Forward genetics identifies Kdf1/1810019J16Rik as an essential regulator of the proliferation–differentiation decision in epidermal progenitor cells

Cell fate decisions during embryogenesis and adult life govern tissue formation, homeostasis and repair. Two key decisions that must be tightly coordinated are proliferation and differentiation. Overproliferation can lead to hyperplasia or tumor formation while premature differentiation can result in a depletion of proliferating cells and organ failure. Maintaining this balance is especially important in tissues that undergo rapid turnover like skin however, despite recent advances, the genetic mechanisms that balance cell differentiation and proliferation are still unclear. In an unbiased genetic screen to identify genes affecting early development, we identified an essential regulator of the proliferation–differentiation balance in epidermal progenitor cells, the Keratinocyte differentiation factor 1 (Kdf1; 1810019J16Rik) gene. Kdf1 is expressed in epidermal cells from early stages of epidermis formation through adulthood. Specifically, Kdf1 is expressed both in epidermal progenitor cells where it acts to curb the rate of proliferation as well as in their progeny where it is required to block proliferation and promote differentiation. Consequently, Kdf1 mutants display both uncontrolled cell proliferation in the epidermis and failure to develop terminal fates. Our findings reveal a dual role for the novel gene Kdf1 both as a repressive signal for progenitor cell proliferation through its inhibition of p63 and a strong inductive signal for terminal differentiation through its interaction with the cell cycle regulator Stratifin.     

The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling

Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2’-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.     

N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.     

Temporary characteristics of the process of stem cell inactivation by allogenic lymphocytes]

The time during which transplated lymphocytes block proliferation and differentiation of non-syngeic stem cells has been determined by retrasplantation of immuno-competent cells from one lethally irradiated recipient to another one. It was established that process of inactivation of CFU by allogeneic lymphocytes proceeds itwo stages. At the first stage, the colonization of recipient's tissues takes place. The colonization of tissues and processes of early recognition are completed during the first hours after transplantation of cell mixtures. At the second stage, the processes of redistribution of injected cells occur and a complete inactivation of stem cells take place. These events are completed in bone marrow and spleen 4-5 days after transplantation of cells mixture, possibly with the participation of lymphocytes sensibilized with the target-cells.     

Patterns of Cell Division,Cell Differentiation and Cell Elongation in Epidermis and Cortex of Arabidopsis pedicels in the Wild Type and in erecta

Plant organ shape and size are established during growth by a predictable, controlled sequence of cell proliferation, differentiation, and elongation. To understand the regulation and coordination of these processes, we studied the temporal behavior of epidermal and cortex cells in Arabidopsis pedicels and used computational modeling to analyze cell behavior in tissues. Pedicels offer multiple advantages for such a study, as their growth is determinate, mostly one dimensional, and epidermis differentiation is uniform along the proximodistal axis. Three developmental stages were distinguished during pedicel growth: a proliferative stage, a stomata differentiation stage, and a cell elongation stage. Throughout the first two stages pedicel growth is exponential, while during the final stage growth becomes linear and depends on flower fertilization. During the first stage, the average cell cycle duration in the cortex and during symmetric divisions of epidermal cells was constant and cells divided at a fairly specific size. We also examined the mutant of ERECTA, a gene with strong influence on pedicel growth. We demonstrate that during the first two stages of pedicel development ERECTA is important for the rate of cell growth along the proximodistal axis and for cell cycle duration in epidermis and cortex. The second function of ERECTA is to prolong the proliferative phase and inhibit premature cell differentiation in the epidermis. Comparison of epidermis development in the wild type and erecta suggests that differentiation is a synchronized event in which the stomata differentiation and the transition of pavement cells from proliferation to expansion are intimately connected.     

The incidence of hematologic tumours: A cellular model for the age dependence

Most hematologic tumors (acute and chronic myelogenous leukemia, chronic lymphatic leukemia, polycythemia vera, and multiple myeloma) exhibit an exponential increase in incidence with advancing age of the host. This age-incidence pattern resembles that for carcinomas and can be explained by the accumulation of harmful mutations in the stem cells of the tissues of tumor origin during the course of normal aging. The age-incidence pattern for acute lymphatic leukemia is unique and complex with a linear increase in incidence from birth to age 3, an exponential decline in incidence form age 3 to 34, a low and constant rate of incidence from age 34 to 60, and a slight increase in incidence at ages greater than 60. We conclude that variation in tumor incidence with host age is determined by the pattern of cell proliferation in the tissue of tumor origin. We suggest that cell proliferation in the tissue of origin of acute lymphatic leukemia occurs in stages: (1) rapid stem cell proliferation from before birth to age 3, (2) random stem cell differentiation from age 3 to 34, and (3) a constant rate of stem cell proliferation and differentiation after age 34.     

Kit ligand actions on ovarian stromal cells: effects on theca cell recruitment and steroid production

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.     

Cell cycling and cell enlargement in developing leaves of Arabidopsis.

Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level.     

APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem

Postembryonic organ formation in higher plants relies on the activity of stem cell niches in shoot and root meristems where differentiation of the resident cells is repressed by signals from surrounding cells. We searched for mutations affecting stem cell maintenance and isolated the semidominant l28 mutant, which displays premature termination of the shoot meristem and differentiation of the stem cells. Allele competition experiments suggest that l28 is a dominant-negative allele of the APETALA2 (AP2) gene, which previously has been implicated in floral patterning and seed development. Expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) genes, which regulate stem cell maintenance in the wild type, were disrupted in l28 shoot apices from early stages on. Unlike in floral patterning, AP2 mRNA is active in the center of the shoot meristem and acts via a mechanism independent of AGAMOUS, which is a repressor of WUS and stem cell maintenance in the floral meristem. Genetic analysis shows that termination of the primary shoot meristem in l28 mutants requires an active CLV signaling pathway, indicating that AP2 functions in stem cell maintenance by modifying the WUS-CLV3 feedback loop.     

MAPK signaling during Müller glial cell development in retina explant cultures

The Müller cell is the only glial cell type generated from the retinal neuroepithelium. This cell type controls normal retina homeostasis and has been suggested to play a neuroprotective role. Recent evidence suggests that mammalian Müller cells can de-differentiate and return to a progenitor or stem cell stage following injury or disease. In vivo exploration of the molecular mechanisms of Müller cell differentiation and proliferation will add essential information to manipulate Müller cell functions. Signal transduction pathways that regulate Müller cell responses and activity are a critical part of their cellular machinery. In this study, we focus on mitogen-activated protein kinase (MAPK) signaling pathway during Müller glial cell differentiation and proliferation. We found that both MAPK and STAT3 signaling pathways are present during Müller glial cell development. Ciliary neurotrophic factor (CNTF)-stimulated Müller glial cell proliferation is associated with early developmental stages. Specific inhibition of MAPK phosphorylation significantly reduced the number of Müller glial cells with or without CNTF stimulation. These results suggested that the MAPK signal transduction pathway is important in the formation of Müller glial cells during retina development.     

Copine1 regulates neural stem cell functions during brain development

Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development.     

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.     

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.