Development of Diagnostic and

Therapeutic Procedures

Program 5

This programme focuses on the

identification of the molecular causes of

diseases and on the development of new

procedures for the prevention and

diagnostics of the diseases.

Genes, Mouse Models, Brain

Development and Pathologies

Nicole Dodd

13th June 2013Laboratory of Molecular Pathogenetics

Institute of Biotechnology AS CR v.v.i.

Overview

• The mouse model

• Protein of interest

• Cerebellum region of the brain

• Structure

• Development

• Rationale

• Preliminary results

• Summary

The mouse model

• Relatively easy to maintain, with the ability to

multiply quickly

• The mouse is closely related to humans

• Genome is organised similarly

• Most human genes have functional mouse

counterparts

• Mutations that cause diseases in humans often

cause similar diseases in mice

• Gene transfer technology is

highly advanced

Genetic manipulation

• Gene knock out

Sequenced but unknown

function

• Cre-lox recombination

Allows genes to be

activated, repressed, or

exchanged

• Transgenic

Introducing an exogenous

gene

Cre recombinase activity indicated by the ROSA26-stop-lacZ reporter

Floxed Neurod1/Atoh1

Cre Isl1 Isl1

LacZ Floxed

STOP β-Galactosidase catalyzes

the hydrolysis of X-Gal

producing a blue precipitate

LoxP

R26R

Promoter

Transgenic cre-lox recombination system

Pax2-Isl1 Transgene

Pax2 8.5-kb regulatory

sequencePax2 promoter Isl1

SalI, AatlI, NotI

pGEM7f

SalI, NotI, AatlI

8.5Size (kb): 1.6 0.8 3.4

lacZ mRNA tag

NotI

Wnt1 poly A

• Islet1 knock-out is lethal in early development

• Generated transgenic mice

Transgenics - pronuclear injection of Pax2-isl1 construct

Pax2 promoter

LIM LIM

Homeodomain

DNA bindingProtein binding

Isl1

FVB

T2

T1

-/+

-/+

• Transcription factor Islet1 (Isl1)

• Protein that binds to DNA to control the flow of

transcription

• Important for development of a number of organs

• Expressed in ganglion neurons

• Required for various aspects of motor neuron

development

• Represses genes involved in neurogenesis

Protein of interest

The Cerebellum

• Represents 10% of

total brain vol.

• Contains more than

half of our neurons

• Functions to fine

tune movement and

balance

• Plays a major role in

the coordination of

muscle activity

Cerebellum location

• Located at the back of the brain, underlying the cerebral

cortex

• Develops from the dorsal region of the posterior neural tube

• Cells arise from two different germinal matrices

• Size and structure are determined by correct number of

neurons (proliferation and apoptosis)

• Correct location of neurons (migration)

• Cell type (differentiation)

Brain (cerebellum) development

Bergman glia

Purkinje layer

External granular layer

Molecular layer

Internal granular layer

Defects in cerebellar neuronal proliferation and

migration lead to tumour and ataxia

Medulloblastoma

• Malignant

medulloblastomas are

the most common

malignant brain

tumour of childhood

• Thought to originate

from granule neuron

precursor cells the

developing cerebellum

Rationale

• Unravel the role of Isl1 in neurosensory development

• Due to early KO lethality, role in motor neuron development

is unclear

• Determine the effects of ectopic expression in the

cerebellum on proliferation, migration & differentiation

• Defects in cerebellar neuronal proliferation and migration

can lead to cancer

0

2

4

6

8

10

12

wt T1 T2

Nu

mb

er

of su

rviv

ing

p

up

s/litte

r

Number of surving pups

P < 0.001

• Significant difference in number of surviving pups

• Transgenic expression of Isl1 affects embryonic

development and postnatal survival of transgenic

pups

Number of surviving pups

Changes in Cerebellum

T2wt

P0

P3

Immunohistochemistry

• By staining sections with fluorescently labelled antibodies we

can identify the cells that are expressing the transgene

T1 P3 mt T1 P3 wt

• Significant changes in

morphology

• Isl1 misexpression

• Mechanism?

• Possible interactions with other

TFs

Summary