Biology 624 - Developmental Genetics

Lecture #8 - Tube Formation I

Tubular Organs

Tube Formation is critical to forming:

1.Lung*2.Kidney*3.Mammary gland4.Blood vessels*5.Fly trachea6.C. elegans excretory system (1 cell!)

From Nelson, 2003

There are three types of tubes:

1.Multicellular2.Unicellular, with autocellular

junction3.Unicellular, seamless

From Lubarsky and Krasnow, 2003

Processes of Tubulogenesis

From Lubarsky and Krasnow, 2003

Processes of Tubulogenesis

From Lubarsky and Krasnow, 2003

Genetic Programs involved in Tube Formation

From Nelson, 2003

MDCK Cells in Collagen Gels

- HGF +HGF

MDCK in collagen starts with a polarized cyst

Gp135 = apical, redb-catenin = basolateral, green

From Pollack et al, 98; Zegers et al, 03

Upon addition of HGF, some cellsform an extension outward on their

basolateral side

Gp135, apicalE-cadherin, basolateral

From Pollack et al, 98

The extended cell divides, and cells in the extension express E-cadherin

but not gp135

E-cadherin, basolateral

Gp135, apical

From Pollack et al, 98

From Pollack et al, 98From Pollack et al, 98

As the lumen begins to reform in theextension, gp135 is re-expressed

on the new apical surface

E-cadherin, basolateral

Gp135, apical

From Pollack et al, 98

As lumen formation continues, cells re-establish basolateral expression of

E-cadherin

E-cadherin, basolateral

Gp135, apical

Model derived from this study:

1.Stimulation of migration is the first step in tubulogenesis

2.Apical/basal polarity is transiently lost and then restored

3.Discontinuous lumens form in tubules

4.Cell-cell contacts are retained throughout the process

Apical membrane biogenesis is importantFor lumen formation in MDCK cells

From Lubarsky and Krasnow, 03

Tube formation requires and stabilizes apical-basolateral polarity

From Bryant and Mostov, 08

From Bryant and Mostov, 08

Tube formation requires and stabilizes apical-basolateral polarity

From Uv et al, 2003

Drosophila Tracheal System

•Simple structure

•Powerful genetics

•Easy observation

Embryonic Tracheal Development

From Uv et al, 2003

I

II

III

IV

Drosophila Trachea Has Four Types of Tubes

Drosophila Trachea Has Four Types of Tubes

From Uv et al, 2003

4-5 µM 1 µM 0.5 µM

2-5 cells compose the lumen circumference

single tube-shaped cellswith AJ encircle the lumen

doughnut shaped cells with no AJ

protrusions from single cells without AJ

1 µM or 4-5 µM

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to

FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching

Specification of Tracheal Cells

From Affolter and Shilo, 2000

Trh = trachealess (bHLH PAS transcription factor)Tgo=Tango (bHLH Pas transcription factor, binding partner for Trh)Vvl/Dfr = Ventral veinless-Drifter (POU domain transcription regulator)

Invagination of Tracheal Placode

EGFR = epidermal growth factor receptorSpitz=EGFR ligandRho = Rhomboid (EGF pathway activator)

From Affolter and Shilo, 2000

Model of Tracheal Cell Invagination

Trh, Vvl

Rho (EGF pathway)

Apical Actin Enrichment

Localized Apical Cell Constriction

Ordered Cell invagination

Brodu V and Casanova J 2006

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to

FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching

DB = dorsal branch

DTa/p = dorsal trunk (anterior/posterior)

VB = visceral branch

SB = spiracular branch

LTa/p = lateral trunk (anterior/posterior)LTp is also called GB or ganglionic branch

Branches of the Drosophila Trachea

From Cabernard et al, 2004

From Affolter M 2002

Determination of branch identity

EGF=Epidermal growth factor : GB and DTWg= wigless: DTDpp = Decapentaplegic (Transforming growth factor β-like): DB, LT

EGF αPS1 integrin

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to

FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching

FGF Signaling Directs Primary Branch Outgrowth

From Cabernard et al, 2004

bnl/FGF = blueBtl/FGFR in Trachea cells = brown

Primary Branching Requires Bnl/Btl Signaling

From Cabernard et al, 2004green = actin-GFP (WT)Red = cells lacking Btl/FGFr

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to

FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching

Dpp Signaling is Required for Dorsal Branch Migration in Addition to FGF Signaling

Affolter M 2002

Dpp (ligand)Pnt (receptor)

From Englund et al, 2002

Branch Migration Requires Slit/Robo Signaling

blue= tracheal lumenBrown =DSRF (GB marker)

From Cabernard et al, 2004

Branch Elongation via Cell Rearrangements

Type I Type II Type IIType I

AJ Remodeling during Intercalation Process

From Ribeiro C et al 2003

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to

FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching

From Uv et al, 2003

Formation of Specialized Tracheal Cells

Fusion Process

E-Cadherin Membrane

Cytoskeleton Lumen

Migration Contact Adhesion Invagination Lumen Formation

Dysfusion is Expressed in Tracheal Fusion Cells

DT

DB

LT

btl-lacZ Dys

dys Misexpression Causes Inhibition of Migration

DysMab 2A12

Wild type

btl-gal4; UAS-dys

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to

FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching

From Uv et al, 2003

I

II

III

IV

VB that has ramified to form dozens of fine terminal branches on the gut

Larval Tracheal System

Ghabrial and Krasnow MA 2003

Terminal branch expansion in response to Hypoxia induced branchless

Jarecki J, Johnson E and Krasnow MA 1999 Type IV tube

Model for Patterning of Terminal Branching by Bnl

Jarecki J, Johnson E and Krasnow MA 1999

Steps of Tracheal Tube Formation

• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode

• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals and branch

identity genes Branch identity dentermination DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation

• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching