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Myosin XV is a negative regulator of signaling filopodia
during long-range lateral inhibition
Rhiannon Clements1*, Tyler Smith1*, Luke Cowart1, Jennifer Zhumi1, Alan Sherrod1,
Aidan Cahill1, Ginger L Hunter1‡,
1Department of Biology, Clarkson University, Potsdam, NY, 13699, USA
*Equally contributing authors. ‡Correspondence: ghunter@clarkson.edu
Abstract
The self-organization of cells during development is essential for the formation of
healthy tissues, and requires the coordination of cell activities at local scales.
Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that
allow cells to engage in contact mediated signaling at a distance. While signaling
filopodia have been shown to support several signaling paradigms during development,
less is understood about how these protrusions are regulated. We investigated the role
of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling
filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly
Drosophila melanogaster. We found that Myosin XV is required for regulating signaling
filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout
the patterning epithelium. We found that Myosin XV is required for limiting the length
and number of signaling filopodia generated by bristle precursor cells. Cells with
additional and longer signaling filopodia due to loss of Myosin XV are not signaling
competent, due to altered levels of Delta ligand and Notch receptor along their lengths.
We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as
promote the ability of signaling filopodia to engage in long-range Notch signaling. Since
Myosin XV is present across several vertebrate and invertebrate systems, this may have
significance for other long-range signaling mechanisms.
Introduction
The formation of robust and reproducible patterns is essential during development.
Initially unordered precursor cells can be organized to form simple spot arrays, stripes,
or more complex 3-dimensional structures like intestinal villi or limbs. The incorrect
spatial or temporal organization of cells during development can lead to catastrophic
defects in organ function or early embryonic lethality. Generally, the patterning of cells
requires input from morphogenetic cues that can be distributed in both a passive and
active manner throughout the tissue. Passive processes include the short range diffusion
of signaling molecules (Stapornwongkul et al., 2020). Active processes include the
distribution of signaling molecules via cytonemes, or signaling filopodia (Kornberg and
Roy, 2014; Zhang and Scholpp, 2019). Cytonemes are long (>2µm), thin (≈200nm),
actin-based projections that support the delivery of signaling molecules (Kornberg,
2017; Ram´ırez-Weber and Kornberg, 1999). Cytonemes are involved in numerous
patterning events during the development of the fruit fly Drosophila melanogaster
including: distributing Dpp/TGFβ(Roy et al., 2014) and Hh (Bischoff et al., 2013;
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Chen et al., 2017; Gonz´alez-M´endez et al., 2020) associated with wing disc development;
distribution of growth factors during mechanosensory bristle development (Peng et al.,
2012); supporting long-range Notch signaling (Huang and Kornberg, 2015; Hunter et al.,
2019); and Hh-mediated stem cell niche maintenance (Rojas-R´ıos et al., 2012). In
vertebrates, cytonemes are known to play a role in the distribution of Shh, Wnt, and
Notch (Eom et al., 2015; Hall et al., 2021; Hamada et al., 2014; Mattes et al., 2018;
Sanders et al., 2013; Stanganello et al., 2015) in developmental processes ranging from
pigment stripe formation, neural plate formation, and limb bud morphogenesis. While
active processes have been the focus of intense research across several signaling
paradigms, how changes in cell morphology affect the ability of cells to send or receive
signal are only beginning to be understood. There is evidence that feedback from
signaling mechanisms supported by cytonemes can stimulate the formation of longer or
additional cytonemes (de Joussineau et al., 2003; Snyder et al., 2015). Disruption of
cytoskeletal components that regulate cell shape can disrupt signaling output in
cytoneme-dependent processes (Cohen et al., 2010; Hunter et al., 2019). However, the
strategies used to disrupt the cytoskeleton are usually not specific to cytonemes, and
broadly affect sub-cellular processes that are dependent on actin and microtubules.
Very few tools to specifically perturb cytonemes exist (Zhang et al., 2021). Further
investigation is needed to gain insight into how the molecular mechanisms that regulate
cell shape changes coordinate with cell-cell signaling processes.
The sensory bristle pattern on the dorsal thorax of Drosophila melanogaster is a
model system for the study of pattern formation mediated by signaling filopodia
(Heitzler and Simpson, 1991; Collier et al., 1996; Cohen et al., 2010; Corson et al., 2017;
Hunter et al., 2019). Over several hours during pupal development, a regular and
well-spaced array of sensory bristle precursor cells is formed from a sheet of bipotential
epithelial cells. The selection of bristle precursor cells occurs through canonical
Notch-mediated lateral inhibition (Bray, 2016; Troost et al., 2015). When
transmembrane Delta ligand and Notch receptor proteins interact in trans-, the Notch
receptor is activated through two proteolytic cleavages that lead to the release and
translocation of the intracellular domain (NICD) to the nucleus. Once in the nucleus,
the NICD plays a context dependent role on transcription with its co-factors, including
Suppressor of Hairless (CBF1 or Lag-1) and Mastermind (Kopan and Ilagan, 2009).
During bristle patterning, cells with low levels of Notch activation allow the expression
of genes associated with a pro-neural fate, and commit to the bristle lineage. Cells with
high levels of Notch activation repress the pro-neural genes and commit to an epithelial
cell fate. In order to achieve the correct density of bristle precursor cells, a long-range
Notch-mediated lateral inhibition signal is required (+1-2 cell diameters)(Cohen et al.,
2010; Collier et al., 1996; Corson et al., 2017; Hadjivasiliou and Hunter, 2022). Long,
actin-rich protrusions are formed on the basal surface of thoracic epithelial cells during
patterning stages (de Joussineau et al., 2003; Cohen et al., 2010; Hunter et al., 2019;
Renaud and Simpson, 2001; Georgiou and Baum, 2010). These protrusions are dynamic
and allow cells to contact neighbors 2-3 cell diameters away. Several regulators of the
actin cytoskeleton are required to form the basal signaling filopodia, including SCAR,
Rac, cdc42, and non-muscle myosin II (Cohen et al., 2010; Georgiou and Baum, 2010;
Hunter et al., 2019). Long-range contact between two or more basal signaling filopodia
carrying Notch receptor or Delta ligand should lead to the activation of cell surface
Notch, however, direct evidence for this mechanism has yet to be obtained.
