Neonatal Lethality Research Articles

Analysis of differential neonatal lethality in cystathionine β-synthase deficient mouse models using metabolic profiling.

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine β-synthase (Cbs-/- ) gene or an inactive human CBS protein (Tg-G307S Cbs-/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs-/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs+/- , Tg-G307S Cbs-/- , and Tg-I278T Cbs-/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs-/- and Tg-I278T Cbs-/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs-/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs-/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.

SNED1: a Novel ECM Protein Regulating Neural Crest Cells Phenotype and Craniofacial Morphogenesis

The extracellular matrix (ECM) is a complex meshwork of proteins that provides structural and biochemical support to cells. We have recently reported that the homozygous ablation of Sned1, which encodes the novel ECM protein SNED1, caused neonatal lethality, and the few knockout (KO) survivors we obtained exhibited craniofacial malformations including shorter snouts and wider neurocrania (Figure 1). Since cranial neural crest cells (NCCs) form the facial skeleton and given the importance of the ECM in guiding NCC phenotype, we hypothesized that SNED1 influences the behavior of this cell population. To test this, we generated an NCC-specific Sned1 KO mouse. While Sned1NCC-/NCC- mice survive, they present craniofacial anomalies resembling the malformations observed in the global Sned1 KOmice (Barqué et al., Dev Dyn, 2020). In order to form craniofacial structures, cephalic NCCs first need to delaminate from the neural tube, then migrate towards the frontonasal region and the pharyngeal arches, and there differentiate into bone and cartilage. Our goal is now to determine precisely which cellular processes along the NCC path are directly controlled by SNED1. To assess this, we are first performing in-vivo assays including in-situ hybridization and immunohistochemical staining on Sned1NCC-/NCC- and control mouse embryos at different embryonic timepoints to assess NCC behaviors. In parallel, we are conducting in-vitro assays using neural tube explants excised from Sned1NCC-/NCC- and control mouse embryos and recapitulating the key steps NCCs take towards the formation of craniofacial structures. These steps include migration, proliferation, and chondrogenic and osteogenic differentiation and can be monitored by tracking the expression of specific molecular markers. Once we identify which cellular processes are controlled by SNED1, we will turn our focus to the identification of the molecular mechanisms by which SNED1 governs NCC phenotype and craniofacial morphogenesis. Sequence analysis of SNED1 revealed two consensus binding sites for integrins, which are known ECM receptors. Thus, we have developed SNED1 constructs containing mutations in the putative integrin-binding sites that we will use to elucidate whether integrins serve as SNED1 receptors. Importantly, deletion of 2q37.3, a region that comprises the SNED1 locus, has been linked to facial dysmorphism in patients. We thus propose that SNED1 contributes to the phenotype of these patients and that our novel mouse model may shed light on some of the features of this syndrome. Moreover, we previously reported SNED1 as a promoter of breast cancer metastasis (Naba et al., eLife, 2014). Since metastatic cells share common features with NCC, our studies will also allow us to better understand the role of SNED1 in metastasis.

Identification of homozygous haplotypes carrying putative recessive lethal mutations that compromise fertility traits in French Lacaune dairy sheep

