Mouse numblike is the second mammalian gene to be isolated that shows significant sequence similarity to Drosophila numb. Numblike protein shows 76% homology to mouse Numb and 63.6% homology to Drosophila Numb. It is known that both m-Numb and Numblike, two murine numb proteins, can physically interact with the intracellular domain of Notch 1. m-Numb and Numblike each have distinct characteristics that suggest that Numblike would be unable to fully substitute for m-Numb. When expressed in dividing neural precursors in Drosophila, Numblike is symmetrically distributed in the cytoplasm, unlike either endogenous Drosophila Numb or expressed m-Numb, both of which are asymmetrically localized to one half of the cell membrane. In Drosophila numb loss-of-function mutant embryos, expression of Numblike allows both daughter cells of a neural precursor to adopt the fate of the cell that normally inherits Drosophila Numb. In mice, numblike mRNA is preferentially expressed in the adult and embryonic nervous system. In the developing neocortex, Numblike is expressed in postmitotic neurons in the cortical plate, but not in progenitors within the ventricular zone where m-Numb and Notch1 are expressed. Numblike appears to be a cytoplasmic protein while m-Numb is a membrane associated protein, as is Drosophila Numb. In dividing cortical progenitors, Notch1 is distributed around the entire membrane, unlike m-Numb, which is asymmetrically localized to the apical membrane. It is concluded that Numblike functions in postmitotic cells, with m-Numb to suppress the residual Notch1 activity in the cell and allow it to fully differentiate into a neuron. In contrast, m-Numb and Numb function to ensure that daughter cells of asymmetric divisions acquire distinct fates. It is proposed that an interplay between cell-intrinsic mechanisms (executed by m-numb and numblike) and cell-extrinsic mechanisms (mediated by Notch1) may be involved in both progenitor cell proliferation and neuronal differentiation during mammalian cortical neurogenesis (Zhong, 1997).

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast and nematodes. EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yield a number of putative EH interactors, all of which possessed NPF motifs that have been shown to be responsible for the interaction. Among these interactors are the human homolog of Numb, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. Coimmunoprecipitation of Eps15 with NUMB and RAB has been demonstrated. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening has established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, it is proposed that EH domains are involved in processes connected with the transport and sorting of molecules within the cell (Salcini, 1997).

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Numb is shown in this study to be an endocytic protein. Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process (Santolini, 2000).

During neurogenesis of the mammalian neocortex, neural progenitor cells divide to generate daughter cells that either become neurons or remain as progenitor cells. The mouse numb (m-numb) gene encodes a membrane-associated protein that is asymmetrically localized to the apical cell membrane of dividing cortical progenitor cells and may be segregated to only the apical daughter cell, which has been suggested to remain as a progenitor cell. To examine m-numb function during neural development, a loss-of-function mutant allele of m-numb was generated. Mice homozygous for this mutation exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain and die around embryonic day 11.5 (E11.5). These findings suggest that m-numb is an essential gene that plays a role in promoting progenitor cell fate during cortical neurogenesis (Zhong, 2000).

Despite being generated precociously, neurons in the mutant neocortex lay outside of the ventricular zone in the external mantle layer where neurons normally reside, suggesting that m-numb mutation has little effect on the placement of early neurons. Most cells in the ventricular zone of the E10.5 mutant neuroepithelium appear to be neural progenitor cells, as indicated by the expression of BF-1 and Pax-6 and staining with an antibody against the Nestin protein, a neural progenitor cell marker. Indeed, BrdUrd-positive nuclei appear after a short pulse of labeling in the outer half of the ventricular zone of both mutant and wild-type embryos; no gross difference in the density or location of these proliferating cells has been observed. Previous studies indicate that of the cell divisions in the neuroepithelium, only a small fraction (<20%) are asymmetric at the beginning of cortical neurogenesis. Therefore, it is not surprising that the majority of the neural progenitor cells that divide symmetrically to produce more progenitor cells appear not to be affected by the m-numb mutation (Zhong, 2000).

Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that, in Drosophila, antagonizes Notch signaling in connection with binary cell fate decisions of certain cell lineages. Although overexpression experiments have suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. Mice with a homozygous inactivation of Numb have been generated. These mice die before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos have an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons is reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons are formed. Trunk neural crest cells migrate and differentiate into sympathetic neurons. In contrast, a selective differentiation anomaly is observed in dorsal root ganglia, where neural crest-derived progenitor cells migrate normally to form ganglionic structures, but fail to differentiate into sensory neurons. Thus, mouse Numb is involved in multiple developmental processes and is required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb (Zilian, 2001).

No direct evidence was found to suggest that Numb antagonizes Notch signaling during murine development. Thus, Hes1 and Hes5, both effectors of Notch signaling in the nervous system, are not upregulated in Numb mutant mice. However, Hes genes might not be obligatory and unique mediators of Notch signaling, since in some cases their overexpression fails to recapitulate Notch responses. Thus, as indicated by its ability to interact with a multitude of target proteins, Numb might well exert Notch-independent functions, and some Notch signaling may be regulated independent of Numb, as has been observed in Drosophila. This is consistent with the finding that the loss of Numb had no general effect on the development of the sympathetic lineage in vivo, even though in vitro, Notch signaling prevents autonomic neurogenesis. The lineage-specific requirement of Numb is unlikely to be due to differential expression in the developing vertebrate nervous system, since Numb is broadly expressed, including in the anlage of sympathetic ganglia (Zilian, 2001).

The Mdm2 oncoprotein is a well-known inhibitor of the p53 tumor suppressor, but it may also possess p53-independent activities. In search of such p53-independent activities, the yeast two-hybrid screen was employed to identify Mdm2-binding proteins. In vitro and in transfected cells, Mdm2 can associate with Numb, a protein involved in the determination of cell fate. This association causes translocation of overexpressed Numb into the nucleus and leads to a reduction in overall cellular Numb levels. Through its interaction with Numb, Mdm2 may influence processes such as differentiation and survival. This could potentially contribute to the altered properties of tumor cells that overexpress Mdm2 (Juven-Gershon, 1999).

The orientation of cell divisions and the distribution of Numb has been studied in the developing rat retina. Whereas most retinal neuroepithelial cells divide with their mitotic spindles oriented parallel to the plane of the neuroepithelium, a substantial minority divides with their spindles oriented perpendicularly. The proportion of these vertically dividing cells changes during development, peaking around the day of birth. Numb appears to be inherited only by the apical daughter cell when a neuroepithelial cell divides vertically. Similarly, in dissociated cell cultures, some retinal neuroepithelial cells divide asymmetrically and distribute Numb to only one of the two daughter cells, suggesting that the dissociated cells can retain their polarity in vitro. Using retinal explant cultures, it has been found that the retinal pigment epithelium apparently promotes vertical divisions in the neural retina. This is the first evidence that asymmetric segregation of cell-fate determinants may contribute to cell diversification in the mammalian retina and that an epithelium controls this process by influencing the plane of division in the adjacent neural retina (Cayouette, 2001).

Siah-1 is shown to interact directly with and promote the degradation of the cell fate regulator Numb

The Drosophila Seven in absentia (Sina) gene product originally was described as a protein that controls cell fate decisions during eye development. Its mammalian homolog, Siah-1, recently was found to be involved in p53-dependent and -independent pathways of apoptosis and G1 arrest. Siah-1 is shown to interact directly with and promote the degradation of the cell fate regulator Numb. Siah-1-mediated Numb degradation leads to redistribution of endogenous cell-surface Notch to the cytoplasm and nucleus and to augmented Notch-regulated transcriptional activity. These data imply that through its ability to target Numb for degradation, Siah-1 can act as a key regulator of Numb-related activities, including Notch signaling (Susini, 2001).

Interaction-mapping experiments have revealed that GST-Numb binds to a minimal region of Siah-1 (Delta10), composed of amino acids 180-211. The region of Delta10 overlaps with the binding sites of other known Siah-1 interactors, including DCC and the recently described beta-catenin-binding proteins. Interestingly, in the corresponding site, two allelic mutations have been identified in Drosophila Sina that affect R7 photoreceptor development. Within the Numb protein, residues 91-400 are sufficient to bind Siah-1. This region includes the C-terminal part of the PTB domain of Numb. PTB domains have been implicated in phosphorylation-dependent and phosphorylation-independent molecular interactions (Susini, 2001).

Numb physically interacts with and inhibits the signaling of Notch1, a cell-surface receptor that promotes cell fate decisions by activating downstream transcription factors of the CSL family. This is achieved by proteolytic cleavage within an intracellular site of Notch that results in the release and subsequent translocation of its cytosolic fragment (NICD) into the nucleus. The consequences of Siah-1 overexpression on Notch subcellular localization were investigated. Confocal microscopy analysis of Notch1 immunofluorescence in control U937 cells has revealed a rim-like staining pattern typical of cell-surface receptors. In striking contrast, Siah-1-overexpressing cells exhibited a redistribution of Notch1 immunofluorescence in the cytoplasm and in the nucleus. Confocal imaging within the Z plane of the nucleus confirms Notch1 nuclear localization. To show that the observed pattern of Notch expression in Siah-1-overpressing U937 cells resembles that of an activation state, U937 vector control cells were analyzed for Notch translocation after EDTA treatment, which mimics the effects of ligand-induced nuclear translocation of Notch. By 30 min after EDTA removal, Notch1 immunofluorescence was visualized in and around the nucleus. Together, these observations suggest further that Siah-1 overexpression promotes Notch1 activation. This conclusion was validated directly by monitoring endogenous NICD activity in MCF-7 cells stably transfected with vector control or pBK-RSV-Siah-1. A luciferase reporter construct whose activation is proportional to NICD translocation to the nucleus was transfected into these cells. The presence of endogenous Notch1 was verified by Western blot analysis. A consistent, >3-fold increase in endogenous NICD activity was observed in MCF-7 cells overexpressing Siah-1, and this increase was reduced by transient transfection of Numb (Susini, 2001).

mNumb protein levels are regulated by ubiquitin-dependent proteolysis and therefore one mechanism to generate asymmetric localization of Numb is through asymmetric or localized degradation

LNX is a RING finger and PDZ domain containing protein that interacts with the cell fate determinant Numb. To investigate the function of LNX, its RING finger domain was tested for ubiquitin ligase activity. The isolated RING finger domain is able to function as an E2-dependent, E3 ubiquitin ligase in vitro and mutation of a conserved cysteine residue within the RING domain abolishes its activity, indicating that LNX is the first described PDZ domain-containing member of the E3 ubiquitin ligase family. Numb has been identified as a substrate of LNX E3 activity in vitro and in vivo. In addition to the RING finger, a region of LNX, including the Numb PTB domain-binding site and the first PDZ domain, is required for Numb ubiquitylation. Expression of wild-type but not mutant LNX causes proteasome-dependent degradation of Numb and can enhance Notch signaling. These results suggest that the levels of mammalian Numb protein and therefore, by extension, the processes of asymmetric cell division and cell fate determination may be regulated by ubiquitin-dependent proteolysis. In mammalian systems, the mechanism that gives rise to asymmetric distribution of Numb is unknown and so far orthologs of Pon and Miranda have not been described. This study shows that mNumb protein levels are regulated by ubiquitin-dependent proteolysis and therefore one mechanism to generate asymmetric localization of Numb is through asymmetric or localized degradation. The presence of multiple PDZ domains in LNX would be consistent with a model in which asymmetric or polarized distribution of LNX could establish asymmetric distribution of Numb protein (Nie, 2002).

The mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor

The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. The mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression results in the degradation of the Notch intracellular domain following activation -- this correlates with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb is required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 is not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement, Numb is shown to interact with the cytosolic HECT domain-containing E3 ligase Itch; Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes (McGill, 2003).

Membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates

The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD (Roncarati, 2002).

Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domains bind to integrin tails

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrin biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction the binding of a series of recombinant PTB domains to a panel of short integrin beta tails was examined. In addition to the known integrin-binding proteins, Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domains bind to integrin tails. Furthermore, Dok-1 physically associates with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirm that these interactions are canonical PTB domain-ligand interactions: (1) the interactions were blocked by mutation of an NPXY motif in the integrin tail; (2) integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. This specificity, and a molecular model of an integrin beta tail-PTB domain interaction, was used to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism (Calderwood, 2003).

Phosphorylation of Numb family proteins: possible involvement of Ca2+/calmodulin-dependent protein kinases

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), affinity chromatography purification was performed using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), two Numb family proteins (Numb and Numbl) were identified from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, the phosphorylation of Numb family proteins was observed in various rat tissue extracts, and the ionomycin-induced phosphorylation of endogenous Numb at Ser264 was observed in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, it was found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein (Tokumitsu, 2005; full text of article).

NUMB localizes in the basal cortex of mitotic avian neuroepithelial cells and modulates neuronal differentiation by binding to NOTCH-1

The importance of lateral inhibition mediated by Notch signaling is well demonstrated to control neurogenesis both in invertebrates and vertebrates. The chicken homolog of Drosophila numb, which suppresses Notch signaling, has been identified. NUMB (c-NUMB) protein is localized to the basal cortex of mitotic neuroepithelial cells, suggesting that c-NUMB regulates neurogenesis by the modification of Notch signaling through asymmetrical cell division. Consistent with this suggestion, it has been shown (1) that c-NUMB interferes with the nuclear translocation of activated c-NOTCH-1 through direct binding to the PEST sequence in the cytoplasmic domain of c-NOTCH-1 and (2) that c-NUMB interferes with c-NOTCH-1-mediated inhibition of neuronal differentiation (Wakamatsu, 1999).

Published studies on the distribution of Numb and Notch in asymmetrically dividing neuroepithelial cells in vertebrates have challenged understanding of Notch function in neurogenesis. Thus, it has been reported that NUMB-IR is localized on the apical side of mitotic neuroepithelial cells in mice. Further, NOTCH-IR has been reported to be localized basally in mitotic neuroepithelial cells in the developing ferret neocortex. Since the basal daughter cells of asymmetrically dividing neuroepithelial cells appear to undergo neuronal differentiation in these vertebrate systems, it has been proposed that apical daughter cells that receive Numb remain undifferentiated. Activation of Notch signaling in the basal daughter cells has also been proposed to be responsible for causing the postmitotic, but nondifferentiated, state of migratory daughter cells. This unprecedented role for Notch in promoting a nondividing, but nondifferentiated, intermediate neuronal phenotype and the implied role of Numb in preventing neuronal differentiation by repressing Notch function in apical cells seem paradoxical and difficult to reconcile with the Drosophila literature. In contrast, the basal localization of Numb that has been observed in the current study suggests a more parsimonious model in which Numb inhibits Notch signaling and thereby permits neuronal differentiation in the basal daughter cells. This model is consistent both with the perceived function of Notch to inhibit neuronal differentiation in vertebrate neurogenesis and with the role of Numb to suppress that inhibition in the development of the Drosophila nervous system. At present, both of these models remain to be tested further. Perhaps, future experiments using m-NUMB targeted loss-of-function mutations will be useful to elucidate the issue. At present the apparent discrepancy between the data demonstrating basal c-NUMB localization and the data reporting apical localization of m-NUMB cannot be explained (Wakamatsu, 1999).

Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis

Avian trunk neural crest cells give rise to a variety of cell types including neurons and satellite glial cells in peripheral ganglia. It is widely assumed that crest cell fate is regulated by environmental cues from surrounding embryonic tissues. However, it is not clear how such environmental cues could cause both neurons and glial cells to differentiate from crest-derived precursors in the same ganglionic locations. To elucidate this issue, expression and function of components of the NOTCH signaling pathway have been examined in early crest cells and in avian dorsal root ganglia. Delta1, which encodes a NOTCH ligand, is expressed in early crest- derived neuronal cells, and NOTCH1 activation in crest cells prevents neuronal differentiation and permits glial differentiation in vitro. NUMB, a NOTCH antagonist, is asymmetrically segregated when some undifferentiated crest-derived cells in nascent dorsal root ganglia undergo mitosis. It is concluded that neuron-glia fate determination of crest cells is regulated, at least in part, by NOTCH-mediated lateral inhibition among crest-derived cells, and by asymmetric cell division (Wakamatsu, 2000).

Expression of NUMB protein was observed in nascent DRGs of stage 22 chicken embryos. NUMB immunoreactivity is present in many but not all the mitotic cells in the periphery of nascent DRGs, as well as in the processes of non-mitotic cells. Importantly, in stage 22 DRGs, nearly 40% of mitotic cells have asymmetrically localized NUMB, in which chromosome orientation would cause NUMB to be inherited unevenly in daughter cells after cytokinesis. In contrast to the basal localization in neuroepithelial cells, however, asymmetry of NUMB localization could not be oriented with respect to any known anatomical landmark within the nascent DRG. At later stages of development, such as stages 25-27, only a few mitotic figures are observed. In those mitotic cells, NUMB localizes diffusely and symmetrically. When crest cells are cultured free from surrounding tissues, NUMB is also seen to be localized asymmetrically in mitotic cells, suggesting that some cell-intrinsic mechanism effects the intracellular localization of NUMB in crest cells. Thus, NUMB is asymmetrically localized, with respect to the cleavage plane in approximately 20%-30% of mitotic cells. In these cells, NUMB would be inherited in high concentration by only one of the daughter cells. The remaining mitotic cells either lack detectable NUMB expression, or appear to segregate NUMB symmetrically. Under these culture conditions, neurogenesis is almost complete by 5 days, and the number of mitotic cells that possess NUMB asymmetrically declines rapidly. In all stages examined, however, NUMB was symmetrically distributed throughout the cytoplasm of mitotic Hu-positive neuronal cells (Hu is a neuron-specific family of RNA binding proteins related to Drosophila ELAV), suggesting the machinery regulating asymmetrical NUMB segregation no longer functions in fate-restricted neuronal cells. NUMB immunoreactivity is enriched in the processes of non-mitotic Hu-negative cells, and consequently sequestered away from the cell body, as also observed in vivo. In non-mitotic Hu-positive neuronal cells, NUMB was observed throughout the cell body and their processes, so that activation of residual NOTCH molecules might be prevented (Wakamatsu, 2000).

Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts

Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. Whether a mouse homolog m-numb has a similar role during mouse cortical development was investigated. Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a ß-tubulin III- progenitor and a ß-tubulin III+ neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse undergo significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb (Shen, 2002).

When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin+ progenitor and a Nestin neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation (Shen, 2002).

At terminal divisions generating two neurons, Numb is symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. A significant association was found between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb are similar and most with asymmetric Numb are different. Developing cortical neurons with Numb have longer processes than those without. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development (Shen, 2002).

aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. This study identified in vivo phosphorylation sites of mammalian Numb, and showed that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions (Smith, 2007).

Numb links the Notch and TCR signaling pathways

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. This possibility was explored by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together these data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process (Anderson, 2005).

Notch signaling in the development of the inner ear: Lessons from Drosophila

The sensory patches in the vertebrate ear can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell-cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. Here, the arguments are reviewed for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and the role of Notch signaling in each system is examined more closely. Using viral vectors to misexpress components of the Notch pathway in the chick ear, it has been shown that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition, is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help understanding of the vertebrate (Eddison, 2000).

The pattern of production of hair cells and supporting cells cannot be determined simply by the pattern of expression of Notch ligands, in the manner proposed by the simple model of lateral inhibition with feedback. The cells that become hair cells are not selected to do so by escape from exposure to Ser1 (they are constantly exposed), Dl1 (its ectopic expression does not change cell fate), or Ser2 (the knockout has only a mild effect). However hair cells contain Numb, which can block Notch activation, supporting the idea that hair cells escape the inhibitory effect of Notch activation not because of lack of ligands from their neighbors, but because they are deaf to the signal delivered by the ligands (Eddison, 2000).

Why are hair cells and supporting cells produced in the observed ratio? This cannot be accounted for simply in terms of the rules of asymmetric inheritance of Numb. If each cell in the developing sensory patch went through a final asymmetric division, yielding one daughter that inherited Numb and one daughter that did not, the result would be a 1:1 ratio of hair cells to supporting cells, whereas the measured ratio (in chick basilar papilla) ranges from 1:1.7 to 1:3.9. The level of Numb in the prospective hair cells as opposed to supporting cells may be controlled in some more complex way or through more complex sequences of cell divisions, or some molecule other than Numb and its asymmetrically located companion proteins may confer immunity to lateral inhibition and serve as the key determinant of cell fate (Eddison, 2000).

Developmental changes in Notch1 and Numb expression mediated by local cell-cell interactions underlie progressively increasing delta sensitivity in neural crest stem cells

Neural stem cells become progressively less neurogenic and more gliogenic with development. Between E10.5 and E14.5, neural crest stem cells (NCSCs) become increasingly sensitive to the Notch ligand Delta-Fc, a progliogenic and anti-neurogenic signal. This transition is correlated with a 20- to 30-fold increase in the relative ratio of expression of Notch and Numb (a putative inhibitor of Notch signaling). Misexpression experiments suggest that these changes contribute causally to increased Delta sensitivity. Moreover, such changes can occur in NCSCs cultured at clonal density in the absence of other cell types. However, they require local cell-cell interactions within developing clones. Delta-Fc mimics the effect of such cell-cell interactions to increase Notch and decrease Numb expression in isolated NCSCs. Thus, Delta-mediated feedback interactions between NCSCs, coupled with positive feedback control of Notch sensitivity within individual cells, may underlie developmental changes in the ligand-sensitivity of these cells (Kubu, 2002).

Characterization of four mammalian Numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. Four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa have been identified that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The two sequence inserts encode an 11-amino acid insert in the PTB region (denoted by PTBi), identical to that found in rat and human, and a novel 49-amino acid insert within the central region of the protein adjacent to the proline-rich region (denoted by PRRi). The endogenous expression pattern of these isoforms, examined using specific antisera, varies in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein reveals that the form of the PTB domain with the alternatively spliced insert constitutively associates with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert is cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties (Dho, 1999).

The region of Numb encompassing the PTB domain insert, and the insert itself, is rich in basic residues, a property often associated with proteins that bind membrane lipids. Furthermore, specific arginine and lysine residues within the PTB domain of SHC are involved in both binding to acidic phospholipids and membrane localization. Therefore, a test was performed to see whether the presence of the four positively charged residues in the PTB insert could increase the relative affinity of that PTB domain for membrane lipids, and hence, its association with membranes could be compared to that without the insert. The ability of the Numb PTB domain isoforms to bind acidic phospholipids was compared: both the PTBi and PTBo (a novel epitope generated by the juxtaposition of amino acids adjacent to the inserts) domains exhibit promiscuous binding to most of the acidic phospholipids tested. However, in both of the assays used, the Numb PTBi domain appears to have a greater affinity for PI(4)P, when compared with PTBo. Given that both PTBi and PTBo bind several abundant membrane phospholipids, it is unlikely that the small difference in PI(4)P binding is sufficient to explain the striking difference in the subcellular localization of these two proteins. Therefore, the localization of the Numb PTBi isoforms may involve more complicated mechanisms, in which phospholipid binding could promote targeting of Numb to a membrane region where it could be retained by a specific protein target (Dho, 1999).

