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).
Cell proliferation in animals must be precisely controlled, but the signaling
mechanisms that regulate the cell cycle are not well characterized. A regulated
terminal mitosis, called the second mitotic wave (SMW), occurs during Drosophila
eye development, providing a model for the genetic analysis of proliferation
control. This study reports a cell cycle checkpoint at the G1-S transition that initiates
the SMW; Notch signaling is required for cells to
overcome this checkpoint. Notch triggers the onset of proliferation by multiple
pathways, including the activation of dE2F1, a member of the E2F transcription
factor family. Delta to Notch signaling derepresses the inhibition of dE2F1 by
RBF, and Delta expression depends on the secreted proteins Hedgehog and Dpp.
Notch is also required for the expression of Cyclin A in the SMW (Baonza, 2005).
This work identifies a new cell cycle checkpoint in the second mitotic wave
and describes how intercellular signaling overcomes this checkpoint. Delta
signaling to Notch triggers a progression from G1 arrest in the morphogenetic
furrow into the S phase of the terminal mitosis. Two
effectors of this Notch requirement have been identified, dE2F1 transcriptional activity and
cyclin A expression. Although the data imply that at least one other
target also exists, this is unidentified. The data preclude this additional factor from being
Cyclin E. Previous work has identified a later SMW checkpoint, at the G2-M
transition. Together, Notch and
the EGFR therefore coordinately provide spatial and temporal control of the cell
cycle in the SMW (Baonza, 2005).
These results led to a
proposal of the following course of events. Notch is activated by the uniform band
of Delta in all cells as they emerge from the morphogenetic furrow. Cells that
are uncommitted thereby enter S phase, whereas cells that are part of the
precluster are blocked from responding and remain in G1. It has been
shown that the G1 arrest of precluster
cells is dependent on EGFR activation, although the details of the mechanism
remain unclear. One of the consequences of EGFR activation in precluster cells
is the upregulation of Delta expression. Cells between the preclusters would
therefore end up initially receiving low-level uniform Delta, later reinforced
by the upregulated Delta in the adjacent preclusters. Together, these provide a
robust and modulated activation of Notch in cells that will enter the SMW (Baonza, 2005).
It is
emphasized that this work uncovers a normal developmental function for Notch
signaling only in the control of a specific terminal mitotic cycle. The fact
that clones of Notch and Delta mutant cells can be generated
implies that they are not required for the earlier, unpatterned proliferation
ahead of the morphogenetic furrow. Similarly, the ability to make clones in
other imaginal discs indicates that there is no requirement for Delta/Notch
signaling in most cell proliferation in Drosophila. Rather, this signal
requirement, and the subsequent EGFR-dependent entry into mitosis, is
superimposed upon normal controls in this regulated terminal mitosis. Moreover,
the ability of Notch signaling to initiate S phase is restricted to a short
period. Notch has other functions later in eye development, and it has been
shown that later ectopic signaling does not lead to additional proliferation.
Nevertheless, Delta-expressing clones in other tissues also hyperproliferate,
suggesting that ectopic Notch activity has a wider ability to trigger
inappropriate proliferation (Baonza, 2005).
The EGFR and Notch signal systems play distinct
roles in regulating the SMW. After completing its preliminary role in
maintaining cells in G1 arrest, EGFR signaling ensures that cells only undergo
mitosis if they are adjacent to developing clusters, thereby matching the number
of cells born with the number that will be required to complete ommatidial
differentiation. In
contrast, Notch initiates the whole process by regulating whether cells emerging
from the morphogenetic furrow enter the SMW or remain arrested in G1 and start
to differentiate (Baonza, 2005).
The secreted protein Hedgehog has a primary
role in the forward movement of the morphogenetic furrow.
