decapentaplegic

Effects of Mutation or Deletion (part 1/3)

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 dpp^d-ho/dpp^hr4 display wild-type wings. The dpp^d-ho mutation is a small deletion in the 3' cis-regulatory region of dpp. dpp^d-ho homozygous flies have wings that are held out laterally from the body axis. The dpp^hr4 mutation is a missense mutation in the protein-coding region of dpp. When homozygous, the dpp^hr4 allele is embryonic lethal. When dpp^d-ho and dpp^hr4 are paired, the wild-type regulatory region of the dpp^hr4 allele appears to act in trans on the wild-type coding region of the dpp^d-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, dpp^d-ho/dpp^hr4 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 dpp^d-ho/dpp^hr4 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 dpp^hr4/dpp^d-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 dpp^d-ho/dpp^hr4 transvection-dependent phenotype. Df(2L)C144 and l(2)a16 were tested for the ability to disrupt this phenotype. Forty-six percent of dpp^d-ho Df(2L) c144 /dpp^hr4 flies had heldout wings; of these flies, 47% were severely heldout. Twenty percent of dpp^d-ho l(2)a16/dpp^hr4 flies had heldout wings; of these flies, 50% were severely heldout. These results are similar to those of DTD46.4. Twenty-six percent of dpp^d-ho DTD46.4/dpp^hr4 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 dpp^d-ho/dpp^hr4 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 lilli^s35 or lilli^xs407. 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 dpp^e87, dpp^hr56, dpp^hr4, and dpp^hr92. No genetic interactions were detected with the weak alleles dpp^e87 and dpp^hr56. However, all lilli alleles tested showed significant dominant maternal enhancement of the strong alleles dpp^hr4 and dpp^hr92. Modest dominant zygotic enhancement of dpp^hr4 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, scw^S12, or gbb¹. However, lilli alleles show dominant maternal enhancement of the recessive lethality of scw^E1. scw^E1 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 dpp^hr56 and scw^E1. 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)2A^161H1) 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 scw^E1 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 Smurf1^15C 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 Smurf1^15C mutant embryos; a lesser but significant expansion of zen was also observed in Smurf1^11R mutant embryos. Later, Smurf1^15C 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 Smurf1^15C phenotype, restoring the embryonic hindgut to a tubular morphology and an interior location. It is noted that the hindgut of Smurf1^15C; 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; Smurf1^15C 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).

Dpp and neurogenesis

Continued: Decapentaplegic Effects of Mutation part 2/3 | part 3/3

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