Knock-in Reporter Mouse Research Articles

Gastrin induces parathyroid hormone-like hormone expression in gastric parietal cells.

Parietal cells play a fundamental role in stomach maintenance, not only by creating a pathogen-free environment through the production of gastric acid, but also by secreting growth factors important for homeostasis of the gastric epithelium. The gastrointestinal hormone gastrin is known to be a central regulator of both parietal cell function and gastric epithelial cell proliferation and differentiation. Our previous gene expression profiling studies of mouse stomach identified parathyroid hormone-like hormone (PTHLH) as a potential gastrin-regulated gastric growth factor. Although PTHLH is commonly overexpressed in gastric tumors, its normal expression, function, and regulation in the stomach are poorly understood. In this study we used pharmacologic and genetic mouse models as well as human gastric cancer cell lines to determine the cellular localization and regulation of this growth factor by the hormone gastrin. Analysis of PthlhLacZ/+ knock-in reporter mice localized Pthlh expression to parietal cells in the gastric corpus. Regulation by gastrin was demonstrated by increased Pthlh mRNA abundance after acute gastrin treatment in wild-type mice and reduced expression in gastrin-deficient mice. PTHLH transcripts were also observed in normal human stomach as well as in human gastric cancer cell lines. Gastrin treatment of AGS-E gastric cancer cells induced a rapid and robust increase in numerous PTHLH mRNA isoforms. This induction was largely due to increased transcriptional initiation, although analysis of mRNA half-life showed that gastrin treatment also extended the half-life of PTHLH mRNA, suggesting that gastrin regulates expression by both transcriptional and posttranscriptional mechanisms.NEW & NOTEWORTHY We show that the growth factor parathyroid hormone-like hormone (PTHLH) is expressed in acid-secreting parietal cells of the mouse stomach. We define the specific PTHLH mRNA isoforms expressed in human stomach and in human gastric cancer cell lines and show that gastrin induces PTHLH expression via transcription activation and mRNA stabilization. Our findings suggest that PTHLH is a gastrin-regulated growth factor that might contribute to gastric epithelial cell homeostasis.

Parathyroid hormone controls paracellular Ca2+ transport in the thick ascending limb by regulating the tight-junction protein Claudin14

Renal Ca2+ reabsorption is essential for maintaining systemic Ca2+ homeostasis and is tightly regulated through the parathyroid hormone (PTH)/PTHrP receptor (PTH1R) signaling pathway. We investigated the role of PTH1R in the kidney by generating a mouse model with targeted deletion of PTH1R in the thick ascending limb of Henle (TAL) and in distal convoluted tubules (DCTs): Ksp-cre;Pth1rfl/fl Mutant mice exhibited hypercalciuria and had lower serum calcium and markedly increased serum PTH levels. Unexpectedly, proteins involved in transcellular Ca2+ reabsorption in DCTs were not decreased. However, claudin14 (Cldn14), an inhibitory factor of the paracellular Ca2+ transport in the TAL, was significantly increased. Analyses by flow cytometry as well as the use of Cldn14-lacZ knock-in reporter mice confirmed increased Cldn14 expression and promoter activity in the TAL of Ksp-cre;Pth1rfl/fl mice. Moreover, PTH treatment of HEK293 cells stably transfected with CLDN14-GFP, together with PTH1R, induced cytosolic translocation of CLDN14 from the tight junction. Furthermore, mice with high serum PTH levels, regardless of high or low serum calcium, demonstrated that PTH/PTH1R signaling exerts a suppressive effect on Cldn14. We therefore conclude that PTH1R signaling directly and indirectly regulates the paracellular Ca2+ transport pathway by modulating Cldn14 expression in the TAL. Finally, systemic deletion of Cldn14 completely rescued the hypercalciuric and lower serum calcium phenotype in Ksp-cre;Pth1rfl/fl mice, emphasizing the importance of PTH in inhibiting Cldn14. Consequently, suppressing CLDN14 could provide a potential treatment to correct urinary Ca2+ loss, particularly in patients with hypoparathyroidism.

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation.

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation.

