2nd Report draft
Abstract
During the development of Drosophila Melanogaster embryo, the product of Decapentaplegic (Dpp) gene plays an essential role in the final organization of dorsal-ventral pattern. The Dpp is a homologue of Bone Morphogenetic Proteins 2 and 4 (BMP 2/4) and acts as an extracellular morphogen to form a gradient with the effect of another BMP homologue Screw (Scw) and some other regulators including short gastrulation (Sog), twisted gastrulation (Tsg) and Tolloid (Tld). Among these Dpp regulators, Sog and Tsg are products of zygotic genes, and Tld is metalloprotease. Moreover two type IV collagens Dcg1 and Viking (Vkg) may participate in the BMP signalling as well. In our project, we checked the interaction between Tsg, Vkg, Sog and Dpp by GST pull down assay, and tried to find out the exact region where the interaction occurs. We confirmed the enhancement of Sog to Tsg on affinity to Dpp, and found that Sog reduces the binding activity of Vkg C terminus part to Dpp. We also performed in situ hybridization on Vkg and Dcg1 mutated embryos and wild type embryos to compare the expression patterns of Race and U-shaped (Ush) genes which are related to the expression of Dpp. And we found that there are obvious differences between the wild type and mutated embryos.
Introduction
As kind of insect, Drosophila Melanogaster undergoes gastrulation germ layer differentiation and then forms endoderm, mesoderm, ectoderm and their suborganelles during embryo development. This differentiation process rises from the different spatial positions of the cells in the embryo and is mostly controlled by morphogens. (Ashe and Briscoe, 2006)
During the early stages of embryo Drosophila Melanogaster development, the product of spätzle gene acts as a morphogen to form the Dorsal (Dl) activity gradient and thus establishes the dorsal-ventral pattern (Morisato and Anderson, 1994; Raftery and Sutherland, 2003). Then, the control is taken away by other morphogens. In previous studies, it has been reported that two homologs of Bone Morphogenetic Proteins 2 and 4 (BMP 2/4), Decapentaplegic (Dpp) and Screw (Scw) which are members of TGF-β superfamily, are the control factors for the formation of dorsoal ectoderm in Drosophila Melanogaster. Further researches indicated that Dpp plays the main role since all dorsal cells assume ventral lateral fates and the mutant phenotype of the zygotically required genes appears the most severely with the lack of Dpp (Arora and Nusslein-volhard, 1992; Ferguson and Anderson, 1992a). Based on these discoveries, the scientist put emphasis on the Dpp. Later experiments showed that after the dorsal-ventral (DV) axes is patterned, the effect of the morphogenetic Dpp depends on the local concentration level. In detail, high Dpp level leads to the fate of being amnioserosa whilst lower levels make the cell become dorsal ectoderm tissue (Ashe et al., 2000; Shimmi and O'Connor, 2003). The Dpp/Scw functions by binding to a receptor complex which is supposed to be composed of type I and type II transmembrane serine/threonine kinases (Hogan, 1996; Nakayama et al., 2000). It has been elucidated that in Drosophila, the type I receptor Thick veins (Tkv) interact with the Dpp molecule and the type II receptor Punt (Put) to form a heteromeric complex. The active Put within this complex phosphorylate the Tkv consequently and thus activates the associated type I kinases which will phosphorylate the cytoplasmic protein Mothers against Dpp (Mad). The Mad which belongs to the Smad superfamily then function inside of the cell and lead to the differentiation and in this way the signal of Dpp is transduced. Moreover, in the signal transduction of Scw, it seems that another type I receptor Saxophone (Sax) takes the place of Tkv in Dpp signal transduction. However experiment also showed that high level of Tkv is able to compensate the loss of Sax, and indicates that these two morphogens shares a similar intracellular pathway. (Affolter et al., 2001)
Based on the knowledge above, the regulation of dorsal ectoderm and amnioserosa formation by BMP signalling pathway is suggested to be achieved by the gradient distribution of Dpp. However, an interesting and indispensable phenomenon of the gradient of Dpp is that it is not smoothly distributed. So far, we are able to observe the visualized the Dpp distribution in Drosophila embryos and the results showed that Dpp activity has a peak level at the 8-10 dorsalmost cells which will later form the amnioserosa, and there is a sharp fall of Dpp level at the edge of the dorsalmost cells where cells on the different sides of the confine receive high and very low BMP signal (Shimmi and O'Connor, 2003). This step gradient specifies the very small region to amnioserosa and ensures the normal development of amnioserosa and dorsal epidermis.
According to the model of morphogen distribution which concerns the Dpp and Scw alone, this phenomenon is unable to be explained. In the classical morphogen gradient formation model, specified cell or region produces the morphogen and the morphogen diffuses to the tissue nearby via extracellular or intracellular pathway. Thus a morphogen concentration gradient which peaks at the secreting cell and smoothly deduced along with the increasing distance to the secreting cell (Ashe and Briscoe, 2006). The step gradient of Dpp activity is incompatible with this model, thus the formation of this gradient should be created by another mechanism with some other factors.
As far as we know, BMP signalling activity is regulated by the products of two zygotic genes short gastrulation (Sog) and twisted gastrulation (Tsg) , a metalloprotease Tolloid (Tld) (Ashe, 2005). And just recently two type IV collagens Viking (Vkg) (Yasothornsrikula et al., 1997) and Dcg1 (Blumberg et al., 1988) are thought to be playing a role in the BMP signalling pathway.
It is known that Sog gene is a homologue of Xenopus Chordin (Chd) gene which produces antagonist of BMPs both structural and functional. (Ferguson and Anderson, 1992b; Piccolo et al., 1996). And later studies have discovered that Tsg is also a secreted protein which acts as extracellular antagonists of BMP (Chang et al., 2001; Ross et al., 2001). These works gave the evidence that Sog and Tsg bind to BMP directly and inhibit the interaction between BMP and its receptor (Tkv and Put) to form a complex in order to block the signal transduction (Ashe, 2005). However, these interactions will not destroy the BMP molecule itself, and the formed complex can be divided into the original single molecular to regain its function as well. In Drosophila, this cleavage process is operated by the protease Tld which was first recognized as an activator of Dpp (Ferguson and Anderson, 1992a). As far as we know, these three regulators (Sog, Tsg and Tld) of BMP signalling pathway are suggested to play essential part in the mechanism which determined the peak level and the step gradient distribution of BMPs since the loss of any one of these three regulators will lead to the loss of the step gradient.
The Dpp protein itself is nonautonomous and is able to diffuse under a narrow limitation. The Dpp direct visualization result shows that there is an even level of Dpp throughout the dorsal 40% of Drosophila embryo before the late cellularisation stage. Though this uniform distribution will lost in later stages of normal embryo development, it can be maintained by Scw mutation. The activity pattern of Smad observed in the embryo also supports this Dpp distribution. (Raftery and Sutherland, 2003; Shimmi et al., 2005; Wang and Ferguson, 2005) Though we are still not able to detect and visualize the intracellular concentration of Dpp or Scw directly, the phenomenon observed gives the hypothesis that the Dpp may be evenly secreted in the dorsal part of the embryo after the patterning of DV axes. Moreover, it also been reported that the Dpp and Scw function much more effectively by forming a Dpp-Scw heterodimer which is able to induce a 10 to 100 times higher phosphorylated Mad accumulation than Dpp or Scw homodimer do within the cells exposed to these molecules (Shimmi et al., 2005). However, there is a controversy between the reports of Wang and Ferguson group (Wang and Ferguson, 2005) and Shimmi et al. (Shimmi et al., 2005) group. The previous group found that with the lack of Scw the interpreted results of their experiments support the mechanism that Dpp and Scw homodimers function cooperatively whilst the report from the latter one gave evidence for the existence of Dpp-Scw heterodimer.
