HomeodomainMediated CateninDependent Switching Events Dictate CellLineage Determination

1
Homeodomain-Mediated ß-Catenin-Dependent
Switching Events Dictate Cell-Lineage
Determination

• Lorin E. Olson1,8,9, Jessica Tollkuhn1, 2, 8,
  Claudio Scafoglio1, 3, Anna Krones1, Jie Zhang1,
  Kenneth A. Ohgi1, Wei Wu1, Makoto M. Taketo4,
  Rolf Kemler5, Rudolf Grosschedl5, Dave Rose6, Xue
  Li7, and Michael G. Rosenfeld1,
• 1Howard Hughes Medical Institute, Department and
  School of Medicine, University of California, San
  Diego, La Jolla, CA 92093, USA2Biomedical
  Sciences Graduate Program, University of
  California, San Diego, La Jolla, CA 92093,
  USA3Dottorato di Ricerca in Oncologia Medica e
  Chirurgica ed Immunologia Clinica (XVIII Ciclo),
  Seconda Università degli Studi di Napoli, Napoli
  80138, Italy4Department of Pharmacology,
  Graduate School of Medicine, Kyoto University,
  Yoshida-Konoé-cho, Sakyo Kyoto 606-8501,
  Japan5Max-Planck-Institute of Immunobiology,
  Stubeweg 51, 79108 Freiburg, Germany6Division of
  Endocrinology and Metabolism, School of Medicine,
  University of California, San Diego, La Jolla, CA
  92093, USA7Department of Surgery/Urology,
  Department of Pathology, and Children's Hospital,
  Harvard Medical School, Boston, MA 02115, USA

Cell 125, 593-605, May 5, 2006
2
Wnt/b-Catenin Pathway

• Embryogenesis
• Tumorigenesis
• Stem cell development

• b-Cat
• Acts as a nuclear co-activator
• Recruits P300/CBP
• Interacts with Lef/TCF Family

3
Anterior Pituitary Gland

• Good model for studying signaling
• Progresses from a primodium of pluripotent cells
  to a complex organ containing distinct hormone
  producing cells

• Cell Types (5)
• Corticotropes - ACTH
• Lactotropes Prl
• Somatotropes GH
• Thyrotropes - TSH
• Gonadotropes FSH and LH

4
Pituitary Cell Differentiation

• Pit-1
• Plays roles in production of
• Somatotropes
• Lactotropes
• Thyrotropes
• Regulation of Pit-1
• Positive Prophet of Pit-1 (Prop-1)
• Negative Hesx-1

• Summary
• Wnt/b-Cat pathway helps in determining cell
  lineages by the interaction of b-Cat and Prop-1
  in the activation of the Pit-1 gene

5
Wnt
?
b-Cat Prop-1
?
Pit-1
Cell Differentiation
6

• A. In situ hybridization
• Lef1 a co-activator
• Axin2 a Wnt target
• B. ChIP on microdissected Rathkes Pouches at
  e12.5
• b-cat found on Axin2 and Lef1 promoters
• C. Cre-expressing transgenic lines
• EX3 (CA-Cat) generates a stable for of b-Cat
• Ex2-6 (KO-Cat) deletes critical b-Cat coding
  region
• b-Cat Genotypes (FITC)
• D. Coordinated Expression of repressors and
  activators

Fig. 1
7

• A. Sections of Embryonic Pituitary
• KO-Cat had normal development
• B. In situ hybridization and qPCR
• Pit1 expression absent
• CD. GH, TSH, LH, PRL hormone markers for Pit1
  dependent cell types
• Markers completely absent
• E. Later stages of development
• Normal hormonal levels by PO

Fig. 2
8
How do Lef1 and Axin2 play roles?

• A. b-Cat KO Mice
• Axin2 and Lef1 expression
• B. Expression comparison of Lef1 and Pit1
• CD. Lef1 deleted mice
• E. ChIP Analysis
• Pit1 gene
• F. Active Pit1 gene

Fig. 3
9
Are a complex of Prop1 and b-Cat required for
Pit1 activation?

• A. Prop1 KO mice
• Loss of Pit1 expression
• B. Prop1 and b-Cat interaction in double
  heterozygous embryos
• Diminished Pit1 expression
• C. In vitro biochemical interaction
• Interaction between b-Cat and Prop1

Fig. 4
10
Are in vitro results true for cellular context?

• D. Co-IP
• b-Cat IP with Prop1
• E. PrdQ site consensus Prop1 binding site
• b-Cat IP with PrdQ
• F. Pit1 promoter activity
• Prop1 and b-Cat work synergistically
• G. KO of b-Cat
• J. Inhibitors of b-Cat
• Chibby, ICAT
• KL. Excess Lef1 impairs recruitment of Prop1 to
  PrdQ site of b-Cat

Fig. 4
11

• ChIP Assays
• A. e11.5
• Inhibitors Hesx1 and TLE are bound
• No Pit1 expression
• B. e12.5
• Prop1 occupies EE
• TLE still present in small amount
• Dimethylation of EE
• C. e13.5
• Prop1 and b-Cat occupy EE
• Promoter is Di- and Trimethylated
• D. Adult
• Both LE and EE are methylated
• Full activation of Pit1

Fig. 5
12
Does b-Cat inhibit Hesx1?

• A. In situ hybridization
• Hesx1 persistence
• B. b-Cat activation
• Absent pituitary at e13.5
• C. LHx3 Rathkes Pouch Marker
• LHx3 expression normal
• Hesx1 not expressed
• D. LHx3 absent by e10.5
• E. Lef1 and Axin2 are Wnt/b-Cat targets
• Both upregulated
• G. Transfection assay
• Prop1 repression of Hesx1
• H. ChIP Hesx1 gene
• Prop1 and b-Cat present on Hesx1 gene regulatory
  region
• IJ. Prop1 reduces activity of Hesx1

Fig. 6
13

• b-Cat Control of Cell Lineage Determination

Fig. 6