A long non-coding RNA Leat1 mediates the hormone responsiveness of EfnB2 during male urogenital development.

The novel long non-coding RNA (lncRNA) Leat1 is extraordinarily conserved in both its location (syntenic with EfnB2, an essential gene in anogenital patterning) and sequence. Here we show that Leat1 is upregulated following the testosterone surge from the developing testis and directly interacts with EfnB2, positively regulating its expression. Leat1 expression is suppressed by estrogen, which in turn suppresses the expression of EfnB2. Moreover, the loss of Leat1 leads to reduced EfnB2, resulting in a severe hypospadias phenotype. The human LEAT1 gene is also co-expressed with EFNB2 in the developing human penis suggesting a conserved function for this gene in urethral closure. Together our data identify Leat1 as a novel molecular regulator of urethral closure and implicate it as a target of endocrine disruption in the etiology of hypospadias.

PGC1α is required for the renoprotective effect of lncRNA Tug1 in vivo and links Tug1 with urea cycle metabolites.

lncRNA taurine-upregulated gene 1 (Tug1) is a promising therapeutic target in the progression of diabetic nephropathy (DN), but the molecular basis of its protection remains poorly understood. Here, we generate a triple-mutant diabetic mouse model coupled with metabolomic profiling data to interrogate whether Tug1 interaction with peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) is required for mitochondrial remodeling and progression of DN in vivo. We find that, compared with diabetic conditional deletion of Pgc1α in podocytes alone (db/db; Pgc1αPod-f/f), diabetic Pgc1α knockout combined with podocyte-specific Tug1 overexpression (db/db; TugPodTg; Pgc1αPod-f/f) reverses the protective phenotype of Tug1 overexpression, suggesting that PGC1α is required for the renoprotective effect of Tug1. Using unbiased metabolomic profiling, we find that altered urea cycle metabolites and mitochondrial arginase 2 play an important role in Tug1/PGC1α-induced mitochondrial remodeling. Our work identifies a functional role of the Tug1/PGC1α axis on mitochondrial metabolic homeostasis and urea cycle metabolites in experimental models of diabetes.

Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1.

Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.

A novel long non-coding RNA, Leat1, causes reduced anogenital distance and fertility in female mice.

Defective anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. Only a few molecular cues have been identified in early formation of the female urogenital system. Here we describe a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is syntenic with EfnB2 (which encodes EphrinB2) and expressed during embryonic development of the genital tubercle. While lncRNAs have varied functions, many are known to regulate their neighbouring genes. Eph/Ephrin bidirectional signaling molecules mediate many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 and its possible regulation of EphrinB2 during development of the female reproductive tract. We show that a loss of Leat1 leads to reduced EfnB2 expression in the developing female genital tubercle, reduced anogenital distance and decreased fertility.

Real-time in vivo mitochondrial redox assessment confirms enhanced mitochondrial reactive oxygen species in diabetic nephropathy.

While increased mitochondrial reactive oxygen species have been commonly implicated in a variety of disease states, their in vivo role in the pathogenesis of diabetic nephropathy remains controversial. Using a two-photon imaging approach with a genetically encoded redox biosensor, we monitored mitochondrial redox state in the kidneys of experimental models of diabetes in real-time in vivo. Diabetic (db/db) mice that express a redox-sensitive Green Fluorescent Protein biosensor (roGFP) specifically in the mitochondrial matrix (db/dbmt-roGFP) were generated, allowing dynamic monitoring of redox changes in the kidneys. These db/dbmt-roGFP mice exhibited a marked increase in mitochondrial reactive oxygen species in the kidneys. Yeast NADH-dehydrogenase, a mammalian Complex I homolog, was ectopically expressed in cultured podocytes, and this forced expression in roGFP-expressing podocytes prevented high glucose-induced increases in mitochondrial reactive oxygen species. Thus, in vivo monitoring of mitochondrial roGFP in diabetic mice confirms increased production of mitochondrial reactive oxygen species in the kidneys.

