Functions In Adult Tissues Research Articles

The DNA methyltransferase DMAP1 is required for tissue maintenance and planarian regeneration

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's function in adult tissues.

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy.

Dormancy is a key feature of stem cell function in adult tissues as well as in embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for preimplantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in the loss of pluripotent cells. Through the integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network, and the perturbation of the network compromises embryo survival in diapause. We further identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic MTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.

FRI291 Musashi1 And Musashi2 Mark Distinct Pituitary Stem Cell Populations

Abstract Disclosure: Y. Rahmatallah: None. J. Banik: None. K. Bronson: None. M.C. MacNicol: None. The cell plastic state of the pituitary is required to meet the changing demands upon pituitary function and may also result in these cells being more vulnerable to malignant transformation. The mechanisms that control pituitary cell plasticity are unknown. The RNA binding protein Musashi (MSI) is expressed in embryonic stem cells, and in cancer stem cells, where MSI functions to promote stem cell plasticity and to oppose premature cell maturation. MSI is also expressed at high levels in adult gonad and adult pituitary. The function of MSI in adult tissues is not fully understood, and the mechanisms that control the pro-proliferative and pro-malignant aspects of MSI function in adult tissues are not known. The two evolutionarily conserved MSI proteins, Musashi1 (MSI1) and Musashi2 (MSI2), are predicted to function redundantly, however, regulatory distinctions have been reported for MSI1 and MSI2. Towards a characterization of MSI1 and MSI2 in pituitary, we analyzed transcriptomics data from public datasets of single-nucleus and single-cell and bulk-tissue gene expression data from physiological and pathophysiological human pituitary tissue. We performed bioinformatics analysis utilizing the single-cell genomics analysis tool, Seurat in R and the pseudotime trajectory analysis tool Monocle and additional genomics and statistics analyses tools in R. We identify the MSI1-expressing pituitary cell population as immature stem cells and the distinct MSI2-expressing pituitary cell population as more mature, committed progenitor cells. Pituitary from human fetal tissue contains multiple distinct MSI1-expressing cell populations while, in contrast, pituitary from human adult tissue contains multiple distinct MSI2-expressing cell populations. Pituitary from human pediatric tissue demonstrates an intermediate state with both distinct MSI1-expressing and distinct MSI2-expressing cell populations. We identify increased MSI1 expression but not increased MSI2 expression in adult pituitary adenoma, and a high level of variation in the MSI1 to MSI2 expression ratio between distinct pituitary adenomas. Based upon this re-analysis of reposited datasets from other pituitary investigators, we hypothesize that the increase that we observe, in MSI1 to MSI2 expression ratio in adult pituitary tumors is consistent with a reversion of mature cells to an earlier immature state. Adult pituitary may contain the mechanisms that regulate MSI2 function but may not be equipped to properly control high levels of re-expressed MSI1. An understanding of the physiological functions and mechanisms of regulation of the distinct MSI proteins in pituitary may provide insight into potential mechanisms that could be recruited to increase physiological pituitary cell growth and differentiation in regenerative therapy and to oppose pathological cell growth in pituitary tumor. Presentation: Friday, June 16, 2023

Microphysiological Neurovascular Barriers to Model the Inner Retinal Microvasculature.

Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood–brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular tissues and iBRB modeling. Recent BBB in vitro models using human cells of the neurovascular unit (NVU) showed physiological morphology and permeability values, and reproduced brain neurological disorder phenotypes that could be applicable to modeling the iBRB. Here, we describe similarities between iBRB and BBB properties, compare existing neurovascular barrier models, propose leverage of MPS-based strategies to develop new iBRB models, and explore potentials to personalize cellular inputs and improve pre-clinical testing.

