Development 143: doi:10.1242/dev.130906: Supplementary information
Figure S1: Additional cut mutant analysis
(A) Wild-type ddaB MARCM. Inset: wild-type ddaB neurons do not express Pdm1/2. AntiPdm1/2 staining (magenta) shows labeled dmd1 nucleus adjacent to the unlabeled clone nucleus.
Arrowhead indicates the axon in this and subsequent panels.
Development • Supplementary information
Supplementary Material
Development 143: doi:10.1242/dev.130906: Supplementary information
(B) cutc145 mutant ddaB clone showing compact, stunted dendritic arbor (arrows). B' shows
clone (green) in relation to nearby cells and structures labeled by HRP (magenta). The yellow
arrow indicates a dendrite branch that is growing away from the epidermis along dmd1 toward
the ISN (asterisks).
(C) Example of a wild-type ddaC axon projection. Top: Confocal projection of an individual
ddaC axon (green) in relation to FasII labeled longitudinal fascicles (magenta). The axon enters
the VNC and forms medial anterior, posterior, and midline crossing terminal projections, as
previously described (Grueber et al., 2007). Arrow indicates position of the transverse slice
shown in bottom panel. Bottom: The transverse view reveals the ventral positioning of the axon
terminations, near the VM fascicles.
(D) Class IV ventral neuropil axon targeting is unchanged in cut mutants. Example of a cutc145
ddaC axon projection. Top: Confocal projection shows cutc145 mutant ddaC axons retain the
ventromedial positioning of the anterior and posterior terminal branches, but often fail to extend
a contralateral branch (12/15 cut ddaC clones lack contralateral projections). Bottom: Transverse
view at the level of the arrow shows the remaining branches are still positioned ventrally, near
the VM fascicle.
(E) cutc145 mutant ddaB clone (green) co-labeled with anti-Pdm antibody (magenta). E' shows
Pdm channel alone. Labeled nuclei include the ddaB cut clone (arrow), dmd1, and dbd.
(F) Wild-type ddaC (class IV) labeled with anti-GFP and anti-Pdm1/2 is not immunoreactive for
Pdm1/2 (inset).
(G) ddaC cut clones do not show severely stunted dendrites like the dorsal class II and class III
neurons and lack Pdm1/2 immunoreactivity.
Scale bars = 50 µm (A, B, E-H, inset nuclei zoomed 2X); 10 µm (C-D)
Development • Supplementary information
(H) Class I cut clones retain a wild-type morphology and lack Pdm1/2 immunoreactivity (ddaD:
n=7; ddaE n=3. ddaD cut clone is pictured.
Figure S2: Additional Pdm loss-of-function analysis
(A-A''') Dmd1 nub1 MARCM clones do not show morphological defects. nub1 is an allele of
pdm1 (Flybase: nubbin) that does not affect pdm2. (A) Confocal projection of dorsal cluster
neurons labeled with HRP (red) including a nub1 dmd1 clone (green). (A') GFP channel alone.
(A'') side view projection of A showing dmd1 dendrites still extend away from the epidermis to
the muscle layer. (A''') GFP channel alone.
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Development 143: doi:10.1242/dev.130906: Supplementary information
Development 143: doi:10.1242/dev.130906: Supplementary information
(B-B''') Loss of Pdm2 alone does not cause morphological defects. pdm2E46 is a null allele of
pdm2. The Df(2L)ED773 deficiency removes both pdm1 and pdm2. In trans, this combination
results in an animal completely lacking functional Pdm2, and retaining only 1 copy of pdm1. (B,
B') Confocal projection of dorsal cluster neurons in a pdm2E46/Df(2L)ED773 larva. Dmd1 and
dbd (green) are labeled using the clh8-Gal4 driver; HRP (red) labels all dorsal cluster neurons.
(B') GFP channel alone. Dbd retains a normal bipolar morphology, and dmd1 dendrites project
away from the epidermis towards the muscle layer, as shown in side view projections of panels
B'' and B'''.
Genotype: w1118/w1118; Df(2L)ED773/pdm2E46; clh8-Gal4/UAS-mCD8::Cherry
(C-C''') Homozygous nubR5 clones lack any detectable Pdm1/2 expression. (C) Dorsal cluster
containing a nubR5 dbd clone (green) along with HRP (blue) and anti-Pdm2 staining (red), which
can recognize both Pdm1 and Pdm2. The channels are separated in C'-C''' to illustrate that there
is no anti-Pdm staining in the homozygous dbd clone. As expected there is still anti-Pdm
staining visible in the dmd1 cell body, which is only heterozygous for the deficiency.
(D) Pdm1/2 does not reciprocally repress Cut in nubR5 clones. Confocal projection of a nubR5
dmd1 clone (green). Anti-Cut staining is shown in blue.
(E) Anti-Cut channel alone shows no Cut staining in the dmd1 cell body (outlined). Surrounding
Cut+ nuclei of other da neurons are labeled.
(F) Wild-type ddaC MARCM clone.
(H) Additional example of dbd dendrite overgrowth in nubR5/Df(2L) ED773 heteroallelic 1st
instar animal. Cells were live-imaged using clh8-Gal4, UAS-mCD8::GFP, which labels dbd,
dmd1, ddaE, and ddaD. Labeling of cells other than dbd is sporadic at early stages with this
driver, so we focused on dbd for our analysis. Abnormalities in dbd morphology included
branching of its longitudinal dendrites (white arrows) and growth of dorsally-extending dendrites
that arborize on the epidermis (red arrows). These morphological defects are qualitatively
similar to dbd nubR5 MARCM clones. Dmd1 is visible in this image and also displays abnormal
dendrite outgrowth and branching.
