Mouse Gene Research Articles

THBS1 is a new autosomal recessive non-syndromic hearing impairment gene

BackgroundPrelingual hearing impairment (HI) is genetically highly heterogenous. Early diagnosis and intervention are essential for psychosocial development. In this study we investigated a consanguineous family from Pakistan with autosomal recessive (AR) non-syndromic sensorineural HI (NSHI).MethodsA DNA sample from an HI member of a consanguineous Pakistani family segregating ARNSHL underwent exome sequencing. Using Sanger sequencing select variants were validated and tested for segregation using DNA samples from additional family members. We further investigated RNA expression data for the candidate gene in mouse and human inner ear and human inner ear organoids using data obtained from the gene Expression Analysis Resource.ResultsWe identified thrombospondin 1 (THBS1) as a new NSHI gene. A homozygous frameshift variant [c.1470del: p.(Ile491Serfs*45)] was observed in the three hearing-impaired and in the heterozygous state in three unaffected family members. Unlike for most ARNSHI, hearing-impaired individuals had audiograms with a sloping pattern, showing more pronounced HI in the mid and high frequencies (ranging from moderate to profound) compared to the low frequencies. RNA expression data indicates THBS1 is expressed during human inner ear development. Additionally, THBS1 is expressed in the cochlear epithelium and supporting cells of the mouse inner ear during embryonic and postnatal stages. Previously, THBS1 was demonstrated to affect hearing in knockout mice by influencing the formation and function of afferent synapses in the inner ear.ConclusionsOur findings highlight THBS1 as a potential novel candidate gene for human HI characterized by a sloping high-frequency audio profile. This discovery enhances our understanding of the genetic etiology of HI and will aid in advancing molecular diagnosis.

Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.

Accumulation of free α-globin is a critical factor in the pathogenesis of β-thalassemia. Autophagy plays a crucial role in clearing toxic free α-globin, thereby reducing disease severity. However, the impact of natural mutations in autophagy-related genes (ATGs) on the phenotypic variability of β-thalassemia remains unclear. In this study, we systematically investigated the relationship between variants in ATGs and disease phenotypes in a cohort of 1,022 patients with β-thalassemia, identifying four missense mutations in the autophagy and beclin 1 regulator 1 (AMBRA1) gene. Disruption of the Ambra1 gene in β-thalassemic mice was found to reduce autophagic clearance of α-globin in red blood cell precursors, exacerbating disease phenotypes. Functional characterization of the AMBRA1 gene and these mutations in patient-derived CD34+ cells, edited HUDEP-2 cells, and engineered HUDEP-2 β-thalassemic cells confirmed that AMBRA1 facilitates the autophagic clearance of free α-globin in human erythroid cells. Functional studies demonstrated that AMBRA1 missense mutants destabilize ULK1 protein, inhibit LC3 lipidation, and subsequently hinder autophagic flux, leading to increased α-globin deposition. Additionally, these mutations were associated with erythrotoxic effects in vitro, including increased intracellular reactive oxygen species levels, higher apoptosis rates, and impaired erythroid differentiation and maturation. This study sheds light on the molecular association between mutations in ATGs and the exacerbation of β-thalassemia, highlighting the potential role of the AMBRA1 gene as a promising diagnostic and therapeutic target for β-hemoglobinopathies.

Intrauterine administration of peripheral blood mononuclear cells helps manage recurrent implantation failure by normalizing dysregulated gene expression including estrogen-responsive genes in mice

