Download Dendritic diversification through transcription factor

© 2016. Published by The Company of Biologists Ltd | Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
RESEARCH ARTICLE
Dendritic diversification through transcription factor-mediated
suppression of alternative morphologies
ABSTRACT
Neurons display a striking degree of functional and morphological
diversity, and the developmental mechanisms that underlie
diversification are of significant interest for understanding neural
circuit assembly and function. We find that the morphology of
Drosophila sensory neurons is diversified through a series of
suppressive transcriptional interactions involving the POU domain
transcription factors Pdm1 (Nubbin) and Pdm2, the homeodomain
transcription factor Cut, and the transcriptional regulators Scalloped
and Vestigial. Pdm1 and Pdm2 are expressed in a subset of
proprioceptive sensory neurons and function to inhibit dendrite
growth and branching. A subset of touch receptors show a capacity
to express Pdm1/2, but Cut represses this expression and promotes
more complex dendritic arbors. Levels of Cut expression are
diversified in distinct sensory neurons by selective expression of
Scalloped and Vestigial. Different levels of Cut impact dendritic
complexity and, consistent with this, we show that Scalloped and
Vestigial suppress terminal dendritic branching. This transcriptional
hierarchy therefore acts to suppress alternative morphologies to
diversify three distinct types of somatosensory neurons.
KEY WORDS: Dendrite, Drosophila, Transcription factor,
Homeodomain, POU domain, Neuronal morphogenesis,
Axon targeting
INTRODUCTION
The morphologies of neurons are highly diverse, fitting with distinct
functions and connectivity. Transcriptional regulators drive
neuronal diversification, as demonstrated by studies in several
systems, including rodents, worms and flies (Allan and Thor, 2015).
Given the enormous cellular diversity in the nervous system,
understanding how multiple transcriptional regulators interact to
drive subclass and individual neuronal features is an important goal.
Studies of the specification of motor neurons in fly and vertebrate
systems have revealed multiple strategies for generating specific
axonal trajectories including combinatorial codes, hierarchical
genetic cascades, and spatial and temporal patterning (Landgraf
and Thor, 2006; Dasen and Jessell, 2009; Enriquez et al., 2015).
Drosophila multidendritic sensory neurons have provided a
powerful model for identifying transcriptional programs that
underlie the morphological diversification of dendritic arbors;
nevertheless, regulators have been studied largely in isolation and
1
Department of Neuroscience, Columbia University Medical Center, 630 W. 168th
2
St. P&S 12-403, New York, NY 10032, USA. Department of Physiology and Cellular
Biophysics, Columbia University Medical Center, 630 W. 168th St. P&S 12-403,
New York, NY 10032, USA.
*Present address: Department of Neurobiology, University of Massachusetts
Medical School, 364 Plantation Street, LRB 740, Worcester, MA 01605, USA.
the strategies for diversification are poorly understood (Corty et al.,
2009; Jan and Jan, 2010). Here, we examine transcriptional
strategies for the diversification of multiple morphologically and
functionally distinct types of somatosensory neurons.
Drosophila sensory neurons are segregated into morphological
classes distinguished by sensory dendrite branching pattern, end
organ innervation, and axonal targeting in the central nervous system
(Bodmer and Jan, 1987; Merritt and Whitington, 1995; Grueber et al.,
2002, 2007). A subset of md neurons, the dendritic arborization (da)
neurons, extend sensory dendrites across the body wall to cover large
territories. The da neurons have been further divided into classes I-IV
based on dendritic morphology (Grueber et al., 2002) (Fig. 1A).
Dendritic morphology correlates with sensory function, with class I,
dbd and dmd1 neurons functioning as proprioceptors (Hughes and
Thomas, 2007), class III neurons mediating responses to gentle touch
(Yan et al., 2013), and class IV neurons functioning as multimodal
nociceptors (Hwang et al., 2007; Xiang et al., 2010). Class II neurons
are touch sensitive, but a specific functional role has not been
resolved (Tsubouchi et al., 2012).
Multiple transcription factors are known to play a role in sensory
neuron dendrite morphogenesis (Moore et al., 2002; Grueber et al.,
2003; Li et al., 2004; Sugimura et al., 2004; Kim et al., 2006; Hattori
et al., 2007; Jinushi-Nakao et al., 2007; Crozatier and Vincent,
2008; Ye et al., 2011; Ferreira et al., 2014). The transcriptional
regulator Cut is a key factor in the establishment of diverse da
neuron morphologies (Grueber et al., 2003; Jinushi-Nakao et al.,
2007; Iyer et al., 2013). Cut promotes the development of class II, III
and IV da neurons with larger territories and complex branching
patterns. By contrast, class I, dbd and dmd1 neurons with smaller
fields and less complex morphologies lack detectable Cut
expression and do not require Cut for dendritic morphogenesis.
Cut is expressed at different levels in different da neuron classes.
These levels help to specify the development of distinct neuronal
morphologies (Grueber et al., 2003). Cut expression is promoted by
the Longitudinals lacking (Lola) transcription factor in class IV
neurons (Ferreira et al., 2014), but it is still not known how distinct
Cut levels are established in different cell types and, more generally,
how class-specific expression patterns of transcriptional regulators
are generated to diversify sensory neuron morphology.
We addressed these questions by examining links between Cut
and the transcription factors Pdm1/2, Scalloped and Vestigial in
sensory neuron subset diversification. Our results identify a
transcriptional pathway for diversification of sensory neuron
morphology via suppression of distinct alternative morphological
states.
‡
RESULTS
Cut is required for proper dendritic elaboration and axonal
targeting in a subset of da sensory neurons
Received 29 September 2015; Accepted 25 February 2016
The homeodomain transcription factor Cut is expressed in class II
(low expression), III (high expression) and IV (intermediate
Author for correspondence ([email protected])
1351
DEVELOPMENT
Megan M. Corty1,2, *, Justina Tam2 and Wesley B. Grueber1,2,‡
RESEARCH ARTICLE
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
expression) da neurons. Prior studies of Cut in da neuron dendritic
morphogenesis identified two very distinct phenotypes in cut null
mutant MARCM clones. Depending on the specific neuron
examined, cut clones either showed simplified dendritic terminal
branching or a more severe dendritic growth defect (Grueber et al.,
2003). We examined the basis for these qualitatively distinct
phenotypes. Neurons that showed severe growth defects included
the class II and class III neurons ddaA, ddaB, ddaF and ldaB in the
dorsal and lateral clusters (Fig. 1B-F, Fig.2G-K, Fig. S1A,B).
Mutant neurons either extended a single primary dendrite that ended
in a compact, dense arborization a short distance from the cell body,
or adopted a bipolar dendrite morphology (Fig. 1B-D). Stunted
dendrites often targeted to a nearby chordotonal organ or nerve
(Fig. 1F,F′, Fig. S1B′).
1352
Axon morphology and position in the neuropil are also
distinguishing features of sensory cell types. Cut+ touch-sensing
and nociceptive neurons (classes II, III and IV) terminate in the
ventral neuropil, whereas Cut− proprioceptive neurons (class I and
dbd) terminate more dorsally (Merritt and Whitington, 1995;
Schrader and Merritt, 2000; Grueber et al., 2007). We assessed the
positions of afferent terminals relative to Fasciclin 2 (Fas2)-labeled
ventromedial (VM) or dorsomedial (DM) fascicles. In contrast to
the targeting of wild-type neurons to the VM fascicle, the terminals
of a subset of cut mutant MARCM clones were repositioned to
the DM fascicle (n=20; Fig. 1G-J). Notably, axon mistargeting to
the DM fascicle was specific to the neurons (ddaA, ddaB, ddaF and
ldaB) that also showed severe dendritic growth phenotypes upon
loss of Cut (Fig. S1C,D). These data suggest that Cut is required to
DEVELOPMENT
Fig. 1. Cut function in class II and III dorsal
cluster sensory neuron dendrite and axon
morphogenesis. (A) Representative tracings and
descriptions of peripheral sensory neuron subtypes
found in the dorsal cluster of third instar Drosophila
larvae. (B) Wild-type class III neuron ddaA MARCM
clone. Arrowheads indicate axons here and in
subsequent panels. (C) Example of a cutc145
mutant ddaA clone showing stunted dendritic arbor
(arrow). (D) Example of a cutc145 mutant ddaA clone
displaying a bipolar dendritic morphology (arrows).
(E) Wild-type class III ddaF clone. (F,F′) cutc145
mutant ddaF clone (green) showing targeting of
stunted dendrites (white arrows) to the v’ch1
chordotonal organ (labeled with HRP, magenta;
yellow arrows). (G) Wild-type class III ddaA axons
project to the ventral medial (VM) fascicle of the
VNC. (Top) Confocal projection of VNC showing
ddaA axon (arrow) terminating near the medial
fascicles (Fas2, magenta). (Bottom) Transverse
view shows axon termination near the VM fascicle
(arrow). Fascicle labels: L, lateral; I, intermediate;
M, medial. (H) Axon projection of cutc145 mutant
ddaA neuron. Top: Axon (arrow) projects to the
medial fascicles. Bottom: Transverse view shows
aberrant termination near the dorsal medial (DM)
fascicle (arrow). (I) Classification of cut mutant axon
phenotypes. The percentage of ddaA, ddaB, ddaF
and ldaB clones that projected to either dorsal or
ventral neuropil regions is shown (classes
displayed as group; n=20). (J) Schematic summary
of cut axon phenotypes. Wild-type (WT) ddaA,
ddaF, ldaB and ddaB axons (green) normally
terminated near the VM fascicles. When mutant for
cut the axons of these cells (red) instead terminated
near the DM fascicles. Fas2+ tracts are shown in
magenta. Scale bars: 50 µm in B-F′; 10 µm in G,H.
