Cre Mouse Line Research Articles

Comparative analysis of In vivo endothelial cell translatomes across central nervous system vascular beds

Endothelial cells (ECs) display organ- and tissue-specific heterogeneity. In the eye, the retinal and choroidal vascular beds are distinct networks with different molecular and morphological properties that serve location-specific functions, i.e., the former maintaining a tight barrier and the latter, a permeable fenestrated vasculature. Given that retinal health critically relies on the function of these vascular beds and that their dysfunction is implicated in a variety of retinal diseases, a molecular understanding of both physiological and pathophysiological characteristics of these distinct vasculatures is critical. Given their interspersed anatomic distribution among parenchymal cells, the study of EC gene expression, in vivo, has been hampered by the challenge of isolating pure populations of ocular ECs in sufficient quantities for large-scale transcriptomics. To address this challenge, we present a methodological and analytical workflow to facilitate inter-tissue comparisons of the in vivo EC translatome isolated from choroid, retina, and brain using the Cre-inducible NuTRAP flox construct and two widely-used endothelial Cre mouse lines: constitutive Tie2-Cre and tamoxifen-inducible Cdh5-CreERT2. For each Cre line, inter-tissue comparison of TRAP-RNAseq enrichment (TRAP-isolated translatome vs input transcriptome) showed tissue-specific gene enrichments with differential pathway representation. For each mouse model, inter-tissue comparison of the EC translatome (choroid vs brain, choroid vs retina, and brain vs retina) showed over 50% overlap of differentially expressed genes (DEGs) between the three paired comparisons, with differential pathway representation for each tissue. Pathway analysis of DEGs in the Cdh5-NuTRAP vs Tie2-NuTRAP comparison for retina, choroid, and brain predicted inhibition of processes related to myeloid cell function and activation, consistent with more specific targeting of ECs in the Cdh5-NuTRAP than in the Tie2-NuTRAP model which also targets hematopoietic progenitors giving rise to immune cells. Indeed, while TRAP enriches for EC transcripts in both models, myeloid transcripts were also captured in the Tie2-NuTRAP model which was confirmed using cell sorting. We suggest experimental/analytical considerations should be taken when selecting Cre-lines to target ECs.

Dysregulation of kappa opioid receptor neuromodulation of lateral habenula synaptic function following a repetitive mild traumatic brain injury

Mild traumatic brain injury (mTBI) increases the risk of affective disorders, anxiety and substance use disorder. The lateral habenula (LHb) plays an important role in pathophysiology of psychiatric disorders. Recently, we demonstrated a causal link between mTBI-induced LHb hyperactivity due to excitation/inhibition (E/I) imbalance and motivational deficits in male mice using a repetitive closed head injury mTBI model. A major neuromodulatory system that is responsive to traumatic brain injuries, influences affective states and also modulates LHb activity is the dynorphin/kappa opioid receptor (Dyn/KOR) system. However, the effects of mTBI on KOR neuromodulation of LHb function are unknown. Here, we first used retrograde tracing in male and female Cre mouse lines and identified several major KOR-expressing and two prominent Dyn-expressing inputs projecting to the mouse LHb, highlighting the medial prefrontal cortex (mPFC) and the ventromedial nucleus of the hypothalamus (VMH) as the main LHb-projecting Dyn inputs that regulate KOR signaling to the LHb. We then functionally evaluated the effects of in vitro KOR modulation of spontaneous synaptic activity within the LHb of male and female sham and mTBI mice at 4 week post-injury. We observed sex-specific differences in spontaneous release of glutamate and GABA from presynaptic terminals onto LHb neurons with higher levels of presynaptic glutamate and GABA release in females compared to male mice. However, KOR effects on the spontaneous E/I ratios and synaptic drive ratio within the LHb did not differ between male and female sham and mTBI mice. KOR activation generally suppressed spontaneous glutamatergic transmission without altering GABAergic transmission, resulting in a significant but sex-similar reduction in net spontaneous E/I and synaptic drive ratios in LHb neurons of sham mice. Following mTBI, while responses to KOR activation at LHb glutamatergic synapses remained intact, LHb GABAergic synapses acquired an additional sensitivity to KOR-mediated inhibition where we observed a reduction in GABA release probability in response to KOR stimulation in LHb neurons of mTBI mice. Further analysis of percent change in spontaneous synaptic ratios induced by KOR activation revealed that independent of sex mTBI switches KOR-driven synaptic inhibition of LHb neurons (normally observed in sham mice) in a subset of mTBI mice toward synaptic excitation resulting in mTBI-induced divergence of KOR actions within the LHb. Overall, we uncovered the sources of major Dyn/KOR-expressing synaptic inputs projecting to the mouse LHb. We demonstrate that an engagement of intra-LHb Dyn/KOR signaling provides a global KOR-driven synaptic inhibition within the mouse LHb independent of sex. The additional engagement of KOR-mediated action on LHb GABAergic transmission by mTBI could contribute to the E/I imbalance after mTBI, with Dyn/KOR signaling serving as a disinhibitory mechanism for LHb neurons of a subset of mTBI mice.