(I) Wild-type dbd axon projection from a FRT40A; GMR11F05-Gal4, UAS-CD8::GFP
MARCM background. Top: Confocal projection of an individual dbd axon (green) entering from
the right in relation to FasII labeled longitudinal fascicles (magenta). Dashed line indicates the
position of the transverse slice shown in the bottom panel. Bottom: The transverse view shows
the dorsal positioning of the dbd axon termination, near the DM fascicle.
(J) Example of two nubR5 dbd MARCM clone axon projections. Top: Confocal projection shows
the dbd axons retain a normal morphology and trajectory. Bottom: Transverse view at the level
of the dashed line shows the positions of the axon terminals near the DM fascicle, also
unchanged from wild-type.
(K) Quantification of nubR5 MARCM axon targeting data. All nubR5 dbd and dmd1 clones
examined retained normal dorsal neuropil terminations.
Scale bars = 50 µm (A-C, F, G); 10 µm (D, E, I, J); 25 µm (H)
Development • Supplementary information
(G) nubR5 ddaC MARCM clone shows less dense higher order branching.
Figure S3: Inhibition of dendrite growth in class II and IV neurons upon overexpression of
Pdm1 or Pdm2
(A) Wild-type arbor of dorsal cluster class II neuron ddaB, visualized using the FLP-out
approach with 109(2)80-Gal4 as a driver of UAS-FRT-rCD2-FRT-mCD8::GFP.
(A') Class II ddaB neuron misexpressing UAS-Pdm1 driven by 109(2)80-Gal4.
(A'') Class II ddaB neuron expressing UAS-Pdm2 driven by 109(2)80-Gal4.
(B) Wild-type arbor of dorsal cluster class IV neuron ddaC, visualized using the FLP-out
approach with 109(2)80-Gal4 as a driver of UAS-FRT-rCD2-FRT-mCD8::GFP. (Full
arborization extends beyond borders of image.)
(B') Class IV ddaC neuron misexpressing UAS-Pdm1 driven by 109(2)80-Gal4 shows greatly
reduced dendrite growth compared to wild-type.
Development • Supplementary information
Development 143: doi:10.1242/dev.130906: Supplementary information
Development 143: doi:10.1242/dev.130906: Supplementary information
(B'') Class IV ddaC neuron misexpressing UAS-Pdm2 driven by 109(2)80-Gal4 shows greatly
reduced dendrite growth compared to wild-type.
(C-E) Quantification of Pdm overexpression phenotypes for ddaB. (C) Total total dendrite
length was significantly reduced upon either Pdm1 (p<0.0001) or Pdm2 (p<0.0001)
overexpression. (D) Branch points were also significantly reduced with overexpression of Pdm1
(p=0.0008) or Pdm2 (p=0.0006). (E) Branch points per micron was not significantly different
between wild-type and Pdm1 (p=0.9934) or Pdm2 (p=0.3249) expression
Significance determined by one-way ANOVA with Tukey’s post hoc test; multiplicity adjusted p
values reported.
Development • Supplementary information
Scale bars = 50 µm
Figure S4. Further analysis of Scalloped and Vestigial expression and function in larval
sensory neurons
(A-B) HRP labeling of dendritic process at dorsal midline showing that transformation of ddaB
neurons to a class III morphology in sd MARCM clone is associated with selective loss of
characteristic midline ddaB processes.
(C) Control, wild-type FRT 19A clone of lateral class II neuron ldaA.
Development • Supplementary information
Development 143: doi:10.1242/dev.130906: Supplementary information
Development 143: doi:10.1242/dev.130906: Supplementary information
(D) sdΔB clone of class II neuron ldaA retains normal class II morphology.
(E) Quantification of main arbor length (total dendrite length minus total length of terminal
branches) for wild-type and sdΔB clones: ddaA (p>0.9999), ddaB (p=0.9696), and ddaF
(p>0.9999). Significance in determined by Kruskal-Wallis with Dunn’s multiple comparisons
test. (F) ykiB5 mutant ddaB neuron is indistinguishable from wild-type ddaB morphology.
(G) HRP (red), Vg::mCitrine (green), and Cut (blue) co-labeling. Vg:mCitrine labeling is
observed in ddaF (high Cut-expressing) and ddaB (low Cut-expressing; arrows) and an external
sensory bristle neuron between these cells.
(H) Sd:GFP and Vg are expressed together in ddaB and ddaF. Sd:GFP (green) and anti-Vg
staining (red) are coexpressed in two dorsal cluster nuclei corresponding to ddaF and ddaB as
seen in the HRP (blue) channel.
(I) Quantification of changes in main arbor length of ddaF in tub>sd, UAS-cut experiments.
There are no significant changes in main arbor length among any of the conditions. (Control vs.
tub>sd: p > 0.9999; control vs UAS-cut: p =0.1894; control vs. tub>sd, UAS-cut: p = 0.3573;
tub>sd vs. tub>sd, UAS-cut: p = 0.3324). Significance determined by one-way ANOVA with
Tukey’s post hoc test.
(J) Quantification of main arbor length of ddaA in tub>sd experiments. There is no significant
difference between conditions for ddaA (p = 0.1142 )
(K) Quantification of main arbor length of ddaB in tub>sd experiments. There is no significant
difference between conditions for ddaB (p = 0.0692).
Scale bars = 25 µm (A-B), 50 µm (C-G).
Development • Supplementary information
Significance in (J, K) determined by unpaired t-test with Welch’s correction.