A Novel CD48-Based Analysis of Sepsis-Induced Mouse Myeloid-Derived Suppressor Cell Compartments.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous subset of cells that expands dramatically in many disease states and can suppress T-cell responses. MDSCs mainly include monocytic and granulocytic subpopulations that can be distinguished in mice by the expression of Ly6G and Ly6C cell surface markers. This identification system has been validated in experimental tumor models, but not in models of inflammation-associated conditions such as sepsis. We challenged growth factor independent 1 transcription repressor green fluorescent protein (Gfi1:GFP) knock-in reporter mice with cecal ligation and puncture surgery and found that CD11b+Ly6GlowLy6Chigh MDSCs in this sepsis model comprised both monocytic and granulocytic MDSCs. The evidence that conventional Ly6G/Ly6C marker analysis may not be suited to study of inflammation-induced MDSCs led to the development of a novel strategy of distinguishing granulocytic MDSCs from monocytic MDSCs in septic mice by expression of CD48. Application of this novel model should help achieve a more accurate understanding of the inflammation-induced MDSC activity.

Expression of Noggin and Gremlin1 and its implications in fine-tuning BMP activities in mouse cartilage tissues.

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1, BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin-LacZ and Gremlin1-LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin-LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1-LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671-1682, 2017.

Defining C3aR expression in myeloid and lymphoid cells using a novel Td-Tomato-C3aR reporter knockin mouse

Defining C3aR expression in myeloid and lymphoid cells using a novel Td-Tomato-C3aR reporter knockin mouse

Asynchronous digital activation of the germline Iγ1 and Iɛ promoters in single cells reveals that temporal control determines the fate of switching to IgE in B cells

Abstract Immunoglobulin IgE is a key effector in the adaptive immune response to parasitic infestations and a potent mediator of allergy and asthma. Switching to IgE is linked to the activation of the germline promoter (Iɛ) that drives transcription through the region (Sɛ) immediately upstream of the ɛ constant region and the recruitment of Activation Induced Deaminase (AID) to induce double strand breaks. The molecular basis of AID recruitment to Sɛ and thus the specificity of switching to IgE remains elusive given that the same stimuli that activate Iɛ also initiate transcription from the Iγ1 promoter upstream of the γ1 constant region. We have used knock-in fluorescent reporter mice for the endogenous Iγ1 and Iɛ promoters to monitor the kinetics of in vitro I-promoter activation by cytokines and isotype switching. While the activation of I-promoters is biallelic, a significant number of activated cells (2/3) displays asynchronous activation, with activation of a single Iγ1 allele followed by activation of the second allele over a period of 12–24 hrs in vitro. Intriguingly, early activation of the Iγ1 promoter is unaffected by the rearrangement status of the cis VDJ region. Single-cell transcriptomic and qPCR analyses on activated B cells reveal enormous heterogeneity in I-promoter activation within individual cells, identifying a distinct early population of cells in which only the Iɛ promoter becomes active, which is correlated with enhanced switching to IgE. Heterogeneity of allele activation in single cells is emerging as a general property of transcriptional programming. In B cells, the intrinsic temporal heterogeneity of I-promoter activation in individual cells is crucial for the control of AID targeting and the levels of IgE switching.

The complement receptor C5aR1 contributes to renal damage but protects the heart in angiotensin II-induced hypertension.

Adaptive and innate immune responses contribute to hypertension and hypertensive end-organ damage. Here, we determined the role of anaphylatoxin C5a, a major inflammatory effector of the innate immune system that is generated in response to complement activation, in hypertensive end-organ damage. For this purpose, we assessed the phenotype of C5a receptor 1 (C5aR1)-deficient mice in ANG II-induced renal and cardiac injury. Expression of C5aR1 on infiltrating and resident renal as well as cardiac cells was determined using a green fluorescent protein (GFP)-C5aR1 reporter knockin mouse. Flow cytometric analysis of leukocytes isolated from the kidney of GFP-C5aR1 reporter mice showed that 28% of CD45-positive cells expressed C5aR1. Dendritic cells were identified as the major C5aR1-expressing population (88.5%) followed by macrophages and neutrophils. Using confocal microscopy, we detected C5aR1 in the kidney mainly on infiltrating cells. In the heart, only infiltrating cells stained C5aR1 positive. To evaluate the role of C5aR1 deficiency in hypertensive injury, an aggravated model of hypertension was used. Unilateral nephrectomy was performed followed by infusion of ANG II (1.5 ng·g(-1)·min(-1)) and salt in wild-type (n = 34) and C5aR1-deficient mice (n = 32). C5aR1-deficient mice exhibited less renal injury, as evidenced by significantly reduced albuminuria. In contrast, cardiac injury was accelerated with significantly increased cardiac fibrosis and heart weight in C5aR1-deficient mice after ANG II infusion. No effect was found on blood pressure. In summary, the C5a:C5aR1 axis drives end-organ damage in the kidney but protects from the development of cardiac fibrosis and hypertrophy in experimental ANG II-induced hypertension.

