the adpkd genes pkd1a/b and pkd2 regulate extracellular...

RESEARCH ARTICLE

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INTRODUCTIONMutations in the polycystin genes PKD1 and PKD2 are responsiblefor autosomal dominant polycystic kidney disease (ADPKD), themost common heritable human disease (Sutters and Germino,2003). The proteins encoded by the PKD genes function inmechanosensory ion channel complexes (Sutters and Germino,2003) that regulate calcium influx via the primary cilium (Nauli etal., 2003) or calcium release from intracellular calcium stores(Koulen et al., 2002). In addition to cystic pathology of the kidney,liver and pancreatic duct (Sutters and Germino, 2003), mutationsin polycystins are also linked to abdominal hernia (Morris-Stiff etal., 1997) and intracranial aneurysm (van Dijk et al., 1995); both ofthese disorders are commonly associated with altered integrity ofthe extracellular matrix (ECM). Vascular defects are also observedin Pkd1 hypomorphic mouse mutants (Kim et al., 2000; Hassaneet al., 2007), where basement membrane thickening is the earliestpathology associated with dissecting aneurysm (Hassane et al.,2007). Other extra renal manifestations of ADPKD that mayinvolve matrix structural defects include gastrointestinal cysts,cardiac valvular defects and pericardial effusion (Hossack et al.,1988; Qian et al., 2007). An altered ECM has long been known tobe associated with ADPKD pathology in human tissue (Candianoet al., 1992; Somlo et al., 1993), and in ADPKD animal and cellculture models (Schafer et al., 1994). Matrix genes figureprominently in the lists of upregulated mRNAs in expressionprofiling studies of human cystic tissue and cystic animal models(Riera et al., 2006). Although matrix defects occur in ADPKD tissue,and mutations in matrix genes have been shown to be sufficient

for cyst formation (Shannon et al., 2006), it has been difficult toestablish a link between the function of polycystin1 and polycystin2as regulators of calcium signaling, and to establish a direct role forpolycystins in regulating matrix production.

Our group and others have shown that ADPKD can be modeledin zebrafish by mutation or knockdown of zebrafish polycystin2(pkd2), also known as polycystic kidney disease 2 (Sun et al., 2004;Bisgrove et al., 2005; Obara et al., 2006; Schottenfeld et al., 2007; Fuet al., 2008). pkd2 loss of function results in left-right asymmetrydefects, kidney cysts, hydrocephalus, and pronounced dorsal axiscurvature. The mechanisms of cyst formation in pkd2morphants/mutants are distinct from other zebrafish cyst mutantssince, as is seen in mouse Pkd2 mutants, cilia function appears normal(Obara et al., 2006; Schottenfeld et al., 2007). Pronephric cystformation is instead associated with altered morphogenesis of thedistal pronephric nephron and absence of a patent pronephricopening (Obara et al., 2006). Cell culture studies and studies in PKDmutant mouse models point clearly to a role for polycystins inregulating intracellular calcium (Koulen et al., 2002; Nauli et al., 2003);however, how altered calcium signaling might lead to ADPKD tissuedefects remains poorly understood. Here, we have sought to extendour understanding of the role of the polycystins in the cell biologyof ADPKD by cloning and characterizing the function of zebrafishpolycystin1 (pkd1). We find that two paralogous pkd1 genes exist inzebrafish and that simultaneous disruption of both pkd1 paralogsresults in a multi-organ phenotype that is essentially identical to thepkd2 loss-of-function phenotype. Focusing on the most commonand penetrant phenotype, dorsal axis curvature, we provide evidencethat both pkd1a/b and pkd2 regulate collagen mRNA and proteinexpression, and that collagen overexpression can account for dorsalaxis curvature defects. Our results suggest that polycystins mayparticipate directly in regulating ECM production and that the matrix

Disease Models & Mechanisms 3, 354-365 (2010) doi:10.1242/dmm.003194© 2010. Published by The Company of Biologists Ltd

The ADPKD genes pkd1a/b and pkd2 regulateextracellular matrix formationSteve Mangos1, Pui-ying Lam1, Angela Zhao1, Yan Liu1, Sudha Mudumana1, Aleksandr Vasilyev1, Aiping Liu1 andIain A. Drummond1,*

SUMMARY

Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associatedwith vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combinedknockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephriccyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combinedpkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagenoverexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficientembryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limitscollagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicatingpolycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulationof col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression.Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defectunderlying ADPKD tissue pathologies.

1Massachusetts General Hospital, Department of Medicine, Nephrology Division,and Harvard Medical School Department of Genetics, Charlestown, MA 02129, USA*Author for correspondence ([email protected])

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Disease Models & Mechanisms 355

Polycystins regulate matrix production RESEARCH ARTICLE

defects seen in ADPKD patients may be a central feature of polycystinloss of function.

RESULTSCloning and expression of zebrafish orthologs to human PKD1Polycystin1 is present as two paralogous genes in zebrafish, pkd1aand pkd1b, based on the finding that both zebrafish pkd1 genesare most closely related to mammalian PKD1 genes, and not tomammalian polycystin 1 like (PKD1L) genes or mammalian REJ(receptor for egg jelly) orthologs (Fig. 1A,B). In situ hybridizationrevealed that pkd1a is expressed broadly in chondrogenic tissues(Fig. 1C-H), whereas pkd1b is expressed primarily in the nervoussystem (Fig. 1I-N). pkd1a is expressed diffusely throughout theembryonic axis at the 1-somite stage (Fig. 1C), and in cellssurrounding Kupffer’s vesicle, an embryonic organ associated with

left-right asymmetry (Fig. 1C, inset). By 24 hours post-fertilization(hpf), pkd1a expression becomes concentrated in the brain andventral head tissue (Fig. 1D), and by 48 hpf, pkd1a expression isstrongest in the forming pharyngeal arches (Fig. 1E) and in thenotochord (Fig. 1F). At 72 hpf, pkd1a expression in the jaw regionis concentrated in perichondrial mesenchyme (Fig. 1G) and is alsodetectable in cells surrounding cartilage in the fins (Fig. 1H). pkd1bis expressed in the caudal neural plate at the 6-somite stage (Fig.1I), and later, by the 10-somite stage, it is uniformly expressedthroughout the neural plate (Fig. 1J). Uniform expression of pkd1bin the spinal cord at the 18-somite stage (Fig. 1K) is followed byan anterior-to-posterior downregulation of expression, seen at 24hpf (Fig. 1L). In free-swimming larvae (Fig. 1M,N), pkd1bexpression is restricted to ependymal cells of the ventral neural tube(medial floor plate) (Fig. 1N).

