Multipotent versus differentiated cell fate selection in the developing Drosophila airways

Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway. DOI: http://dx.doi.org/10.7554/eLife.09646.001


Introduction
Multipotent stem cells are essential for both growth and generation of cell diversities in developing organs. They also serve as reservoir to replace damaged or aged cells during physiological or pathological tissue homeostasis. The discovery of a small number of transcription factors that can induce pluripotent cells (Takahashi and Yamanaka, 2006) has fueled major research efforts to reveal the mechanisms biasing the choice between pluripotent/multipotent and more differentiated cell fates in vivo (O'Brien and Bilder, 2013). Using the Drosophila airways (tracheal system) (Manning and Krasnow, 1993;Samakovlis et al., 1996b) (Figure 1A), we studied how the initial selection of different potencies within an organ becomes first predisposed and then regionally confined.
By mid-embryogenesis, Drosophila embryos are metamerically divided along the anterior-posterior (AP) axis (Ingham, 1988;Sanson, 2001). Each unit (segment or parasegment) is also subdivided along the dorso-ventral (DV) embryo axis (Anderson et al., 1985b;Wharton et al., 1993). Generation of the airway primordia is spatially restricted along these AP or DV axis, by the local concentrations of repressing and activating signals (Bier et al., 1989;Isaac and Andrew, 1996;Perrimon et al., 1991;Wilk et al., 1996). For example, Wingless (Wg) expressed in stripes along the AP axis and Decapentaplegic (Dpp) expressed in the dorsal ectoderm ( Figure 1B) repress the airway primordia specification (Isaac and Andrew, 1996;Wilk et al., 1996). As a result, 10 metameric groups of cells expressing a master gene, Trachealess (Trh) are specified on each side of the lateral ectoderm ( Figure 1A-B). Invagination of each primordial cluster transforms the two-dimensional (2D) shape of each cluster to 3D tubes ( Figure 1C) (Campos-Ortega and Hartenstein, 1985;Turner and Mahowald, 1977). Within the invaginated primitive sacs/tubes, the proximal cells form a narrow cord, the spiracular branch/SB (P-fate) ( Figure 1C, K) and stay multipotent to later proliferate and to generate many parts of the pupal/adult airways (Manning and Krasnow, 1993;Pitsouli and Perrimon, 2010;Weaver and Krasnow, 2008). Within the remaining distal cells (Dfate), the more distal parts achieve a series of morphogenetic events, extending six primary branches, fusing with branches from neighboring metameres and supplying oxygen directly to the target cells (Manning and Krasnow, 1993;Samakovlis et al., 1996a;Samakovlis et al., 1996b), while the more proximal parts (transverse connectives/TC) connect SB with the primary branches ( Figure 1C, K).
The primary branches constitute D-fate cells located distally from TC, and their morphological diversification depends on the activation of various signaling pathways. For example, rhomboid (rho) is expressed in each primordium (Bier et al., 1990) and activates dEGFR (also called torpedo or faint little ball) (Price et al., 1989;Schejter and Shilo, 1989) by generating the secreted active ligand Spitz (s-Spi) Golembo et al., 1996;Schweitzer et al., 1995b;Urban et al., 2001;Wappner et al., 1997). Activated dEGFR instructs the cytoskeletal changes that coordinate invagination (Brodu and Casanova, 2006;Kondo and Hayashi, 2013;Llimargas and Casanova, 1999;Nishimura et al., 2007). Branchless (Bnl)/dFGF is expressed in patches of surrounding tissues to guide primary branching  by activating Breathless (Btl)/dFGFR on the airway cells (Klambt et al., 1992;Lee et al., 1996). Apart from receptor tyrosine kinase (RTK) signaling, Decapentaplegic (Dpp)/BMP, Wingless (Wg)/WNT, and Hedgehog (Hh) signals combinatorially establish different primary branch identities (Affolter et al., 1994;Chen et al., 1998;Chihara and Hayashi, 2000;Glazer and Shilo, 2001;Llimargas, 2000;Llimargas and Lawrence, 2001;Matsuda et al., 2015;Vincent et al., 1997). Although many studies focused on the differentiation and subsequent morphogenesis of the D-fate cells (Beitel and Krasnow, 2000;Cabernard et al., 2004;Ghabrial et al., 2011;Hosono et al., 2015), generation of the P-fate has been underexplored. Our current dissection of P/D fate establishment in the embryo, together with metamerespecific control programs of differentiation/de-differentiation in the larval airways (Djabrayan et al., 2014;Guha et al., 2008;Sato et al., 2008;Weaver and Krasnow, 2008) may broaden our understanding of the strategies for stem cell selection and maintenance in vivo. eLife digest Many organs are composed of tubes of different sizes, shapes and patterns that transport vital substances from one site to another. In the fruit fly species Drosophila melanogaster, oxygen is transported by a tubular network, which divides into finer tubes that allow the oxygen to reach every part of the body.
