diluted in RNase free water with 0.1% phenol red as an injection tracer

and injected (1 nl solution / per injection) into fertilized embryos at 1-4 cell stages.

Results

First, we isolated an approximately 1 kb upstream sequence of the slc12a3 gene and

found that this promoter fragment drove expression of mCherry reporter transgene

in the DL of the pronephros (Fig. 1A), similar to the DL restricted expression of

endogenous slc12a3 as revealed by whole-mount in situ hybridization (Fig. 1B).

Furthermore, mCherry expression was visible in the distal portion of adult

mesonephric nephrons. Interestingly, in the adult zebrafish kidney, transgene

expression was observed not only in DL but also in collecting ducts (CD), which are

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running as rather thick tubules perpendicular to the DL (Fig. 1C). Nevertheless, the

transgene expression in CDs does not reflect NCC abundance as revealed by

immunostaining of isolated tubules. In these isolated tubules, NCC abundance is

restricted to the DL, but absent from the CD (suppl. Fig. 1). During microdissection,

the mCherry+ CDs were easily separated from the mCherry+ DLs due to their distinct

morphology, their larger diameter and their specific localization in the middle of the

mesonephroi. Subsequently, RNAs were extracted both from microdissected

mCherry+ DL segments and mCherry- structures (Fig. 1D). In order to validate the

distal specificity of the mCherry+ fractions, prior to RNA-seq, RT-PCR was first

performed on cDNAs reverse-transcribed from the extracted RNAs using primer pairs

for slc12a3 and podocin. These experiments demonstrated the presence of a PCR

product for slc12a3 in mCherry+ samples while it was absent in mCherry- samples.

The inverse expression pattern was seen for podocin (Fig. 1E), which is a podocyte-

specific but not DL expressed gene [17]. The weak band for podocin in the mCherry+

sample may have resulted from a slight contamination by glomeruli. Next, RNA-seq

analysis was performed on the mCherry+ and mCherry- fractions. Hierarchical

clustering of the samples indicated that all three mCherry+ samples and mCherry-

samples were grouped together (Fig. 1F).

The genes known to be specific in the distal segments in the pronephros, such as

slc12a3 and the chloride channel K (clcnk), were found enriched in mCherry+ samples

while no enrichment was detected for glomerular, proximal tubule, thick ascending

limb, and collecting duct markers, such as podocin, slc20a1a, trpm7, slc12a1 and

myosin6a, respectively (suppl. table 3). The data confirm that our approach allowed a

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successful isolation of DL from the zebrafish mesonephroi though they do not fully

exclude a certain contamination by other cell types. We listed the top 181 genes

(adjusted p-value < 0.01) as DL enriched gene products (suppl. table 1). We used p-

value instead of false detection rate (FDR) to set the threshold since, according to the

Zebrafish Model Organism Database (ZFIN), a number of genes with an FDR greater

than 10 % exhibit DL specific expression in the DL of the zebrafish pronephros [36].

Though contamination by other cell types cannot be fully excluded, the collected

evidence suggests that our approach allows a successful isolation of DL from the

zebrafish mesonephroi. Consistent with an efficient isolation of DL segments, the

zebrafish slc12a3 was found among the top 50 of gene products enriched in the

analyzed tubules according to the log2 fold change. Likewise, a non-mammalian and

teleost-specific NCC homologue, slc12a10.3, was highly enriched in the DL. Next, we

compared the DL transcriptome to our previously obtained list of 339 gene products

enriched in the DCT in the mouse kidney and isolated 13 gene products enriched in

both zebrafish DL and mouse DCT including the known DCT gene products slc12a3,

clcnk and barttin (bsnd) (Fig. 2).

To analyze the regulation of zebrafish NCC (zNCC), we developed three antibodies

against total zNCC (t-zNCC) and two phospho-forms of zNCC (pT49-zNCC and pT62-

zNCC). The immunoblot with t-zNCC antibody on lysates from HEK cells expressing

zNCC showed several bands (Fig. 3A). The bands did not appear in preparations from

control cells transfected with EGFP indicating that they represent zNCC. The

immunoblot with pT49-zNCC antibody showed a strong band at about 150 kDa in the

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zNCC transfected cells. Although no signal was detected with the pT62-zNCC antibody

under control conditions, when HEK cells were treated with the protein phosphatase

1 inhibitor, Calyculin A, to block dephosphorylation of zNCC, a weak band at about

150 kDa appeared (Fig. 3B). As this band was absent in preparations from

untransfected control cells treated with 20 nM Calyculin A, it likely represents

phospho-zNCC. Whole-mount immunostaining with these antibodies yielded a

staining pattern that overlapped with that of the in situ hybridization staining of zNCC

transcripts (Fig. 3C). Na-K-ATPase was ranked as one of the most enriched gene

products in the RNA-seq data. Consistent with this, whole-mount immunostaining

revealed very high expression levels of the Na-K-ATPase in the DL segment. Also in

mouse, rat and rabbit, the DCT has the highest Na-K-ATPase activity of all nephron

segments [15], underlining the high ion transport capacity of this segment, which is

apparently relevant not only for the mammalian but also for the teleost kidney.

