Embryonic Stem Cell Clones Research Articles

A Novel Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia Locates the Erythroid Defect at CFU-E / Proerythroblast Transition.

Abstract 178Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Presently, many different DBA disease genes are known that all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). In order to study pathophysiology and to evaluate novel therapies, DBA animal models are needed. Since RNA interference -mediated RPS19 down regulation has been shown to result in a DBA phenotype in human cells in vitro, we decided to use the short hairpin RNA (shRNA) technology to create an RPS19-deficient mouse model for DBA. We designed miR30 -styled shRNAs against RPS19 and introduced them into mouse embryonic stem (ES) cells downstream of the collagen A1 locus using site-specific recombination. The resulting ES cell clones contain a single RPS19-targeting shRNA under the control of a doxycycline-responsive promoter. We have generated and characterized two mouse models expressing different RPS19-targeting shRNAs (shRNA-B and shRNA-D). In general, this system allows an inducible and dose-dependent regulation of shRNA expression providing an ideal tool to study conditions like DBA that are caused by haploinsufficient expression of a protein. To induce the expression of RPS19-targeting shRNAs mice were fed with doxycycline administered in drinking water. Induction of the shRNA-B construct had no effect on the erythrocyte level, hemoglobin concentration or hematocrit, although we saw a gradual elevation in mean corpuscular volume (MCV) and a decrease in platelet number. However, after 25–35 days of doxycycline treatment the mice homozygous for the shRNA-B underwent severe weight loss accompanied with a reduction in erythrocyte number and ultimately died. In contrast, shRNA-D mice exhibited decreased erythrocyte number, hemoglobin and hematocrit already after a 10 day doxycycline treatment. When the induction was kept on longer, the homozygous mice developed a more severe anemia and died around day 20, while the heterozygous mice were able to compensate the blood indices back to normal level. In spite of the differences in blood phenotypes, both models had a similar FACS phenotype revealing a profound decrease in the number of proerythroblasts, while the levels of erythroid and bipotential megakaryocytic-erythroid (MegE) progenitors were normal or increased. We also saw an accumulation of the late erythroid precursors. Proliferative potential of erythroid progenitors was evaluated at a clonal level in vitro. When MegE or CFU-E progenitors from induced mice were cultured in presence of doxycycline, the number and size of the erythroid clones were decreased compared to controls. However, when RPS19 expression was restored by culturing the cells without doxycycline, the observed proliferation defect of MegE clones was completely restored while the rescue of CFU-E clones was only partial. We also noticed that RPS19-deficient megakaryocytes appeared smaller in size compared to controls. Importantly, transduction of RPS19-deficient cells with a lentiviral vector overexpressing sequence-modified RPS19 cDNA rescued the proliferation and colony-forming defects in vitro demonstrating that the erythroid phenotype is specifically due to down regulation of RPS19. In summary, we have generated two novel mouse models for RPS19-deficient DBA that recapitulate the key erythroid phenotype seen in patients based on both FACS analysis and single-cell proliferation assays. These models will serve as a good tool to determine the molecular mechanisms responsible for DBA and also to test gene replacement therapies. Disclosures:No relevant conflicts of interest to declare.

Analysis of Blood Stem Cell Activity and Cystatin Gene Expression in a Mouse Model Presenting a Chromosomal Deletion Encompassing Csta and Stfa2l1

The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del16qB3Δ/16qB3Δ) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del16qB3Δ/16qB3Δ animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del16qB3Δ/16qB3Δ hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions.

Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.

Counting Chromosomes in Embryonic Stem (ES) Cells

INTRODUCTIONAneuploidy in embryonic stem (ES) cells is the major cause of failure in obtaining contributions to all tissues of chimeras, including the germ line. This protocol describes a simple method for counting chromosomes from metaphase spreads in ES cells. ES cell clones showing euploidy on 70%-80% of spreads are used for chimera production.

