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other defects. In some instances, the position of the terminal cell within the specimen did not allow analysis,
and these cells were excluded from counts.

Microscopy. Larvae were imaged using direct fluorescence and Brightfield optics using a Leica DM5500 B
upright widefield epifluorescence microscope (Leica Microsystems). Images were acquired using a Leica
DFC360FX camera. Z-stacks were captured and processed by deconvolution using Leica Advanced
Fluorescence Application Suite (Leica Microsystems). For wing hairs, wings mounted in Euparal were
examined with brightfield optics using a Leica DM6000 inverted microscope, and images were captured
using a Hammamatsu Orca-R2 Digital CCD camera (C10600, Hamamatsu Photonics).

RESULTS
We previously identified a requirement for the CCM3-GCKIII signaling pathway in the Drosophila tracheal
system . Importantly, the CCM3-GCKIII tube morphogenesis program shares common components with
[20]
[2]
an orthologous pathway in the human vascular system (reviewed in Riolo et al. ), which is partially
conserved from yeast [Figure 1]. Prior to our work, Tricornered (Trc), the most downstream kinase in the
cascade, was best known for its role in the morphogenesis of other tissues [28,31,41] . We decided to test whether
regulation of Trc by CCM3-GCKIII was tissue-specific, or instead might be a general feature of Trc
regulation. To do so, we turned to the Drosophila wing, a system widely used for studying developmental
signaling pathways and planar cell polarity [42-47] .

CCM3-GCKIII pathway regulates wing hair morphogenesis
In flies, wing epithelia possess planar polarity that is easily read out in each cell by the position and
orientation of actin-based cellular protrusions called wing hairs. In wild type wing epithelial cells, a single
hair that tapers to a point extends from the posterior vertex of the cell, points distally, and is aligned with
the hairs of neighboring cells [Figure 2A]. In tricornered and furry mutant cells, the organization of the
actin-based hairs appears to be disrupted such that the hairs split, giving rise to multiple hairs [Figure 2B
and E] and/or hairs with split ends [24,28] . Although prior studies suggest no role for Mo25 in wing hair
[30]
morphogenesis , we decided to determine if disruption of ccm3 and GckIII activity would give rise to wing
hair defects characteristic of trc-fry [26,27] , and we examined wings expressing a dominant negative GCKIII
isoform (Figure 2C and F, Gck T167A [21] ), as well as ccm3 mosaic wings [Figure 2G]. In both cases, we found
that the affected wing cells developed multiple wing hairs with disruption of hair orientation.

Furry is required in tracheal terminal cell tube morphogenesis
Furry has been identified genetically and biochemically as a partner for Trc [24,26,27] , and has been shown to
function together with Trc in the shaping of actin-based cellular projections (wing hairs, etc.) in epithelial
[23]
cells, as well as the polarized deposition of basement membrane . In neurons, Fry has been shown to
function with Trc in the morphogenesis of dendrites . A role for Fry in tubulogenesis has not been
[31]
examined, although fryl mutants in mice have been reported to have kidney defects attributed to a role in
the regulation of a microRNA [35,36] . Because Furry appears to be required in all other contexts where Trc
function has been described as necessary for morphogenesis, we tested if furry was also required in the
tracheal system. For this analysis we characterized the tracheal terminal cell phenotype of 5 independent
mutant alleles of furry, derived from 3 different genetic screens [25,34,39] .

As has been the case for other genes in the pathway (tao, ccm3, GckIII [20,21] ; Figure 3A and B), and is also true
for Mo25 [Figure 3C], zygotic loss of fry appeared to uniquely disrupt tube morphogenesis in terminal cells,
but not other tracheal cell subtypes. In homozygous fry terminal cells [Figure 3D], we observed a range of
phenotypes, running from cells with tubes indistinguishable from wild type, to cells with multiple transition