BackgroundHomozygous recessive deleterious mutations can cause embryo/fetal or neonatal lethality, or genetic defects that affect female fertility and animal welfare. In livestock populations under selection, the frequency of such lethal mutations may increase due to inbreeding, genetic drift, and/or the positive pleiotropic effects of heterozygous carriers on selected traits.ResultsBy scanning the genome of 19,102 Lacaune sheep using 50 k single nucleotide polymorphism (SNP) phased genotypes and pedigree data, we identified 11 Lacaune deficient homozygous haplotypes (LDHH1 to LDHH11) showing a highly significant deficit of homozygous animals ranging from 79 to 100%. These haplotypes located on chromosomes 3, 4, 13, 17 and 18, spanned regions from 1.2 to 3.0 Mb long with a frequency of heterozygous carriers between 3.7 and 12.1%. When we compared at-risk matings (between carrier rams and daughters of carrier rams) and safe matings, seven of the 11 haplotypes were associated with a significant alteration of two fertility traits, a reduced success of artificial insemination (LDHH1, 2, 8 and 9), and/or an increased stillbirth rate (LDHH3, 6, 8, 9, and 10). The 11 haplotypes were also tested for a putative selective advantage of heterozygous carrier rams based on their daughter yield deviation for six dairy traits (milk, fat and protein yields, fat and protein contents and lactation somatic cell score). LDHH1, 3, 4, 5, 7, 9 and 11 were associated with positive effects on at least one selected dairy trait, in particular milk yield. For each haplotype, the most probable candidate genes were identified based on their roles in lethality of mouse knock-out models and in mammalian genetic disorders.ConclusionsBased on a reverse genetic strategy, we identified at least 11 haplotypes with homozygous deficiency segregating in French Lacaune dairy sheep. This strategy represents a first tool to limit at-risk matings in the Lacaune dairy selection scheme. We assume that most of the identified LDHH are in strong linkage disequilibrium with a recessive lethal mutation that affects embryonic or juvenile survival in sheep but is yet to be identified.

Metabolic impact of pathogenic variants in the mitochondrial glutamyl‐tRNA synthetase EARS2

Glutamyl-tRNA synthetase 2 (encoded by EARS2) is a mitochondrial aminoacyl-tRNA synthetase required to translate the 13 subunits of the electron transport chain encoded by the mitochondrial DNA. Pathogenic EARS2 variants cause combined oxidative phosphorylation deficiency, subtype 12 (COXPD12), an autosomal recessive disorder involving lactic acidosis, intellectual disability, and other features of mitochondrial compromise. Patients with EARS2 deficiency present with variable phenotypes ranging from neonatal lethality to a mitigated disease with clinical improvement in early childhood. Here, we report a neonate homozygous for a rare pathogenic variant in EARS2 (c.949G>T; p.G317C). Metabolomics in primary fibroblasts from this patient revealed expected abnormalities in TCA cycle metabolites, as well as numerous changes in purine, pyrimidine, and fatty acid metabolism. To examine genotype-phenotype correlations in COXPD12, we compared the metabolic impact of reconstituting these fibroblasts with wild-type EARS2 versus four additional EARS2 variants from COXPD12 patients with varying clinical severity. Metabolomics identified a group of signature metabolites, mostly from the TCA cycle and amino acid metabolism, that discriminate between EARS2 variants causing relatively mild and severe COXPD12. Taken together, these findings indicate that metabolomics in patient-derived fibroblasts may help establish genotype-phenotype correlations in EARS2 deficiency and likely other mitochondrial disorders.

486 Systemic collagen VII protein therapy for treatment of advanced RDEB

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin blistering disease associated with progressive multi-organ fibrosis. RDEB is caused by biallelic COL7A1 mutations leading to reduced production or functionality of the extracellular matrix protein, collagen VII (C7), which is necessary for epidermal-dermal adherence. C7 is not simply a structural protein but also has multiple functions, including regulation of TGFβ bioavailability and the inhibition of skin scarring after wounding. The multi-organ involvement of RDEB calls for systemic treatment. Intriguingly, intravenous (IV) administration of recombinant C7 (rC7) improves the clinical phenotype of newborn C7 deficient mice and rescues them from neonatal lethality. The effect on established RDEB in adult animals has not been determined. Here, we used small and large adult RDEB animal models to investigate the disease-modulating abilities of rC7 on established RDEB. In adult RDEB mice, IV-injected rC7 accumulated dose-dependently at the dermal-epidermal junction of wounded skin and oral mucosa. Bi-weekly IV injections of rC7 for 7 weeks in adult RDEB mice reduced new skin blisters and effectively reduced progression of the fibrotic mitten deformities of their paws. The inhibition of fibrosis was mediated through reduction of TGFβ signaling. IV infusion of rC7 in adult dogs with RDEB also improved disease by promoting the healing of blister-induced skin erosions without excessive scarring. In both models, IV C7 was well tolerated. These preclinical studies suggest that repeated IV administration of rC7 is an efficacious systemic treatment for established adult RDEB.