Abstract 17346: Loss of Neuropilin 2 Impairs Lymphangiogenesis and Exacerbates Lymphedema

Dysfunctional lymphatic networks can lead to lymphatic diseases such as lymphedema as well as aggravate cardiovascular diseases such as atherosclerosis and hypertension. Some primary lymphedema patients have mutations in the Neuropilin 2 ( NRP2 ) gene, which encodes a transmembrane receptor that acts with VEGFR3 for the ligands VEGF-C and VEGF-D. Nrp2 has been shown to be a key regulator of lymphatic vessel development in the neonate as global Nrp2 -deficient mice suffer from reduced lymphatic density. However, Nrp2 protein expression is dramatically downregulated after birth and its role in adult lymphangiogenesis or its relevance in lymphedema is less clear. Our hypothesis is that Nrp2 expression is necessary for optimal lymphangiogenesis and proper lymphatic drainage function in the adult, and that loss of Nrp2 , either genetically or transcriptionally via down-regulation by specific transcription factors, may worsen lymphedema. In acute and chronic mouse inflammation models, we found that loss of Nrp2 specifically in the lymphatic endothelium ( prox1-cre ERT2 ;Nrp2 f/f ) resulted in prolonged swelling and reduced lymphatic drainage compared to control mice. Using the VEGFC-induced corneal lymphangiogenesis model and the tail wound-induced secondary lymphedema model, LEC-Nrp2-iKO mice showed diminished lymphangiogenesis and reduced lymphatic drainage compared to littermate controls. Furthermore, primary mouse dermal LEC isolated from Nrp2-iKO mice reveal suppressed VEGFR3 activation, signaling, and proliferation following VEGF-C stimulation, compared to LEC isolated from control mice. Collectively, our results identify Nrp2 as a critical mediator of lymphangiogenesis and homeostatic lymphatic drainage function in adult tissues.

Abstract A43: Identification of novel YAP/TAZ regulators in metastatic cancer

Abstract Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the downstream effectors of the Hippo tumor inhibition pathway. Studies from us and others show that inappropriate activation of YAP and/or TAZ promotes tumor growth and metastasis. Increased YAP and/or TAZ expression and nuclear localization is observed in many human cancers and often predict poor prognosis. Mutations in Hippo-YAP/TAZ pathway exist but are not common enough to explain the frequently observed increase of YAP/TAZ expression and activity in cancer. Therefore, we hypothesize that other cancer-associated pathways cause aberrant YAP/TAZ activation in cancer cells, and identifying such pathways would provide targets for treatment of YAP/TAZ-driven cancers. Furthermore, transcriptional cofactors are often difficult to target and YAP/TAZ have important functions in adult tissues. Therefore, targeting YAP/TAZ regulators may provide a nonsystemic path of targeting YAP/TAZ that could avoid severe adverse effects. To identify novel YAP/TAZ regulators in metastatic cancer cells, we screened a lentiviral shRNA library that targets 124 cancer-associated genes in A375 MA2 (MA2) cells using a YAP/TAZ-TEAD (TEA domain family members) transcriptional reporter assay. We found that 62 genes are able to regulate YAP/TAZ activity in MA2, 24 of which are already established YAP/TAZ regulators, which supports the effectiveness of our approach. The other 38 genes have not been reported previously, which are potential novel YAP/TAZ regulators. We subsequently validated these candidate YAP/TAZ regulators by examining the expression of the YAP/TAZ target gene CTGF by qPCR and phosphorylation of YAP and TAZ by Western blot in MA2 and other metastatic cell lines. We are currently pursuing several of these novel YAP/TAZ regulators to determine the mechanism through which they regulate YAP/TAZ activity and test their role in metastasis. Our results suggest that YAP/TAZ regulators are cell type and context specific and may also functionally compensate for each other. Therefore, it is critical to identify a “core” group of YAP/TAZ regulators that are important in multiple cancer types. This could provide new strategies to more effectively inhibit YAP/TAZ activation and treat metastatic cancer. Citation Format: Yuxuan Xiao, Emily Norton, Janine Warren, John Lamar. Identification of novel YAP/TAZ regulators in metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A43.

