Cre Mouse Line Research Articles

P10.02.B The fragile balance between proliferation and neoplasia in neural stem cells: role of DIAPH3 in glioblastoma

Abstract Background DIAPH3 belongs to the formin family of proteins that are master regulators of the cell cytoskeleton. DIAPH3 plays a key role in cytokinesis as well karyokinesis. Hence, in the mouse brain, DIAPH3-deficient neural stem cells exhibit abnormal cell division leading to aneuploidy and apoptosis of cells progeny. Given the importance of aneuploidy in cancer development and progression, we asked whether DIAPH3 loss could be involved in the genesis and the aggressiveness of brain tumors. Material and Methods We co-inactivated Diaph3 and Trp53 genes in the mouse cerebral cortex using the Emx1-Cre mouse line (Diaph3/Trp53 dcKO). As a control, we inactivated only Trp53 gene using the same Cre mouse line (Trp53 cKO). Mouse survival was analyzed, and tumors were collected for histological analysis as well as molecular characterization. Additionally, we measured DIAPH3 expression in human glioblastoma samples and studied its impact on patient overall survival. Results Diaph3/Trp53 dcKO develop brain tumors earlier compared to Trp53 cKO resulting in a decrease of mouse survival. Hematoxylin-eosin staining, as well as GFAP, OLIG2 and KI67 immunostainings strongly suggest that the tumors are glioblastomas. Quantitative reverse transcription PCR performed on biopsies from 73 patients uncover variable DIAPH3 expression levels in glioblastoma samples. Interestingly, multivariate Cox proportional-hazards analysis shows that low DIAPH3 expression predicts a worse overall survival, independently from other known prognosis factors. Conclusion On one hand, our study shows that DIAPH3 loss favors glioblastoma development in mouse, strongly suggesting a tumor-suppressor role for DIAPH3. On the other hand, the clinical study shows that DIAPH3 low expression has a negative impact on patient overall survival indicating that DIAPH3 loss may favor resistance to chemotherapy and/or radiotherapy.

Characterisation of deep dorsal horn projection neurons in the spinal cord of the Phox2a::Cre mouse line.

Projection neurons belonging to the anterolateral system (ALS) underlie the perception of pain, skin temperature and itch. Many ALS cells are located in laminae III-V of the dorsal horn and the adjacent lateral white matter. However, relatively little is known about the excitatory synaptic input to these deep ALS cells, and therefore about their engagement with the neuronal circuitry of the region. We have used a recently developed mouse line, Phox2a::Cre, to investigate a population of deep dorsal horn ALS neurons known as “antenna cells”, which are characterised by dense innervation from peptidergic nociceptors, and to compare these with other ALS cells in the deep dorsal horn and lateral white matter. We show that these two classes differ, both in the density of excitatory synapses, and in the source of input at these synapses. Peptidergic nociceptors account for around two-thirds of the excitatory synapses on the antenna cells, but for only a small proportion of the input to the non-antenna cells. Conversely, boutons with high levels of VGLUT2, which are likely to originate mainly from glutamatergic spinal neurons, account for only ∼5% of the excitatory synapses on antenna cells, but for a much larger proportion of the input to the non-antenna cells. VGLUT1 is expressed by myelinated low-threshold mechanoreceptors and corticospinal axons, and these innervate both antenna and non-antenna cells. However, the density of VGLUT1 input to the non-antenna cells is highly variable, consistent with the view that these neurons are functionally heterogeneous.

A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping

BackgroundThe functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production.ResultsBriefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle.ConclusionsThe lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII–PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line

A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50–60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.

Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis

Angiogenesis is a dynamic process that involves expansion of a preexisting vascular network that can occur in a number of physiological and pathological settings. Despite its importance, the origin of the new angiogenic vasculature is poorly defined. In particular, the primary subtype of endothelial cells (capillary, venous, arterial) driving this process remains undefined. Endothelial cells were fate-mapped with the use of genetic markers specific to arterial and capillary cells. In addition, we identified a novel venous endothelial marker gene (Gm5127) and used it to generate inducible venous endothelium-specific Cre and Dre driver mouse lines. Contributions of these various types of endothelial cells to angiogenesis were examined during normal postnatal development and in disease-specific setting. Using a comprehensive set of endothelial subtype-specific inducible reporter mice, including tip, arterial, and venous endothelial reporter lines, we showed that venous endothelial cells are the primary endothelial subtype responsible for the expansion of an angiogenic vascular network. During physiological angiogenesis, venous endothelial cells proliferate, migrating against the blood flow and differentiating into tip, capillary, and arterial endothelial cells of the new vasculature. Using intravital 2-photon imaging, we observed venous endothelial cells migrating against the blood flow to form new blood vessels. Venous endothelial cell migration also plays a key role in pathological angiogenesis. This was observed both in formation of arteriovenous malformations in mice with inducible endothelium-specific Smad4 deletion mice and in pathological vessel growth seen in oxygen-induced retinopathy. Our studies establish that venous endothelial cells are the primary endothelial subtype responsible for normal expansion of vascular networks, formation of arteriovenous malformations, and pathological angiogenesis. These observations highlight the central role of the venous endothelium in normal development and disease pathogenesis.

Molecular cell identities in the mediodorsal thalamus of infant mice and marmoset.

The mediodorsal thalamus (MD) is a higher-order nucleus located within the central thalamus in many mammalian species. Emerging evidence from MD lesions and tracer injections suggests that the MD is reciprocally connected to the prefrontal cortex (PFC) and plays an essential role in specific cognitive processes and tasks. MD subdivisions (medial, central, and lateral) are poorly segregated at the molecular level in rodents, leading to a lack of MD subdivision-specific Cre driver mice. Moreover, this lack of molecular identifiers hinders MD subdivision- and cell-type-specific circuit formation and function analysis. Therefore, using publicly available databases, we explored molecules separately expressed in MD subdivisions. In addition to MD subdivision markers, we identified several genes expressed in a subdivision-specific combination and classified them. Furthermore, after developing medial MD (MDm) or central MD (MDc) region-specific Cre mouse lines, we identified diverse region- and layer-specific PFC projection patterns. Comparison between classified MD marker genes in mice and common marmosets, a nonhuman primate model, revealed diverging gene expression patterns. These results highlight the species-specific organization of cell types and their projections in the MD thalamus.

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease.

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/– mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/– mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/– mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.

Development of a Novel Tetracycline-Inducible Kiss1-Cre Mouse Line for Temporally Controlled Gene Deletion in Kisspeptin Neurons

The tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgenic expression in mice. Previously, we generated a GnRH-CreTeR mouse model, using a first-generation Tet-inducible system to temporally induce expression of Cre recombinase in GnRH neurons. Recently, the Tet-inducible system has undergone several modifications to significantly reduce previous limitations that include leaky background expression and lower sensitivity to tetracycline induction. Therefore, we have developed a novel mouse model bearing a Tet-inducible kisspeptin-Cre allele (iKiss-Cre mouse) that will enable temporal control over the selective deletion of genes from Kiss1 neurons. This temporally controlled gene deletion will eliminate a longstanding technical limitation of conventional steroid receptor knockout models in which steroid regulation of the axis is confounded by steroid developmental and organizational effects in the reproductive axis. Two mouse lines were generated. The first line targets kisspeptin neurons with a third generation Tet-inducible reverse tetracycline transactivator (rtTA, Tet-On 3G) expressed under the control of the Kiss1 allele. Using CRISPR-Cas9 technology, we inserted a cassette containing an internal ribosome entry site (IRES) sequence followed by the rtTA downstream of the Kiss1 coding region as was previously done using Cre recombinase. Transcription of the recombinant Kiss1 allele yields a bicistronic messenger RNA, from which both kisspeptin and rtTA are independently translated. The second mouse line, TRE-Cre mice, was constructed to express Cre recombinase under control of the PTRE3G promoter. The PTRE3G promoter is bidirectional to allow simultaneous monitoring of Cre expression and a second florescent protein reporter (ZsGreen1). The Kiss1-rtTA only binds to and activates the PTRE3G promoter in the presence of doxycycline (an analog of tetracycline). Offspring from breeding of these mouse lines, iKiss-Cre, result in a system capable of generating Tet-induced expression of Cre recombinase in kisspeptin neurons. In order to document specificity and sensitivity of this system, we performed immunofluorescent staining and observed colocalization of kisspeptin with Cre recombinase in brain sections of iKiss-Cre mice only after doxycycline treatment. We will use this model to investigate negative feedback actions of E2 on the adultpulse generator after E2 exerts its organizational actions on maturing kisspeptin neurons. We expect that this mouse model will become a major tool used by the neuroendocrine community and serve as proof of principle for development of similar inducible knockout models employing the current generation of inducible methodologies.

