Flox Allele Research Articles

Analysis of Brain, Blood, and Testis Phenotypes Lacking the Vps13a Gene in C57BL/6N Mice.

The Vps13a gene encodes a lipid transfer protein called VPS13A, or chorein, associated with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondria-endosomes, and lipid droplets. This protein plays a crucial role in inter-organelle communication and lipid transport. Mutations in the VPS13A gene are implicated in the pathogenesis of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder characterized by chorea, orofacial dyskinesias, hyperkinetic movements, seizures, cognitive impairment, and acanthocytosis. Previous mouse models of ChAc have shown variable disease phenotypes depending on the genetic background. In this study, we report the generation of a Vps13a flox allele in a pure C57BL/6N mouse background and the subsequent creation of Vps13a knockout (KO) mice via Cre-recombination. Our Vps13a KO mice exhibited increased reticulocytes but not acanthocytes in peripheral blood smears. Additionally, there were no significant differences in the GFAP- and Iba1-positive cells in the striatum, the basal ganglia of the central nervous system. Interestingly, we observed abnormal spermatogenesis leading to male infertility. These findings indicate that Vps13a KO mice are valuable models for studying male infertility and some hematological aspects of ChAc.

β‐catenin C‐terminal Domain/Sphingosine‐1‐Phosphate Receptor 1 Axis is a Potential Therapeutic Target in Vascular Remodeling

Vascular smooth muscle cells (SMCs), in part via activation of the Wnt/β‐catenin signaling pathway, contribute importantly to vascular remodeling. While the C‐terminal domain of β‐catenin links the pathway to gene expression, how this domain and related molecular mechanisms are involved in vascular remodeling is unknown. Inhibitors targeting specifically the C‐terminal domain of β‐catenin have been developed, but their use in obstructive vascular disease is not yet justified because of this gap of knowledge. Our group has previously shown that β‐catenin C‐terminal domain in SMCs is essential for arterial wall assembly during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Therefore, the objective of this study was to define the importance of β‐catenin C‐terminal domain in SMCs during vascular remodeling and the underlying molecular mechanisms.To test the hypothesis that β‐catenin C‐terminal domain in SMCs is required for vascular remodeling, mice bearing a mutant β‐catenin allele (ΔC) that prevents the transcription of the C‐terminal domain were crossed with the SMC‐specific SMA‐CreERT2 line to generate mice bearing one β‐catenin flox allele and one knockin mutant (Ctnnb1ΔC/flox) or wt (Ctnnb1wt/flox) allele. In this system, tamoxifen administration removes the flox allele, resulting in mice bearing the mutant allele (SMβCΔC mice) or the wt allele (SMβCwt/‐ mice) as the only sources of β‐catenin in SMCs. To induce vascular remodeling, we performed carotid artery ligation one week after tamoxifen injection in male and female adult mice. Carotid arteries from both male and female SMβCΔC mice showed reduced neointima formation after injury induced by ligation compared to SMβCwt/‐ mice. Arteries from SMβCΔC mice also showed reduced SMC proliferation and increased SMC apoptosis and differentiation markers. We also isolated mouse aortic SMCs (MASMCs) from Ctnnb1ΔC/flox mice, and transduced these cells with GFP‐ or Cre‐expressing adenovirus to obtain control (βCControl) or cells expressing β‐cateninΔC(βCΔC) and found that βCΔC MASMCs show reduced growth compared to βCControl. To gain further insight on the mechanisms underlying the effects of β‐catenin C‐terminus, we performed whole‐transcriptome analysis by RNA‐seq of βCΔC and βCControl MASMCs and found a downregulation of the sphingosine‐1‐phosphate receptor 1 (S1pr1) gene in βCΔC MASMCs. This receptor is known to be important in vascular remodeling, but its regulation is not fully elucidated. Interestingly, we found decreased S1PR1 expression in SMCs of injured arteries from SMβC∆C mice. Consistent with this observation, βCΔC MASMCs showed reduced S1PR1 mRNA and protein levels and decreased S1pr1 promoter activity, which were all rescued by the transfection of a constitutively active form of β‐catenin. By using a S1PR1lox/stop/lox conditional gain‐of‐function mouse line, we found that overexpression of S1PR1 in SMCs rescues the injury‐induced neointima formation in SMβC∆C mice. Consistently, overexpression of S1PR1 in SMCs restored βCΔC MASMC growth towards βCControl levels. Taken together, our results define an essential SMC β‐catenin C‐terminal domain–S1PR1 axis that drives neointima formation after arterial injury, and identify multiple points for potential therapeutic intervention to control vascular remodeling in disease.

