Cre-Lox Basics - Generating Knockout Mice

ECM and Mechanical Signals

How to build a blood vessel MOVIES Origins of Vascular Smooth Muscle Cells

Wnt1Cre/R26R

Majesky, Arterioscler. Thromb. Vasc. Biol. 27:1248 (2007) Jiang et al., Develop. 127:1607 (2000) Cre-loxP

Cre (“causes recombination”) Recombinase: Catalyzes recombination between two LoxP sites

loxP site: 34 base pair DNA sequence Location and orientation of loxP sites determines: • Deletion (same chromosome, same orientation) • Inversion (same chromosome, opposite orientation) • Translocation (different chromosomes) Cre-loxP: Gene Deletion

Cre-Lox Basics - Generating Knockout Mice. The Jackson Laboratory Download from class website FLP-FRT System

Flipase recombinase (FLP) recognizes a FRT sequences Inducible Cre Models: CreER

CreERT2

Kohan, Kidney Int 74:427 (2008) Doxycyline-regulated Gene Expression

Kohan, Kidney Int 74:427 (2008) Mfap2 Knockout Allele Lineage Tracing Reporter Line and Cre Drivers

mTmG reporter mouse line

CRE Drivers

Smooth Muscle Cells

Secondary Heart Field

Neural Crest

Endothelial Cells

Endothelial Cells Sm22αCre-mediates recombination in VSMC

Sm22αCre;Elnf/+ Sm22αCre;Elnf/+

P14

AscAo mT/mG DescAo

SM22+ (SMC) lineage Cdh5-Cre & Tie2-Cre mediate recombination in EC

Cdh5-Cre;Elnf/+

AscAo DescAo

mT/mG

Endothelial Cell lineage Isl1-Cre (Secondary Heart Field)

Isl1Cre;Elnf/+

P20

AscAo

SMC (SHF) lineage Cells from SHF lineage are major contributors to neointima

Isl1-Cre;Elnf/f

SMC (SHF) lineage Three Requirements of Elastic Vessels

• Highly resilient wall

• Nonlinear Elasticity

• Low hysteresis Elastic Artery! Elastic Artery!

Muscular Artery!

Establishment of SMC Layers is Complete by ~E14 in the Mouse Aorta

In Adult Mouse Ascending Aorta: ~8 SMC layers Descending Aorta: ~5 SMC layers

~E9-10: Heart begins to beat ~E12: Heart is chambered-- Pulsatile ﬂow ~E14: Separation of systemic and E12 pulmonary circulations Physiology and Gene Expression Analysis

Blood pressure, vascular compliance, and • Gene Expression analysis in aortic tissue. cardiac output on mice from E14-Adult . • Extracellular Matrix Expression in the Vessel Wall

• Early stages (proteins (eg., ﬁbronectin).

• As SMCs begin to form tight layers, enhanced expression of proteoglycanases and basement membrane proteins.

• With the onset of pulsatile ﬂow and increases in blood pressure, expression of structural matrix proteins (elastin, ﬁbrillar collagens, etc.) predominates. Correlation Between Changes in Blood Pressure and ECM Expression

Wagenseil and Mecham, Physiol. Rev. 2009 Elastic Modulus as a Function of Blood Pressure

Shadwick, J. Exp. Biol. 202:3305 (1999)

Elastic Modulus = ~0.4 (0.3- 1.0) MPa for all vessels Characteristic wave velocity = ~2-4 m/s Unanswered Questions

• How do cells sense mechanical forces?

• How do cells know which matrix molecules to produce and when/where to secrete them?

• How do cells secrete and assemble the complex biopolymers of the ECM?

• How do cells interpret ECM complexity to regulate gene expression and cellular differentiation? Learning Objectives

• Cell movement and differentiation is greatly inﬂuenced by the ECM (chemotaxis and haptotaxis)

• Know how Cre-loxP systems work: • 1) to inactivate/activate genes • 2) to lineage trace cells in tissues • 3) as conditional and inducible systems • 3) with FLP-FRT system

• Understand why the extracellular environment is important in regulating gene expression.

• Know that cells sense mechanical forces in their microenvironment.

• Know which ECM molecules cells use to impart needed mechanical properties to tissues