Different parts of the fruit fly's airways develop from different groups of tracheal precursor cells. P-fate cells form the most 'proximal' tubes (which are found next to the outer layer of the fly). These cells are 'multipotent' stem cells, and have the ability to specialize into many different types of cells during metamorphosis. The more 'distal' branches that emerge from the proximal tubes develop from D-fate cells. These are cells that generally acquire a narrower range of cell identities.
By performing a genetic analysis of fruit fly embryos, Matsuda et al. have now identified several proteins and signaling molecules that control whether tracheal precursor cells become D-fate or Pfate cells. For example, several signaling pathways work with a protein called Ventral veinless to cause D-fate cells to develop instead of P-fate cells. However, molecules that prevent signaling occurring via these pathways help P-fate cells to form. Different amounts of the molecules that either promote or hinder these signaling processes are present in different parts of the fly embryo; this helps the airways of the fly to develop in the correct pattern.
This work provides a comprehensive view of how cell types with different developmental potentials are positioned in a complex tubular network. This sets a basis for future studies addressing how the respiratory organs -and indeed the entire organism -are sustained.

Results and discussion
The proximo-distal cell fate organization in the Drosophila airway tree Every cell in the main airways derives from 20 primordial cell clusters expressing the master regulator TF Trh ( Figure 1A-C, K) (Isaac and Andrew, 1996;Perrimon et al., 1991;Wilk et al., 1996). However, the expression of several other genes is enriched either distally or proximally in the airways. rho is expressed preferentially in the central and distal regions of the airway primordia before and during invagination (Figure 1-figure supplement 1A-E). btl/dFGFR is also expressed preferentially in the distal part ( Figure 1D-E), and its expression is upregulated by Bnl/dFGF in the most distal leading cells of the primary branch tips ( Figure 1E) Ohshiro and Saigo, 1997). mAb2A12 detecting the putative chitin-biding protein Gasp (Tiklova et al., 2013) preferentially labels the distal airways during stages 14-17 ( Figure 1F) . On the other hand, unpaired (upd), a ligand activating the JAK/STAT signaling is expressed only in the proximal SB cells from stage 12 on ( Figure 1G-I) . From stage 13 onwards, the SB cells also show stronger expression of the P0144 enhancer trap marker ( Figure 1E,F,I) (http://flyview.uni-muenster.de). Compared to the anterior metameres, the 10 th tracheal primordium (Tr10) does not establish the P0144-postive P-fate due to the high level of a Hox protein Abdominal-B (AbdB) in this part of the embryo ( Figure 1F, J), Matsuda et al., 2015). In trh mutants, both the D-fate marker (Chung et al., 2011;Jin et al., 2001;Ohshiro and Saigo, 1997;Zelzer and Shilo, 2000) and the P-fate marker expressions (Figure 1-figure supplement 1U, V) are lost.
To define the origins of the proximo-distal organization of the airway tree, we labeled cell groups during the 2D primordial stage and recorded their fates in the 3D tree. pointed (pnt) is a general transcriptional target and mediator of RTK signaling (Brunner et al., 1994;Klambt, 1993;O'Neill et al., 1994). rho induces pnt expression in the central parts of the airway primordia  (Carroll et al., 1988;Hooper et al., 1989;Zhan et al., 2010) is confined to the dorso-central part ( Figure 1-figure supplement 1P) and its reporter (h-lacZ) becomes active in most of the DB, VB, and DT cells but not in TC, SB nor in ventral branches ( Figure 1K, Figure 1-figure supplement 1Q) . In contrast, salm labels the dorsal part of the airway primordia ( Figure 1-figure supplement 1R), (Kuhnlein and Schuh, 1996) and salm-gal4 driven UAS-GFP marks DB and DT, and parts of TC and SB in the airways ( Figure 1K, Figure 1-figure supplement 1S-T). These results suggest that the dorso-peripheral sector of each primordium, which is positive for salm but lacking h, composes half of the proximal SB cells in the airway tree. The other half may originate from the ventro-peripheral sector of the primordia. Consistently, around the completion of invagination, the P-fate marker upd is enriched in a horseshoe-like peripheral ring ( Figure 1G) , which eventually marks the SB cells at stage 12 ( Figure 1H,I,K). Collectively, the gene expression analysis suggests a fate map of the airway primordia, where the centro-peripheral axis inversely correlates both with the ordered pattern of tracheal cell invagination (Brodu and Casanova, 2006;Nishimura et al., 2007) and the PD axis of airway branching ( Figure 1C,K).
