Ptpn20 Deletion in H-tx Rats Facilitates Cotransporter-mediated Water Transport in the Choroid Plexus: Evidence of Genetic Risk for Hydrocephalus in an Experimental Study

Hanbing Xu Juntendo University Graduate School of Medicine Masakazu Miyajima (  mmasaka@juntendo.ac.jp ) Juntendo Tokyo Koto Geriatric Medical Center Madoka Nakajima Juntendo University Graduate School of Medicine Ikuko Ogino Juntendo University Graduate School of Medicine Kaito Kawamura Juntendo University Graduate School of Medicine Chihiro Akiba Juntendo Tokyo Koto Geriatric Medical Center Chihiro Kamohara Juntendo University Graduate School of Medicine Koichiro Sakamoto Juntendo University Graduate School of Medicine Kostadin Karagiozov Juntendo University Graduate School of Medicine Eri Nakamura Juntendo University Graduate School of Medicine Nobuhiro Tada Juntendo University Graduate School of Medicine Hajime Arai Juntendo University Graduate School of Medicine Akihide Kondo Juntendo University Graduate School of Medicine


Introduction
Hydrocephalus represents excess accumulation of cerebrospinal uid (CSF) in the cerebral ventricles and subarachnoid space. In humans, congenital hydrocephalus occurs with a high frequency of 0.5-1.5 per 1000 births, representing the most common neurological disorder requiring surgery in children [1][2][3]. Despite the high prevalence of congenital hydrocephalus, knowledge about the pathophysiological mechanisms leading to this disorder remains extremely limited. A large body of evidence suggests that congenital hydrocephalus is an inheritable disorder and characterization at the molecular level should greatly increase the understanding of the disease and lead to new therapies. However, relatively few genes in humans have been linked with hydrocephalus to date, including L1CAM, MPDZ, and CCDC88C [4][5][6]. Less than 5% of mutations in these genes account for primary congenital hydrocephalus cases [7].
As animal models of inherited disease provide opportunities to identify potential genetic causes of disease in humans, a variety of genes causing hydrocephalus in rodents have been identi ed and characterized over the past decades in transgenic animal models, including the Hydrocephalic-Texas (H-Tx) rat, the Wistar-Lewis rats, the hydrocephalus-1 mouse, the hydrocephalus with hop gait mouse, and the TGF-beta1 mouse [8][9][10][11][12][13][14].
Among these, the H-Tx rat strain, rst described in 1981, is widely used as a model of congenital hydrocephalus resulting from a spontaneous mutation to study the pathogenesis of the disease [15]. Hydrocephalus in H-Tx rats develops in early gestation via a complex mode of inheritance and with a prevalence of 30-50% [16]. Ventricular dilatation starts to occur from days 17-18 of gestation and is associated with aqueductal stenosis [17,18]. Pups with severe hydrocephalus are detected at birth by a domed head and die at 4-6 weeks of age. Breeding data from crosses between H-Tx rats and Sprague-Dawley (SD) rats have indicated that hydrocephalus in H-Tx rats is a single autosomal recessive gene disease [19], but a series of studies by Jones et al indicated that the phenotypic expression of congenital hydrocephalus in H-Tx rats is controlled by a combination of genetic and epigenetic factors [16, [20][21][22][23][24].
The genetic abnormality causing congenital hydrocephalus in H-Tx rats remains only partially understood. In this study, we performed copy number analysis to investigate candidate genes potentially responsible for hydrocephalus in H-Tx rats. Under this approach, we identi ed a copy number loss in the Ptpn20 gene in hydrocephalic H-Tx rats and generated Ptpn20-knockout mice, then tested for genotypic and phenotypic differences from wild-type (WT) mice. Based on our ndings, we propose a pathophysiological mechanism to explain the formation of hydrocephalus and a possible therapeutic target for its treatment.

