An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors

Rodent incisors grow throughout adult life, but are prevented from becoming excessively long by constant abrasion, which is facilitated by the absence of enamel on one side of the incisor. Here we report that loss-of-function of sprouty genes, which encode antagonists of receptor tyrosine kinase signaling, leads to bilateral enamel deposition, thus impeding incisor abrasion and resulting in unchecked tooth elongation. We demonstrate that sprouty genes function to ensure that enamel-producing ameloblasts are generated on only one side of the tooth by inhibiting the formation of ectopic ameloblasts from self-renewing stem cells, and that they do so by preventing the establishment of an epithelial-mesenchymal FGF signaling loop. Interestingly, although inactivation of Spry4 alone initiates ectopic ameloblast formation in the embryo, the dosage of another sprouty gene must also be reduced to sustain it after birth. These data reveal that the generation of differentiated progeny from a particular stem cell population can be differently regulated in the embryo and adult.     

IL-17 and FGF signaling involved in mouse mesenchymal stem cell proliferation

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin 17 (IL-17) and basic fibroblast growth factor (bFGF), on BM-MSCs growth and elucidated the signaling pathways involved. The results revealed that IL-17 increased the frequency of colony-forming units fibroblast (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-17 and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-17 and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.     

Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon

Multipotent, self-renewing neural stem cells reside in the embryonic mouse telencephalic germinal zone. Using an in vitro neurosphere assay for neural stem cell proliferation, we demonstrate that FGF-responsive neural stem cells are present as early as E8.5 in the anterior neural plate, but EGF-responsive neural stem cells emerge later in development in a temporally and spatially specific manner. By separately blocking EGF and FGF2 signaling, we also show that EGF alone and FGF2 alone can independently elicit neural stem cell proliferation and at relatively high cell densities separate cell nonautonomous effects can substantially enhance the mitogen-induced proliferation. At lower cell densities, neural stem cell proliferation is additive in the presence of EGF and FGF2 combined, revealing two different stem cell populations. However, both FGF-responsive and EGF-responsive neural stem cells retain their self-renewal and multilineage potential, regardless of growth factor conditions. These results support a model in which separate, lineage-related EGF- and FGF-responsive neural stem cells are present in the embryonic telencephalic germinal zone.     

FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development

FGF10 plays an important role in the morphogenesis of several tissues by control of mesenchymal-to-epithelial signaling. In the pancreas, mesenchymal FGF10 is required to maintain the Pdx1-expressing epithelial progenitor cell population, and in the absence of FGF10 signaling, these cells fail to proliferate. Ectopic expression of FGF10 in the pancreatic epithelium caused increased proliferation of pancreatic progenitor cells and abrogation of pancreatic cell differentiation of all cell types. A hyperplastic pancreas consisting of undifferentiated cells expressing Pdx1, Nkx6.1, and cell adhesion markers normally characterizing early pancreatic progenitor cells resulted. Differentiation was attenuated even as proliferation of the pancreatic cells slowed during late gestation, suggesting that the trophic effect of FGF10 was independent of its effects upon cell differentiation. The FGF10-positive pancreatic cells expressed Notch1 and Notch2, the Notch-ligand genes Jagged1 and Jagged2, as well as the Notch target gene Hes1. This activation of Notch is distinct from the previously recognized mechanism of lateral inhibition. These data suggest that FGF10 signaling serves to integrate cell growth and terminal differentiation at the level of Notch activation, revealing a novel second role of this key signaling system during pancreatic development.     

Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

Fibroblast growth factor 10 (FGF10) is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multipotent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothelial markers, uroplakin II, III, and cytokeratin 8, were monitored by RT-PCR, immunocytochemistry, and Western blot analysis. Co-cultured stem cells began to express uroplakin II, III, and cytokeratin 8. Targeted FGF10 gene knockdown from bladder cancer cells abolished the directed differentiation. In addition, when FGF10 downstream signaling was blocked with the Mek inhibitor, the co-culture system lost the capacity to induce urothelial differentiation. Exogenous addition of recombinant FGF10 protein promoted stem cell differentiation into urothelium cell lineage. Together, this report suggests that paracrine FGF10 signaling stimulates the differentiation of human stem cell into urothelial cells. Current study provides insight into the potential role of FGF10 as a lead growth factor for bladder regeneration and its therapeutic application for bladder transplantation.     

FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development

FGF receptor 2 isoform IIIb (FGFR2b), originally discovered as a receptor for FGF7, is known to be an important receptor in vertebrate morphogenesis, because FGFR2b null mice exhibit agenesis or dysgenesis of various organs, which undergo budding and branching morphogenesis. Since FGF7 null mice do not exhibit marked defects in organogenesis, it has been considered that other FGF(s) than FGF7 might function as a major ligand for FGFR2b during organogenesis. One of the candidate ligands is FGF10, because FGF10 binds to FGFR2b with high affinity and the formation of the limb and lung is arrested in FGF10 null mice as found in FGFR2b-deficient mice. Previous analyses of FGF10 null mice revealed that FGF10 is required for limb and lung development. To elucidate the role of FGF10 in wide-range organogenesis, we further analyzed the phenotypes of the FGF10 knockout mice. We found diverse phenotypes closely related to those for FGFR2b-deficient mice, which includes the absence of thyroid, pituitary, and salivary glands, while minor defects were observed in the formation of teeth, kidneys, hair follicles, and digestive organs. These results suggest that FGF10 acts as a major ligand for FGFR2b in mouse multi-organ development.     

FGF10 maintains distal lung bud epithelium and excessive signaling leads to progenitor state arrest,distalization, and goblet cell metaplasia

Background  

Interaction with the surrounding mesenchyme is necessary for development of endodermal organs, and Fibroblast growth factors have recently emerged as mesenchymal-expressed morphogens that direct endodermal morphogenesis. The fibroblast growth factor 10 (Fgf10) null mouse is characterized by the absence of lung bud development. Previous studies have shown that this requirement for Fgf10 is due in part to its role as a chemotactic factor during branching morphogenesis. In other endodermal organs Fgf10 also plays a role in regulating differentiation.     

myc maintains embryonic stem cell pluripotency and self-renewal

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.     

Residual radiation effect in the murine hematopoietic stem cell compartment

Stem cells surviving radiation injury may carry defects which contribute to long-term effects. The ratio of 125-iododeoxyuridine (IUdR) uptake into spleens of lethally irradiated recipient mice between day 3 and day 5 after cell transfusion revealed reduced proliferative ability (PF) of spleen seeding cells in parallel with reduced CFU-S content of donors throughout the study period of one year after 5 Gy gamma irradiation. Additional data aided in evaluating possible mechanisms of PF reduction. Within the range of the graft sizes used, PF was independent of the numbers of cells or CFU-S transfused. Radiation-induced increase in loss of label between days 3 and 5 and prolonged doubling time of proliferating cells indicated enhancement of cell maturation and increase in mitotic cycle time. Increased IUdR uptake per transfused CFU-S suggested extra divisions of transit cells due to insufficiency in the stem cell compartment. It is concluded that persisting defects in surviving stem cells interfere in a complex way with cell proliferation in the hemopoietic system.     

The null cell compartment of the mouse spleen

Null lymphocytes were defined as lymphocytes without detectable T- or B-cell markers using a battery of techniques. The null cell compartment was divided into pre-T cells, pre-B cells, and other null cells based upon their acquisition of membrane markers when incubated with ubiquitin. The null cell subpopulations were remarkably consistent in spleen cell suspensions from young adult mice of various strains. Commitment to T- or B-cell differentiation took place at the null cell stage and did not require thymic input. Pre-T cells, but not pre-B cells, were steroid sensitive. Pre-T cells accumulate with congenital thymic deficiency. This differed from senescent thymodeprivation where the outstanding finding was an accumulation of uninducible null cells. Neonatal mouse spleens were deficient in pre-T and pre-B cells but had an accumulation of uninducible cells.     

Reporting live from the epidermal stem cell compartment!

Hair follicle regeneration is controlled by an intricate relationship between epidermal stem cells and their microenvironment. A recent report in Nature by Rompolas et?al. (2012) uses two-photon live imaging to interrogate the spatial organization and cellular requirements for hair follicle regeneration by epidermal stem cells and their immediate progeny.     