A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells.

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knock-in reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this ‘imprinting reporter mouse’ can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals the role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease.

Identification of the Origin of Eosinophils

Eosinophil granulocytes participate in parasite protection and also play a part in asthma and allergy. Histology staining shows a characteristic red granular stain, which separates the eosinophils from neutrophil and basophil granulocytes. Eosinophils can be characterized by SiglecF and CCL3 surface expression, and differentiate from bone-marrow CD34+ Granulocyte Monocyte Progenitors (GMP). Isolating GMP and stimulating with IL-3, GM-CSF and IL-5 leads to eosinophil colony forming units (CFU) in methylcellulose. Eosinophil precursors are currently are isolated by IL-5ra expression in GMP and IL-5 is critical for eosinophil differentiation and expansion both in vivo and in vitro. Gata1 is a transcription factor necessary for erythrocyte and megakaryocyte formation. Previously, a Gata1-GFP transgenic reporter mouse was used to show that all eosinophil CFU capacity within the GMP came from those cells which induce Gata1 expression. Apart from that, little is known about the required transcriptional activity for eosinophil development. We performed single cell RNA seq using the Fluidigm C1 system on murine hematopoietic stem and progenitor cells to characterize myeloid differentiation and development. While primarily studying neutrophil granulocyte differentiation programs within the GMP, we noticed progenitors that expressed eosinophil genes. Gfi1 is a known granulocyte transcription factor, which is critical for neutrophil granulocytes in mice and man. We utilized a Gfi1-GFP knock in reporter mouse to exclude Gfi1-GFPhigh GMP (which we find are neutrophil precursors). Unbiased hierarchical clustering of single-cell RNA Seq from Gfi1-GFPlow GMP identified four clusters. To identify uniquely expressed transcripts within each cluster, we utilized the MarkerFinder algorithm in a bioinformatics analysis platform called AltAnalyze (p<0.05 and fold >2). One cluster expressed transcripts like Fos, Jun, Gata1 and Gata2, indicating a stem-like progenitor. A second cluster, lost the stem-progenitor signature but expressed Elane, Mpo and Csf3r, suggesting a neutrophil progenitor signature. A third cluster lost Fos and Jun expression while gaining Il5ra, Epx and Prg2, suggesting that this cluster contained eosinophil-primed progenitors. Interestingly, although part of the stem-progenitor gene expression was lost, this cluster retained Gata1 expression. Thus, we have identified rare Gata1-expressing GMP populations with either a stem-progenitor or an eosinophil gene expression program. To biologically test whether Gfi1-GFPlow GMP have eosinophil potential, we performed CFU assays with or without IL-5 and then analyzed the colonies for eosinophil markers. We found that IL-5 stimulation of Gfi1-GFPlow GMP led to a significant CFU-G eosinophil-like colony formation, whereas IL-5 stimulation of Gfi1-GFPhigh GMP did not result in any eosinophil-like colonies. Flow cytometric analysis showed SiglecF and CCL3 surface expression in IL-5 treated Gfi1-GFPlow GMP. Furthermore, we observed increased IL-5ra, Epx and Prg2 transcript levels in IL-5 treated Gfi1-GFPlow GMP. Histology stain showed characteristic eosinophilic granules in the IL-5 treated condition. While earlier studies posited the induction of Gata1 expression in GMP as part of the program leading to eosinophil development, our data reveal a rare Gata1-expressing stem-progenitor population which gives rise to eosinophil progenitors as these cells differentiate along the granulocyte-monocyte pathway. Thus, single-cell RNA Seq, combined with conventional methodology can be used to define previously rare and intermediary progenitor populations to understand hematopoietic development. DisclosuresNo relevant conflicts of interest to declare.