Animals
All experimental animals were group-housed with 2-5 animals per cage and maintained in a temperatureand humidity-controlled facility (23 ± 1°C, 55 ± 5% humidity) on a 12-h light/dark cycle at our animal care facility in the Center for Experimental Medicine of Juntendo University, Japan. All procedures involving animals were approved by the Ethics Review Committee for Animal Experimentation of the Juntendo University School of Medicine (rats: approval no. 288; mice: approval no. 1337) and followed the Principles of Laboratory Animal Care as outlined by the National Institutes of Health.
Animals were decapitated for dissection of the brain after induction of deep anesthesia using intraperitoneal pentobarbital (50 mg/kg body weight). Each brain sample was immediately stored in RNAlater ® solution (AM7021; Thermo Fisher Scienti c, Waltham, MA, USA) for DNA, RNA and protein extractions.

Identi cation of genetic risk in H-Tx rats
Comparative genomic hybridization array DNA was extracted from the brain of H-Tx (+) rats and H-Tx (-) rats at E18, and genome-wide DNA copy number analysis was performed using a SurePrint G3 Rat Comparative genomic hybridization array (CGH) Microarray kit 1x1M SG13464375 (ID-027065; Agilent Technologies, Santa Clara, CA, USA) according to the protocol from the manufacturer. Data were extracted using Feature Extraction version 10.1.1.1 software (Agilent Technologies), and analyses were performed using Agilent Genomic Workbench Standard Edition version 5.0.14 software (Statistical algorithm ADM-2, threshold of 6.0, fuzzy zero correction). Non-redundant copy number abnormalities were called as a minimum of three consecutive probes and log2 ratio > 0.3 for gains and < -0.3 for losses were considered signi cant.

Real-time PCR of copy number
Copy number quanti cation was performed by TaqMan ® quantitative polymerase chain reaction on the brain tissues of E18 H-Tx (+) and H-Tx (-) rats using an ABI7500 real-time PCR system (Applied Biosystems, Thermo Fisher Scienti c). TaqMan Copy Number assays (Ptpn20 exon 6-7, Custom ID: CC70L9K, CCLJ15L) were run simultaneously with TaqMan Copy Number Reference assays (Rnase11 (Rn02349567_s1), Ftmt (Rn01492073_s1) and Hus1 (Rn02115575_s1)) (Applied Biosystems) according to the instructions from the manufacturers. Copy number variations were analyzed using ABI7500 software (Applied Biosystems) and Copy Caller version 2.0 software (Applied Biosystems).
Real-time PCR of Ptpn20 mRNA Total RNA (500 ng) was converted into single-stranded cDNA using SuperScript™ IV VILO™ (SSIV VILO) Master Mix (Invitrogen, Thermo Fisher Scienti c). The ABI 7500 Real-time PCR System (Applied Biosystems) and TaqMan ® Gene Expression Assays (Applied Biosystems) were used according to the instructions from the manufacturer to quantify gene expression. Assay IDs are listed in additional le 5: Table S2.
Expressions of target genes were standardized to the expression of Actb. The presence of a single PCR amplicon was con rmed by melting curve analysis. Expressions of each gene in each sample were analyzed in triplicate.
Immuno uorescence of the CP Animal brains were removed and post xed with 4% paraformaldehyde in 0.01-M phosphate buffer (pH 7.2). Para n-embedded sections (4 µm) and cryosections (30 µm) were blocked with 5× SEA BLOCK TM blocking buffer (37527; Thermo Fisher Scienti c) and 1% donkey serum in Phosphate buffered saline (PBS; AJ9P003; TaKaRa, Shiga, Japan) for 30 min, incubated in primary antibody overnight at 4°C and secondary antibodies for 60 min at room temperature. Vibratome sections (300 µm) were blocked using the same blocking buffer with 0.05% TritonX and 1% donkey serum in PBS for 30 min, then incubated in primary antibody for 2 days at 4°C and secondary antibodies for 2-h at room temperature. Primary and secondary antibodies are listed in additional le 7: Table S4. Nuclei in all sections were counterstained with ProLong Gold and SlowFade Gold Antifade Reagent with DAPI (4′,6-diamidino-2-phenylindole; P36935; Molecular Probes ® , Invitrogen). Images were acquired with a confocal scanning microscope (TCS-SP5; Leica Microsystems, Wetzlar, Germany). Leica Application Suite Advanced Fluorescence Lite (Leica Microsystems) was used for image acquisition and processing.
