Xenopus Delta homologs

The Xenopus homolog of Delta mediates lateral inhibition in neural development (Chitnis, 1995).

A zebrafish Delta homolog (delta D) is a 717 amino acid protein with all the characteristic features of this family of proteins: a signal peptide, a transmembrane domain, and an extracellular region comprising the DSL domain and eight EGF-like repeats. The gene is transcribed in a complex pattern in the developing nervous system as well as in the hypoblast. For example, three longitudinal expression domains are present in the neural plate and deltaD is transcribed within the primordia of fore-, mid- and hindbrain. Overexpression of this gene following mRNA injections leads to a reduction in the number of islet-I positive cells, which are assumed to be primary neurons, and to various defects in the adaxial mesoderm, as well as in the somites and myotomes. This suggests that delta D, and the Notch signaling pathway are involved in the differentiation of primary neurons within the neural plate, as well as in somite development (Dornseifer, 1997).

Recently, a model to explain the mechanism of Xenopus tail bud formation has been proposed. The NMC model proposes that three regions around the late blastopore lip are required to initiate tail formation. These are the posterior-most neural plate, fated to form tail somites (M), the neural plate (N), immediately anterior to M, and the underlying caudal notochord (C). To initiate tail formation, C must underlie (and presumably signal to) the junction of N and M, which subsequently forms the tip of the tail. During normal development, the NMC interaction leading to specification of the tail bud occurs at the end of gastrulation. Outgrowth of the tail bud commences much later, becoming clearly visible by stage 30 (Beck, 1998 and references).

Several domains of the Xenopus tail bud are defined by two phases of gene expression. The first group of genes are already expressed in the tail bud region before its determination at stage 13 and are subsequently restricted in the extending tail bud by stage 30. This group, the early genes, includes the Notch ligand X-delta-1, the lim domain homeobox factor Xlim1, the T-box factor Xbra, and the homeobox factor Xnot2 and Xcad2, a member of the caudal family. X-delta-1 is expressed specifically in the posterior wall of the neuroenteric canal but is excluded from the chordoneural hinge at stage 30, thus maintaining its earlier expression in the lateral and ventral blastopore lips. Xim1 is expressed in the notochord and dorsal blastopore lip at the end of gastrulation, and is maintained in the chordoneural hinge and posterior tip of the differentiated notochord in later stages. Xnot2 is expressed in the ventral neural tube and chordoneural hinge, but not in the posterior notochord. The posterior notochord therefore represents a novel tail bud region by stage 30, marked by Xlim but not Xnot transcripts, whereas the posterior ventral neural tube is marked by Xnot but not Xbra or Xlim1. Xbra is expressed in the chordoneural hinge and posterior wall. Xcad3 expression in the posterior neural plate is later maintained in the posterior wall and posterior dorsal neural tube. Xpo is expressed in all tissues of the tail bud with the exception of the chordoneural hinge, and is expressed in the fin and epidermis (Beck 1998).

Neurogenin is a candidate as a vertebrate neuronal determination gene. Neurogenin shows 67% identity to NeuroD and is closely related to other mammalian bHLH proteins including MATH2/Nex-1 and MATH1. MATH proteins are related to the Drosophila protein Atonal, while Neurogenin is distantly related to Atonal. Neurogenin has been cloned from both the mouse and Xenopus. Mouse neurogenin and NeuroD are sequentially expressed in overlapping regions. In the ventral spinal cord, for example, ngn mRNA is expressed throughout the ventricular zone, in regions where uncommitted progenitors are located, while NeuroD transcripts are expressed at the lateral border of the ventricular zone that contains migrating neuroblasts (Ma, 1996).

Expression of the Xenopus Neurogenin protein, Xenopus Neurogenin-related-1 (X-NGNR-1) and XNeuroD show a similar spatial overlap with temporal displacement. X-NGNR-1 induces ectopic neurogenesis and ectopic expression of X NeuroD mRNA, but not vice-versa. X-ngnr-1 expression precedes expression of X-Delta-1, and X-NGNR-1 can serve to activate expression of X-Delta-1. Expression of the intracellular domain of XNotch-1 inhibits both the expression and function of X-ngnr-1. Thus endogenous X-ngnr-1 expression becomes restricted to subsets of cells by lateral inhibition mediated by X-Delta-l and X-Notch. The properties of X-NGNR-1 are thus analogous to those of the Drosophila proneural genes, suggesting that it functions as a vertebrate neuronal determination factor (Ma, 1996).

