Delta
See the embryonic expression pattern of Dl at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Maternal 5.4 and 4.5 kb Delta transcripts are uniformly distributed throughout the embryo during the first nine nuclear divisions of the syncytial blastoderm (Kopczynski, 1989). Zygotic transcripts begin to accumulate at the start of cellularization (early stage 5), and are localized in lateral bands bordering the prospective mesoderm. After gastrulation Delta is found in ventral neurogenic region and in a pair-rule-like banded structure in the ectoderm, distributed in a ventral to dorsal gradient (Haenlin, 1990).
By stage 6, there is accumulation in the anterior gnathal segments [Images] and procephalic neurogenic ectoderm From stage 8 through 11, Delta transcripts accumulate in endodermal derivatives (anterior and posterior midgut). By stage twelve, Delta transcripts are reduced throughout the ventral epidermis. Delta accumulates at high levels in the mesoderm by stage 10 (Kopczynski, 1989).
Big brain and Delta proteins colocalize. In the prospective mesoderm just before gastrulation, Bib
protein disappears from the plasma membrane and is present in punctate cytoplasmic structures basal to the
nucleus. The Delta protein is expressed in a similar manner. To address whether Bib and Dl colocalize,
embryos were simultaneously labeled with Bib and Dl antibodies.
The Bib and Dl proteins did in fact colocalize in the plasma membrane and in the punctate cytoplasmic structures of prospective mesoderm cells, although the intensity
of the two signals was not always similar. By immunoelectron microscopy, it was
found that bib is associated with the plasma membrane and concentrated in apical
adherens junctions as well as in small cytoplasmic vesicles (Doherty, 1997).
For information about the role of Delta (reviewed by Hartenstein, 1992) in the development of specific tissues see:
Notch signalling is an evolutionarily conserved cell interaction mechanism; its role in controlling cell fate choices has been studied extensively. Recent studies in both vertebrates and invertebrates have revealed additional functions of Notch in proliferation and apoptotic events. Evidence suggests an essential role of the Notch signalling pathway during morphogenetic cell movements required for the formation of the foregut-associated proventriculus organ in the Drosophila embryo. The activation of the Notch receptor occurs in two rows of boundary cells in the proventriculus primordium. The boundary cells delimit a population of foregut epithelial cells that invaginate into the endodermal midgut layer during proventriculus morphogenesis. Notch receptor activation requires the expression of its ligand Delta in the invaginating cells and apical Notch receptor localization in the boundary cells. The movement of the proventricular cells is dependent on the short stop gene that encodes the Drosophila plectin homolog of vertebrates and is a cytoskeletal linker protein of the spectraplakin superfamily. short stop is transcriptionally activated in response to the Notch signalling pathway in boundary cells, and it has been shown that the localization of the Notch receptor and Notch signalling activity depend on short stop activity. These results provide a novel link between the Notch signalling pathway and cytoskeletal reorganization controlling cell movement during the development of foregut-associated organs (Fuss, 2004).
The proventriculus is a multiply folded, cardia-shaped organ that functions as a valve to regulate food passage from the foregut into the midgut of Drosophila larvae. It is derived from the stomodeum, which gives rise to the foregut tube and to parts of the anterior midgut in the early embryo. Cell shape changes are initiated at stage 12 when cell proliferation has been completed within the proventriculus primordium. Anti-Forkhead (Fkh)/anti-Defective proventriculus (Dve) double immunostainings which specifically visualize ectodermal and endodermal cells, respectively, reveal that the first step of proventriculus morphogenesis involves the formation of a ball-like evagination at the ectoderm/endoderm boundary of the posterior foregut tube. The formation of this evagination is initiated by a local constriction of apical membranes at the ectoderm/endoderm boundary leading to an accumulation of membrane-associated markers such as Arm towards the luminal (apical) side. It is notable that the ectodermal part of the ball-like evagination localizes in a mesoderm-free region, whereas the surrounding cells of the developing foregut and the midgut are covered by visceral mesoderm. At stage 14, a constriction forms at the boundary of the ectodermal and the endodermal cells. This results in the formation of the 'keyhole' structure. From stage 14 onward, cells from the anterior portion of the ectodermal keyhole part (in the mesoderm-free area) begin to move inward into the endodermal part of the keyhole and a heart-like structure is formed. The ectodermal keyhole cells continue to move inward until late stage 17 and give rise to the recurrent layer of the proventriculus; it links the outer endodermal layer (derived from the endodermal keyhole cells) and the inner layer of the proventriculus which is a continuation of the esophagus. The cells at the tip of the invaginating ectodermal keyhole cells thatderive from the most anterior region of the keyhole, are not covered by visceral mesoderm. It has been observed before that these cells assume a stretched appearance with long cytoplasmic extensions (Fuss, 2004 and references therein).
