TCF and the wingless pathway
The apical ectodermal ridge (AER) is an essential structure for vertebrate limb development. Wnt3a is expressed during the induction of the chick AER, and misexpression of Wnt3a induces ectopic expression of AER-specific genes in the limb ectoderm. The genes beta-catenin and Lef1 can mimic the effect of Wnt3a, and blocking the intrinsic Lef1 activity disrupts AER formation. Hence, Wnt3a functions in AER formation through the beta-catenin/LEF1 pathway. In contrast, neither beta-catenin nor Lef1 affects the Wnt7a-regulated dorsoventral polarity of the limb. Thus, two related Wnt genes elicit distinct responses in the same tissues by using different intracellular pathways (Kengaku, 1998).
Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with ß-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a was genetically examined. Analysis of the expression of Brachyury in Lef1-/-:Tcf1-/- mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicates that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. The expression of an activated form of LEF-1, containing the ß-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation (Galceran, 2001).
The Wnt/beta-catenin signaling pathway plays multiple roles during embryonic development, only a few of which have been extensively characterized. Although domains of Wnt expression have been identified throughout embryogenesis, anatomical and molecular characterization of responding cells has been mostly unexplored. A transgenic zebrafish line has been generated that expresses a destabilized green fluorescent protein (GFP) variant under the control of a beta-catenin responsive promoter. Early zygotic expression of this transgene (TOPdGFP) mirrors known domains of Wnt signaling in the embryo. Loss of Lef1 activity results in decreased reporter expression and posterior defects, while loss of Tcf3 (Headless, Hdl) activity does not alter reporter expression, even though it results in loss of forebrain structures. In addition, ectopic Wnt1 expression can activate the reporter. In older embryos, a number of transgene-expressing cell populations have been identified as novel sites of beta-catenin signaling. It is concluded that the TOP-dGFP reporter line faithfully illustrates domains of beta-catenin activity and enables the identification of responsive cell populations (Dorsky, 2002).
The Bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/ß-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Wnt signaling, in acting upstream of Pitx2, directly induces Pitx2 gene expression, based on the recruitment of LEF1 to evolutionary-conserved sites in the Pitx2 gene 5'-regulatory regions, with a regulated exchange of HDAC1 for ß-catenin occurring on these Pitx2 sites. Regulated exchange of HDAC1/ß-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction (Kioussi, 2002).
Activation of the Wnt pathway results in rapid recruitment of the Pitx2 gene and binding of Pitx2 to promoters of specific growth control genes. This linkage between the Wnt pathway and Pitx2 gene expression provides an insight into the molecular mechanisms of cell type-specific proliferation, based on the required actions of Pitx2 to activate specific, critical growth-control gene targets acting in G1. Based on in vivo studies, as well as the actions in pituitary and muscle cell models, Pitx2 is required for normal proliferation when expressed in heterologous cells; Pitx2 can actually inhibit proliferation. It is speculated that this may occur by squelching coregulatory factors required by DNA binding transcription factors that exert analogous functions to Pitx2. To subserve its proliferative effects, Pitx2 must bind to its cognate DNA sites and requires an N-terminal activation domain, but not the C terminus. Together, these data suggests that three independent events underlie Pitx2-dependent activation of cell type-specific proliferation: Wnt-dependent activation of Pitx2; Wnt and growth factor-dependent relief of Pitx2 repression function; and serial recruitment of a series of specific coactivator complexes that act in a promoter-specific manner, analogous to effects of β-catenin on LEF1 (Kioussi, 2002).
β-catenin-dependent or canonical Wnt signals are fundamental in animal development and tumor progression. Using Xenopus laevis, it is reported that the BTB/POZ zinc finger family member Kaiso directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1, and c-Myc) in conjunction with TCF/LEF (TCF). Analogous to β-catenin relief of TCF repressive activity, it is shown that p120-catenin relieves Kaiso-mediated repression of Siamois. Furthermore, Kaiso and TCF coassociate, and the combination of Kaiso and TCF derepression results in pronounced Siamois expression and increased β-catenin coprecipitation with the Siamois promoter. The functional interdependency is underlined by Kaiso suppression of β-catenin-induced axis duplication and by TCF-3 rescue of Kaiso depletion phenotypes. These studies point to convergence of parallel p120-catenin/Kaiso and β-catenin/TCF signaling pathways to regulate gene expression in vertebrate development and possibly carcinogenesis (Par, 2005).
Wnt signaling has been implicated in stem cell (SC) biology, but little is known about how stabilized ß-catenin functions within native SC niches. This was addressed by defining the impact of ß-catenin stabilization on maintenance, proliferation, and lineage commitment of multipotent follicle SCs when in their native niche and in culture. Gain of function mutations and inducible loss of function mutations were employed to demonstrate that ß-catenin stabilization is essential for promoting the transition between SC quiescence and conversion to proliferating transit amplifying (TA) progeny. Purified SCs isolated directly from wild-type and elevated ß-catenin follicles were transcriptionally profiled in both resting and activated states to uncover the discrete set of genes whose expression in native SCs is dependent upon ß-catenin stabilization. Finally, the underlying mechanism was addressed; in the SC niche, Wnt signaling and ß-catenin stabilization transiently activates Lef1/Tcf complexes and promote their binding to target genes that promote TA cell conversion and proliferation to form the activated cells of the newly developing hair follicle. These changes precede subsequent Wnt signals that impact on the TA progeny to specify the differentiation lineages of the follicle (Lowry, 2005).
Pontin and Reptin (See Drosophila Reptin) are nuclear beta-catenin interaction partners that antagonistically modulate beta-catenin transcriptional activity. Hint1/PKCI, a member of the evolutionary conserved family of histidine triad proteins, was characterised as a new interaction partner of Pontin and Reptin. Pull-down assays and co-immunoprecipitation experiments show that Hint1/PKCI directly binds to Pontin and Reptin. The Hint1/PKCI-binding site was mapped to amino acids 214-295 and 218-289 in Pontin and Reptin, respectively. Conversely, Pontin and Reptin bind to the N-terminus of Hint1/PKCI. Moreover, by its interaction with Pontin and Reptin, Hint1/PKCI is associated with the LEF-1/TCF-beta-catenin transcription complex. In this context, Hint1/PKCI acts as a negative regulator of TCF-beta-catenin transcriptional activity in Wnt-transfected cells and in SW480 colon carcinoma cells as shown in reporter gene assays. Consistent with these observations, Hint1/PKCI represses expression of the endogenous target genes cyclin D1 and axin2 whereas knockdown of Hint1/PKCI by RNA interference increases their expression. Disruption of the Pontin/Reptin complex appears to mediate this modulatory effect of Hint1/PKCI on TCF-beta-catenin-mediated transcription. These data now provide a molecular mechanism to explain the tumor suppressor function of Hint1/PKCI recently suggested from the analysis of Hint1/PKCI knockout mice (Weiske, 2005).