A key factor in the generation of actin-based cell protrusions such as cytonemes are
the actin binding proteins that organize the cytoskeleton. Among the actin-binding
myosin motors that play a role in regulating cellular protrusions are the unconventional
class of MyTH4-FERM domain containing myosins, including Myosin VIIa, VIIb, X,
and XV (Sellers, 2000; Weck et al., 2017). Mutations in the human genes that encode
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Myosin VIIa and XV are associated with congenital forms of deafness (Hasson et al.,
1995; Wang et al., 1998; Weil et al., 1995). Mammalian Myosin VIIa and Myosin XV
play roles in maintaining the function of stereocilia: VIIA maintains the integrity of the
tip complex to help organize the characteristic ‘staircase’ organization of inner ear hair
cells (Boeda, 2002; Self et al., 1998), where as XV trafficks actin regulators to stereocilia
tips that help control the structure’s length (Belyantseva et al., 2005; Manor et al.,
2011). Mammalian Myosin VIIb is involved in the maintenance and formation of
microvilli (Weck et al., 2016), while Myosin X is closely associated with the formation
and function of filopodia (Berg and Cheney, 2002; Heimsath et al., 2017). Notably,
Myosin X has also been shown to play a role in the trafficking of signaling molecules in
cytonemes, as well as in the formation cytonemes (Hall et al., 2021; Snyder et al., 2015).
The Drosophila melanogaster genome encodes three unconventional Myosins: VIIa,
VIIb, and XV. In the fly, loss of Myosin VIIa causes deafness (Todi et al., 2005) and
disrupts the formation of other actin-based projections during development (Sallee et
al., 2021). It is currently unknown what effect the loss of Myosin VIIB has on cell
morphology and development (encoded by the gene myosin 28B1, FBgn0040299). Loss
of Drosophila Myosin XV leads to defects in cell sheet migration during embryogenesis
(Liu et al., 2007), as well as defects in the elongation of sensory bristles, which require
parallel bundles of actin filaments (Rich et al., 2021). Interestingly, the Drosophila
genome lacks a gene that encodes Myosin X. Despite a lack of Myosin X, which
supports cytoneme signaling in vertebrates, there are many morphogenetic events that
rely on the activity of cytonemes and filopodia in Drosophila (Bischoff et al., 2013;
Cohen et al., 2010; Huang et al., 2019; Huang and Kornberg, 2015; Hunter et al., 2019;
Millard and Martin, 2008; Ram´ırez-Weber and Kornberg, 1999). This suggests that, in
the fly, there are alternative pathways to supporting the formation of, and trafficking
within, these cellular protrusions.
In this study, we investigate the mechanisms that regulate the morphology and
dynamics of signaling filopodia and how these behaviors contribute to the progression of
Notch-mediated spot patterning. We show that the formation of basal signaling
filopodia in the notum requires the activity of Myosin XV. We further investigate the
requirement for Myosin XV in the sub-cellular distribution of Notch and Delta in bristle
precursor cells. To understand better the mechanisms by which Myosin XV promotes
signaling filopodia length, we investigate which of the motor domains are required for
full-length filopodia formation and Delta localization. Together, our data sheds light on
the mechanisms that regulate the activity and formation of signaling filopodia as well as
a new role for the unconventional myosin motor Myosin XV.
Materials and methods
Fly husbandry
Drosophila stocks were maintained on standard Drosophila food (JazzMix, Fisher
Scientific), at 18°C with 24 hour light cycle. Crosses are maintained at room
temperature (23-25°C). White pre-pupae were screened against balancers and then aged
at 18°C for 24 hours in humidified chambers. Pupae aged to 12-14 hours after
pupariation were then dissected for live or fixed imaging protocols.
Immunofluorescence
Dissected nota of 12-14 hAP pupae were fixed for 20 minutes in 4%
paraformaldehyde/1X PBS solution. Tissues were then blocked in 1:1 blocking buffer
(5% w/v BSA, 3% FBS in 1X PBS) in 1X PBST at room temperature for 1 hour.
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Tissues were then incubated in primary antibody with 5% blocking buffer in 1X PBST
for 2 hours at room temperature or 4°C overnight. Tissues were washed twice in 1X
PBST for 10 minutes each at room temperature, followed by incubation with secondary
antibody in 1X PBST containing fluorescently-labeled phalloidin and DAPI, for 2 hours
at room temperature or 4°C overnight. Tissues were washed twice in 1X PBST for 10
minutes each, then equilibrated in 50% glycerol overnight. Nota were then mounted on
coverslips and sealed with nail varnish for storage at 4°C until imaged. The following
primary antibodies were used in this study: chicken anti-GFP (EDMillipore 1:1000),
mouse anti-NotchECD (C458.2H, DSHB, 1:250), mouse anti-Delta (C594.9B, DSHB,
1:250). The following secondary antibodies were used in this study: AlexaFluor 488
donkey anti-chicken (Jackson Immunological, 1:2000), AlexaFluor 647 anti-mouse. The
following stains were also used: ActiStain Phalloidin (Phdh1, Cytoskeleton Inc, 1:500),
DAPI (Thermoscientific, 1:1000).
Imaging
Samples were imaged on a either a Lieca DMi8 SPE confocal microscope using LASX
software, or Nikon C2+ or EclipseTi confocal microscopes using NIS Elements. The
pupal cases of live 12-14 hAP pupae were removed, exposing the head and thorax. A
coverslip coated with a thin film of Halocarbon 27 oil (Sigma) was placed on top of
spacers such that only the dorsal thorax contacted the coverslip (as previously published
(Loub´ery and Gonz´alez-Gait´an, 2014)). Live pupae were imaged using a x40 (0.8 NA)
air objective (SPE) or or x60 (1.4 NA) oil objective (EclipseTi). Fixed tissues were
imaged on the SPE using a x40 (1.15NA), x63 (1.3 NA) oil objectives, and on the Nikon
C2+ using a x40 (1.3 NA) oil objective.
Quantification and statistical analysis
All image analysis was performed in FIJI/ImageJ. Time-lapse images of SOP cell
signaling filopodia were acquired and lengths were measured using FiloQuant and
Trackmate plugins in FIJI/ImageJ (Jacquemet et al., 2019). Number of filopodia per
cell over either short windows of time or in freeze frames were quantified manually.
