Developmental vascular remodeling defects and postnatal kidney failure in mice lacking Gpr116 (Adgrf5) and Eltd1 (Adgrl4)

GPR116 (ADGRF5) and ELTD1 (ADGRL4) belong to different subfamilies of the adhesion G-protein-coupled receptor group but are both expressed in endothelial cells. We therefore analyzed their functions in mice lacking these receptors. While loss of GPR116 or ELTD1 alone had no obvious effect on cardiovascular or kidney function, mice lacking both, GPR116 and ELTD1, showed malformations of the aortic arch arteries and the cardiac outflow tract leading to perinatal lethality in about 50% of the mutants. In addition to cardiovascular malformations, surviving mice developed renal thrombotic microangiopathy as well as hemolysis and splenomegaly, and their lifespan was significantly reduced. Loss of GPR116 and ELTD1 specifically in endothelial cells or neural crest-derived cells did not recapitulate any of the phenotypes observed in GPR116-ELTD1 double deficient mice, indicating that loss of GPR116 and ELTD1 expressed by other cells accounts for the observed cardiovascular and renal defects.


Introduction
G-protein-coupled receptors (GPCRs) constitute the largest receptor family in eukaryotes [1]. They consist of five families in mammals, among which the adhesion GPCR family is the second largest with 33 members in humans and 31 members in mice [2]. Many adhesion GPCRs have long N-terminal extracellular regions that contain various domains with adhesive functions. The extracellular N-terminal part of the protein can be autoproteolytically cleaved at the GPCR proteolysis site (GPS) motif, which is highly conserved and located close to the first transmembrane helix [3,4]. The GPS motif is part of a much larger domain, the GPCR a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Mice with null or floxed alleles of Gpr116 and Eltd1 were generated after gene targeting in embryonic stem (ES) cells. In V6.5 (C57BL/6 x 129S4/SvJae) ES cells (Novus Biologicals), exon 8 of Gpr116 or exon 5 of Eltd1 was replaced by a cassette carrying the neomycin resistance gene (flanked by FRT recombination sites) via homologous recombination. Correct targeting was verified by Southern blotting and PCR. Highly chimeric males obtained from targeted ES cell clone injection were bred onto C57BL/6 background. F1 generation mice carrying targeted allele were mated either with flp recombinase expressing mice [22] resulting in removal of the neo cassette, to generate Gpr116-or Eltd1-floxed mice, or were mated with EIIa-Cre mice [23] to generate a null allele of Gpr116 or Eltd1 by recombining loxP sites in the early embryonic stage. Deletion of the targeted exon was confirmed by both quantitative RT-PCR (qPCR) and RT-PCR. Animals were back-crossed with C57BL/6N mice for at least 8 generations. Gpr116/Eltd1 double knockout (Gpr116 -/-;Eltd1 -/-) mice were generated by crossing Gpr116 -/animals with Eltd1 -/animals. To generate conditional double knockout mice, Gpr116 flox/flox ;Eltd1 flox/flox mice were crossed with vascular endothelial cadherin (VE-Cad)-Cre mice [24] or Pax3-Cre mice [25].
Mice were housed under a 12-h light-dark cycle with free access to food and water and under specific pathogen-free conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Regierungspräsidia Karlsruhe and Darmstadt. Immunofluorescence microscopy 8 μm cryo-sections were fixed in 4% PFA for 10 min, washed three times in PBS, incubated for 30 min with 5% BSA in PBS + 0.1% Triton for blocking and permeabilization, and sections were then incubated overnight at 4˚C with primary antibodies (anti-CD31, 1:200, Cat.550274, BD Bioscience; anti-Podocin, 1:600, Cat.P0372, Sigma-Aldrich; anti-Desmin, 1:400, Cat. D10033, Sigma-Aldrich; anti-Prosurfactant Protein C, 1:300, Cat.AB3786, Millipore). Slices were washed three times in PBS and then incubated for 1 h at room temperature with secondary antibodies (1:200, Life Technologies) and DAPI (4',6-diamidino-2-phenylindole) (1:10000, Invitrogen). After washing three times in PBS, slices were mounted and analyzed using the Leica TCS SP5 or Zeiss Axio Observer.Z1 microscope.

