Developmental Biology 399 (2015) 129–138

Contents lists available at ScienceDirect

Developmental Biology

journal homepage: www.elsevier.com/locate/developmentalbiology

A requirement for Gch1 and in embryonic development

Gillian Douglas a,b,n, Ashley B. Hale a,b, Mark J Crabtree a,b, Brent J. Ryan c, Alex Hansler d, Katrin Watschinger e, Steven S Gross d, Craig A. Lygate a,b, Nicholas J. Alp a,b, Keith M. Channon a,b a BHF Centre of Research Excellence, Division of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK b Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK c Oxford Parkinson's Disease Centre, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK d Department of Pharmacology, Weill Cornell Medical College, NY, USA e Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria article info abstract

Article history: Introduction: GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis Received 28 July 2014 of the enzymatic , tetrahydrobiopterin (BH4). Loss of function in the GCH1 lead to Received in revised form congenital neurological diseases such as DOPA-responsive and . How- 19 December 2014 ever, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular Accepted 20 December 2014 whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH Available online 31 December 2014 deficiency in the developing embryo. Keywords: Methods and results: Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2–3 GCH of the Gch1 gene. Gch1fl/fl mice were bred with Sox2cre mice to generate mice with global Gch1 Development deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and Embryo GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient Heart / fi Tetrahydrobiopterin maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1 embryos were de cient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1/ embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and L-DOPA. Conclusion: These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4. & 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction required for (NO) production, catabolism, synthesis of the , , epi- GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting nephrine and , and for the of ether lipids. reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin Genetic deficiency in GTPCH, due to loss of function mutations in (BH4). BH4 is essential for several with critical physiologic GCH1, cause dopa-responsive and hyperphenylalaninemia, and metabolic functions, including the three nitric oxide synthases resulting from deficiency in BH4. Clinically, GTPCH deficiency is (NOS1–3; EC 1.14.13.39), the aromatic hydroxylases (phe- characterised by low levels of BH4, particularly in the cerebrospinal nylalanine (EC 1.14.16.1), tyrosine (EC 1.14.16.2) and tryptophan (EC fluid (Furukawa et al., 1998), leading to dopamine and serotonin 1.14.16.4) hydroxylases) and alkylglycerol mono-oxygenase (AGMO; depletion in the CNS, with or without hyperphenylalaninemia due to EC 1.14.16.5) (Werner et al., 2011). Through these enzymes, BH4 is insufficiency in hepatic phenylalanine metabolism (Opladen et al., 2012). GTPCH deficiency can have a mild to severe phenotype and is associated with a wide range of symptoms including muscle hypo/ n Corresponding author at: BHF Centre of Research Excellence, Division of hypertonia, dystonia, movement disorders without dystonia, convul- Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford OX3 9DU, UK. Fax: þ44 1865 222077. sions and autonomic symptoms (Opladen et al., 2012). Recommen- E-mail address: [email protected] (G. Douglas). ded treatment strategies for patients with GTPCH deficiency include http://dx.doi.org/10.1016/j.ydbio.2014.12.025 0012-1606/& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 130 G. Douglas et al. / Developmental Biology 399 (2015) 129–138

þ/ / þ / supplementation of neutrotransmitters, with L-DOPA and 5-HT, and Gch1 mice were mated to generate Gch1 , Gch1 and BH4 replacement therapy. In addition to the critical role of BH4 in Gch1þ/ þ littermate embryos. Dams were treated with sepiapterin, these severe genetic diseases, BH4 also plays a role in the pathogenesis (twice daily subcutaneous dosing, 5 mg/kg in PBS) and/or L-DOPA of common, acquired diseases. These include vascular disease states (drinking water, 1 mg/ml with 0.25% ascorbic acid) from E7.5 until such as high blood pressure, atherosclerosis and pulmonary hyperten- either E13.5 or E15.5. Embryos were harvested from sacrificed sion, diabetes, neuropathic pain, depression and sickle cell disease dams at either E13.5 or E15.5. At harvest embryos were examined (Bendall et al., 2013; Costigan et al., 2012; Cunnington and Channon, for the presence of a heart beat and gross abnormalities. 2010; Katusic et al., 2009). Although GTPCH deficiencies are treatable conditions, delayed Embryonic heart rate analysis diagnosis and treatment can result in significant neurocognitive impairment (Opladen et al., 2012), and early treatment does not result At E10.5 and E11.5; dams were anesthetised with isoflurane in normal neurocognitive development in all patients (Ponzone et al., (1–1.5%) in 100% oxygen and placed on a homoeothermic mat, the 1990). A recent study suggested that prenatal treatment with L-DOPA abdomen was shaved and embryonic heart rates were quantified in may improve outcomes (Brüggemann et al., 2012), indicating that utero using a Visualsonics Vevo 2100 with a 22–55 MHz transducer. targeting BH4 deficiency in utero may be beneficial. However, little is Maternal body temperature was maintained between 36 and known about how GTPCH and BH4 affect embryonic development in 37.5 1C and maternal heart rate was maintained between 400 and utero, and in particular whether metabolic replacement or supple- 500 bpm. An initial scan was made and a uterine map drawn of the mentation in pregnancy is sufficient to rescue genetic BH4 deficiency location of each embryo. Embryonic heart rates were measured by in the developing embryo. This is a critical question to devise new counting waveforms in pulse wave Doppler mode over a 30 s treatment strategies. period. Embryos were harvested from the dam under general Previous studies of the importance of BH4 have anaesthetic and each embryo was assigned an identification num- used systemic pharmacologic inhibitors of GTPCH (Cotton, 1986; ber corresponding to their location on the scan to allow for genetic Mitchell et al., 2003), or the hph-1 hyperphenylalaninemic mouse, identification of the scanned embryos. generated by ENU mutagenesis, that has moderate systemic BH4 deficiency due to reduced from the GCH1 Histology and immunohistochemistry (Cosentino et al., 2001; Khoo et al., 2004). However, in the hph-1 mouse, variable BH4 deficiency in different cell and tissue types, Embryo morphology was assessed in paraffin-embedded embryo and the moderate reduction in GTPCH activity, results in no gross sections stained with haematoxylin and eosin (Merck, Germany). abnormality in foetal development or survival, and limit the Whole embryos were sectioned, stained and all section assessed for interpretation of the role in GTPCH in development (McDonald abnormalities. Whole mount PECAM staining was performed on and Bode, 1988). Indeed, no genetic mouse model has tested the embryosasdescribedbefore(Smart et al., 2010), using rat anti- requirement for GTPCH and de novo BH4 biosynthesis in vivo.To mouse PECAM. Embryos were then subjected to DAB substrate (Vector test this hypothesis we generated a novel mouse model with Labs) and cleared (50% glycerol). Histological quantification was genetic deletion of Gch1, and assessed embryonic development carried out on digitised microscopic images using Image-Pro Plus. and the effectiveness of metabolic supplementation during preg- nancy to treat embryonic BH4 deficiency. RNA

