Cre Activation Research Articles

Role of Kctd13 in modulating AR and SOX9 expression in different penile cell populations.

Micropenis is a condition with significant physical and psychological implications caused mainly by decreased androgen action in penile development. Kctd13-knockout (Kctd13-KO) mice have micropenis, cryptorchidism, and fertility defects because of reduced levels of androgen receptor (AR) and SOX9. We hypothesized that normalizing the levels of AR and SOX9 in the Kctd13-KO penis could help us to understand the mechanism of action of these signaling pathways on penile development. We generated transgenic mice lacking Kctd13 and conditionally expressing AR in the urethral mesenchyme after Cre activation with Twist2cre (Kctd13-KO; AR-CMV; Twist2cre; herein called AR+), and Sox9 in the urethral epithelium after Cre activation with Shhcre (Kctd13-KO; Sox9-CAG; Shhcre; herein called SOX9+). Mice penile morphology, fertility, and the effect of KCTD13 on AR and SOX9 ubiquitination were evaluated. Kctd13-KO micropenis phenotype was rescued after increasing levels of penile AR or SOX9 as transgenic AR+ and SOX9+ mice have longer penile lengths than Kctd13-KO mice and are comparable to WT mice. In addition, male-urogenital-mating-protuberance and the baculum were significantly shorter and narrower in Kctd13-KO mice compared with transgenic AR+ and SOX9+ mice. The position of the urethral meatus was similar and orthotopic in location in Kctd13-KO, AR+, SOX9+, and WT penises indicating that none of these mice had hypospadias. The subfertility of AR+ and SOX9+ mice was improved. The ectopic expression of KCTD13 in HEK293 cells strongly reduced AR ubiquitination which is abolished when the proteasome pathway is inhibited and this process is mediated by the ubiquitin ligase, STUB1. The effect of KCTD13 on SOX9 ubiquitination is minimal. KCTD13 regulates AR ubiquitination by modulating STUB1 binding to AR. Penile restoration of AR and SOX9 improved penile development in Kctd13-KO mice allowing us to discern the contribution from individual signaling pathways and cell types in penile development.

A new Prdm1-Cre line is suitable for studying the second heart field development

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

Tamoxifen exerts direct and microglia-mediated effects preventing neuroinflammatory changes in the adult mouse hippocampal neurogenic niche.

Tamoxifen-inducible systems are widely used in research to control Cre-mediated gene deletion in genetically modified animals. Beyond Cre activation, tamoxifen also exerts off-target effects, whose consequences are still poorly addressed. Here, we investigated the impact of tamoxifen on lipopolysaccharide (LPS)-induced neuroinflammatory responses, focusing on the neurogenic activity in the adult mouse dentate gyrus. We demonstrated that a four-day LPS treatment led to an increase in microglia, astrocytes and radial glial cells with concomitant reduction of newborn neurons. These effects were counteracted by a two-day tamoxifen pre-treatment. Through selective microglia depletion, we elucidated that both LPS and tamoxifen influenced astrogliogenesis via microglia mediated mechanisms, while the effects on neurogenesis persisted even in a microglia-depleted environment. Notably, changes in radial glial cells resulted from a combination of microglia-dependent and -independent mechanisms. Overall, our data reveal that tamoxifen treatment per se does not alter the balance between adult neurogenesis and astrogliogenesis but does modulate cellular responses to inflammatory stimuli exerting a protective role within the adult hippocampal neurogenic niche.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Exploring Aging Dependent Adaptations of Splicing Factor Mutations in Clonal Hematopoiesis