Developmental roles of EGFL7: Vasculogenesis and male germ cell differentiation

EGFL7 is expressed in mouse embryonic stem cells (ESC), endothelial cells (EC), primordial germ cells (PGC), and recently this lab has shown expression in spermatids and spermatozoa. Our aim was to investigate the role of EGFL7 during vasculogenesis. Egfl7 was knocked‐down in mouse ESCs using lentiviral constructs with siRNA and eGFP sequences. Using the in vitro embryoid body model we found that Egfl7 knock‐down (k/d) was associated with sheet‐like EC structures, consistent with studies of mouse aortic ring, and zebrafish k/d, models. Eight chimeric males derived from k/d ESC clones were crossed with WT females. Unexpectedly, transmission of the k/d construct was not seen in pups, embryos, or blastocysts. Further investigation revealed absence of eGFP+ maturing sperm cells in chimeras, suggesting that k/d of Egfl7 prevents germ cell maturation past the PGC stage. In testicular tissue presence of the Egfl7 k/d construct in ECs was associated with atrophy in 15% of the parenchyma. These results suggest a developmental role for EGFL7 in EC migration and cord‐formation during angiogenesis, and a second role during the differentiation of male germ cells. Funding provided by NIH grant R21 HL082098 to HS.

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.

E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks

Hereditary cases of breast and ovarian cancer are often attributed to germ-line mutations of the BRCA1 tumor suppressor gene. Although BRCA1 is involved in diverse cellular processes, its role in the maintenance of genomic integrity may be a key component of its tumor suppression activity. The protein encoded by BRCA1 interacts in vivo with the related BARD1 protein to form a heterodimeric complex that acts as a ubiquitin E3 ligase. Because the enzymatic activity of the BRCA1/BARD1 heterodimer is conserved over a broad phylogenetic range, it is thought to be critical for the central functions of BRCA1. To test this hypothesis, we have generated isogenic clones of embryonic stem cells that do or do not express an enzymatically proficient Brca1 polypeptide. Surprisingly, cells lacking the ubiquitin ligase activity of BRCA1 are viable and do not accumulate spontaneous cytogenetic rearrangements. Gene targeting efficiencies are modestly reduced in these cells, and chromosomal rearrangements arise at elevated rates in response to genotoxic stress. Nonetheless, cells lacking Brca1 enzymatic activity are not hypersensitive to the DNA cross-linking agent mitomycin C. They also form Rad51 focus in response to ionizing radiation and repair chromosome breaks by homologous recombination at wild-type levels. These results indicate that key aspects of BRCA1 function in genome maintenance, including its role in homology-directed repair of double-strand DNA breaks, do not depend on the E3 ligase activity of BRCA1.

Role of ornithine decarboxylase antizyme inhibitor in vivo

Ornithine decarboxylase (ODC) antizyme inhibitor (AZI) has been shown to regulate ODC activity in cell cultures. However, its biological functions in an organism remain unknown. An embryonic stem (ES) cell clone was established, in which the Azin1 gene was disrupted by the gene trap technique. To identify the function of Azin1 gene in vivo, a mutant mouse line was generated using these trapped ES cells. Homozygous mutant mice died at P0 with abnormal liver morphology. Further analysis indicated that the deletion of Azin1 in homozygous mice resulted in the degradation of ODC, and reduced the biosynthesis of putrescine and spermidine. Our results thus show that AZI plays an important role in regulating the levels of ODC, putrescine and spermidine in mice, and is essential for the survival of mice.

Neurological phenotype and reduced lifespan in heterozygous Tim23 knockout mice, the first mouse model of defective mitochondrial import

The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.

Defining the in Vivo Role for Cytochrome b5 in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50–90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

Neuregulin-1 promotes cardiomyocyte differentiation of genetically engineered embryonic stem cell clones

Embryonic stem (ES) cell-derived cardiomyocytes (ESCMs) must be specifically purified in order to prevent teratoma formation, and this confusing issue has hampered their clinical application. We therefore investigated a technique to generate pure labeled ESCMs for possible use in cardiac repair. We generated transgenic ES cell lines expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Differentiated EGFP-positive ES cells displayed characteristics of CMs. Furthermore, neuregulin-1 (NRG-1) upregulated the expression of the cardiac-restricted transcription factors Nkx2.5 and GATA-4, as well as differentiated CM factors (alpha-MHC, beta-MHC). Immunohistochemistry demonstrated that NRG-1 increased expression of cardiac-specific troponin T in the beating foci of the embryoid bodies. This work revealed a potential method for specifically labeling and enriching ESCMs by combining genetically-engineered ES cell clones and exogenous growth factor treatment.