The roles of FGF signaling in germ cell migration in the mouse

Fibroblast growth factor (FGF) signaling is thought to play a role in germ cell behavior. FGF2 has been reported to be a mitogen for primordial germ cells in vitro, whilst combinations of FGF2, steel factor and LIF cause cultured germ cells to transform into permanent lines of pluripotent cells resembling ES cells. However, the actual function of FGF signaling on the migrating germ cells in vivo is unknown. We show, by RT-PCR analysis of cDNA from purified E10.5 germ cells, that germ cells express two FGF receptors: Fgfr1-IIIc and Fgfr2-IIIb. Second, we show that FGF-mediated activation of the MAP kinase pathway occurs in germ cells during their migration, and thus they are potentially direct targets of FGF signaling. Third, we use cultured embryo slices in simple gain-of-function experiments, using FGF ligands, to show that FGF2, a ligand for FGFR1-IIIc, affects motility, whereas FGF7, a ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-of-function allele of FGFR2-IIIb. In FGFR2-IIIb(-/-) embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.     

FGF signaling in the developing endochondral skeleton

Mutations in fibroblast growth factor receptors (Fgfrs) are the etiology of many craniosynostosis and chondrodysplasia syndromes in humans. The phenotypes associated with these human syndromes and the phenotypes resulting from targeted mutagenesis in the mouse have defined essential roles for FGF signaling in both endochondral and intramembranous bone development. In this review, I will focus on the role of FGF signaling in chondrocytes and osteoblasts and how FGFs regulate the growth and development of endochondral bone.     

Granule cell raphes in the developing mouse cerebellum

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.     

Heterogeneity within the hematopoietic stem cell compartment: evidence for a marrow-seeding stem cell distinct from CFU-s

Using a chromosome marker within a syngeneic system, we investigated the seeding characteristics of murine hematopoietic stem cells after transplantation to irradiated hosts. The chromosome-marked test cells were allowed to compete with normal marrow cells in repopulating the spleen and marrow of irradiated mice. Although the seeding behavior of normal marrow could be predicted from the number of colony-forming units-spleen (CFU-s) transplanted, the marrow seeding of melphalan-treated marrow was 7-fold greater than expected. Repopulation of marrow by spleen cells was less effective than expected from the CFU-s content, while the reverse was true after repopulation by fetal liver cells. These differences were emphasized after treatment of cell donors with melphalan. The results were due primarily to differences in the lodging properties of the transplanted cells, those seeding in the marrow were less sensitive to melphalan than CFU-s. In some instances marrow-repopulating ability could be separated from peak CFU-s activity on a density gradient, suggesting a marrow-repopulating cell exists that is distinct from CFU-s.     

The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype

Glioblastomas are highly lethal cancers that contain cellular hierarchies with self-renewing cancer stem cells that can propagate tumors in secondary transplant assays. The potential significance of cancer stem cells in cancer biology has been demonstrated by studies showing contributions to therapeutic resistance, angiogenesis, and tumor dispersal. We recently reported that physiologic oxygen levels differentially induce hypoxia inducible factor-2α (HIF2α) levels in cancer stem cells. HIF1α functioned in proliferation and survival of all cancer cells but also was activated in normal neural progenitors suggesting a potentially restricted therapeutic index while HIF2α was essential in only in cancer stem cells and was not expressed by normal neural progenitors demonstrating HIF2α is a cancer stem cell specific target. We now extend these studies to examine the role of hypoxia in regulating tumor cell plasticity. We find that hypoxia promotes the self-renewal capability of the stem and non-stem population as well as promoting a more stem-like phenotype in the non-stem population with increased neurosphere formation as well as upregulation of important stem cell factors, such as OCT4, NANOG, and c-MYC. The importance of HIF2α was further supported as forced expression of non-degradable HIF2α induced a cancer stem cell marker and augmented the tumorigenic potential of the non-stem population. This novel finding may indicate a specific role of HIF2α in promoting glioma tumorigenesis. The unexpected plasticity of the non-stem glioma population and the stem-like phenotype emphasizes the importance of developing therapeutic strategies targeting the microenvironmental influence on the tumor in addition to cancer stem cells.