Mothers against dpp
The single 2.6-kb MAD mRNA is detected in all developmental stages, although it appear most abundant in early embryos, pupae, and adult females. In 0 to 4 hour embryos, which contain maternally loaded mRNA as well as early zygotic transcripts, a minor transcript that is slightly smaller than 2.6 kb is also detected. The abundance of the MAD transcript decreases throughout embryonic and larval development and then returns to high levels in pupae and adult females. A larger transcript is detected in total RNA from early pupal stages and from adult females (Sekeley, 1995)
The BMP pathway patterns the dorsal region of the
Drosophila embryo. Using an antibody recognizing
phosphorylated Mad (pMad), signaling was followed
directly. In wild-type embryos, a biphasic activation pattern
is observed. At the cellular blastoderm stage, high pMad
levels are detected only in the dorsal-most cell rows that
give rise to amnioserosa. This accumulation of pMad
requires the ligand Screw (Scw), the Short gastrulation
(Sog) protein, and cleavage of their complex by Tolloid
(Tld). When the inhibitory activity of Sog is removed, Mad
phosphorylation is expanded. In spite of the uniform
expression of Scw, pMad expansion is restricted to the
dorsal domain of the embryo where Dpp is expressed.
This demonstrates that Mad phosphorylation requires
simultaneous activation by Scw and Dpp. Indeed, the early
pMad pattern is abolished when either the Scw receptor
Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or
Dpp are removed. After germ band extension, a uniform
accumulation of pMad is observed in the entire dorsal
domain of the embryo, with a sharp border at the junction
with the neuroectoderm. From this stage onward,
activation by Scw is no longer required, and Dpp suffices
to induce high levels of pMad. In these subsequent phases
pMad accumulates normally in the presence of ectopic Sog,
in contrast to the early phase, indicating that Sog is only
capable of blocking activation by Scw and not by Dpp (Dorfman, 2001).
Thus two distinct phases of pMad
activation have been identified. The early phase requires
activation by both Scw and Dpp ligands, while the second
phase depends only on Dpp. Signaling is first detected in the cellular blastoderm embryo. While activation is observed within the dorsal-most 8-10 cell
rows, the sensitivity of the detection method fails to monitor
signaling in the rest of the dorsal domain. High signaling levels
are induced by Scw, and give rise to amnioserosa. Within the
domain where pMad is observed, graded
activation is detected, which may have the capacity to induce more than
one cell fate in the region (Dorfman, 2001).
The cardinal players in the generation of the early pMad
gradient are Scw, Tld and Sog. Tld has been suggested to generate
a sink for the active ligand, by cleaving the Sog/ligand complex. The similarity between the pMad pattern of scw and tld mutants suggests that Tld is primarily
involved in the release of Scw from the complex with Sog.
Absence of Scw, Tld or Sax abolished the early pMad
pattern while retaining the second phase, indicating that the
second phase relies only on Dpp signaling.
Similarly, overexpression of Sog eliminated only the early but
not the subsequent pMad patterns. This suggests that
Sog preferentially associates with Scw, in agreement with
previous biological assays of Sog activity.
Generation of graded patterning in the dorsal region does
not rely on restricted gene expression within this domain.
Rather, expression of genes confined to the neuroectoderm
may lead to graded distribution of their gene products within
the dorsal domain. The essential component for generation of
graded patterning appears to be Sog, which is produced only
in the neuroectoderm, but is capable of diffusing to the dorsal
region. Disruption of the normal distribution of Sog by uniform
misexpression, abolishes the early pMad activation profile (Dorfman, 2001).
This suggests that normally Sog may form a graded
distribution in the dorsal region, which is essential for
patterning. When the Sog/Scw complex is cleaved by Tld, Scw
is released and can bind either Sog or Sax. The data suggest
that in regions closer to the neuroectoderm, the levels of Sog
are high and titrate the free ligand. In the dorsal-most region
however, where Sog levels are low, the released Scw has a
greater probability of binding and activating the Sax receptor,
rather than being trapped again by Sog. Thus, the graded
distribution of Sog is critical for generating the reciprocal
distribution of Scw, and the ensuing activation profile (Dorfman, 2001).
In sog mutant embryos an expansion of the
early pMad pattern is observed. In the absence of Sog, a uniform
distribution of Scw is expected, and hence the activation level
should be lower than the maximal level in wild-type embryos.
The staining levels in wild-type and sog
mutant embryos have been quantitated. While the pattern of staining is reproducible
in all wild-type embryos, variations in the absolute levels of up
to threefold between embryos were observed in any given
staining reaction. It is thus difficult to compare reliably the
wild-type level to the absolute staining levels of sog mutants.
Nevertheless, the impression is that the expanded pMad in sog
mutant embryos is comparable in levels to the maximal
signaling levels in wild-type embryos. In spite of this expanded
pMad activation pattern, amnioserosa cell fates are abolished
in sog mutants. This result
suggests that in addition to the role of Sog in determining the
graded distribution of Scw, Sog or its cleavage products may
provide an additional signal facilitating the induction of
amnioserosa cell fates (Dorfman, 2001).
Activation of Tkv by Dpp is essential for the appearance of the
early pMad pattern, corresponding to the future amnioserosa
cells. At this stage, distinct cell fates are also induced in the
dorsolateral cells, as reflected by expression of pnr and
repression of msh expression. It is assumed that low
levels of activation that may be induced by Dpp alone, but not
detected by pMad antibodies, are responsible for these fates.
Elimination of Dpp or Tkv leads to complete absence of
early, as well as late, pMad patterns. Thus, Scw is not
sufficient for the early activation phase, and the presence of
Dpp is crucial. Cooperativity between Scw and Dpp occurs at
the level of receptor activation. One possibility is that the
observed pMad levels reflect only an additive effect of Scw and
Dpp signaling. Indeed, the number of dpp copies
has a profound effect on signaling levels and the shape of the
early pMad distribution. Alternatively, it is possible
that there is a synergistic interaction between Scw and Dpp
signaling. In this case, the requirement of both ligands for the
production of the early pMad pattern may indicate that synergy
occurs at the level of receptor activation. Phosphorylation of
Mad may require the formation of heterotetrameric receptors,
containing both Sax/Put and Tkv/Put pairs. Cross linking
experiments of the vertebrate receptors support this model (Dorfman, 2001).
Scw is required for generating the pMad pattern only in the
early phase. All subsequent patterns rely only on Dpp. This
feature may be explained differently by each of the above two
models. If Scw and Dpp are required additively in the early
phase, higher levels of Dpp may suffice to induce the pMad
pattern at later stages. The autoregulatory effects of Dpp on its
transcription may account for the
elevation in Dpp levels. Alternatively, if Scw and Dpp
signaling is synergistic, why is such a synergism
necessary only in the early phase? In the early embryo, a
maternal transcript encoding an inhibitor of BMP signaling
may be translated, to block signaling by Sax/Put or Tkv/Put
dimers. Such inhibitor(s) may be displaced only in ligand-bound
heterotetrameric receptor complexes. The maternal
transcripts of the inhibitor(s) may diminish by stage 9, to allow
pMad production by activation of Tkv/Put alone (Dorfman, 2001).
By stage 8/9, Dpp/Tkv activation is sufficient to induce
detectable levels of phosphorylated Mad. The second phase of
activation does not rely on execution of the early phase, and is
detected in scw, tld or sax mutants. A uniform pattern of pMad
is observed at this stage within the entire dorsal domain, in
accordance with the pattern of autoregulated dpp expression.
In the neuroectoderm, brinker (brk) is expressed to suppress
Dpp autoregulation. The uniform
pMad pattern corresponds to the resulting expression pattern
of genes like pannier (pnr) at stage 9,
indicating that this second phase of activation is indeed
instructive for induction of target genes in the entire dorsal
domain. Once cell intercalation leading to germ band extension
has been completed, it may be necessary to induce, within the
dorsal region, such a uniform activation of Dpp target genes (Dorfman, 2001).
In the second phase, sharp borders of pMad localization are
observed, with no detectable activation in the neuroectoderm. Dpp is a diffusible ligand, as indicated by the induction of pMad several cell rows away from the dorsal
row of cells expressing Dpp at stage 11. Direct
visualization of Dpp in the wing disc has also demonstrated its
diffusion capacity over many cell rows. How are the sharp pMad borders
generated at stage 9, in view of the diffusability of Dpp? It is suggested that the neuroectoderm cells may produce an inhibitor
that prevents activation of the pathway by Dpp molecules that
could diffuse from the adjacent dorsal region. Alternatively, the
neuroectoderm cells may express cell surface proteins that
would block the diffusion of Dpp into the neuroectoderm. When Dpp is expressed ectopically at physiological
levels in perpendicular stripes, no pMad activation is observed
in the neuroectoderm outside the stripes of Dpp expression. Thus, lower levels of Dpp are not capable of activating
the pathway in the neuroectoderm at stage 9 (Dorfman, 2001).
Wing and leg precursors of Drosophila are recruited from
a common pool of ectodermal cells expressing the
homeobox gene Dll. Induction by Dpp promotes this cell
fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes
the wing-promoting function of Dpp and allows
recruitment of leg precursor cells from uncommitted
ectodermal cells. By monitoring the spatial distribution of
cells responding to Dpp and Egfr, it has been shown that nuclear
transduction of the two signals peaks at different positions
along the dorsoventral axis when the fates of wing and leg
discs are specified and that the balance of the two signals
assessed within the nucleus determines the number of cells
recruited to the wing. Differential activation of the two
signals and the cross talk between them critically affect this
cell fate choice (Kubota, 2000).
The spatial distribution of cells responding to
Dpp and its relationship to Egfr signals was studied. To this end, an
antibody specific to phosphorylated C-terminal sequence of
Mad was produced. The
phosphorylated sequence corresponds to the site at which the
type I BMP receptor phosphorylates SMad1. The antibody detects an antigen
distributed in a pattern similar to, but broader than, that of
DPP mRNA. This
immunoreactivity is dependent on Dpp signaling, as it is
absent in stage 11 mutants of thick veins encoding type
I Dpp receptor and in dpp
mutants. This indicates that other extant
TGFbeta-related signaling molecules present in Drosophila
embryos do not
substitute for Dpp to induce this immunoreactivity.
Conversely, ectopic expression of Dpp results in high
accumulation of this immunoreactivity. These results suggest that the antibody detects a Dpp-specific
signaling event, most likely the phosphorylation and
nuclear transport of Mad. Hereafter, the
immunoreactivity detected by this antibody is called pSSVS (Kubota, 2000).
pSSVS is found mainly localized in the nucleus and
distributed in regions a few cells wider in diameter than those
of dpp-expressing cells. These properties are
consistent with the previous findings that Mad transduces the
Dpp signal to the nucleus. Double labeling of pSSVS and DLL
mRNA shows that pSSVS expression is higher in the dorsal
region of Dll-expressing cells. Combined with the
double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr
overlap, but the peak of the responses are shifted. Such
differential distribution of the two signals results in an
arrangement of cells responding to a different strength of Dpp
and Egfr along the dorsoventral axis (Kubota, 2000).
The increase in the number of wing disc cells in rho mutants
resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might
prevent wing disc development by negatively regulating Dpp
signaling. Such a cross talk could occur at several levels
including the following: (1) regulation of dpp transcription, (2)
signal transduction from Dpp receptors to the nucleus, and (3)
transcriptional regulation of downstream target genes. The
analyses excluded the first two possibilities for two reasons. (1) The
expression pattern of DPP mRNA is unaffected by the
mutation of rho. A previous report showing an expansion of dpp expression in Egfr
mutants probably reflects the global patterning role of Egfr
in the earlier stage. (2) pSSVS expression around limb
primordia does not change in rho mutants. Conversely,
the expression pattern of dpMAPK is not changed by a null
mutation of tkv. These results suggest that the
differential distribution of cells responding to Dpp and Egfr
is set up independently of each other's activity (Kubota, 2000).
dad is an immediate transcriptional target gene of Dpp, the expression of which closely
parallels that of pSSVS expression in embryos and
is inducible by Dpp. dad expression is not affected
in Egfr or rho mutants. Furthermore, elevated
dad expression induced by Dpp is not affected by sSpi, suggesting that at least one of the immediate
transcriptional responses to Dpp is unaffected by elevated
Egfr signaling (Kubota, 2000).
The antagonism between Dpp and Egfr during wing disc
development raises a question: what is the default state of
the wing and leg primordia in the absence of the two signals?
Double mutant phenotypes of Dpp and
Egfr signaling were examined. tkv mutants lack wing discs and their leg discs
are malformed. This
phenotype reflects a disc cell autonomous requirement for Dpp
signaling, because the phenotype is reproduced by the disc-specific
inhibition of Dpp signaling by dad, which inhibits
Mad. The phenotype of either tkv;rho
or tkv;Egfr double mutants is a simple addition of each
mutation, in which wing discs are lost completely and leg
discs are severely reduced. Since Dll-expressing
limb primordial cells are present in tkv;Egfr double mutants in
stage 11, it has been concluded that these cells fail to
differentiate as wing discs and their ability to differentiate as
leg discs is also compromised. A few Esg-positive cells
remain at the position of the leg, and it is speculated that this
reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely
required for wing disc development irrespective of the activity
of Egfr (Kubota, 2000).
