Progenitor outgrowth from the niche in Drosophila trachea is guided by FGF from decaying branches

Abstract

Although there has been progress identifying adult stem and progenitor cells and the signals that control their proliferation and differentiation, little is known about the substrates and signals that guide them out of their niche. By examining Drosophila tracheal outgrowth during metamorphosis, we show that progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. The embryonic tracheal inducer branchless FGF (fibroblast growth factor) is expressed dynamically just ahead of progenitor outgrowth in decaying branches. Knockdown of branchless abrogates progenitor outgrowth, whereas misexpression redirects it. Thus, reactivation of an embryonic tracheal inducer in decaying branches directs outgrowth of progenitors that replace them. This explains how the structure of a newly generated tissue is coordinated with that of the old.

Fig. 1. Progenitor outgrowth during Drosophila tracheal metamorphosis

(A) Air-filled trachea (reflected light) (left) and schematics (right) in L3 larva and pupa ~13 hours after puparium formation. Tr, tracheal metamere; DB, dorsal branch; LT, lateral trunk; VB, visceral branch. Circles denote spiracles. Tracheal branches in the posterior (Tr6 to Tr10) are lost (dashed lines) during metamorphosis and are replaced by PAT from Tr4 and Tr5. (B to E) Fluorescence micrographs and schematics of Tr5 of ppk4-Gal4>UAS-GFP; btl-RFP-moe larva of the indicated ages stained to show activated tracheal progenitors (anti-RFP, red), larval tracheal cells (anti-GFP, green), and nuclei (4′,6-diamidino-2-phenylindole, blue). BPF, approximate time before puparium formation; W3L, wandering third-instar larva. (B) Quiescent progenitors in a Tr5 SB niche. Progenitors are also present in the SB niche of Tr2 to Tr4 and Tr6 to Tr9, but only those in Tr4 and Tr5 are activated to form PAT. P6 to P10, progenitors 6 to 10; the other five progenitors (P1 to P5) are shown in fig. S1A. NE, niche exit site (SB-TC junction). (C) Activated progenitors proliferating in the niche. (Inset) Progenitors near the niche exit site (dash), which have begun expressing btl-RFP-moe (red). (D) Progenitors exiting the niche. btl-RFP-moe expression increases and ppk4>GFP decreases as progenitors leave the niche and migrate dorsally along TC branches. Arrowhead, progenitor migration front. (E) Progenitors paused at the DT (arrowhead). Several progenitors have extended onto the VB (arrow). (F to J) Schematics and micrographs [as in (B) to (E)] of trachea (Tr4 to Tr9) of pupa of indicated ages APF. Paused progenitors (F) move onto the DT and turn posteriorly (G), extending along the DT (H and I) before ramifying into the PAT (J). Posterior tracheal branches start to collapse 9 hours APF and are fully collapsed and no longer conduct air by 13 hours APF. The larval cells die during metamorphosis (10), although we did not detect the apoptosis marker anticleaved caspase-3 during collapse (fig. S9). Scale bars: 100 μm, (A) and (F) to (J); 50 μm, (B) to (E).

Fig. 2. Progenitor out-growth requires breathless FGFR

(A) PAT progenitor migration in a control esg^P127-Gal4, UAS-GFP/act5c>Y>Gal4, UAS-GFP; UAS-FLP pupa 6 hours APF at 18°C with progenitors marked with GFP (green) and tracheal lumens stained (red). Brackets show position of progenitor migration front in the four phenotypic classes (0, I, II, III) scored in control and in esg^P127-Gal4, UAS-GFP/act5c>Y>Gal4, UAS-GFP; UAS-FLP/UAS-DN-btl pupae, in which dominant-negative breathless (DN-btl) is selectively expressed in progenitors. Examples of each phenotypic class can be found in fig. S5, A to D. Arrowhead, progenitor migration front. The graph at right shows quantification. n, number of branches. (B) PAT formation in control (top) and DN-btl pupae (bottom) as above, except reared for 1 to 2 additional days to allow formation of mature, air-filled pupal tracheae (reflected light). No PAT have formed in the DN-btl pupa. The graph shows quantification, with phenotypes classified as in fig. S5, E to H. Scale bars, 100 μm.

Fig. 3. Expression and requirement of branchless FGF during progenitor outgrowth

A) branchless reporter expression during progenitor outgrowth visualized by GFP immunostaining (white) of UAS-GFP; bnl-Gal4 NP2211/btl-RFP-moe larvae and pupae of indicated ages. Reporter expression dynamically expands along the progenitor outgrowth path, initially turning on in isolated larval cells in each area. Arrowheads denote furthest detected reporter expression. (B and C) Quantification (as in Fig. 2) of progenitor migration 3 hours APF (B) and PAT formation (C) phenotypes in control (ppk4-Gal4, UAS-GFP; btl-RFP-moe) and ppk4-Gal4, UAS-GFP; btl-RFP-moe/UAS-bnl RNAi pupae, in which bnl was inactivated in larval tracheal cells. For examples of the phenotypic classes, see fig. S7, A and B. (D) Frames at indicated times (hours:minutes) APF from live imaging of an act5c>Y>Gal4, UAS-GFP/UAS-FLP; prd-Gal4, btl-RFP-moe/UAS-bnl RNAi pupa in which bnl expression was inactivated in a DT patch (brackets). Tr5 progenitors (btl-RFP-moe) exit the niche (dashes) and move onto the DT but never pass the patch. Scale bars, 100 μm.

Fig. 4. Effect of ectopic bnl on progenitor migration

Tracheal progenitors (red, anti-RFP) in Tr4 or Tr5 [(A) to (F)] and Tr8 [(G) and (H)] of control (dfr-FLP/act5c>Y>Gal4, UAS-GFP; btl-RFP-moe) and experimental (dfr-FLP/act5c>Y>Gal4, UAS-GFP; btl-RFP-moe/UAS-bnl) wandering third-instar larvae showing control clones of larval tracheal cells expressing GFP alone (green) [(A) and (G)] or experimental clones expressing GFP and ectopic bnl FGF [(B to F) and (H)]. Blue, tracheal lumen (Alexa Fluor 350-conjugated wheat germ agglutinin). Arrowheads denote progenitor migration fronts. (A) Progenitors have exited the niche and reached the TC-DT junction (arrowhead). Control clones (green) have no effect. (B) Some progenitors have exited the niche in the wrong direction along TC branches (open arrowhead), extending toward the bnl-expressing clone ventral to the niche exit. Other progenitors (solid arrowhead) have exited the niche normally toward DT. (C) Progenitors have prematurely moved onto DT, extending toward a single larval DT cell expressing bnl. (D) Inappropriate anterior migration of progenitors along DT (arrowhead) toward the bnl-expressing clone. (E) Bifurcation of progenitor cluster (arrowheads) toward a pair of bnl-expressing clones located anterior and posterior to the TC-DT junction. (F) Bifurcation (arrowheads) where progenitors extend to fill the shape of a large bnl-expressing clone (open arrowhead). (G) Progenitors in Tr8 (as well as Tr3 and Tr6 to Tr9) normally remain within the niche and are unaffected by control clones. (H) Tr8 progenitors exit the niche toward a clone of bnl-expressing cells on TC branches. Scale bars [(A) to (H)], 100 μm. (I) Model of progenitor outgrowth guided by a signal produced by decaying tissue (green). Progenitors (red) are attracted to and form new tissue at the site of decay.