Immunohistochemical analysis demonstrates that the ligands of the Notch
receptor, Delta and Serrate are expressed in the ectodermal keyhole cells that invaginate into the endodermal cell layer during proventriculus development. Their expression becomes downregulated in the anterior and posterior boundary cells in which the Notch receptor is elevated and in which the Notch signalling pathway is activated, as demonstrated by the Notch-dependent Gbe-Su(H)m8-lacZ reporter construct. Whereas there is no proventricular phenotype in Ser mutants, the invagination movement of the ectodermal keyhole cells is defective in mutants of other components of the Notch signalling pathway, such as Notch, Delta, fng or Su(H). This strongly suggests that the boundary cells play a crucial role for cell movement during proventriculus development. It is not known whether
the cell movements are driven by the anterior boundary cells, dragging the
esophageal cells behind or whether the major force for the inward movement is contributed by the ectodermal foregut cells changing their shapes from a cuboidal to a more stretched appearance. The latter is known to occur during mid and late stages of embryogenesis when the foregut and the hindgut elongate dramatically increasing their size by two- to threefold. It has been shown for dorsal closure that multiple forces contribute to cell sheet morphogenesis. A similar scenario may apply for proventriculus morphogenesis. Genetic mosaic studies have revealed that the activity of the Notch receptor occurs in cells that are adjacent to the ligand-expressing cells. Therefore, the downregulation of Delta in the boundary cells may be a prerequisite for Notch signalling and cell movement, which would be consistent with the observation that a Notch-like proventriculus phenotype is induced when Delta expression is maintained in the anterior and posterior boundary cells (Fuss, 2004).
Delta and Notch are required for partitioning of vein and intervein cell fates within the provein during Drosophila metamorphosis. Partitioning of these fates is dependent on Delta-mediated signaling from 22 to 30 hours after puparium formation at 25 degrees C. Within the provein, Delta is expressed more highly in central provein cells (presumptive vein cells) and Notch is expressed
more highly in lateral provein cells (presumptive intervein cells). Accumulation of Notch in presumptive intervein cells is dependent on
Delta signaling activity in presumptive vein cells; constitutive Notch receptor activity represses Delta accumulation in presumptive
vein cells. When Delta protein expression is elevated ectopically in presumptive intervein cells, complementary Delta and Notch
expression patterns in provein cells are reversed, and vein loss occurs because central provein cells are unable to stably adopt the vein
cell fate. These findings imply that Delta-Notch signaling exerts feedback regulation on Delta and Notch expression during metamorphic
wing vein development, and that the resultant asymmetries in Delta and Notch expression underlie the proper specification of vein and
intervein cell fates within the provein (Huppert, 1997).
Notch activation at the midline plays an essential role both
in promoting the growth of the eye primordia and in regulating eye patterning. Specialized cells are established along the dorsal-ventral midline
of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by Wingless expression. Both Eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four jointed (fj), is also conserved. four jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).
During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).