BackgroundIntrauterine peripheral blood mononuclear cell (PBMC) therapy for recurrent implantation failure (RIF) has been reported to improve embryo implantation by acting on the endometrium. However, the exact mode of action of PBMC on the endometrium of patients with RIF remains unclear. In addition, the differences in the therapeutic effects of PBMC therapy with and without human chorionic gonadotropin (hCG) are unknown. Therefore, in this study, we investigated the changes in the endometrium during the implantation phase induced by PBMC administration and the differences in the efficacy of this therapy with and without hCG using a mouse model of implantation failure (IF).MethodsIF model was established by the subcutaneous administration of low-dose RU486. Pregnant mice were randomly divided into five groups: control, IF, culture medium, PBMC, and PBMC-hCG (the latter three groups were IF model mice with intrauterine administration). The pregnancy rate and the number and size of implantation sites were recorded during early pregnancy (day 7.5). Uteri from the preimplantation phase (evening of day 3.5) were collected and analyzed using RNA-sequencing (RNA-seq).ResultsThe pregnancy rate, the number of implantation sites, and the number of normal-sized implantation sites were significantly decreased in the IF model and were improved in the medium, PBMC, and PBMC-hCG groups. RNA-seq data showed that PBMC treatment normalized the expression of the majority of dysregulated genes in the endometrium during the preimplantation phase in the IF model, especially the overexpression of estrogen-activated genes. In addition, PBMC treatment increased the expression of local glucocorticoid receptors and suppressed the expression of inflammation-related genes, whereas no significant changes in blood estradiol and glucocorticoid levels were observed. These changes were more pronounced in the PBMC-hCG group and were consistent with the pregnancy outcomes.ConclusionsIntrauterine administration of PBMC before embryo implantation promoted embryo implantation in the IF mouse model, and hCG enhanced pregnancy outcomes. PBMC modulated steroid receptor expression and suppressed inflammation and excessive estrogen action.

Rapid effects of valproic acid on the fetal brain transcriptome: implications for brain development and autism

There is an increased incidence of autism among the children of women who take the anti-epileptic, mood-stabilizing drug, valproic acid (VPA) during pregnancy; moreover, exposure to VPA in utero causes autistic-like symptoms in rodents and non-human primates. Analysis of RNA-seq data obtained from E12.5 fetal mouse brains 3 hours after VPA administration to the pregnant dam revealed that VPA rapidly and significantly increased or decreased the expression of approximately 7,300 genes. No significant sex differences in VPA-induced gene expression were observed. Expression of 399 autism risk genes was significantly altered by VPA as was expression of 258 genes that have been reported to modulate fetal brain development but are not otherwise linked to autism. Expression of genes associated with intracellular signaling pathways, neurogenesis, and excitation-inhibition balance as well as synaptogenesis, neuronal fate determination, axon and dendritic development, neuroinflammation, circadian rhythms, and epigenetic modulation of gene expression was dysregulated by VPA. Notably, at least 40 genes that are known to regulate embryonic neurogenesis were dysregulated by VPA. The goal of this study was to identify mouse genes that are: (a) significantly up- or downregulated by VPA in the fetal brain and (b) associated with autism and/or known to play a role in embryonic neurodevelopmental processes, perturbation of which has the potential to alter brain connectivity and, consequently behavior, in the adult. The genes meeting these criteria provide potential targets for future hypothesis-driven studies to elucidate the proximal causes of errors in brain connectivity underlying neurodevelopmental disorders such as autism.

Multi-omics mechanical analysis of gut microbiota, carboxylic acids, and cardiac gene expression interaction triggering diabetic cardiomyopathy.

Our research results clearly link the changes in heart genes of T2DM and normal mice with changes in serum metabolites and gut microbiota, indicating that some genes in biological processes are closely related to the reduction of protective microbiota in the gut microbiota. This study provides a theoretical basis for investigating the mechanism of DCM and may provide preliminary evidence for the future use of gut microbiota therapy for DCM.

Retraction: Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice.

Retraction: Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice.

