decapentaplegic
Components of the Dpp pathway CrebA is thought to be epistatic to known dorsal/ventral patterning genes. Epistasis tests were done with the decapentaplegic gene and the spitz gene. In dpp/CrebA double mutants the entire cuticle is lateralized, while in dpp mutants the cuticle is ventralized. In spitz/CrebA double mutants narrower denticle bands are seen with some fusion of denticles between segments; however the denticles have the same morphology found in CrebA mutants alone. It is thought that near the end of both the Dpp- and Spi-signaling cascades, CrebA functions
to translate the corresponding extracellular signals into changes in gene expression. The only determinant tested that shows altered expression patterns in CrebA mutants was Dsc73, a secreted protein expressed at late embryonic stages in the epidermal cells that produce denticles and hairs. There was a decrease in the levels of Dsc73 on the dorsal and ventral surfaces compared to levels of Dsc73 in lateral positions, which appear unchanged (Andrew, 1997).
This paper describes the genetic and molecular characterization of a new Drosophila zygotic lethal locus, vrille (vri). vri alleles act not only as dominant maternal enhancers of embryonic dorsoventral patterning defects caused by easter and decapentaplegic
(dpp) mutations, but also as dominant zygotic enhancers of dpp alleles for phenotypes in wing. The vri gene encodes a new member of the bZIP family of transcription factors closely related to gene 9 of Xenopus laevis, induced by thyroid hormone during the tadpole tail resorption program, and NF-IL3A, a human T cell transcription factor that transactivates the
interleukin-3 promoter. NF-IL3A shares 93% similarity and 60% identity with Vri for a stretch of 68 amino acids that includes the bZIP domain. A uniformly distributed Vrille maternal mRNA is present at the preblastoderm stage. From stage 10, the transcripts begin to localize and can be seen at higher levels in the primordium of the foregut. At stage 13, transcripts are present in the hypopharyngal groove, at the ventral opening of the stomodeum, and also found in the foregut, the proventriculus primordium, the hindgut, anal pads and posterior spiracles. At stage 14, transcripts are located in stripes along the epidermis in the anterior part of each segment. At stage 16, the transcripts are still present in the stomodeum, anal pads and a network of lateral and dorsal cells probably corresponding to the tracheal trunk. Although all the alleles tested behave like antimorphs, the dominant enhancement is also seen with a nonsense mutation allele that prevents translation of the bZIP domain. Because of the strong dominant enhancement of dpp
phenotypes by vri alleles in both embryo and wing, and also the similarity between the wing vein phenotypes caused by the vri and shortvein dpp alleles, it is postulated that vri interacts either directly or indirectly with certain components of the dpp signal transduction pathway (George, 1997).
Mechanisms that regulate the activity of TGF-beta family members and mediate the biological response to such proteins remain incompletely understood. A genetic approach has been taken to identify factors required for TGF-beta function in Drosophila by testing for genetic interactions between mutant alleles of dpp and a collection of chromosomal deficiencies. This survey has identified two deficiencies that act as maternal enhancers of recessive embryonic lethal alleles of dpp. The enhanced individuals die with weakly ventralized phenotypes. These phenotypes are consistent with a mechanism whereby the deficiencies deplete two maternally provided factors required for dpp's role in embryonic dorsal-ventral pattern formation. One of these deficiencies also appears to delete a factor required for dpp function in wing vein formation. These deficiencies remove material from the 54F-55A and 66B-66C polytene chromosomal regions, respectively. As neither of these regions has been previously implicated in dpp function, it is proposed that each of the deficiencies removes a novel factor or factors required for dpp function (Nicholls, 1998).
p38 mitogen-activated protein kinase (p38) has been extensively studied as a stress-responsive kinase, but its
role in development remains unknown. D-p38's inhibit antimicrobial peptide production in cultured cells. D-p38's are also activated by stress-inducing and inflammatory stimuli, such as UV irradiation, high osmolarity, heat, serum starvation, H202 and LPS. Drosophila has two p38 genes: D-p38a, which maps to 95E4-to-95F1,
and D-p38b, which maps to the 34D region. To elucidate the developmental function of the Drosophila p38's, various genetic and
pharmacological manipulations were used to interfere with their functions: expression of a dominant-negative form of
D-p38b; expression of antisense D-p38b RNA; reduction of the D-p38 gene dosage, and treatment with the
p38 inhibitor SB203580. Expression of a dominant-negative D-p38b in the wing imaginal disc causes a
decapentaplegic-like phenotype and enhances the phenotype of a dpp mutant. Inhibition of D-p38b function also causes the
suppression of the wing phenotype induced by constitutively active Tkv (TkvCA). Mosaic analysis reveals
that D-p38b regulates the Tkv-dependent transcription of the optomotor-blind (omb) gene in
non-Dpp-producing cells, indicating that the site of D-p38b action is downstream of Tkv. Furthermore, forced
expression of TkvCA induces an increase in the phosphorylated active form(s) of D-p38(s). These results
demonstrate that p38, in addition to its role as a transducer of emergency stress signaling, may function to
modulate Dpp signaling. It is hypothesized that a D-p38b cascade exists that can be involved in Dpp signaling and that includes D-MKK3 and a homolog of TGF-beta-activated kinase 1 (TAK1: see TGF-ß activated kinase 1). While Mad is directly phosphorylated by Tkv, the activation of D-p38 by TkvCA may be indirect. It is also possible that one or more transcription factors functioning in the Dpp response requires prior phosphorylation mediated by the D-p38 cascade, a process that is regulated independent of Tkv (Adachi-Yamada, 1999).
A genetic screen was carried out for dpp signaling pathway components that exploits transvection effects at the dpp locus. This screen has identified
lilliputian, the only member of the Fragile-X/Burkitt's lymphoma family of transcription factors in Drosophila, as a potnetial dpp pathway component. Transvection, or pairing-dependent intragenic complementation between two alleles of a gene, is seen at a number of loci. As a result of transvection, trans-heterozygous individuals of the genotype dppd-ho/dpphr4 display wild-type wings. The dppd-ho mutation is a small deletion in the 3' cis-regulatory region of dpp. dppd-ho homozygous flies have wings that are held out laterally from the body axis. The dpphr4 mutation is a missense mutation in the protein-coding region of dpp. When homozygous, the dpphr4 allele is embryonic lethal. When dppd-ho and dpphr4 are paired, the wild-type regulatory region of the dpphr4 allele appears to act in trans on the wild-type coding region of the dppd-ho allele to generate viable adults with wild-type wings (Su, 2001).
During transvection, the respective regions (regulatory and coding) must be in close physical proximity. A chromosomal rearrangement that physically moves a dpp allele to another part of the chromosome disrupts transvection. Rather than having wild-type wings, dppd-ho/dpphr4 flies with chromosomal rearrangements have heldout wings. Analyses of polytene chromosomes from rearrangement genotypes show asynapsis at the dpp locus. These rearrangements are referred to as normal dpp transvection-disruptors (normal DTDs). Trans-heterozygous dppd-ho/dpphr4 flies will also display a heldout phenotype if they contain a rearrangement with a breakpoint in a gene required for dpp function (e.g., Mad). This type of rearrangement, one that generates heldout phenotypes in trans-heterozygous flies without asynapsis at the dpp locus, is referred to as an exceptional DTD (Su, 2001).
To determine if a DTD is normal or exceptional, an unknown DTD is paired with a previously characterized normal DTD. If the unknown DTD is a normal DTD, trans-heterozygous flies will display wild-type wings. Two normal DTDs (even those with very different rearrangements) have the ability to arrange themselves in such a way that synapsis occurs at the dpp locus. If the unknown DTD is an exceptional DTD, trans-heterozygous flies will display heldout wings. The presence of a normal DTD cannot suppress a heldout phenotype that is due to a mutation in a gene required for dpp function. Mutations that act as exceptional DTDs are therefore candidates for components of the dpp signaling pathway (Su, 2001).
