Cre Transgenic Lines Research Articles

Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping.

Decoding the functional connectivity of the nervous system is facilitated by transgenic methods that express a genetically encoded reporter or effector in specific neurons; however, most transgenic lines show broad spatiotemporal and cell-type expression. Increased specificity can be achieved using intersectional genetic methods which restrict reporter expression to cells that co-express multiple drivers, such as Gal4 and Cre. To facilitate intersectional targeting in zebrafish, we have generated more than 50 new Cre lines, and co-registered brain expression images with the Zebrafish Brain Browser, a cellular resolution atlas of 264 transgenic lines. Lines labeling neurons of interest can be identified using a web-browser to perform a 3D spatial search (zbbrowser.com). This resource facilitates the design of intersectional genetic experiments and will advance a wide range of precision circuit-mapping studies.

ZEB2 and LMO2 Drive Immature T-Cell Lymphoblastic Leukemia Via Distinct Oncogenic Mechanisms

ZEB2, a E-box binding homeobox transcription factor, is able to induce epithelial-to-mesenchymal transition in the context of solid tumors. As such, its expression is correlated with cancer cell invasion, dissemination and metastasis, but also with the acquisition of cancer stem cell properties and therapy resistance.Using conditional knockout mice, we previously have shown that ZEB2 is an essential hematopoietic transcription factors, indispensable for normal embryonic(1) and adult(2) hematopoiesis. In addition, using conditional overexpressing mice we have demonstrated that ZEB2 is a bona fide driver of an immature subtype of T-ALL with increased leukemia-initiating potential(3).Here, we investigated the effects of ZEB2 overexpression on normal T-cell differentiation. Already from young age, ZEB2-overexpressing mice have a smaller thymus, with increased percentages of early T-cell precursors. Detailed flow cytometric analysis indicate a T-cell differentiation delay at the beta-selection checkpoint, with accumulation of DN3 (CD4- CD8- CD44- CD25+) cells. Using in vitro differentiation assays on OP9-DL1 stromal cells, we demonstrate that the perturbed T-cell differentiation is independent of the thymic micro-environment. Lowering the dose of exogenous interleukin-7 levels in the culture media can partially rescue this phenotype, which suggests that ZEB2-overexpressing thymocytes are delayed in T-cell differentiation due to their inability to downregulate the interleukin-7 receptor (IL7R) levels.Both the perturbed T-cell differentiation, and the latency/penetrance of immature subtype of the T-ALL formed in ZEB2-overexpressing mice is similar to what has been described for LMO2-overexpressing mice. Based on this phenocopy, we hypothesized that ZEB2 and LMO2 induce immature T-ALL via converging oncogenic pathway and/or via regulating similar crucial genes for the T-cell differentiation program. However, in contrast to LMO2, ZEB2-overexpresssing pre-leukemic thymocytes do not display aberrant self-renewal potential as demonstrated by transplantation experiments in sublethal irradiated syngeneic hosts. In addition, by using multiple Cre-transgenic lines, allowing the expression of ZEB2 at different stages of T-cell differentiation, we demonstrate that the oncogenic potential of ZEB2 is independent of the perturbed T-cell differentiation observed during the early stage of the disease.Based on these results we conclude that ZEB2 and LMO2 both drive the development of an immature subtype of T-ALL, but via distinct oncogenic mechanisms.(1) Goossens S. et al., Blood 2011 May 26; 117(21):5620-30.(2) Li J. et al., Blood 2017 Jan 26; 129(4):460-472.(3) Goossens S. et al., Nat Commun. 2015 Jan 7;6:5794. DisclosuresNo relevant conflicts of interest to declare.

Generation of an inducible RPE-specific Cre transgenic-mouse line.

The retinal pigment epithelium (RPE) is an epithelial monolayer in the back of the vertebrate eye. RPE dysfunction is associated with retinal degeneration and blindness. In order to fully understand how dysregulation affects visual function, RPE-specific gene knockouts are indispensable. Since the currently available RPE-specific Cre recombinases show lack of specificity or poor recombination, we sought to generate an alternative. We generated a tamoxifen-inducible RPE-specific Cre transgenic mouse line under transcriptional control of an RPE-specific Tyrosinase enhancer. We characterized the Cre-mediated recombinant expression by crossing our RPE-Tyrosinase-CreErT2 mouse line with the tdTomato reporter line, Ai14. Detected fluorescence was quantified via high-content image analysis. Recombination was predominantly observed in the RPE and adjacent ciliary body. RPE flatmount preparations revealed a high level of recombination in adult mice (47.25–69.48%). Regional analysis of dorsal, ventral, nasal and temporal areas did not show significant changes in recombination. However, recombination was higher in the central RPE compared to the periphery. Higher levels of Cre-mediated recombinant expression was observed in embryonic RPE (~83%). Compared to other RPE-specific Cre transgenic mouse lines, this newly generated RPE-Tyrosinase-CreErT2 line shows a more uniform and higher level of recombination with the advantage to initiate recombination in both, prenatal and postnatal animals. This line can serve as a valuable tool for researches exploring the role of individual gene functions, in both developing and differentiated RPE.