Expression and role of Ptpn20 in mice Immunohistochemistry Brain and six other tissues (testis, kidney, heart, pancreas, liver and spleen) of 4-week-old C57BL/6J mice were xed in 4% paraformaldehyde xative (33111; Muto Pure Chemicals Co., Tokyo, Japan) for at least 1 week, embedded in para n, and cut into 6-µm sections. Endogenous peroxidase was blocked by incubation of brain sections with 0.3% hydrogen peroxide for 30 min. Sections were blocked with 5× SEA BLOCK Blocking Buffer and 1% donkey serum in PBS at 25°C for 30 min. Sections were incubated with rabbit PTPN20A antibody (CSB-PA199334, 1:50 dilution; CUSABIO, Houston, TX, USA) overnight at 4°C. The following day, sections were incubated with EnVision™ System Labeled Polymer (DAKO, Glostrup, Denmark) as the secondary antibody for 30 min at room temperature. Finally, sections were stained with 3,3'-diaminobenzidine and counterstained with Mayer's hematoxylin, dehydrated, cleared, and mounted.
Both sgRNA and Cas9 protein were injected into the cytoplasm of fertilized one-cell eggs using continuous pneumatic pressure. Eggs were cultured in modi ed Whitten's medium for approximately 24 h and developed to 2-cell stage embryos. Two-cell stage embryos were transferred into each oviduct of pseudopregnant Institute of Cancer Research (ICR) recipient females that had been mated to vasectomized ICR male mice. Embryo transfer to pseudopregnant females was performed on the day the vaginal plug was detected. After birth, genomic DNA was extracted from tail tips of mutant F0 mice and subjected to PCR using primers. The band was evaluated for amounts of mismatch-digestion fragments.
Cycling conditions consisted of an initial 12-min denaturation step at 95°C followed by 35 cycles of 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s, then nal extension at 72°C for 7 min. PCR products were sequenced on an ABI 3500 Genetic Analyzer using the BigDye Terminator Cycles Sequencing Kit v3.1 (Thermo Fisher Scienti c). Samples were analyzed using Seq Scanner version 2 (Thermo Fisher Scienti c) and compared with the public sequence in GenBank (NC_000080.6: Ptpn20).
Transmission electron microscopy of Ptpn20-knockout mice Fresh brains from 8-week-old WT and Ptpn20 -/mice were perfused with 4% paraformaldehyde. Materials were immersed in 2.5% glutaraldehyde solution after being cut into smaller fragments for TEM (1´2 mm 2 ). Samples were washed with PBS, post-xed in 2% osmium tetroxide for 2-h at 4°C and dehydrated in graded concentrations of ethanol. For TEM, samples were placed in resin for 4 days at 60°C. Ultrathin sections were observed under an HT7700 electron microscope (Hitachi High-Technologies, Tokyo, Japan).

Measurement of the lateral ventricle
Fresh brains 8-week-old WT and Ptpn20 -/mice were post xed in 4% paraformaldehyde for 72-h, equilibrated in 30% sucrose solution for 24-h at 4°C, then embedded in embedding matrix and frozen at -80°C for further use. Coronal sections of 30 μm were obtained using a freezing microtome and stained with HE according to standard methods. We viewed and photographed sections using the E800 microscope, and images were captured with an AxioCam 506 color Digital Camera using AxioVison Rel version 4.7.2.0 image-processing software.