A total of 44,000 dpphr4/dppd-ho flies were screened and 321 DTD mutations were isolated. Of these mutations, 30 were exceptional DTDs. All exceptional DTDs were cytologically mapped. If an exceptional DTD chromosome appeared cytologically normal, the DTD mutation was mapped by recombination. DTD46.4 is a recessive lethal strain obtained in the screen that has a T(2;3) 23C; 93F rearrangement. To determine which translocation breakpoint results in the recessive lethality, DTD46.4-bearing flies were mated to flies with deletions spanning one of the two breakpoints. DTD46.4 complemented Df(3R)e-N19, a deletion of 93B-94. DTD-46.4 failed to complement Df(2L)JS17, a deletion spanning cytological region 23C-D that includes Mad. Mad is known to act as a dpp transvection disrupter, so it was suspected that DTD46.4 might be a new allele of Mad. To test this hypothesis DTD46.4 was further characterized. Complementation tests were conducted with a number of deficiencies and other mutations in the 23C-D cytological region. The DTD46.4 chromosome failed to complement the deficiencies Df(2L)C144, Df(2L)DTD52xD51, and Df(2L)JS17 and an EMS-induced loss-of-function mutation l(2)a16. These five strains are referred to as the 23C complementation group. However, the DTD46.4 chromosome was viable over Mad6, Mad11, and Mad12 and the small deletion Df(2L)C28 that uncovers Mad. These results place the recessive lethality of DTD46.4 distal to Mad in 23C1-2. Polytene in situ hybridization studies utilizing a variety of probes have demonstrated that the Drosophila Genome Project P1 clones DS00906 and DS07149 span the 23C1-2 breakpoint (Su, 2001).
It was necessary to determine if the 23C1-2 breakpoint of DTD46.4 is also responsible for disrupting the dppd-ho/dpphr4 transvection-dependent phenotype. Df(2L)C144 and l(2)a16 were tested for the ability to disrupt this phenotype. Forty-six percent of dppd-ho Df(2L) c144 /dpphr4 flies had heldout wings; of these flies, 47% were severely heldout. Twenty percent of dppd-ho l(2)a16/dpphr4 flies had heldout wings; of these flies, 50% were severely heldout. These results are similar to those of DTD46.4. Twenty-six percent of dppd-ho DTD46.4/dpphr4 flies had heldout wings; of these flies, 53% were severely heldout. It was concluded that the site of DTD46.4 recessive lethality in 23C1-2 is also the site that disrupts the dppd-ho/dpphr4 transvection-dependent phenotype (Su, 2001).
Other studies had identified a new gene located in cytological region 23C1-2. This gene, lilliputian (lilli), was identified in two screens for Ras/Mitogen-activated protein kinase (MAPK) signal transduction pathway components. Complementation tests showed that both DTD46.4 and l(2)a16 failed to complement either lillis35 or lillixs407. It was concluded that members of the 23C1-2 complementation group are alleles of lilli. In addition, a screen for genes that interact with dRaf, another component of MAPK signaling pathways, identified a locus in 23C1-2. Loss-of-function mutations in Su(Raf)2A suppress gain-of-function dRaf phenotypes. It seems likely that Su(Raf)2A mutations are also allelic to DTD46.4 and lilli (Su, 2001 and references therein).
Four lilli alleles were tested for dominant maternal enhancement of dpp recessive embryonic lethality. Df(2L)JS17 was excluded because it uncovers Mad. The lilli alleles were tested with dppe87, dpphr56, dpphr4, and dpphr92. No genetic interactions were detected with the weak alleles dppe87 and dpphr56. However, all lilli alleles tested showed significant dominant maternal enhancement of the strong alleles dpphr4 and dpphr92. Modest dominant zygotic enhancement of dpphr4 was also detected. Thus, lilli alleles that disrupt a dpp transvection-dependent phenotype are also dominant enhancers of dpp recessive embryonic lethality (Su, 2001).
The same alleles of lilli were tested for genetic interactions with other genes that function in dpp signaling. lilli alleles do not enhance the recessive lethality of the loss-of-function mutations Mad12, Med1, sax1, tkv8, scwS12, or gbb1. However, lilli alleles show dominant maternal enhancement of the recessive lethality of scwE1. scwE1 is a gain-of-function allele that is itself a dominant zygotic enhancer of dpp recessive embryonic lethality (Su, 2001).
The stage of lethality for the lilli loss-of-function mutation l(2)a16 was determined. lilli mutant individuals [l(2)a16/Df(2L)C144] were identified using the dominant visible marker Black cells (Bc). When l(2)a16/In(2LR)Gla Bc males were mated with Df(2L)C144/In(2LR)Gla Bc females, only Bc larvae were recovered. Bc is not visible in first instar larvae, suggesting that lilli mutants die as embryos or as first instar larvae. Examination of lilli mutant embryos revealed a partially ventralized phenotype. This phenotype is also seen in zygotic mutant embryos of dpphr56 and scwE1. Several of the hallmarks of this phenotype are a herniated head, internalized filzkorper, and disorganized/expanded denticle bands. Embryos derived from germline clones of weak Su(Raf)2A mutations (e.g., Su(Raf)2A161H1) also show this partially ventralized phenotype (Su, 2001).
Three results from genetic tests suggest that lilli is a strong candidate for a new component of the dpp signaling pathway. (1)lilli mutations enhance dpp heldout phenotypes and embryonic recessive lethality. The enhancement of dpp embryonic lethality by lilli mutations is not as strong as that of Mad or Med mutations. Mutations in Mad or Med enhance weak dpp alleles while lilli mutations do not. (2) lilli mutations enhance the recessive embryonic lethality of a gain-of-function allele of the TGF-ß family member scw. scw augments dpp signaling in embryonic dorsal-ventral patterning. To date, tests for interactions between scwE1 and other dpp pathway components such as Mad or Med have not been reported. lilli mutations do not enhance the recessive lethality of mutations in genes that encode Dpp signal transduction proteins (sax, tkv, Mad, or Med). (3) lilli homozygous mutant embryos have dorsal-ventral patterning defects similar to zygotic mutant embryos of dpp and scw. Utilizing these genetic criteria, lilli has as strong a connection to dpp signaling as Mad and Med (Su, 2001).
Dpp and dorsal/ventral polarity Among genes affecting dorsal-ventral polarity, decapentaplegic has the strongest mutant phenotype: in the absence of dpp, all cells in the dorsal and dorsolateral regions of the embryo adopt fates characteristic of more ventrally derived cells. tolloid is required for dorsal, but not dorsolateral, pattern. Extragenic suppressors of tolloid mutations have been isolated that prove to be mutations that elevate dpp activity. The function of tolloid is to increase dpp activity. Like tolloid, the phenotypes of mutant embryos lacking shrew gene function are suppressed by elevated dpp, indicating that shrew also acts upstream of dpp to increase dpp activity. In contrast, increasing the number of copies of the dpp gene enhances the short gastrulation (sog) mutant phenotype, causing ventrolateral cells to adopt dorsal fates. This indicates that SOG gene product normally blocks dpp activity ventrally (Ferguson, 1992).
Mutations at five loci delete specific pattern elements in the
dorsal half of the embryo and cause partial ventralization. Mutations in the genes zerknüllt and shrew affect cell division only in the dorsalmost cells corresponding to the [Image], while the genes tolloid, screw and decapentaplegic affect divisions in both the prospective amnioserosa
and the dorsal epidermis. In each of these mutants dorsally placed mitotic
domains are absent. This effect is correlated with an expansion and dorsal shift in the position of more ventral domains. The correlation between phenotypic strength and the progressive loss of dorsal pattern elements in the ventralized mutants, suggests that one of these gene products, perhaps DPP, may provide positional information in a graded manner (Arora, 1992).
Dorsal-ventral patterning within the embryonic ectoderm of Drosophila requires two type I TGFbeta
receptors, Tkv and Sax, as well as two TGFbeta ligands, Dpp and Scw. In embryos lacking dpp signaling, increasing the level of Tkv activity promotes progressively more dorsal cell types, while
activation of Sax alone has no phenotypic consequences. However, Sax activity synergizes with Tkv activity to promote dorsal development.