Mouse Cre Models for the Study of Bone Diseases.

Transgenic Cre lines are a valuable tool for conditionally inactivating or activating genes to understand their function. Here, we provide an overview of Cre transgenic models used for studying gene function in bone cells and discuss their advantages and limitations, with particular emphasis on Cre lines used for studying osteocyte and osteoclast function. Recent studies have shown that many bone cell-targeted Cre models are not as specific as originally thought. To ensure accurate data interpretation, it is important for investigators to test for unexpected recombination events due to transient expression of Cre recombinase during development or in precursor cells and to be aware of the potential for germ line recombination of targeted genes as well as the potential for unexpected phenotypes due to the Cre transgene. Although many of the bone-targeted Cre-deleter strains are imperfect and each model has its own limitations, their careful use will continue to provide key advances in our understanding of bone cell function in health and disease.

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Neuronal Organization in the Inferior Colliculus Revisited with Cell-Type-Dependent Monosynaptic Tracing.

The inferior colliculus (IC) is a critical integration center in the auditory pathway. However, because the inputs to the IC have typically been studied by the use of conventional anterograde and retrograde tracers, the neuronal organization and cell-type-specific connections in the IC are poorly understood. Here, we used monosynaptic rabies tracing and in situ hybridization combined with excitatory and inhibitory Cre transgenic mouse lines of both sexes to characterize the brainwide and cell-type-specific inputs to specific neuron types within the lemniscal IC core and nonlemniscal IC shell. We observed that both excitatory and inhibitory neurons of the IC shell predominantly received ascending inputs rather than descending or core inputs. Correlation and clustering analyses revealed two groups of excitatory neurons in the shell: one received inputs from a combination of ascending nuclei, and the other received inputs from a combination of descending nuclei, neuromodulatory nuclei, and the contralateral IC. In contrast, inhibitory neurons in the core received inputs from the same combination of all nuclei. After normalizing the extrinsic inputs, we found that core inhibitory neurons received a higher proportion of inhibitory inputs from the ventral nucleus of the lateral lemniscus than excitatory neurons. Furthermore, the inhibitory neurons preferentially received inhibitory inputs from the contralateral IC shell. Because IC inhibitory neurons innervate the thalamus and contralateral IC, the inhibitory inputs we uncovered here suggest two long-range disinhibitory circuits. In summary, we found: (1) dominant ascending inputs to the shell, (2) two subpopulations of shell excitatory neurons, and (3) two disinhibitory circuits.SIGNIFICANCE STATEMENT Sound undergoes extensive processing in the brainstem. The inferior colliculus (IC) core is classically viewed as the integration center for ascending auditory information, whereas the IC shell integrates descending feedback information. Here, we demonstrate that ascending inputs predominated in the IC shell but appeared to be separated from the descending inputs. The presence of inhibitory projection neurons is a unique feature of the auditory ascending pathways, but the connections of these neurons are poorly understood. Interestingly, we also found that inhibitory neurons in the IC core and shell preferentially received inhibitory inputs from ascending nuclei and contralateral IC, respectively. Therefore, our results suggest a bipartite domain in the IC shell and disinhibitory circuits in the IC.

Increased NK cell immunity in a transgenic mouse model of NKp46 overexpression

Natural Killer (NK) cells employ activating receptors like the Natural Cytotoxicity Receptors (NCRs: NKp30, NKp44 and NKp46), of which only NKp46 has a mouse orthologue (Ncr1), to eliminate abnormal cells. NKp46/Ncr1 is considered a selective marker for NK cells, although it is also found on a subset of ILCs, where it appears to be without function. The influenza virus hemagglutinin (HA) was the first ligand identified for Ncr1/NKp46 followed by other viral, bacterial and even fungal ligands. NKp46/Ncr1 also recognizes unknown self and tumor ligands. Here we describe the generation of a transgenic mouse where the Ncr1 gene is expressed in the Rosa locus, preceded by a floxed stop sequence allowing Ncr1/NKp46 expression in various tissues upon crossing with Cre transgenic mouse lines. Surprisingly, while several crossings were attempted, Ncr1 overexpression was successful only where cre recombinase expression was dependent on the Ncr1 promoter. Ncr1 overexpression in NK cells increased NK cell immunity in two hallmark Ncr1 related pathologies, influenza virus infection and B16 melanoma. These data suggest that increasing NK cell cytotoxicity by enforced NKp46/Ncr1 expression serves as a potential therapeutic opportunity for the treatment of various pathologies, and in immunotherapy.