A sensitive red/far-red photoswitch for controllable gene therapy in mouse models of metabolic diseases

Red light optogenetic systems are in high demand for the precise control of gene expression for gene- and cell-based therapies. Here, we report a red/far-red light-inducible photoswitch (REDLIP) system based on the chimeric photosensory protein FnBphP (Fn-REDLIP) or PnBphP (Pn-REDLIP) and their interaction partner LDB3, which enables efficient dynamic regulation of gene expression with a timescale of seconds without exogenous administration of a chromophore in mammals. We use the REDLIP system to establish the REDLIP-mediated CRISPR-dCas9 (REDLIPcas) system, enabling optogenetic activation of endogenous target genes in mammalian cells and mice. The REDLIP system is small enough to support packaging into adeno-associated viruses (AAVs), facilitating its therapeutic application. Demonstrating its capacity to treat metabolic diseases, we show that an AAV-delivered Fn-REDLIP system achieved optogenetic control of insulin expression to effectively lower blood glucose levels in type 1 diabetes model mice and control an anti-obesity therapeutic protein (thymic stromal lymphopoietin, TSLP) to reduce body weight in obesity model mice. REDLIP is a compact and sensitive optogenetic tool for reversible and non-invasive control that can facilitate basic biological and biomedical research.

Early life lipid overload in Native American Myopathy is phenocopied by stac3 knockout in zebrafish

Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving musculoskeletal muscle performance, such as restoring the functional integrity of the cytoskeleton. SH3 and cysteine-rich domain 3 (STAC3) are proteins involved in nutrient regulation and are an essential component of the excitation–contraction (EC) coupling machinery for Ca2+ releasing. A mutation in STAC3 causes debilitating Native American Myopathy (NAM) in humans, while loss of this gene in mice and zebrafish (ZF) results in premature death. Clinically, NAM patients demonstrated increased lipids in skeletal muscle, but it is unclear if neutral lipids are associated with altered muscle function in NAM. Using a CRISPR/Cas9 induced stac3-/- knockout (KO) zebrafish model, we determined that loss of stac3 leads to delayed larval hatching which corresponds with muscle weakness and decreased whole-body Ca2+ level during early skeletal development. Specifically, we observed defects in the cytoskeleton in F-actin and slow muscle fibers at 5 and 7 days post-fertilizations (dpf). Myogenesis regulators such as myoD and myf5, mstnb were significantly altered in stac3-/- larvae. These muscle alterations were associated with elevated neutral lipid levels starting at 5 dpf and persisting beyond 7 dpf. Larva lacking stac3 had reduced viability with no larva knockouts surviving past 11 dpf. This data suggests that our stac3-/- zebrafish serve as an alternative model to study the diminished muscle function seen in NAM patients. The data gathered from this new model over time supports a mechanistic view of lipotoxicity as a critical part of the pathology of NAM and the associated loss of function in muscle.

Immune cell-enriched single-cell RNA sequencing unveils the interplay between infiltrated CD8+ T resident memory cells and choroid plexus epithelial cells in Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurological disorder and the leading cause of dementia. Despite significant efforts, treatment strategies targeting amyloid-β have been less successful than anticipated. Recently, the role of neuroinflammation and adaptive immune response in AD pathogenesis has gained attention. Here, we performed immune cell-enriched single-cell RNA sequencing of brain parenchymal cells from 12-month-old 5xFAD, an AD mouse model. We analyzed 11,587 single cells and found distinct differences in T cell and choroid plexus cell populations between 5xFAD mouse and littermate control. Subsequent sub-clustering of T cells in the 5xFAD mouse revealed distinct subtypes, with CD8+ resident memory T cells (TRM) being the most prevalent T cell type. In addition, we observed an increase in T cell exhaustion markers, including Pdcd1, Ctla4, and Havcr2, with a particularly significant elevation of PD-1 and TIM-3 in CD8+ TRM in 5xFAD mouse. Furthermore, choroid plexus (ChP) epithelial cells showed altered gene expression patterns, with higher expression of MHC class I and Type I IFN-stimulated genes in 5xFAD mouse compared to the control mouse, suggesting an association with clonal expansion of AD-specific T cells in the brain. Through single-cell RNA sequencing (scRNA-seq) analysis, our study highlights the potential role of resident memory CD8+ T cell and their possible interactions with ChP epithelial cells. This study provides an exploration of the brain microenvironment landscape in AD, revealing critical insights into its underlying mechanisms.

