Notch and leg morphogenesis (part 2/2) The expression of the Serrate and Delta genes patterns the
segments of the leg in Drosophila by a combination of their
signaling activities. Coincident stripes of Serrate and Delta
expressing cells activate Enhancer of split expression in
adjacent cells through Notch signaling. These cells form a
patterning boundary from which a putative secondary
signal leads to the development of leg joints. Elsewhere in
the tarsal segments, signaling by Dl and N is
necessary for the development of non-joint parts of the leg.
It is proposed that these two effects result from different
thresholds of N activation, which are translated into
different downstream gene expression effects. A general mechanism is proposed for creation of boundaries by Notch
signaling (Bishop, 1999).
N mutant flies
show a marked reduction in leg length with all areas of the leg
segments being affected. Joints are completely lost
but also often apical bristles. The overall length of the
segments, and especially of the tarsal region, is more reduced
than in Ser or Dl mutants because both joint and interjoint
tissue is missing. Thus, the N mutant phenotype looks
like a composition of the Ser and Dl mutant phenotypes. When
the expression of disco is revealed in N mutants, a combination
of Ser and Dl phenotypes is also seen. disco stripes do not
resolve properly, as in Dl mutants, and then they are
subsequently lost, as in Sermutants. Expressing a dominant-negative form of N in
the interjoint regions results in shortened legs, due to the loss of interjoint tissue,
but the joints are still present and sometimes fused.
These leg phenotypes thus resemble those produced in
interjoint regions by loss of Dl function.
Expression of a truncated and constitutively activated form
of N in the fourth tarsal segment
becoming hyper-jointed: double ball joints are formed. In addition, the interjoint region is reduced, either
as a consequence of its conversion to extra joint tissue, or to
an inability to develop the interjoint cell fates that have low,
but not high, levels of N activation.
Altogether these results suggest that the overlap of Ser and
Dl expression and requirements at the joints are mediated by N
activation. As a marker of N activity, the
expression of members of the E(spl) complex has been monitored. Using reporter constructs with the regulatory regions of
E(spl), which
reproduce the endogenous E(spl) expression in the leg discs, it can be seen that E(spl)m8 expression
is related to joints while m5 and presumably m6 are not.
Expression of the E(spl)m8 reporter construct in third instar
discs is initially strong in regions undergoing PNS
development. In the legs, these correspond to the chordotonal
organs in the femur and the tibia. In addition, expression near the presumptive joints is
seen to appear, and then resolve in the pupa into one-cell wide
stripes proximal to the leg constrictions, in positions
that correlate with cells with maximum levels of disco
expression. A similar although much weaker pattern of
expression of E(spl)mdelta is seen as revealed by the mAb323
antibody. Another marker
of N activity is the expression of N itself, which becomes
upregulated in cells where N signaling is being received. Using an anti-N antibody,
upregulated expression of N is seen immediately proximal to
constrictions in pupal legs. This upregulation is restricted to a single
row of cells at this position, thus confirming that
Ser and Dl are triggering N signaling in these cells (Bishop, 1999).
The results presented suggest a model in which the co-expression
of Ser and high levels of Dl in a stripe of cells
proximal to the future presumptive joints activate N in cells
adjacent but distal to this stripe. Could this
specificity be due to the presence of other factors that
would be interfering with Ser and Dl signaling in cells located
inside the Ser-Dl stripe or proximal to it? In the DV boundary
of the wing, the membrane protein encoded by the gene fringe
(fng) has been postulated to modulate N signaling by
interfering with Ser signaling. In the developing legs, fng is expressed
in stripes or rings around the positions of
presumptive joints. Using a UAS-fng
construct, fng was misexpressed.
Uniform tibial and tarsal fng expression only affects the joints, which are reduced
or disappear, a phenotype reminiscent of Ser
mutants. Thus, fng activity in the leg
seems to be restricted to a repression of joint
development around presumptive joint areas. It is
possible that fng expression in the wild type is
repressing N signaling in cells located in the Ser-Dl
stripe or proximal to it, providing the polarity in
the joint-promoting function of Ser and Dl (Bishop, 1999).
These results suggest a model in which the co-expression
of Ser and high levels of Dl in a stripe
of cells activate N in cells adjacent but distal to this
stripe. Activation of N promotes expression of members of the
E(spl) complex and leads to joint formation and disco
expression.
Loss of Dl eliminates first the regions between disco/Ser-expressing
rings, but also, secondly, joints. Since loss of
interjoint regions is also seen both in N mutants and following
expression of a dominant-negative form of N, it is postulated that
Dl expression in the interjoint regions produces low levels of
activation of N that do not lead to E(spl) expression but which
allow cell survival and/or cell proliferation.
