Download Dr David Hipfner lecture notes

3/24/17
Branching Morphogenesis
David Hipfner
Epithelial Cell Biology Research Unit
IRCM
What is morphogenesis?
Gastrulation and neurulation in Xenopus laevis (15 h elapsed time)
http://faculty.virginia.edu/shook/ShowMovies/Xenopus_Gastrulation.mov
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What is morphogenesis?
Dorsal closure and head involution in Drosophila
http://www.celldynamics.org/celldynamics/gallery/liveLabel.html
What is morphogenesis?
Imaginal disc eversion in Drosophila
Ward, R.E. et al. (2003). Dev. Biol., 256: 389-402.
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What is branching morphogenesis?
“The restructuring of epithelial tissues to complex but highly
organized tubular networks that transport gases and/or fluids”
Affolter et al., 2003
-  the most common structural design for organs is a branched
tubular network
Many organs are composed of branching tubes
Lungs
Salivary gland
Kidney
Vasculature
Mammary glands
Drosophila trachea
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Why branch?
Branching morphogenesis is a fundamental evolutionary
adaptation to increasing organism size:
- enables targetted delivery of nutrients (e.g. vasculature, trachea)
-  allows efficient packing into a minimal volume (e.g. secretory glands)
-  maximizes surface area for metabolic exchange (e.g. vasculature,
lung, kidney)
Example - the lung
Without branching: ~0.5 m2 surface area for gas exchange with the blood
With branching (300 million alveoli): ~100 m2 surface area for gas exchange
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Goal of today’s lecture
o 
Provide general background on how epithelial cells and tissues are shaped
o 
Illustrate key conserved principles of branching morphogenesis, using
two well characterized models:
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Drosophila trachea
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mammalian kidney
At the end, you should be able to answer questions like:
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What mechanisms are used to shape epithelial cells?
How do epithelial cells form tubes?
What aspects of branching morphogenesis are conserved across systems/species?
What aspects are different?
What role does signalling from mesenchyme to epithelia play?
What role does signalling from epithelia to mesenchyme play?
How are different types of branches formed by reiterative use of one signalling pathway?
Epithelial versus mesenchymal cells
Mesenchyme:
- connective tissue
- derives from embryonic mesoderm
- consists of loosesly packed, unspecialized cells embedded
in extracellular matrix - e.g. fibroblasts
Epithelia:
- line nearly all surfaces
- most derives from embryonic ectoderm or endoderm
- consists of highly organized layers of tightly adherent cells
with a shared apical-basal polarity
- rest on basement membrane
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Epithelial cell have apical-basal polarity
APICAL
Drosophila hindgut
epithelium
BASOLATERAL
JUNCTIONS
http://mcb.berkeley.edu/labs/bilder/Polarity.html
Adherens junctions
-  provide cell-cell adhesion; provide anchor point for actin cytoskeleton; join cytoskeletons of neighbouring cells, allowing contractility across the epithelial layer
from Alberts et al., “Molecular Biology of the Cell”
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Epithelial and mesenchymal cells can be
interconverted
mesenchymal-to-epithelial
transition (MET)
MESENCHYMAL
CELL
EPITHELIAL
CELL
epithelial-to-mesenchymal
transition (EMT)
What kinds of (cellular) processes drive
branching morphogenesis?
Time-lapse video of developing embryo with tracheal cells expressing GFP
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Identifying genes required for
morphogenesis
A. GENETIC SCREENS
Identifying genes required for
morphogenesis
Wild-type
punt
(Type II TGF-β
receptor)
canoe
(Afadin)
kayak
(Fos)
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Morphogenesis relies upon a
conserved signaling cascade
+ve regulators
Rho GTPase
ROCK
Myosin II
TENSION
-ve regulators
Quintin et al. (2008). Trends in Genetics 24:221-230
Actomyosin-based contractility
muscle Myosin II
Vale and Milligan, Science (2000)
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Regulation of non-muscle Myosin II by
RLC phosphorylation
Heavy chain rod domain
Regulatory light chain
Essential light chain
Heavy chain head domain
adapted from Vicente-Manzanares et al.,
Nature Reviews (2000)
RLC phosphorylation promotes Myosin
antiparallel filament formation
“Actin bundling”
adapted from Vicente-Manzanares et al.,
Nature Reviews (2000)
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RLC phosphorylation stimulates actinactivated ATPase activity
adapted from Vicente-Manzanares et al.,
Nature Reviews (2000)
Rho GTPases act as “molecular switches”
to control actin cytoskeleton dynamics
“OFF”
“ON”
GAP = “GTPase activating protein
GEF = “GDP:GTP exchange factor”
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Rho-GTPase signaling regulates MRLC through
the protein kinase ROCK
Epithelial cells can change their
SHAPE and ARRANGEMENT
Quintin et al. (2008). Trends in Genetics 24:221-230
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How do epithelial cells form tubes?