In earlier studies, there was a good hypothesis considering the combined effect of BMP inhibitors (Sog and Tsg), the cleaver (Tld) to explain the step gradient of Dpp activity (Shimmi and O'Connor, 2003). Nevertheless, the controversy mentioned above gives the birth of a new hypothesis which counts the controversy in whilst interpreting the step gradient of BMPs at the same time. This new hypothesis inherited the basic mechanism of step gradient formation from the earlier one.
Start from Sog, it is produced at the presumptive neuroectoderm and the distribution of Sog in Drosophila embryos had been directly shown to be in a graded fashion and limited by Tld in dorsal region (Srinivasan et al., 2002). The distribution of Sog protein agrees with the classical passive morphogen distribution model since the level of Sog reach the highest at the producing region and falls whilst the distance to the presumptive neuroectoderm increases. However, the Sog detected is thought to be in the complex including Sog and Dpp and Sog single molecule itself will be degraded via endocytosis. Thus it is also suggested that the shaping of the lowest level of Sog at the dorsalmost region is associated by the protease Tld which is able to cleave the complex and release Sog to be degraded. The interaction between Sog and Dpp here not only contributes to the formation of Sog gradient, but also help the diffusion of Dpp. As mentioned, either the Dpp homodimer or Dpp-Scw heterodimer has the activity to bind the receptor on cell membrane and then are trapped and lost the diffusion ability. Thus, the binding between Sog and Dpp on the one hand is an inhibition on Dpp, on the another hand is a protection of Dpp signalling activity and helps the diffusion of Dpp. Since the Tld restores the activity of Dpp by cleaving the complex when the complex reaches that region, a long-range enhancement and local inhibition of Dpp activity by Sog is then created. (Ashe, 2002; Ashe and Levine, 1999)
A notable fact is that Tld is distributed evenly in the embryo (Shimell et al., 1991), thus the cleavage rate of the Sog-Dpp complex is the same throughout the embryo. However, only in the dorsalmost region where is lack of Sog, the Dpp released by Tld cleavage is able to bind to the receptor before forming the complex with the Sog protein again.
Later studies demonstrated that Tsg is another inhibitor of Dpp activity, its role is generally recognized to be similar to Sog. Tsg and Sog are enhancers to each other since the loss of either Tsg or Sog reduces the efficiency of Dpp inhibition in the embryo and they show strong Dpp affinity when existing together. Thus the mechanism of the enhancement is suggested to be the formation of a Tsg-Sog-Dpp complex (Chang et al., 2001; Shimmi and O'Connor, 2003). The knowledge on the complex is further advanced by the recent discoveries mentioned above that gave evidence for the existence of Tsg-Sog-Dpp-Scw complex. It is also discovered that the affinity of Sog/Tsg for Dpp and Scw homodimer is weaker than Dpp-Scw heterodimer.
What is novel in the new hypothesis is that it suggests there are three kinds of receptors on the cell membrane for the BMPs which are able to specially bind to Dpp homodimer, Scw homodimer or Dpp-Scw heterodimer. There is a positive feedback with unclear mechanism between the interaction of Dpp-Scw heterodimer and the Dpp-Scw-receptor. Combining the potent signalling of Dpp-Scw heterodimer and the effect of Sog, Tsg and Tld, this feedback helps the formation of peak BMPs signalling level in the dorsalmost cells (Ashe, 2005; Wang and Ferguson, 2005). In contrary, in other regions where the Sog/Tsg level is higher, the free BMPs are thought to be mostly Dpp and Scw homodimers and thus the signalling strength is much weaker.
This hypothesis is reasonable and satisfaction. However, two proteins, Dcg1 and Vkg, may have to be put into the BMP signalling pathway in Drosophila embryos according to our investigation. Vkg and Dcg1 are type IV collagens which are the components of extracellular matrix and may limiting the diffusion of Dpp/Scw. In our study, we detected the marker gene Race (Tatei et al., 1994) and U-shaped (Ush) (Cubadda et al., 1997) using in situ hybridization method in the Vkg and Dcg1 mutated Drosophila embryos. The result showed that both of them are essential for the BMP signalling, but the exact role for them is unclear yet. To get deeper understanding of the BMP signalling pathway, we also further investigated the interaction among Tsg, Sog and Dpp by GST pull down assay. And we tried to split the Dpp and Sog protein into smaller fragments and performed GST pull down assay on them consequently to investigate which is the interaction region on these proteins. The interaction analyse was carried out on Vkg fragment and Dpp as well. Moreover, we compared the type IV collagens in different species to find out whether there is a conserved site which may related to the BMP signalling pathway. Based on our knowledge, we suggest that there may be a competition between Dcg1/Vkg and Tsg/Sog to bind the BMPs.
Materials & Methods
Mutant Drosophila Melanogaster
The yw67c23 Drosophila Melanogaster from ___ is used as the wild type. Vkgk00236 and Dcg1k00405 flies (gift from Xiaomeng Wang, University of Manchester) are used as Vkg and Dcg1 mutants. (details needed) These two mutants have weaker Vkg or Dcg1 expression, because the completely disappearance of functional Vkg or Dcg1 leads to flies unable to lay eggs.
Embryo collection
The embryos from the wild type, Vkg mutant and Dcg1 mutant flies were collected every 2 hours at 25 ºC and left to develop for another period equal to 2 hours at 25 ºC before fixation. The embryos were fixed with fixing buffer and hepaton and stored in ethanol at -20 ºC. Thus the embryos collected were at the development stages between 2-4 hours. And a small number of overnight embryos were also fixed and stored with the 2-4 hours embryos.
In situ hybridization
In situ hybridization was performed with the embryos collected described above. Race and Ush digoxigenin-labelled RNA probes were used to visualize the distribution of Race and Ush gene. (copy the protocol here or cite the book?) The slides were observed under ___ microscope and images were taken at the same time.
Plasmids
GST protein expression plasmid pGEX4T-3 (reference) and pGEX4T-1 (reference) was used for GST fusion protein construction cloning. The splitting of Dpp and Sog proteins were based on the Drosophila cell expression constructs Dpp-HA ____ (reference here) and Sog-Myc ____ (reference here).
The Dpp protein will be cleaved in cytosol and the CDS region which is 402 base pairs long is localized at the very end of the full Dpp DNA sequence. In the ___(Dpp-HA) construct, a triple HA tag region was inserted after the 90th base pair in the CDS region and is 195 base pairs long. And in the ___ (Sog-Myc) construct, the Myc tag is inserted in front of the Sog DNA sequence.
Cloning
Phusion (purchased from Finnzyme) polymerase was used for PCR. And all primers were ordered from Sigma-Aldrich. (Should write the primer sequences here?)
All restriction digestion enzymes used in cloning were products of New England Biolabs except EcoRV which is purchased from Rosche.
T4 Ligase used was produced by Rosche.
T-easy ligation kit (purchased from Promega) was used in some of the cloning work.
DH5α E.Coli cell strain was used for transformation for plasmid amplification and selection.