Numb regulates somatic cell lineage commitment during early gonadogenesis in mice

During early gonadogenesis, proliferating cells in the coelomic epithelium (CE) give rise to most somatic cells in both XX and XY gonads. Previous dye-labeling experiments showed that a single CE cell could give rise to additional CE cells and to both supporting and interstitial cell lineages, implying that cells in the CE domain are multipotent progenitors, and suggesting that an asymmetric division is involved in the acquisition of gonadal cell fates. This study found that NUMB (see Drosophila Numb) is asymmetrically localized in CE cells, suggesting that it might be involved. To test this hypothesis, Numb was conditionally deleted on a Numb-like mutant background just prior to gonadogenesis. Mutant gonads showed a loss of cell polarity in the surface epithelial layers, large interior cell patches expressing the undifferentiated marker LHX9, and loss of differentiated cells in somatic cell lineages. These results indicate that NUMB is necessary for establishing polarity in CE cells, and that asymmetric divisions resulting from CE polarity are required for commitment to differentiated somatic cell fates. Surprisingly, germ cells, which do not arise from the CE, were also affected in mutants, which may be a direct or indirect effect of loss of Numb (Lin, 2017).

A switch in numb isoforms is a critical step in cortical development

Loss of numb function suggests that numb maintains progenitors in an undifferentiated state. This study demonstrates that numb1 and numb3 are expressed in undifferentiated cortical progenitors, whereas numb2 and numb4 become prominent throughout differentiation. To further assess the role of different numb isoforms in cortical neural development, a Numb-null state was created with antisense morpholino, followed by the re-expression of specific numb isoforms. The re-expression of numb1 or numb3 resulted in a significant reduction of neural differentiation, correlating with an expansion of the cortical progenitor pool. In contrast, the expression of numb2 or numb4 resulted in a reduction of proliferating progenitors and a corresponding increase in mammalian achete-scute homologue (MASH1) expression, concurrent with the appearance of the microtubule-associated protein-2-positive neurons. Of interest, the effect of numb isoforms on neural differentiation could not be directly related to Notch, because classic canonical Notch signaling assays failed to uncover any differences in the four isoforms to inhibit the Notch downstream events. This finding suggests that numb may have other signaling properties during neuronal differentiation in addition to augmenting notch signal strength (Bani-Yaghoub, 2007).

Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage

Drosophila Numb is a signaling adapter protein with two protein-protein interaction domains: a phosphotyrosine-binding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. There are at least four human NUMB isoforms and these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human Numb protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the Notch receptor during mammalian neurogenesis (Verdi, 1999).

To isolate human Numb homologs, a human neuronal precursor NT2 cDNA library was screened at reduced stringency. Analysis of 10 cDNA clones containing both predicted initiation, and termination codons has revealed the existence of four classes of alternatively spliced transcripts of the hNUMB gene. The alternative splicing generates variant ORFs that affect the two regions of NUMB, which direct interactions with signaling pathway components: the PTB and the SH3-binding domain defined by a PRR. Two of the four classes of transcripts contain a 33-nt (11-codon) insert in the PTB domain-encoding region relative to Drosophila Numb, whereas two have a 144-nt (48-codon) insert in the PRR-encoding region. Differential mRNA splicing leads to the production of NUMB isoforms representing all four possible combinations: hNUMB1 PTBinsert(+) PRRinsert(+) = PTBL PRRL; hNUMB2 PTBinsert(+) PRRinsert(-) = PTBL PRRS; hNUMB3 PTBinsert(-) PRRinsert(+) = PTBS PRRL, and hNUMB4 PTBinsert(-) PRRinsert(-) = PTBS PRRS (Verdi, 1999).

Transcripts encoding hNUMB PRRS and PRRL are differentially expressed in adults. Northern blot analysis of multiple adult human tissues and cancer cell lines has revealed that transcripts encoding hNUMB isoforms without insertions into the PRR domain (PRRS) are ubiquitously expressed; this includes all human tumors examined. However, transcripts encoding isoforms with insertions into the PRR (PRRL) were detectable in significantly lower levels than PRRS and in only a subset of the tissues examined (prostate, testis, and intestine). The levels of both hNUMB isoforms were significantly elevated in the colon-rectal carcinoma sw480 cell line (Verdi, 1999).

That transcripts encoding hNUMB PRRL-isoforms are rarer than those encoding PRRS-isoforms was confirmed by reverse transcription-PCR (RT-PCR) analysis of human adult brain cDNA by using primers that flank the PRR-coding portion of the ORF. Further, a rat multiple tissue Northern blot was probed by using the 191-bp PRRL-specific insert from the human cDNA. Numb transcripts containing PRRL were detectable after a 1-wk exposure in adult brain, liver, testis, and kidney, but were absent in skeletal muscle, spleen, and heart, even after prolonged exposure (Verdi, 1999).

To examine the expression of Numb transcripts during brain development, RT-PCR analysis of RNA from developing rat neural tissue was conducted by using primers that flank the potential PRRL insertion. PRRL-encoding transcripts are expressed at low levels throughout early neuronal development peaking at embryonic day 10 and decreasing thereafter. PRRL transcripts are undetectable after embryonic day 14. In contrast, PRRS transcript levels remain constant in developing and adult brain. The developmental profile of PRRL transcripts in mice was assayed by Northern blot analysis, confirming the rat brain RT-PCR studies showing expression from very low levels at embryonic day 7 to modest levels at day 11. Numb transcripts encoding PRRL were not detectable after embryonic day 13. Together, these results demonstrate that transcripts encoding the PRRL vs. PRRS hNUMB isoforms are differentially expressed during neural development. In particular, only transcripts encoding the PRRL isoforms are dynamically expressed, peaking during the stages at which neuronal precursor cell proliferation is occurring and then decreasing to undetectable levels in adult brain (Verdi, 1999).

hNUMB PRRS but not PRRL isoforms mediate neuronal cell fate choice in Drosophila. Functional differences were examined by ectopic expression of hNUMB isoforms in transgenic Drosophila. Misexpression of rat NUMB (PTBL PRRS) at the developing wing margin by using the GAL4C96 driver results in transformation of the sensory organ precursor lineage throughout the entire margin toward neuronal fate. For the present assays, because hNUMB4 (PTBS PRRS) is structurally homologous to Drosophila NUMB, hNUMB4 to hNUMB2 (PTBL PRRS) (in order to uncover differences in PTB domain function) and hNUMB4 to hNUMB3 (PTBS PRRL) were compared (to uncover differences in PRR domain function). Insertions within the hNUMB PRR dramatically abrogate the ability of hNUMB to direct neuronal cell fate choice, whereas insertions into the PTB domain had no detectable effect. Ectopic expression of hNUMB4 (PTBS PRRS) and hNUMB2 (PTBL PRRS) causes the strongest transformation toward neuronal fate (>87% loss of hairs from each wing margin, whereas hNUMB3 (PTBS PRRL) causes a significantly weaker transformation (<20% loss of hairs from each wing margin). It is concluded that, in the Drosophila assay, both types of PTB-domains are equally functional, but the insert into the PRR (PRRL) reduces the ability to direct both the IIa to IIb transformation and neuronal fate specification (Verdi, 1999).

hNUMB PRRS-containing isoforms promote differentiation, whereas PRRL-containing isoforms promote proliferation during mammalian neurogenesis. The murine P19 embryonic carcinoma cell line serves as an excellent tissue culture model for mammalian neurogenesis. To confirm functional differences between PRRL and PRRS isoforms, the hNUMB isoforms were overexpressed in P19 cells. Overexpression of a rat NUMB (mNUMB = PTBL PRRS) dramatically biases undifferentiated P19 cells toward neuronal fate, whereas overexpression of a dominant-negative form of NUMB (only the PTB domain) biases cells away from neuronal fate. Pooled stable lines of P19 cells overexpressing type I (PTBL PRRS or PTBS PRRS) human NUMB isoforms show a dramatic increase in the number of neurofilament-positive cells after 4 d of aggregation and retinoic acid treatment (2.6- and 2.2-fold). In contrast, pooled stable lines of P19 cells expressing type II isoforms (PTBS PRRL or PTBL PRRL) show an increase in the total number of cells, although the fraction of cells bearing neuronal processes was similar to controls. This observation confirms that hNUMB PRRS-containing isoforms bias cells toward neuronal fate. Unexpectedly, however, the data also suggest that hNUMB isoforms containing insertions into the PRR (PRRL) either increase the survival of undifferentiated cells or increase the proliferation rate of undifferentiated cells (Verdi, 1999).

To test whether PRRL-containing hNUMB isoforms promote proliferation, BrdU incorporation assays were conducted on unaggregated P19 cell lines harboring each of the four hNUMB isoforms. No differences in BrdUrd incorporation were observed from control values for cells not undergoing overt cellular differentiation. However, on differentiation (aggregation in the presence of retinoic acid) P19 lines expressing type II isoforms (PTBL PRRL and PTBS PRRL) show a 1.5-fold increase in BrdUrd-positive cells after an 8-hr pulse. BrdU incorporation into lines expressing the type I isoforms (PTBS PRRS and PTBL PRRS) show no increase in proliferation, and perhaps even a slight decrease, relative to controls. These results suggest distinct developmental functions for PRRL- vs. PRRS-containing hNUMB isoforms: PRRS-containing isoforms promote neuronal differentiation. In contrast, PRRL-containing isoforms do not direct differentiation, but rather promote cell proliferation (Verdi, 1999).

To verify the distinct functions of PRRS- and PRRL-containing hNUMB isoforms, were expressed in an immortalized neural crest stem cell line (MONC-1) and in primary neural crest stem cells. Neural crest stem cells offer a wider possibility of phenotypic cell fate choices in culture (neurons, glia, and smooth muscle) than do P19 cells (neurons and glia). Moreover, the development of individual cells can be followed during the assay to monitor the effects on proliferation, differentiation, and apoptosis. As in the previous analyses of mNUMB (rat PTBL PRRS), overexpression in MONC-1 cells of PRRS-containing hNUMB isoforms forces the majority of the resulting clones into a 'neuron only' phenotype. However, when PRRL-containing hNUMB isoforms are expressed, there is no difference relative to control clones (i.e., there is no bias toward or away from neuronal differentiation). MONC-1 and primary neural crest stem cells overexpressing the same isoforms also show a strong bias toward the neuronal lineage when carrying PRRS-containing hNUMB isoforms, whereas no neuronal bias is seen when PRRL-containing hNUMB isoforms were expressed. Strikingly, the resulting terminal clone size in both MONC experiments and primary crest experiments for PRRL hNUMB expressing clones was two to three times greater than that of PRRS hNUMB-expressing cells or control clones. These results confirm that PRRL-containing hNUMB isoforms promote mitosis of undifferentiated progenitors (Verdi, 1999).

Thus the PRRS- and the PRRL-containing hNUMB isoforms are likely to implement distinct functions during mammalian neurogenesis by promoting either neuronal differentiation (PRRS) or proliferation (PRRL). The recent observation that hNUMB is translocated into the nucleus and interacts with a critical member of the mitotic index machinery MDM2 (Juven-Gershon, 1998; Freedman, 1999) is consistent with this conclusion. What is the mechanism underlying these distinct hNUMB functions? The Drosophila Numb protein is known to interact with Drosophila Notch through the Numb PTB domain, thus inhibiting Notch function. If a similar hNUMB-NOTCH interaction occurs in mammalian cells, then PRRS and PRRL could serve to link NOTCH to distinct SH3 domain-containing proteins. The PRRS class would promote neuronal differentiation, whereas the PRRL class would direct proliferation. At present, no data is available that addresses whether additional modulation of NOTCH signaling might be accomplished through differential NOTCH binding of the PTBS and the PTBL domains. Because there are at least four vertebrate NOTCH isoforms, it is possible that distinct hNUMB isoforms interact with distinct NOTCH isoforms to signal either differentiation or proliferation during neurogenesis as well as in other lineages (Verdi, 1999 and references therein).