Loss of Neuropilin 2 in Adult Lymphatic Endothelium Promotes Lymphedema

The lymphatic system is essential in mediating tissue fluid balance. Lymphatic diseases including lymphedema occur as a result of dysfunctional lymphatic networks and are a significant public health issue with no known cure. Our long‐term goal is to understand the cellular and molecular processes that control lymphatic function in order to better treat lymphatic diseases. Some primary lymphedema patients have mutations in the neuropilin‐2 gene (NRP2). NRP2 is a transmembrane receptor found in lymphatic endothelial cells (LEC) that is unique in its ability to transmit both pro‐lymphangiogenic signals from VEGF‐C/D ligands as well as suppressive signals from Semaphorin‐3F (SEMA3F). Previously, we observed elevated tissue swelling in constitutive Nrp2‐deficient mice compared to wild‐type littermates following acute inflammatory reactions. However, it was unclear whether tissue swelling was due to increased vascular leakage (edema) or decreased lymphatic drainage (lymphedema) or both. We hypothesize that Nrp2 regulates lymphatic drainage function in adult tissues and that loss of Nrp2 in adult lymphatic vessels may aggravate lymphedema and swelling. In order to more carefully dissect the molecular signaling events in adult LEC that may contribute to lymphedema, we created inducible, LEC‐specific Nrp2 knockout mice (prox1‐creERT2;Nrp2f/f) and examined lymphangiogenesis and lymphedema models after tamoxifen treatment in adult mice. Compared to appropriate littermate controls, mice lacking Nrp2 in LEC are poorly lymphangiogenic in VEGFC‐induced corneal lymphangiogenesis models and display massive swelling due to increased interstitial fluid and reduced lymphatic drainage in acute and chronic delayed‐type hypersensitivity models and secondary lymphedema models in vivo. Further, culture of primary dermal mouse LEC (mLEC) isolated from Prox1‐iNrp2‐KO mice following tamoxifen treatment reveal suppressed VEGFR3 activation compared to mLEC isolated from control mice. Taken together, our new data indicate that Nrp2 is a critical mediator of homeostatic lymphatic function and overall fluid balance in adult tissues. Our data suggests that the Nrp2 axis may be a pivotal pathway to stimulate lymphangiogenesis and lymphatic drainage in lymphedema conditions resulting from a paucity of lymphatic vessels.Support or Funding InformationR01 HL141858, R01 HL133216, R01 HL137229

TTBK2 and primary cilia are essential for the connectivity and survival of cerebellar Purkinje neurons.

Primary cilia are vital signaling organelles that extend from most types of cells, including neurons and glia. These structures are essential for development of many tissues and organs; however, their function in adult tissues, particularly neurons in the brain, remains largely unknown. Tau tubulin kinase 2 (TTBK2) is a critical regulator of ciliogenesis, and is also mutated in a hereditary neurodegenerative disorder, spinocerebellar ataxia type 11 (SCA11). Here, we show that conditional knockout of Ttbk2 in adult mice results in degenerative cerebellar phenotypes that recapitulate aspects of SCA11 including motor coordination deficits and defects to Purkinje cell (PC) integrity. We also find that the Ttbk2 conditional mutant mice quickly lose cilia throughout the brain. We show that conditional knockout of the key ciliary trafficking gene Ift88 in adult mice results in nearly identical cerebellar phenotypes to those of the Ttbk2 knockout, indicating that disruption of ciliary signaling is a key driver of these phenotypes. Our data suggest that primary cilia play an integral role in maintaining the function of PCs in the adult cerebellum and reveal novel insights into mechanisms involved in neurodegeneration.

SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis

When properly employed, targeted therapies are effective cancer treatments. However, the development of such therapies requires the identification of targetable drivers of cancer development and metastasis. The expression and nuclear localization of the transcriptional coactivators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are increased in many human cancers, and experimental evidence indicates that aberrant YAP or TAZ activation drives tumor formation and metastasis. Although these findings make YAP and TAZ appealing therapeutic targets, both have important functions in adult tissues, so directly targeting them could cause adverse effects. The identification of pathways active in cancer cells and required for YAP/TAZ activity could provide a way to inhibit YAP and TAZ. Here, we show that SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) is an important driver of YAP/TAZ activity in human breast cancer and melanoma cells. SRC activation increased YAP/TAZ activity and the expression of YAP/TAZ-regulated genes. In contrast, SRC inhibition or knockdown repressed both YAP/TAZ activity and the expression of YAP/TAZ-regulated genes. We also show that SRC increases the activity of YAP and TAZ by repressing large tumor suppressor homolog (LATS), and we identify the GTPase-activating protein GIT ArfGAP 1 (GIT1) as an SRC effector that regulates both YAP and TAZ. Importantly, we demonstrate that SRC-mediated YAP/TAZ activity promotes tumor growth and enhances metastasis and that SRC-dependent tumor progression depends, at least in part, on YAP and TAZ. Our findings suggest that therapies targeting SRC could help manage some YAP/TAZ-dependent cancers.