To determine the interrelationship between the signaling pathways downstream of the Tkv and Sax receptors, an assay was carried out of
the phenotypic consequences of activating each signaling pathway separately in embryos that lack dpp expression. Increasing levels of activation of Tkv signaling recapitulate embryonic dorsal-ventral pattern, as measured by
the dosage-dependent production of dorsal epidermal and amnioserosal cell fates. In contrast, activation of the Sax
signaling pathway alone does not promote formation of any dorsal structures. However, the activated Sax receptor
synergizes with the activated Tkv receptor in production of both dorsal epidermis and amnioserosal cell fates. From these
data it is concluded that, while the functions of both receptors are necessary for in vivo patterning, elevation of Tkv signaling
can bypass the requirement for Sax signaling. Furthermore, the data indicate that Sax signaling is dependent on Tkv
signaling for phenotypic consequences and that Sax signaling elevates the biological response to a given level of Tkv signaling (Neul, 1998).
Functional experiments suggest the two receptors have different ligands: Dpp acts through Tkv, and Scw acts through Sax. Furthermore, Sog, a
negative regulator of this patterning process, preferentially blocks Scw activity. To establish functional interactions between the Scw ligand and the Sax receptor, use was made of the ability of scw
mutant embryos to produce amnioserosa in response to injection of either DPP or SCW mRNAs. Injection of mRNA encoding a dominant-negative Sax receptor is able to block the biological activity of injected SCW
mRNA but is unable to block the activity of injected DPP mRNA. These findings were extended by showing that scw
function is required for the ability of a chimeric receptor containing the extracellular domain of Sax fused to the
intracellular domain of Tkv to rescue a tkv mutant. Taken together, these results strongly suggest that Scw is an obligate
component of the Sax ligand. Furthermore, because ventral expression of scw in cells that do not express dpp is
sufficient to rescue a scw mutant, Scw-DPP heterodimers appear not to be essential for the generation of wild-type pattern,
raising the possibility that Scw homodimers are the in vivo ligand for the Sax receptor (Neul, 1998).
Injection of SOG mRNA blocks the biological response of scw mutants to injection of SCW
mRNA, but not to injection of DPP mRNA. These results strongly suggest that Sog, which has been genetically
characterized as a negative regulator of Dpp activity, functions primarily to modulate Scw activity over the dorsal-ventral
axis. These data thus suggest that an activity gradient of dpp results from the differential spatial modulation of Scw activity by
Sog. This could happen by either of two mechanisms. One possibility is that the existence of a local ventral source for
Sog and the presence of a 'sink' for Sog in the dorsal regions of the embryo (the cleavage of Sog by Tld) could result
in a ventral-to-dorsal gradient of Sog. The binding of Sog to Scw could thereby result
in the formation of a reciprocal dorsal-to-ventral gradient of scw activity. A second model for the action of Sog
posits that Sog facilitates the directional diffusion of the Scw ligand from the lateral to the
dorsal regions of the embryo. Specifically, Sog binding to Scw shields the ligand from binding to its ubiquitously
localized receptors and thereby allows the Scw-Sog complex to diffuse in the perivitelline space. Dorsally located Tolloid
then cleaves Sog, releasing the Scw ligand from the inhibitor. The action of Sog would thus lead to increased dorsal
localization of Scw and increased activity of the Sax pathway, ultimately resulting in formation of amnioserosa.
This facilitated diffusion model implies that one function of Sog is to elevate Dpp/Scw signaling dorsally. This model
would directly explain the reduction in amnioserosa observed in sog mutants and would
account for the cell nonautonomous function of Scw, revealed by ventral injections of SCW mRNA.
Moreover, this model could also provide an explanation for a puzzling aspect of the phenotype of embryos that lack the
nuclear gradient of dorsal gene product. Such dorsalized embryos have a pattern of zygotic gene expression around the
embryonic circumference that is similar to that of the most dorsal cells in the wild-type embryo. However, only a small
number of cells in dorsalized embryos differentiate as amnioserosa; the great majority of cells in these embryos differentiate
as dorsal ectoderm. An increase in dpp gene dosage in dorsalized
embryos is sufficient to increase the number of amnioserosal cells. Thus, it appears that despite the pattern of gene
expression in dorsalized embryos, the level of dpp/scw signaling is not sufficient to fate amnioserosa. Dorsalized
embryos do not express sog; thus, the lack of 'facilitated diffusion' of the Scw ligand mediated by Sog could be the
cause of this phenotype (Neul, 1998 and references).
It is proposed that the original function of Dpp might have been to mediate
dose-independent cell fate decisions. The ability of Dpp
to function in a dose-dependent manner was acquired evolutionarily by the recruitment of a second signaling system whose output could
modulate Tkv activity, but whose biological function was dependent on Dpp. The genetic compartmentalization inherent
within this circuitry would have ensured the increased evolutionary capacity of such a patterning system. Specifically, genetic
alterations in components of the modulatory signaling pathway could lead to significant phenotypic variability without
disruption of the original cell fate choice mediated by Dpp. Thus, this genetic circuitry could have been a component in the
generation of diverse body plans (Neul, 1998).
In a dpp null mutant, all dorsal cell fates are missing and the embryos are completely ventralized. In contrast, embryos mutant for scw are partially ventralized and lack amnioserosa but
differentiate a reduced dorsal ectoderm. The relative severity of the dpp and
scw mutant phenotypes does not correlate with their expression patterns, since scw is transcribed uniformly at the syncitial
blastoderm stage and dpp expression is restricted to the dorsal side of the embryo. One explanation for the different efficacies of the two ligands could be that they differ in
abundance or have different affinities for their receptors. Alternatively, the ligands could evoke qualitatively different
responses, perhaps by acting through different receptors.
To distinguish between these alternatives, the ability of SCW mRNA to restore dorsal pattern in dpp null
embryos was assayed. If the difference in the scw and dpp mutant phenotypes simply reflects their effective concentrations, excess
Scw protein should compensate for the loss of dpp function. Injected Scw protein fails to restore amnioserosa in embryos that lack
dpp function. This suggests that Scw and Dpp act in qualitatively distinct ways. While it had been postulated that dimerization between Scw and Dpp potentiates Dpp signaling by the formation of a potent Scw/Dpp dimer, this has been shown not to be the case. Expression of Scw in ventral cells in which Dpp is absent, rescues a scw mutant phenotype. Because Scw/Dpp dimers are likely to form intracellularly, these results
strongly argue that formation of Scw/Dpp heterodimers is not a prerequisite for the biological activity of Scw in the
embryo (N. Nguyen, 1998).
To understand the basis for the differential response of the embryo to Scw and Dpp signaling, the interaction
of the ligands with the two type I receptors Sax and Tkv was examined. Using dominant-negative forms of the type I receptors Sax and Tkv, it is demonstrated that Sax mediates
the Scw signal, while Tkv is required for both Dpp and Scw activity. While Dpp/Tkv signaling is obligatorily required, Scw/Sax
activity is necessary but not sufficient for dorsal patterning. Tkv function is required for the response to both
ligands, while the ability of Sax-DN to interfere specifically with Scw and not Dpp signaling strongly argues that Sax
preferentially mediates the response to Scw.
Sax and Tkv act synergistically, suggesting a mechanism for integration of the Scw and
Dpp signals. Further, it is shown that the extracellular protein Sog can antagonize Scw, thus limiting its ability to augment Dpp signaling in a graded
manner (N. Nguyen, 1998).
Genetic and phenotypic studies have established that sog and dpp exert opposing influences on dorsal patterning, leading
to the suggestion that Sog functions as an antagonist of Dpp activity. Levels
of Sog that do not affect Dpp signaling can block the ability of Scw to promote dorsal cell fates. The ability of
Sog to specifically interfere with Scw does not conflict with previous studies showing a genetic antagonism of dpp
activity by sog. Since Scw augments Dpp signaling, the inhibition of Scw activity by Sog is equivalent to antagonism of
Dpp. In fact, results from earlier studies support the assertion that Sog preferentially targets Scw activity in the embryo. Thus, it is proposed that one way by which Sog mediates
its negative effect on dorsal patterning is by antagonizing Scw function (N. Nguyen, 1998).
