extra macrochaetae

Emc homologs and differentiation

In humans, uterine attachment requires that the placenta's specialized epithelial cells, termed cytotrophoblasts, aggressively invade the uterine lining. Other than the fact that these cells stop proliferating and invasion is limited to a circumscribed area (the decidualized endometrium and the inner third of the myometrium), this process is more akin to tumorigenesis than to organogenesis. The method whereby cytotrophoblasts initiate maternal blood flow to the placenta is equally unusual. During invasion, a subpopulation of cells opens the termini of uterine blood vessels. Subsequently, these fetal cells replace the resident maternal endothelium and portions of the smooth muscle wall, thereby creating a hybrid vasculature that is particularly evident on the arterial side of the circulation. Viewed from a developmental perspective, these unusual attributes are the end result of cytotrophoblast differentiation along the invasive pathway. The cytotrophoblast stem cells form a polarized epithelium that is attached to the basement membrane that surrounds the stromal cores of chorionic villi. During differentiation/invasion, cytotrophoblasts leave this basement membrane to form columns of unpolarized cells that attach to, then penetrate, the uterine wall. The ends of the columns terminate within the superficial endometrium, where they give rise to invasive cytotrophoblasts. During interstitial invasion, a subset of these cells, either individually or in small clusters, commingles with resident decidual, myometrial and immune cells. During endovascular invasion, masses of cytotrophoblasts migrate into the vessels before the lumina eventually recanalize. Together, the two components of cytotrophoblast invasion anchor the placenta to the uterus and permit a steady increase in the supply of maternal blood that is delivered to the developing fetus (Janatpour, 2000).

Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop- helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. The importance of this family of factors in the human placenta was examined. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain [(Inhibitor of DNA binding proteins (Id-1 to Id-4)]. During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, Id expression was characterized in cytotrophoblast cells. The cells express Id-3 constitutively; Id-2 is downregulated, at the mRNA and protein levels, as the cells differentiate in culture and in situ, respectively. In cases when cytotrophoblast differentiation is compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression is maintained. To assess the functional relevance of these correlations, an adenovirus vector was used to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retain characteristics of undifferentiated cells: a1 integrin expression is low and cyclin B expression is retained. Furthermore, invasion through Matrigel is partially inhibited and migration is strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development (Janatpour, 2000).

Vertebrate neural crest cells, derived from the neural folds, generate a variety of tissues, such as cartilage, ganglia, and cranial (intramembranous) bone. The chick homolog of the helix-loop-helix transcriptional regulator Id2 is expressed in cranial but not trunk neural folds and subsequently in some migrating cranial neural crest cells. Ectopic expression of Id2 with recombinant retroviruses converts ectodermal cells to a neural crest fate, demonstrating that proper regulation of Id2 is important for sustaining epidermal traits. In addition, overexpression of Id2 results in overgrowth and premature neurogenesis of the dorsal neural tube. These results suggest that Id2 may allocate ectodermal precursors into neural rather than epidermal lineages (Martinsen, 1998).

Transcription factors with a basic helix-loop-helix (HLH) motif have been shown to be crucial for various cell differentiation processes during development of multicellular organisms. Id proteins inhibit the functions of these transcription factors in a dominant-negative manner by suppressing their heterodimerization partners through the HLH domains. Members of the Id family also promote cell proliferation, implying a role in the control of cell differentiation. Id2 is indispensable for normal development of mice. Id2-/- mice lack lymph nodes and Peyer's patches. However, their splenic architecture is normal, exhibiting T-cell and B-cell compartments and distinct germinal centers. The cell population that produces lymphotoxins, essential factors for the development of secondary lymphoid organs, is barely detectable in the Id2-/- intestine. Furthermore, the null mutants show a greatly reduced population of natural killer (NK) cells, which is due to an intrinsic defect in NK-cell precursors. These results indicate that Id2 has an essential role in the generation of peripheral lymphoid organs and NK cells (Yokota, 1999).

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements, inhibits development of the transduced cells into TCRalphabeta and gammadelta cells in a fetal thymic organ culture (FTOC). Overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalphabeta but not into TCRgammadelta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalphabeta and gammadelta cells. Cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes develop from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells have properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint (Blom, 1999).