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 signaling. In the zebrafish ear rudiment, homologs of Notch are widely expressed, while the Delta homologs 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 (see Drosophila Mind bomb) mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signaling: 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 homolog, 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 signaling controls the pattern of sensory cell differentiation in the ear. Although the molecular nature of the mib gene remains to be discovered, the mutant provides a way to test the role of the Notch signaling pathway in the various tissues of the cell (Haddon, 1998b).

Delta is a candidate for regulating progenitor competence in generating the variety of mature neurons and glia found in the developing retina. For example, in the Xenopus retina, the last cells to express X-Notch-1, a Delta receptor, assume the last-generated fate in the retina, that of Müller glia, and cells expressing a dominant-activated form of X-Notch-1 do not differentiate. To test the ability of Delta to alter retinal cell fates, Delta messenger RNA was misexpressed in Xenopus retina. Delta-misexpressing cells adopt earlier fates, most often becoming ganglion cells and cone photoreceptors. Progenitors transfected with Delta later in development also produce rod photoreceptors, but not the latest-generated cell types, demonstrating the importance of timing in Delta function. It is concluded that Delta signaling in the vertebrate retina is a basic regulatory mechanism that can be used to generate neuronal diversity (Dorsky, 1997).

The posteriorizing agent retinoic acid can accelerate anterior neuronal differentiation in Xenopus laevis embryos. To elucidate the role of retinoic acid in the primary neurogenesis cascade, an investigation was carried out to see whether retinoic acid treatment of whole embryos can change the spatial expression of a set of genes known to be involved in neurogenesis. Retinoic acid expands the N-tubulin, X-ngnr-1, X-MyT1, X-Delta-1 and Gli3 domains and inhibits the expression of Zic2 and sonic hedgehog in the neural ectoderm, whereas a retinoid antagonist produces the opposite changes. In contrast, sonic and banded hedgehog overexpression reduce the N-tubulin stripes, enlarge the neural plate at the expense of the neural crest, downregulate Gli3 and upregulate Zic2. Thus, retinoic acid and hedgehog signaling have opposite effects on the prepattern genes Gli3 and Zic2 and on other genes acting downstream in the neurogenesis cascade. In addition, retinoic acid cannot rescue the inhibitory effect of NotchICD, Zic2 or sonic hedgehog on primary neurogenesis. These results suggest that retinoic acid acts very early, upstream of sonic hedgehog, and a model is proposed for regulation of differentiation and proliferation in the neural plate, showing that retinoic acid might be activating primary neurogenesis by repressing sonic hedgehog expression (Franco, 1999).

Because X-Delta-1 appears to be expressed in the future primary neurons themselves, they should be the source of the inhibitory signal that activates X-Notch-1 in the neighboring cells, thus preventing them from undergoing neuronal differentiation, inhibiting their own X-Delta-1 expression and decreasing their ability to inhibit the original signaling cell. This would generate a feedback loop that reinforces contrasts between adjacent cells. Here it is shown that RA treatment enhances the density of X-Delta-1-positive cells and it is presumed that, in this way, it impairs the developing distinction between adjacent cells, allowing more precursors to become neurons. Since X-ngnr-1 overexpression leads to X-Delta-1 overproduction, RA could be activating X-Delta-1 expression through X-ngnr-1 induction (Franco, 1999).

Xath3 encodes a Xenopus neuronal-specific basic helix-loop-helix transcription factor related to the Drosophila proneural factor Atonal. Xath3 acts downstream of X-ngnr-1 (another Atonal homolog) during neuronal differentiation in the neural plate and retina and its expression and activity are modulated by Notch signaling. X-ngnr-1 activates Xath3 and NeuroD by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm, X-ngnr-1 and Xath3 have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina, X-ngnr-1 and Xath3 overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1 and Xath3 regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity. X-ngnr-1 promotes lateral inhibition by inducing X-Delta-1 expression. To determine whether Xath3 plays a role in the induction or maintenance of X-Delta-1 expression, which starts from stage 12 onwards, Xath3 was overexpressed in embryos and these embryos were analyzed for X-Delta-1 expression by in situ hybridization. In 38/41 cases, an induction of X-Delta-1 expression was seen on the injected side, indicating that Xath3 is indeed an activator of X-Delta-1 (Perron, 1999).