Joint loss in Dl mutants is presumably
less severe than interjoint loss because Ser and Dl expression
could be synergistic and partially redundant. The combined and potentially
synergistic effects of Ser and Dl would produce a high level of
activation of N that would lead to expression of members of
the E(spl) complex, upregulation of N expression, and to joint
development and disco expression. Thus, it is believed that
combinations of signaling by Ser and Dl could produce
different levels of activation of N, which in turn are translated
into different downstream effects. As noted in other systems these downstream effects of N signaling
should be mediated by more factors than just E(spl), since
E(spl) mutant legs have been reported as having a wild-type
phenotype (Bishop, 1999 and references).
The width of the final joint region is wider than the single
row of cells activated by the membrane-tethered Ser and Dl
proteins and visualised by E(spl) expression. In principle it is
possible that the cells of the whole final joint all descend from
the E(spl) expressing cells, but previous studies have shown
that only one or two cell divisions occur in the legs after
puparium formation. Thus it is
likely that in the E(spl) expressing cells another cell signaling
molecule is activated, which in a secondary event would define
a wider joint presumptive region, just as N-induced expression
of the secreted signaling wingless protein defines the
presumptive wing margin. A reflection of this putative second signaling event in
the joints can be seen in the expression of disco. disco
expression is dependent on Ser but it is wider than the single
row of cells where N is activated and thus it cannot be directly
reflecting N signaling at the joint. However, the 'bell-shaped'
distribution of disco might reflect this putative secondary
signaling event, with a maximum in cells at the edge of the
Ser-Dl stripe. The nature of the joint-promoting putative
secondary signal is unknown at the moment, but one possible
component is the product of the four-jointed (fj) gene. The fj
protein is a putative signaling molecule that is expressed and
required at the joints. fj expression
has recently been shown to depend on fng and N signaling
during eye development, and it
is lost in N mutant legs (Bishop, 1999 and references).
An
autonomous negative effect of Dl and Ser does not explain why cells adjacent
but proximal to the Ser-Dl stripe do not seem to be signaled.
A possible explanation would be either an asymmetric
distribution of Ser and Dl, forming gradients like those seen
in the late third instar wing margin and
in ectopic expression situations, or a downregulation of N expression as has been
noted in the developing wing veins. The
Ser and Dl stripes in legs show no apparent asymmetry but
N distribution, although ubiquitous and initially uniform, becomes upregulated in cells distal to the Ser-Dl
stripes. Low availability of N protein could have an effect on
the intensity of N signaling, but since upregulation of N is in
itself a consequence of N signaling,
some other factor must polarize the signaling initially. Another
explanation would rely on the action of a repressor acting upon
cells proximal to the stripe. The phenotypes obtained after
ectopic expression of fng are consistent with such a role for
fng, as postulated in the wing. The expression of fng in the leg, which has been
described as complementary to that of E(spl), that is, present
in non-signaled cells but excluded from joint forming ones, is also consistent with this hypothesis. Such
a function of fng could also repress Ser and Dl signaling in
the stripe without recourse, or in addition, to putative
autonomous dominant negative effects of Ser and Dl. However,
other factors could also be involved, such as the cell polarity
pathway. Mutant phenotypes for dsh
and other members of the cell polarity pathway produce
ectopic joints with reversed polarity, which
appear just proximal to the position of Ser and Dl
stripes. Furthermore, in dsh mutants ectopic N
activation is seen proximal to the Ser-Dl stripe. Since the Dsh protein
has been shown to interact with N, and Dsh has been postulated
to inhibit N signaling in this manner,
the cell polarity pathway could be involved in repressing Ser
and Dl signaling to cells proximal to the Ser and Dl stripe (Bishop, 1999 and references).
The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and
sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well
as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream
of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).
fng mutant clones also result in fused joints and shortened legs. fng is required with the formation of all joints except the tibia-tarsal (ta1: basitarsus) joint. In most cases, the formation of the joints appears to be an autonomous property of wild type cells, while the failure to form joint structures is an autonomous property of cells mutant for Notch, Dl, Ser or fng. However, some exceptions have been observed in which joint formation is inhibited within wild type cells that border mutant clones or mutant cells appear to contribute to joint structure (Rauskolb, 1999).