The nuclear transduction of the Dpp
signal, as visualized by the distribution of pSSVS and
expression of dad, is unaffected by Egfr. The
results suggest that the antagonistic effect of Egfr on Dpp
signaling occurs after transduction into the nucleus. Therefore,
the mechanism of SMad inhibition by direct phosphorylation
by MAP kinase does not play
a major role in this case (Kubota, 2000).
The finding that Egfr is activated in the limb primordium and
prevents wing disc formation suggests that Egfr is a key
factor in the diversification of the wing and leg fate. It is
proposed that the differential activation of Dpp and Egfr, and
the dorsal cell migration brings a subset of limb primordial
cells out of the range of Egfr signaling, and thereby allows
Dpp to induce wing development. It follows that dorsally
migrating cells acquire the wing cell identity only after the
separation from leg-promoting signals. Consistent with this
idea, expression of wing-specific markers Vg and Sna, start
only after the separation of the two primordia. Mechanisms that
promote the dorsal cell migration remain to be identified.
Given that the basic genetic components for the induction of
the wing and leg have been identified in the model organism
Drosophila, it can now be asked how the genetic
mechanism of wing and leg specification has evolved by
comparing the expression and function of these genes in limb
primordial cells of primitive insects (Kubota, 2000).
Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Patterns have been compared of two related cellular responses - signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Nuclear accumulation of Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (Sog) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm (Sutherland, 2003).
These in vivo studies validate the molecular model for signal-dependent
nuclear accumulation of Medea. Nuclear accumulation of Medea requires both
competence to oligomerize and MAD. Nuclear accumulation is signal dependent, requiring both BMP ligands, Dpp and Scw. Conversely, all cells
accumulated nuclear Medea in the presence of constitutively active Tkv
receptor. At these stages, any independent contribution from activin-like
signals is below the detection limit (Sutherland, 2003).
In wild-type embryos, two transitions in the distribution of BMP activity are evident. Many cellular blastoderm embryos lack detectable levels of nuclear Medea, but a few have low levels of nuclear Medea in a broad dorsal domain, with little gradation. From the proportion of cellular blastoderm embryos with this pattern, the duration of nuclear Medea appears to be brief. These data parallel reports of broad, weak PMad staining during mid-cellularization, except that nuclear Medea is detected later and in a broader pattern. The time lag between the earliest reported detection of PMad and detection of nuclear Medea probably stems from a combination of technical differences and the time necessary for nuclear accumulation. In sum, initial BMP activity is weak and distributed broadly in dorsal regions. Low BMP activity at this phase is required to maintain the early phase of zen expression (Sutherland, 2003).
Onset of gastrulation is associated with a dramatic change in the domain
of nuclear Medea, which narrows to a tight midline stripe of cells while
staining levels intensify. PMad shows a similar transition to a narrower domain, but earlier. Thus, lateral SMAD responses became undetectable just as a steep activity gradient forms along the dorsal midline (Sutherland, 2003).
A third response pattern arises during mid-gastrulation: dorsolateral
domains of cells exhibit low levels of nuclear Medea. Response levels
remain high in the dorsal-most cells, even as they move laterally during
gastrulation. Levels fall off
rapidly over a few cells on either side, with a sharp transition to flanking
plateaus of weak responses. The subcellular distribution of Medea is
unchanging in ventral and ventrolateral cells. The full BMP response domain
does not extend as far ventrally as it does during blastoderm, even though many dorsal cells move laterally during germband extension. Thus, the lateral-most cells with responses at blastoderm
have decreased responses during gastrulation (Sutherland, 2003).
Mutants with expanded ventral ectoderm show reduced SMAD responses during
the first phase of BMP activity. PMad was not detected in blastoderm
tld embryos. Homozygotes for moderate dpp alleles have lower
PMad levels during blastoderm. Conversely, sog embryos have a slightly expanded PMad response during blastoderm, and a slight expansion of dorsal ectoderm. Thus, BMP activity during blastoderm positions the boundary between dorsal and ventral ectoderm (Sutherland, 2003).
BMP activity in the dorsal ectoderm does not end with germband extension.
During stage 9, PMad is detected throughout the dorsal ectoderm and
amnioserosa, and might finalize determination of dorsal ectoderm fates. Dpp
expression within the dorsal ectoderm contributes to combinatoral regulation
of gene expression patterns in subsets of dorsal ectodermal cells. However,
the ventral boundary of dpp expression in the stage 9 dorsal ectoderm
must be defined by earlier events (Sutherland, 2003).
The BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those observed in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth (McCabe, 2003).
In the case of the BMP signal described in this study, the finding that a high accumulation of P-Mad is detectable in motoneuron nuclei when Gbb is resupplied to nerve terminals from the postsynaptic muscle cell implies that a retrograde signal likely contributes to P-Mad nuclear localization. Consistent with this view is the observation that blocks in the dynein/dynactin motor complex also disrupt P-Mad accumulation similar to what has been reported for transport of activated Trks. Since Mad and Medea mutants also display NMJ defects that are very similar to those exhibited by receptor and ligand mutants, it seems likely that the majority of these defects result from the lack of the retrograde signal itself as opposed to some being caused by the lack of a hypothetical local signal. As is the case for Trks, a signaling endosome consisting of activated heteromeric receptor complexes containing Gbb, Wit, Tkv, and Sax might be transported back to the cell body where these complexes would phosphorylate cytoplasmic Mad, resulting in its translocation to the nucleus. Alternatively, nonphosphorylated Mad may first be transported anterogradely to the nerve. Subsequent to phosphorylation at the NMJ, it may then be selectively transported in a retrograde fashion back to the cell body (McCabe, 2003).
Although loss-of-function and rescue experiments clearly demonstrate that Gbb is required for proper synaptic development at the NMJ, it is not certain that it is the only TGF-β-type ligand or indeed the primary ligand that regulates this process. The electrophysiological and ultrastructure defects observed in gbb mutant synapses are not as severe as those found in wit null mutants. This could simply reflect an inability to produce true null animals that survive to the third instar stage, or it may indicate that another ligand also provides a signal. In support of this view is the observation that P-Mad accumulation is not totally eliminated in gbb1/gbb2 null mutant embryos as it is in wit mutants. In addition, it is noted that while overexpression of Gbb in the CNS only weakly rescued P-Mad accumulation in the CNS, the pattern of accumulation does not appear to change. That is, P-Mad still seems to be found primarily in motoneurons. Thus, other neurons do not appear to be competent to respond to BMP-type ligands, perhaps because a specific cosignal is absent or because they do not express the right combination of receptors. It is interesting to note that in several other developmental contexts in Drosophila, it appears that at least two BMP ligands provide regulatory inputs into a common process (McCabe, 2003).
Smad signal transducers are required for transforming growth factor-ß-mediated developmental events in many organisms including humans. However, the roles of individual human Smad genes (hSmads) in development are largely unknown. It was hypothesized that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad). Six hSmad and four dSmad transgenes were expressed in Drosophila using the Gal4/UAS system and their phenotypes were compared. Phylogenetically related human and Drosophila Smads induce similar phenotypes supporting the hypothesis. In contrast, two nearly identical hSmads generate distinct phenotypes. When expressed in wing imaginal discs, hSmad2 induces oversize wings while hSmad3 induces cell death. This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles. These observations also suggest new roles for the dSmads, Medea and Dad, in Drosophila Activin signaling (see Drosophila Activins Activin-ß and Activin Like Protein at 23B; the Drosophila Activin receptor is Baboon) and in potential interactions between these family members. Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families (Marquez, 2001).
hSma1 and Mad can transduce Dpp/BMP signals. hSmad4 and possibly Med can transduce signals for both TGF-ß subfamilies. hSmad4 forms complexes with hSmad1 and Med forms complexes with Mad. These relationships suggest that these Smads will produce similar phenotypes. One copy of UAS.Mad or UAS.hSmad1 does not generate many phenotypes. Strains containing two copies of these transgenes were then used. UAS.Mad and UAS.hSmad1 induced similar wing and leg phenotypes. For example, UAS.Mad/ptc.Gal4 genotypes have ectopic vein tissue between L3 and L5. This vein phenotype is consistent with two previous results: clonal analysis shows a role for Mad in vein formation and ptc.Gal4 expression in wing disc cells that eventually reside between L3 and L4. In UAS.Mad/ptc.Gal4 wings the distance between L3 and L5 appears reduced. This may be due to the smaller size of vein cells vs. intervein cells. A comparison of wing surface areas shows that UAS.Mad/ptc.Gal4 wings are 22% smaller than wild type. UAS.hSmad1/ptc.Gal4 wings have ectopic vein tissue in roughly the same region. UAS.hSmad1/ptc.Gal4 wings are 15% smaller than wild type. UAS.Mad and UAS.hSmad1 expression appears to mimic dpp's role in vein formation. UAS.Mad and UAS.hSmad1 expression does not appear to mimic dpp's other roles in wing development (cell proliferation and/or cell survival and anterior/posterior patterning) (Marquez, 2001).
UAS.Medea/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 wings have ectopic vein tissue in the same region as UAS.Mad/ptc.Gal4 and UAS.hSmad1/ptc.Gal4 wings. This suggests that Med forms complexes with Mad during vein formation. UAS.Med/ptc.Gal4 wings are 11% larger and UAS.hSmad4/ptc.Gal4 wings are 6% larger than wild type. The larger, rather than smaller, size of UAS.Med/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 wings suggests that multi-subfamily signaling Smads can influence wing size and vein formation (Marquez, 2001).
UAS.Mad/ptc.Gal4 flies have an ectopic leg on the ventral side of a normal limb. The ectopic leg has several segments and terminates in a set of tarsal claws. During leg development, wingless normally represses dpp expression on the ventral side of the limb. In limbs expressing ectopic dpp an additional leg develops on the ventral side of the limb. The similarity between phenotypes that result from ectopic dpp expression and UAS.Mad expression suggests that UAS.Mad is capable of simulating Dpp signals in leg patterning. UAS.hSmad1/ptc.Gal4 flies also have an ectopic leg on the ventral side of a normal limb. The ectopic leg consists of a single segment. UAS.Med/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 flies have ectopic legs of a different type. Legs from these genotypes have short, abnormally wide tibia that lead to duplicated tarsi of one or more segments. The abnormal tibia of UAS.Med/ptc.Gal4 flies has an additional patterning defect: severe bristle overgrowth (Marquez, 2001).
Overall, these four Smads generated comparable vein and leg phenotypes. The phenotypes of phylogenetically related Smads show the greatest similarity. The phenotypes suggest that UAS.hSmad1 and UAS.hSmad4 can also simulate Dpp signaling in Drosophila limb development. These findings are consistent with cell culture studies noted above and further support the view that these hSmads transduce BMP signals during human development. The size of UAS.Med and UAS.hSmad4 wings suggests a role for these multi-subfamily signaling Smads not shared with the Dpp/BMP signaling Smads UAS.Mad and UAS.hSmad1 (Marquez, 2001).
In summary, this analysis of hSmad and dSmad transgenes supports the hypothesis that phylogenetically related Smads fulfill developmental roles that are conserved between humans and Drosophila. The results also suggest a number of new hypotheses regarding roles for human and Drosophila Smads in pattern formation, cell proliferation, and cell death. The data suggest that a small number of amino acid differences between two very similar Smads in the same species can confer distinct activities. Overall, this study demonstrates that transgenic methods in Drosophila can provide new information about mammalian members of developmentally important multigene families (Marquez, 2001).
Advances in image acquisition and informatics technology have led to organism-scale spatiotemporal atlases of gene expression and protein distributions. To maximize the utility of this information for the study of developmental processes, a new generation of mathematical models is needed for discovery and hypothesis testing. A data-driven, geometrically accurate model has been developed of early Drosophila embryonic bone morphogenetic protein (BMP)-mediated patterning. Nine different mechanisms for signal transduction with feedback, eight combinations of geometry and gene expression prepatterns, and two scale-invariance mechanisms were tested for their ability to reproduce proper BMP signaling output in wild-type and mutant embryos. It was found that a model based on positive feedback of a secreted BMP-binding protein, coupled with the experimentally measured embryo geometry, provides the best agreement with population mean image data. The results demonstrate that using bioimages to build and optimize a three-dimensional model provides significant insights into mechanisms that guide tissue patterning (Umulis, 2010).
