Lineage tracing using Wnt2b-2A-CreERT2 knock-in mice reveals the contributions of Wnt2b-expressing cells to novel subpopulations of mesothelial/epicardial cell lineages during mouse development.

Mesothelial and epicardial cells give rise to various types of mesenchymal cells via epithelial (mesothelial)-to-mesenchymal transition during development. However, the genes controlling the differentiation and diversification of mesothelial/epicardial cells remain unclear. Here, we examined Wnt2b expression in the embryonic mesothelium and epicardium and performed lineage tracing of Wnt2b-expressing cells by using novel Wnt2b-2A-CreERT2 knock-in and LacZ-reporter mice. Wnt2b was expressed in mesothelial cells covering visceral organs, but the expression was restricted in their subpopulations. Wnt2b-expressing cells labeled at embryonic day (E) 10.5 were distributed to the mesothelium and mesenchyme in the lungs, abdominal wall, stomach, and spleen in Wnt2b2A-CreERT2/+;R26RLacZ/+ mice at E13.0. Wnt2b was initially expressed in the proepicardial organ (PEO) at E9.5 and then in the epicardium after E10.0. Wnt2b-expressing PEO cells labeled at E9.5 differentiated into a small fraction of cardiac fibroblasts and preferentially localized at the left side of the postnatal heart. LacZ+ epicardium-derived cells labeled at E10.5 differentiated into a small fraction of fibroblasts and smooth muscle cells in the postnatal heart. Taken together, our results reveal novel subpopulations of PEO and mesothelial/epicardial cells that are distinguishable by Wnt2b expression and elucidate the unique contribution of Wnt2b-expressing PEO and epicardial cells to the postnatal heart.

Bone or Tooth dentin: The TGF-ß signaling is the key.

To investigate the cell linkage between tooth dentin and bones, we studied TGF-ß roles during postnatal dentin development using TGF-ß receptor 2 (Tgfßr2) cKO models and cell lineage tracing approaches. Micro-CT showed that the early Tgfßr2 cKO exhibit short roots and thin root dentin (n = 4; p<0.01), a switch from multilayer pre-odontoblasts/odontoblasts to a single-layer of bone-like cells with a significant loss of ~85% of dentinal tubules (n = 4; p<0.01), and a matrix shift from dentin to bone. Mechanistic studies revealed a statistically significant decrease in odontogenic markers, and a sharp increase in bone markers. The late Tgfßr2 cKO teeth displayed losses of odontoblast polarity, a significant reduction in crown dentin volume, and the onset of massive bone-like structures in the crown pulp with high expression levels of bone markers and low levels of dentin markers. We thus concluded that bones and tooth dentin are in the same evolutionary linkage in which TGF-ß signaling defines the odontogenic fate of dental mesenchymal cells and odontoblasts. This finding also raises the possibility of switching the pulp odontogenic to the osteogenic feature of pulp cells via a local manipulation of gene programs in future treatment of tooth fractures.

Inhibition of EZH2 Reduces Aging-Related Decline in Interstitial Cells of Cajal of the Mouse Stomach.

BACKGROUND & AIMS: Restricted gastric motor functions contribute to aging-associated undernutrition, sarcopenia, and frailty. We previously identified a decline in interstitial cells of Cajal (ICC; gastrointestinal pacemaker and neuromodulator cells) and their stem cells (ICC-SC) as a key factor of gastric aging. Altered functionality of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is central to organismal aging. Here, we investigated the role of EZH2 in the aging-related loss of ICC/ICC-SC. METHODS: klotho mice, a model of accelerated aging, were treated with the most clinically advanced EZH2 inhibitor, EPZ6438 (tazemetostat; 160 mg/kg intraperitoneally twice a day for 3 weeks). Gastric ICC were analyzed by Western blotting and immunohistochemistry. ICC and ICC-SC were quantified by flow cytometry. Gastric slow wave activity was assessed by intracellular electrophysiology. Ezh2 was deactivated in ICC by treating KitcreERT2/+;Ezh2fl/fl mice with tamoxifen. TRP53, a key mediator of aging-related ICC loss, was induced with nutlin 3a in gastric muscle organotypic cultures and an ICC-SC line. RESULTS: In klotho mice, EPZ6438 treatment mitigated the decline in the ICC growth factor KIT ligand/stem cell factor and gastric ICC. EPZ6438 also improved gastric slow wave activity and mitigated the reduced food intake and impaired body weight gain characteristic of this strain. Conditional genomic deletion of Ezh2 in Kit-expressing cells also prevented ICC loss. In organotypic cultures and ICC-SC, EZH2 inhibition prevented the aging-like effects of TRP53 stabilization on ICC/ICC-SC. CONCLUSIONS: Inhibition of EZH2 with EPZ6438 mitigates aging-related ICC/ICC-SC loss and gastric motor dysfunction, improving slow wave activity and food intake in klotho mice.