While the biological roles of canonical Wnt/beta-catenin signaling in development and disease are well documented, understanding the molecular logic underlying the functionally distinct nuclear transcriptional programs mediating the diverse functions of beta-catenin remains a major challenge. This study reports an unexpected strategy for beta-catenin-dependent regulation of cell-lineage determination based on interactions between beta-catenin and a specific homeodomain factor, Prop1, rather than Lef/Tcfs. beta-catenin acts as a binary switch to simultaneously activate expression of the critical lineage-determining transcription factor, Pit1, and to repress the gene encoding the lineage-inhibiting transcription factor, Hesx1, acting via TLE/Reptin/HDAC1 corepressor complexes. The strategy of functionally distinct actions of a homeodomain factor in response to Wnt signaling is suggested to be prototypic of a widely used mechanism for generating diverse cell types from pluripotent precursor cells in response to common signaling pathways during organogenesis (Olson, 2006).
Wnt signaling orchestrates multiple aspects of central nervous system development, including cell proliferation and cell fate choices. In this study, gene transfer was used to activate or inhibit canonical Wnt signaling in vivo in the developing eye. The expression of Wnt2b or constitutively active (CA) ß-catenin inhibited retinal progenitor gene (RPG) expression and the differentiation of retinal neurons. In addition, Wnt signal activation in the central retina is sufficient to induce the expression of markers of the ciliary body and iris, two tissues derived from the peripheral optic cup (OC). The expression of a dominant-negative (DN) allele of Lef1, or of a Lef1-engrailed fusion protein, leads to the inhibition of expression of peripheral genes and iris hypoplasia, suggesting that canonical Wnt signaling is required for peripheral eye development. It is proposed that canonical Wnt signaling in the developing optic vesicle (OV) and OC plays a crucial role in determining the identity of the ciliary body and iris. Because wingless (wg) plays a similar role in the induction of peripheral eye tissues of Drosophila, these findings indicate a possible conservation of the process that patterns the photoreceptive and support structures of the eye (Cho, 2006).
These findings provide an additional link between the development of the vertebrate and invertebrate eye. In Drosophila, photoreceptor cells are surrounded at the periphery with a non-neural cuticular structure. wg, the Drosophila homolog of the Wnt genes, is expressed in the margin of the eye imaginal disc, which is the anlage of peripheral eye tissues. Activation of wg, or armadillo, the Drosophila ß-catenin, in the eye imaginal disc promotes head cuticle formation at the expense of ommatidia, and has been proposed to act as a morphogen to pattern the peripheral structures (Cho, 2006).
Wnt signaling thus promotes the development of the non-neural, peripheral support structures in both Drosophila and chicks. The similarity of wg/Wnt expression and function in eye development provides an additional line of evidence that strengthens the proposed evolutionary conservation of the vertebrate and invertebrate eyes. The modern version of this model originated with the observation of a conserved expression and activity for the eyeless/Pax6 gene. The fact that wg/Wnt appears to play a role in patterning the central and peripheral eye structures suggests that the visual structure of the last common ancestor of flies and vertebrates had not only a photoreceptive component, but a support structure as well. A conserved unit of neural and non-neural eye tissues has also been suggested by the observation of a single-celled dinoflagellate that has several of the support structures of an eye, including pigment, a lens, a cornea and a photoreceptor. The fact that Pax6 plays a role in the development of not only the NR, but also the supporting tissues, such as the lens, cornea, iris and RPE, might also be seen as being in keeping with this model (Cho, 2006).
In the mouse, Cdx1 is essential for normal anteroposterior vertebral patterning through regulation of a subset of Hox genes. Retinoic acid (RA) and certain Wnts have also been implicated in vertebral patterning, although the relationship between these signaling pathways and the regulation of mesodermal Hox gene expression is not fully understood. Prior work has shown that Cdx1 is a direct target of both Wnt and retinoid signaling pathways, and might therefore act to relay these signals to the Hox genes. Wnt and RA are believed to impact on Cdx1 through an atypical RA-response element (RARE) and Lef/Tcf-response elements (LRE), respectively, in the proximal promoter. To address the roles of these regulatory motifs and pathways, mice mutated for the LRE or the LRE plus the RARE were produced. In contrast to RARE-null mutants, which exhibit limited vertebral defects, LRE-null and LRE+RARE-null mutants exhibited vertebral malformations affecting the entire cervical region that closely phenocopied the malformations seen in Cdx1-null mutants. Mutation of the LRE also greatly reduced induction of Cdx1 by RA, demonstrating a requirement for Wnt signaling in the regulation of this gene by retinoids. LRE and LRE+RARE mutants also exhibited vertebral fusions, suggesting a defect in somitogenesis. As Wnt signaling is implicated in somitogenesis upstream of the Notch pathway, it is conceivable that Cdx1 might play a role in this process. However, none of the Notch pathway genes assessed was overtly affected (Pilon, 2007).
Wnt ligands have pleiotropic and context-specific roles in embryogenesis and adult tissues. Among other effects, certain Wnts stabilize the beta-catenin protein, leading to the ability of beta-catenin to activate T-cell factor (TCF)-mediated transcription. Mutations resulting in constitutive beta-catenin stabilization underlie development of several human cancers. Genetic studies in Drosophila highlighted the split ends (spen) gene as a positive regulator of Wnt-dependent signaling. This study has assessed the role of SHARP, a human homologue of spen, in Wnt/beta-catenin/TCF function in mammalian cells. SHARP gene and protein expression were found to be elevated in human colon and ovarian endometrioid adenocarcinomas and mouse colon adenomas and carcinomas carrying gene defects leading to beta-catenin dysregulation. When ectopically expressed, the silencing mediator for retinoid and thyroid receptors/histone deacetylase 1-associated repressor protein (SHARP) protein potently enhances beta-catenin/TCF transcription of a model reporter gene and cellular target genes. Inhibition of endogenous SHARP function via RNA inhibitory (RNAi) approaches antagonized beta-catenin/TCF-mediated activation of target genes. The effect of SHARP on beta-catenin/TCF-regulated genes is mediated via a functional interaction between SHARP and TCF. beta-Catenin-dependent neoplastic transformation of RK3E cells is enhanced by ectopic expression of SHARP, and RNAi-mediated inhibition of endogenous SHARP in colon cancer cells inhibits their transformed growth. These findings implicate SHARP as an important positive regulator of Wnt signaling in cancers with beta-catenin dysregulation (Feng, 2007).
LEF-1/TCF, development and differentiation
An effector of intercellular adhesion, beta-catenin also functions in Wnt signaling, associating with Lef-1/Tcf DNA-binding proteins to form a functional transcription factor. This pathway operates in keratinocytes: mice expressing a stabilized beta-catenin controlled by an epidermal promoter undergo a process resembling de novo hair morphogenesis. The new follicles form sebaceous glands and dermal papilla, normally established only in embryogenesis. As in embryologically initiated hair germs, transgenic follicles induce Lef-1, but follicles are disoriented and defective in sonic hedgehog polarization. Additionally, proliferation continues unchecked, resulting in two types of tumors also found in humans. These findings suggest that transient beta-catenin stabilization may be a key player in the long-sought epidermal signal leading to hair development and implicate aberrant beta-catenin activation in hair tumors (Gat, 1998).