N
sfGFP
nuclear fluorescence was measured from 12h AP to nuclear envelope breakdown
(NEBD), as previously described (Hunter et al., 2016). Briefly, an ROI was drawn in
the nuclei of an NsfGFP expressing cell, and the average fluorescence intensity within
that ROI was reported for each time frame of the time-lapse. At the point of NEBD, a
nucleus is no longer distinguishable using NsfGFP and measurements are stopped.
Statistical analysis was performed using GraphPad Prism. Specific statistical tests are
described in the figure legends.
Drosophila stocks
The following stocks used in this study are available at the Bloomington Drosophila
Stock Center (BDSC, Bloomington, Indiana): UAS-Myosin XV RNAi, w1118,
UAS-white RNAi, UAS-Myosin VIIa RNAi, UAS-Myosin VIIb RNAi. We also used the
Myosin XV allele syph21J/FM7Ci,Act5c-GFP (Rich et al., 2021). The following GAL4
background lines were used, that were previously published elsewhere: NsfGFP,
neuralized::H2BmRFP/CyO-GFP; neuralized-GAL4/TM6B (Hunter et al., 2016).
shotgunGFP, neur-GFP-moesinCA/CyO-GFP; pannier-GAL4/TM6B and
neuralized-GAL4, UAS- GFP-moesinCA/TM6B (Cohen et al., 2010). The following
UAS lines were also used, that were previously constructed and published in (Rich et al.,
2021): Sp/CyO; UAS-syphGFP ∆FERM(2)/TM6B, UAS-syphRFP, UAS-syphGFP
∆FERM(1)/CyOGFP, UAS-syphGFP
R213A, G434A
/CyOGFP, UAS-syphGFP/CyOGFP,
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UAS-syphGFP ∆MyTH4(2)/CyOGFP, UAS-syphGFP ∆motor/TM6BTb,
UAS-syphGFP ∆MyTH4 (1, 2), UAS-syphGFP ∆cargo, UAS-syphGFP ∆FERM(1)-
∆MyTH4(2)- ∆FERM(2), UAS-syphGFP ∆MyTH4(2)-∆FERM(2), and UAS-syphGFP
∆MyTH4(1).
Results
Loss of Myosin XV leads to increased bristle density.
Disruptions to the morphology or dynamics of signaling filopodia have been shown to
lead to changes in the overall density and organization of bristle precursor cells at the
end of bristle pattern formation (Cohen et al., 2010; Hadjivasiliou et al., 2016; Hunter et
al., 2019). Specifically, conditions that generate shorter or longer signaling filopodia are
predicted to lead to more or less dense bristle patterns, respectively. The unconventional
MyTH4-FERM myosins function as plus-end directed motors on bundled actin
structures (Sellers, 2000). This class of myosins are implicated in both trafficking of
proteins along protrusions, as well as in their formation (Fitz et al., 2023; Weck et al.,
2017). To determine if any of these myosins are important for signaling filopodia
activity, we used an RNAi-based strategy to knockdown their expression throughout the
central notum region using pannier-GAL4 (pnr-GAL4). We targeted the expression of
Myosin VIIa, Myosin VIIb and Myosin XV using UAS-inducible shRNA transgenics
(RNAi). The Drosophila genome does not contain Myosin X, which is the fourth major
myosin of this group in mammalian organisms. The thorax bristle pattern is complete
by 24 hours after pupariation (hAP). We hypothesized that pnr-GAL4 mediated
knockdown of any MyTH4-FERM myosin that functions in signaling filopodia behaviors
would disrupt the density of the final bristle pattern. We found that loss of Myosin XV
leads to an increase in the density of bristle precursor cells by 24 hAP (Figure 1A-B).
Fig 1. The MyTH4-FERM Myosin XV plays a role in bristle density. (A)
RNAi targeting Myosin XV (MyoXV), Myosin VIIa (MyoVIIa), Myosin VIIb
(MyoVIIb) or white (control) were expressed in the central notum using the
pannier-GAL4 driver. Tissues also express E-cadherin GFP to label all cell boundaries
and neuralized-GFP-moesin
CA
to label bristle precursor cells. Images are from 24 hAP
pupae. Scale bar, 50 µm. Anterior (A), Posterior (P). Midline is labeled in control (pink
dashed line) and all images are oriented similarly. (B) Quantification of bristle density
in RNAi-expressing 24 hAP pupae. One-way ANOVA with multiple comparisons
(Dunnett’s) was performed. Ns = not significant. 7 ROI were analyzed across a
minimum of 4 pupae for each genotype. Individual data points (circles) with mean ±
SD (overlay) shown.
We did not observe any changes in the density of bristles in Myosin VIIa or VIIb
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RNAi expressing tissues (Figure 1A-B). Therefore, we focused on the role of Myosin XV
in long-range lateral inhibition. In Drosophila, Myosin XV is an approximately 330kDa
protein encoded by the gene myo10a which is also called sisyphus (syph). Note, 10a
refers to the cytogenetic position of the gene on the X-chromosome.
Loss of Myosin XV disrupts the development of the bristle
pattern.
The formation of the notum bristle pattern takes several hours over pupal stages (Cohen
et al., 2010; Corson et al., 2017). We next asked how the development of pattern at
earlier stages (14 hAP) in Myosin XV RNAi expressing tissues was disrupted. We
visualized the selection of bristle precursor cells using neuralized promoter driven
GFP-moesinCA, which drives an GFP-tagged moesin based F-actin reporter (GMCA;
Edwards et al., 1997) in bristle precursor cells only (Figure 2A-A’). We simultaneously
used pnr-GAL4 to express Myosin XV RNAi throughout the central notum epithelium.
We found that decreased Myosin XV throughout the nota leads to an over-production of
GFP-positive cells compared to controls (Figure 2B-B’). We also observe that the
development of bristle rows 2-4 are disorganized relative to controls. Since neuralized is
primarily expressed in cells that are Notch inactive, we interpret this to mean that loss
of Myosin XV leads to a disruption in timely Notch signaling.
