C. elegans Delta homologs

C. elegans protein Lag-2 is a ligand for Lin-12 and Glp-1, homologs of Notch. Lag-2 has EGF repeats. Mutational studies show that a single amino acid change in an EGF repeat alters the ligand properties (Tax, 1994).

Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates. In C. elegans, two DSL genes, lag-2 and apx-1, influence different cell fate decisions during development. APX-1 can fully substitute for LAG-2 when expressed under the control of lag-2 regulatory sequences. In addition, truncated forms lacking the transmembrane and intracellular domains of both LAG-2 and APX-1 can also substitute for endogenous lag-2 activity. Moreover, these truncated forms are secreted and able to activate LIN-12 and GLP-1 ectopically. Expression of a secreted DSL domain alone may enhance endogenous LAG-2 signaling. These data suggest ways that activated forms of DSL ligands in other systems may be created (Fitzgerald, 1995).

During the 4-cell stage of C. elegans embryogenesis, the P2 blastomere provides a signal that allows two initially equivalent sister blastomeres, called ABa and ABp, to adopt different fates. Preventing P2 signalling in wild-type embryos results in defects in ABp development that are similar to those caused by mutations in the glp-1 and apx-1 genes, which are homologs of the Drosophila genes Notch and Delta, respectively. Previous studies have shown that GLP-1 protein is expressed in 4-cell stage embryos in both ABa and ABp. APX-1 protein is expressed in the P2 blastomere. A temperature-sensitive apx-1 mutant has a temperature-sensitive period between the 4-cell and 8-cell stages. It is propose that APX-1 is part or all of the P2 signal that induces ABp to adopt a fate different than ABa (Mickey, 1996).

Cell-cell interactions mediated by LIN-12 and GLP-1, members of the LNG (LIN-12, Notch, GLP-1) family of receptors, are required to specify numerous cell fates during development of the nematode Caenorhabditis elegans. Maternally expressed glp-1 participates in two of at least four sequential inductive interactions that specify the fates of early embryonic descendants of the AB founder cell. GLP-1 and LIN-12, and apparently their ligand, LAG-2, as well as a downstream component, LAG-1, are required in the latter two inductions. lag-2 is expressed in the signaling cells and lin-12 is expressed in cells receiving the inductions, consistent with their proposed respective roles as ligand and receptor. Maternal GLP-1 activity is required (1) to repress early zygotic lag-2 expression and (2) to activate zygotic lin-12 expression in the early embryo. The patterning of both receptor and ligand expression by maternal GLP-1 signaling establishes competence for the zygotic LNG-mediated cellular interactions and localizes these interactions to the appropriate cells. It is proposed that activation of maternal GLP-1 regulates zygotic lin-12 and lag-2 expression by a regulatory mechanism analogous to that described for the post-embryonic gonad (Moskowitz, 1996).

The C. elegans intestine is a simple tube consisting of a monolayer of epithelial cells. During embryogenesis, cells in the anterior of the intestinal primordium undergo reproducible movements that lead to an invariant, asymmetrical 'twist' in the intestine. The development of this twist has been examined to determine how left-right and anterior-posterior asymmetries are generated within the intestinal primordium. The twist requires the LIN-12/ Notch-like signaling pathway of C. elegans. All cells within the intestinal primordium initially express LIN-12, a receptor related to Notch; however, only cells in the left half of the primordium contact external, nonintestinal cells that express LAG-2, a ligand related to Delta. LIN-12 and LAG-2 mediated interactions result in the left primordial cells expressing lower levels of LIN-12 than the right primordial cells. It is proposed that this asymmetrical pattern of LIN-12 expression is the basis for asymmetry in later cell-cell interactions within the primordium that lead directly to intestinal twist. Like the interactions that initially establish LIN-12 asymmetry, the later interactions are mediated by LIN-12. The later interactions, however, involve a different ligand related to Delta, called APX-1. The anterior-posterior asymmetry in intestinal twist involves the kinase LIT-1, which is part of a signaling pathway in early embryogenesis that generates anterior-posterior differences between sister cells (Hermann, 2000).

The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Ten genes have been computationally identified that encode potential ligands for LIN-12; three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. Transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays (Chen, 2004).

Sequence analysis indicates that three DSL proteins have highly probable predicted transmembrane domains; these are encoded by the three genes that had been identified previously, lag-2, apx-1, and arg-1. Two of the proteins identified by computational analysis, DSL-2 and DSL-6, may also have transmembrane domains, although the potential transmembrane domains were assessed as lower probability in prediction programs. Surprisingly, the five remaining genes (dsl-1, dsl-3, dsl-4, dsl-5, and dsl-7) encode DSL proteins that are predicted to lack transmembrane domains and hence are likely to be secreted (Chen, 2004).

In terms of VPC specification, the cell biology of lateral signaling offers a rationale for why a secreted or peripheral membrane protein ligand might be a component of the lateral signal. The VPCs are polarized epithelial cells: they have an apical region and a basolateral domain, separated by adherens junctions. Since the apical regions of adjacent VPCs appear to be in contact only in the vicinity of the cell junctions, and LIN-12 is distributed over the whole apical surface, a transmembrane ligand on the surface of one VPC might have access to LIN-12 on the apical surface of its neighbor only in a relatively limited area. A ligand that can diffuse may be available to activate LIN-12 over a greater region of the apical domain, affording one solution to such a topographical problem (Chen, 2004).

The Notch signaling pathway controls growth, differentiation and patterning in divergent animal phyla; in humans, defective Notch signaling has been implicated in cancer, stroke and neurodegenerative disorders. Despite its developmental and medical significance, little is known about the factors that render cells to become competent for Notch signaling. This study shows that during vulval development in the nematode C. elegans the HOX protein LIN-39 and its EXD/PBX-like cofactor CEH-20 are required for LIN-12/Notch-mediated lateral signaling that specifies the 2° vulval cell fate. Inactivation of either lin-39 or ceh-20 resulted in the misspecification of 2° vulval cells and suppresses the multivulva phenotype of lin-12(n137) gain-of-function mutant animals. Furthermore, both LIN-39 and CEH-20 are required for the expression of basal levels of the genes encoding the LIN-12/Notch receptor and one of its ligands in the vulval precursor cells, LAG-2/Delta/Serrate, rendering them competent for the subsequent lin-12/Notch induction events. These results suggest that the transcription factors LIN-39 and CEH-20, which function at the bottom of the RTK/Ras and Wnt pathways in vulval induction, serve as major integration sites in coordinating and transmitting signals to the LIN-12/Notch cascade to regulate vulval cell fates (Takács-Vellai, 2007).