Altered sacral neural crest development in Pax3 spina bifida mutants underlies deficits of bladder innervation and function

Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.

Generation and validation of novel conditional flox and inducible Cre alleles targeting fibroblast growth factor 18 (Fgf18).

Fibroblast growth factor 18 (FGF18) functions in the development of several tissues, including the lung, limb bud, palate, skeleton, central nervous system, and hair follicle. Mice containing a germline knockout of Fgf18 (Fgf18 -/- ) die shortly after birth. Postnatally, FGF18 is being evaluated for pathogenic roles in fibrosis and several types of cancer. The specific cell types that express FGF18 have been difficult to identify, and the function of FGF18 in postnatal development and tissue homeostasis has been hampered by the perinatal lethality of Fgf18 null mice. We engineered a floxed allele of Fgf18 (Fgf18 flox ) that allows conditional gene inactivation and a CreERT2 knockin allele (Fgf18 CreERT2 ) that allows the precise identification of cells that express Fgf18 and their lineage. We validated the Fgf18 flox allele by targeting it in mesenchymal tissue and primary mesoderm during embryonic development, resulting in similar phenotypes to those observed in Fgf18 null mice. We also use the Fgf18 CreERT2 allele, in combination with a conditional fluorescent reporter to confirm known and identify new sites of Fgf18 expression. These alleles will be useful to investigate FGF18 function during organogenesis and tissue homeostasis, and to target specific cell lineages at embryonic and postnatal time points.

Differential CRE Expression in Lhrh-cre and GnRH-cre Alleles and the Impact on Fertility in Otx2-Flox Mice

There is an increasing trend in studies utilizing cell-specific deletion of genes through conditional gene deletion by CRE recombination. Despite numerous advantages, this strategy also has limitations such as ectopic CRE-expression and germline recombination. Two commonly used gonadotropin-releasing hormone (Gnrh)-driven CRE-expressing mice both target GnRH neurons. However, a direct comparison of the cells targeted and their phenotypic outcome have not yet been presented. To compare where recombination takes place, we crossed the Gnrh-cre and Lhrh-cre lines with the Rosa26-LacZ reporter mouse. Lhrh-cre allowed recombination of the Rosa26-LacZ gene in ∼700 cells, which is comparable to the GnRH neuronal population. Surprisingly, there were > 20 times more LacZ expressing cells in the adult Gnrh-cre:Rosa26-LacZ than the Lhrh-cre:Rosa26-LacZ brain. The greatest differences in targeting of the Gnrh-cre and Lhrh-cre lines were found in the septum, the suprachiasmatic nucleus, and the septohypothalamic area. This difference in cells targeted was present from embryonic day 12. A prior study using the Gnrh-cre to delete the transcription factor Otx2 found fewer GnRH neurons, leading to male and female subfertility. To recapitulate this study, we performed a fertility assay in Otx2:Lhrh-cre mice. We confirmed the requirement for Otx2 in GnRH neuron development, fertility and correct gonadotropin hormone release in Otx2:Lhrh-cre males, but the subfertility was more modest than in Otx2:Gnrh-cre and absent in female Otx2:Lhrh-cre. This suggests that ectopic expression of Gnrh-cre contributes to the reproductive phenotype observed. Finally, the Cre alleles caused germline recombination of the flox allele when transmitted from either parent, generating embryonic lethal knock-out offspring, producing smaller live litters.

Functional interplay between c-Myc and Max in B lymphocyte differentiation.

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.