Sclt1 deficiency causes cystic kidney by activating ERK and STAT3 signaling.

Ciliopathies form a group of inherited disorders sharing several clinical manifestations because of abnormal cilia formation or function, and few treatments have been successful against these disorders. Here, we report a mouse model with mutated Sclt1 gene, which encodes a centriole distal appendage protein important for ciliogenesis. Sodium channel and clathrin linker 1 (SCLT1) mutations were associated with the oral-facial-digital syndrome (OFD), an autosomal recessive ciliopathy. The Sclt1-/- mice exhibit typical ciliopathy phenotypes, including cystic kidney, cleft palate and polydactyly. Sclt1-loss decreases the number of cilia in kidney; increases proliferation and apoptosis of renal tubule epithelial cells; elevates protein kinase A, extracellular signal-regulated kinases, SMAD and signal transducer and activator of transcription 3 (STAT3) pathways; and enhances pro-inflammation and pro-fibrosis pathways with disease progression. Embryonic kidney cyst formation of Sclt1-/- mice was effectively reduced by an anti-STAT3 treatment using pyrimethamine. Overall, we reported a new mouse model for the OFD; and our data suggest that STAT3 inhibition may be a promising treatment for SCLT1-associated cystic kidney.

Dynamic imaging and quantitative analysis of cranial neural tube closure in the mouse embryo using optical coherence tomography.

Neural tube closure is a critical feature of central nervous system morphogenesis during embryonic development. Failure of this process leads to neural tube defects, one of the most common forms of human congenital defects. Although molecular and genetic studies in model organisms have provided insights into the genes and proteins that are required for normal neural tube development, complications associated with live imaging of neural tube closure in mammals limit efficient morphological analyses. Here, we report the use of optical coherence tomography (OCT) for dynamic imaging and quantitative assessment of cranial neural tube closure in live mouse embryos in culture. Through time-lapse imaging, we captured two neural tube closure mechanisms in different cranial regions, zipper-like closure of the hindbrain region and button-like closure of the midbrain region. We also used OCT imaging for phenotypic characterization of a neural tube defect in a mouse mutant. These results suggest that the described approach is a useful tool for live dynamic analysis of normal neural tube closure and neural tube defects in the mouse model.

Long noncoding RNA Tug1 regulates mitochondrial bioenergetics in diabetic nephropathy.

The regulatory roles of long noncoding RNAs (lncRNAs) in transcriptional coactivators are still largely unknown. Here, we have shown that the peroxisome proliferator-activated receptor γ (PPARγ) coactivator α (PGC-1α, encoded by Ppargc1a) is functionally regulated by the lncRNA taurine-upregulated gene 1 (Tug1). Further, we have described a role for Tug1 in the regulation of mitochondrial function in podocytes. Using a murine model of diabetic nephropathy (DN), we performed an unbiased RNA-sequencing (RNA-seq) analysis of kidney glomeruli and identified Tug1 as a differentially expressed lncRNA in the diabetic milieu. Podocyte-specific overexpression (OE) of Tug1 in diabetic mice improved the biochemical and histological features associated with DN. Unexpectedly, we found that Tug1 OE rescued the expression of PGC-1α and its transcriptional targets. Tug1 OE was also associated with improvements in mitochondrial bioenergetics in the podocytes of diabetic mice. Mechanistically, we found that the interaction between Tug1 and PGC-1α promotes the binding of PGC-1α to its own promoter. We identified a Tug1-binding element (TBE) upstream of the Ppargc1a gene and showed that Tug1 binds with the TBE to enhance Ppargc1a promoter activity. These findings indicate that a direct interaction between PGC-1α and Tug1 modulates mitochondrial bioenergetics in podocytes in the diabetic milieu.

Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice.

After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2-3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.

miR-93 regulates Msk2-mediated chromatin remodelling in diabetic nephropathy.