Widths of the brain and lateral ventricles were measured on sections at 0.5 cm anterior to the bregma. The ratio of ventricular width to brain width (VBR) was used to determine the degree of ventricular dilatation. [22] Data were calculated as mean ± standard deviation for WT. Differences between WT and Ptpn20 -/mice were evaluated using the Mann-Whitney U test.

Ventricular injection of dye
Eight-week-old WT and Ptpn20 -/mice were deeply anesthetized by pentobarbital sodium (100 mg/kg; Kyoritsu Seiyaku, Tokyo, Japan). The head was xed into a stereotaxic apparatus (Narishige Scienti c Instrument Laboratory, Tokyo, Japan), and a small burr hole was drilled in the cranium on the right, 1 mm lateral to the bregma. The needle of a microsyringe (MS-10; Ito Corporation, Shizuoka, Japan) was slowly inserted into the lateral ventricle 4 mm from the brain surface through the hole and Evans blue dye (6 µl; FUJIFILM, Wako Pure Chemical Corporation, Osaka, Japan) was injected at 2 µl/min. Ten minutes after injection, mouse brains were harvested, xed in 4% paraformaldehyde for 72-h, then cut into 1-mm-thick coronal slices.
Real-time PCR, immunoblot and immuno uorescence These three methods are similar to those used in the rst part, but the results in the second part of the real-time PCR were quanti ed using the 2 -ΔΔCt method. Reagents are listed in additional le 5-7: Tables S2-4.

Statistical analysis
All experimental results are presented as mean ± standard deviation. Real-time PCR was performed as at least ve independent experiments. Signi cant differences among groups were determined using Student's t-test. Statistical differences of the lateral ventricle between WT and Ptpn20 -/mice were assessed by the Mann-Whitney U test. Results were considered statistically signi cant for values of P<0.05.

Identi cation of causative gene for hydrocephalus in H-Tx rats
The SurePrint G3 Rat Genome CGH Microarray kit was used to compare the copy numbers of genes in the brains of non-hydrocephalic H-Tx (H-Tx (-)) rats and hydrocephalic H-Tx (H-Tx (+)) rats. Among 30,584 genes tested, we found 47 genes with copy number variations in the brains. Although we did not detect any copy number variations that were present in all H-Tx (+) rats and not in H-Tx (-) rats among these 47 genes, a 14-kbp loss from chromosome region 16p16 was found in all four H-Tx (+) rats (copy number loss), and six of eight H-Tx (-) rats showed no copy number loss encompassing the Ptpn20 gene (Fig. 1A, Additional le 4: Table S1). We further con rmed and quanti ed the alteration of copy numbers for the Ptpn20 exon in twelve H-Tx (-) rats and six H-Tx (+) rats by real-time polymerase chain reaction (PCR). H-Tx (+) rats exhibited an approximately 2.4-fold decrease in exon 6 (P < 0.01) and 1.6-fold decrease in exon 7 (P < 0.001) in copy numbers of Ptpn20 relative to H-Tx (-) rats (Fig. 1A). After this, we con rmed differential expression of Ptpn20 mRNAin the brains of H-Tx (+) rats, H-Tx (-) rats, SD rats and Wistar rats by real-time PCR. H-Tx (+) rats showed almost half the amount of Ptpn20 mRNA expression compared to other rat lineages and H-Tx (-) rats (Fig. 1B). Subsequently, we detected Ptpn20 proteinexpression in H-Tx (-) and H-Tx (+) rats using immunoblot, which showed immunoreactivity to a 50-kDa Ptpn20 protein to be approximately absent or lower in the choroid plexus (CP) of H-Tx (+) rats (Fig. 1C). Moreover, immuno uorescent staining (IF) also showed Ptpn20 immunoreactivity in CP epithelial cells of H-Tx (-) and SD rats, while immunoreactivity was low in H-Tx (+) rats (Fig. 1D).