These data are also inconsistent with a central role for sog in modulating Dpp activity in late development. Ectopic expression
of Sog in the wing disc using a variety of GAL4 drivers causes no significant phenotypic defects. This is quite striking given the prominent role of Dpp in organizing pattern along the
anterior-posterior axis in the wing disc. It is worth noting that the loss of posterior crossveins caused by expression of Sog
is similar to the defect caused by Sax-DN, rather than Tkv-DN. An explanation for the failure
of Sog to target Dpp could be that Dpp is bound to extracellular matrix components or forms a high-affinity complex with
its receptor. Alternatively, the observation that Xenopus Noggin can severely ventralize Drosophila embryos raises the possibility that a Noggin-like factor may be the functionally relevant Dpp antagonist (N. Nguyen, 1998).
If Sog primarily blocks Scw activity during embryogenesis, the role of Tolloid may be to potentiate Scw signaling by
releasing it from an inhibitory complex. Scw can promote Tld-dependent cleavage of Sog.
This may explain why the loss of tld function results in a partially ventralized phenotype similar to that of scw- mutants,
rather than the complete ventralization typical of dpp null embryos. The observation that
embryos lacking both scw and tld function do not display a more severe phenotype is also compatible with this view (N. Nguyen, 1998).
In the visceral mesoderm, dpp is expressed in parasegment (ps) 7 under the control of the homeotic
gene Ultrabithorax (Ubx). In this cell layer, dpp stimulates its own expression
and the expression of Ubx. dpp also
stimulates the expression of wingless (wg), an extracellular signaling molecule of the Wnt family, in
the neighbouring ps8. wg in turn feeds back to stimulate Ubx and dpp expression in ps7. Thus, dpp is part
of a parautocrine feedback loop by which Ubx maintains its own expression indirectly through
controlling dpp and wg. Dpp also diffuses from its mesodermal source through the
endodermal cell layer of the embyonic midgut, where it stimulates the expression of D-Fos and of the homeotic gene labial. These inductive steps ultimately specify the differentiation of
distinct cell types in the larval midgut epithelium. In order to understand the mechanism by which dpp stimulates transcription, a short enhancer fragment of Ubx, called Ubx B, has been characterized that contains response sequences for
dpp and wg signaling in the embryonic midgut. The dpp response sequence
of this enhancer is bipartite, consisting of a tandem repeat of Mad binding sites and a cAMP response
element (CRE). The presence of the latter raised the
question whether the co-activator CBP (CREB-binding protein, binding to CREs) might participate in
Dpp-induced transcriptional activation (Waltzer, 1999).
Drosophila CBP loss-of-function mutants show specific defects that mimic those seen
in mutants that lack the extracellular signal Dpp or its effector Mad. CBP loss severely compromises the ability of Dpp
target enhancers to respond to endogenous or exogenous Dpp. CBP binds to the C-terminal domain of Mad. These results
provide evidence that CBP functions as a co-activator during Dpp signaling, and they suggest that Mad may recruit CBP to effect the
transcriptional activation of Dpp-responsive genes during development (Waltzer, 1999).
The embryonic midgut of nejire (nej) mutants (whose CBP function is reduced) show phenotypes related to
wg gain-of-function phenotypes: increased labial expression in the endoderm, and derepression
of the Ubx B enhancer in the visceral mesoderm. These phenotypes do not resemble those seen in dpp or Mad mutants: in Mad mutants,
labial expression is strongly reduced, and so is the beta-galactosidase
(lacZ) staining mediated by the Ubx B enhancer in the middle midgut. However, the narrow band of lacZ staining normally visible in the visceral mesoderm of the gastric
caeca (in ps3) is absent in nej mutant embryos. Indeed, closer inspection reveals that the gastric caeca frequently fail to elongate in these
mutants. A similar phenotype is observed in Mad and in dpp mutants. Thus nej, like dpp, is required for the formation of the gastric caeca,
and also for the activity of the Ubx B enhancer in the caecal primordia. The activity of this
enhancer in these primordia coincides with Dpp expression and depends on dpp function. The formation of the first midgut constriction is often impeded. While this could reflect overactive Wg signaling, it also
mimics loss of glass bottom boat (gbb) signaling: Gbb is a Dpp homolog expressed in the visceral mesoderm and whose function is required for the formation of the first midgut constriction (Waltzer, 1999).
The hypothesis that CBP is a co-activator of dpp-induced transcription was tested by
examining the Dpp response of the Ubx enhancer in nej mutants. Because it was expected that the repressive
effect of CBP on this enhancer would mask a possible activating effect of CBP in cells in which the
enhancer is stimulated by Wg signaling, a mutant
version of Ubx B, called B4, was used whose positive response to Wg is abolished.
B4 activity in the midgut is reduced compared with the wild-type enhancer; however, B4 still contains a
fully functional dpp-response sequence and can be efficiently stimulated by ectopic Dpp. B4 can thus be used to selectively monitor the stimulation of Ubx by Dpp in the visceral
mesoderm. The activity of Ubx B4 is significantly reduced in nej mutants. LacZ staining is particularly weak in ps6/7 (near the Dpp source), but also in ps10, and is
barely detectable in the gastric caeca. Furthermore, in nej mutant embryos
derived from nej mutant germlines (nej), lacZ staining mediated by B4 is even weaker than in
the zygotic nej mutants: although these nej GLC embryos are somewhat variable in
terms of their phenotypes the most severely mutant embryos show
lacZ staining in only a few cells in the ps8 region. Similarly, in Mad12 mutant embryos, lacZ staining is
much reduced, with some staining remaining in ps6 and ps8. This implies that CBP, like
Mad, is required for the Dpp response of the Ubx B4 enhancer (Waltzer, 1999).
The response of B4 to GAL4-mediated ectopic Dpp was examined in nej mutant embryos. If Dpp
is expressed throughout the mesoderm, B4-mediated lacZ staining is increased and detectable
throughout the midgut mesoderm. In nej mutants, this response
of B4 to ectopic Dpp is strikingly disabled: there is barely any lacZ staining
in the anterior midgut, and only a moderate increase of lacZ staining in the ps8/9 region, indicating a
residual Dpp response in this region. These results strongly support the conclusion that CBP is required
for the transcriptional response of the Ubx enhancer to Dpp signalling. They argue that CBP functions
downstream of the Dpp signal (Waltzer, 1999).
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).
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).
Drosophila Smurf1 is a negative regulator of signaling by the BMP2/4 ortholog Decapentaplegic during embryonic dorsal-ventral patterning. Smurf1 encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog in Drosophila Mothers against dpp (Mad) and likely promotes its proteolysis. The essential function of Drosophila Smurf1 is restricted to its action on the Dpp pathway. Smurf1 has two distinct, possibly mechanistically separate, functions in controlling Dpp signaling. Prior to gastrulation, Smurf1 mutations cause a spatial increase in the Dpp gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, Smurf1 mutations cause a temporal delay in downregulation of earlier Dpp signals, resulting in a lethal defect in hindgut organogenesis. The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, and may have additional functions in regulating the levels of Dpp receptors (Podos, 2001).
In wild-type embryos at the onset of gastrulation, a stripe of P-Mad staining is visible in a dorsal subset of dpp-expressing cells and in the cells at either pole of the embryo. In Smurf115C mutant embryos, there is a small but statistically significant increase in the width of the dorsal P-Mad stripe as well as a nonquantitated increase in the intensity of staining. In wild-type embryos at this stage, the Dpp target genes zen and Race are activated by high levels of Dpp signaling in the presumptive amnioserosa, while the intermediate threshold target gene u-shaped (ush) is activated in a broader domain by lower levels of Dpp activity. All three transcriptional domains showed significant lateral expansion in Smurf115C mutant embryos; a lesser but significant expansion of zen was also observed in Smurf111R mutant embryos. Later, Smurf115C mutant embryos differentiate a nearly 2-fold excess of amnioserosa cells compared to wild-type. A 2-fold increase in dpp gene dosage effects a similar expansion of zen transcription and a comparable increase in amnioserosa cell number. These observations indicate that disruption of Smurf1 gene activity elicits an expansion of multiple Dpp signaling thresholds in the early embryonic ectoderm comparable to the phenotype caused by a doubling of dpp gene dosage (Podos, 2001).
