Animal Models of Pediatric Combined Pituitary Hormone Deficiency Diseases

1. Ia 2 3 4 5 6 Ib HindIII 3' 5' BamHI BamHI BamHI BamHI BamHI BbvCI BbvCI BbvCI BbvCI BbvCI BbvCI Neo loxP loxP W227Ter 55 12 70 73 107 121 150 132 HD LIM 1 LIM 2 M 7 15kb E17.5 WT TSH E12.5 WT LHX3 (LIM3) E17.5 WT LH 4.9kb Possible Outcomes • 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 LHX3function in nervous system development NLI ISL1 D3 D4 D2 D1 LHX3 NeuroM + E47 Adapted from Lee and Pfaff, Neuron 38, 731 (2003) 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 Abstract LHX3a W224Ter Patients 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 • 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 LHX3a mRNA ΔT =g.159delT neuroectoderm C→T = A210V G→A = W224ter 23bp del • Monitor animal morphology, growth, fertility, and to examine pituitary morphology and gene expression. LHX3 gene deletion A→T = K50ter • Functional data suggest that this protein may retain some residual function17 GC→TCCT = E173ter A→G = Y111C Lhx3 -/- Mice oral ectoderm • 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. 3088bp intragenic deletion • 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 LHX3a WT Zn Zn Zn Zn 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. W224ter Zn Zn Zn Zn Introduction – Pituitary Nervous system? Pituitary? THE ANTERIOR PITUITARY Activation of the Hb9 motor neuron (MNE) enhancer 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 1998;95(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 2002;110(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 1999;397(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 2000;275(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 2005;94(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 Feb;265-266:190-5. • 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 May;92(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 15;17(14):2150-9 Generating Lhx3W227Ter Mice Hormones Mutations in HumanLHX3 • Growth • Metabolism • Reproduction • Stress response • Lactation • 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: “Frozen” pouch Rathke’s pouch TRANSCRIPTIONAL REGULATION OF ANTERIOR PITUITARY DEVELOPMENT pp AP Identification of Correctly Targeted Clones neuroectoderm • Used PCR and Southern Blotting to: • Confirm the presence of Neo • Confirm the presence of the point mutation LH FSH Rathke’s Pouch TSH PRL ACTH GH Hesx1 Sheng et al., Science, 1997 LHX4 Missing LHX3 • 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. Tpit • Both PCR and Southern blot confirm the presence of the point mutation and the Neo cassette. Time Prop1 SF1 Pit-1 LHX3a W224Ter Patients KI=4.9kb WT=15kb HindIII Ia Ib II III VI V IV 5' 3' LHX3 pituitary target genes2,9,10,11 Neo loxP loxP Probe A W227Ter BamHI LH FSH GH ACTH TSH PRL Ia Ib II III VI V IV 5' 3' Lactotrope PRL Pit-1 Somatotrope Pit-1 Gonadotrope Alpha GSU FSH beta GnRH-R Thyrotrope Alpha GSU TSH beta Pit-1 Corticotrope Adapted from Colvin et al., 2007 16