Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells (Itoh, 2003).
Precise regulation of Notch signaling activity is critical for development of many different tissues. The zebrafish insertional mutation Hi904 attenuates Notch signaling, and is allelic to mind bomb. Mind bomb protein displays E3 ubiquitin ligase activity in vitro, and it is associated with Delta and enhances its ubiquitination and internalization in transfected cells. By functional analysis of three conserved regions of Mind bomb, it is shown that the N-terminal half is required for Delta association, the ankyrin repeats are important for Delta internalization, and the ring fingers are required for Delta ubiquitination. Thus, the three functionally distinct modules of Mind bomb work cooperatively to regulate Notch signaling by associating with, ubiquitinating, and internalizing Delta (Chen, 2004).
Somitogenesis is a highly controlled process that results in segmentation of the paraxial mesoderm. Notch pathway activity in the presomitic mesoderm is fundamental for management of synchronized gene expression which is necessary for regulation of somitogenesis. An embryonic lethal mutation, SBU2, was isolated that causes somite formation defects very similar to Notch pathway mutants. SBU2 mutants generate only 6-7 asymmetrically arranged somites. However, in contrast to Notch pathway mutants, these mutants do not maintain previously formed somite boundaries and by 24 hpf, almost no somite boundaries remain. Other developmental processes disrupted in SBU2 mutants include tail morphogenesis, muscle fiber elongation, pigmentation, circulatory system development and neural differentiation. These defects are the result of a nonsense mutation within the spt6 gene (see Drosophila Spt6). spt6 encodes a transcription elongation factor that genetically interacts with the Paf-1 chromatin remodeling complex. SBU2 mutant phenotypes could be rescued by microinjection of spt6 mRNA and microinjection of spt6 morpholinos phenocopied the mutation. Real-time PCR analysis revealed that Spt6 is essential for the transcriptional response to activation of the Notch pathway. Analysis of sbu2;mib double mutants indicates that Spt6 deficiency suppresses the neurogenic effects of the mib. Altogether, these results demonstrate that Spt6 is critical for somite formation in zebrafish and suggest that some defects observed in spt6 mutants result from alterations in Notch signaling. However, additional Spt6 mutant phenotypes are likely caused by vital functions of Spt6 in other pathways (Kok, 2007).
Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells, forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle development suggest that this pattern could be generated through lateral inhibition mediated by Notch signalling. In the zebrafish ear rudiment, homologues of Notch are widely expressed, while the Delta homologues deltaA, deltaB and deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein, would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signalling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene expression. Similar misregulation of delta genes is seen in the ear, along with misregulation of a Serrate homologue, serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signalling controls the pattern of sensory cell differentiation in the ear (Haddon, 1998).
Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analyzing mutant zebrafish embryos, it has been found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, it was also learned that constitutive Notch activity promotes formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. These data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate (Park, 2003).
Because mouse embryos that are homozygous for null mutations of Delta or Notch genes die at early stages ofeural development, there is little information that addresses the requirement of Notch signaling for vertebrate CNS glial specification. This limitation can be circumvented through analysis of mice in which Notch1 is conditionally inactivated in the cerebellum. These mice prematurely express neuronal markers and have reduced number of mutant cerebellar cells that express the glial marker GFAP. In an alternative approach, neurospheres can derived from Delta-like 1 mutant mice. After culturing, mutant neurospheres produce excess neurons and a deficit of oligodendrocytes and astrocytes compared with controls. Additionally, retinas of mice that are homozygous for a mutation of Hes5, which encodes a downstream effector of Notch signaling, have fewer Müller glia than the wild type. These observations are consistent with the idea that Delta-Notch signaling regulates neuronal-glial fate decisions (Park, 2003).
Several lines of evidence point toward a role for Delta-Notch signaling in regulating specification of motoneuron and oligodendrocyte fates in zebrafish. (1) Prospective primary motoneurons are usually replaced when they are removed at the 11-somite stage. This is similar to observations that ablated neuroblasts were replaced by neighboring cells in grasshoppers and raises the possibility that primary motoneurons, like grasshopper neuroblasts, inhibit neighboring precursors from adopting the same fate. (2) Prospective primary motoneurons expressed higher levels of the two Delta-related genes dla and dld than neighboring cells, indicating that Notch ligands are present at the right time and place to regulate specification of cells that arise in close proximity to primary motoneurons. (3) Mutant zebrafish that had reduced levels of Notch signaling had excess primary motoneurons and a concomitant deficit of later-born secondary motoneurons, showing that Delta-Notch signaling regulates specification of neural precursors for different neuronal fates. Finally, medial neural plate cells, which occupy ventral spinal cord upon completion of neurulation, give rise to primary motoneurons and oligodendrocytes. Thus, Delta proteins expressed by primary motoneurons can regulate specification of nearby cells for oligodendrocyte fate (Park, 2003).