Xenopus X-Serrate-1 encodes a transmembrane protein with a Delta/Serrate/LAG-2 domain, 16 epidermal growth factor-like repeats and a cysteine-rich region. Expression of X-Serrate-1 is found ubiquitous from unfertilized egg to tadpole, but an upregulation occurs in the tailbud stage embryo. Adult expression is found in eye, brain, kidney, heart, spleen and ovary. Organ-related expression occurs in eye, brain, heart and kidney from an early stage of rudiment formation. Overexpression of X-Serrate-1 leads to a reduction of primary neurons, whereas an intracellularly deleted form of X-Serrate-1 increases the number of primary neurons. Although the function of X-Serrate-1 in primary neurogenesis is quite similar to that of X-Delta-1, expression of X-Serrate-1 and X-Delta-1 do not affect each other. Co-injection experiments have shown that wild-type X-Serrate-1 and X-Delta-1 suppress overproduction of primary neurons induced by dominant-negative forms of X-Delta-1 and X-Serrate-1, respectively. These results suggest that X-Serrate-1 regulates the patterning of primary neurons in a complementary manner with X-Delta-1-mediated Notch signaling (Kiyota, 2001).

Notch signaling in Drosophila requires a RING finger (RF) protein encoded by neuralized. The Xenopus homolog of neuralized (Xneur) is expressed where Notch signaling controls cell fate choices in early embryos. Overexpressing XNeur or putative dominant-negative forms in embryos inhibits Notch signaling. As expected for a RF protein, XNeur fulfills the biochemical requirements of ubiquitin ligases. Wild-type XNeur decreases the cell surface level of the Notch ligand, XDelta1, while putative inhibitory forms of XNeur increase it. Evidence is provided that XNeur acts as a ubiquitin ligase for XDelta1 in vitro. It is proposed that XNeur plays a conserved role in Notch activation by regulating the cell surface levels of the Delta ligands, perhaps directly, via ubiquitination (Deblandre, 2001).

Different cell types that occupy the midline of vertebrate embryos originate within the Spemann-Mangold or gastrula organizer. One such cell type is hypochord, which lies ventral to notochord in anamniote embryos. Hypochord precursors arise from the lateral edges of the organizer in zebrafish. During gastrulation, hypochord precursors are closely associated with the Brachyury homolog no tail-expressing midline precursors and paraxial mesoderm; these mesoderm cells also express deltaC and deltaD. Loss-of-function experiments have revealed that deltaC and deltaD are required for her4 expression in presumptive hypochord precursors and for hypochord development. Conversely, ectopic, unregulated Notch activity blocks no tail expression and promotes her4 expression. It is proposed that Delta signaling from paraxial mesoderm diversifies midline cell fate by inducing a subset of neighboring midline precursors to develop as hypochord, rather than as notochord (Latimer, 2002).

How might Delta signals induce hypochord development? One key might be regulation of ntl expression. ntl mutant embryos lack notochord and rostral hypochord and have excess floor plate. It has been proposed that ntl regulates a midline precursor fate decision by promoting notochord and inhibiting floor-plate development. It is further proposed that modulation of ntl expression within midline precursors by Delta-Notch signaling is required for hypochord development. In this model, ntl promotes formation of a population of midline precursors that have the potential to develop either as notochord or hypochord. Activation of Notch in a subset of precursors by Delta ligands expressed by neighboring paraxial mesoderm cells induces her4 and represses ntl expression. Consistent with this, constitutive Notch activity can cell-autonomously drive ectopic her4 expression. In the absence of Notch activity, her4 expression is not induced, and excess midline cells express ntl. Thus, Notch activity diverts midline precursors from notochord to hypochord fate (Latimer, 2002).

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates -- among them are neural plate formation and neural patterning. The Xenopus Iro (Xiro) function in primary neurogenesis has been studied in detail. Misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, XGadd45-gamma has been identified as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. It is concluded that during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons (de la Calle-Mustienes, 2002).