The four-jointed gene is expressed in a gradient during early eye development, with a peak of expression along the D-V midline. Together with Ser and Dl, Fj serves as a molecular marker of midline fate. Ubiquitous expression of Fng during early eye development, generated by placing fng under the control of an eyeless enhancer, eliminates detectable expression of Ser and Dl along the midline. Conversely, misexpression of Fng in clones of cells, can result in ectopic expression of Ser and fj that is centered along novel borders of Fng expression in the dorsal eye. Ectopic Ser and fj expression can also be detected along the borders of fng mutant clones in the ventral eye. These observations show that Fng expression borders play an essential and instructive role in establishing a distinct group of cells along the D-V midline of the developing eye. Animals with reduced fng activity have small eyes. Moreover, ubiquitous fng expression also results in a dramatic loss of tissue. Tissue loss is detectable in the developing imaginal disc, before the morphogenetic furrow moves across the eye. Moreover, eye loss is observed when fng is ectopically expressed during early development, but not when fng is ectopically expressed behind the furrow. These observations indictate that a Fng expression border is required for eye growth, specifically during early eye development (Papayannopoulos, 1998).
Fng differentially modulates the action of Notch ligands in the eye just as it does in the wing.
Clones of cells ectopically expressing Dl can induce Ser expression in ventral, Fng-expressing cells, but not in dorsal cells. Fng alone can induce Ser expression in dorsal cells, but only near the D-V midline. When Fng and Dl are co-misexpressed, Ser expression can be induced in dorsal cells even when the clones are far from the D-V midline. Clones of cells ectopically expressing Ser are able to induce increased expression of Dl in dorsal cells but not in ventral, Fng expressing cells. However, if Ser is ectopicallly expressed in fng mutant animals, it can induce Dl expression in ventral cells (Papayannopoulos, 1998).
Notch function is also necessary for normal D-V midline cell fate. The ability of Ser and Dl to induce one another's expression indicates that the expression of either one is a marker for Notch activation in the eye. Analysis of loss-of-function mutants of Notch and its ligands, as well as ectopic expression studies, indicate that Notch activation also regulates eye growth. Several observations indicate that the D-V midline is the focus of Notch activation required for growth. Moreover, the midline corresponds to a fng expression border, which is essential for growth and modulates Notch signaling during early eye development. Because local activation of Notch has long-range effects on growth and four-jointed expression, it is inferred that Notch induces the expression of a diffusible growth factor at the midline. Notch activation influences ommatidial chirality. fng mutant clone borders within the ventral eye can be associated with reversals of ommatidial chirality, whereas mutant clones that cross the D-V midline disrupt the normal equator. The equatorial bias in the influence of ectopic Notch activation implies that the equator is the normal source of a Notch-dependent, chirality-determining signal (Papayannopoulos, 1998).
In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).
Comparison between Mad Su(H) ci cells and Su(H) ci cells
shows that Dpp signaling is sufficient to initiate eye differentiation in its
normal location in the absence of Hh or N signals, but such differentiation is delayed. The normal timing of differentiation is restored by combined Dpp and N signals (in ci clones). This is the basis for the ectopic
differentiation on co-expression of Dpp and Dl ahead of the furrow (Fu, 2003).
Dl, Hh and Dpp are generally thought to signal over very different
distances. How can signals of such different range substitute for one another
to permit normal eye development? Dpp is
transcribed in response to Hh signaling and is produced where Ci155 levels are highest. Dl is regulated by Hh indirectly through Ato and
Ato-dependent Egfr activity in differentiating cells. Hh is
expressed most posteriorly of the three, in differentiating photoreceptors (Fu, 2003).
Eye differentiation uses Hh to progress through cells unable to respond to
Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion
depends in part on the shape of the morphogenetic furrow cells. The Dpp
that drives differentiation through ci-mutant cells unable to respond
to Hh must diffuse from outside the ci clones because Dpp synthesis
is Hh dependent. Large ci clones develop normally so Dpp diffusion
cannot be limiting (dpp-mutant clones offer no information about the
range of Dpp because they express and differentiate in response to Hh).
Instead, the rate of progression in response to Dpp is controlled by Dl. Dl
signals over (at most) one or two cell diameters at the morphogenetic furrow (Fu, 2003).