The global gene expression profile of the secondary transition during pancreatic development

Pancreas organogenesis is a highly dynamic process where neighboring tissue interactions lead to dynamic changes in gene regulatory networks that orchestrate endocrine, exocrine, and ductal lineage formation. To understand the spatio-temporal regulatory logic we have used the Forkhead transcription factor Foxa2-Venus fusion (FVF) knock-in reporter mouse to separate the FVF+ pancreatic epithelium from the FVF− surrounding tissue (mesenchyme, neurons, blood, and blood vessels) to perform a genome-wide mRNA expression profiling at embryonic days (E) 12.5–15.5. Annotating genes and molecular processes suggest that FVF marks endoderm-derived multipotent epithelial progenitors at several lineage restriction steps, when the bulk of endocrine, exocrine and ductal cells are formed during the secondary transition. In the pancreatic epithelial compartment, we identified most known endocrine and exocrine lineage determining factors and diabetes-associated genes, but also unknown genes with spatio-temporal regulated pancreatic expression. In the non-endoderm-derived compartment, we identified many well-described regulatory genes that are not yet functionally annotated in pancreas development, emphasizing that neighboring tissue interactions are still ill defined. Pancreatic expression of over 635 genes was analyzed with the mRNA in situ hybridization Genepaint public database. This validated the quality of the profiling data set and identified hundreds of genes with spatially restricted expression patterns in the pancreas. Some of these genes are also targeted by pancreatic transcription factors and show active chromatin marks in human islets of Langerhans. Thus, with the highest spatio-temporal resolution of a global gene expression profile during the secondary transition, our study enables to shed light on neighboring tissue interactions, developmental timing and diabetes gene regulation.

A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice.

BackgroundThe GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs.ResultsWe have generated a Gata6H2B-Venus knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution.ConclusionsExpression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6H2B-Venus/H2B-Venus homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0086-5) contains supplementary material, which is available to authorized users.

Melanocortin-4 receptor expression in the cuneiform nucleus is involved in modulation of opioidergic signaling.

Substantial evidence has suggested that deep brain stimulation of the cuneiform nucleus has become a remarkable treatment option for intractable pain, but the possible mechanism is poorly understood. Using a melanocortin-4 receptor (MC4R)-green fluorescent protein (GFP) reporter knockin mouse, we showed that a large number of MC4R-GFP-positive neurons were expressed in the cuneiform nucleus. Immunofluorescence revealed that approximately 40%-50% of MC4R-GFP-positive neurons expressed mu opioid receptors, indicating that they were opioidergic signaling. Our findings support the hypothesis that MC4R expression in the cuneiform nucleus is involved in the modulation of opioidergic signaling.

Abstract 980: Encoding immortality: Transcriptional control of telomerase in stem cells in vivo

Abstract One of the invariant features of human cancer is unlimited proliferation, a hallmark conferred by telomerase in 90% tumors. Somatic mutations in the telomerase reverse transcriptase (TERT) gene promoter are highly recurrent in human cancers. Telomerase is also critically important in human stem cells, as evidenced by mutations in telomerase, which contribute to degenerative diseases. Despite the importance of telomerase in tissue maintenance, the identity of telomerase-positive cells has remained elusive, owing to low levels of the core telomerase components. The ability to isolate TERT-positive cells in vivo would significantly advance our understanding of telomerase regulation, tissue function and carcinogenesis. To address these issues, we created knock-in transcriptional reporters of TERT expression by replacing the TERT open reading frame with the red fluorescent protein, TdTomato. Among mouse tissues, telomerase activity is most strongly expressed in testis, a tissue in which resident stem cells fuel the continuous generation of male gametes. In human sperm, telomere lengths are preserved with age, although how this is achieved, in contrast to the age-dependent telomere shortening seen in somatic tissues, remains unresolved. Using TERTTdTomato/+ knock-in reporter mice, we found that only a rare subset of cells in mouse testis expresses high levels of TERT. By double immunostaining, these TERTHigh cells were synonymous with undifferentiated spermatogonia, the primitive cell population in which male germline stem cells reside. By FACS of the germ cells in testis, TERTHigh cells and TERTLow cells represent discrete populations that were further studied using additional markers. The undifferentiated spermatogonia in the TERTHigh population were further fractionated into GFRalpha+ and GFRalpha- populations. Cells in the TERTLow population were nearly all cKit+, consistent with their identification as differentiated spermatogonia. We characterized these populations in molecular and functional terms. Using RNAseq, we established a hierarchy among these populations according to which the TERTHigh GFRalpha1+ cells give rise to TERTHigh GFRalpha1- cells, which in turn yield TERTLow cKit+ cells. Surprisingly, in transplantation studies, TERTHigh GFRalpha1+ cells and TERTHigh GFRalpha1- cells possess comparable stem cell activity. These data suggest the existence of stem cell plasticity according to which cells in either primitive population retain stem cell potential. In contrast, TERTLow cKit+ cells fail to reconstitute spermatogenesis in transplantation experiments and therefore lack stem cell activity. These studies reveal marked transcriptional regulation of telomerase in vivo and show a strong concordance between stemness and telomerase levels in rare subsets of tissue stem cells in vivo. These findings indicate the existence of innate signaling pathways controlling TERT expression over a surprising dynamic range. Citation Format: Matthew Pech, Alina Garbuzov, Meena Sukhwani, Berenice Benayoun, Shengda Lin, Anne Brunet, Kyle Orwig, Steven E. Artandi. Encoding immortality: Transcriptional control of telomerase in stem cells in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 980. doi:10.1158/1538-7445.AM2015-980