Hedgehog also has an important function in
initiating and coordinating the onset of the SMW, specifically the initiation of
S phase. Hh and Dpp together lead to the expression of Delta
in the furrow. Furthermore, Hh is essential
for the expression of cyclin D and cyclin E in the morphogenetic
furrow, whereas Cyclin E
is the main cyclin that regulates S phase onset. These data imply that Hedgehog
signaling activates several independent branches of the pathway that lead to
the onset of S phase in the SMW. Incidentally, the observation that Cyclin E
accumulates in Notch mutant clones, which lack dE2F1 activity, indicates
that, at least in this context, Cyclin E is not sufficient to inhibit RBF and
thereby activate dE2F1 activity (Baonza, 2005).
Cyclin A is best characterized as
a mitotic cyclin, and its destruction is a key step in the completion of
mitosis. An additional function in the onset of S phase in Drosophila
remains enigmatic. Mammalian Cyclin A and its associated kinase Cdk2 can drive
G1 cell extracts into S phase, and anti-Cyclin A antibodies can block S phase in injected cells.
But, in Drosophila, S phase can proceed normally in the absence of Cyclin A and Cyclin A
does not bind Cdk2. Nevertheless, when overexpressed, Cyclin A can overcome the lack of
Cyclin E and allow cells to enter S phase. Furthermore, overexpression of the
Cyclin A inhibitor Roughex blocks entry into S phase in embryos, and
roughex mutants show precocious S phase entry in the SMW.
Ectopic BrdU incorporation is observed in the eye disc when Cyclin A is misexpressed.
The data indicating that Cyclin A is one of the targets of Notch
signaling further support the idea that Cyclin A is part of the machinery that
controls the onset of S phase in the SMW (Baonza, 2005).
Notch signaling in mammals, as in
flies, is pleiotropic and context dependent. This is highlighted in human
cancer, where Notch is oncogenic in a number of cases, particularly in
hematopoietic neoplasms, but in other contexts has tumor suppressor functions.
Moreover, although the current work highlights a proliferative function, it has been shown that
Notch inhibits proliferation in the wing disc. Notwithstanding this caveat, it
is striking that Notch activity can be hyperproliferative in humans and in
Drosophila, and little is known about this proliferative response. It has
recently been shown in the developing Drosophila central nervous system
that Notch activity can maintain cells in a proliferative state by antagonizing
the p21/p27 homolog Dacapo, thereby maintaining Cyclin E expression. Similarly,
the dacapo gene is downregulated in response to Notch in the
mitotic-to-endocycle transition in Drosophila follicle cells. This work
describes a different mechanism: Notch signaling overcomes a G1-S checkpoint via
the activation of universally conserved cell cycle components, RBF1, dE2F1, and
possibly Cyclin A. Although tempting to speculate that these data may provide
some insight into oncogenic mechanisms, it will be important to ascertain
whether the particular relationships between Notch and the core machinery that
triggers S phase is indeed conserved. In fact, the data imply that Notch
probably also influences the mitotic cycle at other points. If the only role of
Notch were to advance cells into S phase, they would simply arrest at the next
checkpoint, G2-M. The fact that Notch activity leads to overgrowth therefore
implies that Notch can also, directly or indirectly, drive cells through the
subsequent G2-M checkpoint (Baonza, 2005).
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).
The Drosophila protein kinase Par-1 is expressed throughout Drosophila development, but its function has not been extensively characterized because of oocyte lethality of null mutants. This report characterizes the function of Par-1 in embryonic and post-embryonic epithelia. Par-1 protein is dynamically localized during embryonic cell polarization, transiently restricted to the lateral membrane domain, followed by apicolateral localization. Maternal and zygotic par-1 was depleated by RNAi and a requirement was revealed for Par-1 in establishing cell polarity. Par-1 restricts the coalescing adherens junction to an apicolateral position and prevents its widespread formation along the lateral domain. Par-1 also promotes the localization of lateral membrane proteins such as Delta. These activities are important for the further development of cell polarity during gastrulation. By contrast, Par-1 is not essential to maintain epithelial polarity once it has been established. However, it still has a maintenance role since overexpression causes severe polarity disruption. Additionally, a novel role is found for Par-1 in Notch signal transduction during embryonic neurogenesis and retina determination. Epistasis analysis indicates that Par-1 functions upstream of Notch and is critical for proper localization of the Notch ligand Delta (Bayraktar, 2006).