RTK signaling drives the D-fate selection at the expense of the P-fate As activation of RTKs propels several morphogenetic programs in the D-cells, we first assessed their effects on the discrimination of the P-and the D-fate. In btl/dFGFR mutants, where active branch extension does not occur, apoptotic staining in the distal cells increases from stage 12 (  Thus, dEGFR and Btl/ dFGFR cooperate to suppress apoptosis in the airway cells. To analyze the effects of RTK signaling in the P/D fate choice independently of cell survival, we introduced Df(H99) into some of the genotypes used in the analysis described below.
In bnl/dFGF mutants, the feedback upregulation of the D-fate marker btl/dFGFR is lost in the tips of the primary branches ( Figure 2A,B) . However, the distal part of the invaginated stump retains the mab2A12 signal  and the basal btl/dFGFR expression ( Figure 2B). Similarly, expression of the P-fate markers (upd and P0144-lacZ) in btl/dFGFR mutants remains largely comparable to the wild type ( Figure 2E-F, I-J, M). These results suggest that Btl/ dFGFR signaling does not play a major role in the P-D discrimination. In rho mutants, however, the refinement of upd expression to the primordia periphery is retarded ( Correspondingly, the domain of btl-expressing cells is decreased ( Figure 2C) suggesting that the rho-mediated dEGFR signaling drives the D-fate selection at the expense of the P-fate. We investigated the possible redundant functions of the two RTKs in D-fate definition. In rho bnl/dFGF double mutants, btl expression is further reduced or even lost in some segments ( Figure 2D). Correspondingly, in rho btl/dFGFR double mutants, another D-fate marker mab2A12/Gasp becomes reduced or undetectable in some segments, accompanied by a concomitant increase of the proximal P0144-lacZ expression domain ( Figure 2L, N). Consistent with the phenotypes of rho btl/dFGFR mutants, P0144-lacZ expression is also increased in pnt mutants, ( Figure  vvl drives the D-fate selection at the expense of the P-fate The expression of two key components of RTK signaling, rho in the central part of the airway primordia and btl in the extending distal airways, partly depends on the POU-domain transcription factor vvl (Anderson et al., 1996;Llimargas and Casanova, 1997). We confirmed that in vvl mutants, expression of the D-fate markers rho and btl is reduced ( Figure 3A, B, E, F). Moreover, mAb2A12 staining is almost diminished ( Figure 3N, O) in the residual distal branches, which is restored by UAS-vvl driven with btl-gal4 (Figure 3-figure supplement 1T). By contrast, the expression of the Pfate marker upd is expanded to the distal branches at stage 12 ( Figure 3J-K) and the P0144-lacZ domain is significantly increased at stage 15 in vvl mutants ( Figure 2N, 3O). This indicates that vvl promotes the D-fate at the expense of the P-fate, and a part of this function may be achieved through vvl-dependent expression of rho and btl. In wild type, vvl expression is enriched in the distal trachea, indicating that vvl itself is a D-fate marker (  the D-cells while P0144-lacZ is strongly detected in the P-cells (F, arrowheads). At stage 11 (G), upd transcript is detected at the peripheral area of each primordium (arrowheads). At stage 13 (H, I), upd is expressed in the Pcells that express P0144-lacZ (arrowheads). Note that compared to the control, where upd and P0144-lacZ are not expressed in Tr10 (arrows in F, H), the P-fate (P0144-lacZ) is established in Tr10 of AbdB mutants (asterisks in J). Scale bar is 2 mm in the enlarged panels of E-F. Scale bar in the remaining panels is 50 mm. (K) summarizes typical gene expression patterns of various marker genes in the airway primordium and in the mature airways. See text for details. DOI: 10.7554/eLife.09646.003 The following figure supplement is available for figure 1: and mab2A12 (J) and the P-fate markers upd (F) and P0144-lacZ (J) appears similar. Note, however, that upregulation of btl in a pan-tip pattern is abolished in bnl mutants (B) . In rho mutants (C, G) or Df(H99) rho mutants (K), the expression area of the P-fate markers upd (G) and P0144-lacZ (K, arrowheads) is increased, leading to the decreased area of the D-fate marker expression (C, K). In rho bnl double mutants (D), rho btl double mutants (H) or Df(H99) rho btl triple mutants (L), the P-fate area is further expanded with concomitant reduction of the D-fate markers. Note that in D,L expression of the D-fate markers becomes reduced very much in a few segments (asterisk). Scale bar is 2 mm in the enlarged panels of