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Antwi-Adjei et al. Vessel Plus 2021;5:35 https://dx.doi.org/10.20517/2574-1209.2021.48 Page 3 of 10

[23]
(FRY and FRYL) have been implicated in several processes , including tubulogenesis in the kidney [35,36] , but
have not yet been linked to vascular biology.

METHODS
Wings. Young adult flies (newly eclosed up to 1 day old) were anesthetized on CO pads and then
2
transferred into 100% ethanol in glass dissection dishes. Using microdissection spring scissors and Dumont
forceps (Fine Science Tools, Foster, CA, USA), the wings were clipped off at their attachment site. Wings
were transferred to a drop of Euparal (BioQuip, 6372A), and a coverslip was applied. Weights were placed
on the coverslip to flatten the specimens, and slides were allowed to dry at room temperature for 1 day or
more before imaging. Images were captured at the wing margin and just distal to the cross-vein connecting
122
wing vein 3 and 4. For ccm3 mosaic wings, virgin FRT ccm3/TM6B flies were crossed with y, w, hsFLP ;
82B
FRT males, and the progeny (embryos and larvae) were subjected to heat shocks (38.5 °C) for 1-2 h
82B
starting at 6 h after egg lay (a.e.l.). Heat shocks were repeated every day through the end of the 3rd larval
instar. Heat shock induction of Flipase (FLP), a site-specific recombinase, resulted in mitotic recombination
between the homologous chromosomes at a centromere proximal FLP Recombinase Target (FRT) site
(FRT ). As a consequence, genetic mosaic animals were produced with most cells being heterozygous, but
82B
with clones of homozyougs wild type and homozygous mutant cells. Mosaic adults were easily recognized
by eye color mosaicism, as well as by an unevenness of the wing surfaces (mosaic wings appeared somewhat
crinkled rather than the wild type flat appearance that characterized the wings of their TM6B siblings). For
GckIII T167A wing analysis, the wing specific nubGAL4 driver flies were crossed to the UAS-GckIII T167A F2 flies
(dominant negative, non-phosphorylatable GCKIII ).
[21]

Mosaic analysis of larval trachea. To test the cell autonomous requirement for furry and Mo25 in tracheal
O98
O41
cells we generated genetic mosaic animals using FLP-FRT approaches [37,38] . The alleles fry , fry and fry
O31
were gifts from Dr. Sally Horne-Badovinac and encode truncated Fry proteins (Q1008term, W394term and
G666term, respectively). The fry allele carries a 1 bp deletion causing a frame-shift after aa 403. Lastly, the
1
[25]
fry allele comes from an EMS/X-ray screen for modifiers of sinaGMR.PN eye phenotype . Except for
s308
[39]
80B
s308
fry , all fry alleles were induced on chromosomes carrying FRT . For these alleles, mosaic larvae were
generated using the MARCM strategy. For fry , we generated recombinant chromosomes carrying both
s308
2A
the fry mutation and FRT , and generated mosaic larvae using a MARCM-related approach we had
previously developed, substituting a GFP RNAi transgene for the Tubulin-GAL80 transgene. For Mo25, the
[40]
s308
D8-2 allele was used . Except for analysis of fry , virgins of the genotype y, w, hsFLP122; btl-GAL4, UAS-
GFP, UAS-DsRED2nls; Tub-GAL80 FRT80B were crossed to males carrying fry mutant alleles on FRT80B
chromosomes in trans to TM3Sb, Twist>GFP. For the fry analysis, virgins of the genotype y, w, hsFLP122;
s308
s308
btlGAL4, UAS-GFP, UAS-DsRED2nls; UAS-GFP RNAi FRT2A were crossed to males of the genotype fry
FRT2A/TM3, Sb. Crosses were established with 40 virgins and 20-40 males. After 4 h at 25 °C, adults were
transferred to fresh vials, and the 0-4 h a.e.l. embryos were subjected to 45-60 min heat shock. The embryos
were then cultured for an additional 5 days at 25 °C and analyzed at the third larval instar. Mosaic larvae
were identified based on the presence of individual GFP expressing cells in the tracheal system (marking the
homozygous mutant clones). The mosaic larvae were then heat killed (~10 s at 70 °C) in a drop of 50%
glycerol on a slide; a cover slip was added and fluorescent microscopy was used to identify mosaic animals.
Mutant terminal cells were analyzed by brightfield microscopy to score the presence and shape of gas-filled
tubes. Cells were also analyzed by fluorescent microscopy, with the black space in GFP labeled cells
revealing tube lumens, including the lumens of tubes that were not gas-filled (and thus not detectable by
brightfield microscopy). Larvae were examined using 20× and 40× objectives on a Leica compound
fluorescent microscope. Z-stacks were captured for each GFP positive terminal cell using Leica software.
Unidentified images were then scored independently for the presence of transition zone tube dilations and