Convergent intercellular signals must be precisely coordinated in order to elicit specific biological responses. The C. elegans vulva provides an excellent experimental microcosm for studying how cell fate is specified according to the combined effects of different signaling pathways. This paper has studied the role of the Hox gene lin-39 and the Exd ortholog ceh-20 in vulval development. Genetic and molecular evidence is presented that the HOX protein LIN-39 and its putative cofactor CEH-20 are required for basal expression levels of lin-12 and lag-2 in the VPCs prior to vulval induction; this regulation may be important to render the VPCs competent for the subsequent lin-12/Notch induction events at the L3 larval stage. Identifying transcriptional regulators of lateral signaling in C. elegans vulval development will be essential for understanding how the Notch signaling pathway specifies cell fate in divergent animal species, and how compromised Notch signaling leads to human diseases (Takács-Vellai, 2007).

LIN-39 and CEH-20 are both required at the first larval stage to prevent fusion of the VPCs to the surrounding hypodermis. The data lead to the attractive possibility that LIN-39 and its putative cofactor CEH-20 regulate the competence of the VPCs to respond to any of the patterning signals during vulval formation. Along this line, it is challenging to speculate that, besides regulating lin-12 and lag-2 expression, they might also promote the expression of components of the inductive pathway (such as let-23) or other Notch pathway genes in the VPCs (Takács-Vellai, 2007).

It has been shown that CEH-20 binds in vitro, together with LIN-39, to the promoter of the twist transcription factor ortholog hlh-8 to regulate its expression in postembryonic mesodermal cells. ChIP experiments demonstrate that LIN-39 associates with the lag-2promoter, suggesting that the regulation of lag-2 expression by LIN-39 may be direct. It is proposed that LIN-39 forms a heterodimer with CEH-20 to promote the basal transcription of lag-2 and lin-12 in the VPCs. Based on their different expression pattern in the Pn.p lineages, ceh-20 is assumed to have some functions that are independent of lin-39. Indeed, mab-5 has been shown to be expressed in the descendants of the posterior VPCs, P7.p and P8.p, and to prevent them from adopting an induced vulval fate. Thus, it is possible that CEH-20 also interacts and functions with MAB-5 in controlling certain aspects of vulval fate specification. Furthermore, it is noted that ceh-20(ay9) mutant animals sometimes displayed a dual AC phenotype, whereas lin-39 mutants never did. RNAi-mediated depletion of mab-5 sometimes resulted in 2 ACs, suggesting that the correct AC specification requires the combined activity of mab-5 and ceh-20 (Takács-Vellai, 2007).

Finally, CEH-20 has been shown to be required as a cofactor for autoregulatory expression of the anterior Hox paralog (labial-like) ceh-13 in embryonic cells. Because ceh-13 is expressed all along the anteroposterior body axis in the ventral mid-line during the L1–L4 larval stages and a few percent of the ceh-13(sw1) mutant animals that are able to develop into fertile adults exhibit various defects in vulval formation, it is possible that CEH-13 acts with CEH-20 to control cell fate in the anterior VPC lineages. The future analysis of a potential role of ceh-13 in vulval development would help to establish the role of all of the major body Hox genes in this important process (Takács-Vellai, 2007).

Delta in spiders

Early neurogenesis in the spider is characterized by a stereotyped pattern of sequential recruitment of neural cells from the neuroectoderm, comparable with neuroblast formation in Drosophila. However, in contrast to Drosophila, where single cells delaminate from the neuroectoderm, groups of cells adopt the neural fate and invaginate into the spider embryo. This raises the question of whether Delta/Notch signaling is involved in this process, since this system normally leads to a singling out of individual cells through lateral inhibition. Homologs of Delta and Notch have been cloned from the spider Cupiennius salei and their expression and function have been studied. The genes are indeed expressed during the formation of neural cells in the ventral neuroectoderm. Loss of function of either gene leads to an upregulation of the proneural genes and an altered morphology of the neuroectoderm that is comparable with Delta and Notch mutant phenotypes in Drosophila. Thus, although Delta/Notch signaling appears to be used in the same way as in Drosophila, the lateral inhibition process produces clusters of invaginating cells, rather than single cells. Intriguingly, neuroectodermal cells that are not invaginating seem to become neural cells at a later stage, while the epidermal cells are derived from lateral regions that overgrow the neuroectoderm. In this respect, the neuroectodermal region of the spider is more similar to the neural plate of vertebrates, than to the neuroectoderm of Drosophila (Stollewerk, 2002).

In the development of most arthropods, the caudal region of the elongating germ band (the growth zone) sequentially produces new segments. Previous work with the spider Cupiennius salei suggested involvement of Delta-Notch signaling in segmentation. This study reports that, in the spider Achaearanea tepidariorum, the same signaling pathway exerts a different function in the presumptive caudal region before initiation of segmentation. In the developing spider embryo, the growth zone becomes morphologically apparent as a caudal lobe around the closed blastopore. Preceding caudal lobe formation, transcripts of a Delta homolog, At-Delta, are expressed in evenly spaced cells in a small area covering the closing blastopore and then in a progressively wider area of the germ disc epithelium. Cells with high At-Delta expression are likely to be prospective mesoderm cells, which later express a twist homolog, At-twist, and individually internalize. Cells remaining at the surface begin to express a caudal homolog, At-caudal, to differentiate as caudal ectoderm. Knockdown of At-Delta by parental RNA interference results in overproduction of At-twist-expressing mesoderm cells at the expense of At-caudal-expressing ectoderm cells. This condition gives rise to a disorganized caudal region that fails to pattern the opisthosoma. In addition, knockdown of Notch and Suppressor of Hairless homologs produces similar phenotypes. It is suggested that, in the spider, progressive activation of Delta-Notch signaling from around the blastopore leads to stochastic cell fate decisions between mesoderm and caudal ectoderm through a process of lateral inhibition to set up a functional caudal lobe (Oda, 2007).

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration. In bilaterians, there are at least 12 subfamilies of Wnt genes. Wnt3 and Wnt8 are required for somitogenesis in vertebrates and are thought to be involved in posterior specification in deuterostomes in general. Although TCF and β-catenin have been implicated in the posterior patterning of some short-germ insects, the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. This study investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. These results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, it is proposed that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes (McGregor, 2008).

The posterior truncation phenotypes resulting from pRNAi against Wnt8 in the spider are at least superficially similar to those observed when Wnt8 and/or Wnt3 are perturbed in vertebrate embryos. Removal or blocking Wnt8 and/or Wnt3 in Xenopus, zebrafish, and mouse results in truncated embryos with only a few anterior somites and no tail bud. Although analysis of TCF and β-catenin in Oncopeltus and Gryllus, respectively, indicated that Wnt signaling might be involved in the development of the growth zone and posterior segments in arthropods, the current data show that in fact the same ligand, Wnt8, is employed in posterior development in both vertebrates and arthropods (McGregor, 2008).