An intracellular timer is thought to help control the timing of oligodendrocyte differentiation. The expression of the helix-loop-helix gene Id4 in oligodendrocyte precursor cells decreases in vivo and in vitro with a time course expected if Id4 is part of the timer. Id4 expression decreases prematurely when the precursor cells are induced to differentiate by mitogen withdrawal. Both Id4 mRNA and protein decrease together under all of these conditions, suggesting that the control of Id4 expression is transcriptional. Enforced expression of Id4 stimulates cell proliferation and blocks differentiation induced by either mitogen withdrawal or treatment with thyroid hormone. These findings suggest that a progressive fall in Id4 transcription is part of the intracellular timer that helps determine when oligodendrocyte precursor cells withdraw from the cell cycle and differentiate (Kondo, 2000).

Id proteins are thought to be negative regulators of cell differentiation and positive regulators of cell proliferation. Mammary glands of Id2-/- female mice reveal severely impaired lobulo-alveolar development during pregnancy. Id2-/- mammary epithelia show no precocious maturation, but instead exhibit intrinsic defects in both cell proliferation and cell survival, implying that the role of Id2 in pregnant mammary epithelia is mainly stimulation of cell proliferation and support of cell viability. Expression studies of genes required for mammary gland development suggest Id2 to be a downstream or parallel factor of these genes. A decrease in the DNA binding activity of Stat5 was also observed in Id2-/- mammary glands at 7 days post-coitus. These results indicate an indispensable role of Id2 in mammary glands of pregnant mice (Mori, 2000).

Compared to neurons, the intracellular mechanisms that control glial differentiation are still poorly understood. Oligodendrocyte lineage cells express the helix-loop-helix proteins Mash1 and Id2. Although Mash1 has been found to regulate neuronal development, in the absence of Mash1 oligodendrocyte, differentiation occurs normally. In contrast, it was found that overexpression of Id2 powerfully inhibits oligodendrocyte differentiation; Id2 normally translocates out of the nucleus at the onset of differentiation, and that absence of Id2 induces premature oligodendrocyte differentiation in vitro. These findings demonstrate that Id2 is a component of the intracellular mechanism that times oligodendrocyte differentiation and point to the existence of an as yet unidentified MyoD-like bHLH protein necessary for oligodendrocyte differentiation (Wang, 2001).

Emc homologs, the suppression of cell growth, and apoptosis

The helix-loop-helix transcription factor E2A plays several important developmental roles: not only does it promote cellular differentiation, but it also suppresses cell growth. Id proteins, inhibitors of E2A, have the opposite effects on cell differentiation and growth. To understand the mechanisms by which E2A suppresses cell growth, the role of E2A was examined in regulating the expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1/SD11 (See Drosophila Dacapo), which when overexpressed, prevents cell cycle progression. Overexpression of E2A can transcriptionally activate the p21 gene. Out of the eight putative E2A-binding sequences (E1 to E8) in the promoter, the E1 to E3 sequences located close to the transcription start site have been found to be essential. Loss of the E boxes in the promoter also reduces p21 expression without cotransfection with E2A in HIT pancreatic cells, where the endogenous E2A-like activity is high. Overexpression of E2A in 293T cells activates expression of the endogenous p21 gene at both the mRNA and protein levels. In correlation with the finding that E47 overexpression leads to growth arrest in NIH 3T3 cells, it has been shown that Id1 overexpression in NIH 3T3 cells accelerates cell growth and inhibits p21 expression. Taken together, these results provide insight into the mechanisms by which E2A and Id proteins control cell growth (Prabhu, 1997).

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators that function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. Nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. Disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life (Yan, 1997).

The Id family of helix-loop-helix proteins function as negative regulators of cell differentiation and as positive regulators of G1 cell cycle control. Enforced overexpression of the Id3 gene suppresses the colony-forming efficiency of primary rat embryo fibroblasts. Cotransfection with the antiapoptotic Bcl2 or BclXL genes alleviates this suppression and leads to cell immortalization. Consistent with this, enforced expression of Id genes in isolation is found to be a strong inducer of apoptosis in serum-deprived fibroblast cells. Id3-induced apoptosis is mediated at least in part through p53-independent mechanisms and can be efficiently rescued by Bcl2, BclXL, and the basic helix-loop-helix protein E47, which is known to oppose the functions of Id3 in vivo through the formation of stable heterodimers. Enforced overexpression of Id proteins has previously been shown to promote the cell cycle S phase in serum-deprived embryo fibroblasts. The extent of apoptosis induced by loss- and gain-of-function Id3 mutants and by wild-type Id3, either alone or in combination with the Bcl2, BClXL, and E47 genes, is invariably correlated with the relative magnitude of cell cycle S phase promotion. Id3-transfected cell populations displaying apoptosis and those in S phase are largely coincident in different experiments. These findings highlight the close coupling between the G1 progression and apoptosis functions of Id proteins and hint at a common mechanism for this family of transcriptional regulators in cell determination (Norton, 1998).