Abstract A055: Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming

Abstract Aggressive human prostate tumors traverse a contractile smooth muscle pseudo-capsule, in a process termed extracapsular extension (ECE), defining the pT3 pathologic stage associating with increased biochemical recurrence, bone metastases, and cancer-specific mortality. While T3 lesions can be detected by non-invasive mpMRI imaging, how the tumor invades into and through the contractile muscle is unknown. Using a mouse xenograft model system of ECE, we investigated the transcriptomic consequences of human tumor invasion into and through the contractile smooth muscle of the mouse diaphragm. Both human and mouse gene transcripts were documented and analyzed. Tumor cells were intraperitoneally injected into male NSG mice and 6 weeks later, three tumor compartments (pre-invasive, muscle-invasive, and super-invasive cells that had reached the superior diaphragm surface) were analyzed for differential bulk human and mouse gene expression. Whole genome transcriptomic sequencing and GO pathway analysis revealed that approximately 414 human genes were differentially expressed between the non- invasive, muscle-resident, and super-invasive tumor populations and at least 5 enriched pathways were involved. The unique expression gene patterns of muscle-resident tumor cells were reversible when compared to the pre-invasive and super-invasive expression patterns. Significant increases in g-H2AX and nuclear deformation were observed in the muscle-resident tumor clusters as compared to the non-invasive tumor mass. No differences in tumor cell proliferation, as detected by Ki67 staining, were found between the tumor clusters in the three compartments. Immunohistochemistry staining for a damage response cytokeratin (KRT6A) and integrin (CD49f) revealed heterogeneity within the muscle resident tumors as compared to the non- invasive cells. Taken together, these data suggest a hostile contractile muscle environment elicits specific responses by the invading tumor. Single cell analysis within each of the tumor compartments is currently underway to define the spatial heterogeneity of gene expression in invasive tumor cell clusters within the contractile muscle. A dynamic reciprocity of the tumor/muscle microenvironment is suggested by the analysis of the bulk transcriptomic sequencing demonstrating a significant increase in mouse muscle bio-synthetic gene transcription as a consequence of the human tumor penetrating the tissue. Taken together, these data indicate that human tumors, during the act of traversing the contractile muscle layer, respond to this unique microenvironment by transiently altering transcription, while sustaining nuclear damage which results in the reprogramming of the muscle. This new information suggests that novel tumor or muscle biomarkers might indicate early muscle invasion events and assist new high-resolution image analysis for precision diagnostic and/or therapeutic decisions. (Supported in part by P30 CA23074; F30 CA143924, UACC Team Science Award). Citation Format: Kendra D Marr, Beatrice S Knudsen, Rafael Sainz, Kelvin W Pond, Noel E Warfel, Belinda E Sun, Anne E Cress. Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A055.

Mitochondria-Targeted DNA Repair Glycosylase hOGG1 Protects Against HFD-Induced Liver Oxidative Mitochondrial DNA Damage and Insulin Resistance in OGG1-Deficient Mice.

8-oxoguanine DNA glycosylase-1 (OGG1) is a DNA glycosylase mediating the first step in base excision repair which removes 7,8-dihydro-8-oxoguanine (8-oxoG) and repairs oxidized nuclear and mitochondrial DNA. Previous studies showed that OGG1 deficiency results in an increased susceptibility to high-fat diet (HFD)-induced obesity and metabolic dysfunction in mice, suggesting a crucial role of OGG1 in metabolism. However, the tissue-specific mechanisms of how OGG1 deficiency leads to insulin resistance is unknown. Thus, in the current study, we used a hyperinsulinemic-euglycemic clamp to evaluate in-depth glucose metabolism in male wild-type (WT) mice and Ogg1-/- (Ogg1-KO) mice fed an HFD. Ogg1-KO mice fed HFD were more obese, with significantly lower hepatic insulin action compared to WT/HFD mice. Targeting human OGG1 to mitochondria protected against HFD-induced obesity, insulin resistance, oxidative mitochondrial DNA damage in the liver and showed decreased expression of liver gluconeogenic genes in Ogg1-KO mice, suggesting a putative protective mechanism. Additionally, several subunits of oxidative phosphorylation protein levels were noticeably increased in Ogg1-KO/Tg compared to Ogg1-KO mice fed an HFD which was associated with improved insulin signaling. Our findings demonstrate the crucial role of mitochondrial hOGG1 in HFD-induced insulin resistance and propose several protective mechanisms which can further direct the development of therapeutic treatment.