It might not be that Par-1 simply defines the limits of the AJ. Rather, it might also act positively to specify the basolateral domain. Par-1 is required for localization of Delta to the basolateral region. This positive effect of Par-1 on Delta is not due to preventing the AJ from inhibiting Delta. Delta co-localizes with the AJ both normally and when Par-1 is depleted. So how does Par-1 guide membrane regionalization during cellularization? Par-1 protein is distributed along the lateral membrane immediately basal to the incipient AJ. Based on this, three models suggest themselves. Par-1 could assemble a diffusion barrier that physically blocks movement of SAJs into the lateral region and limits movement of Delta into the apicolateral region. If par-1(RNAi) disrupts such a barrier, then other mechanisms must maintain apical Crb restriction from the lateral region. An alternative is that Par-1 has a role in the polarized targeting of transport vesicles carrying SAJ and Delta proteins. In this model, Par-1 might interact with the 'exocyst', a secretory targeting apparatus involved in polarized segregation of transmembrane proteins. Data from yeast Par-1 indicate that it directly associates with a t-SNARE, a membrane-bound component of the exocyst. Moreover, Par-1 phosphorylation of the t-SNARE protein triggers its release from the cell membrane. If Drosophila Par-1 also interacts with the exocyst, then it might selectively block the fusion of SAJ exocytic vesicles to the basolateral membrane. In support of this model, punctate intracellular staining of Par-1 can be seen during cellularization and is reminiscent of vesicles. Par-1 might also stimulate targeting of other cargo, such as Delta-loaded vesicles, to the basolateral membrane. Consistent with this notion, Par-1-depleted ectoderm cells accumulate Delta-positive cytoplasmic vesicles. Finally, Par-1 could differentially affect the stability of proteins in the lateral domain, by de-stabilizing some and stabilizing others. This could occur through degradation or rapid recycling via endocytosis (Bayraktar, 2006).
How directly would Par-1 participate in these mechanisms? This is unclear at present. None of the known substrates for Drosophila Par-1 kinase include Delta or AJ components. In ovarian follicle cells, Par-1 phosphorylation of Baz prevents Baz association with Par-6-aPKC. Baz and Par-6 are among the earliest acting proteins in polarization of blastoderm. During cellularization, Baz associates with the apicolateral membrane, whereas Par-6 is localized to the apical cortex. If either Baz or Par-6 is mutated, the apical AJ proteins do not coalesce but disperse along the lateral membrane. Thus, Baz could be a candidate for mediating the effects of Par-1 in the blastoderm. However, several observations indicate that Par-1 phosphorylation of Baz is not necessarily essential to establish AJ localization. First, Par-1 does not have a polarized distribution during early cellularization and is detected in the apicolateral regions where Baz and Par-6 are already localized. A similar co-distribution is seen by the time the ectoderm has reached mid-gastrula stage. Second, although Baz is required for apical localization of Crb and Patj, Par-1 has no significant effect on their apical localization. Third, establishment of the AJ in follicle cells is not dependent upon Par-1 phosphorylation of Baz (Bayraktar, 2006).
Par-1 plays a curious role in maintenance of polarity of imaginal disc epithelia that derive directly from ectoderm. Par-1 is localized to the apical and marginal zones of imaginal disc cells but is not essential for their polarity. Possibly, redundant mechanisms operate in the absence of Par-1. This idea is supported by overexpression experiments. When Par-1 is overexpressed, the AJ and apical domain are disorganized, and cells are compromised for differentiation, growth and death. This result argues that Par-1 normally plays a role in maintaining cell polarity that is sensitive to its activity level. By contrast, Par-1 is essential to maintain polarity in follicle cell epithelia surrounding adult egg chambers, suggesting that redundancy is restricted to imaginal discs (Bayraktar, 2006).