In class II and III At-Wnt8pRNAi embryos exhibiting fused L4 limb buds, it also appeared that the most ventral part of this segment is missing. This phenotype shows similarities to the phenotype when short-gastrulation is knocked down in this spider. It suggests that, in addition to A-P patterning, At-Wnt8 is involved in D-V patterning in the spider, a role Wnt8 genes also perform in vertebrates (McGregor, 2008).

There is evidence that Wnt signaling acts upstream of Delta/Notch in vertebrate somitogenesis. Although the expression of Wnt3a and Wnt8 is not cyclical during somitogenesis in vertebrates, some downstream components of Wnt signaling, such as Axin2, are cyclically expressed in mice and possibly are integral to the Delta/Notch-dependent segmentation clock. However, recent experiments have shown that Axin2 and components of the Delta/Notch pathway continue to oscillate in the presence of stabilized β-catenin, which suggests that in mice, Wnt signaling may be permissive for the segmentation clock rather than instructive. Similarly, in zebrafish it is thought that Wnt8 may act to maintain a precursor population of stem cells in the PSM and tailbud rather than directly regulate the segmentation clock. It is proposed that the same ligand, Wnt8, could play a similar permissive role for segmentation in the growth zone of the spider by establishing and possibly maintaining a pool of cells that develop into the opisthosomal segments. When At-Wnt8 activity is reduced, cells are ectopically used in L3/L4 or internalized, depleting the putative growth-zone pool. This depletion manifests as a smaller opisthosoma, separated clusters of cells that give rise to separate irregular germbands, or even no opisthosoma (McGregor, 2008).

It was previously shown that Delta/Notch signaling is also involved in posterior development in the spiders Cupiennius. These new results reveal that in the spider, Wnt8 is required for the clearing of Dl and h expression in the posterior and that this is necessary for repression of twi, activation of cad, and establishment of the growth zone (McGregor, 2008).

The involvement of Wnt8, Delta/Notch signaling, and cad in the posterior development of other arthropods has also been directly demonstrated by functional analysis or inferred from expression patterns, and in vertebrates, Wnt3a and Wnt8 probably act upstream of Delta/Notch and cad during somitogenesis. Taken together, this suggests that a regulatory genetic network for posterior specification including Wnt8, Delta/Notch signaling, and cad could have been present in the last common ancestor of protostomes and deuterostomes, but has subsequently been modified in some lineages. For example, in Drosophila, Delta/Notch signaling is not involved in segmentation, and although the Drosophila Wnt8 ortholog, WntD, is required for D-V patterning, it is not involved in posterior development. Segments arise almost simultaneously in Drosophila, rather than sequentially from a growth zone, so this may suggest that the role of Wnt8 in posterior development was not required for this mode of development and therefore was lost during the evolution of these insects (McGregor, 2008).

These results suggest that Wnt8 regulates formation of the posterior growth zone and then maintains a pool of undifferentiated cells in this tissue required for development of the opisthosoma. Wnt signaling thus regulates the establishment and maintenance of an undifferentiated pool of posterior cells in both vertebrates and spiders and in fact the same Wnt ligand, Wnt8, is used in both phyla. Therefore, Wnt8 could be part of an ancient genetic regulatory network, also including Dl, Notch, h, and cad, that was used for posterior specification in the last common ancestor of deuterostomes and protostomes (McGregor, 2008).

Delta in myriapods

Molecular data suggest that myriapods are a basal arthropod group and may even be the sister group of chelicerates. To find morphological indications for this relationship neurogenesis has been analyzed in the myriapod Glomeris marginata (Diplopoda). Groups of neural precursors, rather than single cells as in insects, invaginate from the ventral neuroectoderm in a manner similar to that in the spider: invaginating cell groups arise sequentially and at stereotyped positions in the ventral neuroectoderm of Glomeris, and all cells of the neurogenic region seem to enter the neural pathway. As in the spider, 30-32 invaginating cell groups are arranged in a regular pattern of seven rows consisting of four to five invaginaton sites each. However, analysis of serial transverse sections reveals that up to 11 cells contribute to an individual invagination site, while in the spider only five to nine cells were counted. Furthermore, in contrast to the spider, the ventral neuroectoderm has a multi-layered structure: the apical region covered by a single invagination site seems to be larger and the spacing between the individual invagination sites is narrower than in the spider. The reason for these morphological features is that the invaginating cell groups are located closer together and because of limited space come to lie over and above each other. The invaginating cells of a group do not all occupy a basal position as in the spider, but they also form stacks of cells. Since more cells contribute to an invagination site and the cell processes of the invaginating cells are not as constricted as in the spider, the apical region occupied by an individual invagination site is larger than in the spider (Dove, 2003).

Homologs for achaete-scute, Delta and Notch have been identified in Glomeris. The genes are expressed in a pattern similar to that found in spider homologues and show more sequence similarity to the chelicerates than to the insects (Dove, 2003).

The data support the hypothesis that myriapods are closer to chelicerates than to insects. The spider and the millipede share several features that cannot be found in equivalent form in the insects: in both the spider and the millipede, about 30 groups of neural precursors invaginate from the neuroectoderm in a strikingly similar pattern. Furthermore, in contrast to the insects, there is no decision between epidermal and neural fate in the ventral neuroectoderm of both species analysed. It is concluded that the myriapod pattern of neural precursor formation is compatible with the possibility of a chelicerate-myriapod sister group relationship (Dove, 2003).

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, the functions of the Delta (Gb'Delta) and Notch (Gb'Notch) genes were analyzed in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. Gb'Delta and Gb'Notch were expressed in developing legs, and RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. The results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, Gb'Delta was found to be expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries (Mito, 2011).

Sea urchin Delta homolog and the induction of mesoderm

Signals from micromere descendants play a critical role in patterning the early sea urchin embryo. Previous work has demonstrated a link between the induction of mesoderm by micromere descendants and the Notch signaling pathway. These micromere descendants express LvDelta, a ligand for the Notch receptor. LvDelta is expressed by micromere descendants during the blastula stage, a time when signaling has been shown to occur. By a combination of embryo microsurgery, mRNA injection and antisense morpholino experiments, it has been shown that expression of LvDelta by micromere descendants is both necessary and sufficient for the development of two mesodermal cell types, pigment cells and blastocoelar cells. LvDelta is expressed by macromere descendants during mesenchyme blastula and early gastrula stages. Macromere-derived LvDelta is necessary for blastocoelar cell and muscle cell development. Expression of LvDelta is sufficient to endow blastomeres with the ability to function as a vegetal organizing center and to coordinate the development of a complete pluteus larva (Sweet, 2002).

Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a grid-like organisation of distinct cell identities in the ascidian neural plate

The ascidian neural plate has a grid-like organisation, with six rows and eight columns of aligned cells, generated by a series of stereotypical cell divisions. Unique molecular signatures have been defined for each of the eight cells in the posterior-most two rows of the neural plate - rows I and II. Using a combination of morpholino gene knockdown, dominant-negative forms and pharmacological inhibitors, the role of three signalling pathways was tested in defining these distinct cell identities. Nodal signalling at the 64-cell stage is required to define two different neural plate domains - medial and lateral - with Nodal inducing lateral and repressing medial identities. Delta2, an early Nodal target, then subdivides each of the lateral and medial domains to generate four columns. Finally, a separate signalling system along the anteroposterior axis, involving restricted ERK1/2 activation, was found to promote row I fates and repress row II fates. These results reveal how the sequential integration of three signalling pathways -- Nodal, Delta2/Notch and FGF/MEK/ERK -- defines eight different sub-domains that characterise the ascidian caudal neural plate. Most remarkably, the distinct fates of the eight neural precursors are each determined by a unique combination of inputs from these three signalling pathways (Hudson, 2007).

All three of these signalling pathways are involved in a myriad of cell fate specification events during vertebrate development. Within the vertebrate neural tube, Nodal is required for induction of the floor plate, the ventral-most structure of the neural tube, at least in zebrafish. This is at odds with the role for Nodal in promoting lateral fates at the expense of medial (including floor plate) fates in ascidians and suggests that the role of Nodal in neural tube patterning is not conserved among chordates. By contrast, in the vertebrate neural tube, Delta/Notch signalling has been implicated in the formation of both extreme dorsal (neural crest) and ventral (floor plate) cell types. In Ciona, Delta2/Notch is involved in both the specification of lateral and medial fates within the neural plate. Interestingly, there are some differences in the mode of action of Delta/Notch signalling in Ciona, because Ci-MRF, Ci-COE and Ci-Ngn, which all encode HLH proteins, are activated by Delta2/Notch, whereas expression of these transcription factors is generally negatively regulated by Notch signals. Later, during neurogenesis, Notch signalling is involved in the selection of neurones in neurogenic regions of the developing neural plate, a process known as lateral inhibition. Although this role has not been addressed in the CNS of Ciona, it is involved in the selection of epidermal sensory neurones within the dorsal and ventral midline neurogenic regions of the larval tail epidermis. In vertebrates, FGF, together with Wnt, signalling is required during late gastrula stages to impose a posterior identity on neural tissue. This is reminiscent of the situation in ascidians, in which FGF/MEK/ERK signalling is required for posterior identities in the neural plate. Thus, the role of this signalling pathway during posteriorisation might represent a core evolutionary strategy to generate posterior cell types within neural tissue (Hudson, 2007).

Endomesoderm is the common progenitor of endoderm and mesoderm early in the development of many animals. In the sea urchin embryo, the Delta/Notch pathway is necessary for the diversification of this tissue, as are two early transcription factors, Gcm and FoxA, which are expressed in mesoderm and endoderm, respectively. This study provides a detailed lineage analysis of the cleavages leading to endomesoderm segregation, and examines the expression patterns and the regulatory relationships of three known regulators of this cell fate dichotomy in the context of the lineages. Endomesoderm segregation was seen to first occur at hatched blastula stage. Prior to this stage, Gcm and FoxA are co-expressed in the same cells, whereas at hatching these genes are detected in two distinct cell populations. Gcm remains expressed in the most vegetal endomesoderm descendant cells, while FoxA is downregulated in those cells and activated in the above neighboring cells. Initially, Delta is expressed exclusively in the micromeres, where it is necessary for the most vegetal endomesoderm cell descendants to express Gcm and become mesoderm. These experiments show a requirement for a continuous Delta input for more than two cleavages (or about 2.5 hours) before Gcm expression continues in those cells independently of further Delta input. Thus, this study provides new insights into the timing mechanisms and the molecular dynamics of endomesoderm segregation during sea urchin embryogenesis and into the mode of action of the Delta/Notch pathway in mediating mesoderm fate (Croce, 2010).

Zebrafish Delta

In Drosophila, cells are thought to be singled out for a neural fate through a competitive mechanism based on lateral inhibition mediated by Delta-Notch signalling. In tetrapod vertebrates, nascent neurons express the Delta1 gene and thereby deliver lateral inhibition to their neighbours, but it is not clear how these cells are singled out within the neurectoderm in the first place. Four Delta homologs have been found in the zebrafish, twice as many as reported in any tetrapod vertebrate. Three of these deltaA, deltaB and deltaD are involved in primary neurogenesis, while two deltaC and deltaD appear to be involved in somite development. In the neural plate, deltaA and deltaD, unlike Delta1 in tetrapods, are expressed in large patches of contiguous cells, within which scattered individuals expressing deltaB become singled out as primary neurons. By gene misexpression experiments, it has been shown that: (1) the singling-out of primary neurons, including the unique Mauthner cell on each side of the hindbrain, depends on Delta-Notch-mediated lateral inhibition; (2) deltaA, deltaB and deltaD all have products that can deliver lateral inhibition and (3) all three of these genes are themselves subject to negative regulation by lateral inhibition. These properties imply that competitive lateral inhibition, mediated by coordinated activities of deltaA, deltaB and deltaD, is sufficient to explain how primary neurons emerge from proneural clusters of neuroepithelial cells in the zebrafish (Haddon, 1998a).

The vertebrate spinal cord consists of a large number of different cell types in close proximity to one another. The identities of these cells appear to be specified largely by information acquired from their local environments. Local cell-cell interactions, mediated by zebrafish homologs of the Drosophila neurogenic gene Delta, regulate specification of diverse neuronal types in the ventral spinal cord. A novel zebrafish Delta gene, deltaA, has been identified that is expressed specifically in the nervous system. In zebrafish, cells that give rise to primary neurons of the trunk begin to exit the mitotic cycle once gastrulation is completed. The first of these cells known to differentiate are primary sensory Rohon Beard neurons (RBs) and primary neurons, which arise from lateral and medial regions of the neural plate, respectively. deltaA expression is initiated in the epiblast prior to completion of gastrulation. At the 2- to 3- somite stage (10.5 hours) low levels of deltaA RNA are distributed throughout the trunk CNS, with cells expressing higher levels found in the medial and lateral regions of the neural plate. These regions correspond to the positions at which primary motoneurons and RBs originate. Cells expressing high levels of deltaA RNA do not form contiguous domains. Rather, single cells or small clusters of several cells showing high expression are interspersed with cells having lower expression. deltaA is expressed in cells specified for neuronal fates such as presumptive primary motoneurons and presumptive RB neurons. By expressing a dominant negative form of Delta protein in embryos, it has been shown that Delta proteins mediate lateral inhibition in the zebrafish spinal cord. Delta function is important for specification of a variety of spinal cord neurons, suggesting that lateral inhibition serves to diversify neuronal fate during development of the vertebrate spinal cord (Appel, 1998).