Submucosal glands (SMGs) secrete fluid, mucous and bacteriocidal proteins, which are important in maintaining normal lung function. SMGs are thought to play an important role in the pathogenesis of a number of hypersecretory lung diseases such as cystic fibrosis (CF), chronic bronchitis and asthma. Transcription of the lymphoid enhancer binding factor 1 (Lef1) gene is upregulated in submucosal gland progenitor cells just prior to gland bud formation in the developing ferret trachea. Several animal models have been utilized to functionally investigate the role of LEF1 in initiating and supporting gland development in the airway. Studies on Lef1-deficient mice and antisense oligonucleotides in a ferret xenograft model demonstrate that LEF1 is functionally required for submucosal gland formation in the nasal and tracheal mucosa. To determine whether LEF1 expression is sufficient for the induction of airway submucosal glands, two additional model systems were utilized. In the first, recombinant adeno-associated virus was used to overexpress the human LEF1 gene in a human bronchial xenograft model of regenerative gland development in the adult airway. Human bronchial xenografts were reconstituted using primary human bronchial cells infected with recombinant AV.LEF1/GFP. Denuded rat tracheas were seeded with infected primary airway epithelial cells and the xenograft cassettes were implanted subcutaneously into nude mice. In a second model, the LEF1 gene was ectopically overexpressed under the direction of the proximal airway-specific CC10 promoter in transgenic mice. In both of these models, morphometric analyses reveal no increase in the number or size of airway submucosal glands, indicating that ectopic LEF1 expression alone is insufficient to induce submucosal gland development. In summary, these studies demonstrate that LEF1 expression is required, but in and of itself is insufficient, for the initiation and continued morphogenesis of submucosal glands in the airway (Duan, 1999).
In mammals, embryonic skin epithelial cells are pluripotent; that is, they are able to choose between epidermal and hair follicle cell fates. Commitment to follicle formation occurs when an underlying mesenchymal cue instructs overlying ectoderm to commit to forming an appendage. As the epithelium thickens, forming first a placode and then a small downgrowth or germ, it transmits a message to the underlying mesenchyme, stimulating their condensation into a dermal papilla. A second dermal message is then transmitted back to the adjacent epithelial cells, instructing them to proliferate. As development proceeds, the epithelial cells differentiate, producing first an outer and inner root sheath (ORS and IRS, respectively), and then near or at birth, a hair shaft at the center of the follicle. The epithelial cells forming a cloak surrounding the dermal papilla are called matrix cells, which proliferate transiently. As matrix cells withdraw from the cell cycle, they differentiate into upwardly moving cells. At the center, matrix cells differentiate into precortical cells, which subsequently give rise to the cortex, medulla and cuticle of the hair shaft. A surrounding concentric ring of matrix cells gives rise to the IRS, which in turn is surrounded by the ORS. Near the skin surface, the IRS degenerates, freeing the hair shaft to push outward as matrix cells proliferate and differentiate at the base. Once established, follicles proceed through cycles of active periods of hair growth (anagen), regression and shortening (catagen) and rest (telogen). Only the lower epithelial portion of the follicle actually cycles. At the base of the permanent portion of the follicle is a region known as the bulge, thought to contain a population of self-renewing epithelial stem cells (DasGupta, 1999).
During the hair cycle, when matrix cells lose their proliferative capacity, the follicle ceases growth and the lower epithelial portion regresses, bringing the dermal papilla cells upward to the bulge. At the transition between telogen and the initiation of the next hair cycle, a signal, perhaps from the dermal papilla, converts one or more epithelial stem cells to proliferating matrix and ORS cells, which now move downward and differentiate. Once the lower follicle is fully formed, proliferation becomes restricted to the base where matrix cells maintain contact with the dermal papilla. The inductive signals exchanged among epithelial components within the follicle and between follicular epithelia and mesenchyme are largely unknown; however, recent studies suggest that Wnt signaling pathways might be involved (DasGupta, 1999).
LEF/TCF DNA-binding proteins act in concert with activated beta-catenin, the product of Wnt signaling, to transactivate downstream target genes. To probe the role of activated LEF/TCF transcription factor complexes in hair follicle morphogenesis and differentiation, mice harboring TOPGAL, a beta-galactosidase gene was engineered under the control of a LEF/TCF and beta-catenin inducible promoter. In mice, TOPGAL expression is directly stimulated by a stabilized form of beta-catenin, but is also dependent upon LEF1/TCF3 in skin. During embryogenesis, TOPGAL activation occurs transiently in a subset of LEF1-positive cells of pluripotent ectoderm and underlying mesenchyme. Downgrowth of initiated follicles proceeds in the absence of detectable TOPGAL expression, even though LEF1 is still expressed. While proliferative matrix cells express the highest levels of Lef1 mRNAs, LEF1 concentrates in the precursor cells to the hair shaft, where TOPGAL expression is co-induced with hair-specific keratin genes containing LEF/TCF- binding motifs. LEF1 and TOPGAL expression ceases during catagen and telogen, but reappears at the start of the postnatal hair cycle, concomitant with precortex formation. In contrast to hair shaft precursor cells, postnatal outer root sheath express TCF3, but not TOPGAL. TCF3 is also expressed in the putative follicle stem cells, and while TOPGAL is generally silent in this compartment, it is stimulated at the start of the hair cycle in a fashion that appeared to be dependent upon stabilization of beta-catenin. Taken together, these findings demonstrate that LEF1/TCF3 is necessary but not sufficient for TOPGAL activation, revealing the existence of positive and negative regulators of these factors in the skin. Furthermore, these findings unveil the importance of activated LEF/TCF complexes at distinct times in hair development and cycling when changes in cell fate and differentiation commitments take place (DasGupta, 1999).
A fascinating feature of hair matrix cells is that they are able to select morphologically and biochemically distinct differentiation pathways leading to the IRS and hair shaft. How matrix cells select these programs of differentiation has always been a mystery. The present studies are among the first to provide biochemical insights into this process. Thus, while most if not all matrix cells express Lef1 mRNAs and some LEF1 protein, only cells at the center of the bulb appear to accumulate LEF1 and activate downstream target genes concomitant with commitment to differentiate. The TOPGAL-expressing cells give rise to the hair shaft, while the surrounding cells choose an alternative pathway of differentiation and give rise to the IRS. These findings suggest that induction of Lef1 expression is a characteristic of undifferentiated matrix cells, and that utilizing LEF1 to activate downstream target genes is a feature of matrix cells that become committed to a hair shaft differentiation program. The finding that LEF1 activation plays a role in hair shaft differentiation provides an explanation for why so many hair-specific keratin genes possess LEF1-binding motifs in their upstream regulatory sequences (DasGupta, 1999).
Lef1 and other genes of the LEF1/TCF family of transcription factors are nuclear mediators of Wnt signaling. The expression pattern and functional importance of Lef1 in the developing forebrain of the mouse has been studied. Lef1 is expressed in the developing hippocampus, and LEF1-deficient embryos lack dentate gyrus granule cells but do contain glial cells and interneurons in the region of the dentate gyrus. In mouse embryos homozygous for a Lef1-lacZ fusion gene, which encodes a protein that is not only deficient in DNA binding but also interferes with beta-catenin-mediated transcriptional activation by other LEF1/TCF proteins, the entire hippocampus including the CA fields is missing. Thus, LEF1 regulates the generation of dentate gyrus granule cells, and together with other LEF1/TCF proteins, the development of the hippocampus (Galceran, 2000).