Early B-cell Factor1 (Ebf1) promotes early osteoblast differentiation but suppresses osteoblast function.

Early B cell factor 1 (Ebf1) is a transcription factor that regulates B cell, neuronal cell and adipocyte differentiation. We and others have shown that Ebf1 is expressed in osteoblasts and that global deletion of Ebf1 results in increased bone formation in vivo. However, as Ebf1 is expressed in multiple tissues and cell types, it has remained unclear, which of the phenotypic changes in bone are derived from bone cells. The aim of this study was to determine the cell-autonomous and differentiation stage-specific roles of Ebf1 in osteoblasts. In vitro, haploinsufficient Ebf1+/- calvarial cells showed impaired osteoblastic differentiation indicated by lower alkaline phosphatase (ALP) activity and reduced mRNA expression of osteoblastic genes, while overexpression of Ebf1 in wild type mouse calvarial cells led to enhanced osteoblast differentiation with increased expression of Osterix (Osx). We identified a putative Ebf1 binding site in the Osterix promoter by ChIP assay in MC3T3-E1 osteoblasts and showed that Ebf1 was able to activate Osx-luc reporter construct that included this Ebf1 binding site, suggesting that Ebf1 indeed regulates osteoblast differentiation by inducing Osterix expression. To reconcile our previous data and that of others with our novel findings, we hypothesized that Ebf1 could have a dual role in osteoblast differentiation promoting early but inhibiting late stages of differentiation and osteoblast function. To test this hypothesis in vivo, we generated conditional Ebf1 knockout mice, in which Ebf1 deletion was targeted to early or late osteoblasts by crossing Ebf1fl/fl mice with Osx- or Osteocalcin (hOC)-Cre mouse lines, respectively. Deletion of Ebf1 in early Ebf1Osx-/- osteoblasts resulted in significantly increased bone volume and trabecular number at 12weeks by μCT analysis, while Ebf1hOC-/- mice did not have a bone phenotype. To conclude, our data demonstrate that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. However, Ebf1 inhibits bone accrual in the Osterix expressing osteoblasts in vivo but it is redundant in the maintenance of mature osteoblast function.

Utility of progesterone receptor-ires-Cre to generate conditional knockout mice for uterine study.

In mice, the conditional knockout strategy using the Cre‐loxP system is useful for various types of research. The Cre mouse line with progesterone receptor promoter (Pgr Cre ) has been widely used to produce specific uterine gene‐deficient mice, but in the Cre line, endogenous Pgr gene is replaced by Cre recombinase gene, which makes the breeding of homozygous mice (Pgr Cre/Cre ) difficult because they are infertile. Yang et al. (2013, https://10.1016/j.cell.2013.04.017) reported the generation of another Pgr iresCre mouse line that still has endogenous Pgr gene, and they inserted Cre recombinase downstream of the Pgr gene via an internal ribosome entry site (IRES). It is possible that this new Pgr iresCre line would be useful for uterine research as the mice can be bred as homozygotes (Pgr iresCre/iresCre ). Herein, we confirmed the Pgr iresCre mice effectively directed recombination in the female reproductive tract and was capable of genetic alteration in the endometrium that enables the studies of its uterine function. Our findings demonstrate that the new Pgr iresCre mouse line is also useful for the generation of uterine‐specific knockout mice. The findings using Pgr iresCre mouse will contribute to the understanding of reproductive systems and diseases in humans and domestic animals.