Cell transfection, expression & harvesting
S2 R+ cell line used for protein expression was incubated at 25 ºC in FCS medium (medium detail here) until it reaches the cell density suitable for transfection. Transfectgene transfection kit (purchased from QIAGEN) was used in transfection following the manual. CuSO4 (detail quantity) was added 24 hours after transfection to induce the expression of transfected plasmid DNA, and then the transfected cells were incubated for another 72 hours. The cell cultures were harvested afterwards. Cells and medium were first separated by centrifugation, and the cell pellets were lysed subsequently. Both the supernatant and pellet lysate were checked by western blotting and were stored in -80 ºC freezer.
GST fusion protein expression & purification
For GST fusion protein expression, BL21 E.Coli cell strain was used for transformation. Single colony was picked to set up start culture and the start culture was later seeded into more medium to make expression culture. These cultures were all incubated shaking at 37 ºC. 50 µL of 0.2M IPTG was added to the expression culture to induce protein expression when the OD600 reached 0.6, and the culture was moved to 25 ºC shaker. After 3 hours of incubation, the cells were harvested by centrifugation. Then the cells were processed with lysozyme and sonication, and proteins were collected by GSH-sepherase beads.
The purified protein was checked on SDS gel and was stored at 4 ºC.
(copy the detail protocol here?)
GST pull down assay
Interactions between GST fusion proteins and proteins expressed in Drosophila cells were tested by GST pull down assay.
At the beginning the quantity of the GST fusion proteins were estimated on SDS gel and the amount of GST fusion proteins using was neutralized. Then GST fusion proteins bound to the GSH-sephorose beads and the product of S2 R+ cell harvesting mentioned above were mixed together in pull down buffer (PD buffer). 2x Laemmli buffer equal to the amount of the GST fusion protein was added at the same time.
The mixture was kept at 4 ºC on rotator for 2 hours and was washed by PD buffer for 4 times consequently, thus the protein which is not able to bind to the GST fusion protein on the beads was washed away. Finally, the protein left was analyzed by western blotting.
Western blotting
First antibodies used in western blotting including monoclonal anti-HA antibody produced in mouse (___) and monoclonal anti-Myc antibody produced in mouse (___), all worked by using 1:2,000 dilution.
Sencondary antibody used was anti-mouse antibody (___), worked by using 1:10,000 dilution.
Results
In situ hybridization
Photos of the embryos with targeted mutated gene at the stage of dorsal ectoderm formation were taken from the top view. Photos of wild type embryos were also taken for comparison. The photos are shown in Figure 1.
It is obvious from the photos that the Race and Ush staining is much weaker in Vkgk00236 and Dcg1k00405 embryos than the wild type ones.
GST fusion protein construction
Plasmids pGEX4T-3 and pGEX4T-1 described in material sector was used. A pGEX4T-1 plasmid with Vkg protein DNA sequence of the C terminus part inserted between the ___ and ___ restriction digestion sites within Multi Cloning Site (MCS) was a gift from Xiaomeng Wang and is named GST-VkgC construct. Tsg DNA sequence taken from SKAsc2-Tsg (gift from ___) which is originated from cDNA bank was inserted between the EcoRI and XhoI restriction digestion sites within the MCS of pGEX4T-3 and was checked by sequencing. The restriction sites used were chosen according to the plasmid and Tsg DNA sequence. The primers used were: Tsg prime primer 5’-___-3’ and Tsg reverse primer 5’-___-3’. Note that a 5’-tag-3’ stop codon was added in the prime primer.
This plasmid was named GST-Tsg.
GST fusion protein purification
The purification of GST, GST-VkgC and GST-Tsg proteins were performed as described. The products were check and neutralized simultaneously on 10% SDS resolving gel. The gel is shown in Figure 2.
Construction of plasmid for Drosophila cell expression
Since the full sequence of the ___(Dpp-HA) and ___(Sog-Myc) construct is unknown to us, restriction digestion test was first performed on them to find out the usable restriction enzymes which do not cut the constructs. From the results it can be found that for ___(Dpp-HA) construct, BglII restriction digestion enzyme can be used and for ___(Sog-Myc) construct EcoRV is the only available one to be used.
As mentioned, the triple HA within the ___ (Dpp-HA) construct is localized at the front of the Dpp CDS region. Thus to split it into two fragments without losing the HA tag, the first 285 base pairs in the ___ (Dpp-HA) CDS region was kept in both fragments, and the latter 312 base pairs was evenly spitted into two parts which are Dpp N terminus and Dpp C terminus in our cloning. Primers were designed to amplify the whole plasmid except the small part which should be deleted and restriction digestion sites were added at the end of the PCR product. The cloning strategy for this work can be checked on Figure 3. The primers used are: _____. (should write here?) Note that 5’-tag-3’ stop codon was added in the prime primer for Dpp N and not the one for Dpp C. The PCR products were digested by the BglII restriction digestion enzyme, linked using T4 ligase, and subsequently transformed to be selected on plates.
For the ___ (Sog-Myc) construct, the Sog DNA sequence was divided into three fragments in order to express C terminus, N terminus and the central part of the Sog protein since mature Sog protein is much larger than Dpp – the Sog DNA sequence is 4818 base pairs in length. Similar to the ___ (Dpp-HA), there is a transmembrane signal region localized at 1327-1383 base pairs and this region should be kept in all fragments to maintain the transmembrane ability. Thus the region before the 1384th base pair was shared in all three fragments. Moreover, the very end region of Sog DNA sequence is too AT rich, and because that the sequence outside of the Sog DNA region is unknown; the very end part of the Sog DNA was kept as a common part in the fragments as well. (See Figure 4 for the model graph of this cloning work.) Primers were designed according to these conditions. The PCR products were processed following standard protocol consequently. However, the self ligation of the Sog central part cloning is very serve and the expected product have not been got so far.
The final products of the cloning works were sequenced and proved to be correct.
Drosophila S2 R+ cell protein expression
After testing of expression efficiency using Sus529, S2 R+ and Flag-Mad cell lines, S2 R+ was chosen for its high expression efficiency.
The transfection and harvesting were performed as described, ___ (Dpp-HA), Dpp N, Dpp C, Sog N and Sog C constructs were transfected. The western blotting results for the expression showed that Dpp-HA and Dpp N was expressed perfectly. However there was no expression of Sog N and Sog detected, and Sog C which supposed to be in supernatant was in fact be detected in the pellet lysate. The western blotting results are shown in Figure 5 (Sog-Myc data not included).
GST pull down assay result
The following combinations were tested using GST pull down assay, all Dpp-HA used was supernatant (group A was probed using anti-HA antibody which detects Dpp and Group B was probed using anti-Myc antibody which detects Sog).
Group A:
GST-Tsg + Dpp-HA
GST-Tsg + Sog-Myc + Dpp-HA
GST-Tsg + Dpp N (supernatant)
GST-Tsg + Dpp N (pellet lysate)
GST-Tsg + Dpp C (pellet lysate)
GST-VkgC + Dpp-HA
GST-VkgC + Dpp N (supernatant)
GST-VkgC + Dpp N (pellet lysate)
GST-VkgC + Dpp C (pellet lysate)
GST-VkgC + Dpp-HA + Sog C (pellet lysate)
Group B:
GST-VkgC + Sog-Myc
GST-VkgC + Sog C (pellet lysate)
GST-Tsg + Sog-Myc
GST-Tsg + Sog C (pellet lysate)
The western blotting photo of Group A based on GST-VkgC is shown in Figure 6 and the photos of Group A based on GST-Tsg are shown in Figure 7. No signal is detected in the Group B tests (data not shown). GST protein was also tested with the proteins prepared from S2 R+ cells as control for these tests and the results were all blank which proved that there was no interaction between GST protein and the proteins tested (data not shown).