Urine analysis
Mouse urine samples were collected and centrifuged for 5 min at 900 x g to remove cell debris. 45 μl of urine were mixed with 15 μl Laemmli (4x) and incubated for 5 min at 99˚C, and 10 μl of the mixture was loaded onto an 8% SDS-PAGE. Bovine serum albumin (BSA) (New England BioLabs) was used as control. After protein separation, the gel was fixed in a fixation buffer (25% (v/v) isopropanol and 10% (v/v) acetic acid) for 30 min, subsequently stained in Coomassie staining buffer (0.2% (w/v) Coomassie Brilliant Blue R-250 (Cat. 27816, Sigma-Aldrich), 40% methanol and 10% acetic acid) for 1 h and finally in destaining buffer (40% (v/ v) methanol and 10% (v/v) acetic acid) for 2-4 h. The gel was scanned using HP Scanjet G4050.

Magnetic resonance imaging (MRI)
All MRI experiments were performed on a 7.0T superconducting magnet (Bruker Biospin, Pharmascan, 70/16, Ettlingen, Germany) equipped with an actively shielded imaging gradient field of 760 mT/m operating at frequencies of 300.1 MHz [27]. Mice were measured under volatile isoflurane (1.5-2.0% in oxygen and air with a flow rate of 1.0 L/min) anesthesia; the body temperature was maintained 37˚C by a thermostatically regulated water flow system during the entire imaging protocol.

Vascular corrosion casting and Micro-CT imaging
Mice were deeply anesthetized with pentobarbital and perfused via the left ventricle, first with physiological 0.9% (w/v) NaCl containing heparin (25.000 U/l) followed by 4% (v/v) PFA at 4 ml/min. Both solutions were kept at 35˚C. Immediately thereafter, the polymer PU4ii (vasQtec, Switzerland) was infused at the same rate. After resin curing (2 days, room temperature), soft tissue was macerated in 7.5% (w/v) KOH for 24 h at 0050˚C and thereafter washed in tap water. Vascular casts were scanned using a Quantum GX cone-beam microCT imaging system (Rigaku Co., Tokyo, Japan). The system uses a CMOS flat-panel detector and a microfocus Xray source mounted on a gantry with continuous rotation. 6,424 projection images were acquired over a 36-mm field of view (FOV) with the X-ray source set to 90 kV and 80 μA over a complete 360-degree rotation for a total scan time of 14 minutes. 2352 X 2944 detector elements were read and images were reconstructed using a modified filtered back-projection Feldkamp algorithm selecting 512 X 512 X 512 matrix size with a reconstructed isotropic voxel size of 20 μm. Image processing software Osirix 6.0 (Pixmeo, Switzerland) was used to generate 3D video and images.

Blood analysis
Blood samples were collected in EDTA (10 mM final concentration). Blood smears were stained with Giemsa solution (Cat.32884, Sigma-Aldrich). Blood and plasma parameters were determined by IDEXX Laboratories, Ludwigsburg, Germany.

Isolation of endothelial cells from the lung and real-time qPCR
Isolation of endothelial cells from the lung was reported previously [28]. Briefly, animals were killed, perfused with PBS and lungs were dissected, minced and enzymatically digested for 60 min while shaking at 37˚C in a digestion mix containing collagenase II (2 mg/ml; Worthington), elastase-I (0.04 mg/ml; Sigma), DNase1 (5 U/ml; New England Biolabs) and dispase II (1.2 U/ml; Sigma). Cell suspensions were serially filtered through 70 and 40 μm cell strainers. Endothelial cells were enriched using the autoMACS (Milteny Biotec) after labelling with rat anti-mouse CD31-PE antibody (Cat.MCA2388PE, Serotec) and anti-PE magnetic microbeads (130-105-639, Milteny Biotec). Immune cells were labelled by rat anti-mouse CD45-FITC antibody (Cat.553079, BD Biosciences). Subsequently, using flow cytometric gating strategies to detect live single cells, the endothelial cells were identified and sorted based on CD31 + /CD45 -/ DAPIexpression. Endothelial cells (20000 cells per mouse) were directly sorted in lysis buffer for RNA isolation. RNA was prepared using RNAeasy Micro Kit (74004, Qiagen). First-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase (M0368, New England Bio-Labs). The same reaction was performed without reverse transcriptase as non-RT control to test for genomic DNA contamination. The real-time qPCR was performed using the LightCycler 480 SYBR Green I Master System (Roche). Relative expression levels were obtained by normalization to Actb mRNA. Following real-time qPCR primers (intron-spanning) were used: Gpr116 (forward):

Statistics
Data are presented as mean values ± SD. Statistical analyses were performed using GraphPad Prism 5 software. Paired t test was used in MRI study. For other studies unpaired t test was used to compare two groups. Differences were considered statistically significant at P 0.05.

