Correction: Tagger-A Swiss army knife for multiomics to dissect cell type-specific mechanisms of gene expression in mice.

[This corrects the article DOI: 10.1371/journal.pbio.3000374.].

Tagger-A Swiss army knife for multiomics to dissect cell type-specific mechanisms of gene expression in mice.

A deep understanding of how regulation of the multiple levels of gene expression in mammalian tissues give rise to complex phenotypes has been impeded by cellular diversity. A handful of techniques were developed to tag-select nucleic acids of interest in specific cell types, thereby enabling their capture. We expanded this strategy by developing the Tagger knock-in mouse line bearing a quad-cistronic transgene combining enrichment tools for nuclei, nascent RNA, translating mRNA, and mature microRNA (miRNA). We demonstrate that Tagger can capture the desired nucleic acids, enabling multiple omics approaches to be applied to specific cell types in vivo using a single transgenic mouse line.

Inactivation of ceramide synthase 2 catalytic activity in mice affects transcription of genes involved in lipid metabolism and cell division.

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life.

Ceramide Synthase 5 Is Essential to Maintain C16:0-Ceramide Pools and Contributes to the Development of Diet-induced Obesity.

Ceramides are bioactive sphingolipids, which are composed of sphingoid bases carrying acyl chains of various lengths. Ceramides are synthesized by a family of six ceramide synthases (CerS) in mammals, which produce ceramides with differentN-linked acyl chains. Increased ceramide levels are known to contribute to the development of obesity and insulin resistance. Recently, it has been demonstrated that the ceramide acylation pattern is of particular importance for an organism to maintain energy homeostasis. However, which of theCerSfamily members are involved in this process is not yet completely known. Using newly developedCerS5knock-out mice, we show here thatCerS5is essential to maintain cellular C16:0sphingolipid pools in lung, spleen, muscle, liver, and white adipose tissue. Glycerophospholipid levels inCerS5-deficient mice were not altered. We found a strong impact of CerS5-dependent ceramide synthesis in white adipose tissue after high fat diet feeding. In skeletal muscle, liver, and spleen, C16:0-ceramide levels were altered independent of feeding conditions. The loss ofCerS5is associated with reduced weight gain and improved systemic health, including maintenance of glucose homeostasis and reduced white adipose tissue inflammation after high fat diet challenge. Our findings indicate that reduction of endogenous C16:0-ceramide by genetic inhibition ofCerS5is sufficient to ameliorate obesity and its comorbidities.

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice.

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.

Characterization of Panglial Gap Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique Population of Glial Cells.

The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions.

Ceramide synthase 4 deficiency in mice causes lipid alterations in sebum and results in alopecia.

Five ceramide synthases (CerS2-CerS6) are expressed in mouse skin. Although CerS3 has been shown to fulfill an essential function during skin development, neither CerS6- nor CerS2-deficient mice show an obvious skin phenotype. In order to study the role of CerS4, we generated CerS4-deficient mice (Cers4-/-) and CerS4-specific antibodies. With these biological tools we analysed the tissue distribution and determined the cell-type specific expression of CerS4 in suprabasal epidermal layers of footpads as well as in sebaceous glands of the dorsal skin. Loss of CerS4 protein leads to an altered lipid composition of the sebum, which is more solidified and therefore might cause progressive hair loss due to physical blocking of the hair canal. We also noticed a strong decrease in C20 1,2-alkane diols consistent with the decrease of wax diesters in the sebum of Cers4-/- mice. Cers4-/- mice at 12 months old display additional epidermal tissue destruction due to dilated and obstructed pilary canals. Mass spectrometric analyses additionally show a strong decrease in C20-containing sphingolipids.

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases.

Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.

The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29).

The potential gap junction forming mouse connexin29 (Cx29) protein is concomitantly expressed with connexin32 (Cx32) in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47) in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harboring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29-mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

Inactivation of ceramide synthase 6 in mice results in an altered sphingolipid metabolism and behavioral abnormalities.

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for ß-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.

Ablation of neuronal ceramide synthase 1 in mice decreases ganglioside levels and expression of myelin-associated glycoprotein in oligodendrocytes.

Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.

Dual reporter approaches for identification of Cre efficacy and astrocyte heterogeneity.

Gene inactivation reporters are powerful tools to circumvent limitations of the widely used Cre/loxP system of conditional mutagenesis. With new conditional transgenic mouse lines expressing the enhanced cyan fluorescent protein (ECFP) instead of connexin43 (Cx43) after Cre-mediated recombination, we demonstrate dual reporter approaches to simultaneously examine astrocyte subpopulations expressing different connexins, identify compensatory up-regulation within gene families, and quantify Cre-mediated deletion at the allelic level. Analysis of a newly generated Cx43 knock-in ECFP mouse revealed an unexpected heterogeneity of Cx43-expressing astrocytes across brain areas.