Distinct functions of human numb isoforms revealed by misexpression in the neural stem cell lineage in the Drosophila larval brain

Mammalian Numb (mNumb) has multiple functions and plays important roles in the regulation of neural development, including maintenance of neural progenitor cells and promotion of neuronal differentiation in the central nervous system (CNS). However, the molecular bases underlying the distinct functions of Numb have not yet been elucidated. mNumb, which has four splicing isoforms, can be divided into two types based on the presence or absence of an amino acid insert in the proline-rich region (PRR) in the C-terminus. It has been proposed that the distinct functions of mNumb may be attributable to these two different types of isoforms. In this study, the outer optic anlage (OOA) of the Drosophila larval brain was used as an assay system to analyze the functions of these two types of isoforms in the neural stem cells, since the proliferation pattern of neuroepithelial (NE) stem cells in the OOA closely resembles that of the vertebrate neural stem/progenitor cells. They divide to expand the progenitor cell pool during early neurogenesis and to produce neural precursors/neurons during late neurogenesis. Clonal analysis in the OOA allows one to discriminate between the NE stem cells, which divide symmetrically to expand the progenitor pool, and the postembryonic neuroblasts (pNBs), which divide asymmetrically to produce neural precursors (ganglion mother cells), each of which divides once to produce two neurons. In the OOA, the human Numb isoform with a long PRR domain (hNumb-PRRL), which is mainly expressed during early neurogenesis in the mouse CNS, promotes proliferation of both NE cells and pNBs without affecting neuronal differentiation, while the other type of hNumb isoform with a short PRR domain (hNumb-PRRS), which is expressed throughout neurogenesis in the mouse embryonic CNS, inhibits proliferation of the stem cells and promotes neuronal differentiation. It was also found that hNumb-PRRS, a functional homologue of Drosophila Numb, more strongly decreases the amount of nuclear Notch than hNumb-PRRL, and can antagonize Notch functions probably through endocytic degradation, suggesting that the two distinct types of hNumb isoforms contribute to different phases of neurogenesis in the mouse embryonic CNS (Toriya, 2006).

Progenitor cell maintenance requires numb and numblike during mouse neurogenesis

Neurons in most regions of the mammalian nervous system are generated over an extended period of time during development. Maintaining sufficient numbers of progenitors over the course of neurogenesis is essential to ensure that neural cells are produced in correct numbers and diverse types. The underlying molecular mechanisms, like those governing stem-cell self-renewal in general, remain poorly understood. Mouse numb and numblike (Nbl), two highly conserved homologs of Drosophila numb, play redundant but critical roles in maintaining neural progenitor cells during embryogenesis, by allowing their progenies to choose progenitor over neuronal fates. In Nbl mutant embryos also conditionally mutant for mouse numb in the nervous system, early neurons emerge in the expected spatial and temporal pattern, but at the expense of progenitor cells, leading to a nearly complete depletion of dividing cells shortly after the onset of neurogenesis. These findings show that a shared molecular mechanism, with mouse Numb and Nbl as key components, governs the self-renewal of all neural progenitor cells, regardless of their lineage or regional identities (Petersen, 2002).

In Drosophila, Numb is a membrane-associated signaling protein that allows two daughter cells to adopt different fates after an asymmetric division. It does this by localizing to only one half of the cell membrane in dividing precursor cells, so that it is segregated primarily to one cell. On the basis of studies in the developing mouse neocortex, it has been postulated that mouse Numb segregates to, and promotes the fate of, progenitor cells in asymmetric divisions that generate a neuron and a daughter progenitor cell during mammalian neurogenesis. This view, however, has been controversial; others have postulated instead that vertebrate Numb proteins promote the neuronal fate in such divisions. numb mutant mice exhibit severe defects in cranial neural tube closure and die around embryonic day (E) 11.5, but neurogenesis abnormalities are limited, and insufficient to resolve the controversy. Nbl homozygous mutant mice have been generated that are viable, fertile and exhibit no obvious phenotypes. Low levels of Nbl expression is found in E8.5 embryos, including in neural progenitor cells. Moreover, embryos mutant for both mouse numb and Nbl die around E9.5 with more widespread defects than single mutants (Petersen, 2002).

Therefore, Cre-loxP mediated gene targeting was used to examine numb and Nbl function in neurogenesis. A transgenic line (NesCre8) was generated with Cre expression controlled by a nestin promoter that is active in neural progenitor cells and somites. Within the nervous system, Cre-mediated recombination is readily detectable in a majority of the progenitor cells at E8.5 and becomes nearly complete by E12.5. Conditional knockout (cKO) using NesCre8 and a floxed mouse numb allele did not cause embryonic lethality or defects in neural tube closure. In fact, cKO mice are viable, fertile and indistinguishable from their wild-type littermates. As expected, immunoblots show mouse Numb protein level is already greatly reduced in E9.5 conditional mutant embryos. Most of the residual mouse Numb protein probably comes from tissues where Cre is not active (Petersen, 2002).

However, mouse numb cKO in the Nbl homozygous mutant background (conditional double-knockout, or cDKO) results in embryonic lethality. cDKO mice were never recovered postnatally, whereas those with other allelic combinations, in particular cKO in Nbl heterozygous background, were viable and exhibited no gross morphological or behavioral defects. cDKO embryos are indistinguishable from their littermates at E9.5, but become completely necrotic by E12.5. Those recovered at E11.5 are considerably smaller than the littermates, suggesting that cDKO embryos die around this stage (Petersen, 2002).

At E10.5, cDKO embryos were consistently recovered. They were 80%-90% the size of their wild-type or single-mutant littermates, although many are within the range of variation seen in wild-type litters. cDKO embryos are morphologically appropriate for their age with the expected somite numbers, but have significantly reduced telencephalic vesicle and undulating spinal cord, the combination of which can be reliably used to identify them. Histological analysis reveals that E10.5 cDKO embryos have severe thinning of the neural tube, from the most rostral telencephalon to caudal spinal cord, including optic discs. In 82% of the mutants, the neuroepithelium is only one-quarter to one-half the thickness of that in the littermates, with frequent buckling of the surface (Petersen, 2002).

E10.5 wild-type neuroepithelium consists mainly of progenitor cells that make up the wider, inner ventricular zone, with a much smaller number of neurons forming the outer mantle zone. Neurons could be detected in E10.5 cDKO embryos, in a region-specific pattern similar to that in control littermates, wild-type or other allelic combinations, using two general neuronal markers: anti-HuC/D or Neurofilament (NF). Dll1, a marker for newborn, migrating neurons in the ventricular zone, is also expressed throughout the cDKO nervous system, including the forebrain, which has few Hu- or NF-positive neurons. In fact, the cDKO neuroepithelium frequently contains large patches of Dll1-positive cells, unlike in control embryos where such cells are invariably discrete (Petersen, 2002).

To assess the severity of the neural progenitor cell loss in E10.5 cDKO embryos, an antibody against phospho-Histone H3 (P-H3) was used to identify mitotic cells. In wild-type embryos, neural progenitor cells within the neural tube undergo S phase (DNA synthesis) when their nuclei are in the outer half of the ventricular zone. The nuclei then translocate towards the ventricular surface where cells undergo mitosis. Accordingly, P-H3-positive cells form a near continuous outline of the ventricular surface. In the cDKO nervous system, however, there is a dramatic reduction of P-H3-positive cells. The loss in the forebrain, the hindbrain (at the level of otic vesicle), and the spinal cord (at cervical levels), ranges from about 80% to nearly 100% (Petersen, 2002).

Bromodeoxyuridine (BrdU) labelling experiments were performed. BrdU is incorporated into DNA by S-phase cells and, therefore, the number of cells labelled during a short pulse reflects the number of cells still proliferating but not in mitosis. There is little variation among control littermates in the pattern and percentage of neural progenitor cells labelled by BrdU. In forebrain, hindbrain and cervical spinal cord sections, BrdU-labelled cells account for about 49%, 33% and 40% of the ventricular zone cells, respectively. In contrast, only a few BrdU-positive cells are present in the cDKO nervous system, indicating a loss of over 99% of the S-phase cells (Petersen, 2002).

The nearly complete absence of neural progenitor cells is based on the analysis of, and invariably observed in, embryos with a neural tube that is less than half the normal thickness, a group representing 82% of the cDKO embryos recovered at E10.5. Furthermore, BrdU-labelled cells in many non-neural tissues are also reduced in numbers. Whether this is a secondary effect or shows a direct requirement for mouse numb and Nbl in non-neural tissues remains to be determined (Petersen, 2002).

What causes the near-absence of neural progenitor cells in E10.5 cDKO embryos? At E9.5, such embryos show no significant defects in cell proliferation. Consistently, the overall neural tube thickness in E9.5 and E10 cDKO embryos is comparable to that in control littermates, although the mutant neuroepithelium, particularly the ventral spinal cord, is sometimes punctuated by thinner regions. At E10, however, S-phase cells in the nervous system are reduced in numbers, particularly in regions with more active neurogenesis, indicating that the absence of progenitor cells at E10.5 is probably due to defects in self-renewal rather than smaller or defective founding populations. There is, however, significant variation in the severity of such loss among cDKO embryos, probably due to the perdurance effect of the mouse Numb protein and variations in the onset of Cre expression. Consequently, E10.25 cDKO embryos show varying degrees of neural tube thinning and reduction of BrdU-labelled cells, ranging from a near-complete loss to about 60% (Petersen, 2002).

Between E10 and E10.5, mostly neurons are being generated in wild-type embryos. cDKO embryos show no ectopic expression of glia markers like Sox10 or Olig2, indicating an absence of premature gliogenesis. Neuron production was examined in E10 and E10.25 cDKO embryos to ascertain whether the inability of neural progenitor cells to self-renew results from their progenies all adopting neuronal fates, which would cause an initial overproduction of neurons and, more importantly, a significant increase of their percentage within the neuroepithelium owing to a depletion of progenitor cells. Other causes, such as progenitor cells becoming quiescent or defective in cell-cycle progression, or undergoing programmed cell death, should affect neuron production negatively, resulting in fewer neurons, both in absolute numbers and as a percentage of the neuroepithelium (Petersen, 2002).

E10.25 cDKO embryos were analysed in which neural tube thinning is not yet apparent. Three lines of evidence show unambiguously that a diminished self-renewal capability among cDKO neural progenitor cells indeed results from over-differentiation of their progenies. (1) Reductions in the number of BrdU-labelled cells are accompanied by a similar decrease in cells expressing progenitor marker Hes5. (2) Within progenitor domains marked by Hes5, a higher percentage of cells express Dll1, a marker for newborn neurons. (3) Most important, there is a significant expansion of, proportional to the decrease of progenitor domains, cells expressing neuronal marker Hu. In these cDKO embryos, there are patches of Hes5-positive progenitor cells at the most lateral positions of the neuroepithelium. It remains to be determined whether they have migrated aberrantly, or whether their nuclei are physically prevented from translocating medially owing to neuron overproduction near the ventricular surface (Petersen, 2002).

Neurogenesis was analyzed in more detail in the ventral spinal cord, where motor neurons emerge shortly after E9 and are continuously generated until E13.5. At E9.5, cDKO and control embryos show little difference in Olig2 and Isl1 expression, which marks motor progenitors and motor neurons, respectively. By E10, less than two cell cycles later, there are dramatic differences. At cervical levels in control embryos, wild-type or other allelic combinations, which are indistinguishable, Isl1-positive neurons colonize the lateral half of the motor domain, whereas Olig2-positive progenitor cells occupy the medial half. On average, only 25.6% of the Olig2-expressing cells are doubly positive for Isl1, representing newborn motor neurons. In contrast, Isl1-expressing cells frequently span the entire width of the cDKO neuroepithelium. As expected, more Olig2-positive cells in the mutant co-express Isl1, ranging from 40.6% to as high as 68.7%. Even in regions where motor neurons appear to have only colonized the lateral positions, Olig2-positive cells are also absent from the ventricular zone and many, consistent with their position, co-express Isl1 (Petersen, 2002).