Taste buds are not derived from neural crest in mouse, chicken, and zebrafish

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreERT2/tdT mouse, GFP+ neural fold transplantation to GFP− chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreERT2 specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP+/GFP− chicken chimera model, GFP+ cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish.

Cocaine-Dependent Acquisition of Locomotor Sensitization and Conditioned Place Preference Requires D1 Dopaminergic Signaling through a Cyclic AMP, NCS-Rapgef2, ERK, and Egr-1/Zif268 Pathway.

Elucidation of the mechanism of dopamine signaling to ERK that underlies plasticity in dopamine D1 receptor-expressing neurons leading to acquired cocaine preference is incomplete. NCS-Rapgef2 is a novel cAMP effector, expressed in neuronal and endocrine cells in adult mammals, that is required for D1 dopamine receptor-dependent ERK phosphorylation in mouse brain. In this report, we studied the effects of abrogating NCS-Rapgef2 expression on cAMP-dependent ERK→Egr-1/Zif268 signaling in cultured neuroendocrine cells; in D1 medium spiny neurons of NAc slices; and in either male or female mouse brain in a region-specific manner. NCS-Rapgef2 gene deletion in the NAc in adult mice, using adeno-associated virus-mediated expression of cre recombinase, eliminated cocaine-induced ERK phosphorylation and Egr-1/Zif268 upregulation in D1-medium spiny neurons and cocaine-induced behaviors, including locomotor sensitization and conditioned place preference. Abrogation of NCS-Rapgef2 gene expression in mPFC and BLA, by crossing mice bearing a floxed Rapgef2 allele with a cre mouse line driven by calcium/calmodulin-dependent kinase IIα promoter also eliminated cocaine-induced phospho-ERK activation and Egr-1/Zif268 induction, but without effect on the cocaine-induced behaviors. Our results indicate that NCS-Rapgef2 signaling to ERK in dopamine D1 receptor-expressing neurons in the NAc, but not in corticolimbic areas, contributes to cocaine-induced locomotor sensitization and conditioned place preference. Ablation of cocaine-dependent ERK activation by elimination of NCS-Rapgef2 occurred with no effect on phosphorylation of CREB in D1 dopaminoceptive neurons of NAc. This study reveals a new cAMP-dependent signaling pathway for cocaine-induced behavioral adaptations, mediated through NCS-Rapgef2/phospho-ERK activation, independently of PKA/CREB signaling.SIGNIFICANCE STATEMENT ERK phosphorylation in dopamine D1 receptor-expressing neurons exerts a pivotal role in psychostimulant-induced neuronal gene regulation and behavioral adaptation, including locomotor sensitization and drug preference in rodents. In this study, we examined the role of dopamine signaling through the D1 receptor via a novel pathway initiated through the cAMP-activated guanine nucleotide exchange factor NCS-Rapgef2 in mice. NCS-Rapgef2 in the NAc is required for activation of ERK and Egr-1/Zif268 in D1 dopaminoceptive neurons after acute cocaine administration, and subsequent enhanced locomotor response and drug seeking behavior after repeated cocaine administration. This novel component in dopamine signaling provides a potential new target for intervention in psychostimulant-shaped behaviors, and new understanding of how D1-medium spiny neurons encode the experience of psychomotor stimulant exposure.

Advantages and Limitations of Cre Mouse Lines Used in Skeletal Research.

The Cre-LoxP technology permits gene ablation in specific cell lineages, at chosen differentiation stages of this lineage and in an inducible manner. It has allowed tremendous advances in our understanding of skeleton biology and related pathophysiological mechanisms, through the generation of loss/gain of function or cell tracing experiments based on the creation of an expanding toolbox of transgenic mice expressing the Cre recombinase in skeletal stem cells, chondrocytes, osteoblasts, or osteoclasts. In this chapter, we provide an overview of the different Cre-LoxP systems and Cre mouse lines used in the bone field, we discuss their advantages, limitations, and we outline best practices to interpret results obtained from the use of Cre mice.