Fig 2. Decreased expression of Myosin XV leads to defects in bristle
precursor organization during patterning. (A) Control pupae (pannier-GAL4 >
UAS-white RNAi) expressing neuralized-GFP-moesin
CA
to label bristle precursor cells,
14 hAP. Scale bar, 25 µm. Midline is labeled in control (pink dashed line), and all
images are oriented similarly. Anterior, left; Posterior, right. Bristle rows are labeled
1-4. (A’) Control pupae with same genotype and timing as (A), zoomed in to visualize
cell morphology. Scale bar 10 µm. (B) Myosin XV RNAi pupae (pannier-GAL4 >
UAS-myosin XV RNAi) expressing neuralized-GFP-moesin
CA
to label bristle precursor
cells, 14 hAP. Scale bar, 50 µm. (B’) Pupae with same genotype and timing as (B),
zoomed in to visualize cell morphology. Scale bar, 10 µm. (C) Hemizygous males
carrying the syph
21J
allele, and neuralized-GAL4
>
UAS-GFP-moesin
CA
to label bristle
precursor cells, 14 hAP. Scale bar, 50 µm. (C’) Pupae with same genotype and timing
as (C), zoomed in to visualize cell morphology. Scale bar 10 µm. (D) Quantification of
24 hAP bristle density in hemizygous male escapers carrying the syph
21J
allele (21J/Y),
compared to controls (neuralized-GAL4 >UAS-GFP-moesinCA). n = 6 ROIs across 3
21J/Y animals and 8 ROIs across 4 control animals. Individual data points (circles) with
mean ±SD (overlay) shown. Significance was determined by unpaired student’s t-test.
To verify our findings with Myosin XV RNAi, we next observed patterns in pupae
carrying the FRT-mediated knockout allele of Myosin XV, syph21J (Rich et al., 2021).
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The entire coding region of Myosin XV is removed in this mutant. We co-expressed
neur-GAL4, UAS-GMCA to label bristle precursor cells, imaged live male pupae
hemizygous for the syph21J mutation, and observed their bristle patterns at 14 hAP
(Figure 2C-C’). Both RNAi knockdown and the null allele have severe developmental
defects that lead to death during larval stages (data not shown)(Rich et al., 2021).
Compared to control males, GFP-positive cells in syph21Jmutant pupae are often
grouped along rows rather than isolated (Figure 2C). Those cells that are isolated are
closer to each other than controls (21.0 ±8.0 µm across n =55 syph21J cell pairs,
compared to 29.3 ±8.4 µm across n = 43 control cell pairs; p<0.0001 by student’s
t-test). Consistent with the observation that knockdown of Myosin XV by RNAi leads
to increased bristle density, hemizygous syph21J pupae also have increased bristle
density relative to controls (Figure 2D). The signaling filopodia of syph
21J
mutant cells
appear indistinguishable from wildtype (Figure 2C’). In contrast to Myosin XV RNAi
expressing pupae, we find that syph21J pupae sometimes have developmental delays
such that entire bristle rows have not yet appeared by 14 hAP (Figure 2C). Altogether,
our analysis of tissue-wide RNAi-mediated knockdown or a null allele of Myosin XV
show that wildtype Myosin XV plays a role in the long-range lateral inhibition
mechanism that regulates the well-spaced array of sensory bristles on the dorsal thorax.
Decreased expression of Myosin XV in bristle precursors
increases filopodia length and number.
The spacing of bristle precursor cells depends on a dynamic length determinant, in part
facilitated by the dynamics of basal signaling filopodia (Cohen et al., 2010; Corson et
al., 2017; Hadjivasiliou et al., 2016). We next asked whether loss of Myosin XV caused
defects specifically associated with the morphology or behavior of signaling filopodia.
Based on our quantification of bristle density, we hypothesized that decreased Myosin
XV expression leads to shorter and fewer signaling filopodia. Since syph
21J
mutants and
pnr-GAL4 driven RNAi pupae do not produce large amounts of samples due to
increased lethality at earlier stages, we next used neuralized-GAL4 to express
UAS-Myosin XV RNAi in bristle precursor cells only, while co-expressing UAS-GMCA
to visualize cell shape and filamentous actin structures. We observe that signaling
filopodia in bristle precursor cells expressing Myosin XV RNAi appear morphologically
indistinguishable from control signaling filopodia (Figure 3A-B).
To quantify the formation and dynamics of signaling filopodia over time, we used
FiloQuant to automatically track and measure filopodia in bristle precursor cells
(Jacquemet et al., 2019). We found that signaling filopodia in Myosin XV knockdown
cells are longer (8.7 ±2.6 µm, n = 272 filopodia) compared to control cells (7.1 ±1.9
µm, n = 38 filopodia)(Figure 3D). We also observed more filopodia in Myosin XV
knockdown cells (27.2 ±5.4 filopodia per cell, n = 10 cells, N = 4 pupae) compared to
control cells (17.9 ±2.6 filopodia per cell, n = 8 cells, N = 3 pupae) (Figure 3E).
Consistent with this result, at 14 hAP syph
21J
mutant bristle precursors also have more
filopodia (12.0 ±2.8 filopodia per cell, n = 11 cells, N = 3 pupae) relative to controls
(7.3 ±2.1 filopodia per cell, n = 14 cells, N = 3 pupae)(Figure 3F) Given these results,
we hypothesized that over-expression of Myosin XV could be sufficient to generate fewer
or shorter filopodia. Surprisingly, we found that over-expression of full length Myosin
XV does not change the length (7.9 ±2.7 µm, n = 36 filopodia) or number of signaling
filopodia (16.4 ±3.6 filopodia per cell, n = 9 cells, N = 3 pupae) (Figure 3C-E). Taken
together, these results suggest that Myosin XV is a negative regulator of signaling
filopodia length and formation.