RESEARCH ARTICLE
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
repress morphological characteristics of proprioceptive neurons in a
subset of dorsal cluster touch-sensing neurons.
Loss of Cut leads to ectopic Pdm1/2 expression in neurons
showing stunted dendritic growth
Cut has been shown to repress Pdm1 [Nubbin (Nub) – FlyBase] in
embryonic sensory neurons (Brewster et al., 2001), prompting us
to study this factor and the closely related transcription factor
Pdm2 (collectively Pdm1/2) in more detail. Pdm1/2 are normally
co-expressed in dbd and dmd1 sensory neurons, both of which are
Cut− and predicted to function as proprioceptors (Billin et al., 1991;
Dick et al., 1991; Brewster et al., 2001; Hughes and Thomas, 2007).
dbd resides along a connective tissue strand and extends dendrites in
a simple bipolar fashion to span each segment (Schrader and
Merritt, 2007). We found that dmd1 resides on the body wall and
extends a compact dendrite bundle away from the body wall to the
intersegmental nerve (ISN) (Fig. 2A,B). Thus, in contrast to da
neurons, Pdm1/2+ neurons extend simple dendrites that are not
associated with an epidermal substrate. dbd and dmd1 axons both
terminate near the DM fascicle, consistent with a proprioceptive
1353
DEVELOPMENT
Fig. 2. Morphology of wild-type Pdm1/2-expressing cells and cell-autonomous suppression of Pdm1/2 by Cut in dorsal cluster neurons. (A) Wild-type
morphology of dmd1 and dbd in third instar larvae as shown by labeling with mCD8::GFP driven by pdm1-Gal4 (green). Anti-HRP (magenta) labels all neuronal
membranes. (A′) GFP channel only. Arrow indicates axon. Dorsal is to the top and anterior is to the left in all panels. (B) Orthogonal view shows dmd1 dendrites
(arrowhead) extending away from the epidermis to the intersegmental nerve (ISN). (B′) GFP channel only. (C) Axon projection of wild-type dmd1 MARCM clone
extends to the VNC medial fascicle (arrow; fascicles labeled in magenta). (D,D′) Orthogonal views at the positions indicated by the dashed lines in C show
termination near the DM fascicle (D, arrow) and the trajectory through the neuropil (D′, dashed arrow). (E) Cut and Pdm2 immunoreactivity in embryonic md
neurons (labeled by the E7-2-36-lacZ enhancer trap). In cut heterozygotes, Pdm2 is limited to dmd1 and dbd. (F) Pdm2 immunoreactivity is expanded to
additional dorsal da neurons in cut homozygous mutant embryos. (G) Control ddaA MARCM clone labeled with anti-GFP (green) lacks Pdm2 expression
(magenta). Inset is a magnified view of the Pdm2 channel. (H) cutc145 ddaA MARCM clone shows ectopic Pdm2 expression. Inset is a magnified view of the Pdm2
channel. (I) Wild-type ldaB (class III) clone does not show immunoreactivity for Pdm2 (inset). (J) cutc145 ldaB clone showing reduced terminal branching but intact
primary scaffold does not show immunoreactivity for Pdm2 (inset). (K) cutc145 ldaB clone with a stunted primary outgrowth shows immunoreactivity for Pdm2
(inset). (L) Quantification of Pdm1/2 expression in cutc145 clones reveals a correlation between dendritic transformation and the misregulation of Pdm1/2. (M) Wildtype dorsal cluster. Md sensory neurons are labeled with 109(2)80-Gal4, UAS-mCD8::GFP (green) and anti-Pdm1 antibody (magenta). (M′) Anti-Pdm1-labeling
is seen exclusively in the dmd1 and dbd neurons. (N) Dorsal cluster in which UAS-cut and UAS-mCD8::GFP are driven by 109(2)80-Gal4. (N′) Absence of antiPdm1 labeling in dmd1 and dbd when UAS-cut is driven in these cells. (O) Wild-type morphology of dmd1 FLP-out clone. (P) dmd1 FLP-out clone expressing
UAS-cut shows dendritic overgrowth. Arrows indicate dendritic overgrowth along the epidermis. Scale bars: 50 µm in A,G-K,M-P; 10 µm in C-F.
function, but arrive via distinct paths, with dbd axons traveling
through the dorsal neuropil (Schrader and Merritt, 2000) and dmd1
axons taking a ventral-to-dorsal route (Fig. 2C,D). Thus, the normal
dendritic and axonal projections of Pdm1/2-expressing dbd and
dmd1 resembled those of the transformed cut clones described
above.
Using antibodies against Pdm1 and Pdm2, we confirmed that
Pdm1/2 expression was expanded in cut mutant embryos to
include extra da neurons, which we assigned by position as ddaA,
ddaB and ddaF (Fig. 2E,F). To determine whether Cut represses
Pdm1/2 expression cell-autonomously we assessed Pdm1/2
expression in cut MARCM clones. We found strong expression
of Pdm1/2 in cutc145 clones of ddaA, ddaB and ddaF (Fig. 2G,H,
L, Fig. S1E) but not in class IV or class I neurons (Fig. S1F-H).
Thus, ectopic Pdm1/2 expression was observed only in neurons
that showed dendritic or axonal transformations. The strong
correlation is exemplified by phenotypes observed for the lateral
class III ldaB neuron. For this cell, the dendritic transformation
phenotype was observed in only a subset of MARCM clones
(transformation n=12/15), and ectopic expression of Pdm1/2 was
observed only in transformed clones (Fig. 2I-L). Ectopic
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
expression of Cut was sufficient to suppress Pdm1/2 expression
(Brewster et al., 2001) and promote ectopic dendritic branching in
dmd1 and dbd neurons (Fig. 2M-P). Together, these data point to
a role for Cut in suppressing Pdm1/2 expression in dorsal/lateral
class II and III neurons.
Loss-of-function evidence that Pdm1/2 suppress dendritic
growth
We next examined the role of Pdm1/2 in specifying dbd and dmd1
morphology. Mutation of either pdm1 or pdm2 individually caused
no morphological defects and thus each is separately dispensable for
dendritogenesis (Fig. S2A,B), consistent with similar redundant
function in CNS neuroblasts (Yeo et al., 1995; Grosskortenhaus
et al., 2006). We characterized a lethal allele of pdm1, nubR5,
generated by imprecise P-element excision (Terriente et al., 2008),
and found that homozygous embryos and MARCM clones lacked
detectable expression of both Pdm1 and Pdm2, indicating that this
deletion is likely to be a Pdm1/2 protein null (Fig. S2C). MARCM
analysis revealed a dendritic overgrowth phenotype in nubR5 dmd1
and dbd clones. Most dmd1 clones projected dendrites across the
epidermis rather than the ISN (Fig. 3A-D). Most dbd clones retained
Fig. 3. Genetic deletion of the pdm1/2 region leads to dendritic overgrowth. (A) Wild-type dmd1 MARCM clone. Dendrites project to the ISN (yellow arrow).
Arrowhead in this and subsequent panels indicates the axon. (B) Dendrites of nubR5 dmd1 MARCM clone arborize on the epidermis (white arrows). (C) Tracings
of a wild-type (boxed, top left) and mutant nubR5 dmd1 MARCM clones. Red arrow indicates normal dendritic pattern and black arrows indicate ectopic
growth along the epidermis. (D) Classification of nubR5 dmd1 dendrite phenotypes (see Materials and Methods for classification criteria). (E) Wild-type dbd
MARCM clone showing simple bipolar morphology (yellow arrows). (F) Aberrant growth and branching of dendrites (white arrows) in a nubR5 dbd MARCM clone.
Bipolar dendrites are still seen (yellow arrows). (G) Tracings of a wild-type (top) and nubR5 dbd clones. Red arrows indicate typical branches and black arrows
indicate extra branches. (H) Classification of nubR5 dbd dendrite phenotypes (see Materials and Methods for classification criteria). (I) Control dorsal cluster from a
nubR5/+ first instar larvae. Animals were live imaged using clh8-Gal4, UAS-mCD8::GFP. Arrows indicate the normal bipolar morphology of dbd. Asterisks mark
cell bodies of other labeled neurons. (J) Tracing of cells in panel I to illustrate normal morphology. dbd displays a characteristic bipolar morphology in control
animals. (K,L) Examples of dbd dendrite overgrowth in nubR5/Df(2L) ED773 heteroallelic animals. Abnormalities in dbd morphology included branching of
longitudinal dendrites (white arrows) and growth of dorsally extending arborized dendrites (red arrow). Scale bars: 50 µm in A-C,E-G; 25 µm in I-L.