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice.

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.

Loss of TANGO1 Leads to Absence of Bone Mineralization.

ABSTRACT TANGO1 (transport and Golgi organization‐1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER‐Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild‐to‐moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near‐total bone loss and phenocopies the situation of Mia3 −/− mice. Whole‐exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4‐base pair (4‐bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1‐related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe‐to‐lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder

Autophagy is an intracellular degradation system, but its physiological functions in vertebrates are not yet fully understood. Here, we show that autophagy is required for inflation of air-filled organs: zebrafish swim bladder and mouse lung. In wild-type zebrafish swim bladder and mouse lung type II pulmonary epithelial cells, autophagosomes are formed and frequently fuse with lamellar bodies. The lamellar body is a lysosome-related organelle that stores a phospholipid-containing surfactant complex that lines the air-liquid interface and reduces surface tension. We find that autophagy is critical for maturation of the lamellar body. Accordingly, atg-deficient zebrafish fail to maintain their position in the water, and type-II-pneumocyte-specific Fip200-deficient mice show neonatal lethality with respiratory failure. Autophagy suppression does not affect synthesis of the surfactant phospholipid, suggesting that autophagy supplies lipids and membranes to lamellar bodies. These results demonstrate an evolutionarily conserved role of autophagy in lamellar body maturation.

GPRASP/ARMCX Protein Family: Potential Involvement in Health and Diseases Revealed by their Novel Interacting Partners.

GPRASP (GPCR-associated sorting protein)/ARMCX (ARMadillo repeat-Containing proteins on the X chromosome) family is composed of 10 proteins, whose genes are located on a small locus of the X chromosome except one. They possess at least two armadillo-like repeats on their carboxylterminal homologous sequence, but they can be subdivided on specific sequence features. Subfamily 1 (GPRASP1, GPRASP2, GPRASP3, ARMCX4 and ARMCX5) displays additional repeated motifs while a mitochondrial targeting transmembrane domain is present in subfamily 2 (ARMC10, ARMCX1, ARMCX2, ARMCX3 and ARMCX6). Although their roles are not yet fully understood, the recent identification of several interacting partners has shed new light on the processes in which GPRASP/ARMCX proteins are implicated. Among the interacting partners of proteins from subfamily 1, many are GPCRs. GPRASP1 binds trafficking proteins, such as Beclin2 and the Dysbindin-HRS-Gαs complex, to participate in GPCR post-endocytic sorting. Moreover, in vitro as well as in vivo experiments indicate that GPRASP1 is a critical player in the adaptive responses related to chronic treatments with GPCR agonists. GPRASP2 seems to play a key role in the signaling of the hedgehog pathway in the primary cilium through a Smoothened-GPRASP2-Pifo complex. Identified small compound inhibitors of this complex could treat drug-resistant smoothened derived cancer forms. Deletion of GPRASP2 in mice causes neurodevelopmental alteration and affects mGluR5 regulation, reflected by autism-like behavior. Several members of subfamily 2, in complex with TRAK2 and MIRO, are involved in the trafficking of mitochondria in axons and in the regulation of their size and division, influencing the cell cycle. The essential role of GPRASP/ARMCX proteins in cellular physiology is supported by human cases of deletions, causing male neonatal lethality by pulmonary delayed development, dysmorphic face, and psychiatric and intellectual impacts in females.

Loss of Protein C in Zebrafish Results in Impaired Neutrophil Migration after Tissue Injury