Total RNA was obtained from homogenised embryos or adult Materials and methods tissue by Trizol extraction. Reverse was carried out using the QuantiTect reverse transcription kit (Qiagen, UK) on 1 μg fl/fl – Generation of the GCH1 mouse and GCH1 global knock out mouse total RNA. Quantitative real-time RT PCR was performed with an iCycler IQ real-time detection system (BioRad Laboratories, USA) A plasmid containing a FRT-PGK-promoter and a neomycin-FRT- using primers and probes from the TaqMan Gene Expression Assay LoxP positive selection cassette was inserted in the intron 3 sequence system (Applied Biosystems, UK). of Gch1. An additional LoxP site was inserted into intron 1 to flox exon 2–3. The Gch1 floxed mouse was generated using homologous re- LC-MS-based Metabolomic screening combination in ES cells. Two chimeric male offspring with 90–100% chimerism were selected for breeding, with one chimera generating a E11.5 embryos were exposed to three subsequent rounds of founder with germline transmission. Flp-mediated excision of the homogenisation in cold (80 1C) methanol (80%) to rapidly quench neomycin cassette was carried out by breeding the founder male with metabolism and extract small molecule metabolites. The supernatant a Flp deleter female. These pups were further bred with C57BL6/J mice from each round was pooled and dried in a speed-vac. assays to backcross 7, to generate pure lines of GCH1fl/fl mice. were carried out on the tissue pellet to allow for normalisation. In order to generate mice globally deficient in BH4, we crossed Metabolite extracts were resuspended in 70% acetonitrileþ0.2% GCH1fl/fl mice with Sox2Cre mice. The progeny of this cross were bred ammonium hydroxide to their appropriate protein concentrations. to generate mice with heterozygous deletion of Gch1 (Gch1 þ /), Resuspended metabolite samples were spun to remove residual withouteitheraCreoraGch1 floxed allele. debris, transferred to 250 mL conical polypropylene vials, capped, and loaded into the LC autosampler for subsequent LC-MS analysis. 3 mL of each sample was injected into the LC-MS platform. Further Timed matings and dosing studies details regarding the LC-MS platform, data analysis and the identi- fication of sample metabolites are described in detail within the þ/ Timed matings were carried out using Gch1 male and female Supplementary Material. mice. Identification of a mucosal plug was considered day 0.5 gesta- tion, embryos were harvested and either snap frozen and stored at BH4, Phenylalanine and catecholamine analysis 80 1C until analysis or processed for histological staining. Embryo- nic yolk sacs were divided into two and the genotype of the embryos BH4 levels in embryos and adult tissue was determined by HPLC determined by standard PCR techniques. followed by electrochemical and fluorescent detection, as previously G. Douglas et al. / Developmental Biology 399 (2015) 129–138 131 described (Crabtree et al., 2009). Briefly, embryos were homogenised test the differences between categorical variables. A value of Po0.05 in phosphate-buffered saline (50 mM), pH 7.4, containing dithioery- was considered statistically significant. Statistical analysis was carried thritol (1 mM) and EDTA (100 μM), centrifuged (15 min at 13,000 rpm out using GraphPad Prism version 6 (GraphPad Software, USA). and 4 1C), and the supernatant removed and the protein precipitated withicecoldphosphoricacid(1M),TCA(2M),anddithioerythritol (1 mM) and centrifuged. Samples were injected onto an isocratic HPLC Results system and quantified using sequential electrochemical (Dionex Coulochem III; Thermoscientific, Buckinghamshire, UK) and fluores- Generation of a Gch1 knock out mouse cence (Jasco, Essex, UK) detection. HPLC separation was performed using a 250-mm, ACE C-18 column (Hichrom, Berkshire, UK) and a For conditional deletion of the Gch1 gene, exons 2–3wereflanked mobile phase comprising of sodium acetate (50 mM), citric acid with loxP sites, as these exons encode amino acids involve in the (5 mM), EDTA (48 μM), and dithioerythritol (160 μM)(pH5.2)ata GTPCHactivesite(Fig. 1 AandB).Wefirst confirmed that insertion of flow rate of 1.3 ml/min. Background currents of þ500 and 50 μA loxP sites in the Gch1 gene at these positions did not result in a were used for the detection of BH4 on electrochemical cells E1 and E2, hypomorphic allele. There was no difference in BH4 levels between þ /þ fl/þ fl/fl respectively. Quantification of BH4 was done by comparison with Gch1 , Gch1 and Gch1 mice in heart, liver or lung tissues, authentic external standards, processed identically. indicating that the floxed Gch1 allele leads to normal levels of BH4 For L-DOPA analysis, embryos were homogenised in PBS containing synthesis in gene-targeted mice (Supplementary Fig. 1). 