1354
DEVELOPMENT
RESEARCH ARTICLE
their longitudinal dendrites, but sprouted ectopic branches that grew
along the epidermis (Fig. 3E-H). We did not observe Cut
immunoreactivity in dmd1 or dbd nubR5 clones, indicating that
Pdm1/2 does not reciprocally repress Cut (Fig. S2D,E). ddaC nubR5
clones (class IV, normally Pdm1/2−) showed reduced growth (n=8;
Fig. S2F,G), although we noted no obvious defects in class III
arborization patterns (n=3). Thus, one or more genes within the
nubR5 deficiency might promote complex dendritic branching in
class IV da neurons.
To narrow the region of the nubR5 deficiency responsible for
the overgrowth phenotypes we analyzed dbd morphology in nubR5/
Df(2L)ED773 animals. This combination of deficiencies eliminates
Pdm1 and Pdm2 expression and putatively disrupts only two other
genes in the region, RNA and export factor binding protein 2 (Ref2;
coding region deleted) and bunched (bun; loss of all upstream
sequence and part of its 5′ sequence). We visualized dbd
morphology using live imaging in surviving early first instar
larvae labeled with clh8-Gal4, UAS-mCD8::GFP and observed
abnormal morphologies with excessive dendritic growth and
branching (n=6; Fig. 3I-L, Fig. S2H), consistent with the
overgrowth phenotypes seen in dbd nubR5 MARCM clones.
In contrast to the overgrowth and targeting defects observed in
dendrites, axons in nubR5 dmd1 and dbd MARCM clones had
grossly normal morphologies and retained their terminations near
the DM fascicle (Fig. S2I-K). These loss-of-function studies,
together with misexpression experiments presented below, point to
a role for Pdm1/2 in restricting dendritic growth, but not in directing
axonal targeting, of dmd1 and dbd neurons.
Ectopic Pdm1/2 expression restricts dendritic growth
To test whether Pdm1/2 expression is sufficient to restrict dendritic
growth we drove expression of each gene with Gal4-109(2)80 and
resolved dendritic morphologies using the FLP-out method (Struhl
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
and Basler, 1993). Expression of either gene in class II, III or IV
neurons dramatically reduced dendritic growth and branching
(Fig. 4, Fig. S3). Misexpression did not cause targeting of
dendrites to the ISN or to connective tissue, as seen in wild-type
Pdm1/2+ neurons. Thus, although Pdm1/2 can strongly suppress
growth and branching, Pdm1/2 misexpression is not able to shift
dendrite substrate preference or terminal targeting. Growth and
targeting might be under separate transcriptional control in Pdm1/2expressing neurons, or the timing of Pdm1/2 expression might be
critical for dendritic targeting.
Scalloped is expressed in a subset of da neurons
The above data demonstrate a role for Cut in directing dorsal
cluster class II and III da neurons toward a complex dendritic
morphology via suppression of a default Pdm1/2-dependent
program of proprioceptor morphogenesis. Diversification of class
II and class III sensory terminal dendrites depends at least in part
on differential Cut expression levels, with high levels in class III
neurons and low levels in class II neurons (Grueber et al., 2003).
We explored the basis of differential Cut expression as a route to
further dendritic diversification. From a collection of GFP trap
lines (Morin et al., 2001; Buszczak et al., 2007; Quinones-Coello
et al., 2007) we identified prominent expression of the TEAD/
TEF-1 transcription factor Scalloped (Sd) (Campbell et al., 1992)
in the class II neuron ddaB and the class III neuron ddaF in third
instar larvae (Fig. 5A). Very low GFP expression could
occasionally be observed in the class I neuron ddaE and in the
class III neuron ddaA. We used an antibody against Sd that had
previously revealed its expression in sensory neurons of the
embryonic PNS (Guss et al., 2013), and confirmed Sd expression
in ddaB and ddaF neurons in third instar larvae consistent with the
GFP trap (Fig. 5B). Expression of Sd in a subset of Cut+ da
neurons and the interaction between Sd and Cut in wing
Fig. 4. Overexpression of Pdm1 or Pdm2 inhibits dendritic growth and branching in class III neurons. (A) Wild-type class III neuron ddaA, visualized
using the FLP-out approach with 109(2)80-Gal4, UAS-FRT-rCD2-FRT-mCD8::GFP. (B,C) Class III ddaA neuron FLP-out clones expressing (B) UAS-pdm1 or (C)
UAS-pdm2. (D,E) Total dendrite length (D) and total branch point number (E) were significantly reduced upon Pdm1 (***P<0.0001) or Pdm2 (***P<0.0001)
overexpression. (F) Branch points per total dendrite length (µm) was significantly reduced upon Pdm1 (**P=0.003) but not Pdm2 (P=0.0838) overexpression. n.s.,
not statistically significant. Scale bars: 50 µm.
1355
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
Fig. 5. Sd is required in ddaB to repress a class III-like morphology. (A) Labeling of third instar larvae with anti-HRP (red), anti-GFP (green) and anti-Cut
(blue). Sd::GFP expression is strong in the class II neuron ddaB and the class III neuron ddaF in larval stages (arrows). (B) Labeling of third instar larvae
with anti-Cut (green), anti-Sd (red) and anti-Elav (blue). Anti-Sd labeling is seen in the class II neuron ddaB and class III neuron ddaF (arrows). (C) Dendrites of
wild-type ddaB MARCM clone (arrowheads). (D) sdΔB ddaB MARCM clone showing extensive terminal branching. (E) Enlarged views of the arborization patterns
of wild-type ddaB (from C), sdΔB ddaB (from D), and wild-type ddaA MARCM clones. (F) Quantification of branch points per total dendrite length. sdΔB ddaA
(P>0.9999) and sdΔB ddaF (P=0.0714) were not significantly different from wild-type clones. sdΔB ddaB clones showed significant (***P<0.0001) increases in
branches per length, rendering them statistically indistinguishable from ddaA on this measure. (G) Quantification of total branch points for wild-type and sdΔB ddaA
(P=0.9555), ddaB (***P<0.0001) and ddaF (*P=0.0224) clones. Scale bars: 25 μm in A,B,E; 50 μm in C,D.
Sd suppresses a class III-like terminal branching pattern in
ddaB
To examine whether Sd regulates sensory neuron morphology, we
generated homozygous mutant sd MARCM clones. We did not
observe any obvious phenotypes for class I or IV neurons. We found
no clones with a typical ddaB morphology (0/90). By contrast,
among wild-type clones ddaB made up 12% (14/119) of the total.
These data suggested that Sd could be required for either the
production, survival or proper differentiation of ddaB. Using the
md-neuron marker E7-2-36-lacZ, we found no difference in the
number of md neurons in the dorsal cluster in sdΔB embryos [8.0
±0.0 cells (±s.d.), n=53 clusters] compared with the sd+/− control
(8.0±0.24 cells, n=6 clusters). These data argue that Sd is not
required for the survival or initial generation of ddaB, and raised the
possibility of a role in morphogenesis.
We investigated whether sd mutant ddaB clones show
morphological transformations. Examination of sdΔB clones
revealed mutant neurons in the typical location of ddaB that had
developed a highly branched morphology with the short fine
terminal dendrite ‘spikes’ that are a hallmark of class III neurons. We
inferred the identity of these clones by verifying the presence of
ddaA as well as the absence of the ddaB-characteristic dendritic arbor
using HRP labeling (Fig. S4A,B). Quantification of the dendrites of
these clones revealed they were statistically indistinguishable from
wild-type class III ddaA clones in each of the parameters examined
(Fig. 5C-G, Fig. S4E). Relative to ddaB wild-type clones, sd clones
1356
showed dramatically increased terminal branching (Fig. 5F,G).
Similar dendritic terminal phenotypes were observed with the sdΔC
allele (n=3). A modest, but significant increase in total dendritic
branch points was also observed for ddaF, which also expresses Sd,
without a change in main arbor length (Fig. 5F,G). We did not
observe transformations of other class II neurons to a class III-like
morphology (ldaA, n=8; vdaA, n=4; Fig. S4C,D).
We conclude that Sd is required in ddaB to repress elaboration of
a class III-like morphology and in ddaF to limit terminal branching.
Our data also suggest that different genetic programs specify class II
neuron morphology in different regions of the body wall.
Sd and Vestigial suppress Cut expression to low levels
One hallmark of class II versus class III identity is the expression
level of Cut. Class II neurons express low levels of Cut, whereas
class III neurons express high levels that promote extensive terminal
branching. To examine whether sd class II ddaB clones with altered
morphology also show altered Cut expression we quantified the
levels of Cut immunoreactivity in sd −/− ddaB clones relative to
adjacent sd+/− class III ddaA neurons. As controls, we quantified
Cut levels in wild-type ddaB clones relative to their neighboring
ddaA neurons, as well as the levels of Cut in ddaB and ddaA neurons
in sd+/− larvae. In both controls, levels of Cut immunoreactivity in
ddaB neurons were, on average, about half those of ddaA neurons
(mean CutddaB:CutddaA ratio=0.41; Fig. 6A,I). By contrast, sd
mutant ddaB clones showed high levels of Cut that were
indistinguishable from those of ddaA (mean CutddaB:CutddaA
ratio=0.98; Fig. 6B,I). These results suggest that Sd is required in
ddaB to limit the Cut expression level.
