Notch
To date, very little information is available about the regulation of Notch transcription. Suppressor of Hairless exhibits allele specific interaction with Notch, indicating that the Notch pathway may regulate Notch transcription by means of Su(H) (Fortini, 1994).
Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor
organs. ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are
required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated
in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function,
regulating arborization during larval and pupal development by interacting with Notch (Hassan, 2000).
Since Ato is expressed in adult brains, ato-Gal4/UAS-lacZ adult brains were stained with anti-beta-galactosidase (anti-beta-gal) and the data were analyzed with confocal microscopy. In adult flies, ato-Gal4 is expressed in a DC and in two ventral clusters (VCs: VLC and VBC) of neurons. Some axons of the DC project ipsilaterally over the lobula. However, most axons of the DC form a bundle that is a component of the dorsal commissure and project contralaterally toward the lobula complex and the medulla. These neurites fan out over the lobula complex and the inner chiasm. Ten to twelve tracts cross the outer chiasm, toward the medulla, in a ladder-like pattern. Over the medulla, the fibers branch and appear to contact one another to form a 'grid-like' lattice. No fibers cross the lamina (Hassan, 2000).
What might the function(s) of ato be in the central brain? To address this question, the consequences of the loss and gain of ato function in the DC in L3 and adult brains was examined using the ato-Gal4 line as a marker and a driver. Surprisingly, all three clusters (DC, VBC, VLC) are present in brains homozygous for the ato1 allele as well as in brains transheterozygous for ato1 and a deficiency that uncovers the ato region. However, several defects are obvious in ato mutant brains. While heterozygous control L3 brains show a normal DC, mutant L3 brains show severe defects in DC position and organization. In addition, probably as a result of the loose morphology of the cluster, the descending axon bundles are defasiculated. These defects were observed with about 15% penetrance and suggest a weak or partially redundant differentiation requirement for ato in the precursors of the DC lineage. Note, however, that the DC axons form a commissural tract, strongly suggesting that their basic identity as commissural neurons is not affected by the ato mutation (Hassan, 2000).
To investigate if ato is required in the postmitotic DC neurons, as the reinitiation of its expression suggests, the morphology of the axonal projections of the DC neurons of adult ato mutant brains was examined. The DC forms a stereotypical axonal pattern, making it simple to detect aberrations that may be caused by the ato mutation. In adult brains, in addition to the aberrant positioning of the cluster seen in L3, the arborization pattern of the DC over the lobula is severely impaired in ato deficiency flies. Most axons enter the lobula either ventrally or dorsally and show very limited branching, failing to form a proper 'fan-shaped' pattern. Since ato mutant brains have a severely reduced medulla, the medullar part of the pattern could not be examined. While these data suggest a role for ato in axon arborization, alternative interpretations are possible: (1) the observed axonal defects may be caused by the significant loss of optic lobe structures associated with loss of ato function (no lamina or medulla, reduced lobula); (2) it is possible that the defects are a reflection of the improper differentiation of the DC cells rather than a reflection of a specific role for ato in arborization (Hassan, 2000).
To understand the mechanism by which ato functions in axonal development, the role of Notch was examined in the development of the DC axonal pattern. Two observations make Notch a logical candidate. (1) ato and Notch interact in an antagonistic fashion during CHO development. For example, in the leg femoral CHO, gain of Notch function reduces the number of precursors selected from the ato-expressing proneural cluster. (2) Notch has been shown to be required for axon guidance, perhaps mediating axon–substrate interactions, in both the PNS and CNS. If ato and Notch act antagonistically in arborization, it is expected that Notch activity levels would be relatively low within the DC, where its function is repressed by ato, and relatively high in the substrate cells, where ato does not antagonize it. Thus, after ato expression is reinitiated in the DC neurons, a differential in Notch activity levels may occur between the arborizing axon and the substrate cells. Perturbations of this imbalance, either by raising Notch activity levels in the DC or by reducing them in the surrounding cells, may result in defective arborization patterns. This model allows for three specific predictions: (1) the generalized loss of Notch function may result in excessive arborization of the DC neurons, whereas the DC-specific loss of function would cause no significant defects; (2) the gain of Notch in the DC neurons is expected to inhibit arborization; (3) if the activation of ato in the DC neurons serves to antagonize Notch activity, then it is expected that the gain of Notch function will be epistatic to the gain of ato function (Hassan, 2000).
To determine the requirements for Notch in DC axon development, two Notch alleles were examined: a temperature-sensitive allele (Nts) and a viable hypomorphic allele (facet notchoid [Nnd3]). In the first set of experiments, Nts;ato-Gal4,UAS-lacZ larvae were raised in a cycling incubator delivering a 30 min, 34°C heat shock every 8 hr from late L1 through wandering L3. L3 brains were examined for DC defects. Reducing Notch activity during larval development has no effects on the number, morphology, or position of the DC neurons or on the formation of the commissure. In contrast, defects in axon branching out of the commissure into the optic lobe were observed. Specifically, excessive branching and defasiculation of the axon bundles entering the optic lobe were observed. Importantly, these defects were not rescued by a wild-type copy of Notch driven by the ato enhancer in the DC (Nts; UAS-N+;ato-Gal4,UAS-lacZ), suggesting that the requirement for Notch in DC axon arborization is nonautonomous in contrast to the requirement for ato. Larvae reared under the cycling heat shock paradigm or at a consistent 28°C temperature between either L1 and adult or L3 and adult do not produce homozygous Nts flies. Therefore, to examine the adult DC innervation pattern in a background of reduced Notch activity, Nnd3;ato-Gal4,UAS-lacZ male flies were used. These flies show excessive branching in the medulla, resulting in an aberrant innervation pattern similar to that caused by ato overexpression in the DC neurons. The disruption is milder due to the fact that Nnd3 is a weak allele of Notch (Hassan, 2000).