Genetic Ablation of V2a Ipsilateral Interneurons Disrupts Left-Right Locomotor Coordination in Mammalian Spinal Cord

The initiation and coordination of activity in limb muscles are the main functions of neural circuits that control locomotion. Commissural neurons connect locomotor circuits on the two sides of the spinal cord, and represent the known neural substrate for left-right coordination. Here we demonstrate that a group of ipsilateral interneurons, V2a interneurons, plays an essential role in the control of left-right alternation. In the absence of V2a interneurons, the spinal cord fails to exhibit consistent left-right alternation. Locomotor burst activity shows increased variability, but flexor-extensor coordination is unaffected. Anatomical tracing studies reveal a direct excitatory input of V2a interneurons onto commissural interneurons, including a set of molecularly defined V0 neurons that drive left-right alternation. Our findings imply that the neural substrate for left-right coordination consists of at least two components; commissural neurons and a class of ipsilateral interneurons that activate commissural pathways.

Large-scale gene trapping in C57BL/6N mouse embryonic stem cells

We report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Scalable Selection of Hepatocyte- and Hepatocyte Precursor-Like Cells from Culture of Differentiating Transgenically Modified Murine Embryonic Stem Cells

Potential therapeutic applications of embryonic stem cell (ESC)-derived hepatocytes are limited by their relatively low output in differentiating ESC cultures, as well as by the danger of contamination with tumorigenic undifferentiated ESCs. To address these problems, we developed transgenic murine ESC clones possessing bicistronic expression vector that contains the alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live" fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under established culture conditions these clones allowed for both monitoring of differentiation and for puromycin selection of hepatocyte-committed cells in a suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]). When plated on fibronectin, the selected eGFP-positive cells formed colonies, in which intensely proliferating hepatocyte precursor-like cells gave rise to morphologically differentiated cells expressing alpha-1-antitrypsin, alpha-fetoprotein, and albumin. A number of cells synthesized glycogen and in some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte marker genes were expressed in the culture, along with the gene and protein expression of stem/progenitor markers, suggesting the features of both hepatocyte precursors and more advanced differentiated cells. When cultured in suspension, the EB-derived puromycin-selected cells formed spheroids capable of outgrowing on an adhesive substrate, resembling the behavior of fetal mouse hepatic progenitor cells. The established system based on the highly efficient selection/purification procedure could be suitable for scalable generation of ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a potential in vitro source of cells for transplantation therapy of liver diseases, tissue engineering, and drug and toxicology screening.

AIP1 Is Critical in Transducing IRE1-mediated Endoplasmic Reticulum Stress Response

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

Instability of Retroviral DNA Methylation in Embryonic Stem Cells

The epigenetic status of pluripotent stem cells has been demonstrated to be extremely unstable. In our current study, we have attempted to further investigate the epigenetic dynamics of the stem cell genome by monitoring the expression of the murine stem cell virus (MSCV) retroviral vector in embryonic stem (ES) cells. Although MSCV is progressively silenced by proviral DNA methylation in ES cells, a substantial number of MSCV-transduced ES cell clones do show variegated proviral expression. This expression profile is due in part to the transient and reversible properties of MSCV silencing. However, the spontaneous reactivation rates of the silenced proviruses differ significantly between these variegated clones, indicating that the reversibility of silencing is dependent on the proviral integration site. Our current data suggest that the fidelity of DNA methylation among the genomic sequences that flank the proviral integration sites may be the determinant of this reversibility of MSCV silencing. Given that the adjoining epigenome environment affects the epigenetic regulation of proviral DNA, the reversible MSCV silencing effect is thus likely to reflect a unique and interesting feature of ES cell epigenome regulation that has not previously been revealed.