Analysis of Ptpn20 expression in mice
To assess Ptpn20 expression and distribution in C57BL/6J mice, we examined the expression of Ptpn20 mRNA in different regions of the brain as well as several tissues (heart, lung, spleen, kidney, liver, and testis) from 4-week-old mice using real-time PCR. Ptpn20 mRNA was expressed mainly in brain and testis. In the brain, Ptpn20 mRNA was expressed in the CP, hippocampus, and cerebral cortex. Ptpn20 mRNA expression levels were 2.73-and 1.92-fold higher in the CP than in the cerebral cortex and hippocampus, respectively. However, Ptpn20 mRNA showed its highest expression in the testis, at 415fold higher than in the CP. In contrast, noexpression was detected in other tissues (Fig. 2A). These results were further validated in immunohistochemical staining of murine heart, lung, spleen, pancreas, liver, testis, and CP using Ptpn20 antibody. The staining showed extremely strong immunoreactivity in the testis and CP epithelial cells, but no change in other tissues (Fig. 2B, Additional le 2: Fig. S2).

Generation of Ptpn20-knockout mice
To investigate whether deletion of Ptpn20 represents a risk factor for hydrocephalus, we generated Ptpn20-knockout (Ptpn20 -/-) mice using CRISPR/Cas9 technology. We targeted and generated a sgRNA against exon 4 of the Ptpn20 mouse gene. DNA sequencing results con rmed the targeted 104-bp deletion mutations in Ptpn20 -/mice (Fig. 3A). For immunoblot analysis, Ptpn20 protein was almost completely absent in the CP of Ptpn20 -/mice (Fig. 3B). We then performed IF using anti-Ptpn20 and anti-F-actin antibodies. Ptpn20 was detected in the cytoplasm of CP epithelial cells in WT mice, but not in ependymal cells, and expression was completely abolished in Ptpn20 -/mice. F-actin staining showed no obvious difference between WT and Ptpn20 -/mice (Fig. 3C). Transmission electron microscopy (TEM) applied to detect substantial structural differences revealed no obvious differences between the CP epithelial cells of WT and Ptpn20 -/mice (Fig. 3D).
Ptpn20 -/mice develop communicating hydrocephalus Ptpn20 -/mice seemed to have normal appearance and lifespan, because almost all lived to over 18 months. To elucidate the morphology of the brain and ventricles of Ptpn20 -/mice, we analyzed brain slices from 8-week-old mice by hematoxylin and eosin (HE)-staining. Mean relative ventricle-to-brain ratio (VBR) was 0.11 ± 0.01 for WT mice and 0.14 for Ptpn20 -/mice (P < 0.01). In Ptpn20 -/mice, VBR exceeding the mean WT value by 2 standard deviations was de ned as indicating hydrocephalus. Of these 29 Ptpn20 -/mice, 16 mice met the criteria for hydrocephalus, and 13 mice did not develop hydrocephalus, while some of the hydrocephalic mice exhibited very mild ventriculomegaly (Fig. 4A). To establish whether an anatomical defect resulted in hydrocephalus, we injected Evans blue in the lateral ventricle of Ptpn20 -/mice. Blue dye appeared in the aqueduct, the fourth ventricle and the atlantooccipital subarachnoid space in all cases, suggesting the entire CSF drainage pathway was patent (Fig. 4B).