Expression of Gpr116 and Eltd1 in mice
To analyze the expression of Gpr116 in mice, we generated a bacterial artificial chromosome (BAC)-based transgenic mouse line expressing mCherry under the control of the Gpr116 promoter and enhancer elements (S1 Fig). Expression of Gpr116 co-localized with the endothelial cell marker CD31 and could be detected in microvessels in different organs including the heart, pancreas, adipose tissue ( Fig 1A) and lung ( Fig 1B). Gpr116 was also detected in alveolar epithelial type II cells of the lung (Fig 1B), which is consistent with previous studies showing an important role of Gpr116 in the alveolar surfactant homeostasis [14][15][16][17]. In the kidney, Gpr116 expression exclusively co-localized with the endothelial cell marker but not with desmin or podocin indicating mesangial cells or podocytes, respectively ( Fig 1C). A knock-in mouse line expressing the lacZ gene under the control of the Eltd1 promoter showed Eltd1 expression in microvessels of various organs including the brain, heart, kidney and liver (Fig 2A). In the kidney, Eltd1 expression exclusively co-localized with endothelial cells but not with mesangial cells or podocytes ( Fig 2B).
Expression of Gpr116 and Eltd1 was also observed in the early stage of embryonal development (E10-12) and co-localized with endothelial cell marker CD31 in the developing heart, aortic arch arteries as well as other vessels (Fig 3A and 3B). At embryonic day 18.5, we also confirmed expression of Gpr116 and Eltd1 in endothelial cells in multiple organs including the heart, lung and skeletal muscle (S2 Fig).

Perinatal lethality in mice lacking Gpr116 and Eltd1
In order to study the functions of both receptors in vivo, mice with global and conditional deficiency of Gpr116 and Eltd1 were generated (S3 and S4 Figs). As described before, we did not observe any obvious phenotypical abnormalities in Gpr116 -/and Eltd1 -/mice except a defect in lung surfactant homeostasis in Gpr116 -/mice [12,[14][15][16][17]. Given the strong overlap in expression of Gpr116 and Eltd1 in endothelial cells, we generated Gpr116/Eltd1 double deficient (dKO) mice by crossing Gpr116 -/animals with Eltd1 -/animals. In the offspring of Gpr116 -/-; Eltd1 -/+ or Gpr116 -/+ ;Eltd1 -/mice we found significantly reduced number of Gpr116 -/-;Eltd1 -/mice than expected. Double-deficient mice showed a reduced life expectancy with half of the animals surviving the first postnatal month and none of the animals surviving the third postnatal month ( Fig 4A). To further analyze the time point of early death of Gpr116 -/-;Eltd1 -/mice, we set up timed matings and analyzed litters before birth (E18.5) as well as after birth (P0). As shown in Fig 4D, normal ratios of mice lacking both Gpr116 and Eltd1 were seen alive at embryonic day 18.5; however, half of the Gpr116 -/-;Eltd1 -/mice were found dead shortly after birth. This indicates that about 50% of mice lacking both Gpr116 and Eltd1 died perinatally ( Fig 4A). The surviving 50% developed significant growth impairment (Fig 4B and 4C) and started to die from the third week after birth. More than 80% of Gpr116 -/-;Eltd1 -/mice had died at 8 weeks of age ( Fig 4A).