I-L where the P-fate cells are bracketed. Scale bar in the remaining panels is 50 mm. Source data 1. Source data for Figure 2M. DOI: 10.7554/eLife.09646.006 Source data 2. Source data for Figure 2N. DOI: 10.7554/eLife.09646.007 Figure 2 continued on next page mutant embryos, the expression of the D-fate marker btl is initiated at stage11 but is not maintained by stage 12 (Figure 3-figure supplement 1G,H). Consistently, the mab2A12 signal is undetectable in vvl rho btl mutants ( Figure 3J-L). In contrast, expression of the P-fate markers upd and P0144-lacZ appears to be expressed in all tracheal cells ( Figure 2N, Figure 3-figure supplement 1I, L). These results suggest that the two RTK pathways act both downstream of and in parallel to vvl to drive the D-fate selection at the expense of the P-fate.

Hh and Vvl drive the D-fate selection
Hh is expressed just anteriorly to the airway primordia (Glazer and Shilo, 2001) and is proposed to enhance the primordial vvl expression (Llimargas and Casanova, 1997). We confirmed that in hh mutants, vvl expression is variably decreased or lost (Figure 3-figure supplement 1D). Consistently, expression of the D-fate markers rho and btl is also variably decreased or lost in hh mutants ( Figure 3C,G, in 6 out of 26 embryos for btl expression). In contrast, expression of the P-fate marker upd is expanded and sometimes occupies the whole trachea in some segments of hh mutants ( Figure 3L). A similar effect is observed on the expression of a different set of the P/D fate markers, mab2A12/Gasp and P0144-lacZ ( Figure 3P). These results suggest that Hh signals from the anterior border of the airway primordia to orient the P/D fate selection toward the D-fate direction. Similar to the cooperative activities of rho, btl, and vvl, hh could synergize with vvl to drive the D-fate. Indeed, in the absence of both hh and vvl, although expression of the D-fate markers rho and btl is variably detected in the primordia ( Figure 3D,H), btl expression is completely absent later ( Figure 3I). Correspondingly, the expression of P-fate markers expands to all tracheal cells ( Figure 3M,Q). A similar trend was observed in mutants for hh and RTK signaling components (Figure 3-figure supplement 1M-S). Taken together, we conclude that Hh and Vvl cooperatively drive the D fate selection, partly through the activation of dEGFR and btl/dFGFR signaling in the central part of the primordium and in the distal branches.

Grn promotes the P-fate selection
The identification of several signaling pathways converging in the initiation and establishment of the D-fate in the airways prompted us to interrogate what promotes the P-fate. We revealed the GATAfamily TF Grn (Brown and Castelli-Gair Hombria, 2000;Garces and Thor, 2006;Lin et al., 1995) as the P-fate promoting factor. grn is preferentially expressed in the peripheral parts of the airway primordia from stage 11 on ( Grn overexpression results in the upregulation of the P-fate marker P0144-lacZ in the more distal TC branches ( Figure 4A-C). Conversely, in grn mutants, expression of the P-fate markers upd and P0144-lacZ is lost ( Figure 4D-F, Figure 4-figure supplement 1J), resulting in mAb2A12-positive tubes directly connecting to the epidermis. Counting the Trh-positive cells in Tr5 and in TC5/SB5 reveals that around 10 cells are selectively lost from the TC/VB region ( Figure 4G). As halving of the airway cell number in CycA mutants (Beitel and Krasnow, 2000) does not abolish the P-fate ( Figure 4H, Figure 4-figure supplement 1K), we suggest that reduction of cell number does not account for the loss of the P-fate in grn mutants. Also, arm-gal4-mediated overexpression of Trh in grn mutants does not restore the P-fate ( Figure 4H, Figure 4-figure supplement 1L). These results suggest that grn functions in two ways, namely to establish Trh expression in the P-region and to induce the P-fate marker expression ( Figure 4K). Accordingly, we designate grn a master regulator of the Pfate. , the P-fate marker expression is not robustly restored. In grn vvl double mutants, the trh expression area appears to be very much reduced ( Figure 5F), probably because the P-fate cells depend on grn for trh expression. However, in this reduced area of the distal region, we  We conclude that the regulators of the D-and P-fate cooperatively promote and maintain the airway cell identity defined by trh expression. Below, we first describe roles of the embryo axis determinants on P/D-fate selection and then proceed to dissect the function of D-factors by gain-offunction approaches.