Lentiviral vectors for retrograde delivery of recombinases and transactivators.

Lentiviral vectors pseudotyped with the rabies virus (RV) envelope glycoprotein efficiently infect via axon terminals to stably deliver transgenes to distant neurons projecting to an injection site, but the resulting expression levels are too low and variable for most neuroscientific applications. If used to deliver recombinases or transactivators, however, lentiviral vectors are excellent means of targeting projection neurons when used in reporter mice or in combination with a second virus to express "payload" transgenes at high levels. For retrograde infection of significant numbers of neurons, high virus titers are critical. Here we present reagents and a protocol for generating high-titer supernatants that can be concentrated 1000-fold for final titers in excess of 10(10) infectious units per milliliter. We demonstrate the usefulness of these vectors by selectively targeting corticothalamic and corticotectal neurons for high-level expression of a fluorophore in knock-in reporter mice.

ID4 controls mammary stem cells and marks breast cancers with a stem cell-like phenotype.

Basal-like breast cancer (BLBC) is a heterogeneous disease with poor prognosis; however, its cellular origins and aetiology are poorly understood. In this study, we show that inhibitor of differentiation 4 (ID4) is a key regulator of mammary stem cell self-renewal and marks a subset of BLBC with a putative mammary basal cell of origin. Using an ID4GFP knock-in reporter mouse and single-cell transcriptomics, we show that ID4 marks a stem cell-enriched subset of the mammary basal cell population. ID4 maintains the mammary stem cell pool by suppressing key factors required for luminal differentiation. Furthermore, ID4 is specifically expressed by a subset of human BLBC that possess a very poor prognosis and a transcriptional signature similar to a mammary stem cell. These studies identify ID4 as a mammary stem cell regulator, deconvolute the heterogeneity of BLBC and link a subset of mammary stem cells to the aetiology of BLBC.

Monitoring and cell-specific deletion of C5aR1 using a novel floxed GFP-C5aR1 reporter knock-in mouse.

Many of the biological properties of C5a are mediated through activation of its receptor (C5aR1), the expression of which has been demonstrated convincingly on myeloid cells, such as neutrophils, monocytes, and macrophages. In contrast, conflicting results exist regarding C5aR1 expression in dendritic cells (DCs) and lymphoid lineage cells. In this article, we report the generation of a floxed GFP-C5aR1 reporter knock-in mouse. Using this mouse strain, we confirmed strong C5aR1 expression in neutrophils from bone marrow, blood, lung, and spleen, as well as in peritoneal macrophages. Further, we show C5aR1 expression in lung eosinophils, lung- and lamina propria-resident and alveolar macrophages, bone marrow-derived DCs, and lung-resident CD11b(+) and monocyte-derived DCs, whereas intestinal and pulmonary CD103(+) DCs stained negative. Also, some splenic NKT cells expressed GFP, whereas naive NK cells and B2 cells lacked GFP expression. Finally, we did not observe any C5aR1 expression in naive or activated CD4(+) Th cells in vitro or in vivo. Mating the floxed GFP-C5aR1 mouse strain with LysMCre mice, we were able to specifically delete C5aR1 in neutrophils and macrophages, whereas C5aR1 expression was retained in DCs. In summary, our findings suggest that C5aR1 expression in mice is largely restricted to cells of the myeloid lineage. The novel floxed C5aR1 reporter knock-in mouse will prove useful to track C5aR1 expression in experimental models of acute and chronic inflammation and to conditionally delete C5aR1 in immune cells.

Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium.

The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.

Gata2 expression dynamics correlates with distinct stage specific waves in ES cell hematopoietic differentiation

In the mouse embryo the production of the hematopoietic system occurs in several spatiotemporally distinct waves. Adult repopulating hematopoietic stem cells (HSCs) are generated during the final definitive wave of hematogenesis. HSC production begins at E10.5 in the aorta-gonads-mesonephros (AGM) region and is tightly controlled by a combination of transcription and extrinsic factors.The Gata2 transcription factor has a pivotal role in hematopoiesis. Germline Gata2 deletion results in severe hematopoietic defects and embryonic lethality at E10.5 (Tsai, 1994). Conditional knockouts reveal that Gata2 is required for HSC generation in the embryo and HSC survival in the adult (de Pater, 2013; Gao, 2013). We developed a Gata2-Venus reporter knockin mouse model in which Gata2 expressing cells can be prospectively isolated and examined for hematopoietic function, and found that all HSCs in vivo are Gata2 expressing. To study the role of Gata2 in the early stages of HSC development, we use the novel Gata2-Venus ES cell (ESC) line for embryoid body (EB) differentiation. EB cultures are known to recapitulate early hematopoietic development. Our analysis of Gata2 expression dynamics shows that ESC hematopoietic differentiation occurs in several waves, producing progeny with stage-specific potential. These waves are similar to those observed in vivo. The first wave of Gata2 expressing EBs recapitulates primitive hematopoiesis, whereas gene expression analysis and hematopoietic progenitor assays suggest that cells initiating Gata2 expression later serve as precursors for definitive multipotent progenitors. To initiate further waves/stages of hematogenesis we have developed a novel chimeric ESC differentiation model where AGM derived stromal cells are exploited to create a hematopoiesis-supportive niche within the EBs. This results in a significant increase in Gata2 expressing cells and is accompanied by the generation of multipotent hematopoietic progenitor cells. Thus, by tracking Gata2 expression we have established a culture system that enhances definitive hematopoietic progenitor production and may allow for HSC generation. In the mouse embryo the production of the hematopoietic system occurs in several spatiotemporally distinct waves. Adult repopulating hematopoietic stem cells (HSCs) are generated during the final definitive wave of hematogenesis. HSC production begins at E10.5 in the aorta-gonads-mesonephros (AGM) region and is tightly controlled by a combination of transcription and extrinsic factors. The Gata2 transcription factor has a pivotal role in hematopoiesis. Germline Gata2 deletion results in severe hematopoietic defects and embryonic lethality at E10.5 (Tsai, 1994). Conditional knockouts reveal that Gata2 is required for HSC generation in the embryo and HSC survival in the adult (de Pater, 2013; Gao, 2013). We developed a Gata2-Venus reporter knockin mouse model in which Gata2 expressing cells can be prospectively isolated and examined for hematopoietic function, and found that all HSCs in vivo are Gata2 expressing. To study the role of Gata2 in the early stages of HSC development, we use the novel Gata2-Venus ES cell (ESC) line for embryoid body (EB) differentiation. EB cultures are known to recapitulate early hematopoietic development. Our analysis of Gata2 expression dynamics shows that ESC hematopoietic differentiation occurs in several waves, producing progeny with stage-specific potential. These waves are similar to those observed in vivo. The first wave of Gata2 expressing EBs recapitulates primitive hematopoiesis, whereas gene expression analysis and hematopoietic progenitor assays suggest that cells initiating Gata2 expression later serve as precursors for definitive multipotent progenitors. To initiate further waves/stages of hematogenesis we have developed a novel chimeric ESC differentiation model where AGM derived stromal cells are exploited to create a hematopoiesis-supportive niche within the EBs. This results in a significant increase in Gata2 expressing cells and is accompanied by the generation of multipotent hematopoietic progenitor cells. Thus, by tracking Gata2 expression we have established a culture system that enhances definitive hematopoietic progenitor production and may allow for HSC generation.

Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age.

Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.