Primary and secondary motoneurons are born beginning about 9-10 and 14-15 hours, respectively. Zebrafish shh expression is initiated in presumptive dorsal mesoderm at about 7 hours. In cyclops; floating head double mutant embryos, which lack differentiated notochord and floorplate, shh expression is initiated normally but notmaintained after gastrulation; primary (but not secondary) motorneurons develop. Thus, early shh signaling may induce development of primary, but not secondary motorneurons. In chick, motoneuronal induction requires exposure to Shh for 1-2 cell cycles. The zebrafish cell cycle length during early neurogenesis is about 4 hours. Thus, secondary motoneurons are born 1-2 cell cycles after initiation of shh expression. Together, these observations suggest that secondary motoneurons are equivalent to chick motoneurons in their requirement for Shh signaling, but that primary motoneurons require only brief exposure. In the absence of lateral inhibition, too many primary motoneurons develop concomitant with the loss of secondary motoneurons. Thus it is proposed that Delta-Notch signaling specifies primary and secondary motoneuronal fates by regulating how neural precursor cells respond to Shh signaling. In this model, some cells of the neural plate respond to Shh by immediately developing as primary motoneurons. DeltaA is expressed at high levels in these cells and inhibits neighboring cells from responding to Shh in the same way. Later, as DeltaA is downregulated in primary motoneurons, neighboring cells are released from lateral inhibition and respond to Shh by adapting secondary neuronal fate. Alternatively, secondary motoneurons may be specified by a late-arising signal that acts with, or subsequent to, Shh (Appel, 1998 and references).

Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures (the floorplate in the ventral neural tube, the notochord and the dorsal endoderm) occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. One of four known zebrafish homologs of Drosophila Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. A reverse genetic strategy was used to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos have reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos have excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produces a similar effect. In contrast, overexpression of dlA has the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. These results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord (Appel, 1999).

During vertebrate embryonic development, the paraxial mesoderm becomes subdivided into metameric units known as somites. In the zebrafish embryo, genes encoding homologs of the proteins of the Drosophila Notch signaling pathway are expressed in the presomitic mesoderm and expression is maintained in a segmental pattern during somitogenesis. This expression pattern suggests a role for these genes during somite development. Various zebrafish genes of this group were misexpressed by injecting mRNA into early embryos. RNA encoding a constitutively active form of NOTCH1a (notch1a-intra) and a truncated variant of deltaD [deltaD(Pst)], as well as transcripts of deltaC and deltaD, the hairy-E(spl) homologs her1 and her4, and groucho2 were tested for their effects on somite formation, myogenesis and on the pattern of transcription of putative downstream genes. In embryos injected with any of these RNAs, with the exception of groucho2 RNA, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment correctly. Activation of Notch results in ectopic activation of her1 and her4. This misregulation of the expression of her genes might be causally related to the observed mesodermal defects, since her1 and her4 mRNA injections led to effects similar to those seen with notch1a-intra. deltaC and deltaD seem to function after subdivision of the presomitic mesoderm, since the her gene transcription pattern in the presomitic mesoderm remains essentially normal after misexpression of delta genes. Whereas Notch signaling alone apparently does not affect myogenesis, zebrafish groucho2 is involved in differentiation of mesodermal derivatives (Takke, 1999).

Results regarding the effect of misexpressing wild-type deltaD have suggested a function for this gene in somite development. Misexpression of a dominant negative variant of deltaD, wild-type deltaC, or an activated form of NOTCH1a, or misexpression of the hairy-E(spl) homolog her1 or her4, leads in all cases to considerable disruption of somitogenesis. However, whereas the mesodermal effects of perturbing deltaC and deltaD activity are similar, those observed following either Notch activation or coinjection of her1 and her4 appear to have a different basis. In the former case, patterning defects are evident, but the presomitic mesoderm seems to be subdivided into somitomeres, as incomplete somite borders are visible; in the latter case, somites apparently do not form, because no somite borders can be seen. Since both her1 and her4 are ectopically activated by notch1a-intra within the presomitic mesoderm and misexpression of both her genes causes defects similar to those seen with notch1a-intra mRNA, it is proposed that both her genes are targets of Notch during somitogenesis. It follows then that, during normal development, Notch-mediated activation of her genes may be causally related to the initial subdivision of the paraxial mesoderm into somitomeres. In contrast, in the case of deltaC or deltaD misexpression the defects seem to be independent of the activity of the two her genes because it fails to perturb the transcription pattern of her1 and her4. The same applies to misexpression of a truncated variant of DELTAD. This result is surprising and leads to two important corollaries: (1) it suggests that neither DELTAC nor DELTAD acts as a ligand to trigger NOTCH-dependent activation of her genes, and (2), it suggests that the DELTAC/D-dependent somitic defects do not depend directly on the activity of her genes. Accordingly, the delta function in somitogenesis that appears to operate downstream of the component of Notch function was assayed; namely, her gene activity. Double in situ hybridizations with her1 or her4 and MyoD probes following misexpression of delta variants suggest that the latter act within the somites once the presomitic mesoderm has been subdivided into somitomeres. By analogy to the situation in Drosophila, where the Notch regulatory network is required for maintenance of the epithelial state in several different instances, as well as for the formation of borders in the wing disc, it is proposed that DELTAC and DELTAD act during the definition and/or maintenance of somitic borders in zebrafish embryos (Takke, 1999).

At least three important questions remain open in this scenario: (1) whereas the proposed function of Delta in controlling boundary development may rely on a mechanism similar to that operating in the wing margin of Drosophila, the mechanism by which Notch contributes to subdivide the presomitic mesoderm is unclear. (2) The ligand that activates the NOTCH1a receptor (and, consequently, the her genes) during the subdivision of the presomitic mesoderm is unknown. Although there are no less than four delta genes in the zebrafish, only deltaC and deltaD are expressed in the mesoderm and apparently neither one is capable of activating her genes under these experimental conditions. Therefore, there is no obvious candidate for this function. (3) The receptor required for the DELTAC/D-mediated function during later stages of somite development is also unknown (Takke, 1999).

Somitogenesis has been linked both to a molecular clock that controls the oscillation of gene expression in the presomitic mesoderm (PSM) and to Notch pathway signaling. The oscillator, or clock, is thought to create a prepattern of stripes of gene expression that regulates the activity of the Notch pathway that subsequently directs somite border formation. The zebrafish gene after eight (aei) that is required for both somitogenesis and neurogenesis encodes the Notch ligand DeltaD. Additional analysis has revealed that stripes of hairy relate her1 expression oscillate within the PSM and that aei/DeltaD signaling is required for this oscillation. aei/DeltaD expression does not oscillate, indicating that the activity of the Notch pathway upstream of her1 may function within the oscillator itself. Moreover, her1 stripes are expressed in the anlage of consecutive somites, indicating that her1 expression pattern is not pair-rule. Analysis of her1 expression in aei/DeltaD, fused somites (fss), and aei;fss embryos has uncovered a wave-front activity that is capable of continually inducing her1 expression de novo in the anterior PSM in the absence of the oscillation of her1. The wave-front activity, in reference to the clock and wave-front model, is defined as such because it interacts with the oscillator-derived pattern in the anterior PSM and is required for somite morphogenesis. This wave-front activity is blocked in embryos mutant for fss but not aei/DeltaD. Thus, this analysis indicates that the smooth sequence of formation, refinement, and fading of her1 stripes in the PSM is governed by two separate activities (Holley, 2000).