Transgenic Targeting of Fcrls Creates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity.

Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines can achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specific Fcrls gene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis in Fcrls-2A-Cre mice. Crossing Fcrls-2A-Cre mice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border-associated macrophages are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line, Cx3cr1-CreM Fcrls-2A-Cre mice are immediately available (JAX #036591) and serve as a valuable addition to the community's microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable.

Generation of tamoxifen-inducible Tfap2b-CreERT2 mice using CRISPR-Cas9.

Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreERT2 knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreERT2 transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.

Molecular signatures define subtypes of auditory afferents with distinct peripheral projection patterns and physiological properties.

Type I spiral ganglion neurons (SGNs) are the auditory afferents that transmit sound information from cochlear inner hair cells (IHCs) to the brainstem. These afferents consist of physiological subtypes that differ in their spontaneous firing rate (SR), activation threshold, and dynamic range and have been described as low, medium, and high SR fibers. Lately, single-cell RNA sequencing experiments have revealed three molecularly defined type I SGN subtypes. The extent to which physiological type I SGN subtypes correspond to molecularly defined subtypes is unclear. To address this question, we have generated mouse lines expressing CreERT2 in SGN subtypes that allow for a physiological assessment of molecular subtypes. We show that Lypd1-CreERT2 expressing SGNs represent a well-defined group of neurons that preferentially innervate the IHC modiolar side and exhibit a narrow range of low SRs. In contrast, Calb2-CreERT2 expressing SGNs preferentially innervate the IHC pillar side and exhibit a wider range of SRs, thus suggesting that a strict stratification of all SGNs into three molecular subclasses is not obvious, at least not with the CreERT2 tools used here. Genetically marked neuronal subtypes refine their innervation specificity onto IHCs postnatally during the time when activity is required to refine their molecular phenotype. Type I SGNs thus consist of genetically defined subtypes with distinct physiological properties and innervation patterns. The molecular subtype-specific lines characterized here will provide important tools for investigating the role of the physiologically distinct type I SGNs in encoding sound signals.

Spatiotemporal control of genome engineering in cone photoreceptors.

BACKGROUND: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. RESULTS: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. CONCLUSIONS: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.

Pericyte-derived cells participate in optic nerve scar formation.

Introduction: Pericytes (PCs) are specialized cells located abluminal of endothelial cells on capillaries, fulfilling numerous important functions. Their potential involvement in wound healing and scar formation is achieving increasing attention since years. Thus, many studies investigated the participation of PCs following brain and spinal cord (SC) injury, however, lacking in-depth analysis of lesioned optic nerve (ON) tissue. Further, due to the lack of a unique PC marker and uniform definition of PCs, contradicting results are published. Methods: In the present study the inducible PDGFRß-P2A-CreERT2-tdTomato lineage tracing reporter mouse was used to investigate the participation and trans-differentiation of endogenous PC-derived cells in an ON crush (ONC) injury model, analyzing five different post lesion time points up to 8 weeks post lesion. Results: PC-specific labeling of the reporter was evaluated and confirmed in the unlesioned ON of the reporter mouse. After ONC, we detected PC-derived tdTomato+ cells in the lesion, whereof the majority is not associated with vascular structures. The number of PC-derived tdTomato+ cells within the lesion increased over time, accounting for 60-90% of all PDGFRß+ cells in the lesion. The presence of PDGFRß+tdTomato- cells in the ON scar suggests the existence of fibrotic cell subpopulations of different origins. Discussion: Our results clearly demonstrate the presence of non-vascular associated tdTomato+ cells in the lesion core, indicating the participation of PC-derived cells in fibrotic scar formation following ONC. Thus, these PC-derived cells represent promising target cells for therapeutic treatment strategies to modulate fibrotic scar formation to improve axonal regeneration.

