BIOLOGICAL OVERVIEW

Mothers against dpp was discovered (Raftery, 1995) in a search for elements of the decapentaplegic signaling pathway in Drosophila (for review, see Raftery, 1999). When given the problem of identifying elements in a biochemical pathway, a geneticist will usually take a genetic approach, one that has worked in previous attempts in other systems. The tactic is to disclose enhancers which exacerbate known mutations. An enhancer is a mutation in the sought-for gene that engenders a more severe phenotype than that caused by the mutation of a single, already characterized gene. Thus a search was carried out for mutations that produce a more severe phenotype (an "enhancement") when combined with known dpp mutations. Mad mutations can be placed in an allelic series based on the relative severity of the maternal effect enhancement of weak dpp alleles, thus explaining the name Mothers against dpp..

Two types of experiments were carried out. The first was designed to reveal mutations in genes expressed in zygotes that exacerbate the phenotype of embryos that have a limiting amount of DPP. Mutations that act as enhancers of such DPP limited embryos cause embryonic lethality. The possibility that some of the gene products involved in DPP signaling might be supplied during oogenesis necessitated a second experiment looking for failure to recover progeny (that is, embryonic lethals) from mothers exhibiting a limiting amount of DPP. The two experiments yielded several kinds of enhancers: 1) new dpp alleles, which along with an already limited quantity of DPP cause embryonic lethality; 2) mutations in tolloid, a gene whose product is involved in DPP processing; 3) mutations in screw whose protein product is a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo; 4) mutations in Media, another gene involved in DPP signaling, and 5) mutations in Mad. Mutations in Mad, when interacting with limiting DPP levels, produce a defective amnioserosa (see also [Image]), the extra-embryonic membrane comprising the dorsal-most cells in early embryos (Raftery, 1995).

Mad mutations are also enhancers of mutant dpp appendage phenotypes. Thus Mad mutants produce a further reduction in wing blade size, a slight reduction in the eye, and loss of tarsal claws. These Mad mutant phenotypes exhibit a close correspondence to dpp mutant phenotypes. Homozygous Mad mutant larvae also show midgut defects and a greatly reduced gastric caecae. DPP signaling from visceral mesoderm to midgut endoderm is required for proper extension of the gastric caecae in parasegment 4 and for the induction of the homeotic gene labial in the adjacent endoderm of parasegment 7. Homozygous Mad mutant embryos lack labial expression and have defects in midgut constriction engendered by labial expression. Other imaginal disc derived structural defects are evident in homozygous Mad mutants, including heldout wings, split notum, loss of distal leg segments, duplications of the third antennal segment and defects in female genitalia (Sekelsky, 1995).

To date there is no indication that the Drosophila MAD protein is nuclear: antibody staining experiments indicate a cytoplasmic localization. Neverless there is clear indication that a human MAD homolog enters the nucleus upon BMP2 signaling (Hoodless, 1996).

A simple experiment was carried out to see if MAD acts upstream or downstream of DPP. DPP was ubiquitously expressed in Mad mutants. If Mad acts upstream of DPP, then ubiquitous expression of DPP should result in labial induction in the midgut endoderm independently of Mad. In fact labial does not get induced in Mad mutants even when dpp is expressed ubiquitously, indicating that MAD acts downstream of dpp. When MAD is artifically expressed in mesoderm, it fails to rescue labial induction in embryos otherwise deficient in MAD, but artificial expression of MAD in endoderm does rescue labial induction (Newfeld, 1996). Thus MAD appears to be a component in DPP signaling acting downstream of dpp in cells that are the recipients of DPP signaling.

Daughters against dpp (Dad), whose transcription is induced by Dpp shares, weak homology with Drosophila Mad, a protein required for transduction of Dpp signals. Dad is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule, and in fact ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad blocks Dpp activity. Dpp target gene optomotor blind is absent in Dad-overexpressing cells. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by either protein alone. Dad can also antagonize the activity of a vertebrate homolog of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. It is concluded that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).

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 (the mammalian homolog of Mad) 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 (Wisotzkey, 1998).

GENE STRUCTURE

Bases in 5' UTR -346

Bases in 3' UTR - 937

PROTEIN STRUCTURE

Amino Acids - 455

Structural Domains

MAD and its homolog in vertebrates (Smad1) are essential for signaling in DPP and BMP-2/4 pathways and can elicit biological responses characteristic of BMP-2/4 (Newfeld, 1996). SMAD proteins share a high degree of homology in their amino-terminal MH1 (Mad homology) and carboxy-terminal MH2 domains. The MH2 domain is considered the effector domain, whose activity is opposed by its physical interaction with the MH1 domain. SMAD4 is a central signaling molecule of several TGFbeta-related pathways. In contrast to the pathway-restricted SMADs, SMAD4 rapidly associates with both SMAD1 in response to BMPR-1 signaling and SMAD2 in response to TbetaR-I and ActR-IB signaling. Unlike SMAD4, SMAD1 and SMAD2 contain consensus phosphorylation sites for receptor type I Ser/Thr kinases within their MH2 domains. The model emerging from recent biochemical and crystallographic studies implies that phosphorylation of the receptor-regulated SMADs relieves them of the MH1 inhibitory effect, allowing their interaction with SMAD4 and subsequent translocation to the nucleus (Sirard, 1998 and references).

Signal transduction specificity in the transforming growth factor-beta (TGF-beta) system is determined by ligand activation of a receptor complex, which then recruits and phosphorylates a subset of SMAD proteins, including Smads 1 and 2. In vertebrates, Smad1, and presumably its close homologs Smad5 and Smad8, are phosphorylated by BMP receptors and mediate BMP responses. Smad2 and its close homolog Smad3 are phosphorylated by TGF-beta receptors and mediate TGF-beta and activin responses. In Drosophla, Mad (a close homolog of Smad1) mediates the effects of the BMP-like factor, Dpp. After phosphorylation by receptors, Smads 1 and 2 associate with Smad4 and move into the nucleus where they regulate transcription. A discrete surface structure has been identified in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C-terminal domain. The L3 loop sequence is invariant among TGF-beta and bone morphogenetic protein (BMP)-activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF-beta receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF-beta receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping these two amino acids while, in addition, swapping four amino acids near the C-terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD-receptor interactions, and indicate that the L3 loop, together with the C-terminal tail, specifies SMAD activation (Lo, 1998).

date revised: 21 APR 97 

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