DEVELOPMENT
development (Morcillo et al., 1996) led us to examine a possible
role in dendritic diversification.
RESEARCH ARTICLE
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
Either of two transcriptional intermediary factors, Vestigial (Vg)
or Yorkie (Yki), interact with Sd in different tissues (Halder et al.,
1998; Paumard-Rigal et al., 1998; Simmonds et al., 1998; Goulev
et al., 2008; Wu et al., 2008; Zhang et al., 2008). yki MARCM
clones showed normal ddaB dendritic morphology (n=6; Fig.
S4F). By contrast, vg ddaB clones showed extra terminal
branching and short fine terminal processes similar to class III
neurons and sd mutant ddaB clones (n=5; Fig. 6C,D,F-G). As with
sd mutants, Cut immunoreactivity in vg ddaB clones was
significantly increased (Fig. 6E,H,J). In addition, we found that
a Vg::mCitrine fusion protein is expressed in ddaB and ddaF and
that anti-Vg immunolabeling overlapped with Sd::GFP expression
in those neurons (Fig. S4G,H). These results suggest that Sd and
Vg function together in ddaB to repress class III characteristics,
including a high Cut expression level and extensive terminal
branching.
1357
DEVELOPMENT
Fig. 6. Sd and Vg are required for low Cut
expression levels in ddaB. (A) Anti-HRP
labeling of control dorsal cluster. Individual da
neurons are named. (A′) Anti-Cut labeling of
dorsal cluster neurons in grayscale (left) and
pseudo-color image (look-up table, LUT; right).
ddaA (arrowhead) and ddaB (arrow) neurons are
outlined by a green box. (B) Anti-HRP labeling of
dorsal cluster. ddaB neuron is mutant for sd.
(B′) Anti-Cut labeling of dorsal cluster neurons in
grayscale (left) and pseudo-color image (right).
ddaA (arrowhead) and ddaB (arrow) neurons are
outlined by a green box. (C) Wild-type MARCM
clone of ddaB (arrow) showing location of cell
body relative to the class III neuron ddaA
(arrowhead). Clone is labeled by anti-GFP and
neurons are labeled by anti-HRP. (D) Tracing of
clone showing wild-type ddaB neuron (green,
arrow) and terminal extensions of class III
neurons (magenta, arrowhead). (E) Anti-Cut
labeling of wild-type clone in C showing
immunoreactivity in ddaB (arrow) and ddaA
(arrowhead). Lower panel shows pseudocoloring of anti-Cut label. (F) vg − ddaB MARCM
clone (arrow) shows a class III-like branching
morphology. (F′) Arbors of vg clone show
numerous fine terminal processes. (G) Tracing
of vg− clone (green, arrow) and the class III
neuron ddaA (magenta, arrowhead). (H) Cut
labeling of vg − ddaB MARCM clone (arrow) and
ddaA (arrowhead). Lower panel shows pseudocoloring of anti-Cut label. (I) Quantification of Cut
labeling in wild-type and sd ddaB neurons.
Ratios of ddaB:ddaA Cut labeling in control
genotypes (sd+/+ ddaB:sd+/+ ddaA, and sd+/−
ddaB:sd+/− ddaA) were statistically equivalent.
The ratio of ddaB:ddaA Cut labeling between
sd −/− ddaB clones and sd+/− ddaA was
significantly increased compared with both wildtype controls (**P=0.0017) and heterozygous
controls (***P=0.0004). Significance was
determined by Kruskal–Wallis with Dunn’s
multiple comparisons test. (J) Quantification of
Cut labeling in wild-type and vg ddaB neurons.
The ratio of ddaB:ddaA Cut labeling is
significantly increased for vg ddaB clones
(***P=0.0006). Scale bars: 50 μm in A-D,F,G;
10 μm in E,H.
Sd is sufficient to suppress terminal branching in dorsal
class III neurons
To examine whether Sd is sufficient to suppress branching in
high Cut-expressing class III neurons we performed ectopic
expression experiments. We examined the consequences of ectopic
sd expression under the control of the tubulin (αTub84B) promoter
(tub>HA-sd). tub>HA-sd rescues lethality and wing phenotypes of sd
mutants and thus can functionally substitute for sd. Furthermore, in an
sd+ background tub>HA-sd does not cause lethality or generate the
overexpression phenotypes caused by UAS-based overexpression,
such as wing scalloping (M. Zecca, personal communication).
We again visualized individual neurons using the FLP-out
technique. Class III ddaF neurons in a tub>HA-sd background
showed highly simplified dendrites characterized by the loss of fine
terminal branches (Fig. 7A,B,F,I), while the main arbor length of
these cells was unchanged (Fig. S4I). The class III neuron ddaA also
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
showed a significant decrease in branch points per total dendrite
length and reduced branch number, whereas ddaB showed no
dendritic defects (Fig. 7D,E,G,H, Fig. S4J-K). Thus, forced Sd
expression can reduce dendritic branching of class III neurons.
These data also suggest that either the level or the timing of
endogenous Sd expression is not sufficient to strongly suppress
terminal branching in ddaF. UAS-cut expression provided
significant rescue of ddaF branch points per length in a tub>HAsd background but did not restore arbors to wild-type morphology
and did not restore total branch number in this cell (Fig. 7C,F,I).
Altogether, the partial restoration of dendritic complexity in
tub>HA-sd by UAS-cut, together with our loss-of-function data,
suggest that Sd regulates dendritic morphology in part by specifying
low levels of Cut expression. These data also raise the possibility
that Sd can interfere with dendritic branching programs that operate
downstream of, or in parallel to, Cut.
Fig. 7. Sd driven by the tubulin promoter simplifies ddaF dendrites and this effect is partially reversed by overexpression of Cut. (A) Wild-type proximal
arbor of a dorsal cluster class III neuron ddaF, visualized using the FLP-out approach with 109(2)80-Gal4, UAS-FRT-rCD2-FRT-mCD8::GFP. Terminal branches
are indicated by arrows. (B) A ddaF arbor in tub>HA-sd larva. Terminal branches are indicated by arrows. A branch from the ddaA neuron, which is less affected
than ddaF, is visible at the bottom (arrowhead). (C) A ddaF arbor when UAS-cut is expressed using 109(2)80-Gal4 together with tub>HA-sd. (D) Quantification of
the effects of tub>HA-sd on branch points per total dendrite length for ddaA (*P=0.0108). (E) Quantification of effects of tub>HA-sd on branch points per total
dendrite length for ddaB (P=0.278). (F) Quantification of effects of tub>HA-sd and UAS-cut on branch points per total dendrite length for ddaF. Branch points per
total dendrite length was unchanged in UAS-cut compared with control (P=0.7015), but significantly decreased in tub>HA-sd compared with control
(***P<0.0001). Expression of UAS-cut together with tub>HA-sd significantly increased branch density compared with tub>HA-sd alone (*P=0.0126) but did not
restore wild-type density (***P<0.0001). (G) Quantification of effects of tub>HA-sd on total number of branch points in ddaA (*P=0.0351). (H) Quantification of
effects of tub>HA-sd on total number of branches in ddaB (P=0.8431). (I) Quantification of effects of tub>HA-sd and UAS-cut on total number of branch points in
ddaF. Compared with wild-type, branch points were significantly reduced in tub>HA-sd larvae (***P<0.0001) and in tub>HA-sd, UAS-cut larvae (*P=0.0213).
Expression of UAS-cut with tub>HA-sd did not significantly affect branch point number compared with tub>HA-sd alone (P>0.9999). Significance in panel I was
determined by Kruskal–Wallis with Dunn’s multiple comparisons test. Scale bars: 50 μm.
1358
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
DISCUSSION
We have studied how three distinct somatosensory neuron
morphologies are established in Drosophila. Together, our results
reveal a repressive strategy for the diversification of dendritic arbor
morphology involving the transcription factors Cut, Pdm1/2, Sd and
Vg (Fig. 8).
Repression of alternative developmental programs promotes
sensory neuron morphological diversification
Cut/Cux transcription factors have conserved roles in dendritic
morphogenesis, including control of dendritic elaboration of da
neurons (Grueber et al., 2003; Jinushi-Nakao et al., 2007), dendritic
targeting of Drosophila olfactory projection neurons (Komiyama
and Luo, 2007), and control of dendritic morphogenesis of upper
layer cortical neurons in vertebrates (Cubelos et al., 2010). Previous
studies showing that Cut levels impact class-specific complexity of
da neuron dendritic arbors (Grueber et al., 2003) raised several
questions, including how Cut activity establishes divergent sensory
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
neuron morphologies, how different levels of Cut are specified, and
whether Cut controls axonal targeting as well as dendritic
branching in da neurons. Our data begin to address these
questions by characterizing regulatory interactions with Pdm1/2,
Sd and Vg, and by identifying a role for Cut in da neuron axon
targeting (Fig. 8).