Phosphorylation of NKCC1 is maintained in CP epithelial cells of Ptpn20 -/mice
To further explore connections between the pathogenesis of hydrocephalus and CP in Ptpn20 -/mice, we compared aquaporin 1 (AQP1), sodium-potassium adenosine triphosphatase (Na + /K + -ATPase), and Na-K-Cl cotransporter (NKCC1) expressions in the CP, as proteins involved in the secretion or absorption of CSF. No signi cant differences in the mRNA expressions of AQP1, Na + /K + -ATPase, or NKCC1 were detected in the CP by real-time PCR (Fig. 5A). We then used immunoblotting techniques to investigate the expression of phosphorylated NKCC1 (pNKCC1) in the CP, which correlates with the phosphorylation of Thr212/Thr217 in its N-terminal domain. Ptpn20 -/mice showed differential increase in levels of pNKCC1 expression from 8 weeks until 72 weeks compared with WT (Fig. 5B, Additional le 3: Fig. S3A). Furthermore, consistent with our immunoblotting results, IF analysis of CP sections labeled with pNKCC1 antibody showed signi cantly increased expression of pNKCC1 in the apical membrane of CP epithelial cells in Ptpn20 -/mice, while no signi cant differences were seen there for unphosphorylated NKCC1, AQP1 or Na + /K + -ATPase compared with the WT (Fig. 5C).

Discussion
This study identi ed copy number loss of Ptpn20 in the brain of H-Tx (+) rats. Ptpn20 was enriched in the CP of non-hydrocephalic rats, but signi cantly decreased in the CP of H-Tx (+) rats. We then generated Ptpn20-de cient mice to investigate whether the deletion of Ptpn20 is a risk for hydrocephalus. Ptpn20 -/mice do not exhibit the characteristics of congenital hydrocephalus observed in H-Tx rats after birth.
However, further analysis in adult Ptpn20 -/mice revealed ventriculomegaly in more than half of these mice without signi cant blockage of CSF circulatory pathways, thus indicating that Ptpn20 -/mice had developed communicating hydrocephalus. Since hydrocephalus is not associated with stenosis of the cerebral aqueduct, we further investigated the CP, which is mainly affected by Ptpn20 deletion. Previous studies have shown that structural or functional abnormalities of CP may affect CSF production and thus lead to the development of hydrocephalus [25][26][27]. To evaluate possible CP abnormalities, we analyzed the ultrastructure of the CPs of the lateral and third ventricles in Ptpn20 -/mice using TEM. A normal appearance of the CP epithelium and intact junctions between cells suggest that passive diffusion of molecules from the CP vasculature into the CSF is unlikely. However, in analyzing the channels and transporters involved in ion and water transport for CP epithelial cells, we found that expression of pNKCC1 protein was mainly at the apical surface of CP epithelial cells and was signi cantly increased in about half of Ptpn20 -/mice as compared with WT mice. This result may provide useful insights into the pathogenesis of hydrocephalus, and the individual differences in the degree of pNKCC1 expression seem to explain why only half of the knockout mice developed hydrocephalus. NKCC1 is located on the basolateral membrane and is involved in active transportation of Na + , K + , and Cl -, which play important roles in uid absorption and secretion in most epithelial tissues. However, NKCC1 in the brain is highly expressed in the apical membrane of CP epithelial cells [28]. Several studies have shown that inhibition of NKCC1 by bumetanide leads to decreased CSF production in the CP [29][30][31][32]. Ex vivo and in vivo studies have shown that NKCC1 contributes approximately half of the CSF production that is independent of the osmotic gradient [31]. In addition, previous studies have shown that increased NKCC1 activity is associated with direct phosphorylation of NKCC1 [33], suggesting that enhanced or continued NKCC1 phosphorylation may result in greater CSF production by the CP. A previous study showed that in a rat model of posthemorrhagic hydrocephalus, in ammation-induced phosphorylation of NKCC1 caused hypersecretion of CSF [34].
Ptpn20 is a member of the PTPs family, which, together with protein tyrosine kinases, is responsible for regulating the phosphorylation state of intracellular protein [35]. The function of Ptpn20 is not yet completely understood. Limited studies have shown that Ptpn20 is expressed in a variety of cell lines, showing a dynamic subcellular distribution in response to various extracellular stimuli targeting sites of actin polymerization [36]. Previous studies have shown that inhibition of actin polymerization with cytochrome D increases NKCC1 activity in colonic cells [37,38]. However, our results did not show obvious differences in F-actin staining (Fig. 3C), suggesting that Ptpn20 correlates with actin, but has no signi cant effects on actin polymerization or depolymerization.