Lef1 is expressed in the proliferating precursors of granule cells in the ventricular zone and in the migratory secondary proliferative population. Consistent with this expression pattern, LEF1-deficient embryos lack molecular markers of dentate granule cells (Prox1 and Calretinin) in the region of the dentate gyrus. In addition, the dentate gyrus remnant of LEF1-deficient embryos contains an approximately 2.5-fold reduced number of proliferating cells. BrdU-pulse experiments suggest that granule cells, or their precursors, do not arrive at the dentate gyrus. Therefore, the observed decrease in the number of proliferating cells may reflect the absence of dentate granule cells. The residual proliferating cells are likely to be glia, which are present in the region of the dentate gyrus remnant and are known to be mitotically active in the SSP and migratory stream. The absence of a specific cell type, the dentate gyrus granule cells, in LEF1-deficient mice provides insight into the generation of granule cells, glia and interneurons. It is hypothesized that the hippocampal ventricular zone adjacent to the fimbria, which expresses the highest levels of LEF1, contains the granule cell progenitors. In LEF1-deficient embryos, these cells are incapable of differentiating into granule cells as evidenced by the loss of Prox1 expression in the dentate gyrus remnant. In contrast, the ventricular zone flanking this region is able to produce pyramidal cells of the CA field. In addition, GFAP-expressing glia cells continue to be produced in the mutant mice, consistent with the model that these cells are derived from the fimbria, which does not express Lef1. Finally, the distribution of Dlx2-expressing immature interneurons is not affected by the LEF1-deficient mutation, consistent with the model that cortical (including hippocampal) interneurons are derived from the basal ganglia (Galceran, 2000).
During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, evidence in vitro and in vivo is provided that Cdx1 is a direct transcriptional target of the Wnt/beta-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/beta-catenin complexes and mediate beta-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/beta-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development (Lickert, 2000).
In skin, multipotent stem cells generate the keratinocytes of the epidermis, sebaceous gland, and hair follicles. Tcf3 and Lef1 control these differentiation lineages. In contrast to Lef1, which requires Wnt signaling and stabilized ß-catenin to express the hair-specific keratin genes and control hair differentiation, Tcf3 can act independent of its ß-catenin interacting domain to suppress features of epidermal terminal differentiation, in which Tcf3 is normally shut off, and promote features of the follicle outer root sheath (ORS) and multipotent stem cells (bulge), the compartments which naturally express Tcf3. These aspects of Tcf3's action are dependent on its DNA binding and Groucho repressor-binding domains. In the absence of its ß-catenin interacting domain, Lef1's behavior (DeltaNLef1) seems to be markedly distinct from that of DeltaNTcf3. DeltaNLef1 does not suppress epidermal differentiation and promote ORS/bulge differentiation, but rather suppresses hair differentiation and gives rise to sebocyte differentiation. Taken together, these findings provide powerful evidence that the status of Tcf3/Lef complexes has a key role in controlling cell fate lineages in multipotent skin stem cells (Merrill, 2001).
Lef1 behaves as a transcriptional activator and promotes hair follicle differentiation in response to Wnt signals that stabilize ß-catenin. Inhibition of the Wnt response blocks hair differentiation and promotes sebaceous cell differentiation, underscoring the importance of Wnt signaling and ß-catenin stabilization to the pilosebaceous unit of the skin (Merrill, 2001).
In contrast, Tcf3 inhibits certain features of epidermal differentiation while promoting other differentiation features characteristic of the bulge and ORS. In ORS and bulge that normally express Tcf3, transgenic Tcf3 had no obvious morphological effects, despite yielding nuclear localization of ß-catenin in these cells. In striking contrast to Lef1, Tcf3's behavior does not rely on Wnt signaling and ß-catenin stabilization: DeltaNTcf3 and Tcf3 render similar phenotypes. These data suggest that the status of Tcf3 activity in the epidermis, ORS, and bulge influences these pathways of skin epithelial differentiation and in these cells, Tcf3-regulated genes may not require Wnt signaling (Merrill, 2001).
In keratinocytes in vitro, Tcf3 seemed to act as a transcriptional repressor in the presence of the appropriate cofactors. The transdifferentiation effects of Tcf3 in the epidermis in vivo appear to be dependent on the ability of Tcf3 to bind DNA and to interact with the Groucho family of repressor proteins. These effects are not dependent on the carboxy-terminal domain of Tcf3, thought to associate with CtBP repressor proteins, and as indicated above, they do not rely on the amino terminus of Tcf3, known to bind ß-catenin. Based on these findings, the data support the argument that in vivo as in vitro, Tcf3 acts as a repressor in keratinocytes. A role for Tcf3 as a transcriptional repressor in mammals is consistent with its function in other model organisms and the involvement of Groucho proteins in this action is consistent with the fact that mammalian Groucho proteins can interact with histone deacetylases. Given the parallels between the differentiation seen in the epidermis of K14-Tcf3 transgenic mice and that which occurs in the bulge and ORS of naturally Tcf3-positive cells, it seems likely that both in the wild-type hair follicle and in the K14-Tcf3 transgenic epidermis, Tcf3 functions similarly (Merrill, 2001).
Although in vitro mutagenesis studies did not support a role for CtBP in Tcf3-mediated repression, they did uncover a role for a potential interaction between proteins that associate with Grg and CtBP domains that may be similar to that seen for the Hairy protein, required for embryonic segmentation in Drosophila. Hairy protein physically interacts with both dCtBP and Groucho proteins, and whereas Groucho mutations enhance defects in hairy mutants, reduced dCtBP activity suppresses defects in hairy mutants. In mammalian keratinocytes, the Grg-interaction domain of Tcf3 was required for repression, and the CtBP-interaction domain is required for ß-catenin mediated activation of TOPFlash (Tcf Optimal Promoter + luciferase). Interestingly, the converse is also observed, namely that the CtBP-interaction domain is not required for repression, and the Grg-interaction domain is not required for ß-catenin-mediated activation. These data suggest a model in which CtBP moderates a competition between ß-catenin and Grg proteins to determine whether Tcf3 will activate or repress target genes. Consistent with this model is the finding that CtBP can act either as a coactivator or as a corepressor when tethered to DNA, depending on the cell type in which it is expressed. Therefore, differences in CtBP levels/activity could explain why in culture the behavior of Tcf3 is markedly influenced by ß-catenin levels (Merrill, 2001).
Drosophila ovo/svb is required for epidermal cuticle/denticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of ovo, movo1, is required for the proper formation of hair, a mammalian epidermal appendage. Evidence is provided that movo1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those bound by Ovo, further supporting the notion that mOvo1a and Ovo are genetically and biochemically homologous proteins. Additionally, the movo1 promoter is shown to be activated by the lymphoid enhancer factor 1 (LEF1)/beta-catenin complex, a transducer of wnt signaling. Collectively, these findings suggest that movo1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg/wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies (Li, 2002).