Cardiovascular defects in Gpr116/Eltd1 double deficient mice
To understand the reason of the perinatal death of dKO newborns, E18.5 dKO embryos and their littermates were dissected and analyzed. In 21 out of 30 analyzed Gpr116/Eltd1 dKO embryos, we could identify various obvious defects in the heart and the large vessels, whereas Gpr116 and Eltd1 single deficient embryos were indistinguishable from wild-type embryos ( Table 1). Among the most frequently observed malformations were ventricle septum defects (VSDs) (Fig 5A). In about 20% of E18.5 dKO mice, the right subclavian artery originating from the innominate artery was missing ( Fig 5B). As reported before in other mouse mutants [29,30], the right subclavian artery instead originated from the descending aorta ( Fig 5C and  S5 Fig). About 10% of the dKO embryos showed interrupted aortic archs (Fig 5C and 5D), double outlet right ventricles (DORVs) (Fig 5E) or double aortic archs (Fig 5C and 5F). Several of the malformations found at E18.5 were likely to result in perinatal death.
To see whether Gpr116/Eltd1-dKO mice survived the perinatal period also had malformations of the cardiovascular system, we analyzed 4-week-old Gpr116/Eltd1 dKO mice. These animals showed increased relative spleen and heart weights ( Fig 6A). Histological analysis revealed a concentric cardiac hypertrophy in dKO mice, which at the same time showed a decreased heart and body size (Fig 6B and S1 and S2 Movies). Magnetic resonance imaging (MRI) analysis of hearts in 4-and 8-week-old mice confirmed an increase in left ventricular (LV) mass (Fig 6C). While the stroke volume and ejection fraction were not significantly different between dKO and littermate control mice at 4 weeks of age, dKO mice exhibited not only a further increase in LV mass, but also severely reduced stroke volume and ejection fraction compared to littermate control animals at 8 weeks of age (Fig 6C and S1  Given that both Gpr116 and Eltd1 are highly expressed in endothelial cells, we wondered whether the phenotype observed in global double deficient mice is due to Gpr116/Eltd1 deficiency in endothelial cells. We therefore crossed Gpr116 flox/flox ;Eltd1 flox/flox with mice expressing Cre under the control of the vascular endothelial cadherin (VE-Cad) promoter (VE-Cadherin-Cre) to generate endothelial cell-specific dKO mice. Endothelial cells were isolated from the lung from endothelial cell-specific dKO mice and control mice). Real-time qPCR was performed to identify expression levels of Gpr116 and Eltd1. Endothelial cell-specific dKO mice showed almost 90% reduction in endothelial levels of RNAs encoding Gpr 116 and Eltd1 compared with Cardiac neural crest cells also contribute to cardiac outflow tract septation and aortic arch artery patterning [31][32][33]. To elucidate whether the phenotype observed in global dKO mice is due to Gpr116/Eltd1 deficiency in neural crest cells, we crossed the Gpr116 flox/flox ;Eltd1 flox/flox mice with Pax3-Cre mice [25] to generate neural crest cell-specific dKO mice. However

Hemolysis and kidney defects in mice lacking Eltd1 and Gpr116
Upon hematological analysis of mice lacking Eltd1 and Gpr116, we observed a large number of misshapen erythrocytes of which many were identified as fragmentocytes (Fig 7A). Erythrocytes numbers, packed cell volume as well as mean corpuscular volume (MCV) were significantly reduced in dKO mice compared to Gpr116 and Eltd1 single deficient mice or wild-type animals (Fig 7B). The occurrence of fragmentocytes was accompanied by reduced hemoglobin and increased lactate dehydrogenase (LDH) plasma levels ( Fig 7B) indicating increased hemolysis. In addition to changes in erythrocyte size and shape, we observed massive amounts of protein in the urine of mice lacking Gpr116 and Eltd1, but not in littermate controls. In addition, plasma urea levels were significantly elevated in dKO animals indicating kidney failure (Fig 7C). To identify the cause of protein leakage in the kidney, histological and ultrastructural analyses of kidneys from 4-week-old mice were performed. Light microscopy showed massive red blood cell fragments being entrapped in glomerular capillary lumen ( Fig 7D) and varying degrees of mesangial matrix expansion (Fig 7E). Transmission electron microscopy (EM)  revealed an accumulation of fragmented red blood cells (schistocytes) in the expanded subendothelial zone, decreased capillary lumen and a damaged glomerular filtration barrier including loss of endothelial fenestration and fusion of podocytes foot processes (Fig 7F). The observed microscopic defects of the kidney indicated glomerular thrombotic microangiopathy. Interestingly, no renal defects were observed in mice with constitutive endothelium-specific deficiency of Gpr116 and Eltd1 (S6 Fig), indicating that loss of Gpr116 and/or Eltd1 in endothelial cells is not responsible for the glomerulopathy observed in global dKO mice. When we analyzed urine of newborn mice, we observed a minor albuminuria in dKO pups compared to Gpr116 and Eltd1 single deficient animals (S7 Fig), whereas no pathological lesions after PASstaining or upon ultrastructural analysis could be seen at that stage in kidneys of dKO animals (S7 Fig). This indicates that a functional glomerulopathy was already present in dKO mice at birth.