Wg/WNT and Dpp/BMP signaling converge on the P/D-fate selection
A key aspect of the P/D-fate selection in the Drosophila airways is the separation of the trh positive airway primordia into a D-fate circle (vvl, rho and btl positive) surrounded by a concentric P-fate ring (grn and upd positive). To investigate the origins of this rough setup, we examined the effects of Wg/WNT and Dpp/BMP ( Figure 1B), two major axis determinants along the AP or DV axis, respectively (DiNardo et al., 1994;Wharton et al., 1993).  In wg/WNT mutants, expression of the D-fate markers vvl (de Celis et al., 1995), mab2A12 (Llimargas, 2000, and btl expands along the AP axis to cover most, if not all lateral parts of the embryos as a single band ( Figure 6A-B, I, Figure 6-figure supplement 1B). On the other hand, expression of the P-fate markers grn, upd, and P0144-lacZ also expands along the AP axis, but this time, approximately forms two narrow stripes, dorsal and ventral, sandwiching the D-fate ( Figure 6C, I, Figure 6-figure supplement 1A-B). These results suggest that Wg/WNT confines both the D-and P-fates along the AP axis ( Figure 6I).
The dorsal and ventral stripes of the proximal markers surrounding the expanded airways in wg/ WNT mutants suggest that there are cues driving the P/D fate selection along the DV axis. Dpp/ BMP presents a top candidate for such a signal (Isaac and Andrew, 1996;Wilk et al., 1996). In the absence of dpp, trh expression expands to the dorsal midline ( Figure 6-figure supplement 2A, D) (Isaac and Andrew, 1996) and the cells initiate btl expression (Figure 6-figure supplement 2B). However, neither the D-fate nor the P-fate is established (Figure 6-figure supplement 2A-E), suggesting that Dpp also functions as a positive regulator of early airway development, in addition to its repressive effect on the earliest trh expression. In dpp hypomorphs ( Figure 6D-F, Figure 6-figure supplement 2F-J), trh expression is expanded to the dorsal midline and P/D specification is confined near the dorsal midline, depending on the allele severities or the residual Dpp/BMP activity in the hypomorphic conditions. In the dorsally expanded primordia of dpp hr92 homozygotes, expression of the D-factors vvl and btl is detected as stripes straddling the dorsal midline ( Figure 6D-E), and P-fate marker expression typically encircles the entire airway primordium except for its anterior border ( Figure 6F, I, Figure 6-figure supplement 2J). The loss of the P-fate markers at the anterior border in dpp hr92 homozygotes could be due to the activity of the D-fate inducer Hh, which is expressed just anterior to the airway primordia ( Figure 1B) (Glazer and Shilo, 2001). Consistent with the Hh-dependent anterior bias of the D-fate selection, the expression of the dorsal-distal marker h-lacZ, is lost in hh mutant ( Figure 6-figure supplement 3A-B). Similarly, expression of h-lacZ is lost in rho and pnt mutants but not in btl mutants (Figure 6-figure supplement 3A, C, D). On the other hand, overactivation of Hh signaling in wg/WNT mutants expands h expression along the AP axis ( Figure 6-figure supplement 1N-Q). Collectively, this analysis suggests that the graded Dpp/BMP activity controls the P/D fate selection at the dorsal border of the airway primordia. Wg/WNT signals impinge on the P/D-fate selection along the AP axis, while Hh modulates P/D differentiation from the anterior border. Consistent with the predominant roles of Dpp/BMP and Wg/WNT in airway primordia specification, in Df(wg) dpp hr92 double mutants, the whole dorsal area of the trunk/abdominal parts of the embryo takes the airway cell fate ( Figure 6G, I). In this situation, the dorsal part of the airway primordia takes the D-fate and only the ventral periphery takes the P fate ( Figure 6H, I), supporting our model that Dpp/BMP and Wg/WNT are key regulators of the P/D fate selection.
