Introduction: Chondrocranial-derived components of the craniofacial skeleton are thought to exert a morphogenetic influence on surrounding intramembranous-derived skeletal structures. In our previous study (Foster and Holton, 2016), we found that the ontogenetic relationship between the nasal septum (derived primarily from the chondrocranium) and surrounding intramembranous-derived facial skeleton was highly variable in BL6 and C3H mice. The purpose of our present study was to examine samples of BL6 and C3H mice from 3 to 15 weeks of age to determine whether the variable interaction noted previously extends to chondrocranial- and intramembranous-derived components throughout the cranium. Methods: Cross-sectional samples of male C57BL/6J (BL6) and C3H/HeJ (C3H) at 3 (n=18) and 15 (n=30) weeks of age were studied. All mouse crania were scanned using a Skyscan 1272 microCT scanner, and a series of three-dimensional traditional and semilandmark coordinate landmarks were collected. Geometric morphometric techniques were utilized for data analysis. Results: During the period of 3-15 weeks of age, there was considerable variation in the ontogeny of chondrocranial- and intramembranous-derived craniofacial regions. Conclusions: During the period of 3-15 weeks of age, C3H mice were characterized by a relative enlargement of the chondrocranial-derived craniofacial skeleton. The chondrocranial morphology of the C3H mice may be a causal factor in the increased magnitude of variation seen in the C3H intramembranous-derived facial skeleton. Developmental variation within specific components of the craniofacial complex may account for taxonomic variation in facial height that accompanies anteroposterior facial skeletal reduction characteristic of taxa such as anthropoid primates.

Bovine horns develop primarily after birth and the presence or absence of horns is due to a single gene. It has been reported that the horn bud appears in the bovine embryo at d 60 of gestation. Our hypothesis is that the gene that determines the presence of horns is expressed in osteoprogenitor cells of the early fetus and will affect the expression of RUNX2, MSX1, MSX2, and/or TWIST1. To test this hypothesis, bovine fetal samples were collected from commercial females at the Caviness Packing Company in Hereford, Texas. Fetuses ranged from d 28 to d 80 of gestation. A survey of the expression of genes from the region on bovine chromosome 1 known to contain the locus that causes horns (IFNAR1 to SOD1), was conducted using qualitative and quantitative RT-PCR, and in situ hybridization. Genes with known roles in osteogenesis and chrondrogenesis (MSX1, TWIST1, RUNX2 and SOX9) were included as positive controls. With the exception of OLIG1, which was only expressed in the brain, all of the genes investigated were expressed in fetal frontal and parietal bones by qualitative RT-PCR. The level of expression of C21orf59, C21orf66, IL10RB, and SFRS15 increased in the frontal bone of horned samples from d 55 to d 70 of gestation. At d 60 of gestation, a change in the shape of the frontal bone was observed, which has been reported to be the developmental stage when the horn bud appears. At this time point, MSX1, TWIST1, RUNX2 and SOX9 were detected in frontal bone, in cells from the osteoblast lineage, as expected. Furthermore, C21orf59, C21orf62, C21or66 and SFRS15 from the polled interval were localized to developing mesenchyme, osteoblasts and/or osteoclasts of the frontal bone, suggesting that each of these genes has a role in intramembranous bone formation. In addition, gradients of expressed C21orf66 and SFRS15 were detected in developing endochondral bone. There was evidence of an antisense transcript of C21orf66 expressed in the same cell types as the sense transcript. Further characterization of this antisense transcript demonstrated that it covered the entire sense transcript. Based on observed expression in the mesenchyme, rather than just in mature osteoblasts or osteoclasts, C21orf66 and/or its antisense transcript become the most likely candidates for the polled locus.

The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.

The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.

No description available.