TET1 Alternative Isoform Regulates Oscillatory Shear Stress Induced Endothelial Dysfunction

Oscillatory shear stress (OSS) is one of the major risk factors related to endothelial (EC) dysfunction, which contributes to atherosclerosis. Our previous study indicated that inhibitor of DNA binding1 (Id1) play vital role in the regulation of OSS mediated EC function related to atherosclerosis. However, the initiation mechanism during this process remains to be elucidated. Ten-eleven Translocation protein 1 alternative isoform (Tet1s) is a newly reported protein that may have function in adult tissue. Here, we investigate the role of Tet1s in regulating OSS mediated endothelial dysfunction and its underlying mechanism. First, physical interaction between Tet1s and Id1 was found and proved by immunoprecipitation. By using carotid partial ligation mice model in vivo and OSS applied on human umbilical venous endothelial cell (HUVEC) in vitro, we found that EC proliferation rate and adhesion molecule expression were upregulated in the local area with OSS characteristics. Compared to the grater curvature (laminar shear stress), a lower Tet1s expression level in atheroprone lesser curvature (OSS) suggested Tet1s regulate the EC function under OSS. This notion is supported by the decline of Tet1s expression in cell culture model. In order to explore the Tet1s expression regulated mechanism, the potential binding sites in Tet1s promoter region for CEBPB was identified by in silico analysis. By using PKA/CEBPB inhibitor H89, we found that H89 inhibited Tet1s expression. HUVEC cell proliferation, proinflammation gene expression as well as monocytes adhesion were enhanced after knockdown of Tet1s by specific siRNA. And overexpression of Tet1s eliminated OSS induced HUVEC proliferation and inflammation. Further studies revealed Tet1s negatively regulated the expression of Id1. Meanwhile, knockdown of Tet1s induced nucleocytoplasmic shuttling of Id1. Our finding indicates a significant role of Tet1s in regulating OSS mediated endothelial dysfunction with respect of abnormal proliferation and inflammation though Id1-dependent pathway.

300 - Conditional deletion of thioredoxin 1 in the adult mouse causes multiple tissue failures and early death

Although thioredoxin 1 (Trx1) is ubiquitously expressed, necessary for development, and has modified activity during pathogenesis of numerous oxidative diseases, very little is known about the role of this oxidoreductase in maintenance of adult tissue function. To determine physiological functions of Trx1, we developed doxycycline-inducible Trx1 conditional knockout (KO) mice. Administration of doxycycline to adult mice resulted in death or moribund conditions requiring euthanasia in 13-25 days post treatment. By treatment day 10, KO mice lost 4.2±1.5% and control mice gained 5.2±1.9% of initial body weight (P

Organ-specific lymphatic vasculature: From development to pathophysiology.

Recent discoveries of novel functions and diverse origins of lymphatic vessels have drastically changed our view of lymphatic vasculature. Traditionally regarded as passive conduits for fluid and immune cells, lymphatic vessels now emerge as active, tissue-specific players in major physiological and pathophysiological processes. Lymphatic vessels show remarkable plasticity and heterogeneity, reflecting their functional specialization to control the tissue microenvironment. Moreover, alternative developmental origins of lymphatic endothelial cells in some organs may contribute to the diversity of their functions in adult tissues. This review aims to summarize the most recent findings of organotypic differentiation of lymphatic endothelial cells in terms of their distinct (patho)physiological functions in skin, lymph nodes, small intestine, brain, and eye. We discuss recent advances in our understanding of the heterogeneity of lymphatic vessels with respect to the organ-specific functional and molecular specialization of lymphatic endothelium, such as the hybrid blood-lymphatic identity of Schlemm's canal, functions of intestinal lymphatics in dietary fat uptake, and discovery of meningeal lymphatic vasculature and perivascular brain lymphatic endothelial cells.