This may be due to redundancy between different Gadd45 proteins. The spatial and temporal patterns of expression of Gadd45-gamma and the Notch ligand XDl1 largely coincide. Moreover, both XGadd45-gamma and XDl1 are positively regulated by proneural genes and negatively controlled by Notch signaling. According to the lateral inhibition model, activation of the Notch pathway within a cell, by signaling from neighboring cells, maintains the cell's mitotic potential and prevents its differentiation. In contrast, a cell that expresses high levels of Notch ligands and signals strongly, escapes lateral inhibition, exits the cell cycle and differentiates. XGadd45-gamma may provide a link between Notch signaling, cell-cycle arrest and differentiation. Thus, in the neural plate, cells with high levels of proneural genes have also high levels of XDl1 and XGadd45-gamma. The first allows them to escape lateral inhibition, and the second to exit the cell cycle. These cells can then differentiate. Mitotic arrest mediated by XGadd45-gamma probably occurs through interaction with cyclin and inhibitors of cyclin-dependent kinases. In neighboring cells, the Notch pathway is activated, proneural genes and XGadd45-gamma are downregulated, and cell-cycle arrest and differentiation cannot occur. It is of interest that induction of Gadd45 genes in cell culture stops the cell cycle in G1 phase. This phase is compatible with exiting the cell cycle, a requirement for terminal neuronal differentiation. Cells that differentiate outside the neural plate may resort to genes different from the proneural ones to accumulate Notch ligands and XGadd45-gamma (de la Calle-Mustienes, 2002).

This study compares the effects of overexpressing either Xiro1, -2 or -3 in neural development. To make comparisons more meaningful, equivalent constructs were prepared in the pCS2MT plasmid. The overexpression of each Xiro gene causes similar effects, although Xiro3 was approximately five to ten times more potent. Paradoxically, the overexpressions activated Xngnr1 and repressed neuronal differentiation. This may be explained at least in part by the finding that Xiro upregulates the neuronal repressors XHairy2A and XZic2. Indeed, it has been shown that XZic2 antagonizes development of Xngnr1-promoted ectopic neurons. XZic2 antagonizes Xngnr1-promoted XGadd45-gamma and XDl1 expression. Consistently with these findings, in wild type embryos the intermediate stripes of expression of XHairy2A and XZic2 are within the Xiro1 domains. Also in accordance with these results, in the prospective spinal chord, the Xiro1 domain is contained within the broader Xngnr1 domain and neurons arise at the border of the Xiro1 domain. Taken together, these observations suggest that Xiro proteins simultaneously participate in the activation of Xngnr1 and of genes that antagonize primary neuron formation (de la Calle-Mustienes, 2002).

Overexpressions of Xiro genes represses both XGadd45-gamma and XDl1 in territories where primary neurons arise. Consistently, in wild type embryos, XGadd45-gamma and XDl1 are expressed at the borders of Xiro domains. Moreover, XDl1 is activated in embryos expressing a Xiro1 chimera that converts the Xiro1 repressor into an activator (HD-GR-E1A). This activation occurs even in the absence of protein synthesis. Thus, XDl1 is probably directly repressed by Xiro. However, XGadd45-gamma is repressed by HD-GR-E1A, probably because Xngnr1 is also downregulated. Indeed, coinjection of HD-GR-E1A and Xngnr1 mRNAs rescues the expression of XGadd45-gamma. Thus, Xiro-mediated repression of XGadd45-gamma is probably indirect and may take place, at least in part, by Xiro-upregulated neuronal repressors. In this case, interference with Xiro function would suppress neuronal repressors, but would also downregulate Xngnr1, which is needed for XGadd45-gamma expression (de la Calle-Mustienes, 2002).

A model is proposed for the function of Xiro in neural patterning that integrates the above data. Xiro proteins, as well as other factors, participate in the activation of Xngnr1. Within the Xiro domains, Xngnr1 does not activate XDl1 or XGadd45-gamma, and cannot promote differentiation of primary neurons due to the upregulation by Xiro of neuronal repressors, such as XHairy2A and XZic2. In addition, Xiro probably represses XDl1 directly. Outside the Xiro domains, other factors, such as the Gli proteins, activate Xngnr1, which in turn promotes the expression of XDl1 and XGadd45-gamma in those cells that will become primary neurons. XDl1 switches on the lateral inhibition mechanism by which the Notch signaling pathway is activated in neighboring cells. This pathway downregulates proneural genes, XDl1 and XGadd45-gamma. As a consequence, these cells keep dividing and do not differentiate. In contrast, cells with high levels of Xngnr1, XDl1 and XGadd45-gamma escape lateral inhibition, exit the cell cycle (in part due to the presence of XGadd45-gamma) and differentiate as primary neurons. This differentiation is triggered by a genetic program activated by Xngnr1. Thus, Xiro proteins may help coordinate cell cycle and differentiation (de la Calle-Mustienes, 2002).

