BIOLOGICAL OVERVIEW

Medea was originally identified as a genetic modifier of decapentaplegic, employing the same maternal effect screen that had identified Mothers against decapentaplegic (Mad) (Raftery, 1995, for review, see Raftery, 1999). Drosophila Mad is the founding member of the Smad family of signal transducers, all of which function in TGF-beta signaling pathways. Despite being highly related to one another, particular Smad proteins function in distinct pathways. For instance, Smad1 (a Mad homolog) mediates BMP2/4 signals, whereas Smad2 and Smad3 function in TGF-beta/activin pathways. Several of these Smad proteins are phosphorylated in response to activation of specific pathways. Phosphorylation is directly mediated by the cognate type I ser/thr kinase receptor and occurs on the last two serines of a conserved SSXS motif at the carboxy terminus of the Smad protein. Once phosphorylated, Smad proteins translocate to the nucleus where they appear to function as transcriptional regulators through their interactions with DNA binding proteins (Wisotzkey, 1998 and references).

Drosophila Medea encodes a homolog of Smad4. Smad4 is relatively divergent from other vertebrate Smads and does not appear to be regulated by signal-dependent phosphorylation. However, overexpression of vertebrate Smad4 stimulates TGF-beta and activin responses. Smad4 associates with Smad1 in response to BMP2/4 or with Smad2 in response to TGF-beta, and dominant negative Smad4 blocks both BMP and activin responses. These observations have generated a model in which Smad4 is essential for signal transduction by all TGF-beta family members through its interaction with phosphorylated receptor-regulated Smads. Medea functions downstream of Dpp; complete removal of the Medea gene product causes the same embryonic phenotype as dpp null mutations. Mad undergoes signal-dependent translocation to the nucleus in the absence of Medea; in contrast, Medea is localized in the cytoplasm and requires Mad in order to accumulate in the nucleus. Specific mutations identified in strong alleles of Medea disrupt either Medea interaction with Mad or nuclear translocation of the Mad/Medea complex. Thus, interaction with Mad and nuclear import are critical for Medea function. However, unlike Mad, Medea is not required for expression of all Dpp-dependent genes and in its absence intracellular Dpp signaling rapidly attenuates with distance from the Dpp source. It is propose that the presence of Medea in heteromeric nuclear complexes with Mad modifies or enhances Dpp signaling. These studies provide a model for the biological function of Smad4 in vertebrate TGF-beta family signaling (Wisotzkey, 1998).

The role of Medea in transmission of the Dpp signal is exemplified by the finding of a position-specific requirement for Medea in wing development. dpp is expressed in a line along the anterior-posterior compartment boundary; In comparison, Thick veins and Mad are required throughout the wing primordium or pouch for cell proliferation and expression of the gene optomotor blind (omb). A second Dpp type I receptor gene, saxophone, is dispensable for proliferation, but is required throughout the wing pouch to boost the final level of the Dpp signal for pattern formation. Therefore, the requirement for Medea was examined in these two aspects of wing development (Wisotzkey, 1998).

Clones of cells homozygous for either Medea 8 or Medea 2 were generated. Medea mutant clones in the wing pouch do not survive when induced during the first larval instar (96-72 hours before pupariation), although mutant clones are recovered elsewhere in the wing disk. A similar position-dependent requirement for proliferation in the wing disk has been found for tkv. However, Medea mutant clones of variable size are recovered in the wing pouch when induced in the middle of the second larval instar (66 hours before pupariation). In contrast, clones of cells homozygous for null alleles of Mad cannot be recovered in the wing imaginal disk when they are induced at this stage of development. Thus, there is a weaker requirement for Medea in cell proliferation than for Mad. Analysis of the expression of omb reporter within Medea mutant clones indicates that loss of Medea function does not simply mimic the phenotype of clones homozygous for a weak Mad allele. Most Medea mutant clones show a strong reduction in omb expression; in all cases omb expression is altered only in mutant cells. Thus, Medea is required in cells responding to Dpp. Within the wing pouch, the reduction in omb expression depends on where the clone is located. Clones that fall along the anterior-posterior midline, where dpp is expressed, show only a slight reduction. Clones distant from the midline displayed the greatest reductions in expression, many having undetectable levels of omb expression. In contrast to the position-dependent effects on omb expression in Medea clones, all clones homozygous for a leaky allele of Mad fail to express omb. Similarly, omb expression is lost in tkv clones induced 24 hours before pupariation, while Medea clones induced at this stage have little effect (Wisotzkey, 1998).

Together, these observations indicate that Medea is not required for all Dpp-dependent signaling; instead, the requirement for Medea varies with its position in the wing pouch. Since omb expression is only weakly affected in Medea mutant clones located at the anterior-posterior midline of the wing pouch, and given that dpp is expressed in this vicinity, it is possible that the requirement for Medea in maintaining omb expression can be bypassed in the presence of stable, high level expression of Dpp. These observations suggest that while Mad is absolutely required for Dpp signaling, Medea enhances or modifies the signal (Wisotzkey, 1998).

It is suggested that Medea uniquely strengthens responses to Dpp and that in its absence, intracellular Dpp signaling rapidly attenuates in response to changes in Dpp levels. This model reconciles the absolute requirement for Medea in dorsal-ventral patterning of the embryonic ectoderm with the variable requirement for Medea in the wing primordium. In contrast to wing patterning, dorsal-ventral patterning is highly sensitive to perturbations in the level of Dpp, due to the low level and transient expression of the gene. Thus, even at the highest levels of Dpp signal in the early embryo, Dpp protein levels are limiting and Medea is essential to specify dorsal fates (Wisotzkey, 1998).

GENE STRUCTURE

Bases in 5' UTR - 696

Exons - 9

Bases in 3' UTR - 303

PROTEIN STRUCTURE

Amino Acids - 711

Structural Domains

Comparison of Medea with other Smad proteins indicates that Medea is most closely related to human Smad4, with an overall identity of 59%. The regions of greatest identity between Medea and Smad4 include the two MAD homology domains, MH1 and MH2, with 76 and 80 % identity, respectively. Both proteins share an insert in the MH2 domain of 30 and 34 amino acids, respectively. The linker regions between the MH1 and MH2 domains display little sequence conservation; however, like all Smad family members, this domain is rich in proline residues. Additional sequences within the linker region of Medea, including the presence of several unusual poly-glutamine repeats, account for the protein's larger size relative to Smad4. Sequence comparison of Smad family members indicates that they can be divided into three groups: the Smad4 subgroup, which comprises Medea, Smad4 and the C. elegans sma4; a second group, which includes those Smads most closely related to Mad and a third group comprising the inhibitory Smads (for example, Drosophila Dad). Within the MH1 and MH2 domains there are amino acids conserved in most positively-acting Smad proteins and amino acids that are conserved only within a sub-group. Of note, a conserved SSXS motif present at the carboxy-terminus of all MAD subgroup proteins is absent in the Smad4 subgroup. Since serine residues in this motif are targets for type I receptor-mediated phosphorylation (see Punt and Thickveins), the absence of these residues raises questions regarding the regulation and function of Medea in Dpp signaling (Wisotzkey, 1998 and Hudson, 1998).

date revised: 18 April 98

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