Abstract 4133040: Cardiac Favored Transcription of the Exogenous Gene in Mice after A Single Systemic Delivery of AAV9-cBIN1

Introduction: Heart failure (HF) is associated with high mortality and morbidity, and there are limited therapeutic options available to improve cardiac muscle function. Recent studies suggest that the impairment of cardiomyocyte T-tubule microdomains is a key factor in HF pathophysiology. AAV9-cBIN1 gene therapy effectively addresses this impairment. However, the kinetics and biodistribution of AAV9-cBIN1 have not been explored, limiting the knowledge of the transcription efficiency and organ specificity of cBIN1 gene therapy when delivered systemically. Aim: This study aims to elucidate the kinetics and biodistribution of AAV9-cBIN1 in a murine model. Method: Adult male C57BL/6J mice (n=20) were administered a single retro-orbital injection of AAV9-CMV-cBIN1-V5 at a dose (2x10^12 vg/kg) known to effectively transduce the majority of cardiomyocytes. Mice were sacrificed at pre-determined time points: prior to injection (Day 0, n=2) and post-injection at Days 3, 7, 14, 28, 56, and 84 (n=3/group). Tissues were harvested from the heart, lung, liver, kidney, skeletal muscle, and spleen. Genomic DNA was extracted and analyzed using qPCR to determine vector genome (vg) copies. For transcriptional analysis of the AAV9-transduced exogenous gene, mRNA was extracted and converted to cDNA for qRT-PCR quantification of cBin1-V5 , normalized to the housekeeping gene Hprt1 . Results: All mice survived to their designated sacrifice times. Kinetic analysis demonstrated a time-dependent expression of the exogenous cBin1-V5 gene, with peak expression observed at 4 weeks in both the heart and liver, with expression declining in the liver after this peak (Figure 1A). Genomic DNA analysis showed a gradual increase and plateau of viral genome distribution in the heart between 2-4 weeks, while in the liver, distribution peaked sharply between Days 3-7 and then decreased. When correcting the gene transcription of cBin1-V5 (ΔCq of V5/Hprt1 ) for vector DNA vg copies in the tissue, the transcription efficiency in the heart was higher than in the liver (Figure 1B). Outside of the liver and heart, cBin1-V5 mRNA expression was undetectable in all other tissues by Day 56. Conclusion: In adult male mice, a single systemic administration of AAV9-cBIN1 results in robust transduction and higher transcription efficiency in the heart compared to the liver. AAV9-cBIN1 holds promise for targeted gene therapy in cardiac tissues, with minimal off-target expression in other organs.

Effects of the fruits on antioxidant capacity and modulation of ARE associated gene in mice

Effects of the fruits on antioxidant capacity and modulation of ARE associated gene in mice

Detection and Annotation of Unique Regions in Mammalian Genomes.