A knock-in allele of Hand2 expressing Dre recombinase.

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed “Cre-only” Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

Dietary potassium effects on renal calcium transport at single nucleus resolution

Calcium homeostasis is maintained by the coordination of the intestines, bones, and kidneys. Dietary calcium is absorbed in the intestines, enters the bloodstream, and eventually finds its place in the bone reservoir or undergoes filtration by the glomerulus before being reabsorbed throughout the nephron. The late distal convoluted tubule (DCT2) and connecting tubule (CNT) determine the final rate of urinary calcium excretion, since no calcium reabsorption takes place beyond the connecting tubule (CNT). When excess calcium is excreted in the urine, also known as hypercalciuria, it contributes to the development of osteoporosis and the formation of kidney stones. High dietary potassium intake is strongly associated with a lower risk of kidney stone formation. Yet, the precise mechanism by which potassium intake modulates calcium excretion is not clear. To examine the transcriptional changes linking high dietary potassium intake and calcium transport, we applied single-nucleus RNA-sequencing (snRNA-Seq) in enriched distal nephron cells. To achieve the enrichment, we crossed a Calbindin 1 (Calb1)-driven Cre mouse line with the INTACT (for Isolation of Nuclei TAgged in Specific Cell Types) reporter line, which allows the GFP reporter to be expressed at the nuclear envelope of all calbindin 1-expressing cells. As the Calb1-Cre is constitutively expressed, all cells that have calbindin 1 expression at any time point during development will express the reporter. We examined the GFP expression by immunofluorescence, and found that it is expressed along the entire distal nephron from the distal convoluted tubule (DCT), CNT, to the collecting duct (CD). Male Calb1-Cre-INTACT mice were provided either a normal (NK, 1.05%) or a high (HK, 2%) potassium diet for 4 days and kidneys were snap-frozen for targeted snRNA-Seq using 10X Chromium (n=3 mice per group, targeting 10,000 nuclei per mouse). Our snRNA-seq dataset showed 6 major clusters, including DCT (DCT1 and DCT2), CNT (3 subclusters), CD principal cells (3 subclusters), ɑ-intercalated cells (2 subclusters), β-intercalated cells (2 subclusters), and proliferating cells. We curated a calcium score from the expression of known calciotropic genes ( Slc8a1, Vdr, Trpv5, Calb1, S100g, Ryr2, Trpv6) and used it as an index of calcium handling capacity. The calcium score is the highest in DCT2 and CNT along the distal nephron. In all CNT subclusters of the HK-treated animals, the calcium score is higher compared to the NK-treated animals, suggesting more calcium transport in CNT. Given that HK stimulates aldosterone and causes CNT hypertrophy, we compared the results with mice subjected to low dietary potassium (LK) treatment and metolazone (thiazide) treatment. The aim was to delineate the effects of dietary potassium and aldosterone A comprehensive summary of plasma potassium levels, urinary calcium excretion, aldosterone levels, calcium scores, and CNT morphology has been presented in the table below. Notably, the comparative analysis reveals a significant correlation between aldosterone levels and CNT size, both of which exhibit an inverse relationship with urinary calcium excretion: [Formula: see text] Our results suggest that DCT2 and CNT are the major sites for calcium transport. Yet, CNT exhibit more heightened adaptability in response to physiological or pharmacological perturbations at both transcriptional and morphological levels. Calcium homeostasis coincides with the regulation of aldosterone levels and CNT remodeling. DK51496 and DK133220 to DHE; K01DK121737 and AHA 20CDA35320169 to JWN. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines.