Hemostasis is a natural protective process that developed to retain a circulating blood system, conferred by a complicated yet sophisticated balance of factors. Disturbances of this network result in thrombosis or hemorrhage. Among many well-characterized coagulation factors, protein C (PC) exhibits multifunctional roles including anticoagulant, cytoprotective, and anti-inflammatory activities. The importance of PC has been demonstrated not only by the increased risk of venous thrombosis in individuals with heterozygous deficiency, but also the observed neonatal lethality in patients. Knockout mice exhibit similar neonatal lethality, which has made it difficult to further study complete deficiency. The zebrafish is a vertebrate organism that is characterized by a powerful genetic system, prolific breeding, rapid and transparent development, and a well described and highly conserved coagulation cascade. Here we utilize genome editing to generate a null allele of the PC gene (proc) in zebrafish and discover that its loss not only impairs hemostatic balance, but also affects neutrophil recruitment to sites of tissue injury. Through examination of publicly available zebrafish genome sequence, we determined that the proc locus is duplicated in tandem, resulting in two closely adjacent copies with nearly identical sequences. We used CRISPR/Cas9 with two single guide RNAs flanking the entire locus to produce a 17.3 kilobase deletion that knocks out both copies of proc to produce a complete null mutation, verified by sequencing and quantitative PCR. proc-/- mutants survived well into adulthood, with ~50% lethality by seven months of age. The embryonic survival and accessibility enabled us to perform intravital microscopy to evaluate the hemostatic effects of PC deficiency. We used laser-induced endothelial injury on the posterior cardinal vein (PCV) at 3 days post fertilization (dpf), which typically results in rapid formation of an occlusive fibrin-rich thrombus. proc-/- mutants had an average time to occlusion of 60 seconds versus 13 seconds in controls (p < 0.0001), consistent with a consumptive coagulopathy, as previously seen in antithrombin III (at3) mutants. A transgenic background with fluorescently labeled fibrinogen showed that more than 95% of proc-/- mutants had spontaneous thrombi in the PCV, which was not present in controls. To assess the role of PC in inflammation, we used two different injury strategies, non-vascular tail transection and chemical treatment (copper sulfate), on 3 dpf zebrafish larvae. Staining for neutrophil granules revealed homing to the site of injury within 60-75 minutes. In proc-/- mutants we found an average 50% reduction in the number of neutrophils recruited to the site of injury yet counts in the caudal hematopoietic tissue (the site of larval hematopoiesis) were unchanged. Since protein S (PS) is a cofactor for PC anticoagulant function, we hypothesized that the consumptive coagulopathy, but not the neutrophil recruitment, would be PS-dependent. We used genome editing to disrupt the PS gene (pros1) and found that loss of PS also results in a mild consumptive coagulopathy, but spontaneous thrombus formation was less common in the PCV (25%) and was often in the heart instead (80%). Neutrophil recruitment was unaffected in pros1 mutants, and evaluation of double proc/pros1 mutants revealed no synergy in any of the phenotypes. In conclusion, PC and PS deficiency in zebrafish show some similarity to our previously reported model of AT3 deficiency, but the effects are less potent, allowing robust survival that enables in vivo analyses. Our data suggest that the thrombotic phenotypes of PC and PS deficiency are not identical, and display tissue-specific phenotypes. We also found evidence for PS-independent functions of PC in neutrophil migration. We speculate this is due to the role that PC plays in inflammation and signaling but cannot exclude a role in neutrophil extracellular trap (NET) formation. This model of complete proc-/- deficiency in an accessible organism will facilitate further in vivo study of PS-dependent and independent functions of PC, as well as interplay between the two factors. Disclosures Shavit: Bayer: Consultancy; Taked: Consultancy.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3−/−) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3−/− embryos. Slc52a3−/− mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Knockout of the gene encoding the extracellular matrix protein SNED1 results in early neonatal lethality and craniofacial malformations.

The extracellular matrix (ECM) is a fundamental component of multicellular organisms that orchestrates developmental processes and controls cell and tissue organization. We previously identified the novel ECM protein SNED1 as a promoter of breast cancer metastasis and showed that its level of expression negatively correlated with breast cancer patient survival. Here, we sought to identify the roles of SNED1 during murine development. We generated two novel Sned1 knockout mouse strains and showed that Sned1 is essential since homozygous ablation of the gene led to early neonatal lethality. Phenotypic analysis of the surviving knockout mice revealed a role for SNED1 in the development of craniofacial and skeletal structures since Sned1 knockout resulted in growth defects, nasal cavity occlusion, and craniofacial malformations. Sned1 is widely expressed in embryos, notably by cell populations undergoing epithelial-to-mesenchymal transition, such as the neural crest cells. We further show that mice with a neural-crest-cell-specific deletion of Sned1 survive, but display facial anomalies partly phenocopying the global knockout mice. Our results demonstrate requisite roles for SNED1 during development and neonatal survival. Importantly, the deletion of 2q37.3 in humans, a region that includes the SNED1 locus, has been associated with facial dysmorphism and short stature.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

Deficiency and overexpression of Rtl1 in the mouse cause distinct muscle abnormalities related to Temple and Kagami-Ogata syndromes.