Introduction: It has been shown that leukemia-associated mutations can be detected in the blood of healthy individuals and their clonal expansion in hematopoietic stem and progenitor cell (HSPC) populations is referred to as Clonal Hematopoiesis (CH). In one study, a population analysis of a cohort of 17,182 patients performed by Bick et al. [PMID7944936] selected without prior screening for hematological malignancies, indicated that advancing age is directly associated with an increase in CH. When the mutant clone accounts for 4% of the nucleated blood cells, it is then referred to as Clonal Hematopoiesis of Indeterminant Potential (CHIP). CHIP has been classified by the World Health Organization as a premalignant state and risk criteria for neoplastic development. CHIP is highly associated with adverse outcomes including auto-immune or auto-inflammatory conditions, cardiovascular disease, cytopenia, cancers in general, and other hematological neoplasms. The associated hematopoietic neoplasms include myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and acute myeloid leukemia (AML). Population studies reveal splicing factor mutations to be among the most common mutations in CHIP and are highly correlated with age and show a high hazard ratio for risk of developing secondary neoplasms including AML. RNA splicing factors U2AF1, SRSF2, and SF3B1 make up core components of the spliceosome and are critical to prevent aberrant splicing. Mutations in these factors have widespread effects implicating a multitude of cellular processes. There are several established mutant splicing factor mouse models that can be utilized; however, many employ non-specific or leaky Cre drivers. Methods: To avoid non-specific and leaky Cre activation during aging, we used a Cre/lox-mediated U2af1(Q157R) conditional knock-in mouse model that does not contain an internal Cre driver. This mouse has a floxed wild-type U2af1 cDNA insert that expresses wild-type U2AF1 under normal circumstances. However, upon the exogenous addition of recombinant Cre the U2af1 cDNA is excised and the U2af1(Q157R) splicing factor mutation in exon 6 is expressed instead. This mouse model also possesses lox-stop-lox-YFP, leading to expression of YFP along with mutant U2AF1(Q157R) when Cre is introduced. We then isolated bone marrow from 6 week (young) and 6 months (aged) floxed Q157R/YFP mice and treated lineage-depleted bone marrow with recombinant Cre protein for two hours, followed by transplantation into mildly conditioned 6 week (young) and 18 month (old) wild-type mice. The percentage of YFP in the peripheral blood is quantified every 3 weeks. Full analyses, including RNA-seq, of the bone marrow progenitor populations after 3 months is in progress. Results and Discussion: Using the previously mentioned bone marrow transplant model our work shows that when a U2AF1 mutation is introduced into young and old recipient mice, there is significant preferential outgrowth when the donor mice are aged (Figure 1). This is indicated by an increase in the ratio of YFP, which represents the U2AF1(Q157R) mutant, to CD45.1 events, which represent an internal engraftment control. This would suggest that the expansion of U2AF1(Q157R) splicing mutant cells seen in clinical data may be the result of intrinsic factors from the aging cell population. Comprehensive analysis of the effects of alternative splicing in this model is ongoing, however, these data provide support for further investigation of the intrinsic effects of splicing factor mutations in an aged context.

Abstract 18828: Generation and Characterization of a Novel Myh11 Knock-In Smooth Muscle Cell-Specific CreEr T2 Mouse Model

Background: MYH11 is the most specific and robust marker for differentiated smooth muscle cells (SMCs) and its proximal promoter has been used as a SMC Cre-driver to investigate SMC-specific roles of floxed target genes. Two distinct Myh11-CreER T2 ( Myh11-CreER T2 -Off and -RAD ) mouse lines have been used widely for inducible SMC-specific Cre/loxP-mediated recombination in conditional mutagenesis or lineage tracing. However, both mouse lines were generated using bacterial artificial chromosome (BAC), which were randomly integrated into the genome, potentially resulting in multiple copies of the transgene and the unpredictable disruption of endogenous genes. An additional limitation of the Myh11-CreER T2 -Off mouse is its only works on the male mice. To address these limitations, we generated and characterized a new Myh11-CreER T2 mouse ( Myh11-CreER T2 -KI ), which results from integration of a CreER T2 cassette into the endogenous Myh11 gene locus, enabling faithful SMC-specific lineage tracing or specific gene knockout in SMC in vivo in both sexes. Method: A tamoxifen-inducible, CreER T2 cassette was inserted into one of the Myh11 gene alleles after the initiation codon of Myh11 . To validate the Myh11 CreERT2/+ -mediated gene recombination in SMCs, Myh11-CreER T2 -KI mice were crossed with mTmG dual reporter mice or the lncRNA Carmn GFP knock-in reporter/knockout mice to trace SMC lineages or generate SMC-specific inducible Carmn knockout mice. The recombination specificity in SMCs was analyzed by GFP fluorescence analysis. Results: The CreER T2 cassette inserted into the Myh11 gene locus negligibly affects endogenous MYH11 protein expression. In mTmG / Myh11-CreER T2 -KI reporter mice, GFP expression was detected specifically in both vascular and visceral SMCs in various tissues after tamoxifen mediated Cre activation. Moreover, Myh11-CreER T2 -KI -mediated Carmn SMC-specific deletion in adult female mice recapitulated the lethal intestinal pseudo-obstructive phenotype observed in male Myh11-CreER T2 -Off Carmn KO mice. Conclusions: Our new Myh11-CreER T2 -KI mouse model provides an additional genetic tool using the fidelity of an endogenous SMC gene locus to trace SMC lineages and delete genes in the SMC of both sexes of mice.