Three genetic criteria indicate that defects in Dpp signaling directly cause the hindgut phenotype in Smurf1 mutant embryos: (1) the hindgut defects are not observed in Smurf1 mutant embryos that lack one copy of dpp; (2) a complete loss of zen function substantially suppresses the Smurf115C phenotype, restoring the embryonic hindgut to a tubular morphology and an interior location. It is noted that the hindgut of Smurf115C; zen double mutant embryos often fails to adopt the normal hook-shaped trajectory, suggesting that the deregulation of other target genes also contributes to the Smurf1 phenotype. (3) The hindgut defect was also suppressed in sog; Smurf115C double mutant embryos. While Sog antagonizes BMP signaling in the ventrolateral ectoderm, a positive activity of Sog is also required at the onset of gastrulation to promote the Dpp-dependent specification of amnioserosa at the dorsal midline. It is proposed that the blastoderm-specific Dpp signaling in the dorsal-most region of sog;Smurf1 embryos is reduced to a level that, even in the absence of temporal downregulation of P-Mad, does not elicit the observed Smurf1 hindgut defect (Podos, 2001).
The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, the necessity of which has been supported by previous genetic observations. Although not normally haploinsufficient, the Mad gene is rendered so when the activities of other components of the Dpp pathway, including dpp, zen, and sog, are reduced. More generally, limiting amounts of Smad protein might be an essential feature of all graded TGF-ß superfamily signaling systems. Cytoplasmic Smad pools are similarly limiting in Xenopus embryos, according to quantitative studies of activin signaling. Experimental elevations in Smad2 concentration cause proportionate increases in Smad activation, as represented by both nuclear Smad2 import and transcriptional readout. Therefore, it is predicted that Smurf enzymes will prove to be essential to maintain Smad proteins at limiting concentrations to ensure appropriate responses to all graded BMP and activin/TGF-ß signals (Podos, 2001).
This study showS that cell fate decisions in the dorsal and lateral mesoderm of Drosophila depend on the antagonistic action of the Gli-like transcription factor Lame duck (Lmd) and the zinc finger homeodomain factor Zfh1. Lmd expression leads to the reduction of Zfh1 positive cell types, thereby restricting the number of Odd-skipped (Odd) positive and Tinman (Tin) positive pericardial cells in the dorsal mesoderm. In more lateral regions, ectopic activation of Zfh1 or loss of Lmd leads to an excess of adult muscle precursor (AMP) like cells. It was also observed that Lmd is co-expressed with Tin in the early dorsal mesoderm and leads to a reduction of Tin expression in cells destined to become dorsal fusion competent myoblasts (FCMs). In the absence of Lmd function, these cells remain Tin positive and develop as Tin positive pericardial cells although they do not express Zfh1. Further, it was shown that Tin repression and pericardial restriction in the dorsal mesoderm facilitated by Lmd is instructed by a late Decapentaplegic (Dpp) signal that is abolished in embryos carrying the disk region mutation dppd6 (Sellin, 2009).
Loss of Lame duck (Lmd) leads to an increase of pericardial cells and adult muscle precursor like cells: In embryos lacking Lmd function, staining for zinc finger homeodomain factor 1 (Zfh1) expression reveals a pericardial hyperplasia phenotype and a general excess of Zfh1 positive mesodermal cells. In wild type embryos, three types of pericardial cells (PCs) have been described: Tin positive (TPCs), Odd positive (OPCs) and Eve positive (EPCs) pericardial cells, all of which express Zfh1 and the handC- GFP reporter. Closer inspection of the pericardial cells in lmd mutant embryos revealed that the number of TPCs and OPCs is dramatically increased, while the number of EPCs is normal. All OPCs co-express the handC- GFP reporter and Zfh1 in wild type and lmd mutant embryos. In contrast, a considerable number of ectopic Tin positive cells, though positive for handC- GFP, do not express Zfh1 in lmd mutant embryos. The absence of β3Tubulin expression in these cells is consistent with earlier reports in which a normal set of cardioblasts was described in lmd mutant embryos. To decide whether the Zfh1 negative/Tin positive cells are atypical pericardial cells or dorsal mesodermal cells that fail to differentiate, a triple staining waa conducted for Tin, Zfh1 and Pericardin (Prc), a collagen that is secreted by differentiated pericardial cells. Prc protein was observed surrounding all Tin positive/Zfh1 negative cells, suggesting that they are ectopic pericardial cells. However, due to the fact that Prc is a secreted protein the possibility cannot be ruled out that there might be occasional Tin positive/Zfh1 negative cells in lmd mutant embryos which do not express Prc themselves, but remain in an uncommitted, dorsal mesodermal state (Sellin, 2009).
For further analysis of the ectopic pericardial cells, the number of OPCs in stage 16-17 embryos was counted. An average of 206.3 OPCs was observed in lmd mutant embryos as compared to 97.8 in wild type embryos, thereby representing a ~2-fold increase. It has been reported that the Odd subgroup of pericardial cells (OPCs) originates from two different lineages: a symmetric lineage (two OPCs from one precursor) and an asymmetric lineage (two OPCs from two precursors), adding up to a total of four OPCs per hemisegment. Of note, the siblings of the asymmetrically derived OPCs, the Seven-up (Svp) positive cardioblasts, are normal in lmd mutants, thus suggesting that the asymmetrically derived OPCs do not contribute to the lmd phenotype. Since the two different types of OPCs can not be distinguished directly because the anti-Svp antibody stains the precursor cells and the cardioblast siblings, but not the final PCs at later stages, their abundance was measured in lmd mutants indirectly. The fact was utilized that in inscutable (insc) mutants, asymmetric cell division fails, and all siblings of the asymmetric OPC lineage become Svp positive cardioblasts. The difference in OPC number between insc; lmd and lmd mutant embryos therefore corresponds to the number of asymmetrically derived OPCs in lmd mutant embryos. A loss of ~45 OPCs was observed in insc; lmd double mutant embryos as compared to lmd mutant embryos. This number is reasonably close to the number of ~38 OPCs that are lost in insc mutant embryos when compared to wild type embryos. In addition, the number of Svp positive precursors, which give rise to the asymmetric Odd lineage, is normal in lmd mutant embryos at early stage 13. Altogether, these data strongly support the initial hypothesis that there is the normal amount of asymmetrically derived OPCs in lmd mutant embryos and the phenotype is not caused by a failure of asymmetric cell division (Sellin, 2009).
An excess of Zfh1 positive cells was also observed in the lateral mesoderm of lmd mutant embryos, where it is normally expressed in the adult muscle precursor cells (AMPs). These imaginal myogenic cells retain Twist (Twi) expression, but do not express any other myogenic genes in the embryo. Instead, they are maintained in a less differentiated state during embryogenesis and are dormant until metamorphosis, when they start to differentiate and give rise to the adult musculature of the fly. In the embryo, they are arranged as groups of cells in the thoracic segments, while six solitary cells (one dorsal, two dorsolateral, two lateral and one ventral) are present in the abdominal hemisegments. It was reported earlier that too many Twi positive cells persist in the lateral mesoderm of lmd mutant embryos. Together with the fact that both Zfh1 and Twi are present in AMPs in the wild type, it appeared likely that both factors are also co-localized in embryos mutant for lmd. Indeed, double staining for Zfh1 and Twi showed a complete overlap in the lateral mesoderm and confirmed that both populations of ectopic cells are identical. They also express the gene holes in muscles (him) which is another marker specific for AMPs. For further characterization, the expression patterns were analyzed of several myogenic markers in lmd mutant embryos. No expression was detected of the muscle specific genes myocyte enhancing factor 2 (Mef2), β3 Tubulin or the reporter rP298 (Duf-lacZ) in Twi/Zfh1/Him positive cells (Sellin, 2009).