In many systems, spatially patterned cellular differentiation is regulated by signaling molecules called morphogens, which initiate spatiotemporal patterns of gene expression in a concentration-dependant manner. In early Drosophila embryos, a morphogen composed of a heterodimer of Decapentaplegic (Dpp) and Screw (Scw), two members of the bone morphogenetic protein (BMP) family. Unlike classical morphogen systems that rely on the slow spreading of a molecule from a localized source to establish a gradient, BMPs in the early Drosophila embryo are secreted from a broad region making up the dorsal-most 40% of the embryo circumference. Subsequently, they are dynamically concentrated into a narrow region centered about the dorsal midline that makes up only 10% of the embryo circumference (Umulis, 2010).
A number of extracellular regulators contribute to the dynamics and localization of BMP signaling. Laterally secreted Short gastrulation (Sog) and dorsally secreted Twisted gastrulation (Tsg) diffuse from their regions of expression and form a heterodimer inhibitor (Sog/Tsg) that binds to Dpp-Scw, preventing it from binding to receptors. The cell matrix may mediate the formation of this complex, as it has recently been shown that collagen can bind both BMPs and Sog, thereby facilitating their association (Wang, 2008). The extracellular binding reactions lead to a gradient of inhibitor-bound Dpp-Scw that is high laterally and low at the dorsal midline, and an opposing gradient of free Dpp-Scw that is high at the dorsal midline. The dorsally secreted metalloprotease Tolloid (Tld) processes Sog only when Sog is bound to BMP ligands, and the degradation of Sog by Tld further enhances both the gradient of inhibitor-bound Dpp-Scw and of free Dpp-Scw. Thus, extracellular Dpp-Scw is redistributed by a combination of binding to inhibitor, processing of this complex, and diffusion (Umulis, 2010).
Simultaneously, receptors and other surface-localized binding proteins compete with Sog to bind the available Dpp-Scw. Dpp-Scw activates signaling by binding to and recruiting the Drosophila type I receptors, Thickveins (Tkv) and Saxophone (Sax), into a high-order complex containing two subunits of the type II receptor Punt. The receptor complex phosphorylates Mad (pMad), a member of the Smad family of signal transducers, and phosphorlyated Mad binds to the co-Smad Medea, forming a complex that then accumulates in the nucleus, where it regulates gene expression in a concentration-dependent manner (Umulis, 2010).
Although complex formation and transport favor a net movement of ligand toward the dorsal midline of the embryo, positive feedback in response to pMad signaling is needed to further concentrate the surface-localized Dpp-Scw at the dorsal midline. A loss of extracellular BMP regulators or positive feedback impedes the attenuation of pMad laterally as well as the accumulation of pMad signaling at the dorsal midline. Although feedback, extracellular transport, and signal transduction each provide a specific mode of Dpp-Scw signal regulation, it is the dynamic interaction of these regulatory mechanisms that patterns the dorsal surface of Drosophila embryos. Not only does the mechanism work under optimal laboratory conditions, but dorsal surface patterning appears to be remarkably resilient to nonideal conditions such as temperature fluctuations, reductions in the level of regulatory factors such as Tsg, ectopic gene expression, and other perturbations. These issues illustrate the complexity of the problem and suggest that it is not possilbe to rely solely on genetic and biochemical data to fully explain this rather simple patterning problem (Umulis, 2010).
To address a number of unanswered questions about Dpp-Scw-mediated patterning and to take full advantage of the available data on Drosophila development, a methodology was developed that seamlessly integrates biological information in the form of prepatterns, geometry, mechanisms, and training data into an organism-scale model of the blastoderm embryo that is based on a reaction-diffusion description of patterning. The mathematical model is simulated by using the widely available computational frameworks Comsol and Matlab, which makes extensive use of the model and methodology feasible (Umulis, 2010).
An image analysis protocol was developed to obtain model training and initial condition data and to calculate population statistics for patterns of pMad signaling in wild-type (wt) and mutant D. melanogaster. Both the mean and variability of pMad signaling along the dorsal-ventral (DV) axis depends on anterior-posterior (AP) position and the specific choice of threshold. Using mutations previously considered robust, differences could be detected between mutant and wild-type pMad signaling patterns, which provided an information-rich data set for model training and for testing the contributions of diverse positive-feedback mechanisms and of proteins that concentrate BMPs at the cell surface. Unexpectedly, it was found that geometry also has a large impact on the predicted patterns of BMP-bound receptors, whereas the prepatterned expression of receptors and other modulators of signaling did not greatly affect model-data correspondence. It was found that if the embryo geometry is perturbed slightly in the model, then including the prepattern information greatly enhanced the model's ability to fit the observed pMad patterns, which suggests that the prepatterns may mitigate the effects of slightly misshapen embryos. Conditions in the model were identified that improve the scale invariance of patterning and tested the model predictions by staining for pMad in different species of Drosophila. These studies demonstrate that building a model based on image data and training the three-dimensional (3D) model against multidimensional expression data provide insights into the properties of several important developmental principles, including positive feedback, biological robustness, and scale invariance (Umulis, 2010).
Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord (VNC), the six Tv neurons express the neuropeptide gene FMRFamide (FMRFa). Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain (LIM-HD) gene apterous (ap). The zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this 'code,' by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells (Allan, 2003).
FMRFa is specifically expressed in the six Tv neuroendocrine neurons located bilaterally in the three thoracic (T1-3) segments of the embryonic and larval VNC. apterous is expressed in three interneurons per VNC hemisegment, as well as in a lateral cluster of four neurons (the ap-cluster) in each of the T1-3 hemisegments. One of the four ap-cluster cells is the FMRFa-expressing Tv neuron. All ap interneurons in the VNC, except for the Tv, join a common ipsilateral axon tract termed the ap-fascicle. The Tv axon instead projects to the midline and exits the VNC dorsally to innervate the dorsal neurohemal organ (DNH). The DNH is a club-like neuroendocrine structure formed by two glial cells protruding from the midline of each thoracic segment. Anteriorly, two additional FMRFa-expressing cells are found, denoted SE2 cells. The SE2 cells do not express, nor depend upon, any regulators described in this study for their FMRFa expression. ap is important for the expression of FMRFa in the Tv neurons, but since most ap neurons do not express FMRFa, other regulators are likely needed for FMRFa regulation (Allan, 2003).
Rotund, a zinc finger protein of the C2H2 Krüppel-type belongs to a conserved subfamily of zinc finger proteins together with Drosophila CG5557, C. elegans Lin-29, and rat CIZ. Squeeze is most closely related to Rotund, with identity greater than 90% throughout the zinc finger region; Squeeze is 78% identical to LIN-29 in the conserved zinc finger region. Both rotund and CG5557 are expressed in subsets of cells in the developing CNS. CG5557 has a larval lethal phase. Mutants eclosed at a low frequency as immotile adults that died within 24 hr. Mutant larvae display a motility defect whereby the body wall musculature over-contract radially during the peristaltic wave typical of insect larval motility, apparent as a 'squeezing' of the intestine. Since this motility phenotype is fully penetrant and scored with 100% accuracy (sqzlacZ/sqzDf), CG5557 was renamed squeeze (sqz) (Allan, 2003).
What is the identity of the retrograde FMRFa-inducing signal? Recently, a Drosophila BMP type-II receptor, wishful thinking (wit), was implicated in mediating a retrograde signal from muscles to motor neurons, responsible for presynaptic maturation. Signaling by the TGF-β/BMP superfamily occurs via activation of a receptor complex, consisting of two type I and two type II receptors, leading to phosphorylation and nuclear translocation of a receptor Smad protein. In Drosophila, BMP signaling leads to the phosphorylation and nuclear translocation of the Smad protein Mothers against dpp (Mad), which can be monitored using antibodies specific to phosphorylated Mad (pMad) (Allan, 2003).
Using antibodies to pMad, BMP activation in peptidergic neurons was assayed. Nuclear pMad was detected not only in motor neurons, but also in the Tv, Va, and Vap neurons, demonstrating that peptidergic neurons projecting out of the VNC also show evidence of BMP activation. Accumulation of pMad in the Tv neurons commences during stage 17, immediately following DNH innervation. These results led to a test of whether Tv innervation of the DNH would be critical for pMad accumulation and consequently for FMRFa expression. Indeed, it was found that the absence of the DNH (in tin mutants), Tv axon pathfinding alterations (in apGAL4/UAS-robo and apGAL4/UAS-racV12) and interference with Tv axonal transport (in apGAL4/UAS-GluedDN and apGAL4/UAS-τ-myc) are all accompanied by loss of pMad staining specifically in Tv neurons. The ectopic ap-cluster FMRFa-expressing cell induced by sqz misexpression is also pMad positive. Given the role of sqz in Tv axon pathfinding, this is interpreted as resulting from sqz dominantly altering the projection of one other ap-cluster cell, forcing it to innervate the DNH. Thus, in all genotypes examined, Tv axonal projection to the DNH is critical for pMad accumulation (Allan, 2003).
Since Wit is expressed in a restricted pattern in the developing VNC, attempts were made to address whether the Tv neurons express Wit. However, single-cell resolution could not be obtained with the Wit antibody and Wit could not be definitely localized in Tv cells. However, the wit-dependent pMad accumulation in Tv neurons, the apGAL4/UAS-tkvA, UAS-saxA-mediated rescue of wit mutants, and the UAS-gbb-mediated 'rescue' of UAS-robo misexpression, provide genetic evidence supporting the expression of wit in Tv cells. Previous studies have shown that gbb is expressed in developing endoderm and visceral mesoderm, but it has not been detected in the VNC. By in situ hybridization, no apparent expression was detected in the DNH. Given that the DNH only contains two cell bodies, low-level gbb expression may be beyond detection. Moreover, since the anterior midgut is positioned in very close proximity to the DNHs, it is possible that Gbb diffuses from the visceral mesoderm to the DNH (Allan, 2003).
Inhibition of postsynaptic glutamate receptors at the Drosophila NMJ initiates a compensatory increase in presynaptic release termed synaptic homeostasis. BMP signaling is necessary for normal synaptic growth and stability. It remains unknown whether BMPs have a specific role during synaptic homeostasis and, if so, whether BMP signaling functions as an instructive retrograde signal that directly modulates presynaptic transmitter release. This study demonstrates that the BMP receptor (Wit) and ligand (Gbb) are necessary for the rapid induction of synaptic homeostasis. Evidence is provided that both Wit and Gbb have functions during synaptic homeostasis that are separable from NMJ growth. However, further genetic experiments demonstrate that Gbb does not function as an instructive retrograde signal during synaptic homeostasis. Rather, the data indicate that Wit and Gbb function via the downstream transcription factor Mad and that Mad-mediated signaling is continuously required during development to confer competence of motoneurons to express synaptic homeostasis (Goold, 2007).
These data advance understanding of BMP signaling at the Drosophila NMJ in several important ways. First, it was demonstrated that BMP signaling is essential for the rapid, protein-synthesis-independent, induction of synaptic homeostasis identified at this NMJ. Because expression of UAS-wit in motoneurons restores synaptic homeostasis in the wit mutant and because suppression of Mad-mediated signaling in neurons blocks synaptic homeostasis, it is concluded that BMP signaling acts upon the motoneuron to enable the rapid induction of synaptic homeostasis. Next, it was shown that the requirement for BMP signaling during synaptic homeostasis is separable from BMP-dependent support of synaptic growth and baseline neurotransmission. Finally, the temporal and spatial requirements for BMP signaling was dissected. The data support the conclusion that Mad-mediated signaling is required constitutively, downstream of the Wit receptor, in order to maintain the competence of motoneurons to express homeostatic plasticity. Further, the data argue that Gbb is not the retrograde signal that directly acts upon the presynaptic motoneuron terminal to homeostatically modulate presynaptic release (Goold, 2007).
It has been hypothesized that Gbb could function as a homeostatic retrograde signal at the Drosophila NMJ. According to this model, Gbb would be released in proportion to the perturbation of postsynaptic muscle excitation in a glutamate receptor mutant and, thereby, instruct the degree of homeostatic compensation expressed by the presynaptic motoneuron terminal. In favor of this model, homeostatic compensation observed in a glutamate receptor mutant is blocked by the wit mutation. This study present two lines of evidence that are consistent with the necessity of BMP signaling for homeostatic compensation. First, it was confirmed that the rapid induction of homeostatic compensation following application of a use-dependent glutamate receptor antagonist, Philanthotoxin (PhTx) is blocked by null mutations in both wit and gbb. Furthermore, it was shown that muscle-specific rescue of the gbb null mutation is sufficient to restore the rapid induction of homeostatic compensation (Goold, 2007).
Despite these compelling genetic data, several experiments now argue against the possibility that Gbb functions as an instructive, retrograde signal that directly modulates presynaptic release during synaptic homeostasis. First, it was found that although muscle-specific rescue of the gbb null mutation is sufficient to restore synaptic homeostasis, so is neuron-specific rescue of the gbb null mutation. Thus, homeostatic compensation can occur even in the absence of muscle-derived Gbb. These data argue against a model in which Gbb functions as the instructive retrograde signal that directly modulates presynaptic release during synaptic homeostasis (Goold, 2007).