Different regulation of limb development by p63 transcript variants.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Abstract PR05: Dicer1 deletion: Sufficient for angiosarcoma development and a haploinsufficient tumor suppressor in rhabdomyosarcoma

Abstract In this study, the role of Dicer and microRNAs (miRNAs) is investigated in pediatric and adult soft tissue sarcomas with the hypothesis that understanding miRNAs and their biogenesis may provide biological insights into the pathogenesis of these diseases and identify novel therapeutic targets. In several mouse models of cancer, Dicer1 is thought to function as a haploinsufficient tumor suppressor where loss of a single allele promotes tumorigenesis yet loss of both alleles is detrimental to tumor formation. Heterozygous germline mutations in DICER1 in humans is associated with rare malignant and benign neoplasias including embryonal rhabdomyosarcoma (ERMS). With DICER1 syndrome mutations found in ERMS, a pediatric soft tissue sarcoma resembling skeletal muscle precursors that fail to differentiate, we sought to further understand the role of Dicer and miRNAs utilizing a transgenic mouse model of ERMS combined with conditional Dicer1Flox/Flox null mice. The ERMS mouse model results from activation of a constitutively active SmoM2 allele with Cre recombinase expressed from the adipose protein 2 (aP2) promoter resulting in unrestrained hedgehog signaling in the aP2 lineage. Heterozygous Dicer1 loss led to increased penetrance of and a decrease in latency to onset of tumors. In agreement with the haploinsufficient tumor suppressor model, homozygous Dicer1Flox/Flox mice rarely developed tumors in stark contrast to wild type and heterozygous littermates. Furthermore, tumors that did develop in Dicer1Flox/Flox mice escaped Cre recombination and retained a flox allele indicating functional Dicer1 activity. Serendipitously, aP2-Cre; SmoM2; Dicer1Flox/Flox mice that did not develop ERMS all developed multifocal angiosarcomas. This finding prompted the analysis of Dicer loss alone in the aP2 lineage and remarkably Dicer1Flox/Flox mice similarly developed angiosarcoma whereas Dicer1Flox/+ do not, providing evidence for Dicer loss independent of a cooperating oncogene driving tumorigenesis in a mouse model. This study provides evidence for the role of Dicer and miRNAs in rhabdomyosarcoma and angiosarcomas. Further understanding the molecular mechanisms and genetic drivers of soft tissue sarcomas is critical for the development of novel targeted therapeutics. This is true especially for soft tissue sarcomas where treatment strategies and survival outcomes have not changed in decades despite numerous clinical trials. This abstract is also presented as Poster B06. Citation Format: Jason A. Hanna, Matthew R. Garcia, Jerold Rehg, Mark E. Hatley. Dicer1 deletion: Sufficient for angiosarcoma development and a haploinsufficient tumor suppressor in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR05.

The role of platelet MyD88 in host response during gram‐negative sepsis

Beside their role in hemostasis, platelets serve as sentinel cells in host defense during infection. In sepsis, platelets have been implicated in both beneficial (antibacterial) and detrimental responses (thrombosis and organ damage). Toll-like receptors and their common adaptor, myeloid differentiation factor 88 (MyD88), are essential for pathogen recognition and protective immunity. Platelets express functional Toll-like receptors and MyD88, which participate in platelet responsiveness to bacterial agonists. Considering the pivotal involvement of platelets and MyD88 in the host response to bacteria, we studied the role of platelet MyD88 in gram-negative sepsis using intravenous and airway infections with the common human sepsis pathogen Klebsiella pneumoniae. Platelet-specific Myd88(-/-) mice were generated by crossing mice with a conditional Myd88 flox allele with mice expressing Cre recombinase controlled by the platelet factor 4 promoter. In a reverse approach, full Myd88(-/-) mice were transfused with wild-type platelets. In both settings, platelet MyD88 did not impact on bacterial growth or dissemination. In addition, platelet MyD88 did not influence hallmark sepsis responses such as thrombocytopenia, coagulation or endothelial activation, or distant organ injury. Platelet MyD88 played no role in lung pathology during pneumonia-derived sepsis. Despite known literature, platelet MyD88-dependent TLR signaling does not contribute to the host response during gram-negative sepsis.

