Animal Models of Pediatric

1
Animal 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
  control of pituitary organogenesis. Science 278
  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
  Metab 2006.
• Sharma K, Sheng HZ, Lettieri K, Li H, Karavanov
  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
  Gene Promoter. Mol Endocrinol 2006.
• Thor S, Andersson SG, Tomlinson A, Thomas JB. A
  LIM-homeodomain combinatorial code for
  motor-neuron pathway selection. Nature
  1999397(6714)76-80.
• 13. Savage JJ, Hunter CS, Clark-Sturm SL, Jacob
  TM, Pfaeffle RW, Rhodes SJ. Mutations in the Lhx3
  gene cause dysregulation of pituitary and neural
  target genes that reflect patient phenotypes.
  Gene 2007.
• Parker GE, Sandoval RM, Feister HA, Bidwell JP,
  Rhodes SJ. The homeodomain coordinates nuclear
  entry of the Lhx3 neuroendocrine transcription
  factor and association with the nuclear matrix. J
  Biol Chem 2000275(31)23891-23898.
• Parker GE, West BE, Witzmann FA, Rhodes SJ.
  Serine/threonine/tyrosine phosphorylation of the
  LHX3 LIM-homeodomain transcription factor. J Cell
  Biochem 200594(1)67-80.
• Mullen RD, Colvin SC, Hunter CS, Savage JJ,
  Walvoord EC, Bhangoo AP, Ten S, Weigel J,
  Pfaeffle RW, Rhodes SJ. Roles of the LHX3 and
  LHX4 LIM-homeodomain factors in pituitary
  development. Mol Cell Endocrinol. 2007
  Feb265-266190-5.
• Pfaeffle RW, Savage JJ, Hunter CS, Palme C,
  Ahlmann M, Kumar P, Bellone J, Schoenau E, Korsch
  E, Branswig JH, Stobbe HM, Blum WF, Rhodes SJ.
  Four novel mutations of the LHX3 gene cause
  combined pituitary hormone deficiencies with or
  without limited neck rotation. J Clin Endocrinol
  Metab. 2007 May92(5)1909-19.
• Rajab A, Kelberman D, de Castro SC, Biebermann H,
  Shaikh H, Pearce K, Hall CM, Shaikh G, Gerelli D,
  Grueters A, Krude H, Dattani MT Novel mutations
  in LHX3 are associated with hypopituitarism and
  sensorineural hearing loss. Hum Mol Genet. 2008
  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