Members of the helix-loop-helix (HLH) family of Id proteins have demonstrated roles in the regulation of differentiation and cell proliferation. Id proteins inhibit differentiation by HLH-mediated heterodimerization with basic HLH transcription factors. This blocks their sequence-specific binding to DNA and activation of target genes that are often expressed in a tissue-specific manner. Id proteins can also act as positive regulators of cell proliferation. The different mechanisms proposed for Id-mediated promotion of entry into S phase also involve HLH-mediated interactions affecting regulators of the G1/S transition. Id2 augments apoptosis in both interleukin-3 (IL-3)-dependent 32D.3 myeloid progenitors and U2OS osteosarcoma cells. No similar activity could be detected for Id3. In contrast to the effects of Id2 on differentiation and cell proliferation, Id2-mediated apoptosis is independent of HLH-mediated dimerization. The ability of Id2 to promote cell death resides in its N-terminal region and is associated with the enhanced expression of a known component of the programmed cell death pathway, the proapoptotic gene BAX (Florio, 1998).

The neural crest is a unique population of mitotically active, multipotent progenitors that arise at the vertebrate neural plate border. The helix-loop-helix transcriptional regulator Id3 has a novel role in cell cycle progression and survival of neural crest progenitors in Xenopus. Id3 is localized at the neural plate border during gastrulation and neurulation, overlapping the domain of neural crest induction. Morpholino oligonucleotide-mediated depletion of Id3 results in the absence of neural crest precursors and a resultant loss of neural crest derivatives. This appears to be mediated by cell cycle inhibition followed by cell death of the neural crest progenitor pool, rather than a cell fate switch. Conversely, overexpression of Id3 increases cell proliferation and results in expansion of the neural crest domain. These data suggest that Id3 functions by a novel mechanism, independent of cell fate determination, to mediate the decision of neural crest precursors to proliferate or die (Kee, 2005 ).

Emc homologs and the cell cycle

The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. This activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 (See Drosophila Cyclin E) or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation (Hara, 1997).

The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signaling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle; this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro. An analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or hetero-dimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Whereas unphosphorylated Id3 abrogates an E12 homodimer complex but not the E12-MyoD heterodimer, phosphorylation at serine 5 results in a switch to abrogation of the E12-MyoD heterodimer complex. Compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. The Asp5 Id3 mutant is completely devoid of any activity in promoting S phase, implying that Cdk-dependent phosphorylation inactivates the G1-to-S cell cycle regulatory function of this Id protein. Therefore, Cdk2-dependent phosphorylation provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression: these data establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation (Deed, 1997).

Emc homologs and stem cells

The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Bone morphogenetic proteins act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. It is concluded that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3 (Ying, 2003).

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. This study demonstrates that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca_v1.2/1.3 (L-type) Ca²⁺ channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an 'excitation-neurogenesis coupling rule' within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories (Deisseroth, 2004).

Using an array of approaches, the coupling of excitation to neurogenesis in proliferating adult-derived NPCs was studied both in vitro and in vivo. Adult neurogenesis is potently enhanced by excitatory stimuli and involves Ca_v1.2/1.3 channels and NMDA receptors. These Ca²⁺ influx pathways are located on the proliferating NPCs, allowing them to directly sense and process excitatory stimuli. No effect of excitation was found on the extent of differentiation in individual cells (measured by extent of MAP2ab expression in the NPC-derived neurons) nor were effects observed on proliferative rate or fraction, survival, or apoptosis. Instead, excitation increased the fraction of NPC progeny that were neurons, both in vitro and in vivo, and total neuron number was increased as well. The Ca²⁺ signal in NPCs leads to rapid induction of a proneural gene expression pattern involving the bHLH genes HES1, Id2, and NeuroD, and the resulting cells become fully functional neurons defined by neuronal morphology, expression of neuronal structural proteins (MAP2ab and Doublecortin), expression of neuronal TTX-sensitive voltage-gated Na⁺ channels, and synaptic incorporation into active neural circuits. A monotonically increasing function characterizes excitation-neurogenesis coupling, and incorporation of this relationship into a layered Hebbian neural network suggests surprising advantages for both the clearance of old memories and the storage of new memories. Taken together, these results provide a new experimental and theoretical framework for further investigation of adult excitation-neurogenesis coupling (Deisseroth, 2004).