The neural crest is a population of cells that originates at the interface between the neural plate and non-neural ectoderm. The role that Notch and the homeoprotein Xiro1 play in the specification of the neural crest has been anayzed. Xiro1, Notch and the Notch target gene Hairy2A are all expressed in the neural crest territory, whereas the Notch ligands Delta1 and Serrate are expressed in the cells that surround the prospective crest cells. Inducible dominant-negative and activator constructs of both Notch signaling components and Xiro1 were used to analyze the role of these factors in neural crest specification without interfering with mesodermal or neural plate development (Galvic, 2004).

Activation of Xiro1 or Notch signaling leads to an enlargement of the neural crest territory, whereas blocking their activity inhibits the expression of neural crest markers. It is known that BMPs are involved in the induction of the neural crest and, thus, whether these two elements might influence the expression of Bmp4 was assessed. Activation of Xiro1 and of Notch signaling upregulates Hairy2A and inhibits Bmp4 transcription during neural crest specification. These results, in conjunction with data from rescue experiments, allow a model to be proposed wherein Xiro1 lies upstream of the cascade regulating Delta1 transcription. At the early gastrula stage, the coordinated action of Xiro1, as a positive regulator, and Snail, as a repressor, restricts the expression of Delta1 at the border of the neural crest territory. At the late gastrula stage, Delta1 interacts with Notch to activate Hairy2A in the region of the neural fold. Subsequently, Hairy2A acts as a repressor of Bmp4 transcription, ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region. Finally, the activity of additional signals (WNTs, FGF and retinoic acid) in this newly defined domain induces the production of neural crest cells. These data also highlight the different roles played by BMP in neural crest specification in chick and Xenopus or zebrafish embryos (Galvic, 2004).

In conclusion, Notch signaling activates the expression of Hairy2A in the region of the neural folds, and thereby represses Bmp4 transcription. This effect of Notch signaling is dependent on Xmsx1 activity, since the inhibition of Notch by Su(H)DBMGR can be reversed by Xmsx1, and the effects produced by activating Notch can be blocked by a dominant-negative Xmsx1 construct. These results also provide a possible explanation for the apparent discrepancy in the role played by BMP in chick and Xenopus or zebrafish neural crest induction. At the time of neural crest induction, the levels of BMP at the neural plate border are high in both Xenopus and zebrafish, and low in the chick. If it is assumed that an intermediate level is required to induce neural crest in all these vertebrates, then an increase in BMP levels in the chick would establish similar levels to those generated by a decrease in Xenopus and zebrafish. Thus, because of the initial differences in the levels of BMP in these two groups of organisms, the molecular machinery that induces neural crest formation (e.g. Notch/Delta, Xiro1) must adjust the specific levels of BMP by producing opposing effects on BMP expression. Thus, Notch/Delta signaling induces the neural crest by increasing BMP expression in the chick, and decreasing it in Xenopus (Galvic, 2004).

The early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Evidence has been found that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone (Lopez, 2005).

Chicken Delta homologs

A chick Serrate homologue, C-Serrate-1, is expressed in the central nervous system, as well as in the cranial placodes, nephric epithelium, vascular system, and distal limb-bud mesenchyme. In most of these sites, its expression is associated with expression of C-Notch-1 and C- Delta-1. All three genes are expressed in the ventricular zone of the hindbrain and spinal cord, throughout the period when neurons are being born. Within this zone, C-Delta-1 and C-Serrate-1 are expressed in complementary subsets of nondividing cells that appear to be nascent neurons: C- Serrate-1 expression is restricted to specific locations along the dorsoventral axis, forming narrow bands extending from the anterior hindbrain to the tail. These observations strongly suggest that Delta-Notch signalling delivers lateral inhibition not only early but throughout vertebrate neurogenesis to regulate neuronal commitment, and that Serrate-Notch signalling may act similarly in this process. By analogy with its role in Drosophila wing patterning, C-Serrate-1 may also have a role in organising the dorso-ventral pattern of the neural tube. Signalling via Notch maintains neurogenesis, both in vertebrates and in flies, by keeping a proportion of the neuroepithelial cells in an uncommitted stem-cell-like state (Myat, 1996).

