Faculty Bibliography

Parathyroid hormone (PTH) was the first osteoanabolic hormone for treating osteoporosis. We have previously shown that PTH acts through PTHR1 and protein kinase A (PKA) activation to regulate osteoblastic gene expression. Our study aimed to elucidate the effects of increased PKA activity and better understand the actions of PTH (1-34) in bone. Tamoxifen (1mg/10g) was injected weekly to 1 or 5 month-old C57Bl/6J male col1CRE^ERT/Prkar1a^fl/fl mice or Prkar1a^fl/fl mice as controls for 3-4 weeks to delete the PKA regulatory subunit 1A in osteoblasts and increase PKA activity. At both ages, col1CRE^ERT/Prkar1a^fl/fl mice demonstrated bone pathologies in their skulls, femurs and vertebrae and tumors in their tails (Figure 1). MicroCT showed cortical bone breakdown with apparent trabecular bone in the cortical area in femurs and vertebrae and expansion of bone in skulls. Deletion of Prkar1a increased bone turnover with a huge increase in osteoblast activity shown by serum-P1NP levels (6.5-13 fold), only single fluorescent labeling and a substantial increase in osteoclast activity shown by CTX levels (4.4-12 fold) and TRAP staining. Surprisingly, the col1CRE^ERT/Prkar1a^fl/fl skulls showed thicker calvariae, shown by alizarin red staining and muCT but no changes in mandibles or teeth. Col1CRE^ERT/Prkar1a^fl/fl mice had tumors in their tails evident by an invasion of stromal and osteoclastic cells but with intact growth plate, cartilage and intervertebral discs. In conclusion, high PKA activity in osteoblasts appears to be involved with high bone turnover and pathological events mimicking hyperparathyroidism, Jansen's metaphyseal chondrodysplasia or McCune-Albright syndrome. [Formula presented]
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The musculoskeletal system, which comprises muscles, tendons, and bones, is an efficient tissue complex that coordinates body movement and maintains structural stability. The process of its construction into a single functional and complex organization is unclear. SRY-box containing gene 9 (Sox9) is expressed initially in pluripotent cells and subsequently in ectodermal, endodermal, and mesodermal derivatives. This study investigated how Sox9 controls the development of each component of the musculoskeletal system. Sox9 was expressed in MTJ, tendon, and bone progenitor cells at E13 and in bone at E16. We detected Sox9 expression in muscle progenitor cells using double-transgenic mice and myoblastic cell lines. However, we found no Sox9 expression in developed muscle. A decrease in Sox9 expression in muscle-associated connective tissues, tendons, and bones led to hypoplasia of the cartilage and its attachment to tendons and muscle. These results showed that switching on Sox9 expression in each component (muscle, tendon, and bone) is essential for the development of the musculoskeletal system. Sox9 is expressed in not only tendon and bone progenitor cells but also muscle progenitor cells, and it controls musculoskeletal system development.

The strains of inbred laboratory mice are isogenic and homogeneous for over 98.6% of their genomes. However, geometric morphometric studies have demonstrated clear differences among the skull shapes of various mice strains. The question now arises: why are skull shapes different among the mice strains? Epigenetic processes, such as morphological interaction between the muscles and bones, may cause differences in the skull shapes among various mice strains. To test these predictions, the objective of this study is to examine the morphological association between a specific part of the skull and its adjacent muscle. We examined C57BL6J, BALB/cA, and ICR mice on embryonic days (E) 12.5 and 16.5 as well as on postnatal days (P) 0, 10, and 90. As a result, we found morphological differences between C57BL6J and BALB/cA mice with respect to the inferior spine of the hypophyseal cartilage or basisphenoid (SP) and the tensor veli palatini muscle (TVP) during the prenatal and postnatal periods. There was a morphological correlation between the SP and the TVP in the C57BL6J, BALB/cA, and ICR mice during E15 and P0. However, there were not correlation between the TVP and the SP during P10. After discectomy, bone deformation was associated with a change in the shape of the adjacent muscle. Therefore, epigenetic modifications linked to the interaction between the muscles and bones might occur easily during the prenatal period, and inflammation seems to allow epigenetic modifications between the two to occur.