Neurogenesis in the spinal cord proceeds in a rostral to caudal gradient. More caudally, there are more Olig2 single-positive cells than are found at cervical levels in E10 cDKO embryos. Although the overall reduction of BrdU-labelled cells within the spinal cord is limited, it is much more severe among Olig2-expressing cells, which sometimes show no BrdU incorporation at all. Accordingly, in E10.25 cDKO embryos at similar caudal levels, Isl1-positive motor neurons span the entire width of the neuroepithelium and most of the remaining Olig2-positive cells co-express Isl1 (Petersen, 2002).

Although neurons are initially overproduced in cDKO embryos, there is significant increase in apoptosis among mutant neurons, indicating that many die shortly after birth. This is consistent with the absence of axon tracts underneath the floor plate, and suggests a role for mouse numb and Nbl in later events of neural development, as has been postulated, although neuronal death may be a secondary effect (Petersen, 2002).

It remains possible that cDKO progenitor cells adopt an abnormal developmental pathway and differentiate en masse before the onset of neurogenesis. However, two lines of evidence strongly suggest that they are rapidly depleted as neurogenesis progresses because their daughter cells all adopt neuronal fates instead of self-renewing after division: (1) in cDKO embryos where BrdU-labelled S-phase cells are significantly reduced in numbers but not totally absent, the number of mitotic cells is comparable to that in control littermates; (2) whereas neuron overproduction is observed throughout the cDKO nervous system, it is more pronounced in regions where neurogenesis has been more active. In the control E10.25 forebrain, for example, only a few scattered Hu-positive neurons are present, indicating that neurogenesis was just underway. Neurons are overproduced in the cDKO forebrain, but represent only a small fraction of the neuroepithelium. However, ventral spinal cord in control littermates contains large numbers of neurons. Accordingly, Hu-positive neurons not only are overproduced in the cDKO but also span nearly the entire width of the neuroepithelium. Similar differences in neuron overproduction can be observed in other regions and along the dorsoventral neural axis (Petersen, 2002).

The findings reported here are consistent with the earlier hypothesis, and demonstrate unequivocally that the main function of numb homologs in mouse neurogenesis is to maintain progenitor cells, not promoting neuronal fates, during the initial progenitor versus neuronal fate decision. This is also supported by gain-of-function studies in chick showing an increase in progenitors among neuroepithelial cells over-expressing chick Numb. A near-absence of motor and sensory neurons in mouse numb single mutants at E10.5 due to impaired differentiation has been reported. This phenotype is not seen in single or cDKO mutants and, therefore, is unlikely to be due to a loss of mouse numb function (Petersen, 2002).

These findings are the first direct evidence of a pan-neural program for precursor cells (regardless of their lineage or regional identities) to choose between proliferation and differentiation. There is growing evidence from direct imaging experiments that asymmetric cell division occurs during mammalian neurogenesis. Mouse Numb is asymmetrically localized to the apical membrane in dividing progenitor cells, but how Nbl protein is distributed in these cells is unknown, owing to low levels of expression. Therefore, although an effect on asymmetric division can account for pan-neural progenitor depletion in cDKO embryos, further studies are necessary to ascertain this, in particular the specific effects on multipotential neural stem cells and progenitors with more limited developmental potentials. Similarly, to ascertain if mouse Numb and Nbl act by inhibiting Notch activity like Drosophila Numb, it is necessary to determine first whether Notch signaling plays a generic role in regulating cell fate choices between proliferation and differentiation -- as seen with mouse Numb and Nbl -- or acts differently in different progenitor populations (Petersen, 2002).

Finally, the widespread defects exhibited by mouse numb and Nbl constitutive double mutants at E9.5 raise an interesting possibility that these two genes are involved in stem-cell self-renewal in other tissues. If mouse Numb and Nbl proteins indeed act like their Drosophila counterpart, they would provide an attractive molecular mechanism to integrate lineage or cell-extrinsic cues for progenies of various stem cells to choose between self-renewal and adopting appropriate differentiated fates (Petersen, 2002).

The gamma-secretase-generated intracellular domain of beta-amyloid precursor protein binds Numb and inhibits Notch signaling

The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. This study shows that membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling: positive when cleaving Notch and generating NICD, and negative when processing APP and generating AID, which inhibits the function of NICD (Roncarati, 2002).

Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis

Numb and Numblike, conserved homologs of Drosophila Numb, have been implicated in cortical neurogenesis; however, analysis of their involvement in later stages of cortical development has been hampered by early lethality of double mutants in previous studies. Using Emx1IREScre to induce more restricted inactivation of Numb in the dorsal forebrain of numblike null mice beginning at E9.5, viable double mutants were generated that displayed striking brain defects. It was thus possible to examine neurogenesis during the later peak phase (E12.5-E16.5). Loss of Numb and Numblike in dorsal forebrain results in neural progenitor hyperproliferation, delayed cell cycle exit, impaired neuronal differentiation, and concomitant defects in cortical morphogenesis. These findings reveal novel and essential function of Numb and Numblike during the peak period of cortical neurogenesis. Further, these double mutant mice provide an unprecedented viable animal model for severe brain malformations due to defects in neural progenitor cells (Li, 2004).

To examine the functional requirement of mouse Numb and Numblike, both expressed in progenitors of the central nervous system during embryogenesis, Emx1IREScre was used to inactivate numb in nbl null mice specifically in the dorsal forebrain starting from E9.5. Whereas Nestin-Cre-driven numb inactivation in nbl null mice starting at E8.5 causes near complete depletion of neural progenitors and subsequent embryonic lethality at around E11.5, the Emx1IREScre-mediated numb and numblike double mutants are viable. Given their seemingly mild behavioral abnormalities, it was very surprising to find large cavities in the adult brains, with a near total loss of specific neuronal types in the caudodorsal brain regions. The embryonic mutant cortex exhibits extensive undulation, folding, fusion and formation of neurogenic cellular rosettes, and shedding of cell clumps into the ventricle. These phenotypes are accompanied by increased proliferation and apoptosis of neural progenitors and reduced neuronal differentiation during the later phase of neurogenesis. The remarkable difference between the Nestin-Cre and the Emx1IREScre-induced double mutants indicates that progenitors at different developmental stages might have different requirements for Numb and Numblike. Another possible explanation for the different mutant phenotypes is a potential inhibition of progenitor proliferation by neurons. These two possibilities are not mutually exclusive nor are they the only conceivable explanations (Li, 2004).

During development, there are two phases of neurogesis in mice: the first phase lasts from E8.5 to E10.5, and the second phase commences around E12.5, reaches the peak by E15.5, and then diminishes by E17.5. In the first phase, neural progenitor cells are columnar, connecting the pial surface and the apical surface, and predominantly undergo symmetric cell division to rapidly expand the founder progenitor cell pools. In the second phase, neural progenitors in the pseudostratified neuroepithelium predominantly undergo asymmetric cell division to generate one daughter progenitor and one differentiated neuron in a stem cell-like mode. Loss of both mouse Numb and Numblike in neural progenitors starting at E8.5 decreases neural progenitor cell proliferation and increases neuronal production. These double mutants derived from Nestin-Cre-driven recombination soon become depleted of neural progenitors and die as early embryos before the second phase of neurogenesis begins, suggesting that Numb and Numblike may be required to promote the differentiation of neuroectodermal cells to the founder neural progenitor cells or maintain the founder neural progenitor cells and inhibit premature neuronal differentiation. In contrast, removing both mouse Numb and Numblike from the dorsal forebrain starting from E9.5 increases neural progenitor cell proliferation, delays cell cycle exit, increases apoptosis of neural progenitors, and reduces neuronal differentiation during the second phase of neurogenesis (Li, 2004).

What might be the explanation for these apparently different mutant phenotypes? One possibility is that neural progenitors at different stages of development have different functional requirements for Numb and Numblike. Although neural progenitors in early neurogenesis dwindle upon the removal of Numb and Numblike via Nestin-Cre, neural progenitor cells from the Emx1IREScre-mediated numb and numblike double mutant proliferate more in vivo, exhibiting fewer P/N and N/N divisions but more numerous P/P divisions in culture. Both the in vivo and in vitro mutant phenotypes observed between E12.5 and E16.5 could be explained by supposing that Numb and Numblike control neural progenitor cell number by limiting the extent of proliferation and may be required for neuronal differentiation in the second phase of neurogenesis. It is also worth noting here that potential variability in Cre activation and Numb perdurance in different cells could be one factor that contributes to the range of mutant phenotypes observed (Li, 2004).

If nb and nbl genes act differently in different neural progenitor cells, what might be the mechanism? In vertebrates, there are different isoforms of Numb with different temporal expression and different functions, with Numb-PRRS promoting neuronal differentiation and Numb-PRRL promoting proliferation. Numb has a number of potential interaction partners, including Notch, MDM2, Esp15, NAK, α-adaptin, Lux2, Siah-1, APP and LNX, and E3 ligase Itch; and Numblike binds to both APP and GRIP-1. Given that one known function of Numb is to inhibit Notch activity in postmitotic vertebrate neurons as revealed by in vitro cell culture, it is worth noting that the undulating neuroepithelium and thickening of targeted cortex reported in this study are similar to the convoluted cortical neuroepithelium due to overexpression of activated Notch3. Overexpression of an activated form of the intracellular domain of Xenopus Notch causes the expansion and disorganization of the Xenopus brain, and overexpression of transcription factors Hes1 and Hes5 that mediate Notch signaling increases neural progenitor cell proliferation, retains progenitor cells in the apical surface, and inhibits neuronal differentiation in the mouse brain. These results led to the proposal that activated Notch signaling keeps neural progenitor cells uncommitted and undifferentiated. Furthermore, it has been shown that Numb recruits E3 ligase Itch to promote the ubiquitination of Notch1 for its degradation in cell culture. It is possible that Notch ubiquitination and degradation is reduced in the double mutant, leading to the hyperactivation of Notch signaling pathway. It would be of interest to determine in future studies whether the interaction of Numb with Notch or other proteins, such as Itch and α-adaptin in the ubiquitination-endocytic pathway, is involved in the differentiation of progenitors during neurogenesis (Li, 2004).

A second but not mutually exclusive possibility is that cell non-autonomous effects indirectly contribute to the apparently different behavior of progenitors in the Emx1IREScre conditioned knockout mice. For example, one may envision that the differentiating neurons may exert inhibitory effects over the proliferation of progenitor cells during cortical neurogenesis. The overabundance of neurons in Nestin-Cre conditioned knockout mice may further restrict progenitor proliferation, whereas the sparsity of neurons in Emx1IREScre conditioned knockout mice may lead to excessive proliferation of progenitors. Although in vitro pair-cell analysis of neural progenitors from Emx1IREScre conditioned knockout mutants reveal an increase of divisions that produce more progenitors and thus suggesting a direct role of Numb and Numblike in restricting symmetric divisions that expand progenitors at E13.5 and E14.5, the second possibility of indirect effect of Numb and Numblike on neural progenitor cell behavior cannot be ruled out (Li, 2004).

2009). Mammalian Par3 regulates progenitor cell asymmetric division via notch signaling in the developing neocortex

Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. This study found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex (Bultje, 2009).