The results showed that the existence of Sog enhanced the affinity of Tsg to Dpp but reduced the signal strength of VkgC + Dpp-HA test, VkgC is able to bind Dpp, and Dpp N showed to be the binding region in Dpp.
Alignment of different type IV collagens
The amino acid sequence of type IV collagens ___(list here, give the reference name?) were aligned using bioedit (Hall, 1999), and the results showed a highly conserved site in the type IV collagens. (Give a picture here.)
Discussion
The in situ hybridization experiments gave strong evidence for the involvement of Vkg and Dcg1 in BMP signalling. The C terminus part of Vkg protein was used in the latter experiments because Xiaomeng has reported that the Dpp binding region on Vkg is localized within this part (Wang and Ashe, submitted). The idea of the involvement of Vkg is further advanced by the GST pull down assay results which directly showed that GST-VkgC does bind Dpp. These interactions must occur extracellular since Vkg and Dcg1 are components of extracellular matrix. And this result indicates that at least the Vkg should be put into consideration of the BMP signalling model in the future. An interesting observation of the GST pull down assay is that the existence of Sog reduces the efficiency of Vkg C terminus part binding with Dpp. This may imply a competition of Dpp binding between Sog and Vkg. This finding is an addition to the hypothesis mentioned by Xiaomeng (Wang and Ashe, submitted) which suggests that Sog and Tsg together induce the release of Dpp-Scw heterodimer from Vkg. Nevertheless, the interaction network of Vkg/Dcg1, Dpp/Scw and Tsg/Sog is mostly unknown.
Because the involvement of type IV collagens in the BMP signalling pathway discovered in Drosophila, and both the type IV collagens as components of extracellular matrix and BMPs as morphogens directing the differentiation are essential in the animals, we suggest that this involvement may be common among the animals which undergo germ layer differentiation. The BMPs are highly conserved in different species which implies if the type IV collagens do function in the BMP signalling pathway, the interaction site of these type IV collagens with BMPs should be highly conserved as well. Depending on this conception, we compared the amino acid sequence of type IV collagen proteins (some of these sequences are predicted translation) from the NCBI database. The result is positive: a very small region of 8 amino acids is discovered to be highly conserved in many different species as described in the result. It is not established whether this conserved region is the site that binds to BMPs, but considering that the established BMPs binding protein Vkg and Dcg1 also have this region, it is possible. Some structure analyzes may be helpful to this research and it will be interesting to do some point mutants on this conserved site to see the consequences. Via these work, the function of this region can be investigated. And if this site is confirmed to be the interaction site, the involvement of type IV collagens in the BMP signalling pathway is consequently established.
From the interaction test among GST-Tsg, Dpp and Sog, it can be seen that with the existence of Sog, the Dpp bound by Tsg was increased. This enhancement effect by Sog to Tsg affinity to Dpp was first reported to be established without the direct disturbing of other proteins (though there were other secreted proteins in the supernatant) in vitro. Moreover, the Dpp and Sog protein were produced in Drosophila cell line which implies the normal translation and post translational process and thus improved the reliability of the result. This result also confirmed the basic interaction theory of the step gradient formation hypothesis mentioned in the introduction.
From the GST pull down assays, we also possibly found the interaction region of Dpp with Tsg. As mentioned, Dpp protein was separated into the N terminus part and the C terminus part. Though the C terminus part may be expressed but is not successfully secreted and no binding between Dpp C terminus part and Tsg was found, luckily we observed that the Dpp N terminus part which was normally secreted bound to the GST-Tsg fusion protein. This may be the evidence that clues on the binding region of Dpp to Tsg is on the Dpp N terminus part. The Dpp N terminus part is small enough (the DNA sequence is 441 base pairs in length) to perform point mutants tests and the exact interaction site may be found out in this way. However, this conclusion is not strong. The Dpp C terminus part is suspected to be existed in the pellet lysate since a band of the right size can be found from the western blotting film, but the reason for it not being secreted is unknown. There are two possibilities: first, the band detected may not be the mature form of Dpp credit to an unknown reason, and the second, the proteins along with the Dpp C terminus protein within the pellet lysate may interfere with the interaction between Tsg and Dpp C terminus protein. What to mention here is that there is also a suspicious band detected in the pellet lysate of Dpp N terminus part expression cells which is of the right size of Dpp N terminus part though the band is thinner than in the supernatant. However there is no bound observed between the protein and Tsg in GST pull down assay. The two possibilities may apply on this Dpp N pellet lysate result as well. In sum, the interaction between Dpp C terminus part and Tsg is unsure according to the results, there is no decisively evidence to show that there is no bound between them, thus though the interaction between Dpp N terminus part and Tsg is established, the possible Dpp interaction region range with Tsg is still in doubt.
Similar argument may apply on the GST pull down assay results for Sog fragments. A difficulty we encountered in the experiments related to Sog protein is that normally we can not detect full length Sog by western blotting. It seems that the because of some unknown reasons, the full length Sog-Myc protein expressed in Drosophila cell was arrested in the stacking gel and is hardly entering the resolving gel. There are evidences for the existence of Sog protein: we have once detected a band at the very top of the resolving gel in western blotting which may represent the Sog protein, and the enhancement of Tsg affinity to Dpp by the Sog added gives another example. Concerning the Sog fragments, no signal that can be the candidate for Sog N terminus part was detected by western blotting, but considering that the full length Sog may be trapped in the stacking gel, this may happen to the Sog N terminus part as well. However this is lack of evidence and is doubtful. About the Sog C terminus part, signal of the expected size was only detected in the pellet lysate, and no interaction between this fragment and Tsg or VkgC was observed. Here because we reserved the transmembrane region in all Sog fragments by cloning, it is suspicious whether this Sog C terminus part protein is mature just like the doubt on Dpp C terminus fragment. Also whether there is no interaction between Sog C terminus part and Tsg remains doubtful in the same way as the Dpp C terminus. However, the fact that the existence of Sog C significantly lowered the binding efficiency between VkgC and Dpp proved it to be functional. This point thus needs more exploring. In this research work, the reason for the arresting of Sog by stacking gel is worth investigation. To further advance the understanding on Sog protein, more analyze on the structure of Sog may be necessary, and if the Sog central part cloning can be done in the future, it will give more ideas in this field.
An unsolved problem is the detection of exact distribution of Dpp. There are two ways for us to detect the distribution of Dpp: one is using Dpp probes to visualize Dpp distribution directly, the other one is to detect the distribution of Smad which is the effector of Dpp. The previous method will not distinguish different forms of Dpp and all forms of Dpp including single molecule, Dpp homodimer, Dpp-Scw heterodimer, Dpp-Scw-Tsg-Sog complex and the newly revealed complex formed by Dpp and Vkg/Dcg1 will be visualized. The latter method does not provide the Dpp distribution directly to us, but give the real functional level of Dpp/Scw instead. Some other methods like the detection of marker genes by in situ hybridization we did also gives the function level of Dpp as the same as the Smad detection method. None of these methods is able to give the distribution status of a specialized form of Dpp, or evidence for intracellular Dpp (before secreting or after endocytosis). This problem is partly solved by the visualization of Sog since the combine analyze is able to exclude the Sog-Dpp complex portion in the total visualized Dpp. In this way the active Dpp distribution was recognized though the portion of Dpp/Scw homodimer and Dpp-Scw heterodimer is still unknown. Probes designed aiming at the specific receptor complex or the specific forms of Dpp/Scw may be helpful to this problem as well.
