Summary from Dr. Andrew

1
Summary from Dr. Andrew

• The work to be discussed by Dr. Andrew will focus
  on novel mechanisms for tube size control in two
  tubular organs that form during Drosophila
  embryogenesis the salivary gland and trachea.
  She will first describe experiments revealing the
  role of a novel transcriptional cassette in
  regulating the expression and activities of
  apical membrane proteins to allow tube
  elongation. She will then discuss the
  identification of a new member of a highly
  conserved pathway with previously unknown roles
  in limiting tube elongation.

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Drosophila tracheae develop from ectodermal
placodes
DB
DT
TC
VB
LT
GB
3
Each tracheal unit connects to form a network
4
Signaling molecules regulate direction of branches
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Different tube morphologies
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Type II tubes form by one cell moving distally
and one moving proximally
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Tubes fuse to create type III tubes
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Salivary gland branching involves 2 cell types
ducts and secretory cells
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Salivary gland invaginates and then extends
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Salivary gland migrates over surface of
mesenchymal tissues
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Bradley paper

• Already known rib mutation affects
• Trachea
• Malpighian tubules
• Hindgut
• Dorsal closure
• But how does rib affect development of these
  tissues?
• Is there a common thread?

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rib is required at later stages of tracheal
development
DT
Figure 2 Rachel, Chunyao
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Requirement for rib during early tracheal
branching
Figure 2 Rachel, Chunyao
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Are tracheal defects secondary to other
developmental problems?
DT
VB
LT
GB
Rescue Btl-Gal4 UAS-rib
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Does rib function in a signaling cascade?
btl FGFR
bnl FGF
DPP --gt KNI
Figure 3 Erin, Spencer
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Disagreeing with published data
Wappner et al., 1997

• EGFR specifically affects DT and VB formation
• EGFR maintains sal in DT
• sal keeps DT cells from migrating dorsally
• Loss of DT, reduced LT and GB
• DB and VB not affected

rib1 phenotype
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Does loss of EGFR signaling phenocopy rib
mutation?
DB
DB
DT
gt Site of placode formation
LT
GB
Figure 3 Erin, Spencer
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Is rib involved in the WG cascade?

• wg mutants also lose DT cell migration
• DT cells cluster below DB
• In early stages, wg is required for sal
  expression
• In wg and tkv double mutants, only VB cells
  migrate

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Is rib involved in the WG cascade?
sal is an early target gene of WG
Artificially elevating sal
Figure 4 Jeff, Tomas
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What happens when you combine rib and tkv
mutations?
Figure 4 Jeff, Tomas
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rib affects salivary gland migration
Figure 6 JoAnn
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Does rib affect salivary gland migration directly
or indirectly?
Rescue Fkh-Gal4 UAS-rib
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Expression pattern of rib
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What else in known about rib?rib affects
morphology of hindgut and Malpighian tubules
Malpighian tubules
hindgut
Jack lab, 1997
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rib affects epithelial cell shape
salivary
hindgut
Jack lab, 1998
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rib affects apical constriction
Salivary gland
Jack lab, 1998
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What about branching in other systems?

• Vertebrate lung branching
• Vertebrate salivary gland branching

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Lung branching follows a stereotypical pattern
Bellusci
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Epithelial-mesenchymal interactionslung
budding/branching
Hogan
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Permissive/instructive Mesenchyme in lung
development
Mesenchyme is required for lung and tracheal
development Lung endoderm lung mesenchyme
lung Lung endoderm tracheal mesenchyme
trachea Tracheal endoderm lung mesenchyme lung
trachea
branching lung
no branching
branching
Bellusci
Wessels
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Fibronectin surrounds developing lung
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Inhibiting Wnt and inhibiting FN binding each
decrease lung branching
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FN rescues Wnt inhibition
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Morphogenesis by proliferation
Bellusci, 1997
Lung endoderm has a higher proliferative rate
where it is budding out
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Salivary gland initiates branching by cell
contraction
Cleft formation
Microfilaments contract on one side
Microfilament contraction
Tissue bends
Cytochalasin B
Wash out Cytochalasin B
Spooner and Wessells
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Salivary gland initiates branching by cell
contraction
Cleft formation
Microfilaments contract on one side
Microfilament contraction
Tissue bends
Cytochalasin B
Wash out Cytochalasin B
Spooner and Wessells
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ECM components are deposited differentially in
clefts vs. lobules
Salivary gland
Fibronectin RNA
Fibronectin ISH
Perlican
/
Sakai et al., 2003
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Blocking fibronectin inhibits lobulation in
salivary gland
Buds/gland
control
a-FN
a-FN
Sakai et al., 2003
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Blocking fibronectin expression inhibits
branching in salivary gland
Cy3-siRNA
Sakai et al., 2003
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Adding fibronectin to media enhances branching
So, it does not have to come from the epithelium
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Fibronectin from media gets deposited in forming
clefts
Design
Red-FN
2 h
Green-FN
4 h
Visualize
Yamada lab
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Branching/tubulogenesis summary

• Involves differential migration and shape changes
• Is regulated by growth factors, transcription
  factors and ECM components
• Movement, architecture and proliferation have to
  be precisely orchestrated