ABSTRACTTemple and Kagami-Ogata syndromes are genomic imprinting diseases caused by maternal and paternal duplication of human chromosome 14, respectively. They exhibit different postnatal muscle-related symptoms as well as prenatal placental problems. Using the mouse models for these syndromes, it has been demonstrated that retrotransposon gag like 1 [Rtl1, also known as paternally expressed 11 (Peg11)] located in the mouse orthologous imprinted region is responsible for the prenatal placental problems because it is an essential placental gene for maintenance of fetal capillary network during gestation. However, the causative imprinted gene for the postnatal muscle-related symptoms remains unknown. Here, we demonstrate that Rtl1 also plays an important role in fetal/neonatal skeletal muscle development: its deletion and overproduction in mice lead to neonatal lethality associated with severe but distinct skeletal muscle defects, similar to those of Temple and Kagami-Ogata syndromes, respectively. Thus, it is strongly suggested that RTL1 is the major gene responsible for the muscle defects in addition to the placental defects in these two genomic imprinting diseases. This is the first example of an LTR retrotransposon-derived gene specific to eutherians contributing to eutherian skeletal muscle development.

Two mouse models carrying truncating mutations in Magel2 show distinct phenotypes.

Schaaf-Yang syndrome (SYS) is a neurodevelopmental disorder caused by truncating variants in the paternal allele of MAGEL2, located in the Prader-Willi critical region, 15q11-q13. Although the phenotypes of SYS overlap those of Prader-Willi syndrome (PWS), including neonatal hypotonia, feeding problems, and developmental delay/intellectual disability, SYS patients show autism spectrum disorder and joint contractures, which are atypical phenotypes for PWS. Therefore, we hypothesized that the truncated Magel2 protein could potentially produce gain-of-function toxic effects. To test the hypothesis, we generated two engineered mouse models; one, an overexpression model that expressed the N-terminal region of Magel2 that was FLAG tagged with a strong ubiquitous promoter, and another, a genome-edited model that carried a truncating variant in Magel2 generated using the CRISPR/Cas9 system. In the overexpression model, all transgenic mice died in the fetal or neonatal period indicating embryonic or neonatal lethality of the transgene. Therefore, overexpression of the truncated Magel2 could show toxic effects. In the genome-edited model, we generated a mouse model carrying a frameshift variant (c.1690_1924del; p(Glu564Serfs*130)) in Magel2. Model mice carrying the frameshift variant in the paternal or maternal allele of Magel2 were termed Magel2P:fs and Magel2M:fs, respectively. The imprinted expression and spatial distribution of truncating Magel2 transcripts in the brain were maintained. Although neonatal Magel2P:fs mice were lighter than wildtype littermates, Magel2P:fs males and females weighed the same as their wildtype littermates by eight and four weeks of age, respectively. Collectively, the overexpression mouse model may recapitulate fetal or neonatal death, which are the severest phenotypes for SYS. In contrast, the genome-edited mouse model maintains genomic imprinting and distribution of truncated Magel2 transcripts in the brain, but only partially recapitulates SYS phenotypes. Therefore, our results imply that simple gain-of-function toxic effects may not explain the patho-mechanism of SYS, but rather suggest a range of effects due to Magel2 variants as in human SYS patients.

Glucuronyl C5-epimerase is crucial for epithelial cell maturation during embryonic lung development.