Long unique genomic regions have been reported to be highly enriched for developmental genes in mice and humans. In this paper we identify unique genomic regions using an efficient method based on fast string matching. We quantify the resource consumption and accuracy of this method before applying it to the genomes of 18 mammals. We annotate their unique regions of at least 10 kb and find that they are strongly enriched for developmental genes across the board. We then investigated the subset of unique regions that lack annotations, which we call "anonymous". The longest anonymous unique region in the tasmanian devil spanned 83 kb and contained the gene encoding inositol polyphosphate-5-phosphatase A, which is an essential part of intracellular signaling. This discovery of an essential gene in a unique region implies that unique regions might be given priority when annotating mammalian genomes. Our documented pipeline for annotating unique regions in any mammalian genome is available from the repository github.com/evolbioinf/auger; additional data for this study is available from the dataverse at doi.org/10.17617/3.4IKQAG.

Rescue of the endogenous FVIII expression in hemophilia A mice using CRISPR-Cas9 mRNA LNPs

Gene editing provides a promising alternative approach that may achieve long-term FVIII expression for hemophilia A (HemA) treatment. In this study, we investigated in vivo correction of a mutant factor VIII (FVIII) gene in HemA mice. We first developed MC3-based LNPs for efficient mRNA delivery into liver sinusoidal endothelial cells (LSECs), the major site of FVIII biosynthesis. To target a five base pair deletion in FVIII exon 1 in a specific HemA mouse strain, we injected LNPs encapsulating Cas9 mRNA and specifically designed sgRNAs intravenously for in vivo gene editing of the mutant FVIII. Indel variants generated at the mutant site contained mostly a single base pair deletion , resulting in frame-shift correction of FVIII gene. Sustained endogenous FVIII activity up to 6% was achieved over 26 weeks in treated HemA mice. Sequencing data indicated an average gene editing rate of 15.3% in LSECs. Our study suggests that optimized MC3 LNP formulations, combined with CRISPR/Cas9 technology, can effectively correct the mutant FVIII gene in LSECs and restore FVIII activity for therapeutic treatment of HemA.

Angiopoietin 1 Attenuates Dysregulated Angiogenesis in the Gastrocnemius of DMD Mice.

Duchenne muscular dystrophy (DMD) is a degenerative neuromuscular disease caused by a lack of functional dystrophin. Ang 1 paracrine signalling maintains the endothelial barrier of blood vessels, preventing plasma leakage. Chronic inflammation, a consequence of DMD, causes endothelial barrier dysfunction in skeletal muscle. We aim to elucidate changes in the DMD mouse's gastrocnemius microvascular niche following local administration of Ang 1. Gastrocnemii were collected from eight-week-old mdx/utrn+/- and healthy mice. Additional DMD cohort received an intramuscular injection of Ang 1 to gastrocnemius and contralateral control. Gastrocnemii were collected for analysis after two weeks. Using immunohistochemistry and real-time quantitative reverse transcription, we demonstrated an abundance of endothelial cells in DMD mouse's gastrocnemius, but morphology and gene expression were altered. Myofiber perimeters were shorter in DMD mice. Following Ang 1 treatment, fewer endothelial cells were present, and microvessels were more circular. Vegfr1, Vegfr2, and Vegfa expression in Ang 1-treated gastronemii increased, while myofiber size distribution was consistent with vehicle-only gastrocnemii. These results suggest robust angiogenesis in DMD mice, but essential genes were underexpressed-furthermore, exogenous Ang 1 attenuated angiogenesis. Consequentially, gene expression increased. The impact must be investigated further, as Ang 1 therapy may be pivotal in restoring the skeletal muscle microvascular niche.

Construction and validation of a mouse model for studying severe human adenovirus infections