Wnt signaling pathways have been demonstrated to play important roles in controlling tissue patterning and cell proliferation. In the gastrointestinal tract, mutations that lead to activation of the canonical Wnt pathway through small ß-catenin result in familial and sporadic colon cancers. The downstream transcription factor Tcf4 is required to maintain the proliferative stem cell compartment in the crypts of the small intestine. Activation of TCF-dependent transcription is a good correlate to neoplastic transformation. Despite its association with cancer in the colon, little is known of the role for Wnt signaling during development and patterning of the gut tube. A comprehensive expression screen was constructed for Wnt signaling components during different stages of gut development in the chick. Conserved expression patterns of these genes indicate that they likely play essential roles in gut morphogenesis. Based on the expression profiles of putative components of each pathway, it is possible to postulate specific roles for the various pathways during gut development. Predictions of roles for canonical signaling in the developing gizzard, duodenum, and large intestine in chick were tested by viral misexpression of dominant-negative (DN) forms of the downstream cofactors Tcf4 and Lef1. In the chick, Tcf4 is expressed in the posterior gizzard mesoderm. Misexpression of DN-Tcf4 in the splanchnic mesoderm results in the failure of the gizzard epithelium to form microvilli. Lef1 is expressed in the chick duodenum and large intestine mesoderm. Viral misexpression of DN-Lef1 results in diminished mesoderm and overproliferation of the large intestine endoderm, leading to stenosis of the lumen. The results from these misexpression studies in the chick, together with evidence from colorectal lesions, indicate that the canonical Wnt pathway plays critical roles in balancing cell proliferation versus cell differentiation during gut development. The expression profiles of the Wnt signaling components presented in this paper should prove valuable in deciphering additional roles of the Wnt pathways during patterning of the vertebrate gut tube (Theodosiou, 2003).
The roles of Lef/Tcf proteins in determining cell fate characteristics have been described in many contexts during vertebrate embryogenesis, organ and tissue homeostasis, and cancer formation. Although much of the accumulated work on these proteins involves their ability to transactivate target genes when stimulated by ß-catenin, Lef/Tcf proteins can repress target genes in the absence of stabilized ß-catenin. By ablating Tcf3 function, an important requirement for a repressor function of Lef/Tcf proteins was uncovered during early mouse development. Tcf3-/- embryos proceed through gastrulation to form mesoderm, but they develop expanded and often duplicated axial mesoderm structures, including nodes and notochords. These duplications are preceded by ectopic expression of Foxa2, an axial mesoderm gene involved in node specification, with a concomitant reduction in Lefty2, a marker for lateral mesoderm. By contrast, expression of a ß-catenin-dependent, Lef/Tcf reporter (TOPGal), is not ectopically activated but is faithfully maintained in the primitive streak. Taken together, these data reveal a unique requirement for Tcf3 repressor function in restricting induction of the anterior-posterior axis (Merrill, 2003).
Naïve myogenic cells migrate from the somites into the developing vertebrate limb, where they simultaneously differentiate into myotubes and form distinct anatomical muscles. Limb signals have been hypothesized to direct the pattern of muscles formed, but the molecular nature of these signals and the identity of the cells that produce them have remained unclear. A population of lateral plate-derived limb mesodermal cells in both chick and mouse have been identified that expresses the transcription factor Tcf4 in a muscle-specific pattern independently of the muscle cells themselves. Functional experiments in the chick demonstrate that TCF4 and the Wnt-β-catenin pathway in these limb mesodermal cells are critical for muscle patterning. It is proposed that Tcf4-expressing cells establish a prepattern in the limb mesoderm that determines the sites of myogenic differentiation and thus establishes the basic pattern of limb muscles (Kardon, 2003).
In the embryonic kidney, progenitors in the metanephric mesenchyme differentiate into specialized renal epithelia in a defined sequence characterized by the formation of cellular aggregates, conversion into polarized epithelia and segmentation along a proximal-distal axis. This sequence is reiterated throughout renal development to generate nephrons. This study identified global transcriptional programs associated with epithelial differentiation utilizing an organ culture model of rat metanephric mesenchymal differentiation, which recapitulates the hallmarks of epithelialization in vivo in a synchronized rather than reiterative fashion. Activation of multiple putative targets of β-catenin/TCF/Lef-dependent transcription were observed coinciding with epithelial differentiation. It was shown, in cultured explants, that isolated activation of β-catenin signaling in epithelial progenitors induces, in a TCF/Lef-dependent manner, a subset of the transcripts associated with epithelialization, including Pax8, cyclin D1 (Ccnd1) and Emx2. This is associated with anti-apoptotic and proliferative effects in epithelial progenitors, whereas cells with impaired TCF/Lef-dependent transcription are progressively depleted from the epithelial lineage. In vivo, TCF/Lef-responsive genes comprise a conserved transcriptional program in differentiating renal epithelial progenitors and β-catenin-containing transcriptional complexes directly bind to their promoter regions. Thus, β-catenin/TCF/Lef-mediated transcriptional events control a subset of the differentiation-associated transcriptional program and thereby participate in maintenance, expansion and stage progression of the epithelial lineage (Schmidt-Ott, 2007).
At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos, a highly conserved pattern of Wnt responsiveness was found in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. The consequences of disrupting Wnt signaling was explored, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibit radically altered facial features that culminates in a hyperteloric appearance and a foreshortened midface. A biochemical approach was used to perturb Wnt signaling; in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. The hypothesis was tested that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated (Brugmann, 2007).
Despite the importance of taste in determining nutrient intake, understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/β-catenin signaling pathway. However, the regulation of these factors, including their induction, remains poorly understood. This study identified a crucial role for the Wilms' tumor 1 protein (WT1) in circumvallate (CV) papillae development. WT1 is a transcription factor that is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by E12.5, when the CV taste placode begins to form. In mice lacking WT1, the CV fails to develop normally and markers of early taste development are dysregulated compared with wild type. Expression of the WT1 target genes Lef1, Ptch1 and Bmp4 is significantly reduced in developing tongue tissue derived from Wt1 knockout mice, and, in normal tongue, WT1 was shown to be bound to the promoter regions of these genes. Moreover, siRNA knockdown of WT1 in cultured taste cells leads to a reduction in the expression of Lef1 and Ptch1. These data identify WT1 as a crucial transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes (Gao, 2014).
Environmental signals are important in the development of neural crest, during which process multipotent progenitors must choose from several fates. However, the nature of these environmental signals is unknown. A fate map of zebrafish cranial neural crest shows that lineage-restricted clones of pigment cells arise from medial cells near the neural keel, and that clones of neurons arise from lateral cells farther from the neural keel. Wnt-1 and Wnt-3a are candidate genes for influencing neural crest fate, as they are expressed next to medial, but not lateral, crest cells. The role of Wnt signals in modulating the fate of neural crest has been determined by injecting messenger RNAs into single, premigratory neural crest cells of zebrafish. Lineage analysis of injected cells shows that activation of Wnt signaling by injection of mRNA encoding cytoplasmic beta-catenin promotes pigment-cell formation at the expense of neurons and glia. Conversely, inhibition of the Wnt pathway, by injection of mRNAs encoding either a truncated form of the transcription factor Tcf-3 or a dominant-negative Wnt, promotes neuronal fates at the expense of pigment cells. It is concluded that endogenous Wnt signaling normally promotes pigment-cell formation by medial crest cells and thereby contributes to the diversity of neural crest cell fates (Dorsky, 1998).