Par-1 also regulates Notch signaling and it acts upstream of Notch as determined by epistasis analysis. Two different Notch signaling decisions regulated by Par-1 are detected. The first was in the embryonic ectoderm where Par-1 depletion disables Notch-mediated lateral inhibition. The second is in the eye imaginal disc where Par-1 overexpression disables Notch-mediated eye cell determination. Since Notch is disabled when Par-1 is missing or overactive, it suggests that Par-1 is not playing an instructive role in Notch signaling. Rather, it is probably a permissive effect that is related to cell polarity regulation. Indeed, localization of Delta is dramatically reduced along the basolateral domains of blastoderm and ectoderm cells of par-1(RNAi) embryos. It is reasonable to think that Par-1 acts in Notch signaling by localizing Delta to a region of the membrane where it can make a productive interaction with Notch. This permissive model of Notch signaling is nevertheless specific; other regulators of ectoderm polarity do not affect Notch signaling. Moreover, other signaling pathways active in the ectoderm are unaffected by Par-1. Interestingly, a synergistic interaction between Par-1 and Notch was found in the eye imaginal disc. Disc growth significantly increased when Par-1 was overexpressed with ligand-independent Notch. The extra eye tissue developed photoreceptors, indicating the ectopic cells are properly specified. Since loss of cell polarity is associated with hyperplasia in the eye disc, this supports the notion that Par-1 exerts this effect through perturbation of eye disc cell polarity. The synergistic interaction with Notch may be useful in the future for screening of genes involved in tumor formation or progression to a cancerous state (Bayraktar, 2006).
In vertebrates and invertebrates, spatially defined proneural gene expression is an early and essential event in neuronal patterning. In this study, the mechanisms involved in establishing proneural gene expression were investigated in the primordia of a group of small mechanosensory bristles (microchaetae), which on the legs of the Drosophila adult are arranged in a series of longitudinal rows along the leg circumference. In prepupal legs, the proneural gene achaete (ac) is expressed in longitudinal stripes, which comprise the leg microchaete primordia. Periodic ac expression is partially established by the prepattern gene, hairy, which represses ac expression in four of eight interstripe domains. This study identifies Delta (Dl), which encodes a Notch (N) ligand, as a second leg prepattern gene. Hairy and Dl function concertedly and nonredundantly to define periodic ac expression. The regulation of periodic hairy expression was explored. In prior studies, it was found that expression of two hairy stripes along the D/V axis is induced in response to the Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) morphogens. This study shows that expression of two other hairy stripes along the orthogonal A/P axis is established through a distinct mechanism which involves uniform activation combined with repressive influences from Dpp and Wg. These findings allow formulation of a general model for generation of periodic pattern in the adult leg. This process involves broad and late activation of ac expression combined with refinement in response to a prepattern of repression, established by Hairy and Dl, which unfolds progressively during larval and early prepupal stages (Joshi, 2006).
Patterning of the leg imaginal disc along its circumference axis is controlled by the Hh, Dpp and Wg morphogens. This study sought to elucidate the molecular mechanisms through which these signals give rise to specific morphological features of the leg, the mechanosensory microchaetae. Patterning of leg mechanosensory microchaetae was shown to requires spatially defined expression of the proneural gene ac and its repressor Hairy. Expression of hairy in two pairs of longitudinal stripes, the D/V-hairy and A/P-hairy stripes, is directed by separate enhancers that are Hh-, Dpp- and Wg-responsive. The D/V-hairy and A/P-hairy stripes are differentially regulated by Dpp and Wg and distinct mechanisms are utilized to control hairy expression along the A/P and D/V axes. D/V-hairy expression is locally induced near the A/P compartment boundary by Hh signaling. In addition, Dpp and Wg positively influence expression of the dorsal and ventral components of the D/V-hairy stripes, respectively, by acting together with Hh to define the register of these stripes relative to the compartment boundary. In contrast, the A/P-hairy stripes, which are expressed orthogonal to the D/V-hairy stripes and A/P compartment boundary, are not activated via local induction. Rather, it appears that they are broadly activated along the leg circumference and repressed by Dpp dorsally and Wg ventrally to define their dorsal and ventral boundaries. This model for A/P-hairy regulation is supported by the observations that hairy is ectopically expressed in dorsal, but not ventral, clones lacking tkv or Mad function and that A/P-hairy expression is compromised by elevation of Dpp signaling. Furthermore, ventral, but not dorsal, clones lacking dsh function also ectopically express hairy and high-level Wg signaling results in loss of A/P-hairy expression (Joshi, 2006).