Osteoblast-derived FGF9 regulates skeletal homeostasis

FGF9 has complex and important roles in skeletal development and repair. We have previously observed that Fgf9 expression in osteoblasts (OBs) is regulated by G protein signaling and therefore the present study was done to determine whether OB-derived FGF9 was important in skeletal homeostasis. To directly test this idea, we deleted functional expression of Fgf9 gene in OBs using a 2.3kb collagen type I promoter-driven Cre transgenic mouse line (Fgf9OB−/−). Both Fgf9 knockout (Fgf9OB−/−) and the Fgf9 floxed littermates (Fgf9fl/fl) mice were fully backcrossed and maintained in an FBV/N background. Three month old Fgf9OB−/− mice displayed a significant decrease in cancellous bone and bone formation in the distal femur and a significant decrease in cortical thickness at the TFJ. Strikingly, female Fgf9OB−/− mice did not display altered bone mass. Continuous treatment of mouse BMSCs with exogenous FGF9 inhibited mouse BMSC mineralization while acute treatment increased the proliferation of progenitors, an effect requiring the activation of Akt1. Our results suggest that mature OBs are an important source of FGF9, positively regulating skeletal homeostasis in male mice. Osteoblast-derived FGF9 may serve a paracrine role to maintain the osteogenic progenitor cell population through activation of Akt signaling.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification.

Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events.

Germline recombination in a novel Cre transgenic line, Prl3b1-Cre mouse.

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc.

Generation of a Conditional Allele of the Transcription Factor Atonal Homolog 8 (Atoh8).

Atonal Homolog 8 (Atoh8) is a basic helix-loop-helix (bHLH) transcription factor that is highly conserved across species and expressed in multiple tissues during embryogenesis. In the developing pancreas, Atoh8 is expressed in endocrine progenitors but declines in hormone-positive cells, suggesting a role during early stages of the endocrine differentiation program. We previously generated a whole-body Atoh8 knockout but early lethality of null embryos precluded assessment of Atoh8 functions during organ development. Here we report the generation of a conditional Atoh8 knockout mouse strain by insertion of two loxP sites flanking exon 1 of the Atoh8 gene. Pancreas-specific Atoh8 knockout (Atoh8 Δpanc) mice were obtained by mating this strain with a Pdx1-Cre transgenic line. Atoh8 Δpanc mice were born at the expected mendelian ratio and showed normal appearance and fertility. Pancreas weight and gross pancreatic morphology were normal. All pancreatic cell lineages were present, although endocrine δ (somatostatin) cells were modestly augmented in Atoh8 Δpanc as compared to control neonates. This increase did not affect whole-body glucose tolerance in adult knockout animals. Gene expression analysis in embryonic pancreases at the time of the major endocrine differentiation wave revealed modest alterations in several early endocrine differentiation markers. Together, these data argue that Atoh8 modulates activation of the endocrine program but it is not essential for pancreas formation or endocrine differentiation in the mouse. Given the ubiquitous expression pattern of Atoh8, the availability of a mouse strain carrying a conditional allele for this gene warrants further studies using temporally regulated Cre transgenic lines to elucidate time or cell-autonomous functions of Atoh8 during development and in the adult.

Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties.

Over-Expression of Ephrin-A5 in Mice Results in Decreasing the Size of Progenitor Pool through Inducing Apoptosis.

Eph receptors and their ligands, ephrins, mediate cell-to-cell contacts in a specific brain region and their bidirectional signaling is implicated in the regulation of apoptosis during early brain development. In this report, we used the alpha(α)-Cre transgenic line to induce ephrin-A5 over-expression in the distal region of the neural retina. Using this double transgenic embryo, we show that the over-expression of ephrin-A5 was responsible for inducing massive apoptosis in both the nasal and temporal retinas. In addition, the number of differentiated retinal neurons with the exception of the bipolar neuron was significantly reduced, whereas the laminar organization of the mature retina remained intact. Consistent with this finding, an analysis of the mature retina revealed that the size of the whole retina— particularly the nasal and temporal regions—is markedly reduced. These results strongly suggest that the level of ephrin-A5 expression plays a role in the regulation of the size of the retinal progenitor pool in the neural retina.

The Macrophage-Specific Promoter mfap4 Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish.

Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, in vivo analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the mpeg1 promoter. However, mpeg1 transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including Salmonella typhimurium and Mycobacterium marinum. Here, we describe mfap4 as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of mfap4, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.

Col2-Cre and tamoxifen-inducible Col2-CreER target different cell populations in the knee joint

Collagen type 2 (Col2)-Cre or tamoxifen-inducible Col2-CreER transgenic mouse lines have been used for studies to explore the cellular and molecular pathogenesis of osteoarthritis (OA). The purpose of this study is to investigate whether the targeted cells are the same or different in the two mouse lines. We crossed tamoxifen inducible Col2-CreER and Col2-Cre mice with Rosa tdTomato reporter mice and analyzed the labeling patterns at different time points. In the Col2-CreER mice, 90.8 [95% confidence interval (CI) (88.3, 93.2)] and 82.8 (77.4, 88.3) % of the articular surface cells are Tomato positive when tamoxifen was administered at 2 and 2.5 weeks of age and strong activity was observed even 4.5 months after injection. However, 46.0 (32.8, 59.1) and 22.2 (11.7, 32.6) % of the surface cells were Tomato positive when tamoxifen was administered at 3 and 4 weeks of age, respectively. Little to no Tomato activity in the articular surface cells was observed when tamoxifen was administered at 8 weeks of age. At any stage of tamoxifen injection, the Tomato activity was detected in growth plate and epiphyseal bone in addition to articular chondrocytes, but little in endosteum and not in the synovium and ligament. In contrast, the targeted tissues in the Col2-Cre mouse line were articular cartilage, growth plate, meniscus, endosteum, ligament, bone and synovium. This study demonstrates that the pattern of targeted cells in the inducible Col2-CreER mice are partially overlapping with but different from that of targeted cells in Col2-Cre mice and the pattern varies dependent on when tamoxifen is administered.

Abstract 2280: A novel c-Myc transgenic model for human cancers

Abstract Androgen-deprivation therapy (ADT) is a standard therapy for advanced and metastatic prostate cancer (PCa). However, most PCa will relapse and become the lethal castration-resistant PCa (CRPC). Chemotherapy such as Docetaxel, only modestly extends patient survival. To overcome castration resistance and chemoresistance, it is crucial to delineate the underlying molecular mechanisms. c-Myc is the most significantly amplified oncogene in human PCa, and its overexpression is very common in PCa as early as PIN. The Hi-Myc model uses an enhanced probasin promoter to drive c-Myc overexpression in prostate epithelia. Hi-Myc mice develop invasive prostate carcinomas that share molecular features with human PCa. However, since the probasin promoter activity is androgen-dependent, Hi-Myc tumors lose c-Myc expression upon castration. Therefore, the tumor regression represents the mixed effects of both an artificial direct effect from loss of c-Myc oncogene expression and a potential real effect from tumor cellular response to androgen-ablation that is relevant to human PCa. Finally, Hi-Myc mice do not develop CRPC tumors. To study CRPC and chemoresistance of CRPC, we have developed a novel transgenic model to express the c-Myc oncogene in Any tissue in an inducible manner (Ai-Myc model). After crossing with PB-Cre4 mice expressing Cre in prostate epithelia, this model allows us to specifically turn on the expression of c-Myc along with Luc2 (an enhanced luciferase for BLI imaging) in prostate. In addition, once turned on, the expression of c-Myc and Luc2 are no longer dependent on androgen. The PB-Cre4/Ai-Myc mice developed early onset of mPIN, but with much delayed prostate tumor progression. Only a fraction of mice developed significant prostate tumors after one-year of age. Since c-Myc oncogene may induce p53 tumor suppressor activation, and loss or mutation of p53 is a frequent event in late-stage human PCa, we further crossed these mice with p53loxP/loxP mice to generate the PB-Cre4/Ai-Myc/p53loxP/loxP model. Loss of p53 greatly enhanced prostate tumor progression; about 40% of mice developed significant prostate tumors within 4-5 months when all mice have to be sacrificed due to the unexpected growth of large tumors of the epididymis, a rare cancer in human. These epididymis tumors are positive for c-Myc and Luc2, confirming the previously reported off-target activity of PB-Cre4 mice. The nature of these epididymis tumors remains to be determined. We are exploring using several approaches, including early castration together with implantation of androgen pellets, to overcome this problem, which will allow us to concisely study the c-Myc signaling pathway in castration response, castration and chemo-resistance of prostate tumors. Finally, different tissue / organ-specific Cre transgenic mouse line will allow our Ai-Myc model to be used to study different types of human cancers and/or other diseases. Citation Format: Wei Wang, Jiangong Ren, Bingning Dong, Michael M. Ittmann, David D. Moore, Feng Yang. A novel c-Myc transgenic model for human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2280. doi:10.1158/1538-7445.AM2015-2280