0.1 M perchloric acid solution (PCA), homogenised and centrifuged In order to generate mice with global deletion of Gch1,wefirst fl/fl þ/ þ /þ (13,000 g for 15 min at 4 1C). L-DOPA levels were quantified as crossed Gch1 mice with Sox2Cre mice. Gch1 mice from Gch1 þ / previously described (Ryan et al., 2014). Briefly, samples were injected x Gch1 matingswerebornintheexpectedratiowithnoobvious þ / onto an isocratic HPLC system and quantified using a Decade SDC defects. Adult Gch1 mice had a significant reduction in Gch1 mRNA þ /þ detector and a working electrode held at þ0.7 V vs a Ag/AgCl expression in the heart compared to Gch1 littermates (Fig. 1C). As reference electrode (Antec). HPLC separation was performed using a expected, due to the decrease in mRNA expression there was a 250-mm Microsorb C18 reverse-phase column (Agilent) and a mobile concomitant reduction in GTPCH activity and BH4 levels in the hearts þ/ þ /þ phase comprising methanol (13% v/v), NaH2PO4 (120 mM), EDTA of adult Gch1 mice compared with Gch1 littermates (Fig. 1D (0.8 mM), and sodium octane sulphonate (3.2 mM), pH 3.27, and the and E). A significant reduction in BH4 levels was also observed in the þ / þ/þ flow rate was fixed at 1 ml/min. L-DOPA content was determined lung and spleen of adult Gch1 mice compared with Gch1 relative to authentic, freshly prepared standards and normalised to littermate controls. However, no difference was observed in liver or sample protein content. plasma BH4 levels in these mice (Supplementary Fig. 2). / For phenylalanine and tyrosine analysis embryos were homoge- In order to generate homozygous Gch1 knockout mice (Gch1 ) þ / nised in PBS, deproteinated by the addition of PCA (0.6 mol/L) in a we mated Gch1 mice. However, this mating did not yield any / ratio of 1:1 and centrifuged (13,000 rpm for 15 min at 4 1C). The Gch1 pups from more than 20 litters. To determine the onset of þ / þ / supernatant was injected onto a Jasco Plus HPLC system and quanti- embryonic lethality, embryos from Gch1 Gch1 matings were fied using UV spectrophotometric detection (wavelength 210 nm). harvested after timed matings, for genotyping and analysis. Up to / HPLC separation was performed using a Aeris PEPTIDE C18 column E10.5, Gch1 embryos were present at the expected Mendelian / (3.6 mm, 150 4.6 mm, Phenomenex) and a mobile phase comprising ratio, however, from E11.5 onwards alive Gch1 embryos were acetonitrile (2.5%), octylamine (10 uL/L) and perchloric acid (0.8 mL/L), significantly underrepresented. At E11.5 and E12.5 only 55% and 39% of / / pH 2, at a flow rate of 0.75 ml/min. Quantification of phenylalanine Gch1 embryos were alive. By E13.5, all Gch1 embryos were / and tyrosine was done by comparison with external standards and either absent or dead and by E15.5 dead Gch1 embryos were / normalised to sample protein content. under represented in the litter with only 4 Gch1 embryos present compared with 18 GCHþ /þ and 23 Gch1þ / embryos indicating Measurement of GTPCH activity significant reabsorption (Fig. 1F). At E11.5, surviving Gch1/ embryos appeared normal with no GTPCH activity was assessed by the quantification of the conver- obvious oedema or blood spots present (Fig. 3A). There were no sion of GTP to 7,8-dihydroneopterin. Embryos and adult tissue was differences in either crown to rump length or placenta weights of / þ / þ /þ homogenised in 300 mlTris–HCl (100 mM), KCl (300 mM), EDTA surviving Gch1 embryos compared with Gch1 or Gch1 (2.5 mM), phenylmethanesulfonyl fluoride (100 mM), and glycerol littermates (Fig. 3BandC). (10% v/v), pH 7.8. After centrifugation (5 min at 13,000 rpm and 4 1C) the supernatant was incubated with 10 mM GTP (final volume Embryonic Gch1 deletion abolishes GTPCH activity and reduces both 300 ml) for 1 h at 37 1C. Samples, or neopterin standards, were BH4 and L-DOPA, but without global metabolomic derangement subsequently incubated with 10 mlHCl(1M)and10mlI2/KI solution (1% w/v I in 2% KI) and incubated for 1 h. Samples were centrifuged Gch1 mRNA expression increased significantly with the age of (2 min at 13,000 rpm) and 10 ml ascorbate (100 mM), 10 mlNaOH the embryos from E9.5 to E13.5 (Fig. 2A). Gch1/ embryos had no (1 M), and calf intestinal alkaline phosphatase (0.32U) were added to detectable Gch1 mRNA expression, whereas Gch1 mRNA was the supernatant (final volume 340 ml). Samples were centrifuged and maintained in Gch1þ / embryos. A significant reduction in GTPCH then assessed using the electrochemical/fluorescence HPLC activity was observed in Gch1þ / compared to Gch1þ / þ embryos system detailed above. and this was further reduced in Gch1-/- embryos to almost non- detectable levels (Fig. 2B). Surprisingly, given the lack of GTPCH Statistical Analysis activity in Gch1/ embryos a significant reduction in BH4 levels was not detected until E11.5 (Fig. 2C-E). The maintenance of Data are presented as mean7SEM. Groups were compared using normal BH4 levels in Gch1/ embryos at E9.5–10.5 might be the Mann-Whitney U test for non-parametric data or the Student's indicative of maternal transfer of BH4. However, at E11.5 where two-sided t-test for parametric data. When comparing multiple the embryo is larger, maternal transfer appears insufficient to groups data were analysed by analysis of variance (ANOVA) with maintain Gch1þ / þ levels of BH4 in the Gch1/ embryos. Newman–Keuls post-test for parametric data or Kruskal–Wallis test The reduction in BH4 levels at E11.5 resulted in a concurrent / with Dunns post-test for non-parametric data. Chi Square was used to decrease in L-DOPA levels in Gch1 embryos (Fig. 2F). There was no 132 G. Douglas et al. / Developmental Biology 399 (2015) 129–138