Somite formation is thought to be regulated by an unknown oscillator mechanism that causes the cells of the presomitic mesoderm to activate and then repress the transcription of specific genes in a cyclical fashion. These oscillations create stripes/waves of gene expression that repeatedly pass through the presomitic mesoderm in a posterior-to-anterior direction. In both the mouse and the zebrafish, it has been shown that the notch pathway is required to create the stripes/waves of gene expression. However, it is not clear if the notch pathway comprises part of the oscillator mechanism or if the notch pathway simply coordinates the activity of the oscillator among neighboring cells. In the zebrafish, oscillations in the expression of a hairy-related transcription factor, her1 and the notch ligand deltaC precede somite formation. This study focuses on how the oscillations in the expression of these two genes areaffected in the mutants aei/deltaD and des/notch1, in 'morpholino knockdowns' of deltaC and her1 and in double 'mutant' combinations. This analysis indicates that these oscillations in gene expression are created by a genetic circuit comprised of the notch pathway and the notch target gene her1. A later function of the notch pathway can create a segmental pattern even in the absence of prior oscillations in her1 and deltaC expression (Holley, 2002).

Both aei/deltaD and des/Notch1 are necessary to promote the expression of the oscillating genes her1 and deltaC. Meanwhile, her1 regulates deltaC expression and functions, directly or indirectly, in a negative feedback loop to repress its own transcription. Thus, the notch pathway functions upstream of her1 to promote the transcription of her1 mRNA, and her1 functions upstream of the Notch pathway to create the oscillating pattern of deltaC transcription. This identifies a rudimentary genetic loop (notch pathway > her1 > notch pathway) that functions within the PSM. Further, fused somites (fss) functions downstream of the notch pathway but upstream of her1 in the anterior PSM, and the notch pathway and fss function downstream of her1 slightly later in the anteriormost PSM. Therefore, the regulatory circuit consisting of her1 and the notch pathway exists throughout the PSM. Because this genetic circuit comprises genes that are required to create the oscillations in gene expression, these findings suggest that her1 and the notch pathway have cyclical functions at the center of the somitogenesis oscillator (Holley, 2002).

The genetic analysis of her1 and the notch pathway suggest a model in which these genes somehow generate the oscillations in gene expression. The initiation of the oscillations may be coupled to the commitment to become paraxial mesoderm. The expression of each of these genes (her1, deltaC, aei/deltaD and des/notch1) is initiated at the tip of the tailbud as cells subduct to form the paraxial mesoderm. The subsequent activities of these proteins could then initiate the interactions that create the oscillations in gene expression. deltaC, aei/deltaD and des/notch1 signaling would activate the transcription of her1 and deltaC. The subsequent increase in Her1 protein would then act to block the transcription of her1. Since the hairy proteins typically function as transcriptional repressors, an increase in Her1 should result in an increase in repressive activity, and the gradual degradation of this protein would produce a gradual decrease in this repressive activity. Therefore, the anterior progression/activation of a stripe of gene expression could be driven by the gradual loss of a repressive activity generated during the previous somite cycle. The positive regulation via notch could also display a cyclical variation, but ultimately the re-initiation of her1 and deltaC transcription would not occur until the level of Her1 drops below a specific threshold. In essence, this model suggests that the anterior progression of a stripe of gene expression is, at least in part, driven by the degradation of an existing, repressive activity (Her1), as opposed to the de novo synthesis of an activating component (Holley, 2002).

The analysis of deltaC expression in her1mo embryos uncovers an additional Notch-dependent patterning activity in the anterior PSM. This activity can create a segmental pattern of gene expression in the absence of any evidence of oscillations in her1 and deltaC expression: a smooth domain of deltaC expression is refined anteriorly to create stripes of expression that persist in the somitic mesoderm. This refinement requires the activity of fss, aei/deltaD, des/notch1, deltaC and beamter (bea), indicating that each of these genes has an additional function in the anterior-most PSM, downstream of her1. This is consistent with the fact that aei/deltaD, deltaC and des/notch1 are each transcribed within the PSM and later in the somitic mesoderm. In fact, this refining pattern is likely to be revealed only within the her1mo embryos because her1 is the only one of these cloned genes whose expression is restricted to the PSM. Ultimately, this indicates that the phenotypes observed in aei/deltaD and des/notch1 embryos are composites of defects that occur both upstream and downstream of her1 (oscillator) function. It has been shown that notch pathway signaling is involved in establishing the anteroposterior pattern within each somite. The late activity of the notch pathway described here probably represents this same anteroposterior patterning function. What is remarkable is that this late function can create a segmental pattern in the absence of prior oscillations in her1 and deltaC expression (Holley, 2002).

The role of Delta signaling was examined in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced Delta signaling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development (Cornell, 2000).

Neural crest is usually considered a defining feature of vertebrates, distinguishing them from chordate sister groups, cephalochordates and urochordates. The evolutionary appearance of neural crest is associated with the transition to predatory behavior and increased success of vertebrates relative to other chordates. Shared features between vertebrates and cephalochordates are assumed to have existed in the chordate precursor from which both of these modern groups evolved. While amphioxus, a commonly studied cephalochordate, lacks neural crest derivatives, it has dorsal spinal sensory neurons that may be homologous to RBs, suggesting this cell type predates neural crest. It has been proposed that, during neural crest evolution, a subset of RBs migrated from the neural tube and became extramedullary sensory neurons similar to modern DRG neurons. Thus the earliest neural crest derivative may have been a sensory neuron, which then diversified to form the many modern neural crest derivatives. The phenotypes of the dlA mutant, supernumerary RBs at the expense of neural crest, and the narrowminded mutant, lacking both RBs and neural crest, together demonstrate that the regulation of RB and neural crest fates are tightly linked and thus potentially evolutionarily related. A population of cells in the amphioxus neural plate expresses snail, a gene related to zebrafish sna2, and might be homologous to vertebrate neural crest. It will be interesting to learn whether dorsal spinal sensory neurons arise from this population (Cornell, 2000).

With respect to this model, why should Delta signaling regulate specification of trunk, but not cranial neural crest? Perhaps cranial neural crest originated from a class of cranial sensory neurons that has been lost. It has been suggested that the vertebrate precursor had dorsal sensory neurons throughout the neuraxis but that these cells were lost from the hindbrain of jawed vertebrates. Indeed, dorsal sensory neurons are present in the head of amphioxus, but it is unclear what relationship these cells have to RBs. Interestingly, jawed vertebrates, including zebrafish, have dorsal sensory neurons in a hindbrain nucleus, mesencephalic V; these may be homologous to RBs. It will be important to learn whether Delta signaling regulates these neurons and whether they are derived from an equivalence group that includes neural crest (Cornell, 2000).

