Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding

Al Alam, Denise

Bennett, Daimark

Bower, Dan J

Bower, Danielle V

Connell, Marilyn G

Featherstone, Neil C

Fernandez, G Esteban

Fraser, Scott E

Frey, Mark R

Jesudason, Edwin C

Lansdale, Nick

Navarro, Sonia

Trinh, Le A

Truong, Thai V

Warburton, David

Biology Open

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca^(2+) reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca^(2+) levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca^(2+) differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca^(2+) in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca^(2+) signaling, established by SERCA, in iterative budding

Bower, Danielle V.

Truong, Thai V.

Bower, Dan J.

Featherstone, Neil C.

Connell, Marilyn G.

Frey, Mark R.

Trinh, Le A.

Fernandez, G. Esteban

Fraser, Scott E.

Jesudason, Edwin C.

Caltech Authors - Main

 Supplementary Figures
 Fig. S1. Cell shape change, migration, and proliferation are unicellular behaviors adopted 
for multicellular branching 
Multicellular tissues across species employ different combinations of cell shape change, cell 
migration, and proliferation to undergo branching morphogenesis. For each branching 
structure in the table, + indicates the presence of a specific behavior and – indicates its 
absence.  
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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      Fig. S2. serca RNAi disrupts Drosophila larval air sac budding after depleting serca mRNA 
and Ca2+ stores 
  (A) Each configuration of da-GAL4 driven serca RNAi reduced serca mRNA levels relative to 
18S when compared to wild-type (w1118). 
 (B) Cells were dissociated from stage 16 wild-type and da> serca RNAi Drosophila embryos. 
 (C) The air sacs are visualized by btl-driven GFP expression. Compared to the control ‘leaf and 
Compared to wild-type Drosophila cells (left), RNAi cells (right) have deficient SERCA 
protein function by late embryogenesis featuring a subnormal rise in intracellular Ca2+ 
(measured by Ca2+ indicator fluorescence intensity) upon thapsigargin challenge. 
stalk’ type structure, btl-GAL4 driven serca RNAi +/- dicer induced a range of phenotypes, 
from no budding to very stunted and dysmorphic buds without the typical stalk. Combining 
RNAi constructs (4474 and 107446) to enhance the knockdown further increased the 
frequency of severely deranged air sac morphogenesis (Scale bar: 25 µm). Bottom panels are 
three representative examples. Control image is repeated from Fig. 1A.
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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     Fig. S3. serca RNAi does not affect apoptosis rate in larval air sac primordia 
Confocal micrographs show third instar larval imaginal wing discs, with tracheal system in 
green (actin-GFP) and apoptotic cells labeled by cleaved Caspase-3 in magenta. Scale bars: 
20 µm. 
     (A) Wild-type control air sac primordia.  
         (B) Mutant serca RNAi air sac primordia, arrow indicates one apoptotic cell outside the air sac.         
    (C) Positive control showing Caspase-3-positive cells in the wing disc border. 
         (D) Table shows number of air sac primordia imaged for each genotype, and total number of 
cleaved Caspase-3-positive cells detected within the air sacs across all samples: no apoptotic 
cells were seen in wild-type air sacs, and only a single apoptotic cell was seen in one of 22 
mutant air sacs. 
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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7 
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 Fig. S4. RNAi is not sufficient to disrupt SERCA protein levels or function during early  
Drosophila embryogenesis 
  (A)    qRT-PCR of stage 11 wild-type embryos (w1118) or embryos ubiquitously expressing RNAi 
constructs (4474, 107446, 4474+dicer2, or 4474+107446) using the da-Gal4 driver to knock 
down serca does not deplete serca mRNA relative to 18S mRNA. (Error bars indicate +/- 1 
SEM.) 
 (B) In da-Gal4 X sercaRNAi4474 / CyO-twi>GFP embryos, when GFP is not expressed, the RNA 
construct against serca is ubiquitously expressed. Confocal micrographs show ubiquitous 
expression of SERCA protein (red, fluorescent thapsigargin) in controls (left side) and with 
RNAi construct expression (right side), at stage 11. 
 (C) Cells were dissociated from stage 11 wild type and da> serca RNAi Drosophila embryos. 
Compared to wild type Drosophila cells (left), RNAi cells (right) have normal SERCA 
protein function (mean +/- SD) with a normal rise in intracellular Ca2+ (measured by Ca2+ 
indicator fluorescence intensity) upon thapsigargin challenge.  
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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   Fig. S5 SERCA inhibition with CPA creates physical gaps in tracheal trunks, but these are 
          not due to cell death, and tracheal structure is rescued by PKC activation 
   Tracheal cells are genetically labeled by Breathless (Btl) driving eGFP fused to actin. The dorsal
          trunk is outlined with dotted yellow lines in each panel. Scale bar: 50 µm throughout. 
      (A)  Control embryos treated with DMSO. 
    (B) Incubation with SERCA inhibitor CPA results in physical gaps in the dorsal trunk where 
tracheal cells are absent (arrowhead) and tenuous cytoplasmic connections between segments
(arrow). 
(C) Removal of CPA at stage 12 and further incubation without the inhibitor results in fewer 
gaps (solid arrowhead) but exuberant sprouting (open arrowheads) and undulating branches. 
Sometimes an extranumerary lateral trunk partially or completely forms (arrows). 
   (D)  Treatment with PKC activator PMA alone results in normal tracheal structure. 
   (E) Co-treatment with SERCA inhibitor and PKC activator rescues tracheal structure. 
   (F) In live embryos, fluorescently labeled nuclei of tracheal cells were tracked in controls treated 
with DMSO, and in CPA-treated embryos at segments where a continuous lateral trunk failed 
to form. From stages 14-16 during the time the lateral trunk should have formed, there was 
no tracheal cell death observed in either CPA- or DMSO-treated embryos. All labeled nuclei 
remained present throughout; at affected segments in the CPA-treated embryos, the cells 
simply did not migrate to form the trunk.  
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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 Fig. S6. SERCA blockade with CPA elevates overall Ca2+ level of Drosophila tracheal cells 
     (A-C) For DMSO-treated (A), CPA-treated (B), and CPA+PMA-treated (C) embryos, three 
representative pairs of leader (blue) and trailer (magenta) cells are plotted together. Ca2+ level 
is consistently higher in leader cells in A and C, while this differential is reversed in CPA-
treated embryos (B). Also, the overall Ca2+ levels of the tracheal cells in the CPA-treated 
embryos are higher than in the DMSO- and CPA+PMA-treated embryos, consistent with 
blockade of the SERCA pump. This is alleviated by co-treatment with PKC-activator PMA 
(C).  
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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 Fig. S7. SERCA inhibition induces Drosophila neural phenotypes that are rescued by PMA 
      (A-D) Permeablized embryos are seen at stage 15-16 after incubation with (A) DMSO (control), 
 (E) Normal and disrupted regions of the Drosophila nerve tracts intersperse following SERCA 
inhibition. Three examples of permeablized Drosophila embryos treated with 20 µM CPA and 
immunostained with antibody against FasII display a range of neural phenotypes. Aberrant 
neural branching revealed by midline crossings, merged longitudinal tracts, or discontinuities 
(red arrowheads) occur adjacent to sections of the nerve tracts and peripheral nerve projections 
that display relatively normal structure (white arrowheads).  This abrupt juxtaposition of 
normal and disrupted branching, and the ability to perturb all parts of the nerve cord when 
multiple embryos are considered collectively, support a cell-autonomous role for SERCA in 
neural guidance.  This is consistent with disrupted neural pathfinding in cells that take up a 
sufficient amount of SERCA inhibitor, and unperturbed pathfinding in cells that happen to 
acquire minimal inhibitor.  Scale bar: 40 µm.  
(B) 20 µM CPA, (C) 20 µM CPA with washout and (D) 20 µM CPA and 100 nM PMA. 
Neural structure is demonstrated by FasII immunostaining. SERCA-inhibited embryos (B) 
frequently display discontinuities and merged or aberrantly spaced FasII tracks (red 