0.0014 0.012 10 )

* 0.0007 * 0.006 5

(delta Ct) (delta * GTPCH activity Gch1 G 4 (pmol/mg protein mol/mg protein/min) mol/mg m (f BH4 0.0000 0.000 0 GCH+/+ GCH+/- GCH+/+ GCH+/- GCH+/+ GCH+/-

120

80

40 % Survival

0 29 48 28 43 80 40 50 88 27 26 52 11 29 64 0 18 23 0 44 73 0 E9.5 E10.5 E11.5 E12.5 E13.5 E15.5 Term +/+ +/- -/- +/+ +/- -/- +/++/- -/- +/++/- -/- +/++/- -/- +/++/- -/- +/++/- -/-

Fig. 1. A, Schematic showing the targeting of the murine Gch1 locus with loxP sites flanking the exons encoding the of the GTPCH protein. B, Genomic DNA was probed for the presence of loxP sites, a predicted 1056 bp band was observed in GCHfl/fl mice. Sox2 Cre mediated excision results in the presence of a knockout allele, a predicted 1332 bp product was observed in Gch1/ mice, Gch1þ / mice had both a knockout and wild type band. C, Gch1 mRNA levels were significantly reduced (*¼Po0.05, n¼6 per group) in heart tissue from adult Gch1þ / mice compared with Gch1þ /þ littermates. D, GTPCH activity was significantly reduced (*¼Po0.05, n¼6 per group) in heart tissue from adult Gch1þ / mice compared with Gch1þ / þ littermates. E, BH4 levels were significantly reduced (*¼Po0.05, n¼6 per group) in heart tissue from adult Gch1þ/ mice compared with Gch1þ /þ littermates. F, Percentage embryo survival of each genotype from E9.5 to birth, at E11.5 and 12.5 live Gch1/ are significantly under-represented and completely absent after E12.5 (Po0.05, n¼35–10 litters per group; number of alive embryos for each group is written in the bar). difference in phenylalanine levels between Gch1 þ/þ and Gch1/ 150 compounds, generated with pure chemical standards, known to embryos (Fig. 2G), which could be due to the low expression levels of be implicated in BH4 biology and related metabolic processes. Thirty PAH at this time point (Supplementary Fig. 2). No difference was eight of these compounds (listed in supplementary Table 1)were observed in tyrosine levels between Gch1þ /þ and Gch1/ embryos observed in at least 80% of samples from at least one experimental (Fig. 2H).TherewasalsonocompensatoryupregulationofmRNAof group. This targeted approach yielded similar results to the untargeted other BH4 dependent enzymes, or critical enzymes in BH4 biosynth- metabolomics screen, with no significant difference in any of the esis downstream of GTPCH (Supplementary Fig. 3). observed metabolites between the two genotypes (P40.05) and no Since BH4 is a critical co-factor for a number of important distinct separation by genotype with unsupervised hierarchical analy- metabolic enzymes, we next sought to determine whether global sis (Fig. 2J). These experiments indicate that Gch1/ embryo lethality BH4 deficiency altered the metabolic profile of the embryos. Untar- observed at E11.5 is not due to gross disruption in metabolic pathways. geted metabolomics analysis was used to compare metabolic profiles between surviving E11.5 Gch1 þ /þ and Gch1/ embryos. We quanti- Embryonic Gch1 deletion does not cause structural developmental fied the relative abundance of 590 features (metabolites) that were defects, but results in foetal bradycardia detected in at least 80% of samples from either Gch1þ/þ and Gch1/ embryos. However, no significant differences were observed in Since embryonic deficiency in Gch1 did not lead to any difference metabolite levels between the two groups (P40.05). Furthermore, in metabolic profile at the whole embryo level, we reasoned that Gch1 unsupervised principal component analysis (data not shown) and deficiency could result in cell specific defects in tissues that require de hierarchical clustering, based on these 590 compounds, did not result novo BH4 synthesis and may not be capable of BH4 uptake. As the in separation or clustering of the embryos by genotype (Fig. 2I). embryonic cardiovascular system is critical for foetal survival beyond We further analysed potential metabolomic differences between E9.5, we determined whether Gch1 deficiency leads to defects in the Gch1 þ /þ and Gch1/ embryos, using a curated metabolite library of cardiovascular system. At E11.5 no excessive pericardial fluid was G. Douglas et al. / Developmental Biology 399 (2015) 129–138 133

0.0010 1.0 +/+ +/- -/- ivity t 0.0005 0.5 GCH (delta Ct)

GCH ac * (pmol/embryo/min) 0.0000 * 0.0 910111213 +/+ +/- -/- Days

100 E9.5100 E10.5 100 E11.5 ) yo) mbr e 50 50 50

mol/ * (p 4 H B BH4 (pmol/embryo BH4 (pmol/embryo) 0 0 0 +/+ +/- -/- +/+ +/- -/- +/+ +/- -/-

30 10 8 n) i e e tein) n rot rotein) p alani 15 5 gp 4 l yrosine /m l L-DOPA T eny o m Ph µ µ (pmol/mg pro ( * (mol/mg 0 0 0 +/+ +/- -/- +/+ +/- -/- +/+ +/- -/-

Fig. 2. A, Whole embryo mRNA expression of Gch1 in Gch1 þ/þ , Gch1þ / and Gch1/ embryos Gch1 expression increased with development (*¼Po0.05 of 4–6embryos)inGch1þ /þ and Gch1þ / embryos whereas in Gch1/ embryos Gch1 was not detected at any time point. B, Whole embryo Gch1 activity in E11.5 embryos, activity was significantly reduced in Gch1þ / and further reduced in Gch1-/- embryos (*¼Po0.05, n¼8–6 per group). C, BH4 levels in E9.5 embryos, D, E10.5 embryos and E, E11.5 embryos, no difference in embryonic BH4 / levels between genotypes at either E9.5 or E10.5. At E11.5 there was a significant reduction in BH4 levels in Gch1 embryos (Po0.05, n¼7–8pergroup).F,L-DOPA levels in E11.5 embryos were significantly reduced in Gch1/ embryos compared with Gch1þ /þ and Gch1þ/ embryos (*¼Po0.05, n¼8–6 per group). G, Phenylalanine levels were not different between any groups at E11.5. H, Whole embryos tyrosine levels were not different between Gch1þ /þ, Gch1þ / and Gch1/ embryosatE11.5.I,Hierarchicalclusteringofanunbiased metabolomics screen of 590 compounds and J, 38 detected compounds from a targeted screen of 150 common metabolites in E11.5 Gch1 þ/þ (blue) and Gch1/ (red) embryos. No significant difference were observed between groups and unsupervised clustering did not result in separation of embryos by genotype (P40.05, n¼8–9 per group). 134 G. Douglas et al. / Developmental Biology 399 (2015) 129–138

+/+ +/

10 50 mm)

5 25 Weight (mg) Weight Embryo length (m lengthEmbryo 0 0 +/++/--/- +/++/--/-+/++/--/- E10.5 E11.5

+/+

-/-

+/+ -/-

E9.5

E10.5

Fig. 3. A, Representative photographs of E11.5 Gch1þ /þ , Gch1þ / and Gch1/ embryos. B, Embryos crow to rump length at E11.5, no differences between groups was observed. C, Placenta weights of Gch1þ / þ, Gch1þ / and Gch1/ embryos at E10.5 and E11.5, no significant difference was observed between groups. D, Representative images of the heart, outflow tracks and dorsal aorta from E11.5 Gch1þ / þ and Gch1/ embryos. E, Representative images of whole mount PECAM stained E9.5 and E10.5 Gch1þ / þ and Gch1/ embryos. G. Douglas et al. / Developmental Biology 399 (2015) 129–138 135