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard (RB) cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion (DRG) sensory neurons; however, RB neurons and DRG neurons are not necessarily derived from the same precursor cell. It is proposed that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons (Cornell, 2002).

deltaD is one of the four zebrafish Delta homologs presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. The organization of the regulatory region of the deltaD gene has been analysed using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organized in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation (using a minigene comprising 10.5 kb of this genomic DNA attached to the 3' end of a full-length cDNA clone) rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eightAR33. Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition (Hans, 2002).

Transgenic analysis of the deltaD locus has revealed six distinct cis-regulatory regions, five upstream and one downstream of the transcription start site, that direct gene expression in neuroectodermal and mesodermal subdomains of the embryo. The upstream region of the mouse Dll1 locus shows a similar organization. It is proposed that both promoters are organized in five modules, of which at least three are phylogenetically conserved. Two of these modules correspond to regions HI and HII, identified on the basis of their high sequence similarity; in addition, both regions are located in the same relative positions and in the same orientation in both species. However, there is a difference in the pattern of expression driven by these elements in zebrafish and mouse. In stably transformed mouse embryos, HI coupled to the minimal Dll1 promoter is able to direct reporter gene expression primarily to the ventral tube and some derivatives of the neural crest, such as dorsal root and spinal ganglia. By contrast, transformants bearing HII fused to the minimal Dll1 promoter direct expression in the marginal zone of the dorsal region of the neural tube. In zebrafish, the expression patterns of HI and HII do not exhibit a restriction to dorsal or ventral regions of the neural tube. It seems probable that this apparent difference is due to the different expression of neurogenin 1 and Mash1 (Ascl1 -- Mouse Genome Informatics), and neurogenin 1 and zash1a/b, in mouse and zebrafish, respectively. Indeed, in the mouse embryo the expression patterns of neurogenin 1 and Mash1 show a similar restriction in the neural tube, and it appears that there is a complete overlap in the expression patterns of HI and HII with neurogenin 1 and Mash1. Thus, the regulatory network appears to be conserved in zebrafish and mouse, although the expression domains of the corresponding proneural genes have changed during evolution (Hans, 2002).

With respect to the three mesodermal modules, two appear to be conserved whereas the other has diverged. In both species, one mesodermal element in the region immediately proximal to the minimal promoter is able to direct reporter gene expression in the presomitic mesoderm and nascent somites. However, sequence comparison of these two regions in zebrafish and mouse revealed only minor stretches of similarity, the significance of which still remains to be tested. A second module is represented by the putative silencer of transcription in the presomitic mesoderm at –1.8 to –1.3, flanked by enhancer elements. In mouse, negative regulators have been described flanked by positive regulators of expression in the presomitic mesoderm, i.e., a similar organization to that in zebrafish. Unfortunately, again in this case the comparison of both DNA sequences has failed to show any similarity in this region. The third mesodermal module identified in the mouse Dll1 promoter is located within the two elements HI and HII. This module is not present in the zebrafish deltaD and might be responsible for the difference in the expression in mature somites: zebrafish deltaD is expressed in the anterior halves of the mature somites, whereas mouse Dll1 is expressed in the posterior halves. Therefore, all these considerations reveal a great deal of phylogenetic conservation in the organization of the regulatory regions of deltaD and Dll1 expression in zebrafish and mouse (Hans, 2002).

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 of neural 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; see Drosophila Mind bomb] 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).

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).

Suppressor of Hairless [Su(H)] codes for a protein that interacts with the intracellular domain of Notch to activate the target genes of the Delta-Notch signalling pathway. The zebrafish homolog of Su(H) has been cloned and characterized and its function has been analyzed by morpholino mediated knockdown. While there are at least four notch and four delta homologs in zebrafish, there appears to be only one complete Su(H) homolog. The function of Su(H) in the somitogenesis process was analyzed and its influence on the expression of notch pathway genes was examined, in particular her1, her7, deltaC and deltaD. The cyclic expression of her1, her7 and deltaC in the presomitic mesoderm is disrupted by the Su(H) knockdown mimicking the expression of these genes in the notch1a mutant deadly seven. deltaD expression is similarly affected by Su(H) knockdown like deltaC but shows in addition an ectopic expression in the developing neural tube. The inactivation of Su(H) in a fss/tbx24 mutant background leads furthermore to a clear breakdown of cyclic her1 and her7 expression, indicating that the Delta-Notch pathway is required for the creation of oscillation and not only for the synchronization between neighboring cells. The strongest phenotypes in the Su(H) knockdown embryos show a loss of all somites posterior to the first five to seven. This phenotype is stronger than the known amorphic phenotypes for notch1 (des) or deltaD (aei) in zebrafish, but mimicks the knockout phenotype of RBP-Jkappa gene in the mouse that is the homolog of Su(H). This suggests that there is some functional redundancy among the Notch and Delta genes. This fact that the first five to seven somites are only weakly affected by Su(H) knockdown indicates that additional genetic pathways may be active in the specification of the most anterior somites (Sieger, 2003).

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).

Somitogenesis is the process by which the segmented precursors of the skeletal muscle and vertebral column are generated during vertebrate embryogenesis. While somitogenesis appears to be a serially homologous, reiterative process, there are differences between the genetic control of early/anterior and late/posterior somitogenesis. Point mutations can cause segmentation defects in either the anterior, middle, or posterior somites in the zebrafish. Mutations in zebrafish integrinα5 disrupt anterior somite formation, giving a phenotype complementary to the posterior defects seen in the notch pathway mutants after eight/deltaD and deadly seven/notch1a. Double mutants between the notch pathway and integrinα5 display somite defects along the entire body axis, with a complete loss of the mesenchymal-to-epithelial transition and Fibronectin matrix assembly in the posterior. The data suggest that notch- and integrinα5-dependent cell polarization and Fibronectin matrix assembly occur concomitantly and interdependently during border morphogenesis (Julich, 2005).

Notch signalling by the ligand Delta-like 4 (Dll4) is essential for normal vascular remodelling, yet the precise way in which the pathway influences the behaviour of endothelial cells remains a mystery. Using the embryonic zebrafish, it has been shown that, when Dll4-Notch signalling is defective, endothelial cells continue to migrate and proliferate when they should normally stop these processes. Artificial overactivation of the Notch pathway has opposite consequences. When vascular endothelial growth factor (Vegf) signalling and Dll4-Notch signalling are both blocked, the endothelial cells remain quiescent. Thus, Dll4-Notch signalling acts as an angiogenic 'off' switch by making endothelial cells unresponsive to Vegf (Leslie, 2007).

