Persistent expression of stabilized beta-catenin delays maturation of radial glial cells into intermediate progenitors

Transgenic mice expressing stabilized beta-catenin in neural progenitors develop enlarged brains resulting from increased progenitor expansion. To more precisely define beta-catenin regulation of progenitor fate, we employed a conditional transgenic approach to delete the beta-catenin regulatory domain from neural progenitors, resulting in expression of stabilized protein from its endogenous promoter in these cells and their progeny. An increased fraction of transgenic cortical cells express the progenitor markers Nestin and LewisX, confirming a relative expansion of this population. Sustained beta-catenin activity expands RC2 and Pax6 expression in the developing cortex while postponing the onset of Tbr2 expression, suggesting a delay in maturation of radial glia into intermediate progenitors. Furthermore, transgenic cortical cells fail to either upregulate ErbB4 or develop a mitogenic response to epidermal growth factor, changes that normally accompany the acquisition of an intermediate fate. Likewise, transgenic brains do not develop a distinct subventricular zone or superficial cortical layers, and overexpression of stabilized beta-catenin by in utero electroporation caused a relative reduction of upper layer vs. lower layer cortical neurons, indicating that persistent beta-catenin activity interferes with the generation of progenitors responsible for the production of upper layer cortical neurons. Collectively, these findings demonstrate that beta-catenin functions to maintain the radial glial population, and suggest that downregulation of beta-catenin signaling may be critical to facilitate the transition to an intermediate progenitor phenotype.     

Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. Our data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure.     

Hypoxia requires notch signaling to maintain the undifferentiated cell state

In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. Here, we show that the latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process.     

Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.     

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7(+) MyoD(-) undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis.     

Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.     

Mind bomb 1 is essential for generating functional Notch ligands to activate Notch

The Delta-Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for cell fate specification. Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. We now report that mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulated in the plasma membrane, while it was localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling.     

Beta1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination

Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and beta1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of beta1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in beta1 integrin-null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in beta1 integrin-null nerves improves sorting. Thus, increased activation of Rac1 by beta1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.     

T/NK bipotent progenitors in the thymus retain the potential to generate dendritic cells

We have previously shown that the earliest thymic progenitors retain the potential to generate T and NK cells and that they lose the bipotentiality to give rise to unipotent T and NK progenitors during the progression of intrathymic developmental stages. The present study examines the ability of these thymic progenitors for generation of dendritic cells (DC) with a new clonal assay that is capable of determining the developmental potential for DC in addition to T cells and NK cells. We found that the large majority of the T/NK bipotential progenitors in the earliest population of fetal thymus was able to generate DC. Although the DC potential is lost with the progression of the differentiation stage, some of the T/NK bipotential progenitors still retain their DC potential even at the CD44(+)CD25(+) stage.     

Noncanonical notch signaling modulates cytokine responses of dendritic cells to inflammatory stimuli

Dendritic cell (DC)-derived cytokines play a key role in specifying adaptive immune responses tailored to the type of pathogen encountered and the local tissue environment. However, little is known about how DCs perceive the local environment. We investigated whether endogenous Notch signaling could affect DC responses to pathogenic stimuli. We demonstrate that concurrent Notch and TLR stimulation results in a unique cytokine profile in mouse bone-marrow derived DCs characterized by enhanced IL-10 and IL-2, and reduced IL-12 expression compared with TLR ligation alone. Unexpectedly, modulation of cytokine production occurred through a noncanonical Notch signaling pathway, independent of γ-secretase activity. Modulation required de novo protein synthesis, and PI3K, JNK, and ERK activity were necessary for enhanced IL-2 expression, whereas modulation of IL-10 required only PI3K activity. Further, we show that this γ-secretase-independent Notch pathway can induce PI3K activity. In contrast, expression of the canonical Notch target gene Hes1 was suppressed in DCs stimulated with Notch and TLR ligands simultaneously. Thus, our data suggest that Notch acts as an endogenous signal that modulates cytokine expression of DCs through a noncanonical pathway and therefore has the potential to tailor the subsequent adaptive immune response in a tissue- and/or stage-dependent manner.     

