Analyzing Signaling Dynamics in Developing Salivary Glands Using a
Multiplexing Approach
Charles Manhardt1, Michael Lazare1,2, Musodiq Bello2, Dierdre Nelson1, Alex Corwin2, Albert Santamaria-Pang2, Swami Manickam2, Elise Gervais1, Sean Dinn2,
Meghan Rothney2, Fiona Ginty2, Brion Sarachan2, Michael J. Gerdes2, and Melinda Larsen1
1University at Albany, State University of New York, Albany, NY 12222 2GE Global Research Center, Niskayuna, NY 12309
Abstract
Construction of a Tissue Microarray
Epithelium 1
Donor block
Cores removed and placed
into acceptor block
E17
Adhesive
slide
A tissue array maker (Beecher) was used to implant cores from donor blocks containing the tissue of
interest into one receptor block. 5 µm paraffin sections were transferred from the donor block onto a
glass slide to create a tissue microarray (TMA) specific for mouse submandibular gland development,
covering from embryonic day 13 (E13) through adult. D1, post natal day 1; AdF, adult female; FL, female
lactating; FP, female pregnant; M, male; Cells, pellets derived from cell lines.
Multiplexed IHC
Multiple signaling pathways are implicated in salivary gland
development. A subset of intracellular signaling molecules is shown
with their critical phosphorylated residues.
Channel:
DAPI
Cy3
Bleach
Tyr458
Image
Probe C + D
Cy5
Bleach
ΥΥ
Cy3
DAPI
Ser 9
Ser 2448
Image
Prior to the first round of direct IHC, tissues are stained with the nuclear intercalator dye, DAPI. In round
1, the tissues are subjected to direct IHC with two multiplexing probes (one Cy3 and one Cy5 – probe A
and B). Images are captured, and the fluorophores are subsequently chemically inactivated, or bleached.
DAPI is not affected. Images are captured of the bleached tissue. In round 2, the same slide is re-probed
with two different multiplexing probes (C and D), imaged, bleached, and imaged again. This process of
multiplexed direct IHC alleviates both species and spectral limitations imposed by indirect IHC and
conventional fluorescent microscopes.
PAK1
DAPI
A. Multiple epithelial markers were compared to identify the optimal combination to identify
epithelium. A combination of E-cadherin and pan-cytokeratin were used to create the Epithelial Mask. B.
A combination of pan-cadherin and Na+/K+-ATPase staining was used to identify epithelial membranes
(Super Membrane). C. An Segmented Epithelium image was developed by computationally combining
the nuclei, cytoplasm, Epithelial mask and Super Membrane to identify membrane, cytoplasm, and
nuclei.
GSK3β
pGSK3β
PI3K (p85)
Figure 6. “Virtual H&E” images are computationally
constructed from immuofluorescent images
A
Actual H&E stained tissue
B
Virtual H&E
Before image registration
After image registration
Overlapping sequential rounds- To
overlap images from different rounds of
staining we use the nuclear intercalator,
DAPI (Ex 358 nm, Em 461 nm), which is
unaffected by the bleaching procedure,
to register each round of images with
the subsequent round.
round 2
Na+/K+-ATPase
E-cadherin
DAPI
S6 kinase
Bleached tissue
• With these markers we have been able to recognize and computationally segment epithelial tissue
into multiple sub-compartments at multiple developmental stages of the mouse submandibular
salivary gland.
• The active (phosphorylated) forms of multiple signaling proteins show regionally distinct
expression patterns within the epithelium and mesenchyme compartments.
Segmentation
markers under
development
Future Directions
SMα-actin
vimentin
β3- tubulin
collagen IV
Web-based data viewer for SGDAtlas.albany.edu
Differentiation
markers and
signaling proteins
Overlay of round 1 and round 2
Auto-fluorescence removal
Before AF removal
A. Actual hematoxilin and eosin
(H&E) stained slide of adult salivary
gland tissue. Hematoxylin (purple)
stains nuclei and eosin (pink) stains
the cytoplasm. B. “Virtual” H&E
image of adult salivary gland tissue
computationally constructed by GEGRC using fluorescent
immunostaining patterns. (Scale, 25
um).
• We have validated 40 multiplexing probes that can be used as segmentation markers to identify
specific cell types, cell sub-compartments, and signaling proteins (both total and active forms).
FAK
After AF removal
Tissue auto-fluorescence is
computationaly removed by
subtracting the bleached
image from the prior round
from each image. Staining for
claudin 3 in E18 tissue. Scale,
10 µm.