Next, it was demonstrated that homeostatic signaling is blocked by expression of DN-Glued in neurons, which disrupts retrograde axonal transport. In this experiment, Gbb signaling at the NMJ should, in theory, persist. Furthermore, it was established that an intact motor axon is not required for the rapid induction of synaptic homeostasis. Thus, it can be concluded that trans-synaptic Gbb signaling from muscle to nerve is not sufficient for the rapid induction of synaptic homeostasis (Goold, 2007).
Given that Wit and Gbb are necessary for synaptic homeostasis, how do they participate in the process if Gbb is not the instructive retrograde signal? This study demonstrates that Mad is necessary for synaptic homeostasis, and evidence is providied that Mad-mediated signaling is required in the motoneuron. In addition, neuronal expression of UAS-Gbb restores homeostatic compensation in the presence of the DN-Glued transgene. These results suggest that the reason DN-Glued disrupts synaptic homeostasis is because it interferes with the retrograde axonal transport of P-Mad downstream of the Wit receptor. This is consistent with the prior demonstration that neuronal expression of Gbb can restore nuclear P-Mad in the presence of UAS-DN-Glued. Because the induction of synaptic homeostasis does not require the motoneuron soma, it is concluded that Gbb does not function as an acute, retrograde signal. Rather, Gbb may be a muscle-derived signal that acts developmentally to confer the competence of motoneurons to express synaptic homeostasis. Thus, the identity of the homeostatic retrograde signal at the NMJ remains unknown. It remains possible that other TGF-β superfamily signaling molecules could function at the NMJ in this capacity, including myoglianin and maverick, though it has been shown that synaptic homeostasis is intact in the baboon receptor mutant (Goold, 2007).
There are several possible ways in which BMP signaling could confer competence for motoneurons to express homeostatic plasticity. One possibility is that the BMPs control a transcriptional program that is necessary for synaptic homeostasis. For example, BMPs are potent regulators of cell fate during embryonic development. Perhaps the ability of motoneurons to express synaptic homeostasis is related to the maintenance of their cellular or electrical identity. An alternate possibility is that BMPs control the expression of essential presynaptic proteins that are required for synaptic homeostasis. For example, it has been shown in other systems that target-dependent TGF-β signaling can modulate neuronal ion channel expression. It has been demonstrated that CaV2.1 calcium channels are required for synaptic homeostasis at the Drosophila NMJ. However, it is considered unlikely that BMPs control synaptic homeostasis through the regulation of CaV2.1 channel expression because there is not a strong correlation between altered baseline synaptic transmission and the expression of synaptic homeostasis. Furthermore, overexpression of a GFP-tagged CaV2.1 calcium channel (cacophony-GFP) is unable to restore synaptic homeostasis when coexpressed with UAS-dad. Finally, BMP signaling could influence the expression of synaptic homeostasis by targeting the rate of spontaneous miniature release. Spontaneous release events that persist in the absence of evoked neurotransmission are sufficient to induce homeostatic compensation at the Drosophila NMJ. However, no strong correlation is found between baseline mEPSP frequency and whether or not a mutant NMJ is able to express synaptic homeostasis. Although the wit mutants show a severe decrease in mEPSP rate compared to wild-type, the expression of UAS-dad or UAS-DN-Glued both block synaptic homeostasis without severely impairing baseline mEPSP rate. Ultimately, continued forward genetic investigation of homeostatic signaling may be required to identify the BMP-dependent mechanisms that control the expression of synaptic homeostasis (Goold, 2007).
BMP signaling is required for NMJ growth, baseline neurotransmission, and NMJ stability in addition to being required for synaptic homeostasis. It is a challenge, therefore, to determine whether BMP signaling has a specific function during synaptic homeostasis versus a more general role during synapse development. This study presents several lines of evidence that BMP signaling may have a separable function during synaptic growth versus synaptic homeostasis. First, it was demonstrated that synaptic homeostasis can occur at BMP mutant synapses that show severely impaired synaptic growth. For example, the gbb hypomorphic mutant has a decrease in bouton number that is just as severe as the gbb null mutant, but the gbb hypomorphic mutant shows normal homeostatic compensation. As another example, animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in bouton number but normal homeostatic compensation. Thus, it is concluded that normal BMP-dependent synaptic growth is not required for the expression of synaptic homeostasis (Goold, 2007).
It was also possible to dissociate BMP-dependent baseline transmission from both synaptic growth and synaptic homeostasis. (1) Muscle-specific rescue of the gbb null mutation significantly restores synaptic growth and rescues synaptic homeostasis, but baseline transmission remains at levels observed in the null mutant. (2) Motoneuron-specific rescue of the wit mutation (OK371-GAL4) similarly rescues bouton number and synaptic homeostasis, although baseline transmission remains severely impaired. (3) Animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in baseline transmission but normal homeostatic compensation. (4) Results were obtained that show the converse effect. When UAS-dad is expressed for 1.5 days at the end of larval development, both synaptic homeostasis and baseline transmission are significantly impaired, but synaptic bouton numbers remain wild-type. From these data it is concluded that impaired synaptic homeostasis is not a secondary consequence of BMP-dependent functional NMJ development. It also appears that there may be distinct effects of BMP signaling on the anatomical versus functional development of the NMJ. One possibility, consistent with BMPs being a classical morphogen, is that different levels of the ligand could initiate specific transcriptional programs with distinct effects on bouton number, baseline transmission, and homeostatic plasticity. It is also possible that the site of action of BMP signaling will play an important role in specifying signaling outcome (Goold, 2007).
It has been speculated that synaptic homeostasis might function, over the course of development, to ensure that the muscle cell is normally depolarized by the NMJ. How can one explain the observation that csp and syx/+ mutations have decreased baseline neurotransmitter release but normal acute synaptic homeostasis in response to PhTx application, or other genotypes explored in this study that show impaired baseline transmission and normal acute synaptic homeostasis? It has been demonstrated that the acute induction of synaptic homeostasis is independent of evoked neurotransmission. Thus, synaptic homeostasis may not function to modulate the absolute amplitude of evoked neurotransmitter release. Rather, synaptic homeostasis might be a rapid system to offset acute perturbations of postsynaptic receptor function. In this case, developmental programs that specify NMJ anatomy and active zone addition would achieve the reproducible development of the NMJ. Alternatively, the mechanisms of acute homeostatic compensation following PhTx application may be separable, either temporally or molecularly, from the other potential mechanisms that monitor and homeostatically control evoked EPSP amplitudes (Goold, 2007).
The data also suggest a possible link between the expression of homeostatic plasticity and the mechanisms of neuromuscular degenerative disease. Genetic mutations that impair retrograde axonal transport have been shown to cause familial amyotrophic lateral sclerosis. It has also been shown that, in Drosophila and mice, mutations that disrupt dynein-dynactin complex function lead to neuromuscular synapse degeneration. It is hypothesized that impaired retrograde axonal transport deprives motoneurons of muscle-derived trophic support leading to motoneuron degeneration. This study has demonstrated that impaired retrograde axonal transport blocks the expression of homeostatic plasticity at the NMJ. This deficit can be restored by expression of BMPs in the central nervous system, bypassing retrograde axonal transport as the source of BMPs to the motoneuron cell body. It is tempting to speculate that impaired synaptic homeostasis at the NMJ may play a role in the progression of motoneuron disease associated with impaired retrograde axonal transport (Goold, 2007).
Finally, the data could have relevance to the sustained expression of homeostatic plasticity in regions of the adult nervous system. BMPs and downstream signaling proteins such as the Smads continue to be expressed in the adult nervous system. In particular, BMPs are secreted into the cerebral spinal fluid at concentrations that are relevant for neuronal signaling. It is, therefore, interesting to speculate that circulating levels of BMPs might sustain the competence of neurons to express homeostatic plasticity without driving morphological plasticity in the adult nervous system (Goold, 2007).
The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila. The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments. A new site of dpp expression has been found in the posterior wing compartment that appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity that is independent of that originating in the anterior compartment. This posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. It was further shown that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, it is suggested that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs (Foronda, 2009).
This study was triggered by a consistent observation of a small region in the P compartment of the wing disc that appeared to be active in dpp transcription. This P compartment expression of dpp has not been properly analysed in previous works about Dpp function in wing disc. dpp expression was carefully examined in third instar wing and haltere discs by in situ hybridization and also with a P-element insertion (P10638) at the disk region of the dpp gene. Transcriptional activity was identified close to and anterior to the A/P border. In addition, dpp transcripts were found in a proximal region of the posterior compartment. A homologous zone of dpp expression was also found in the posterior region of the haltere disc. This dpp posterior transcriptional domain appears during the third larval period; wing discs from early 3rd larval instar do not show it (Foronda, 2009).
According to the fate map, the posterior region containing dpp expression gives rise to proximal adult wing structures, including the alula and the axillary cord. This domain in adult wings was delimited by X-gal staining freshly emerged flies carrying the dpp-lacZ insertion. The area with lacZ activity corresponds mostly to the axillary cord (Foronda, 2009).
To test whether the posterior dpp expression activates the Dpp transduction pathway, use was made of an antibody raised against the phosphorylated (active) form of Mad, an indicator of Dpp pathway activity. As expected from previous work there are high levels of pMad in the centre of the disc, but in addition a domain of pMad activity was observed in the posterior compartment, which includes the dpp expression domain. The zone expressing pMad is bigger than that expressing dpp, consistent with the formation of a diffusion gradient of Dpp activity (Foronda, 2009).
In addition to pMad levels, whether other elements of the Dpp pathway are expressed in the posterior dpp domain was also examined. The gene daughters against dpp (dad) is a target that requires moderate levels of Dpp signalling. dad is expressed in the posterior wing compartment in the same region where pMad is active. Other Dpp-target genes, like optomotor blind and spalt, are also expressed in this region (Foronda, 2009).
The brinker (brk) gene is a special case as it is negatively regulated by Dpp activity. Therefore, it is expressed in the lateral regions of the wing disc where the levels of Dpp activity are lower. The domain of Dpp activity in the proximal posterior compartment should therefore repress brk activity in that region. The comparison of pMad and brk activities in this region clearly shows they occupy mutually exclusive domains, supporting the idea that brk is repressed by the posterior pMad activity. The staining of adult wings of brk-lacZ genotype also argues in the same direction because the region of brk expression does not coincide but abut with that containing Dpp activity. Although the haltere disc was not analyzed with the same detail, there is also a posterior region of pMad activity in the zone where dpp is expressed (Foronda, 2009).
pMad and brk expression were examined in wing discs from second instar larvae. These exhibit the central domain of pMad activity but there is no detectable staining in the posterior compartment. This result is consistent with the late appearance of the posterior dpp transcription domain and indicates that this domain functions only during the second half of the proliferation phase of the disc (Foronda, 2009).
Having shown that the posterior dpp expression domain acts as a source of morphogen the question was addressed whether it has a functional role. Previous experiments analysing dpp mutant clones did not report significant alterations in the posterior wing. However, given the diffusible nature of the Dpp product it is possible that the lack of Dpp in the mutant clones could be rescued by Dpp emanating from neighbour wildtype cells (Foronda, 2009).
An experiment was designed in which all cells in the posterior compartment would be homozygous for the dppd12 mutation, which eliminates dpp activity in the wing disc without affecting embryonic or pupal expression. In discs of genotype dppd12 ck FRT40A/M (2)24F arm-Z FRT40A; hh-gal4/UAS-Flp the high levels of flipase generated by the hh-gal4 driver would induce FRT-mediated mitotic recombination in virtually all the cells in the posterior compartment. The dpp− M+ clones produced will have proliferation advantage and are expected to fill the posterior compartment. They can be identified in the disc because they lose the arm-lacZ transgene, and in the adult wing because they are homozygous for the cuticular marker crinkled (ck) (Foronda, 2009).
These clones entirely fill the posterior compartment. Staining for pMad activity shows the normal pattern in the centre of the disc, but the posterior pMad domain is completely absent, in clear contrast with wildtype discs (Foronda, 2009).
Since the genotype used allows good viability of the flies containing dpp− M+ clones, a large number of adult wings was examined. In nearly every case the entire posterior compartment is marked with ck, indicating that it lacks dpp expression. The pattern and size of these wings is normal except in the proximal posterior region: the alula does not form and the pattern that appears in its place resembles that of a more distal region, which is specified by Dpp of anterior origin. The axillary cord is much reduced in size and has lost all its characteristic long hairs; some sclerites are also missing. This loss of structures does not appear to be due to death of proximal cells; no indication of caspase activity was found in this region. Moreover, the addition of the apoptosis inhibitor P35 in the posterior compartment does not modify the phenotype of the dpp− M+ compartments (Foronda, 2009).