arrowheads) throughout the central nervous system. Merging of tracks frequently seen at the 
level of A4-A5 (red arrow in B) may also reflect perturbed cell migration during germ band 
retraction, since migration problems typically manifest in these segments. Severity of neural 
perturbation at stage 15-16 is reduced by washout of CPA at stage 12 (C) and rescued by co-
incubation with PMA (D).  n > 30 per treatment group. Scale bar: 40 µm. 
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Fig. S8. Inhibition of Ca2+-dependent mechanotransduction neither reprises nor alleviates 
the SERCA inhibition phenotype 
Mammalian lung epithelial tips isolated from E12.5 murine lungs bud in Matrigel with FGF10 
(control) (A, image repeated from Fig. 6G leftmost panel). Lung tips were likewise cultured 
without or with SERCA inhibitor (10 µM CPA) and in the presence of specific inhibitors of: 
(B) PKC (2.2 µM Bisindolylmaleimide I Hydrochloride), (C) PLC (10 µM L108), (D) ROCK 
(20 µM Y-26732), or (E) RAC (12.5 µM NSC 23766). None of these inhibitors recreated or 
mitigated the phenotype of SERCA inhibition with CPA. Scale bars: 100 μm.
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Fig. S9.  SERCA inhibition does not disrupt epithelial geometry 
Confocal images of phalloidin-stained epithelia from mouse lung or Drosophila air sac were 
skeletonized computationally to calculate cell areas and perimeters and for scoring cell 
sidedness. 
   (A) Square root of cell area vs perimeter for cells in murine epithelial lung tips cultured with 10 
µM CPA (red) or without (+). 
(B)  Frequency distributions show no differences in the number of sides for wild-type epithelial tip 
cells compared to those treated with 10 µM CPA +/- 100 nM PMA. 
 (C) Square root of cell area vs perimeter for cells in Drosophila air sac with targeted serca RNAi 
(red) or without (+). 
 (D) Frequency distributions show no differences in the number of sides for epithelial cells in 
wildtype Drosophila air sacs compared to those in serca RNAi air sacs. 
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Movies
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Movie 1
Movie 1b
Notes on Movies 
Movies 3‐11: Dynamic Ca2+ imaging of Drosophila tracheogenesis. Two‐photon light‐sheet microscopy  
was used to image permeablized Drosophila embryos expressing the Ca2+ indicator GCamP3, from 
stage 13 to 16 (timepoints 3 seconds apart), during treatment with DMSO, 20 μM CPA, or 20 μM CPA + 
100 nM PMA. A summative projection of the images was made for each timepoint. Movies are 50x  
faster than real time.  To quantify the Ca2+ level in lateral trunk cells, 3D summative projections were 
reconstructed to include the lateral trunk but exclude autofluorescence from the gut. Representative 
clips are shown. Full movies are available upon request. For some embryos, the dorsal and ventral 
tracheal branches had to be excluded in order to exclude gut autofluorescence. In such cases, separate 
reconstructions were generated to visualize the more complete tracheal network, albeit only for 
qualitative purposes and with more gut autofluorescence. 
Movies 9‐11: High magnification view of cells displayed in Fig. 3 whose Ca2+ levels were quantified over 
time. An image from the complete dataset was selected every 2 minutes for demonstration purposes. 
The blue and magenta markers track the leader and follower cells, respectively, to show where Ca2+ 
intensity measurements were made. 
Movies 1 & 2: SERCA loss stalls cell migration during Drosophila tracheogenesis. Permeablized embryos 
from the transgenic line w; Btl:Gal4, UAS-dsRed-NLS, UAS-actin-GFP were treated with DMSO (Movie 1)*  
or 20 μM CPA (Movie 2) and imaged dynamically. Actin‐GFP is shown in cyan, nuclear dsRed in magenta. 
Movies show 3D reconstructions from approximately stage 14 during Drosophila tracheogenesis, when 
cells migrate to form the lateral trunk. Spots label the cell nuclei of interest in chosen segments, the 
squiggly tails indicate their path over the previous 10 time steps. Scale bars: 30 μm.
Movie 1: In the representative DMSO-treated control embryo, cells from adjacent segments converge to 
form the lateral trunk.
Movie 2: In the representative CPA-treated mutant embryo, these lateral trunk cells continue moving in 
