Transcript Animal Models of Pediatric Combined Pituitary

Animal Models of Pediatric
Combined Pituitary Hormone Deficiency Diseases
Stephanie C.
1,2
Colvin ,
and Simon J.
1
Rhodes
1Department
of Cellular and Integrative Physiology, Indiana University School of Medicine,
2Department of Biology, Indiana University-Purdue University Indianapolis
Abstract
Introduction – LHX3
LHX3 is a LIM homeodomain transcription factor that has essential roles in
pituitary and nervous system development in mammals. Through in vitro and
in vivo experimentation, we have elucidated roles for LHX3 in pituitary
development. At least two mRNAs are transcribed from the LHX3 gene from
which three protein isoforms are translated (LHX3a, LHX3b, and M2-LHX3),
each with distinct biochemical properties. In collaboration with hospitals
worldwide, including University Children’s Hospital, Leipzig, Germany and
Riley Hospital for Children in Indianapolis, we have been investigating the
molecular consequences of novel LHX3 mutations in pediatric patients.
Patients with mutations in the gene encoding this regulatory protein present
with combined pituitary hormone deficiency characterized by the absence of
multiple anterior pituitary hormones. Some LHX3 patients present with
additional neural defects, including a characteristic limited head rotation.
However, of the seven LHX3 mutations that have been published, only one of
these mutations does not induce the characteristic limited head rotation. This
W224ter mutation introduces a premature stop codon predicted to cause loss
of the carboxyl terminus of the LHX3 protein. The phenotype of patients with
this mutation supports the hypothesis that the actions of LHX3 in the pituitary
and nervous system are functionally separable, perhaps mediated by the
different domains of the protein, and that the carboxyl terminus of LHX3 is
essential for pituitary development. To further investigate this hypothesis, a
knock-in mouse model of this human disease is being generated so the
molecular/cellular effects of this particular mutation can be studied throughout
development, an approach that is not feasible with the human patients.
LHX3
LHX3a W224ter Family Pedigree
•LIM homeodomain transcription factor expressed in developing spinal cord,
medulla oblongata, pineal gland, lungs, and pituitary gland1.
Patients have a milder form of
hormone deficiency, a normal pituitary
morphology, and a normal neck
phenotype17
Role of Lhx3 in developing pituitary
neuroectoderm
LH
FSH
TSH
Sheng et al., Science, 1997
PRL
GH
Missing
•Lhx3 -/- mice die within 24 hours after birth, a definitive pouch forms, but four
of the five hormone-secreting cell types are missing3,4.
Role of Lhx3 in developing nervous system
•Ventral motor neurons develop normally in Lhx3 -/- mice and in Lhx4 -/- mice7.
•In Lhx3 -/-/Lhx4 -/- double knockouts, no ventral motor neurons develop7.
•V2 interneuron specification requires tetrameric complex of NLI and Lhx38,12.
•Motor neuron specification requires hexameric complex of NLI, Isl1, and
Lhx38,12.
E
Activation of the Hb9 motor neuron (MN ) enhancer
NLI
ISL1
LHX3
NeuroM
+ E47
Research Design and Methods
TRANSCRIPTIONAL REGULATION OF
ANTERIOR PITUITARY DEVELOPMENT
2
Ib
3
4 5
6
3'
5'
neuroectoderm
Rathke’s
Pouch
HD
LIM 2
LHX3a mRNA
ΔT
=g.159delT
LHX4
LHX3 gene deletion
GC→TCCT
= E173ter
A→G
= Y111C
LHX3
Ia
Ib
II
5'
Time
Prop1
SF1
Pit-1
8.
III
IV
VI
3'
12.
13.
14.
7
12
55
107
121
132
150
70
73
M
Neo
V
loxP
•All LHX3 mutations to date are homozygous
•Patients present with combined pituitary hormone deficiency (CPHD).
Deficient in GH, TSH, LH, FSH, and PRL.
•Variable pituitary morphology.
•Some patients present with a rigid cervical spine leading to limited head rotation.
7.
11.
3088bp intragenic
deletion
recessive5,6,17,18
6.
10.
loxP
Tpit
5.
9.
•Used PCR and Southern Blotting to:
•Confirm the presence of Neo
•Confirm the presence of the point mutation
C→T
G→A
23bp del = A210V = W224ter
A→T
= K50ter
4.