Fig. 1. Zebrafish pkd1 structure and expression. (A)Domain structure of the predicted zebrafish Pkd1a and Pkd1b proteins compared with human PKD1.(B)Phylogenetic analysis of zebrafish pkd1a and pkd1b genes reveals that the two zebrafish polycystin1 paralogs are most closely related to human PKD1.Multiple sequence alignment was performed using ClustalW and output dendrograms were drawn using Drawtree (PHYLIP 3.65). (C-N)Expression of pkd1a andpkd1b during embryogenesis. pkd1a is expressed uniformly at the tail bud stage (C) and is observed in cells adjacent to Kupffer’s vesicle (C, inset). At 24 hpf,pkd1a is expressed in the head and brain (D). (E)At 52 hpf, pkd1a expression in the head is strongest in chondrogenic tissue associated with the pharyngealarches (arrowhead). (F)Expression in the notochord progressed to the tail (arrowhead). (G)Histological section of the head of a 72-hpf embryo shows strongexpression in perichondrial mesenchyme surrounding condensed chondrocytes (arrows) (e, eye; fb, forebrain). (H)At 72 hpf, expression in jaw-forming regionsremains strong and a new area of expression is observed in the chondrogenic tissue of the pectoral fins (arrowheads). At the 6-somite stage, pkd1b is expressedin the neural plate, caudal to somite 1 (I). Histological section of a 6-somite embryo (J), showing neural plate expression of pkd1b. At the 18-somite stage (K),expression is observed in the neural tube and in lateral mesoderm (arrowheads). By 24 hpf (L), expression in the neural tube diminishes in a rostral to caudalprogression. (M)Dorsal view of an 84-hpf larva showing midline pkd1b expression in neural tube ependymal cells (floor plate). (N)A section of the embryo in M;the arrowhead points to an ependymal cell. Bars, 10 mm (G,J,N).

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Polycystins regulate matrix productionRESEARCH ARTICLE

pkd1a knockdown results in pleiotropic craniofacial, axial andkidney defectsTo assess the function of polycystin1 paralogs, we designedantisense morpholino oligonucleotides targeting the translationstart site and the exon 8 splice donor site of pkd1a, and the exon44 and exon 45 splice donor sites of pkd1b. Reverse transcription(RT)-PCR on single morphant embryos revealed that pkd1a exon8 morpholino (exon8MO) embryos (Fig. 2A) display a smallerpkd1a RT-PCR product, reflecting a partial deletion of exon 8 andout-of-frame fusion with exon 9, which generates a premature stopcodon in the mRNA encoding the N-terminal extracellular domainof Pkd1a (Fig. 2B). Disruption of pkd1b mRNA processing resultedin in-frame deletions of the predicted Pkd1b transmembranedomains tm10 (exon44dMO; supplementary material Fig. S1) ortm11 (exon45dMO; Fig. 2C,D). Both of the pkd1b internal deletionsare predicted to alter the transmembrane topology of Pkd1b andforce the C-terminal tail, a domain that is essential for polycystin1function, into the extracellular space. pkd1a exon8MO and pkd1b

exon45MO splice disruption completely eliminated wild-typetranscripts (Fig. 2A,C) and were used as primary morpholinos forsubsequent analyses.

Knockdown of pkd1a alone using either ATG or exon 8morpholinos resulted in hydrocephalus, jaw defects and kidneycysts (Fig. 2E). Hydrocephalus (Fig. 2E,F) was observed in allmorphant embryos, whereas kidney cysts (Fig. 2F) were present inonly 10-20% of morphant embryos (three out of 31 embryos in arepresentative experiment) despite the complete absence of wild-type pkd1a mRNA (Fig. 2A). pkd1a knockdown also resulted inshort jaw defects (Fig. 2H) compared with control embryos at fivedays of age (Fig. 2G). Surprisingly, disrupting the expression ofpkd1b with morpholinos targeted to either exon44 or exon45 didnot result in a visible phenotype (data not shown). Control invertedsense and randomized sequence morpholinos for both pkd1a andpkd1b did not cause any developmental abnormalities, indicatingthat the observed phenotypes were specific to polycystin loss offunction.

Fig. 2. pkd1a and pkd1b knockdown and phenotypes of pkd1a/b-deficient embryos. (A)RT-PCR products generated from pkd1a exon 8 morpholino(exon8MO)-injected embryos demonstrate an absence of wild-type mRNA and a deletion induced by blocking the pkd1a exon 8 splice donor. (B)Diagram ofaltered pkd1a splicing in morphants. pkd1a ex8dMO splices exon 9 to a cryptic donor in exon 8, resulting in out-of-frame fusion and truncation of the Pkd1aprotein. (C)RT-PCR products generated from pkd1b exon45MO-injected embryos demonstrate a deletion caused by blocking the pkd1b exon 45 splice donor.(D)Diagram of altered pkd1b splicing. pkd1b ex45dMO splices exon 46 to a cryptic donor in exon 45, causing an in-frame deletion of transmembrane domain 11and deletion of 41 amino acids in the C-terminal cytoplasmic tail of Pkd1b. (E)pkd1a ATG morpholino injection results in body axis curvature, kidney cysts andhydrocephalus, shown at higher magnification in F (arrow in F: swollen brain ventricle; arrowhead in F: kidney cysts). (G)Alcian Blue staining of 5-day larval jawcartilage. (H)pkd1a ATG morpholino knockdown results in a short jaw phenotype. (I)Combined knockdown of pkd1a and pkd1b (pkd1a/b morphants) results instrong dorsal axis curvature (arrowheads) and hydrocephalus (small arrow), which is very similar to the phenotype of pkd2 morphants (J). (K)col1a1:EGFPtransgenic larvae at 3.5 dpf show GFP fluorescence in the stacked chondrocytes of the jaw mandible. (L)pkd1a/b morphants show shorter jaws composed ofmore rounded cells (arrowheads). (M)Calcein staining of 5-dpf larvae reveals ossification of the forming head bones in ventral views of the head, with anterior tothe top. Ventral bones are identified on the left and dorsal bones on the right. max, maxilla; den, dentary; qu, quadrate; ra, retroarticular; ch, ceratohyal; bsrp,branchiostegal ray; cl, cleithrum; op, opercle (Kimmel et al., 2003). (N)In pkd1a/b morphants at five days of age, only the opercle (op) and the cleithrum (cl) arepositive for calcein fluorescence. Bars, 10 mm (K,L); 20 mm (M,N).