In the hippocampal formation, neural stem cells exist either within the adjacent ventricular zone or within the subgranular zone proper at the margin between the granule cell layer and the hilus, where proliferative activity is most robust. These cells do not express neuronal markers but proliferate and produce dividing progeny that incrementally commit to differentiated fates (such as the neuronal lineage) over successive cell divisions. Native NPC populations in vivo are therefore heterogenous with regard to lineage potential, and markers are not available that distinguish between the multipotent stem cell and the subtly committed yet proliferative progenitor cell. Excitation may therefore act on either or both types of proliferating precursor, in vitro and in vivo. The functional consequences of coupling excitation to insertion of new neurons for the neural network, however, is independent of which precursor cell types respond to excitation (Deisseroth, 2004).

The enhancement of hippocampal neurogenesis by behavioral stimuli such as environmental enrichment and running may, at least in part, be implemented at the molecular level by excitation-neurogenesis coupling. Notably, running and environmental enrichment increase adult neurogenesis in the hippocampus but not in the subventricular zone. Of course, not every neurogenic region in the brain need follow the excitation-neurogenesis coupling rule outlined here. An activity rule appropriate for the unique information processing or storage function of that brain region might be expected to operate. In this context, it is interesting to note that, while subventricular zone/olfactory bulb precursor neurogenesis is not enhanced by behavioral activity, proliferation and survival in this system can be influenced by olfactory sensory stimuli. This suggests that a different form of activity rule, appropriate for that local circuit, may govern olfactory bulb neurogenesis (Deisseroth, 2004).

The mechanisms that determine whether a precursor cell re-enters the cell cycle or exits and differentiates are crucial in determining the types and numbers of cells that constitute a particular organ. Id4 is required for normal brain size, and regulates lateral expansion of the proliferative zone in the developing cortex and hippocampus. In its absence, proliferation of stem cells in the ventricular zone (VZ) is compromised. In early cortical progenitors, Id4 is required for the normal G1-S transition. By contrast, at later ages, ectopically positioned proliferating cells are found in the mantle zone of the Id4^-/- cortex. These observations, together with evidence for the premature differentiation of early cortical stem cells, indicate that Id4 has a unique and complex function in regulating neural stem cell proliferation and differentiation (Yun, 2004).

A role for Id2 in regulating photic entrainment of the mammalian circadian system

Inhibitor of DNA binding genes (Id1-Id4) encode helix-loop-helix (HLH) transcriptional repressors associated with development and tumorigenesis, but little is known concerning the function(s) of these genes in normal adult animals. Id2 has been identified in DNA microarray screens for rhythmically expressed genes, and further analysis revealed a circadian pattern of expression of all four Id genes in multiple tissues including the suprachiasmatic nucleus. To explore an in vivo function, deletion mutations of Id2 and of Id4 were generated and characterized. Id2^-/- mice exhibit abnormally rapid entrainment and an increase in the magnitude of the phase shift of the pacemaker. A significant proportion of mice also exhibit disrupted rhythms when maintained under constant darkness. Conversely, Id4^-/- mice did not exhibit a noticeable circadian phenotype. In vitro studies using an mPer1 and an AVP promoter reporter revealed the potential for ID1, ID2, and ID3 proteins to interact with the canonical basic HLH clock proteins BMAL1 and CLOCK. These data suggest that the Id genes may be important for entrainment and operation of the mammalian circadian system, potentially acting through BMAL1 and CLOCK targets (Duffield, 2009).

Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. During development, progeny of multipotent progenitors in the thymus transit through four subsets as CD4^-CD8^- [double negative (DN)] cells, before expressing both CD4 and CD8 at the double positive (DP) stage. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo β-selection, during which cells expressing functionally rearranged TCRβ proliferate and differentiate into CD4+CD8+ progeny, Notch1 signaling is abruptly down-regulated. This report investigated the mechanisms that control Notch1 expression during thymopoiesis. Notch1 and E2A directly regulate Notch1 transcription in pre-β-selected thymocytes. Following successful β-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-β-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. These findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions (Yashiro-Ohtani, 2009).

Notch1 controls multiple essential functions during thymocyte development. Notch1 signals initiate the generation of the earliest intrathymic T cells from multipotent hematopoietic progenitors. Subsequently, Notch1 is required for αα T-cell development through β-selection, an important checkpoint during which immature thymocytes expressing functionally rearranged TCRα proliferate and then differentiate into quiescent CD4⁺CD8⁺ cells. Conditional inactivation of Notch1, Rbpj, or inhibition of Notch signaling by dominant-negative Mastermind-like 1 ((DNMAML) arrests T-cell development at the DN3 stage, prior to β-selection. In vitro studies using OP9 feeder cells have shown that both Notch1 and pre-TCR signals are required to traverse the β-selection checkpoint; Notch1 provides important differentiation, survival, proliferation, and metabolic signals during this juncture in T-cell development (Yashiro-Ohtani, 2009).

Following β-selection, Notch signaling and Notch1 expression are abruptly down-regulated. CD27 expression can be used to separate DN3 cells into two distinct populations, DN3a and DN3b. The pre-β-selection CD27^-DN3a population is Notch-dependent, whereas post-β-selection CD27⁺DN3b cells do not require Notch signals for further intrathymic differentiation or survival. Significantly, Notch1 expression is high in DN3a cells and low in DN3b cells (Yashiro-Ohtani, 2009).

Although the mechanism of Notch1 down-regulation in β-selected cells is poorly understood, high levels of Notch signaling post-β-selection may be oncogenic. For example, expression of the Notch1 intracellular domain (ICN1) driven by either a retroviral vector or a Lck transgene allows sustained Notch activity past the DN3 stage that is associated with increased proliferation and survival, a developmental block, and acute lymphoblastic T-cell leukemia (T-ALL). These findings emphasize the importance of precise control of Notch1 signaling at the β-selection checkpoint (Yashiro-Ohtani, 2009).

E-proteins, which include E12, E47, E2-2, and HEB in mammals, encode a class of widely expressed basic helix-loop-helix (bHLH) transcription factors that are critical for B-cell development and play important roles in thymocyte development. E12 and E47 (collectively termed E2A) are encoded by one gene, Tcfe2a, and are generated through alternative splicing, whereas E2-2 and HEB are encoded by distinct genes. The primary E-protein complex in thymocytes is a E47/HEB heterodimer. The functions of E-proteins in thymocyte development have been revealed through several loss-of-function approaches. E2A knockout mice exhibit an incomplete block in early T-cell development at the DN1 stage, whereas HEB knockout mice display reduced thymic cellularity and increased immature single positive (ISP) cells. Expression of a HEB dominant-negative protein causes a more severe decline in thymocyte numbers and an earlier block in T-cell development than HEB knockout mice, as this antagonist prevents compensation by other E-proteins. Enforced expression of the E-protein antagonist Inhibitor of DNA binding 3 (Id3) in human T-lineage precursor cells blocks T-cell lineage differentiation from CD34⁺ progenitors. Like Notch, E2A activity is dynamically regulated during thymocyte development. E2A is active prior to β-selection, whereupon pre-TCR signals up-regulate Id3 expression to reduce the DNA-binding activity of E2A in DP or DN thymocytes (Yashiro-Ohtani, 2009).

Emerging data suggest cross-talk between E2A and Notch signals during T-cell development. Expression of several genes that are important in T-cell development, such as Hes1 and pTα, are coregulated by Notch and E2A, and both Notch1 and Notch3 mRNA levels are decreased in E47-deficient fetal thymocytes. Furthermore, retroviral ICN1 expression in E2A^-/- fetal thymocyte progenitors rescues the developmental arrest caused by E2A deficiency. Although they provide synergistic functions, the precise nature of the interactions between Notch and E2A have not been determined (Yashiro-Ohtani, 2009).