Do the defects observed in Notch mutants reflect an independent function for Notch in arborization? It is possible that the conditions used in this study result in mild neurogenic phenotypes generating extra DC target cells. This, in turn, would cause the DC neurons to arborize excessively to innervate new targets. To rule out this possibility, area density (number of cells per unit area) and optic lobe cortex volume (volume occupied by optic lobe cell bodies) analyses were performed on Nts L3 brains and Nnd3 adult brains. By both criteria, there are no significant differences between wild-type and mutant brains. Therefore, these data support a model in which ato generates the branching pattern by antagonizing Notch activity in the DC (Hassan, 2000).
To show that Notch function is not required within the DC neurons themselves, a dominant-negative form of Notch (UAS-NEC) was expressed using ato-Gal4. NEC has no effect on the formation of the larval or adult axonal patterns. In contrast, NEC expression in imaginal discs results in strong loss of Notch function phenotypes and pupal lethality, demonstrating that the construct is active. This shows that while Notch function is required specifically for arborization of the DC neurons, its requirement is nonautonomous. The prediction that ato represses Notch activity in the DC cells implies that gain of Notch function within the DC would result in inhibition of axonal branching, a phenotype similar to that of loss of ato function. The membrane-bound, wild-type form of Notch (N+) is known to be required for the rescue of axonal defects associated with the loss of Notch function. However, in all cases in which ato and Notch appear to interact in the PNS, it is the nuclear form of Notch that is thought to be involved. To evaluate the effects of the gain of Notch function, both forms of Notch were overexpressed in the DC neurons: the membrane-bound N+ and the nuclear form, Nintra. Overexpression of N+ has no effect on the axonal pattern, whereas overexpression of Nintra results, in comparison with controls, in a severe inhibition of axonal branching over the lobula and a complete failure of innervation of the medulla. These data suggest that the nuclear form of Notch, but not the membrane-bound form, affects the arborization pattern of the DC axons. Finally, if ato suppresses Notch signaling within the DC, gain of Notch function should be epistatic to the gain of ato function, placing Notch genetically downstream of ato. Therefore, the combined overexpression of ato and Nintra should result in the same phenotype as the overexpression of Nintra alone. Brains in which both ato and Nintra are overexpressed using ato-Gal4 have a phenotype identical to that of Nintra overexpression alone. Taken together, the data presented above support the hypothesis that ato acts to suppress Notch signaling within the DC and that this suppression is essential for the generation of the proper arborization pattern of the DC axons (Hassan, 2000).
Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely
unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings
are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation,
the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).
The expressions of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) protein lacking a basic motif, and Notch (N), were examined because both genes have been shown to be involved in the control of cell proliferation in the wing. In third instar larvae, emc is expressed at a low level throughout the wing disc and at a higher level in two stripes of cells corresponding to the prospective A margin and the AP organizer. Unmodified at the A margin, emc expression is completely lost from the AP organizer cells in either col1 or col1/kn1 mutant discs, showing that Col is required for emc transcription in the L3-L4 intervein primordium. Levels of N protein are high in intervein regions and low in presumptive vein territories in late third instar. In col1 mutants, N is downregulated in the L3m provein domain. col requirement for emc and N upregulation in the AP organizer cells is consistent with the reduced cell number in the central region of col1 mutant discs (Crozatier, 2002).
Notch signaling, provided by the ligands Delta and Serrate, is carried to the nucleus by Suppressor of Hairless protein.
The Notch pathway is required for Enhancer of split expression during neurogenesis (Jennings, 1994). Whether or not Suppressor of Hairless, and indirectly, Notch, have other targets has not yet been established. Possible Notch targets are suggested by the results of Notch overexpression in the wing disc. Notch is locally activated at the wing margin, as demonstrated by the restricted expression
of the Enhancer of split proteins in dorsal and ventral cells abutting the D/V boundary throughout
the third larval instar. Notch gain-of-function alleles
in which Notch activity is not restricted to the dorsoventral boundary cause mis-expression of cut
and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation
for wing development (de Celis, 1996a).
Notch targets tramtrack. Experimental evidence suggests that there is an alteration of ttk expression due to reduction or overexpression of Notch. ttk is normally expressed in the sheath cell, one of the products of the sensory organ precursor lineage, but not in the neural cell, the sister of the sheath cell. In Notch mutants, extra neurons have detected resulting from a transformation of sheath cells into neurons. ttk is expressed in most cells in the epidermis of the mutant embryo, but not in neurons, including the supernumerary neurons derived from transformation of sheath cells. Thus Notch function is required not only to specify the sheath cell but also to express ttk in this daughter cell of an asymmetric division. In a reciprocal experiment, overexpression of Notch turns on ttk expression in cells that normally do not express ttk. It is concluded that Notch targets ttk, presumably downstream of Numb (Guo, 1996).
The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm (Cowden, 2002).
Neural precursors (or neuroblasts) divide in a stem
cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair
of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only
once to generate two neurons. A screen was carried out for genes expressed in the MP2 neuroblast and its
progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. A
P-element insertion line was identified that expresses the reporter gene, tau-beta-galactosidase, in the MP2 precursor
and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene,
mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its
sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the
MP2 lineage transforms dMP2 to vMP2. Notch signaling requires Mastermind, suggesting that
Mastermind acts downstream of Notch to determine the vMP2 cell fate. Mastermind
plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies
the pCC and RP2sib fates, respectively. This suggests that Notch signaling through Mastermind plays
a wider role in specifying neuronal identity in the Drosophila central nervous system. Notch is expressed in both MP2 progeny. Notch signaling is blocked by Numb, which segregates exclusively to dMP2 when the MP2 precursor divides. Numb interacts directly with the intracellular domain of Notch. By antagonizing Notch, Numb promotes the dMP2 cell fate. Thus it is likely that Numb antagonism of Notch signaling in dMP2 confines Mastermind function, acting downstream of Notch, to the vMP2 neuron (Schuldt, 1998).
Cell proliferation in the excretory organs of Drosophila, the Malpighian tubules (MT), is under the control of a neural tip cell. This
unique cell is singled out from equivalent MT primordial cells in response to Notch signalling. The gene Kruppel (Kr),
best known for its segmentation function in the early embryo, is under the control of the Notch-dependent signalling process.
