Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization (Bi et al. 2001)
Campomelic dysplasia (CD)
is an autosomal dominant condition that is characterized by bone malformations
such as bowing of the limbs and other skeletal malformations. In addition to some other non-skeletal
abnormalities, ¾ of patients are found to be transgender (genetically male, but
looks like a female). There also is high
incidence of postnatal death shortly after birth. Since this disease is relatively common, and
little was known about the pathogenesis of the disease they thought that it
would be important to find a mouse model of the disease. So they genetically engineered mice to lack
one copy of a gene called Sox9 and looked at the phenotype of the mice compared
to normal. They did this in the hopes
that insights in mice would result in important finds for human campomelic
dysplasia patients.
As expected, the Sox9+/- mice died shortly after birth with gross morphological defects similar to CD patients. They also found that protein levels of Sox9 in the cartilage cells (chondrocytes) was drastically reduced compared to normal (wildtype) levels. The next thing the investigators did was to look at the newborn (or just before birth: embryonic day 18.5 -or- E18.5) phenotype of the Sox9+/- mice compared to normal. They did this by staining the skeletal elements for alizarin red (bone) and alican blue (cartilage). Upon doing this, they found that the skeletal phenotype of the Sox9+/- mice was very similar to what is found in humans with CD… In that all skeletal elements that were derived from a process called endochondral ossification (bone that developed from cartilage that was there first) all had hypoplasia. The long bones also were found to have some degree of bowing, with the ulna being the most severe.
Next, they looked at the skeletal element’s development earlier in development (E14.5 to E15.5), and they found that the cartilaginous elements were smaller and thinner than those in the control. They thought that the newborn phenotype could be caused by these defective cartilage precursors. This made them want to look earlier (E12.5) to see how the precursors (mesenchymal cells) were developing. Upon staining the E12.5 embryos with alican blue, they saw that these precartilaginous skeletal elements were smaller and were stained to a lesser degree than the wildtype embryos. They also saw histologically, that these progenitors looked different from normal. Upon looking at this data, the investigators concluded that development of the cartilage primordia was delayed and smaller, which was likely due to not having enough Sox9 to correctly do these processes.
Finally, the investigators wanted to look at why there seemed to be more calcified regions than cartilage regions in the E18.5 embryos. They looked histologically (tissue sections to see cell organization under the microscope) at E15.5 at the ossification centers in the vertebrae, and found that the ossification came earlier and was larger than control. Hence making them think that Sox9+/- probably accelerated differentiation of the chondrocytes. This induced them to look at the growth plates in newborns and E18.5 embryos, and they found that the “hypertrophic zone” of the growth plate was enlarged, while the zones around them looked normal. This was confirmed by looking at gene expression of important known developmental genes (Col10a1, PTHrP, and Ihh). The data of the Sox9+/- premature mineralization and enlarged hypertrophic zones of the growth plate made the authors suggest that Sox9 probably regulates the rate of hypertrophic chondrocye differentiation. All caused by this “haploinsufficientcy” of Sox9.
Given these conclusions, the authors wanted to compare the differences between these mutant mice and humans with CD. They found that, in general, they mimicked each other. The only differences (see table below) were probably caused by effects of muscles on the bones, since the musculature on these areas are slightly different in mice and humans. This was a pivotal study in the field of limb/skeletal development because they found that Sox9 was involved in chondrocyte differentiation, which is being utilized now for studies of Embryonic Stem Cells to differentiate into cartilage cells in vitro for possible therapies of replacing cartilage in patients with diseases such as Arthritis (personal communication with Andrew Handorf, Department of Orthopedics at UW).
As expected, the Sox9+/- mice died shortly after birth with gross morphological defects similar to CD patients. They also found that protein levels of Sox9 in the cartilage cells (chondrocytes) was drastically reduced compared to normal (wildtype) levels. The next thing the investigators did was to look at the newborn (or just before birth: embryonic day 18.5 -or- E18.5) phenotype of the Sox9+/- mice compared to normal. They did this by staining the skeletal elements for alizarin red (bone) and alican blue (cartilage). Upon doing this, they found that the skeletal phenotype of the Sox9+/- mice was very similar to what is found in humans with CD… In that all skeletal elements that were derived from a process called endochondral ossification (bone that developed from cartilage that was there first) all had hypoplasia. The long bones also were found to have some degree of bowing, with the ulna being the most severe.
Next, they looked at the skeletal element’s development earlier in development (E14.5 to E15.5), and they found that the cartilaginous elements were smaller and thinner than those in the control. They thought that the newborn phenotype could be caused by these defective cartilage precursors. This made them want to look earlier (E12.5) to see how the precursors (mesenchymal cells) were developing. Upon staining the E12.5 embryos with alican blue, they saw that these precartilaginous skeletal elements were smaller and were stained to a lesser degree than the wildtype embryos. They also saw histologically, that these progenitors looked different from normal. Upon looking at this data, the investigators concluded that development of the cartilage primordia was delayed and smaller, which was likely due to not having enough Sox9 to correctly do these processes.
Finally, the investigators wanted to look at why there seemed to be more calcified regions than cartilage regions in the E18.5 embryos. They looked histologically (tissue sections to see cell organization under the microscope) at E15.5 at the ossification centers in the vertebrae, and found that the ossification came earlier and was larger than control. Hence making them think that Sox9+/- probably accelerated differentiation of the chondrocytes. This induced them to look at the growth plates in newborns and E18.5 embryos, and they found that the “hypertrophic zone” of the growth plate was enlarged, while the zones around them looked normal. This was confirmed by looking at gene expression of important known developmental genes (Col10a1, PTHrP, and Ihh). The data of the Sox9+/- premature mineralization and enlarged hypertrophic zones of the growth plate made the authors suggest that Sox9 probably regulates the rate of hypertrophic chondrocye differentiation. All caused by this “haploinsufficientcy” of Sox9.
Given these conclusions, the authors wanted to compare the differences between these mutant mice and humans with CD. They found that, in general, they mimicked each other. The only differences (see table below) were probably caused by effects of muscles on the bones, since the musculature on these areas are slightly different in mice and humans. This was a pivotal study in the field of limb/skeletal development because they found that Sox9 was involved in chondrocyte differentiation, which is being utilized now for studies of Embryonic Stem Cells to differentiate into cartilage cells in vitro for possible therapies of replacing cartilage in patients with diseases such as Arthritis (personal communication with Andrew Handorf, Department of Orthopedics at UW).