Ventricular septal defects in mice targeted deletion of versican in vascular smooth muscle cells

Purpose: To determine the function of versican (Vcan) in the cardiovascular system in vivo, we generated the versican conditional knockout mice, in which the Vcan gene was selectively ablated in vascular smooth muscle cells (SMCs). Versican is a large secreted chondroitin sulfate proteoglycan, which is important for cardiac development, and is also implicated in adult cardiovascular disease. Four splice variants of versican (V0, V1, V2, V3) have been identified, each containing two invariant domains, an N-terminal G1 domain that interact with hyaluronan, and a C-terminal G3 domain that interact with other extracellular matrix (ECM) molecules. As it has been reported that versican knockout mice died of cardiac defects at E10.5, the role of versican in cardiac development and disease has not been completely elucidated. Methods: SM22-Cre+:Vcanflox/flox conditional knockout mice were generated by combining Cre/loxP with a Flp/FRT system. In brief, a targeting vector harboring the Vcan flox allele was constructed by franking exon2 including start codon with loxP sites in the combination of a PGK-neoR cassette flanked by the FRT sequence. After obtaining Vcanflox/flox mice, they crossbred with SM22-Cre Tg mice, and the Vcan gene was selectively ablated in SMCs. The expression of different cardiac markers and ECM molecules were analyzed using histological and biochemical methods. Results: SM22-Cre+:Vcanflox/flox mice were viable and fertile, and they can reach their lifespan. The hearts in SM22-Cre+:Vcanflox/flox newborn mice showed normal cardiac looping; however, exhibited dilated heart and ventricular septal defects (VSD). The expressions of the a-actinin in their ventricular wall, which is a marker of cardiac muscle cell, decreased compared to wild type. Furthermore, though their arterial walls were thinner than wild type, elastic lamina was formed. Conclusions: Previously we generated the versican mutant mice (VcanΔ3/Δ3) whose versican lacked the A-subdomain of the G1 domain. The VcanΔ3/Δ3 mice died at neonatal period day 0, exhibiting the dilated heart and VSD. In this study, the targeted deletion of versican in SMCs leads to moderate cardiovascular defects with the dilated heart and VSD. SMCs transduce a variety of signals from a highly structured ECM that modulate cellular differentiation, migration, and proliferation. Our results suggest that the versican expression from SMCs is important for the cardiovascular development, and this study can lead to the treatment for congenital cardiovascular disease.

Abstract 2716: Oncogenic K-ras mutation associates with GLUT1 expression in mice and human colorectal cancer.

Abstract Background and aims: Somatic APC defects are present in 75% of sporadic colorectal cancer. K-ras gene mutations found in 40-50%, occur early in the progression to carcinoma, but their functional contribution remains incompletely understood. Previous study reported that GLUT1, encoding glucose transporter-1, was one of the genes consistently up-regulated in colorectal cancer cell lines with K-RAS or BRAF mutations. In this study, we have analyzed tumors from genetically modified mice carrying either mutation in the Apc gene or in combination with another mutation in the K-ras G12D allele. Subsequently, we evaluated whether K-RAS gene mutations associate GLUT1 expression in human colorectal cancer. Methods: Mice carrying transgenes regulated by CDX2 homeobox gene promoter (CDX2P9.5) showed tightly restricted transgene expression in the colon in adult tissues. Cre recombinase transgenes with 22 guanine nucleotides (G22Cre) introduced downstream of the initiating ATG codon, and CDX2P9.5-G22Cre transgenic mice homozygous for the Apc flox allele lived only for 10-27 d after birth due to the tumor in the proximal colon. Mice carrying loxP-flanked Apc alleles homozygously (ApcloxP/loxP, 580S) were intercrossed with Lox-Stop-Lox K-ras conditional mice strain (referred to as LSL-K-ras G12D) to generate CDX2P9.5-G22Cre;Apcflox/flox;LSL-K-ras G12D with K-ras G12D expression (CPC;Apc 580D;Lox-K-ras G12D). Gene expression analyses on the 3 samples from CPC;Apc 580D;Lox-K-ras G12D mice and CPC;Apc 580D;K-ras WT mice were performed with high-density oligonucleotide arrays (GeneChip Mouse Genome 430 2.0 Array). Results: Glut1 was found to be up-regulated significantly in CPC;Apc 580D;Lox-K-ras G12D mice. Immunohistochemical staining revealed that Glut1 protein was up-regulated in CPC;Apc 580D;Lox-K-ras G12D mice. 47 samples of colorectal cancer patients with K-RAS mutations in 18 patients (38.3%) showed GLUT1 expression in 12 patients of 18 K-RAS mutated patients and 8 patients of 29 K-RAS wild-type patients. Conclusions: Using mice carrying conditional solely mutant in the Apc gene or in combination with the K-ras G12D allele, we assessed the relevance of oncogenic K-ras with Glut1 in colorectal cancer. Microarray analysis, quantitative PCR, and immunohistochemistry suggested the association of the K-ras with the Glut1 (Glut1). Our clinical analysis showed K-RAS gene mutations significantly associate GLUT1 expression in human colorectal cancer. Citation Format: Yasuo Kawaguchi, Takao Hinoi, Tatsunari Sasada, Tomohiro Adachi, Yasufumi Saito, Shinji Miguchi, Hiroaki Niitsu, Hideki Ohdan. Oncogenic K-ras mutation associates with GLUT1 expression in mice and human colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2716. doi:10.1158/1538-7445.AM2013-2716