/ observed in surviving Gch1 embryos. Histological analysis con- rescued by maternal treatment with BH4 and catecholamine. L-DOPA firmed no cardiovascular development abnormalities in the heart or (10 mg/kg), administered to pregnant dams in the drinking water, / outflow tract and well organised trabeculas in the hearts of Gch1 resulted in a significant increase in embryonic L-DOPA levels in E11.5 embryos, with no difference observed in trabeculated area between Gch1 þ /þ embryos (Fig. 5A). Maternal treatment with a BH4 precursor the two groups (0.05670.005 vs. 0.06470.007 Gch1þ/þ vs. Gch1/, sepiapterin (5 mg/kg twice daily by subcutaneous injection) signifi- po0.05; n¼5 per group). No blood pooling was observed in any cantly increased embryo BH4 levels, which peaked at 6 h and declined tissues, the dorsal aortas appeared normal and blood was contained back to baseline 14 h after administration (Fig. 5B). Based on these within the artery (Fig. 3A and D). Whole mount PECAM staining at observations, and to ensure continuous embryonic supplementation E9.5 and E10.5 did not reveal any abnormality in the embryonic with BH4, dams were dosed twice daily with sepiapterin until E11.5. vasculature. Blood vessels of the intersomitic regions were normal and Quantification of BH4 in embryos, harvested 14 h hours after maternal an intact vascular network was observed in the head region of both BH4 injection (i.e. at trough level) confirmed that BH4 levels in Gch1-/- Gch1þ /þ and Gch1/ embryos suggesting that lack of embryonic embryos were significantly elevated compared with embryos from Gch1 did not affect vasculogenesis (Fig. 3E). untreated dams, and achieved levels of embryonic BH4 similar to We next examined if the embryo lethality could in part be due to Gch1 þ /þ embryos. Indeed, in embryos from dams treated with an altered heart rate, as deletion of tyrosine hydroxylase which sepiapterin there was no difference in whole embryo BH4 between reduces catecholamine synthesis results in embryonic bradycardia. Gch1 þ /þ , Gch1 þ / or Gch1/ embryos (Fig. 5C), indicating effective Conversely a deficiency in Gch1 may lead to an indirect failing of the supplementation of maternal BH4 that is able to restore the biochem- heart. We assessed heart rate in utero using ultrasound imaging and ical deficiency in BH4 in Gch1/ embryos. demonstrated no difference in embryonic heart rate between Gch1 þ/þ To determine whether biochemical rescue of embryonic BH4 and Gch1/ embryos at E10.5 (Fig. 4A).AtE11.5,7outof12Gch1/ deficiency would prevent embryonic lethality due to Gch1 deletion, embryos were already dead and the remaining alive Gch1-/- had a we treated Gch1 þ/ dams, mated with Gch1 þ / males, with either þ/þ significantly decreased in heart rate compared to their Gch1 and L-DOPAþsepiapterin, or sepiapterin alone. Treatment with sepiapterin Gch1þ / littermates (8374vs.7074inGch1 þ /þ vs. Gch1/; alonedidnotresultinasignificant increase in Gch1/ embryo Fig. 4B). survival at E13.5, with only one Gch1/ embryo alive compared with 13 dead. However, supplementation with both L-DOPA and sepiapterin Embryonic Gch1 deficiency is not rescued by maternal resulted in a significant increase in embryonic survival, with 8 out of / supplementation with BH4 18 Gch1 embryos alive at E13.5, compared with no surviving Gch1/ embryosoutof16incontroldamstreatedwithsaline Post-natal lethality observed in other genetic models of BH4 (Fig. 5D). We next tested whether the partial rescue in embryo survival deficiencies can be rescued by post-partum supplementation with at E13.5 with sepiapterin and L-DOPA treatment would be sustained. / neurotransmitters and/or BH4. Accordingly, we next sought to estab- At E15.5 we observed some surviving Gch1 embryos (4 alive out of / lish whether embryonic lethality due to Gch1 deletion could be a total of 9), but in both the control and treated group Gch1 embryos were significantly under represented compared with the expected Mendelian ratio, with only 4 embryos alive representing only 100 o fi / E10.5 8% of the litter (P 0.02), indicating a signi cant loss of Gch1 embryos prior to E15.5, despite continued maternal supplementation with sepiapterin and L-DOPA (Fig. 5E). min)

50 Discussion

In this study we report the first gene targeted mouse model of Gch1 deficiency. We show that deletion of Gch1 causes loss of e eart rate (beats/m rate eart embryonic BH4 synthesis and embryonic lethality by E13.5. Further- H more, lethality cannot be rescued with BH4 supplementation, indicat- 0 ing a requirement for Gch1 in embryonic development. Embryonic +/+ +/- -/- lethality in Gch1 deletion is not associated with any gross perturbation in the metabolic pathways or with any gross organogenesis defects, 100 E11.5 but causes embryonic bradycardia. Our study provides novel insights into the role of Gch1 in develop- ment. Clinically, loss of function mutations in the Gch1 gene lead to rare * congenital neurological diseases such as dystonia, DOPA-responsive

ats/min) dystonia and hyperphenylalaninemia. The majority of mutations are 50 heterozygous autosomal dominant with a smaller number of auto- somal recessive mutations (Thöny and Blau, 2006). These disorders are characterised by low levels of BH4 and decreased GTPCH activity (Furukawa et al., 1998)(Opladen et al., 2012, 2011). Infants with GTPCH fi

Heart rate (bea rate Heart de ciency are born normally but develop muscle hypotonia and movement disorders usually within the first year of life, with more 0 fi +/+/ +/- / -/- / severe BH4 de ciency leading to symptoms at an earlier age (Opladen et al., 2012). In our study global deficiency of Gch1 resulted in embryo Fig. 4. Gch1-/- embryos were significantly bradycardic at E11.5 A, In utero heart rate lethality at mid-gestation. Clinically, non-functional mutations resulting þ / þ þ / / at E10.5 in Gch1 , Gch1 and Gch1 , embryos no significant differences in complete loss of BH4 synthesis have not been described, suggesting were observed between groups (n¼6–9 per group from 6 independent litters). B, In that complete loss of Gch1 in humans is also likely to be embryonically utero heart rates at E11.5, Gch1 / embryos had a significant reduction in heart rate þ /þ þ / fi compared with Gch1 and Gch1 littermates (*¼Po0.05, n¼5–9 per group of lethal. Unlike the eNOS KO mouse, which shows signi cant growth 10 independent litters). restriction at E17.5 (Hefler et al., 2001; van der Heijden et al., 2005), 136 G. Douglas et al. / Developmental Biology 399 (2015) 129–138

60 150 60 +/+ ) ) ) o +/-+/ -/- * 30 75 30 L-DOPA (pmol/mg protein) BH4 (pmol/embryo

BH4 (pmol/embryo * 0 0 0 L-DOPA Saline 0h 1h 6h 14h saline 14h Sep

Alive E13.5 Alive E15.5 Dead E13.5 Dead E15.5

100 * 100 * s os o

50 50 vs. dead embry vs. deadvs. embryos 18 39 16 17 33 18 10 22 14 18 23 4 15 24 9 % alive % alive % alive 0 0 +/+ +/- -/- +/+ +/- -/- +/+ +/- -/- +/+ +/- -/- +/+ +/- -/- saline L-DOPA + sepiapterin saline L-DOPA + sepiapterin sepiapterin