Neurons of the vertebrate central nervous system (CNS) are generated sequentially over a prolonged period from dividing neuroepithelial progenitor cells. Some cells in the progenitor cell population continue to proliferate while others stop dividing and differentiate as neurons. The mechanism that maintains the balance between these two behaviors is not known, although previous work has implicated Delta-Notch signaling in the process. In normal development, the proliferative layer of the neuroepithelium includes both nascent neurons that transiently express Delta-1 (Dl1), and progenitor cells that do not. Using retrovirus-mediated gene misexpression in the embryonic chick retina, it has been shown that where progenitor cells are exposed to Dl1 signaling, they are prevented from embarking on neuronal differentiation. A converse effect is seen in cells expressing a dominant-negative form of Dl1, Dl1(dn), which renders expressing cells deaf to inhibitory signals from their neighbours. In a multicellular patch of neuroepithelium expressing Dl1(dn), essentially all progenitors stop dividing and differentiate prematurely as neurons, which can be of diverse types. Thus, Delta-Notch signaling controls a cell's choice between remaining as a progenitor and differentiating as a neuron. It is concluded that nascent retinal neurons, by expressing Dl1, deliver lateral inhibition to neighbouring progenitors; this signal is essential to prevent progenitors from entering the neuronal differentiation pathway. Lateral inhibition serves the key function of maintaining a balanced mixture of dividing progenitors and differentiating progeny. It is proposed that the same mechanism operates throughout the vertebrate CNS, enabling large numbers of neurons to be produced sequentially and adopt different characters in response to a variety of signals. A similar mechanism of lateral inhibition, mediated by Delta and Notch proteins, may regulate stem-cell function in other tissues (Henrique, 1997).

Neural crest is induced at the junction of epidermal ectoderm and neural plate by the mutual interaction of these tissues. BMP4 has been shown to pattern the ectodermal tissues, and BMP4 can induce neural crest cells from the neural plate. Epidermally expressed Delta1, which encodes a Notch ligand, is required for the activation and/or maintenance of Bmp4 expression in this tissue, and is thus indirectly required for neural crest induction by BMP4 at the epidermis-neural plate boundary. Notch activation in the epidermis additionally inhibits neural crest formation in this tissue, so that neural crest generation by BMP4 is restricted to the junction (Endo, 2002).

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) results in a luminal differentiation, while forced activation of Notch signaling promotes the specification of immature glandular cells, but prevents the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus (Matsuda, 2005).

Ligand-dependent Notch signaling strength orchestrates lateral induction and lateral inhibition in the developing inner ear

During inner ear development, Notch exhibits two modes of operation: lateral induction, which is associated with prosensory specification, and lateral inhibition, which is involved in hair cell determination. These mechanisms depend respectively on two different ligands, jagged 1 (Jag1) and delta 1 (Dl1), that rely on a common signaling cascade initiated after Notch activation. In the chicken otocyst, expression of Jag1 and the Notch target Hey1 correlates well with lateral induction, whereas both Jag1 and Dl1 are expressed during lateral inhibition, as are Notch targets Hey1 and Hes5. This study shows that Jag1 drives lower levels of Notch activity than Dl1, which results in the differential expression of Hey1 and Hes5. In addition, Jag1 interferes with the ability of Dl1 to elicit high levels of Notch activity. Modeling the sensory epithelium when the two ligands are expressed together shows that ligand regulation, differential signaling strength and ligand competition are crucial to allow the two modes of operation and for establishing the alternate pattern of hair cells and supporting cells. Jag1, while driving lateral induction on its own, facilitates patterning by lateral inhibition in the presence of Dl1. This novel behavior emerges from Jag1 acting as a competitive inhibitor of Dl1 for Notch signaling. Both modeling and experiments show that hair cell patterning is very robust. The model suggests that autoactivation of proneural factor Atoh1, upstream of Dl1, is a fundamental component for robustness. The results stress the importance of the levels of Notch signaling and ligand competition for Notch function (Petrovic, 2014).

A novel function of DELTA-NOTCH signalling mediates the transition from proliferation to neurogenesis in neural progenitor cells

A complete account of the whole developmental process of neurogenesis involves understanding a number of complex underlying molecular processes. Among them, those that govern the crucial transition from proliferative (self-replicating) to neurogenic neural progenitor (NP) cells remain largely unknown. Due to its sequential rostro-caudal gradients of proliferation and neurogenesis, the prospective spinal cord of the chick embryo is a good experimental system to study this issue. This study reports that the NOTCH ligand DELTA-1 is expressed in scattered cycling NP cells in the prospective chick spinal cord preceding the onset of neurogenesis. These Delta-1-expressing progenitors are placed in between the proliferating caudal neural plate (stem zone) and the rostral neurogenic zone (NZ) where neurons are born. Thus, these Delta-1-expressing progenitors define a proliferation to neurogenesis transition zone (PNTZ). Gain and loss of function experiments carried by electroporation demonstrate that the expression of Delta-1 in individual progenitors of the PNTZ is necessary and sufficient to induce neuronal generation. The activation of NOTCH signalling by DELTA-1 in the adjacent progenitors inhibits neurogenesis and is required to maintain proliferation. However, rather than inducing cell cycle exit and neuronal differentiation by a typical lateral inhibition mechanism as in the NZ, DELTA-1/NOTCH signalling functions in a distinct manner in the PNTZ. Thus, the inhibition of NOTCH signalling arrests proliferation but it is not sufficient to elicit neuronal differentiation. Moreover, after the expression of Delta-1 PNTZ NP continue cycling and induce the expression of Tis21, a gene that is upregulated in neurogenic progenitors, before generating neurons. Together, these experiments unravel a novel function of DELTA-NOTCH signalling that regulates the transition from proliferation to neurogenesis in NP cells. It is hypothesized that this novel function is evolutionary conserved (Hammerle, 2007).