Wnt/\u03b2-catenin activation cooperates with loss of p53 to cause adrenocortical carcinoma in mice

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with limited therapeutic options. The lack of mouse models that recapitulate the genetics of ACC has hampered progress in the field. We analyzed The Cancer Genome Atlas (TCGA) dataset for ACC and found that patients harboring alterations in both p53/Rb and Wnt/β-catenin signaling pathways show a worse prognosis compared with patients that harbored alterations in only one. To model this, we utilized the Cyp11b2(AS)Cre mouse line to generate mice with adrenocortical-specific Wnt/β-catenin activation, Trp53 deletion, or the combination of both. Mice with targeted Wnt/β-catenin activation or Trp53 deletion showed no changes associated with tumor formation. In contrast, alterations in both pathways led to ACC with pulmonary metastases. Similar to ACCs in humans, these tumors produced increased levels of corticosterone and aldosterone and showed a high proliferation index. Gene expression analysis revealed that mouse tumors exhibited downregulation of Star and Cyp11b1 and upregulation of Ezh2, similar to ACC patients with a poor prognosis. Altogether, these data show that altering both Wnt/β-catenin and p53/Rb signaling is sufficient to drive ACC in mouse. This autochthonous model of ACC represents a new tool to investigate the biology of ACC and to identify new treatment strategies.

A Whole-Brain Cell-Type-Specific Sparse Neuron Labeling Method and Its Application in a Shank3 Autistic Mouse Model.

Single neurons, as the basic unit of the brain, consist of a cell body and processes, including dendrites and axons. Even neurons of the same type show various subtle process characteristics to fit into the diverse neural circuits. Different cell types of neurons form complicated circuits in the brain. Therefore, detailed neuronal morphology is required to understand normal neuronal function and pathological mechanisms, such as those that occur in autism. Here, we developed a strategy to sparsely label the same type of neurons throughout the whole brain and tested its application in an autistic animal model—Shank3 knockout (KO) mice. To achieve this, we designed an adeno-associated virus (AAV) that expresses Cre recombinase-dependent regular and membrane-targeted enhanced green fluorescent protein (EGFP) under a human synapsin 1 promoter and verified it in several Cre transgenic mice. We could sparsely label the projection neurons in multiple brain areas by retro-ocular injection of the virus into CaMKIIα-Cre mice. Then, we analyzed the morphology of the projection neurons in Shank3 KO mice with this method. We found differential dendritic complexity and dendritic spine changes in projection neurons in Shank3 KO mice crossed with CaMKIIα-Cre mice compared with littermate control mice in the striatum, cortex, and hippocampus. By combining this method with various Cre mouse lines crossed with mouse models of disease, we can screen the morphological traits of distinct types of neurons throughout the whole brain that will help us to understand the exact role of the specific cell types of neurons not only in autism spectrum disorder (ASD) mouse models but also in other psychiatric disorder mouse models.

Generation of a conditional transgenic mouse model expressing human Phospholipase A2 Receptor 1

The Phospholipase A2 Receptor 1 (PLA2R1) was first identified for its ability to bind some secreted PLA2s (sPLA2s). It belongs to the C-type lectin superfamily and it binds different types of proteins. It is likely a multifunctional protein that plays a role i) in inflammation and inflammatory diseases, ii) in cellular senescence, a mechanism participating in aging and age-related diseases including cancer, and iii) in membranous nephropathy (MN), a rare autoimmune kidney disease where PLA2R1 is the major autoantigen. To help study the role of PLA2R1 in these pathophysiological conditions, we have generated a versatile NeoR-hPLA2R1 conditional transgenic mice which will allow the specific expression of human PLA2R1 (hPLA2R1) in relevant organs and cells following Cre recombinase-driven excision of the NeoR-stop cassette flanked by LoxP sites. Proof-of-concept breeding of NeoR-hPLA2R1 mice with the ubiquitous adenoviral EIIa promoter-driven Cre mouse line resulted in the expected excision of the NeoR-stop cassette and the expression of hPLA2R1 in all tested tissues. These Tg-hPLA2R1 animals breed normally, with no reproduction or apparent growth defect. These models, especially the NeoR-hPLA2R1 conditional transgenic mouse line, will facilitate the future investigation of PLA2R1 functions in relevant pathophysiological contexts, including inflammatory diseases, age-related diseases and MN.