The previous view of eye patterning was influenced by the morphogen
function of Hh and Dpp in other discs. It was thought that domains of Ato and Hairy expression reflected increasing concentrations of Hh and Dpp. The data shows that, in the eye, the combination of signals is important. Differentiation is triggered where Dl and/or Hh synergize with Dpp, regardless of where the source of Dpp is. The additional requirements limit Dpp to initiating differentiation at the same locations that Hh does (Fu, 2003).
> Epsin is part of a protein complex that performs endocytosis in eukaryotes. Drosophila epsin, Liquid facets (Lqf), was identified because it is essential for patterning the eye and other imaginal disc derivatives. Previous work has provided only indirect evidence that Lqf is required for endocytosis in Drosophila. Epsins are modular and have an N-terminal ENTH (epsin N-terminal homology) domain that binds PIP2 at the cell membrane and four different classes of protein-protein interaction motifs. The current model for epsin function in higher eukaryotes is that epsin bridges the cell membrane, a transmembrane protein to be internalized, and the core endocytic complex. This study shows directly that Drosophila epsin (Lqf) is required for endocytosis. Specifically, Lqf is essential for internalization of the Delta (Dl) transmembrane ligand in the developing eye. Using this endocytic defect in lqf mutants, a transgene rescue assay has been developed and a structure/function analysis of Lqf has been performed. When Lqf is divided into two pieces, an ENTH domain and an ENTH-less protein, each part retains significant ability to function in Dl internalization and eye patterning. These results challenge the model for epsin function that requires an intact protein (Overstreet, 2003).
To test for endocytosis defects in lqf− mutants, the localization of the transmembrane receptor Dl was monitored in developing eyes. Dl normally undergoes endocytosis in the eye, and as the internalized protein is not degraded rapidly, internalized Dl can be detected in vesicles (Overstreet, 2003).
The Drosophila eye, composed of 800 identical 22-cell ommatidia, or facets, develops in the larval and pupal stages in a monolayer epithelium called the eye imaginal disc. Eye development occurs as a wave, where the morphogenetic furrow forms at the posterior of the disc, and moves anteriorly into the monolayer of undifferentiated cells. Rows of ommatidia assemble stepwise posterior to the furrow one or two cells at a time, starting with the eight photoreceptors (R1-R8) (Overstreet, 2003).
In wild-type, Dl is detected exclusively as intracellular dots within developing ommatidial clusters throughout the eye disc. In larval eye discs homozygous for lqfFDD9, a weak, viable mutant allele, Dl is detected mainly at the membranes of cells just posterior to the furrow. In clones of cells homozygous for lqfARI, a strong, lethal mutant allele, similar membrane localization of Dl is observed. It is concluded that lqf+ is required for Dl internalization (Overstreet, 2003).
All epsins have an amino-terminal ENTH domain that binds PIP2 at the cell membrane and three or four types of protein-protein interaction motifs, whose copy numbers vary among different epsins. The ubiquitin interaction motifs (UIMs) bind ubiquitin (Ub) noncovalently. There are also clathrin binding motifs (CBMs), DPW motifs that bind the core endocytic adaptor complex, AP-2, and NPF motifs that bind Eps15, another accessory factor (Overstreet, 2003).
A step toward understanding the role of Lqf in endocytosis is the identification of the modules of Lqf protein that are required. In yeast, there are straightforward assays for the function of the two epsins (Ent1 and Ent2). Structure/function analyses have demonstrated that the ENTH domain of Ent1 is necessary and sufficient to rescue the lethality of ent1Δent2Δ double mutants. Moreover, the ENTH domain and to a lesser extent the UIMs have been shown to be required for endocytosis. Because there are mechanistic differences between endocytosis in yeast and higher eukaryotes, the yeast epsins might function somewhat differently from vertebrate epsins and Drosophila Lqf. The major difference between these systems is that the AP-2 core adaptor complex in yeast has no known function in endocytosis, and, accordingly, the yeast epsins lack DPW motifs. As in yeast, structure/function analyses of epsins in vertebrate cell culture have pointed to the importance of the ENTH domain. These assays, however, rely on dominant-negative effects of mutant epsin proteins on endocytosis, and their interpretation is difficult (Overstreet, 2003).