A potential caveat to this model for regulation of A/P-hairy expression is that conclusions were drawn from analysis of endogenous hairy expression rather than by examining expression directed by isolated A/P-hairy enhancer(s). Hence, it is possible that the ectopic hairy expression seen in tkv, Mad and dsh mutant clones is a result of expansion of D/V-hairy rather than A/P-hairy expression. However, several lines of evidence argue against this interpretation. First, through genetic and molecular analyses of D/V-hairy enhancer function, it has been demonstrated that Dpp and Wg positively regulate D/V-hairy expression, an observation that is inconsistent with the suggestion that D/V-hairy is ectopically expressed in clones unable to respond to Dpp or Wg signaling. Furthermore, in 3rd instar and early prepupal leg discs, stages at which the A/P-hairy stripes are not expressed, ectopic hairy expression is not observed in tkv mutant clones. Second, it was found that the D/V-hairy stripes can only be expressed in anterior compartment cells near the A/P boundary, which are the cells that receive and respond to Hh signal. Thus, it is unlikely that ectopic hairy expression observed in clones at distance from the compartment boundary, which receive little or no Hh signal, and in the posterior compartment, in which cells do not respond to Hh signal, corresponds to D/V-hairy expression. Finally, it was found that elevation of Dpp or Wg signaling specifically disrupts A/P-hairy but not D/V-hairy expression. Taken together, these findings are consistent with the conclusion that A/P-hairy rather than D/V-hairy is expressed in clones compromised in their response to Dpp and Wg signaling (Joshi, 2006).
This study identified Dl as a second prepattern gene that functions together with hairy to establish ac expression in the leg microchaete proneural fields. Several lines of evidence are are presented that support this conclusion. First, it was found that, beginning at 4 h APF, Dl expression is up-regulated in domains overlapping the microchaete proneural fields. This distribution of Dl is similar to that, in the notum, where Dl has been shown to regulate proneural ac expression. Second, it is shown that ac expression is expanded in legs with reduced Dl function. Third, it was found that elevated N signaling throughout the tarsus results in severely reduced ac expression. Finally, activation of N signaling was observed within the hairy-OFF interstripes (ac interstripes that do not express hairy), in agreement with the genetic requirement for Dl/N signaling in these domains. Based on these results, it is proposed that ac expression is activated broadly during mid-prepupal leg development but is confined to the microchaete proneural fields by a previously generated prepattern of repression, established by Hairy and Dl/N signaling. This hypothesis is supported by analysis of cis-regulatory elements that direct ac expression in the leg microchaete proneural fields (Joshi and Orenic, unpublished cited in Joshi, 2006). By generating rescue and reporter constructs, an enhancer has been identified that specifically controls expression of ac in the microchaete proneural fields. Unlike the hairy leg enhancers, no modular organization of the cis-regulatory elements that control expression of ac stripes in different regions of the leg is observed. Rather, preliminary analyses suggest that there is one enhancer consisting of an activation element that directs broad expression of ac along the leg circumference and two repression elements, which are N- or Hairy-responsive. This finding is consistent with genetic studies and the model for regulation of ac expression in the leg microchaete proneural fields (Joshi, 2006).
hairy and Dl function to repress ac expression in complementary domains. hairy encodes a transcriptional repressor which has been previously shown to directly repress ac expression in the wing by binding a specific site in the ac promoter. It is likely that Hairy acts through a similar site to repress ac expression in the leg. Dl represses ac expression via a different mechanism: presumably, cells of the microchaete proneural fields, which express high levels of Dl, signal to adjacent cells to activate N. This suggestion is supported by the observation that expression of two N-responsive reporters is specifically activated in cells corresponding to the hairy-OFF interstripes. One of the reporters used in this study, E(spl)mβ-CD2, and other similar reporters recapitulate endogenous E(spl)mβ-CD2 expression in wing and leg imaginal discs. E(spl)mβ is one of seven genes in the E(spl)-C that encode bHLH repressors related to Hairy. Hence, it appears that ac expression in the leg microchaete proneural fields may be established by a prepattern of periodically expressed bHLH repressors (Joshi, 2006).
