Stereocilia defects in the sensory hair cells of the inner ear in mice deficient in integrin alpha8beta1

The mammalian inner ear contains organs for the detection of sound and acceleration, the cochlea and the vestibule, respectively. Mechanosensory hair cells within the neuroepithelia of these organs transduce mechanical force generated by sound waves or head movements into neuronal signals. Defects in hair cells lead to deafness and balance defects. Hair cells have stereocilia that are indispensable for mechanosensation, but the molecular mechanisms regulating stereocilia formation are poorly understood. We show here that integrin alpha8beta1, its ligand fibronectin and the integrin-regulated focal adhesion kinase (FAK) co-localize to the apical hair-cell surface where stereocilia are forming. In mice homozygous for a targeted mutation of Itga8 (encoding the alphabeta8 subunit), this co-localization is perturbed and hair cells in the utricle, a vestibular subcompartment, lack stereocilia or contain malformed stereocilia. Most integrin alpha-8beta1-deficient mice die soon after birth due to kidney defects. Many of the survivors have difficulty balancing, consistent with the structural defects of the inner ear. Our data suggest that integrin alpha8beta1, and potentially other integrins, regulates hair-cell differentiation and stereocilia maturation. Mutations affecting matrix molecules cause inherited forms of inner ear disease and integrins may mediate some effects of matrix molecules in the ear; thus, mutations in integrin genes may lead to inner-ear diseases as well.     

Notch1 functions as a tumor suppressor in mouse skin

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.     

Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs

During embryogenesis, multipotent progenitors within the single-layered surface epithelium differentiate to form the epidermis and its appendages. Here, we show that microRNAs (miRNAs) have an essential role in orchestrating these events. We cloned more than 100 miRNAs from skin and show that epidermis and hair follicles differentially express discrete miRNA families. To explore the functional significance of this finding, we conditionally targeted Dicer1 gene ablation in embryonic skin progenitors. Within the first week after loss of miRNA expression, cell fate specification and differentiation were not markedly impaired, and in the interfollicular epidermis, apoptosis was not markedly increased. Notably, however, developing hair germs evaginate rather than invaginate, thereby perturbing the epidermal organization. Here we characterize miRNAs in skin, the existence of which was hitherto unappreciated, and demonstrate their differential expression and importance in the morphogenesis of epithelial tissues within this vital organ.     

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.     

PTEN-deficient intestinal stem cells initiate intestinal polyposis

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector beta-catenin. Akt phosphorylates beta-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of beta-catenin.     

Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch

The epidermis is a highly organized structure, the integrity of which is central to the protection of an organism. Development and subsequent maintenance of this tissue depends critically on the intricate balance between proliferation and differentiation of a resident stem cell population; however, the signals controlling the proliferation-differentiation switch in vivo remain elusive. Here, we show that mice carrying a homozygous missense mutation in interferon regulatory factor 6 (Irf6), the homolog of the gene mutated in the human congenital disorders Van der Woude syndrome and popliteal pterygium syndrome, have a hyperproliferative epidermis that fails to undergo terminal differentiation, resulting in soft tissue fusions. We further demonstrate that mice that are compound heterozygotes for mutations in Irf6 and the gene encoding the cell cycle regulator protein stratifin (Sfn; also known as 14-3-3sigma) show similar defects of keratinizing epithelia. Our results indicate that Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch and that Irf6 and Sfn interact genetically in this process.     

Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.     

p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation

Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38alpha mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38alpha positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38alpha controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38alpha leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-Ras(G12V)-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38alpha has a key role in the regulation of lung cell renewal and tumorigenesis.     

Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2-/- mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.     

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL-induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.     

Bmi1 is expressed in vivo in intestinal stem cells

Bmi1 plays an essential part in the self-renewal of hematopoietic and neural stem cells. To investigate its role in other adult stem cell populations, we generated a mouse expressing a tamoxifen-inducible Cre from the Bmi1 locus. We found that Bmi1 is expressed in discrete cells located near the bottom of crypts in the small intestine, predominantly four cells above the base of the crypt (+4 position). Over time, these cells proliferate, expand, self-renew and give rise to all the differentiated cell lineages of the small intestine epithelium. The induction of a stable form of beta-catenin in these cells was sufficient to rapidly generate adenomas. Moreover, ablation of Bmi1(+) cells using a Rosa26 conditional allele, expressing diphtheria toxin, led to crypt loss. These experiments identify Bmi1 as an intestinal stem cell marker in vivo. Unexpectedly, the distribution of Bmi1-expressing stem cells along the length of the small intestine suggested that mammals use more than one molecularly distinguishable adult stem cell subpopulation to maintain organ homeostasis.     

Lgr5 marks cycling, yet long-lived, hair follicle stem cells

In mouse hair follicles, a group of quiescent cells in the bulge is believed to have stem cell activity. Lgr5, a marker of intestinal stem cells, is expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles. Here we show that Lgr5(+) cells comprise an actively proliferating and multipotent stem cell population able to give rise to new hair follicles and maintain all cell lineages of the hair follicle over long periods of time. Lgr5(+) progeny repopulate other stem cell compartments in the hair follicle, supporting the existence of a stem or progenitor cell hierarchy. By marking Lgr5(+) cells during trafficking through the lower outer root sheath, we show that these cells retain stem cell properties and contribute to hair follicle growth during the next anagen. Expression analysis suggests involvement of autocrine Hedgehog signaling in maintaining the Lgr5(+) stem cell population.