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Fig 3. Myosin XV negatively regulates signaling filopodia length and
number. (A-C) Stills from movies used to quantify data in (D-E). (A) Myosin XV
RNAi = neur-GAL4 >UAS-GMCA, UAS-Myosin XV RNAi pupae, at 14 hAP. (B)
Control = neur-GAL4
>
UAS-GMCA, UAS-white RNAi pupae, at 14 hAP. (C) Myosin
XV GFP = neur-GAL4 >UAS-GMCA, UAS- GFP full length Myosin XV pupae, at
14h AP. Scale bars on all images, 10 µm. (D) Quantification of filopodia length
according to genotypes as in (A-C). (E) Quantification of filopodia produced per cell
over 15 minutes, according to genotypes as in (A-C). One-way ANOVA with multiple
comparisons (Dunnett’s) was performed. Ns = not significant. (F) Quantification of
filopodia per cell (1 frame), in hemizygous syph21J and control pupae. n = 11 cells
across 3 21J/Y animals and 14 cells across 3 control animals. Significance was
determined by unpaired student’s t-test. For (D-F), individual data points (circles) with
mean ±SD (overlay) shown. Minimum of 3 pupae were imaged and analyzed per
genotype.
Myosin XV localizes along the lengths of signaling filopodia.
Our results thus far are consistent with Myosin XV playing a role in the formation and
dynamics of signaling filopodia during notum patterning. We next wanted to determine
the sub-cellular localization of Myosin XV in bristle precursor cells. Mammalian Myosin
XV plays a major role in the formation and maintenance of mechanosensory stereocilia
and localizes to the tips of these bundled actin structures (Belyantseva et al., 2005;
Manor et al., 2011). In elongating sensory bristles (≈33 hAP), Drosophila Myosin XV
localizes along the length and at the tips of developing bristles (Rich et al., 2021). In
embryos and in insect cell culture, Myosin XV localizes along the length and at the tips
of filopodia (Liu et al., 2007). Since no antibody currently exists to visualize endogenous
Drosophila Myosin XV, we instead over-expressed RFP-tagged full length Myosin XV in
live bristle precursor cells. We co-expressed GMCA in these cells to simultaneously
visualize F-actin. We observed localization of Myosin XV along signaling filopodia, as
well as their tips (arrowheads, Figure 4). This result is consistent with our knockdown
data, suggesting that Myosin XV plays a role in signaling filopodia dynamics.
Decreased expression of Myosin XV dampens Notch signaling
activity.
The specification of bristle precursor cells in the initially unpatterned notum is driven
by Notch-mediated lateral inhibition. Long-range Notch signaling is facilitated by the
activity of signaling filopodia. Based on our results showing that decreased Myosin XV
leads to altered filopodia length and number, and that the overall patterning is altered
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Fig 4. Localization of Myosin XV in bristle precursor cell signaling filopodia.
(A-D) Stills from four individual bristle precursor cells across 3 pupae expressing
neuralized-GAL4
>
UAS-GMCA, UAS-RFP-Myosin XV. Scale bars on all images, 5 µm.
Arrowheads indicate RFP puncta within a cellular projection.
in these tissues, we hypothesized that decreased levels of Myosin XV in signal sending
cells (bristle precursors) would disrupt Notch activation in neighboring epithelial cells,
relative to controls. To test this hypothesis, we expressed Myosin XV RNAi in bristle
cells only using neuralized-GAL4, in a notum epithelium that expresses the NsfGFP
Notch transcriptional reporter (Figure 5A). This reporter expresses a nuclear localized,
PEST-tagged, superfolder GFP under the control of a Notch responsive promoter
(Hunter et al., 2016). We then tracked sfGFP fluorescence in nuclei of cells adjacent to
or one cell diameter distant from the nearest bristle precursor cell. We term these cells
adjacent or distant epithelial cells, respectively (see Figure 5B-B’ insets). Adjacent cells
can engage in Notch signaling with a bristle precursor via both large cell-cell interfaces
and signaling filopodia. In contrast, distant cells can only engage in Notch signaling
with a bristle precursor via signaling filopodia. Importantly, by using neuralized-GAL4,
all adjacent and distant epithelial cells express wildtype levels of Myosin XV; only
bristle precursor cells see Myosin XV knockdown. We tracked nuclear fluorescence levels
from 12 hAP until nuclear envelope breakdown (NEBD), which occurs between 15-20
hAP. Loss of Myosin XV in bristle cell precursors alone leads to a decreased Notch
response in all wildtype neighboring epithelial cells (Figure 5B-B’). These results are
consistent with our observation of increased GFP-positive cells during patterning stages
(Figure 2B), as an increase in cells with activation of neuralized expression indicates a
decrease in Notch activation. Together, this data shows that Myosin XV plays role in
the activation of robust Notch signaling both in adjacent and distant cells, both of
which may signal to bristle precursor cells via signaling filopodia.
Myosin XV plays a role in the localization of Delta and Notch to
signaling filopodia.
Our results support a model in which Myosin XV indirectly supports long-range
signaling through the regulation of filopodia number and length. However, it remains
unclear why the formation of more and longer signaling filopodia would lead to a
decrease in Notch response in signal receiving cells, and increased bristle density. We
hypothesized that although more signaling filopodia are being formed in Myosin XV
RNAi expressing cells, these signaling filopodia are not necessarily competent to signal.
In other filopodia and stereocilia, Myosin XV plays a role in trafficking proteins along
bundled actin structures towards the tip (Belyantseva et al., 2005; Manor et al., 2011;
Weck et al., 2017). It is currently unknown if Myosin XV directly interacts with Notch
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Fig 5. Myosin XV is required for robust Notch signaling response. (A)
Montage from a control pupae expressing the Notch transcriptional reporter
NsfGFP(grayscale). Anterior, left; Posterior, right. Yellow line in 12h AP image
indicates the animal midline. Notch response in epithelial cells peaks 14-16 hAP,
immediately followed by mitosis 16-18 hAP, and termination of Notch signaling by 20-24
hAP. Scale bar, 50 µm. (B-B’) Nuclear GFP fluorescence in signal receiving cells (B)
adjacent to bristle precursor cells or (B’) distant from bristle precursor cells. Inset
cartoons illustrate cell position (*) relative to bristle precursor cell (b) for each group.
GFP signal is normalized to initial fluorescence level. All time-lapse measurements were
aligned such that time = 0 minutes at nuclear envelope breakdown (entry into mitosis).
Myosin XV RNAi (orange): 50 nuclei of each position (adjacent or distant) were
measured across 3 pupae. Control (black): 20 nuclei of each position (adjacent or
distant) were measured across 3 pupae. Mean ±SEM shown.
or Delta.