The favored interpretation of the wing phenotype is that in absence of the organising activity of the posterior Dpp, the proximal region is only patterned by Dpp of anterior provenance. It is interesting to note that even though dpp is not expressed in the alula cells, this structure is affected. This result illustrates the role of the posterior Dpp as an organizer, since it affects patterning in a non-autonomous manner (Foronda, 2009).
One intriguing question was if this posterior Dpp could form these proximal structures without Dpp of anterior origin. A transgenic strain carrying UAS-shmiR-dpp2 construct was used which has been shown to degrade the mRNA of Dpp. The smhiR-dpp over-expression in the anterior Dpp domain (by using ptc-gal4 or dppblnk-gal4) is able to silence efficiently Dpp gene activity, as shown by absence of pMad staining and the extended brk expression domain. Neither the posterior dpp expression nor the formation of alula and axillary cord are affected in both size and morphology. It is concluded that Dpp of posterior origin is necessary and sufficient to pattern these structures (Foronda, 2009).
dpp transcription in the P compartment is an intriguing finding and suggests a novel mode of dpp activation. The normal activation of dpp in the A compartment requires Hh signalling, which is blocked in the P compartment. Alternatively, it was possible that a local diminution of en activity in the proximal region of the P compartment would allow Hh and dpp activation by the standard mechanism. This question was addressed by examining in this region the expression of Cubitus interruptus (ci), a transcription factor that is essential for Hh signalling, and of patched (ptc), a Hh target gene. It was found that neither ci nor ptc are expressed in the posterior. However, there was the possibility that these two genes were expressed at low, undetectable levels. To test this possibility in full, Dpp activity was examined in clones of cells mutant for the smoothened (smo) gene, which would be unable to transduce the Hh signal. The result demonstrates that the posterior expression of dpp does not require Hh signalling and must therefore be activated by a different mechanism (Foronda, 2009).
The approach taken to identify the factor/s behind this posterior Dpp expression was to look for candidate genes or signalling pathways which are expressed in the corresponding place in the posterior compartment. The first one was vein, a ligand of EGFR signalling pathway, which coexpresses with Dpp in late 3rd instar wing disc. vein is the only EGFR ligand required for a proper wing development, so it was a good candidate. The elimination of all posterior vein function has effect neither on posterior Dpp function nor on hinge morphology. Other genes were tested based on expression and/or mutant phenotype in the alula, i.e., homothorax, Zfh2 and empty spiracles, among others. None of them affected Dpp expression (Foronda, 2009).
Another likely candidate was mirror, a member of the Iroquois complex (iro-C), for which a role in alula and axillary cord formation has been described. mirr expression was examined using the mirr-lacZ line and it was found that mirr is expressed in the presumptive alula and axillary cord region (Foronda, 2009).
M+ mirr− clones were made to generate posterior compartments that were wholly mirr−. They show an adult phenotype more extreme than that of dpp− compartments: the alula and the axillary cord are entirely missing. In the discs dpp expression (shown by pMad staining) in the posterior compartment is lost, and consequently brk expression is up-regulated in the presumptive alula region. This result indicates that mirr is necessary for posterior dpp expression. In contrast, Dpp is not required for mirr expression, since the lack of posterior Dpp does not have an effect on mirr-LacZ transcription (Foronda, 2009).
Since the preceding results might suggest a mirr-mediated dpp activation gain of function clones of mirr were generated and whether they gave rise to ectopic Dpp activity was checked. NI significant up-regulation of dpp associated with those clones was detected. These experiments demonstrate that mirr activity is necessary for posterior dpp expression, but it is not sufficient to induce it. Therefore there must be other factors involved in posterior dpp activation (Foronda, 2009).
These results report a novel organizer role of Dpp that occurs during the third instar and is necessary and sufficient to pattern the proximal posterior region of the wing. This is achieved by an hh-independent, mirr-dependent activation of dpp in the posterior compartment (Foronda, 2009).
These findings provide a more complete picture of the development of the wing disc. There is evidence that the three signalling Dpp, Wg and Hh pathways are necessary for normal wing pattern and originate at compartment borders. The results indicate an unforeseen complexity of the function of the Dpp pathway. dpp becomes active in two different body domains, which have independent temporal and spatial regulation. The anterior domain is required for distal wing growth and patterning, whereas the P compartment domain is responsible for the formation of the posterior-proximal structures of the wing. Moreover, the source of Dpp in the posterior domain does not appear to be a compartment border, since no lineage restriction has been reported in the region. A comparable situation has been described for leg development, in which the Dpp and Wg signals originate at the A/P border, but the source of the EFGR signal is not a lineage border (Foronda, 2009).
The fact that this new dpp expression domain is common to wings and halteres may have some evolutionary significance, since this may be an attribute of dorsal thoracic appendages. These results suggest that the posterior tier of dpp expression may have appeared before the mesothoracic and metathoracic appendages diverged. It is therefore, possible that the new model of dorsal appendage development proposed may occur in other species of insects (Foronda, 2009).
Steroid hormones are required in Drosophila for progression of oogenesis during late stages of egg maturation. This study shows that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay switching on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed (König, 2011).
This study shows that in Drosophila ecdysone signalling regulates differentiation of a GSC daughter and modulates ovarian stem cell niche size. The delay in GSC progeny differentiation correlates with reduced expression levels of TGF-β pathway components. Based on expression patterns it appears that germarial somatic cells, niche and ECs are the critical sites of ecdysteroid action and a co-activator of ecdysone receptor, Taiman is the spatially restricted regulator of ecdysone signalling in soma. During adulthood the ecdysone pathway has a specific role in EC differentiation and soma-germline cell contact establishment. In addition, during development the ecdysone signalling pathway has a role in somatic niche formation (König, 2011).
Ecdysteroids in general control major developmental transformations such as metamorphosis and morphogenesis in Drosophila. Different tissues and even different cell types within the same tissue respond to this broad signalling in a specific fashion and in a timely manner. In the developing Drosophila ovary steroid hormone receptors are expressed in a well-timed mode, high levels coinciding with proliferative and immature stages and low levels preceding reduced DNA replication and differentiation. Mutations in ecdysone pathway components affect ovarian morphogenesis, including heterochronic delay or acceleration in the onset of terminal filament differentiation. During the niche establishment the levels of both ecdysone receptors, EcR and USP are greatly downregulated in anterior somatic cells that will contribute to the niche per se. This study shows that perturbation of ecdysone signalling in pre-adult ovarian soma leads to the formation of enlarged niches. The specific response to systemic hormonal signalling in niche precursors is achieved by a specific function of the ecdysone receptor co-activator Taiman. When timely regulation of ecdysone signalling does not occur, more cells are recruited to become niche cells resulting in enlarged niches that are capable to host more stem cells. These data first show that ecdysone steroid hormonal signalling regulates the formation of the adult stem cell niche and suggest that a developmental tuning of ecdysone signalling controls the number of anterior somatic cells that will differentiate into cap cells (König, 2011).
It is logical that stem cell division and germline differentiation are regulated by some systemic signalling depending on the general state of the organism, which depends on age, nutrition, environmental conditions and so on. Hormones are great candidates for this type of regulation as they act in a paracrine fashion and their levels are changing in response to ever-changing external and internal conditions. Steroid binding to nuclear receptors in vertebrates triggers a conformational switch accompanied by increased histone acetylation that permits transcriptional co-activators binding and the transcription initiation complex assembly. In Drosophila, the trithorax-related protein, a histone H3 methyltransferase that like Taiman belongs to the p160 class of co-activators, and an ISWI-containing ATP-dependent chromatin remodelling complex (NURF), that regulates transcription by catalysing nucleosome sliding, both bind EcR in an ecdysone-dependent manner, showing that chromatin modifications can mediate response to this general signalling. Transcriptional regulation has a key role in GSC maintenance and differentiation, for example, the TGF-β ligand Dpp secreted by niche cells induces phosphorylation of the transcription factor Mad in GSCs that in turn suppresses transcription of the differentiation factor Bam. In addition, it has been shown recently that in Drosophila adult GSC ecdysone modulates the strength of TGF-β signalling through a functional interaction with the chromatin remodelling factors ISWI and Nurf301, a subunit of the ISWI-containing NURF chromatin remodelling complex (Ables, 2010). Therefore, it is plausible that ecdysone regulates Mad expression cell autonomously via chromatin modifications. Since pMad directly suppresses a differentiation factor Bam, it is expected that Bam would be expressed in pMad-negative cells. Interestingly, the findings show that ecdysone deficit decreases amounts of phosphorylated Mad in GSCs and also cell non-autonomously suppresses Bam in SSCs. As SSCs that express neither pMad nor Bam are accumulated when the ecdysone pathway is perturbed it suggests that there should be an alternative mechanism of Bam regulation. Even though eventually this still can be done on the level of chromatin modification, the data suggest that the origin of this soma-generated signal may be associated with cell adhesion protein levels. Further understanding of the nature of this signalling is of a great interest (König, 2011).
The progression of oogenesis within the germarium requires cooperation between two stem cell types, germline and somatic (escort) stem cells. In Drosophila, reciprocal signals between germline and escort (in female) or somatic cyst (in male) cells can inhibit reversion to the stem cell state and restrict germ cell proliferation and cyst growth. Therefore, the non-autonomous ecdysone effect can be explained by the necessity of two stem cell types that share the same niche (GSC and ESC) to coordinate their division and progeny differentiation. This coordination is most likely achieved via adhesive cues, as disruption of ecdysone signalling affects turnover of adhesion complexes and cytoskeletal proteins in somatic ECs: mutant cells exhibited abnormal accumulation of DE-Cadherin, β-catenin/Armadillo and Adducin (König, 2011).
Cell adhesion has a crucial role in Drosophila stem cells; GSCs are recruited to and maintained in their niches via cell adhesion. Two major components of this adhesion process, DE-Cadherin and Armadillo/β-catenin, accumulate at high levels in the junctions between GSCs and niche cells, while in the developing cystoblasts and escort cells levels of these proteins are strongly reduced. Levels of DE-Cadherin in GSCs are regulated by various signals, for example, nutrition activation of insulin signalling or chemokine activation of STAT, and this study shows that in ESCs it is regulated by steroid hormone signalling. Possibly, these two stem cell types respond to different signals but then differentiation of their progeny is synchronised via cell contacts. While hormones, growth factors and cytokines certainly manage stem cell maintenance and differentiation, the evidence also reveals that the responses to hormonal stimuli are strongly modified by adhesive cues (König, 2011).
Specificity to endocrine signalling can be achieved via availability of co-factors in the targeted tissue. Tai is a spatially restricted co-factor that cooperates with the EcR/USP nuclear receptor complex to define appropriate responses to globally available hormonal signals. Tai-positive regulation of ecdysone signalling can be alleviated by Abrupt via direct binding of these two proteins that prevents Tai association with EcR/USP (Jang, 2009). Abrupt has been shown to be downregulated by JAK/STAT signalling (Jang, 2009). Interestingly, JAK/STAT signalling also has a critical role in ovarian niche function and controls the morphology and proliferation of ESCs as well as GSCs. JAK/STAT signalling may interact with ecdysone pathway components in ECs to further modulate cell type-specific responses to global endocrine signalling. A combination of regulated by different signalling pathway factors that are also spatially and timely restricted builds a network that ensures the specificity of systemic signalling (König, 2011).
Knowledge of how steroids regulate stem cells and their niche has a great potential for stem cell and regenerative medicine. The current findings open the way for a detailed analysis of a role for steroid hormones in niche development and regulation of germline differentiation via adjacent soma (König, 2011).
The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, BMP signaling was analyzed in the FCs of 16 Drosophila species that span 45 million years of evolution. The spatial patterns of BMP signaling in the FCs were found to be dynamic and exhibit a range of interspecies variation. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. These results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning (Niepielko, 2011).
In FCs of D. melanogaster, dynamics of BMP signaling are regulated by the dynamics of tkv. In most Drosophila species, a correlation between tkv expression and BMP signaling dynamics was found. Remarkably, ectopic expression/depletion of tkv was sufficient to diversify BMP signaling in D. melanogaster. These perturbations successfully transformed the pattern of BMP signaling found in D. melanogaster into the diverse patterns of BMP signaling naturally found across Drosophila species, supporting the claim that tkv plays a major role in specifying the distribution of BMP signaling in the FCs. In D. melanogaster, a similar mechanism restricts the distribution of BMP signaling in wing and haltere imaginal discs (Niepielko, 2011).
In D. melanogaster, the dynamics of tkv are regulated jointly by BR and BMP signaling. Thus, the late patterns of BR, TKV, and P-MAD are observed in a similar group of cells. Surprisingly, in some species, the pattern of P-MAD correlated with the pattern of tkv; however, these patterns did not fully overlap the BR domain. Specifically, in addition to overlapping the BR domain, tkv was expressed in the adjacent cells that lacked BR expression. It is proposed that, in addition to being regulated by BR and BMP signaling, tkv is also regulated by transcription factors that are expressed in the adjacent domain such as Jra and Fos/Kayak (Niepielko, 2011 and references therein).