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Specificity and efficiency of tamoxifen-mediated Cre induction is equivalent regardless of age

Temporally controlling Cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report early post-natal/juvenile (<2 m.o.) Tam induction, but age-related neurodegeneration and aging studies can require Cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam-mediated Cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare Cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at early and late ages. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each Cre and flox mouse line should be independently validated, however, these findings demonstrate that Tam-mediated Cre induction can be performed even into older mouse ages and should be generalizable to other inducible Cre models.

Abstract 16402: Pitx2c-Conditional Knockout Mice May Become a Useful Model to Elucidate the Pathogenesis of Post-Capillary Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is an intractable disease that causes cardiopulmonary dysfunction due to elevated pulmonary arterial pressure. The pathogenesis of PH varies greatly depending on whether the primary lesion is upstream or downstream of the pulmonary capillaries, and the latter, post-capillary PH (pc-PH) caused by pulmonary vein stenosis/obstruction or left heart disease, currently has not an appropriate animal models to study its pathophysiology and pharmacological effects. Hypothesis: We hypothesized that a tissue-specific deletion of a gene involved in the formation of pulmonary veins could create a pathological mouse model of pc-PH. Methods: Focusing on the homeobox transcription factor Pitx2c, which has been reported to be indispensable for the formation of pulmonary veins, we generated Pitx2c-conditional knockout (cKO) mice specific for pulmonary vein and atrial myocardium by crossing Pitx2c-floxed mouse lines with Cre mouse lines for sarcolipin, a sarcoplasmic reticulum membrane protein. Then, we examined the cardiac morphology and hemodynamics of the Pitx2c-cKO mice. Results: Tissue weights of 8-week-old Pitx2c-cKO mice showed that the relative weights of both atria (especially the right atrium), right ventricle and lungs were significantly increased in Pitx2c-cKO mice, while the relative weight of the left ventricle was decreased. Furthermore, echocardiography performed on 3-month-old Pitx2c-cKO mice revealed that the right ventricle was markedly enlarged in Pitx2c-cKO mice, with enlarged pulmonary artery annulus diameter and increased velocity time integral (VTI) of blood flow through the right ventricular outflow tract. Pitx2c-cKO mice also had significantly decreased left ventricular end-diastolic diameter (LVDd), end-systolic diameter (LVDs), and mass (LVM). On the other hand, there was no difference in left ventricular fractional shortening (%FS) or tricuspid annular plane systolic excursion (TAPSE), indicating no cardiac dysfunction in Pitx2c-cKO mice. Cardiac catheterization also revealed elevated right ventricular pressure in some Pitx2c-cKO mice. Conclusion: These primary findings suggest that the Pitx2c-cKO mice may be a model for understanding the pathogenesis of pc-PH.