Lack-of-function and gain-of-function experiments demonstrate that Kr activity determines the neural fate of tip cells by acting as a
direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. A unique cis-acting element has been identified that mediates all spatial and temporal aspects of Kr gene expression during MT
development. This element contains functional binding sites for the restricted proneural bHLH factors and Fork head protein which
is expressed in all MT cells. These results suggest a mechanism in which these transcription factors cooperate to set up a unique cell
fate within an equivalence group of cells by restricting the activity of the developmental switch gene Kr in response to Notch
signalling (Hoch, 1998).
Notch signaling may regulate the restricted expression of buttonless. buttonless is expressed in dorsal median cells, mesodermal cells that are arranged as a single pair within each segment along the dorsal midline, just above the central nervous system.
Dorsal midline cells in Notch mutant cells differentiate with approximately twice the wild-type number. The phenocritical period for hypertrophy of the DM cells in the temperature sensitive Notch mutant occurs between 4 and 6 hours after fertilization (Chiang, 1994 and Hartenstein, 1992).
lame duck expression was examined in wg and N mutant embryos to determine the relative position of lmd within the genetic hierarchy that controls somatic muscle specification. In wgcx4 mutant embryos, lmd RNA expression is not detectable in dorsolateral and lateral somatic mesodermal cells although there is residual expression in cells located in the ventral region. Thus, activation of lmd expression is mediated via wg-dependent and wg-independent pathways. Significantly, lmd expression in the somatic mesoderm is completely abolished in N5419 mutant embryos, indicating that activation of lmd expression in presumed fusion-competent myoblasts requires active Notch signaling. By contrast, founder cell formation is promoted in the absence of Notch function (Duan, 2001).
What is the function of Sparkling in the differentiation of bristles? All bristles or mechanosensory organs of the adult fly arise in a simple, stereotyped manner by three consecutive asymmetric divisions during which a single SOP gives rise to a neuron and three support cells. The first division generates different siblings, one of which by subsequent division produces the cells that form the socket (tormogen) and shaft (trichogen); the other sibling produces the neuron and glial cell (thecogen). Sparkling appears to be required at various stages during bristle development. Initially, it is observed in SOPs and all four bristle precursor cells (Fu, 1997). However, by mid-pupal stages it is no longer expressed in the tormogen and neuron, but continues to be expressed in the thecogen and is strongly elevated in the trichogen. If thecogen and trichogen, in response to opposite Notch (N) signaling, arise from their precursors by analogous asymmetric divisions, Sparkling expression must also react to N signaling in these cells in opposite ways. For example, while N signaling may activate sparkling through Su(H) in glial cells, it may repress sparkling in socket cells. Comparison of Spa protein levels in the developing eye bristles of svn (svn is an allele of sparkling) mutants with those of wild type suggests that its expression needs to be maintained at least to mid-pupal stages for proper differentiation of trichogens and the formation of a shaft of normal size. However, trichogen cells are able to differentiate to some extent in hypomorphic svn mutants that express Spa during early pupal stages. If the levels of Spa are further reduced, the shafts are also shortened or completely missing. In that case, the shaft is replaced by a second socket, which indicates that the trichogen is no longer able to differentiate properly but becomes a tormogen. It appears therefore that Sparkling protein is not only required during differentiation of a shaft cell, but also serves at an earlier time to specify the fate of shaft versus socket cell. This would be consistent with Sparkling being a target of the N signaling pathway. The roles that Sparkling plays in developing cone and primary pigment cells or in bristle cells, which manifest themselves in the various enhancer mutant alleles of spa and sv, are probably not the only functions of Sparkling as evident from several additional locations where Spa protein can be detected during embryonic and larval development. The expression of Sparkling in the embryonic PNS may indicate that Sparkling plays a function in the development of larval sense organs analogous to that of sv in the development of the mechanosensory bristles of the adult (Fu, 1998).
The adult peripheral nervous system of Drosophila includes
a complex array of mechanosensory organs (bristles) that
cover much of the body surface of the fly. The four cells
(shaft, socket, sheath, and neuron) that compose each of
these organs adopt distinct fates as a result of cell-cell
signaling via the Notch (N) pathway. However, the specific
mechanisms by which these cells execute their conferred
fates are not well understood. shaven, called in this paper D-Pax2,
the Drosophila homolog of the vertebrate Pax2 gene, has an
essential role in the differentiation of the shaft cell. In flies
bearing strong loss-of-function mutations in shaven, shaft structures specifically fail to
develop. Consistent with this, Shaven protein
is expressed in all cells of the bristle lineage during the
mitotic (cell fate specification) phase of bristle
development, but becomes sharply restricted to the shaft
and sheath (glial) cells in the post-mitotic (differentiative) phase.
Three cell types, early on the pIIA secondary precursor and later the tormogen,
and the thecogen, are responsive to the N-mediated signals sent by
their sister cells, but pIIB, the trichogen, and the neuron are made
resistant to the reciprocal signal by N pathway antagonists such as
Numb and Hairless. Interestingly, while the tormogen/socket and thecogen/glial/sheath cells are recipients of Notch signals, it is the trichogen/shaft and thecogen that express Shaven (Kavaler, 1999).
An anti-Shaven antiserum was used to
examine the temporal and spatial pattern of Shaven
accumulation in the pupal notum during microchaete
development. Shaven expression is first apparent in the
SOP nucleus before division (14 hours after puparium
formation (APF). After SOP division (16 hours APF),
Shaven is present at similar levels in the nuclei of the two
daughter cells, the secondary precursors pIIA and pIIB. Following the completion of the pIIA and pIIB divisions
(18 hours APF), all four cells of the microchaete lineage
express Shaven at fairly comparable levels, though one cell
(the presumptive trichogen/shaft) is regularly
distinguishable at this stage by its slightly elevated
accumulation of the protein. Subsequently, the
pattern of Shaven expression is refined so that by
32 hours APF, Shaven protein is present in only two cells, one
containing a large polyploid nucleus (either the trichogen or
the tormogen/socket) and one
containing a small nucleus (either the neuron or thecogen/glia). By double-labeling nota with anti-Shaven antibody and the neuron/shaft marker mAb 22C10, the two cells that express
Shaven at 32 hours APF could be identified. Microchaete
neurons (clearly identifiable by their 22C10-positive cell
bodies and axons) are Shaven-negative, but are positioned
adjacent to small, Shaven-positive nuclei in 22C10-negative
cells, which by inference are thecogens. This
interpretation is confirmed by a second double-labeling
experiment using anti-Elav (a specific nuclear marker for post-mitotic
neurons) and anti-D-Shaven antibodies at 32 hours APF. In both
macrochaetes and microchaetes,
the small nucleus that labels with anti-Shaven is clearly
distinct from the small nucleus that labels with anti-Elav. Thus, the
two bristle cells that exhibit specific nuclear expression of Shaven
at 32 hours APF are the thecogen and trichogen.