Of particular interest are species with three DAs' eggshell, due to the absence of tkv from the BR domain. It is suggested that this signaling pattern provides an example for decoupling of the regulation of tkv by BR from its regulation by BMP signaling. A model is proposed by which tkv is regulated by BMP signaling (anterior); however, it lost its regulation by BR. Of note, the mechanism governing tkv patterning is still unknown, and the established tkv enhancer trap in the Drosophila wing failed to recapitulate the patterns of tkv in the FCs. Thus, the proposed modifications in tkv regulation remain to be experimentally validated (Niepielko, 2011).
In D. melanogaster, early BMP signaling appears as an anterior stripe, reflecting the anterior secretion of the ligand DPP that signals through a uniformly expressed tkv receptor. Thus, in species from the virilis repleta groups, the uniform expression of tkv could account for the anterior stripe domain of late BMP signaling. Indeed, ectopic expression of tkv in all FCs prevented the dorsal anterior repression of BMP signaling in D. melanogaster, which was consistent with the pattern found in flies from the virilis repleta groups (Niepielko, 2011).
In the first three patterning classes, spatial modifications in the late patterns of tkv provided a reasonable explanation for the diversity in late patterns of BMP signaling. In the virilis repleta groups, late expression of tkv was uniform in all FCs; at the same time, BMP signaling was patterned. While the possibility cannot be excluded that a second copy of tkv is present in the non-sequenced species, it is proposed that in these species other BMP components have evolved to gain control of BMP signaling dynamics. In D. melanogaster, the disruption of saxophone (sax), a type I BMP receptor, deformed operculum size and DAs' morphologies. Thus, SAX is a potential regulator of the BMP signaling dynamics in the virilis repleta groups. Also, additional mechanisms were shown to regulate BMP signaling across animals including intracellular and extracellular inhibitors, co-receptors, levels of ligand expression, combinations of ligands, and interactions with other signaling pathways. These mechanisms should be studied systematically in order to determine which BMP component controls the DV phase of signaling in these species (Niepielko, 2011).
In D. melanogaster, the early phase of BMP signaling prevents the anterior domain of the follicle cells from acquiring DA cell fate. This mechanism is based on the inhibition of br expression by the anterior BMP signaling. Thus, it is not surprising that disruption of early BMP signaling is associated with eggshell deformations including modifications in the numbers and shapes of DAs'. Due to the high sensitivity of the eggshell's structures to changes in BMP signaling, it is speculated that small differences in early BMP signaling could guide the natural variations in numbers and shapes of DAs found across Drosophila species (Niepielko, 2011).
In D. melanogaster, the late phase of BMP signaling is associated with the repression of br mRNA in the dorsolateral patches; however, its role in eggshell morphology was not explicitly explored. Depletion of BMP signaling from the BR domain did not affect early BR patterning and operculum size; however, this perturbation deformed DAs' morphology possibly due to late migration of BR cells. Interestingly, similar morphologies were found by disrupting Cad74A, a cadherin gene regulated by BMP signaling that was found to be important for proper eggshell morphology. BMP signaling regulates cadherins in the pupal retina of D. melanogaster and in the human renal epithelial cells. It is proposed that late BMP signaling is involved in the morphological processes of DAs' formation by affecting cell adhesion molecules. Other cadherin genes are expressed or repressed in the DAs' forming cells, and it will be interesting to study how their regulation by BMP signaling affects DAs' morphogenesis (Niepielko, 2011).
Mothers against dpp is required for dpp function.
Mad was identified in screens to identify genes that interact with dpp. Mad loss-of-function mutations interact with dpp
alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects,
suggesting a role for MAD in mediating some aspect of DPP function. In support of this,
homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk
development and embryonic dorsal-ventral patterning reminiscent of dpp mutant
phenotypes (Sekelsky, 1995).
Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex
combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when Scr is
expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr
is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins
encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland
genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic
prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland
gene activation have been identified: two known receptors (the type I receptor encoded by the
thick veins gene and the type II receptor encoded by the punt gene); two of the four known
Drosophila members of the Smad family of proteins which transduce signals from the receptors to the
nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription
factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and
dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999).
To determine roles for Medea during larval development, clones mutant for Medea have been examined in the eye. Dpp has an important role in the initiation and progression of the
morphogenetic furrow. Mad clones in the posterior of the eye result in the loss of eye
structures, which are instead replaced by head cuticular
structures. These clones showed the ectopic expression of wingless (wg), a gene that is normally repressed by dpp signaling and is required at the lateral margins of the
eye disk to regulate the proper timing of furrow initiation and
progression. Hence clones of Mad mutant cells were unable to transduce the Dpp signal and are
unable to initiate the morphogenetic furrow. Clones of the strong Medea alleles, Med 1 and Med 26 were induced, and these clones gave very similar phenotypes to
Mad clones, such as loss of eye tissue. Such clones are
observed only at the margins of the eye, most commonly the
posterior margin, where the furrow initiates. This indicates that
Medea has overlapping functions with Mad in dpp signaling
during furrow initiation. Clonal analysis with Medea has also revealed abnormalities
in other tissues, in keeping with its involvement in dpp
signaling. For example, partial duplications
of the leg are observed, a phenotype reported in the dorsal regions of the leg for clones of
the Dpp receptor, punt. These analyses strongly suggest a
closely related function for Medea and dpp during imaginal
disc development (Das, 1998).
Since Mad and Medea are separately mutable, it is expected
that they function non-redundantly and cannot substitute for each other. Consistent with this model, ubiquitous expression of Mad (Ubi-Mad) cannot rescue Medea lethality. To further examine the relationship between these two Smads, a sensitized assay system was used. This assay
utilizes the dominant maternal effect lethality of Mad and
Medea with dpp. The extent of this lethality depends
on the strength of the Mad or Medea allele and the dpp allele with which it
is crossed. Crossing a strong, hypomorphic dpp allele,
dpp hr27, to the strongest available alleles of Mad, (Mad 12) or
Medea (Med 1), results in 100% lethality of both dpp classes
among the progeny. As expected, Ubi-Mad can rescue the
maternal effect lethality of Mad 12, and Ubi-Medea rescues that of Med 1.
The effects of introducing Ubi-Medea or Ubi-Mad from Mad/+ or Medea/+ females, respectively, were examined. Interestingly, a Ubi-Medea transgene can reduce the maternal
effect lethality of Mad 12 /+ females with dpp from 100% to
12%, while Ubi-Mad reduces that of Med 1/+ females to 68%. To assay whether this rescue is simply due to increased levels of dpp pathway components, a Ubi-tkv
line was usedin the same assay system. While this line is able to
rescue a tkv mutant, it cannot rescue
the maternal effect lethality associated with Mad or Medea. The lower extent of Ubi-Mad rescue of Medea
maternal effect lethality, may be due to the fact that Med 1 may
be an antimorphic allele. Alternatively, this may be indicative
of an important aspect of Smad function.
While it is clear that Mad and Medea cannot substitute for
each other, the genetic data argue that a reduction in one class
of Smads can be at least partially compensated by augmenting
the dosage of the other Smad class. This compensation may be
a Smad-specific feature, as elevated levels of tkv do not yield
the same results. The simplest explanation for these genetic
observations is that increased levels of one class of Smads may
enhance the ability of the other class to signal (Das, 1998).
Stem cells are thought to occupy special local environments, or niches, established by neighboring cells that give them the capability for self-renewal. Each ovariole in the Drosophila ovary contains two germline stem cells surrounded by a group of differentiated somatic cells that express hedgehog and wingless. The BMP2/4 homolog decapentaplegic (dpp) is specifically required to maintain female germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Overexpressing dpp for 3 days after eclosion produces tumorous germaria. Large germline cells filling germarial regions 1 and 2a contain spectrosomes but showed no
evidence of cyst formation. In regions 2b and 3, 16-cell cysts are observed that probably derive from
differentiated cystoblasts, or cysts that had formed before the first heat shock. This phenotype is very similar to that of bag of marbles (bam) and benign gonial cell
neoplasm (bgcn) mutants. These results suggest that ectopic Dpp inhibits cystoblast differentiation but does not block cyst formation and
maturation (Xie, 1998).
Mutations in dpp or its receptor (saxophone) accelerate stem cell loss and retard stem cell division. Mutant germline stem cell clones were constructed to show that the dpp signal is directly received by germline stem cells. punt, thickveins, mad, Medea and Dad are all shown to be required cell-autonomously for germline stem cell
maintenance; punt, tkv, mad, and Med are shown to be required cell-autonomously to stimulate germline stem cell division. During aging, the number and activity of stem cells is thought to be reduced. The level of
dpp signaling is shown to control the life span and division rate of germline stem cells. Reduced dpp signaling causes
premature stem cell loss. Perhaps more surprising is the observation that putative increases in signaling, caused
by removal of Dad activity from stem cells, causes them to be maintained longer. This finding suggests that dpp
signaling not only is necessary, but may sometimes be rate limiting for stem cell maintenance. This is the first
example where stem cell life span has been extended in an intact organism. Thus, dpp signaling helps define a niche that controls germline stem cell proliferation (Xie, 1998).
Mad acts as a dominant enhancer of vrille phenotypes in wing. About 10% of Mad6+/+ vri2 flies show a wing phenotype. The L5 vein is shortened and sometimes the posterior cross vein is also shortened and extra vein material is observed along the L2 vein. The same phenotype is observed with vri1 whereas with the other alleles the effect is weaker. Since this phenotype in not observed in the Mad/+ and vri/+ controls, it is concluded that it is due to the association of both genes. In order to investigate a possible interaction between vri and dpp in wing, a dominant effect of vri was sought in a dpp- context. The dpphr4/dppd6 phenotype consists of a reduction of wing to about one half of the wild type size and no defects in eyes or legs. When one dose of vri is associated with this genotype in dpphr4 vri2/dppd6 + flies, a further reduction in wing size is observed with reduction of veins. Furthermore, eyes are smaller with a rough aspect and legs are truncated. The enhancement of dpp phenotypes by vri2 is always observed, although the strength of the enhancement is variable. The same phenotypes are observed with vri1 (George, 1997).
TGF-beta comprise a superfamily of secreted proteins with diverse functions in patterning and cell division control. TGF-beta signaling has been implicated in synapse assembly and plasticity in both vertebrate and invertebrate systems. wishful thinking, a Drosophila gene that encodes a protein related to BMP type II receptors, has been shown to be required for the normal function and development of the neuromuscular junction (NMJ). These findings suggest that a TGF-beta-related ligand activates a signaling cascade involving type I and II receptors and the Smad family of transcription factors to orchestrate the assembly of the NMJ. This study demonstrates that the TGF-beta type I receptor Saxophone and the downstream transcription factor Mothers against dpp (Mad) are essential for the normal structural and functional development of the Drosophila NMJ, a synapse that displays activity-dependent plasticity (Rawson, 2003).
Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic and Screw. short gastrulation, which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Sog has a second function, which is to promote BMP signaling on the dorsal side of the embryo. A weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and this lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of Sog is directed against Scw, data are presented that suggest that the positive activity of Sog is directed towards Dpp. Chordin shares the same apparent ligand specificity as does Sog, preferentially inhibiting Scw but not Dpp activity. However, in Drosophila assays, Chordin does not have the same capacity to elevate BMP signaling as does Sog, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates (Decotto, 2001).
Morphogen gradients, once a purely theoretical concept, are now viewed as central players in the establishment of cell identity in a broad range of developmental processes. However, the exact biological mechanisms used to establish and maintain a morphogen gradient vary, depending on the biological context. In the Drosophila embryo, while Dpp can act in a dose-dependent fashion to specify different cell fates along the DV axis, in vivo its activity is modulated spatially by other components of the patterning system. In particular, Sog, a diffusible BMP-binding protein, has been shown to inhibit BMP signaling ventrally by preventing ligand access to the BMP receptors. A novel aspect of Sog’s function has been characterized in this study. Specifically, Sog functions cell non-autonomously to elevate BMP signaling on the dorsal side of the embryo. Thus, the interpretation of any experiment to elucidate the role of Sog in the control of dorsoventral patterning must take into account the two apparently opposing functions of the protein (Decotto, 2001).
Loss-of-function mutations in Mad or Medea have been identified as dominant enhancers of a weak homozygous-viable sog mutation, and the enhanced embryos have been shown to have defects in amnioserosa specification. Furthermore, synthetic lethality between weak homozygous-viable alleles of sog and zen has been demonstrated, indicating that both are required for maximal production of amnioserosa. Lastly, there was a dramatic decrease in the level of zen transcription in sogP129D embryos that were derived from Mad/+ females, compared to the level of zen transcription in either genotype alone. Taken together, these results unambiguously demonstrate that the positive action of Sog is exerted before gastrulation to attain the maximal expression of a direct BMP target gene (Decotto, 2001).
In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).