dla-/-;dld-/- and mib-/- embryos [mind bomb (mib)] encodes a ubiquitin ligase necessary for efficient Notch signaling] do not produce OPCs or premyelinating oligodendrocytes. Additionally, neural precursors prematurely exit the cell cycle and differentiated as neurons in these embryos. Since secondary motoneurons and oligodendrocytes arise after primary motoneurons, one interpretation of the data is that Notch signaling prevents a subset of ventral spinal cord precursors from developing as primary motoneurons, enabling them to take later neuronal or oligodendrocyte fates. In this view, downregulation of delta gene expression during primary motoneuron differentiation would result in a decrease of Notch activity in neighboring precursors. A release from Notch-mediated inhibition soon after primary motoneuron specification might allow a cell to develop as a secondary motoneuron, whereas a later release might result in oligodendrocyte development. Thus, temporal regulation of Notch signaling might underlie the temporal switch in production of primary motoneurons to secondary motoneurons to oligodendrocytes (Park, 2003).
A switch between production of neurons and glial cells has been proposed to be regulated by bHLH proteins. In the ventral spinal cord, motoneuron and oligodendrocyte precursors expressed Olig bHLH proteins, which are structurally similar to proneural Ngns. During the period of motoneuron production, a subset of Olig+ cells expressed Ngns. Later, Ngn expression subsides, coincident with the time at which oligodendrocytes are thought to be specified. These observations, coupled with various functional tests, led to the proposal that Ngn and Olig proteins create a simple bHLH protein code in which Ngn and Olig expression together specify motoneuron development and Olig alone, upon Ngn downregulation, specifies oligodendrocyte development (Park, 2003).
The data provide evidence supporting the importance of a bHLH protein code to motoneuron and oligodendrocyte specification and show that Delta-Notch signaling is required to establish the code. The failure to restrict ngn1 expression to a subset of medial neural plate cells in Notch signaling deficient zebrafish embryos correlates with formation of excess neurons, consistent with observations that Notch signaling inhibits proneural genes expression and neuronal development in vertebrate and invertebrate embryos. Furthermore, dla-/-;dld-/- and mib-/- embryos fail to maintain a proliferative population of olig2+ cells. This is interpreted to mean that, in the absence of Delta-Notch mediated inhibition, uniformly high levels of Ngns cause all olig2+ neural precursors to stop dividing and differentiate as neurons at the expense of oligodendrocytes. Thus, in normal embryos, high levels of Notch activity prevents ngn gene expression in a subset of olig2+ neural precursors, reserving them to produce other cell types, such as oligodendrocytes, at a later time. In this view, Delta-Notch signaling might play a purely permissive role in neural cell fate diversification, by regulating the ability of neural precursors to respond to other instructive signals (Park, 2003).
Failure of Notch signaling in zebrafish mind bomb mutants results in a neurogenic phenotype where an overproduction of early differentiating neurons is accompanied by the loss of later-differentiating cell types. The hindbrain phenotype of mib mutants has been characterized in detail. Hindbrain branchiomotor neurons (BMNs) are reduced in number but not missing in mib mutants. In addition, BMN clusters are frequently fused across the midline in mutants. Mosaic analysis indicates that the BMN patterning and fusion defects in the mib hindbrain arise non-cell autonomously. Ventral midline signaling is defective in the mutant hindbrain, in part due to the differentiation of some midline cells into neural cells. Interestingly, while early hindbrain patterning appears normal in mib mutants, subsequent rhombomere-specific gene expression is completely lost. The defects in ventral midline signaling and rhombomere patterning are accompanied by an apparent loss of neuroepithelial cells in the mutant hindbrain. These observations suggest that, by regulating the differentiation of neuroepithelial cells into neurons, Notch signaling preserves a population of non-neuronal cells that are essential for maintaining patterning mechanisms in the developing neural tube (Bingham, 2003).