Oit3, a promising hallmark gene for targeting liver sinusoidal endothelial cells

Liver sinusoidal endothelial cells (LSECs) play a pivotal role in maintaining liver homeostasis and influencing the pathological processes of various liver diseases. However, neither LSEC-specific hallmark genes nor a LSEC promoter-driven Cre mouse line has been introduced before, which largely restricts the study of liver diseases with vascular disorders. To explore LSEC-specific hallmark genes, we compared the top 50 marker genes between liver endothelial cells (ECs) and liver capillary ECs and identified 18 overlapping genes. After excluding globally expressed genes and those with low expression percentages, we narrowed our focus to two final candidates: Oit3 and Dnase1l3. Through single-cell RNA sequencing (scRNA-seq) and analysis of the NCBI database, we confirmed the extrahepatic expression of Dnase1l3. The paired-cell sequencing data further demonstrated that Oit3 was predominantly expressed in the midlobular liver ECs. Subsequently, we constructed inducible Oit3-CreERT2 transgenic mice, which were further crossed with ROSA26-tdTomato mice. Microscopy validated that the established Oit3-CreERT2-tdTomato mice exhibited significant fluorescence in the liver rather than in other organs. The staining analysis confirmed the colocalization of tdTomato and EC markers. Ex-vivo experiments further confirmed that isolated tdTomato+ cells exhibited well-differentiated fenestrae and highly expressed EC markers, confirming their identity as LSECs. Overall, Oit3 is a promising hallmark gene for tracing LSECs. The establishment of Oit3-CreERT2-tdTomato mice provides a valuable model for studying the complexities of LSECs in liver diseases.

A simple, rapid fluorescent reporter-based method for detection of ectopic cre recombinase expression in presumed retinal cell type-targeted mouse lines

Although cell type-specific Cre recombinase-expressing mouse lines are commonly used to generate conditional knockout of genes of interest, germline recombination and ectopic “leakiness” in Cre recombinase expression in non-specific cell types has been observed in several neuronal and glial-specific Cre lines. This often leads to inadvertent loss of conditional mouse lines, requiring rederivation. It is therefore imperative to be able to monitor and validate cell type-specific Cre recombinase-mediated gene editing. Herein, we describe a simple, inexpensive, rapid ZsGreen fluor-reporter-based strategy for genotype-free identification of ectopic leakiness using a custom-designed, 3-D blue LED light box. We assessed cell type-specific expression in several allegedly specific Cre recombinase mouse lines commonly used in vision research: retinal pigment epithelium (RPE)-specific (VMD2 (Best1) Cre, RPE65 Cre); astrocyte-specific (GFAP Cre); as well as photoreceptor-bipolar progenitor cell-specific (CRX Cre). Our standardized workflow allows facile, rapid identification of ectopic and non-specific Cre recombinase expression in any presume specific Cre mouse line, without the need for genotyping and without causing animal distress.

A new Hoxb8FlpO mouse line for intersectional approaches to dissect developmentally defined adult sensorimotor circuits.

Improvements in the speed and cost of expression profiling of neuronal tissues offer an unprecedented opportunity to define ever finer subgroups of neurons for functional studies. In the spinal cord, single cell RNA sequencing studies support decades of work on spinal cord lineage studies, offering a unique opportunity to probe adult function based on developmental lineage. While Cre/Flp recombinase intersectional strategies remain a powerful tool to manipulate spinal neurons, the field lacks genetic tools and strategies to restrict manipulations to the adult mouse spinal cord at the speed at which new tools develop. This study establishes a new workflow for intersectional mouse-viral strategies to dissect adult spinal function based on developmental lineages in a modular fashion. To restrict manipulations to the spinal cord, we generate a brain-sparing Hoxb8FlpO mouse line restricting Flp recombinase expression to caudal tissue. Recapitulating endogenous Hoxb8 gene expression, Flp-dependent reporter expression is present in the caudal embryo starting day 9.5. This expression restricts Flp activity in the adult to the caudal brainstem and below. Hoxb8FlpO heterozygous and homozygous mice do not develop any of the sensory or locomotor phenotypes evident in Hoxb8 heterozygous or mutant animals, suggesting normal developmental function of the Hoxb8 gene and protein in Hoxb8FlpO mice. Compared to the variability of brain recombination in available caudal Cre and Flp lines, Hoxb8FlpO activity is not present in the brain above the caudal brainstem, independent of mouse genetic background. Lastly, we combine the Hoxb8FlpO mouse line with dorsal horn developmental lineage Cre mouse lines to express GFP in developmentally determined dorsal horn populations. Using GFP-dependent Cre recombinase viruses and Cre recombinase-dependent inhibitory chemogenetics, we target developmentally defined lineages in the adult. We show how developmental knock-out versus transient adult silencing of the same ROR𝛃 lineage neurons affects adult sensorimotor behavior. In summary, this new mouse line and viral approach provides a blueprint to dissect adult somatosensory circuit function using Cre/Flp genetic tools to target spinal cord interneurons based on genetic lineage.