þ /þ Fig. 5. A, L-DOPA levels in Gch1 E11.5 embryos from L-DOPA treated dams (drinking water 1 mg/ml in 0.25% ascorbic acid, E7.5-E11.5) a significant increase in embryonic þ /þ L-DOPA levels was observed in treated dams (*¼Po0.05). B, BH4 levels in Gch1 E11.5 after sepiapterin treatment (2 5 mg/kg, SC, E7.5-E11.5) a significant increase in BH4 levels was observed 1 and 6 h after treatment (*¼Po0.05, n¼3–4 per group of 3 independent litters). C, BH4 levels in Gch1þ /þ E11.5 embryos 14 h after SC injection with either sepiapterin (2 5 mg/Kg SC, E7.5-E11) or saline, the significant decrease in BH4 levels observed in Gch1/ embryos was abolished 14 h after maternal sepiapterin treatment (*¼Po 0.05, n¼4–9 per group of 4 independent litters). D, Embryonic survival after maternal supplementation with either sepiapterin (2 5 mg/kg/ day) or sepiapterin with L-DOPA (drinking water, 1 mg/ml in 0.25% ascorbic acid) from E7.5-E13.5, maternal treatment with sepiapterin and L-DOPA but not saline or sepiapterin alone resulted in a significant increase in alive Gch1/ embryos at E13.5 (*¼Po0.05, from 10–16 independent litters; total number of embryos aliveþdead for each group written in the bar). E, Embryonic survival after maternal supplementation with sepiapterin (2 5 mg/kg/day) and L-DOPA (drinking water 1 mg/ml in 0.25% ascorbic acid) or saline from E7.5-E15.5, in both groups Gch1/ were significantly under represented (*¼Po0.05 from 8–10 independent litters; total number of embryos aliveþdead for each group written in the bar). there was no difference in embryonic size or placenta weights between metabolic analysis carried out at the whole embryo level does not Gch1/ and Gch1 þ/þ littermates at E11.5, indicating that up to this exclude the possibility that cell or tissue specificdifferencesinthe time point embryo growth was not retarded. As there were no metabolic profile may in part account for embryonic lethality. apparent histological differences in heart or vascular structure between Other mouse models with genetic deletion of the downstream the Gch1/ and Gch1 þ /þ littermates, it is unlikely the lethality was enzymes involved in the synthesis of BH4 have been previously due to structural abnormalities. Both catecholamine-deficient embryos investigated. PTPS knockout mice are born alive with the expected and eNOS/ embryos show signs of an altered development of the Mendelian ratio but die within 48 h of birth (Elzaouk et al., 2003; vasculature and the myocardium, with blood congestion and thinning Sumi-Ichinose et al., 2001). It had previously been thought that of the myocardium (Thomas et al., 1995; Zhou et al., 1995)(Liu et al., there was no alternative pathway for the synthesis of BH4 in the 2012). However, these defects predominantly present at E12.5, by absence of PTPS. However, if this were true then it would be which time significantly lethality in Gch1/ has already occurred. expected that PTPS knock out mice would have the same pheno- GTPCH catalyses the first and rate limiting step in the synthesis of type as Gch1-/- mice. The finding of a more severe phenotype in BH4, a critical co-factor for a number of important metabolic enzymes. Gch1-/- mice suggests that either an alternative pathway for the At E11.5 Gch1/ embryos were significantly deficient in both BH4 synthesis of BH4 exists, or that GTPCH has a role in embryonic and L-DOPA, we hypothesise that as the embryos appeared structurally development that is independent of BH4 biosynthesis. Studies in normal that the lethality was in part due to a gross perturbation of rats have indicated that GTPCH could act as a chaperone, and/or metabolicpathways.Weusedatargetedanduntargetedmetabolo- interact with other (Du et al., 2012), such that loss of mics screen approach to look for candidate pathways which may have these functions could be a contributing mechanism to the embryo- been altered due to Gch1 deficiency. Surprisingly, given the role of BH4 nic lethality of Gch1 deletion. in multiple pathways there was no difference in the metabolic profile In Gch1 þ /þ embryos Gch1 mRNA and BH4 levels were detectable between Gch1/ and Gch1 þ/þ littermates, suggesting that any meta- from E9.5 and increased markedly at E11.5 onwards, indicating a bolic disturbance can be effectively buffered by maternal metabolism, growing role for BH4 as the embryo develops beyond this gestational and indicating that the embryonic lethality in Gch1 deficiency is not stage. Despite having no detectable Gch1 mRNA and minimal GTPCH due to gross alteration in metabolic pathways. Nevertheless, the enzymatic activity, Gch1-/- mice did not become deficient in BH4 until G. Douglas et al. / Developmental Biology 399 (2015) 129–138 137