Discussion
Adhesion GPCRs are a large group of transmembrane proteins whose functions are in most cases still poorly understood. Studies describing their cellular role are often limited since most receptors lack a defined ligand and downstream signaling mechanism [3]. Another approach which has helped to describe functions of adhesion GPCRs is the generation of mice lacking particular members of this receptor group [6]. GPR116 and ELTD1 are two adhesion GPCRs from different families. Previous studies as well as our own expression analysis demonstrated that both receptors are highly expressed in the microvascular endothelium of multiple organs [19], yet Gpr116 or Eltd1 single deficient mice do not show a basic vascular phenotype except a moderate vascular leakage in the brain of animals lacking Gpr116 [17]. This raised the question whether Gpr116 and Eltd1 have redundant vascular functions. In mice lacking Gpr116 and Eltd1 we observed increased perinatal and postnatal lethality. This was accompanied by various cardiovascular malformations occurring with variable penetrance and involving defects in the septation of the cardiac outflow tract such as ventricular septum defects and double outlet right ventricle and defects in the development and/or remodeling of pharyngeal arch arteries including doubled aortic arch, interrupted aortic arch and aberrant right subclavian artery.
These complex morphogenetic processes of pharyngeal arch arteries remodeling and cardiac outflow tract septation requires coordinated communications between cells of mesoderm, endoderm, surface ectoderm, and the neural crest [31,34,35]. Defects in the development and/or remodeling of pharyngeal arch arteries result in various malformations such as an interrupted aortic arch, a right-sided aortic arch and an aberrant origin of the right subclavian artery in humans [36]. In mouse models, formation and remodeling of pharyngeal arch arteries has been shown to involve multiple signaling molecules including integrins, endothelins, of age. (D) Genotype of offspring from intercrosses of Gpr116 -/-;Eltd1 +/and Gpr116 +/-;Eltd1 -/mice at E18. 5  Mice lacking Gpr116 (Adgrf5) and Eltd1 (Adgrl4)  [29,[37][38][39][40][41]. Among these signaling molecules, α5 integrin appears to be of particular interest as its ablation in the anterior mesoderm resulting in abnormalities very similar to those observed in Gpr116/Eltd1 deficient mice including ventricular septum defects and aortic arch malformations [41]. The anterior mesoderm gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo, affected also differentiation of neural crest-derived cell into VSMCs in the 4 th and 6 th pharyngeal arch [41]. A very similar phenotype was observed in mice lacking α5 and αv integrin, but not α5 integrin alone, in the endothelium indicating that α5 integrin expressed by endothelial cells can also contribute to the differentiation of neural crest cells and the development of the cardiac outflow tract and aortic arch vessels [38]. In our study, deletion of Gpr116/Eltd1 in neural crest cells using Pax3-Cre knock-in mice exhibited no cardiovascular development abnormalities suggesting that the observed phenotypes in dKOs were not due to functions of Gpr116 and Eltd1 in neural crest cells. The similarity of the cardiovascular developmental defects in α5 integrin mutant and Gpr116/ Eltd1 deficient mice may suggest a functional link between these transmembrane proteins. Although α5ß1 integrin has been identified as ligand for another adhesion GPCR, CD97, [42] which shares with Eltd1 the presence of an EGF and a calcium-binding EGF domain in their N-terminal part [3], no interaction between Gpr116 or Eltd1 and integrins has been reported so far. Since neural crest cell and endothelial cell specific loss of Gpr116 and Eltd1 did not result in the phenotype observed in global Gpr116/Eltd1-deficient mice, it remains unclear in which cells function of the two receptors is required for normal formation of large arteries and cardiac outflow tract. It is possible that during development Gpr116 and Eltd1 are expressed in particular cells during defined periods of time which have escaped our expression analysis. Alternatively, both receptors may be expressed in different cell types. It is also conceivable that a small fraction of endothelial cells, in which Gpr116/Eltd1 function is critical, did not undergo recombination in VE-cadherin-Cre transgenic mice.