To address our hypothesis, we investigated the levels and localization of Delta ligand
and Notch receptor in cells with wildtype or decreased expression of Myosin XV. In the
notum, apical Delta and Notch are localized primarily to the apical cell-cell junctions
(Bellec et al., 2020; Benhra et al., 2010). Cytoplasmic puncta staining positive for Delta
or Notch can also be observed in bristle precursor cells. The cytoplasmic puncta may
represent: (1) endocytosed Delta ligand and trans-endocytosed Notch extracellular
domain (NECD) post-cleavage, or (2) Delta and Notch being trafficked to the bristle
precursor cell surface. Additionally, all cells in the notum are expected to express at
least low levels of both ligand and receptor (Collier et al., 1996). On the basal surface,
Delta and Notch puncta are associated with signaling filopodia, as discrete puncta,
often at the tips of signaling filopodia (Figure 6A-B) (Hunter et al., 2019). In control
cells, we observe Delta positive puncta within the cell body and, to a lesser extent,
within filopodia (Figure 6A). NECD positive puncta are also visible both in the cell
body and within filopodia (Figure 6B). We do not observe any difference between the
number of Delta (5.2 ±2.0 puncta, n = 11 cells) and Notch (5.1 ±2.3 puncta, n = 34
cells; n.s. by student’s t-test) positive puncta in control bristle precursor cells.
Importantly, this result does not distinguish whether the Delta or Notch positive puncta
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are a result of signaling between adjacent neighbors or distant neighbors.
Fig 6. Myosin XV is required for the balance of Delta and Notch in
filopodia. (A-D) Example of fixed images used to generate data in (E-H). Control
pupae of the genotype neur-GAL4 >UAS-GMCA, UAS white RNAi stained with
anti-GFP (green) and (A) anti-Delta antibody (white) or (B) anti-Notch extracellular
domain (NECD, white). Myosin XV knockdown pupae of the genotype neur-GAL4 >
UAS-GMCA, UAS Myosin XV RNAi stained with anti-GFP (green) and (C) anti-Delta
antibody (white) or (D) anti-NECD (white). Magenta arrowheads indicate Delta or
NECD puncta on signaling filopodia. Scale bars for all images, 5 µm. (E)
Quantification of Delta puncta on signaling filopodia in control (0.42 ±0.5 puncta, n =
48 filopodia in 13 cells across 4 pupae) and Myosin XV RNAi (0.21 ±0.5 puncta, n =
72 filopodia in 16 cells across 4 pupae) expressing bristle precursor cells. (F)
Quantification of NECD puncta on signaling filopodia in control (0.32 ±0.5 puncta, n
= 62 filopodia in 15 cells across 4 pupae) and Myosin XV RNAi (0.54 ±0.6 puncta, n
= 59 filopodia in 17 cells across 4 pupae) expressing bristle precursor cells. (G) Total
Delta puncta per cell in control (5.2 ±2.0 puncta, n = 11 cells across 4 pupae) and
Myosin XV RNAi (8.9 ±3.6 puncta, n = 37 cells across 11 pupae) expressing bristle
precursor cells. (H) Total NECD puncta per cell in control (5.1 ±2.3 puncta, n = 34
cells across 12 pupae) and Myosin XV RNAi (6.6 ±2.3 puncta, n = 24 cells across 8
pupae) expressing bristle precursor cells. For (E-H), individual data points shown
(circles). Mean ±SD is overlain in (G-H). Mean ±SD reported in legend above, and
significance was determined by unpaired student’s t-test.
To determine if the expression level of Myosin XV plays a role in the localization of
ligand and receptor in bristle precursor cells, we performed immunofluorescence staining
for Delta or NECD in tissues with bristle precursor cells labeled with GFP
(neuralized-GAL4, UAS-GMCA) and co-expressing UAS-Myosin XV RNAi (Figure
6C-D). Consistent with our live filopodia tracking results, we observe an increase in the
average number of signaling filopodia in bristle precursor cells expressing Myosin XV
RNAi (8.6 ±2.7 filopodia per cell, n = 61 cells) compared to controls (6.5 ±2.1
filopodia per cell, n = 45 cells; p
<
0.0001 by student’s t-test). Signaling filopodia were
also longer in Myosin XV RNAi cells (7.0 ±2.6 µm, n = 110 filopodia) compared to
controls (6.4 ±1.9 µm, n = 131 filopodia; p = 0.03 by student’s t-test), although both
were shorter than measured in live cells, which may be in part due to fixation methods.
Bristle precursor cells expressing Myosin XV RNAi exhibited a misbalance in Delta
and NECD positive puncta throughout the cell body and filopodia (Figure 6E-H). First,
we analyzed the number of Delta or Notch puncta along signaling filopodia. We observe
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fewer Delta positive puncta in Myosin XV RNAi filopodia compared to controls (Figure
6E), and more Notch extracellular domain positive puncta in Myosin XV RNAi
filopodia compared to controls (Figure 6F). We find that control filopodia contain
roughly the equal numbers of Delta and NECD puncta along their lengths (n.s. by
student’s t-test). However we find that Delta puncta are underrepresented in Myosin
XV RNAi filopodia compared to NECD puncta in the same cell genotype (p=0.0005 by
student’s t-test). Next we compared the levels of Delta and NECD puncta throughout
the cell body (including filopodia). Myosin XV RNAi expressing cells exhibit more
Delta or NECD positive puncta throughout the cell body compared to controls (Figure
6G-H). Control bristle cells show no significant difference (student’s t-test) between
number of Delta or NECD puncta throughout the cells, that is, control cells have equal
amounts of Delta and NECD puncta throughout the cell body. We observe that Delta
puncta are over-represented in the Myosin XV RNAi expressing cell body compared to
NECD positive puncta (p = 0.006, student’s t-test). These data suggest that the
balance of Delta ligand mediated Notch activation is disrupted in bristle precursor cells
that express Myosin XV RNAi. The finding that there is decreased Delta puncta on
filopodia is also consistent with our findings that Notch signaling is decreased in
neighboring epithelial cells. Our interpretation of these data are that decreased Myosin
XV expression leads to the formation of increased, but not necessarily signaling
competent, signaling filopodia.