Either the ENTH domain alone, or an ENTH-less Lqf protein, rescues the patterning and Dl endocytosis defects in lqfFDD9 homozygous eyes. Since experimental results in yeast and in vertebrates have emphasized the importance of the ENTH domain, the most remarkable result is that an ENTH-less Lqf protein can function. The simplest interpretation of the rescue results is that LqfΔENTH can function independently of the ENTH domain (Overstreet, 2003).
Transgenes that express Rat epsin1 or human epsin 2b in Drosophila with pRO, each as full-length proteins or without the ENTH domain, rescue the eye defects in lqfFDD9 homozygotes. Thus, there is unlikely to be a significant functional difference between the Drosophila and vertebrate epsins in the region C-terminal to the ENTH domain. In addition, the ENTH domains of Lqf and yeast epsin are functionally similar. It was shown previously that expression of the ENTH domain of Ent1, but not the complementary portion of the protein, restores viability to ent1Δent2Δ yeast. Similarly, expression of full-length Lqf or LqfENTH rescues ent1Δent2Δ lethality but LqfΔENTH expression does not (Overstreet, 2003).
Thus Drosophila epsin, Lqf, is essential for endocytosis of Dl in the developing eye. Moreover, the ENTH domain alone and an ENTH-less Lqf protein each retain significant function. The prevailing model in vertebrates is that epsin functions like a bridge, where the ENTH domain links the membrane to clathrin, a cell surface protein to be internalized, and to AP-2. Since this model requires an intact epsin protein, the results presented here suggest that the prevailing model cannot be the whole story. Moreover, the observation that either the ENTH domain or the remainder of the protein, which are functionally distinct, can be deleted without destroying Lqf function completely suggests that each fragment of Lqf may be partially redundant with another Drosophila endocytic protein. Candidates for the other endycotgic protein include the other ENTH domain protein in Drosophila, Epsin-2 and the Drosophila homolog of AP180, which, like the ENTH-less Lqf protein, binds clathrin and AP-2 (Overstreet, 2003).
The role of scabrous in the evenly spaced bristle pattern of Drosophila has been explored. Loss-of-function of sca results in development of an excess of
bristles. Segregation of alternately spaced bristle precursors and epidermal
cells from a group of equipotential cells relies on lateral inhibition mediated
by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit
the neural fate of neighboring cells, causing them to adopt the epidermal fate.
Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signaling has been increased. scabrous acts nonautonomously, and evidence is presented that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signaling are discussed. It is also shown that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and these structures may play an active role during signaling (Renaud, 2001).
sca functions in the inhibition of the neural fate, since in its absence an excess of neural precursors form. The sca mutant phenotype is very similar to that of hypomorphic N and Dl mutants and indeed Notch and Dl are known to be the main components of the signaling pathway regulating the spaced pattern of bristles. Thus, Sca is likely to positively modulate N signaling. During bristle precursor selection, like Dl, sca acts nonautonomously and is not required for reception of the signal that places its activity upstream of N. What are the respective roles of Dl and sca in the segregation of bristle precursors? Both genes are expressed in proneural domains and then at high levels in the bristle precursors, and their products act nonautonomously on neighboring presumptive epidermal cells. Both proteins associate with N, but so far only Dl has been shown to be an activating ligand. Scabrous is a secreted molecule, whereas data accumulated to date suggest that active Dl is membrane-bound. In the complete absence of Dl, all cells adopt the neural fate and thus bristle precursors arise adjacent to one another. In the complete absence of Sca, there is an excess of bristle precursors but they are never adjacent and are always separated by at least one epidermal cell. This indicates that sca is not needed for the bristles to be spaced apart by a short distance, and that Dl, which is expressed in sca mutants, is able to inhibit cells immediately adjacent to the precursors without any help from Sca. In contrast, in the absence of Sca, Dl is unable to inhibit those cells not in direct contact with the precursor. This suggests a role for Sca in the inhibition of cells not adjacent to the precursor. These two, possibly separable events, are referred to as 'short' and 'long' range signaling (Renaud, 2001).