Generation and characterization of an inducible renal proximal tubule-specific CreERT2 mouse.

Protein reabsorption in renal proximal tubules is essential for maintaining nutrient homeostasis. Renal proximal tubule-specific gene knockout is a powerful method to assess the function of genes involved in renal proximal tubule protein reabsorption. However, the lack of inducible renal proximal tubule-specific Cre recombinase-expressing mouse strains hinders the study of gene function in renal proximal tubules. To facilitate the functional study of genes in renal proximal tubules, we developed an AMN CreERT2 knock-in mouse strain expressing a Cre recombinase-estrogen receptor fusion protein under the control of the promoter of the amnionless (AMN) gene, a protein reabsorption receptor in renal proximal tubules. AMN CreERT2 knock-in mice were generated using the CRISPR/Cas9 strategy, and the tissue specificity of Cre activity was investigated using the Cre/loxP reporter system. We showed that the expression pattern of CreERT2-mEGFP in AMN CreERT2 mice was consistent with that of the endogenous AMN gene. Furthermore, we showed that the Cre activity in AMN CreERT2 knock-in mice was only detected in renal proximal tubules with high tamoxifen induction efficiency. As a proof-of-principle study, we demonstrated that renal proximal tubule-specific knockout of Exoc4 using AMNCreERT2 led to albumin accumulation in renal proximal tubular epithelial cells. The AMN CreERT2 mouse is a powerful tool for conditional gene knockout in renal proximal tubules and should offer useful insight into the physiological function of genes expressed in renal proximal tubules.

Two novel CreERT2 transgenic mouse lines to study melanocytic cells in vivo.

The skin of adult mammals protects from radiation, physical and chemical insults. While melanocytes and melanocyte-producing stem cells contribute to proper skin function in healthy organisms, dysfunction of these cells can lead to the generation of malignant melanoma-the deadliest type of skin cancer. Addressing cells of the melanocyte lineage in vivo represents a prerequisite for the understanding of melanoma on cellular level and the development of preventive and treatment strategies. Here, the inducible Cre-loxP-system has emerged as a promising tool to specifically target, monitor, and modulate cells in adult mice. Re-analysis of existing sequencing data sets of melanocytic cells revealed that genes with a known function in neural cells, including neural stem cells (Aldh1L1 and Nestin), are also expressed in melanocytic cells. Therefore, in this study, we explored whether the promoter activity of Nestin and Aldh1L1 can serve to target cells of the melanocyte lineage using the inducible CreERT2 -loxP-system. Using an immunohistochemical approach and different time points of analysis, we were able to map the melanocytic fate of recombined stem cells in the adult hair follicle of Nestin-CreERT2 and Aldh1L1-CreERT2 transgenic mice. Thus, we here present two new mouse models and propose their use to study and putatively modulate adult melanocytic cells in vivo.

Heterogeneity and genomic loci of ubiquitous transgenic Cre reporter lines in zebrafish.

BACKGROUND: The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. RESULTS: Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of the two additional Switch reporters actb2:BFP-DsRed and actb2:Stop-DsRed. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. CONCLUSIONS: Our data documents the heterogeneity among lox-based Switch transgenes toward informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

Transcriptional and Distributional Profiling of Microglia in Retinal Angiomatous Proliferation.

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.

Retinoic Acid Receptor Alpha Is Essential in Postnatal Sertoli Cells but Not in Germ Cells.