Myosin XV motor activity is required to promote signaling
filopodia formation.
Finally, we investigated how Myosin XV regulates both the morphology of signaling
filopodia and the localization of Delta. Full length Myosin XV comprises the following
domains: motor, IQ motifs, two FERM (band 4.1, ezrin, radixin, moesin) domains and
two MyTH4 (Myosin tail homology 4) domains (Rich et al., 2021)(Figure 7A). Notably,
Drosophila Myosin XV lacks the large N-terminal extension found in mammalian
Myo15. In order to determine the domains of Myosin XV are required for signaling
filopodia formation, we over-expressed GFP-tagged truncated Myosin XV constructs in
bristle precursor cells expressing neur-GAL4, UAS-GMCA. We analyzed the number of
filopodia formed by a bristle precursor cell in pupae at 14 hAP. Over-expression of the
domain deletion constructs does not lead to any gross morphological defects in bristle
precursor cell shape or the ability to form basal cytonemes (Figure S1). We did however
observe that expression of the ∆motor construct leads to nuclear GFP localization
(Figure 7C). Despite this, ∆motor expressing cells are still able to form signaling
filopodia, perhaps due to the presence of endogenously expressed Myosin XV. When we
analyzed the number of filopodia formed by cells over-expressing different Myosin XV
deletion domain constructs, we found that only expression of the point mutant Myosin
XVR213A, G434A led to the overproduction of signaling filopodia compared to control
cells over-expressing full length Myosin XV (Figure 7D-E). Myosin XV
R213A, G434A
is a
motor dead construct that changes key amino acid residues in switch I and II of the
head domain to Alanine (Rich et al., 2021). This construct may bind to filamentous
actin, but is unable to move along filaments. We did not observe any nuclear GFP
signal in Myosin XVR213A, G434A expressing cells, which may partially explain why the
two conditions have different phenotypes. Therefore, this finding is consistent with our
data showing that loss of Myosin XV expression increases filopodia number (Figure
3E-F). Over-expression of Myosin XV truncation mutants lacking all or parts of the
cargo domain did not affect the number of filopodia formed (Figure 7E).
We also analyzed whether over-expression of domain deletion constructs could
disrupt the number of Delta puncta in bristle precursor cell filopodia. We hypothesized
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Fig 7. Myosin XV motor activity is required for its effects on filopodia
length. (A) Organization of Myosin XV protein domains. (B-D) Example images used
to generate data in (D). See Supplement Figure S1 for additional panels.
Neuralized-GAL4 was used to co-express GFP tagged deletion domain or mutant
constructs listed, e.g., neur-GAL4 >UAS-GMCA, UAS-GFP- ∆motor-Myosin XV.
Pupae are stained with anti-GFP antibody. Scale bars for all images, 5 µm. (E)
Number of filopodia per bristle precursor cell overexpressing the listed Myosin XV
construct was quantified in fixed tissues. n = number of cells analyzed, in a minimum of
3 pupae per genotype. Individual data points shown (circles), with mean ±SD overlay.
One-way ANOVA with multiple comparisons (Dunnett’s) was performed. Individual
comparisons to full length Myosin XV were n.s. unless indicated.
that loss of specific cargo domains would lead to decreased Delta puncta in filopodia
due to failure to form trafficking complexes. However, we did not observe any
differences in the number of Delta puncta along filopodia relative to filopodia in cells
expressing full length Myosin XV control (Figure S1). One possibility for this result is
the presence of sufficient endogenous Myosin XV to traffick cargo to or within filopodia.
Altogether our results with the domain deletion constructs indicate that the motor
activity of Myosin XV is required for the negative regulation of signaling filopodia.
Discussion
Cell morphology plays an important role in the ability of cells in tissues to send and
receive signals during development. The formation and activity of cytonemes, or
signaling filopodia, are particularly interesting as they play an important role in the
active distribution of morphogens, and have been implicated in the development of
several tissues in vertebrates and invertebrates (Kornberg, 2017). Therefore it is
important to understand how changes in cell morphology affect the ability of cells to
send or receive signal. Here we identified a role for the Myosin XV in the regulation of
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signaling filopodia dynamics and Notch mediated lateral inhibition during bristle
patterning.
Of the three unconventional myosins present in the Drosophila genome, we identified
Myosin XV as a key regulator of bristle pattern density. Loss of Myosin XV expression
led to an increased bristle density, whereas loss of Myosin VIIa and VIIb did not. Based
on previous mathematical models (Cohen et al., 2010; Hadjivasiliou et al., 2016), we
anticipated that loss of Myosin XV was associated with shorter signaling filopodia
because decreasing the length scale of Notch signaling leads to patterns that are more
dense. We were then surprised to find that Myosin XV negatively regulates filopodia
length and number in bristle precursor cells during lateral inhibition stages. How might
decreased levels of Myosin XV lead to longer and increased numbers of signaling
filopodia? The best studied Myosin XV is mammalian Myo15, which localizes to
stereocilia in hair cells of the cochlea (Belyantseva et al., 2003; Fang et al., 2015).
Myo15, along with its binding partners Whirlin and Eps8, is associated with the
plus-ends of the bundled actin filaments at the tips of stereocilia (Belyantseva et al.,
2005; Manor et al., 2011; Mauriac et al., 2017; Moreland et al., 2021). Loss of Myo15 is
associated with shorter and increased numbers of stereocilia structures (Moreland et al.,
2021). Why loss of Myo15 leads to increased numbers of stereocilia is unclear. The
effect of Myosin XV on the elongation of signaling filopodia could be direct or indirect.
Mammalian Myo15 has been shown to directly participate in the nucleation of F-actin
filaments in vitro (Gong et al., 2022) and the association of the Myo15 motor domain
with the plasma membrane is sufficient to stimulate the formation of filopodia in cell
culture (Fitz et al., 2023). It is currently unknown if Drosophila Myosin XV has a
similar ability. In that case however, we would expect that loss of Myosin XV would
shorten signaling filopodia. A recent study showed that Drosophila Myosin XV
associates with MICAL, an enzyme that promotes the disassembly of filamentous actin
in elongating bristles (Rich et al., 2021). If Myosin XV were involved in the trafficking
of negative regulators to the tips of the filamentous actin bundles inside cytonemes,
then we might expect to see increased length when Myosin XV levels are decreased.