Formally, there are three possible mechanisms for 'long' range signaling. The first would be that sca is part of a relay mechanism whereby cells immediately adjacent to the precursor are inhibited by Dl and then relay the signal farther out by means of Sca. This hypothesis is very unlikely, however, because one would expect sca to be expressed in cells adjacent to the precursor following activation of N by Dl. In fact, sca protein is only detectable in the precursor itself, although sca is earlier expressed at low levels in the proneural domain. Furthermore, sca is expressed in the absence of Dl and therefore does not require a prior signaling event mediated by Dl (Renaud, 2001).
A second possible mechanism is that there may be two independent signals, one acting at a 'short' range (Dl) and another at a 'long' range (Sca). Two observations suggest that this, too, is unlikely: (1) in the absence of Dl, all cells adopt the neural fate and so the process of bristle spacing is completely abolished. scabrous is expressed in cells mutant for Dl so this result indicates that Sca alone is unable to repress the neural fate; (2) the results indicate that sca is not involved in the process of lateral inhibition whereby a pattern of alternating neural and epidermal cells is generated. Along a border between wild-type cells and cells mutant for Dl, the precursors are nearly always selected from the pool of wild-type cells, rather than from the mutant cells. This is thought to be because the mutant cells produce little or no signal and are inhibited by the Dl-producing wild-type cells. Along sca mosaic borders, the mutant cells can adopt the neural fate, indicating that they are not defective in the production of the activating ligand Dl. Furthermore, since wild-type precursors also form along the mosaic border, neural precursors can be chosen from cells of either genotype. Thus, cells choose the neural or epidermal fate regardless of whether or not they express sca (Renaud, 2001).
This would explain the fact that precursors are never adjacent in sca mutants and is not inconsistent with the observed sca phenotype of excess bristles. During segregation of both the normal component of precursors, as well as the additional precursors, adjacent cells have to choose between epidermal and neural fates. Any failure of this process would result in the presence of adjacent bristle precursors, a phenotype characteristic of N and Dl mutants, but not sca mutants. Segregation of single neural precursors surrounded by epidermal neighbors is thus probably mediated by Dl alone, which would explain why bristle spacing is completely abolished in the absence of Dl, even though Sca is present (Renaud, 2001).
The third possibility is that 'long' range signaling requires both Dl and Sca. Under this hypothesis, Dl would be the activating ligand in 'long' range signaling, but would require Sca in order to inhibit cells not directly in contact with the precursor. This could be the case regardless of whether the signal originates exclusively in the precursor or additionally in proneural cells. One observation in favour of this hypothesis is afforded by examination of flies mosaic for Dl. Along the edges of Dl mutant clones, mutant cells are able to differentiate as epidermis under the influence of an inhibitory signal from neighboring wild-type cells. This 'rescue,' due to expression of Dl in the wild-type neighbours, can extend up to four cell diameters. If there is no relay mechanism and no other signal, then Dl must somehow be able to activate N several cell diameters away from the cell in which it is produced. Scabrous is of course present in both the Dl+ and the Dl- cells, but is unable to effect any rescue of cells mutant for Dl that are situated more than about four cells away from the wild-type Dl-expressing cells. Examination of Dl mutant clones in a mutant sca background would indicate the range of Dl signaling in the absence of Sca (Renaud, 2001). Clones doubly mutant for sca and Dl, however, fail to differentiate the cuticular components of the bristles (for all allelic combinations tested) and so are uninformative (Renaud, 2001).
