Title: Animal Models of Pediatric
1Animal Models of Pediatric Combined Pituitary
Hormone Deficiency Diseases Stephanie C.
Colvin1,2, and Simon J. Rhodes1 1Department of
Cellular and Integrative Physiology, Indiana
University School of Medicine, 2Department of
Biology, Indiana University-Purdue University
Indianapolis
Introduction LHX3
Analysis of the Disease Model
LHX3a W224Ter Patients
Abstract
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 Childrens
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
- 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.
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
- Monitor animal morphology, growth, fertility, and
to examine pituitary morphology and gene
expression.
- Functional data suggest that this protein may
retain some residual function17
- 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.
- 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.
Hypothesis
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.
References
Research Design and Methods
- 1. Hunter CS and Rhodes SJ LIM-homeodomain genes
in mammalian development and human disease. Mol
Biol Rep 32 67-77, 2005. - 2. Savage JJ, Yaden BC, Kiratipranon P and Rhodes
SJ Transcriptional control during mammalian
anterior pituitary development. Gene 319 1-19,
2003. - 3. Sheng HZ, Zhadanov AB, Mosinger B, Jr., Fujii
T, Bertuzzi S, Grinberg A, Lee EJ, Huang SP,
Mahon KA and Westphal H Specification of
pituitary cell lineages by the LIM homeobox gene
Lhx3. Science 272 1004-7, 1996. - 4. Sheng HZ, Moriyama K, Yamashita T, Li H,
Potter SS, Mahon KA and Westphal H Multistep
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1809-12, 1997. - 5. Netchine I, Sobrier ML, Krude H, Schnabel D,
Maghnie M, Marcos E, Duriez B, Cacheux V, Moers
A, Goossens M, Gruters A and Amselem S Mutations
in LHX3 result in a new syndrome revealed by
combined pituitary hormone deficiency. Nat Genet
25 182-6, 2000. - 6. Bhangoo AP, Hunter CS, Savage JJ, Anhalt H,
Pavlakis S, Walvoord EC, Ten S and Rhodes SJ A
Novel LHX3 Mutation Presenting as Combined
Pituitary Hormonal Deficiency. J Clin Endocrinol
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A, Potter S, Westphal H, Pfaff SL. LIM
homeodomain factors Lhx3 and Lhx4 assign subtype
identities for motor neurons. Cell
199895(6)817-828. - Thaler JP, Lee SK, Jurata JW, Gill GN, Pfaff SL.
LIM factor Lhx3 contributes to the specification
of motor neuron and interneuron identity through
cell-type-specific protein-protein interactions.
Cell 2002110(2)237-249. - McGillivray SM, Bailey JS, Ramezani R, Kirkwood
BJ and Mellon PL Mouse GnRH receptor gene
expression is mediated by the LHX3 homeodomain
protein. Endocrinology 146 2180-5, 2005. - 10. West BE, Parker GE, Savage JJ, Kiratipranon
P, Toomey KS, Beach LR, Colvin SC, Sloop KW and
Rhodes SJ Regulation of the follicle-stimulating
hormone beta gene by the LHX3 LIM-homeodomain
transcription factor. Endocrinology 145 4866-79,
2004. - 11. Granger A, Bleux C, Kottler ML, Rhodes SJ,
Counis R and Laverriere JN The LIM-homeodomain
Proteins Isl-1 and Lhx3 act with Steroidogenic
Factor-1 to Enhance Gonadotrope-specific Activity
of the Gonadotropin-Releasing Hormone Receptor
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TM, Pfaeffle RW, Rhodes SJ. Mutations in the Lhx3
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target genes that reflect patient phenotypes.
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Rhodes SJ. The homeodomain coordinates nuclear
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Serine/threonine/tyrosine phosphorylation of the
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Jul 1517(14)2150-9
Generating Lhx3W227Ter Mice
Mutations in Human LHX3
- 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
Identification of Correctly Targeted Clones
- Used PCR and Southern Blotting to
- Confirm the presence of Neo
- Confirm the presence of the point mutation
- All LHX3 mutations to date are homozygous
recessive5,6,17,18 - 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.
- Both PCR and Southern blot confirm the presence
of the point mutation and the Neo cassette.
LHX3a W224Ter Patients