The distalizing factors hh and vvl dominantly promote invagination of the airway progenitors.

(A) A schema showing that invagination transforms the centro-peripheral patterning of the primordium into the proximo-distal patterning of the tubes. Extrinsic Hh and intrinsic Vvl dominantly promote the distal gene expression program (dark green domain) whereas rho and btl/FGFR are the distal factors that are known to distalize differentiation along both gene expression and morphology. The proximal factors including grn promote the proximal gene expression program (light green domain). (B-C) Single sections of lateral views of stage 14 embryos stained with Trh (a pan airway marker) and P0144-LacZ (a proximal marker). In the control (B), both the distal as well as the proximal airway progenitors (yellow arrowheads) invaginate to form a tube network whereas in H99 hh vvl mutants (H99 hh13C vvlutH599 P0144 homozygotes) (C), Trh+ cells express the proximal marker P0144-LacZ and many of them stay at the epidermal layer (yellow arrowheads). Scale bar is 50 μm

Expression of rho and trh in hh vvl double mutants, hhAC vvlutH599/hh13C Df(vvl) transheterozygotes.

(A) Weak and sporadic expression of rho is detected at early stage 11 in hh vvl double mutants (yellow arrowheads). (B) The airway progenitors frequently fail invagination in hh vvl double mutants as well and those cells retain trh expression at stage 12 (yellow arrowheads). Scale bar is 50 μm

Medium Dpp/BMP activity promotes trh expression and the airway progenitors.

(A) A schema showing that the embryonic trunk is divided into discrete domains along the D-V axis. Out of the 5 domains (amnioserosa, dorsal ectoderm, ventral ectoderm, mesectoderm and mesoderm), mesoderm invaginates during gastrulation. Graded activities of a TF dorsal orchestrates the ventral domains whereas Dpp/BMP signaling orchestrates the dorsal domains. Differential expression of TFs subdivides both the dorsal and the ventral ectoderm into medial, intermediate and lateral columns. EGFR is dynamically activated to establish the ventro-intermediate column (Yagi et al. 1998). Expression of TFs marks the column of dorso-medial (pannier), dorso-intermediate (araucan/ara, caupolican/caup), ventrol-lateral (Dr/msh), ventro-intermediate (Dicheaete/D, intermediate neuroblasts defective/ind), ventro-medial (D, ventral nervous system defective/vnd). (B-E) Distribution of trh and vvl transcripts relative to the ectodermal subdivision along the D-V axis. (B-C) Ventral and lateral views stage 10 embryos. The ventral limit of trh expression abuts the dorsal limit of D-LacZ expression (small yellow arrows) which marks the ventro-medial and the ventro-intermediate columns. Large arrows show trh expression in the salivary gland primordia. (D-E) vvl expression at stage 10 straddles the border between the dorsal ectoderm and the Dr/msh-LacZ positive ventro-lateral ectoderm (D, yellow arrows) whereas the ventral limit of vvl expression is some cells away from the ventral limit of Dr/msh-LacZ expression (D, yellow arrowheads). An enhancer fragment of Dr/msh active in the ventro-lateral column marks the ventral parts of the airway tubes at stage 11 (E, yellow arrows). (F-I) Expression of trh-LacZ and trh transcripts in allelic series of dpp mutants. Dorsal views of embryos where the presumptive dorsal midline are marked with blue arrows. In dpp null mutants (F, G), trh expression detected with trh-LacZ or trh RNA expands to the dorsal midline (blue arrows). However at later stages, trh RNA is not maintained though trh-LacZ positive cells remain (G). Note that in dpp null mutants the body is twisted. In the milder condition (H), trh RNA is sporadically maintained near the dorsal midline whereas in dpp hypomorph homozygotes (I), trh RNA is detected in many of the expanded progenitor areas. Scale bar is 50 μm

The proximal airways and the ectodermal subdivision.

(A-B) Lateral views of stage 13 embryos. The spiracular branch cells (trh-LacZ, yellow arrowheads) are several cells ventrally to the caup RNA expressing cells (A). Grn-GFP expresses not only in the spiracular branches but also in the dorso-lateral ectodermal cells that reside ventrally to the iroquois-LacZ (iro-LacZ)/araucan-LacZ) positive dorso-intermediate column (B, yellow arrows). (C-D) Lateral views of stage 11 embryos. trh-LacZ positive cells that lose trh RNA are pronounced in the ventral parts of the control (C). This becomes more pronounced in the dpp hypomorhps (D). Yellow arrowheads mark the ventral limits of trh-LacZ expression. A, C and D are single sections. Scale bar is 50 μm

Maintenance of trh expression does not follow changes in tissue architecture Lateral views of embryos stained with trh-LacZ and trh transcripts.