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Combined knockdown of pkd1a and pkd1b results inchondrogenic, body axis and kidney cystic defectsThe existence of two pkd1 paralogs in zebrafish and the apparentlack of effect of pkd1b knockdown suggested that zebrafish pkd1genes may have partially overlapping or redundant functions. Toreveal the full pkd1 loss-of-function phenotype, we knocked downboth pkd1a and pkd1b (hereafter referred to as pkd1a/b morphants)simultaneously by injecting a combination of the pkd1a exon 8splice donor MO and the pkd1b exon 45 splice donor MO. Doublepkd1a/b morphants exhibited a strong and fully penetrant dorsalaxis curvature (Fig. 2I), in contrast to the variable axis curvatureseen with knockdowns of pkd1a alone (Fig. 2E). As seen with pkd1aknockdown alone, pkd1a/b morphants developed hydrocephalus(Fig. 2I), which was confirmed in histological sections (data notshown). Both hydrocephalus and axis curvature phenotypes werestrikingly similar to phenotypes observed in pkd2 morphantembryos (Fig. 2J). Jaw development was assessed in col1a1:EGFP(enhanced green fluorescent protein) transgenic embryos.Compared with wild-type embryos (Fig. 2K), pkd1a/b doublemorphants exhibited defects in jaw morphogenesis (Fig. 2L) thatwere similar to pkd1a morphants (Fig. 2H). Chondrocytes ofMeckel’s cartilage persisted in a rounded immature morphologyand appeared disorganized (Fig. 2L) when compared with theordered stacking of the flattened differentiated chondrocytes in thejaws of control embryos (Fig. 2K). Calcification of jaw bones wasassessed by calcein staining and also appeared delayed or inhibitedin pkd1a/b morphants (Fig. 2N) compared with controls (Fig. 2M).

The frequency of kidney cysts was slightly increased in pkd1a/bdouble morphants but never exceeded 20% of the injected embryos.Approximately 40% (in one experiment 18 of 45 injected embryos)of the pkd1a/b morphants showed severe edema at 5 days postfertilization (dpf), which is a sign of kidney failure.

pkd1a/b and pkd2 act in a common axial morphogenesis pathwayWe chose to focus on the dorsal axis curvature phenotype since(1) it was the most penetrant and consistent pkd1a/b-deficiencyphenotype in zebrafish and (2) it was a quantitative trait, withembryos showing dose-dependent severity of curvature.Surprisingly, kidney cyst formation was poorly penetrant (10-20%of knockdown embryos), suggesting that this phenotype has a highthreshold and may only be manifest in the most severely affectedembryos. Axis curvature in pkd1a/b morphants was strikinglysimilar to the phenotype seen in pkd2 mutants and morphants (Fig.2J) (Obara et al., 2006; Schottenfeld et al., 2007), presenting anopportunity to assess whether pkd1a/b and pkd2 function in acommon pathway affecting axial morphogenesis. Previous studieshave shown that polycystin 1 and polycystin 2 physically interact(Qian et al., 1997; Tsiokas et al., 1997) and that this heterodimericassembly produces a non-selective cation channel (Hanaoka et al.,2000). To test for pkd1a/b: pkd2 genetic interactions in zebrafish,we generated partial loss-of-function embryos for both pkd1a/band pkd2 using threshold doses of morpholinos and then assessedwhether the combined low doses of pkd1a/b: pkd2 morpholinosproduced a phenotype stronger than either alone. Embryos weresorted as having a curvature of less than 90 degrees (mild), morethan 90 degrees (moderate), and full curvature with the tail crossingthe body axis (severe) (Fig. 3). Reducing the dose of injectedpkd1a/b and pkd2 morpholinos to a tenth of the full knockdowndose resulted in the majority of embryos having mild axis curvatureand very few having severe dorsal axis curvature (20% of pkd1a/bmorphants and 10% of pkd2 morphants) (Fig. 3). Knockdown ofpkd1a/b and pkd2 together using sub-threshold doses ofmorpholinos revealed a striking interaction in pkd1a/b and pkd2activity; combined knockdown resulted in a synergistic increase inthe fraction of embryos displaying a severe axis curvature (56%)(Fig. 3), indicating that pkd1a/b and pkd2 function in the samemolecular pathway regulating axis formation.

Axis curvature in polycystin morphants is linked to increased ECMdepositionAxial elongation in chordate embryos is dependent on theformation of the notochord and two additional rigid structures:the floor plate and the hypochord, which are dorsal and ventral tothe notochord, respectively (reviewed in Scott and Stemple, 2005).pkd1a/b-deficient embryos often displayed an undulatingnotochord phenotype at 24 hpf (Fig. 4B) and 48 hpf (Fig. 4C)compared with control embryos (Fig. 4A), suggesting that alterednotochord structure could be a primary defect in pkd1a/bmorphants. Notochord structure is dependent on the formationof a surrounding basement membrane or sheath consisting ofmultiple ECM proteins including laminin 5 and collagen types II,VIII and IX (Scott and Stemple, 2005). Expression of the mostabundant notochord collagen protein, collagen type II alpha 1(Col2a1), was significantly increased in the notochord sheath byknockdown of pkd1a/b (Fig. 4E) or pkd2 (Fig. 4F) compared with

Fig. 3. pkd1a/b and pkd2 interact in axial morphogenesis. Top panels: therange of severity in dorsal axis curvature that was used for scoring. Mild: <90degrees; moderate >90 degrees; severe: tail tip crossing the body axis. Thechart represents the percentage of embryos with mild, moderate or severecurvature; the knockdown conditions were as noted in the legend. The datarepresent the averages of three separate experiments; error bars representstandard error of the mean (s.e.m.).

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control embryos (Fig. 4D). These results raised the possibility thatdorsal axis curvature in polycystin morphants could be the resultof collagen overexpression in the notochord sheath.

To assay directly whether misregulation of notochord collagengene expression occurred owing to pkd1a/b or pkd2 deficiency,we performed in situ hybridization using probes for col2a1, col9a2and col27a1. The col2a1 gene is strongly expressed early duringnotochord formation at 20 hpf and is progressively downregulatedas the notochord matures, such that, by 48 hpf, only cells of thehypochord and floor plate show col2a1 expression and, by 96 hpf,axial col2a1 expression in the notochord is minimal or absent(supplementary material Fig. S2). A comparison of notochordcol2a1 expression in control injected embryos with pkd1a/b orpkd2 morphants revealed that polycystin knockdown results inabnormally persistent col2a1 mRNA expression in the notochord(Fig. 4G-I). Expression of the col9a2 and col27a1 mRNAs wasalso abnormally upregulated in pkd1a/b and pkd2 morphants (Fig.4J-O), indicating that polycystin knockdown had pleiotropiceffects on the expression of multiple collagen genes. Interestingly,expression of col27a1 in the distal end of the pronephric nephron(red arrowheads in Fig. 4M-O) was enhanced in pkd2 morphantsbut diminished in pkd1a/b morphants, suggesting that althoughboth pkd1a/b and pkd2 affect collagen expression and can beshown to interact, they may have unique effects on collagen generegulation in different tissues. Abnormally persistent col2a1expression was also observed in the pkd2 retroviral insertionalmutant pkd2hi4166Tg (Fig. 4P,Q), indicating that our results usingpolycystin morpholino knockdowns were not the result of non-specific developmental delay. To further exclude the potentialeffects of non-specific developmental delay, we examined theexpression of sox9a, a transcription factor that is responsible forthe early activation of collagen expression in the notochord. sox9awas strongly expressed in the notochord at 24 hpf, andsubsequently decayed to baseline levels by 72 hpf in wild-typeembryos (supplementary material Fig. S3A-C), pkd1a/bmorphants (supplementary material Fig. S3D-F) and pkd2morphants (supplementary material Fig. S3G-I). The normal rateof sox9a downregulation in polycystin morphants excludes generaldevelopmental delay as an explanation for the observedphenotypes, and argues for a specific role for polycystins incollagen gene expression and maturation of the notochord.