A Pipeline for the Rapid Production and Dissemination of Mouse Intersectional Genetic Alleles for Functional, Molecular, and Anatomical Neural Circuit Mapping. (mouseintersectionalgenetics.org)

Here we describe an effort to build and distribute a community library, based on public input, of more than 100 mouse intersectional genetic alleles to facilitate developmental, anatomical, molecular, and functional characterizations of neural circuit organization in behavior and physiology. Even within narrowly defined cell types, significant diversity is found at multiple levels including genetic and molecular signatures, activity patterns, and synaptic connectivity. Current challenges now center on tools to identify, access, and study cell populations with increasing precision. Intersectional genetics offers exceptionally high resolution to consistently delineate distinct cell types in the embryo and adult mouse for functional, molecular and anatomical studies. Intersectional genetics utilizes a ubiquitously expressed conditional allele that is activated by Cre and Flp site specific recombinases. Upon activation, the intersectional genetic alleles may express any number of Genetically Encodable Effector Molecules (GEEMS), such as channelrhodopsins for neural activity perturbations or an L10‐GFP ribotrap fusion to affinity purify translating mRNAs. The intersectional allele is activated by overlapping expression of both Flp and Cre recombinases in the same cell. Traditionally, these recombinases have been deployed as gene knock‐in or transgenic alleles that are designed to express in a cell or gene specific fashion. The use of two selectors (Cre and Flp) to define a cellular population enables high specificity and modularity in combining any set of Cre, Flp, and intersectional alleles to fit an experiment. The number of Cre and Flp recombinase mouse lines are constantly growing, giving greater access to increasing numbers of cell types. Additionally, these recombinases are the focus of multiple efforts to deploy them in ways that select cells based on other unique properties such as neuronal activity and synaptic connectivity. To make this powerful genetic approach more accessible to as many research laboratories as possible, we are building a production pipeline to create a suite of resources for anyone to easily make their own intersectional mouse line, to produce over 100 targeted ES cell lines that can be developed into mouse lines, as well as 10–15 high demand mouse lines all for public distribution. Priority for the GEEMS to be intersectionally deployed will be based on a public input collected at mouseintersectionalgenetics.org. All plasmids, cell lines, and mouse lines developed under this effort will be distributed without restriction to non‐profit research organizations (Addgene and Mutant Mouse Resource and Research Center; MMRRC).Support or Funding InformationR21OD025327Figure 1

An inducible Cre mouse line to sparsely target nervous system cells, including Remak Schwann cells

Nerves of the peripheral nervous system contain two classes of Schwann cells: myelinating Schwann cells that ensheath large caliber axons and generate the myelin sheath, and Remak Schwann cells that surround smaller axons and do not myelinate. While tools exist for genetic targeting of Schwann cell precursors and myelinating Schwann cells, such reagents have been challenging to generate specifically for the Remak population, in part because many of the genes that mark this population in maturity are also robustly expressed in Schwann cell precursors. To circumvent this challenge, we utilized BAC transgenesis to generate a mouse line expressing a tamoxifen-inducible Cre under the control of a Remak-expressed gene promoter (Egr1). However, as Egr1 is also an activity dependent gene expressed by some neurons, we flanked this Cre by flippase (Flpe) recognition sites, and coinjected a BAC expressing Flpe under control of a pan-neuronal Snap25 promoter to excise the Cre transgene from these neuronal cells. Genotyping and inheritance demonstrate that the two BACs co-integrated into a single locus, facilitating maintenance of the line. Anatomical studies following a cross to a reporter line show sparse tamoxifen-dependent recombination in Remak Schwann cells within the mature sciatic nerve. However, depletion of neuronal Cre activity by Flpe is partial, with some neurons and astrocytes also showing evidence of Cre reporter activity in the central nervous system. Thus, this mouse line will be useful in mosaic loss-of-function studies, lineage tracing studies following injury, live cell imaging studies, or other experiments benefiting from sparse labeling.

Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations.

Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent “helper” adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.