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.

Many adult stem cells reside in specific anatomical locations, or niches, and are activated during tissue homeostasis and after injury (1–4). Although considerable effort has been made to identify factors that control stem cell proliferation and differentiation, how stem or progenitor cells move out of the niche and how they form new tissue are not well understood (4–6). Tissue formation in mature animals faces challenges not present in the embryo (7, 8). The new cells migrate longer distances and navigate around and integrate into a complex milieu of differentiated tissues. In this work, we investigated the substratum and signals that guide Drosophila tracheal imaginal progenitor cells into the posterior during metamorphosis to form the pupal abdominal tracheae (PAT) that replace the posterior half of the larval tracheal system (tracheal metameres Tr6 to Tr10), which decays at this time (9, 10) (Fig. 1A).

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).

The PAT extend from the transverse connective (TC) branches in Tr4 and Tr5 (Fig. 1A). Each PAT consists of a multicellular stalk with many secondary branches, each of which has dozens of terminal cells that form numerous fine terminal branches (tracheoles) (10). There are two known tracheal progenitor populations at metamorphosis: dedifferentiated larval tracheal cells and spiracular branch (SB) imaginal tracheal cells set aside during embryonic tracheal development (11–14). Lineage tracing showed that PAT derive from imaginal progenitors (fig. S1, B and C).

To determine how progenitors in Tr4 and Tr5 reach the posterior, we used a btl-RFP-moe transgene (15) (RFP, red fluorescent protein) to label activated progenitor cells, and ppk4>GFP (16) (GFP, green fluorescent protein) to label larval tracheal branches (fig. S2A). Before metamorphosis, there are 7 to 10 quiescent progenitor cells in each SB niche (Fig. 1B and fig. S1A) (11, 13). In early third larval instar (L3), progenitors proliferate but remain in the niche (Fig. 1C). Later in L3, progenitors leave the niche, moving onto the larval TC branches toward the dorsal trunk (DT) (Fig. 1D), while progenitors within the niche continue to proliferate (13). Progenitors in other metameres also proliferate but do not move out of the niche (fig. S2B). Migrating progenitors in Tr4 and Tr5 crawl along the basal surface of larval tracheal cells, with cytoplasmic projections emanating from cells at the leading edges of the progenitor cluster (fig. S3C). Progenitors maintain epithelial polarity and a lumen continuous with the SB and TC branches, forming a saclike structure (fig. S3, A and B) (9). By wandering L3, progenitors reach the DT (Fig. 1E), where they pause (~12 hours) until the onset of puparium formation (Fig. 1F).

Around 1 hour after puparium formation (APF), progenitors move onto the DT and turn posteriorly (Fig. 1G). Posterior migration continues for 9 hours, extending half the animal’s length (~0.8 mm) past Tr9 (Fig. 1, H to J). Live imaging showed that progenitors move at ~1.7 μm/min, crawling along and wrapping around the DT as they migrate (movie S1).

Differentiation begins as progenitors migrate. At the beginning of puparium formation (0 hours APF, Fig. 1F), a subset of progenitors that have exited the niche begins to express the terminal cell master regulator Pruned SRF (serum response factor) (17), initiating cell specialization (fig. S4A). As progenitors migrate along the DT, budlike structures composed of Pruned-expressing cells are detected at the tips of progenitor clusters, whereas Pruned-negative cells form the stalks of new trachea (fig. S4B). By 6 hours APF, Pruned-expressing progenitors in the tips adopt an elongated and differentiated morphology (fig. S4, C and D), flattening along the DT as they extend further posteriorly (movie S1). Around 13 hours APF, the PAT mature and fill with gas as posterior tracheal branches collapse (Fig. 1, A and J).

