Homeobox Genes and Muscle Development
Melissa Calhoun
Dr. Chrissa Kioussi
Department of
Biochemistry & Biophysics
Homeodomain Transcription Factors
Pattern Formation
Cell Fate Determination
Organogenesis
Human Disorders
Pitx2 / RIEG / Otlx2 / ARP1
Rieger Syndrome (PITX2, 4q25)
Expressed in the left lateral plate mesoderm
Expressed in muscles
Target of the human acute leukemia ALL1
R
L
Pitx2 in Humans, Rieger Syndrome
Autosomal Dominant Disorder
Developmental defects in eyes,
tooth and heart
Mental Retardation
Phenotype of Pitx2 Gene-deleted Mice
Visceral and Axial Turning
Abnormalities
Right Pulmonary Isomerism
lung
+/+
+/+
-/-
Organ Proliferation Defects
+/-
-/-
-/-
Mouse Pitx2 Isoforms
Exon I
Pitx2a
+
Pitx2b
+
Pitx2c
Exon II
Exon III
+
+
Exon IV
HD
Exon V
HD
+
+
+
+
+
+
Pitx2c controls left right asymmetry in organs.
Pitx2 Over-expression Plasmids
Pitx2c
Cloning Procedure
Amplify insert by PCR, gel purify.
Plasmid
Cut both insert and vector with restriction enzyme(s).
E.coli
Ligate insert and vector (Quick Ligase, New England
BioLabs).
Restriction
Sites
Restriction Enzyme
Gene of
interest
Transform ligation product into E.coli and grow on Amp
plate.
Restriction Enzyme
Perform mini prep, screen colonies for insert using PCR and
RE digestions
Cohesive Ends
Ligation
PCR diagnosis
Identify clones by gel electrophoresis (by size) and
sequencing.
Drd I Restriction Enzyme Digestions
M 1
2
3 4
5
6
2,658
1,432
920
717
Myogenesis
Pax3
Pitx2
MyoD
Myogenin
Myosin Heavy Chain
C2C12 Transfection/Differentiation Assays
C2C12 cells grow in serum
Grow to 40%
No Serum
With Serum
48 hours
Transfections
Transfections
24 hours
A
24 hours
C
ICC
No Serum
48 hours
B
ICC
ICC
C2C12 Myoblast Transfections
Immunocytochemistry
Fix cells onto glass coverslips with 4%
PFA
Incubate with primary antibodies
(LacZ, Pitx2, MyoD, Pax3, Myogenin,
Myosin Heavy Chain).
Wash coverslips with PBS
Incubate with fluorescence conjugated
secondary antibody
Wash coverslips with PBS, dehydrate,
and fix onto slides.
Confocal Microscopy
Pitx2 Overexpression Inhibits MyoD in C2C12
Myoblasts
A. CMV-Pitx2a
Future Experiments
Photograph and Analyze transfected cells with
CMV-Pitx2a, CMV-Exon III, CMV in all A, B, C conditions
with all molecular markers in order to study the function of
Pitx2 during muscle proliferation/differentiation.
Acknowledgements
Dr. Chrissa Kioussi
Dr. Michael Gross
Dr. Kevin Ahern
Jack Loflin
Hung Ping Shih
Giao Bui
Chris Barnhart
Meg Christ
Howard Hughes Medical Institute