Here we examined Meckel s cartilage of
Here, we examined Meckel's cartilage of mice where Setdb1 was knocked out only in neural crest derived npy receptor with the purpose of investigating the roles of Setdb1 in Meckel's cartilage, a supportive tissue for mandible formation. We observed enlarged Meckel's cartilage that was a result of increased proliferation and hypertrophy of chondrocytes in this Setdb1 mutant. Furthermore, Meckel's cartilage of Setdb1 mutant exhibited calcium deposition, which is not present in normal Meckel's cartilage. These changes were likely attributable to the excessive BMP signals.
Material and methods
Results Since conventional knockout of Setdb1 was embryo-lethal at the implantation stage (∼E5.5) , the role of Setdb1 in organogenesis during the late embryonic development was unknown. To solve this problem, we used Cre-LoxP system to knockout Setdb1 allele conditionally. To elucidate the role of Setdb1 in craniofacial development, we crossed Setdb1 with Setdb1 mice. Setdb1 (Setdb1 CKO) mice displayed obvious decrease in craniofacial dimensions, especially in the mandible just after the birth (Sup Fig. 1). Next we analyzed the factors responsible for the phenotypes in the mandible and the frontal sections of the Setdb1 CKO were observed. To ensure that the sections compared were similar, frontal sections that go through the lower molar in each of the samples were selected. In all stages that were examined from E13.5 to E18.5, Meckel's cartilage of Setdb1 CKO mice was remarkably larger than in the control animals (Fig. 1 A–J). In general, Meckel's cartilage disappeared or degenerated at later stages of development with sustained the perichondrium morphology (Fig. 1A,C,E,G,I) , . However, in Setdb1 CKO mice, the Meckel's cartilage was unchanged or even increased in size until E16.5. Interestingly at E16.5 and E18.5, part of perichondrium was disrupted and Alcian blue positive area was reduced (Fig. 1H,J; arrowhead). Enhanced BMP signaling in Meckel's cartilage showed similar phenotype of enlarged Meckel's cartilage. In addition, the mice showed endochondral ossification in Meckel's cartilage which usually degenerates in wild type mice , . To examine whether Meckel's cartilage of Setdb1 mutant also showed similar trend, we have checked Alizarin red staining at E18.5. As shown in Fig. 1N, Alizarin red positive area was observed in some parts of the Meckel's cartilage of Setdb1 CKO (Fig. 1N arrow) mice, but not in the control embryo (Fig. 1M). Thus, Setdb1 CKO led to enlarged Meckel's cartilage and mineralization of some of the Meckel's cartilage, which was very similar to the result of enhanced BMP signaling in mice. Next we examined whether the enlarged Meckel's cartilage in Setdb1 CKO mice was due to the increase in the size of the chondrocytes and/or the increase in the number of chondrocytes. Hypertrophy of chondrocytes has been observed during normal development of Meckel's cartilage . However, as shown in Fig. 1L, chondrocytes of Setdb1 CKO mice were more hypertrophic compared to those of the control mice. To test whether the enlargement of Meckel's cartilage was attributable to the increased cell proliferation in Setdb1 CKO mice, we intraperitoneally administered BrdU to pregnant mice at E14.5 stage, 2 h before dissecting the embryos. Chondrocyte proliferation rate in Meckel's cartilage was higher in Setdb1 CKO mice (Fig. 2A–C). Furthermore, we knocked down Setdb1 using siRNA in ATDC5 cells and examined the number of BrdU-positive cells. The results were similar to those in Setdb1 CKO mice (Fig. 2D–F). During the development of Meckel's cartilage, chondrogenesis that occurs via mesenchymal proliferation and chondrocyte differentiation are regulated by the orchestrated expression of the chondrogenic genes . To investigate the effect of Setdb1 on chondrocytes, we knocked down Setdb1 in the mouse chondrogenic cell line ATDC5, using siRNA. mRNA levels of chondrogenic genes, Sox9, Collagen II, Aggrecan, and MMP13 were significantly lower in Setdb1 siRNA treated samples compared to those in the control cells treated with scramble siRNA. In contrast, mRNA levels of Axin2 were upregulated upon Setdb1 siRNA treatment (Fig. 3). This suggests that chondrogenic differentiation is delayed in the Setdb1 knockdown ATDC5 cells.