Retinoic acid signaling is indispensable for the completion of spermatogenesis. It is known that loss of retinoic acid nuclear receptor alpha (RARA) induces male sterility due to seminiferous epithelium degeneration. Initial genetic studies established that RARA acts in Sertoli cells, but a recent paper proposed that RARA is also instrumental in germ cells. In the present study, we have re-assessed the function of RARA in germ cells by genetically ablating the Rara gene in spermatogonia and their progenies using a cell-specific conditional mutagenesis approach. We show that loss of Rara in postnatal male germ cells does not alter the histology of the seminiferous epithelium. Furthermore, RARA-deficient germ cells differentiate normally and give rise to normal, living pups. This establishes that RARA plays no crucial role in germ cells. We also tested whether RARA is required in Sertoli cells during the fetal period or after birth. For this purpose, we deleted the Rara gene in Sertoli cells at postnatal day 15 (PN15), i.e., after the onset of the first spermatogenic wave. To do so, we used temporally controlled cell-specific mutagenesis. By comparing the testis phenotypes generated when Rara is lost either at PN15 or at embryonic day 13, we show that RARA exerts all of its functions in Sertoli cells not at the fetal stage but from puberty.

A vascular smooth muscle-specific integrin-α8 Cre mouse for lymphatic contraction studies that allows male-female comparisons and avoids visceral myopathy.

Introduction: The widely-used, tamoxifen-inducible, smooth muscle (SM)-specific Cre, Myh11-CreERT2 , suffers from two disadvantages: 1) it is carried on the Y-chromosome and thus only effective for gene deletion in male mice, and 2) it recombines in both vascular and non-vascular SM, potentially leading to unwanted or confounding gastrointestinal phenotypes. Here, we tested the effectiveness of a new, SM-specific Cre, based on the integrin α8 promoter (Itga8-CreERT2 ), that has been recently developed and characterized, to assess the effects of Cav1.2 deletion on mouse lymphatic SM function. Methods: Cav1.2 (the L-type voltage-gated calcium channel) is essential for lymphatic pacemaking and contraction and its deletion using either Myh11-CreERT2 or Itga8-CreERT2 abolished spontaneous lymphatic contractions. Mouse lymphatic contractile function was assessed using two ex vivo methods. Results: Myh11-CreERT2 ; Cav1.2 f/f mice died of gastrointestinal obstruction within 20 days of the first tamoxifen injection, preceded by several days of progressively poor health, with symptoms including weight loss, poor grooming, hunched posture, and reduced overall activity. In contrast, Itga8-CreERT2 ; Cav1.2 f/f mice survived for >80 days after induction and were in normal health until the time of sacrifice for experimental studies. Cav1.2 deletion was equally effective in male and female mice. Discussion: Our results demonstrate that Itga8-CreER T2 can be used to effectively delete genes in lymphatic smooth muscle while avoiding potentially lethal visceral myopathy and allowing comparative studies of lymphatic contractile function in both male and female mice.

A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development.

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

Distribution of Aldh1L1-CreERT2 Recombination in Astrocytes Versus Neural Stem Cells in the Neurogenic Niches of the Adult Mouse Brain.

In the adult central nervous system, neural stem cells (NSCs) reside in two discrete niches: the subependymal zone (SEZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Here, NSCs represent a population of highly specialized astrocytes that are able to proliferate and give rise to neuronal and glial progeny. This process, termed adult neurogenesis, is extrinsically regulated by other niche cells such as non-stem cell astrocytes. Studying these non-stem cell niche astrocytes and their role during adult neuro- and gliogenesis has been hampered by the lack of genetic tools to discriminate between transcriptionally similar NSCs and niche astrocytes. Recently, Aldh1L1 has been shown to be a pan-astrocyte marker and that its promoter can be used to specifically target astrocytes using the Cre-loxP system. In this study we explored whether the recently described Aldh1L1-CreERT2 mouse line (Winchenbach et al., 2016) can serve to specifically target niche astrocytes without inducing recombination in NSCs in adult neurogenic niches. Using short- and long-term tamoxifen protocols we revealed high recombination efficiency and specificity in non-stem cell astrocytes and little to no recombination in NSCs of the adult DG. However, in the SEZ we observed recombination in ependymal cells, astrocytes, and NSCs, the latter giving rise to neuronal progeny of the rostral migratory stream and olfactory bulb. Thus, we recommend the here described Aldh1L1-CreERT2 mouse line for predominantly studying the functions of non-stem cell astrocytes in the DG under physiological and pathological conditions.