Abstract LB-132: Adult lineage-restricted CNS progenitors specify distinct glioblastoma subtypes

Abstract A central question in glioblastoma multiforme (GBM) research is the hierarchy of tumor-initiating cells, and its contribution to the malignant phenotype and genomic make-up of GBM. We examine the potential of adult lineage restricted central nervous system (CNS) progenitors to form malignant gliomas. We show that targeting of Nf1,p53 and Pten mutations in adult CNS progenitors but not stem cells gives rise to fully penetrant GBM. We identify two phenotypically and molecularly distinct GBM subtypes that arise from different adult progenitor lineages. Using multiple inducible cre transgenic lines, we demonstrate that murine GBMs are molecularly separable based on the cell of origin. The oligodendrocyte progenitor cell-derived murine tumors have parallels with a subset of human high-grade gliomas with oligodendrocytic component, tumors that have been ascribed better prognosis compared to classic malignant astrocytomas. These studies indicate that two independent sources of GBM-initiating adult progenitors are susceptible to identical mutations and point to the cell of origin as a major determinant of GBM subtype. Citation Format: Sheila R. Alcantara Llaguno, Zilai Wang, Daochun Sun, Jian Chen, Jing Xu, Euiseok Kim, Kimmo Hatanpaa, Jack Raisanen, Dennis Burns, Jane Johnson, Luis F. Parada. Adult lineage-restricted CNS progenitors specify distinct glioblastoma subtypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-132. doi:10.1158/1538-7445.AM2015-LB-132

Generation of BAF53b-Cre transgenic mice with pan-neuronal Cre activities.

Molecular and functional studies of genes in neurons in mouse models require neuron-specific Cre lines. The current available neuronal Cre transgenic or knock-in lines either result in expression in a subset of neurons or expression in both neuronal and non-neuronal tissues. Previously we identified BAF53b as a neuron-specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b-Cre is largely neuron-specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b-Cre functioned in primary cultures in a pan-neuron-specific manner. Thus, BAF53b-Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies.

Astrocytic β1-integrin affects cellular composition of murine blood brain barrier in the cerebral cortex

The blood brain barrier (BBB) is composed of endothelial cells, astrocytes, and pericytes and maintains functional homeostasis by regulating transport of ions, fluid and cells between blood and neural tissue. The cellular and molecular pathways that contribute to the formation of the BBB in the developing brain have not been fully deciphered. β1-integrin (β1-itg) within endothelial cells is known to play a critical role in vasculogenesis. However, the role of astrocytic β1-itg in BBB development is not known. Our study used a mouse glial fibrillary acidic protein (GFAP)-cre transgenic line to selectively ablate β1-itg within astrocytes. We found that deletion of astrocytic β1-itg had a striking effect on the different cell types that form the BBB. Mutant mice had a decreased density of aquaporin-4 immunoreactivity within the perivascular astrocytic end-feet. We also found decreases in immunoreactivity for vimentin and CD-31 within endothelial cells. These changes were not accompanied by functional changes in BBB under physiological conditions as assessed by extravasation of large and small molecular weight molecules. However, mutant mice had an increased incidence of severe cystic injury in response to neonatal hypoxia. Our findings show that astrocytic β1-itg has an important role in defining cellular properties of the blood brain barrier in the cerebral cortex.

Targeted Ablation, Silencing, and Activation Establish Glycinergic Dorsal Horn Neurons as Key Components of a Spinal Gate for Pain and Itch

SummaryThe gate control theory of pain proposes that inhibitory neurons of the spinal dorsal horn exert critical control over the relay of nociceptive signals to higher brain areas. Here we investigated how the glycinergic subpopulation of these neurons contributes to modality-specific pain and itch processing. We generated a GlyT2::Cre transgenic mouse line suitable for virus-mediated retrograde tracing studies and for spatially precise ablation, silencing, and activation of glycinergic neurons. We found that these neurons receive sensory input mainly from myelinated primary sensory neurons and that their local toxin-mediated ablation or silencing induces localized mechanical, heat, and cold hyperalgesia; spontaneous flinching behavior; and excessive licking and biting directed toward the corresponding skin territory. Conversely, local pharmacogenetic activation of the same neurons alleviated neuropathic hyperalgesia and chloroquine- and histamine-induced itch. These results establish glycinergic neurons of the spinal dorsal horn as key elements of an inhibitory pain and itch control circuit.