Several other cues including inducers of the airway fate (Brown et al., 2001) are expected to contribute to the P/D fate selection. At the ventral border for example, apart from the dorso-ventral gradient of Dpp/BMP, the ventro-dorsal gradient of dEGFR signaling Golembo et al., 1996;Raz and Shilo, 1993) and another cue originating from the ventro-lateral gradient of the TF Dorsal (Anderson et al., 1985a;Roth et al., 1991;Zhao and Skeath, 2002) may orient the P/D fate selection. The exact mechanisms by which Wg/WNT, Dpp/BMP, and these other cues impinge on P/D fate selection await future research and the development of appropriate cellspecific gene inactivation methods.

Overactivation of the D-fate determinants abrogates the P-fate selection
In order to investigate how the P-and D-fates are balanced during airway cell differentiation, we analyzed the effects of overactivation of the D-fate determinants, namely RTK signaling, hh signaling and vvl. argos (aos) is a secreted negative feedback regulator of dEGFR signaling (Schweitzer et al., 1995a) while anterior open (aop) encodes an ETS TF that antagonizes RTK signaling (Rebay and Rubin, 1995). Upon activation of RTK signaling, aos is induced, while Aop is excluded from the nucleus and becomes degraded. In the airway primordia, aos expression gradually expands from the center to the periphery (Figure 7-figure supplement 1A-E). At early stage 12, both aos and Aop are preferentially detected in the proximal airways (Figure 7-figure supplement 1E-F). In aop mutants, the number of cells expressing P-fate markers is very much reduced ( Figure 7A, Figure 7-  (A,B), and form a single lateral band (bracket) with occasional interruptions before the initiation of primordia invagination (A, stage 10). After invagination (B,C, stage 12), some D-fate cells remain exposed at the embryo surface to form a lateral band (B). The P-fate markers form a dorsal and a lateral band of expression at the ectoderm (C, arrowheads). In dpp hr92 mutants (D-F), the airway primordia expand to the dorsal midline (D, stage 10). Concomitantly, both the D-fate (D,E) and the P-fate (F) expand to the dorsal midline. Note that the P-fate encircles the D-fate (arrowheads) and that the anterior cells often do not express the P-fate marker upd in (F, stage 11/12), probably due to the distalization by Hh that is expressed in the anterior border of each primordium. In dpp hr92 Df(wg) double mutants (G,H), the whole dorsal area of the trunk/abdominal body parts become Trh positive at stage 11 (G). At stage 14/15 (H), the P-fate (arrowheads) is specified abutting the D-fate only at the  Figure 7D) or s-spi ( Figure 7E) with btl-gal4 or trh-gal4 in an aos mutant background. In double mutants of aos and Gap1, a GTPase-activating protein for Ras85D (Gaul et al., 1992), expression of the P-fate markers upd and grn is variably reduced or abolished ( Figure 7G,H). The expression of another P fate marker P0144-lacZ is also lost ( Figure 7F, Figure 7- figure supplement 2A-B). Concomitantly, the distal mAb2A12/Gasp marker staining appears on the embryo outer surface ( Figure 7E-F). These results suggest that aos, Gap1, and aop are parts of a negative regulatory mechanism balancing the D-fate inducing activity of RTK signaling to assure P-fate selection.
The loss of P0144-lacZ expression in aos Gap1 double mutants is partially suppressed by overexpression of the P fate determinant Grn ( Figure 7I, Figure 7-figure supplement 2A). Reversion of the aos Gap1 mutant phenotypes is also detected by introducing vvl, hh, rho, Ras85D (Simon et al., 1991) or pnt mutations but not by btl or by single-minded (sim) mutations (Figure 7-figure supplement 2A-I). sim governs the ventral midline cell fate, which is one source of dEGFR signaling before stage 10 (Golembo et al., 1996;Mayer and Nusslein-Volhard, 1988). Thus, these results illustrate the essential roles of vvl, hh, and RTK signaling components as D-fate determinants and suggest that neither the CNS midline nor btl/dFGFR are involved in the RTK overactivation observed in the aos Gap1 double mutants. Similarly, arm-gal4 mediated, ubiquitous overexpression of secreted active spitz (s-spi) results in loss of the P-fate marker P0144-lacZ (Figure 7-figure supplement 2J-K). This defect is also suppressed by mutations of vvl, pnt, or Ras85D but not by btl, rho, or hh mutations (Figure 7-figure supplement 2J-P). This suggests that vvl has an additional essential role in dEGFR signaling other than facilitating active dEGFR ligand production. In contrast to s-spi, bnl overexpression does not abolish P0144-lacZ expression (Figure 7-figure supplement 1Q). However, simultaneous overexpression of an activated form of btl/dFGFR and its downstream mediator downstream of FGFR (dof) (Imam et al., 1999;Michelson et al., 1998;Vincent et al., 1998) reduced P0144-lacZ expression (Figure 7-figure supplement 1R) suggesting that dEGFR and btl/ dFGFR share the downstream signaling pathway for P/D fate selection.