There has been rapid progress recently in the identification of signaling pathways regulating tooth development. It has become apparent that signaling networks involved in Drosophila development and the development of structures such as limbs are also used in tooth development. Teeth are epithelial appendages formed in the oral region of vertebrates; their early developmental anatomy resembles that of other strucures, such as hairs and glands. The neural crest origin of tooth mesenchyme has been confirmed and recent evidence suggests that specific combinations of homeobox genes expressed in the neural crest cells may regulate the types of teeth and their patterning. Signaling molecules in the Shh, FGF, BMP and Wnt families appear to regulate the early steps of tooth morphogenesis. Certain transcription factors associated with these pathways have been shown to be necessary for tooth development. Sonic hedgehog is expressed in dental epithelium as several stages starting in the early epithelial thickenings, then reappears in the enamel knot and subsequently is expressed in the ameloblast cell lineage. Lef-1, involved in the transduction of the Wnt signal, is expressed throughout tooth development: its expression is not restricted to either epithelial or mesenchymal tissues, although it is needed only in epithelium during early development. Lef-1 appears to be involved in the regulation of an epithelial signal acting on dental mesenchyme during the bud stage of tooth morphogenesis. Several Wnt genes are expressed during tooth development, including the Wnt-10 gene, which is expressed in early dental epithelium. Several of the conserved signals are also transiently expressed in the enamel knots in the dental epithelium. The enamel knots are associated with the characteristic epithelial folding morphogenesis, which is responsible for the development of tooth shape. It is currently believed that the enamel knots function as signaling centers, regulating the development of tooth shape. Enamel knots constitute a specific ectodermal cell lineage; it has been proposed that enamel knots determine the site of the first cusp of teeth and that they regulate the formation of other cusps in molar teeth (Thesleff, 1997).
LEF1 is a cell-type-specific transcription factor and mediates Wnt signaling pathway by association with its co-activator β-catenin. Wnt signaling is known to be critical for the specification of cranial neural crest (CNC) cells and may regulate the fate diversity of the CNC during craniofacial morphogenesis. Loss of Lef1 results in arrested tooth development at the late bud stage and LEF1 is required for a relay of a Wnt signaling to a cascade of FGF signaling activities to mediate the epithelial-mesenchymal interaction during tooth morphogenesis. It remains unclear, however, what is the cellular mechanism of LEF1 signaling in regulating tooth morphogenesis. To test the hypothesis that LEF1 signaling regulates the fate of the dental epithelial and the CNC-derived mesenchymal cells during tooth morphogenesis, the cellular migration, proliferation, and apoptotic activity within the tooth germ were investigated and compared between the wild-type and Lef1 null mutant mice. Using the Wnt1-Cre/R26R transgenic system for indelibly marking the progenies of CNC cells, it was shown that there is no CNC migration defect in the Lef1 null mutant mice, indicating that the arrest in tooth development is not the result of shortage of the CNC contribution into the first branchial arch in the Lef1 mutant. Furthermore, there is no alteration in cell proliferation or condensation of the CNC-derived dental mesenchyme in the Lef1 null mutant, suggesting that LEF1 may not affect the cell cycle progression of the multipotential CNC cells during tooth morphogenesis. Importantly, apoptotic activity is significantly increased within the dental epithelium in the Lef1 null mutant mice. As the result of this increased cell death, the bud stage tooth germ fails to advance to the cap stage in the absence of Lef1. Inhibition of apoptotic activity by FGF4 rescues the tooth development in the Lef1 null mutant. These studies suggest that LEF1 is a critical survival factor for the dental epithelial cells during tooth morphogenesis (Sasaki, 2005).
LEF-1/TCF and brain development
After the primary anterior-posterior patterning of the neural plate, a subset of wnt signaling molecules including Xwnt-1, Xwnt-2b, Xwnt-3A, Xwnt-8b are still expressed in the developing brain in a region spanning from the posterior part of the diencephalon to the mesencephalon/metencephalon boundary. In this expression field, they are colocalized with the HMG-box transcription factor XTcf-4. Using antisense morpholino loss-of-function strategies, it was demonstrated that the expression of this transcription factor depends on Xwnt-2b, which itself is under the control of XTcf-4. Marker gene analyses reveal that this autoregulatory loop is important for proper development of the midbrain and the isthmus. Staining for NCAM reveals a lack of dorsal neural tissue in this area. This reduction is caused by a reduced proliferation rate as shown by staining for PhosphoH3 positive nuclei. In rescue experiments, it was demonstrated that individual isoforms of XTcf-4 control the development of different parts of the brain. XTcf-4A restores the expression of the mesencephalon marker genes pax-6 and wnt-2b but not the isthmus marker gene en-2. XTcf-4C, in contrast, restores en-2, but has only weak effects on pax-6 and wnt-2b. Thus, autoregulation of canonical Wnt signaling and alternative expression of different isoforms of XTcf-4 is essential for specifying the developing CNS (Kunz, 2004).
Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. This study shows that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of beta-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex (Kuwahara, 2010).
Wnt signaling and its downstream target N-Myc play a key role in the production of basal progenitors. Expression of N-myc or stabilized β-catenin increases, while conditional gene deletion of N-myc decreases the numbers of basal progenitors found in the developing neocortex, as determined by the numbers of Tbr2-positive cells and non-surface dividing cells. The increase in basal progenitors by the Wnt-N-myc axis can be ascribed to either: (1) differentiation of apical progenitors into basal progenitors; or (2) proliferation (and survival) of basal progenitors, or both. The observation that retroviral expression of stabilized β-catenin or N-myc in the neocortex reduced the number of apical progenitors while increasing that of the basal progenitors supports a role for the former mechanism (Kuwahara, 2010).
Members of the Myc family have been reported to be involved in differentiation processes in other cell types, including epithelial, neural crest and hematopoietic stem cells, although previous reports have not directly demonstrated that Myc is involved in fate commitment by a lineage-tracing analysis. In this study, the clonal analysis suggests that N-myc instructs commitment of NPC fate into the neuronal lineage at the expense of the glial lineage and reduces multipotent neurosphere-forming NPCs. This function of N-myc is similar to the reported function of Wnt signaling (Kuwahara, 2010).
It is not known what transcriptional targets of N-myc are involved in instructing neurogenesis. Possible candidates include the proneural gene Ngn1, as deletion of N-myc was observed to cause a decrease in the level of Ngn1 mRNA in the developing neocortex. As Ngn1 is also a direct target of the β-catenin/Tcf transcription complex, it would be interesting to examine the interaction between N-myc and these transcription factors on the Ngn1 promoter. The Myc family has also been shown to function in the regulation of the global chromatin state, in addition to its function as a classical transcription factor; thus it is possible that mechanisms other than direct target gene activation are also involved in N-myc regulation of neurogenesis and proliferation of NPCs (Kuwahara, 2010).