These results distinguish two phases
of Shaven expression in the bristle lineage: an early stage, in
which the protein appears at similar levels in the SOP and all
of its progeny as cell division proceeds, and a
late stage, in which Shaven expression is restricted to the
postmitotic trichogen (shaft) and thecogen (sheath) cells (Kavaler, 1999).
Two lines of evidence described here indicate that shaven
expression and function is at least in part downstream of
cell fate specification mechanisms such as N signaling.
(1) The lack of late shaven expression in the
socket cell (the sister of the shaft cell) is controlled by N
pathway activity; (2) loss of shaven
function is epistatic to the socket-to-shaft cell fate
transformation caused by reduced N signaling.
Overexpression of H after the
completion of the microchaete cell divisions results in the
expression of the shaft cell fate by both the normal trichogen
and the transformed tormogen; that is, reduction of N pathway function
promotes the expression of the shaft differentiation program.
Loss of shaven function has the opposite effect: the
stronger shaven mutant genotypes cause a broad failure of
shaft development while permitting the normal or
nearly normal expression of the other bristle cell fates. The epistatic relationship between excess
H activity and loss of shaven function was examined by combining these two
conditions and observing the effect on shaft differentiation. Homozygosity for even the comparatively
weak shaven allele effectively
suppresses the double shaft phenotype of heat-treated Hs-H
transgenic flies. Thus, in the combined genotype
large territories devoid of external bristle structures are
observed. This effect may be explained as follows: excess
H activity alone causes both the normal trichogen and the
transformed tormogen to adopt the shaft fate, but reduction
of shaven function prevents the expression of this fate by
either cell, so no external cuticular structures are produced by
the double mutant bristles. In other words, loss of shaven
activity is epistatic to the effects of reduced N pathway
function on the expression of the shaft differentiation program.
This result strongly suggests that shaven acts at least in part
downstream of the N-dependent trichogen/tormogen cell fate
decision in the bristle lineage, and plays a major role in the
differentiation phase of shaft cell development.
Additional experiments show that misexpression of shaven is sufficient to induce
the production of ectopic shaft structures. From these
results, it is proposed that Shaven is a high-level
transcriptional regulator of the shaft cell differentiation
program, and acts downstream of the N signaling pathway
as a specific link between cell fate determination and cell
differentiation in the bristle lineage (Kavaler, 1999).
atonal is a proneural gene for the development of Drosophila chordotonal organs and photoreceptor cells. atonal expression is controlled by modular enhancer elements located 5' or 3' to the atonal-coding sequences. During chordotonal organ development, the 3' enhancer directs expression in proneural clusters; whereas successive modular enhancers located in the 5' region drive tissue-specific expression in chordotonal organ precursors in the embryo and larval leg, wing and antennal imaginal discs. Similarly, in the eye disc, the 3' enhancer directs initial expression in a stripe anterior to the morphogenetic furrow. These atonal-expressing cells are then patterned through a Notch-dependent process into initial clusters, representing the earliest patterning event yet identified during eye morphogenesis. A distinct 5' enhancer drives expression in intermediate groups and R8 cells within and posterior to the morphogenetic furrow. Both enhancers are required for normal atonal function in the eye. The 5' enhancer, but not the 3' enhancer, depends on endogenous atonal function, suggesting a switch from regulation directed by other upstream genes to atonal autoregulation during the process of lateral inhibition. The regulatory regions identified in this study can thus account for atonal expression in every tissue and essentially in every stage of its expression during chordotonal organ and photoreceptor development (Sun, 1998).
Restriction of proneural gene expression from
proneural clusters to SOPs is usually Notch (N)
dependent. During eye development, N is known to
function within and posterior to the MF in restricting
ato expression to R8 cells within intermediate groups. Anterior to
the morphogenetic furrow, N has been shown to
promote ato expression. To test the function of N in the formation of the ato prepattern
anterior to the MF and in regulating the 3' enhancer, lacZ expression was examined from the 3' enhancer-lacZ reporter gene in a temperature-sensitive N mutant
background. When larvae carrying the temperature sensitive N allele and the 3'
enhancer-lacZ fusion gene are shifted to the restrictive temperature for 2 hours, the 3' enhancer-directed lacZ expression anterior to the MF becomes continuous and appears broader and stronger than that in wild type, and the initial clusters normally seen within the initial stripe fail to form. The
endogenous ato gene responds similarly to N inactivation
in the initial stripe. It is concluded that N is involved in refining ato expression anterior to the MF from a continuous band to patterned initial clusters, which prefigure the future ommatidia (Sun, 1998).
The adult external sense organ precursor (SOP) lineage is
a model system for studying asymmetric cell division. Adult
SOPs divide asymmetrically to produce IIa and IIb
daughter cells; IIa generates the external socket (tormogen)
and hair (trichogen) cells, while IIb generates the internal
neuron and sheath (thecogen) cells. The
expression and function of prospero has been examined in the adult SOP
lineage. Although Prospero is asymmetrically localized in
embryonic SOP lineage, this is not observed in the adult
SOP lineage: Prospero is first detected in the IIb nucleus; during IIb division, it is cytoplasmic and inherited by
both neuron and sheath cells. Subsequently, Prospero is
downregulated in the neuron but maintained in the sheath
cell. Loss of prospero function leads to double bristle sense
organs (reflecting a IIb-to-IIa transformation) or single
bristle sense organs with abnormal neuronal
differentiation (reflecting defective IIb development).
Conversely, ectopic prospero expression results in duplicate
neurons and sheath cells and a complete absence of
hair/socket cells (reflecting a IIa-to-IIb transformation).