EndoC-βH5 cells are storable and ready-to-use human pancreatic beta cells with physiological insulin secretion

ObjectivesReadily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family. MethodsEndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays. ResultsReady to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation. ConclusionsEndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.

Abstract 2601: Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development

Abstract LRIG1, leucine-rich repeats and immunoglobulin-like domains protein 1, is deleted or downregulated in many cancer types and has been reported to function as a tumor suppressor. LRIG1 is preferentially expressed in adult stem/progenitor cells and regulates their biological properties and quiescence by repressing the ERBB signaling. We recently reported LRIG1 to be overexpressed in human prostate cancer and to function as a feedback tumor suppressor. Human LRIG1 transgenically expressed in the mouse prostate inhibited the development and growth of both Hi-Myc and TRAMP tumors. The relationship between Lrig1-expressing adult stem/progenitor cells and cells-of-origin for cancer remains poorly understood. To address this, we deleted the Pten tumor suppressor gene in Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with Ptenfl/fl animals followed by Cre activation via tamoxifen administration. This knockout of Pten in Lrig1-expressing cells allowed us to assess the tumor suppressive function of Lrig1 as Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated. We also generated a Lrig1-lineage tracing mouse model to label endogenous Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with CAG-tdTomato animals. Cre recombinase in these animals was activated by tamoxifen and epithelial tissues were harvested and analyzed for tomato signal by fluorescent microscopy. Lrig1-lineage tracing model showed that Lrig1-expressing cells in mouse prostate and oral epithelia are of CK5+ and CK8- phenotype suggesting their basal lineage. Targeted deletion of Pten in the Lrig1-expressing cellular compartments resulted in hyperplasic lesions in oral mucosa as early as 4 weeks and prevalent papilloma by 8 weeks. Pten deletion in the mouse prostate led to hyperplasia and high-grade prostate intraepithelial neoplasia (HGPIN) in a time-dependent manner. Consistent with these hyperplastic and tumor phenotypes, we observed elevated Ki-67 and pAkt staining indicating increased cell proliferation. Since Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated, our data suggest that even one allelic loss of Lrig1 promotes more aggressive tumor phenotypes compared to those reported earlier with Pten deletion in CK5+ basal cells (CK5-CreERT2:Ptenfl/fl) in which similar lesions in oral mucosa and prostate tissues developed only after 20-24 weeks of Pten deletion. These results suggested that LRIG1 is a haplo-insufficient tumor suppressor as mono-allelic deletion of Lrig1 is sufficient to promote the tumorigenic process in mouse epithelial tissues induced by Pten loss. Our study also suggests that the Lrig-1 expressing cells can function as the cells-of-origin of cancer. We propose that Lrig1 may be used as a prognostic marker in cancers of epithelial origin and higher levels may portend a better prognosis. Citation Format: Moyi Wang, Qiuhui Li, Amanda Tracz, Jason Kirk, Anmbreen Jamroze, Rahul Kumar, Dean G. Tang. Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2601.

Activation of β-catenin in mesenchymal progenitors leads to muscle mass loss.

Loss of muscle mass is a common manifestation of chronic disease. We find the canonical Wnt pathway to be activated in mesenchymal progenitors (MPs) from cancer-induced cachectic mouse muscle. Next, we induce β-catenin transcriptional activity in murine MPs. As a result, we observe expansion of MPs in the absence of tissue damage, as well as rapid loss of muscle mass. Because MPs are present throughout the organism, we use spatially restricted CRE activation and show that the induction of tissue-resident MP activation is sufficient to induce muscle atrophy. We further identify increased expression of stromal NOGGIN and ACTIVIN-A as key drivers of atrophic processes in myofibers, and we verify their expression by MPs in cachectic muscle. Finally, we show that blocking ACTIVIN-A rescues the mass loss phenotype triggered by β-catenin activation in MPs, confirming its key functional role and strengthening the rationale for targeting this pathway in chronic disease.