Role of Egfl7 During Vascular Development

Epidermal growth factor domain‐like 7, Egfl7, encodes a 29kDa protein with an early and endothelial‐restricted expression profile. In vivo, Egfl7 mRNA is detected at mouse embryonic day 7.5. During development, expression is restricted to sites of blood island formation, the vascular endothelium, and endocardium. In adults, Egfl7 is down‐regulated except in the lung, pregnant uterus, and regenerating endothelium. We have used loss‐ and gain‐of‐function to determine the role of Egfl7 during vasculogenesis and angiogenesis. We are using a lentiviral siRNA‐mediated approach to establish Egfl7 knock‐down in ESC clones and transgenic mice. Embryoid bodies with Egfl7 knock‐down appear to have less organized vascular cords compared with controls. Tie2‐Egfl7 transgenic mice, with forced expression of the gene in endothelial cells, have been generated for gain‐of‐function analysis, and these display increased vessel lumen diameter, hemorrhaging, and other vascular abnormalities. We will report on analyses of these ESC and transgenic mice studies, and describe how they relate to the role of Egfl7 during embryogenesis. In addition, we will report on a second site of Egfl7 expression in embryonic germ cells (EGC) and embryonic stem cells (ESC), primordial stem cells, and during adult spermatogenesis.Research Support: NIH HL082098, Tri‐Institutional Stem Cell Initiative (H.S.)

Neurotrophin Receptor-Mediated Death of Misspecified Neurons Generated from Embryonic Stem Cells Lacking Pax6

Pax6-positive radial glial (RG) cells are the progenitors of most glutamatergic neurons in the cortex, a lineage that can be recapitulated in vitro using embryonic stem (ES) cells. We show here that ES cells lacking Pax6, a transcription factor long known to be essential for cortical development, generate Mash1-positive RG cells that differentiate in GABAergic neurons. These neurons express high levels of the neurotrophin receptor p75NTR causing their rapid death. Pax6 function was also investigated following transplantation of ES cells in the developing chick telencephalon and in mice lacking both Pax6 and p75NTR. Taken together, our results indicate that reliable predictions can be made with cultured ES cells when used to explore the role of genes impacting early aspects of mammalian neurogenesis. They also provide a novel opportunity to compare the molecular constituents of glutamatergic with those of GABA-ergic neurons and to explore the mechanisms of their generation.

Mice Lacking α/β Subunits of GlcNAc-1-Phosphotransferase Exhibit Growth Retardation, Retinal Degeneration, and Secretory Cell Lesions

Mucolipidosis II and III (ML II; ML III) are lysosomal storage diseases characterized by a deficiency in GlcNAc-1-phosphotransferase. Patients with ML III have retinal disease, but in cases of the more clinically severe ML II, human ophthalmic studies are limited. In this study, retinal function and overall disease were assessed in mice lacking GNPTAB, the gene mutated in patients with ML II. Mice deficient in GNPTAB were generated from Omnibank, a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones as part of a large-scale effort to knock out, phenotypically screen, and thereby validate pharmaceutically tractable genes for drug development. Routine diagnostics, expression analysis, histopathology, and ERG analyses were performed on mice lacking GNPTAB. In addition, measurements of serum lysosomal enzymes were performed. Severe retinal degeneration was observed in mice deficient in GNPTAB. Heterozygous mice were phenotypically normal and in situ hybridization showed expression across the neural retina. Compared to wild-type mice, the GNPTAB homozygous mice were smaller, had elevated levels of serum lysosomal enzymes, exhibited cartilage defects, and had cytoplasmic alterations in secretory cells of several exocrine glands. Mice deficient in GNPTAB exhibited severe retinal degeneration. Additional features observed in patients with ML II, a lysosomal storage disease, are also present in these mice. Understanding underlying mechanisms of this gene in the eye will increase its therapeutic potential for the treatment of retinal diseases.