Loss-of-function and gain-of-function experiments indicate that
Pdm1 and Pdm2 inhibit dendritic growth. Cut represses the action of
Pdm1/2 and, by extension, derepresses a program for extensive
dendritic elaboration (Fig. 8). Complementing level-dependent
functions for Cut, this function of Cut is likely to be level
independent as both low (class II) and high (class III) levels appear
sufficient to repress Pdm1/2 expression. We suggest that a minimum
level of Cut is required for Pdm1/2 repression in dorsal cluster class
II and III neurons, allowing for the establishment of an expansive
epidermal territory. Higher levels of Cut can promote increased
higher-order branching and further diversify sensory neuron
morphology. POU domain transcription factors are important
regulators of neuronal morphogenesis in both vertebrate and
invertebrate sensory systems (Komiyama et al., 2003; Badea
et al., 2009) and it would be interesting to determine whether
repression of alternative or default states is a more general
mechanism by which transcriptional programs involving POU
proteins generate diversity in neuronal morphology.
In the fly somatosensory system, sensory neuron subtype is
correlated with the morphology of dendrites in the periphery and the
positioning of sensory afferents centrally. While the mechanisms that
differentiate dendrite versus axon development are beginning to be
uncovered (Ye et al., 2007, 2011; Satoh et al., 2008; Zheng et al.,
2008), the mechanisms that coordinate md neuron dendrite and axon
morphology have not been established. Our results suggest that Cut
fulfills such a role in a subset of somatosensory neurons. Loss of Cut
led to the development of proprioceptive dendritic features and to a
shift in axon targeting to regions where proprioceptive neurons
terminate. Still unknown are the mechanisms by which Cut regulates
this switch, as Pdm1/2 appear not to affect axon terminal positioning
of da neurons. Prior studies have shown that axon positioning along the
dorsoventral axis is controlled in part by Semaphorin-Plexin signaling
(Zlatic et al., 2009), and it will be important to examine whether Cut
directly or indirectly regulates this pathway to control axonal targeting.
Fig. 8. Cut, Pdm1/2, Sd and Vg interact to diversify sensory neuron
morphology. (A) Summary of the proposed regulatory interactions between
Cut, Pdm1/2, Sd and Vg that regulate morphogenesis of dorsal cluster
neurons. Multiple neurons have a capacity to express Pdm1/2, but expression
is blocked by Cut (see Fig. 2). Pdm1/2 limit dendrite growth in dbd and dmd1
(see Fig. 3). Cut promotes growth by repressing Pdm1/2 and by promoting
dendritic growth and branching. At high levels, Cut promotes the development
of fine terminal branches characteristic of class III da neurons (Grueber et al.,
2003). Sd and Vg repress Cut to low levels in ddaB and prevent acquisition of
class III features (see Figs 5 and 6). Unlike in ddaB, Cut expression remains
high in the presence of Sd and Vg in ddaF neurons. These cells develop class
III-like terminal branches, although Sd limits the number of terminal branches
(not shown in model; see Fig. 5). In addition to acting through Cut regulation,
Sd may also act in parallel to, or downstream of, Cut to restrict terminal
branching (see Fig. 7). (B) The regulatory interactions depicted in A promote
specific transcription factor expression patterns as determined by antibody
labeling (see Figs 2, 5, Fig. S4). (C) The interactions summarized in A
contribute to the mature dendritic morphology of individual sensory neurons.
Different levels of transcriptional regulators can affect neuronal
morphogenesis (Grueber et al., 2003; Chen et al., 2006), but how
specific levels are established is not understood. We found that Sd and
Vg establish low Cut levels and repress a more highly branched class
III morphology in ddaB (Fig. 8). Sd and Vg are also expressed in ddaF,
which develops a class III-like branching pattern, and our loss-offunction experiments suggest that Sd has a modest effect on branching
in this cell as well. Our results also raise the possibility (not
schematized in Fig. 8) that Sd might also act via a parallel pathway to
set an upper limit on the branch-promoting activity of Cut, perhaps by
blocking pathways that are required downstream of Cut for
dendrogenic activity. In C. elegans sensory neuron specification, the
AHR-1 transcription factor operates by controlling both a transcription
factor and a subset of its downstream targets (Smith et al., 2013). Duallevel regulatory strategies might add robustness and range to
transcription factor encoding of specific morphological features or
identity. The role of class III terminal branches in mediating
responsiveness to gentle touch (Tsubouchi et al., 2012; Bagley et al.,
2014) raises the possibility that Sd regulation of morphology could
fine-tune mechanoreceptor sensitivity.
1359
DEVELOPMENT
Transcriptional control of Cut levels
Sd and Cut interact during wing development (Morcillo et al.,
1996), and during adult development Vg suppresses Cut levels in a
subset of muscle precursors (Sudarsan et al., 2001). Sd and Vg
therefore operate in several contexts to regulate Cut levels and
promote cell diversification, and our results identify roles in
differentiating neurons in the control of dendrite morphology.
Similarly, in C. elegans, the sd homolog egl-44 suppresses late stage
ectopic branching in multidendritic PVD neurons (Smith et al.,
2010). Given these roles in dendritic branching in invertebrate
neurons, and the broad expression of Tead1 (the mouse homolog of
Sd) in the mouse brain (Lein et al., 2007), it will be interesting to
examine whether TEAD transcription factors also regulate dendritic
branching in vertebrate neurons.
Possible implications for the diversification of neuronal
morphology during evolution
Our findings provide an example of how linked repressive
interactions among transcriptional regulators can diversify
dendritic morphology. In Drosophila sensory neurons, it appears
that a broad competence to produce dbd/dmd1-type morphologies in
dorsal regions of the larva is restricted by the action of Cut (Fig. 8).
This switch generates at least two types of sensory neurons: Cut+/
Pdm− (which perform tactile functions) and Cut−/Pdm+ (which
function as proprioceptors) (Fig. 8). Further morphological
diversification of Cut+/Pdm− neurons is achieved by modulation
of Cut expression levels (Fig. 8). The partial, rather than complete,
repression of Cut levels in ddaB becomes important in this context,
since it allows for the maintenance of Pdm1/2 repression, as well as
the development of a third distinct Cut-dependent morphology
(Fig. 8). High-level Cut expression might represent a default state for
the dorsal cluster class II neuron that is refined by Sd and Vg (Fig. 8).
Repression of alternative identities and morphologies by
transcriptional regulators is an important driver of neuronal
diversification during the development of sensory and motor
systems (Jung et al., 2010; Li et al., 2013; Philippidou and Dasen,
2013; Gordon and Hobert, 2015). Transcription factors may suppress
alternative programs of differentiation and promote neuronal diversity
by direct repression of other transcription factors, repression of their
downstream target genes, or by competitive binding of common cofactors to block transcription factor function (Smith et al., 2013;
Borromeo et al., 2014; Gordon and Hobert, 2015). Differential
expression levels of the same transcription factor can also drive
distinct morphology, identity and connectivity to further increase
diversity in the nervous system (Grueber et al., 2003; Chen et al.,
2006; Dasen et al., 2008; Smith et al., 2013). It has been proposed that
such transformations from one cell type, or cell characteristic, to
another by mutations in terminal selector genes might provide a basis
for the evolutionary diversification of cell types in the nervous system
(Arlotta and Hobert, 2015). One speculative scenario is that, in the
Drosophila PNS, the suppression of default programs in subsets of
equivalent neurons by the action of transcriptional regulators provided
a mechanism for the evolutionary diversification of somatosensory
cell types without disruption of crucial existing modalities.
MATERIALS AND METHODS
Fly stocks
Animals were reared using standard methods. We used pdm1-Gal4 [nubGal4 (Calleja et al., 2000)], clh8-Gal4 (Hughes and Thomas, 2007),
GMR11F05-Gal4 (Pfeiffer et al., 2008; Li et al., 2014), 109(2)80-Gal4 (Gao
et al., 1999) and clh201-Gal4 (Hughes and Thomas, 2007). tubP>HA-sd
and FRT42D vg83b27R were provided by Drs M. Zecca and G. Struhl
(Columbia University), vg::mCitrine by Dr G. Struhl (unpublished), and
sdΔB and sdΔC alleles were from Dr J. Jiang (UT Southwestern) (Zhang et al.,
1360
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
2008). FRT42D ykiB5 was from Dr Duojia Pan (Johns Hopkins). cutc145 and
cutdb3 are null alleles (Jack, 1985; Blochlinger et al., 1988). cutdb3 was
combined with the md neuron marker E7-2-36 lacZ for studies of embryonic
stages. The nubR5 deletion is the result of P-element excision (Terriente
et al., 2008) and was provided by Dr F. Diaz-Benjumea (Universidad
Autónoma de Madrid, Spain). PCR mapping revealed that the nubR5
deletion removes ∼224 kb of genomic material spanning 11 protein-coding
regions, which is about half the number of genes deleted in Df(2L)ED773,
which also deletes both pdm1 and pdm2 (Ryder et al., 2004, 2007;
Grosskortenhaus et al., 2006). Other stocks were Df(2L)ED773
(Bloomington Stock Center), pdm2E46 (Bloomington Stock Center), Sd::
GFP (CA07575; Buszczak et al., 2007), full-length UAS-cut (Ludlow et al.,
1996; Grueber et al., 2003), UAS-pdm1 (Neumann and Cohen, 1998), UASpdm2 (Grosskortenhaus et al., 2006), UAS-mCD8::GFP and UAS-mCD8::
Cherry (Bloomington Stock Center). Animals were analyzed as third instar
larvae, except when noted (i.e. embryos or first instar).