Previous reports have suggested the existence of a negativefeedback signaling mechanism that monitors the formation of thenotochord collagen sheath (Anderson et al., 2007; Gansner et al.,2007). Inhibition of collagen crosslinking by copper chelation, directinhibition of lysyl oxidase, or knockdown of lysyl oxidase mRNAsresults in a characteristic kinked notochord and abnormallypersistent expression of col2a1, similar to what we observe inpolycystin morphants (Anderson et al., 2007; Gansner et al., 2007).To better define the nature of the feedback signals regulatingnotochord collagen expression, we knocked down col2a1 andassayed the expression of the remaining notochord collagens,col9a2 and col27a1. col27a1 (Fig. 4R,S), as well as col9a2(supplementary material Fig. S4), were expressed ectopically incol2a1 morphants. Reciprocal experiments knocking down col9a2and assaying the expression of col2a1 mRNA resulted in a similarectopic expression of col2a1 (data not shown), indicating thatnotochord cells regulate collagen genes as a cohort and

Fig. 4. Notochord defects and collagen expression in pkd1a/b and pkd2morphants. (A)Wild-type trunk at 24 hpf. (B,C)pkd1a/b morphant at 24 hpf (B)and 48 hpf (C) showing undulations in notochord structure (arrowheads).(D-F)Confocal image projections of anti-Col2a1-stained (red) vibratome sectionsof the notochord; nuclei are stained with DAPI (blue). pkd1a/b morphants (E) andpkd2 morphants (F) show significantly enhanced expression of type II collagen inthe notochord sheath compared with wild-type embryos (D). (G-S)Abnormalpersistence of collagen gene expression in polycystin morphants. (G)Wild-typecol2a1 expression at 48 hpf highlights cells of the floor plate and hypochord, butthe notochord is negative for col2a1 expression. At 48 hpf, pkd1a/b (H) and pkd2

(I) morphants show abnormally persistent expression of col2a1, particularly in themost posterior curved tail (arrowheads). Similarly, at 48 hpf, pkd1a/b (K) and pkd2

(L) morphants show abnormally persistent expression of col9a2 in the mostposterior curved tail (arrowheads) compared with wild-type embryos (J). col27a1

expression is also persistent in pkd1a/b (N) and pkd2 (O) morphants at 48 hpfcompared with wild-type embryos (M). The red arrowheads in M-O highlight thecloaca, also shown in the insets in M-O. Notochord col2a1 expression is enhancedin insertional mutant pkd2hi4166Tg homozygotes (Q) compared with wild-typesiblings (P); the arrowheads in P and Q mark the anterior extent of col2a1

notochord expression. Strong col2a1 expression in the jaw is also noted (redarrowhead in Q). By 24 hpf, notochord col27a1 is downregulated in wild-typeembryos (R), but persists in col2a1 morphants (S), suggesting a general feedbackcontrol of collagen gene expression. Bars, 10 mm (D-F).

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downregulate collagen expression only after a complete matrix hasformed.

Attenuated collagen expression or crosslinking rescues polycystinaxis curvatureOverexpression of the col2a1 mRNA and protein in polycystin-deficient notochords suggested that excessive collagen depositioncould underlie notochord axial defects. We therefore tested whetherknocking down col2a1 expression would rescue polycystin axisdefects. A morpholino oligonucleotide targeting the col2a1 exon1 splice donor effectively eliminated normal col2a1 mRNA andgenerated two mis-spliced products that were predicted to truncatethe Col2a1 protein immediately after the exon 1 coding sequence(supplementary material Fig. S5). As shown above, morpholinoknockdown of either pkd1a/b (Fig. 5B) or pkd2 (Fig. 5C) resultedin severe dorsal axis curvature compared with control embryos (Fig.5A). Knockdown of col2a1 alone caused a mild ventral body axiscurvature (Fig. 5D). Strikingly, combined knockdown of col2a1 and

pkd1a/b (Fig. 5E) or pkd2 (Fig. 5F) completely rescued the dorsalaxis curvature defect caused by polycystin knockdown alone (Fig.5B,C). The notochord sheath collagen structure can also bemodified by inhibition of lysyl oxidase, an enzyme responsible forcollagen crosslinking. Prevention of collagen crosslinking using thesmall molecule 3-aminopropionitrile (bAPN) also prevented thedorsal axis curvature in pkd1a/b (Fig. 5H) and pkd2 morphants(Fig. 5I). The results demonstrate that the polycystin axis curvaturephenotype is dependent on the abnormal persistence of embryoniccollagen gene expression, and further suggests that polycystinscould play a direct role in regulating axial matrix assembly ordeposition.

polycystin deficiency and matrix misassembly interactsynergistically to cause persistent collagen gene expressionThe upregulation of multiple collagen genes in polycystinmorphants suggested that polycystins could function as part of anegative feedback system that detects completely assembled,crosslinked ECM and that generates signals that repress collagengene expression. To examine the relationship between polycystinfunction and matrix synthesis and assembly, we assayed whetherregulation of collagen gene expression was synergistically affectedby disruption of collagen crosslinking (lysyl oxidase inhibition) andpolycystin knockdown. MCP (2-mercaptopyridine N-oxide) is apotent inhibitor of lysyl oxidase, an enzyme that is required forcollagen crosslinking and notochord matrix assembly (Andersonet al., 2007). MCP-treated embryos fail to downregulate col2a1mRNA expression by 48 hpf and exhibit an undulating notochordphenotype (Anderson et al., 2007). As expected, embryos treatedwith MCP alone showed overexpression of col2a1 at 48 hpf (datanot shown) (Anderson et al., 2007). However, by 4 days (96 hpf),col2a1 expression had returned to baseline (Fig. 5J), despite thefact that the notochord remained structurally distorted (Fig. 5M).By contrast, pkd2 (Fig. 5K,N) and pkd1a/b (Fig. 5L,O) morphantstreated with the same dose of MCP failed to suppress col2a1expression by 96 hpf. The results suggest that polycystins arerequired for negative feedback control of embryonic collagen geneexpression and could play a role in signaling the completion ofmorphogenesis.