Abstract A054: Activating transcription factor 3 promotes KRAS-mediated pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) has ~10% survival 5 years after diagnosis often due to lack of chemotherapeutic response. Unlike many other tumors, pancreatic tumors have a high stromal content and cancer associated fibroblasts (CAFs) play a role in response. To improve patient outcome, we need to determine how tumor and non-tumor cell interactions contribute to PDAC progression. Our laboratory recently showed global deletion of Atf3 in mice expressing oncogenic KRAS (Atf3-/-Ptf1acreERT/+KRASG12D; referred to as APK) reduced preneoplastic lesion progression. However, APK mice also showed increased fibrosis and altered immune cell infiltration and single cell RNA-seq revealed ATF3 expression in tumor and non-tumor cells. These findings suggest ATF3 may affect multiple cell types in PDAC. The goal of this study was to determine the epithelial-specific role ATF3 plays in PDAC initiation. We hypothesize that ATF3 promotes PDAC initiation and progression through epithelial and non-epithelial cell mechanisms. Methods: Mice allowing cre recombinase-inducible KRASG12D activation with (Ptf1acreERT/+KRASG12D) or without Atf3 (APK mice) or combined with Atf3 deletion specific to pancreatic acinar cells (AacinarPK mice) were generated. 2-4 month-old mice from all genotypes were gavaged with tamoxifen to induce cre-mediated recombination. Ten and twelve days following cre activation, pancreatic injury was induced by cerulein. Two weeks post injury, general morphology, tissue histology, and gene expression was compared. In some cases, cells were isolated from pancreatic tissue and prepared for 3-dimensional organoid cultures. Results: Histological analysis indicated AacinarPK mice had fewer high-grade PanIN lesions compared to both APK or Ptf1acreERT/+KRASG12D mice with reduced lesion size, based on the PanIN marker CK19, and pERK accumulation, a downstream mediator of KRAS. In addition, Ptf1acreERT/+KRASG12D, APK, and AacinarPK show differences in stromal makeup. While analysis of the stromal compartment shows no change in overall CAF accumulation, IHC for a-SMA showed reduced accumulation of myCAFs in APK tissue. There is also a reduction in immune cell infiltration when ATF3 is deleted, based on CD45 staining. Analysis of organoid cultures showed loss of ATF3 reduced compact morphology of organoid structures and reduced mesenchymal cell transformation. Conclusion and Future Directions: This analysis supports an epithelial-specific role for ATF3 in KRASG12 -mediated PDAC. Since APK and AacinarPK mice do not show the same phenotype, our data also suggests ATF3 has additional roles in nonepithelial cells. Therefore, cell-specific targeting needs to be considered in treating PDAC. Future experiments will examine ATF3-regulated pathways in the various cell populations found in PDAC. Citation Format: Mickenzie B. Martin, Ye Shen, Christopher Pin. Activating transcription factor 3 promotes KRAS-mediated pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A054.