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During the development of Drosophila Melanogaster embryo, the product of Decapentaplegic (Dpp) gene plays an essential role in the final organization of dorsal-ventral pattern. The Dpp is a homologue of Bone Morphogenetic Proteins 2 and 4 (BMP 2/4) and acts as an extracellular morphogen to form a gradient with the effect of another BMP homologue Screw (Scw) and some other regulators including short gastrulation (Sog), twisted gastrulation (Tsg) and Tolloid (Tld). Among these Dpp regulators, Sog and Tsg are products of zygotic genes, and Tld is metalloprotease. Moreover two type IV collagens Dcg1 and Viking (Vkg) may participate in the BMP signalling as well. In our project, we checked the interaction between Tsg, Vkg, Sog and Dpp by GST pull down assay, and tried to find out the exact region where the interaction occurs. We confirmed the enhancement of Sog to Tsg on affinity to Dpp, and found that Sog reduces the binding activity of Vkg C terminus part to Dpp. We also performed in situ hybridization on Vkg and Dcg1 mutated embryos and wild type embryos to compare the expression patterns of Race and U-shaped (Ush) genes which are related to the expression of Dpp. And we found that there are obvious differences between the wild type and mutated embryos.
Introduction
As kind of insect, Drosophila Melanogaster undergoes gastrulation germ layer differentiation and then forms endoderm, mesoderm, ectoderm and their suborganelles during embryo development. This differentiation process rises from the different spatial positions of the cells in the embryo and is mostly controlled by morphogens. (Ashe and Briscoe, 2006)
During the early stages of embryo Drosophila Melanogaster development, the product of spätzle gene acts as a morphogen to form the Dorsal (Dl) activity gradient and thus establishes the dorsal-ventral pattern (Morisato and Anderson, 1994; Raftery and Sutherland, 2003). Then, the control is taken away by other morphogens. In previous studies, it has been reported that two homologs of Bone Morphogenetic Proteins 2 and 4 (BMP 2/4), Decapentaplegic (Dpp) and Screw (Scw) which are members of TGF-β superfamily, are the control factors for the formation of dorsoal ectoderm in Drosophila Melanogaster. Further researches indicated that Dpp plays the main role since all dorsal cells assume ventral lateral fates and the mutant phenotype of the zygotically required genes appears the most severely with the lack of Dpp (Arora and Nusslein-volhard, 1992; Ferguson and Anderson, 1992a). Based on these discoveries, the scientist put emphasis on the Dpp. Later experiments showed that after the dorsal-ventral (DV) axes is patterned, the effect of the morphogenetic Dpp depends on the local concentration level. In detail, high Dpp level leads to the fate of being amnioserosa whilst lower levels make the cell become dorsal ectoderm tissue (Ashe et al., 2000; Shimmi and O'Connor, 2003). The Dpp/Scw functions by binding to a receptor complex which is supposed to be composed of type I and type II transmembrane serine/threonine kinases (Hogan, 1996; Nakayama et al., 2000). It has been elucidated that in Drosophila, the type I receptor Thick veins (Tkv) interact with the Dpp molecule and the type II receptor Punt (Put) to form a heteromeric complex. The active Put within this complex phosphorylate the Tkv consequently and thus activates the associated type I kinases which will phosphorylate the cytoplasmic protein Mothers against Dpp (Mad). The Mad which belongs to the Smad superfamily then function inside of the cell and lead to the differentiation and in this way the signal of Dpp is transduced. Moreover, in the signal transduction of Scw, it seems that another type I receptor Saxophone (Sax) takes the place of Tkv in Dpp signal transduction. However experiment also showed that high level of Tkv is able to compensate the loss of Sax, and indicates that these two morphogens shares a similar intracellular pathway. (Affolter et al., 2001)
Based on the knowledge above, the regulation of dorsal ectoderm and amnioserosa formation by BMP signalling pathway is suggested to be achieved by the gradient distribution of Dpp. However, an interesting and indispensable phenomenon of the gradient of Dpp is that it is not smoothly distributed. So far, we are able to observe the visualized the Dpp distribution in Drosophila embryos and the results showed that Dpp activity has a peak level at the 8-10 dorsalmost cells which will later form the amnioserosa, and there is a sharp fall of Dpp level at the edge of the dorsalmost cells where cells on the different sides of the confine receive high and very low BMP signal (Shimmi and O'Connor, 2003). This step gradient specifies the very small region to amnioserosa and ensures the normal development of amnioserosa and dorsal epidermis.
According to the model of morphogen distribution which concerns the Dpp and Scw alone, this phenomenon is unable to be explained. In the classical morphogen gradient formation model, specified cell or region produces the morphogen and the morphogen diffuses to the tissue nearby via extracellular or intracellular pathway. Thus a morphogen concentration gradient which peaks at the secreting cell and smoothly deduced along with the increasing distance to the secreting cell (Ashe and Briscoe, 2006). The step gradient of Dpp activity is incompatible with this model, thus the formation of this gradient should be created by another mechanism with some other factors.
As far as we know, BMP signalling activity is regulated by the products of two zygotic genes short gastrulation (Sog) and twisted gastrulation (Tsg) , a metalloprotease Tolloid (Tld) (Ashe, 2005). And just recently two type IV collagens Viking (Vkg) (Yasothornsrikula et al., 1997) and Dcg1 (Blumberg et al., 1988) are thought to be playing a role in the BMP signalling pathway.
It is known that Sog gene is a homologue of Xenopus Chordin (Chd) gene which produces antagonist of BMPs both structural and functional. (Ferguson and Anderson, 1992b; Piccolo et al., 1996). And later studies have discovered that Tsg is also a secreted protein which acts as extracellular antagonists of BMP (Chang et al., 2001; Ross et al., 2001). These works gave the evidence that Sog and Tsg bind to BMP directly and inhibit the interaction between BMP and its receptor (Tkv and Put) to form a complex in order to block the signal transduction (Ashe, 2005). However, these interactions will not destroy the BMP molecule itself, and the formed complex can be divided into the original single molecular to regain its function as well. In Drosophila, this cleavage process is operated by the protease Tld which was first recognized as an activator of Dpp (Ferguson and Anderson, 1992a). As far as we know, these three regulators (Sog, Tsg and Tld) of BMP signalling pathway are suggested to play essential part in the mechanism which determined the peak level and the step gradient distribution of BMPs since the loss of any one of these three regulators will lead to the loss of the step gradient.
The Dpp protein itself is nonautonomous and is able to diffuse under a narrow limitation. The Dpp direct visualization result shows that there is an even level of Dpp throughout the dorsal 40% of Drosophila embryo before the late cellularisation stage. Though this uniform distribution will lost in later stages of normal embryo development, it can be maintained by Scw mutation. The activity pattern of Smad observed in the embryo also supports this Dpp distribution. (Raftery and Sutherland, 2003; Shimmi et al., 2005; Wang and Ferguson, 2005) Though we are still not able to detect and visualize the intracellular concentration of Dpp or Scw directly, the phenomenon observed gives the hypothesis that the Dpp may be evenly secreted in the dorsal part of the embryo after the patterning of DV axes. Moreover, it also been reported that the Dpp and Scw function much more effectively by forming a Dpp-Scw heterodimer which is able to induce a 10 to 100 times higher phosphorylated Mad accumulation than Dpp or Scw homodimer do within the cells exposed to these molecules (Shimmi et al., 2005). However, there is a controversy between the reports of Wang and Ferguson group (Wang and Ferguson, 2005) and Shimmi et al. (Shimmi et al., 2005) group. The previous group found that with the lack of Scw the interpreted results of their experiments support the mechanism that Dpp and Scw homodimers function cooperatively whilst the report from the latter one gave evidence for the existence of Dpp-Scw heterodimer.