Depletion of endoplasmic reticulum calcium stores, or inhibitionof PI3K signaling, phenocopy the effects of polycystin on collagengene regulationTo identify signaling pathways that might control collagen synthesisin the context of polycystin function, we screened chemicalinhibitors that had previously been implicated in both polycystinsignaling and collagen gene regulation. Polycystins function in ionchannel complexes that regulate calcium release from intracellularcalcium stores (Koulen et al., 2002; Nauli et al., 2003; Sutters andGermino, 2003), and maintain calcium store levels in the lumen ofthe endoplasmic reticulum (ER). Non-toxic doses (50 mM) of theSERCA [sarco(endo)plasmic reticulum Ca2+ ATPase] calciumpump inhibitor cyclopiazonic acid phenocopied polycystinknockdown with respect to col2a1 overexpression (Fig. 6C).Epidermal growth factor receptor (EGFR), JAK/STAT andAKT/PI3K have also been implicated as kinase signaling pathwaysthat mediate polycystin function. Although inhibition of EGFR orJAK/STAT had little effect on col2a1 expression, treating embryos

Fig. 5. Inhibition of col2a1 expression or crosslinking rescues polycystinaxis curvature. (A-C)Wild-type embryo (A), pkd1a/b morphant (B), and pkd2

morphant (C) at 48 hpf. (D)col2a1 morphant at 48 hpf with slight ventralcurvature. (E,F)col2a1 knockdown in pkd1a/b (E) and pkd2 (F) morphantsrelieves the dorsal axis curvature. (G)Wild-type 48 hpf embryo treated with 10mM bAPN, a lysyl oxidase inhibitor, shows notochord distortion associatedwith a failure to generate a rigid, crosslinked notochord collagen sheath.Blocking collagen crosslinking relieves dorsal axis curvature in pkd1a/b (H) andpkd2 (I) morphants. (J-O)polycystin knockdown disrupts the feedbackregulation of collagen gene expression. Embryos treated with 200 nM MCPand allowed to develop to 96 hpf (4 days) successfully repress col2a1

expression (J), but exhibit kinked notochords when stained with Alcian Blue(M). pkd2 (K) and pkd1a/b (L) morphants treated with 200 nM MCP fail todownregulate col2a1 expression in the notochord. (N,O)Histological sectionsof the embryos in K and L, respectively, confirm the aberrant notochordexpression of col2a1. Bars, 10 mm (N,O).

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from 14 hpf to 44 hpf with the phosphoinositide 3-kinase (PI3K)inhibitors wortmannin and LY294002 resulted in a dose-dependentenhancement of notochord col2a1 expression (Fig. 6D-I), similarto what we observed in pkd1a/b and pkd2 morphants. Despite theupregulation of collagen gene expression, PI3K inhibition did notcause dorsal axis curvature, suggesting that these inhibitors mayhave more pleiotropic effects on embryogenesis. Our resultsindicate that maintenance of intracellular calcium stores and PI3Ksignaling are required for the developmental regulation of col2a1expression in the notochord.

DISCUSSIONThe identification of the PKD1 and PKD2 genes as the cause ofADPKD provided important leads in determining the cellular basisof cystic disease (Hughes et al., 1995; Mochizuki et al., 1996).However, the cellular mechanisms maintaining normal tissuearchitecture that are controlled by polycystins remain unclear. Ourfinding that pkd1a/b knockdown results in pleiotropic defects inthe kidney, cartilage and brain are consistent with knockout modelsof polycystin1 in the mouse. In addition to cystic kidneys, weobserve cartilage defects similar to the undulating spinal chord andshort jaw phenotypes that have been reported in mouse Pkd1knockouts (Boulter et al., 2001; Kolpakova-Hart et al., 2008).Expression of Pkd1 in the mouse is observed in the notochord andfloor plate, as well as in other chondrogenic tissues (Boulter et al.,2001), similar to what we observe in zebrafish. Expression ofzebrafish pkd1 in perichondrial mesenchyme in the head parallelsthe expression seen in long bone perichondrial mesenchyme in themouse (Lu et al., 2001). The delayed ossification of the skull andspinal chord in Pkd1-deficient mice (Lu et al., 2001) is consistentwith defects in ossification (calcein staining) that we observe in theforming skull and jaw bones in zebrafish pkd1a/b knockdowns. Wealso present genetic evidence that pkd1 and pkd2 interact in

zebrafish as they do in mammalian kidneys (Wu et al., 2002). Theseresults argue that the zebrafish is a useful model to further pursuethe cellular mechanisms of polycystin function.

Somewhat surprisingly, pronephric kidney cyst formation wasnot highly penetrant in pkd1a/b morphants. Although we coulddemonstrate complete knockdown of pkd1a and pkd1b mRNA,kidney cysts formed in only 20% of injected embryos. Our resultsmay be most relevant to conditional knockout of Pkd1 in juvenilemouse models (after postnatal day 14) where cyst formation is alsopoorly penetrant and slow to develop (Piontek et al., 2007; Takakuraet al., 2008). In this context, cyst formation may require additionalkidney insults beyond PKD1 loss of function (Takakura et al., 2008).Loss of pkd1a and pkd1b may sensitize embryos to othermorphogenesis defects or injury, but by itself may not be sufficientto cause kidney cysts at high frequency. To analyze the cellular basisof pkd1 function, we focused instead on the role of polycystins inchondrogenesis and fully penetrant axial morphogenesis defects.The requirement for double pkd1a/b knockdown for full expressionof axis curvature (compared with single gene knockdown in pkd2morphants) probably reflects the cell type-specific expression ofpkd1a in the notochord and pkd1b in the floor plate, whereas pkd2is expressed ubiquitously (Obara et al., 2006). In this view, pkd1a/baxis curvature is a compound phenotype involving defects in boththe notochord and floor plate. Electron microscopic examinationof the notochord sheath did not reveal strong qualitative differencesin collagen structure between pkd1a/b morphants and controls;collagen fibril periodicity and diameter did not appear different(data not shown). It is therefore unlikely that pkd1a/b deficiencycaused collagen crosslinking defects in the notochord like thoseobserved with bAPN. The notochord undulations that were seenduring early development in pkd1a/b morphants probably arise bya different mechanism involving overproduction of collagen.

In mouse models of Pkd1 deficiency, raising or lowering theamount of polycystin1 protein expression is sufficient to causepolycystic kidney disease (PKD) kidney phenotypes (Pritchard etal., 2000; Lantinga-van Leeuwen et al., 2004), indicating that thePkd1 gene dosage is crucial for normal tissue architecture. For bothzebrafish pkd1a/b and pkd2, we found that the axial curvaturephenotype is highly sensitive to gene dosage and provides aquantitative readout of polycystin function. The overt similarityand genetic interaction that we demonstrate in pkd1a/b and pkd2axis curvature defects substantiates the idea that pkd1 and pkd2interact in an underlying cellular process or signaling pathway thatensures proper morphogenesis (Wu et al., 2002).