Furthermore, in immunohistochemistry on sections of the zebrafish pronephros,

pT49-zNCC antibody showed a strong immunofluorescent signal on the apical surface

of the pronephros while the Na-K-ATPase localized to the basolateral plasma

membrane, recapitulating the localization patterns of mammalian NCC and Na-K-

ATPase in the DCT (Fig. 3D).

In previous studies on the mouse, we identified the protein phosphatase 1 inhibitor

1 gene, Ppp1r1a, as a DCT-enriched transcript that significantly regulates the

phosphorylation of mouse NCC (26). This prompted us to check if ppp1r1a is also a

gene product enriched in the zebrafish DL. Interestingly, ppp1r1a is not a DL-enriched

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transcript. However, a closely related homologue, namely ppp1r1b, was found among

the list of DL-enriched gene. Consistent with previous results [30], in situ

hybridization confirmed the DL-specific expression of ppp1r1b in the zebrafish

pronephros (Fig. 4A). To determine whether this gene product is involved in

regulation of NCC phosphorylation in the zebrafish DL just like I1 in the mouse

kidney, we performed morpholino knockdown experiments on ppp1r1b. Whole-

mount immunostaining of ppp1r1b morphants with the phospho-antibodies

demonstrated reductions in the staining intensity of phospho-zNCC compared to that

of controls while no apparent difference in staining intensity was detected by in situ

hybridization for the ppp1r1a mRNA transcripts (Fig. 4B).

Discussion

In the present study, we isolated 13 commonly enriched DCT genes between the

zebrafish and the mouse by the comparative transcriptomic approach. In addition to

slc12a3, two other solute carrier family members, slc16a7 and slc5a3b were also

found DL-enriched. Furthermore, we found two transcription factors, transcription

factor AP-2 b (tfap2b) and estrogen related receptor b (esrrb) among the list. Mice

lacking Tfap2b die early postpartum due to polycystic kidney disease with renal

failure, hypercalcemia, hyperphosphatemia and hyperuremia [24]. This indicates a

critical role of this transcription factor for renal development, differentiation, and

function. Unlike to what the name suggests, the Esrrb does not bind estrogens and

therefore presumably does not mediate the stimulatory effects of estrogens on NCC

[7]. However, Esrrb was implicated in the regulation of NKCC2 function [16] and was

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linked to the control of stem cell renewal and differentiation [10], which may

contribute to the remarkable epithelial plasticity of the DCT [14]. Future work will

need to define the roles of these gene products in the DCT.

To our surprise, the zebrafish orthologues for the major regulators of NCC (e.g. WNKs,

SPAK) in mammals are absent in the list of DL-enriched genes [29]. The specific

regulation of NCC through WNKs and SPAK in the DCT may have evolved in higher

vertebrates only. Interestingly, however, the DL does express, with log2 fold changes

higher than 1, the STE20-like kinase b (slkb) and serine/threonine kinase 38b

(stk38b), which are similar to SPAK. Similarly, the orthologue of Kelch-like 3

homologue, which regulates NCC in mammals, is not DL enriched but another

homologue, klhl23, is present in the list. Therefore, it is tempting to speculate that for

the same conserved cellular processes, teleosts and mammals may have deployed

analogous but different genes during the evolution.

Consistent with this idea, we found ppp1r1b, a close homologue of I1, to regulate

phophorylation of NCC in the zebrafish pronephros. Although the orthologue of I1 in

the zebrafish is ppp1r1c, this gene was not listed in the DL-enriched genes. Instead,

ppp1r1b was found specifically expressed in the DL segment of the pronephros, of

which functional knockdowns led to reduced abundance of NCC in the zebrafish

pronephros. In mammalian kidneys, Ppp1r1b encoding for DARPP-32 is highly

expressed in the thick ascending limb (TAL) [11]. NKCC2 is a close homologue of NCC

localized to the TAL. It remains to be determined whether DARPP-32 regulates

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NKCC2 activity in the TAL, analogous to the NCC regulation by I1 in the DCT, but our

data indicates that the regulatory mechanism of ion transporters by protein

phosphatases may have already existed in a common ancestor, from which teleosts

and mammals have descended.