These studies further show that Notch activation is both necessary and sufficient to promote leg growth. Target genes regulated both positively and negatively downstream of Notch signaling have been identified that are required for normal leg development. The nubbin gene (nub) encodes a POU-domain protein that is expressed in a series of concentric rings in late discs. The strongest mutant alleles, which are not null, result in shortened and gnarled legs. Notch mutant clones cause loss of Nub expression. Conversely, ectopic expression of Nub is induced within clones of cells expressing activated Notch. These observations indicate that nub is positively regulated downstream of Notch activation in the leg. Although in most cases the influence of Notch signaling on nub appears to be autonomous, exceptions to this have been observed. These exceptions indicate that regulation of nub expression by Notch signaling may be indirect. In addition, other factors must modulate the ability of Notch to induce nub expression, because tarsal segments 1-4 do not express Nubbin, and ectopic Notch activation in this region fails to induce Nub (Rauskolb, 1999).
When fng is ectopically expressed, Nub expression can be induced along the inside of clone borders. Similarly, joint structures in the adult can be induced along fng-expression borders and can be inhibited within patches of fng-expressing cells. These observations are consistent with prior studies of fng action during Drosophila wing and eye development, in that Notch activation is positioned along the borders of fng expression (Rauskolb, 1999).
The four-jointed (fj) gene encodes a type 2 transmembrane protein and is also expressed in concentric rings within the developing leg imaginal disc. In fj mutants, growth of the femur, tibia, and first three tarsal segments is reduced, and the ta2-ta3 segment border is absent. The rings of fj expression in leg imaginal discs are complementary to the rings of Nub expression. Consistent with this complementarity, fj expression is inhibited in cells expressing activated Notch; in cells neighboring ectopically expressing Ser or Dl, and in cells along the borders of ectopic fng expression. By contrast, fj expression is activated within cells expressing Ser or Dl. These observations indicate that fj is negatively regulated downstream of Notch signaling in the leg. Thus, Notch signaling subdivides each leg segment into distinct domains of gene expression (Rauskolb, 1999).
While the requirements for odd-skipped (odd) function during leg development have not yet been described, this gene is of special interest because it is required for embryonic segmentation in Drosophila and is expressed in a segmentally repeated pattern both in the embryo and in leg discs. odd expression, like nub, is induced within clones of cells expressing activated Notch in many regions of the leg disc, though not in ta1-ta4. The observation that three different genes expressed in segmentally repeated patterns all respond to Notch signaling, together with the severe effects of Notch mutant clones, indicates that Notch acts at a crucial step in a leg segmentation hierarchy. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues (Rauskolb, 1999).
Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. Strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). This study shows that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement (Tajiri, 2011).
To examine how the combination of cell differentiation and cell movement evolved to create the ball-and-socket tarsal joint, the tarsal joint morphologies of various insect species was compared (see Tarsal joint morphologies). The ball-and-socket morphology was not found in the tarsal joints of the primitive insects Apterygota and Paleoptera. In the distal tarsal joint of the bristletail (Apterygota) and in all the tarsal joints of the mayfly (Paleoptera), the joint cavity was covered by a uniform, continuous cuticle. Electron microscopy revealed no ball-socket distinction within the cuticle of the mayfly joint. These findings suggested that all the cells in the invaginated region produced a single type of cuticle during tarsal joint development, instead of differentiating into two distinct (ball-producing and socket-producing) populations (Tajiri, 2011).
The proximal tarsal joint of the bristletail, the tarsal joints of the firebrat (Apterygota) and those of the damselfly and dragonfly (Paleoptera) consisted of two pieces of hard cuticle that lined the cavity and were positioned side by side, without one enwrapping the other. As the enwrapment of the ball by the socket in the Drosophila tarsal joint is achieved by cell movement during cuticle secretion, this side-by-side morphology is likely to represent joint formation without cell movement (Tajiri, 2011).
All three types of tarsal joints (ball-and-socket, side-by-side and uniform) were found in the Polyneoptera. For example, the cave cricket contains all three types within a single tarsus. In the Paraneoptera and Holometabola, the ball-and-socket type was often found, for example in the stink bug (Hemiptera), stag beetle (Coleoptera) and ant (Hymenoptera), with some exceptions (Tajiri, 2011).
Given these findings, it is proposed that multiple, separate gains and losses of the two essential processes for ball-and-socket joint morphogenesis, (1) differentiation of ball-producing cells versus socket-producing cells and (2) cell movement, underlie the evolution of tarsal joint morphologies. The two processes may be considered to be 'building blocks' or 'modules' for different joint structures (Tajiri, 2011).