Glucuronyl C5-epimerase (Hsepi) is a key enzyme in the biosynthesis of heparan sulfate that is a sulfated polysaccharide expressed on the cell surface and in the extracellular matrix of alveolar walls and blood vessels. Targeted interruption of the Hsepi gene, Glce, in mice resulted in neonatal lethality, which is most likely due to lung atelectasis. In this study, we examined the potential mechanisms behind the defect in lung development. Histological analysis of the lungs from embryos revealed no difference in the morphology between wild-type and mutant animals up to E16.5. This suggests that the initial events leading to formation of the lung primordium and branching morphogenesis are not disturbed. However, the distal lung of E17.5-18.5 mutants is still populated by epithelial tubules, lacking the typical saccular structural characteristic of a normal E17.5 lung. Immunostaining revealed strong signals of surfactant protein-C, but a weaker signal of T1α in the mutant lungs in comparison to WT littermates, suggesting differentiation of type I alveolar epithelial cells (AT1) is impaired. One of the parameters contributed to the failure of AT1 maturation is reduced vascularization in the developing lungs.

Influence of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 on epithelial differentiation and organization during lung development.

Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1-/- mutant mice. Mating of Aimp1+/- produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1-/- pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1-/- lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1-/- mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.

ApoC2 deficiency elicits severe hypertriglyceridemia and spontaneous atherosclerosis: A rodent model rescued from neonatal death

RationaleApoC2 is an important activator for lipoprotein lipase-mediated hydrolysis of triglyceride-rich plasma lipoproteins. ApoC2-deficient patients display severe hypertriglyceridemia (sHTG) and recurrent acute pancreatitis. However, due to embryonic lethality in ApoC2 deleted mouse extensive understanding of ApoC2 function is limited in mammalian species. ObjectiveWe sought to generate an animal model with ApoC2 deficiency in a rodent with some human-like features and then study the precise effects of ApoC2 on lipid and glucose homeostasis. Methods and resultsUsing CRISPR/Cas9, we deleted Apoc2 gene from golden Syrian hamster and the homozygous (−/−) pups can be born in matured term but exhibited neonatal lethality. By continuous iv administration of normal hamster serum the ApoC2−/− pups could survive till weaning and displayed severe HTG in adulthood on chow diet. A single iv injection of AAV-hApoC2 at birth can also rescue the neonatal death of ApoC2−/− pups. Adult ApoC2−/−hamsters exhibited a unique phenotype of sHTG with hypoglycemia, hypoinsulinemia and spontaneous atherosclerosis. The sHTG in ApoC2−/− adult hamsters could not be corrected by various lipid-lowering medications, but partially ameliorated by medium chain triglyceride diet and completely corrected by AAV-hApoC2. ConclusionsOur study provides a novel ApoC2-deleted mammalian model with severe hypertriglyceridemia that was fully characterized and highlights a potential therapeutic approach for the treatment of ApoC2 deficient patients.

Cathelicidin represents a new target for manipulation of skin inflammation in Netherton syndrome

Netherton syndrome (NS) is a severe ichthyosis caused by inactivating mutations in the SPINK5 gene encoding the serine protease inhibitor LEKTI. Spink5−/− mice recapitulate NS and die perinatally from extensive dehydration as a result of a severe defect of the epidermal barrier. We showed that deletion of Klk5 in Spink5−/− rescues neonatal lethality (Furio et al., 2015). However, Spink5−/−Klk5−/− mice developed skin shedding and inflammation during the first week from birth and the majority (70%) succumbed on P7. The remaining mice lived short (i.e. mean survival was 5 months) indicating alternative inflammatory pathways. Since cathelicidin is increased in Spink5−/− epidermis, we investigated whether it could be implicated in NS pathology. Ablation of Camp in Spink5−/− suppressed epidermal inflammation and restored abnormal epidermal differentiation, nevertheless, it failed to inhibit overdesquamation and Spink5−/−Camp−/− succumbed perinatally due to skin barrier defect, similarly to Spink5−/−. Joint invalidation of Klk5 and Camp significantly extended survival of Spink5−/−Klk5−/−Camp−/− mice. We provide evidence that cathelicidin is implicated in NS-associated skin inflammation in vivo. Therefore, marketed products that are known to reduce cathelicidin expression could be repurposed for the management of NS.