It is concluded that (1) despite the absence of asymmetric
protein localization, prospero expression is restricted to the
IIb cell but not its IIa sibling; (2) prospero promotes IIb cell
fate and inhibits IIa cell fate, and (3) prospero is required
for proper axon and dendrite morphology of the neuron
derived from the IIb cell. Thus, prospero plays a
fundamental role in establishing binary IIa/IIb sibling cell
fates without being asymmetrically localized during SOP
division. Finally, in contrast to previous studies, the IIb cell is found to divide prior to the IIa cell in the SOP lineage (Manning, 1999).
What mechanisms lead to prospero expression in the IIb
cell but not in the IIa cell? Specification of IIa/IIb cell fates
is determined by the relative activity of Notch signaling.
Productive Notch signaling results in IIa cell fate;
asymmetric localization of Numb protein into the IIb cell
blocks Notch signaling and results in the IIb cell fate. It is proposed that
productive Notch signaling prevents prospero expression in
the IIa cell, whereas lack of Notch signaling allows
prospero expression in the IIb cell. Consistent with this
model, SOP lineages with unregulated Notch signaling
produce a pair of IIa cells that both fail to express prospero,
while SOP lineages lacking Notch function produce two IIb
cells that both express prospero (Reddy,
1999). One effector of Notch signaling in the IIa cell is the
zinc-finger transcriptional repressor Tramtrack, which may
directly or indirectly repress prospero expression. Interestingly, prospero is expressed in the R7 neuron
during eye development and tramtrack mutants have supernumerary R7
neurons, while tramtrack misexpression reduces R7
differentiation. Thus, a
similar Notch-, tramtrack-dependent pathway may repress
prospero expression in both the R7 photoreceptor neuron and
the IIa cell. It should be noted that a somewhat different
mechanism must be involved in repressing prospero in the
neuron but not the sheath cell; in this case, Notch signaling is
required for sheath cell fate, the cell that maintains prospero
expression. The lack of Notch-mediated repression of
prospero expression in the sheath cell may reflect the fact
that Notch signaling is SuH-dependent in the IIa cell, but
SuH-independent in the sheath cell.
prospero is essential for distinguishing IIa and IIb
cell fates (Manning, 1999 and references).
A role for prospero in establishing different
IIa/IIb cell fates has been demonstrated based on both loss-of-function and
misexpression experiments. A significant fraction of the SOP
lineages lacking prospero function show a duplication of the
external bristle (a progeny of the IIa cell) and
a loss of the neuron (a progeny of the IIb cell) (Reddy, 1999). Socket
cell fate could not be adequately scored, because multiple socket cells can generate a single,
fused socket structure. The simplest interpretation of the
double bristle prospero minus sense organs is that the IIb cell has
become partially or fully transformed into a IIa cell, resulting
in duplicate hair/sockets and loss of neuron/sheath cell. It is unlikely, but it cannot be rule out, that the
neuron is transformed into a duplicate hair cell and the sheath
cell is unaffected. In both notum and eye, however, there are
still many single bristle sense organs that have an associated
neuron and, in these sense organs, the IIb cell must have been
specified relatively normally. Thus prospero is not strictly
necessary for IIb cell specification, but its function is important
for the high-fidelity specification of IIb cell fate.
While the presence of prospero in the IIb cell is important
for reliable IIb cell specification, the absence of prospero
from the IIa cell is absolutely essential for IIa cell
specification. Misexpression of prospero in the IIa cell and
its progeny results in a fully penetrant loss of a socket cell
marker (SuH) as well as the morphological external socket
and hair structures; there is a corresponding increase in the
internal Elav+ neurons and BarH1+ sheath cells. The
misexpression experiments show that absence of Prospero in
the IIa cell is required for normal IIa development, and that the
presence of Prospero in the IIa transforms it partially or fully
to the IIb cell fate. Thus, differential expression of prospero
between IIa and IIb siblings is essential for normal SOP
development. Similar results were observed using transient
heat-shock-induced misexpression of prospero, although in
these experiments a very low frequency of
double and triple bristle sense organs at the borders of the
bald areas was observer. The cell lineage of these rare sense organs is
unknown (Manning, 1999).
It is interesting to consider the different mechanisms by which
prospero acts to distinguish sibling cell fate. During embryonic
neuroblast cell division, localization of Prospero into the
daughter GMC is necessary for GMC development, but
exclusion of Prospero from the neuroblast is relatively
unimportant for neuroblast development (this is because neuroblast
development is fairly normal in miranda mutants where
Prospero remains in the neuroblast; Chris Doe, unpublished
results cited in Manning, 1999). In contrast, during the adult SOP lineage, it appears
equally important to remove Prospero from the IIa cell as well
as provide it to the IIb cell. Another key difference between
the adult SOP lineage and the embryonic SOP and neuroblast
lineages is the timing of cell divisions. There are several hours
between each cell division in the adult SOP lineage,
considerably longer than the 40-60 minutes cell cycle of
embryonic neuroblasts and SOPs. The shorter cell cycles of the
embryonic lineages may require asymmetric localization of
Prospero for efficient specification of sibling cell fate, whereas
the longer adult SOP cell cycles may provide time for the
action of other regulatory mechanisms (e.g. Notch-mediated
repression of prospero expression) (Manning, 1999).
In single bristle prospero minus sense organs, a single
neuron was observed with profound defects in neurite outgrowth. The
defects in axon and dendrite outgrowth and connectivity
could be due to lack of prospero function in the IIb cell, a
non-autonomous effect due to lack of prospero function in
the sheath cell, or the absence of prospero function in the
neuron itself. The first possibility is unlikely
because axon outgrowth defects can be observed in R7
neurons, which do not arise from a Prospero+ precursor cell.
The second possibility is unlikely because lack of
sheath cells (in glial cells missing embryos) does not
generate similar axon outgrowth defects.