Inducible Rbpms-CreERT2 Mouse Line for Studying Gene Function in Retinal Ganglion Cell Physiology and Disease.

Retinal ganglion cells (RGCs) are the sole output neurons conveying visual stimuli from the retina to the brain, and dysfunction or loss of RGCs is the primary determinant of visual loss in traumatic and degenerative ocular conditions. Currently, there is a lack of RGC-specific Cre mouse lines that serve as invaluable tools for manipulating genes in RGCs and studying the genetic basis of RGC diseases. The RNA-binding protein with multiple splicing (RBPMS) is identified as the specific marker of all RGCs. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused in-frame to the C-terminus of endogenous RBPMS, allowing for the co-expression of RBPMS and CreERT2. The inducible Rbpms-CreERT2 mice exhibited a high recombination efficiency in activating the expression of the tdTomato reporter gene in nearly all adult RGCs as well as in differentiated RGCs starting at E13.5. Additionally, both heterozygous and homozygous Rbpms-CreERT2 knock-in mice showed no detectable defect in the retinal structure, visual function, and transcriptome. Together, these results demonstrated that the Rbpms-CreERT2 knock-in mouse can serve as a powerful and highly desired genetic tool for lineage tracing, genetic manipulation, retinal physiology study, and ocular disease modeling in RGCs.

The astrocytic Na+ -HCO3 - cotransporter, NBCe1, is dispensable for respiratory chemosensitivity.

The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO2 /H+ is exquisitely sensitive, with convergent roles proposed for chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na+ -HCO3 - cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO2 -induced local extracellular acidification or purinergic signalling). We tested these NBCe1-centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP-Cre;Slc4a4fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1-mediated current. Despite disrupted NBCe1 function in RTN-adjacent astrocytes from these conditional knockout mice, CO2 -induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO2 -stimulated breathing, were indistinguishable from NBCe1-intact littermates; hypoxia-stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice. Again, there was no difference in effects of CO2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1-deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1-independent mechanisms. KEY POINTS: The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO2 /H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing. We used two different Cre mouse lines for cell-specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis. In both mouse lines, Slc4a4 was depleted from RTN-associated astrocytes but CO2 -induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO2 or O2 were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.

Comparison of Kindlin-2 deficiency-stimulated osteoarthritis-like lesions induced by Prg4CreERT2 versus AggrecanCreERT2 transgene in mice