Overactivation of hh signaling, either by mutation of the inhibitory receptor ptc (Chen and Struhl, 1996;Ingham et al., 1991) or by arm-gal4-mediated hh overexpression, reduces the number of cells expressing P-fate markers ( Figure 7J-K). Although overactivation of Hh signaling is expected to increase the expression of Wg/WNT (DiNardo et al., 1994), a negative regulator of trh expression (Wilk et al., 1996), reduction of the P-fate cell number upon hh overactivation still occurs in wg/ WNT mutant backgrounds ( Figure 7M-N). Moreover, driving a repressor form of Cubitus interruptus (Ci rep ), a mediator TF of Hh signaling (Hepker et al., 1997;Methot and Basler, 2001) at the dorsal ectoderm with salm-gal4 in ptc wg mutants locally increases the P-fate cell number in the dorsal side Figure 6 continued ventral edge. Note that in the posterior part, the P-fate is not established as it is not established in the wild type. Scale bar: 50 mm. (I) Interpretation of data in each genotype regarding specification of the airway field (left parts) and P/D-fate selection (right parts). Expression domains of dpp and wg are colored pink and orange, respectively. In wg mutants, the airway field (light blue) expands along the AP axis (arrows) and the P-fate is established approximately as a dorsal and a ventral stripes (light green). In dpp hypomorphs (dpp hr92 ), the airway field expands dorsally to the dorsal midline and the P-fate is established in the periphery except for the anterior and dorsal margin. In double mutants of wg and dpp hypomorph, the airway field expands both dorsally and along the AP axis, and the P-fate is only established in a ventral stripe. DOI: 10.7554/eLife.09646.018 The following figure supplements are available for figure 6:  Figure 7N). These results imply that overactivation of hh signaling autonomously represses the Pfate selection independent of its effect on wg/WNT.
Overexpression of vvl with arm-gal4, like s-spi overexpression reduces or abolishes the expression of the P-fate markers, upd or P0144-lacZ ( Figure 7O-P), and this occurs independently of the presence of wg/WNT (Figure 7-figure supplement 3A-C). Collectively, the analysis indicates that expansion of the D-fate-inducing activities of RTKs, Hh or Vvl is deleterious to the P-fate selection and that the negative regulators of RTK signaling (aos, aop or Gap1) and hh signaling (ptc) ensure the selection or maintenance of the P-fate.

Differential competence of the primary branches and the TC
The hitherto analysis indicates that aos sensitizes the circuit of P/D-fate selection. We noticed that crossing of a weaker driving strain of UAS-Ras V12 with arm-gal4 causes loss of P0144-lacZ expression only in the aos mutant background while the stronger UAS-Ras V12 strain is sufficient to eliminate P0144-lacZ expression on its own (Figure 8-figure supplement 1A-D). Using the weaker UAS-Ras V12 strain, we demonstrate that within the D-fate group, the cells of the TC and the remaining primary branches have differential competence (Figure 8-figure supplement 1E). In wild-type embryos, Kni and Knrl are induced in a subset of the distal primary branches (DB, LT and GB) in response to Dpp/BMP Vincent et al., 1997). arm-gal4-mediated overexpression of Dpp/BMP or the active form of its co-receptor tkv QD (Nellen et al., 1996) is sufficient to induce ectopic Kni in additional branches ( Figure 8A,B) . However, Kni levels in the TC cells is weaker compared to the more distal cells, suggesting that TC cells are less competent in inducing the D-fate marker Kni in response to Dpp/BMP signaling. When both UAS-dpp and the weaker line of UAS-Ras V12 are simultaneously driven by arm-gal4, kni expression becomes homogenously induced in both the TC cells and the more distal cells ( Figure 8C). This suggests that graded activity of the D-fate inducers, like RTKs may act to generate three different cell states along the PD axis of the airways. The most distal part (the primary branches) is established in response to the highest activity, the intermediate domain (TC) requires weaker activity, whereas the level of the D-factors needs to be low in the most proximal part (Figure 8-figure supplement 1E). Slight expansion of the P-fate only in the TC region upon Grn overexpression ( Figure 4A-C) may also reflect this differential competence of TC and the remaining primary branches.