We have observed a strong increase in cardiac weight in Gpr116/Eltd1-deficient mice already shortly after birth, which was much more pronounced than the increase in heart weight observed in Gpr116 single deficient animals [17]. We think it is most likely that the cardiac hypertrophy in mice lacking both receptors is due to altered hemodynamics caused by defects in the structure of large arteries and the cardiac outflow tract. The hemolysis in addition may contribute to an increased cardiac load. Since none of these defects have been arteries is shown below. Arrow points to the position where the right subclavian artery usually branches off. 1, Aortic arch; 2, Innominate artery; 3, Right subclavian artery; 4, Left common carotid artery; 5, Ascending thoracic aorta; 6, pulmonary artery. (C) Schematic of the embryonic remodeling process of the branchial arch arteries into the aortic arch and the great vessels in normal configuration (E10. 5  reported in Gpr116 mice, it remains unclear to what degree the cardiac phenotypes of Gpr116 and Gpr116/Eltd1 deficient mice share a common mechanism.
Global Gpr116/Eltd1 dKO mice developed renal thrombotic microangiopathy, which is indicated by loss of regular glomerular endothelial phenotype with loss of fenestrae, detachment from basement membrane and enlarged subendothelial zones with accumulated schistocytes and damaged glomerular filtration barrier [43]. In addition, dKO mice exhibited fragmented red blood cells, decreased hemoglobin and elevated plasma LDH levels indicating hemolysis. Taken together, these findings are typical clinical and pathological indications of an atypical hemolytic uremic syndrome, which is a form of thrombotic microangiopathy and manifests predominately in the kidney [43]. The atypical hemolytic uremic syndrome is caused by genetic or acquired factors that lead to uncontrolled activation of the alternative complement pathway on the endothelial surface, which results in endothelial cell injury and consequently thrombus formation especially in the kidney [44]. Endothelial damage of capillary vessels can generate abnormally high levels of shear stress, which may be responsible for the fragmentation and hemolysis of erythrocytes [44]. While newborn Gpr116/Eltd1 deficient mice showed no obvious pathological lesions in their kidneys, they already had minor albuminuria indicating a glomerular filtration barrier dysfunction. After birth, dKO mouse showed rapid development of renal thrombotic microangiopathy with severe glomerulopathy at 4 weeks of age. Mutations in several genes, most of them encoding regulatory proteins involved in complement signaling, have been shown to cause atypical hemolytic uremic syndrome [45,46]. In addition, deficiency of diacylglycerol kinase ε has been suggested to lead to excessive arachidonic acid-containing diacylglycerol, which through activation of protein kinase C exert potent pro-thrombotic effects in endothelial cells and platelets, leading to thrombotic microangiopathy [47]. Hence, atypical hemolytic uremic syndrome can be caused by uncontrolled complement pathway or alteration of the anti-thrombogenic properties of the endothelium. Interestingly, no renal defects were observed in mice with constitutive endothelium-specific deficiency of Gpr116 and Eltd1. Therefore, it is reasonable to speculate that Gpr116 and Eltd1 play a role in an unknown cell type related to regulation of the complement system.
We report here about the unexpected finding that loss of the adhesion GPCRs GPR116 and ELTD1 results in malformation of the cardiac outflow tract and the aortic arch arteries as well in the postnatal development of a thrombotic microangiopathy. This indicates that both receptors function together in the development and homeostasis of the vascular system. This work also provides the foundation for exploring the functions of Gpr116 and Eltd1 in the development and remodeling of the cardiac outflow tract and large arteries as well as their potential role in the development of thrombotic microangiopathies. A better understanding of the celltype specific functions of both receptors and the identification of receptor ligands and downstream signaling mechanisms will be critical for further progress in understanding the role Gpr116 and Eltd1 play in vascular development and function.