The ball-and-socket type in the Neoptera but not in relatively primitive insect species or other arthropod subphyla (Myriapoda, Crustacea and Chelicerata). Tarsal joints of this type might have evolved in the Neoptera through the acquisition of cell movement during cuticle secretion. Among the ball-and-socket joints, the 'ball' morphology varies substantially, ranging from literally ball-like globular shapes in Drosophila and the ant to thin, stick-like ones in the stink bug and the book lice. Even in the side-by-side joints, the morphology of the distal cuticle varied, from a rod shape in the damselfly to a branched structure in the distal-most joint of the cave cricket. The ball-and-socket and side-by-side joint types might have evolved more than once, giving rise to these distinct structures. Alternatively, the ball morphology might have been differentially modified in different lineages; for example, it could have undergone additional swelling in Drosophila and the ant, or thinning in the stinkbug. If so, the regulation of ball morphology might be another module of joint morphology evolution, acting in parallel with cell differentiation and cell movement (Tajiri, 2011).
An important evolutionary question is how the different morphologies of tarsal joints affect organism fitness. As the number of tarsomeres appears to be uncorrelated with the class(es) of tarsal joint morphologies in each species, it is difficult to assess how the flexibility of individual joints contributes to the mobility of the tarsus. It is suspected that tarsal joint morphology has minimal impact on whole-leg motions, such as walking and jumping, because larger, muscle-containing segments, including the tibia and the femur, are more likely to effect these motions (Tajiri, 2011).
Intriguingly, the ventral surfaces of tarsomeres in many insects have adhesive pads and sensilla. The ball-and-socket joint, which is presumably more flexible than the other types, might permit more tarsomeres of a single limb to fit to curved or jagged surfaces, allowing better substrate attachment and sensing. However, the uniform and side-by-side joint types might have different advantages. The appearance of side-by-side joints in some Holometabolous insects and the co-existence of different types within a single tarsus in some Polyneoptera suggest that tarsal joint morphology has not evolved in a linear fashion from uniform to side-by-side to ball-and-socket. Rather, different ecology- and physiology-dependent selective pressures seem to have resulted in the evolution of any or all three joint types in individual species (Tajiri, 2011).
The Notch signaling pathway has a central role in the segmentation of the arthropod leg. In Drosophila melanogaster, loss of Notch function that begins in the larval period impairs the initial invagination, and blocks the development of joint structure. In an attempt to 'freeze' joint morphogenesis at different phases, Notch function was knocked down at different times during pupa, using a temperature-sensitive allele of Notch (Nts). Mutants were reared at 18°C (permissive temperature), shifted to 32°C (restrictive) at different times during the pupal period and maintained at 32°C until adulthood (Tajiri, 2011).
Mutants raised at 18°C throughout development or shifted to 32°C after the onset of cuticle secretion had normal tarsal joints, and a shift to 32°C during the prepupal period sometimes blocked invagination, consistent with Notch's known essential role in inducing invagination. Shifts at intermediate stages resulted in a range of joint phenotypes, as follows (Tajiri, 2011).
Flies shifted around or shortly after pupation showed a uniform, continuous cuticle that covered the entire joint cavity, which was strikingly reminiscent of the 'primitive' tarsal joints seen in the mayfly. Electron microscopy confirmed the uniform nature of this cuticle. Notably, none of the uniform cuticle showed distinct layers, which is a hallmark of the ball cuticle. Additionally, the lubricant was severely reduced. In some cases, the uniform cuticle had a projection on the ventral side. Later shifts resulted in an almost-normal ball-and-socket morphology, except that the ball and socket abutted each other instead of being separated by lubricant (Tajiri, 2011).
These observations indicated that, in Drosophila, Notch function during the early pupal period is required for the correct ball-socket distinction in the cuticle morphology and ultrastructure (Tajiri, 2011).
In normal Drosophila joint morphogenesis, elongation of the ball 'lip' and the socket coincides with the movement of the cells secreting the components that form them. It was hypothesized that the occasional lip-like projection on the uniform cuticle in Nts mutants represented cell movement during cuticle secretion. A subset of cells was labeled with a membrane-tethered GFP and whether their movement correlated with cuticle morphology was examined. In controls, the apices of the labeled cells continuously contacted the ends of the ball-and-socket cuticles, moving from the original dorsal position to the final ventral position. In mutant uniform joints with a projection, the labeled cells likewise moved their apical surfaces along the cuticular projection. In uniform joints without a projection, the apical surfaces of the labeled cells remained in the dorsal-proximal region (Tajiri, 2011).
These results show that Notch activity in the early pupal period is required for cell motility. The occurrence of cell movement regardless of impairment of the ball-socket distinction indicates that the two processes can be uncoupled. They are both controlled by Notch signaling, but probably through independent pathways. Taken together with a previous finding that cell movement proceeds even when the cuticle structure is severely disrupted, it is proposed that Notch signaling activates a cell-intrinsic mechanism that drives the movement of cell apical surfaces. The uncoupling of the ball-socket distinction and cell movement supports the hypothesis that the two steps have served as evolutionary 'modules' to permit variation in joint structures (Tajiri, 2011).