What guides tracheal progenitors on their stereotyped path along specific branches of the larval tracheal system? Expression of breathless (btl) FGFR (fibroblast growth factor receptor) is induced in PAT progenitors (11, 13), as shown by the btl-RFP-moe reporter (Fig. 1, B to J). We tested whether the Btl pathway, which directs tracheal branch outgrowth in embryos (18–20) and larvae (21) and induces adult air-sac primordium formation (22), is involved. Expression of dominant-negative Btl FGFR (19) in the progenitors and their descendants (23) blocked migration and diminished or eliminated PAT formation (Fig. 2 and fig. S5). To determine the source of the only known Btl ligand, Branchless (Bnl) FGF (20), we used a bnl reporter, bnl-Gal4 enhancer trap line NP2211 (24) driving UAS-GFP. Unlike previously described examples of tracheal outgrowth (20–22), bnl was not expressed in surrounding tissue. Instead, it was expressed within the tracheal system, specifically by larval tracheal cells along which progenitors migrate. The expression pattern is dynamic and precise, almost perfectly matching the positions and timing of progenitor migration (Fig. 3A and fig. S6A). In L3 animals, when progenitors are observed along the TC branches, bnl>GFP was expressed in TC larval cells in Tr4 and Tr5, but not in other metameres. Shortly after puparium formation, when PAT progenitors turn to migrate toward the posterior, DT larval cells in the segment just posterior to PAT progenitors express bnl>GFP. As progenitors continue along the DT, DT larval cells activate bnl>GFP expression one segment at a time from anterior to posterior, matching progenitor movement.

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.

This dynamic bnl expression along the migration path is required for progenitor outgrowth. Knockdown of bnl expression by RNA interference (RNAi) in larval tracheal cells blocked migration and resulted in diminished or absent PAT (Fig. 3, B and C; fig. S7, A to C; and movie S2). Mosaic expression of bnl RNAi in small patches along the path (23) also arrested migration, so long as the patch encompassed the full DT circumference (Fig. 3D; fig. S7, D and E; and movies S3 and S4). Thus, Bnl is required all along the migration path, and the signal does not cross even short gaps.

Ectopic bnl expression in GFP-labeled clones of larval tracheal cells induced by dfr-FLP (23) redirected progenitor migration. Depending on the location of the clones, ectopic bnl caused incorrect exit from the niche, premature entry onto the DT, or wrong turns on the DT (Fig. 4, B to D). Dual clones induced bifurcation with groups of progenitors moving toward each ectopic bnl source (Fig. 4E). Clones in Tr3 and posterior metameres caused progenitors in these regions to leave the niche, even though they do not normally do so (Fig. 4, G and H, and fig. S8, D and E). When there was a large clone, progenitors migrated throughout the clone (Fig. 4F), implying that progenitors do not require a gradient and will spread to cover an entire region of cells expressing bnl at equivalent levels. When bnl-expressing clones failed to induce migration, the clones appeared to be too far from the progenitors or there was competition from another clone close by (fig. S8, A and B). Ectopic bnl expression within the progenitor cluster arrested migration (fig. S8C).

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.

The results show that the embryonic tracheal inducer Bnl FGF guides tracheal progenitors out of the niche and into the posterior during tracheal metamorphosis. The source of Bnl is the larval tracheal branches destined for destruction, which serve both as the source of the chemoattractant and as the substratum for progenitor migration. Several days earlier in embryos, these larval tracheal branches were themselves induced by Bnl provided by neighboring tissues. But after embryonic development, most tracheal cells, including those in the decaying larval branches, down-regulate btl FGFR expression (fig. S2A) and thus do not respond to (or sequester) the Bnl signal they later express. One of the most notable aspects of this larval Bnl is its exquisitely specific pattern in decaying larval branches, which presages progenitor outgrowth. It is unclear how Bnl expression is controlled, though it does not appear to require signals from migrating progenitors because the bnl reporter expression front progressed normally when progenitor outgrowth was stalled by a tracheal break (fig. S6C). Perhaps expression of Bnl involves gradients in the tracheal system or spatial patterning cues established during embryonic development in conjunction with temporal signals mediated by molting hormones.

Because the signal guiding progenitor migration is provided by tracheae destined for destruction, progenitors become positioned along the larval branches they replace (Fig. 4I). Perhaps during tissue repair and homeostasis, recruitment of adult stem or progenitor cells from the niche is similarly guided by signals from decaying tissue, thereby ensuring that new tissue is directed to the appropriate sites.