Our results suggest that both pkd1a/b and pkd2 are required fornotochord cells to transition to a mature differentiated phenotype.In the absence of polycystins, notochord cells persistently expressembryonic collagen genes at high levels, resulting in overproductionof the ECM and distortion of the embryonic axis. These resultssupport the idea that polycystin1 may act as a sensor of differentiationto repress embryonic programs of gene expression. Similar evidencefor a role for polycystin1 as a sensor of differentiation is found inthe persistent overexpression of embryonic matrix genes andreceptors (collagens type I, III and IV, laminins, heparan sulfateproteoglycan, b4 integrin), cell surface proteins (erb-B2, b2 Na/KATPase subunit), and secreted proteins (periostin) in ADPKD tissue(Ebihara et al., 1988; Haverty and Neilson, 1988; Ebihara et al., 1995;Joly et al., 2003; Wallace et al., 2008). These results have implications

Fig. 6. Inhibition of PI3K or SERCA phenocopies polycystin loss offunction. Control untreated (A) or DMSO-treated (B) embryos at 48 hpf showno col2a1 expression in the notochord. (C)Treatment with 50mMcyclopiazonic acid, an inhibitor of the ER calcium uptake ATPase SERCA, resultsin elevated expression of col2a1 in the notochord. (D-F)The PI3K inhibitorwortmannin causes a dose-dependent increase in the expression of col2a1 inthe notochord. (G-I)Similarly, the PI3K inhibitor LY294002 causes a dose-dependent increase in the expression of col2a1 in the notochord.

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for understanding the fibrotic defects that are often associated withPKD (Cuppage et al., 1980; Wilson et al., 1992), as well asunderstanding other Pkd1 matrix-associated phenotypes, includingintracranial aneurysm (Brooke et al., 2003; Ruigrok et al., 2005);vascular fragility and maintenance of the aortic wall ECM (Kim etal., 2000; Hassane et al., 2007); overlapping connective tissue disorders(Somlo et al., 1993; Kaplan et al., 1997); abdominal wall hernia(Morris-Stiff et al., 1997); and increased pericardial complianceassociated with pericardial effusion in ADPKD patients (Qian et al.,2007). Our finding that a reduction in matrix gene expression bycol2a1 knockdown can rescue polycystin axis curvature defectssuggests that abnormalities in matrix composition or integrity arelikely to be developmental defects linked directly to polycystinfunction, as opposed to secondary consequences of tissue damageor deformity. This view of polycystins as sensors of mature ECMmay be analogous to ‘outside-in’ integrin signaling (Arnaout et al.,2005). A role for polycystin1 as a matrix sensor (Fig. 7) is supportedby findings that (1) polycystin1 can bind to multiple matrix molecules,including collagen types I, II and IV (Weston et al., 2001); (2)polycystin1 is localized, in part, to focal complexes in both muscleand epithelial cells (Wilson and Burrow, 1999; Qian et al., 2003a);(3) polycystin1 interacts with focal adhesion proteins (Wilson et al.,1999); and (4) polycystin1 can recruit focal adhesion signalingmolecules to alter cell adhesiveness and migration (Wilson et al.,1999; Joly et al., 2006).

Cells sense their local environment by both ligation-inducedsignaling, which depends on cell-ECM biochemical interactions,

and by traction-induced signaling, which depends on cellular forcegeneration between points of cell-ECM contact (Arnaout et al.,2005; Giannone and Sheetz, 2006). In our experiments with collagencross-linker inhibitors and polycystin knockdowns, we find noevidence for the absence of a matrix ligand; indeed, we observe thatmore type II collagen protein is synthesized from increasedexpression of col2a1 mRNA. Nonetheless, the embryos appear tolack a negative feedback signal that represses collagen geneexpression. This would argue that ligation-induced signaling isunlikely to initiate negative feedback signaling. Inhibition ofcollagen crosslinking produces a non-rigid ECM that wouldinterfere with the ability of the cell to generate traction forces,cluster adhesion receptors, and initiate focal adhesion kinasecascade signaling (Wozniak et al., 2004; Giannone and Sheetz,2006). The idea that polycystin1 participates in traction-inducedsignaling remains to be tested.

Whatever the nature of the initiating signal, our studies suggestthat negative feedback regulation of collagen gene expressioninvolves activation of PI3K and calcium signaling. Consistent withour results, cultured mammalian epithelial cells overexpressingpolycystin1 activate PI3K and the downstream kinase Akt (Bocaet al., 2007). G-protein inhibition with pertussis toxin was sufficientto downregulate PI3K activity in Pkd1-overexpressing cells,indicating that polycystin1 may activate Gi as part of its signalingcascade (Calvet and Grantham, 2001; Boca et al., 2007).Alternatively, PI3K is also known to be activated by focal adhesionkinase (FAK) (Hanks et al., 2003). Interestingly, a small C-terminalfragment of polycystin1 is sufficient to recruit FAK (Joly et al., 2006),and FAK recruitment to focal complexes containing polycystin1does not occur in ADPKD (polycystin mutant) cells (Wilson et al.,1999). Together, these findings suggest that, in both fish andmammalian cells, polycystin1 may signal the state of theirenvironment by activating PI3K.

Immunolocalization studies have revealed that polycystin1 canfunction in several different subcellular domains including theapical cilium, focal adhesions, desmosomes, or intracellular ERmembranes (Wilson et al., 1999; Scheffers et al., 2000; Xu et al.,2001; Yoder et al., 2002; Grimm et al., 2003; Qian et al., 2003a). Itis currently not known which of these locations is involved in theregulation of axial morphogenesis in zebrafish. Expression of amutant form of polycystin2 that is retained in the ER has beenreported to partially rescue zebrafish pkd2 morphant body axiscurvature, suggesting that polycystin2 regulation of ER calciumstores or calcium release may be an important site of polycystin2signaling in axis morphogenesis (Fu et al., 2008). Our finding thattreating embryos with cyclopiazonic acid, an inhibitor of the ERcalcium ATPase, phenocopies polycystin loss of function in termsof the notochord defects and the persistence of collagen geneexpression is consistent with this idea. These results suggest analternative model of polycystin function in the ER to ensure propercollagen processing in the secretory pathway (Fig. 7). Bothpolycystin1 and polycystin2 are known to be required to maintainproper ER lumenal calcium levels (Koulen et al., 2002; Grimm etal., 2003; Nauli et al., 2003; Qian et al., 2003b; Qian et al., 2003a;Hooper et al., 2005; Anyatonwu et al., 2007; Xu et al., 2007; Weberet al., 2008). It is possible that, in addition to a role as a matrixsensor, polycystin maintenance of ER calcium may also be requiredfor proper collagen crosslinking and assembly.

Fig. 7. A model of the negative feedback control of developmental geneexpression by polycystins. Polycystin1, functioning in the same pathwaywith polycystin2, generates a negative feedback signal on developmentalcollagen gene expression when the crosslinked mature ECM has beenassembled. Alternatively, the polycystins, acting to maintain normal levels ofER calcium, could play a role in ensuring the proper folding, crosslinking andsecretion of collagen subunits, ultimately controlling the rate of matrixmaturation.