The development of cells mutant for all three transcription factors, Mad, Su(H), and ci is a helpful starting
point, since they may reflect a 'ground state' of eye development that requires extracellular signals to differentiate. Mad Su(H) ci cells fail to express the atonal or senseless genes that initiate R8
differentiation, and, consequently, fail to support retinal differentiation.
This shows that the absence of Ci75 is not sufficient for differentiation. Dpp alone can induce Ato [e.g., in Su(H) ci clones], but N and Dpp
signaling together are required to activate Atonal with normal kinetics, as
occurs in ci-mutant cells. N signaling alone (in tkv ci
clones) is insufficient. In the presence of Ci, prompt differentiation
requires Hh to downregulate Ci75, and differentiation is delayed in
Smo clones that lack this input. The normal role of Hh is not just
to remove Ci75 thus permitting Dpp and N to work, because Atonal is turned on
normally in Mad Su(H) clones that do not respond to Dpp or N signals.
Such differentiation depends exclusively on Hh yet progresses normally, except that a neurogenic phenotype reflects dependence of lateral inhibition on Su(H). Hh depends positively on ci to drive differentiation
in Mad Su(H) cells and, therefore, requires Ci155. The positive role
of ci can also be inferred from the delayed differentiation of
Su(H) ci clones in comparison with Su(H) clones (Fu, 2003).
Hairy is downregulated redundantly by Hh and N signaling.
Prolonged Hairy expression is not sufficient to block differentiation
completely but it does antagonize it (e.g., in Su(H) ci clones).
Downregulation of Hairy in response to Hh as well as N explains why both
ci and Su(H) mutant clones can differentiate promptly, and
why N enhances differentiation in response to Dpp but is not required for
differentiation in response to Hh (Fu, 2003).
Comparison between Mad Su(H) ci cells and Su(H) ci cells
shows that Dpp signaling is sufficient to initiate eye differentiation in its
normal location in the absence of Hh or N signals, but such differentiation is delayed. The normal timing of differentiation is restored by combined Dpp and N signals (in ci clones). This is the basis for the ectopic
differentiation on co-expression of Dpp and Dl ahead of the furrow (Fu, 2003).
Superficially, these results differ from previous ectopic expression studies that concluded that Dpp signaling alone was not sufficient to induce ectopic differentiation in all locations. This discrepancy is probably explained by the baseline repressor activity of Su(H) protein.
Previous work shows that without N signaling, repressor activity of Su(H)
protein retards differentiation. Dpp signaling is sufficient for differentiation in
the experiments where the Su(H) gene has been deleted. In the
presence of the Su(H) gene, Dpp may be most effective at locations
where there is little Su(H) repressor activity, such as close to the
morphogenetic furrow where N signaling is active (Fu, 2003).
Comparison between Mad Su(H) ci cells, which do not differentiate,
and Mad ci or tkv ci cells, which differentiate slowly or
not at all, shows that Notch signaling alone is insufficient for
differentiation. Premature differentiation reported when N is activated
ectopically ahead of the furrow must reflect endogenous Dpp signaling at such
locations (Fu, 2003).
These experiments reveal an outline of the mechanisms of Hh, Dpp and N
redundancy. First,
the results show that Mad and Ci independently reinforce differentiation,
presumably through the transcription of target genes because Mad is sufficient
for differentiation in the absence of Ci, and vice versa. The results show
unequivocally that the transcriptional activator Ci155 activates
differentiation in addition to Ci75 antagonizing differentiation (Fu, 2003).
It was surprising to find that Dpp stabilizes Ci155 in the absence of Smo,
which suggests Dpp input into Hh signal transduction. Although the
requirement for smo-dependent input through fused makes it
unlikely that Ci155 is functional in smo clones,
Ci155 accumulation might be associated with reduced Ci75 levels. Ci75 is shown to repress differentiation in smo clones because smo ci
clones differentiation normally. Ci155 stabilization cannot be due to an
indirect effect of Dpp signaling on Hh, Ptc or Smo expression levels because
the effect is detected in the absence of smo, and, therefore,
reflects an effect on Hh signal transduction components downstream of Smo. One idea is that Dpp signaling (or Dpp-induced differentiation) may replace
SCFSlimb processing of Ci (which cleaves Ci155 to Ci75) with
Cullin3-mediated Ci degradation, just as normally occurs posterior to the
morphogenetic furrow. In a smo clone, Ci155 would accumulate because Smo is required for Cullin3 to degrade Ci. However, the
SCFSlimb-to-Cullin3 switch may not be the only effect of Dpp on Ci
processing, because Tkv slightly enhances Ci155 accumulation even when
smo is present (Fu, 2003).
Finally, downregulation of Hairy by N requires the Su(H) gene. N
also overcomes baseline repressor activity of Su(H) protein to promote
progression of differentiation. This role of N must be independent of Hairy (Fu, 2003).
Dl, Hh and Dpp are generally thought to signal over very different
distances. How can signals of such different range substitute for one another
to permit normal eye development? Dpp is
transcribed in response to Hh signaling and is produced where Ci155 levels are highest. Dl is regulated by Hh indirectly through Ato and
Ato-dependent Egfr activity in differentiating cells. Hh is
expressed most posteriorly of the three, in differentiating photoreceptors (Fu, 2003).
Eye differentiation uses Hh to progress through cells unable to respond to
Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion
depends in part on the shape of the morphogenetic furrow cells. The Dpp
that drives differentiation through ci-mutant cells unable to respond
to Hh must diffuse from outside the ci clones because Dpp synthesis
is Hh dependent. Large ci clones develop normally so Dpp diffusion
cannot be limiting (dpp-mutant clones offer no information about the
range of Dpp because they express and differentiate in response to Hh).
Instead, the rate of progression in response to Dpp is controlled by Dl. Dl
signals over (at most) one or two cell diameters at the morphogenetic furrow (Fu, 2003).
The previous view of eye patterning was influenced by the morphogen
function of Hh and Dpp in other discs. It was thought that domains of Ato and Hairy expression reflected increasing concentrations of Hh and Dpp. The data shows that, in the eye, the combination of signals is important. Differentiation is triggered where Dl and/or Hh synergize with Dpp, regardless of where the source of Dpp is. The additional requirements limit Dpp to initiating differentiation at the same locations that Hh does (Fu, 2003).
Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. Signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential Dpp receptor, loses the ability to clonally populate a niche. Therefore, Dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches (Zhu, 2003).
This demonstrates how an adult GSC niche is populated with stem cells in the Drosophila ovary. Before niche formation, all PGCs proliferate as pre-stem cells and are undifferentiated. As niche formation starts, PGCs divide into two distinct subpopulations: anterior PGCs adjacent to cap cells start to acquire stem cell identity, and the remaining PGCs directly proceed to differentiation. GSCs in one niche can come from one PGC. Dpp is likely involved in stimulating clonal expansion of GSCs during niche formation. This study suggests that signals from newly formed niches are important for expanding GSCs and most likely for populating nascent adult GSC niches (Zhu, 2003).
How stem cell identity is established initially remains elusive even in the well-studied stem cell systems -- Drosophila ovary and testis. In the primitive female gonads before the pupal stage, PGCs appear to undergo symmetric division to generate germ cells with the identical pre-stem cell fate. Several studies suggest that GSCs are established at the early pupal stage. At the early pupal stage, there are 136 germ cells on average in each gonad. The adult ovary, which is composed of 12-16 ovarioles with two or three GSCs per ovariole (average of 2.5), contains about 30 to 40 GSCs. Therefore, at the most, 20%-30% of PGCs in the early pupal gonad are recruited to niches and turn into GSCs (Zhu, 2003).
How is a particular germ cell selected and recruited to niches, and how
does it become a GSC? Positional information is known to be very important for
cell-fate determination in various developmental processes. In this study, a developmental approach was taken to investigate when key niche components form, and how PGCs are subdivided into GSCs and differentiated germ cells. The
expression of bam is associated with germ cell differentiation in the adult ovary. Using bam expression as an indicator for germ cell differentiation, it has been shown that no PGCs in late third instar larval gonads have differentiated. In early pupal gonads (about 0-4 hours after pupation), all the PGCs that are not in contact with TFs/cap cells are differentiated; therefore, the PGCs that contact newly formed cap cells remain undifferentiated and become GSCs. Possibly, newly formed TFs/cap cells directly prevent the most anterior PGCs from differentiation when an unknown developmental signal triggers PGC differentiation around the larval-pupal transition stage. This study demonstrates that the stem cell fate of PGCs is determined by their position, i.e. juxtaposition to TFs/cap cells (Zhu, 2003).
The next important question is how these anterior PGCs populate niches. In this study, it has been show that the PGCs in contact with newly formed cap cells at the early pupal stage divide more frequently than the rest of the PGCs. The division patterns are very interesting: one division pattern generates two daughters that are both in contact with cap cells; the other pattern generates only one daughter that is in contact with cap cells. As in the adult ovary, two daughters that are in contact with cap cells can both become GSCs. This is
verified by the observation that one marked PGC in the gonad at the late
third-instar larval stage can generate two or three GSCs in a niche. The
results also indicate that the stem cells in a niche can come from multiple PGCs. Whether GSCs in a niche come from one or multiple PGCs probably depends on whether one or multiple PGCs directly contact cap cells within the developing niche. If only one PGC contacts cap cells, it probably has an opportunity to generate two or three germ cells that contact cap cells and become GSCs. This study shows that newly formed niches do not simply recruit existing PGCs and turn them into GSCs, but also stimulate PGCs to proliferate and produce more GSCs (Zhu, 2003).
The clonal expansion of GSCs in a niche clearly requires the newly
established stem cell to divide rapidly and generate a daughter that occupies the same niche, which further prevents other neighboring precursor cells from entering it. Consistent with this prediction, it was observed that the anterior row of germ cells at the early pupal stage is more mitotically active than the rest of the germ cells based on the BrdU incorporation assay. dpp is known to be important for maintaining GSCs and stimulating their division
in the adult ovary. dpp is expressed in TFs/cap cells and
other anterior somatic cells, and PGCs close to cap cells are capable of
responding to dpp. Furthermore, overexpressing dpp promotes PGC proliferation. To demonstrate the necessity of dpp signaling in stimulating GSC clonal expansion, it has been shown that a PGC mutant for tkv, an essential dpp receptor, fails to clonally populate a niche. All these results demonstrate that dpp is probably a signal for stimulating GSC clonal expansion (Zhu, 2003).
As in the adult ovary, hh is also expressed in terminal filaments and cap cells in developing female gonads. Hh has been shown to play a minor role in modulating GSC division. Wingless
(Wg) protein is expressed in terminal filaments and cap cells. Its expression in developing female gonads has not been
examined. Because wg, dpp and hh often work together to
regulate many developmental processes in Drosophila, it is possible that hh and wg could also cooperate with dpp to regulate PGC proliferation and modulate GSC clonal expansion in niches (Zhu, 2003).
PGCs in the gonad do not show any signs of differentiation until the
larval-to-pupal transition. At the early pupal stage, only the PGCs in the
anterior row remain undifferentiated, but the rest have already
differentiated. It seems that a developmental signal(s) starts to appear and then induces the differentiation of PGCs during the transition from larva to pupa. Such a developmental signal could be mediated by a steroid-like hormone ecdysone. Interestingly, during most of the third instar larval stage, the ecdysteroid levels are very low but begin to rise and peak just before pupation. The ecdysteroid peak could be potentially responsible for the initial differentiation of germ cells in the gonad of the larva ready for pupation. It is also possible that the hormone is not a direct signal but controls the production of the signal(s). Somehow, the signals from the anterior somatic cells antagonize the differentiating signals and thus prevent the anterior row
of the PGCs from differentiation. One of the signals that prevent PGCs from differentiation could be encoded by dpp. Dpp is known to prevent GSCs from differentiation in the adult ovary. In
this study, 2.5% of the marked tkv mutant PGCs and none of the marked mad mutant PGCs before the third instar larval stage were recruited to niches or were maintained as GSCs before adulthood. The failure of tkv and mad mutant GSCs to be maintained in niches could be explained by the role of dpp in preventing PGCs from differentiation. It could also be explained by other possibilities, such as defects in the formation of adherens junctions between cap cells and GSCs. Whether dpp is a signal for maintaining the undifferentiated state of PGCs during early ovarian development remains undetermined. Therefore, the signals that maintain the undifferentiated state of PGCs from TFs/cap cells remain to be identified (Zhu, 2003).
Thick veins is likely to target p38b, a MAP kinase implicated in Dpp signal transduction. Two Drosophila homologs of p38, Mpk2 (also known as p38a or simply p38) and p38b, have been identified on the basis of their homology to mammalian p38 and to one another. p38b is maternally expressed and is present ubiquitously during embryonic development (Han, 1998). The chromosomal region
around the p38b locus has been well characterized
genetically. However, a
p38b transgene was unable to rescue any of
the known mutations mapping to this region.
Likewise, attempts to isolate a mutant of p38b were unsuccessful.