This study also provides evidence that N-myc is directly regulated by the β-catenin/Tcf transcription complex and mediates the functions of Wnt signaling to stimulate neocortical NPC proliferation and differentiation: (1) Wnt3a treatment and stabilized β-catenin expression induced N-myc expression, whereas expression of a dominant-negative form of Tcf3 reduced N-myc expression in NPC cultures; (2) misexpression of stabilized β-catenin in the ventral telencephalon induced ectopic N-myc expression in vivo; (3) N-myc is expressed in the developing neocortex in a pattern similar to that of a Tcf reporter transgene; (4) Tcf3 directly binds to a Tcf-consensus site 1.6 kb upstream of the N-myc gene; (5) Wnt stimulation of proliferation and differentiation in NPC cultures was abrogated by deletion of the N-myc gene. These results provide evidence that N-myc is a key downstream mediator of Wnt-β-catenin signaling in the developing neocortex. It is of note that N-myc is not the only downstream target responsible for the functions of Wnt signaling in the neocortex (Kuwahara, 2010).
The Wnt-β-catenin pathway exerts multiple functions in a context-dependent manner. For instance, persistent expression of stabilized β-catenin in NPCs results in overproliferation of apical progenitors and horizontal/tangential expansion of the cortex in addition to the reduction of Tbr2-positive basal progenitors. However, when the same stabilized β-catenin was expressed by retroviral infection in a small proportion of NPCs located at the VZ, it had the opposite effect: increasing the numbers of basal progenitors and decreasing the number of apical progenitors. This difference does not appear to be due to the differential requirement of N-myc, as N-myc gene deletion rescued both proliferative and differentiating effects of activation of β-catenin. This difference might be rather due to the aberrant brain architecture generated in the β-catenin-δEx3 mice (mutant for β-catenin), to other non-cell autonomous effects of β-catenin or to differences in the levels or timing of active β-catenin expression. Indeed, different levels of active β-catenin expression result in different outcomes in hair follicle stem cells (Kuwahara, 2010).
Although it has previously been postulated that β-catenin exerts its different functions via distinct targets, this study observed that both the proliferating and neurogenic functions of Wnt-β-catenin signaling in the developing neocortex are mediated in common by N-myc. It is noteworthy that c-Myc can also exert distinct functions depending on its expression levels, such as in epithelial stem cells, raising the possibility that the levels of N-myc might determine the cellular output. Importantly, heterozygous mutation of N-MYC (MYCN) in humans causes Feingold syndrome, comprising several defects including microcephaly, supporting the notion that the levels of N-myc in the nervous system are crucial for determining the neuronal number and brain size. It is also possible that N-myc alters its function in a developmental-stage-dependent manner. This possibility is consistent with a previous finding that canonical Wnt signaling promotes proliferation of neocortical neural precursor cells at a relatively early stage (E10.5) but promotes their differentiation at a relatively late stage (E13.5) (Kuwahara, 2010).
Which Wnt ligands are responsible for the activation of N-myc and consequent regulation of basal progenitors in the developing brain? Wnt7a is expressed in NPCs at the VZ and might be important for increase in cells localized in the SVZ. Wnt7b, which is expressed in the deep-layer neurons (neurons at the layer VI), might elicit a feed-forward signal to increase the number of basal progenitors that in turn contribute to the generation of the upper-layer neurons. It is plausible that extracellular signals other than Wnt ligands are also involved in the activation of N-myc and regulation of basal progenitors. N-myc is induced by Shh signaling in cerebellar granule cells, and a recent report shows that Shh protein is localized in the IMZ of the neocortex and contributes to the production of basal progenitors. Growth factors expressed in NPCs such as Fgf2 and epidermal growth factor (Egf) might also participate in the activation of N-myc. Growth factor receptors activate the PI3K (Pik3r1 - Mouse Genome Informatics) pathway, which induces phosphorylation and stabilization of N-myc protein. In addition, Egfr as well as Frs2, an adaptor of Fgfr/Egfr, have been shown to regulate the production of basal progenitors. The RNA-binding protein HuC/D is another candidate that could regulate N-myc function in basal progenitors, as it binds to and stabilizes N-myc mRNA and is localized in the SVZ (Kuwahara, 2010).
As a mechanism of neocortical expansion during animal evolution, the increase of basal progenitors is considered to be a key event, given that basal progenitors increase the number of neurons from a given number of apical progenitors through extra cell division and that the number of basal progenitors dramatically increases during animal evolution. The observation in this study that N-myc deletion decreases Tbr2-positive cells and non-surface dividing cells without marked reduction of Pax6-positive cells supports the notion that Wnt signaling, via N-myc, promotes differentiation from apical progenitors to basal progenitors and promotes indirect neurogenesis. It would be interesting to investigate possible roles of this signaling pathway in the neocortical expansion during animal evolution in future studies (Kuwahara, 2010).
During neural tube development, Shh signaling through Gli transcription factors is necessary to establish five distinct ventral progenitor domains that give rise to unique classes of neurons and glia that arise in specific positions along the dorsoventral axis. These cells are generated from progenitors that display distinct transcription factor gene expression profiles in specific domains in the ventricular zone. However, the molecular genetic mechanisms that control the differential spatiotemporal transcriptional responses of progenitor target genes to graded Shh-Gli signaling remain unclear. The current study demonstrates a role for Tcf/Lef repressor activity in this process. Tcf3 and Tcf7L2 (Tcf4) were shown to be required for proper ventral patterning and function by independently regulating two Shh-Gli target genes, Nkx2.2 and Olig2, which are initially induced in a common pool of progenitors that ultimately segregate into unique territories giving rise to distinct progeny. Genetic and functional studies in vivo show that Tcf transcriptional repressors selectively elevate the strength and duration of Gli activity necessary to induce Nkx2.2, but have no effect on Olig2, and thereby contribute to the establishment of their distinct expression domains in cooperation with graded Shh signaling. Together, these data reveal a Shh-Gli-independent transcriptional input that is required to shape the precise spatial and temporal response to extracellular morphogen signaling information during lineage segregation in the CNS (Wang, 2011).
Tissue regeneration relies on resident stem cells (SCs), whose activity and lineage choices are influenced by the microenvironment. Exploiting the synchronized, cyclical bouts of tissue regeneration in hair follicles (HFs), this study investigated how microenvironment dynamics shape the emergence of stem cell lineages. Employing epigenetic and ChIP-seq profiling, this study uncovered how signal-dependent transcription factors couple spatiotemporal cues to chromatin dynamics, thereby choreographing stem cell lineages. Using enhancer-driven reporters, mutagenesis, and genetics, this study showed that simultaneous BMP-inhibitory and WNT signals set the stage for lineage choices by establishing chromatin platforms permissive for diversification. Mechanistically, when binding of BMP effector pSMAD1 is relieved, enhancers driving HF-stem cell master regulators are silenced. Concomitantly, multipotent, lineage-fated enhancers silent in HF-stem cells become activated by exchanging WNT effectors TCF3/4 for LEF1. Throughout regeneration, lineage enhancers continue reliance upon LEF1 but then achieve specificity by accommodating additional incoming signaling effectors. Barriers to progenitor plasticity increase when diverse, signal-sensitive transcription factors shape LEF1-regulated enhancer dynamics (Adam, 2018).