Construct a tissue
microarray
Claudin 3
aquaporin 5
claudin 3
β-catenin
FAK
Multiplexed IHC showing expression patterns for 12 proteins sequentially detected in a single tissue
section of adult female SMG. Top row: Current cell segmentation markers. Middle row: Segmentation
markers under development for cell types: smooth muscle α-actin, (myoepithelial cells), vimentin
(mesenchyme), β3-tubulin (nerves), and collagen IV (basement membrane). Bottom row: Differentiation
markers and signaling proteins: aquaporin 5 (acinar cell), claudin 3 (tight junctions), β-catenin (signaling
protein) and focal adhesion kinase (signaling protein) (Scale bar, 25µm).
Image processing
Develop compartment
and expression scores
Develop web-based
database:
SGDAtlas.albany.edu
Figure 4. Identification of expression profiles throughout
developmental time-points
Results
Figure 1. Cellular segmentation
DAPI
Aquaporin 5
E-cadherin
Epithelial sub-cellular compartments
A.
DAPI
E-Cadherin
Pan Cadherin
Na+/K+-ATPase
S6 kinase
E15
• SGDAtlas.albany.edu will be developed as a tool for the research community to view multiplexed
datasets and quantitative profiles.
• Sixty additional multiplexing probes are currently under development.
Development of multiplexing probes
• We will use signaling profiles as a starting point to understand the contribution of signaling
pathways to both morphogenesis and cellular differentiation.
Nuclei
ΥΥΥ
Υ Υ
ΥΥ Υ
ΥΥ
Cy3 or Cy5 dye
AKT
Total and active proteins
(phosphorylated forms)
were detected sequentially
in SMG epithelial tissue
using multiplexing probes.
Regional differences within
the epithelium were
detected, with pErk and
pGSK3β expressed more
strongly in the outer
columnar cells than in the
inner polymorphic cells,
pEGFR
whereas the total protein
is more widely distributed.
EGF-R pAKT and total AKT were
expressed more strongly in
the mesenchyme than in
the epithelium. The p85
subunit of PI3K is
expressed broadly in the
epithelium. pmTOR is
pAKT expressed more in the
outer two layers of cells
than inside the bud. E13
SMG organ explants were
cultured for 24 hr prior to
IHC. EGFR was detected in
E15 SMG tissue. Scale, 20
µm (top row), Scale, 10 µm
pMTOR (bottom two rows).
Conclusions
Current cell
segmentation
markers
Claudin 3
Pure IgG
pERK
Figure 3. Multiplexed IHC of 12 proteins in a single tissue
section
Image registration
round1
Directly conjugate
Cy3/Cy5 fluorophore
to purified antibody to
create multiplexing
probe
ERK
Cytoplasm
Image
Thr 202 Tyr 204
Collect tissue from all
SMG developmental
stages
ΥΥ
Round 2
Ser 473
Overview: Using multiplexed IHC as a systems approach to profile
developmental signaling pathways
Apply
multiplexing
probes and
capture images
Na+/K+-ATPase
Pan-cadherin
Nuclei
Probe A + B
Methods
Validate antibody
staining pattern
20x
E-EGfR
cadherin
Image
To develop a high-resolution in situ proteomics-based atlas, which categorizes and quantifies active signaling
protein expression levels within specific cell types and subcellular compartments throughout all key developmental
time-points of mouse submandibular salivary glands.
Select potential
specific antibodies
Super Membrane
Key:
Nuclei
Cytoplasm
Epithelial Membrane
Cy5
Tyr 1068
Overall Project Goal:
Inactivate cyanine
flourophores and
capture bleached
images
Membrane 2
Phosphorylated sites
To understand organ morphogenesis, it is critical to know the temporal and spatial expression and activation of
proteins during progressive developmental stages. To understand differentiation of specific cell-types, it is
necessary to identify the cell-type expression, subcellular localization, and activation of specific signaling pathways
at a sub-cellular level.
Multiplexed IHC
pan-cytokeratin
Membrane 1
C
Round 1
Signaling
proteins Function
initiate several signal transduction
cascades leading to DNA synthesis
Serous Acini
EGFR
and cell proliferation
cell growth, proliferation,
differentiation, motility, survival and
PI3K
intracellular trafficking
glucose metabolism, cell
proliferation, apoptosis, transcription
Mucous
AKT
and cell migration
Acini
regulation of meiosis, mitosis, and
Gartner, L.P. and J.L. Hiatt, 2005. Color Atlas of Histology.