Abstract 938: Characterization of a murine pancreatic ductal adenocarcinoma (PDAC) model with tamoxifen inducible pancreas-specific blockade of transforming growth factor-β (TGF-β) signaling with KrasG12D expression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a five-year overall survival of less than 10%. The lack of effective therapeutic strategies for PDAC necessitates the development of models to better understand the disease. Transforming growth factor-β (TGFβ)-SMAD4 signaling plays a critical role in PDAC development with more than half of all patients either have mutated SMAD4 (loss of function) or altered type II TGF TGFβ -receptor (Tgfbr2) gene. Here we present a murine model of PDAC with Tamoxifen induced conditional deletion of the TGFβ-receptor 2 (Tgfbr2) gene and expression of an activating KrasG12D mutation in pancreatic acinar cells. LSL-KrasG12D/+; Tgfbriifl/fl; Ptf1a-CreER mice were generated by crossing Ptf1aCre-ERTM mice with LSL-KrasG12D mice in which expression of KrasG12D is restrained by a lox-stop-lox cassette until Cre activation by tamoxifen injection. These mice were then bred with Tgfbr2fl/fl mice with loxP sites flanking exon 4 of the Tgfbr2 allele to block TGFβ-SMAD4 signaling. 18Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography was performed to detect tumors and measure their metabolic volumes and standard uptake values over time. This model showed a latency of approximately 16-20 weeks following 5 i.p. injections of tamoxifen (~75 mg/kg in corn oil). Follow-up on the survival of the mice post tamoxifen injections showed the median survival of 252 days in male and 234 days in female mice. Histological evaluation showed multifocal nodules of neoplasia composed of tubules and acini with cuboidal to polygonal cells. Carcinoma cells were positive for cytokeratin 19 and for mucins identified by Alcian blue staining, both confirming the ductal phenotype of these tumors. Picrosirius red staining demonstrated dense stromal desmoplasia which recapitulates human disease. Flow cytometric evolution of the immune microenvironment was also done to evaluate the infiltration of immune cells. Additional characterization of our novel mouse model of pancreatic cancer is underway to understand PDAC progression and develop effective therapeutic approaches. Citation Format: Sanjay Pandey, Brett Bell, Claudia G. Chavez, Wade Koba, Talicia Savage, Patrik Asp, Indranil Basu, Chandan Guha. Characterization of a murine pancreatic ductal adenocarcinoma (PDAC) model with tamoxifen inducible pancreas-specific blockade of transforming growth factor-β (TGF-β) signaling with KrasG12D expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 938.

229-LB: G6PC2, a GWAS Locus for Fasting Glucose and HbA1c, Regulates Glucose-Suppression of Glucagon Secretion in a-Cells

The G6PC2 locus, encoding the catalytic subunit of the islet-specific glucose-6-phosphatase enzyme, is an established risk locus for HbA1c and fasting glucose levels. A critical role of glycolytic flux in regulating glucagon secretion was established through manipulation of the activity and expression of the glycolytic enzyme glucokinase (GCK) which controls the setpoint for glucose-suppression of glucagon secretion (GSGS) . G6PC2 opposes the action of GCK by hydrolyzing glucose-6-phosphate back to glucose and creating a futile substrate cycle. Previous studies from germline and β-cell-specific G6PC2 null mice have demonstrated that G6PC2 ablation reduces glucose-6-phosphatase activity, glucose cycling, and glycolytic flux, resulting in a leftward shift of dose-response curve for glucose-stimulated insulin secretion. Through cell type specific chromatin accessibility maps of sorted human islet cells, we established that G6PC2 risk SNPs are in open chromatin regions in human α-cells, suggesting that they might not only affect β-cell but also α-cell expression of this critical gene. To test whether α-cell G6PC2 impacts glycemic control, we derived a new mouse line that induces ablation of the first three exons of G6PC2 after Cre activation – resulting in a null allele (G6pc2 loxP/loxP; Gcg-CreERT2; referred to as “α-G6PC2KO”) . Assessment of glucagonemia during glucose tolerance tests revealed a decrease in glucagon levels in α-G6PC2KO mice relative to controls. Intriguingly, fasting blood glucose levels were significantly lower in α-G6PC2KO mice. α-G6PC2KO mice recovered more slowly from hypoglycemia during an insulin tolerance test, and presented lower glucagon levels relative to control mice following the insulin bolus. α-G6PC2KO islets secreted less glucagon relative to control islets when incubated in low-glucose media, independent of alterations in insulin secretion or islet hormone content. Collectively, our data demonstrate that the G6PC2 locus impacts glycemic control via its action in α-cells. Disclosure V. Bahl: None. C. L. May: None. Human pancreas analysis program: n/a. K. H. Kaestner: None.

Expression of Cre recombinase in chondrocytes causes abnormal craniofacial and skeletal development.