E11.5, and even by E11.5 the embryonic deficiency in BH4 was which were rescued to term with catecholamine supplementation, proportionately much less than the observed loss of both Gch1 Gch1/ embryos were significantly underrepresented in the litter expression and GTPCH enzymatic activity. These findings support (8% of the litter at E15.5) indicating that an additional factor other previous indications that BH4 can cross the placenta (Thomas et al., than catecholamine deficiency was responsible for the lethality. 1995; Vásquez-Vivar et al., 2009), and indicate that up to E10.5 Indeed, the bradycardia observed in untreated E11.5 Gch1þ / maternal transfer of BH4 across the placenta is sufficient to maintain embryos could also be an indirect consequence of Gch1deficiency normal BH4 levels in the embryo, and beyond E10.5 remains sufficient resulting from a failing heart due to an alternative consequence of to substantially mitigate the effects of Gch1 deletion on BH4 levels. It is Gch1 deficiency. possible that BH4 has a physiological role in embryo development Recently, AGMO has been identified as a BH4 dependent before E10.5 which is masked in Gch1/ embryos by maternal . AGMO is the only enzyme known to cleave the ether supplementation of BH4. Interestingly, the finding that sepiapterin bond of alkylgycerols and lyso-alkylglycerol phospholidids, includ- supplement Gch1/ embryos had whole embryo BH4 levels compar- ing lyso-platelet activation factor (Watschinger et al., 2010; able to WT littermates at trough when BH4 levels in supplemented Watschinger and Werner, 2013). In our study only minimal AGMO Gch1þ /þ embryos were no different from control Gchþ /þ embryos expression was observed in Gch1þ / þ embryos up to E11.5 with a raises the possibility that BH4 in the Gch-/- embryosisbeingactively marked increase in expression observed from E12.5, hence it is maintained. unlikely that loss of AGMO function due to BH4 deficiency is the Since some BH4 can cross the placenta, we hypothesised that cause of embryo lethality observed between E10.5 to E12.5 in this Gch1-/- embryos could be rescued by maternal supplementation with study. However, further research into potential roles for AGMO in BH4. Indeed, administration of sepiapterin significantly increased embryo development will help to answer this question. embryonic BH4 such that, even at trough levels, Gch1-/- embryos In conclusion, we have demonstrated a requirement for Gch1 in had comparable BH4 levels to thoseobservedinWTlittermates. embryonic development. lethality in Gch1/ embryosisnotasso- However, despite this biochemical restoration of BH4 levels at the level ciated with major metabolic disturbance, nor embryonic structural of the whole embryo, embryonic lethality could not be rescued. It is abnormalities, but causes embryonic bradycardia. Gch1 deletion is not possible that despite reaching super-physiological levels of BH4 for at rescued by BH4 supplementation (sepiapterin 5 mg/Kg twice daily), least 6 h post-injection and maintain WT levels of BH4 in Gch1/ and is only minimally impacted by catecholamine supplementation. embryos at trough that key BH4-requiring cells in the embryo did not Thus, cellular requirements for Gch1 in embryonic development have reach WT levels. Additional experiments with alternative dosing important implications for understanding the pathogenesis and treat- regimens may help to answer this question. ment of genetic BH4 deficiencies, and for identifying new potential In common with previous observations in the PTPS knock out roles for BH4. mouse, we found no gross organ defects but a significant brady- cardia (Sumi-Ichinose et al., 2001), indicating that the cause of lethality in Gch1/ embryos is physiological rather that anatomi- Funding cal. The observed bradycardia in Gch1/ embryos is most likely due to a deficiency in catecholamines, as tyrosine hydroxylase (TH) This work was supported by the British Heart Foundation (RG/10/ knockout mice (leading to deficient L-DOPA, dopamine, noradrena- 15/28578 and RG/07/003/23133), the NIHR Oxford Biomedical Research line and ) also show significant bradycardia at E12.5 Centre, and the Wellcome Trust (090532/Z/09/Z; gr0744287ma). (Zhou et al., 1995). In common with the PTPS knock out mice and Gch1-/- embryos, both TH and dopamine β-hydroxylase KO embryos show no gross organ abnormalities but display embryo lethality Acknowledgements between E16.5-E18.5 (Kobayashi et al., 1995; Sumi-Ichinose et al., 2001; Thomas et al., 1995; Zhou et al., 1995). Catecholamines are The authors would like to thank Dr Nandi Manasi and Dr Lamia released by the embryo in response to in utero stress such as Heikal for their help in carrying out the phenylalanine and hypoxia which acts to increase heart rate via the β-adrenergic tyrosine assay. receptor (Portbury et al., 2003). Post-partum lethality in PTPS pups can be rescued with BH4 and replacement ther- apy (Elzaouk et al., 2003; Sumi-Ichinose et al., 2001). Both TH and Appendix A. Supporting information dopamine β-hydroxylase KO embryos can be rescued to term by Supplementary data associated with this article can be found in maternal supplementation with L-DOPA (Zhou et al., 1995)orDOPS / the online version at http://dx.doi.org/10.1016/j.ydbio.2014.12.025. (Thomas et al., 1995). In Gch1 embryos L-DOPA levels were significantly decreased compared with Gch1þ /þ littermates. The maternal supplementation of embryos observed in this study References indicates that BH4 can cross cell membrane. However, the mechan- ism of this transport is unclear and it has yet to be established if all Bendall, J.K., Douglas, G., McNeill, E., Channon, K., Crabtree, M.J., 2013. Tetrahy- cells are capable of taking up BH4 to the same extent and thus some drobiopterin in cardiovascular health and disease. Antioxid. Redox Signal. fi fi Brüggemann, N., Spiegler, J., Hellenbroich, Y., et al., 2012. BEneficial prenatal BH4 requiring cells may still be signi cantly de cient. If catechola- levodopa therapy in autosomal recessive cyclohydro- mine producing cells in the embryo were unable to uptake BH4 this lase 1 deficiency. Arch. Neurol. 69, 1071–1075. would account for the failure of BH4 supplementation alone to Cosentino, F., Barker, J.E., Brand, M.P., Heales, S.J., Werner, E.R., Tippins, J.R., West, N., rescue Gch1/ embryos. At E13.5 supplementation with BH4 and Channon, K.M., Volpe, M., Luscher, T.F., 2001. Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. / L-DOPA appeared to part rescue the Gch1 embryos with 8/18 Arterioscler Thromb. Vasc. Biol. 21, 496–502. Gch1 / embryos alive at harvest, indicating that the lethality Costigan, M., Latremoliere, A., Woolf, C.J., 2012. Analgesia by inhibiting tetrahy- – observed in Gch1 / embryos was in part due to a catecholamine drobiopterin synthesis. Curr. Opin. Pharmacol. 12, 92 99. fi Cotton, R.G.H., 1986. A model for hyperphenylalaninaemia due to tetrahydrobiop- de ciency. As dams were treated with both sepiapterin and L-DOPA terin deficiency. J. Inherit. Metab. Dis. 9, 4–14. it is likely that the combination of these two agents was required Crabtree, M.J., Tatham, A.L., Al-Wakeel, Y., Warrick, N., Hale, A.B., Cai, S., Channon, K.M., for the partial rescue, reflected in the slight delay in embryonic Alp, N.J., 2009. Quantitative regulation of intracellular endothelial (eNOS) coupling by both tetrahydrobiopterin-eNOS stoichiometry and lethality. However, unlike mice with genetic deletion of either TH biopterin redox status: insights from cells with tet-regulated GTP cyclohydrolase I (Zhou et al., 1995)ordopamineβ-hydroxylase (Thomas et al., 1995), expression.J.Biol.Chem.284,1136–1144. 138 G. Douglas et al. / Developmental Biology 399 (2015) 129–138