N signaling is not activated within ac-expressing cells, even though these cells express high levels of Dl. This could be explained by a dominant-negative effect of Notch ligands on N signaling, which has been previously observed in the wing. In the wing, it has been shown that N signaling is not activated within cells expressing high levels of Dl and Ser but, rather, that these cells signal to adjacent cells to activate N signaling within the wing margin. Consistent with the hypothesis of a potential dominant-negative function for Dl in the leg microchaete proneural fields is the observation that over-expression of Dl along the leg circumference results in expansion of ac expression into the hairy-OFF interstripes, which would be expected if N signaling was disabled. Over-expression of N ligand expression has been shown to exert a similar effect in other tissues (Joshi, 2006).
A curious observation of this study is that, as suggested by genetic evidence and the expression of two N-responsive reporters, N signaling, with one exception, is not activated within the hairy-ON interstripes, even though each Hairy stripe is straddled on either side by a Dl stripe. This suggests either that Dl signals asymmetrically or that there is an asymmetric response to N signaling and raises questions regarding the underlying mechanism of asymmetric activation of N-target gene expression. A potential mechanism for asymmetric signaling by Dl is suggested by studies in the notum, in which it has been shown that the N receptor is distributed in a pattern complementary to Dl. If N levels were higher within the hairy-OFF vs. the hairy-ON interstripes in the leg, this could allow for preferential signaling within these domains. However, N expression was assayed in prepupal legs and it was found that N appears to be uniformly distributed along the leg circumference. Hence, either there is an asymmetric response to N or alternative mechanisms are responsible for establishing the directionality of Dl signaling in the leg, such as post-translational modification N signaling pathway components. For example, glycosylation of N by the Fringe glycosyltransferase influences its interactions with its ligands (Joshi, 2006).
Another intriguing finding is the overlap of N signaling with the V-Hairy stripe. This result was surprising because it would suggest redundancy between hairy and Dl/N signaling in this region. However, an absolute requirement was observed for hairy function in the ventral leg. An explanation for this puzzling finding is suggested by the specific loss of the V-Gbe+Su(H)m8-lacZ stripe in hairy mutant legs, which indicates that Dl/N signaling or responsiveness in the ventral leg is dependent on hairy function. The specific loss of N signaling in the ventral leg could be a result of the expansion of Dl expression in hairy mutant legs, which as explained earlier might have a dominant-negative effect on N signaling. This proposal is corroborated by the expansion of ac expression along the circumference of legs ectopically expressing Dl throughout the tarsus. The overlap of hairy and Dl/N signaling in the ventral leg raises questions regarding the function of Dl/N signaling in this domain. It was observed that V-hairy and Gbe+Su(H)m8-lacZ expression overlap only partially, suggesting that combined function of Dl and Hairy in the ventral leg could serve to establish a broader domain of repression in this region in comparison to other interstripe domains. This idea is supported by the morphology of the adult leg tarsus in which the spacing of bristles is most pronounced along the ventral midline. However, the function of N in the ventral leg is not as yet clear. It is plausible that there is a role for Dl/N signaling in the ventral leg that is unrelated to regulation of ac expression (Joshi, 2006).