A third model is favored, in which prospero has a direct
function in the neuron, because many neurons with different
origins (CNS, PNS, eye) transiently express prospero and all
show a similar prospero mutant phenotype: stunted and
misrouted axons (Manning, 1999).
hibris is regulated by Notch and Ras in a Toll10b mutant background. This regulation was confirmed in vivo in wild-type embryos. hbs expression was examined in Notch and Ras loss-of-function embryos and embryos overexpressing activated forms of Notch and Ras in the mesoderm. A dominant negative Ras construct activates hbs expression in the somatic mesoderm. Zygotic null Notch embryos show lower hbs transcription. Conversely, an activated form of Notch upregulates hbs in the mesoderm, while an activated form of Ras almost completely inhibits hbs expression. These results argue that, upon stimulation, Notch activates hbs, while Ras acts as a negative signal, and predicts that hbs expression in the somatic mesoderm would be restricted to fusion-competent cells (Notch dependent) and excluded from founder cells (Ras dependent). It is not known whether this regulation is direct, that is, Notch or Ras effectors act directly on the hbs promoter, or indirect, that is, Notch/Ras converts cell fate, which in turn would lead to hbs upregulation/downregulation by some other effector (Artero, 2001).
The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).
To investigate the role of the genes expressed in the large intestine, lack- and gain-of-function studies were performed. In amorphic Notch and Delta mutant embryos, kni/knrl, rho, and high levels of Crb expression on the apical plate are absent in the large intestine, and the boundary cell fate is not established. In contrast, ventral cell morphologies are normal in Notch or Delta mutant embryos, and En expression and dorsal cell fates are unchanged. This indicates that Notch signaling is required to establish the boundary cells but not for dorsal or ventral cell fates. To further test this, gain-of-function experiments were performed using the UAS/Gal4 system. As driver lines, the G445.2 or the 14-3-fkhGal4 strains were used -- they mediate ubiquitous gene expression in the developing hindgut from the extended germ band stage onward until late stage 16. In order to ectopically activate the Notch signaling pathway, flies carrying the Notch intracellular domain fragment, Nicd, under the control of UAS sequences were used. Expressing Nicd ubiquitously in the hindgut results in an ectopic induction of kni and of rho. In addition, the cellular localization of the Crb protein is affected in these embryos. In dorsal and ventral cells of the large intestine of wild-type embryos, Crb is localized to the apical cell margins, whereas it is localized to the entire apical plates of the boundary cells. In the embryos, in which Nicd is ectopically expressed, Crb protein is found on the apical plates of all the hindgut cells; in addition, it is found in high concentrations in vesicles, especially on the baso/lateral sides of the cells. A similar but less intensive ectopic expression of Crb can also be induced if both Kni and Rho are coexpressed in all the hindgut cells, suggesting that crb may be a downstream effector gene of Kni/Knrl and Rho activities. This is consistent with the analysis of rho7M; Df(3L) riXT1 mutants [Df(3L) riXT1 is a deficieny encompassing the kni and knrl transcription units] in which the expression of crb in the boundary cells is strongly reduced. In summary, these results suggest that rho, kni/knrl, and Crb are target genes which are activated in response to Notch signaling in the boundary cells (Fusse, 2002).
In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).
These results suggest that the activation of the Notch receptor in the boundary cells of the hindgut is triggered by the binding of Delta, which is expressed at high levels in adjacent ventral cells. If Delta levels are uniform and this boundary condition is lost, as in enE mutants, Notch signaling fails to occur. To further obtain insight into how the spatial control of Notch receptor activation is mediated, the localization of the receptor was determined using antibody stainings to Notch. In ventral and dorsal cells, Notch is expressed in the apical cell margins, as can be demonstrated using coimmunostainings with Neurexin IV. However, in the boundary cells, the Notch receptor is positioned to the entire apical plate where it is colocalized with Crb or Discs-lost. To test whether the apical localization of the receptor is necessary for its signaling activity, amorphic crb mutants were studied in which the sorting of proteins to the apical domain of the cells is affected. In these mutants, a strong reduction of the number of boundary cells was found, although hindgut morphogenesis is only slightly affected. In addition, the remaining boundary cells are mislocalized, and two rows of cells are often found instead of a single row as is found in wild-type. Anti-Notch/anti-Kni double immunostainings of crb mutants demonstrate the reduction of apical Notch receptor localization in crb mutants. Furthermore, in cells in which the Notch receptor is not localized along the apical plate of the cells, the activation of Notch target genes fails to occur. These results indicate that apical localization of the receptor may be important for boundary cell fate determination (Fusse, 2002).
These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).
These results provide evidence that Notch signaling in the Drosophila hindgut controls the fate of a single row of boundary cells separating the dorsal and ventral halves of the gut tube. Activation of the Notch receptor in the boundary cells is mediated by its ligand Delta that is expressed in adjacent ventral cells. The induction of Notch target genes activate the expression of the apical polarity organizer Crb, which may be required, in turn, for apical Notch receptor localization. These findings identify a simple mechanism that controls the specification of a single row of DV boundary cells in an animal gut (Fusse, 2002).
Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive
cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined.
Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).
This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation
gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2
divides asymmetrically to give rise to two founder cells, one
specific for a pair of persistently Eve-positive heart-associated
or pericardial cells (EPCs) in every hemisegment
and a second of previously undetermined identity. This
second founder coexpresses Eve along with the gap gene
Runt, with Eve levels rapidly fading but Runt persisting as
development proceeds. By the time that Eve is
evident in the EPCs, Runt labels a single somatic muscle,
dorsal oblique muscle 2 (DO2). Runt is also detected
in the muscle DO2 precursor during germband retraction (Carmena, 2002).
The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably
expressed marker for it is unavailable (Carmena, 2002).