The results presented here demonstrate that the evolutionarily conserved cell polarity protein mPar3 and the Notch signaling pathway act together to regulate the asymmetric cell division of radial glial progenitor cells in the developing neocortex. Mammalian Par3 is not statically restricted to the apical membrane domain of radial glial cells; instead, its distribution is dynamic depending on the cell cycle progression. It is selectively localized to the ZO-1- expressing lateral membrane domain in the ventricular endfeet during interphase and then becomes dispersed during mitosis. This dynamic distribution of mPar3 can lead to asymmetric inheritance of mPar3 by the two daughter cells, which results in differential Notch signaling activation that depends on Numb/Numb-like and distinct daughter cell fate specification. While the daughter cell that inherits a greater amount of mPar3 develops high Notch signaling activity and remains a radial glial cell, the daughter cell that inherits less mPar3 harbors low Notch signaling activity and adopts either a neuronal or an intermediate progenitor cell (IPC) fate (Bultje, 2009).

The dynamic nature of mPar3 subcellular localization in radial glial progenitor cells has not been shown previously. In fact, the distribution of mPar3 in dividing radial glial progenitor cells has not been rigorously examined. A recent study suggests that the mPar protein promotes the proliferation of progenitor cells. However, it is unclear whether the mPar protein regulates asymmetric radial glial cell division. Precisely determining the subcellular distribution of mPar3 in dividing radial glial cells is of critical importance to understanding its function and the molecular control of asymmetric cell division. Given the enrichment of mPar3 in interphase radial glial cells at the luminal surface of the VZ, where the cell bodies of scarce dividing radial glial cells are located, it is rather challenging to distinguish mPar3 in the cell bodies of dividing radial glial cells from that in the ventricular endfeet of interphase radial glial cells. To overcome this difficulty, advantage was taken of the phospho-Vimentin antibody, which selectively labels radial glial cells in mitosis. Moreover, the cytoplasmic labeling seen with this antibody helps to define the cell contour and its cleavage furrow, thereby facilitating the determination of the precise distribution of mPar3 and the cleavage plane of individual dividing radial glial cells. It was found that at E14.5 in about half of radial glial cells with a defined cleavage plane (i.e., in anaphase/telophase), mPar3 shows asymmetric distribution and the axis of the mPar3 asymmetry is perpendicular to the cleavage plane; this would result in a preferential segregation of mPar3 into one of the two future daughter cells (Bultje, 2009).

Previous studies showed that about half of the divisions in the VZ of the developing mouse cortex at this developmental stage are asymmetric and neurogenic. Although the current analysis of mPar3 asymmetry in dividing radial glial cells is likely an underestimation, these data suggest that the subcellular distribution of mPar3 (i.e., symmetric versus asymmetric) may be critical for determining the mode of division of radial glial cells. Indeed, it was found that disrupting mPar3 asymmetry in radial glial cells either by depletion or by ectopic expression of mPar3 prevents asymmetric cell division and promotes symmetric cell division. While the precise mechanisms underlying the establishment of the mPar3 asymmetry remain to be uncovered, the findings strongly suggest that mPar3 and its subcellular distribution regulate the mode of radial glial cell division and daughter cell fate specification in the developing neocortex (Bultje, 2009).

Interestingly, while both suppression of mPar3 expression and ectopic mPar3 expression impair asymmetric radial glial cell division, their effects on daughter cell fate specification are rather different. Ectopic mPar3 expression promotes radial glial cell fate, whereas suppression of mPar3 expression facilitates neuronal production. These results indicate that the inheritance level of mPar3 influences daughter cell fate specification, although mPar3 itself being an unlikely cell fate determinant. Intriguingly, it was found that the expression level of mPar3 affects Notch signaling activity, a key cell fate regulator required for proper neocortical neurogenesis. While a high level of mPar3 expression leads to high Notch signaling activity, a low level of mPar3 expression results in low Notch signaling activity. Previous studies have shown that Notch signaling activity is high in radial glial progenitor cells, but low in differentiating cells such as neurons. However, it is unclear how differential regulation of Notch signaling activity is initialized in the daughter cells of dividing radial glial progenitors. This study found that asymmetric segregation of mPar3 can lead to differential Notch signaling activity in the two daughter cells (Bultje, 2009).

In Drosophila neuroblasts, the asymmetric localization of Numb, a negative regulator of Notch signaling, is fundamental for differential Notch signaling activity and cell fate diversity in the central nervous system. Furthermore, this asymmetry in Numb distribution depends on the asymmetric segregation of Bazooka, the mPar3 ortholog in Drosophila. In mammals there are two Numb homologs, Numb and Numb-like. Previous studies suggest that Numb is essential for the proper development of the mammalian brain. However, the correlation between Numb protein segregation and asymmetric daughter cell fate specification has not been definitively established. In addition, recent studies suggest that Numb is involved in trafficking and proper localization of the junctional protein cadherin in radial glial cells and thereby functions in maintaining the tissue architecture of the developing neocortex. This study found that mPar3 acts through Numb and Numb-like in regulating Notch signaling activity. Moreover, the data suggest that a direct interaction between mPar3 and Numb is critical. Despite that it is unclear whether Numb is asymmetrically distributed in dividing radial glial progenitor cells, these findings suggest that asymmetric inheritance of mPar3, which interacts with Numb/Numb-like, results in differential activation of Notch signaling in the two daughter cells of asymmetrically dividing radial glial progenitors in the developing neocortex. Moreover, a recent study showed that removal of Cdc42 in the developing neocortex leads to mislocalization of mPar3 and defects in neocortical neurogenesis. Given that mPar3 and activated Cdc42 interact with each other, the findings coupled with these observations suggest that the mPar protein complex and its interacting proteins, such as Cdc42 and Lgl, likely represent an essential molecular pathway that regulates Notch signaling activity and asymmetric cell division of radial glial progenitor cells in the mammalian neocortex (Bultje, 2009).

Targeted deletion of numb and numblike in sensory neurons reveals their essential functions in axon arborization

Mouse Numb homologs antagonize Notch1 signaling pathways through largely unknown mechanisms. Conditional mouse mutants with deletion of numb and numblike in developing sensory ganglia show a severe reduction in axonal arborization in afferent fibers, but no deficit in neurogenesis. Consistent with these results, expression of Cre recombinase in sensory neurons from numb conditional mutants results in reduced endocytosis, a significant increase in nuclear Notch1, and severe reductions in axon branch points and total axon length. Conversely, overexpression of Numb, but not mutant Numb lacking alpha-adaptin-interacting domain, leads to accumulation of Notch1 in markedly enlarged endocytic-lysosomal vesicles, reduced nuclear Notch1, and dramatic increases in axonal length and branch points. Taken together, these data provide evidence for previously unidentified functions of Numb and Numblike in sensory axon arborization by regulating Notch1 via the endocytic-lysosomal pathways (Huang, 2005).

While the findings support a lack of function of numb and nbl in mouse sensory neurogenesis, these data are in direct contrast to that reported in earlier studies, in which deletion of numb alone was reported to result in a severe reduction in the expression of neuronal markers NeuroD and NF160 and an up-regulation of glial marker ErbB3 in DRG of E10.5 embryos. Several possible explanations could account for such discrepancies. (1) The effects of Numb in determining cell fate could occur before the closure of neural tube and emigration of neural crest cells. In this scenario, cell fate determination for cells in the sensory nervous system could be determined long before the emigration of neural crest from neural tube. Indeed, cell lineage tracing studies in mouse embryos have indicated that commitment to cranial and trunk neural crests is established before closure of the neural tube. Since Wnt1Cre is only active after the closure of neural tube, this could in part account for the lack of neurogenesis phenotype in the sensory ganglia of Wnt1Cre/+;fnb/fnb;nbl-/- mice. (2) Defects in the sensory ganglia could be an indirect consequence of a more general developmental defect in the mutant embryos. Indeed, numb mutants in the EIIaCre genetic background show defects in the developing blood vessels in head and trunk, failure in the closure of neural tube, and a near complete absence of the developing spinal cord at E10.5, which could have had an adverse effect on neural crest migration and the initial formation of sensory ganglia (Huang, 2005).

In addition to the well-documented roles of Notch in cell fate determination, there is emerging evidence that Notch can also regulate the differentiation of post-mitotic neurons. In Drosophila, combinations of Notch and abl mutations result in synergistic genetic interactions leading to lethality and defects in axonal outgrowth in the absence of any alteration in neuronal identity. It has been proposed that such regulatory roles of Notch could be mediated through interactions between the intracellular domain of Notch and PTB domain of Disabled (Dab), which can potentially recruit Abl to regulate organization of actin cytoskeleton via a sequential or parallel pathway. In mice, Notch1 protein can be detected in neurites and nuclei of embryonic and post-natal cortical neurons. Overexpression of Notch1 ICD inhibits neurite outgrowth and dendritic branching in cortical neurons and in cultured sensory neurons. Most intriguingly, the inhibitory effects of Notch1 ICD can be antagonized by Numb and Nbl, suggesting that key regulators of the Notch signaling pathway also play essential roles in maintaining neuronal differentiation (Huang, 2005).

In addition to these data, recent evidence also supports an independent function of Numb and Nbl in regulating neurite outgrowth. For instance, loss of numb has been shown to result in a drastic reduction of neurite length in cultured cortical neurons. Furthermore, cortical neurons in nestinCre/+;fnb/fnb;nbl–/– or Emx-Cre/+;fnb/fnb;nbl–/– mutants show severe deficits in dendritic growth and branching. Since the development of sensory neurons and spinal interneurons are not affected in Wnt1Cre/+;fnb/fnb;nbl–/– mutants, abnormalities in axonal growth and collateral branching in these mutants most likely are caused by cell-autonomous roles of numb and nbl in regulating sensory axonal branching. Indeed, deletion of numb using HSV-iCre in sensory neurons from conditional mutants (fnb/fnb; nbl–/–) leads to a significant reduction in axonal branching and total length (Huang, 2005).

How might Numb and Nbl regulate axonal growth and collateral branching in sensory neurons? These results indicate that Numb and Notch1 show extensive colocalization in both axons and cytoplasm. Consistent with these findings, Numb has been shown to colocalize with alpha-adaptin and internalized EGFR and transferrin receptors in the endocytic vesicles in nonneuronal cells. Furthermore, Numb interacts with the ear appendage of alpha-adaptin via the two consecutive tripeptide sequences, Asp-Pro-Phe and Asn-Pro-Phe, in the C terminus. Interestingly, these sequences are highly conserved in mammalian and Drosophila Numb, suggesting that the interaction between Numb and alpha-adaptin may be highly conserved in evolution. Indeed, Drosophila Numb also interacts with the ear domain of Drosophila alpha-adaptin, and Drosophila Numb is required for asymmetric localization of alpha-adaptin in SOP during mitosis. Most important, mutations in alpha-adaptin lead to cell fate transformation phenotype that is similar to, albeit weaker than, numb mutants. Although epistatic analyses suggest that Drosophila numb acts upstream of alpha-adaptin and that Notch functions upstream or in parallel to alpha-adaptin, detailed mechanisms of the interactions among these three molecules remain unclear (Huang, 2005).

The extensive colocalization of Numb and Notch1 in endosomal vesicles of axons and cell body of sensory neurons suggests that Numb may regulate retrograde transport of Notch in post-mitotic neurons. Consistent with this notion, loss of numb results in a dramatic reduction in the number of endosomal vesicles. In contrast, overexpression of Numb, but not Nbl, leads to the formation of markedly enlarged vesicular structures with characteristics of late endosome and lysosome. The ability of Numb to induce such abnormal changes in the endosomal pathway requires the alpha-adaptin-interacting domain in the C terminus as deletion of the two tripeptide sequences in Numb (EYFP-numbDelta557-593) completely abolishes this effect. Compared with Numb, Nbl contains only one tripeptide sequence (569DPF) required for alpha-adaptin interaction, it is less abundant in the axons, and shows more diffuse distribution in the cytoplasm. Although overexpression of Nbl does not have similar effects as Numb, in loss-of-function analyses, Nbl shows partial compensatory effect in regulating axonal branching. It is possible that the redundancy between Numb and Nbl could be due to the fact that both proteins act in a sequential or parallel fashion to regulate the same pathway (Huang, 2005).