Supplementary Material

Supplementary Material

Figure S1. Visualizing and lineage-tracing tracheal progenitors that migrate out of the spiracular branch niche. (A) Cells in the Tr5 spiracular branch (SB) niche (dotted line) before progenitor activation. A portion of tracheal metamere Tr5 is shown from a ppk4>GFP; btl-RFP-moe second instar larva (L2) stained for GFP to show tracheal cells (green), for RFP to show activated progenitors (red), and labeled with DAPI to show cell nuclei (blue). The 10 progenitor cells (P1 – P10) in the spiracular branch (SB) niche are labeled, as are six larval tracheal cells (L1–L6) in the neighboring transverse connective (TC). Progenitors P9 and P10 connect to L5 and L6 and the rest of the larval tracheal system at the SB-TC junction, which we refer to as the niche exit (NE) and indicate with a dash. P1 and P2 connect to the epidermis. Progenitors in tracheal metameres Tr2 – Tr4 and Tr6 – Tr9 (not shown) appear similar. DT, dorsal trunk; VB, visceral branch. (B, C) escargot>FLP lineage trace showing imaginal progenitors give rise to pupal abdominal trachea (PAT). Fluorescent micrographs of differentiated PAT stalk (B) and terminal (C) cells in an esg^P127-Gal4, UAS-GFP/act5c>Y>Gal4, UAS-GFP; UAS-FLP pupa approximately 24 hr after puparium formation (APF) at 18°C. SB and other esg^P127-lineage (imaginal) progenitors and their descendants are labeled with GFP (green), and air-filled tracheae and terminal branches (tracheoles) are visualized by reflected light (white). Dashed line, PAT stalk; arrowheads, individual terminal cells, each of which has formed many tracheoles. Bars, 50 μm.

Figure S2. Selective expression of a breathless FGFR reporter in outgrowing progenitors. (A) Fluorescent micrograph of a ppk4>GFP; btl-RFP-moe wandering third instar (W3L) larva stained for GFP to show ppk4>GFP expression and for RFP to show btl-RFP-moe expression as in Fig. 1E. Close-ups of metamere Tr5 (boxed) are shown in A′, with nuclei stained with DAPI (blue). Note that larval tracheal cells along the dorsal trunk (DT), transverse connectives (TC), lateral trunk (LT), visceral branch (VB) and posterior (Tr6 to Tr10) dorsal branches (DB) express ppk4>GFP but little or no btl-RFP-moe, which must have been downregulated after embryonic development (15). Conversely, progenitors have turned off the ppk4>GFP reporter, and Tr4 and Tr5 progenitors that are exiting the niche (arrowheads) highly express the btl reporter. Dotted lines, SB niche. Dashes, SB niche exit (NE). De-differentiating cells in anterior (Tr2 to Tr5) dorsal branches (DB) (11) also express the btl-RFP-moe reporter (A′) though at lower levels. (B) Posterior SB niche after progenitor activation. A portion of tracheal metamere Tr9 of the larva in A is shown with nuclei stained with DAPI (blue). Progenitors have proliferated and those near the niche exit express low but detectable btl-RFP-moe (red). However, progenitors do not exit the niche, as they do in Tr4 and Tr5. (C) Close up of progenitors growing out of the SB niche in an esg^P127-Gal4, UAS-GFP/act5c>Y>Gal4, UAS-GFP; UAS-FLP/btl-RFP-moe pupa 6 hr APF at 18°C stained for esg^P127 lineage-trace progenitors (anti-GFP, green), activated progenitors (anti-RFP, red), and nuclei (DAPI, blue). Note that all progenitors that have exited the SB niche (arrowheads) express the btl reporter but those still in the niche (dotted line) do not. *, larval fusion cells that also express esg. Dash, SB niche exit (NE). Bars, 100 μm (A, C) and 25 μm (B).

Figure S3. Progenitor outgrowth morphology. Portion of tracheal metamere Tr4 in btl-RFP-moe/crumbs::GFP early third-instar (L3) (A) and wandering third instar (W3L) (B) larvae stained for activated tracheal progenitors (anti-RFP, red) and apical tracheal surface and lumen (anti-GFP, green). In early L3 larvae (A), the SB contains a lumen and forms a junction with the larval transverse connective (TC) at the niche exit (NE) site. In wandering L3 larvae (B), the outgrowing progenitors have formed a monolayer-epithelial sac extending from the SB niche (arrowhead). The lumen of the expanding progenitor sac is continuous with the SB lumen and larval TC at the NE. Inset (B′) shows an optical z-section of the boxed region. Note Crumbs::GFP and RFP-moesin co-localization on apical progenitor cell surfaces. (C) Close-up of migrating progenitors in Fig. 1D. Progenitors migrate on the basal surface of larval tracheal cells; cytoplasmic extensions emanate from the leading progenitors (arrowheads), indicating active migration. Bars, 50 μm (A, B) and 5 μm (C).

