(A) Drawing of a L3 wing disc and associated tracheal branches showing the adepithelial myoblasts (brown), Bnl/FGF-expressing columnar epithelial cells (red), and tracheal cell nuclei (green). Black line shows approximate position of thin sections assembled into the montage. The section transects the transverse connective, ASP and myoblasts (M), revealing that these tracheal branches are underneath the disc BL. The thickness of the lamina densa (ld) overlying the ASP (arrows) is less than other regions (arrowhead). (B–C) Higher magnification views of the disc-associated trachea and ASP in (A) showing that the disc-associated trachea (B) has an apical taenidium (T, arrow) that is absent in the ASP (C). (D–F) Sections 5 m apart showing the trachea outside (B) and underneath (C, D) the disc BL. The space between the tracheae and myoblasts is not uniformly electron-dense. (G) Drawing showing the disc-associated trachea within the lamina densa of disc. (H) Collagen IV:GFP (green) illuminates the BL in the wing, haltere and leg discs and in associated tracheae (arrows in all panels indicate points of entry and exit). Tracheal cells (btl-RFP, red). (I, J) High magnification views showing the points at which the transverse connective enters (I) and exits (J) the disc BL. (J) A unicellular tracheole whose nucleus is outside the disc, is encapsulated by BL only where is outside the disc. (K) Confocal-derived XZ (bottom) and YZ (right) reconstructions of Collagen IV:GFP fluorescence (green, and black and white) and tracheal cells (red, btl-Cherry) in a late L3 show disc-associated tracheae and ASP located underneath a Collagen IV:GFP layer. Levels of Collagen IV:GFP above the ASP (arrow in YZ reconstruction shown in the right panel) are lower than in other regions of the notum. Scale bar: 90 um (L) Drawing illustrating invasive coupling of the disc with thinner BL over the trachea contacting the disc.

Clones expressing ectopic Bnl/FGF (dark areas) attract outgrowths of the trachea from a region outside the BL (green; GFP). Region of disc in (A) delineated by white box is shown at high magnification in (B) to reveal the multicellular composition of the outgrowth. (C) Gap:GFP expressed in tracheal cells illuminates the ASP and, from an adjacent tunneled region, an ectopic outgrowth (arrow) that extends toward Bnl-expressing cells (encircled by white line). (D) Collagen IV:GFP marks enlarged ASP and ectopic outgrowth (encircled by red lines). Arrowheads indicate “points of entry/exit” through the disc BL. (E) Collagen IV:GFP fluorescence (arrows), evidence of an independent BL of extensions at the tips of the outgrowth that bridge the outgrowth of the transverse connective and the disc. (F, upper) Drawing showing normal association of transverse connective (green) with the wing disc (gray), and identifyling Bnl/FGF-expressing cells (red) and adepithelial myoblasts (blue). BL (brown) encapsulates both trachea and disc except regions of invasive coupling. (F, lower) Drawing showing ectopic outgrowths from transverse connective, penetrating the disc BL to contact disc clones expressing Bnl/FGF.

Collagen IV:GFP fluorescence marks ECM in wing disc and trachea in preparations isolated from early L3 larvae prior to ASP induction (left panels, A, B) and at successively later stages (middle and right panels). Red: Cherry expressed in btl domain in trachea. (A) Arrowheads indicate “points of entry/exit” through the disc BL; arrows indicate regions of dynamic changes in Collagen IV levels. (B) Confocal images with adjacent XZ (bottom) and YZ (right) reconstructions showing that the abundance of Collagen IV:GFP around the trachea and ASP (solid lines) is lower (arrows) than in other regions (dashed lines). Number of preparations examined: pre-ASP: 5; early: 6; late 5.

(A–C) In contrast to normal animals (A, ASP outlined in white), btl-TIMP (B) and ap-TIMP (C) specimens have elevated Collagen IV:GFP levels at edges of disc-associated transverse connective and ASP (arrows). (D) XZ (bottom) and YZ (right) reconstructions of Collagen IV:GFP distribution in ap-TIMP disc showing that Collagen IV:GFP accumulates around the distal tip of the ASP (XZ, arrow) and that disc-associated trachea and ASP extrude from the disc (YZ, arrows) (E–G) Ultrastructure of ap-TIMP discs reveals defects in the ECM. Trachea is under the lamina densa (arrow), but ectopic lamina densa is detected around tunneled trachea. (F) Higher resolution views of left boxed region in (E) showing disc lamina densa (arrow) and ectopic lamina densa (arrowheads), and extrusion from the disc (G, right boxed region in (E)). (H) RNAi knockdown of mmp2 in the trachea recapitulates the phenotypes in (B) and (C). In mmp2^W307 (I, J), ectopic Collagen IV:GFP is detected around disc-associated tracheae. Note both extrusion of the trachea (J, XZ arrows) and accumulation of Collagen-GFP along the lateral margins (right arrow in XZ, arrows in YZ) of the ASP (K, L) Ultrastructure of mmp2^W307 discs at two positions along the disc - trachea interface showing the trachea underneath the disc lamina densa but extruded from the disc proper. (M) Diagram depicting defects in disc - tracheal association and lamina densa in ap-TIMP and Mmp2^W307 animals. (N) Diagram showing the distribution and levels of Collagen IV and Perlecan in specimens with wild type and reduced Mmp2 activity. Scale bar: 75 um.

(A) Disc from WT animal (48–50 hr, L3) stained with α-Dlg. Similar preparations from ap-TIMP (B) and Mmp2^W307 (C) animals (48–50 hr, L3) revealed ASP induction, but no directional growth. A requirement for Mmp2 expression in the trachea was revealed in preparations from btl- Mmp2^RNAi – expressing animals (D). Mmp2 knockdown in the disc (ap-Mmp2^RNAi) resulted in hyperplastic ASP growth (E). Knockdown of Mmp2 did not diminish Bnl/FGF expression (arrow in (F)) in the disc (F–I) or FGF signaling in the ASP (K–O). Knockdown of Mmp2 in the disc (ap-Mmp2^RNAi) resulted in expansion of the domain of Bnl/FGF expression (arrows, J). (P) Expression of mmp2GAL4 (Mmp2-nls GFP) was detected at higher levels at the distal edge of the ASP. (Q) Animals in which FGF expression was induced by heat shock showed hypertrophy of the ASP (outlined with a white line) and increased levels of Mmp2-nls GFP expression. (R) Model for the role of Mmp2 in ASP morphogenesis. Proliferation and growth of the ASP is accompanied by ECM remodeling (black arrows), mediated, in part, by FGF-dependent expression of Mmp2 in the ASP and consequent modulation of Collagen IV/Perlecan levels (red arrows).

Red lines outline the medioscuteal (upper) and scutellar (lower) air sacs in the thorax of adults imaged with bright field optics by mounting flies in halocarbon oil. Arrows indicate macrochaete. Medioscutal and scutellar lobes are stunted or missing in animals expressing TIMP in tracheal (btl domain) cells.

Two generic possibilities are that both the invasively coupled trachea and the disc have distinct Collagen IV-containing layers that are regulated independently by tracheal- or disc-generated Mmp2 (above), or that there is a single layer that both enzymes proteolyze (below).