The cranial base synchondroses are growth centers that drive cranial and upper facial growth. The intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) are two major synchondroses located in the middle of the cranial base and are maintained at early developmental stages to sustain cranial base elongation. In this study, we report unexpected premature ossification of ISS and SOS when Cre recombinase is activated in a chondrocyte-specific manner. We used a Cre transgenic line expressing Aggrecan enhancer-driven, Tetracycline-inducible Cre (ATC), of which expression is controlled by a Col2a1 promoter. Neonatal doxycycline injection or doxycycline diet fed to breeders was used to activate Cre recombinase. The premature ossification of ISS and/or SOS led to a reduction in cranial base length and subsequently a dome-shaped skull. Furthermore, the mice carrying either heterozygous or homozygous conditional deletion of Tsc1 or Fip200 using ATC mice developed similar craniofacial abnormalities, indicating that Cre activity itself but not conditional deletion of Tsc1 or Fip200 gene, is the major contributor of this phenotype. In contrast, the Col2a1-Cre mice carrying Cre expression in both perichondrium and chondrocytes and the mice carrying the conditional deletion of Tsc1 or Fip200 using Col2a1-Cre did not manifest the same skull abnormalities. In addition to the defective craniofacial bone development, our data also showed that the Cre activation in chondrocytes significantly compromised bone acquisition in femur. Our data calls for the consideration of the potential in vivo adverse effects caused by Cre expression in chondrocytes and reinforcement of the importance of including Cre-containing controls to facilitate accurate phenotype interpretation in transgenic research.

Correction to ‘Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM’

Correction to ‘Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM’

Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM.

Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Cell-autonomous retinoic acid receptor signaling has stage-specific effects on mouse enteric nervous system.

Retinoic acid (RA) signaling is essential for enteric nervous system (ENS) development, since vitamin A deficiency or mutations in RA signaling profoundly reduce bowel colonization by ENS precursors. These RA effects could occur because of RA activity within the ENS lineage or via RA activity in other cell types. To define cell-autonomous roles for retinoid signaling within the ENS lineage at distinct developmental time points, we activated a potent floxed dominant-negative RA receptor α (RarαDN) in the ENS using diverse CRE recombinase–expressing mouse lines. This strategy enabled us to block RA signaling at premigratory, migratory, and postmigratory stages for ENS precursors. We found that cell-autonomous loss of RA receptor (RAR) signaling dramatically affected ENS development. CRE activation of RarαDN expression at premigratory or migratory stages caused severe intestinal aganglionosis, but at later stages, RarαDN induced a broad range of phenotypes including hypoganglionosis, submucosal plexus loss, and abnormal neural differentiation. RNA sequencing highlighted distinct RA-regulated gene sets at different developmental stages. These studies show complicated context-dependent RA-mediated regulation of ENS development.

WWTR1(TAZ)-CAMTA1 gene fusion is sufficient to dysregulate YAP/TAZ signaling and drive epithelioid hemangioendothelioma tumorigenesis.

Epithelioid hemangioendothelioma (EHE) is a genetically homogenous vascular sarcoma that is a paradigm for TAZ dysregulation in cancer. EHE harbors a WWTR1(TAZ)-CAMTA1 gene fusion in >90% of cases, 45% of which have no other genetic alterations. In this study, we used a first of its kind approach to target the Wwtr1-Camta1 gene fusion to the Wwtr1 locus, to develop a conditional EHE mouse model whereby Wwtr1-Camta1 is controlled by the endogenous transcriptional regulators upon Cre activation. These mice develop EHE tumors that are indistinguishable from human EHE clinically, histologically, immunohistochemically, and genetically. Overall, these results demonstrate unequivocally that TAZ-CAMTA1 is sufficient to drive EHE formation with exquisite specificity, as no other tumor types were observed. Furthermore, we fully credential this unique EHE mouse model as a valid preclinical model for understanding the role of TAZ dysregulation in cancer formation and for testing therapies directed at TAZ-CAMTA1, TAZ, and YAP/TAZ signaling.

Inducible TDG knockout models to study epigenetic regulation.

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.

Retina-specific targeting of pericytes reveals structural diversity and enables control of capillary blood flow.

Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. The specificity and pattern of cre activation varied significantly across the three lines, under either the PDGFRβ or NG2 promoter (Pdgfrβ-CreRha, Pdgfrβ-CreCsln, and Cspg4-Cre). In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRβ line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficits, a common pathology in numerous diseases.