Tube formation is driven by:
CELL MIGRATION
CELL REARRANGEMENT
CELL SHAPE CHANGE
* In mammals, CELL PROLIFERATION is also essential
for forming branched organs*
Shaping epithelial cells into tubes
Multicellular tubes
e.g. largest primary trachea branches, larger
blood vessels, most branched vertebrate organs
from Uv et al., 2003
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How are tubular cell shapes generated?
Multicellular tubes are usually formed from existing epithelia
from Lubarsky and Krasnow, 2003
Tube formation by apical constriction
shrink or
“constrict”
apical membrane
surface area
both involve apical constriction to generate “bud”
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Shaping epithelial cells into tubes
Unicellular tubes
e.g. smaller primary trachea branches, smaller
blood vessels
from Uv et al., 2003
How are tubular cell shapes generated?
Unicellular tubes are formed from multicellular tubes
Cells “reach around” each other to form autocellular junction
from Kerman et al., 2006
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Shaping epithelial cells into tubes
Intracellular terminal tubes
e.g. tracheal terminal branches
from Uv et al., 2003
How are tubular cell shapes generated?
De novo formation of intracellular terminal tubes
- intracellular tubes can be created by synthesis of apical membrane
vesicles followed by targetted vesicle fusion
from Lubarsky and Krasnow, 2003
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Early studies of the branching process
1950s-1970s
mesenchyme
epithelial primordium
+
.45µm filter
Inductive signals for branching morphogenesis
-  epithelial branching process is induced by signals from neighbouring
mesenchyme
embryonic lung epithelium
stripped of mesenchyme and
grown in culture on .45µm filter
embryonic lung epithelium
stripped of mesenchyme and
grown in culture on .45µm filter
opposite a piece of lung mesenchyme
Shannon, J.M. and Hyatt, B.A. (2004). Annu. Rev. Physiol. 66:625-45
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Inductive signals for branching morphogenesis
-  mesenchyme responds to inductive signals from neighbouring
epithelia
Taderera, J.V. (1967). Dev Bio16:489-512
Complex Epithelial - Mesodermal Signaling
MESODERM
EPITHELIUM
-  mesoderm signals to epithelium to induce budding and branching
-  triggers signals within the epithelium that:
-  alter the way that the epithelium responds to mesodermal signals
-  signal back to the mesoderm to pattern the mesoderm (in
mammals, not in flies!)
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Drosophila as a model system for
branching morphogenesis
- much of our knowledge about the cellular mechanisms involved in forming
branched organs stems from research on Drosophila trachea development
- remains by far the best characterized branching process
•  genetic tractability - more than 50 genes required for normal branching morphogenesis
have been identified in forward genetic screens; minimal functional redundancy
•  relative simplicity - the whole tracheal system is composed of only ~1600 cells
•  ease of visualization - e.g. fluorescence markers, time lapse video microscopy
*many of the mechanisms, both molecular and cellular, are conserved in mammals*
The Drosophila life cycle
ADULT
EMBRYONIC
stages
1 day
PUPAL stage
5 days
L1
L3
G
R
L2
O
W
H
T
LARVAL stages
4 days
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The Drosophila larval tracheal system
-  air-filled network of interconnected epithelial tubes containing some
10,000 branches
-  transports oxygen and other gases from outside to essentially every cell
of each internal tissue
- bilaterally symmetrical, segmentally repeated pattern
25 µm
(lateral view of Drosophila embryo)
adapted from Ghabrial et al. 2003
Timelapse imaging of tracheal development
Time-lapse video of developing embryo with tracheal cells expressing GFP
http://flymove.uni-muenster.de/
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General scheme for branching morphogenesis
from Affolter et al., 2003
The basic process of branching:
-  specification of a competence zone/primordium
-  epithelium invagination/budding into mesenchyme
-  branch initiation
-  branch outgrowth
Specifying the tracheal primordium
~ 5 h after egg laying (AEL):
- the trachea arise from 20 “placodes” on the embryonic surface ectoderm
Trh
-  each placode consists of ~ 20 cells
-  first morphological sign of the placodes is expression of the transcription
factor Trachealess (Trh)
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Signalling - Specifying tracheal primordia
- the Trachealess (Trh) bHLH-PAS domain transcription factor is the earliest
expressed trachea-specific gene; expressed throughout tracheal development
-  functions as a heterodimer together with Tango, a more broadly expressed
bHLH-PAS domain transcription factor
- shortly after Trh, Ventral veinless (Vvl), a POU domain transcription factor,