How the kidney responds to the metabolic cues from the environment remains a central question in kidney research. This question is particularly relevant to the pathogenesis of diabetic nephropathy (DN) in which evidence suggests that metabolic events in podocytes regulate chromatin structure. Here, we show that miR-93 is a critical metabolic/epigenetic switch in the diabetic milieu linking the metabolic state to chromatin remodelling. Mice with inducible overexpression of a miR-93 transgene exclusively in podocytes exhibit significant improvements in key features of DN. We identify miR-93 as a regulator of nucleosomal dynamics in podocytes. miR-93 has a critical role in chromatin reorganization and progression of DN by modulating its target Msk2, a histone kinase, and its substrate H3S10. These findings implicate a central role for miR-93 in high glucose-induced chromatin remodelling in the kidney, and provide evidence for a previously unrecognized role for Msk2 as a target for DN therapy.

Primary Ovarian Insufficiency Induced by Fanconi Anemia E Mutation in a Mouse Model.

In most cases of primary ovarian insufficiency (POI), the cause of the depletion of ovarian follicles is unknown. Fanconi anemia (FA) proteins are known to play important roles in follicular development. Using random insertional mutagenesis with a lentiviral transgene, we identified a family with reduced fertility in the homozygous transgenic mice. We identified the integration site and found that the lentivirus had integrated into intron 8 of the Fanconi E gene (Fance). By RT-PCR and in situ hybridization, we found that Fance transcript levels were significantly reduced. The Fance homozygous mutant mice were assayed for changes in ovarian development, follicle numbers and estrous cycle. Ovarian dysplasias and a severe lack of follicles were seen in the mutant mice. In addition, the estrous cycle was disrupted in adult females. Our results suggest that POI has been induced by the Fance mutation in this new mouse model.

Live four-dimensional optical coherence tomography reveals embryonic cardiac phenotype in mouse mutant.

Efficient phenotyping of developmental defects in model organisms is critical for understanding the genetic specification of normal development and congenital abnormalities in humans. We previously reported that optical coherence tomography (OCT) combined with live embryo culture is a valuable tool for mouse embryo imaging and four-dimensional (4-D) cardiodynamic analysis; however, its capability for analysis of mouse mutants with cardiac phenotypes has not been previously explored. Here, we report 4-D (three-dimensional+time) OCT imaging and analysis of the embryonic heart in a Wdr19 mouse mutant, revealing a heart looping defect. Quantitative analysis of cardiac looping revealed a statistically significant difference between mutant and control embryos. Our results indicate that live 4-D OCT imaging provides a powerful phenotyping approach to characterize embryonic cardiac function in mouse models.

Human adipose tissue as a reservoir for memory CD4+ T cells and HIV.

OBJECTIVE: The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. DESIGN: We examined memory CD4(+) T cells and HIV DNA in adipose tissue-stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4(+) T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4(+) T cells. METHODS: AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4(+) T-cell activation and HIV production were measured by flow cytometry and ELISA. RESULTS: AT-SVF CD3(+) T cells were activated (>60% CD69(+)) memory CD4(+) and CD8(+) T cells in uninfected and HIV-infected persons, but the AT-SVF CD4(+)/CD8(+) ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4(+) T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-α1ß1. Adipocytes also enhanced T-cell viability. CONCLUSION: Adipose tissues of ART-treated patients harbour activated memory CD4(+) T cells and HIV DNA. Adipocytes promote CD4(+) T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.

Mutant cohesin in premature ovarian failure.

Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.

The candidate splicing factor Sfswap regulates growth and patterning of inner ear sensory organs.

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.

A transgenic insertion on mouse chromosome 17 inactivates a novel immunoglobulin superfamily gene potentially involved in sperm-egg fusion.

Fertilization is the process that leads to the formation of a diploid zygote from two haploid gametes. This is achieved through a complex series of cell-to-cell interactions between a sperm and an egg. The final event of fertilization is the fusion of the gametes' membranes, which allows the delivery of the sperm genetic material into the egg cytoplasm. In vivo studies in the laboratory mouse have led to the discovery of membrane proteins that are essential for the fusion process in both the sperm and egg. Specifically, the sperm protein Izumo1 was shown to be necessary for normal fertility. Izumo1-deficient spermatozoa fail to fuse with the egg plasma membrane. Izumo1 is a member of the Immunoglobulin Superfamily of proteins, which are known to be involved in cell adhesion. Here, we describe BART97b, a new mouse line with a recessive mutation that displays a fertilization block associated with a failure of sperm fusion. BART97b mutants carry a deletion that inactivates Spaca6, a previously uncharacterized gene expressed in testis. Similar to Izumo1, Spaca6 encodes an immunoglobulin-like protein. We propose that the Spaca6 gene product may, together with Izumo1, mediate sperm fusion by binding an as yet unidentified egg membrane receptor.

Histone posttranslational modifications and cell fate determination: lens induction requires the lysine acetyltransferases CBP and p300.

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(-/-) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens.

Novel frem1-related mouse phenotypes and evidence of genetic interactions with gata4 and slit3.

The FRAS1-related extracellular matrix 1 (FREM1) gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organ systems. In humans, recessive mutations in FREM1 cause eye defects, congenital diaphragmatic hernia, renal anomalies and anorectal malformations including anteriorly placed anus. A similar constellation of findings-microphthalmia, cryptophthalmos, congenital diaphragmatic hernia, renal agenesis and rectal prolapse-have been described in FREM1-deficient mice. In this paper, we identify a homozygous Frem1 missense mutation (c.1687A>T, p.Ile563Phe) in an N-ethyl-N-nitrosourea (ENU)-derived mouse strain, crf11, with microphthalmia, cryptophthalmos, renal agenesis and rectal prolapse. This mutation affects a highly conserved residue in FREM1's third CSPG domain. The p.Ile563Phe change is predicted to be deleterious and to cause decreased FREM1 protein stability. The crf11 allele also fails to complement the previously described eyes2 allele of Frem1 (p.Lys826*) providing further evidence that the crf11 phenotype is due to changes affecting Frem1 function. We then use mice bearing the crf11 and eyes2 alleles to identify lung lobulation defects and decreased anogenital distance in males as novel phenotypes associated with FREM1 deficiency in mice. Due to phenotypic overlaps between FREM1-deficient mice and mice that are deficient for the retinoic acid-responsive transcription factor GATA4 and the extracellular matrix protein SLIT3, we also perform experiments to look for in vivo genetic interactions between the genes that encode these proteins. These experiments reveal that Frem1 interacts genetically with Gata4 in the development of lung lobulation defects and with Slit3 in the development of renal agenesis. These results demonstrate that FREM1-deficient mice faithfully recapitulate many of the phenotypes seen in individuals with FREM1 deficiency and that variations in GATA4 and SLIT3 expression modulate some FREM1-related phenotypes in mice.

Optical coherence tomography for live phenotypic analysis of embryonic ocular structures in mouse models.

Mouse models of ocular diseases provide a powerful resource for exploration of molecular regulation of eye development and pre-clinical studies. Availability of a live high-resolution imaging method for mouse embryonic eyes would significantly enhance longitudinal analyses and high-throughput morphological screening. We demonstrate that optical coherence tomography (OCT) can be used for live embryonic ocular imaging throughout gestation. At all studied stages, the whole eye is within the imaging distance of the system and there is a good optical contrast between the structures. We also performed OCT eye imaging in the embryonic retinoblastoma mouse model Pax6-SV40 T-antigen, which spontaneously forms lens and retinal lesions, and demonstrate that OCT allows us to clearly differentiate between the mutant and wild type phenotypes. These results demonstrate that OCTin utero imaging is a potentially useful tool to study embryonic ocular diseases in mouse models.

A spontaneous Fatp4/Scl27a4 splice site mutation in a new murine model for congenital ichthyosis.

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3'-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.