On the other hand, NKCC1 activity is known to be regulated by phosphorylation of its speci c Serine/Threonine residues [39]. Although tyrosine residues have not been identi ed in NKCC1, NKCC1 can be tyrosine-phosphorylated by tyrosine kinase and inhibition of tyrosine kinase decreased NKCC1 activity by around 50% [40,41]. This nding suggests that NKCC1 phosphorylation is regulated by a pathway that can be activated by tyrosine kinases. This also suggests that NKCC1 might be dephosphorylated by the tyrosine phosphatase encoded by Ptpn20. Deletion of Ptpn20 may thus cause NKCC1 to fail to dephosphorylate and so persistently remain in the phosphorylated state, and our results showing increased expression of pNKCC1 in Ptpn20 -/mice from 8 to 72 weeks support this concept. This change would result in a continuous excess of CSF entering the ventricular system. In humans, CSF hypersecretion secondary to CP hyperplasia or CP papilloma disturbs the homeostasis of CSF production and absorption in the brain, leading to hydrocephalus [42,43], and higher secretion rates correlate with more severe hydrocephalus [44]. We therefore consider that CSF hyperproduction may be a direct cause of hydrocephalus in this study. To further con rm this hypothesis, we also examined pNKCC1 expression in H-Tx rats, and we found that pNKCC1 expression was signi cantly increased in the choroid plexus of H-Tx rats compared to SD rats (Additional le 3: Fig. S3B). This result suggests that increased expression of pNKCC1 due to Ptpn20 de ciency is a potential factor contributing to hydrocephalus in both rats and mice.
However, Ptpn20 -/mice did not exhibit severe hydrocephalus after birth like H-Tx rats, and we hypothesize that this may be because Ptpn20 -/mice did not develop aqueductal stenosis during development or that the overproduction of CSF caused by NKCC1 in the genetic background of C57BL/6J was insu cient to cause severe hydrocephalus. Aqueductal stenosis is associated with abnormal development and dysfunction of the subcommissural organ (SCO) in H-Tx rats. The SCO is a secretory gland of epithelial cells dorsal to the aqueduct of the brain, and in H-Tx rats with congenital hydrocephalus, immunoreactivity of the SCO and glycoproteins of midbrain epithelial cells is decreased starting from embryonic day 16, before ventricular enlargement. Abnormal secretion of glycoprotein is known to interfere with the formation of Reissner's ber, obstructing the cerebral aqueduct and leading to ventricular enlargement on embryonic day 17 [45]. This nding is consistent with other animal models regarding the link between SCO function and hydrocephalus [46]. Although the gene for hydrocephalus in H-Tx rats has not been identi ed, quantitative trait analysis suggests that the loci associated with the hydrocephalus phenotype are present on chromosomes 9, 10, 11, and 17 [24]. In contrast, the Ptpn20 gene, located on chromosome 16, may not be involved in the developmental process of SCO.
Furthermore, in a study of L1-de cient mice, signi cantly enlarged ventricles were observed only in mutant mice backcrossed to the C57BL/6J genetic background, whereas the ventricular system appeared normal when the same mutant mouse strain was backcrossed to the 129 genetic background [47]. In addition, Cai et al. [19] found a lower than expected incidence of hydrocephalus and milder disease than in H-Tx rats when they cross-mated hydrocephalic H-Tx rats, and suggested that this result might be due to the effects of gene modi cations in different genetic backgrounds. Similarly, the role of the Ptpn20 gene may be enhanced in the genetic background of H-Tx rats and diminished in C57BL/6J mice. In addition, another study on L1 mutants showed that ventricular enlargement preceded the onset of aqueductal stenosis, and that massive enlargement of the ventricles caused deformation of the brain, which in turn compressed the aqueduct, leading to severe hydrocephalus [48]. Based on this hypothesis, we consider that although phosphorylation of NKCC1 may produce excess CSF, its production rate might be relatively mild and constant, and so may be insu cient to create an unbalanced pressure differential within the ventricular system that would result in brain deformation. As a result, only moderate hydrocephalus developed.