Twist expression is normally present during early stages of somatic muscle development in myoblasts that are not yet differentiated. Zfh1, which has been implicated in the repression of mef2, might help in keeping AMPs in the undifferentiated state until metamorphosis. The gene him, which is also expressed in AMPs, was recently reported to be involved in maintaining cells in an undifferentiated state by inhibiting the myogenic signal provided by Mef2 function. Consequently, the ectopic Zfh1/Twist/Him positive cells in the lmd mutant embryos are likely to be cells with myogenic potential, as are the endogenous AMPs, and hence can be considered to be ectopic AMP like cells. To assess if enhanced proliferation is also involved in generating an increased amount of cells in lmd mutant embryos, staining was oerfirned for phosphorylated Histone 3 (pH3), which specifically marks dividing cells. Over-proliferation in the dorsal and lateral mesoderm was not observed in lmd mutant embryos when compared to wild type embryos. Although there is a considerable number of the additional, AMP like cells that persist until the end of embryogenesis, their number is reduced between stage 13 and 16/17. Staining with Nile Blue A revealed a general excess of dying cells during these stages in lmd mutant embryos as compared to wild type, suggesting that not all ectopic cells survive until the end of embryogenesis (Sellin, 2009).
The supernumerary PCs and AMPs originate from the population of fusion competent myoblasts: While no general myogenic genes are expressed in the ectopic AMP like cells, it was however possible to show co-localization of Zfh1 and lmd mRNA in the somatic mesoderm of lmd mutant embryos, which is not observed in wild type embryos. In the wild type, lmd is expressed in fusion competent myoblasts (FCMs), which fail to differentiate in the absence of Lmd function. lmd mRNA is transcribed in a normal pattern in lmd mutant embryos. In situ hybridization with a lmd specific riboprobe therefore allowed visualization of the population of cells destined to become FCMs, although they do not express any other FCM specific genes in lmd mutant embryos. Since the ectopic Zfh1 positive cells co-express lmd mRNA, it is concluded that the ectopic AMP like cells in lmd mutant embryos originate from the FCM population and adopt AMP like characteristics instead. They are therefore generated by cell fate conversions, which is consistent with the observation that there are no additional cell divisions in lmd mutant embryos (Sellin, 2009).
In the dorsal mesoderm of lmd mutant embryos, the additional Zfh1 positive cells express Tin or Odd and Prc, indicating differentiation as pericardial cells. Pericardial cells usually develop from the dorsal cardiac mesoderm specified by Tin expression, while the somatic musculature is situated more laterally and is characterized by prolonged Twi expression. To address the question of whether a conversion of FCMs into PCs could also account for the pericardial hyperplasia phenotype in lmd mutant embryos in an analogous fashion to the ectopic AMP like cells, staining was carried out for Tin and lmd mRNA. It was reasoned that the ectopic Tin cells should also express lmd mRNA if they originate from the pool of mis-specified FCMs. Indeed, there is co-expression of lmd mRNA and Tin in ectopic pericardial cells in stage 13 lmd mutant embryos, indicating that cell fate conversions from FCM to ectopic PC fate are responsible for the observed pericardial hyperplasia phenotype (Sellin, 2009).
Of note, there is a distinct overlap of lmd mRNA and Tin expression in the dorsal mesoderm of stage 12 embryos, both in wild type and lmd mutant background. This observation is consistent with the observed cell fate switch from FCM to PC fate and indicates that in wild type embryos, dorsal FCMs are specified in the dorsal, Tin positive mesoderm rather than the Twi positive somatic mesoderm. Indeed, dorsal muscle phenotypes can be observed in embryos mutant for tin, consistent with the conclusion that dorsal muscle cell types (i.e., FCMs) develop from the early dorsal mesoderm specified by Tin expression: If this domain is not specified, it can not generate dorsal FCMs (or other dorsal mesodermal derivatives, like heart or visceral mesoderm) (Sellin, 2009).
Co-expression of Tin and lmd mRNA is no longer detectable after germ band retraction (stage 13) in wild type embryos, but persists in lmd mutant embryos until the lmd mRNA signal fades (at about stage 14-15). Thus, it seems that repression of Tin in the dorsal mesoderm depends on the presence of Lmd protein. To substantiate this observation, Lmd was overexpressed in the whole mesoderm with the twi-Gal4 driver to assess its influence on Tin expression. Indeed, a reduction was observed of Tin expression in stage 12 embryos overexpressing Lmd compared to wild type, further confirming a negative influence of Lmd on Tin expression in the dorsal mesoderm. At later stages, the number of TPCs (and OPCs) remains reduced, while the cardioblasts are not affected. A model is therefore proposed in which the initial dorsal mesoderm specified by Tin expression is subdivided by Lmd into cardiac mesoderm and dorsal musculature by repression of Tin in lateral regions and induction of a myogenic differentiation program instead. During this process, Tin expression is maintained only in the cells that are destined to become pericardial cells (or cardioblasts), while Tin is repressed by Lmd in the dorsally localized FCMs. Loss of Lmd function consistently leads to an increased amount of Tin positive cells in the dorsal mesoderm from stage 13 onwards, which then can differentiate as ectopic pericardial cells as indicated by the expression of Prc. Taken altogether, these data suggest that, in the absence of Lmd function, the pool of unspecified FCMs can develop as ectopic PCs in the Tin-positive dorsal mesoderm and as AMP-like cells in the lateral and ventral mesoderm. However, increased cell death, and the possibility that a small number of ectopic Tin positive cells might exist without Prc/Zfh1 expression as mentioned earlier, suggest the possibility that not all cells of the FCM population follow alternative cell fates. Instead, some cells might remain in an uncommitted mesodermal state in lmd mutant embryos (Sellin, 2009).
Normally, instructive Dpp signals from the ectoderm are responsible for the specification of cardiac cell types by maintaining Tin expression solely in the dorsal mesoderm, while Twist activity in the lateral and ventral mesoderm leads to the development of the somatic musculature. To test if reduced Dpp signaling has a similar effect on PC number as overexpression of Lmd, by reducing the size of the Tin domain, embryos carrying the mutation mad1-2 were examined. mad1-2 is a weak hypomorphic allele of the Dpp effector Mad and causes larval lethality, thereby allowing observation of late stages of embryogenesis. Indeed, a decreased number of OPCs and TPCs was observed in mad1-2 mutant embryos, without any effect on cardioblast number, as is the case when overexpressing Lmd. Of note, the number of OPCs is decreased to a similar extent in mad1-2; lmd double, as compared to lmd single mutant embryos. Therefore, it is concluded that in the presence of the hypomorphic mad1-2 mutation, the dorsal mesoderm that is specified by Dpp-dependent Tin expression is reduced, resulting in a reduction of PCs in a Lmd independent manner. However, Lmd further restricts the number of PCs in the mad1-2 mutant background, as revealed by an increased number of PCs and the presence of TPCs without Zfh1 expression in mad1-2; lmd double mutants when compared to mad1-2 single mutants (Sellin, 2009).
Pericardial cells share their developmental origin with the myogenic cardioblasts in a similar fashion as AMPs with founder cells in the somatic musculature. During lateral inhibition, Notch activation promotes myogenic FCM fate as opposed to the progenitors of founder cells in the lateral mesoderm or cardiogenic progenitors in the dorsal mesoderm. Subsequently, during the process of asymmetric cell division, Notch activation renders the daughter cell always non-myogenic (PC or AMP fate). Although the AMPs have the potential to develop into muscle cells during metamorphosis, they are considered non-myogenic in this context because they do not yet express any myogenic genes, such as mef2, lmd or muscle structural genes in the embryo. In the case of pericardial cells, there is surprisingly little data available about their physiological role. While it is known that the OPCs contribute to the population of nephrocytes in postembryonic stages, TPCs and EPCs are not correlated with any function at all, and their developmental fate after embryogenesis is still unknown. A recent study described the development of adult muscular structures, the so called wing hearts, from a specialized subset of EPCs. This is the first hint that some pericardial cells might be considered as imaginal myogenic cells in an analogous fashion to AMPs, and it highlights the necessity to further characterize pericardial cells (Sellin, 2009).
It is currently known that PCs and AMPs have in common a dependency on active Notch signaling although they stem from different cell lineages and mesodermal primordia (Tin vs. Twi domain). FCMs, which adopt AMP or PC like characteristics in lmd mutant embryos, also need active Notch signaling. In fact, Lmd is a downstream target of N signaling and induces the FCM differentiation program. The observed lmd phenotype could be explained if, in the absence of Lmd, Notch activity always promoted AMP or PC (non-myogenic) fate, but not FCM fate, independently of the original pathway that is involved (lateral inhibition or asymmetric cell division). To assess this hypothesis, double mutants for lmd and genes involved in the Notch pathway were established. For this analysis kuzbanian and mastermind alleles were chosen because loss of either gene causes lethality only late in embryogenesis due to a maternal component, thereby allowing the analysis of later events in heart and muscle development. Both genes have also been well studied with respect to their molecular function and developmental implications. Kuzbanian (Kuz) is an ADAM metalloprotease that is known to process the Notch receptor following ligand binding. Zygotic loss of function mutations lead to defects in both lateral inhibition and asymmetric cell division in heart and muscle development, although the phenotype is far weaker than in embryos carrying N loss of function alleles. mastermind (mam) is involved in transducing the Notch signal and displays a stronger heart phenotype than kuz and a mild Notch-like muscle phenotype. Staining was perfomed for expression of Krüppel (Kr) and him mRNA, which are specific for a subset of muscle founders and AMPs/ PCs, respectively, and an increase of Notch negative cell types, corresponding to founders, was observed in the somatic mesoderm of kuz mutant embryos. This is accompanied by a reduction of AMPs, confirming the expected function of Kuz in facilitating N function in muscle cell differentiation. Furthermore, the number of FCMs as marked by Lmd expression is strongly reduced in kuz mutants, although the effect is not as complete as in N loss of function alleles (Sellin, 2009).
In kuz; lmd double mutant embryos, the increase of AMPs is milder than in lmd mutant embryos, which is consistent with a failure in lateral inhibition and a concomitant reduction of FCMs that are available for conversion to AMPs. The number of Kr-positive founder cells is increased to comparable levels in kuz and kuz; lmd mutant embryos, suggesting that Notch inactive cell fates (muscle founders and cardioblasts) are not influenced by the absence of Lmd, and that Notch acts as a permissive signal to allow the cell fate switch in lmd mutant embryos. mam; lmd double mutant embryos display a similar phenotype. Altogether, these findings suggest that in the double mutants, a general reduction of cell types with Notch activity (i.e. FCMs) occurs, followed by the conversion of the remaining potential FCMs to AMP or PC fate under the influence of N signaling in the absence of Lmd. Lame duck is present in stages 12-14, which is later than the period during which Notch activity is involved in facilitating cell fate decisions within the musculature. Hence, it appears that Notch can promote AMP or PC fate at a relatively late time point in the absence of Lmd (Sellin, 2009).
It was of interest to know if the endogenous set of AMPs, which develop through asymmetric cell divisions of muscle progenitors, is specified correctly in lmd mutant embryos. For example, the lateral AMPs are the siblings of the segment border muscle founder (SBM), and share with the latter the expression of the identity factor Ladybird early (Lbe). To discern ectopic cells and endogenous AMPs in lmd mutant embryos, co-staining was performed for Lbe and Twi expression. Indeed, the normal number of lateral AMPs, as marked by Lbe expression, is present in lmd mutant embryos, while far too many Twi-positive cells was observed in general. The latter are the ectopic AMP like cells that are presumed to be recruited from the FCM population. This observation further confirms that individual mesodermal lineages, such as the asymmetrically derived OPCs or individual AMPs, are not influenced by the loss of Lmd function (Sellin, 2009).
The proposed model of cell fate switches from myogenic FCM fate to non-myogenic AMP like or PC fate, but not myogenic fates (cardioblasts or founder cells), is consistent with the observation that Notch signaling is often employed to delay or inhibit the differentiation of stem cells or progenitor cells, especially in myogenesis. In vertebrates, Notch signaling prevents satellite cells (muscle stem cells) from entering a myogenic differentiation program in cell culture as well as in vivo, and impaired upregulation of its ligand Delta-like 1 in satellite cells has been correlated with a decreased capacity of aging muscle tissue to regenerate. While the data are consistent with the general function of Notch in preventing cells to enter the myogenic differentiation program by promoting the AMP or PC fate, they also highlight the special and unusual properties of Lmd - as a target of Notch signaling - in Drosophila muscle development. Although it is activated by Notch, it has the ability to induce myogenic differentiation. The data strongly suggest that the AMP or PC fate is the default consequence of Notch signaling in Drosophila myogenesis and that Lmd function overrules this signal to induce the FCM differentiation program in lateral or dorsal competence domains. It was shown that N has a biphasic function in heart differentiation analogous to the situation in the somatic mesoderm. At an early phase, N activity restricts the number of the sum of CBs and PCs, reflecting a function in the definition of early cardiac progenitors, likely by lateral inhibition. Subsequently, N activity is needed to promote pericardial cell fates in asymmetric cell division of the early progenitors. Although the last division step is in many cases a symmetric division seem to indicate that the majority of cardiac cell types is generated by asymmetric cell divisions segregating cardiac and pericardial fates. This might occur in some cases at one of the earlier division steps of the progenitor(s). Since these data indicate the generation of FCMs from the dorsal mesoderm, as reflected by co-expression of Tin and Lmd in stage 12 embyos, it might be suggested that dorsal FCMs originate from dorsal competence domains which also give rise to the above mentioned cardiac progenitors. These progenitors divide asymmetrically to generate CBs and PCs analogous to FC/AMP sibling pairs from more lateral competence fields, while it ia proposes that all or some of the remaining cells of the competence domains begin to express Lmd and generate FCMs under instructive influence of N signaling. In the absence of Lmd function (either in wild type in the N active daughter cells of the progenitors, or in lmd mutant embryos in all N active cells of the competence domains), the N signal promotes non-myogenic cell fates according to the mesodermal context (i.e., dorsal vs. lateral mesoderm). This would then result in the differentiation of the non-segregating population (normally developing as FCMs) as PCs in the Tin domain and AMPs in the somatic mesoderm (Sellin, 2009).
Lame duck and Zfh1 act antagonistically in mesodermal cell fate decisions: While loss of Lmd function results in an increased number of Zfh1-positive cell types, overexpression of Lmd leads to the opposite phenotype. The pan-mesodermally active twi-Gal4 driver line was used to induce Lmd expression in the whole mesoderm, and a reduction of OPCs, TPCs and AMPs was observed. To assess whether pericardial cell reduction might be a secondary effect of the early Tin repression caused by ectopic Lmd activity, the later and cardiac specific handCA-Gal4 driver, which is active in the heart from stage 12 onwards, was used. At this time point, the OPC precursors are already specified and are no longer expressing Tin. Since hand>Lmd overexpression severely reduces the number of all pericardial cells, it is concluded that their reduction is not only a secondary effect of the narrower Tin domain in embryos overexpressing Lmd. To further confirm this conclusion, the phenotype of zfh1 mutant embryos was compared with that of embryos overexpressing Lmd. The number of OPCs and TPCs is also reduced in zfh1 mutant embryos quite similarly to embryos overexpressing Lmd, although the early Tin expression pattern is normal in the absence of Zfh1 function. It is therefore unlikely that Lmd acts negatively on Zfh1 expression only by reducing Tin expression, but rather also independently of Tin function (Sellin, 2009).
There are however important differences in the phenotypes of twi > Lmd and zfh1 mutant embryos. Zfh1 appears to be involved in maintaining, but not in specification of OPCs, because it has been observed that loss of Zfh1 does not affect the number of OPC precursors at stage 13, but rather leads to a decrease of OPCs at later stages. This is in contrast to a reduced number of OPC precursors in stage 13 embryos overexpressing Lmd. Therefore, Zfh1 repression alone can not account for the loss of PCs in embryos ectopically expressing Lmd. Instead, it might be that the reduction of the dorsal Tin domain by ectopic Lmd expression results in the specification of fewer OPC precursor cells, followed by further reduction of the remaining OPCs by the negative effect of ectopic Lmd on Zfh1 expression. Consistently, a much stronger reduction of OPCs was observed after ectopic expression of Lmd as compared to the loss of OPCs in zfh1 mutant embryos. The observation that loss of Lmd function leads to the appearance of TPCs that do not express Zfh1, but Prc as a marker of pericardial differentiation, is another hint that both effects occur independently of each other and that pericardial differentiation can be accomplished in the absence of Zfh1 in lmd mutant embryos (Sellin, 2009).