Ball-producing cells express big brain (bib), a positive readout of Notch signaling activity. Consistent with this, bib expression coincided with the strong expression of Notch Response Element-lacZ (NRE-lacZ), and was significantly diminished in the Nts mutant at 32°C. As mentioned above, the absence of ball-like characteristics in the uniform cuticle of Nts indicated that the ball-producing activity was compromised. It was therefore hypothesized that Notch activity contributes to the distinction between the ball and the socket by promoting ball production. To test this, Notch signaling activity was manipulated in a cell-specific manner and the effects on ball/socket formation was examined(Tajiri, 2011).
When a constitutively active form of Notch (Nact) was expressed using bib-GAL4 (in the ball-producing cells and neighboring socket-producing cells), the socket cuticle was considerably shorter than normal, but the ball cuticle retained its normal morphology. Expression of Nact with neur-GAL4 (only in socket-producing cells) resulted in a similar phenotype. Electron micrographs confirmed the absence of the socket cuticle in the lateral and ventral regions. Thus, excess Notch activity within the cells that would normally produce the socket interferes with socket production, but it does not cause any significant abnormality in the ball-producing cells (Tajiri, 2011).
Expression of an RNAi against Notch driven by bib-GAL4 caused a phenotype resembling the uniform cuticle of the Nts mutant. A small portion of the cuticle exhibited a layered organization in electron micrographs, which is assumed to be trace ball cuticle. In some cases, a small, incomplete ball cuticle formed and fused with the socket. These observations suggested that ball production was significantly reduced, as in the Nts mutant. When the RNAi was driven by neur-GAL4, the socket cuticle was thinner than normal, indicating that a certain level of Notch signaling is required in the socket-producing cells (Tajiri, 2011).
These results are compatible with the model (see Differential Notch activities promote the ball-socket distinction): high levels of Notch signaling promote ball production, and lower levels are required for socket production. It is presently unclear at which phase Notch signaling regulates these activities. It might be during the fate specification of ball-producing versus socket-producing cells, and/or during their differentiation and maintenance, in which Notch might direct the expression of specialized cuticular components and regulators of cell movement, etc. It is noted that the expression of Nact in neur-expressing cells did not convert them into ball-producing cells; electron micrographs showed that the presumptive neur-expressing cells (judged by their location) did not contact the ball cuticle through the plasma membrane plaques, as ball-producing cells would do. Thus, Notch signaling seems to act in more ways than as a simple binary cell-fate switch (Tajiri, 2011).
The expression of Delta (Dl), a Notch ligand, and of Notch itself were examined during and shortly after the onset of invagination (the sensitive period in the Nts experiment). Anti-Notch immunostaining revealed a sharp boundary between the distal cells, with high Notch levels, and the proximal cells with low Notch levels; this pattern was corroborated by the expression of Notch-lacZ. Delta accumulated at high levels in the most proximal row of Notch-lacZ-expressing cells, which coincided with the proximal end of the bib expression domain (high Notch-signaling levels). Thus, Notch is activated in cells distal to the Dl-expressing cells (corresponding to future ball-producing cells), whereas the proximal cells (corresponding to socket-producing cells) have lower Notch-signaling levels (Tajiri, 2011).
This spatial relationship between ligand expression and Notch signaling activity is identical to that reported for leg discs at prepupal stages, when fringe, frizzled and dishevelled are proposed to repress Notch signaling in cells proximal to the ligand-expressing ones. The same mechanism might maintain the differential Notch signaling in the distal versus proximal cells at the pupal stage. In addition, different levels of Notch expression itself (see Fig. S3A in the supplementary material), probably resulting from positive feedback, might contribute to the differential signaling levels (Tajiri, 2011).
In conclusion, this study has shown that, in Drosophila, the two essential processes for ball-and-socket joint morphogenesis, differentiation of ball-producing cells versus socket-producing cells and cell movement, are regulated by Notch signaling through separate pathways. The variety of tarsal joint morphologies among insects is likely to have been generated by independent evolutionary acquisition and/or loss of these processes. These results provide a framework for clarifying the cellular and molecular mechanisms underlying the developmental regulation of joint morphology, and for understanding how those mechanisms have served evolutionarily as building blocks for their variations. In future studies it will be important to investigate how, and to what extent, the levels and downstream cascades of Notch signaling have been modulated in the individual tarsal joints of other insects, and to determine how this modulation is linked to the evolution of joint morphologies (Tajiri, 2011).
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Notch:
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