The early development of the airway primordia includes two essential aspects: first, the selection and maintenance of the airway field, and second, the selection of the P/D-fates, both of which originate from the AP and DV axis determinants of the embryo (Figure 9). More specifically, we identified a positive role of Dpp/BMP and the P/D fate factors in the establishment of the airway field. In the absence of Dpp/BMP or both P/D fate regulators, the airway field is lost. The latter phenotype could be explained in the following way in terms of P/D fate selection. Without the D-factors, the airway field is expected to uniformly take the P-fate that is promoted by grn. In this situation, grn becomes indispensable for maintenance of trh expression in the main airways. However, Wg/WNT, Hh, Dpp/BMP and other cues generate a centrally enriched expression field of the D-fate determinants vvl, rho, and btl, which then cooperate to repress the P fate, leaving the grn-trh regulation operative only in the P-region. Restoration of the P-fate in grn vvl double mutants suggests that the proximal multipotent fate is the default state for airway cells or that there is another factor that promotes the P-fate in the absence of grn and vvl.
Intriguingly, the Drosophila legs and the trachea are supposed to have evolved form common ancestor appendages (Franch-Marro et al., 2006). The proximo-distal patterning mechanisms of these two organs are similar in that RTK activation distalizes the field (Campbell, 2002;Galindo et al., 2002) while initiation of the distal identity is conferred by the same set of signaling molecules (Dpp/BMP, Wg/WNT, and Hh) from the outside (for the airways) or the inside (for the legs) (Estella et al., 2012). The proximal location of multipotent cells in the Drosophila airway tree resembles the conspicuous proximal localization of multipotent tracheal basal cells in the mouse lung (Rock et al., 2009;Rock et al., 2010) Given the prominent roles of FGFR signaling in branching morphogenesis in both flies  and mice (Min et al., 1998;Sekine et al., 1999), the identification of a genetic mechanism for P-and D-fate selection in flies may aid future studies aiming to identify the mechanisms that spatially confine multipotent cell selection in the embryonic vertebrate lung.   (Sanson, 2001), an airway primordium is specified just posterior to the hh expression domain (a posterior compartment of a segment, P). This is combinatorially controlled along the DV and AP axis. Dpp/BMP expressed in the dorsal region functions as both a repressor (Isaac and Andrew, 1996;Wilk et al., 1996) and an activator of the airway field (this study) while Wg/WNT functions as a repressor along the AP axis. (B) Initiation of the P/D-fate selection by Dpp/BMP, Wg/WNT, and Hh. Each airway primordium is roughly subdivided into two regions, anterior-central (D-fate, dark green) and the peripheral (P-fate, light green). The patterning cues along the AP and DV embryo axis roughly set up this radial patterning. Along the DV axis, Dpp/BMP expressed in the dorsal region functions to discriminate the P/D-fates, at least at the dorsal edge. Dpp/BMP may also discriminate the P/ D-fates at the ventral edge. Alternatively, ventral cues dependent on the Dorsal TF gradient may discriminate the P/D-fates at the ventral edge. Along the AP axis, Wg/WNT expressed in transverse stripes may discriminate the P/ D-fates at each edge. Hh from the anterior border stimulates the D-fate. (C) Establishing the P/D-fates by the P/D- Figure 9 continued on next page Figure 9 continued factors. The ordered Invagination of the primordia converts the centro-peripheral patterning to the proximo-distal organization along the airway tree. By completion of invagination, D-factors establish the D-fate at the expense of the P-fate. Within the D-fate, there are two groups of cells having different responsiveness to overexpression of Dpp/BMP or Grn, e.g., TC (green) and the primary branches (dark green). See text for details. AP, anteriorposterior; DV, dorso-ventral. DOI: 10.7554/eLife.09646.031