Cunnington, C., Channon, K.M., 2010. Tetrahydrobiopterin: pleiotropic roles in Portbury,A.L.,Chandra,R.,Groelle,M.,McMillian,M.K.,Elias,A.,Herlong,J.R.,Rios,M., cardiovascular pathophysiology. Heart 96, 1872–1877. Roffler-Tarlov, S., Chikaraishi, D.M., 2003. Catecholamines act via a β-adrenergic Du, J., Teng, R.J., Lawrence, M., Guan, T., Xu, H., Ge, Y., Shi, Y., 2012. The protein receptor to maintain foetal heart rate and survival. Am. J. Physiol. – Heart Circ. partners of GTP cyclohydrolase I in rat organs. PLoS One 7, e33991. Physiol. 284, H2069–H2077. Elzaouk, L., Leimbacher, W., Turri, M., Ledermann, B., Bürki, K., Blau, N., Thöny, B., Ryan, B.J., Lourenço-Venda, L.L., Crabtree, M.J., Hale, A.B., Channon, K.M., Wade- fi 2003. Dwar sm and low insulin-like growth factor-1 Due to dopamine Martins, R., 2014. α-Synuclein and mitochondrial bioenergetics regulate tetra- –/– depletion in Pts mice rescued by feeding neurotransmitter precursors and hydrobiopterin levels in a human dopaminergic model of Parkinson disease. – H4-biopterin. J. Biol. Chem. 278, 28303 28311. Free Rad. Biol. Med. 67, 58–68. Furukawa, Y., Kish, S.J., Bebin, E.M., Jacobson, R.D., Fryburg, J.S., Wilson, W.G., Smart, N., Dube, K.N., Riley, P.R., 2010. Identification of Thymosin beta4 as an Shimadzu, M., Hyland, K., Trugman, J.M., 1998. Dystonia with motor delay in effector of Hand1-mediated vascular development. Nat. Commun. 1, 46. compound heterozygotes for GTP-cyclohydrolase I gene mutations. Ann. Sumi-Ichinose,C.,Urano,F.,Kuroda,R.,Ohye,T.,Kojima,M.,Tazawa,M.,Shiraishi,H., Neurol. 44, 10–16. Hagino, Y., Nagatsu, T., Nomura, T., Ichinose, H., 2001. Catecholamines and serotonin Hefler, L.A., Reyes, C.A., O'Brien, W.E., Gregg, A.R., 2001. Perinatal development of are differently regulated by tetrahydrobiopterin: a study from 6-Pyruvoyl- endothelial nitric oxide synthase-deficient mice. Biol. Reprod. 64, 666–673. – Katusic, Z.S., d'Uscio, L.V., Nath, K.A., 2009. Vascular protection by tetrahydrobiop- tetrahydropterin synthase knockout mice. J. Biol. Chem. 276, 41150 41160. terin: progress and therapeutic prospects. Trends Pharmacol. Sci. 30, 48–54. Thomas, S.A., Matsumoto, A.M., Palmiter, R.D., 1995. Noradrenaline is essential for – Khoo, J.P., Nicoli, T., Alp, N.J., Fullerton, J., Flint, J., Channon, K.M., 2004. Congenic mouse foetal development. Nature 374, 643 646. mapping and genotyping of the tetrahydrobiopterin-deficient hph-1 mouse. Thöny, B., Blau, N., 2006. Mutations in the BH4-metabolizing GTP cyclohy- Mol. Genet. Metab. 82, 251–254. drolase I, 6-pyruvoyl-tetrahydropterin synthase, , carbi- Kobayashi, K., Morita, S., Sawada, H., Mizuguchi, T., Yamada, K., Nagatsu, I., Hata, T., nolamine-4a-dehydratase, and dihydropteridine reductase. Hum. Mutat. 27, Watanabe, Y., Fujita, K., Nagatsu, T., 1995. Targeted disruption of the tyrosine 870–878. hydroxylase locus results in severe catecholamine depletion and perinatal van der Heijden, O.W.H., Essers, Y.P.G., Fazzi, G., Peeters, L.L.H., De Mey, J.G.R., lethality in mice. J. Biol. Chem. 270, 27235–27243. van Eys, G.J.J.M., 2005. Uterine artery remodeling and reproductive perfor- Liu, Y., Lu, X., Xiang, F.-L., Poelmann, R.E., Gittenberger-de Groot, A.C., Robbins, J., mance are impaired in endothelial nitric oxide synthase-deficient mice. Biol. Feng, Q., 2012. Nitric oxide synthase-3 deficiency results in hypoplastic Reprod. 72, 1161–1168. coronary arteries and postnatal myocardial infarction. Eur. Heart J. Vásquez-Vivar, J., Whitsett, J., Derrick, M., Ji, X., Yu, L., Tan, S., 2009. Tetrahydro- McDonald, J.D., Bode, V.C., 1988. Hyperphenylalaninemia in the hph-1 mouse biopterin in the prevention of hypertonia in hypoxic foetal brain. Ann. Neurol. mutant. Pediatr. Res. 23, 63–67. 66, 323–331. Mitchell, B.M., Dorrance, A.M., Webb, R.C., 2003. GTP cyclohydrolase 1 inhibition Watschinger, K., Keller, M.A., Golderer, G., Hermann, M., Maglione, M., Sarg, B., – attenuates vasodilation and increases blood pressure in rats. Am. J. Physiol. Heart Lindner, H.H., Hermetter, A., Werner-Felmayer, G., Konrat, R., Hulo, N., Werner, – Circ.Physiol.285,H2165 H2170. E.R., 2010. Identification of the gene encoding alkylglycerol monooxygenase Opladen, T., Hoffmann, G., Blau, N., 2012. An international survey of patients with defines a third class of tetrahydrobiopterin-dependent enzymes. Proc. Natl. tetrahydrobiopterin deficiencies presenting with hyperphenylalaninaemia. Acad. Sci. USA 107, 13672–13677. J. Inherit. Metab. Dis. 35, 963–973. Watschinger, K., Werner, E.R., 2013. Alkylglycerol monooxygenase. IUBMB Life 65, Opladen, T., Hoffmann, G., Hörster, F., Hinz, A.-B., Neidhardt, K., Klein, C., Wolf, N., 366–372. 2011. Clinical and biochemical characterization of patients with early infantile Werner, E.R., Blau, N., Thony, B., 2011. Tetrahydrobiopterin: biochemistry and onset of autosomal recessive GTP cyclohydrolase I deficiency without hyper- pathophysiology. Biochem. J. 438, 397–414. phenylalaninemia. Mov. Dis. 26, 157–161. Zhou, Q.-Y., Quaife, C.J., Palmiter, R.D., 1995. Targeted disruption of the tyrosine Ponzone, A., Blau, N., Guardamagna, O., Ferrero, G.B., Dianzani, I., Endres, W., 1990. Progression of 6-pyruvoyl-tetrahydropterin synthase deficiency from a periph- hydroxylase gene reveals that catecholamines are required for mouse foetal – eral into a central phenotype. J. Inherit. Metab. Dis. 13, 298–300. development. Nature 374, 640 643.