These failures are possibly due to the functional redundancy of the two p38 homologs. Various alternative methods were therefore use to interfere with endogenous p38(s) in order to investigate its function. A
dominant-negative allele of p38b, designated
D-p38bDN, was generated by replacing the Thr-183
of the MAPKK target site with Ala, analogous to the change in ERK2
that produces a dominant-negative allele (Adachi-Yamada, 1999).
Two lines were prepared which express D-p38bDN at different levels: D-p38bDN-S
(Strong), which expresses high levels, and D-p38bDN-W
(Weak), which expresses low levels. When two copies of the
D-p38bDN-S transgene are expressed in the wing, a certain fraction of adult flies that escape death
exhibit ectopic vein fragments around the end of the longitudinal
vein L2 and a reduction in the distance between L4 and L5. Both of these features have also been
observed with some mutant alleles of decapentaplegic and thick veins. This wing
phenotype is rescued by coexpression of the wild type
p38b+ transgene.
When two copies of the D-p38bDN-S
transgene are weakly expressed in the wing of a dpp mutant, the vein
phenotype of dpp is strongly enhanced. These
phenotypes suggest the involvement of Drosophila p38(s) in Dpp function in the
early and late stages of wing pattern development. Dpp is known to play
a dual role during wing development, acting as a morphogen and mitogen at early stages, while
activating vein differentiation at later stages (Adachi-Yamada, 1999).
To examine whether p38(s) functions in the Dpp
signaling pathway, the genetic interaction was examined between p38(s)
and a constitutively active mutant of Tkv (TkvCA). Two classes of tkvCA
insertions, tkvCA-S (Strong) and
tkvCA-W (Weak), were used. When
tkvCA-S is expressed, normal wing venation is severely distorted
and extensive production of fragments of vein material is observed. The
abdominal-cuticle pattern also appears irregular. This
wing phenotype suggests that TkvCA may influence Dpp
action during vein formation. Ectopic coexpression of
dpp+ and tkv+ causes similar phenotypes, indicating that these TkvCA-induced
aberrations are indeed the result of an increase in Dpp
signaling. It was thus expected that reducing the levels of downstream components would suppress tkvCA. In fact,
reducing by one-half the gene dosage of Mothers against dpp
(Mad), a well-documented Dpp-signaling factor, significantly suppresses the
tkvCA wing phenotype (Adachi-Yamada, 1999).
To investigate whether p38b is activated by Tkv signaling, a
preliminary biochemical characterization of p38b was carried out. Immediately after
heat treatment of flies, the amount of p38b immunoprecipitated by
anti-p-Tyr antibody was found to increase considerably, demonstrating that p38b is
tyrosine phosphorylated following heat shock, like mammalian p38. The site of tyrosine phosphorylation is expected
to be in the 'activation loop' region recognized by MAPKK, as is
the case in mammalian p38. Thus, a test was performed to see whether
an anti-phospho-p38 (anti-p-p38) antibody raised against a phosphorylated peptide from the activation loop of
mammalian p38 could cross-react with p38b. This anti-p-p38
antibody detects a protein with a calculated size of 42 kDa whose
amount increases immediately after heat shock.
This protein is also more abundant in the flies overproducing p38b
regardless of heat treatment. Therefore, it has been
concluded that anti-phospho-p38 can cross-react with the phosphorylated from
of p38b and can be used to assay recombinant p38b phosphorylation
in vitro. Treatment of p38b with recombinant human MKK6, a MAPKK that activates p38, causes a marked increase
in the level of p38b, as detected with anti-phospho-Tyr and anti-p-p38
antibodies, and a drastic increase in the level of Drosophila p38-dependent
phosphorylation of recombinant human activating transcription factor 2 (ATF2), a physiological substrate for
mammalian p38. The correlation between the
phosphorylation state and kinase activity of p38b indicates that the
anti-p-p38 antibody recognizes the active form of p38b. This allowed
activation of p38b by TkvCA to be examined in vivo. The
amount of active p38b was found to be slightly but significantly
higher in larvae carrying ectopically expressed tkvCA relative to that in wild-type Canton-S larvae. However, it has been reported that p38a protein expressed in
yeast, which was presumed to have the same molecular mass as p38b, is
also recognized by anti-p-p38 antibody. It is therefore
possible that p38b, or both D-p38's, may be activated by Tkv
signaling in vivo (Adachi-Yamada, 1999).
The continuous and steady supply of transient cell types such as skin, blood and gut depends crucially on the controlled proliferation of stem cells and their transit amplifying progeny. Although it is thought that signaling to and from support cells might play a key role in these processes, few signals that might mediate this interaction have been identified. During spermatogenesis in Drosophila, the asymmetric division of each germ line stem cell results in its self-renewal and the production of a committed progenitor that undergoes four mitotic divisions before differentiating while remaining in intimate contact with somatic support cells. TGF-ß signaling pathway components punt and schnurri have been shown to be required in the somatic support cells to restrict germ cell proliferation. This study showns, by contrast, that the maintenance and proliferation of germ line stem cells and their progeny depends upon their ability to transduce the activity of a somatically expressed TGF-ß ligand, the BMP5/8 ortholog Glass Bottom Boat. TGF-ß signaling represses the expression of the Bam protein, which is both necessary and sufficient for germ cell differentiation, thereby maintaining germ line stem cells and spermatogonia in their proliferative state (Shivdasani, 2003).
In order to test the requirement for TGF-β signaling in the germ line, the behavior of marked germ line clones lacking the activity of various TGF-β signaling pathway components was investigated. Germ line stem cells mutant for tkv or put (a type II TGF-β receptor) and spermatocytes lacking the activity of tkv, put, or mad (a transcription factor required for the regulation of TGF-β target genes) were generated but did not persist to the same extent as wild-type clones, as evidenced by assessing the ratio of the number of testes containing at least one germ line clone to the number of testes containing wild-type control clones. Sporadically (approximately 4% of cases), cysts containing eight, rather than 16, spermatocytes were observed, implying that the fourth spermatogonial division had not been complete. Such a scenario might have arisen due to the transient persistence of Tkv, Mad, or Put protein after the respective wild-type allele was lost. Together, these clonal analysis data suggest that TGF-β signaling is required for both germ line stem cell maintenance and spermatogonial proliferation. No requirement was found for schnurri (shn), the product of which is frequently required in Dpp signaling, in the germ line for these processes (Shivdasani, 2003).
Juvenile hormone (JH) biosynthesis in the corpus allatum (CA) is regulated by neuropeptides and neurotransmitters produced in the brain. However, little is known about how these neural signals induce changes in JH biosynthesis. This study reports a novel function of TGFβ signaling in transferring brain signals into transcriptional changes of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis. A Drosophila genetic screen identified that Tkv and Mad are required for JH-mediated suppression of broad (br) expression in young larvae. Further investigation demonstrated that TGFβ signaling stimulates JH biosynthesis by upregulating jhamt expression. Moreover, dpp hypomorphic mutants also induces precocious br expression. The pupal lethality of these dpp mutants is partially rescued by an exogenous JH agonist. Finally, dpp is specifically expressed in the CA cells of ring glands, and its expression profile in the CA correlates with that of jhamt and matched JH levels in the hemolymph. Reduced dpp expression was detected in larvae mutant for Nmdar1, a CA-expressed glutamate receptor. Taken together, it is concluded that the neurotransmitter glutamate promotes dpp expression in the CA, which stimulates JH biosynthesis through Tkv and Mad by upregulating jhamt transcription at the early larval stages to prevent premature metamorphosis (Huang, 2011).
The functions of the TGFβ superfamily and other morphogens in regulating insect metamorphosis are rarely reported. In two independent genetic screens, it was discovered that Drosophila TGFβ signaling controls two different aspects of insect metamorphosis. In a previous study, it was found that Baboon (Babo) and dSmad2-mediated TGFβ signaling regulates larval neuron remodeling, which is part of the insect central nervous system metamorphosis induced by 20E during the pupal stage. Further investigation revealed that Babo/dSmad2-mediated TGFβ signaling controls larval neuron remodeling through regulating the expression of EcR-B1, a specific isoform of the 20E receptor (Huang, 2011).
This paper reports several findings. First, br is precociously expressed in 2nd instar tkv and Mad mutant larvae. Second, the precocious br expression phenotype in tkv and Mad mutant larvae can be suppressed by exogenous JH agonist (JHA). Third, Tkv and Mad repressed br expression in a non-cell-autonomous manner. Fourth, the presence of Mad in the CA is sufficient to repress br expression in the fat body (FB). Fifth, jhamt mRNA levels and JHAMT activity were significantly reduced in the Mad-deficient larvae. These results demonstrate that Tkv- and Mad-mediated signaling is required in the CA to activate jhamt expression and thus JH biosynthesis, which in turn controls insect metamorphosis (Huang, 2011).
The Drosophila genome encodes two TGFβ type II receptors, Punt (Put) and Wishful thinking (Wit). The genetic screen failed to identify a role for either of these receptors in the regulation of JH biosynthesis. Put and Wit are most probably functionally redundant in this biological event, as in the case of TGFβ-mediated mushroom body neuron remodeling (Huang, 2011).
Dpp is a key morphogen that controls dorsal/ventral polarity, segmental compartment determination and imaginal disc patterning. Dpp function usually depends on its gradient distribution. In an attempt to identify the ligand for Tkv/Mad-mediated TGFβ signaling in the CA, a novel, gradient-independent role for Dpp was discovered that controls JH biosynthesis. Dpp is the ligand of Tkv, which regulates jhamt transcription. Loss of Dpp, even RNAi reduction of Dpp in the CA specifically, causes precocious br expression at the early larval stages, which phenocopies tkv and Mad mutants. Phenotypes of dpp, including precocious br expression and lethality, are at least partially rescued by JHA treatment or ectopic jhamt expression in the CA. Notably, dpp-lacZ is strictly expressed in the CA cells, but not in the other two types of endocrine cells in the ring gland: the prothoracic gland and corpus cardiacum cells. The developmental expression profile of dpp in the CA is always consistent with that of jhamt. Finally, dpp expression in the CA may be directly controlled by neurotransmitter signals in the brain, which is supported by reduced dpp and jhamt transcription levels in the Nmdar1 mutant wandering larvae (Huang, 2011).
Several lines of evidence suggest that Met is a crucial regulator at or near the top of a JH signaling hierarchy, possibly acting as a JH receptor. However, null Met mutants of Drosophila are completely viable, which is unexpected if Met is a JH receptor. A recent investigation indicated that another Drosophila bHLH-PAS protein, Germ cell-expressed (Gce), which has more than 50% homology to Met, may function redundantly to Met in transducing JH signaling (Baumann, 2010). Because Met is on the X chromosome in the fly genome, it was not covered by the genetic screen. The br protein in the FBs of a Met null allele, Met27, was tested at the 2nd instar larval stage, and precocious br expression was observed. Importantly, this precocious br expression phenotype could not be suppressed by exogenous JHA. This result not only supports the previous reports regarding the function of Met in transducing JH signaling but also suggests that the precocious br expression is a more sensitive indicator for the reduced JH activity in Drosophila compared with precocious metamorphosis, lethality and other phenotypes (Huang, 2011).
Kr-h1 was reported to act downstream of Met in mediating JH action. Studies in both Drosophila and Tribolium reveal that, at the pupal stages, exogenous JHA induces Kr-h1 expression, which in turn upregulates br expression. The genetic screen successfully identified that Kr-h1 is cell-autonomously required for the suppression of br expression at young larval stages. Precocious br expression occurred in the FBs of Kr-h1 mutants and was not suppressed by JHA treatment. Therefore, these studies further suggest that Kr-h1 functions as a JH signaling component in mediating insect metamorphosis. However, the finding shows that, at the larval stages of Drosophila, the JH-induced Kr-h1 suppresses, rather than stimulates, br expression. This result is consistent with the facts that Kr-h1 functions to prevent Tribolium metamorphosis and Br is a crucial factor in promoting pupa formation (Huang, 2011).
In summary, this study has found a novel function of Dpp, Tkv and Mad-mediated TGFβ signaling in controlling insect metamorphosis. As summarized in a model, the brain sends neurotransmitters, such as glutamate, to the CA through neuronal axons. Glutamate interacts with its receptor (NMDAR) on the surface of CA cells to induce dpp expression. Dpp protein produced and secreted by CA cells forms a complex with TGFβ type I receptor (Tkv) and type II receptor on the membrane of CA cells, followed by phosphorylation and activation of Tkv. Activated Tkv in turn phosphorylates Mad, which is imported into the nucleus together with co-Smad and stimulates jhamt expression. JHAMT in CA cells transforms JH acid into JH, which is released into hemolymph. The presence of JH in young larvae prevents premature metamorphosis through Met/Gce and Kr-h1 by suppressing the expression of br, a crucial gene in initiating insect metamorphosis (Huang, 2011).
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Mothers against dpp:
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