Human adenoviruses (HAdVs) are highly contagious pathogens with various genotypes implicated in acute respiratory disease (ARD) and linked to mortality, especially in immunosuppressed patients, young children, and military recruits. Currently, no vaccines or specific drugs are approved for clinical use. The hosts of adenoviruses are strictly species-specific, which strongly limits the development of vaccines and drugs against HAdVs. In this study, immunocompetent BALB/c mice were challenged with different doses of human adenovirus type 5 (HAdV-5) via tail intravenous injection (i.v.). All mice challenged with a high dose of HAdV-5 (3.2×1010 TCID50/kg) died within 3 to 5 days, while those receiving a low dose of HAdV-5 (8×109 or 4×109 TCID50/kg) survived. Interestingly, among the mice receiving a medium dose of HAdV-5 (1.6×1010 TCID50/kg), 60% (n = 3/5) of male mice died, while all female mice survived. This suggests that male mice may be more susceptible to HAdV-5 infection than female mice, consistent with clinical findings in children. HAdV-5 DNA was mainly distributed in the liver, followed by the spleen and lungs. Pathological changes were observed in the lungs, liver, and spleen, with severity increasing in correlation with the virus challenge dosage. Transcriptome and qPCR analyses of the liver indicated that the down-regulated expression of the H2-Aa, H2-Ea-ps, CD74, and H2-Eb1 genes in male mice, as well as the AHR gene in female mice, may contribute to the observed higher mortality rates in male mice. Therefore, this effective, feasible, and cost-efficient mouse model could serve as a candidate for evaluating HAdV vaccines and anti-adenovirus therapeutics.

Combined effects of genetic background and diet on mouse metabolism and gene expression

Combined effects of genetic background and diet on mouse metabolism and gene expression

Beyond genomic studies of congenital heart defects through systematic modelling and phenotyping.

Congenital heart defects (CHDs), the most common congenital anomalies, are considered to have a significant genetic component. However, despite considerable efforts to identify pathogenic genes in patients with CHDs, few gene variants have been proven as causal. The complexity of the genetic architecture underlying human CHDs likely contributes to this poor genetic discovery rate. However, several other factors are likely to contribute. For example, the level of patient phenotyping required for clinical care may be insufficient for research studies focused on mechanistic discovery. Although several hundred mouse gene knockouts have been described with CHDs, these are generally not phenotyped and described in the same way as CHDs in patients, and thus are not readily comparable. Moreover, most patients with CHDs carry variants of uncertain significance of crucial cardiac genes, further complicating comparisons between humans and mouse mutants. In spite of major advances in cardiac developmental biology over the past 25 years, these advances have not been well communicated to geneticists and cardiologists. As a consequence, the latest data from developmental biology are not always used in the design and interpretation of studies aimed at discovering the genetic causes of CHDs. In this Special Article, while considering other in vitro and in vivo models, we create a coherent framework for accurately modelling and phenotyping human CHDs in mice, thereby enhancing the translation of genetic and genomic studies into the causes of CHDs in patients.

Long Noncoding RNAs Expressed in Mouse Pituitary Development and Mature Hormone-Producing Cells.

Mammalian genomes contain thousands of genes for long noncoding RNA (lncRNAs), some of which have been shown to affect protein coding gene expression through diverse mechanisms. The lncRNA transcripts are longer than 200 nucleotides and are often capped, spliced, and polyadenylated, but not translated into protein. Nuclear lncRNAs can modify chromatin structure and transcription in trans or cis by interacting with the DNA, forming R-loops, and recruiting regulatory proteins. Not much is known about the role of lncRNA in pituitary gland differentiation and function. We mined transcriptome data from mouse pituitary glands collected at embryonic days 12.5 and 14.5 and identified over 200 different lncRNA transcripts. To develop a research resource for the study of lncRNA, we used pituitary cre transgenes to tag pituitary cell types in adult mice with fluorescent markers, and enriched for thyrotropes, gonadotropes, and somatotropes using fluorescence-activated cell sorting. We determined the transcriptome of each cell population using RNA sequencing and mined the data for lncRNA. We detected hundreds of lncRNAs in adult pituitary cells; a few were located immediately nearby genes that encode pituitary hormones or lineage-specific transcription factors. The location of these lncRNAs suggests the possibility of a cis-acting regulatory role in pituitary development or function, and we observe coordinated expression of 2 of them with their putative target genes in transgenic mice. This research resource sets the foundation for examining the actions of lncRNAs on their putative target genes and determining whether they have roles during development and in response to physiological demand.