The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells

The EGF-like homeotic gene Dlk1 appears to function as an inhibitor of adipogenesis. Overexpression of Dlk1 prevents adipogenesis of 3T3-L1 cells. Dlk1-deficient mice are obese; however, adipose tissue still develops in Fc-dlk1 transgenic mice, suggesting that Dlk1 is not a strict inhibitor of adipogenesis. To clarify the role of Dlk1 in adipogenesis, we studied whether Dlk1 could act differently on this process depending upon the differentiation state of the precursor cells. We found that Dlk1 is a potentiator of adipogenesis for mesenchymal C3H10T1/2 cells. This potentiating effect can be triggered by overexpressing the entire protein or the extracellular EGF-like-containing region, but not by overexpressing the intracellular dlk1 sequence. In addition, coculture of C3H10T1/2 cells with other cells expressing Dlk1, but not with cells lacking Dlk1 expression, enhances their adipogenic response. Potentiation of adipogenesis by Dlk1 was associated with changes in the activation of ERK1/2 after IGFI/insulin induction. Finally, as reported with other cells, dlk1 functioned as a Notch signaling inhibitor in C3H10T1/2 cells, but inhibition of Notch1 expression prevented the potentiating effects of Dlk1 in adipogenesis. These data suggest that Dlk1 may potentiate or inhibit adipogenesis depending upon the cellular context, and that Notch1 expression and activation are important factors in this context.     

Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors

Radial glia are among the first cells that develop in the embryonic central nervous system. They are progenitors of glia and neurons but their relationship with restricted precursors that are also derived from neuroepithelia is unclear. To clarify this issue, we analyzed expression of cell type specific markers (BLBP for radial glia, 5A5/E-NCAM for neuronal precursors and A2B5 for glial precursors) on cortical radial glia in vivo and their progeny in vitro. Clones of cortical cells initially expressing only BLBP gave rise to cells that were A2B5+ and eventually lost BLBP expression in vitro. BLBP is expressed in the rat neuroepithelium as early as E12.5 when there is little or no staining for A2B5 and 5A5. In E13.5-15.5 forebrain, A2B5 is spatially restricted co-localizing with a subset of the BLBP+ radial glia. Analysis of cells isolated acutely from embryonic cortices confirmed that BLBP expression could appear without, or together with, A2B5 or 5A5. The numbers of BLBP+/5A5+ cells decreased during neurogenesis while the numbers of BLBP+/A2B5+ cells remained high through the beginning of gliogenesis. The combined results demonstrate that spatially restricted subpopulations of radial glia along the dorsal-ventral axis acquire different markers for neuronal or glial precursors during CNS development.     

pdx-1 function is specifically required in embryonic beta cells to generate appropriate numbers of endocrine cell types and maintain glucose homeostasis

The pdx1 gene is essential for pancreatic organogenesis in humans and mice; pdx1 mutations have been identified in human diabetic patients. Specific inactivation of pdx1 in adult beta cells revealed that this gene is required for maintenance of mature beta cell function. In the following study, a Cre-lox strategy was used to remove pdx1 function specifically from embryonic beta cells beginning at late-gestation, prior to islet formation. Animals in which pdx1 is lost in insulin-producing cells during embryogenesis had elevated blood glucose levels at birth and were overtly diabetic by weaning. Neonatal and adult mutant islets showed a dramatic reduction in the number of insulin(+) cells and an increase in both glucagon(+) and somatostatin(+) cells. Lineage tracing revealed that excess glucagon(+) and somatostatin(+) cells did not arise by interconversion of endocrine cell types. Examination of mutant islets revealed a decrease in proliferation of insulin-producing cells just before birth and a concomitant increase in proliferation of glucagon-producing cells. We propose that pdx1 is required for proliferation and function of the beta cells generated at late gestation, and that one function of normal beta cells is to inhibit the proliferation of other islet cell types, resulting in the appropriate numbers of the different endocrine cell types.     

Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia

Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not.     

The notch signaling pathway is required to specify muscle progenitor cells in Drosophila.

Organization and function of the Notch signaling pathway in Drosophila are best understood with respect to its role in the process of selection of neural progenitor cells. However, there is evidence that, besides neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these cells is increased in neurogenic mutants, and it has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Here, I present evidence for the participation of additional elements of the Notch signaling pathway in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells.     

Reelin induces a radial glial phenotype in human neural progenitor cells by activation of Notch-1

Background  

Reelin and Notch-1 signaling pathways have been recently found to be necessary to induce the expression of brain lipid binding protein (BLBP) and to promote the process extension and the maturation of the neuronal progenitors, the radial glial cells. In this study, we report the cross talk between these two pathways.     

Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.