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The Odd-Skipped Family Transcription Factors Osr1 and Osr2 Have Equivalent Biochemical Activity and Play Critical but Partially Redundant Roles during Organogenesis

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Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2010.
Osr1 and Osr2 are the only mammalian homologs of the Drosophila odd-skipped family developmental regulators. The Osr1 protein contains three zinc-finger mo- tifs whereas Osr2 exists in two isoforms, containing three (Osr2B) and five (Osr2A) zinc-finger motifs respectively, due to alternative splicing of the transcripts. Dur- ing mouse embryonic development and organogenesis, Osr1 and Osr2 exhibit dis- tinct as well as partially overlapping expression patterns. Targeted null mutations in these genes in mice resulted in distinct phenotypes, with heart and urogeni- tal developmental defects in Osr1 −/− mice and with cleft palate, supernumerary teeth and open eyelids at birth in Osr2 −/− mice. Whereas the early embryonic lethality of Osr1 −/− mutant mice precluded direct analysis of the roles of Osr1 in many developmental processes in those mutants, the correlation of developmental defects in the Osr2 −/− mutants to specific tissues that normally do not express Osr1 and the lack of phenotypes in Osr2 −/− mutants in many tissues where Osr1 and Osr2 are normally co-expressed suggest that Osr1 and Osr2 function partly redundantly during mouse embryonic development. I first tested possible functional redundancy between Osr1 and Osr2 during limb development. Osr1 and Osr2 are expressed in partially overlapping patterns in the developing limb, but no limb defect was identified during initial charac- terization of mice lacking either Osr1 or Osr2. Using the Cre/loxP-mediated tissue-specific gene inactivation strategy, I showed that deletion of both Osr1 and Osr2 from the early developing limb mesenchyme caused multiple synovial joint fusions. I found that Osr1 and Osr2 expression was activated highly specifically in the presumptive joint cells prior to any morphological signs of joint forma- tion. Molecular marker studies indicate that Osr1 and Osr2 are involved in the earliest stage of joint morphogenesis, i.e, during the formation of interzones at prospective joint sites. In the absence of both Osr1 and Osr2, joint progenitor cells failed to downregulate Col2a1 and to maintain expression of Gdf5, Wnt4 and Wnt9a, which are all critical for joint formation. Furthermore, Osr1 and Osr2 are necessary for inducing cell apoptosis in the developing joint, which is a key step in joint cavitation. Lubrication is mandatory for normal joint function. Prg4 is a marker for differentiating articular chondrocytes and encodes a proteoglycan essential for joint lubrication. Prg4 expression was dramatically downregulated in the Osr1 fneo/fneoOsr2 −/−Prx1cre mutant joint cells. While the above data indicate functional redundancy between Osr1 and Osr2 during joint development, it is possible that Osr1 and Osr2 may have distinct biochemical functions since Osr1 contains three zinc finger motifs but Osr2A has five zinc finger motifs. To investigate whether the distinct mutant phenotypes in Osr1 −/− and Osr2 −/− mutants are due to differences in their protein structure or to differential expression patterns, we generated mice in which the endoge- nous Osr2 coding region was replaced with either Osr1 cDNA or Osr2A cDNA. The knockin alleles recapitulated endogenous Osr2 mRNA expression patterns in most tissues and rescued palate, tooth and cranial skeletal developmental de- fects of Osr2 −/− mice. Mice hemizygous or homozygous for either knockin allele exhibited open-eyelids at birth, which correlated with differences in expression patterns between the knockin allele and the endogenous Osr2 gene during eyelid development. Molecular marker analyses in Osr2 −/− and Osr2 Osr1ki/Osr1ki mice revealed that Osr2 controls eyelid development through regulation of the Fgf10- Fgfr2 signaling pathway and that Osr1 rescued Osr2 function in maintaining Fgf10 expression during eyelid development in Osr2 Osr1ki/Osr1ki mice. Another transcription factor that plays an essential role in palate and tooth de- velopment is Pax9. Pax9 is a member of the paired box transcription factor family that is characterized by the paired DNA-binding domain. It was reported that homozygous Pax9 -deficient mice have a cleft secondary palate with an abnormally broadened shape and lack characteristic indentations at the lateral sides of the palatal shelves at E13.5. In the absence of Pax9, tooth development was arrested at early bud stage, with down-regulated mesenchymal expression of Bmp4, Msx1, and Lef1. Since recent study from our lab indicated that Osr2 suppressed the Msx1-Bmp4 pathway during tooth development, we investigated the mechanisms underlying the cleft palate and tooth developmental defects in Pax9 null mutant mice and whether Osr2 was involved. We found that Pax9 mRNA exhibited a unique differential pattern of expression along the anterior-posterior axis of the de- veloping secondary palate, with restricted expression in the medial mesenchyme in the anterior region and in a lateral-medial gradient in the posterior region. Molecular marker analyses demonstrated that the boundary between anterior and posterior regions of the secondary palate was disrupted and the anteriorward out- growth of the secondary palate delayed in the Pax9 null mutant embryos. In addition, the levels and patterns of expression of Osr2, Fgf10, and Bmp2 in the developing palatal mesenchyme were significantly altered in the Pax9 mutant em- bryos in comparison with control littermates. These changes correlated well with significantly altered patterns of cell proliferation in the palate mesenchyme in the mutant embryos. A dramatic decrease in cell proliferation was also found in the lateral epithelium of the secondary palate. I have also found a significant decrease in cell proliferation rate in the developing tooth mesenchyme in Pax9 null mutant embryos. In collaboration with a colleague, I am investigating whether changes in the expression of these genes also occur during tooth development in Pax9 null mutant embryos. In summary, Osr1 and Osr2 act redundantly in synovial joint development, by regulating interzone formation, joint cavitation and joint cell differentiation. The distinct roles of Osr1 and Osr2 during mouse development result from evolution- ary divergence of their cis regulatory sequences rather than distinct biochemical activities of their protein products. Pax9 regulates the expression of Osr2, Fgf10, and Bmp2 in palate mesenchyme lingual to the developing tooth bud and coordi- nates cell proliferation in the epithelium and mesenchyme to ensure normal palate morphogenesis and tooth development.
Contributor(s):
Yang Gao - Author

Rulang Jiang - Thesis Advisor

Primary Item Type:
Thesis
Language:
English
Subject Keywords:
Odd-Skipped ; Organogenesis
Sponsor - Description:
National Institutes of Health (NIH) - NIDCR R01 DE013681
Date will be made available to public:
2013-01-01   
License Grantor / Date Granted:
Susan Love / 2011-02-14 08:46:49.786 ( View License )
Date Deposited
2011-02-14 08:46:49.786
Date Last Updated
2012-09-26 16:35:14.586719
Submitter:
Susan Love

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