Mosaic analysis
For MARCM experiments (Lee and Luo, 1999) the following fly stocks
were used: (1) cutc145 FRT19A/FM7, (2) sdΔB FRT19A, (3) FRT19A;;ry, (4)
tub-Gal80, hs-FLP, FRT19A; 109(2)80-Gal4, UAS-mcD8::GFP, (5) ywhsFLP122; nubR5 FRT40A/CyO, (6) nub1 FRT40A/CyO, (7) FRT40A/CyO, (8)
hs-FLP, elav-Gal4, UAS-mCD8::GFP; tub-Gal80 FRT 40A, (9) FRT42D
ykiB5, (10) FRT42D vg83b27R, (11) w; FRT 42D, (12) hs-FLP, elav-Gal4,
UAS-mCD8::GFP; FRT 42D tub-Gal80, (13) tub-Gal80 FRT 40A/CyO;
GMR11F05-Gal4, UAS-mCD8::GFP, (14) ywhs-FLP122; FRT 40A/CyO.
MARCM clones were generated as previously described (Matthews et al.,
2007). For overexpression experiments, we crossed hs-FLP; 109(2)80Gal4; UAS>rCD2>mCD8::GFP to either w1118, w1118; UAS-pdm1/CyO,
Dfd-YFP, w1118; UAS-pdm2/CyO, Dfd-YFP or tub>HA-sd. FLP-out clones
were generated using a 30 min heat shock at 38°C to induce mosaic
expression of rCD2 and mCD8::GFP.
Immunohistochemistry
Immunohistochemistry was performed according to published methods
(Grueber et al., 2002). Antibodies used were rabbit anti-Vg (1:20; Halder
et al., 1998; courtesy of S. Carroll, University of Wisconsin), guinea pig
anti-Sd (1:500; Guss et al., 2013), guinea pig anti-Vg (1:10; Zecca and
Struhl, 2010; courtesy of G. Struhl), mouse anti-Cut (1:20; DSHB #2B10),
goat anti-HRP (1:200; Jackson ImmunoResearch #123-005-021), chicken
anti-GFP (1:1000; Abcam ab13970), rabbit anti-β-gal (1:200; Cappell
#55976), rat anti-ELAV (1:10, DSHB #7E8A10, mouse anti-Fas2 (1:10;
DSHB #1D4); rabbit anti-Pdm1 (1:1000; Yeo, 1995), and rat anti-Pdm2
(1:10; Grosskortenhaus et al., 2006). Secondary antibodies (Jackson
ImmunoResearch) were used at 1:200. Following antibody labeling,
animals were dehydrated in an ethanol series, cleared in xylenes, and
mounted in DPX (Electron Microscopy Sciences) as described (Grueber
et al., 2002). For the experiments depicted in Fig. S2I-K, animals were
mounted in Vectashield (Vector Laboratories).
Image acquisition and analysis
Imaging was performed on a Zeiss 510 Meta confocal microscope
with LSM software (Zeiss) using 40× Plan Neofluar and 63× objectives
for all images except those in Fig. 3I-L and Fig. S2H-J, which were
acquired using a 3i Everest spinning disk confocal microscope with
Slidebook 6.0 (3i) imaging software and a Zeiss Plan-Apochromat 40× oil
objective. Arbors were scanned as single frames or as partially overlapping
frames that were assembled in Photoshop CS2 (Adobe Systems) or
Neurolucida (MBF Bioscience). For quantitative analysis, neurons were
traced as confocal stacks using Neurolucida and quantified using
Neurolucida Explorer. Whole image adjustments of brightness and
contrast were applied to aid in visualization of fine dendritic processes in
figure images.
Quantification of Cut levels was performed similarly to prior studies
(Grueber et al., 2003). Briefly, ddaA and ddaB Cut-labeled nuclei were
identified in confocal stacks. Mean pixel intensity values for each nucleus
were obtained from each successive confocal section in which the nucleus
was visible using the Measure command in Fiji (Schindelin et al., 2012).
DEVELOPMENT
RESEARCH ARTICLE
The highest mean pixel value obtained for each analyzed nucleus was used
to compute ddaB:ddaA ratios. Confocal settings were identical among the
different imaging sessions for data collected for comparison between
groups. Cut values were quantified from raw values for each image without
adjustment of brightness, contrast, or levels. To aid visualization and
comparison in figure panels, levels were adjusted uniformly across some
images from (min-max) of 20-148 to 10-232 (Fig. 6A′) and 11-95 to 10-229
(Fig. 6B′). These changes were made following quantification.
Classification of nubR5 phenotypes: dmd1 clones were classified as
‘normal’ if all dendrites project to the ISN; ‘mild’ if some dendrites project to
the ISN with some dendrites that do not grow towards the target; or ‘severe’ if
there was no ISN targeting and/or substantial growth of dendrites on the
epidermis. dbd clones were classified as ‘normal’ if normal unbranched
bipolar morphology is maintained; ‘mild’ if there is abnormal branching or
mistargeting of at least one of the main dendrites; or ‘severe’ if there is an
additional, branched dorsal dendrite and/or a lack of bipolar morphology.
Statistical analysis
Statistical analysis was performed in R (R Project for Statistical Computing)
and GraphPad Prism. Data were tested for normality using a Shapiro-Wilk
test. Nonparametric analyses (Kruskal–Wallis with Dunn’s multiple
comparisons test) were used for datasets that deviated from a normal
distribution as noted (Fig. S4E, Fig. 6I, Fig. 7I). For all other datasets
statistical significance was determined using a Student’s t-test with Welch’s
correction for all comparisons between two groups and one-way ANOVA
with Tukey’s post-hoc test for all comparisons with three or more groups. n
values refer to individual cells (i.e. MARCM or FLP-out clones) except
where noted in the text. No statistical methods were used to predetermine
sample size. The sample sizes used are consistent with those found in similar
studies of da neuron morphogenesis. Animals were assigned to groups based
on genotype, and researchers were not blind to conditions. Clones were
excluded from analysis if arbors were damaged during processing. Datasets
presented as box and whisker plots are as follows: quartiles Q1-Q3 (25%75%, box), median (thick line), whiskers span minimum to maximum
values. All individual data points are displayed as open circles. Multiplicityadjusted P-values are given for all ANOVA/Tukey results. In figures,
*P<0.05, **P<0.01 and ***P<0.001.
Acknowledgements
We thank Dr M. Zecca for generously sharing fly stocks prior to publication;
Drs J. Bell, S. Carroll, W. Chia, S. Cohen, F. Diaz-Benjumea, C. Doe, K. Guss,
J. Jiang, B. Ohlstein and G. Struhl for generous sharing of fly stocks and antibodies;
Dr J. Dodd for comments on the manuscript; N. Ghani for input on branching
analysis; and Dr O. Hobert and members of the W.B.G. lab for discussions. M.M.C.
thanks Dr M. Freeman for support during preparation of the manuscript. The antiCut, anti-ELAV, and anti-Fas2 antibodies were obtained from the Developmental
Studies Hybridoma Bank (DSHB), created for the NICHD of the NIH and maintained
at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.
Competing interests
The authors declare no competing or financial interests.
Author contributions
M.M.C., J.T. and W.B.G. conceived and performed the experiments and analyzed
the data. M.M.C. and W.B.G. wrote the manuscript with input from J.T.
Funding
This work was supported in part by a National Science Foundation Graduate
Research Fellowship (M.M.C.), the Searle Scholars Program, an Irma T. Hirschl/
Monique Weill-Caulier Trust Career Award, and the National Institutes of Health
[NS061908] (to W.B.G.). Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.130906/-/DC1
References
Allan, D. W. and Thor, S. (2015). Transcriptional selectors, masters, and
combinatorial codes: regulatory principles of neural subtype specification. Wiley
Interdiscip. Rev. Dev. Biol. 4, 505-528.
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
Arlotta, P. and Hobert, O. (2015). Homeotic transformations of neuronal cell
identities. Trends Neurosci. 38, 751-762.
Badea, T. C., Cahill, H., Ecker, J., Hattar, S. and Nathans, J. (2009). Distinct roles
of transcription factors brn3a and brn3b in controlling the development,
morphology, and function of retinal ganglion cells. Neuron 61, 852-864.
Bagley, J. A., Yan, Z., Zhang, W., Wildonger, J., Jan, L. Y. and Jan, Y. N. (2014).
Double-bromo and extraterminal (BET) domain proteins regulate dendrite
morphology and mechanosensory function. Genes Dev. 28, 1940-1956.
Billin, A. N., Cockerill, K. A. and Poole, S. J. (1991). Isolation of a family of Drosophila
POU domain genes expressed in early development. Mech. Dev. 34, 75-84.
Blochlinger, K., Bodmer, R., Jack, J., Jan, L. Y. and Jan, Y. N. (1988). Primary
structure and expression of a product from cut, a locus involved in specifying
sensory organ identity in Drosophila. Nature 333, 629-635.
Bodmer, R. and Jan, Y. N. (1987). Morphological differentiation of the embryonic
peripheral neurons in Drosophila. Roux’s Arch. Dev. Biol. 196, 69-77.
