Regulatory roles of presenilin-1 and nicastrin in neuronal differentiation during in vitro neurogenesis

Presenilin (PS) in association with nicastrin (NICA) forms a gamma-secretase complex that plays a crucial role in facilitating intramembranous processing of Notch, a signaling receptor that is essential for neuronal fate specification and differentiation. Loss of function studies have implicated a role for PS1 in regulating neuronal differentiation in association with the down-regulation of Notch signaling during neurogenesis. By using a system for stable, as well as tetracycline-inducible expression of interfering RNAs (RNAi), we studied the functions of PS1 during neuronal differentiation in the murine pluripotent p19 embryonic carcinoma cell line. After retinoic acid (RA) treatment and in the absence of doxycycline, neuronal progenitor cells in the p19 clone were found to extend their processes towards the neighboring colony to form network-like connections, as revealed by neuron-specific microtubule-associated protein 2 staining and laser scanning confocal microscopy. However, doxycycline-induced expression of PS1 small interfering RNA (siRNA) in the p19 clone resulted in a severe defect in the formation of network-like connections. Expression of the NICA and Notch down-stream effector genes Hes1 and Hes5 was unaffected in p19 cells expressing doxycycline-induced PS1 siRNA. In contrast to PS1, constitutive inactivation of NICA by siRNA in p19 cells resulted in premature and partial differentiation without RA treatment. In these NICA siRNA-expressing p19 cells the expression of the Notch1 down-stream effector Hes1 gene was substantially reduced. After RA treatment the NICA siRNA clone failed to differentiate completely into networks of neurons. These results taken together provide direct evidence that PS1 and NICA may participate in neuronal differentiation during neurogenesis in vitro.     

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.     

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.     

Notch signaling regulates neural precursor allocation and binary neuronal fate decisions in zebrafish

Notch signaling plays a well-described role in regulating the formation of neurons from proliferative neural precursors in vertebrates but whether, as in flies, it also specifies sibling cells for different neuronal fates is not known. Ventral spinal cord precursors called pMN cells produce mostly motoneurons and oligodendrocytes, but recent lineage-marking experiments reveal that they also make astrocytes, ependymal cells and interneurons. Our own clonal analysis of pMN cells in zebrafish showed that some produce a primary motoneuron and KA' interneuron at their final division. We investigated the possibility that Notch signaling regulates a motoneuron-interneuron fate decision using a combination of mutant, transgenic and pharmacological manipulations of Notch activity. We show that continuous absence of Notch activity produces excess primary motoneurons and a deficit of KA' interneurons, whereas transient inactivation preceding neurogenesis results in an excess of both cell types. By contrast, activation of Notch signaling at the neural plate stage produces excess KA' interneurons and a deficit of primary motoneurons. Furthermore, individual pMN cells produce similar kinds of neurons at their final division in mib mutant embryos, which lack Notch signaling. These data provide evidence that, among some postmitotic daughters of pMN cells, Notch promotes KA' interneuron identity and inhibits primary motoneuron fate, raising the possibility that Notch signaling diversifies vertebrate neuron type by mediating similar binary fate decisions.     

Dicer is required for the maintenance of notch signaling and gliogenic competence during mouse retinal development

MicroRNAs (miRNAs) are 19-25 nucleotide RNAs that regulate messenger RNA translation and stability. Recently, we performed a conditional knockout (CKO) of the miRNA-processing enzyme Dicer during mouse retinal development and showed an essential role for miRNAs in the transition of retinal progenitors from an early to a late competence state (Georgi and Reh [2010]: J Neurosci 30:4048-4061). Notably, Dicer CKO progenitors failed to express Ascl1 and generated ganglion cells beyond their normal competence window. Because Ascl1 regulates multiple Notch signaling components, we hypothesized that Notch signaling is downregulated in Dicer CKO retinas. We show here that Notch signaling is severely reduced in Dicer CKO retinas, but that retinal progenitors still retain a low level of Notch signaling. By increasing Notch signaling in Dicer CKO progenitors through constitutive expression of the Notch intracellular domain (NICD), we show that transgenic rescue of Notch signaling has little effect on the competence of retinal progenitors or the enhanced generation of ganglion cells, suggesting that loss of Notch signaling is not a major determinant of these phenotypes. Nevertheless, transgenic NICD expression restored horizontal cells, suggesting an interaction between miRNAs and Notch signaling in the development of this cell type. Furthermore, while NICD overexpression leads to robust glial induction in control retinas, NICD overexpression was insufficient to drive Dicer-null retinal progenitors to a glial fate. Surprisingly, the presence of transgenic NICD expression did not prevent the differentiation of some types of retinal neurons, suggesting that Notch inactivation is not an absolute requirement for the initial stages of neuronal differentiation.     

Notch signaling coordinates the patterning of striatal compartments

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation.     

