An altered phenotype in a conditional knockout of Pitx2 in extraocular muscle.
OBJECTIVE
To determine the temporal and spatial expression of Pitx2, a bicoid-like homeobox transcription factor, during postnatal development of mouse extraocular muscle and to evaluate its role in the growth and phenotypic maintenance of postnatal extraocular muscle.


METHODS
Mouse extraocular muscles of different ages were examined for the expression of Pitx2 by RT-PCR, q-PCR, and immunostaining. A conditional mutant mouse strain, in which Pitx2 function is inactivated at postnatal day (P)0, was generated with a Cre-loxP strategy. Histology, immunostaining, real-time PCR, in vitro muscle contractility, and in vivo ocular motility were used to study the effect of Pitx2 depletion on extraocular muscle.


RESULTS
All three Pitx2 isoforms were expressed by extraocular muscle and at higher levels than in other striated muscles. Immunostaining demonstrated the presence of Pitx2 mainly in extraocular muscle myonuclei. However, no obvious expression patterns were observed in terms of anatomic region (orbital versus global layer), innervation zone, or muscle fiber types. The mutant extraocular muscle had no obvious pathology but had altered muscle fiber sizes. Expression levels of myosin isoforms Myh1, Myh6, Myh7, and Myh13 were reduced, whereas Myh2, Myh3, Myh4, and Myh8 were not affected by postnatal loss of Pitx2. In vitro, Pitx2 loss made the extraocular muscles stronger, faster, and more fatigable. Eye movement recordings found saccades to have a lower peak velocity.


CONCLUSIONS
Pitx2 is important in maintaining the mature extraocular muscle phenotype and regulating the expression of critical contractile proteins. Modulation of Pitx2 expression can influence extraocular muscle function with long-term therapeutic implications.
Zhou
Yuefang
Y
Department of Neurology and Psychiatry, Saint Louis University, St. Louis, Missouri 63104, USA.
Cheng
Georgiana
G
Dieter
Lisa
L
Hjalt
Tord A
TA
Andrade
Francisco H
FH
Stahl
John S
JS
Kaminski
Henry J
HJ
D000818:Animals; D005133:Eye Movements / Q000502:physiology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000235:genetics; D009801:Oculomotor Muscles / Q000254:growth & development / Q000378:metabolism*; D010641:Phenotype; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*; D000097577:Homeobox Protein PITX2
D018398:Homeodomain Proteins; D009218:Myosins; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2