This study had several limitations. First, since accurately determining whether H-Tx rats at P18 have hydrocephalus based solely on appearance is di cult, hydrocephalic H-Tx rats may have been present among the current non-hydrocephalus samples, masking other genetic risk factors. Future studies will be needed, with larger sample sizes and improved sensitivity of the results, to identify other possible causative genes. Eventually, paired or multiple knockout models can be established to further clarify the molecular mechanisms underlying hydrocephalus. On the other hand, since reliable methods to Page 13/22 accurately measure CSF production remain lacking, it is not possible to test the hypothesis of CSF overproduction in Ptpn20 -/mice. In addition, the lack of imaging tools makes the degree of ventricular dilatation impossible to determine directly from live animals. Isolated or dehydrated brain tissue may result in varying degrees of morphological and structural alteration of the ventricles, which may in term have affected the accuracy of our results.

Conclusion
Our strategy of identifying potential pathogenic genes in hydrocephalic H-Tx rats and elucidating mechanisms in transgenic mouse models revealed the unexpected pathological relevance of hydrocephalus to Ptpn20 de ciency. Ptpn20 makes a substantial contribution to CSF production and provides a novel approach for diagnosing hydrocephalus. Furthermore, inhibition of the phosphorylation pathway of NKCC1 by protein tyrosine phosphatase may represent a therapeutic target for the management of hydrocephalus in the future. EN and NT generated knockout mice; and MM, MN, CA, CK, KS, HA and AK were responsible for critical review.
D. Immunoblot analysis of Ptpn20 protein from the CP of P1 H-Tx (-) and H-Tx (+) rats. The 50-kDa bands correspond to Ptpn20 and nonspeci c (n.s.) bands, respectively. Ptpn20 protein shows low or approximately abolished expression in H-Tx (+) rats. Actin was used as a loading control (n = 5 rats per group).
E. Immuno uorescence images of the CP in P1 SD, H-Tx (-) and H-Tx (+) rats are immunolabeled with anti-Ptpn20 (green) antibody and with DAPI (blue). Ptpn20 appears enriched in the CP epithelium of SD and H-Tx (-) rats, with low expression in H-Tx (+) rats, Scale bar = 20 µm.   Generation of Ptpn20 -/mice.
A. Schematic illustration of Ptpn20 locus. Ptpn20 gene was targeted at exon 4. The sgRNA sequence is underlined in yellow. The protospacer adjacent motif sequence is indicated in the red dotted box.
Electropherograms of the 104-bp deletion mutations in exon 4 are indicated in the black dotted box (sgRNA = single guide RNA).
B. Expression levels of Ptpn20 protein in CP were veri ed by immunoblotting. The 50-kDa bands correspond to Ptpn20 and nonspeci c bands. Ptpn20 protein shows almost completely absence in the CP Figure 5 pNKCC1 expression is increased in the CP of Ptpn20 -/mice.
A. Expressions of Ptpn20, water and ion transporters in the CP of Ptpn20 -/mice as analyzed by real-time PCR. Relative fold change is calculated using the 2 −ΔΔCt method. Data are normalized to β-actin and fold change is expressed relative to the expression level of Ptpn20 in the CP of WT mice set as 1. We did not nd signi cant differences in expressions of NKCC1, AQP1 or Na + /K + -ATPase in CP between 8-week-old WT and Ptpn20 -/mice. Values are as follows: NKCC1 = 1.18; AQP1 = 1.01; ATP1a1 (Na + /K + -ATPase) = 0.83 (n = 5 mice per group).