TLD mRNA is first detected at nuclear cycle 10-11. In the central region of the embryo, only dorsally located nuclei express tld, while at the poles, both dorsally and ventrally located nuclei are labelled. The first alternation is a reduction in the staining of the cells located within 10% egg length from each pole. With further reduction, four distinct bands of expression are observed by the end of cellularization. The dorsal-most 10-20% of the embryo's circumference becomes more heavily stained.

During late stage 8, lateral patches of cells located within emerging gnathal segments are stained. During stages 10 and 11, two parallel groups of labelled cells arise. One comprises a continuous stripe of cells located between the dorsal epidermis and the amnioserosa, and a second is compared to segmentally repeated cells near the boundary between dorsal epidermis and the neurogenic region, around the tracheal pits (Schimell, 1991).

Effects of Mutation or Deletion

tld mutants lack a characteristic subset of dorsally derived cuticular structures from the dorsal 40% of the blastoderm fate map. Missing are cuticular specializations of the head and the fitzköpfer in the tail. There is an expansion of the ventral neurogenic ectoderm. Embryos lacking dpp activity have a more severe phenotype (Shimell, 1992).

Doubling the dpp+ gene dosage completely suppresses weak tolloid mutants and partially suppresses the phenotypes of tolloid null mutants. The function of tolloid is to increase dpp activity. tolloid, shrew and short gastrulation genes are required to generate a gradient of dpp activity, which directly specifies the pattern of the dorsal 40% of the embryo (Furguson, 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 amnioserosa, while the genes tolloid, screw and decapentaplegic (dpp) affect divisions in both the prospective amnioserosa and the dorsal epidermis. In each of these mutants dorsally placed mitotic domains are absent and this effect is correlated with an expansion and dorsal shift in the position of more ventral domains (Arora, 1992).

Several mutant tolloid alleles have been generated and have examined their interaction with a graded set of dpp point alleles. Some tolloid alleles act as dominant enhancers of dpp in a trans heterozygote, and are therefore antimorphic alleles. DPP is most closely related to the TGF-beta superfamily members BMP-2 and BMP-4. The common phenotype and genetic interaction data with tolloid suggests a direct physical association between these two proteins. However, a tolloid deficiency shows no such genetic interaction. Antimorphic dominant enhancers of dpp are missense mutations in the protease domain. Most tolloid alleles that do not interact with dpp are missense mutations in the C-terminal EGF and C1r/s repeats, or encode truncated proteins dispersed throughout the length of the protein (Childs, 1994 and Finelli, 1994).

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. In addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Because of the crucial role of dpp in inducing dorsal mesodermal tinman expression and the specification of dorsal mesodermal tissues, it is of interest to determine whether other components known to function in dpp-mediated signaling events during blastoderm are also required for mesoderm induction. Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are all shown to be required to allow full levels of tinman induction during this process. screw, which encodes a second BMP2/4-related molecule, has been proposed to act synergistically with dpp to specify dorsal ectoderm and amnioserosa. Similarly, it has been shown that tolloid, which encodes a BMP1-related metalloproteinase, acts to enhance the activity of the dpp gene product during mesoderm induction. Both scw and tolloid are shown to be required for normal induction of tinman expression in the dorsal mesoderm, and in the absence of either gene activity, tinman expression in the dorsal mesoderm is reduced and segmentally interrupted. Thus scw and tld are necessary for achieving full levels of tinman induction, whereas dpp is obligatory for this event. In addition, unlike dpp mutants, mutants for scw or tld form some residual visceral mesoderm. However, heart formation is more sensitive to the activities of scw and tld and is disrupted to a similar extent as in dpp mutants. schnurri is also necessary for tinman induction in the dorsal mesoderm. The dorsal tinman domain is clearly reduced, as compared to wild-type embryos, although the levels of Tinman mRNA are close to normal. Therefore, shn may be required to enhance dpp signaling during tin induction, but significant levels of tin activation can still occur in the absence of its activity (Yin, 1998).

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).


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tolloid: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 December 2011

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