This study investigated the mechanism underlying the dynamic regulation of Notch1 during β-selection. Prior to β-selection, Notch1 and E2A bind the Notch1 locus and promote Notch1 transcription in DN3 cells. At β-selection, MAPK-dependent pre-TCR signals up-regulate Id3 expression, which inhibits E2A binding to the Notch1 promoter and decreases Notch1 expression. Consistent with this model, loss of Id3 expression enhances Notch1 expression in post-β-selected thymocytes, whereas loss of E2A decreases Notch1 expression in pre-β-selected thymocytes in a dose-dependent manner. Furthermore, enforced Id3 expression inhibits Notch1 expression and Notch1-dependent cell survival in Notch1-dependent T-cell lines. Together, these data reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development and provide new strategies to disable Notch1 expression and signaling (Yashiro-Ohtani, 2009).

Emc Homologs and Transformation

The E2A gene products, E12 and E47, are critical for proper early B-cell development and commitment to the B-cell lineage. Loss of E2A activity results in a partial block at the earliest stage of T-lineage development. This early T-cell phenotype precedes the development of a T-cell lymphoma that occurs between 3 and 9 months of age. The thymomas are monoclonal and highly malignant, displaying a cell surface phenotype similar to that of immature thymocytes. In addition, the thymomas generally express high levels of c-myc. Each of the tumor populations analyzed shows a nonrandom gain of chromosome 15, which contains the c-myc gene. Taken together, the data suggest that the E2A gene products play a role early in thymocyte development that is similar to their function in B-lineage determination. The lack of E2A results in development of T-cell malignancies. It is proposed that E2A inactivation is a common feature of a wide variety of human T-cell proliferative disorders, including those involving the E2A heterodimeric partners tal-1 and lyl-1 (Bain, 1997).

Ids regulate differentiation1 through sequestration of basic helix-loop-helix (bHLH) transcription factors, and the consequent inhibition of their ability to bind DNA. Although all Id proteins are viewed as positive regulators of cell-cycle progression, this role has been firmly established only for one member of the Id family, Id2. Only Id2, and not the other members of the family, Id1 and Id3, is able to disrupt the antiproliferative effects of tumor suppressor proteins of the Rb family (the 'pocket' proteins: Rb, p107 and p130), thus allowing cell-cycle progression. This function correlates with the ability of Id2, but not Id1 and Id3, to associate physically with active, hypophosphorylated forms of the pocket proteins in vitro and in vivo. By inactivating Rb, Id2 is also able to abolish the function of another growth-inhibitory protein, p16, that operates upstream of Rb (Lasorella, 2000).

The Rb-null phenotype is lethal by embryonic day 14.5 because of widespread proliferation, defective differentiation and apoptosis in the nervous system and haematopoietic precursors. Since Id2 is expressed in these cell types at the time that Rb-null embryos die1, it is hypothesized that, if Id2 is a natural target of Rb, manifestation of the Rb-mutant phenotype might require intact Id2 (Lasorella, 2000).

Disruption of the Rb pathway (which also includes cyclin D, cdk4/6 and p16) is a hallmark of cancer and it is widely accepted that normal Rb function must be removed, one way or another, in all human tumors. Therefore, it was of interest to determine whether tumor cells deregulate Id2 to bypass the Rb pathway. Correct expression of Id2 is essential to regulate proliferation and differentiation of the neural crest, thus neural crest precursor cells might be sensitive to inappropriate expression of Id2. In humans, neoplastic transformation of neural crest precursors during embryogenesis causes neuroblastoma. Interestingly, genetic alterations of Rb, cyclin D, cdk4/6 or p16 are absent in neuroblastoma. The genetic hallmark of neuroblastoma is amplification of the gene for a member of the Myc family of proto-oncogenes, N-myc. Resembling enforced expression of Id2, Myc overexpression is sufficient to bypass the Rb-p16 growth-inhibitory pathway, in spite of persistent hypophosphorylated Rb. Consequently, Myc activation may release the pressure to mutate components of the Rb-p16 pathway during tumorigenesis (Lasorella, 2000).

Id2-Rb double knockout embryos survive to term with minimal or no defects in neurogenesis and hematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumor-suppressor function of Rb (Lasorella, 2000).

