Low-Density Lipoprotein Receptor-Related Protein 5-Deficient Rats Have Reduced Bone Mass and Abnormal Development of the Retinal Vasculature.

Humans carrying homozygous loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), develop osteoporosis and a defective retinal vasculature known as familial exudative vitreoretinopathy (FEVR) due to disruption of the Wnt signaling pathway. The purpose of this study was to use CRISPR-Cas9-mediated gene editing to create strains of Lrp5-deficient rats and to determine whether knockout of Lrp5 resulted in phenotypes that model the bone and retina pathology in LRP5-deficient humans. Knockout of Lrp5 in rats produced low bone mass, decreased bone mineral density, and decreased bone size. The superficial retinal vasculature of Lrp5-deficient rats was sparse and disorganized, with extensive exudates and decreases in vascularized area, vessel length, and branch point density. This study showed that Lrp5 could be predictably knocked out in rats using CRISPR-Cas9, causing the expression of bone and retinal phenotypes that will be useful for studying the role of Wnt signaling in bone and retina development and for research on the treatment of osteoporosis and FEVR.

Osteoblast-specific deletion of Hrpt2/Cdc73 results in high bone mass and increased bone turnover.

Inactivating mutations that lead to loss of heterozygosity within the HRPT2/Cdc73 gene are directly linked to the development of primary hyperparathyroidism, parathyroid adenomas, and ossifying fibromas of the jaw (HPT-JT). The protein product of the Cdc73 gene, parafibromin, is a core member of the polymerase-associated factors (PAF) complex, which coordinates epigenetic modifiers and transcriptional machinery to control gene expression. We conditionally deleted Cdc73 within mesenchymal progenitors or within mature osteoblasts and osteocytes to determine the consequences of parafibromin loss within the mesenchymal lineage. Homozygous deletion of Cdc73 via the Dermo1-Cre driver resulted in embryos which lacked mesenchymal organ development of internal organs, including the heart and fetal liver. Immunohistochemical detection of cleaved caspase-3 revealed extensive apoptosis within the progenitor pools of developing organs. Unexpectedly, when Cdc73 was homozygously deleted within mature osteoblasts and osteocytes (via the Ocn-Cre driver), the mice had a normal life span but increased cortical and trabecular bone. OCN-Cre;Cdc73flox/flox bones displayed large cortical pores actively undergoing bone remodeling. Additionally the cortical bone of OCN-Cre;Cdc73flox/flox femurs contained osteocytes with marked amounts of cytoplasmic RNA and a high rate of apoptosis. Transcriptional analysis via RNA-seq within OCN-Cre;Cdc73flox/flox osteoblasts showed that loss of Cdc73 led to a derepression of osteoblast-specific genes, specifically those for collagen and other bone matrix proteins. These results aid in our understanding of the role parafibromin plays within transcriptional regulation, terminal differentiation, and bone homeostasis.

A Dynamic WNT/ß-CATENIN Signaling Environment Leads to WNT-Independent and WNT-Dependent Proliferation of Embryonic Intestinal Progenitor Cells.

Much of our understanding about how intestinal stem and progenitor cells are regulated comes from studying the late fetal stages of development and the adult intestine. In this light, little is known about intestine development prior to the formation of stereotypical villus structures with columnar epithelium, a stage when the epithelium is pseudostratified and appears to be a relatively uniform population of progenitor cells with high proliferative capacity. Here, we investigated a role for WNT/ß-CATENIN signaling during the pseudostratified stages of development (E13.5, E14.5) and following villus formation (E15.5) in mice. In contrast to the well-described role for WNT/ß-CATENIN signaling as a regulator of stem/progenitor cells in the late fetal and adult gut, conditional epithelial deletion of ß-catenin or the Frizzled co-receptors Lrp5 and Lrp6 had no effect on epithelial progenitor cell proliferation in the pseudostratified epithelium. Mutant embryos displayed obvious developmental defects, including loss of proliferation and disruptions in villus formation starting only at E15.5. Mechanistically, our data suggest that WNT signaling-mediated proliferation at the time of villus formation is driven by mesenchymal, but not epithelial, WNT ligand secretion.

Use of Primary Calvarial Osteoblasts to Evaluate the Function of Wnt Signaling in Osteogenesis.

In vitro culture and genetic manipulation of primary calvarial cell cultures is a convenient and robust system to investigate gene function in osteoblast differentiation. We have used this system to study the functions of many genes in the Wnt signaling pathway within osteoblasts. Here, we describe a detailed protocol outlining the establishment and characterization of primary calvarial cells from mice carrying a conditionally inactivatable allele of the Wntless (Wls) gene (Wls (flox/flox)). We previously used this approach to delete the Wntless gene by infecting with a Cre-expressing adenovirus, and to evaluate the effects of Wnt signaling loss on osteogenic potential in osteogenic medium with ascorbic acid. This detailed protocol is adaptable to use with any floxed allele.

WNT signaling in bone development and homeostasis.

The balance between bone formation and bone resorption controls postnatal bone homeostasis. Research over the last decade has provided a vast amount of evidence that WNT signaling plays a pivotal role in regulating this balance. Therefore, understanding how the WNT signaling pathway regulates skeletal development and homeostasis is of great value for human skeletal health and disease.