The potential function of Dl as a regulator of proneural ac expression in the leg was suggested by studies in the notum, on which mechanosensory microchaetae are also organized in longitudinal rows. In the notum, Dl/Notch signaling, rather than Hairy, regulates periodic ac expression. The current studies suggest a distinct mechanism for leg microchaete patterning in which Hairy and Dl act together and nonredundantly to define periodic ac expression. In both the leg and notum, Dl signals to adjacent cells to repress ac expression. However, whereas in the notum Dl activates N signaling in cells on either side of each Dl/Ac stripe, in the leg, N signaling is activated (with one exception) only within the hairy-OFF interstripes. Although the pattern of mechanosensory bristles on the leg and notum is overtly similar, the bristle rows are more precisely aligned in the leg. The more organized pattern on the leg may be a consequence of the combined function of Hairy and Dl which might more precisely define the domains of proneural gene expression (Joshi, 2006).
Dl function is essential for proper patterning of ac expression and it is suggested that accurate positioning of the Dl stripes is necessary for activation of Notch signaling within appropriate domains. Hence, regulation of Dl expression is an important aspect of leg microchaete patterning. In legs lacking hairy function, Dl expression expands into four broad domains and ectopic hairy expression greatly reduces Dl expression, indicating that periodic expression of Dl is regulated in part by hairy. Concomitant with the expansion of Dl expression, there is loss of N signaling in the ventral leg, suggesting that hairy functions to create an apposition of cells expressing high levels of Dl to cells expressing low levels of Dl, which allows for activation of N signaling in the ventral leg. Regulation of Dl expression in proneural fields is not understood. A plausible hypothesis is that, like hairy, Dl expression is established in response to the morphogens that control pattern formation during leg development (Joshi, 2006).
This and previous studies suggest an outline of general genetic pathway for the regulation of ac expression in the leg microchaete proneural fields. This process involves broad and late activation, by an unknown factor, of ac expression along the leg circumference combined with refinement in response to a prepattern of repressors, which is established during larval and early prepupal stages. Hairy and Dl have been identified as the primary prepattern factors that regulate ac expression along the leg circumference. Position-specific expression of both hairy and Dl in longitudinal stripes is essential for proper ac expression. The longitudinal stripes of hairy are established in direct response to the Hh, Dpp and Wg signals, which globally pattern the leg, indicating that hairy acts as an interface between ac and these morphogens. Dl expression is regulated by Hairy, but its regulation is otherwise poorly understood. In addition to elucidating a pathway for establishment of periodic ac expression during leg development, these studies also provide insight into the mechanisms through which morphogens function to generate leg morphology (Joshi, 2006).
Periodic ac expression is established progressively. The first evidence of periodicity is expression of the longitudinal stripes of hairy expression. The D/V-hairy stripes are expressed first in the early 3rd instar leg disc followed by the A/P-stripes between 3 and 4 h APF. Between 4 and 6 h APF, Dl expression within the mechanosensory microchaete primordia is established. Then, ac expression is activated uniformly along the leg circumference. By the time that ac expression is activated, the interstripe domains have been defined by the four Hairy stripes and Dl/N signaling (Joshi, 2006).
The delay of ac expression in the microchaete proneural fields until mid-prepupal stages is likely due to the requirement of ac function for formation of all leg sensory organs. Leg sensory bristles can be grouped into two broad categories based on their time of specification: one group includes the early-specified mechanosensory macrochaetae (large bristles) and chemosensory microchaetae, and the second group includes the more numerous late-specified mechanosensory microchaetae. During the 3rd instar and early prepupal stages, ac is expressed in small clusters of cells that define the primordia of early-specified bristles, while expression of ac in the mechanosensory microchaete primordia is activated later in the mid-prepupal stage. This late expression of ac is activated broadly along the leg circumference and is presumably delayed to allow for expression of the hairy and Dl stripes during earlier stages. Premature expression of this normally late ac expression would likely lead to disturbances in sensory organ patterning, suggesting that temporal control of ac expression is an important aspect of its regulation (Joshi, 2006).
The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1.
In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).
Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).
In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).
The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).
Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).
Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).
Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).
These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).
What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6-7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).