Inhibitor of differentiation 4 drives brain tumor-initiating cell genesis through cyclin E and notch signaling

Cellular origins and genetic factors governing the genesis and maintenance of glioblastomas (GBM) are not well understood. This study reports a pathogenetic role of the developmental regulator Id4 (inhibitor of differentiation 4) in GBM. In primary murine Ink4a/Arf(-/-) astrocytes, and human glioma cells, evidence is provided that enforced Id4 can drive malignant transformation by stimulating increased cyclin E to produce a hyperproliferative profile and by increased Jagged1 expression with Notch1 activation to drive astrocytes into a neural stem-like cell state. Thus, Id4 plays an integral role in the transformation of astrocytes via its combined actions on two-key cell cycle and differentiation regulatory molecules (Jeon, 2008).

The impact was examined of activated Notch signaling on the immature differentiation profiles and neurosphere-forming capacity of Id4-transduced Ink4a/Arf^-/- astrocytes. Notch1, but not cyclin E, knockdown resulted in a marked decrease in the expression of NSC markers, Nestin, Cd133, and Hes1, and correspondingly, NIC overexpression in Ink4a/Arf^-/- astrocytes induced expression of these immature markers. In the neurosphere assay, Notch1 knockdown resulted in a significant decrease in neurosphere number from the Id4-transduced Ink4a/Arf^-/- astrocytes compared with a modest decrease in the cyclin E knockdown cultures. Furthermore, NIC overexpression, but not cyclin E, was comparable with Id4 in promoting neurosphere formation following their transduction into Ink4a/Arf^-/- astrocytes. Pharmacological inhibition of Notch signaling (DAPT, a γ-secretase inhibitor) or Jagged1 knockdown in Id4-transduced Ink4a/Arf^-/- astrocytes resulted in decreased neurosphere-forming capacity and NSC marker expression. Thus, Id4-induced activation of Jagged-Notch axis in Ink4a/Arf^-/- astrocytes plays an essential role in promoting the neural stem cell-like phenotype (Jeon, 2008).

Next, attempts were made to corroborate the murine findings in human glioma cells. shRNA-mediated depletion of Id4 in human LN229 glioma cells (which express the highest levels of Id4) resulted in down-regulation of cyclin E, Jagged1, NIC, Notch-downstream target genes (Hes1, Hey1, and Hey2), and Notch transcriptional activity, as well as a marked decrease in cell proliferation. Conversely, Id4 overexpression in human A172 glioma cells (which express low endogenous levels of Id4) induced up-regulation of Jagged1, NIC, and cyclin E; Notch transcriptional activity; cell proliferation; and neurosphere formation. It was also found that expression levels of Id4, NIC, Jagged1, and cyclin E were markedly increased in the primary human glioma stem cell line, NCI0822, as compared with NHA and HB1.F3 cells (Jeon, 2008).

Furthermore, a Tet-On-inducible gene expression system was used to assess whether Id4 directly leads to induction of cyclin E and Notch signaling. Id4 was markedly increased in the Ink4a/Arf^-/- astrocytes transduced with rtTA and Rev-TRE-Id4 grown in the presence of doxycycline (Dox) for 2 d compared with Dox-untreated counterpart cells. It was also found that Dox-treated cells showed marked elevations in the levels of cyclin E, Jagged1, and NIC. These results strengthen the link between Id4 and the control of cyclin E and Notch signaling in the astrocytes (Jeon, 2008).

In conclusion, Id4 can drive the malignant transformation of astrocytes via disregulation of cell cycle and differentiation control, achieved through the up-regulation of cyclin E and activation of Jagged-Notch1 signaling. These findings of Id4-induced developmental plasticity have implications for both the cellular origins of GBM as well as its prominent renewal potential in experimental and clinical trials setting. Thus, these observations may inform the rational development of anti-Id4 agents that may impede the insuperable nature of GBM recurrence (Jeon, 2008).

The ID1-CULLIN3 axis regulates intracellular SHH and WNT signaling in glioblastoma stem cells

Inhibitor of differentiation 1 (ID1; see Drosophila Extra macrochaetae) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. This study reports that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase (see Drosophila Cullin-3). ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 (see Drosophila Cubitus interruptus) and DVL2 (see Drosophila Dishevelled) and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and beta-CATENIN (see Drosophila Armadillo) effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.

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