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Although our group and others do not detect any structural ormotility defects in pkd1 or pkd2 morphant cilia (data not shown)(Obara et al., 2006; Sullivan-Brown et al., 2008), the primary ciliumcould function as an alternate site of polycystin-initiated signaling.If this were the case, it might be expected that both polycystin andprimary cilium loss-of-function mutants would show similarphenotypes. Instead, we find that zebrafish intraflagellar transportprotein (IFT) mutants that lack functional cilia (ift172, doublebubble; ift88, oval) do not exhibit dorsal axis curvature or persistentcollagen gene expression in the notochord. In addition, the ventralaxis curvature that is exhibited by IFT mutants is not rescued bycol2a1 knockdown (Y.L. and I.A.D., unpublished). In a similar vein,kidney-specific knockout of Pkd1 in mice generates a more severeand early cystic phenotype (Shibazaki et al., 2008) than a similar(using the same ksp-Cre recombinase line) kidney-specificknockout of Kif3a, a gene essential for ciliogenesis (Lin et al., 2003).The increased severity of kidney phenotypes in mutants lackingpolycystin1 versus those specifically lacking cilia suggests that, inaddition to its function in the cilium, polycystin1 is likely to functionat additional sub-cellular locations.

In summary, our results suggest a role for polycystin1 andpolycystin2 in a negative feedback signaling system that detectsmature crosslinked ECM formation and that regulates theexpression of multiple collagen genes accordingly. Although ourwork has focused on the zebrafish notochord, studies with humansand mouse models of cystic kidney disease suggest that the sensormalfunction model for ADPKD may also be applicable to epithelialcells in cystic kidneys (Bissler and Dixon, 2005).

METHODSZebrafish linesWild-type TüAB or WIK zebrafish were maintained and raised, asdescribed previously (Westerfield, 1995). Dechorionated embryoswere kept at 28.5°C in E3 solution with or without 0.003% PTU (1-phenyl-2-thiourea; Sigma) to suppress pigmentation and werestaged according to somite number (som) or hpf (Westerfield,1995). The retroviral insertion line pkd2hi4166Tg was a gift fromZhaoxia Sun (Yale University) and the col1a1:EGFP transgenic linewas a gift from Shannon Fisher.

Cloning polycystins and phylogenetic analysisZebrafish pkd1a cDNA was amplified from total RNA at 48 hpf byRT-PCR using a primer design based on TBLASTN searches ofthe Sanger Center zebrafish genomic sequence using themammalian PKD1 protein sequence. Overlapping 3–4-kb RT-PCRfragments were sequenced directly and the sequence was assembledusing Phred/Phrap (Ewing et al., 1998). pkd1b was isolated as apartial cDNA clone from a 24-hpf whole-embryo zebrafish cDNAlibrary using low-stringency hybridization conditions and thehuman PKD1 C-terminus as a probe. The predicted proteinsequences for each gene were compared with human polycystinsusing ClustalW, and the output was plotted using Drawtree(PHYLIP 3.65) as an unrooted phylogenetic tree.

In situ hybridization and immunohistochemistryIn situ hybridization was performed using standard techniques, asdescribed previously (Drummond et al., 1998). The accessionnumbers for the cDNAs used are: col2a1a (NM_131292), col9a2

(NM_212579) and cb221(col27a1) (BQ169308). The cb221expressed sequence tag (est) represents the 3�-UTR of the predictedtranscribed locus hmm358864 (predicted protein XP_001343932),which encodes the zebrafish homolog of collagen type XXVII, alpha1 (col27a1). Whole-mount immunocytochemistry was performedon embryos fixed in methanol:DMSO (80:20) (anti-chicken collagenII alpha 1 monoclonal antibody II-II6B3, Developmental BiologyHybridoma Bank). Embryos were blocked in 10% normal goatserum (NGS) and then incubated with primary antibody in PBSwith 1% DMSO, 2% NGS and 0.1% Tween-20 overnight at 4°C. Afterwashing in incubation medium, secondary antibodies (Alexa 548anti-mouse; Molecular probes) were used at 1:1000 and the nucleiwere counterstained with DAPI (1:3000, Molecular Probes).Immunostaining of notochord anti-collagen II alpha 1 wasperformed on 100-mm vibratome sections (Leica VT 1000Svibratome) of agarose-embedded embryos. Immunostaining waspreceded by treatment in 6 M urea, 100 mM glycine, pH 3.5, for1 hour at 4°C. Washed tissue sections were cleared in mountingmedia (53% benzyl alcohol, 45% glycerol, 2% N-propyl gallate) andphotographed on a Nikon E800 fluorescence microscope or on aZeiss LSM5 Pascal confocal fluorescent microscope. Thecol1a1:EGFP transgenic and calcein-stained embryos (Du et al.,2001) were imaged on a Zeiss LSM5 Pascal confocal microscopeas image stacks, and then projected using Zeiss software to makethe final image. For the sections, stained embryos were dehydratedand embedded in glycolmethacrylate (JB-4) resin (Polyscience Inc.)following the manufacturer’s instructions and cut at a thickness of1-4 mm.

Morpholino antisense oligonucleotide injectionsWild-type embryos (TüAB or WIK) at the 1- to 2-cell stage were mi-croinjected with 4.6 nl of a 0.05-0.5 mM antisense morpholinooligonucleotide solution (Gene Tools LLC) in 200 mM KCL with0.1% Phenol Red using a nanoject2000 microinjector (WorldPrecision Instruments). The final antisense morpholino oligoconcentrations that were used for the full loss-of-functionexperiments were sufficient to eliminate wild-type mRNA as judgedby RT-PCR. The final amounts of morpholino ranged fromapproximately 1 to 10 ng/embryo. Randomized and invertedsequence morpholinos for pkd1a and pkd1b were used as negativecontrols and showed no effect on development; the sequence of theGene Tools standard negative control MO that was used is: 5�-CCTCTTACCTCAGTTACAATTTATA-3�. The sequence of thetranslation-blocking oligonucleotide for pkd1a was pkd1a MOATG: 5�-GTCTGTTCCTGAGACAGTACCGG-3�, and for cyclops(ndr2) was cyc MO: 5�-GCGACTCCGAGCGTGTGCATGATG-3�(Karlen and Rebagliati, 2001). The splice donor-blockingoligonucleotide sequences were pkd1a MO ex8: 5�-GATCTGAG-GACTCACTGTGTGATTT-3�; pkd1b MO ex45: 5�-ACAT-GATATTTGTACCTCTTTGGTT-3�; pkd1b MO ex44: 5�-TAAAAATACTGTACCATCATGCCTA-3� and col2a1 MO ex1:5�-TGAAAAACTCCAACTTACGGTCATC-3�. Morpholinostargeting pkd2 have been previously described (Obara et al., 2006).Nested RT-PCR primers were designed in flanking exon sequencesto confirm the morpholino oligonucleotide efficacy and tocharacterize altered mRNA splicing products: for pkd1a MO ex8:outside F: 5�-GGCGGAGCTTTCTCTGGTCA-3�, outside R:5�-GAACGTGGCGTGTGAACTGG-3�, inside F: 5�-GGTCAA -

