Table of contents

Targets of DPP in mesoderm: early mesodermal patterning

After gastrulation, progenitor cells of the cardiac, visceral and body wall musculature arise at defined positions within the mesodermal layer of the embryo. An early and important event in the regional subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells. Genetic analysis has shown that this homeobox gene controls the formation of the visceral musculature and the heart from dorsal portions of the mesoderm. An inductive signal from dorsal ectodermal cells is required for activation of tinman in the underlying mesoderm. DPP serves as a signaling molecule in this process. Spatial expression of dpp in the ectoderm determines which cells of the mesoderm become competent to develop into visceral mesoderm and the heart (Frasch, 1995).

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning: it determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. Surprisingly, each of these elements are located at positions downstream of the transcription start site. The early-active enhancer element is a direct target of twist, a gene necessary for tinman activation that encodes a basic helix-loop-helix (bHLH) protein. This 180 bp enhancer includes three E-box sequences that bind Twist protein in vitro and are essential for enhancer activity in vivo. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. This repression requires the function of buttonhead, a head-patterning gene. The second expression domain, restricting tin mRNA expression in the dorsal mesoderm, is triggered by Dpp-mediated induction events (Yin, 1997).

Schnurri, a zinc finger transcription factor, is the first identified downstream component of the signal transduction pathway used by DPP and its receptors. Mutations in shn affect several developmental processes regulated by DPP including induction of visceral mesoderm cell fate, dorsal/ventral patterning of the lateral ectoderm and wing vein formation. Absence of shn function blocks the expanded expression of the homeodomain protein Bagpipe in the embryonic mesoderm caused by ectopic dpp expression, illustrating a requirement for shn function downstream of dpp action (Arora, 1955, Grieder, 1995 and Staehling-Hampton, 1995).

Mothers against dpp is required for dpp function. Mad was identified in screens to identify genes that interact with dpp. Mad loss-of-function mutations interact with dpp alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects, suggesting a role for Mad as a downstream mediator for some aspect of dpp function. In support of this, homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning that are very reminiscent of dpp mutant phenotypes (Sekelsky, 1995).

Dorsal mesoderm induction in arthropods and ventral mesoderm induction in vertebrates are closely related processes that involve signals of the BMP family. In Drosophila, induction by Dpp of visceral mesoderm, dorsal muscles, and the heart is, at least in part, effected through the transcriptional activation and function of the homeobox gene tinman in dorsal mesodermal cells during early embryogenesis. A functional dissection has been carried out of a tinman enhancer that mediates the Dpp response. Mesoderm-specific induction of tinman requires the binding of both activators and repressors. Screens for binding factors yielded Tinman itself and the Smad4 homolog Medea. The binding and synergistic activities of Smad and Tinman proteins are critical for mesodermal tinman induction, whereas repressor binding sites prevent induction in the dorsal ectoderm and amnioserosa. Thus, integration of positive and negative regulators on enhancers of target genes appears to be an important mechanism in tissue-specific induction by TGF-beta molecules (Xu, 1998).

Functional dissection of the tinman gene has identified a 349-bp enhancer in 3'-flanking regions, tin-D, that is strictly active in dorsal portions of the mesoderm of stage 10-11 embryos. The pattern of lacZ reporter gene expression driven by tin-D closely resembles the dpp-dependent pattern of endogenous tinman expression, suggesting that tin-D functions as a Dpp response element. This notion was further supported by the observation that tin-D reporter gene activity is absent in embryos with a dpp null mutant background. Conversely, upon the ectopic expression of a constitutively active DPP type I receptor, TkvQ-D in the entire mesoderm , tin-D reporter gene expression expands into the ventral mesoderm. The observed changes of tin-D activity when the levels and spatial extents of Dpp signaling are altered closely reflect the changes seen for tinman expression under the same conditions. These observations raise the possibility that the tin-D enhancer is receiving direct inputs from the Dpp signal transduction cascade to activate tinman transcription. In addition to its dependence on dpp, dorsal mesodermal tinman expression requires the activity of tinman itself, as tinman mutant embryos show strongly reduced expression. Correspondingly, full activity of the tin-D enhancer depends on the function of tinman as well. Taken together, these results suggest that Dpp signals and autoregulation by tinman cooperate to induce full levels of tin-D enhancer activity and tinman expression in the dorsal mesoderm (Xu, 1998).

To define essential regulatory sequences within the tin-D enhancer, a series of derivatives were created with various deletions of the most strongly conserved sequence blocks and their activity was tested in vivo. Three of these fine deletions do not affect lacZ reporter gene expression in transgenic embryos. This indicates that the deleted sequences (nucleotide 16-47, 205-229, and 244-312) either lack any regulatory potential or contain functionally redundant regulatory sequences. In contrast, two other deletions result in a strong reduction of enhancer activity. One of them encompasses the tandemly repeated CAATGT/GC motifs (deltaD3) and causes an almost complete loss of enhancer activity. The other, deltaD6, which deletes 30 bp from the 3' end of tin-D, also yields strongly reduced activity in the dorsal mesoderm. These results show that the subelements D3 and D6 contain important regulatory sequences for the induction of tinman in the dorsal mesoderm and thus are candidates for target sites of the Dpp signaling cascade (Xu, 1998).