Notch resolves mixed neural identities in the zebrafish epiphysis

Manipulation of Notch activity alters neuronal subtype identity in vertebrate neuronal lineages. Nonetheless, it remains controversial whether Notch activity diversifies cell fate by regulating the timing of neurogenesis or acts directly in neuronal subtype specification. Here, we address the role of Notch in the zebrafish epiphysis, a simple structure containing only two neural subtypes: projection neurons and photoreceptors. Reducing the activity of the Notch pathway results in an excess of projection neurons at the expense of photoreceptors, as well as an increase in cells retaining a mixed identity. However, although forced activation of the pathway inhibits the projection neuron fate, it does not promote photoreceptor identity. As birthdating experiments show that projection neurons and photoreceptors are born simultaneously, Notch acts directly during neuronal specification rather than by controlling the timing of neurogenesis. Finally, our data suggest that two distinct signals are required for photoreceptor fate specification: one for the induction of the photoreceptor fate and the other, involving Notch, for the inhibition of projection neuron traits. We propose a novel model in which Notch resolves mixed neural identities by repressing an undesired genetic program.     

Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate

Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the beta cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4(+) cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the alpha and PP subtypes. In addition, we show that activation of Notch in Pax4(+) progenitors inhibits their differentiation into alpha and beta endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4(+) cells from alpha and beta endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination.     

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.     

Roles for Dicer1 in the patterning and differentiation of the optic cup neuroepithelium

The embryonic ocular neuroepithilium generates a myriad of cell types, including the neuroretina, the pigmented epithelium, the ciliary and iris epithelia, and the iris smooth muscles. As in other regions of the developing nervous system, the generation of these various cell types requires a coordinated sequence of patterning, specification and differentiation events. We investigated the roles of microRNAs (miRNAs) in the development of optic cup (OC)-derived structures. We inactivated Dicer1, a key mediator of miRNA biosynthesis, within the OC in overlapping yet distinct spatiotemporal patterns. Ablation of Dicer1 in the inner layer of the OC resulted in patterning alteration, particularly at the most distal margins. Following loss of Dicer1, this region generated a cryptic population of cells with a mixed phenotype of neuronal and ciliary body (CB) progenitors. Notably, inactivation of Dicer1 in the retinal progenitors further resulted in abrogated neurogenesis, with prolongation of ganglion cell birth and arrested differentiation of other neuronal subtypes, including amacrine and photoreceptor cells. These alterations were accompanied by changes in the expression of Notch and Hedgehog signaling components, indicating the sensitivity of the pathways to miRNA activity. Moreover, this study revealed the requirement of miRNAs for morphogenesis of the iris and for the regulation of CB cell type proliferation and differentiation. Together, analysis of the three genetic models revealed novel, stage-dependent roles for miRNAs in the development of the ocular sub-organs, which are all essential for normal vision.     

Reelin sets the pace of neocortical neurogenesis

Migration of neurons during cortical development is often assumed to rely on purely post-proliferative reelin signaling. However, Notch signaling, long known to regulate neural precursor formation and maintenance, is required for the effects of reelin on neuronal migration. Here, we show that reelin gain-of-function causes a higher expression of Notch target genes in radial glia and accelerates the production of both neurons and intermediate progenitor cells. Converse alterations correlate with reelin loss-of-function, consistent with reelin controlling Notch signaling during neurogenesis. Ectopic expression of reelin in isolated clones of progenitors causes a severe reduction in neuronal differentiation. In mosaic cell cultures, reelin-primed progenitor cells respond to wild-type cells by further decreasing neuronal differentiation, consistent with an increased sensitivity to lateral inhibition. These results indicate that reelin and Notch signaling cooperate to set the pace of neocortical neurogenesis, a prerequisite for proper neuronal migration and cortical layering.     

Notch in the pathway: the roles of Notch signaling in neural crest development

Here, we review recent studies that suggest that Notch signaling has two roles during neural crest development: first in establishing the neural crest domain within the ectoderm via lateral induction and subsequently in diversifying the fates of cells that arise from the neural crest via lateral inhibition. The first of these roles, specification of neural crest via lateral induction, has been explored primarily in the cranial neural folds from which the cranial neural crest arises. Evidence for such a role has thus far only been obtained from chick and frog; results from these two species differ, but share the feature that Notch signaling regulates genes that are expressed by cranial neural crest through effects on expression of Bmp family members. The second of these roles, diversification of neural crest progeny via lateral inhibition, has been identified thus far only in trunk neural crest. Evidence from several species suggests that Notch-mediated lateral inhibition functions in multiple episodes in this context, in each case inhibiting neurogenesis. In the 'standard' mode of lateral inhibition, Notch promotes proliferation and in the 'instructive' mode, it promotes specific secondary fates, including cell death or glial differentiation. We raise the possibility that a single molecular mechanism, inhibition of so-called proneural bHLH genes, underlies both modes of lateral inhibition mediated by Notch signaling.     

Stem-loop binding protein is required for retinal cell proliferation,neurogenesis, and intraretinal axon pathfinding in zebrafish

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3′-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5 dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.