Regulation of neuronal differentiation at the neurogenic wavefront

Signaling mediated by the Delta/Notch system controls the process of lateral inhibition, known to regulate neurogenesis in metazoans. Lateral inhibition takes place in equivalence groups formed by cells having equal capacity to differentiate, and it results in the singling out of precursors, which subsequently become neurons. During normal development, areas of active neurogenesis spread through non-neurogenic regions in response to specific morphogens, giving rise to neurogenic wavefronts. Close contact of these wavefronts with non-neurogenic cells is expected to affect lateral inhibition. Therefore, a mechanism should exist in these regions to prevent disturbances of the lateral inhibitory process. Focusing on the developing chick retina, this study showed that Dll1 is widely expressed by non-neurogenic precursors located at the periphery of this tissue, a region lacking Notch1, lFng, and differentiation-related gene expression. This study investigated the role of this Dll1 expression through mathematical modeling. The analysis predicts that the absence of Dll1 ahead of the neurogenic wavefront results in reduced robustness of the lateral inhibition process, often linked to enhanced neurogenesis and the presence of morphological alterations of the wavefront itself. These predictions are consistent with previous observations in the retina of mice in which Dll1 is conditionally mutated. The predictive capacity of the mathematical model was confirmed further by mimicking published results on the perturbation of morphogenetic furrow progression in the eye imaginal disc of Drosophila. Altogether, it is proposed that Notch-independent Delta expression ahead of the neurogenic wavefront is required to avoid perturbations in lateral inhibition and wavefront progression, thus optimizing the neurogenic process (Formosa-Jordan, 2012).

Notch/Delta signaling represents a major mechanism used by metazoans for cell fate decisions during development, in particular in the nervous system. The classical view, derived from early studies in Drosophila, states that neuronal precursors are formed in equivalence groups, in which cells have equal capacity to become neurons. Precursors expressing high levels of Delta induce Notch-dependent inhibitory signals in the neighboring cells. These inhibitory signals reduce the capacity of these cells to express proneural genes and Delta itself, preventing them from becoming neurons. In turn, the reduced capacity of the these inhibited precursors to trigger inhibitory signals facilitates the differentiation of the high Delta-expressing precursors. This mechanism has been referred to as 'lateral inhibition with feedback' (Formosa-Jordan, 2012).

Neuronal production is often initiated in restricted areas of the neurogenic epithelium, surrounded by non-neurogenic cells. As development proceeds, the neurogenic region expands, forming a wavefront at the boundary with the non-neurogenic tissue. The vertebrate retina represents a paradigmatic example. In this tissue, neurogenesis starts in its central region within a small cell cluster in response to specific signals (Stenkamp, 2003; Martinez-Morales, 2005), and then it gradually spreads to the periphery. Such spreading is largely dependent on the release of Sonic hedgehog (Shh) by the first neurons to be born in this tissue, the differentiated retinal ganglion cells (RGCs). This morphogen-dependent spreading of neurogenesis is reminiscent of the progression of the morphogenetic furrow (MF) in the Drosophila eye imaginal disc (Formosa-Jordan, 2012).

The dynamic pattern of neurogenesis described above has important implications for the process of neuronal differentiation. Specifically, precursors located at the neurogenic wavefront are expected to receive fewer inhibitory signals than those inside the neurogenic region. This is because they are in direct contact with non-neurogenic precursors, which theoretically lack the capacity to trigger lateral inhibition. Therefore, the conditions at the wavefront are expected to have relevant consequences for the final pattern of neuronal differentiation. Although the importance of static boundary conditions at the borders of a pattern-forming tissue have received some theoretical notice, moving wavefronts of lateral inhibition have only recently come to attention. These studies show how a neurogenic wavefront can sweep across a field of identical cells and leave behind a pattern of different cell types. However, the search for biological mechanisms used by metazoans to prevent disturbances in the pattern of neuronal differentiation associated with the existence of this wavefront remains open and represents a question of key importance (Formosa-Jordan, 2012).