Taken altogether, it does not seem likely that Tin and Zfh1 act in an epistatic hierarchy in dorsal mesodermal cell fate decisions. Instead, the data support the conclusion that Lmd regulates OPC and TPC number by two independent mechanisms: (1) Initially, Lmd restricts the cardiac field in general through repression of Tin, which leads to the reduction of early OPC precursors and the elimination of Tin expression in cells that do not express Zfh1 (which can differentiate as TPCs, as indicated by Prc expression, in the absence of Lmd function). (2) Later, it represses Zfh1, thereby reducing further the number of OPCs and TPCs. This is consistent with previous findings which described Zfh1-dependent and Zfh1-independent mechanisms for the regulation of OPC and TPC number (Sellin, 2009).
Of note, it was previously shown that Zfh1 overexpression leads to an increase in pericardial cell number (both OPCs and TPCs) and a concomitant loss of dorsal somatic muscle cells, indicating that overexpression of Zfh1 phenocopies the pericardial hyperplasia in lmd mutant embryos. It was shown further that overexpression of Zfh1 with the twist-Gal4 or 24B-Gal4 driver leads to an increased number of AMP like cells in the dorsal mesoderm although the effect is rather weak when compared to lmd mutant embryos. Zfh1 overexpression does not however alter the pattern of Lmd expression, indicating that Zfh1 does not antagonize Lmd function at the transcriptional level. To verify whether Zfh1 has an influence on Lmd at the posttranscriptional level, the intracellular distribution of Lmd was analyzed in embryos overexpressing Zfh1, because Lmd function has been shown to be modulated by its subcellular localization in wild type embryos. In embryos overexpressing Zfh1, the subcellular localization of Lmd does not appear to be altered, suggesting that Zfh1 does not influence the subcellular distribution of the Lmd protein (Sellin, 2009).
Taken together, these data indicate that Lmd and Zfh1 have generally opposite effects on dorsal mesoderm differentiation: Lmd loss-of-function or Zfh1 gain-of-function leads to increased AMPs or PCs, whereas Lmd gain-of-function and Zfh1 loss-of-function reduce these cell types. Consequently, Lmd and Zfh1 can be considered to be functional antagonists, although their repression is not mutual. One possible explanation for the antagonistic effect of Zfh1 overexpression might be due to its direct negative influence on mef2 expression, thereby counteracting the mef2 activating function of Lmd. The vertebrate functional orthologue of Zfh1, ZEB2 (or Sip1), also inhibits myotube development in culture and represses a number of myogenic genes, and is able to rescue Zfh1 function in Drosophila (Sellin, 2009).
Lmd is instructed to restrict Tin expression by a late, pro-myogenic Dpp signal: While in wild type embryos Tin expression is repressed in cells destined to become dorsal FCMs between stages 12 and 13, there is a prolonged co-localization of Tin and lmd mRNA in cells of the dorsal mesoderm in lmd mutant embryos. As a consequence, dorsal FCMs adopt pericardial cell fates in the absence of Lmd function. Of note, this effect can also be observed in embryos carrying the dppd6 disk region mutation. These embryos lack a late Dpp signal (beginning at about stage 12) that is involved in pericardial restriction. Early Dpp signaling does not seem to be affected since the dorsal mesoderm (characterized by Dpp-dependent Tin expression) is normal in dppd6 mutant embryos. Quite contrary to embryos with otherwise decreased Dpp signaling and a reduced pericardial field, such as mad1-2 embryos, the dppd6 mutant embryos display a pericardial hyperplasia phenotype that resembles in many aspects the phenotype observed in lmd mutant embryos. Too many OPCs, TPCs and atypical TPCs without Zfh1 expression are also detected, although the dppd6 mutant phenotype is milder than the lmd mutant phenotype. This resemblance in phenotypes suggested an epistatic relationship of Lmd and the late Dpp signal. In addition, the accumulation of phosphorylated Mad (pMad) has been traced in PCs and cells within the dorsal musculature that are not positive for founder specific Kr or Eve expression, and hence are likely to be FCMs. Altogether, these findings lead to the hypothesis that Lmd might be a target of the late Dpp signal in FCMs. However, Lmd is expressed in a normal pattern (both at the mRNA and protein levels) in dppd6 mutant embryos, indicating that Lmd expression is independent of Dpp signaling. Nevertheless, co-staining with anti-Tin antibody revealed a prolonged co-localization of Tin and lmd mRNA in dppd6 mutant embryos until stage 14/15, as observed in lmd mutant embryos, suggesting a requirement for late Dpp signaling in the process of pericardial restriction by Lmd. To assess if the restrictive influence of late Dpp signaling on Tin expression is indeed relayed by Lmd in the dorsal mesoderm, or if both negative effects are independent of each other, the late Dpp signal was enhanced in the lmd mutant background. For this purpose, the leading edge driver LE-Gal4 was used to overexpress Dpp, which was shown to reduce the number of OPCs and TPCs in the wild type background. It was reasoned that this effect would be lost in lmd mutant embryos if Lmd is responsible for the restricting effect on PC number. The number of OPCs was counted in LE > Dpp; lmd embryos in comparison to lmd mutant embryos. While overexpression of Dpp with the LE-Gal4 driver in the wild type background led to a reduction of OPCs by ~1.2-fold, no reduction of OPCs was observed in the lmd mutant background, indicating that Lmd is indeed necessary to interpret the late Dpp signal as pro-myogenic. Altogether, these data suggest that the pro-myogenic effect of the late Dpp signal is Lmd dependent, although not by inducing Lmd expression. Instead, the presence of Dpp activity seems to be a prerequisite for the negative influence of Lmd on Tin expression and might act as an instructive signal to modify Lmd activity to allow repression of Tin. If the late Dpp signal is lost -- as is the case in embryos carrying the hypomorphic allele dppd6 - repression of Tin fails even in the presence of Lmd protein, indicating that repressive activity of Lmd is dependent on Dpp signaling (Sellin, 2009).
A model is proposed in which the subdivision of the early Tin positive primordium into pericardial and dorsal muscle tissues is mediated via the antagonistic action of Lmd and Zfh1 under the instructive influence of late Dpp signals. While the early function of Dpp restricts Tin expression to the dorsal mesoderm, subsequent Dpp signaling provides pro-myogenic input to modulate the pericardial field in favor of the dorsal musculature. The present data show that the function of this late Dpp signal requires Lmd activity, strongly suggesting that Lmd is a target of Dpp for establishing the boundary between the dorsal musculature and pericardial field. Repression of Tin also appears to be dependent on Dpp signaling. The previous observation that pMad accumulation occurs in PCs and dorsal muscle cells, which are likely to be FCMs, is consistent with the finding that Lmd is needed to relay the pro-myogenic function of late Dpp signaling. These cells originate from the Tin-expressing dorsal mesoderm, and co-expression of Tin and lmd mRNA in wild type embryos at stage 12 can be observed. In the presence of Lmd protein, this co-expression is not maintained after stage 12 due to a repressive function of Lmd on Tin. Of note, it was previously shown that Lmd function depends on posttranscriptional mechanisms that modulate its specific subcellular localization and activity, and it might be speculated that Dpp signaling is involved in changing Lmd function into a repressive form. However, there is no evidence that the negative influence of Lmd on Tin expression is of a direct nature, or if there are other factors that are involved in the process. In this context, the following explanation for the antagonistic effect of Zfh1 overexpression without repression of Lmd could also be considered. Since the vertebrate homologue ZEB2 was shown to inhibit activation of target genes by Smads, an excess of Zfh1 might antagonize the late Dpp signal by repressing pMad-dependent interaction partners of Lmd, thereby preventing the repression of Tin (and/or other targets) in the dorsal mesoderm. Lmd expression and function would not be affected elsewhere which would be consistent with the observation that Zfh1 is not a general repressor of Lmd (Sellin, 2009).
Dpp and neurogenesis Continued: Decapentaplegic Effects of Mutation part 2/3
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