The dVHL loss-of-function phenotype results in abnormal tracheal structure during embryonic development. (A to J) Stage 15 and 16 embryos were stained with a tracheal lumen-specific antibody 2A12 and anti-GFP antibody. (A) y w stage 16 embryo. Six primary tracheal tubes are marked. DB, dorsal branch; DT, dorsal trunk; GB, ganglionic branch; TC, transverse connector; VB, visceral branch; VT, ventral trunk. (B) dVHL¹ heterozygote. The bracket shows the partially overlapped image of YFP expression from two sides of the embryo. The arrowhead points to a section of the DT that does not connect with the neighboring branch but turns dorsally. The arrow points to an abnormal branching point in the dorsal trunk. (C and D) Two dVHL¹ homozygotes. The sharp arrow in panel C points to isolated fragments of DT. The filled arrows in panels C and D point to ectopic branches, and the empty arrowheads point to convoluted DT. (E) An awd^j2A4 homozygous embryo exhibiting tracheal phenotypes similar to those in panels B to D. The arrowhead points to a section of the DT that turns 90°. The arrow points to an ectopic branch. (F and G) Two independent transgenic lines expressing dVHL duplex (dVHLi) directed by the btl promoter. The arrow in panel F points to an upturned DT tip sprouting abnormal branches (enlarged view). The arrows in panel G point to ectopic branches. (H and I) Two independent transgenic lines expressing full-length dVHL sequences directed from the btl promoter. Empty arrows point to gaps in the DT. These gaps are not accompanied by ectopic branching. (J) Homozygous btl^H82Δ3 embryo showing the typical lack-of-migration phenotype: a gap in the DT without ectopic branching. (K) Quantification of phenotypes. “Moderate” phenotype denotes up to two defects (breaks in the trunk, abnormal branches, etc.) found in the tracheal tree. “Severe” denotes more than two identified defects, including embryos showing severe destruction of the tracheal tree. Bars, 50 μm.

dVHL mutant tracheal cells exhibit ectopic migration phenotypes. Mutant strains were combined with the 1-eve-1-transgenic marker that expresses β-Gal in the trachea (from the trachealess promoter). Stage 15 and 16 embryos were stained with anti-β-Gal and anti-GFP antibodies. (A) Stage 16 1-eve-1 control embryo. (A') Two tracheal subunits from a stage 14 1-eve-1 embryo. Arrows indicate the directional movements of the branches that form the dorsal trunk. (B and C) dVHL¹ heterozygotes. Brackets mark the YFP expressed in the head region from the balancer chromosome. The arrowhead in panel B points to a section of the DT turning 90° ventrally. The further enlarged area shows an isolated cell sprouting multiple filopodia. Arrows in panel C point to ectopic branches. (D and E) Two dVHL¹ homozygous embryos showing severely disrupted tracheal trees with rounded, isolated tracheal cells. Filled arrows in panel D point to ectopic branches. Empty arrowheads in panel E point to abnormal sprouts of filopodia. Sharp arrows point to isolated, rounded cells. The two enlarged views in panel E are different focal planes of the same area. (F) An awd^j2A4 homozygote showing a severely disrupted tracheal tree. The empty arrowhead points to abnormal sprouts of filopodia. The sharp arrow points to rounded cells. (G) A shi² temperature-sensitive mutant grown at a restrictive temperature showing abnormal sprouts (empty arrowheads) and isolated cells (sharp arrows). (H) btl^H82Δ3 homozygote showing the lack-of-migration phenotype with gaps (empty arrows) in the DT. (I) pnt^Δ88 homozygote showing the lack-of-migration phenotype with gaps (empty arrows) in the DT. (J) dVHL¹/+; awd^j2A4/+ transheterozygote showing a severely disrupted tracheal tree with ectopic filopodia (empty arrowheads) and isolated, rounded cells (sharp arrows). Bars, 50 μm.

Surface accumulation of Btl in dVHL mutant cells. (A and B) 1-eve-1 and dVHL¹ mutant late-stage-12 embryos carrying the btl-Gal4; UAS-btl::GFP transgenes were double stained for β-Gal and GFP. (A) The 1-eve-1 embryo shows modest expression of Btl::GFP on the cell surface. (B) The dVHL¹ mutant embryo shows extensive accumulation of Btl::GFP at or near the cell periphery in all tracheal cells. (A') Stage 11 y w embryo expressing GFP (not btl fusion) from the btl promoter. The GFP itself shows strong, diffused expression throughout the cell bodies. (C) 1-eve-1 and dVHL¹ mutant stage 13 embryos carrying the btl-Gal4; UAS-dEGFR::GFP transgenes were double stained for GFP. There is no difference in the expression levels or patterns of dEGFR::GFP. (D) S2 cells are transfected with metallothionein>btl::GFP plasmids and hsp70 vectors expressing randomized small interfering RNA (control), dVHL duplex (dVHLi), or full-length dVHL and processed for staining with anti-GFP and anti-dVHL antibodies. In control cells, the Btl::GFP fusion protein is localized mostly in vesicles also containing dVHL (D'). With dVHL knockdown (dVHLi), Btl::GFP is highly enriched at the cell periphery (arrows). Reduction of surface Btl::GFP expression is observed with dVHL overexpression. (E) Surface biotinylation assay showing surface fractions and total lysates of Btl::GFP. Total cell lysates (left panel) were Western blotted with the indicated antibodies. Surface fractions were probed with anti-GFP. Numbers under the protein bands are the intensities of the signals normalized against the actin control. Red bars, 20 μm; white bars, 5 μm.