Wnt1 regulates neurogenesis and mediates lateral inhibition of boundary cell specification by regulating delta in the zebrafish hindbrain

The formation of localised signalling centres is essential for patterning of a number of tissues during development. Previous work has revealed that a distinct population of boundary cells forms at the interface of segments in the vertebrate hindbrain, but the role of these cells is not known. This study has investigated the function of the Wnt1 signalling molecule, which is expressed by boundary and roof plate cells in the zebrafish hindbrain. Knockdown of wnt1 or of tcf3b, a mediator of Wnt signalling, leads to ectopic expression of boundary cell markers, radical fringe (rfng) and foxb1.2, in non-boundary regions of the hindbrain. Ectopic boundary marker expression also occurs following knockdown of rfng, a modulator of Notch signalling required for wnt1 expression at hindbrain boundaries. The boundary and roof plate expression of wnt1 each contribute to upregulation of proneural and delta gene expression and neurogenesis in non-boundary regions, which in turn blocks ectopic boundary marker expression. Boundary cells therefore play a key role in the regulation of cell differentiation in the zebrafish hindbrain. The network of genes underlying the regulation of neurogenesis and lateral inhibition of boundary cell formation by Wnt1 has a striking similarity to mechanisms at the dorsoventral boundary in the Drosophila wing imaginal disc (Amoyel, 2005).

A potential pathway by which Wnt1 might inhibit ectopic expression of boundary cell markers was suggested by the similarity of gene expression patterns in the zebrafish hindbrain to those adjacent to the dorsoventral boundary of the Drosophila wing imaginal disc. In the wing imaginal disc, expression of Fringe and Serrate in the dorsal compartment, and of Delta in the ventral compartment, leads to a stripe of Notch activation at the dorsoventral boundary, and Notch activation upregulates wg expression. Wg protein acts on adjacent cells in the anterior compartment to upregulate expression of as-c proneural genes, which specify a post-mitotic sensory hair cell fate, and upregulate Delta gene expression. Delta acts cell autonomously to inhibit Notch activation, and because Notch activation is required to activate wg expression, this mediates a lateral inhibition that prevents spreading of the wg expression domain (Amoyel, 2005 and references therein).

After 18 hours of development, expression of ash and ngn proneural genes, and of delta genes, becomes restricted to stripes adjacent to hindbrain boundaries. There is the same regulatory hierarchy in the hindbrain as in the Drosophila wing disc: the modulation of Notch by Rfng upregulates wnt1 in boundary cells; knockdown of wnt1 or tcf3b leads to a major decrease in the number of cells expressing the ash and ngn proneural genes, and knockdown of ash or ngn leads to a decrease in delta gene expression. Finally, it was found that knockdown of ash, ngn or delta gene function leads to spreading of hindbrain boundary marker expression (Amoyel, 2005).

These findings reveal a regulatory loop between boundary cells and non-boundary regions that stabilises the pattern of each cell population via bidirectional lateral inhibition. Rfng-mediated modulation of Notch activation upregulates wnt1 expression in boundary cells. Notch activation also regulates the affinity properties of boundary cells, thus maintaining their segregation to the interfaces of segments. Wnt1 expressed by boundary cells promotes proneural and delta gene expression in non-boundary regions, which enables neuronal differentiation and laterally inhibits the spread of boundary marker expression. In addition, roof-plate expression of Wnt1, which is independent of Rfng function, contributes to the promotion of neurogenesis, but is not sufficient to prevent hindbrain boundary spreading. Delta expression by non-boundary cells activates Notch in boundary cells, and thus laterally inhibits boundary cells from expressing proneural genes and undergoing neuronal differentiation (Amoyel, 2005).

Delta proteins and MAGI proteins: an interaction of Notch ligands with intracellular scaffolding molecules and its significance for zebrafish development

Delta proteins activate Notch through a binding reaction that depends on their extracellular domains; but the intracellular (C-terminal) domains of the Deltas also have significant functions. All classes of vertebrates possess a subset of Delta proteins with a conserved ATEV* motif at their C termini. These ATEV Deltas include Delta1 and Delta4 in mammals and DeltaD and DeltaC in the zebrafish. These Deltas associate with the membrane-associated scaffolding proteins MAGI1, MAGI2 and MAGI3, through a direct interaction between the C termini of the Deltas and a specific PDZ domain (PDZ4) of the MAGIs. In cultured cells and in subsets of cells in the intact zebrafish embryo, DeltaD and MAGI1 are co-localized at the plasma membrane. The interaction and the co-localization can be abolished by injection of a morpholino that blocks the mRNA splicing reaction that gives DeltaD its terminal valine, on which the interaction depends. Embryos treated in this way appear normal with respect to some known functions of DeltaD as a Notch ligand, including the control of somite segmentation, neurogenesis, and hypochord formation. They do, however, show an anomalous distribution of Rohon-Beard neurons in the dorsal neural tube, suggesting that the Delta-MAGI interaction may play some part in the control of neuron migration (Wright, 2004).

Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue

Mind bomb1 (Mib1)-mediated endocytosis of the Notch ligand DeltaD is essential for activation of Notch in a neighboring cell. Although most DeltaD is localized in cytoplasmic puncta in zebrafish neural tissue, it is on the plasma membrane in mib1 mutants because Mib1-mediated endocytosis determines the normal subcellular localization of DeltaD. Knockdown of Notch increases cell surface DeltaA and DeltaD, but not DeltaC, suggesting that, like Mib1, Notch regulates endocytosis of specific ligands. Transplant experiments show that the interaction with Notch, both in the same cell (in cis) and in neighboring cells (in trans), regulates DeltaD endocytosis. Whereas DeltaD endocytosis following interaction in trans activates Notch in a neighboring cell, endocytosis of DeltaD and Notch following an interaction in cis is likely to inhibit Notch signaling by making both unavailable at the cell surface. The transplantation experiments reveal a heterogeneous population of progenitors: in some, cis interactions are more important; in others, trans interactions are more important; and in others, both cis and trans interactions are likely to contribute to DeltaD endocytosis. It is suggested that this heterogeneity represents the process by which effective lateral inhibition leads to diversification of progenitors into cells that become specialized to deliver or receive Delta signals, where trans and cis interactions with Notch play differential roles in DeltaD endocytosis (Matsuda, 2009).

Identification of direct T-box target genes in the developing zebrafish mesoderm

The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. This study surveyed the genome for downstream target genes of the Spt and Ntl T-box transcription factors. Evidence was found for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, target regulatory sequence were sought via a combination of binding motif searches and comparative genomics. Regulatory elements were identified for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, evidence was provided that both are directly regulated by T-box transcription factors. deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process (Garnett, 2009).

Xenopus Delta homologs

Evolutionary homologs - Continued: part 2/3 | part 3/3

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

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