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CTGGGGTGGAGTGG-3�, inside R: 5�-TTTGGCGGTGCAG -GAGTGTA-3�; for pkd1b MO ex44: outside F: 5�-TGGCTTTG-GCAGCTGCTGTTCTA-3�, outside R: 5�-AGCAGCACATAG-GCGTCCCAGTA-3�, inside F: 5�-TCTTAGGCGATGGTTGGT-CATGG-3�, inside R: 5�-ACCCTGTGCGACCCCTTAACAGA-3�;for pkd1b MO ex45: outside F: 5�-TGGCTTTGGCAGCTGCT-GTTCTA-3�, outside R: 5�-GGGCTTAGTGGGGTC CAGC -AGTT-3�, inside F: 5�-TCTTAGGCGATGGTTGGTCATGG-3�,inside R: 5�-CCTGAGAGGATCTGGAGGGTGGA-3�; for col2a1MO ex1: outside F: 5�-GGCGACTTTCACCCCTTAG-3�, outside

R: 5�-GGACTTCCCTTCTCACCCTTA-3�, inside F: 5�-CACCC-CTTAGGACCTGCAT-3�, inside R: 5�-CCTCTTTCTCCACGT-GTTCC-3�.

Drug and small molecule treatmentsCollagen crosslinking was inhibited by treating embryos in E3 withthe lysyl oxidase inhibitors MCP (25-200 nM, as indicated) orbAPN (10 mM) from post-gastrulation (6-8 hpf) to approximately48 hpf (as indicated). The PI3K inhibitors wortmannin andLY294002 were dissolved in DMSO as 10 mM stock solutions andused at the indicated concentrations in E3 egg water containing1% DMSO. Embryos were treated with PI3K inhibitors from 14hpf to 44 hpf, fixed in 4% formaldehyde/0.1 M phosphate buffer,and then processed for in situ hybridization, as describedpreviously.

Histology and cartilage analysisFor histology analysis, embryos were fixed in 1% paraformaldehyde,1.5% glutaraldehyde, 70 mM NaPO4, pH 7.2, 3% sucrose, thenembedded in JB-4 resin (Polyscience, Inc.) and sectioned at athickness of 4 mm. For Alcian Blue staining, embryos were fixed at5.5 dpf in 4% formaldehyde overnight at 4°C, followed by four 20-minute washes in PBST and digestion with proteinase K for 30minutes. Embryos were postfixed for 1 hour in 4% PFA, washedfour times for 20 minute in PBST, and then stained in 0.1% AlcianBlue dissolved in 70% ethanol/30% glacial acidic acid overnight atroom temperature. Embryos were destained using 1% KOH/3%H2O2 and photographed on a Leitz MZ12 dissecting scope with aSpot Insight digital camera (Diagnostic Instruments).ACKNOWLEDGEMENTSThis work was supported by NIH grants DK53093 and DK54711 to I.A.D.; PKDFoundation fellowship grants to S. Mangos and A.Z.; and a PKD Foundation Grantin aid to I.A.D. We also acknowledge the assistance of our colleagues David Beierfor sequencing a pkd1b bac, Richard Sandford for help in cloning pkd1b, ShannonFisher for sharing her col1a1:GFP transgenic, Nathan Hellman and AlexandraPetrova for editing the manuscript, and Amin Arnaout for helpful suggestions.Deposited in PMC for release after 12 months.

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSI.A.D., S. Mangos, P.-y.L. and A.Z. conceived and designed the experiments; S.Mangos, P.-y.L., A.Z., A.V., A.L., Y.L. and I.A.D. performed the experiments; S.Mudumana contributed reagents; I.A.D., A.V. and S. Mangos prepared and editedthe manuscript.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.003194/-/DC1

Received 16 March 2009; Accepted 17 November 2009.

REFERENCESAnderson, C., Bartlett, S. J., Gansner, J. M., Wilson, D., He, L., Gitlin, J. D., Kelsh, R.

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Anyatonwu, G. I., Estrada, M., Tian, X., Somlo, S. and Ehrlich, B. E. (2007).Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2. Proc.Natl. Acad. Sci. USA 104, 6454-6459.

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Bissler, J. J. and Dixon, B. P. (2005). A mechanistic approach to inherited polycystickidney disease. Pediatr. Nephrol. 20, 558-566.

TRANSLATIONAL IMPACT

Clinical issuePolycystic kidney disease (PKD) is the most common heritable disease inhumans. Patients with autosomal dominant polycystic kidney disease (ADPKD)have epithelial cysts in the kidney, liver and pancreas, and often suffer fromabdominal hernias, intracranial aneurysms, gastrointestinal cysts and cardiacvalvular defects. These conditions are often associated with alteredextracellular matrix (ECM) production. Despite a decade of work on theprincipal ADPKD genes, PKD1 and PKD2, questions remain about the etiologyof cystic disease and the role of the ECM in ADPKD pathology.

ResultsThe authors created a zebrafish model with deficiencies in polycystin1 andpolycystin2, similar to those found in humans with ADPKD. The zebrafish is aconvenient model, since multiple genes can be inhibited simultaneously usingantisense morpholino oligonucleotides to produce a large number of rapidlydeveloping embryos in which tissue pathology can be studied. This studyexploits a robust and easily scored axis curvature defect in polycystin-deficientzebrafish embryos to define the role of polycystins in ECM production.

Both polycystin1- and polycystin2-deficient embryos exhibit dorsal curvedbody axis defects that correlate with excess production of type II collagenaround the notochord, the main structural element of the embryonic zebrafishtrunk. Collagen genes, like many genes that encode embryonic structuralproteins, are initially expressed at a high level and then shut off after tissueformation. Excess collagen in polycystin-deficient embryos correlates withabnormally persistent expression of multiple notochord collagen genes,suggesting that, in the absence of polycystins, embryonic genes do not shutoff properly. Distortion of the body axis in polycystin-deficient zebrafishembryos was relieved by concomitant inhibition of collagen production. Othertreatments that prevented collagen crosslinking also relieved the body axisdistortion. This suggests that polycystins provide a feedback sensor of tissueformation, signaling the completion of morphogenesis and suppressingfurther matrix deposition.

Implications and future directionsThis work points to a broader role for polycystins in tissue formation than waspreviously appreciated, and makes direct links between polycystin signalingand matrix gene expression. There is therapeutic potential to restoringpolycystin-mediating signaling (which is common in healthy organs) indiseased polycystin-deficient cells. This will require greater understanding ofthe mechanisms that polycystins use to regulate ECM production. Somesignaling pathways involving intracellular calcium release andphosphoinositide 3-kinase (PI3K) may be involved since disruption of thesesystems is shown to induce similar overexpression of collagen genes in thezebrafish notochord.

This work also establishes a zebrafish model for human ADPKD. It definesthe molecular end points of ADPKD tissue pathology in zebrafish, andsuggests that it is feasible to perform large-scale screening of drugs and smallmolecules for those that could revert the disease phenotype.

doi:10.1242/dmm.005546

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the adpkd genes pkd1a/b and pkd2 regulate extracellular...

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