The transverse connective, ASP and wing imaginal disc are invasively coupled
The ASP originates near a point of bifurcation of the transverse connective and extends over the notum primordium of the wing disc in an oblique, dorsoventral orientation where myoblasts overly the columnar epithelial cells (). Contact between the transverse connective and disc is sufficiently strong that the transverse connective frequently accompanies wing discs when wing discs are dissected from larvae. Although association of the disc and transverse connective can also be observed in intact larva, the nature of the contact between the disc and tracheal tube has not been characterized previously. Our finding that Bnl/FGF signals from the disc to the transverse connective to induce the outgrowth of the ASP (Sato and Kornberg, 2002
) suggested that contact between the transverse connective and the disc has a functional role. We therefore undertook a fine structure analysis to gain insight into the nature of their association.
Invasive coupling of trachea and wing disc
Tracheae are tripartite tubes with an ECM on the outer basal surface, a tube consisting of epithelial cells, and apically, a cuticle (taenidium) that lines the lumen. Serial ultra-thin sections of discs and associated trachea revealed that the ASP differs from other tracheal tubes in two respects (# specimens examined = 2). The ASP lacks a taenidium (compare ). In addition, although the BL that lines segments of the tracheal branch that do not contact the disc has a thick lamina densa that was similar in appearance to the one that encapsulates the disc (), the portions of the transverse connective that are directly apposed to the disc does not (). Instead, they are situated underneath the BL of the disc and were directly juxtaposed to the disc (shown schematically in ). The surfaces of tracheal cells situated underneath the disc BL are associated with lamina densa that is significantly thinner and less uniform than the lamina densa elsewhere in the disc. In some sections (not shown), tracheal cells of the transverse connective are directly juxtaposed to the columnar epithelium, the source of Bnl/FGF. We refer to the placement of the trachea underneath and inside the disc BL as “invasive coupling”.
To further characterize the tracheal-wing disc association, we examined protein trap strains that express GFP in the ECM. We examined a line that tags Collagen IV () and three lines that tag Perlecan (not shown). In specimens dissected from L3 larvae of all four lines, we detected similar patterns of strong fluorescence around the disc and around most of the tracheal branches (). However, we did not detect strong fluorescence from the regions of the trachea that contact the disc (). In addition to the absence of strong fluorescence around the trachea in this region of contact, the intensity of fluorescence in this region was lower than in other regions of the wing imaginal disc. The pattern of fluorescence correlated well with the appearance of the lamina densa in ultrastructural studies – we detected a prominent, thick layer over the disc and around tracheal branches, except the portion of the transverse connective that contacts the disc. In these regions, the lamina densa overlying the trachea was significantly thinner. Confocal microscopy of Collagen IV:GFP expressing discs also showed that both disc-associated trachea and the ASP were underneath the disc BL, directly juxtaposed to the disc (). And both XZ and YZ reconstructions showed the layer of Collagen IV:GFP overlying the ASP, with levels that were significantly were lower than in other regions of the notum (). Epi-fluorescence images clearly revealed the points of tracheal “entry” and “exit” () that define the extent of “invasion”; these points were similar in all specimens we examined. summarizes the disposition of the transverse connective with respect to the disc and BL.
Based on these observations we conclude that the wing disc and trachea are invasively coupled and that the ASP grows out from this invasively coupled tracheal segment. Since we were unable to detect a continuous BL around the tracheal segments that are coupled to the disc (no lamina densa was present at the transverse connective-disc interface), it may be that disc-derived cues suppress BL assembly locally. We note that the unicellular tracheole that emanates from the transverse connective at a point distal to the ASP () has portions both inside and outside the disc BL. Since only the portion of the tube that is outside the disc BL had Collagen IV, BL assembly by this tracheole is not cell autonomous.
The BL is functionally transparent to FGF signaling
Growth of the ASP from a region of the trachea that invasively couples with the disc raises the question: is direct contact necessary for FGF signaling to induce the ASP, would interposition of BL between the disc and tracheal cells abrogate signaling? In order to determine if direct contact is essential for FGF signaling, we induced clones of Bnl/FGF-expressing cells and monitored responses of trachea by examining fluorescence of either btl-GAP: GFP or btl-Collagen IV: GFP-expressing cells. Since we observed association of disc and trachea and detected Collagen IV: GFP in the ECM around these tissues in preparations from first instar larvae (data not shown), induction of Bnl/FGF expression in first and second instar larvae would allow us to address the responses of the trachea after initial BL assembly. Ectopic Bnl/FGF expression induced outgrowths from the transverse connective, both from the portions that had invasively coupled with the disc as well as outlying regions that were outside the disc BL (Fig. A, C, D). The latter type of outgrowths were multi-cellular () and oriented toward Bnl/FGF-expressing disc cells, and some had thin collagenous structures that connected trachea and disc that could be discerned (). Since these ectopic structures suggested that signaling is not blocked by the disc BL (schematized in ), we suggest that direct juxtaposition of disc and trachea is not essential for FGF signaling and tubulogenesis, and that the BL is functionally transparent to FGF signaling.