Lumen phenotypes in the dVHL mutant. (A to I) Late-stage embryos or first-instar larvae were stained with the 2A12 and GFP antibodies. (A) dVHL¹ heterozygote showing a convoluted tracheal trunk (arrow) and ectopic, tortuous secondary branches (arrowheads). (B) A dVHL¹ homozygote showing a dilated dorsal trunk (arrow). (C and D) Two dVHL¹ homozygotes showing a dilated and convoluted dorsal trunk (arrow) and ectopic branches (arrowheads). (E) Embryo expressing dVHL duplex (dVHLi) from the btl promoter showing a convoluted dorsal trunk (arrow) and tortuous branches (arrowhead). (F and G) shi² and awd^j2A4 mutants showing the same lumen phenotypes as in the dVHL¹ mutant, including a dilated dorsal trunk (arrow) and ectopic, tortuous branches (arrowheads). (H) y w 1st-instar larva showing little 2A12 staining in the trachea. (I) dVHL¹ homozygous 1st-instar larva showing strong residual 2A12 staining (arrow). (J and K) 1-eve-1 and dVHL¹; 1-eve-1 embryos were stained with anti-β-Gal antibody and FITC-labeled chitin-binding probe (CBP). At stage 14 (J), both 1-eve-1 and dVHL¹; 1-eve-1 embryos show proper deposition of chitin in the lumen. At stage 15 (K), lumen chitin begins to fade in the 1-eve-1 embryo but remains abundant in the dVHL¹ mutant. In addition, unlike the wild-type lumen chitin, the mutant lumen chitin is filled up against the apical surface of the tracheal cells (compare insets). The arrowhead in the panel K inset points to the small gap between the chitin material and the tracheal cell apical surface in the wild type. (L) A stage 15 embryo from transgenic flies carrying btl-Gal4; UAS-dVHLi was stained with anti-β-Gal antibody and rhodamine-CBP. The tracheal lumen shows abundant chitin accumulation similar to that found in the dVHL¹ mutant. (M to O) Stage 15 embryos from transgenic flies carrying btl-Gal4; UAS-control (as a negative control) or btl-Gal4; UAS-dVHLi were combined with transgenic flies carrying the wild type (Rab5^wt) or the constitutively active (Rab5^CA) or dominant negative (Rab5^DN) variants of UAS-YFP::Rab5. These were stained with anti-GFP antibody and rhodamine-CBP. Rab5^wt cannot rescue (M) but Rab5^CA can reverse (N) the dVHL knockdown phenotypes (increased lumen chitin and a tortuous dorsal trunk). (O) Rab5^DN alone causes delayed chitin clearing and a convoluted dorsal trunk, resembling that of the dVHL¹ mutant. The phenotype is exacerbated when combined with the dVHL duplex. Bars, 50 μm.

Mouse conditional Vhl mutants exhibit dilated kidney tubules and ectopic migration phenotypes. Kidneys from 4-month-old Vhl^loxP/loxP (wild-type) or Hoxb7-Cre/+; Vhl^loxP/loxP (Vhl^−/−) mice were dissected. The kidneys were fixed and sectioned for hematoxylin-eosin staining (A to C) or were processed for tubule cell isolation (D to F). (A) The wild-type kidney shows normal tubules (examples are outlined with dashed lines). G, examples of glomeruli. (B and C) Kidneys from two knockout animals, showing convoluted and dilated tubules. (D) Left panels show cultured primary kidney tubule cells immunostained for VHL and FGFR1. The mutant shows little VHL protein expression and accumulation of FGFR1 on the cell surface at the migrating front (arrows). Bars, 50 μm. The right panel shows quantification of surface FGFR1 versus total cellular FGFR1 levels using the ImageJ photometric analysis program. (E) Lysates from isolated kidney tubule cells were Western blotted for VHL and FGFR signaling mediators: dually phosphorylated ERK (dp-ERK), total ERK, phosphorylated Ets1 (p-Ets1), total Ets1, and actin control. (F) Primary tubule cells were cultured in a 3-dimensional matrix (2% Matrigel) and stained for F-actin (phalloidin), E-cadherin, and DNA (To-Pro3). (a) Wild-type cells form an organized acinus with a defined lumen in the center. (b) Vhl mutant cells form an irregular cluster with outgrowth and a dilated lumen. (c) Another mutant example shows cells migrating away from the irregular cluster with prominent actin fibers in the migrating tips (insets). Bars, 20 μm.