5th Edition. Lippincott, Williams and Wilkins
postmitotic functions in
ERK
differentiated cells
The submandibular salivary gland
involved in energy metabolism,
contains multiple epithelial cell types
neuronal cell development, and body
surrounded by mesenchyme (not shown)
GSKB3
pattern formation
containing parasympathetic nerves, blood
mTOR
cell growth and homeostasis
vessels and smooth muscle cells.
Screen RNA database
(sgmap.nidcr.nih.org)
to identify interesting
targets
E-cadherin
B
Acceptor block
Differentiation
E15
Embryonic
gland
ΥΥ
Striated
duct
E13
TMA
E13 14 15 16 17 18 D1 D5 AdF FL FP M Cells
ΥΥ
Intercalated
duct
E11 E12
Epithelial Mask
Epithelium 2
Segmented Epithelium
Background
The salivary gland undergoes
morphogenesis and differentiation
to achieve its final form and
function in the adult.
A
Tissue section
(5 um) placed
on slide
Morphogenesis
Figure 5. Detection of phosphorylated signaling proteins
in submandibular salivary gland epithelium
Fig. 2. Cellular and sub-cellular image segmentation
Tissue embedded into paraffin blocks
Replicates
The molecular mechanisms governing mammalian organ development, and specifically salivary gland
morphogenesis and differentiation, are not completely understood. We are developing an in situ proteomics atlas
of developing salivary glands to identify the spatio-temporal expression profile of active signaling molecules using
high-throughput methods that allow quantification of image data at a sub-cellular resolution. Phosphorylated
signaling proteins are of particular interest, and currently we are interrogating members of the MAPK pathway
and PI3K pathways, which are implicated in control of branching morphogenesis. Preliminary results demonstrate
that members of these pathways have spatio-temporal specific activation and localization. We are profiling
expression of these and other targets in submandibular salivary gland tissues from embryonic day thirteen
through adult in a tissue microarray (TMA) format. Two antibodies directly conjugated to fluorophores are
simultaneously hybridized to the TMAs each round, images are acquired and then the dyes are inactivated. The
process is repeated such that multiple protein targets can be imaged within a single TMA. Quantification of the
sub-cellular localization of each target is performed as a final step. This dataset will be compiled into an atlas that
will be publically available and facilitate analysis of signaling pathways controlling morphogenesis and
differentiation.
B.
Epithelium
Epithelial subclasses
Aquaporin 5
Cytokeratin 7
Membrane
C.
Membrane
Cytoplasm
Mesenchymal cell types
SM α actin
PECAM
• Multiplexing probes developed for this project will be used to examine changes in expression
patterns and activation of signaling proteins in both mouse models of Sjogren’s syndrome and in
human Sjogren’s syndrome patients relative to normal tissue.
Acknowledgements
Multiplexing probe
Following antibody validation steps, multiplexing probes are produced by directly conjugating either a cyanine 3
(Cy3) (Ex 550 Em 570) or Cy5 (Ex 650 Em 670) fluorophore to each antibody. The optimal dye to protein ratio for
maximal fluorescent signal is determined empirically.
E18
β3 Tubulin
Adult
Acini
Ducts
Blood vessels
Nerves
Myoepithelial
cells
Cellular and subcellular tissue segmentation is accomplished using a combination of immunostaining and
computational algorithms. A. A set of markers is used to identify epithelium, epithelial membrane,
cytoplasm and nuclei. B. Subclasses of epithelial cells can be accomplished using specific markers. C.
Algorithms are being designed at GE GRC to segment additional mesenchymal cell types .
Dr. Sharon Sequeira, William Daley, Sarah Peters, Shayoni Ray, and Riffard Jean-Gilles in the Larsen Lab are
thanked for advice and technical assistance. Anne Shelton at Ualbany Research IT is thanked for IT support.
Chris Hammond (VaudioSoft) is thanked for technical assistance with data management. Denise Hewgley,
Zengyu Pang, Yuchi Huang, Wendy Kan, Deppa Chitre, and Chris Sevinsky, GE Global Research, are thanked for
additional technical support.
Support:
Proteins show differential expression levels and localization throughout development. Expression patterns
for aquaporin5 and E-cadherin are shown at E15, E18, and adult. (Scale, 25 µm)
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