In earlier studies, there was a good hypothesis considering the combined effect of BMP inhibitors (Sog and Tsg), the cleaver (Tld) to explain the step gradient of Dpp activity (Shimmi and O'Connor, 2003). Nevertheless, the controversy mentioned above gives the birth of a new hypothesis which counts the controversy in whilst interpreting the step gradient of BMPs at the same time. This new hypothesis inherited the basic mechanism of step gradient formation from the earlier one.
Start from Sog, it is produced at the presumptive neuroectoderm and the distribution of Sog in Drosophila embryos had been directly shown to be in a graded fashion and limited by Tld in dorsal region (Srinivasan et al., 2002). The distribution of Sog protein agrees with the classical passive morphogen distribution model since the level of Sog reach the highest at the producing region and falls whilst the distance to the presumptive neuroectoderm increases. However, the Sog detected is thought to be in the complex including Sog and Dpp and Sog single molecule itself will be degraded via endocytosis. Thus it is also suggested that the shaping of the lowest level of Sog at the dorsalmost region is associated by the protease Tld which is able to cleave the complex and release Sog to be degraded. The interaction between Sog and Dpp here not only contributes to the formation of Sog gradient, but also help the diffusion of Dpp. As mentioned, either the Dpp homodimer or Dpp-Scw heterodimer has the activity to bind the receptor on cell membrane and then are trapped and lost the diffusion ability. Thus, the binding between Sog and Dpp on the one hand is an inhibition on Dpp, on the another hand is a protection of Dpp signalling activity and helps the diffusion of Dpp. Since the Tld restores the activity of Dpp by cleaving the complex when the complex reaches that region, a long-range enhancement and local inhibition of Dpp activity by Sog is then created. (Ashe, 2002; Ashe and Levine, 1999)
A notable fact is that Tld is distributed evenly in the embryo (Shimell et al., 1991), thus the cleavage rate of the Sog-Dpp complex is the same throughout the embryo. However, only in the dorsalmost region where is lack of Sog, the Dpp released by Tld cleavage is able to bind to the receptor before forming the complex with the Sog protein again.
Later studies demonstrated that Tsg is another inhibitor of Dpp activity, its role is generally recognized to be similar to Sog. Tsg and Sog are enhancers to each other since the loss of either Tsg or Sog reduces the efficiency of Dpp inhibition in the embryo and they show strong Dpp affinity when existing together. Thus the mechanism of the enhancement is suggested to be the formation of a Tsg-Sog-Dpp complex (Chang et al., 2001; Shimmi and O'Connor, 2003). The knowledge on the complex is further advanced by the recent discoveries mentioned above that gave evidence for the existence of Tsg-Sog-Dpp-Scw complex. It is also discovered that the affinity of Sog/Tsg for Dpp and Scw homodimer is weaker than Dpp-Scw heterodimer.
What is novel in the new hypothesis is that it suggests there are three kinds of receptors on the cell membrane for the BMPs which are able to specially bind to Dpp homodimer, Scw homodimer or Dpp-Scw heterodimer. There is a positive feedback with unclear mechanism between the interaction of Dpp-Scw heterodimer and the Dpp-Scw-receptor. Combining the potent signalling of Dpp-Scw heterodimer and the effect of Sog, Tsg and Tld, this feedback helps the formation of peak BMPs signalling level in the dorsalmost cells (Ashe, 2005; Wang and Ferguson, 2005). In contrary, in other regions where the Sog/Tsg level is higher, the free BMPs are thought to be mostly Dpp and Scw homodimers and thus the signalling strength is much weaker.
This hypothesis is reasonable and satisfaction. However, two proteins, Dcg1 and Vkg, may have to be put into the BMP signalling pathway in Drosophila embryos according to our investigation. Vkg and Dcg1 are type IV collagens which are the components of extracellular matrix and may limiting the diffusion of Dpp/Scw. In our study, we detected the marker gene Race (Tatei et al., 1994) and U-shaped (Ush) (Cubadda et al., 1997) using in situ hybridization method in the Vkg and Dcg1 mutated Drosophila embryos. The result showed that both of them are essential for the BMP signalling, but the exact role for them is unclear yet. To get deeper understanding of the BMP signalling pathway, we also further investigated the interaction among Tsg, Sog and Dpp by GST pull down assay. And we tried to split the Dpp and Sog protein into smaller fragments and performed GST pull down assay on them consequently to investigate which is the interaction region on these proteins. The interaction analyse was carried out on Vkg fragment and Dpp as well. Moreover, we compared the type IV collagens in different species to find out whether there is a conserved site which may related to the BMP signalling pathway. Based on our knowledge, we suggest that there may be a competition between Dcg1/Vkg and Tsg/Sog to bind the BMPs.
Materials & Methods
Mutant Drosophila Melanogaster
The yw67c23 Drosophila Melanogaster from ___ is used as the wild type. Vkgk00236 and Dcg1k00405 flies (gift from Xiaomeng Wang, University of Manchester) are used as Vkg and Dcg1 mutants. (details needed) These two mutants have weaker Vkg or Dcg1 expression, because the completely disappearance of functional Vkg or Dcg1 leads to flies unable to lay eggs.
Embryo collection
The embryos from the wild type, Vkg mutant and Dcg1 mutant flies were collected every 2 hours at 25 ºC and left to develop for another period equal to 2 hours at 25 ºC before fixation. The embryos were fixed with fixing buffer and hepaton and stored in ethanol at -20 ºC. Thus the embryos collected were at the development stages between 2-4 hours. And a small number of overnight embryos were also fixed and stored with the 2-4 hours embryos.
In situ hybridization
In situ hybridization was performed with the embryos collected described above. Race and Ush digoxigenin-labelled RNA probes were used to visualize the distribution of Race and Ush gene. (copy the protocol here or cite the book?) The slides were observed under ___ microscope and images were taken at the same time.
Plasmids
GST protein expression plasmid pGEX4T-3 (reference) and pGEX4T-1 (reference) was used for GST fusion protein construction cloning. The splitting of Dpp and Sog proteins were based on the Drosophila cell expression constructs Dpp-HA ____ (reference here) and Sog-Myc ____ (reference here).
The Dpp protein will be cleaved in cytosol and the CDS region which is 402 base pairs long is localized at the very end of the full Dpp DNA sequence. In the ___(Dpp-HA) construct, a triple HA tag region was inserted after the 90th base pair in the CDS region and is 195 base pairs long. And in the ___ (Sog-Myc) construct, the Myc tag is inserted in front of the Sog DNA sequence.
Cloning
Phusion (purchased from Finnzyme) polymerase was used for PCR. And all primers were ordered from Sigma-Aldrich. (Should write the primer sequences here?)
All restriction digestion enzymes used in cloning were products of New England Biolabs except EcoRV which is purchased from Rosche.
T4 Ligase used was produced by Rosche.
T-easy ligation kit (purchased from Promega) was used in some of the cloning work.
DH5α E.Coli cell strain was used for transformation for plasmid amplification and selection.