The data are consistent with the interpretation that Numb regulates endosomal pathway by controlling the delicate balance between the recycling of early endosome and the formation of late endosome/lysosome. One major function of the lysosomal pathway is to dampen signal transduction of membrane receptors by protein degradation, which may contribute to the regulation of Notch1. In the absence of numb and nbl, sensory neurons show a modest increase of Notch1 in the nucleus. Since accumulation of nuclear Notch1 ICD has been shown to regulate gene expression and affect dendritic arborization, it is almost certainly true that a similar effect must regulate axonal branching in the sensory neurons. Conversely, overexpressing Numb leads to accumulation of Notch1 in the abnormally enlarged lysosomes with a concomitant reduction of Notch1 in the nucleus. These findings suggest that at least two pathways are involved in regulating Notch signaling mechanisms. One is the endocytic-lysosomal pathway, in which Numb regulates endocytosis and degradation of Notch, while the other involves transport of Notch ICD to the nucleus through mechanisms that are poorly characterized at the present time. Based on the distribution of Numb and Nbl in sensory neurons, it is possible that these two proteins may regulate intracellular transport of Notch in a sequential or parallel mechanism. These results complement the recent findings that endocytosis down-regulates LIN-12/Notch in C. elegans through a dileucine-containing sorting motif in the cytoplasmic domain of Notch. Moreover, it has also been shown that the Drosophila Notch ligand, Delta, is subject to ubiqutin ligase-dependent internalization and degradation, suggesting that the functions of Notch and Delta might be regulated through evolutionarily conserved endocytosis-mediated pathways. The current results are consistent with the recent data that mammalian Numb promotes the ubiquitination of membrane-bound Notch1 through an E3 ligase-dependent mechanism, which leads to degradation of Notch1 ICD and loss of Notch-dependent transcriptional activation of Hes1. The fact that Numb is involved in the endocytosis of Notch may have additional impacts on the cleavage of membrane-bound Notch. Indeed, recent reports indicate that Numb can also interact with the intracellular domain of ß-amyloid precursor protein (APP) and such interaction is capable of inhibiting Notch signaling through gamma-secretase-mediated pathways. Taken together, these results underscore a previously unidentified function of Numb and Nbl in postmitotic neurons (Huang, 2005).

Numb and growth cone collapse

Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Numb is identified as a CRMP-2-interacting protein. Numb has been shown to interact with alpha-adaptin and to be involved in endocytosis. Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb colocalize. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibits endocytosis of L1 at axonal growth cones and suppresses axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1 (Nishimura, 2003).

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. Rho kinase phosphorylates CRMP-2 during growth cone collapse. CRMP-2 is required for the growth cone collapse of dorsal root ganglion neurons induced by a repulsive guidance cue, semaphorin-3A (Sema3A; also known as collapsin-1). UNC-33, the Caenorhabditis elegans homologue (30% homology), was identified by a mutation resulting in severely uncoordinated movement, abnormalities in axon guidance, and a superabundance of microtubules in neurons. The roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. This study reports that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse (Arimura, 2005).

Knockdown of CRMP-2 in hippocampal neurons has been shown to inhibit both Numb-mediated endocytosis neural cell adhesion molecule L1 and axon axon growth. This study reports that phosphorylated CRMP-2 could not associate with Numb. Thus, it is possible that CRMP-2 phosphorylation also inhibits Numb-mediated L1 endocytosis. However, there have been other reports that growth cone collapse triggered by Sema3A or ephrins was accompanied by enhanced endocytosis; these studies observed the fluorescence-labeled dextran uptake or reorganization of signaling molecules neuropilin 1 (NP1), plexin, and Rac in response to guidance cues. In a reconstituted Sema3A signaling system in COS-7 cells expressing the receptor components NP1 and plexin A1, CRMP and plexin A1 form a physical complex and CRMP accelerates Sema3A-induced cell contraction. In contrast, NGF signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking, as revealed by the increased endocytosis of transferrin. Judging from these reports, endocytosis appears to be selectively regulated for axon outgrowth and growth cone collapse. L1 and L1 recycling are crucial for axon elongation and growth cone motility. Inactivation of CRMP-2 may be required for the selective inhibition of cell adhesion molecules to prevent the growth cone dynamics. Although the exact mechanisms causing growth cone collapse need additional study, the current results imply that ephrin-A5-induced growth cone collapse is enhanced through the inhibition of Numb-mediated endocytosis via CRMP-2 phosphorylation (Arimura, 2005).

Postnatal deletion of Numb/Numblike reveals repair and remodeling capacity in the subventricular neurogenic niche

Neural stem cells are retained in the postnatal subventricular zone (SVZ), a specialized neurogenic niche with unique cytoarchitecture and cell-cell contacts. Although the SVZ stem cells continuously regenerate, how they and the niche respond to local changes is unclear. This study generated nestin-creERtm transgenic mice with inducible Cre recombinase in the SVZ and removed Numb/Numblike, key regulators of embryonic neurogenesis from postnatal SVZ progenitors and ependymal cells. This resulted in severe damage to brain lateral ventricle integrity and identified roles for Numb/Numblike in regulating ependymal wall integrity and SVZ neuroblast survival. Surprisingly, the ventricular damage was eventually repaired: SVZ reconstitution and ventricular wall remodeling were mediated by progenitors that escaped Numb deletion. These results show a self-repair mechanism in the mammalian brain and may have implications for both niche plasticity in other areas of stem cell biology and the therapeutic use of neural stem cells in neurodegenerative diseases (Kuo, 2006).

The mammalian Golgi regulates Numb signaling in asymmetric cell division by releasing ACBD3 during mitosis

Mammalian neural progenitor cells divide asymmetrically to self-renew and produce a neuron by segregating cytosolic Numb proteins primarily to one daughter cell. Numb signaling specifies progenitor over neuronal fates but, paradoxically, also promotes neuronal differentiation. This study reports that ACBD3 (Drosophila homolog; CG14232) is a Numb partner in cell-fate specification. ACBD3 and Numb proteins interact through an essential Numb domain, and the respective loss- and gain-of-function mutant mice share phenotypic similarities. Interestingly, ACBD3 associates with the Golgi apparatus in neurons and interphase progenitor cells but becomes cytosolic after Golgi fragmentation during mitosis, when Numb activity is needed to distinguish the two daughter cells. Accordingly, cytosolic ACBD3 can act synergistically with Numb to specify cell fates, and its continuing presence during the progenitor cell cycle inhibits neuron production. It is proposed that Golgi fragmentation and reconstitution during cell cycle differentially regulate Numb signaling through changes in ACBD3 subcellular distribution and represent a mechanism for coupling cell-fate specification and cell-cycle progression (Zhou, 2007).

Numb and Numbl are required for maintenance of cadherin-based adhesion and polarity of neural progenitors

The polarity and adhesion of radial glial cells (RGCs), which function as progenitors and migrational guides for neurons, are critical for morphogenesis of the cerebral cortex. These characteristics largely depend on cadherin-based adherens junctions, which anchor apical end-feet of adjacent RGCs to each other at the ventricular surface. Mouse numb and numb-like are required for maintaining radial glial adherens junctions. Numb accumulates in the apical end-feet, where it localizes to adherens junction-associated vesicles and interacts with cadherins. Numb and Numbl inactivation in RGCs decreases proper basolateral insertion of cadherins and disrupts adherens junctions and polarity, leading to progenitor dispersion and disorganized cortical lamination. Conversely, overexpression of Numb prolongs RGC polarization, in a cadherin-dependent manner, beyond the normal neurogenic period. Thus, by regulating RGC adhesion and polarity, Numb and Numbl are required for the tissue architecture of neurogenic niches and the cerebral cortex (Rasin, 2007).

These results clarify a number of discrepancies regarding the localization and function of Numb during mammalian neurogenesis. Immuno-electron microscopic analysis showed that the previously reported apical Numb crescent in mitotic cells is likely a misinterpretation of intense immunolabeling in the thin apical end-feet of adjacent interphase cells, which surround the apical pole of the neighboring mitotic cells. During mitosis of RGCs, Numb is distributed throughout the cytoplasm of the basolateral domain. Together with previous reports in Drosophila and chick, tthese findings suggest that the localization of Numb to the basolateral compartment of mitotic neural progenitors is evolutionarily conserved (Rasin, 2007).

Several lines of evidence strongly indicate that this newly described function for Numb in cell adhesion and polarity is independent of its previously described roles in the inhibition of Notch signaling and specification of cell fate during asymmetric cell division. First, Notch activation, which is expected to increase in the absence of Numb and Numbl, blocks cortical neurogenesis during embryogenesis without causing defects in RGC adhesion or polarity. Consistent with this, expression of activated Notch in the postnatal ependymal and SEZ neural progenitor cells does not induce adhesion defects in the ependymal wall. Second, the results indicate that neither inactivation nor overexpression of Numb and Numbl blocks cortical neurogenesis in a manner similar to activated Notch. Moreover, in double KO mice, the astrocytic differentiation of RGCs lacking Numb and Numbl did not occur prematurely, but is increased during the perinatal period when cortical astrocytes are normally generated, and occurs after the appearance of hydrocephalus. Furthermore, by using in utero electroporation to deliver shRNA-expressing plasmids after Cajal-Retzius neurons were generated, the possibility of secondary effects arising from the disruption of Cajal-Retzius neurons, which may indirectly affect RGC morphology and induce rosette formation, was ruled out. In conclusion, this study uncovers an additional function for Numb in regulating cell adhesion and polarity of neural progenitors, indicating that Numb has diverse roles in neural development (Rasin, 2007).

Transitin, a nestin-like intermediate filament protein, mediates cortical localization and the lateral transport of Numb in mitotic avian neuroepithelial cells

Neuroepithelium is an apicobasally polarized tissue that contains neural stem cells and gives rise to neurons and glial cells of the central nervous system. The cleavage orientation of neural stem cells is thought to be important for asymmetric segregation of fate-determinants, such as Numb. This study shows that an intermediate filament protein, transitin, colocalizes with Numb in the cell cortex of mitotic neuroepithelial cells, and that transitin anchors Numb via a physical interaction. Detailed immunohistological and time-lapse analyses reveal that basal Numb-transitin complexes shift laterally during mitosis, allowing asymmetric segregation of Numb-transitin to one of the daughter cells, even when the cell cleavage plane is perpendicular to the ventricular surface. In addition, RNA interference (RNAi) knockdown of the transitin gene reveals its involvement in neurogenesis. These results indicate that transitin has important roles in determining the intracellular localization of Numb, which regulates neurogenesis in the developing nervous system of avian embryos (Wakamatsu, 2007).

Previous studies have reported that Numb localizes in the basal cortex of mitotic NE cells. In this study, it was show that the intermediate filament protein transitin provides an anchor site for Numb in the basal cortex of mitotic NE cells. How transitin initially localizes to the basal cortex of prophase NE cells is not clear, but it has been reported that transitin mRNA is preferentially transported to the basal processes of interphase NE cells. It is therefore possible that locally translated transitin in the basal processes may be transported to the cortex prior to mitosis. Consistent with this idea, in time-lapse analysis it was often observed that Transitin [Trans(1-327)-d1EGFP] in the basal process moved apically prior to the M phase (Wakamatsu, 2007). The regulatory mechanisms of asymmetric cell division have been extensively studied in Drosophila nervous system development, and Numb localizes asymmetrically in mitotic neural cells. Despite many similarities in vertebrates and invertebrates in the regulatory mechanism of development, however, chick and Drosophila now appear to have some differences, because the genome project of Drosophila has shown that the fly does not have cytoplasmic intermediate filaments. Even in vertebrates, the molecular machinery to control Numb localization does not appear to be conserved, because mouse Numb localizes in the apical side of NE cells, whereas avian Numb localizes in the basal cortex. Nestin is the closest relative of transitin; the two proteins are categorized in the same intermediate filament subclass due to the sequence homology in their rod domain, and because they are expressed in NE cells and muscle precursors. However, the sequence of the C-terminal tail, which is responsible for Numb-transitin association, is not conserved in nestin, and, more importantly, nestin is not asymmetrically localized in mitotic NE cells. Although it is not known whether nestin is involved in neurogenesis, nestin does not seem to directly regulate Numb localization in mouse NE cells (Wakamatsu, 2007).