is also expressed in tracheal placode cells and throughout subsequent stages
- the combinatorial activity of Trh/Tango and Vvl triggers early tracheal gene expression
Trh + Tango
tdf
peb
btl
dof
rho
Vvl
tkv
tracheal development
Tracheal pit formation
~ 5-7 h after egg laying (AEL):
-  cells in the placodes invaginate (apical constriction) to form the tracheal pits
-  as they invaginate, each cell undergoes two rounds of cell division,
giving rise to a total of 80 cells per pit
* these are the last cell divisions during formation of the trachea;
after this point, there is no change in tracheal cell number *
antibody staining against trachea lumen protein
from Ghabrial et al. 2003
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Primary branching
~ 7 h AEL:
-  primary branches begin to bud out from tracheal pits
-  form at 6 stereotypical positions (per hemisegment)
-  branches are formed by between 4 and 20 cells
Dorsal branch
Dorsal trunks
Visceral branch
Lateral trunk
Ganglionic branch
from Ghabrial et al. 2003
Primary branching
7-10 h AEL:
-  primary branches lengthen along defined paths
from Ghabrial et al. 2003
-  involves extensive cell rearrangements (cell intercalation) and cell
shape changes
from Affolter et al., 2003
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Fibroblast Growth Factor signalling
drives primary branching
FGF’s
-  a large family of secreted polypeptide growth factors (22 in humans,
3 in Drosophila)
FGF receptors (FGFR’s)
-  receptor tyrosine kinases that are activated upon FGF binding (4 in
humans, 2 in Drosophila)
Implicated in diverse developmental processes including patterning,
differentiation, cell proliferation, cell survival
*FGF’s are potent chemoattractants, stimulate cell migration*
Fibroblast Growth Factor Signalling
IN DROSOPHILA TRACHEA:
FGF = Branchless (Bnl)
FGFR = Breathless (Btl)
adapter = Dof
Transcription factor = Pointed (Pnt)
and Yan
from Thisse and Thisse, 2005
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Tracheal placode cells are “competent”
to respond to FGF
A critical function of the early tracheal transcription factors is to activate
expression of Btl/FGFR and Dof (adapter) in placode cells
Trh + Tango
Vvl
Btl
tdf
peb
btl
dof
rho
tkv
Dof
tracheal development
Klambt et al., 1992
Vincent et al., 1998
results in the establishment of FGF “competence zones”
FGF signaling is required for tracheal branching
ligand mutant
wild-type
adapter mutant
receptor mutant
Ectopic FGF is sufficient to induce tracheal cell migration
ectopic Bnl ligand
ectopic tracheal branch
from Sutherland et al., 1996
Vincent et al., 1998
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FGF induces tracheal cell migration
Bnl/FGF induces migration of two tip cells in each primary branch toward
the source of the ligand
- FGF induces changes in gene expression and cytoskeletal organization
in the tip cells, which are closest to the source of FGF
- Btl/FGFR is a transcriptional target of Bnl/FGF signalling - autoregulation
Remaining cells in each primary branch are “pulled” by tip cells
from Affolter et al., 2003
Dynamic FGF expression drives branching
Bnl/FGF is first expressed in a stereotyped pattern in 6 cell clusters
surrounding each tracheal placode
- presumably under control of global A/P, D/V, segment patterning systems
from Sutherland et al., 1996
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Secondary branching
-  ~ 10 h AEL ~ 24 unicellular secondary branches/hemisegment begin to
sprout out from primary branches, at stereotypical locations
-  from ~10-12 h AEL, fusion cells will fuse
with their partners in neighbouring segments
or on the opposite side of the same segment
to form a continuous network
from Uv et al., 2003
Tertiary branching
-  terminal cells undergo extensive unicellular (tertiary) branching during
larval life
-  make intimate contact with most cells of the internal organs
high magnification view of terminal
trachea branch cell contacting cells
in the mature larval gut (blue nuclei)
from Ghabrial et al. 2003
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Dynamic FGF expression drives
secondary branching
stage 12
stage 13
stage 14
from Sutherland et al., 1996
- when migrating tracheal cells reach the first bnl/FGF sources, they stop
- Bnl/FGF expression is downregulated, then re-initiated in more distant cell
clusters, again in a stereotyped pattern (mechanism unknown…)
-  exposure to this second wave of Bnl/FGF signalling induces secondary
branching (e.g. specification of terminal cells)
Tertiary branch formation also requires Bnl/FGF
-  at later stages, FGF is expressed in response to hypoxia rather than
global patterning cues
- recruits tertiary branches from terminal tracheal cells to cells needing oxygen
from Ghabrial et al. 2003
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How does reiterative Bnl/FGF signalling
produce different responses each time?