It is suggested that bristle spacing may be the result of two signaling events. The first step of lateral signaling involves Dl alone and allows a group of cells to choose a single neural precursor that will inhibit adjacent cells. This step proceeds normally in the absence of Sca. The second step would act to inhibit cells that are not adjacent to the precursor and would require the activity of both Dl and sca. This step is impaired in both sca and Dl mutants. In Dl mutants, in the absence of the inhibitory signal, lateral inhibition fails, leading to adjacent precursors and a loss of epidermal cells. In sca mutants, an excess of precursors form, but they segregate singly and are spaced by at least one epidermal cell through the activity of Dl. Examination of the nascent precursors with neu-lacZ, fail, to reveal two temporally separate waves of precursor formation in sca mutants. Staining of wild-type pupal nota with DE-cadherin, however, may provide a visual correlate of the two groups of target cells. A rosette-like ring of wedge-shaped cells surrounds the bristle precursor; it is reminiscent of the cell preclusters that precede segregation of the R8 photoreceptor during development of ommatidia. These supra-cellular structures may allow more cells to enter into direct contact with the precursor during the first step of inhibitory signaling (Renaud, 2001).
The results demonstrate a requirement for local discontinuities in the levels of Sca between cells during inhibitory signaling. This was also reported to be the case for eye development. Uniform expression of Sca under experimental conditions is unable to rescue the sca mutant phenotype. So the relative quantitative differences in the level of Sca between neighbouring cells is an essential feature of the spacing mechanism. Over-expression of Sca in a wild-type fly causes a phenotype opposite that of the loss-of-function phenotype. The distance between bristles actually increases and there are correspondingly fewer bristles in the domain of over-expression. In these flies, although exogenous Sca is uniformly distributed, regulation of the endogenous gene will provide local differences in levels of the protein. The greater distance between bristles suggests an extension in the range of the inhibitory signal. Scabrous is likely to be present in a gradient of decreasing concentration around each precursor. The molecule has been shown to have a very short half-life. Thus, it would decay rapidly after secretion and this would help maintain a graded distribution from the source. It is postulated that such a gradient could define the range of the inhibitory signal (Renaud, 2001).
A rescue of up to four cell diameters has been observed at the edges of clones of cells mutant for Dl. This suggests a signaling range that exceeds that required in wild-type flies, where bristles are usually only four to five cells apart. It is possible, however, that the signal range is longer than usual in this experimental situation because very large amounts of Sca are likely to be present in these clones due to the hyperplasia of precursors. It is, however, noteworthy that in other drosophilid species, such as D. ararama, the bristles are spaced by eight or nine cells, suggesting a possibly greater signaling range (Renaud, 2001).
One property of Sca is to modulate adhesive parameters of the epithelial cells surrounding the precursor. Discrete changes in the distribution of DE-cadherin, Discs large, and other junctional proteins are seen in the epidermis of sca mutants, that are associated with mislocalization of the adherens and septate junctions and some disruption of epithelial organization. The monolayered nature of the epithelium is retained, consistent with the fact that the morphogenetic changes of metamorphosis are not impaired in sca mutants. This phenotype is only observed in late third instar discs and early pupae, when bristle precursors are forming. If ac-sc activity is removed, the late third instar disc epithelium is wild type. In ac-sc mutants, the absence of Ac and Sc entails a loss of Sca, whose expression is dependent on Ac-Sc (Renaud, 2001).
The epithelial defects resulting from a lack of Sca protein thus coincide with ac-sc expression and the process of lateral inhibition. Scabrous may therefore be required to maintain normal epithelial integrity by counteracting the effects of a protein(s) activated during precursor segregation. It is therefore likely to act through association with a protein(s) whose expression is regulated by Ac and Sc. Notch is ubiquitously expressed in the epithelium, but is activated and probably up-regulated in future epidermal cells surrounding the precursors. Powell (2001) demonstrated that Sca binds N and as a result N is stabilized at the cell surface in S2 cells. Interestingly, in Nts1 mutants at 29°C, where the activity of N is strongly reduced, the distribution of DE-cadherin in the disc epithelium is also discretely altered and the epithelium appears similar, but not identical to that of sca. This suggests that the epithelial defects seen in sca mutants may be the result of a failure to stabilize N protein in epithelial cells. These results are consistent with the idea that Sca acts through N, and with earlier observations linking N to epithelial cell adhesion (Renaud, 2001).
scabrous is not required for inhibition of cells that are adjacent to the precursor. Furthermore, the requirement for sca in the fly is quite restricted: it is not expressed in many other tissues where Notch signaling takes place in its absence. While it is expressed in neural precursors in the embryo, loss of the protein there seems to be without consequence, perhaps because the interactions involve adjacent cells. This leads to the hypothesisis that the effects of Sca on cell adhesion and the stabilization of N may be specifically required when the levels of Dl are limiting (Renaud, 2001).