Borromeo, M. D., Meredith, D. M., Castro, D. S., Chang, J. C., Tung, K.-C.,
Guillemot, F. and Johnson, J. E. (2014). A transcription factor network
specifying inhibitory versus excitatory neurons in the dorsal spinal cord.
Development 141, 2803-2812.
Brewster, R., Hardiman, K., Deo, M., Khan, S. and Bodmer, R. (2001). The
selector gene cut represses a neural cell fate that is specified independently of the
Achaete-Scute-Complex and atonal. Mech. Dev. 105, 57-68.
Buszczak, M., Paterno, S., Lighthouse, D., Bachman, J., Planck, J., Owen, S.,
Skora, A. D., Nystul, T. G., Ohlstein, B., Allen, A. et al. (2007). The carnegie
protein trap library: a versatile tool for Drosophila developmental studies. Genetics
175, 1505-1531.
Calleja, M., Herranz, H., Estella, C., Casal, J., Lawrence, P., Simpson, P. and
Morata, G. (2000). Generation of medial and lateral dorsal body domains by the
pannier gene of Drosophila. Development 127, 3971-3980.
Campbell, S., Inamdar, M., Rodrigues, V., Raghavan, V., Palazzolo, M. and
Chovnick, A. (1992). The scalloped gene encodes a novel, evolutionarily
conserved transcription factor required for sensory organ differentiation in
Drosophila. Genes Dev. 6, 367-379.
Chen, A. I., de Nooij, J. C. and Jessell, T. M. (2006). Graded activity of transcription
factor Runx3 specifies the laminar termination pattern of sensory axons in the
developing spinal cord. Neuron 49, 395-408.
Corty, M. M., Matthews, B. J. and Grueber, W. B. (2009). Molecules and
mechanisms of dendrite development in Drosophila. Development 136, 1049-1061.
Crozatier, M. and Vincent, A. (2008). Control of multidendritic neuron differentiation
in Drosophila: the role of Collier. Dev. Biol. 315, 232-242.
Cubelos, B., Sebastiá n-Serrano, A., Beccari, L., Calcagnotto, M. E., Cisneros,
E., Kim, S., Dopazo, A., Alvarez-Dolado, M., Redondo, J. M., Bovolenta, P.
et al. (2010). Cux1 and Cux2 regulate dendritic branching, spine morphology, and
synapses of the upper layer neurons of the cortex. Neuron 66, 523-535.
Dasen, J. S. and Jessell, T. M. (2009). Hox networks and the origins of motor
neuron diversity. Curr. Top. Dev. Biol. 88, 169-200.
Dasen, J. S., De Camilli, A., Wang, B., Tucker, P. W. and Jessell, T. M. (2008).
Hox repertoires for motor neuron diversity and connectivity gated by a single
accessory factor, FoxP1. Cell 134, 304-316.
Dick, T., Yang, X. H., Yeo, S. L. and Chia, W. (1991). Two closely linked Drosophila
POU domain genes are expressed in neuroblasts and sensory elements. Proc.
Natl. Acad. Sci. USA 88, 7645-7649.
Enriquez, J., Venkatasubramanian, L., Baek, M., Peterson, M., Aghayeva, U.
and Mann, R. S. (2015). Specification of individual adult motor neuron
morphologies by combinatorial transcription factor codes. Neuron 86, 955-970.
Ferreira, T., Ou, Y., Li, S., Giniger, E. and van Meyel, D. J. (2014). Dendrite
architecture organized by transcriptional control of the F-actin nucleator Spire.
Development 141, 650-660.
Gao, F. B., Brenman, J. E., Jan, L. Y. and Jan, Y. N. (1999). Genes regulating
dendritic outgrowth, branching, and routing in Drosophila. Genes Dev. 13,
2549-2561.
Gordon, P. M. and Hobert, O. (2015). A competition mechanism for a homeotic
neuron identity transformation in C. elegans. Dev. Cell 34, 206-219.
Goulev, Y., Fauny, J. D., Gonzalez-Marti, B., Flagiello, D., Silber, J. and Zider, A.
(2008). SCALLOPED interacts with YORKIE, the nuclear effector of the hippo
tumor-suppressor pathway in Drosophila. Curr. Biol. 18, 435-441.
Grosskortenhaus, R., Robinson, K. J. and Doe, C. Q. (2006). Pdm and Castor
specify late-born motor neuron identity in the NB7-1 lineage. Genes Dev. 20,
2618-2627.
Grueber, W. B., Jan, L. Y. and Jan, Y. N. (2002). Tiling of the Drosophila epidermis
by multidendritic sensory neurons. Development 129, 2867-2878.
Grueber, W. B., Jan, L. Y. and Jan, Y. N. (2003). Different levels of the
homeodomain protein cut regulate distinct dendrite branching patterns of
Drosophila multidendritic neurons. Cell 112, 805-818.
Grueber, W. B., Ye, B., Yang, C.-H., Younger, S., Borden, K., Jan, L. Y. and Jan,
Y.-N. (2007). Projections of Drosophila multidendritic neurons in the central nervous
system: links with peripheral dendrite morphology. Development 134, 55-64.
Guss, K. A., Benson, M., Gubitosi, N., Brondell, K., Broadie, K. and Skeath, J. B.
(2013). Expression and function of scalloped during Drosophila development.
Dev. Dyn. 242, 874-885.
1361
DEVELOPMENT
RESEARCH ARTICLE
Halder, G., Polaczyk, P., Kraus, M. E., Hudson, A., Kim, J., Laughon, A. and
Carroll, S. (1998). The Vestigial and Scalloped proteins act together to directly
regulate wing-specific gene expression in Drosophila. Genes Dev. 12, 3900-3909.
Hattori, Y., Sugimura, K. and Uemura, T. (2007). Selective expression of Knot/
Collier, a transcriptional regulator of the EBF/Olf-1 family, endows the Drosophila
sensory system with neuronal class-specific elaborated dendritic patterns. Genes
Cells 12, 1011-1022.
Hughes, C. L. and Thomas, J. B. (2007). A sensory feedback circuit coordinates
muscle activity in Drosophila. Mol. Cell. Neurosci. 35, 383-396.
Hwang, R. Y., Zhong, L., Xu, Y., Johnson, T., Zhang, F., Deisseroth, K. and
Tracey, W. D. (2007). Nociceptive neurons protect Drosophila larvae from
parasitoid wasps. Curr. Biol. 17, 2105-2116.
Iyer, S. C., Ramachandran Iyer, E. P., Meduri, R., Rubaharan, M., Kuntimaddi,
A., Karamsetty, M. and Cox, D. N. (2013). Cut, via CrebA, transcriptionally
regulates the COPII secretory pathway to direct dendrite development in
Drosophila. J. Cell Sci. 126, 4732-4745.
Jack, J. W. (1985). Molecular organization of the cut locus of Drosophila
melanogaster. Cell 42, 869-876.
Jan, Y.-N. and Jan, L. Y. (2010). Branching out: mechanisms of dendritic
arborization. Nat. Rev. Neurosci. 11, 316-328.
Jinushi-Nakao, S., Arvind, R., Amikura, R., Kinameri, E., Liu, A. W. and Moore,
A. W. (2007). Knot/Collier and cut control different aspects of dendrite
cytoskeleton and synergize to define final arbor shape. Neuron 56, 963-978.
Jung, H., Lacombe, J., Mazzoni, E. O., Liem, K. F., Jr., Grinstein, J., Mahony, S.,
Mukhopadhyay, D., Gifford, D. K., Young, R. A., Anderson, K. V. et al. (2010).
Global control of motor neuron topography mediated by the repressive actions of a
single hox gene. Neuron 67, 781-796.
Kim, M. D., Jan, L. Y. and Jan, Y. N. (2006). The bHLH-PAS protein Spineless is
necessary for the diversification of dendrite morphology of Drosophila dendritic
arborization neurons. Genes Dev. 20, 2806-2819.
Komiyama, T. and Luo, L. (2007). Intrinsic control of precise dendritic targeting by
an ensemble of transcription factors. Curr. Biol. 17, 278-285.
Komiyama, T., Johnson, W. A., Luo, L. and Jefferis, G. S. X. E. (2003). From
lineage to wiring specificity. POU domain transcription factors control precise
connections of Drosophila olfactory projection neurons. Cell 112, 157-167.
Landgraf, M. and Thor, S. (2006). Development of Drosophila motoneurons:
specification and morphology. Semin. Cell Dev. Biol. 17, 3-11.
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies
of gene function in neuronal morphogenesis. Neuron 22, 451-461.
Lein, E. S. Hawrylycz, M. J. Ao, N. Ayres, M. Bensinger, A. Bernard, A. Boe, A. F.
Boguski, M. S. Brockway, K. S. Byrnes, E. J. et al. (2007). Genome-wide atlas
of gene expression in the adult mouse brain. Nature 445, 168-176.
Li, W., Wang, F., Menut, L. and Gao, F.-B. (2004). BTB/POZ-zinc finger protein
abrupt suppresses dendritic branching in a neuronal subtype-specific and
dosage-dependent manner. Neuron 43, 823-834.
