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In this paper, the authors try to elucidate the role of a matrix metalloprotease (Mmp-2) which has been implicated by previous research in branching morphogenesis, in the process of FGF-mediated signaling in the developing air sac of Drosophila. The air sac primordium (ASP) is described in late larval stages of the development process in Drosophila from a bud close to the wing imaginal disc, and is a well suited model for the genetic and developmental study of the mechanisms involved in tubule formation, cell migration, invasion and branching morphogenesis in this species. It has been already found that the primary budding as well as the following air sac growth is controlled by the Drosophila FGF homolog Branchless (Bnl) and its receptor, Breathless (Btl). In particular, FGF-signaling controls the development of the growing sac's tips, which are characterized by actin-based cytopodia or cytonemes. Several indicators exist for FGF-signaling activation (phosphorylated ERK) as well as for tip identity (the escargot gene-esg). Matrix metalloproteases on the other hand, are cell-surface enzymes with a proteolytic activity towards the extracellular matrix and a significant role in tissue formation during development. The role of Mmp's in controlling FGF signaling has been proposed by experiments that have demonstrated shedding of mammalian FGF receptors by cells under metalloprotease induced cleavage. Also, mmp-2 knock out Drosophila models have been shown to die with defects in organ tissue histolysis during metamorphosis.
The above show the significant role of matrix metalloproteases in tissue development (histolysis, proteolytic cleavage of redundant tissues in development) in the metamorphosis stage of the Drosophila, a step which is possibly common and conserved in a variety of other species. It can be therefore important in understanding malformations in development and/or abortive behavior. In a more specialized context, the role of these metalloproteases in the controlling of FGF-signaling in development has not been previously shown, and thus is attempted herein.
The experiments for the study are designed in order to elucidate three major goals:
- Highlight the control of FGF-signaling by Mmp-2
- Prove the existence of a feedback loop mechanism in the control of FGF by Mmp
- And Underpin the exact mechanism and location of Mmp control on FGF signaling on epithelial cells.
Methods employed were
- Plasmid reconstruction ( two plasmids :the mmp1-GFP Reporter construct and the UAS-mmp2 RNAi)
- Drosophila strains and genetics: the Gal4/Gal80ts system was used for conditional activation of RNAi or gene expression by crossing btlGal4, UAS-GFP actin, tubulin Gal80. Mmp2-Gal4, UAS-GFPnls and mmp1-GFP reporter were used to 'visualize mmp expression'; esg was used for esg gene expression monitoring.
- MARCM clones were used to conduct clonal analysis on the mmp2 loss of function alleles and FLIP-OUT clones to study the mmp-2 paracrine effect. The latter was done by crossing male carrying hs-bnl and female btl-MRFP moe.
- Immunocytochemistry methods were employed and especially dpERK staining on imaginal wing disks. Confocal microscopy was used to study the images.
- Various cell culture techniques were used on Drosophila S2 strains.
To achieve their experimental goals a series of experiments were performed. First of all the presence of mmp-1 and mmp-2 on the tracheal system during morphogenesis had to be proven. This was achieved by using GFP reporter strains. Both metalloproteases genes were found and are depicted to be active in the larval ASP and to migrate during larval development: mmp-1 to the tubular structures and mmp-2 to the tip cells. Second, to prove that mmp-2 is indispensable for air sac formation, the authors utilized RNAi constructs to produce knock outs (later validated by PCR) that had loss of function (LOF) on the level of air sack extension and formation. Anti-phosphoH3 staining proved that proliferation was unaffected, thus mmp-2 knock outs did not cause a reduction in the initial level of sac development, the tracheoblasts. Further on, the authors monitored the expression of the tip marker (escargot gene-esg) on the LOF mmp-2 knock outs, and found it to be overly expressed. This finding , coupled with the observation that tips in the LOF models were expanded in territory, allowed for the conclusion that mmp-2 is important for the spatial constraint of tip formation. To back this up, secluded over-expression of the esg was induced and an identical formation on tips was observed as with the LOF mmp-2 model.
Following, the role of Mmp-2 on FGF-signaling was experimentally proven on the stalk and tip cells of the ASP. Because tip cell fate and orientation/growth is controlled by the production of FGF, the authors tested the hypothesis that expansion in tip territory in mmp-2 knock outs was otherwise induced by an over expression of FGF activity. This was depicted by dpERK staining. In knock outs, staining was not constricted to the tip cells and conversely a misspecification was observed on tracheoblasts of the stalk. Genetic experiments on Drosophila's Bnl and Btl regions proved that ERK response can be abrogated by coexpression with Mmp2. A catalytically inactive mutant, Mmp-2(E258A), however, had no effect. Hence, the authors conclude that Mmp-2 can interfere Bnl/Btl signaling. Exogenous stimulation of the ERK phosphorylation response is found to be insensitive to the presence or absence of Mmp-2. Thus, Mmp-2 plays a selective role on the FGF signaling pathway. Last, by real-time PCR and western blotting, the FGF is found to induce the production of Mmp-2 in the ASP. By using clonal analysis and random clones of alleles of Mmp-2, the authors find that cells not producing Mmp-2 exert a lateral inhibition on tip cell designation. Moreover, on a model where expressing Mmp-2 clones were inserted, they found areas of diminished ERK phosphorylation near Mmp2-expressing clones, as monitored by dp ERK staining ,suggesting that Mmp-2 activity induces a yes/no decision on neighbor cells, that causes the cell to adopt its fate as tip or stalk. However, this phenomenon was not observed on all neighbor cells.
From the above results, the authors provide with insight on the observation of tip expansion and malformation of sac development in Mmp-2 knock outs, where FGF-signaling is found to be overexerted. Moreover, they hypothesize that the effect Mmp-2 has on the ASP cells is that of an on/off fashion that further decides their fate as tip or subsidiary stalk development. However, the authors on their last conclusion of their results state that not all the cells neighborly to the mmp-2 producing cells exhibited decreased ERK (the FGF marker) activity. The authors acclaim that this finding is of a stochastic nature, since ubiquitous levels of FGF were used in experimental conditions. To the reader's eye however, this constitutes a limitation in being able to state with certainty the lateral inhibition effect Mmp-2 plays on FGF- signaling. In would be wiser to conduct further experiments with varying levels of FGF, to prove this control (on/off) hypothesis.
Strengths of the study include the analytical and extensively tested methods, where the authors seldom rely on one sole observation to conclude their hypothesis (such as in the spatial constraint hypothesis where results were backed with the construction of a further model with esg overexpression and normal Mmp-2 function). Other strengths lie in the authors' ability to employ multiple genetic and immunoassaying techniques, which are extended in variety. They also use most of the previous research experience on the particular subject, revalidating the previous findings and stretching them to extend to the tested hypothesis (such as the use of TIMP - a specific Drosophila Mmp inhibitor found in 1999, the esg gene already known for its role in tip identity and the theories of lateral inhibition in tip development in 2006)
An important weakness observed is mainly that the hypothesis for lateral inhibition and the on/off phenomenon is not sufficiently backed up in the experiments used nor the figures presented. The authors still have an uncertainty about the effect Mmp-2 plays on neighbour cells and this is not adequately stated in the article but instead is discussed in the text, with what the reader feels is of a lesser importance. However, more experiments are needed and one feels that this study does not quite prove that which is stated in its title, but rather provides with indirect evidence/notions of a control on FGF signaling. This is the study's greatest weakness and as such further research is needed to be able to extrapolate this study's indirect findings on other species and even more, mammals.