To test whether the putative tinman binding sites play roles in autoregulation, the activity of a tin-D derivative, tin-D-deltaD1, in which both of these sites were deleted (nucleotides 1-13 and 197-203). Deletion of these sites provoked two interesting effects. The first is a significant reduction of lacZ reporter gene expression in the mesoderm, which indicates that tinman autoregulation is required to achieve full levels of dorsal mesodermal tinman induction through these sequences. A second, more unexpected effect is observed in the ectoderm. Specifically, embryos carrying tin-D-deltaD1 show strong ectopic reporter gene expression in the dorsal ectoderm, which corresponds to the areas of dpp expression at this stage of development . Accordingly, in a dpp mutant background, both the ectodermal and the residual mesodermal activities of this mutant element are absent. These results show that upon deletion of the tinman binding sites, tin-D is still able to respond to dpp, but its response is essentially switched from the target tissue to the signaling tissue. Therefore, it is concluded that in the normal situation, Tinman binding to these sites is required in an autoregulatory fashion for full induction of tinman by the Dpp signals in the dorsal mesoderm. In addition, the Tinman binding sites appear to overlap with binding sites for an unknown repressor that normally prevents induction of tinman in the dorsal ectoderm, and these two mechanisms together apparently ensure the mesoderm-specific response to Dpp (Xu, 1998).

In the normal situation, tinman autoregulation appears to be restricted to the mesoderm, presumably because the early, twist-activated phase of tinman expression is mesoderm specific. To test whether tinman is also able to autoregulate in the ectoderm, tinman was expressed ectopically and tin-D reporter gene expression was examined under these conditions. For this purpose, tinman was expressed with the binary UAS/GAL4 system in ectodermal stripes under the control of an engrailed driver. Ectodermally expressed tinman is capable of activating tin-D in the ectoderm. Interestingly, ectodermal tin-D expression is restricted to dorsal portions of the transverse Tinman stripes, thereby demonstrating that tinman autoregulation can occur both in the mesoderm and in the ectoderm, but only in conjunction with Dpp signaling (Xu, 1998).

Because a combination of tin-D1 and tin-D3 sequences is sufficient to reproduce a virtually normal expression pattern in the dorsal mesoderm, these combined sequences would appear to contain DNA sequences that can bind the essential factors involved in this inductive process. To identify some of these factors molecularly the yeast one-hybrid system was used to screen for Drosophila cDNAs encoding proteins that specifically bind to D1 or D3 sequences. With multimeric D3 sequences as a bait, GAL4 AD fusion cDNAs were isolated containing sequences with strong similarities to DPC4/Smad4 proteins, which are effectors of various TGF-beta signaling processes. Of 54 candidate yeast clones, 8 carried these sequences and were derived from the same gene. Subsequent sequence comparisons showed that these cDNAs correspond to the Medea gene. Conceptual translation and sequence alignments with other members of this protein family indicate that the encoded GAL4 fusion proteins contain the complete amino-terminal portion of Medea but lack the carboxy-terminal portion encoded by sequences 3' to a native NotI site. It is conceivable that the screen selected against full-length clones, because previous reports have shown that the carboxyl terminus of Smad proteins has autoinhibitory activities (Xu, 1998).

To locate the DNA-binding domain in the Medea protein, a series of carboxy-terminal truncation and in-frame fusion constructs of Medea cDNAs with GAL4 AD coding sequences were generated, and their binding activities were tested in the yeast system, using (D3)5/lacZ as a reporter gene. The activity of Medea products increases on removal of the MH2 domain in this assay, indicating that the MH2 domain of Smad4 group proteins has an inhibitory effect on DNA binding, similar to the MH2 domain of Mad group proteins. By removing 10 carboxy-terminal amino acids from the MH1 domain, the activity drops to background levels. The MH2 domain does not display any binding activities in this assay. Thus, it appears that the MH1 domain serves as the DNA-binding domain for Medea, as it does for Mad (Xu, 1998).

DNase I footprinting assays with bacterially expressed GST fusion proteins were used to characterize the binding of Tinman and Medea to sequences of the tin-D element and to test whether Mad is also able to bind. Tinman specifically protects the two D1 sequences that contain NK homeodomain binding sites and are required for autoregulation. The MH1 domains of Medea protect three distinct sequences within tin-D. Importantly, one of them (nucleotide 95-127) overlaps with the D3 sequence that is essential and sufficient for tin-D activity and was used for the isolation of Medea. Mad shows binding to several additional sequences. Two other sites that are protected by Mad, but not Medea under the same conditions, correspond to the 3' portion of D3 and adjacent sequences. Therefore, Mad protects most of the sequence stretch between nucleotides 95 and 160, which has D3 at its core, whereas Medea protects only the 5' two-thirds of D3. Taken together, the DNase I protection data reveal a minimum of eight in vitro binding sites for Medea and Mad in the tin-D element, at least four of which are located in the essential elements D3 and D6. Moreover, it appears that Medea and Mad have overlapping, but not identical, binding specificities to tin-D sequences (Xu, 1998).