BackgroundGenetically modified mice are the most useful tools for investigating the gene functions in articular cartilage biology and the pathogenesis of osteoarthritis. The AggrecanCreERT2 mice are one of the most reported mouse lines used for this purpose. The Prg4 (proteoglycan 4) gene encodes the lubricin protein and is expressed selectively in chondrocytes located at the superficial layer of the articular cartilage. While the Prg4GFPCreERT2 knock-in inducible-Cre transgenic mice were generated a while ago, so far, few studies have used this mouse line to perform gene functional studies in cartilage biology. MethodsWe have recently reported that deleting the Fermt2 gene, which encodes the key focal adhesion protein Kindlin-2, in articular chondrocytes by using the AggrecanCreERT2 transgenic mice, results in spontaneous osteoarthritis (OA) lesions, which highly mimics the human OA pathologies. In this study, we have compared the Kindlin-2 deficiency-caused OA phenotypes induced by Prg4GFPCreERT2 with those caused by AggrecanCreERT2 using imaging and histological analyses. ResultsWe find that Kindlin-2 protein is deleted in about 75% of the superficial articular chondrocytes in the tamoxifen (TAM)-treated Prg4GFPCreERt2/+; Fermt2fl/fl mice compared to controls. At 6 months after TAM injections, the OARSI scores of AggrecanCreERT2/+; Fermt2fl/fl and Prg4GFPCreERt2/+; Fermt2fl/fl mice were 5 and 3, respectively. The knee joints histological osteophyte and synovitis scores were also significantly decreased in Prg4GFPCreERT2/+; Fermt2fl/fl mice compared to those in AggrecanCreERT2/+; Fermt2fl/fl mice. Furthermore, magnitudes of upregulation of the extracellular matrix-degrading enzymes Mmp13 and hypertrophic chondrocyte markers Col10a1 and Runx2 were decreased in Prg4GFPCreERT2/+; Fermt2fl/fl versus AggrecanCreERT2/+; Fermt2fl/fl mice. We finally examined the susceptibility of Prg4GFPCreERT2/+; Fermt2fl/fl mouse model to surgically induce OA lesions. The pathological features of OA in the TAM-DMM model exhibited significant enhancement in cartilage erosion, proteoglycan loss, osteophyte, and synovitis and an increase in OARSI score in articular cartilage compared with those in corn-oil DMM mice. ConclusionKindlin-2 loss causes milder OA-like lesions in Prg4GFPCreERT2/+;Fermt2fl/fl than in AggrecanCreERT2/+; Fermt2fl/fl mice. In contrast, Kindlin-2 loss similarly accelerates the destabilization of the medial meniscus-induced OA lesions in both mice.Translational Potential of this Article: Our study demonstrates that Prg4GFPCreERT2 is a useful tool for gene functional study in OA research. This study provides useful information for investigators to choose appropriate Cre mouse lines for their research in cartilage biology.

A transgenic mouse line for assaying tissue-specific changes in endoplasmic reticulum proteostasis

Maintenance of calcium homeostasis is important for proper endoplasmic reticulum (ER) function. When cellular stress conditions deplete the high concentration of calcium in the ER, ER-resident proteins are secreted into the extracellular space in a process called exodosis. Monitoring exodosis provides insight into changes in ER homeostasis and proteostasis resulting from cellular stress associated with ER calcium dysregulation. To monitor cell-type specific exodosis in the intact animal, we created a transgenic mouse line with a Gaussia luciferase (GLuc)—based, secreted ER calcium-modulated protein, SERCaMP, preceded by a LoxP-STOP-LoxP (LSL) sequence. The Cre-dependent LSL-SERCaMP mice were crossed with albumin (Alb)-Cre and dopamine transporter (DAT)-Cre mouse lines. GLuc-SERCaMP expression was characterized in mouse organs and extracellular fluids, and the secretion of GLuc-SERCaMP in response to cellular stress was monitored following pharmacological depletion of ER calcium. In LSL-SERCaMP × Alb-Cre mice, robust GLuc activity was observed only in the liver and blood, whereas in LSL-SERCaMP × DAT-Cre mice, GLuc activity was seen in midbrain dopaminergic neurons and tissue samples innervated by dopaminergic projections. After calcium depletion, we saw increased GLuc signal in the plasma and cerebrospinal fluid collected from the Alb-Cre and DAT-Cre crosses, respectively. This mouse model can be used to investigate the secretion of ER-resident proteins from specific cell and tissue types during disease pathogenesis and may aid in the identification of therapeutics and biomarkers of disease.