To further substantiate the lineage relationships among
these progenitors and founders, observations
related to RTK signaling dependence of P2 and P15
specification were used: whereas P15 requires the activities of both
Egfr and Htl, only Htl is involved in P2 formation. In
this way, targeted mesodermal expression of a dominant
negative form of Egfr strongly blocks formation of DA1
but not the EPCs. Also, consistent with DO2 and
EPC founders being the progeny of P2, DO2 development,
like that of the EPCs, is not affected by dominant negative
Egfr. Additional support for the sibling
relationship between the DO2 and EPC founders derived
from the analysis of targeted expression of a dominant
negative form of Htl. Under conditions in which early
mesoderm migration is not perturbed, dominant negative
Htl generates an incompletely penetrant phenotype in
which different hemisegments lose derivatives of P2, P15,
or both progenitors. With such
partial inhibition of Htl activity, muscle DO2 and the EPCs
are consistently either both present or both absent from
any given hemisegment; in no cases did one of these cell
types develop without the other, as expected for cells
derived from a common progenitor. In contrast,
muscle DA1 frequently forms in the absence of muscle
DO2 and the EPCs, consistent with its derivation from an
independent progenitor. Taken together, these data
establish that the EPC and DO2 founders are sibling cells of
the P2 division, whereas the other Eve-expressing muscle
founder arises from a different progenitor (Carmena, 2002).
This model differs from one derived on the basis of
clonal analysis in which it was proposed that the two
Eve-positive mesodermal cell types originate from the same
progenitor. This discrepancy may relate to the fact that muscles form by sequential cell fusions involving both founders and fusion-competent cells of potentially different parental cell origins, thereby confounding the interpretation of clonal analysis in which the cytoplasm of a single myotube is labeled by the lineage tracing marker (Carmena, 2002).
Autoregulation of a signal transduction cascade can cause
either enhancement or attenuation of the transduced signal,
depending on whether the feedback loop acts positively or
negatively. Both types of feedback control occur during the Ras- and
N-mediated specification of Eve mesodermal progenitors.
Ras activation leads to increased expression of several
proximal components of both the Fgfr and Egfr pathways
that serve to amplify and/or prolong both fate-inducing
RTK/Ras signals in the emerging Eve progenitors.
A similar amplification of Egfr signaling occurs via induction
of Rho during Drosophila oogenesis and mesothoracic bristle formation, and via upregulation of Egfr expression during
C. elegans vulva development.
The present analysis also uncovers a positive feedback
mechanism for inductive Fgfr signaling, in this case via
increased expression of not only the Htl receptor but also its
specific signal transducer, Heartbroken (Hbr). Interestingly, the
data suggest that the downstream components may respond
to different thresholds of Ras activity since Rho exhibits a less
robust response than either Htl or Hbr to Ras activation (Carmena, 2002).
A negative feedback loop occurs in the Egfr pathway
through autoactivation of the inhibitory ligand, Aos. Aos cooperates with Dl to block the progenitor-inducing Ras signal in both adjacent
and more remote cells of the cluster. Aos could
also exert a late inhibitory effect on the progenitor by
terminating the inductive Egfr signal since Spi levels decrease
following the establishment of cellular identity. Consistent
with this possibility, MAPK activation fades from the
singled out progenitor prior to its asymmetric division,
suggesting that prolonged RTK signaling does not occur (Carmena, 2002).
Positive and negative feedback also occur during N function
in the mesoderm. N activation both downregulates its
ligand Dl and upregulates its own expression, thereby
enhancing the potential for inhibitory signaling in cells not
destined for the progenitor fate. Together, these opposing
changes in Dl and N expression produce a unidirectional
inhibitory signal emanating from the prospective progeni\tor
and directed toward the adjacent nonprogenitor cells. Similar feedback mechanisms regulate the N pathway in the Drosophila embryonic CNS, adult PNS and wing vein-forming cells, and also apply to the N receptor-ligand combinations controlling gonadal and vulval cell fates in C. elegans (Carmena, 2002 and references therein).
Competitive cross-talk between Ras and N is manifest by the ability of
the latter to block the expression of proximal components
of the two RTK pathwaysónamely Htl/Hbr and Rho -- as
well as to prevent the associated activation of MAPK. An
antagonistic relationship between the RTK and N pathways
is also revealed by the strong genetic interaction between
Dl and Egfr, in agreement with previously reported genetic
studies. Collectively,
these results establish that the RTK and N pathways
are not simply acting in parallel to exert opposing influences
on progenitor specification; rather, N must be interfering
with the generation and/or transmission of the inductive
RTK signal. This effect could occur at multiple
levels. The ability of activated N to at least partially block
MAPK activation induced by constitutive Ras argues that N
functions downstream of Ras. An additional direct effect of
N on expression of Ras-responsive target genes cannot be
excluded, particularly since Enhancer of split repressors are
involved in the specification of progenitor cell fates. Such targets could include
eve itself, or, given positive autoregulation of RTK
signaling, one or more RTK pathway components (Carmena, 2002).
During C. elegans vulva development, Lin-12/N inhibits
Egfr activity by stimulating the expression of a MAPK
phosphatase. This is an attractive explanation for the effect of N observed here. However,while stimulation of a MAPK phosphatase could contribute
in part to N inhibition of Ras signaling in the Drosophila
embryonic mesoderm, it cannot be the only explanation
since a constitutively activated form of Pointed is completely
unable to reverse the activity of constitutive N. This is in
marked contrast to the substantial reversal of N exhibited
by activated Ras and occurs even though Pnt is a major Ets domain activator involved in RTK-dependent eve regulation (Carmena, 2002).
To account for the differential abilities of constitutive
Ras and Pnt to compete effectively with constitutive N, the idea is favored that an additional, as yet uncharacterized,
Pnt-independent function of Ras may be a target of N
inhibition. Hence, there exist at least four potential sites of
competitive interaction between these pathways: (1) direct
regulation of target gene enhancers by pathway-specific
transcriptional activators and repressors; (2) regulation of
MAPK phosphorylation; (3) inhibition by N of RTK pathway
component expression; and (4) an additional level of
Pnt-independent cross-talk between Ras and N. These
mechanisms modulate the relative flux through the competing
Ras- and N-dependent processes and determine
which pathway predominates, thereby achieving a critical
threshold for a given cell fate. The importance of
relative activity thresholds is underscored by results
with different combinations of activated Ras and N insertions
in which different numbers of Eve-expressing cells
were induced, presumably reflecting slight fluctuations in
the relative strengths of each pathway in individual cells. Similar levels of control may underlie the antagonistic effects of Ras and N in other
developmental contexts (Carmena, 2002 and references therein).