This study shows that, even if the vertical cleavage plane would result in a horizontal cell division, such cells can still segregate Numb-transitin complexes asymmetrically, because these components, anchored within the basal cortex, shift laterally in late M phase, and thereby allow preferential segregation into one of the two daughter cells. It remains to be studied how the lateral transport of Numb-transitin complexes is regulated. Because, in a third of NE divisions, Trans(1-327)-d1EGFP still remained in the basal cortex and segregated symmetrically, some unknown mechanism(s) probably determines whether the lateral transport during M phase is initiated. It is of note that, at the early phase of mitosis in NE cells, vimentin is phosphorylated, which probably leads to the dissociation of the intermediate filament structure. It has been shown that such phosphorylation-dependent dissociation of intermediate filaments in the M phase is important for cytokinesis and that Aurora B activity, which is strictly regulated during M phase, is involved in this process. Thus, dissociation of rigid intermediate filament structure of vimentin-transitin by phosphorylation of vimentin in the early M phase may permit the transport of transitin. Such dissociation of intermediate filament in M phase is consistent with the fact that cortically-localized Trans(1-327)-d1EGFP becomes cytoplasmic in the late M phase (Wakamatsu, 2007).

Numb has been shown to regulate neurogenesis in mouse embryos, both positively and negatively. The cause of such discrepancy is unclear, but changes in the expression of Numb isoforms during development (Bani-Yaghoub, 2007) might explain the differences observed between mouse knockout lines, at least in part. In any case, the requirement of transitin for the proper intracellular localization of Numb suggests the involvement of transitin in the neurogenesis of avian embryos. Consistently, transitin knockdown causes a depletion of NE cells by reducing proliferation and promoting neuronal differentiation, although how the reduction of transitin expression causes such a phenotype remains elusive. One possibility is that, because transitin stabilizes Numb, transitin knockdown may lead to the reduction of Numb protein, which would otherwise inhibit neurogenesis. This idea is consistent with the observation that Numb-knockout mice show precocious neurogenesis. Alternatively, by losing the transitin anchor, a release of functional Numb in the cytoplasm may promote neurogenesis, possibly by inhibiting Notch signaling. This idea is consistent with the decreased neurogenesis observed in certain Numb-knockout mouse lines. Because it has been suggested that Numb may also influence neurogenesis independently of Notch signaling, it seems important to knockdown Numb in avian system in order to compare the phenotype with that of mouse knockouts. Nevertheless, transitin is unique, because no other intermediate filament protein has been shown to regulate neurogenesis (Wakamatsu, 2007).

Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3

Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. The endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. In cultured mammalian cells, Numb binds to integrin-βs and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, a model is proposed in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge (Nishimura, 2007).

Numb localizes at a part of CCSs and functions in integrin endocytosis as a cargo-selective adaptor. Integrin is thought to be recycled from the tail to the front of migrating cells by endocytosis. However, many focal adhesions or focal complexes formed at the cell front disassemble behind the F-actin-rich lamellipodia. Numb mainly accumulated behind lamellipodia, although a certain population of Numb still remained and colocalized with integrin at the trailing edge. In addition, localization of Numb among CCSs correlated with the position of integrin adhesions, supporting the role of Numb in integrin endocytosis. Talin is a key molecule that tethers integrin to components of focal adhesions and actin stress fibers and is critical for focal-adhesion disassembly. Mutation of a conserved tyrosine residue within the integrin-β3 intracellular domain abolished the binding of both talin and Numb, suggesting that talin and Numb cannot bind to integrin simultaneously. Consistent with these observations, interaction of Numb with talin could not be detected. In addition, the binding of Numb to integrin does not activate the integrin extracellular domain, whereas the binding of talin does. The overexpression or knockdown of Numb does not directly affect cell adhesion. Thus, it appears that Numb does not actively promote focal-adhesion disassembly, but rather recruits free integrins without the components of focal adhesions to the AP-2 complex for internalization. Preferential localization of Numb around focal adhesions at the substratum-facing surface would facilitate recruitment of integrin during focal adhesion disassembly (Nishimura, 2007).

Recent genetic screening isolated Numb as a mutant defective for peripheral glia migration along axons in Drosophila (Edenfeld, 2007). Migration defects of postmitotic neurons have been described in Numb-knockout mice, indicating that Numb regulates particular cell migration in vivo. However, the defects of Numb knockdown on integrin endocytosis and cell migration are less marked than those of AP-2 and clathrin knockdown, suggesting that another adaptor molecule(s) may function in integrin endocytosis. A good candidate is disabled-2 (Dab2), which has a similar domain structure as Numb and binds to both components of clathrin-mediated endocytosis and to integrin-β. Dab2 is expressed in HeLa cells and positively controls cell adhesion and spreading. In contrast to Numb, Dab2 appears to preferentially localize to the apical surface. Thus, Numb and Dab2 may coordinately function in integrin endocytosis in different subcellular compartments for cell motility (Nishimura, 2007).

How does Numb localize at the substratum-facing surface and polarize toward the leading edge? Integrin adhesions could activate several intracellular signaling events and promote protein transport to adhesion sites by targeting microtubules and linking actin stress fibers. The actin cytoskeleton and/or adhesion itself are important for the preferential localization of Numb around adhesions. However, Numb still localized at the substratum-facing surface in the presence of cytochalasin-D, indicating that an additional mechanism may exist. Observations indicate that direct phosphorylation by aPKC may be a part of the regulatory mechanism underlying Numb localization at the substratum-facing surface. In addition, polarized localization of Numb toward the leading edge was lost upon aPKC knockdown. In support of these observations, asymmetric localization of Numb in Drosophila has been shown to be dependent on cortical actomyosin and the polarized localization/function of aPKC and PAR-3. Conclusive evidence will require isolation of the responsible motor(s) and anchor protein(s) for specific Numb localization (Nishimura, 2007).

Numb-full-3A, mutated at three phosphorylation sites, did not function as a constitutively active form that promotes integrin endocytosis and cell migration, but rather inhibited these processes. Similarly, both the phospho-mimic and nonphosphorylated form of μ2-adaptin, which is phosphorylated by AAK1, inhibit transferrin endocytosis, suggesting that clathrin-mediated endocytosis is tightly controlled by cycles of phosphorylation and dephosphorylation. Additional phosphorylation during endocytosis may be required for the dissociation of Numb from the binding proteins, integrin-β, and α-adaptin. Numb is indeed phosphorylated by several PKCs and CaMKs, and phosphatase inhibitor dramatically increases the phosphorylation level of Numb. Thus, local phosphorylation and dephosphorylation seems to allow Numb to localize defined CCSs around adhesion sites (Nishimura, 2007).

Trafficking of internalized integrin is regulated by growth factors and the extracellular matrix through several adaptors/kinases, including PI3-kinase, PKB/Akt, GSK3β, and PKCs. Several growth factors and adhesions indeed promote the recycling of integrins, leading to the upregulation of cell-surface expression, whereas treatment of cells with PDGF does not affect the internalization rate of integrin. The degree of colocalization and interaction of Numb with integrin-β1 was not significantly altered in HeLa cells before and after wounding. These data suggest that Numb functions constitutively in integrin endocytosis, although it is possibile that polarized migration promotes the internalization rate and amount of integrin endocytosis. It might be difficult to detect the changes in the interaction of Numb and integrin during migration due to the nature of rapid cycling of endocytosis and exocytosis and possibly due to the transient interaction. It has been reported that the inhibition of directional membrane trafficking causes membrane extension in all directions. Membrane trafficking controls the directionality of migrating cells. Taking into account the fact that Numb localization becomes polarized coincidently with directional migration, the subcellular region at which integrin is internalized and the subsequent coupling with the recycling processes could be important for efficient cell migration suitable for the particular environment (Nishimura, 2007).

NUMB controls p53 tumour suppressor activity

Numb is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. Numb is also an endocytic protein, and the Notch-Numb counteraction has been linked to this function. There might be, however, additional functions of Numb, as witnessed by its proposed role as a tumour suppressor in breast cancer. This study describes a previously unknown function for human Numb as a regulator of tumour protein p53 (also known as TP53). Numb enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of Numb expression. In primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of Numb expression causes increased activity of the receptor Notch5. Thus, in these cancers, a single event -- loss of Numb expression -- determines activation of an oncogene (Notch) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that Numb-defective breast tumours display poor prognosis. These results uncover a previously unknown tumour suppressor circuitry (Colalucam, 2008).

Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast

During embryonic development, the establishment of the primitive erythroid lineage in the yolk sac is a temporally and spatially restricted program that defines the onset of hematopoiesis. In this report, the embryonic stem cell differentiation system was used to investigate the regulation of primitive erythroid development at the level of the hemangioblast. The combination of Wnt signaling with inhibition of the Notch pathway is required for the development of this lineage. Inhibition of Notch signaling at this stage appears to be mediated by the transient expression of Numb in the hemangioblast-derived blast cell colonies. Activation of the Notch pathway was found to inhibit primitive erythropoiesis efficiently through the upregulation of inhibitors of the Wnt pathway. Together, these findings demonstrate that specification of the primitive erythroid lineage is controlled, in part, by the coordinated interaction of the Wnt and Notch pathways, and position Numb as a key mediator of this process (Cheng, 2008).

Numb family proteins are essential for cardiac morphogenesis and progenitor differentiation

Numb family proteins (NFPs), including Numb and numb-like (Numbl), are cell fate determinants for multiple progenitor cell types. Their functions in cardiac progenitor differentiation and cardiac morphogenesis are unknown. To avoid early embryonic lethality and study NFP function in later cardiac development, Numb and Numbl were deleted specifically in heart to generate myocardial double-knockout (MDKO) mice. MDKOs were embryonic lethal and displayed a variety of defects in cardiac progenitor differentiation, cardiomyocyte proliferation, outflow tract (OFT) and atrioventricular septation, and OFT alignment. By ablating NFPs in different cardiac populations followed by lineage tracing, it was determined that NFPs in the second heart field (SHF) are required for OFT and atrioventricular septation and OFT alignment. MDKOs displayed an SHF progenitor cell differentiation defect, as revealed by a variety of methods including mRNA deep sequencing. Numb regulated cardiac progenitor cell differentiation in an endocytosis-dependent manner. Studies including the use of a transgenic Notch reporter line showed that Notch signaling was upregulated in the MDKO. Suppression of Notch1 signaling in MDKOs rescued defects in p57 expression, proliferation and trabecular thickness. Further studies showed that Numb inhibits Notch1 signaling by promoting the degradation of the Notch1 intracellular domain in cardiomyocytes. This study reveals that NFPs regulate trabecular thickness by inhibiting Notch1 signaling, control cardiac morphogenesis in a Notch1-independent manner, and regulate cardiac progenitor cell differentiation in an endocytosis-dependent manner. The function of NFPs in cardiac progenitor differentiation and cardiac morphogenesis suggests that NFPs might be potential therapeutic candidates for cardiac regeneration and congenital heart diseases (Zhao, 2014).

numb: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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