Cell transfection, expression & harvesting
S2 R+ cell line used for protein expression was incubated at 25 ºC in FCS medium (medium detail here) until it reaches the cell density suitable for transfection. Transfectgene transfection kit (purchased from QIAGEN) was used in transfection following the manual. CuSO4 (detail quantity) was added 24 hours after transfection to induce the expression of transfected plasmid DNA, and then the transfected cells were incubated for another 72 hours. The cell cultures were harvested afterwards. Cells and medium were first separated by centrifugation, and the cell pellets were lysed subsequently. Both the supernatant and pellet lysate were checked by western blotting and were stored in -80 ºC freezer.
GST fusion protein expression & purification
For GST fusion protein expression, BL21 E.Coli cell strain was used for transformation. Single colony was picked to set up start culture and the start culture was later seeded into more medium to make expression culture. These cultures were all incubated shaking at 37 ºC. 50 µL of 0.2M IPTG was added to the expression culture to induce protein expression when the OD600 reached 0.6, and the culture was moved to 25 ºC shaker. After 3 hours of incubation, the cells were harvested by centrifugation. Then the cells were processed with lysozyme and sonication, and proteins were collected by GSH-sepherase beads.
The purified protein was checked on SDS gel and was stored at 4 ºC.
(copy the detail protocol here?)
GST pull down assay
Interactions between GST fusion proteins and proteins expressed in Drosophila cells were tested by GST pull down assay.
At the beginning the quantity of the GST fusion proteins were estimated on SDS gel and the amount of GST fusion proteins using was neutralized. Then GST fusion proteins bound to the GSH-sephorose beads and the product of S2 R+ cell harvesting mentioned above were mixed together in pull down buffer (PD buffer). 2x Laemmli buffer equal to the amount of the GST fusion protein was added at the same time.
The mixture was kept at 4 ºC on rotator for 2 hours and was washed by PD buffer for 4 times consequently, thus the protein which is not able to bind to the GST fusion protein on the beads was washed away. Finally, the protein left was analyzed by western blotting.
Western blotting
First antibodies used in western blotting including monoclonal anti-HA antibody produced in mouse (___) and monoclonal anti-Myc antibody produced in mouse (___), all worked by using 1:2,000 dilution.
Sencondary antibody used was anti-mouse antibody (___), worked by using 1:10,000 dilution.
Results
In situ hybridization
Photos of the embryos with targeted mutated gene at the stage of dorsal ectoderm formation were taken from the top view. Photos of wild type embryos were also taken for comparison. The photos are shown in Figure 1.
It is obvious from the photos that the Race and Ush staining is much weaker in Vkgk00236 and Dcg1k00405 embryos than the wild type ones.
GST fusion protein construction
Plasmids pGEX4T-3 and pGEX4T-1 described in material sector was used. A pGEX4T-1 plasmid with Vkg protein DNA sequence of the C terminus part inserted between the ___ and ___ restriction digestion sites within Multi Cloning Site (MCS) was a gift from Xiaomeng Wang and is named GST-VkgC construct. Tsg DNA sequence taken from SKAsc2-Tsg (gift from ___) which is originated from cDNA bank was inserted between the EcoRI and XhoI restriction digestion sites within the MCS of pGEX4T-3 and was checked by sequencing. The restriction sites used were chosen according to the plasmid and Tsg DNA sequence. The primers used were: Tsg prime primer 5’-___-3’ and Tsg reverse primer 5’-___-3’. Note that a 5’-tag-3’ stop codon was added in the prime primer.
This plasmid was named GST-Tsg.
GST fusion protein purification
The purification of GST, GST-VkgC and GST-Tsg proteins were performed as described. The products were check and neutralized simultaneously on 10% SDS resolving gel. The gel is shown in Figure 2.
Construction of plasmid for Drosophila cell expression
Since the full sequence of the ___(Dpp-HA) and ___(Sog-Myc) construct is unknown to us, restriction digestion test was first performed on them to find out the usable restriction enzymes which do not cut the constructs. From the results it can be found that for ___(Dpp-HA) construct, BglII restriction digestion enzyme can be used and for ___(Sog-Myc) construct EcoRV is the only available one to be used.
As mentioned, the triple HA within the ___ (Dpp-HA) construct is localized at the front of the Dpp CDS region. Thus to split it into two fragments without losing the HA tag, the first 285 base pairs in the ___ (Dpp-HA) CDS region was kept in both fragments, and the latter 312 base pairs was evenly spitted into two parts which are Dpp N terminus and Dpp C terminus in our cloning. Primers were designed to amplify the whole plasmid except the small part which should be deleted and restriction digestion sites were added at the end of the PCR product. The cloning strategy for this work can be checked on Figure 3. The primers used are: _____. (should write here?) Note that 5’-tag-3’ stop codon was added in the prime primer for Dpp N and not the one for Dpp C. The PCR products were digested by the BglII restriction digestion enzyme, linked using T4 ligase, and subsequently transformed to be selected on plates.
For the ___ (Sog-Myc) construct, the Sog DNA sequence was divided into three fragments in order to express C terminus, N terminus and the central part of the Sog protein since mature Sog protein is much larger than Dpp – the Sog DNA sequence is 4818 base pairs in length. Similar to the ___ (Dpp-HA), there is a transmembrane signal region localized at 1327-1383 base pairs and this region should be kept in all fragments to maintain the transmembrane ability. Thus the region before the 1384th base pair was shared in all three fragments. Moreover, the very end region of Sog DNA sequence is too AT rich, and because that the sequence outside of the Sog DNA region is unknown; the very end part of the Sog DNA was kept as a common part in the fragments as well. (See Figure 4 for the model graph of this cloning work.) Primers were designed according to these conditions. The PCR products were processed following standard protocol consequently. However, the self ligation of the Sog central part cloning is very serve and the expected product have not been got so far.
The final products of the cloning works were sequenced and proved to be correct.
Drosophila S2 R+ cell protein expression
After testing of expression efficiency using Sus529, S2 R+ and Flag-Mad cell lines, S2 R+ was chosen for its high expression efficiency.
The transfection and harvesting were performed as described, ___ (Dpp-HA), Dpp N, Dpp C, Sog N and Sog C constructs were transfected. The western blotting results for the expression showed that Dpp-HA and Dpp N was expressed perfectly. However there was no expression of Sog N and Sog detected, and Sog C which supposed to be in supernatant was in fact be detected in the pellet lysate. The western blotting results are shown in Figure 5 (Sog-Myc data not included).
GST pull down assay result
The following combinations were tested using GST pull down assay, all Dpp-HA used was supernatant (group A was probed using anti-HA antibody which detects Dpp and Group B was probed using anti-Myc antibody which detects Sog).
Group A:
GST-Tsg + Dpp-HA
GST-Tsg + Sog-Myc + Dpp-HA
GST-Tsg + Dpp N (supernatant)
GST-Tsg + Dpp N (pellet lysate)
GST-Tsg + Dpp C (pellet lysate)
GST-VkgC + Dpp-HA
GST-VkgC + Dpp N (supernatant)
GST-VkgC + Dpp N (pellet lysate)
GST-VkgC + Dpp C (pellet lysate)
GST-VkgC + Dpp-HA + Sog C (pellet lysate)
Group B:
GST-VkgC + Sog-Myc
GST-VkgC + Sog C (pellet lysate)
GST-Tsg + Sog-Myc
GST-Tsg + Sog C (pellet lysate)
The western blotting photo of Group A based on GST-VkgC is shown in Figure 6 and the photos of Group A based on GST-Tsg are shown in Figure 7. No signal is detected in the Group B tests (data not shown). GST protein was also tested with the proteins prepared from S2 R+ cells as control for these tests and the results were all blank which proved that there was no interaction between GST protein and the proteins tested (data not shown).