Incomplete cre‐mediated excision leads to phenotypic differences between Stra8‐iCre; Mov10l1lox/lox and Stra8‐iCre; Mov10l1lox/Δ mice

In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-codon-improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; gene(lox) (/)(Δ) genotype should be used to evaluate phenotypes instead of Cre; gene(lox/lox) owing to the fact that the latter usually bears incomplete deletion of the flox allele(s).

The E3 Ligase Mind Bomb-1 (Mib1) Modulates Delta-Notch Signaling to Control Neurogenesis and Gliogenesis in the Developing Spinal Cord

The Notch signaling pathway is essential for neuronal and glial specification during CNS development. Mind bomb-1 (Mib1) is an E3 ubiquitin ligase that ubiquitinates and promotes the endocytosis of Notch ligands. Although Mib1 is essential for transmitting the Notch signal, it is still unclear whether it is a primary regulator of Notch ligand activity in the developing spinal cord. In Mib1 conditional knock-out mice, we observed depletion of spinal progenitors, premature differentiation of neurons, and unbalanced specification of V2 interneurons, all of which mimic the conventional Notch phenotype. In agreement with this, the reduction of progenitors in the absence of Mib1 led to a loss of both astrocytes and oligodendrocytes. Late removal of Mib1 using a drug-inducible system suppressed glial differentiation, suggesting that Mib1 continues to play a role in the formation of late progenitors mainly designated for gliogenesis. Finally, misexpression of Mib1 or Mib1 deletion mutants revealed that the ring domain of Mib1 is required for the specification of V2 interneurons in the chick neural tube. Together, these findings suggest that Mib1 is a major component of the signal-sending cells required to provide Notch ligand activity for specifying neurons and glia in the spinal cord.

Suppression of Cardiac L-Type Ca Channel Activity Causes Cardiac Hypertrophy

Increased L type Ca channel (LTCC) activity was proposed to induce Ca dependent hypertrophy and heart failure by activating CaMK and calcineurin (CaN) signaling. Previous experiments showed that upregulation of LTCC activity induced profound cardiomyopathy. However, the enhanced Ca influx did not activate CaN or hypertrophy; but led to necrotic myocyte death. We hypothesized that reducing LTCC by genetic deletion of 50% of Cav1.2 would be cardioprotective like β-adrenergic receptor or LTCC blockers. Surprisingly, this deletion enhanced disease, CaN activation and hypertrophy. These myocytes with reduced Ca current exhibited significantly increased diastolic Ca. To further address these surprising findings we analyzed the Ca dynamics of mice with a standard null allele crossed with a cardiac specific flox allele (α1cf/-Cre), which led to an even greater deletion of the α1c protein in the heart. Here we measured Ca handling dynamics in α1cf/-Cre mice. Twitch amplitudes were significantly reduced (50%) and relaxation time was prolonged (16%). SR Ca load was reduced significantly (∼25%). These findings may be explained by increased diastolic SR Ca leak that we measured in myocytes from α1cf/-Cre mice. Thus, the deletion of the α1c protein in mouse cardiac myocytes leads to an increased basal [Ca]i by increasing Ca leak from the SR. Interestingly, the CaMKII inhibitor KN93 completely reversed the effects of the reduced LTCC: twitch amplitude and relaxation as well as SR Ca load were rescued. These results suggest that resting [Ca]i and the gain in ECC are increased to maintain contractility. This increase might drive hypertrophy by activating CaN and CaMKII.