Gel retardation assays provided additional information on the DNA-binding specificities of Medea and Mad and their binding sites in the tin-D element. Both Medea MH1 and Mad MH1 bind to 32P-labeled D3 probes, and excess of unlabeled D3 DNA can compete for binding. Because D3 contains tandemly repeated CAATGT and GC-rich motifs, a test was carried out to find out which of these two sequence motifs are involved in Medea and Mad binding. Replacement of four GCs in each of the GC-rich motifs by As and Ts renders the mutated D3 sequence unable to compete for Medea and Mad binding to the wild-type D3 sequence. In contrast, in vitro mutagenesis of the CAATGT motifs does not interfere with Medea and Mad binding, as these mutated versions compete equally well as D3 wild-type DNA. These data show that the GC-rich motifs are essential for Mad and Medea binding and likely represent two distinct binding sites for these proteins in the D3 element. In summary, these in vitro DNA binding studies demonstrate that the functionally significant D3 and D6 elements contain at least four GC-rich binding sites for Medea and Mad, although Medea binds with high affinity only to those in D3. In addition, tin-D contains at least four other binding sites for Medea and Mad, all of which include GC-rich stretches (Xu, 1998).

In vitro binding sites of Medea and Mad are essential for the activity of tin-D in vivo. Combinations of single copies of the wild-type and mutated sequences of D3 and D6 similar to the ones used for the in vitro binding assays were tested for their ability to activate reporter gene expression in transgenic embryos. The activity of these sequences was tested in the context of a shortened version of tin-D (tin-D*; nucleotide 1-143 plus Tin binding site 2 and nucleotide 321-349). Tin-D* containing wild-type D3 and D6 sequences produces an almost normal pattern of dorsal mesodermal expression, although expression levels are lower than with the complete tin-D element. In contrast, a version in which both Medea/Mad binding sites in D3 are disrupted by 8 bp exchanges is almost completely inactive in vivo. Similarly, expression is nearly abolished upon disruption of the two Mad binding sites in D6 or of all four Medea/Mad binding sites in D3 and D6. Interestingly, specific disruption of the CAATGT sequences in D3 also results in a complete loss of activity in the dorsal mesoderm. Together, these data demonstrate that each of the pairs of Medea/Mad binding sites in D3 and D6 plays a critical role in the Dpp-induced activity of tin-D in the dorsal mesoderm. Moreover, the CAATGT sequences in D3 appear to be required for the binding of a different factor that is also essential during this process. An interesting difference between tin-D and tin-D* is an ectopic expression in the amnioserosa, which is observed between stage 8 and 11 of embryogenesis. This observation suggests that the region between D3 and D6, which is missing in tin-D*, contains a repressor element for this tissue. The results with mutated versions of tin-D* indicate that the Medea/Mad binding sites in D3 and D6 are necessary for amnioserosa expression, whereas the CAATGT sequences are not required (Xu, 1998).

The absolute requirement for the tandemly repeated CAATGT sequences for the activity of the Dpp response element strongly points to the existence of a second essential coactivator that binds to these sequences. The results with wild-type and mutated versions of the tin-D element predict that this factor is expressed and active in both mesoderm and ectoderm, since disruption of the CAATGT motifs abolishes both mesodermal and ectopic ectodermal induction. The close juxtaposition of these motifs with Smad binding sites in the minimal Dpp response element may suggest that the unknown binding factor also participates in protein-protein interactions with bound Smad proteins. It is interesting to note that this sequence motif is closely related to that of the binding site of Xenopus FAST-1. The forkhead domain protein FAST-1 has been shown to bind to the sequences AAATGT within an activin-response element of the Mix.2 gene and to associate with Smad2 and Smad4 . It is thus conceivable that a related member of the forkhead domain protein family plays a similar role in the tinman Dpp response element, albeit in this case in a complex with DNA-associated Smads (Xu, 1998).

The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut musculature versus body wall musculature. biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature. biniou expression is activated in the trunk visceral mesoderm primordia downstream of dpp, tinman, and bagpipe and is maintained in all types of developing gut muscles (Zaffran, 2001).

bagpipe-expressing domains are defined by the intersecting dorsal activities of dpp/tin, which act positively, and segmentally modulated activities of wg/slp, which have repressing effects. bin also requires tin activity for normal expression in the trunk visceral mesoderm primordia. Whereas bap expression is virtually absent in these cells upon loss of tin activity, residual bin expression is observed in small clusters of cells. To test the possibility that residual expression of bin in tin mutant embryos is due to direct inputs from Dpp, bin expression was examined in embryos in which dpp expression was induced ectopically in the entire mesoderm. Ectopic dpp in a wild-type background, which causes tin expression to be expanded ventrally, results in an analogous expansion of the bin domains. Notably, ventral expansion of the bin domains is also observed upon ectopic dpp expression in the absence of tin activity, although the domains are narrow. Thus, Dpp is able to induce bin in the absence of tin, although tin activity is required for normal expression levels. The residual expression of bin in tin mutant embryos is unstable and not maintained in later stages of development (Zaffran, 2001).

Table of contents

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Developmental Biology | Effect of mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.