Generalized Delta expression is often observed in prospective neural tissues and neurogenic boundary regions. For instance, in the early zebrafish embryo, strong deltaD expression delineates the whole developing retina a few hours before the initiation of neurogenesis in this tissue. In both the avian and murine retina, Delta-like 1 (Dll1) is expressed more peripherally than its homolog Dll4, being detected in a high proportion of mitotically active progenitor cells. In Drosophila, Delta (Dl) expression has been shown to precede achaete protein accumulation in microchaeta proneural stripes. Dl expression has also been described within eye imaginal discs of Drosophila on the surfaces of unpatterned cells ahead of the MF. As in other neural structures, generalized Dl expression ahead of the MF seems to be independent of canonical Notch signaling as hairy (h), encoding a proneural gene repressor, and extra macrochaetae (emc), encoding an antagonist of the proneural gene products, are both expressed in this region. Delta expression in all these areas is often observed in most cells, suggesting that it is not a result of the lateral inhibitory process, but rather it represents a mechanism of mutual inhibition equally affecting all precursors. Overall, these observations suggest that generalized Delta expression ahead of the neurogenic wavefront could be relevant in the process of lateral inhibition during neurogenesis (Formosa-Jordan, 2012).

Using the chick retina as a model system, this study shows that Dll1 becomes expressed initially in its central region, prior to initiation of the neurogenic process. This pre-neurogenic expression of Dll1 is maintained in the peripheral retina at later developmental stages, when active neurogenesis is not yet visualized in this area. From computer simulation results of a mathematical model for the initiation and morphogen-dependent spreading of the neurogenic process, which restricts the dynamics of lateral inhibition to the neurogenic region, it is predicted that the absence of Delta ahead of the neurogenic wavefront results in reduced robustness of the lateral inhibition process. Specifically, the absence of Delta is often linked to enhanced neurogenesis and, surprisingly, morphological alterations of the wavefront itself. These predictions could explain observations made by Rocha (2009) in the retina of mice in which Dll1 is conditionally mutated and Dll4-dependent lateral inhibition remains within the neurogenic region. Based on all this evidence, it is suggested that generalized Delta expression ahead of the wavefront of neurogenesis is required for the avoidance of disturbances in lateral inhibition during the neuronal differentiation process. This might be extrapolated to other organisms and other neural tissues and could therefore be a general control mechanism of differentiation wavefronts (Formosa-Jordan, 2012).

The results raise questions about the mechanism directing Dll1 expression ahead of the neurogenic wavefront. In the chick caudal stem zone, generalized Dll1 expression has been shown to depend on Ascl2, a proneural gene the murine homolog of which is absent from the retina. In the mouse, Ascl1 has been shown to induce Dll1 expression when overexpressed in chick retinal explants, but this effect is likely to be derived from the proneural nature of Ascl1, associated with its expression in Notch-active progenitors. Therefore, the mechanism inducing Dll1 expression in the HH15 chick retina, prior to Ascl1 and Notch1 detection, and in the most peripheral retina at later stages, where Notch1 and lFng are absent, still remains unknown. The uncovering of such a mechanism will facilitate the design of experiments to test the predictions of the model further. One possible experiment to falsify the model would be the creation of knock-in mice in which Dll1 promoter elements specific for Notch-independent Dll1 expression are mutated. This genetic approach would inhibit Dll1 expression in the non-neurogenic region but not in neurogenic precursors undergoing lateral inhibition (Formosa-Jordan, 2012).

Together, the results show that the properties (pattern formed, shape and velocity) of progressing fronts of lateral inhibition, in the current case neuronal differentiation, depend crucially on the conditions ahead of the differentiation front. The observations regarding Dll1 expression point to a mechanism for neurogenic front regulation in the retina, but as the study of the MF in the Drosophila eye shows, it could be an example of a more general developmental mechanism. Ligand expression in front of a lateral inhibition wavefront might act as a key regulator of differentiation processes (Formosa-Jordan, 2012).

Mammalian Delta homologs

Evolutionary homologs - Continued: part 3/3 | back to part 1/3

Delta: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.