Mouse Molecular Genetic Studies of Organogenesis

Richard Behringer University of Texas - M D Anderson Cancer Center

rrb@mdanderson.org

Richard BehringerProgram in Genes & Development

M. D. Anderson Cancer Center

Wee, sleekit, cowrin, tim’rous beastie

Robert Burns, 1785

birds reptiles mammals

monotreme

marsupial

eutherian

Amniotes

House mouse

Mus musculus

gestation: 20 days

weaning: 3 weeks

sexual maturity: 6 weeks

life span: 1-3 years

Karyotype (haploid): 20 chromosomes

Genome size (haploid): 3 x 109 bp

blastocyst cavityblastomereinner cell massmural trophoblastpolar bodypolar trophoblastpronucleuszona pellucida

Mouse preimplantationdevelopment

0.5-3.5 days postcoitus (dpc)ie plug

5.5 days 6.5 days 7.5 days 8.5 days

4.5 days1 day 3 days

Early mouse development

9.5 days*

How big are whale eggs?

Blastocysts without ICMs

Preimplantation embryos 1 meter long

Axis formation before implantation

Human gastrula

Human embryoCarnegie No. 7801Stage 6b ~13 - 13.5 daysInitiation of gastrulation

ys

ac

ep

am

hyp

Why mouse?Eutherian mammal

Mutants (spontaneous & induced)

Chimeras

Transgenics

Embryonic stem (ES) cells

Cloning

Cryopreservation

Genome sequenced, assembled, & annotated (C57Bl/6 inbred)

MutagenesisRadiation

Gene Targeting

Insertion

Retrovirus

Transgene

Transposon

Gene Trap

Chemical

Ethylnitrosourea (ENU)

Sleeping Beauty

Intl. Gene Trap Consort.

Agouti

Sexual development

Sry10.5 - 12.5* dpc

genital ridge

Sry = Testis determining gene

XY Srynull = FemaleXX Sry transgene = Male

• Sry-related gene• HMG transcription factor• Expressed in many tissues,

notably male genital ridge• Evolutionarily conserved

Sox9

Sry testis

Sox9: In vivo evidence

• SOX9 +/- XY human femalescamptomelic dysplasia

• Sox9 +/- XY mice are male

Loss of function

• Correlative evidence suggests that SOX9 is required in a dosage dependent manner for testis formation in humans

• 17q24 duplication XX human male• Odsex XX male mice• Wt1-Sox9 XX male embryos

Gain of function

Sox9: In vivo evidence

Odsex transgenic mice

• Optic degeneration sex reversal• Tyrosinase transgene• One line had no transgenic females• XX tg/+ develop as males• Transgene inserted ~1 Mb 5’ of Sox9• XX tg/+ fetal gonads upregulate Sox9

• Sox9 is sufficient for testis formation in XX mice

• 17q24 duplication XX human male• Odsex XX male mice• Wt1-Sox9 YAC XX male embryos

Gain of function

Sox9: In vivo evidence

Sox9 mutant miceBi et al., 2001

• Sox9 +/- XY form testes• Sox9 +/- mice die at birth with severe skeletal defects, i.e. camptomelic dysplasia

Cre/loxP

• Cre is a DNA recombinase

• Cre specifically recognizes 34 bp loxP sites

Cre Cre

Sox9 conditional null allele

• Sox9 flox allele (Chaboissier and Schedl)

• Sox9del/+ mice are identical to Sox9+/- mice (Akiyama et al., 2002)

loxP loxP

Cre

flox

recombined

Sox9

loxP

Breeding schemeXX Sox9 flox/flox;Zp3-Cre tg/+

Sox9 ΔCreoocyte X

XY Sox9 Δ/Δ (75%)

XY Sox9 flox/flox;Prm1-Cre tg/+

Sox9 ΔCresperm

Sox9 Δ/Δ mutant phenotypeAkiyama et al., 2003

• Embryonic lethal at E11.5• Neural tube, cardiovascular, and other defects• Gonads present but before testis differentiation

Sox9 germline knockout

Absence of testicular cords in Sox9Δ/Δ XY gonads

Sox9Δ/Δ XY gonads are sex reversed

Sox9 represses Sry expression

• Sox9 +/- XY mice have testes• Sox9 -/- XY mice form ovaries• Sry expression not down-regulated• Sox9 is essential for testis formation

Sox9 in mouse testis formation

Sry Sox9 testis

Sry Sox9 testis

Sry Sox9 Mis

Müllerian duct regression

Mullerian duct formation

Lim1-lacZ is expressedin the forming Müllerian duct

Lim1 is expressedin Müllerian duct epithelium

Persistent Müllerian duct syndrome

Persistent Müllerian duct syndrome

ut

tt

ut

vd

t t

vd

t tut

vd

WT XY WT XX

Misr2 -/- XY Bmpr1a flox/- XY

MIS signaling pathway

BMPR-IA

MISR-II

mesenchyme epithelium

Müllerian DuctFetal Testis

MIS

Sertoli cell

Regression

What mediates the MIS signal?

• Wnt4 expressed in Müllerianduct mesenchyme

• Wnt4 mutant mice do not form Müllerian ducts

Absence of Müllerian ducts in Wnt4 mutants

Generation of aWnt4 conditional null allele

exon 2

loxP loxP

Cre

loxP

β-catenin regulated by MIS?

Catnb (β-catenin) conditional null allele

exons 2-6

loxP loxP

Cre

loxP

7 8

7 8

R26R Cre Reporter Mice

pAlacZstopPromoter

pAlacZPromoter

Cre

loxP loxP

R26R/+; Misr2Cre/+

MIS signaling pathway

BMPR-IA

MISR-II

Müllerian mesenchyme

MIS

Catnb

Wnt4Smad

Wnt4

Frizzled

Sox9

• Akio Kobayashi• Soazik Jamin• Guy Eakin

• Benoit de Crombrugghe• Marie Chaboissier• Andreas Schedl

• NIH & NSF

Acknowledgments

Obrigado!

Wee, sleekit, cowrin, tim’rous beastie�House mouseHuman gastrulaWhy mouse?MutagenesisSexual developmentSry = Testis determining geneSox9: In vivo evidenceSox9 mutant mice�Bi et al., 2001Cre/loxPSox9 conditional null alleleBreeding schemeSox9 D/D mutant phenotype�Akiyama et al., 2003Sox9 germline knockoutAbsence of testicular cords in Sox9D/D XY gonadsSox9D/D XY gonads are sex reversedSox9 represses Sry expressionMüllerian duct regressionMullerian duct formationLim1-lacZ is expressed�in the forming Müllerian ductLim1 is expressed�in Müllerian duct epitheliumPersistent Müllerian duct syndromePersistent Müllerian duct syndromeMIS signaling pathwayWhat mediates the MIS signal?Absence of Müllerian ducts in Wnt4 mutantsGeneration of a�Wnt4 conditional null alleleb-catenin regulated by MIS?Catnb (-catenin) �conditional null alleleR26R Cre Reporter MiceR26R/+; Misr2Cre/+MIS signaling pathwayAcknowledgmentsObrigado!