In the head of an embryo, a layer of mesenchyme surrounds the brain underneath the surface ectoderm. This cranial mesenchyme gives rise to the meninges, the calvaria (top part of the skull), and the dermis of the scalp. Abnormal development of these structures, especially the meninges and the calvaria, is linked to significant congenital defects in humans. It has been known that different areas of the cranial mesenchyme have different fates. For example, the calvarial bone develops from the cranial mesenchyme on the baso-lateral side of the head just above the eye (supraorbital mesenchyme, SOM), but not from the mesenchyme apical to SOM (early migrating mesenchyme, EMM). However, the molecular basis of this difference is not fully understood. To answer this question, we compared the transcriptomes of EMM and SOM using high-throughput sequencing (RNA-seq). This experiment identified a large number of genes that were differentially expressed in EMM and SOM, and gene ontology analyses found very different terms enriched in each region. We verified the expression of about 40 genes in the head by RNA in situ hybridization, and the expression patterns were annotated to make a map of molecular markers for 6 subdivisions of the cranial mesenchyme. Our data also provided insights into potential novel regulators of cranial mesenchyme development, including several axon guidance pathways, lectin complement pathway, cyclic-adenosine monophosphate (cAMP) signaling pathway, and ZIC family transcription factors. Together, information in this paper will serve as a unique resource to guide future research on cranial mesenchyme development.

The meninges are membranous layers surrounding the central nervous system. In the head, the meninges lie between the brain and the skull, and interact closely with both during development. The cranial meninges originate from a mesenchymal sheath on the surface of the developing brain, called primary meninx, and undergo differentiation into three layers with distinct histological characteristics: the dura mater, the arachnoid mater, and the pia mater. While genetic regulation of meningeal development is still poorly understood, mouse mutants and other models with meningeal defects have demonstrated the importance of the meninges to normal development of the calvaria and the brain. For the calvaria, the interactions with the meninges are necessary for the progression of calvarial osteogenesis during early development. In later stages, the meninges control the patterning of the skull and the fate of the sutures. For the brain, the meninges regulate diverse processes including cell survival, cell migration, generation of neurons from progenitors, and vascularization. Also, the meninges serve as a stem cell niche for the brain in the postnatal life. Given these important roles of the meninges, further investigation into the molecular mechanisms underlying meningeal development can provide novel insights into the coordinated development of the head.

The calvaria (upper part of the skull) is made of plates of bone and fibrous joints (sutures and fontanelles), and the proper balance and organization of these components are crucial to normal development of the calvaria. In a mouse embryo, the calvaria develops from a layer of head mesenchyme that surrounds the brain from shortly after mid-gestation. The mesenchyme just above the eye (supra-orbital mesenchyme, SOM) generates ossification centers for the bones, which then grow toward the apex gradually. In contrast, the mesenchyme apical to SOM (early migrating mesenchyme, EMM), including the area at the vertex, does not generate an ossification center. As a result, the dorsal midline of the head is occupied by sutures and fontanelles at birth. To date, the molecular basis for this regional difference in developmental programs is unknown. The current study provides vital insights into the genetic regulation of calvarial patterning. First, we showed that osteogenic signals were active in both EMM and SOM during normal development, which suggested the presence of an anti-osteogenic factor in EMM to counter the effect of these signals. Subsequently, we identified Lmx1b as an anti-osteogenic gene that was expressed in EMM but not in SOM. Furthermore, head mesenchyme-specific deletion of Lmx1b resulted in heterotopic ossification from EMM at the vertex, and craniosynostosis affecting multiple sutures. Conversely, forced expression of Lmx1b in SOM was sufficient to inhibit osteogenic specification. Therefore, we conclude that Lmx1b plays a key role as an anti-osteogenic factor in patterning the head mesenchyme into areas with different osteogenic competence. In turn, this patterning event is crucial to generating the proper organization of the bones and soft tissue joints of the calvaria.

Development of the face is regulated by a large number of genes that are expressed in temporally and spatially specific patterns. While significant progress has been made on characterizing the genes that operate in the oral region of the face, those regulating development of the aboral (lateral) region remain largely unknown. Recently, we discovered that transcription factors LIM homeobox (LHX) 6 and LHX8, which are key regulators of oral development, repressed the expression of the genes encoding forkhead box transcription factors, Foxp1 and Foxp2, in the oral region. To gain insights into the potential role of the Foxp genes in region-specific development of the face, we examined their expression patterns in the first pharyngeal arch (primordium for the jaw) of mouse embryos at a high spatial and temporal resolution. Foxp1 and Foxp2 were preferentially expressed in the aboral and posterior parts of the first pharyngeal arch, including the developing temporomandibular joint. Through double immunofluorescence and double fluorescent RNA in situ hybridization, we found that Foxp1 was expressed in the progenitor cells for the muscle, bone, and connective tissue. Foxp2 was expressed in subsets of bone and connective tissue progenitors but not in the myoblasts. Neither gene was expressed in the dental mesenchyme nor in the oral half of the palatal shelf undergoing extensive growth and morphogenesis. Together, we demonstrated for the first time that Foxp1 and Foxp2 are expressed during craniofacial development. Our data suggest that the Foxp genes may regulate development of the aboral and posterior regions of the jaw.

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives.Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57Kip2 (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6-/-;Lhx8-/- mutants. p57Kip2 has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57Kip2 by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57Kip2 via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors.Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57Kip2 expression in the prospective palate area to allow adequate levels of cell proliferation, and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.