Identification of Correctly Targeted Clones
BamHI
Hesx1
LIM 1
•Mice may display a normal phenotype
•If the amino terminus, LIM domains, and the homeodomain are all that are
required for the proper function of LHX3
•Protein may exert a dominant negative effect
•Heterozygotes will be affected
•Mice may display a milder phenotype similar to the human patients
•Delayed growth, thyroid problems, reproductive problems
•Recapitulation of Lhx3 -/- phenotype
•Mice are not viable
•Mice may display a nervous system phenotype
•Carboxyl terminus may have some impact on LHX3 function during
nervous system development
•Mice may not display a nervous system phenotype
•If LIM domains and homeodomain are all that are necessary for proper
LHX3 function in nervous system development
3.
•Used a gene targeting construct and homologous recombination to generate
chimeric mice with the W227Ter mutation knocked-in.
•Bred chimeras to generate heterozygote offspring.
•Crossed heterozygous offspring to EIIA-Cre mice to remove floxed PGK-neo
cassette.
•Heterozygous animals currently being crossed to generate homozygous
animals for analyses.
•Currently, 9 different human mutations have been reported within the
LHX3 gene5,6,17,18:
Ia
Possible Outcomes
2.
Generating Lhx3W227Ter Mice
Mutations in Human LHX3
W227Ter
15.
•Both PCR and Southern
blot confirm the presence
of the point mutation and
the Neo cassette.
16.
15kb
17.
4.9kb
Pit-1
PRL
Pit-1
Zn
Ia
5'
W224ter
Zn
Zn
Zn
Ib
II
III
Zn
IV
Neo
V
VI
3'
loxP
Alpha GSU
TSH beta
Pit-1
Zn
loxP
LHX3 pituitary target
genes2,9,10,11
Alpha GSU
FSH beta
GnRH-R
Zn
BamHI
Zn
Corticotrope Gonadotrope Thyrotrope Somatotrope Lactotrope
BamHI
LHX3a WT
KI=4.9kb
WT=15kb
HindIII
LHX3a W224Ter Patients
PRL
BbvCI
GH
BbvCI
TSH
BamHI
LH
FSH
18.
BbvCI
ACTH
Probe A
Adapted from Colvin et al., 2007 16
Nervous system?
Pituitary?
W227Ter
E17.5 WT TSH
E17.5 WT LH
1.
BamHI
AP
E12.5 WT LHX3 (LIM3)
References
HindIII
pp
•Generated and confirmed the presence of the point mutation and the Neo
cassette in chimeras and heterozygotes using these methods.
•Currently breeding heterozygote mice with the Neo cassette still intact to
generate homozygotes with Neo.
•Also breeding mice in which Neo has been excised by Cre recombinase to
generate homozygotes without the Neo cassette.
• Monitor animal morphology, growth, fertility, and to examine pituitary
morphology and gene expression.
Adapted from Lee and Pfaff, Neuron 38, 731 (2003)
BbvCI
Hormones
 Growth
 Metabolism
 Reproduction
 Stress response
 Lactation
The LHX3 W224Ter human patient symptoms suggest that the
actions of LHX3 in the pituitary and nervous system are
separable, mediated by the different functional domains/motifs
of the protein, and that the carboxyl terminus of LHX3 is
essential for pituitary development.
BbvCI
THE ANTERIOR PITUITARY
Hypothesis
BamHI
Introduction – Pituitary
BbvCI
ACTH
•LHX3a W224Ter retains intact LIM domains and the homeodomain, but lacks
the carboxyl terminus17.
•The carboxyl terminus has been shown to contain critical
activation/repression domains, targets for post-translational modification,
and intracellular targeting signals14,15.
•The patients have normal neck rotation and normal pituitary morphology17.
•The LIM domains and homeodomain of LHX3 are required for motor
neuron development7,8,12 – perhaps this explains the absence of limited
neck rotation in the patients.
•Although less severe and of later onset, patients still present with pituitary
insufficiency17 - the carboxyl terminus is important in pituitary function.
•Functional data suggest that LHX3 W224Ter may retain some function in the
pituitary17.
“Frozen” pouch
Rathke’s pouch
D4
•Functional data suggest that this protein may retain some
residual function17
Lhx3 -/- Mice
*
D3
D2
D1
oral ectoderm
Analysis of the Disease Model
LHX3a W224Ter Patients
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