By unearthing complex differences at the chromatin and gene expression level, and mapping them spatially and temporally, this study was able to dissect progressive chromatin remodeling of hair lineages driven by the collaboration of key lineage transcription factors and external signaling effectors. These analyses exposed hitherto unrecognized distinct, lineage-specific classes of super-enhancer regulatory elements. Armed with newfound knowledge of lineage-specific transcription factors and their spatial and temporal emergence during hair regeneration, major new insights were gained into how lineages are specified and also the regulatory forces that drive lineage restriction (Adam, 2018).
In acquiring this knowledge, it was learned that at each step along the lineage route, transcriptional effectors from distinct signaling pathways converge on lineage-specific regulatory elements driven by lineage-specific master regulators. This concept was first proposed for hematopoietic lineage determination, when it was recognized from ChIP-seq that in erythroid cell lines, pSMAD1 and TCF4 both bind to enhancer elements that are also occupied by master regulator GATA2, while in myeloid cell lines, pSMAD1 and TCF4 shift to enhancer elements bound by C/EBP. These findings led researchers to propose that the regenerative response mediated by BMP and WNT signaling in hematopoiesis is coupled with lineage master regulators to control gene programs that define cellular identity (Adam, 2018).
WNT and BMP signaling play nearly universal roles in dictating stem cell behavior, including HF-stem cells. However, their precise roles vary in a manner that depends upon context, but with unclear mechanistic underpinnings. Previous work showed that reminiscent of the hematopoietic system, WNT effectors, in this case TCF3 and TCF4, bind to the key super-enhancer elements which in HF-stem cells are occupied by master regulators SOX9, LHX2, and NFATc1. This study now finds that pSMAD1 binds adjacent to these TCF3/4 occupied epicenters, following the paradigm set for hematopoietic stem cells. However, by analyzing the next step in the lineage, it was found that both TCF3/4 and pSMAD1 and their associated occupancies are lost rather than shifted as HF-stem cells transition to multi-lineage progenitors. Moreover, as mutagenesis studies demonstrate, this results in silencing of key HF-stem cell identity genes (Adam, 2018).
Adding further intrigue, the activation of WNT signaling and BMP inhibitors and silencing of HF-stem cell super-enhancers leads to Lef1 and Tcf7 expression. LEF1 ChIP-seq and mouse genetic data place LEF1/TCF1 at the helm of the transition from short-term HF-stem cells in the hair germ to multi-lineage progenitors. Moreover, the first activated lineage-fated genes in this progression are regulated by a WNT effector switch: not only were their TAC enhancers silenced and TCF3/4-bound in HF-stem cells, but they became active and LEF1-bound during commitment. This led to the striking realization that within the stem cell, two types of TCF3/4-bound regulatory elements come into play. One binds pSMAD1, TCF3/4, and HF-stem cell master regulators and is active in HF-stem cells. The other lacks pSMAD1 and the HF-stem cell master regulators but still binds TCF3/4; these elements are silenced until TCF3/4 WNT antagonists are replaced by the LEF1 WNT-induced agonist (Adam, 2018).
The findings lend biological meaning and physiological context to the recent finding that in cultured embryonic stem cells, WNT stabilized β-catenin removes TCF3 from DNA, which leads to its degradation. More importantly, these studies illustrate how committed cells establish barriers to cellular plasticity by coupling bidirectional chromatin switches with transcription factors governing alternate fates (Adam, 2018).
During the regeneration process, multipotent progenitors (MPPs) become more restricted in their fates in the progression from activated hair germ to full anagen TACs. The ability to perform chromatin landscaping on small cell numbers has now permitted the dissection of how this happens. This study shows that initially, these WNT-activated, LEF1-bound regulatory elements govern multipotent lineage determination. However, during lineage progression, new, more restricted regulatory elements emerge that still utilize and require LEF1 but now differ in that many IRS regulatory elements contain motifs for NOTCH effector RBPJ, while HS regulatory elements are endowed with pSMAD1 motifs. The findings not only bolster prior genetic studies highlighting the importance of these signaling pathways in HFs, but, in addition, expose how WNT-regulated LEF1-containing enhancer elements can become lineage restricted through binding of additional signaling effectors. Indeed, our mutagenesis studies underscore the functional importance of these new signaling inputs in modifying the chromatin landscapes (Adam, 2018).
In conclusion, the combination of in vivo chromatin and genetic analyses afforded an unprecedented high-resolution precision mapping of the signaling effectors that govern hair lineage determination. This study uncovered a strong link between lineage-restriction and the distinct and ever-changing climates that arise at distinct stages of HF regeneration. Such organization implies the importance of a progenitor's microenvironment in fate decisions, and in this regard is similar to the intimate relation between stem cells and their niche. The findings further indicate that external signals drive chromatin remodeling at each step along the path, so that lineage diversification unfolds in a highly choreographed process of temporal and spatial steps. Finally, it was found that new fates are acquired by layering new signaling effectors on top of previously established chromatin platforms, which keeps stem cell progeny increasingly set in their fates. As future studies are conducted, the multiplicity of external inputs that drive these choices will continue to unfold (Adam, 2018).
ß-catenin and plakoglobin (gamma-catenin) are homologous molecules involved in cell adhesion, linking cadherin receptors to the cytoskeleton. ß-catenin is also a key component of the Wnt pathway by being a coactivator of LEF/TCF transcription factors. To identify novel target genes induced by ß-catenin and/or plakoglobin, DNA microarray analysis was carried out with RNA from cells overexpressing either protein. This analysis revealed that Nr-CAM is the gene most extensively induced by both catenins. Overexpression of either ß-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for its activation by catenins. Retroviral transduction of Nr-CAM into NIH3T3 cells stimulates cell growth, enhances motility, induces transformation, and produces rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression is elevated in human colon cancer tissue and cell lines and in human malignant melanoma cell lines but not in melanocytes or normal colon tissue. Dominant negative LEF-1 decreases Nr-CAM expression and antibodies to Nr-CAM inhibit the motility of B16 melanoma cells. The results indicate that induction of Nr-CAM transcription by ß-catenin or plakoglobin plays a role in melanoma and colon cancer tumorigenesis, probably by promoting cell growth and motility (Conacci-Sorrell, 2002).
Metastasis from lung adenocarcinoma can occur swiftly to multiple organs within months of diagnosis. The mechanisms that confer this rapid metastatic capacity to lung tumors are unknown. Activation of the canonical WNT/TCF pathway is identified here as a determinant of metastasis to brain and bone during lung adenocarcinoma progression. Gene expression signatures denoting WNT/TCF activation are associated with relapse to multiple organs in primary lung adenocarcinoma. Metastatic subpopulations isolated from independent lymph node-derived lung adenocarcinoma cell lines harbor a hyperactive WNT/TCF pathway. Reduction of TCF activity in these cells attenuates their ability to form brain and bone metastases in mice, independently of effects on tumor growth in the lungs. The WNT/TCF target genes HOXB9 and LEF1 are identified as mediators of chemotactic invasion and colony outgrowth. Thus, a distinct WNT/TCF signaling program through LEF1 and HOXB9 enhances the competence of lung adenocarcinoma cells to colonize the bones and the brain (Nguyen, 2009).
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