Li, Q., Ha, T. S., Okuwa, S., Wang, Y., Wang, Q., Millard, S. S., Smith, D. P. and
Volkan, P. C. (2013). Combinatorial rules of precursor specification underlying
olfactory neuron diversity. Curr. Biol. 23, 2481-2490.
Li, H.-H., Kroll, J. R., Lennox, S. M., Ogundeyi, O., Jeter, J., Depasquale, G. and
Truman, J. W. (2014). A GAL4 driver resource for developmental and behavioral
studies on the larval CNS of Drosophila. Cell Rep. 8, 897-908.
Ludlow, C., Choy, R. and Blochlinger, K. (1996). Functional analysis of Drosophila
and mammalian cut proteins in flies. Dev. Biol. 178, 149-159.
Matthews, B. J., Kim, M. E., Flanagan, J. J., Hattori, D., Clemens, J. C., Zipursky,
S. L. and Grueber, W. B. (2007). Dendrite self-avoidance is controlled by dscam.
Cell 129, 593-604.
Merritt, D. J. and Whitington, P. M. (1995). Central projections of sensory neurons
in the Drosophila embryo correlate with sensory modality, soma position, and
proneural gene function. J. Neurosci. 15, 1755-1767.
Moore, A. W., Jan, L. Y. and Jan, Y. N. (2002). hamlet, a binary genetic switch between
single- and multiple- dendrite neuron morphology. Science 297, 1355-1358.
Morcillo, P., Rosen, C. and Dorsett, D. (1996). Genes regulating the remote wing
margin enhancer in the Drosophila cut locus. Genetics 144, 1143-1154.
Morin, X., Daneman, R., Zavortink, M. and Chia, W. (2001). A protein trap strategy
to detect GFP-tagged proteins expressed from their endogenous loci in
Drosophila. Proc. Natl. Acad. Sci. USA 98, 15050-15055.
Neumann, C. J. and Cohen, S. M. (1998). Boundary formation in Drosophila wing:
Notch activity attenuated by the POU protein Nubbin. Science 281, 409-413.
Paumard-Rigal, S., Zider, A., Vaudin, P. and Silber, J. (1998). Specific
interactions between vestigial and scalloped are required to promote wing
tissue proliferation in Drosophila melanogaster. Dev. Genes Evol. 208, 440-446.
Pfeiffer, B. D., Jenett, A., Hammonds, A. S., Ngo, T.-T. B., Misra, S., Murphy, C.,
Scully, A., Carlson, J. W., Wan, K. H., Laverty, T. R. et al. (2008). Tools for
neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. USA 105,
9715-9720.
Philippidou, P. and Dasen, J. S. (2013). Hox genes: choreographers in neural
development, architects of circuit organization. Neuron 80, 12-34.
Quinones-Coello, A. T., Petrella, L. N., Ayers, K., Melillo, A., Mazzalupo, S.,
Hudson, A. M., Wang, S., Castiblanco, C., Buszczak, M., Hoskins, R. A. et al.
1362
Development (2016) 143, 1351-1362 doi:10.1242/dev.130906
(2007). Exploring strategies for protein trapping in Drosophila. Genetics 175,
1089-1104.
Ryder, E., Blows, F., Ashburner, M., Bautista-Llacer, R., Coulson, D.,
Drummond, J., Webster, J., Gubb, D., Gunton, N., Johnson, G. et al. (2004).
The DrosDel collection: a set of P-element insertions for generating custom
chromosomal aberrations in Drosophila melanogaster. Genetics 167, 797-813.
Ryder, E., Ashburner, M., Bautista-Llacer, R., Drummond, J., Webster, J.,
Johnson, G., Morley, T., Chan, Y. S., Blows, F., Coulson, D. et al. (2007). The
DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency
resource. Genetics 177, 615-629.
Satoh, D., Sato, D., Tsuyama, T., Saito, M., Ohkura, H., Rolls, M. M., Ishikawa, F.
and Uemura, T. (2008). Spatial control of branching within dendritic arbors by
dynein-dependent transport of Rab5-endosomes. Nat. Cell Biol. 10, 1164-1171.
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch,
T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B. et al. (2012). Fiji: an
open-source platform for biological-image analysis. Nat. Methods 9, 676-682.
Schrader, S. and Merritt, D. J. (2000). Central projections of Drosophila sensory
neurons in the transition from embryo to larva. J. Comp. Neurol. 425, 34-44.
Schrader, S. and Merritt, D. J. (2007). Dorsal longitudinal stretch receptor of
Drosophila melanogaster larva – fine structure and maturation. Arthropod Struct.
Dev. 36, 157-169.
Simmonds, A. J., Liu, X., Soanes, K. H., Krause, H. M., Irvine, K. D. and Bell,
J. B. (1998). Molecular interactions between Vestigial and Scalloped promote
wing formation in Drosophila. Genes Dev. 12, 3815-3820.
Smith, C. J., Watson, J. D., Spencer, W. C., O’Brien, T., Cha, B., Albeg, A.,
Treinin, M. and Miller, D. M. (2010). Time-lapse imaging and cell-specific
expression profiling reveal dynamic branching and molecular determinants of a
multi-dendritic nociceptor in C. elegans. Dev. Biol. 345, 18-33.
Smith, C. J., O’Brien, T., Chatzigeorgiou, M., Spencer, W. C., Feingold-Link, E.,
Husson, S. J., Hori, S., Mitani, S., Gottschalk, A., Schafer, W. R. et al. (2013).
Sensory neuron fates are distinguished by a transcriptional switch that regulates
dendrite branch stabilization. Neuron 79, 266-280.
Struhl, G. and Basler, K. (1993). Organizing activity of wingless protein in
Drosophila. Cell 72, 527-540.
Sudarsan, V., Anant, S., Guptan, P., VijayRaghavan, K. and Skaer, H. (2001).
Myoblast diversification and ectodermal signaling in Drosophila. Dev. Cell 1,
829-839.
Sugimura, K., Satoh, D., Estes, P., Crews, S. and Uemura, T. (2004).
Development of morphological diversity of dendrites in Drosophila by the BTBzinc finger protein abrupt. Neuron 43, 809-822.
Terriente, J., Perea, D., Suzanne, M. and Dı́az-Benjumea, F. J. (2008). The
Drosophila gene zfh2 is required to establish proximal-distal domains in the wing
disc. Dev. Biol. 320, 102-112.
Tsubouchi, A., Caldwell, J. C. and Tracey, W. D. (2012). Dendritic filopodia,
Ripped Pocket, NOMPC, and NMDARs contribute to the sense of touch in
Drosophila larvae. Curr. Biol. 22, 2124-2134.
Wu, S., Liu, Y., Zheng, Y., Dong, J. and Pan, D. (2008). The TEAD/TEF family
protein Scalloped mediates transcriptional output of the Hippo growth-regulatory
pathway. Dev. Cell 14, 388-398.
Xiang, Y., Yuan, Q., Vogt, N., Looger, L. L., Jan, L. Y. and Jan, Y. N. (2010). Lightavoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature
468, 921-926.
Yan, Z., Zhang, W., He, Y., Gorczyca, D., Xiang, Y., Cheng, L. E., Meltzer, S., Jan,
L. Y. and Jan, Y. N. (2013). Drosophila NOMPC is a mechanotransduction
channel subunit for gentle-touch sensation. Nature 493, 221-225.
Ye, B., Zhang, Y., Song, W., Younger, S. H., Jan, L. Y. and Jan, Y. N. (2007).
Growing dendrites and axons differ in their reliance on the secretory pathway. Cell
130, 717-729.
Ye, B., Kim, J. H., Yang, L., McLachlan, I., Younger, S., Jan, L. Y. and Jan, Y. N.
(2011). Differential regulation of dendritic and axonal development by the novel
Kruppel-like factor Dar1. J. Neurosci. 31, 3309-3319.
Yeo, S. L., Lloyd, A., Kozak, K., Dinh, A., Dick, T., Yang, X., Sakonju, S. and
Chia, W. (1995). On the functional overlap between two Drosophila POU homeo
domain genes and the cell fate specification of a CNS neural precursor. Genes
Dev. 9, 1223-1236.
Zecca, M. and Struhl, G. (2010). A feed-forward circuit linking wingless, fatdachsous signaling, and the warts-hippo pathway to Drosophila wing growth.
PLoS Biol. 8, e1000386.
Zhang, L., Ren, F., Zhang, Q., Chen, Y., Wang, B. and Jiang, J. (2008). The TEAD/
TEF family of transcription factor Scalloped mediates Hippo signaling in organ
size control. Dev. Cell 14, 377-387.
Zheng, Y., Wildonger, J., Ye, B., Zhang, Y., Kita, A., Younger, S. H., Zimmerman,
S., Jan, L. Y. and Jan, Y. N. (2008). Dynein is required for polarized dendritic
transport and uniform microtubule orientation in axons. Nat. Cell Biol. 10,
1172-1180.
Zlatic, M., Li, F., Strigini, M., Grueber, W. and Bate, M. (2009). Positional cues in
the Drosophila nerve cord: semaphorins pattern the dorso-ventral axis. PLoS Biol.
7, e1000135.
DEVELOPMENT
RESEARCH ARTICLE
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.
Development • Supplementary information
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.