Augmentation of BMP Signaling in Cranial Neural Crest Cells Leads to Premature Cranial Sutures Fusion through Endochondral Ossification in Mice.

Craniosynostosis is a congenital anomaly characterized by the premature fusion of cranial sutures. Sutures are a critical connective tissue that regulates bone growth; their aberrant fusion results in abnormal shapes of the head and face. The molecular and cellular mechanisms have been investigated for a long time, but knowledge gaps remain between genetic mutations and mechanisms of pathogenesis for craniosynostosis. We previously demonstrated that the augmentation of bone morphogenetic protein (BMP) signaling through constitutively active BMP type 1A receptor (caBmpr1a) in neural crest cells (NCCs) caused the development of premature fusion of the anterior frontal suture, leading to craniosynostosis in mice. In this study, we demonstrated that ectopic cartilage forms in sutures prior to premature fusion in caBmpr1a mice. The ectopic cartilage is subsequently replaced by bone nodules leading to premature fusion with similar but unique fusion patterns between two neural crest-specific transgenic Cre mouse lines, P0-Cre and Wnt1-Cre mice, which coincides with patterns of premature fusion in each line. Histologic and molecular analyses suggest that endochondral ossification in the affected sutures. Both in vitro and in vivo observations suggest a greater chondrogenic capacity and reduced osteogenic capability of neural crest progenitor cells in mutant lines. These results suggest that the augmentation of BMP signaling alters the cell fate of cranial NCCs toward a chondrogenic lineage to prompt endochondral ossification to prematurely fuse cranial sutures. By comparing P0-Cre;caBmpr1a and Wnt1-Cre;caBmpr1a mice at the stage of neural crest formation, we found more cell death of cranial NCCs in P0-Cre;caBmpr1a than Wnt1-Cre;caBmpr1a mice at the developing facial primordia. These findings may provide a platform for understanding why mutations of broadly expressed genes result in the premature fusion of limited sutures. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization invitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

A novel Oprm1-Cre mouse maintains endogenous expression, function and enables detailed molecular characterization of μ-opioid receptor cells.

Key targets of both the therapeutic and abused properties of opioids are μ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary. Therefore, we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3'UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. The breadth of utility of this new tool will greatly facilitate the study of opioid biology under varying conditions.

Targeting leptin-positive adipocytes by expressing the Cre recombinase transgene under the endogenous leptin gene.

Adipose tissue plays an important role in metabolic physiology through energy storage and endocrine functions. Spatial transcriptomics is revealing the complexity of cell types and their interaction in the adipose tissue with regards to development, homeostasis and disease. Emerging evidence suggests the existence of different subtypes of mature adipocytes that may have distinct functions, the markers of which include leptin (LEP), adiponectin (ADIPOQ), perilipin-1/4 (PLIN), and serum amyloid A (SAA), marking different adipocyte subtypes. Currently, Adipoq-Cre is widely used to study adipocyte biology, however, there is no Cre line that specifically targets LEP+ adipocytes. Here, we report the construction and validation of a Lep-Cre mouse line, which has the endogenous Lep gene edited by the CRISPR-Cas9 technology to generate the Lep-peptide 2A (P2A)-Cre fusion gene. P2A induces an auto-hydrolysis of the fusion protein, leading to expression of the Cre recombinase by the Lep gene activity. The activity of Lep-Cre in different depots of adipose tissues and non-adipose tissues was visualized by the immunofluorescence microscopy in the Lep-Cre Rosa26-loxP-Stop-loxP-tdTomato mice. We showed that Lep-Cre marked white/beige adipose depots extensively, followed by brown adipose depots. Leaky activity was observed in varying degrees among peripheral organs but not in the paraventricular nucleus of the hypothalamus. In summary, we have constructed a new adipocyte-targeting Cre mouse line that would be useful to study the development and physiology of LEP+ adipocytes.