mouse molecular genetic studies of organogenesis · 2018. 11. 21. · richard behringer university...
Post on 02-Feb-2021
1 Views
Preview:
TRANSCRIPT
-
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!
top related