Although the net effect of Ras and N signaling in the present system is the
result of their antagonistic relationship, several forms of
cooperative cross-talk also occur. For example, Ras activation
induces the expression of Dl. Since the Ras signal is
amplified by a positive feedback loop, this has the effect of
biasing Dl expression to the emerging progenitor, thereby
generating a polarized, nonautonomous inhibitory signal
that acts on adjacent cells of the cluster. Aos is also a target
of Ras activation, and Aos acts synergistically with the
neurogenic pathway to block inductive RTK signaling.
Thus, through its effects on the two inhibitory ligands, Dl
and Aos, Ras cooperates with N to ensure that only one cell
segregates as a progenitor from each equivalence group (Carmena, 2002).
Further cooperation is evident in the N-mediated down-regulation
of Dl and Aos in prospective nonprogenitors, a
combination of negative feedback and cross-talk that effectively
prevents neighboring cells from sending an inhibitory
signal to the emerging progenitor. Yan is yet another
Ras-dependent component that reinforces the effect of N:
when MAPK is suppressed in cells in which N is active, Yan is a functional repressor that blocks progenitor fate. Other examples of cooperation between Ras and N signaling include mammalian cell tumorigenesis and photoreceptor specification in the Drosophila eye (Carmena, 2002).
One seemingly paradoxical signaling interaction is N
expression upregulation by Ras. Since Ras output is amplified
in the progenitor, N protein might be expected to
decrease in this cell, thereby restricting lateral inhibition to
the appropriate direction. However, increased N in the Eve progenitor does not actually affect the polarity of lateral inhibition because the
activating ligand, Dl, is downregulated by N in the adjacent
nonprogenitors. Of further relevance, Dl may inhibit N
activity when the two proteins are expressed in the same
cell. Moreover, upregulation of N normally occurs very late in progenitor
specification, as opposed to Dl which increases in
one cell of the cluster at an earlier stage. Lastly, increased N has independent biological significance in progenitors since N is required for an asymmetric division that immediately follows the specification of these cells. In this respect, the response of the N receptor to Ras activation is an efficient, anticipatory 'feed forward' mechanism for insuring that this cell division is appropriately regulated (Carmena, 2002).
A model is presented that summarizes the progressive changes in Ras and N signaling and the cellular events corresponding to each stage. The
model emphasizes that, while clusters of equivalent cells
begin with the same signaling repertoires, they acquire
distinct biochemical states which uniquely determine progenitor
and nonprogenitor identities. Most important,
this complex circuitry drives the reciprocal alterations in
Ras and N activities toward the requisite thresholds that
are essential for determining these fates. What biases one
cell in an equivalence group toward the imbalance in Ras
and N signaling that initiates the entire mechanism remains
an open question. One possibility is that localized
expression of the RTK ligands may provide the initiating
event. Similarities of other developmental systems reinforce
the general relevance of these conclusions (Carmena, 2002).
The reinforcing effects of Aos and Dl are essential since
neither is fully capable of insuring that only one progenitor
segregates from each Eve cluster. Furthermore, simultaneous
loss of both inhibitory pathways leads to the formation
of additional Eve cells within the competence domain
but outside of the normal Eve equivalence groups. This
suggests that the combined actions of both inhibitors prevent
the spreading of the inductive signal beyond the
normal cluster boundaries, as might occur through positive
feedback of Rho expression and the associated increase in
secreted Spi production. Such a remote inhibitory mechanism
is particularly relevant to Aos, which is hypothesized
to act at a longer range than Spi. Of note,
synergistic inhibition by Aos and the N pathway has not
been observed in other systems (Carmena, 2002).
These results also revealed an effect of Aos on Htl- but not
Egfr-dependent C2 cluster development. Although this
could be interpreted as indicating a role for Aos in the
inhibition of the Htl Fgfr, the idea that Aos is
actually blocking basal and/or spontaneous levels of Egfr
activation in C2 cells is favored. This interpretation
is supported by the finding that a dominant
negative form of Egfr suppressed the effect of aos loss-of-function
not only in Egfr-dependent C15, but also in C2,
which does not require Egfr for its specification. In this cluster, the requisite Aos expression is dependent on Htl activity (Carmena, 2002).
Another advantage of combining Aos and Dl relates to
their differing properties. Aos is a secreted inhibitor capable
of acting over several cell diameters,
whereas Dl -- although subject to proteolytic processing -- is generally considered a membrane-bound ligand requiring cell contact for its
activity. If a progenitor
emerges from the center of a cluster such that it is in close
proximity to all of its initially equivalent neighbors, then
Dl alone might be sufficient for the segregation of only one
progenitor. However, if a progenitor forms on the periphery
of a cluster, then the addition of Aos would compensate for
the inability of Dl to inhibit its more distant neighbors.
Thus, two distinct modes of lateral inhibition have complementary
and reinforcing functions (Carmena, 2002).
The involvement of RTK/Ras and N pathways in the
specification of Eve muscle and heart progenitors exemplifies
the complex regulatory interactions that can occur
between two antagonistic signaling pathways acting in
concert. These findings demonstrate RTK/Ras and N signals do not
function independently, converging only at the most distal
step leading to a particular biological response. Rather, their
effects are intertwined at multiple levels to form an integrated
network of cross-talk nodes and feedback loops. The
combination of cell autonomous and nonautonomous components
of both pathways affords the high degree of regulatory
versatility and specificity required to generate the
polarized signaling activities that distinguish progenitors
from their nonprogenitor neighbors. These interactions are
especially remarkable since, once initiated, they propagate
into self-sustaining cascades that differentially drive equipotent
cells to their individual fates. Of further significance,
the mesodermal cells produced by these mechanisms
give rise to the differentiated derivatives that compose the
stereotyped structures of the embryonic heart and body
wall muscles. Thus, the signaling circuitry uncovered here
not only establishes the finely tuned balance between the
inductive and inhibitory influences which coordinately
generate progenitor cell patterns, but also sets the stage for
subsequent morphogenetic events (Carmena, 2002).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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continued:
Biological Overview
| Evolutionary Homologs
| Protein Interactions | Post-transcriptional regulation of Notch mRNA
| Developmental Biology
| Effects of Mutation
| References
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