Figure S4. Migrating progenitors initiate the tracheal morphogenesis program. (A) Portion of tracheal metamere Tr5 in btl-RFP-moe/crumbs::GFP larva at onset of puparium formation (0 hr APF) stained for Pruned SRF (white), tracheal progenitors (anti-RFP, red), and apical surfaces of tracheae (anti-GFP, green). Note that a subset of progenitors in the outgrowing cluster (bracket) has initiated the terminal cell differentiation program as indicated by expression of Pruned. Dotted line, SB niche; dash, SB niche exit (NE). (B, C) Pupae as above at 3 hr (B) and 6 hr (C) APF. Pruned-expressing progenitors are segregated at the tips of the outgrowing PAT (arrowheads), while Pruned-negative cells form the PAT stalk (arrows). (D) Close-up of PAT stalk and terminal cells of the pupa in C also stained for DAPI to show nuclei. Note terminal cells have elongated morphology and have formed intracellular lumens (tracheoles) (outlined by RFP-moesin and Crumbs::GFP) whereas progenitors in the stalk do not express Pruned, do not form tracheoles, and have apical Crumbs::GFP localization. Bars, 50 μm (A), 100 μm (B, C), 25 μm (D).

Figure S5. Effect of dominant negative Breathless on progenitor migration, proliferation, and PAT formation. Dominant-negative breathless (DN-btl) was expressed in SB tracheal progenitors and their progeny, and the animals were analyzed 6 hr APF at 18°C for effects on Tr4 and Tr5 progenitor migration (A–D) as described in Fig. 2A, or for effects on progenitor proliferation by counting the number of progenitors (I), or animals were reared for an additional 1 to 2 days to allow PAT formation (E–H). (A–D) Examples of progenitor migration phenotypes. (A) Normal (Class 0), migration similar to that in control pupae. (B) Partial (Class I), reduced migration along DT. (C) Minimal (Class II), no migration along DT. (D) None (Class III), progenitors do not exit SB niche. Progenitors, green (GFP fluorescence); larval tracheal branches, red (rhodamine-conjugated WGA and rhodamine-conjugated chitin-binding protein); dash, niche exit (NE); *, larval tracheal fusion cells and sporadic larval tracheal cells that also express the progenitor lineage label. (E–H) Examples of PAT formation phenotypes. (E) Normal, extensive PAT formation similar to that in controls. (F) Partial, reduced PAT ramification and extension into posterior. (G) Minimal, limited PAT formation and extension with thin branches at PAT base. (H) None, no PAT formation. White, mature air-filled pupal tracheae; dashed ovals, extent of PAT formation. Similar though less severe progenitor migration and PAT formation phenotypes were observed with expression of a btl RNAi transgene. (I) Quantification of cell number in Tr4 and Tr5 SB niches. The 10 SB progenitors (fig. S1A) proliferate extensively in both control and DN-btl-expressing pupae, but note there are fewer cells in the DN-btl pupae, in which progenitors do not leave the niche. Bars, 50 μm (A–D), 100 μm (E–H).

Figure S6. Expression of branchless (bnl) just ahead of migrating tracheal progenitors. (A) bnl-Gal4 NP2211/UAS-GFP; btl-RFP-moe animals of the indicated ages showing bnl reporter expression (GFP immunostain, white) as in Fig. 3A but co-stained to show relationship to migrating progenitors (RFP immunostain, red). Isolated larval cells initiate bnl reporter expression (arrowheads) ahead of migrating progenitors. Note reporter expression in TC cells ventral to the niche exit that is ignored by migrating progenitors, and absence of reporter expression in the Tr5 DT, along which Tr4 progenitors migrate to meet Tr5 progenitors. Dashes, niche exit (NE). (B) Close up of Tr6 in pupa 4.5 hr APF. Optical sections through dorsal trunk (DT) at planes indicated (s1, s2, s3) are shown in B′ and B″ (bnl reporter expression, white; migrating progenitors, red). Although bnl reporter expression initially appears in individual DT cells (section s1), it expands to cover the entire circumference of the DT (section s3). However, progenitors do not completely envelop the entire circumference (section s3). (C) A 9 hr APF pupa, stained as above, with sporadic break that has separated DT into anterior and posterior regions. Note that progenitors (red, arrow) have not migrated beyond the lesion (*) but bnl reporter expression has expanded beyond lesion into posterior metameres (arrowhead) just as in animals with intact DT (panel A). Bars, 100 μm (A,C), 50 μm (B).

Figure S7. Progenitor migration and PAT formation phenotypes from branchless (bnl) knockdown along migration route. (A) Progenitor migration in a control (ppk4-Gal4/UAS-GFP; btl-RFP-moe) and ppk4-Gal4/UAS-GFP; btl-RFP-moe/UAS-bnl RNAi pupa in which bnl RNAi was expressed in all larval tracheal cells. The effect on progenitor migration 3 hr APF was analyzed as described in Fig. 2A after staining for progenitors (anti-RFP, red), larval tracheal cells in which bnl is knocked down by RNAi (anti-GFP, green), and nuclei (DAPI, blue). Examples of migration phenotypes are indicated, classified as in fig. S5A–D. Dotted line, SB niche; dash, niche exit (NE); arrowheads, extent of progenitor migration. (B) Pupae as in A reared for another 1 to 2 days to allow PAT formation. Air-filled trachea, visualized by reflected light (white). Dashed circles, extent of PAT formation. Examples of PAT formation phenotypes are indicated, classified as in fig. S5E–H. (C) Frames from live imaging (see also Movie S2) at the indicated times APF of control and tracheal bnl RNAi knockdown pupae as in A. Progenitors (white, btl-RFP-moe) migrate along larval DT in control pupa, but they never leave the SB niche (dash) in the tracheal bnl knockdown pupa. Arrowheads, progenitor migration front; *, progenitor migration extends beyond field of view. (D) Frames from live imaging (see Movie S4) at the indicated times APF of a dfr-FLP/act5c>Y>Gal4, UAS-GFP; btl-RFP-moe/UAS-bnl RNAi pupa in which bnl expression was knocked down in a DT patch along the Tr5 DT (bracket). Tr4 progenitors are stalled next to the large patch, whereas Tr5 progenitors are not stalled by the smaller patches of bnl RNAi expression in the Tr6 DT. (E) Confocal fluorescent micrograph of a UAS-FLP/act5c>Y>Gal4, UAS-GFP; prd-Gal4, btl-RFP-moe/UAS-bnl RNAi pupa fixed following 6 hours of live-imaging (see Fig. 3D and Movie S3) and then stained for tracheal progenitors (anti-RFP, red), cells expressing bnl RNAi (anti-GFP, green), and nuclei (DAPI, blue). Optical sections at the planes indicated (s1 – s4) are shown. Progenitors stalled next to the short segment of DT, approximately 2 to 3 larval cells wide, in which paired FLP-out drives a patch of expression (bracket) of UAS-bnl RNAi and UAS-GFP. Optical sections show that bnl RNAi expressing cells encompass full circumference of DT. Bars, 50 μm (A and E), 100 μm (B–D).

Figure S8. Examples of branchless-expressing clones that did not induce ectopic migration of PAT progenitors and clones that induced migration of progenitors that do not normally migrate. GFP-labeled clones of bnl-expressing cells (green) were induced and analyzed in wandering third-instar larvae as in Figure 4. Dash, niche exit (NE); DT, dorsal trunk; TC, transverse connective; DB, dorsal branch; arrowheads, progenitor migration front. (A) A clone (arrow) far from the Tr4 migrating progenitors (red) that did not induce ectopic progenitor migration. (B) A pair of clones (arrow and arrowhead) in which Tr5 progenitors have migrated toward only one of the clones (arrowhead). (C) A clone in the Tr5 progenitor cell cluster. Migration is disrupted and progenitors remain near the SB niche. (D) Control clones in Tr3. Tr3 progenitors normally remain within the SB niche during PAT outgrowth and are unaffected by control clones expressing only GFP. (E) A bnl-expressing clone that has recruited Tr3 progenitors out of the niche to DT. Note that the Tr3 progenitors have reached the DT clone even though there is no endogenous (or ectopic) bnl expression in the TC, perhaps because the Tr3 TC is shorter than those in other metameres. (F) Control clone near Tr5 DB. Progenitors in anterior DBs (Tr2 to Tr5) derived from de-differentiated larval cells express btl-RFP-moe (fig. S2A) and proliferate but normally remain in the DB niche (see also fig. S2A’ and fig. S4B, C). (G) Tr5 DB progenitors (arrowhead) are sometimes recruited onto the DT by clones expressing ectopic bnl. Arrow, Tr5 SB progenitors also recruited by the clone. Dash, DB boundary at DB/DT junction. Bars, 100 μm (A–C), 50 μm (D–G).