Currently, we do not know what proteins interact with Myosin XV in the patterning
notum.
If Myosin XV is a negative regulator of signaling filopodia and length, then why does
decreased Myosin XV expression result in a bristle pattern with increased bristle density
compared to controls? Our data indicate that that although more and longer signaling
filopodia are present, they may not be competent to signal. Several studies support a
role for signaling filopodia in long-range Notch signaling (Cohen et al., 2010; de
Joussineau et al., 2003; Hadjivasiliou et al., 2016). Non-adjacent cells contact each other
using filopodia, which have been shown to carry both Notch receptor and Delta ligand
(Hunter et al., 2019; Renaud and Simpson, 2001). Drosophila Myosin XV is not known
to directly interact with either Delta or Notch. However, a yeast two- hybrid assay for
potential interactors of the Myosin XV C-terminal tail identified Nedd4, an E3
ubiquitin ligase that is required for the internalization and inactivation of Notch (Liu et
al., 2007; Sakata et al., 2004). Our data shows that there are fewer Delta puncta but
more NECD puncta per filopodia in bristle precursor cells. Since bristle precursor cells
are Notch inactive, but are thought to have expressed low levels of Notch (Collier et al.,
1996), one possibility is that decreased Myosin XV levels leads to failure to
down-regulate Notch in bristle precursor cell signaling filopodia. Cis-interactions with
increased levels of Notch along filopodia and remaining Delta along filopodia could lead
to lower levels of Notch signaling in receiving cells due to decreased available surface
ligand (Sprinzak et al., 2011, 2010). Lowering the level of Notch activation in distant
cells, in turn, increases the possibility that those cells will undergo a cell fate switching
event. This would lead to the selection of too many bristle precursor cells, which is
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consistent with our findings that (1) lowered Myosin XV expression leads to a decreased
Notch response in epithelial cells distant from the bristle precursor cell, and (2) bristle
precursor cells are overproduced during active patterning stages.
In bristle precursor cells, we found that Myosin XV localizes at the tips and along
the length of signaling filopodia. The C-terminal tail of MyTH4-FERM domain
containing myosins is known to be important for interactions with cargo, cytoskeleton,
and membrane (Weck et al., 2017). The interactions in the tail domains impact the
ability of a myosin to interact with, and process along, bundled filamentous actin. We
were interested in determining what domains of Myosin XV were essential for the
formation of signaling filopodia and protein localization along their length. When we
over-expressed Myosin XV truncation constructs in bristle precursor cells, we did not
observe changes to levels of Delta puncta along filopodia relative to full length Myosin
XV. One possibility is that endogenous Myosin XV is able to traffick despite
over-expression of truncation constructs. It is not currently known if Myosin XV can, or
needs to, dimerize, like Myosin X (Lu et al., 2012) and other myosins. If Myosin XV
dimerizes, we might expect some of the truncation constructs, especially ∆motor, to act
as dominant negatives, similar to motor-less non-muscle myosin II (Franke et al., 2005).
Interestingly, previous work with these truncation constructs suggested that
over-expression of cargo-binding mutant forms of Myosin XV either have an
over-expression phenotype or function as dominant negatives during the extension of
sensory bristles (Rich et al., 2021). In future work, truncation constructs could be
expressed in Myosin XV mutant tissue clones, given the larval stage lethality associated
with syph21J and RNAi-knockdown. A second possibility is that a separate system is
responsible for distributing Delta into signaling filopodia. Microtubules have been
observed to extend into filopodia in cell culture and during axon guidance (Dent et al.,
2007; Schober et al., 2007), where they can influence filopodia movement. The presence
of microtubules and tip directed kinesin motors within signaling filopodia would be an
alternative to trafficking along bundled actin by tip directed Myosin motors. Previous
findings indicate that microtubules are dispensable for the formation of signaling
filopodia by bristle precursor cells, since treatment with the microtubule inhibitor
colchicine does not disrupt protrusion formation (Georgiou and Baum, 2010). However
this does not rule out a role for microtubules in the distribution of morphogens for
signaling. Further investigations will be needed to determine how Notch and Delta
proteins are targeted to, and trafficked along, signaling filopodia for long-range lateral
inhibition.
Signaling filopodia are an essential active cell mechanism of cell-cell signaling, and
have been shown to be important for several signaling paradigms, across many different
developing tissues, in both vertebrates and invertebrates. Despite the numerous
examples of the role of cytonemes in development, we still do not fully understand how
these structures are formed and regulated. Here, we have shown that Myosin XV plays
a role in the formation of signaling filopodia during lateral inhibition in patterning
epithelia, and that it is required for organization of Delta and Notch within the cell.
The mechanisms by which Myosin XV regulates signaling filopodia dynamics will be the
focus of future research. There is still much to learn about the organization and
dynamics of bundled actin filaments within cytonemes, the interactions of the
projections with their environment, and how this all contributes to the properties that
allow cytonemes to achieve their lengths and signaling specificity.
Supporting information
S1 Fig Overexpression of Myosin XV deletion constructs in bristle
precursor cells (related to Figure 7).
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Author Contributions
The project was conceptualized by GH. Experiments and analysis were carried out by
RC, TS, LC, JZ, AS, AC, and GH. Figures were generated by TS and GH. Original
draft was written by GH. Reviewing and editing was carried out by all authors.
Acknowledgments
We thank the Terman Lab at UTSW for sharing Drosophila reagents. We thank Ed
Giniger (NINDS/NIH) for postdoctoral support. LC and JZ were funded by the McNair
Scholars program. Stocks obtained from the Bloomington Drosophila Stock Center
(NIH P40OD018537) were used in this study. Monoclonal antibodies (as described in
Methods) were obtained from the Developmental Studies Hybridoma Bank, created by
the NICHD of the NIH and maintained at The University of Iowa, Department of
Biology, Iowa City, IA 52242. Work in the Hunter lab is supported by Clarkson
University and the National Institutes of Health (R03NS130395) to GH. We thank
Hunter lab members for their critical reading of this manuscript and comments.
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