Tracheal tunneling does not restrict responsiveness to Bnl/FGF
Matrix metalloproteinase is required during ASP outgrowth for ECM remodeling at the disc:trachea junction
The disc-associated trachea and ASP are situated beneath a layer of Collagen IV:GFP-containing ECM from the earliest stages of ASP induction (). However, during the early stages of growth, Collagen IV-GFP distribution accumulated along the lateral margins of the disc-associated trachea and along the lateral and distal margins of ASP. These bands of fluorescence were variable, but at late stages, most specimens had no apparent pericellular Collagen IV-GFP (). The levels of Collagen IV:GFP in the BL that overlies both the invasively coupled TC and ASP were lower at all stages of growth (). We interpret both the lower levels of Collagen IV:GFP in the BL overlying the ASP and the transient elevation of pericellular Collagen IV:GFP fluorescence along the lateral and distal edges of the ASP at early stages of ASP growth as evidence of active ECM remodeling during growth and extension of the ASP.
ECM dynamics during ASP morphogenesis
The invasive nature of the disc - tracheal association, together with the dynamic changes in the ECM around disc-associated tracheae, suggested that enzymes involved in ECM remodeling might play important roles. The Drosophila genome encodes two MMPs, Dm-Mmp1 and Dm-Mmp2 (Llano et al., 2002
; Llano et al., 2000
; Page-McCaw et al., 2003
), as well as a Tissue Inhibitor of MMPs (TIMP; Brew et al., 2000
). Previous studies reported that Dm-Mmp1 is expressed in the trachea and the ASP, and that Dm-Mmp2 is expressed in the trachea, ASP and wing disc (Llano et al., 2002
; Srivastava et al., 2007
; Uhlirova and Bohmann, 2006
). Drosophila TIMP is thought to inhibit both Dm-Mmp1 and Dm-Mmp2 in the extracellular milieu, although its endogenous distribution and roles are not well understood.
To assess the roles of Mmps, we examined animals in which TIMP was ectopically expressed, mutant animals defective for MMP activity, as well as animals in which RNAi was expressed to reduce Mmp1 and Mmp2 levels. As detailed in the subsequent paragraphs, these approaches led us to focus on the role of Mmp2. While ectopic TIMP expression, which reduces both Mmp1 and Mmp2 activities perturbed disc:tracheal association and ASP growth, the presence of Mmp1RNAi in the btl expression domain had no apparent effect. And although most Mmp1 mutants exhibited early larval lethality, some Mmp1Q273 mutant animals grew slowly and reached the pupal stage, but the imaginal discs, trachea and ASP in these mutants appeared normal. Thus, we found no role for Mmp1 in disc:tracheal association, ASP growth, or ECM remodeling (data not shown).
When TIMP was expressed in the apterous
-GAL4 UAS-TIMP, (ap
-TIMP); # specimens examined = 12) or btl
-GAL4 UAS-TIMP, (btl
-TIMP); # specimens examined = 8), the general level of Collagen IV:GFP fluorescence was comparable to wild type, as was the fluorescence of tracheal branches not in contact with the disc (). However, Collagen IV:GFP fluorescence increased at specific locations at which the invasively coupled transverse connective and the ASP make contact (). ap
-TIMP-expressing discs had a similar pattern of Perlecan distribution, as indicated by Perlecan:GFP fluorescence (see Supplementary Figure
) or immunolabeling with α-Perlecan antibody (not shown). Ectopic basal lamina around the tunneled tracheae was also apparent in serial thin sections of five ap
-TIMP-expressing discs (). In addition to increasing BL where the invasively coupled tracheal tubes and disc make contact, TIMP expression changed the depth at which the disc-associated transverse connective and ASP position within the disc. In wild type, both the transverse connective and ASP tunnel so that the plane of disc BL is relatively flat (, ). In ap
-TIMP animals, however, slight extrusion was apparent both in fluorescence images ( plane) and in thin sections (). These results suggest that MMP is required to limit synthesis of BL that would otherwise form around the tunneled tracheal tubes.