Figure S9. Decaying tracheal branches do not express cleaved Caspase-3. (A, B) Fluorescent micrographs of ppk4>GFP; btl-RFP-moe 15 hr APF pupae immunostained for cleaved Caspase-3 (white), a marker of apoptosis, and for GFP (green) to show tracheal cells. Nuclei are stained with DAPI (blue). Larval tracheal cells in posterior metameres (Tr6 to Tr10) are lost during metamorphosis (10) but are not stained by the cleaved Caspase-3 immunostain, suggesting that they die later in metamorphosis or by other mechanisms. Thin or spotty staining (open arrowheads) is antibody trapped in collapsed posterior tracheal branches (Fig. 1). (C) Fluorescent micrograph of a ppk4-Gal4, UAS-GFP/UAS-rpr; tub-Gal80ts/+ wandering third-instar larva stained as in A and B as a control to show cleaved Caspase-3 expression in apoptotic tracheal cells. Note cleaved Caspase-3 immunostaining (white) in tracheal cells activating expression of the apoptosis inducer reaper (rpr) and marked by GFP (green; arrowheads). Animals were raised at the permissive temperature (18°C) of the GAL80ts repressor to prevent early ectopic rpr expression and allow embryonic tracheal formation, and then transferred to the non-permissive temperature (30°C) to activate rpr-induced apoptosis. Bars, 50 μm.

Figure S10. Effect on the larval tracheal system of tracheal clones expressing branchless FGF. Brightfield (upper) and fluorescence (lower) images of a control Cyo/act5c>Y>Gal4, UAS-GFP; btl-RFP-moe/UAS-bnl wandering third instar larva and a dfr-FLP/act5c>Y>Gal4, UAS-GFP; btl-RFP-moe/UAS-bnl larva in which the GFP-marked clones express ectopic bnl. Note tracheal patterning in the larva with bnl-expressing clones was not globally perturbed, although there are local defects including a sporadic gap (*) in the lateral trunk (LT) and scattered foci of densely-packed tracheoles (arrowhead). DT, dorsal trunk; TC, transverse connective. Bars, 100 μm.

Supplementary Movie 1. Movie S1. Tracheal progenitors migrating along the larval dorsal trunk.

Fluorescence imaging of a live btl-RFP-moe white pupa beginning just after puparium formation (0 hr APF) in which migrating PAT progenitors originating from tracheal metameres Tr4 and Tr5 and expressing RFP-moesin (white; arrowheads) were visualized through the cuticle. Images were acquired every three minutes for seven hours at 25°C. DT, dorsal trunk; TC, transverse connective; dash, SB niche exit. Progenitors move toward the posterior at ~1.7 μm/min, maintaining a close association with the DT and crawling and wrapping around it as they migrate. Tr4 progenitors move onto DT about one hour later than Tr5 progenitors. Note multiple tips of migrating progenitors, each taking slightly different paths along DT into posterior. Bar, 100 μm.

Supplementary Movie 2. Movie S2. Effect of branchless knockdown on progenitors exiting SB niche.

Fluorescence imaging of a live ppk4-Gal4/UAS-GFP; btl-RFP-moe/UAS-bnl RNAi pupa in which bnl was knocked down by RNAi in larval tracheal cells (marked by GFP, pseudo-colored green in merged images). Tracheal progenitors marked by RFP fluorescence and pseudo-colored red in merged images (arrowheads) do not move beyond niche exit (dash). DT, dorsal trunk; TC, transverse connective. Images were acquired every five minutes from 0 hr to 6 hr APF at 25°C. Bar, 100 μm.

Supplementary Movie 3. Movie S3. Effect of branchless knockdown in patch along Tr5 progenitor migration route.

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 portion of Tr6 DT (bracket) by expression of bnl RNAi mediated by paired-Gal4. Tr5 tracheal progenitors move onto DT but stall when they reach DT patch where bnl RNAi is expressed; meanwhile, Tr4 progenitors progress posteriorly. DT, dorsal trunk; TC, transverse connective; dash, SB niche exit; arrowhead, progenitors. Images were acquired every five minutes from 0 hr to 6 hr APF at 25°C. Bar, 100 μm.

Supplementary Movie 4. Movie S4. Effect of branchless knockdown in patch along Tr4 progenitor migration route.

Live imaging of a dfr-flp/act5c>Y>Gal4, UAS-GFP; btl-RFP-moe/UAS-bnl RNAi pupa in which bnl expression was inactivated in a portion of Tr5 DT (bracket) by expression of bnl RNAi. Tr4 tracheal progenitors stall next to DT patch where bnl RNAi is expressed. DT, dorsal trunk; TC, transverse connective; dash, SB niche exit; arrowhead, progenitors. Images were acquired every five minutes from 0 hr to 6 hr APF at 25°C. Bar, 100 μm.