In conclusion, we present a dataset of gene products that are enriched in the zebrafish

DL segment. The dataset per se is an informative and useful resource in

understanding the function of this segment of the zebrafish kidney, an established

model system for human kidney disease. Furthermore, by comparing this dataset to

the corresponding dataset in the mouse kidney, we demonstrated a proof-of-concept

that the cross-species transcriptomic approach between phylogenetically distant

species can aid an identification of evolutionarily conserved potential core players in

nephron segments. With the availability of mammalian segment specific kidney

transcriptomes (e.g. ref. 2, 4, 5, 19, 27) and ease of generation of transgenic zebrafish,

our approach can also be applied to the other nephron segments. Such approaches

would not only help identify critical genes but also give us important insights into the

evolution of the kidney.

Acknowledgment

The authors would like to thank Drs. Jelena Kühn-Georgijevic and Lennart Opitz

(Functional Genomics Center Zurich) for their assistance with RNA-seq and

bioinformatics analysis. Valuable bioinformatics support was also provided by Ville

Kytölä (Genevia Technologies Ltd). We would also like to thank Kerstin Dannenhauer

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and Michèle Heidemeyer for their technical support including an excellent care of the

zebrafish and the careful execution of the HEK293 cell experiments. This work was

supported by the Hartmann Müller Stiftung (to YS), the Forschungskredit from the

Faculty of Medicine at the University of Zurich (to YS), the RiMED Foundation (to

CCC), the Zurich Center for Integrative Human Physiology (to SN, JL), by project

grants (310030_143929/1, 310030_173276) from the Swiss National Science

Foundation (to JL), and the Swiss National Center for Competence in Research

“Kidney.CH” (to JL).

Disclosures

None.

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Figure captions

Fig. 1. (A) mRNA expression of the zebrafish slc12a3 in the distal late (DL) of the

pronephros at 48 hpf. (B) Stable expression of mCherry in the zebrafish DL driven by

the slc12a3 promoter in Tg(slc12a3:mCherry) fish at 48 hpf. (C) Ventral view of the

mesonephric kidney from a 20 months old Tg(slc12a3:mCherry) fish. mCherry

positive distal nephrons (arrows) drain into the large collecting duct (arrowheads).

(D) Representative mCherry-positive (+) DL segments and mCherry-negative (-)

nephron segments isolated by free-hand microdissection. (E) A representative RT-

PCR of slc12a3 and podocin mRNAs from mCherry-positive DL and negative segments

demonstrates a strong enrichment of slc12a3 in the mCherry (+) tubules while

podocin is detectable in these samples. (F) Hierarchical clustering depicts the high

consistency of expression patterns for the 3 different samples for each mCherry (+)

and mCherry (-) sample group. Scale bars = 250 μm.

Fig. 2. Venn-diagram showing the number of zebrafish DL enriched gene products and

mouse DCT enriched gene products. The overlap of the circles indicates the gene

products enriched in zebrafish DL and mouse DCT. Gene names, rate of enrichment

and adjusted p values are given in the table.

Fig. 3. Characterization of the affinity-purified anti-zNCC antibodies by

immunoblotting and immunofluorescence. (A) In lysates from HEK cells transfected

with zNCC, anti-t-zNCC antibody detects several bands that are absent in cell

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homogenates from HEK cells transfected with EGFP only. pT49-zNCC antibody

detects a strong single band in zNCC transfected cell lysates. Detection of actin serves

to confirm equal protein loading. (B) The pT62-zNCC antibody detects several bands

in lysates from cells transfected with zNCC and treated with 20 nM Calyculin A,

including the band corresponding to the single band detected by pT49-zNCC

(arrowhead). The bands are absent in control cell lysates with the exception of one

band at about 130 kDa. (C) Immunofluorescent detection of total (t-zNCC) and

phospho-zNCC (pT49-zNCC and pT62-zNCC) in the DL of the pronephros at 48 hpf

and detection of the Na-K-ATPase in the pronephros at 30 hpf in whole-mount

preparations of zebrafish larvae. Scale bar = 500 μm. (D) Immunostaining reveals

apical localization of phospho-zNCC (red fluorescence) and basolateral localization of

Na-K-ATPase (green fluorescence) in cryotome-made cross-sections through the DL

of the zebrafish larvae at 72 hpf. Scale bar = 20 μm.

Fig. 4. (A) mRNA expression of the zebrafish ppp1r1b in the DL of the pronephros at

48 hpf. (B) Representative immunostainings and in situ hybridizations of control

zebrafish larvae and larvae with morpholino-based knockdown of ppp1r1b. The

knockdown of ppp1r1b decreases the phosphorylation of zNCC as revealed by

immunostainings with the pT49-zNCC and pT62-zNCC antibodies at 72 hpf, while

mRNA expression of slc12a3 is similar for ppp1r1b morphants and control larvae at

48 hpf. Scale bars = 500 μm.

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Figure 1:

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