When Cre-Mediated Recombination in Mice Does Not Result in Protein Loss

Cre/loxP recombination enables cellular specificity and, in the case of inducible systems, temporal control of genomic deletions. Here we used a SM22α tamoxifen-inducible Cre line to inactivate β1 integrin in adult smooth muscle. Interestingly, analysis of two distinct β1 loxP transgenic mice revealed vastly different outcomes after β1 integrin deletion. Lethality occurred 4 weeks postinduction in one Cre/loxP line, while no apparent phenotype was seen in the other line. Genetic analysis revealed appropriate DNA excision in both cases; however, differences were found in the degree of protein loss with absolutely no change in protein levels in the model that lacked a phenotype. Seeking to understand protein persistence despite appropriate recombination, we first validated the flox allele using a constitutive Cre line and demonstrated its ability to mediate effective protein inactivation. We then examined the possibility of heterozygous cell selection, protein turnover, and deletion efficiency with no success for explaining the phenotype. Finally, we documented the presence of the Cre-recombination episomal product, which persisted in tissue samples with no protein loss. The product was only noted in cells with low proliferative capacity. These findings highlight the potential for protein expression from the products of Cre-recombinase excised genes, particularly when deletion occurs in low turnover populations.

Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of a GATA1 Protein Lacking the N-terminal Domain

GATA1 is essential for the differentiation of erythroid cells and megakaryocytes. The Gata1 gene is composed of multiple untranslated first exons and five common coding exons. The erythroid first exon (IE exon) is important for Gata1 gene expression in hematopoietic lineages. Because previous IE exon knockdown analyses resulted in embryonic lethality, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the floxed IE exon in adulthood using an inducible Cre expression system. In this conditional knock-out mouse line, the Gata1 mRNA level was significantly down-regulated in the megakaryocyte lineage, resulting in thrombocytopenia with a marked proliferation of megakaryocytes. By contrast, in the erythroid lineage, Gata1 mRNA was expressed abundantly utilizing alternative first exons. Especially, the IEb/c and newly identified IEd exons were transcribed at a level comparable with that of the IE exon in control mice. Surprisingly, in the IE-null mouse, these transcripts failed to produce full-length GATA1 protein, but instead yielded GATA1 lacking the N-terminal domain inefficiently. With low level expression of the short form of GATA1, IE-null mice showed severe anemia with skewed erythroid maturation. Notably, the hematological phenotypes of adult IE-null mice substantially differ from those observed in mice harboring conditional ablation of the entire Gata1 gene. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and selecting the correct transcription start site of the Gata1 gene.

Targeting the Protein Phosphatase, Shp2, Reduces FLT3-ITD-Induced Hyperproliferation of Murine Hematopoietic Progenitors.

Abstract 827Internal tandem duplications in the FMS-like receptor tyrosine kinase receptor (FLT3-ITDs) are present in approximately 25% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperproliferative cellular growth and promiscuous activation of STAT5; however, the molecular mechanism underlying aberrant activation of these signaling molecules is largely unknown. Upon FLT3 ligand (FL) stimulation, tyrosine 599 (Y599) of WT-FLT3 recruits the protein tyrosine phosphatase, Shp2 and induces cell proliferation and Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73-FLT3, via Y599, contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT-FLT3, N51-FLT3, or N73-FLT3, co-immunoprecipitation assays demonstrated that Shp2 associates with WT-FLT3 in a FL-dependent manner, while Shp2 is constitutively associated with N51-FLT3 and N73-FLT3 independent of FL-stimulation. To investigate the functional role of Shp2 in FLT3-ITD-induced leukemogenesis, Baf3 cells expressing WT-FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III–directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Immunoblot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. To further test the role of Shp2 in FLT3-ITD-induced transformation, Shp2flox/flox mice were crossed with Mx1Cre transgenic mice to produce Shp2flox/flox Mx1Cre+ mice. Shp2flox/floxMx1Cre+ and Shp2flox/floxMx1Cre- pairs were treated with 3 intraperitoneal injections of polyI/polyC to induce recombination of the Shp2 allele and generate a null allele in the Mx1Cre+ animals. PCR and immunoblot analysis revealed ablation of the flox allele and reduction of Shp2 protein, respectively, in Shp2flox/floxMx1Cre+ bone marrow as soon as 2 weeks post injection. Low density mononuclear cells (LDMNCs) isolated from polyI/polyC-treated Shp2flox/floxMx1Cre- and Shp2flox/floxMx1Cre+ mice were transduced with either pMSCV-WT-FLT3 or pMSCV-N51-FLT3. Transduced cells were sorted for EGFP (enhanced green fluorescent protein) expression and subjected to thymidine incorporation assays. Shp2flox/floxMx1Cre+ cells transduced with N51-FLT3 demonstrated significantly reduced proliferation at baseline and in response to FL stimulation compared to Shp2flox/floxMx1Cre- cells expressing N51-FLT3. To pharmacologically examine the role of Shp2 in FLT3-ITD-induced-hyperproliferation and constitutive activation of Stat5, we utilized a Shp2 inhibitor, II-B08, identified from a library of indole salicylic acid derivatives. II-B08 exhibits an IC50 of 5.5 μM for Shp2 and demonstrates excellent cellular activity. Bone marrow LDMNCs retrovirally transduced with WT-FLT3 or N51-FLT3 were incubated in the absence of or in the presence of increasing concentrations of II-B08 and plated into thymidine incorporation assays. A minimal and non-significant reduction in proliferation was observed in WT-FLT3 expressing cells in the presence of II-B08; however N51-FLT3 expressing cells demonstrated a highly significant reduction in cellular proliferation in a concentration-dependent fashion. In addition to these functional analyses, biochemical studies revealed a reduction in Stat5 activation in N51-FLT3 expressing cells treated with II-B08 compared N51-FLT3 expressing cells treated with DMSO in two independent experiments. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3-ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias. Disclosures:No relevant conflicts of interest to declare.