parallel so that the gap between them persists, leading to a fragmented tracheal structure (movies run to 
stage 16).
*Movie 1b follows the embryo in Movie 1 from stage 12 through late 16.
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Movie 2
Movie 3: The reconstruction depicts a representative control embryo treated with DMSO.  Migratory 
leader cells forming the lateral trunk show visibly higher Ca2+ levels than trailing cells. Scale bar: 50 
μm.  
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Movie 4: The reconstruction shows a representative embryo treated with CPA.  The embryo displays 
segments where the lateral trunk cells fail to migrate, and the elevated Ca2+ level seen in leader cells in 
controls is visibly absent. Scale bar: 50 μm. 
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Movie 5: The reconstruction depicts a representative embryo treated with CPA+PMA.  As in the control 
embryo, the migratory leader cells forming the lateral trunk show visibly higher Ca2+ levels compared to 
trailing cells, and the leader cells migrate to form a continuous lateral trunk.  Scale bar: 50 μm. 
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Movie 6: DMSO‐treated control, stage 16. By stage 16, Ca2+ impulses propagate throughout the 
electrically‐coupled tracheal cells of control embryos treated with DMSO. Scale bar: 50 μm. 
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Movie 7: Representative embryo treated with CPA exhibiting extended Ca2+ elevation. In contrast with 
controls, embryos treated with CPA, with or without PMA, featured prolonged elevations of Ca2+. Many 
Ca2+ impulses are of normal duration but at the start of the video, a white arrow marks the start of a 
prolonged Ca2+ impulse, with a grey arrow indicating its return to baseline. Scale bar: 50 μm. 
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Movie 8: Embryo treated with CPA+PMA demonstrates a “hyper‐migration” phenotype. Where 
branching was rescued, either by withdrawing CPA or adding PMA, static imaging revealed some 
embryos with excessive sprouting and even supernumerary tracheal trunks. This movie shows one such 
CPA+PMA‐treated embryo which reveals a “hyper‐migration” phenotype. Arrows mark collections of 
tracheal cells that migrate away from one dorsal branch to join adjacent branches and even form a 
disorganized auxiliary trunk and network. Examples of propagating Ca2+ impulses are also apparent in 
this late‐stage 16 embryo. Scale bar: 50 μm. 
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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Movie 9: In a control embryo treated with DMSO, during tracheal cell migration to form the lateral 
trunk, the leader cell (blue) exhibits a visibly higher level of Ca2+ than the trailing cell (magenta). Scale 
bar: 10 μm. 
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Movie 10: In an embryo treated with CPA, the Ca2+ levels between the leader cell (blue) and trailing 
cell (magenta) are comparable or higher in the trailing cell (magenta), particularly early in the time 
course when the leader cell (blue) should be migrating. Scale bar: 10 μm. 
Movie 11: In an embryo treated with CPA+PMA, just like in the control embryo, the leader cell (blue) 
exhibits a visibly higher Ca2+ level than the trailing cell (magenta).  Scale bar: 10 μm.
Biology Open (2017): doi:10.1242/bio.026039: Supplementary information
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English

SERCA directs cell migration and branching across species and germ layers

Danielle V. Bower

Nick Lansdale

Sonia Navarro

Thai V. Truong

Dan J. Bower

Neil C. Featherstone

Marilyn G. Connell

Denise Al Alam

Mark R. Frey

Le A. Trinh

G. Esteban Fernandez

David Warburton

Scott E. Fraser

Daimark Bennett

Edwin C. Jesudason

Directory of Open Access Journals

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.</jats:p

Denise Al-Alam

Crossref

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.</p

SERCA directs cell migration and branching across species and germ layers

Abstract

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Caltech Authors - Main

Directory of Open Access Journals

Crossref

The University of Manchester - Institutional Repository

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Caltech Authors - Main

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