Quantification of adipocyte numbers following adipose tissue remodeling.

To analyze the capacity of white and brown adipose tissue remodeling, we developed two mouse lines to label, quantitatively trace, and ablate white, brown, and brite/beige adipocytes at different ambient temperatures. We show here that the brown adipocytes are recruited first and reach a peak after 1 week of cold stimulation followed by a decline during prolonged cold exposure. On the contrary, brite/beige cell numbers plateau after 3 weeks of cold exposure. At thermoneutrality, brown adipose tissue, in spite of being masked by a white-like morphology, retains its brown-like physiology, as Ucp1+ cells can be recovered immediately upon beta3-adrenergic stimulation. We further demonstrate that the recruitment of Ucp1+ cells in response to cold is driven by existing adipocytes. In contrast, the regeneration of the interscapular brown adipose tissue following ablation of Ucp1+ cells is driven by de novo differentiation.

IGF1R Deficiency Modulates Brain Signaling Pathways and Disturbs Mitochondria and Redox Homeostasis.

Insulin-like growth factor 1 receptor (IGF1R)-mediated signaling pathways modulate important neurophysiological aspects in the central nervous system, including neurogenesis, synaptic plasticity and complex cognitive functions. In the present study, we intended to characterize the impact of IGF1R deficiency in the brain, focusing on PI3K/Akt and MAPK/ERK1/2 signaling pathways and mitochondria-related parameters. For this purpose, we used 13-week-old UBC-CreERT2; Igf1rfl/fl male mice in which Igf1r was conditionally deleted. IGF1R deficiency caused a decrease in brain weight as well as the activation of the IR/PI3K/Akt and inhibition of the MAPK/ERK1/2/CREB signaling pathways. Despite no alterations in the activity of caspases 3 and 9, a significant alteration in phosphorylated GSK3ß and an increase in phosphorylated Tau protein levels were observed. In addition, significant disturbances in mitochondrial dynamics and content and altered activity of the mitochondrial respiratory chain complexes were noticed. An increase in oxidative stress, characterized by decreased nuclear factor E2-related factor 2 (NRF2) protein levels and aconitase activity and increased H2O2 levels were also found in the brain of IGF1R-deficient mice. Overall, our observations confirm the complexity of IGF1R in mediating brain signaling responses and suggest that its deficiency negatively impacts brain cells homeostasis and survival by affecting mitochondria and redox homeostasis.

Temporal manipulation of KCC3 expression in juvenile or adult mice suggests irreversible developmental deficit in hereditary motor sensory neuropathy with agenesis of the corpus callosum.

Hereditary motor sensory neuropathy (HMSN/ACC) with agenesis of the corpus callosum (ACC) has been documented in the French-derived populations of Charlevoix and Saguenay/Lac St. Jean in Quebec, Canada, as well as a few sporadic families throughout the world. HMSN/ACC occurs because of loss-of-function mutations in the potassium-chloride cotransporter 3 (KCC3). In HMSN/ACC, motor deficits occur early in infancy with rapid and continual deterioration of motor and sensory fibers into juvenile and adulthood. Genetic work in mice has demonstrated that the disease is caused by loss of KCC3 function in neurons and particularly parvalbumin (PV)-expressing neurons. Currently, there are no treatments or cures for HMSN/ACC other than pain management. As genetic counseling in Quebec has increased as a preventative strategy, most individuals with HSMN/ACC are now adults. The onset of the disease is unknown. In particular, it is unknown if the disease starts early during development and whether it can be reversed by restoring KCC3 function. In this study, we used two separate mouse models that when combined to the PV-CreERT2 tamoxifen-inducible system allowed us to 1) disrupt KCC3 expression in adulthood or juvenile periods; and 2) reintroduce KCC3 expression in mice that first develop with a nonfunctional cotransporter. We show that disrupting or reintroducing KCC3 in the adult mouse has no effect on locomotor behavior, indicating that expression of KCC3 is critical during embryonic development and/or the perinatal period and that once the disease has started, reexpressing a functional cotransporter fails to change the course of HMSN/ACC.

Tamoxifen-inducible cardiac-specific Cre transgenic mouse using VIPR2 intron.

Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.