- each exposure to Bnl/FGF changes subsequent response
First exposure
Trh+Tango
Vvl
placode
Btl/FGFR
specification Dof
Second exposure
Third exposure
Bnl/FGF
Bnl/FGF
Bnl/FGF
Btl/FGFR
Dof
Btl/FGFR
Dof
Btl/FGFR
Dof
MAPK
MAPK
MAPK
Pnt
Blistered
1° branching
program
Pnt
2° branching
program
Blistered
(= Serum
response
Factor)
3° branching
program
Sprouty (Spry) controls branch point choice
MESODERM
Bnl/FGF
Btl/FGFR
Dof
MAPK
EPITHELIUM
- loss of Sprouty causes excessive 2° and
terminal branch formation
Pnt
2° branching Sprouty
program
- sprouty is a Bnl/FGF target gene in branching epithelia (conserved)
-  Sprouty functions a general intracellular inhibitor of receptor tyrosine
kinase signalling - negative feedback inhibition
-  in its absence, FGF signalling in tip cells is too strong, causing them to
branch at the wrong time or in the wrong place
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Is it as simple as “follow the FGF”?
No - many other factors feed into branching morphogenesis
Several signalling pathways are involved in defining “branch identity”
(BMP)
(Wnt)
ligand expression pattern
tracheal branching pattern
if tracheal cells unable to
respond to ligand
adapted from Kerman et al., 2006
Other pathfinding cues
-  although Bnl/FGF acts as a chemoattractant, migrating tracheal cells are
dependent on other cues in their physical environment for finding the
correct path:
-  different branches depend on different adhesion molecules (e.g.
integrins) for proper pathfinding
-  some branches follow physical cues - e.g. grooves between muscles, surfaces of other cells - to reach their destination
from Wolf and
Schuh, 2000
blue = mesodermal “bridge” cell
wild-type
mutant lacking
bridge cell
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Branching morphogenesis in mammals
Two major difference between branching morphogenesis
in mammals and flies:
1. Epithelia-to-mesenchyme signalling is essential in mammalian organs
-  Drosophila trachea are simple monolayered epithelial tubes
-  in mammals, the epithelia of branched organs are intimately associated
with mesenchyme-derived tissues (e.g. blood vessels)
- these supporting tissues have to be recruited and organized in a coordinated
manner by the branching epithelial cells
2. Growth of the branching network is driven by cell proliferation; no
direct evidence for cell migration (e.g. filopodia) during the process
in mammals
Kidney structure and function
branching network: ureter-pelvis-major calyx-minor calyx-collecting ducts-nephron
(1)
(1)
(3-5)
(10-20)
(thousands?) (~106)
main functions: filter blood, remove metabolic waste products into urine, homeostasis (regulate pH, ionic concentration, blood volume, blood pressure)
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Visualizing mouse kidney branching
-  studies of branching greatly aided by imaging of developing
kidneys from HoxB7-GFP transgenic mice
time-lapse imaging of
explant grown in culture
“grown” in vivo
from Watanabe and Costantini 2004
The kidney derives from the ureteric bud
from Costantini 2006
from Dressler 2006
- kidney derived from the caudal end of the nephric duct
- E10.5 - ureteric bud invades metanephric mesenchyme
- E11.5 - ureteric bud branches for the first time - T-shape
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Branching in the developing kidney
- ~8 rapid rounds of branching between
E11 and E15.5
-  followed by period of elongation of the
branches formed in rounds 6-8; forms
the long unbranched collecting ducts
-  branching resumes ~ 3 more rounds
of branching in the cortex
from Costantini 2006
-  ~3/4 of branching events are
bifurcations (primary branching at bud tips)
-  ~6% branches originate from trunks
(secondary branching)
The Gdnf/Ret signalling pathway
Glial-derived neurotrophic factor (Gdnf) is a peptide growth factor
GFRα1 is glycosphingolipid-anchored co-receptor that works with Ret
Ret is a receptor tyrosine kinase that signals through the typical RTK
effector pathways (MAPK, PLC-γ, etc.)
