Peter Maye

The Wnt Co-receptor LRP5 Is Essential for Skeletal Mechanotransduction but Not for the Anabolic Bone Response to Parathyroid Hormone Treatment

The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass. Loss-of-function mutations in LRP5 cause the human skeletal disease osteoporosis-pseudoglioma syndrome, an autosomal recessive disorder characterized by severely reduced bone mass and strength. We investigated the role of LRP5 on bone strength using mice engineered with a loss-of-function mutation in the gene. We then tested whether the osteogenic response to mechanical loading was affected by the loss of Lrp5 signaling. Lrp5-null (Lrp5-/-) mice exhibited significantly lower bone mineral density and decreased strength. The osteogenic response to mechanical loading of the ulna was reduced by 88 to 99% in Lrp5-/- mice, yet osteoblast recruitment and/or activation at mechanically strained surfaces was normal. Subsequent experiments demonstrated an inability of Lrp5-/- osteoblasts to synthesize the bone matrix protein osteopontin after a mechanical stimulus. We then tested whether Lrp5-/- mice increased bone formation in response to intermittent parathyroid hormone (PTH), a known anabolic treatment. A 4-week course of intermittent PTH (40 μg/kg/day; 5 days/week) enhanced skeletal mass equally in Lrp5-/- and Lrp5+/+ mice, suggesting that the anabolic effects of PTH do not require Lrp5 signaling. We conclude that Lrp5 is critical for mechanotransduction in osteoblasts. Lrp5 is a mediator of mature osteoblast function following loading. Our data suggest an important component of the skeletal fragility phenotype in individuals affected with osteoporosis-pseudoglioma is inadequate processing of signals derived from mechanical stimulation and that PTH might be an effective treatment for improving bone mass in these patients.

Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation.

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.

Regulation of Gli1 Transcriptional Activity in the Nucleus by Dyrk1

To investigate the cellular role of dual specificity Yak1-related kinase (Dyrk) 1, a nuclear localized dual specificity protein kinase, we examined its effect on transcriptional regulation using reporter gene assays. We found that Dyrk1 can substantially enhance Gli1-dependent, but not LEF-1-, c-Jun-, or Elk-dependent, gene transcription. In part, Dyrk1 does this through retaining Gli1 in the nucleus. However, we also demonstrate that Dyrk1 can enhance the transcriptional activity of Gli1-AHA, a nuclear export mutant, suggesting that Dyrk1 may be more directly involved in regulating the transcriptional activity of Gli1. In addition, Dyrk1 acted synergistically with Sonic hedgehog (Shh) to induce gene transcription and differentiation in mouse C3H10T1/2 cells. The failure of Shh to stimulate Dyrk1 kinase activity suggests that Dyrk1 may not be directly regulated by the Shh signaling pathway but functionally interacts with it. Thus, Gli1 transcriptional activity may be subjected to further regulation in the cell nucleus by a pathway distinct from Shh signaling, one mediated by Dyrk1.

Histological Analysis of GFP Expression in Murine Bone

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a β-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.

Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes.

Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 μM xylenol orange and 30 μM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.

Osterix-Cre Labeled Progenitor Cells Contribute to the Formation and Maintenance of the Bone Marrow Stroma

We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.

Generation and characterization of Col10a1‐mcherry reporter mice

We report here on the generation of a new fluorescent protein reporter transgenic mouse line, Col10a1‐mCherry, which can be used as a tool to study chondrocyte biology and pathology. Collagen, Type X, alpha 1 (Col10a1) is highly expressed in hypertrophic chondrocytes and commonly used as a gene marker for this cell population. The Col10a1‐mCherry reporter line was generated using a bacterial recombination strategy with the mouse BAC clone RP23‐192A7. To aid in the characterization of this animal model, we intercrossed Col10a1‐mCherry mice with Collagen, Type II, alpha 1 (Col2a1) enhanced cyan fluorescent protein (ECFP) reporter mice and characterized the expression of both chondrocyte reporters during embryonic skeletal development from days E10.5 to E17.5. Additionally, at postnatal day 0, Col10a1‐mCherry reporter expression was compared to endogenous Col10a1 mRNA expression in long bones and revealed that mCherry fluorescence extended past the Col10a1 expression domain. However, in situ hybridization for mCherry was consistent with the zone of Col10a1 mRNA expression, indicating that the persistent detection of mCherry fluorescence was a result of the long protein half life of mCherry in conjunction with a very rapid phase of skeletal growth and not due to aberrant transcriptional regulation. Taking advantage of the continued fluorescence of hypertrophic chondrocytes at the chondro‐osseus junction, we intercrossed Col10a1‐mCherry mice with two different Collagen, Type 1, alpha 1, (Col1a1) osteoblast reporter mice, pOBCol3.6‐Topaz and pOBCol2.3‐Emerald to investigate the possibility that hypertrophic chondrocytes transdifferentiate into osteoblasts. Evaluation of long bones at birth suggests that residual hypertrophic chondrocytes and osteoblasts in the trabecular zone exist as two completely distinct cell populations. genesis 49:410–418, 2011.

Isolation of murine bone marrow derived mesenchymal stem cells using Twist2 Cre transgenic mice

While human bone marrow derived mesenchymal stem cells (BMSCs) are of great interest for their potential therapeutic value, their murine equivalent remains an important basic research model that can provide critical insights into the biology of this progenitor cell population. Here we present a novel transgenic strategy that allowed for the selective identification and isolation of murine BMSCs at the early stages of stromal cell culture. This strategy involved crossing Twist2 -Cre mice with Cre reporter mice such as Z/EG or Ai9, which express EGFP or Tomato fluorescent protein, respectively, upon Cre mediated excision of a stop sequence. Using this approach, we identified an adherent fluorescent protein+cell population (T2C+) that is present during the earliest stages of colony formation and by day 5 of culture represents ~20% of the total cell population. Cell surface profiling by flow cytometry showed that T2C+cells are highly positive for SCA1 and CD29 and negative for CD45, CD117, TIE2, and TER119. Isolation of T2C+cells by FACS selected for a cell population with skeletal potential that can be directed to differentiate into osteoblasts, adipocytes, or chondrocytes. We also demonstrated in a calvarial bone defect model that T2C+cells retain a strong efficacy for osteogenic repair and can support a hematopoietic environment. Collectively, these studies provide evidence that the Twist2-Cre x Cre reporter breeding strategy can be used to positively identify and isolate multipotent murine BMSCs.

Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin.

Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule - we named Kartogenin (KGN) - that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFβ superfamily members, particularly TFGβ1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFβ1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFβ1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFβ and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFβ signaling, working in addition to and/or in concert with the filamin A/CBFβ/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.