Macrophage- and Dendritic-Cell-Derived Interleukin-15 Receptor Alpha Supports Homeostasis of Distinct CD8+ T Cell Subsets

Interleukin-15 receptor alpha (IL-15R alpha) is a pleiotropically expressed molecule that chaperones and trans-presents IL-15 to NK and T cells. To investigate whether IL-15R alpha presented by different cells perform distinct physiological functions, we have generated four lines of mice lacking IL-15R alpha in various cell types. We find that IL-15R alpha expression on macrophages but not dendritic cells (DCs) supports the early transition of antigen specific effector CD8(+) T cells to memory cells. After memory CD8(+) T cell differentiation, IL-15R alpha expression on DCs selectively supports central memory CD8(+) T cells, whereas IL-15R alpha expression on macrophages supports both central and effector memory CD8(+) T cells. By contrast, mice lacking IL-15R alpha on macrophages, DCs, or both, exhibit equivalent defects in NK cell homeostasis and activation. These studies define unique roles for macrophage expression of IL-15R alpha and show that NK cells rely upon distinct IL-15R alpha dependent IL-15 signals than memory CD8(+) T cells. Moreover, they demonstrate the diversity, specification, and geographic restriction of cytokine signals.

Mice carrying a conditional Serca2flox allele for the generation of Ca 2+ handling-deficient mouse models

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca 2+ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2 null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2 flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2 flox mice with MLC-2v wt/Cre mice and with αMHC-Cre transgenic mice. In heterozygous Serca2 wt/flox MLC-2v wt/Cre mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2 flox/flox Tg(αMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2 flox mouse is the first murine conditional gene disruption model for the SERCA family of Ca 2+ ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.

Impaired fear response in mice lacking GIT1

G protein-coupled receptor kinase interacting protein 1 (GIT1) belongs to the family of Arf GAP proteins and has been implicated in the regulation of G protein-coupled receptor (GPCR) sequestration, cell migration, synapse formation and dendritic spine morphogenesis in neurons. To extend these cellular studies on GIT1 to an in vivo system, we generated mice with globally inactivated Git1 gene by breeding mice carrying a conditional Git1 flox allele with mice expressing the CMV- Cre transgene. Although many GIT1 knockout (GIT1-KO) animals died shortly after birth, homozygous mutants that survived the early post-partum period developed normally into adulthood and were fertile. Behavioral analyses of adult GIT1-KO mice revealed normal exploratory, anxiety- and depressive-like behaviors. However, GIT1-KO mice show impaired responses to fear conditioning and fear-potentiated startle. Overall, these findings suggest that GIT1 is involved in the regulation of amygdala-mediated experience-based emotional behaviors.