Peripheral lymphangiogenesis in mice depends on ectodermal connexin-26 (Gjb2).

In order to study the specific function of connexin-26 (Cx26, also known as gap junction beta-2 protein; Gjb2), we generated knockin mice that expressed either a floxed lacZ reporter or, after Cre-mediated deletion, connexin-32 (Cx32)-coding DNA, both driven by the endogenous Cx26 promoter. Heterozygous Cx26knock-inCx32 (Cx26KICx32) embryos developed normally until embryonic day 14.5 but died before birth with severe lymphedemas. Although the jugular lymph sacs were normally developed, these embryos had a strongly reduced dermal lymphatic capillary network. By analyses of ß-galactosidase reporter protein expression and lymphatic or blood endothelial-specific marker proteins, we demonstrated that Cx26 expression is temporally closely linked to lymphangiogenesis. No obvious phenotypic abnormalities were observed in Cx26KICx32 mice when Cre-mediated recombination was directed to mesenchyme or blood endothelium using the Prx1-Cre or Tie2-Cre mouse strains, respectively. By contrast, keratin-5-Cre-mediated replacement of Cx26 with Cx32 or deletion of both Cx26 alleles revealed severe lymphedemas similar to the general Cx26KICx32 phenotype. Thus, conditional ablation of Cx26 (loss of function) in ectoderm leads to partial disruption of lymphatic capillaries and embryonic death. We conclude that appropriate development of dermal lymphatic vessels in mice is dependent on the expression of Cx26 in the ectoderm.

Connexin32 can restore hearing in connexin26 deficient mice.

Functional gap junction channels composed of certain connexin proteins are essential for the function of the cochlea. Homozygous deficiency in the Gjb2 (mice) or GJB2 (human) gene coding for connexin26 (Cx26) in the cochlea leads to hearing impairment in mice and humans, respectively. Here we have studied the functional equivalence of Cx26 and connexin32 (Cx32) isoforms in the cochlea. We analyzed a conditional mouse mutant in which the Gjb2 coding DNA was exchanged by LacZ DNA coding for the reporter protein beta-galactosidase. This allowed us to follow the unrestricted and cell type specific expression of Gjb2 promoter activity. After inner ear specific, Otogelin-Cre recombinase mediated deletion of the loxP-site-flanked LacZ coding DNA, transcription of the Gjb1 gene, coding for Cx32 was activated by the Gjb2 promoter. Interbreeding of these mice with conditional Gjb2 null mice resulted in animals in which Cx32 instead of Cx26 protein is expressed in the non-sensory epithelial network of the cochlea. When we analyzed the auditory function in these mice, we found that the expression of Cx32 protein is sufficient to support hearing in the absence of Cx26. Thus Cx32 can functionally replace Cx26 in the mouse cochlea resulting in almost normal hearing.

Pathologic and phenotypic alterations in a mouse expressing a connexin47 missense mutation that causes Pelizaeus-Merzbacher-like disease in humans.

Gap junction channels are intercellular conduits that allow diffusional exchange of ions, second messengers, and metabolites. Human oligodendrocytes express the gap junction protein connexin47 (Cx47), which is encoded by the GJC2 gene. The autosomal recessive mutation hCx47M283T causes Pelizaeus-Merzbacher-like disease 1 (PMLD1), a progressive leukodystrophy characterized by hypomyelination, retarded motor development, nystagmus, and spasticity. We introduced the human missense mutation into the orthologous position of the mouse Gjc2 gene and inserted the mCx47M282T coding sequence into the mouse genome via homologous recombination in embryonic stem cells. Three-week-old homozygous Cx47M282T mice displayed impaired rotarod performance but unchanged open-field behavior. 10-15-day-old homozygous Cx47M282T and Cx47 null mice revealed a more than 80% reduction in the number of cells participating in glial networks after biocytin injections into oligodendrocytes in sections of corpus callosum. Homozygous expression of mCx47M282T resulted in reduced MBP expression and astrogliosis in the cerebellum of ten-day-old mice which could also be detected in Cx47 null mice of the same age. Three-month-old homozygous Cx47M282T mice exhibited neither altered open-field behavior nor impaired rotarod performance anymore. Adult mCx47M282T expressing mice did not show substantial myelin alterations, but homozygous Cx47M282T mice, additionally deprived of connexin32, which is also expressed in oligodendrocytes, died within six weeks after birth and displayed severe myelin defects accompanied by astrogliosis and activated microglia. These results strongly suggest that PMLD1 is caused by the loss of Cx47 channel function that results in impaired panglial coupling in white matter tissue.