During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. A potential role of Notch signaling has been investigated; in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center (Cheng, 2004).
Studies of neurogenesis in the zebrafish hindbrain have shown that differentiation first occurs at rhombomere centers, and only at late stages are neurons formed at the boundaries between rhombomeres. The spatial and temporal pattern of neurogenesis is reflected by the expression of delta genes that mark early neuroblasts: expression is excluded from rhombomere boundaries, and by 24 hr occurs in stripes adjacent to the boundaries. These observations are consistent with Delta mediating a lateral inhibition in a manner analogous to its widely utilized role in the neural epithelium, in which Delta expression by early neuroblasts activates Notch and suppresses neurogenesis and delta expression in adjacent cells. Indeed, ectopic expression of dominant-active Su(H) suppresses delta expression throughout the hindbrain. An important role of the lateral inhibition of neurogenesis is to maintain the progenitor pool of neural epithelial cells that is required for the continued generation of neurons. mind bomb (mib) mutant embryos have a strong Notch pathway deficiency due to mutation of a ubiquitin ligase required for Delta ligand activity. Boundary markers are severely depleted in mib mutant embryos -- this suggests that lateral inhibition maintains the neural epithelium not only in nonboundary regions but also at hindbrain boundaries. Consistent with a role for Notch activation in maintaining boundary cells, following mosaic expression of dominant-active Su(H) in mib mutants, the expressing cells sort to boundaries and boundary marker gene expression is rescued (Cheng, 2004).
These findings reveal that two responses to the activation of Notch are coupled at rhombomere boundaries in the zebrafish hindbrain: the regulation of cell affinity properties of boundary cells and the suppression of neurogenesis. This begs the question of why neurogenesis is delayed at rhombomere boundaries. An attractive possibility is suggested by the observation that signaling centers in the neural epithelium such as the floor plate and roof plate do not undergo neurogenesis and have a low rate of cell proliferation. By enabling the maintenance of a relatively stable number of signaling cells, the suppression of differentiation and proliferation is a simple way to maintain a constant amount of signal. By analogy, the suppression of neurogenesis and proliferation at rhombomere boundaries may reflect that the radical fringe-dependent expression of wnt1 by rhombomere boundary cells is involved in patterning of the zebrafish hindbrain. The regulation by Notch of both cell affinity and the suppression of differentiation at rhombomere boundaries would thus provide a coupling between maintenance of the location and number of signaling cells (Cheng, 2004).
Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, retinas from zebrafish embryos were examined in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. Inactivating Notch-Delta signaling does not prevent differentiation of retinal neurons, but it disrupts spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiate, but their laminar arrangement is disrupted. Photoreceptor differentiation is initiated normally, but its progression is slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors is disrupted: cones of the same spectral subtype are clumped rather than regularly spaced. In contrast to neurons, Muller glia fail to differentiate, suggesting an instructive role for Notch-Delta signaling in gliogenesis (Bernardos, 2005).
The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. Monoclonal antibodies have been generated to identify the absorptive and secretory cells in the epithelium, and these antibodies were used to examine the role that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an after eight (aei) mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium (Crosnier, 2005).
The zebrafish gene mind bomb encodes a protein that positively regulates the Delta-mediated Notch signaling. It interacts with the intracellular domain of Delta to promote its ubiquitination and endocytosis. In a search for the mouse homologue of zebrafish mind bomb, two homologues in the mouse genome were cloned: a mouse orthologue (mouse mib1) and a paralogue, named mind bomb-2 (mib2), which is evolutionarily conserved from Drosophila to human. Both Mib1 and Mib2 have an E3 ubiquitin ligase activity in their C-terminal RING domain and interact with Xenopus Delta (XD) via their N-terminal region. Mib2 is also able to ligate ubiquitin to XD and shift the membrane localization of Delta to intracellular vesicles. Importantly, Mib2 rescues both the neuronal and vascular defects in the zebrafish mibta52b mutants. In contrast to the functional similarities between Mib1 and Mib2, mib2 is highly expressed in adult tissues, but almost not at all in embryos, whereas mib1 is abundantly expressed in both embryos and adult tissues. These data suggest that Mib2 has functional similarities to Mib1, but might have distinct roles in Notch signaling as an E3 ubiquitin ligase (Koo, 2005a).
Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. Mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulates in the plasma membrane, while it is localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. These results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling (Koo, 2005b).
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