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