(A-D) In H99 btl/FGFR mutants (H99 btlΔoh10 /H99 btlΔoh24-1) (A), trh expression is detected both in the distal and the proximal regions whereas in H99 EGFR mutants (topf2 /topf24; H99) (B), trh expression in the proximal regions is significantly reduced (yellow arrowheads). In H99 EGFR btl/FGFR mutants (topf2 /topf24; H99 btlΔoh10/H99 btlΔoh24-1) (C), residual trh expression is detected in parts of the invaginated cells (yellow arrows). Asterisks mark trh expression in the posterior spiracle primordia. A schema in D shows stages and functions of RTK activation in the airway progenitors. (E-L) In the absence of Wg/WNT (E-G), which is expressed in stripes along the A-P axis (H), the airway progenitor areas expand along the A-P axis. trh-LacZ and trh RNA are largely co-expressed since before invagination. Arrowheads in G show that trh is expressed in cells that take the 2D planar configuration. In EGFR wg double mutants (topf24 wgCX4 homozygotes) (I, J), maintenance of trh RNA becomes defective at around the stage of invagination (compare I and J). trh maintenance is restored not by suppression of apoptosis (topf24 wgCX4 ;H99) (K) but by daughterless (da)-Gal4 driven overexpression of RasV12 (topf24 wgCX4 ;da-Gal4/UAS-RasV12) (L). Note that trh RNA is not detected in cells positive for trh-LacZ in J and K (yellow arrowheads). Scale bar is 50 μm

trh expression in aos Gap1 double mutants do not respect tissue architecture.

(A-B) Lateral views of aos Gap1 mutant embryo stained with trh-LacZ and trh transcripts. A projection (A) or a single section (B). trh expression is often detected in planes at the embryo surface (yellow arrows). Scale bar is 50 μm

RTKs require the Ras-Raf/MAPKKK-Dsor1/MAPKK pathway for maintenance of trh expression.

(A) A schema showing that RTK activation involves several downstream signaling branches including the Ras-MAPK pathway and the PI3kinase-PKB pathway. PKB is known to phosphorylate Trh and to upregulate Trh transcriptional activity (black arrow). MAPK may act the same (gray arrow). (B-D) Lateral section views of embryos stained with trh-LacZ and trh transcripts. In the complete absence of Draf/MAPKKK (B) or Dsor1/MAPKK (C), trh RNA is hardly maintained in the cells positive for trh-LacZ, either invaginated or non-invaginated. In the complete absence of Drosophila Ras1/Ras85D, introducing H99 deficiency suppressing apoptosis allows visualization of airway development till later stages (D), demonstrating residual trh expression in parts of the invaginated cells. Yellow arrows in C show trh expression in the salivary gland. Yellow asterisks in D mark trh expression in the posterior spiracle. Scale bar is 50 μm

RasV12 boosts Trh activity toward downstream genes.

(A-F) Lateral views of embryos stained as indicated. Compared to the control (first column), single overexpression of RasV12 (second column) or TrhWT (third column), simultaneous overexpression of RasV12 and TrhWT (fourth column) or single overexpression of TrhS665D (fifth column) induces ectopic expression of btl (A), upd (B), trh-LacZ (C), mab2A12 (Gasp) (D, F) or P0144-LacZ (E, F), which are marked by yellow arrowhead or yellow blankets. Asterisks in A3 marks ectopic progenitors in the anterior and the posterior segments. Scale bar is 50 μm.

A model for specification and priming of the airway progenitors along the three body axes Inputs along the A-P axis and the D-V axis generate radial patterning of the airway progenitors to realize the proximo-distal differences of gene expression and morphology.

(A) A-P axis; Segments and para-segments (PS) are units of Drosophila ectoderm segmentation along the A-P axis. One PS is composed of the posterior of a segment and the anterior of its posterior neighboring segment. Wg/WNT (coloured pink) represses trh and vvl whereas Upd ligands signal through Domeless-JAK-STAT (coloured blue) to induce them, which together generate a graded airway progenitor field along the A-P axis (coloured gray). The upd expression domain is a guess. upd expression is dynamic (Harrison et al. 1998; Sotillos et al. 2010) and it is not known which upd expression is required for inducing the airway primordia. trh expression initiates already at stage 8 (Isaac and Andrew 1996) whereas vvl expression starts at stage 10. Hh (coloured green) positively regulates expression of vvl, thereby distalizes the progenitor field. (B) D-V axis; The ventrally active TF Dorsal (coloured yellow) restricts dpp/BMP expression to the dorsal domain. Dpp/BMP (coloured blue) in turn orchestrates the dorsal parts of the embryos. Midium Dpp/BMP activities promote expression of trh, vvl and grn to generate a graded airway progenitor field along the D-V axis. (C) P-D axis; The radial patterning is realized by Rho mediated activation of EGFR. EGFR activation primes the airway progenitors to commit to the airway differentiation program. At the same time, EGFR distalizes the airway differentiation along both gene expression and morphology to establish the P-D axis of the invaginated tubes. Spreading of rho expression and promotion of trh maintenance may involve EGFR mediated activation of TFs like Trh and Vvl, which boosts their transcriptional activities toward downstream genes including rho and trh. (D) A model of tissue-architecture-independent regulation of trh expression. trh expressing cells (brown coloured nucleus) are composed of the epidermal progenitors (pale brown) and the airway progenitors (brown), the latter of which is further classified into vvl expressing central/distal cells (green cytoplasm) and grn expressing peripheral/proximal cells. Each progenitor type is specified on the 2D cell fields based on the cues along the A-P and the D-V axes (left). Radial EGFR signaling primes the airway progenitors (center, dark brown), to realize morphogenetic and transcriptional differentiation along the P-D axis (right). trh expression in the epidermal progenitors ceases based on the epidermal differentiation programs. Airway progenitor invagination may be aided by segregation of the 2 different cell types whereas invagination processes may finetune the cell type determination processes. Note that grn expression in the epidermal progenitors is omitted.