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Gdnf/Ret signalling in the kidney
Ret is expressed in the ureteric bud
Gfrα1 is expressed in the ureteric bud
and metanephric mesenchyme
Gdnf is expressed in the metanephric
mesenchyme
Sainio et al., 1997
Gdnf promotes sprouting of the ureteric bud
Gdnf signalling is necessary for ureteric budding
wild-type
Gdnf-/-
Gdnf is sufficient to induce ureteric budding
wild-type
misexpressing Gdnf throughout the nephric duct
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Dynamic expression of Gdnf also drives
later branching in the kidney
Ret is a transcriptional target of Gdnf/Ret signalling
from Costantini and Shakya, 2006
Ret becomes restricted to branching tips
Gdnf becomes restricted to undifferentiated mesenchyme at kidney periphery
Ret signalling is required in bud tips
from Shakya et al., 2005
Is Gdnf a chemoattractant in the developing kidney?
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Gdnf doesn’t seem to “guide” branching
from Shakya et al., 2005
Why is Gdnf/Ret signalling required
in tip cells?
Ret is a proto-oncogene… could it be promoting cell proliferation?
BrdU
- bud tip swells by cell proliferation to form “ampulla” prior to branching
- lack of Ret-/- cells in bud tips could reflect their failure to proliferate in the
ureteric bud, prior to budding
from Michael and Davies, 2004
Costantini and Shakya, 2006
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Signaling within the epithelium - Sprouty
function is conserved in kidney
Sprouty1 is a target of Gdnf signalling in the ureteric bud
Sprouty1 mutants show ectopic buds
early and excessive branching later
from Basson et al., 2005 and 2006
Signaling from epithelium to mesenchyme – Bud
tips induce mesenchyme to form nephrons
- ureteric bud gives rise to segments up to collecting ducts
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Importance of physical cues for branching
-  altered branching frequencies/patterns observed upon manipulation of:
•  extracellular matrix molecules
•  matrix receptors
•  matrix remodelling proteins
-  extracellular matrix can play several roles:
Sakai et al., 2003
•  facilitating/resisting branching - e.g. fibronectin in salivary gland)
•  regulating signalling - e.g. ligand co-receptors, sequestering ligands
Good references for those interested
GENERAL: Wang, S., Sekiguchi, R., Daley, W.P., and Yamada, K.M. (2017). Patterned
cell and matrix dynamics in branching morphogenesis. Journal of Cell Biology
216: 559-70.
Quintin, S., Gally, C., and Labouesse, M. (2008). Epithelial morphogenesis
in embryos: asymmetries, motors, and brakes. Trends in Genetics 24:221-230.
Lu, P., Sternlicht, M.D., and Werb, Z. (2006). Comparative Mechanisms of
Branching Morphogenesis in Diverse Systems. J. Mammary Gland Biol.
Neoplasia 11:213-28.
TRACHEA: Ghabrial, A., Luschnig, S., Metzstein, M.M., and Krasnow, M.A. (2003).
Branching Morphogenesis of the Drosophila Tracheal System. Annu. Rev. Cell
Dev. Bio. 19:623-47.
KIDNEY: Short, K.M. and Smyth, I.M. (2016). The contribution of branching morphogenesis to kidney development and disease. Nature Reviews Nephrology 12:
754-67.
Costantini, F. (2006). Renal branching morphogenesis: concepts, questions,
and recent advances. Differentiation 74:402-421.
TUBULOGENESIS: Lubarsky, B. and Krasnow, M. (2003). Tube Morphogenesis:
Making and Shaping Biological Tubes. Cell 112:19-28.
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