Delta is expressed in proneural domains and can still be detected in presumptive epidermal cells after precursor segregation. Nevertheless the amount of Dl remaining in presumptive epidermal cells appears to be insufficient, by itself, to repress the neural fate. In the absence of Sca, or in flies carrying hypomorphic alleles of Dl, the space between bristles is decreased. Furthermore, in epidermal cells, the transcription of Dl progressively declines due to repression of its regulators ac and sc following N activation, whereas in the bristle precursors the levels of Dl increase as the levels of Ac and Sc rise. This leaves two possibilities. One is that all of the signal originates in the precursor cell, in which case Dl must be transported, by some as yet unknown means, from the precursor to cells not in direct contact with the latter. The other, is that the concentration of Dl molecules remaining on the presumptive epidermal cells is insufficient to inhibit by itself, but can do so if helped by Sca. Dl from both groups of cells may participate in the wild type. In either case, stabilization of N may therefore be a means to increase the chances of receptor activation in the presence of limiting amounts of Dl (Renaud, 2001).
Changes in cell adhesion could also be the cause of the abnormal bristle organs seen in sca mutants, where two or more cells of the bristle organ lineage adopt the same fate at the expense of the others. In the wild type, spatial arrangements of the cells of the bristle organs are stereotyped as a result of the nonrandom orientation of the mitotic spindles at each division. In sca mutants, the cells are often randomly arranged and in some cases appear to drift apart from one another. This would be likely to prevent the precise cell-cell interactions, mediated by the N signaling pathway, necessary for the assignment of the correct cell fates (Renaud, 2001).
The precursor cells have a quite distinctive shape, reminiscent of neurons with a number of filopodial-like extensions that fan out in a planar orientation. It is not known whether, during bristle precursor segregation, the epidermal cells of the notum extend similar filopodia. Oriented epidermal outgrowths have been described in the epidermis of other insects and also in the wing pouch and peripodial membrane of Drosophila imaginal discs, where it has been suggested that they may function during signaling. Some of these structures project basally and others extend long, straight and polarized structures. Their morphology differs from that of the extensions observed (Renaud, 2001).
It is not known whether Dl from the precursor cell is able to reach cells that are not adjacent to the Dl-expressing precursor, but one means by which this could occur is via cytoplasmic extensions. This has been suggested for Lag-2 signaling in the nematode germ line. Indeed, the presence of Dl molecules can be detected on the filopodia. Although formation of filopodia will depend on properties of the neural precursor itself, subtle changes in junctional contacts and adhesion between surrounding epithelial cells may help to orient or stabilize these structures. The changes in the bristle density of both wild-type and sca flies that are seen after expression of a dominant negative form of DE-cadherin, suggest a role for adhesion molecules in bristle spacing. Preliminary observations indicate that Sca is not required for the extension of filopods. This is consistent with the nonautonomy of sca mutant cells. If filopodia are the means whereby nonadjacent cells are inhibited, and if Sca were to be required for extension of filopodia from the Sca-producing bristle precursors, then sca would be expected to behave autonomously (Renaud, 2001).
Further studies are necessary to determine the molecular basis of Sca function, but one possibility is that binding of Sca to N leads to discrete modifications in epithelial structure that allow Dl molecules on the cytoplasmic extensions to form stable ligand-receptor complexes. The colocalisation of Sca and Dl in cytoplasmic vesicles may indicate cellular trafficking of protein complexes that include Dl, N, and Sca (Renaud, 2001).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D. Please e-mail comments/corrections to brodyt@codon.nih.gov
Delta:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions
| Effects of Mutation
| References
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