Characterization of the dVHL¹ allele. (A) The left panel shows a schematic representation of the genomic region of the dVHL open reading frame. The two in-frame AUG codons are marked. Arrowheads depict the PCR primers used to distinguish between the wild-type and the deletion mutant fragments. The expected fragment lengths are indicated. The distinction between heterozygous dVHL¹ (dVHL¹/CyO, Dfd-YFP), homozygous dVHL¹, and homozygous balancer (CyO, Dfd-YFP/CyO, Dfd-YFP) is shown in the middle panels. Homozygous balancer (Cy/Cy, containing two copies of Dfd-YFP) expresses an expanded YFP pattern in the head region (thick bracket) compared to that expressed by heterozygotes (dVHL/Cy), which contain only one copy of Dfd-YFP (thin bracket). The visual identification is confirmed by the genotyping (right panel) of hand-sorted embryos. (B) Progeny counts from dVHL¹/CyO; hsp-dVHL parents. The adult progenies were counted based on gender and wing phenotypes. The exogenously expressed dVHL can fully rescue the dVHL¹ lethal phenotype, since the genotypes of the progenies follow the expected Mendelian ratio. (Note: CyO/CyO is lethal at the late embryonic/early larval stages.) No external morphological phenotypes were observed in heterozygous (Cy⁻) or homozygous (Cy⁺) dVHL¹ (right panel). (C) Embryos of the indicated genotypes and stages were stained with rabbit anti-dVHL and mouse anti-GFP antibodies. In y w (representing wild-type) embryos, dVHL protein is ubiquitous but enriched after stage 10 in the tracheal cells (arrowheads in insets). dVHL¹ heterozygous embryos (dVHL¹/CyO, Dfd-YFP) are identifiable by modest expression of YFP in the head region (arrow). Homozygous dVHL¹ embryos exhibit a very low level of dVHL staining. Bars, 50 μm.

dVHL¹ germ line mutants show complete failure in forming trachea. y w embryos and embryos from females containing germ line clones of dVHL¹ ({ovoD1-18}, FRT^G13/dVHL¹, FRT^G13; ovo-FLP/+) crossed with heterozygous dVHL¹ males were stained for Crumbs (Crb) (A and B) or Trachealess (Trh) (C and D). y w embryos show Crb staining on the apical surface of the tracheal tubes facing the lumen (A) and Trh in the nuclei (C). (B) In dVHL¹ germ line mutants, scattered Crb-positive cells are detected, but no tracheal tubes are formed. (D) The identity of the scattered cells is further confirmed by staining for Trh, the tracheal cell-specific transcription factor. Bars, 50 μm.

Elevated Btl signaling in the dVHL mutant. 1-eve-1 (A and C) and dVHL¹ mutant (B and D) embryos were stained for β-Gal and dually phosphorylated MAPK (dp-MAPK; A and B) or Pnt (C and D). In 1-eve-1 embryos, dp-MAPK and Pnt are modestly expressed and localized to the migrating tip cells. In dVHL¹ mutant embryos, dp-MAPK and Pnt expression levels are elevated. They are also ectopically expressed in multiple cells. (E) Quantification of phenotypes showing genetic interactions between dVHL¹ and endocytic pathway components and btl signaling components. Phenotype designations are as described in the legend to Fig. 2K. The pairwise statistical analysis is shown on the left, using a two-sample t test for percentages. *, significant changes (P < 0.05). Bars, 20 μm.

dVHL forms a complex with Awd in vivo. (A) Embryos carrying btl-Gal4; UAS-dVHL::HA were collected at 29°C, and extracts were prepared in RIPA buffer. Extracts (50 μg) were incubated with rabbit polyclonal anti-Awd, rabbit polyclonal anti-HA, or rabbit IgG, as indicated. Immunoprecipitation was performed using protein A-agarose beads. The pulldown fraction was then Western blotted using rabbit anti-dVHL antibody, chicken anti-human Nm23 (which cross-reacts with Awd), or mouse antiactin antibodies. (B) y w embryos or y w embryos containing one copy or two copies of hsp-dVHLi were left untreated or were heat treated twice a day for 30 min each at 37°C for 3 days. Embryonic extracts were Western blotted for the indicated proteins. dVHL levels are knocked down by dVHLi in a dosage-dependent manner, and Awd levels are reduced in step with the reduced dVHL levels. (C) 1-eve-1 or dVHL¹ embryos were processed for immunofluorescence detection of the proteins indicated. The Awd level is diminished in the dVHL mutant. Bars, 50 μm.

VHL disease-related mutations cannot rescue the tracheal phenotypes. (A) There is no rescue of the dVHL phenotypes by either of the dVHL mutations in this assay. (B) Examples of the expressed dVHL transgenes. There is no difference in the cellular expression patterns of the dVHL variants. (C) The Sima::GFP fusion protein was expressed in the btl-directed system, with or without dVHLi-mediated knockdown. Sima::GFP is detected strongly in the posterior portion of the dorsal trunk (green dots). There is no increase in the expression areas or levels of the fusion protein in dVHL knockdown embryos. Bars, 50 μm.