Expression of teneurins is associated with tumor differentiation and patient survival in ovarian cancer.

Teneurins are a family of highly conserved pair-rule proteins involved in morphogenesis and development of the central nervous system. Their function in adult tissues and in disease is largely unknown. Recent evidence suggests a role for dysregulated expression of Teneurins in human tumors, but systematic investigations are missing. Here, we investigated Teneurin-2 and Teneurin-4 expression in various cancer cell lines and in ovarian tumor tissues. Teneurin-2 and Teneurin-4 were expressed in most of the breast cancer cell lines tested. Teneurin-4 was also detected in ovarian cancer cell lines, and throughout ovarian tumors and normal ovary tissue. Ovarian tumors with low Teneurin-4 expression showed less differentiated phenotypes and these patients had shorter mean overall survival. Similarly, Teneurin-2 expression correlated with overall survival as well, especially in patients with serous tumors. In the various cell lines, 5-Aza-cytidine-induced changes in DNA methylation did not alter expression of Teneurin-2 and Teneurin-4, despite the existence of predicted CpG islands in both genes. Interestingly, however, we found evidence for the control of Teneurin-2 expression by the oncogenic growth factor FGF8. Furthermore, we identified multiple transcript splicing variants for Teneurin-2 and Teneurin-4, indicating complex gene expression patterns in malignant cells. Finally, downregulation of Teneurin-4 expression using siRNA caused a cell-type dependent increase in proliferation and resistance to cisplatin. Altogether, our data suggest that low Teneurin-4 expression provides a growth advantage to cancer cells and marks an undifferentiated state characterized by increased drug resistance and clinical aggressiveness. We conclude that Teneurin-2 and Teneurin-4 expression levels could be of prognostic value in ovarian cancer.

A p57 conditional mutant allele that allows tracking of p57-expressing cells.

p57Kip2 (p57) is a maternally expressed imprinted gene regulating growth arrest which belongs to the CIP/KIP family of cyclin-dependent kinase inhibitors. While initially identified as a cell cycle arrest protein through inhibition of cyclin and cyclin-dependent kinase complexes, p57 activity has also been linked to differentiation, apoptosis, and senescence. In addition, p57 has recently been shown to be involved in tumorigenesis and cell fate decisions in stem cells. Yet, p57 function in adult tissues remains poorly characterized due to the perinatal lethality of p57 knock-out mice. To analyze p57 tissue-specific activity, we generated a conditional mouse line (p57FL-ILZ/+ ) by flanking the coding exons 2-3 by LoxP sites. To track p57-expressing or mutant cells, the p57FL-ILZ allele also contains an IRES-linked β-galactosidase reporter inserted in the 3' UTR of the gene. Here, we show that the β-galactosidase reporter expression pattern recapitulates p57 tissue specificity during development and in postnatal mice. Furthermore, we crossed the p57FL-ILZ/+ mice with PGK-Cre mice to generate p57cKO-ILZ/+ animals with ubiquitous loss of p57. p57cKO-ILZ/+ mice display developmental phenotypes analogous to previously described p57 knock-outs. Thus, p57FL-ILZ/+ is a new genetic tool allowing expression and functional conditional analyses of p57.

Quantitative proteomic analysis of age-related subventricular zone proteins associated with neurodegenerative disease.

Aging is characterized by a progressive decline in the function of adult tissues which can lead to neurodegenerative disorders. However, little is known about the correlation between protein changes in the subventricular zone (SVZ) and neurodegenerative diseases with age. In the present study, neural stem cells (NSCs) were derived from the SVZ on postnatal 7 d, 1 m, and 12 m-old mice. With age, NSCs exhibited increased SA-β-gal activity and decreased proliferation and pool size in the SVZ zone, and were associated with elevated inflammatory chemokines and cytokines. Furthermore, quantitative proteomics and ingenuity pathway analysis were used to evaluate the significant age-related alterations in proteins and their functions. Some downregulated proteins such as DPYSL2, TPI1, ALDH, and UCHL1 were found to play critical roles in the neurological disease and PSMA1, PSMA3, PSMC2, PSMD11, and UCHL1 in protein homeostasis. Taken together, we have provided valuable insight into the cellular and molecular processes that underlie aging-associated declines in SVZ neurogenesis for the early detection of differences in gene expression and the potential risk of neurological disease, which is beneficial in the prevention of the diseases.