Adult ceramide synthase 2 (CERS2)-deficient mice exhibit myelin sheath defects, cerebellar degeneration, and hepatocarcinomas.

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22-C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulting mice lack ceramide synthase activity toward C24:1 in the brain as well as the liver and show only very low activity toward C18:0-C22:0 in liver and reduced activity toward C22:0 residues in the brain. In addition, these mice exhibit strongly reduced levels of ceramide species with very long fatty acid residues (>or=C22) in the liver, kidney, and brain. From early adulthood on, myelin stainability is progressively lost, biochemically accompanied by about 50% loss of compacted myelin and 80% loss of myelin basic protein. Starting around 9 months, both the medullary tree and the internal granular layer of the cerebellum show significant signs of degeneration associated with the formation of microcysts. Predominantly in the peripheral nervous system, we observed vesiculation and multifocal detachment of the inner myelin lamellae in about 20% of the axons. Beyond 7 months, the CERS2-deficient mice developed hepatocarcinomas with local destruction of tissue architecture and discrete gaps in renal parenchyma. Our results indicate that CERS2 activity supports different biological functions: maintenance of myelin, stabilization of the cerebellar as well as renal histological architecture, and protection against hepatocarcinomas.

Quality control of astrocyte-directed Cre transgenic mice: the benefits of a direct link between loss of gene expression and reporter activation.

Cre recombinase activity for cell-type restricted deletion of floxed target genes (i.e., flanked by Cre recognition loxP-sites) is often measured by separate matings with recombination-activated reporter gene mice. Using a floxed Gja1 (Cx43) allele, we demonstrate the benefits of a direct link between reporter gene expression and target gene deletion to overcome critical limitations of the Cre/loxP system. The widely used human glial fibrillary acidic protein (hGFAP)-Cre transgene exhibits variable recombination activity and requires postexperimental validation. Such quality control is essential to correlate the extent of Cre-mediated Gja1 ablation with phenotypical alterations and to maintain the activity status of hGFAP-Cre in transgenic mouse colonies. We present several strategies to control for the fidelity of hGFAP-Cre mediated recombination. (c) 2008 Wiley-Liss, Inc.

Role of olivary electrical coupling in cerebellar motor learning.

The level of electrotonic coupling in the inferior olive is extremely high, but its functional role in cerebellar motor control remains elusive. Here, we subjected mice that lack olivary coupling to paradigms that require learning-dependent timing. Cx36-deficient mice showed impaired timing of both locomotion and eye-blink responses that were conditioned to a tone. The latencies of their olivary spike activities in response to the unconditioned stimulus were significantly more variable than those in wild-types. Whole-cell recordings of olivary neurons in vivo showed that these differences in spike timing result at least in part from altered interactions with their subthreshold oscillations. These results, combined with analyses of olivary activities in computer simulations at both the cellular and systems level, suggest that electrotonic coupling among olivary neurons by gap junctions is essential for proper timing of their action potentials and thereby for learning-dependent timing in cerebellar motor control.

A new conditional mouse mutant reveals specific expression and functions of connexin36 in neurons and pancreatic beta-cells.

Connexin36 (Cx36) is the main connexin isoform expressed in neurons of the central nervous system (CNS) and in pancreatic beta-cells, i.e. two types of excitable cells that share - in spite of their different origins - a number of common features. Previous studies on Cx36 deficient mice have documented that loss of Cx36 resulted in phenotypic abnormalities in both the CNS and the pancreas which, however, could not be attributed to specific cell types due to the general deletion nature of the animal model used. Attempts to address this limitation using cell type specific deletions generated by the Cre/loxP strategy have so far been complicated by the lack of Cx36 expression from the floxed allele. We have now generated a conditional Cx36 deficient mouse mutant in which the coding region of Cx36 is flanked by loxP sites, followed by a cyan fluorescent protein (CFP) reporter gene. Here we show that Cx36 was still expressed from the floxed allele in neurons and pancreatic beta-cells. In these cells, a 30-60% decrease of this protein, relative to the expression level of the wildtype allele, did not significantly perturb cell coupling. The deletion of Cx36 by ubiquitously and cell type specifically expressed Cre recombinases revealed that CFP functions as a reliable reporter for Cx36 expression in brain neurons and to some extent in retina neurons, but not in pancreas. Loss of Cx36 by Cre-mediated recombination was documented at transcript and protein levels. Cell type specific deletion of Cx36 in the endocrine pancreas revealed major alterations in the basal as well as the glucose-induced insulin secretion, hence specifically attributing to pancreatic Cx36 an important regulatory role in the control of beta-cell function. Cell type specific deletion of Cx36 in the CNS by suitable Cre recombinases should also help to elucidate the functional role of Cx36 in different neuronal subtypes.