An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Except for Dl upregulation in R3, the mechanism of how Fz/PCP signaling regulates Notch in this context is not understood. The E3-ubiquitin ligase Neuralized (Neur), required for Dl-N signaling, is asymmetrically expressed within the R3/R4 pair. It is required in R3, where it is also upregulated in a Fz/PCP-dependent manner. As is the case for Dl, N activity in R4 further represses neur expression, thus, reinforcing the asymmetry. Neur asymmetry is show to be instructive in correct R3/R4 specification. These data indicate that Fz/PCP-dependent Neur expression in R3 ensures the proper directionality of Dl-N signaling during R3/R4 specification (del Alamo, 2006).
PCP establishment in the eye depends on the specification of photoreceptors R3 and R4 in two steps. First, Fz signaling occurs at higher levels in R3, and second, as a consequence, Dl signaling is directed from R3 to the R4 precursor, where N specifies R4 fate. This study shows that neur is required for proper Dl-N signaling directionality in the R3/R4 pair. In the absence of neur, defects occur in R3/R4 cell-fate specification and PCP. Importantly, neur expression is upregulated in R3 in a Fz/PCP-dependent manner. Finally, this study shows that the asymmetry in neur expression is required for PCP specification (del Alamo, 2006).
Neur is an E3-ubiquitin ligase known to enhance Dl signaling in a variety of Dl-N mediated processes, including lateral inhibition or lateral specification events (e.g., pIIb to pIIa specification in sensory organ development). This study shows that neur is required for lateral specification in R3 for Dl to signal to R4. Analysis of R3/R4 Dl mosaics revealed that the Dl mutant cell always acquires R4 fate, while the wt cell acquires R3 fate. This is consistent with neur analysis showing that in 94.2% of the cases, ommatidia mutant only in R3 showed a PCP defect, indicating that neur is required only in R3, the signal-sending cell (del Alamo, 2006).
There is, nevertheless, a difference between the PCP phenotypes of Dl and neur mosaic ommatidia: Dl mosaics show reversed polarity (chirality flips) when R3 is mutant, while the equivalent neurIF65 mosaics show mostly a symmetric phenotype (89.5% of ommatidia displaying chirality defects). It is likely that the cold-sensitive neurIF65 allele is not null and it is not clear if remaining Dl activity is present in the absence of Neur, accounting for the difference (del Alamo, 2006).
Mib1, another E3-ubiquitin ligase-regulating signaling by Dl and Serrate (Ser, the other N ligand in flies), has no effect on PCP specification. These results are in agreement with data showing that Neur and Mib1 have complementary functions. Taken together, the data indicate that neur but not mib1 is required for R3/R4 specification (del Alamo, 2006).
Previous studies suggested that neur has a permissive role in Dl-N signaling. In lateral inhibition processes, neur is expressed in proneural clusters, whereas in asymmetric cell division, Neur is selectively inherited by one of the daughter cells. In either case, Neur makes the cell in which it is expressed competent for Dl signaling. In the eye, Dl is enriched in R3 as a result of Fz signaling, and this study provides evidence that Neur is enriched in R3 and that this enrichment is also regulated by Fz/PCP signaling. While neur is initially expressed in both cells, the data indicate that Fz/PCP-dependent R3 upregulation of neur is necessary and sufficient for Dl signaling directionality. Since Neur affects Dl activity posttranslationally, Dl is still upregulated in R3 when Neur is misexpressed. This implies that the elimination of the difference in Neur levels between the R3/R4 precursors affects the direction of Dl-N signaling. These data indicate that the Neur expression asymmetry, mediated by Fz/PCP signaling, is instructive for R3/R4 specification (del Alamo, 2006).
The phenotypes resulting from Neur misexpression are relatively mild. Only when both Dl and Neur are coexpressed, chirality defects are induced, suggesting that differential expression of both factors in the R3/R4 cell pair is instructive for cell fate. Furthermore, other factors could also be present in R3/R4 precursors to ensure robustness of the cell-fate decision. These observations suggest a complex network of molecular interactions between Fz/PCP and Notch signaling (del Alamo, 2006).
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