ESE-1 in Early Development: Approaches for the Future.

E26 transformation-specific (Ets) family of transcription factors are characterized by the presence of Ets-DNA binding domain and have been found to be highly involved in hematopoiesis and various tissue differentiation. ESE-1, or Elf3 in mice, is a member of epithelium-specific Ets sub-family which is most prominently expressed in epithelial tissues such as the gut, mammary gland, and lung. The role of ESE-1 during embryogenesis had long been alluded from 30% fetal lethality in homozygous knockout mice and its high expression in preimplantation mouse embryos, but there has been no in-depth of analysis of ESE-1 function in early development. With improved proteomics, gene editing tools and increasing knowledge of ESE-1 function in adult tissues, we hereby propose future research directions for the study of ESE-1 in embryogenesis, including studying its regulation at the protein level and at the protein family level, as well as better defining the developmental phase under investigation. Understanding the role of ESE-1 in early development will provide new insights into its involvement in tissue regeneration and cancer, as well as how it functions with other Ets factors as a protein family.

Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles.

R-spondin proteins are novel Wnt/β-catenin agonists, which signal through their receptors leucine-rich repeat-containing G-protein coupled receptor (LGR) 4/5/6 and substantially enhance Wnt/β-catenin activity. R-spondins are reported to function in embryonic development. They also play important roles in stem cell functions in adult tissues, such as the intestine and mammary glands, which largely rely on Wnt/β-catenin signaling. However, in the skin epithelium and hair follicles, the information about R-spondins is deficient, although the expressions and functions of their receptors, LGR4/5/6, have already been studied in detail. In the present study, highly-enriched expression of the R-spondin family genes (Rspo1/2/3/4) in the hair follicle dermal papilla is revealed. Expression of Rspo1 in the dermal papilla is specifically and prominently upregulated before anagen entry, and exogenous recombinant R-spondin1 protein injection in mid-telogen leads to precocious anagen entry. Moreover, R-spondin1 activates Wnt/β-catenin signaling in cultured bulge stem cells in vitro, changing their fate determination without altering the cell proliferation. Our pioneering study uncovers a role of R-spondin1 in the activation of cultured hair follicle stem cells and the regulation of hair cycle progression, shedding new light on the governance of Wnt/β-catenin signaling in skin biology and providing helpful clues for future treatment of hair follicle disorders.

FBXL12 regulates T-cell differentiation in a cell-autonomous manner.

Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells including embryonic, adult tissue and cancer stem cells. The SCF(FBXL) (12) complex is an authentic ubiquitin ligase that targets ALDH3 for degradation. FBXL12 is essential for the differentiation of trophoblast stem cells into specific cell types in the placenta during mouse embryogenesis, but its physiological functions in adult tissues have remained unknown. We have now investigated the role of the FBXL12-ALDH3 axis in the thymus, in which FBXL12 was most abundant among adult mouse tissues examined. During T-cell differentiation, FBXL12 is most abundant in CD4(+) CD8(+) (DP) cells, with its expression declining as these cells differentiate into CD4(+) CD8(-) or CD4(-) CD8(+) (SP) cells. T cells of FBXL12-null mice manifested a differentiation block at the DP-SP transition that was associated with ALDH3 accumulation in DP cells. This differentiation block was also apparent in wild-type mouse recipients of FBXL12-null bone marrow transplants as well as in FBXL12-null fetal thymic organ culture, suggesting that it is a cell-autonomous phenomenon in the thymus rather than an indirect effect of altered systemic conditions. Our results thus indicate that, in addition to its role in placental development, the FBXL12-ALDH3 axis is required for maturation of undifferentiated thymocytes.

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.

Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila

Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis.