High-Throughput,High-Frequency 3-D Ultrasound for in Utero Analysis of Embryonic Mouse Brain Development

With the emergence of the mouse as the predominant model system for studying mammalian brain development, in utero imaging methods are urgently required to analyze the dynamics of brain growth and patterning in mouse embryos. To address this need, we combined synthetic focusing with a high-frequency (38-MHz) annular-array ultrasound imaging system for extended depth-of-field, coded excitation for improved penetration and respiratory-gated transmit/receive. This combination allowed non-invasive in utero acquisition of motion-free 3-D data from individual embryos in approximately 2 min, and data from four or more embryos in a pregnant mouse in less than 30 min. Data were acquired from 148 embryos spanning 5 d of early to mid-gestational stages of brain development. The results indicated that brain anatomy and cerebral vasculature can be imaged with this system and that quantitative analyses of segmented cerebral ventricles can be used to characterize volumetric changes associated with mouse brain development.     

High-Frequency Chirp Ultrasound Imaging with an Annular Array for Ophthalmologic and Small-Animal Imaging

High-frequency ultrasound (HFU, >20 MHz) is an attractive means of obtaining fine-resolution images of biological tissues for ophthalmologic, dermatological and small-animal imaging applications. Even with current improvements in circuit designs and high-frequency equipment, HFU has two inherent limitations. First, HFU images have a limited depth-of-field (DOF) because of the short wavelength and the low fixed F-number of conventional HFU transducers. Second, HFU is usually limited to shallow imaging because of the significant attenuation in most tissues. In a previous study, a five-element annular array with a 17-MHz center frequency was excited using chirp-coded signals, and a synthetic-focusing algorithm was used to extend the DOF and increase penetration depth. In the present study, a similar approach with two different five-element annular arrays operating near a center frequency of 35 MHz is implemented and validated. Following validation studies, the chirp-imaging methods were applied to imaging vitreous-hemorrhage-mimicking phantoms and mouse embryos. Images of the vitreous phantom showed increased sensitivity using the chirp method compared with a standard monocycle imaging method, and blood droplets could be visualized 4 mm deeper into the phantom. Three-dimensional datasets of 12.5-day-old mouse embryo heads were acquired in utero using chirp and conventional excitations. Images were formed and brain ventricles were segmented and reconstructed in three dimensions. The brain ventricle volumes for the monocycle excitation exhibited artifacts that were not apparent on the chirp-based dataset reconstruction. (E-mail: mamou@rrinyc.org)     

THE ADAPTATION OF UNMODIFIED STRAINS OF YELLOW FEVER VIRUS TO CULTIVATION IN VITRO

1. In a search for suitable tissues for the cultivation of yellow fever virus in vitro, mouse embryos were inoculated with this virus in utero. A titration for virus content of the various organs of the embryos indicated that the virus was present in the brain in greatest concentration. 2. Unmodified strains of yellow fever virus were readily adapted to cultivation in vitro in a medium consisting of minced mouse embryo brain tissue and Tyrode solution containing 10 per cent normal monkey serum. 3. After a continued cultivation in mouse embryo brain tissue cultures for twenty to twenty-five subcultures, these strains were readily adapted to cultivation in whole mouse embryo tissue medium. 4. There is evidence to indicate that a prolonged cultivation of the virus in mouse embryo brain medium increases its neurotropic properties. 5. Attempts to employ monkey tissues for in vitro cultivation of yellow fever virus gave entirely negative results.     

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide–dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.     

40-MHz annular array imaging of mouse embryos

Ultrasound biomicroscopy (UBM) has emerged as an important in vivo imaging approach for analyzing normal and genetically engineered mouse embryos. Current UBM systems use fixed-focus transducers, which are limited in depth-of-focus. Depending on the gestational age of the embryo, regions-of-interest in the image can extend well beyond the depth-of-focus for a fixed-focus transducer. This shortcoming makes it particularly problematic to analyze 3-D data sets and to generate accurate volumetric renderings of the mouse embryonic anatomy. To address this problem, we have developed a five-element, 40-MHz annular array transducer and a computer-controlled system to acquire and reconstruct fixed- and array-focused images of mouse embryos. Both qualitative and quantitative comparisons showed significant improvement with array-focusing, including an increase of 3 to 9 dB in signal-to-noise ratio and an increase of at least 2.5 mm in depth-of-focus. Volumetric-rendered images of brain ventricles demonstrated the clear superiority of array-focusing for 3-D analysis of mouse embryonic anatomy.     

Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1−/− mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1−/− mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.     

Intra-amniotic Transient Transduction of the Periderm With a Viral Vector Encoding TGFβ3 Prevents Cleft Palate in Tgfβ3−/− Mouse Embryos

Cleft palate is a developmental defect resulting from the failure of embryonic palatal shelves to fuse with each other at a critical time. Immediately before and during palatal fusion (E13–E15 in mice), transforming growth factor β3 (TGFβ3) is expressed in the palatal shelf medial edge epithelium (MEE) and plays a pivotal role in palatal fusion. Using Tgfβ3^−/− mice, which display complete penetrance of the cleft palate phenotype, we tested the hypothesis that intra-amniotic gene transfer could be used to prevent cleft palate formation by restoring palatal midline epithelial function. An adenoviral vector encoding Tgfβ3 was microinjected into the amniotic sacs of mouse embryos at successive developmental stages. Transduced Tgfβ3^−/− fetuses showed efficient recovery of palatal fusion with mesenchymal confluence following injection at E12.5 (100%), E13.5 (100%), E14.5 (82%), and E15.5 (75%). Viral vectors injected into the amniotic sac transduced the most superficial and transient peridermal cell layer but not underlying basal epithelial cells. TGFβ3 transduction of the peridermdal cell layer was sufficient to induce adhesion, fusion, and disappearance of the palatal shelf MEE in a cell nonautonomous manner. We propose that intra-amniotic gene transfer approaches have therapeutic potential to prevent cleft palate in utero, especially those resulting from palatal midline epithelial dysfunction.     

Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1^−/−) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations—either directly in brain, or systemic alone —in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the “combined” mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.     

Three-Dimensional High-Frequency Characterization of Cancerous Lymph Nodes

High-frequency ultrasound (HFU) offers a means of investigating biologic tissue at the microscopic level. High-frequency, three-dimensional (3-D) quantitative-ultrasound (QUS) methods were developed to characterize freshly-dissected lymph nodes of cancer patients. Three-dimensional ultrasound data were acquired from lymph nodes using a 25.6-MHz center-frequency transducer. Each node was inked prior to tissue fixation to recover orientation after sectioning for 3-D histologic evaluation. Backscattered echo signals were processed using 3-D cylindrical regions-of-interest to yield four QUS estimates associated with tissue microstructure (i.e., effective scatterer size, acoustic concentration, intercept and slope). QUS estimates were computed following established methods using two scattering models. In this study, 46 lymph nodes acquired from 27 patients diagnosed with colon cancer were processed. Results revealed that fully-metastatic nodes could be perfectly differentiated from cancer-free nodes using slope or scatterer-size estimates. Specifically, results indicated that metastatic nodes had an average effective scatterer size (i.e., 37.1 ± 1.7 μm) significantly larger (p < 0.05) than that in cancer-free nodes (i.e., 26 ± 3.3 μm). Therefore, the 3-D QUS methods could provide a useful means of identifying small metastatic foci in dissected lymph nodes that might not be detectable using current standard pathology procedures. (E-mail: mamou@rrinyc.org)     

DISEASE ACCOMPANYING IN UTERO VIRAL INFECTION : THE ROLE OF MATERNAL ANTIBODY IN TISSUE INJURY AFTER TRANSPLACENTAL INFECTION WITH LYMPHOCYTIC CHORIOMENINGITIS VIRUS

Early, after in utero infection with LCM virus, SWR/J and HA/ICR mice developed manifestations of immune complex disease. Observations based on nursing such mice with virus-infected, immune, or noninfected mouse mothers indicated that maternal antiviral antibody was responsible for the early immune complex glomerulonephritis. Despite comparable viral persistance, in utero-infected offspring failed to develop glomerulonephritis when nursed by noninfected mouse mothers, but did when suckled by virus-infected mouse mothers. Nursing by mouse mothers carrying high titers of anti-LCM viral antibody markedly enhanced the Ig glomerular deposits and the resultant nephritis.     

Pharmacological HIF2α inhibition improves VHL disease–associated phenotypes in zebrafish model

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl^–/– mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl^–/– embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.     

E3 ligase MKRN3 is a tumor suppressor regulating PABPC1 ubiquitination in non–small cell lung cancer

Central precocious puberty (CPP), largely caused by germline mutations in the MKRN3 gene, has been epidemiologically linked to cancers. MKRN3 is frequently mutated in non–small cell lung cancers (NSCLCs) with five cohorts. Genomic MKRN3 aberrations are significantly enriched in NSCLC samples harboring oncogenic KRAS mutations. Low MKRN3 expression levels correlate with poor patient survival. Reconstitution of MKRN3 in MKRN3-inactivated NSCLC cells directly abrogates in vitro and in vivo tumor growth and proliferation. MKRN3 knockout mice are susceptible to urethane-induced lung cancer, and lung cell–specific knockout of endogenous MKRN3 accelerates NSCLC tumorigenesis in vivo. A mass spectrometry–based proteomics screen identified PABPC1 as a major substrate for MKRN3. The tumor suppressor function of MKRN3 is dependent on its E3 ligase activity, and MKRN3 missense mutations identified in patients substantially compromise MKRN3-mediated PABPC1 ubiquitination. Furthermore, MKRN3 modulates cell proliferation through PABPC1 nonproteolytic ubiquitination and subsequently, PABPC1-mediated global protein synthesis. Our integrated approaches demonstrate that the CPP-associated gene MKRN3 is a tumor suppressor.     

Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia

Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.     

Deficiency of FLCN in Mouse Kidney Led to Development of Polycystic Kidneys and Renal Neoplasia

The Birt–Hogg–Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHD^flox/flox/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHD^flox/flox/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD ^flox/+/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome–related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.     

Effect of Ku80 Deficiency on Mutation Frequencies and Spectra at a LacZ Reporter Locus in Mouse Tissues and Cells

Non-homologous end joining (NHEJ) is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs) and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and γH2AX DNA damage foci in Ku80^−/− as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements.     

Targeting the histone demethylase LSD1 prevents cardiomyopathy in a mouse model of laminopathy

LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of Lmna^H222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in Lmna^H222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in Lmna^H222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal Lmna^H222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 Lmna^H222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.