ECM abnormalities in animals expressing TIMP and in Mmp2 mutants
mutant larvae had similar phenotypes. Although their tracheal systems were without detectable structural or cell proliferation defects, mutant wing discs and associated transverse connective and ASP had phenotypes comparable to btl
-TIMP and ap
-TIMP. High levels of both Collagen IV:GFP (; # specimens examined = 8) and Perlecan-GFP (see supplementary figure
) accumulated around the ASP. Confocal images (# specimens examined = 3) and serial ultra-thin sections (# specimens examined =2) of mmp2W307
also revealed invasive coupling of the transverse connective and ASP, and showed that the mutant trachea extruded from the plane of the disc BL throughout the length of their contact ( and summarized in ). Knockdown of Mmp2 in the trachea (btl
)) increased levels of Collagen IV:GFP in patterns that were comparable to ectopic expression of TIMP and Mmp2W307
(; # specimens examined = 5).
In addition to the increases in BL, reduced levels of Mmp2 in ap-TIMP, btl-TIMP, btl-Mmp2RNAi and Mmp2W307 animals stunted ASP growth (; ). These stunted ASP tubes were populated by polarized epithelial cells (), but extension and growth was arrested beyond the stages shown in these figures. Viable adults that expressed TIMP in the trachea (btl-TIMP) lacked normal dorsal air sacs (), showing that Mmp2 function is required to make these adult organs.
Role of Mmp2 in FGF-dependent ASP growth
Dorsal Air Sacs development is inhibited by TIMP
Study of Mmp2 mutant clones revealed the non-autonomy of its function within the ASP. We generated clones of mmp2K07511 and mmp2W307mmp1Q112 cells (# clones = 10 and 8, respectively) in the ASP using the MARCM system, and found no change in the distribution, or reduction in the number or size of the clones when compared with control (# = 20). Thus, our data shows that in the ASP, Mmp2 has a tissue-specific but non-autonomous role.
In sum, reduction of Mmp2 function resulted in three distinctive and consistent mutant phenotypes: 1) ECM proteins Collagen IV and Perlecan accumulated to abnormally high levels at the junctional interfaces of tunneled trachea and disc; 2) tunneled trachea and ASP extruded from the plane of the disc BL; and 3) development of the ASP and dorsal airs sacs were abnormal. As described below, ASP development depends upon Mmp2 function in both the ASP and wing disc.
Autonomy of Mmp2 function for ASP development
The experiments described in the previous section show that reduction of Mmp2 function in trachea alone (btl-Mmp2RNAi) or in both trachea and disc (ap-TIMP and mmp2W307) have similar effects on the the BL distribution, the topology of the trachea within the disc BL and on ASP growth. These results establish that the Mmp2RNAi–induced phenotypes are specific to knockdown of Mmp2 and are not due to off-target effects. Expression of Mmp2RNAi to reduce Mmp2 function in the disc alone (ap-Mmp2RNAi) had an unexpected and strikingly different phenotype. RNAi knockdown of Mmp2 in the ap domain did not affect disc development (not shown), but caused hyperplastic growth of the ASP (). This effect shows that expression of Mmp2 in both disc and ASP is essential for ASP development and that Mmp2 has tissue-specific roles. During normal development presumably, proteolysis of BL components by ASP-produced Mmp2 enables extension and growth of the ASP, whereas proteolysis of BL components by disc-produced Mmp2 suppresses ASP growth.
Bnl/FGF signaling drives Mmp2 expression
Since ASP induction and growth are dependent on Bnl/FGF produced in the disc, we monitored discs with altered levels of Mmp2 for Bnl/FGF expression. In late L3, Bnl/FGF RNA was expressed in a discrete group of cells in the posterior compartment of the disc (). Neither its abundance nor its distribution changed in ap-TIMP, Mmp2W307, and btl-Mmp2RNAi discs (). Discs were also probed for evidence of FGF signal transduction with α-DpERK antibody. We detected nuclear Dp-ERK at the leading edge of the ASP in specimens from all backgrounds (). Although these assays for FGF expression and pathway activation are not quantitative, we conclude that the growth defects were not a consequence of a qualitative change in FGF signaling. In contrast, both the levels and area of Bnl/FGF expression increased in ap-Mmp2RNAi discs (). This increase is consistent with the ASP hyperplasia ().
As shown above, Mmp2 must be expressed in the trachea to promote ASP growth. Previous studies of its expression pattern revealed that it is expressed at elevated levels in the ASP (Llano et al., 2002
; Srivastava et al., 2007
). Since growth of the ASP is dependent upon Bnl/FGF signal transduction (Sato and Kornberg, 2002
), we tested if Bnl/FGF drives Mmp2 expression. We over-expressed Bnl/FGF with a heat shock-Bnl/FGF construct in an Mmp2
GAL4 UAS-nls-GFP background, and observed both increased proliferation and elevated levels of GFP in the ASP (). Elevated Dm-MMP2 expression is consistent with the hypothesis that Dm-MMP2 expression in the ASP is an outcome of Bnl/FGF signaling.