The results showed that the existence of Sog enhanced the affinity of Tsg to Dpp but reduced the signal strength of VkgC + Dpp-HA test, VkgC is able to bind Dpp, and Dpp N showed to be the binding region in Dpp.
Alignment of different type IV collagens
The amino acid sequence of type IV collagens ___(list here, give the reference name?) were aligned using bioedit (Hall, 1999), and the results showed a highly conserved site in the type IV collagens. (Give a picture here.)
Discussion
The in situ hybridization experiments gave strong evidence for the involvement of Vkg and Dcg1 in BMP signalling. The C terminus part of Vkg protein was used in the latter experiments because Xiaomeng has reported that the Dpp binding region on Vkg is localized within this part (Wang and Ashe, submitted). The idea of the involvement of Vkg is further advanced by the GST pull down assay results which directly showed that GST-VkgC does bind Dpp. These interactions must occur extracellular since Vkg and Dcg1 are components of extracellular matrix. And this result indicates that at least the Vkg should be put into consideration of the BMP signalling model in the future. An interesting observation of the GST pull down assay is that the existence of Sog reduces the efficiency of Vkg C terminus part binding with Dpp. This may imply a competition of Dpp binding between Sog and Vkg. This finding is an addition to the hypothesis mentioned by Xiaomeng (Wang and Ashe, submitted) which suggests that Sog and Tsg together induce the release of Dpp-Scw heterodimer from Vkg. Nevertheless, the interaction network of Vkg/Dcg1, Dpp/Scw and Tsg/Sog is mostly unknown.
Because the involvement of type IV collagens in the BMP signalling pathway discovered in Drosophila, and both the type IV collagens as components of extracellular matrix and BMPs as morphogens directing the differentiation are essential in the animals, we suggest that this involvement may be common among the animals which undergo germ layer differentiation. The BMPs are highly conserved in different species which implies if the type IV collagens do function in the BMP signalling pathway, the interaction site of these type IV collagens with BMPs should be highly conserved as well. Depending on this conception, we compared the amino acid sequence of type IV collagen proteins (some of these sequences are predicted translation) from the NCBI database. The result is positive: a very small region of 8 amino acids is discovered to be highly conserved in many different species as described in the result. It is not established whether this conserved region is the site that binds to BMPs, but considering that the established BMPs binding protein Vkg and Dcg1 also have this region, it is possible. Some structure analyzes may be helpful to this research and it will be interesting to do some point mutants on this conserved site to see the consequences. Via these work, the function of this region can be investigated. And if this site is confirmed to be the interaction site, the involvement of type IV collagens in the BMP signalling pathway is consequently established.
From the interaction test among GST-Tsg, Dpp and Sog, it can be seen that with the existence of Sog, the Dpp bound by Tsg was increased. This enhancement effect by Sog to Tsg affinity to Dpp was first reported to be established without the direct disturbing of other proteins (though there were other secreted proteins in the supernatant) in vitro. Moreover, the Dpp and Sog protein were produced in Drosophila cell line which implies the normal translation and post translational process and thus improved the reliability of the result. This result also confirmed the basic interaction theory of the step gradient formation hypothesis mentioned in the introduction.
From the GST pull down assays, we also possibly found the interaction region of Dpp with Tsg. As mentioned, Dpp protein was separated into the N terminus part and the C terminus part. Though the C terminus part may be expressed but is not successfully secreted and no binding between Dpp C terminus part and Tsg was found, luckily we observed that the Dpp N terminus part which was normally secreted bound to the GST-Tsg fusion protein. This may be the evidence that clues on the binding region of Dpp to Tsg is on the Dpp N terminus part. The Dpp N terminus part is small enough (the DNA sequence is 441 base pairs in length) to perform point mutants tests and the exact interaction site may be found out in this way. However, this conclusion is not strong. The Dpp C terminus part is suspected to be existed in the pellet lysate since a band of the right size can be found from the western blotting film, but the reason for it not being secreted is unknown. There are two possibilities: first, the band detected may not be the mature form of Dpp credit to an unknown reason, and the second, the proteins along with the Dpp C terminus protein within the pellet lysate may interfere with the interaction between Tsg and Dpp C terminus protein. What to mention here is that there is also a suspicious band detected in the pellet lysate of Dpp N terminus part expression cells which is of the right size of Dpp N terminus part though the band is thinner than in the supernatant. However there is no bound observed between the protein and Tsg in GST pull down assay. The two possibilities may apply on this Dpp N pellet lysate result as well. In sum, the interaction between Dpp C terminus part and Tsg is unsure according to the results, there is no decisively evidence to show that there is no bound between them, thus though the interaction between Dpp N terminus part and Tsg is established, the possible Dpp interaction region range with Tsg is still in doubt.
Similar argument may apply on the GST pull down assay results for Sog fragments. A difficulty we encountered in the experiments related to Sog protein is that normally we can not detect full length Sog by western blotting. It seems that the because of some unknown reasons, the full length Sog-Myc protein expressed in Drosophila cell was arrested in the stacking gel and is hardly entering the resolving gel. There are evidences for the existence of Sog protein: we have once detected a band at the very top of the resolving gel in western blotting which may represent the Sog protein, and the enhancement of Tsg affinity to Dpp by the Sog added gives another example. Concerning the Sog fragments, no signal that can be the candidate for Sog N terminus part was detected by western blotting, but considering that the full length Sog may be trapped in the stacking gel, this may happen to the Sog N terminus part as well. However this is lack of evidence and is doubtful. About the Sog C terminus part, signal of the expected size was only detected in the pellet lysate, and no interaction between this fragment and Tsg or VkgC was observed. Here because we reserved the transmembrane region in all Sog fragments by cloning, it is suspicious whether this Sog C terminus part protein is mature just like the doubt on Dpp C terminus fragment. Also whether there is no interaction between Sog C terminus part and Tsg remains doubtful in the same way as the Dpp C terminus. However, the fact that the existence of Sog C significantly lowered the binding efficiency between VkgC and Dpp proved it to be functional. This point thus needs more exploring. In this research work, the reason for the arresting of Sog by stacking gel is worth investigation. To further advance the understanding on Sog protein, more analyze on the structure of Sog may be necessary, and if the Sog central part cloning can be done in the future, it will give more ideas in this field.
An unsolved problem is the detection of exact distribution of Dpp. There are two ways for us to detect the distribution of Dpp: one is using Dpp probes to visualize Dpp distribution directly, the other one is to detect the distribution of Smad which is the effector of Dpp. The previous method will not distinguish different forms of Dpp and all forms of Dpp including single molecule, Dpp homodimer, Dpp-Scw heterodimer, Dpp-Scw-Tsg-Sog complex and the newly revealed complex formed by Dpp and Vkg/Dcg1 will be visualized. The latter method does not provide the Dpp distribution directly to us, but give the real functional level of Dpp/Scw instead. Some other methods like the detection of marker genes by in situ hybridization we did also gives the function level of Dpp as the same as the Smad detection method. None of these methods is able to give the distribution status of a specialized form of Dpp, or evidence for intracellular Dpp (before secreting or after endocytosis). This problem is partly solved by the visualization of Sog since the combine analyze is able to exclude the Sog-Dpp complex portion in the total visualized Dpp. In this way the active Dpp distribution was recognized though the portion of Dpp/Scw homodimer and Dpp-Scw heterodimer is still unknown. Probes designed aiming at the specific receptor complex or the specific forms of Dpp/Scw may be helpful to this problem as well.
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