RESEARCH ARTICLE 3991
Development 136, 3991-4000 (2009) doi:10.1242/dev.042150
Two populations of endochondral osteoblasts with differential sensitivity to Hedgehog signalling Christina Lindsey Hammond* and Stefan Schulte-Merker Hedgehog (Hh) signalling has been implicated in the development of osteoblasts and osteoclasts whose balanced activities are critical for proper bone formation. As many mouse mutants in the Hh pathway are embryonic lethal, questions on the exact effects of Hh signalling on osteogenesis remain. Using zebrafish, we show that there are two populations of endochondral osteoblasts with differential sensitivity to Hh signalling. One, formed outside the cartilage structure, requires low levels of Hh signalling and fails to differentiate in Indian hedgehog mutants. The other derives from chondrocytes and requires higher levels of Hh signalling to form. This latter population develops significantly earlier in mutants with increased Hh signalling, leading to premature endochondral ossification, and also fails to differentiate in Indian hedgehog mutants, resulting in severely delayed endochondral ossification. Additionally, we demonstrate that the timing of first osteoclast activity positively correlates to Hh levels in both endochondral and dermal bone.
INTRODUCTION Bone formation in vertebrates is regulated by a balance between the activities of cells that secrete bone matrix, the osteoblasts, and those that reabsorb it, the osteoclasts. The tight regulation of the activities of these two cell types is of crucial importance. Osteoclasts are of haematopoietic origin from the monocyte/ macrophage lineage, and their differentiation is controlled by RANKL (Tnfsf11 – Mouse Genome Informatics) and Csf1 (Kong et al., 1999; Yasuda et al., 1998). Osteoblasts, by contrast, are derived from mesenchymal cells; Runx2 is the master regulator of bone and cartilage cell fate, whereas osterix (Sp7 – Mouse Genome Informatics) is the master regulator of osteoblastogenesis (Nakashima et al., 2002). Additionally there are two types of bone: endochondral bone, in which osteoblasts lead to mineralisation of an existing cartilage matrix; and dermal bone, in which bone is formed de novo. Until recently the majority of the work on bone development has been undertaken either in the mouse, or using mammalian in vitro cell-culture systems; however, as in many other cases in developmental biology, the zebrafish is an excellent model in which to study bone formation, particularly as transgenic lines allow us to follow the behaviour of cells in vivo. Despite some differences, e.g. the appendicular skeleton, zebrafish and the related teleost medaka show remarkable similarities in bone formation to higher vertebrates, particularly in their craniofacial development (Renn and Winkler, 2009; Wagner et al., 2003; Yelick et al., 1996; Yelick and Schilling, 2002). The Hedgehog (Hh) signalling pathway, principally responding to Indian hedgehog, has long been linked to endochondral bone formation. Ihh is expressed by chondrocytes; signals to both chondrocytes and the adjacent perichondral cells and exerts control over the timing of chondrocyte differentiation (St-Jacques et al., 1999; Vortkamp et al., 1996). Analysis of the knockout mouse Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre, 3584CT Utrecht, The Netherlands. *Author for correspondence (
[email protected]) Accepted 22 September 2009
showed that Indian hedgehog regulates chondrocyte proliferation and is required for osteoblast differentiation (St-Jacques et al., 1999). More recently, the Hedgehog receptor protein patched 1 (patched homolog 1, Ptch1 – Mouse Genome Informatics) has been implicated in the regulation of both osteoblast and osteoclast fate in the mouse (Mak et al., 2008a; Ohba et al., 2008). Two papers analysing different Ptch1 mouse mutants reached somewhat different conclusions from their analyses. In mouse, Ptch1 null animals die before bone formation; thus only heterozygous carriers of the Ptch1 gene or conditional knockouts are available for analysis. The Chung group, studying Ptch1 heterozygous animals saw increased bone mass in these animals, which they attributed to increased osteoblast differentiation, through a mechanism whereby reduction of the Gli3 repressor led to increased sensitivity to Runx2 expression (Ohba et al., 2008). By contrast, the Yang group, analysing a conditional knockout, in which Ptch1 was deleted in cells expressing osteocalcin (Bglap1 – Mouse Genome Informatics), i.e. mature osteoblasts, saw reduced bone mass in the adult animals, which they attributed to increased differentiation of osteoclasts under the control of RANKL (Mak et al., 2008a). As many of the murine Hedgehog signalling pathway mutants are embryonic lethal, we chose to undertake studies in the zebrafish, which have a number of advantages that make them ideal for studies of this nature. Importantly, zebrafish carrying null mutations for a number of members of the Hedgehog signalling pathway survive long enough to undertake an analysis of early bone differentiation. These include three mutants thought to lead to activation of the pathway: patched 1, patched 2 and dre (suppressor of fused) (sufu – ZFIN) (Koudijs et al., 2008; Koudijs et al., 2005) and an ihha mutant carrying an early stop, which we believe to be a null mutant. As an independent approach we also made use of drugs that act directly on smoothened to titrate the effects on the pathway. Cyclopamine is a small molecule antagonist that directly binds to smoothened and inhibits its function (Chen et al., 2002). Purmorphamine, by contrast, is a smoothened agonist (Sinha and Chen, 2006), which has been previously observed to induce osteogenic differentiation in cell culture in some studies (Wu et al., 2004), while inhibiting osteoblast differentiation in others (Plaisant et al., 2009). Thus different culture systems can give conflicting results on the effect of activation of
DEVELOPMENT
KEY WORDS: Hedgehog, Chondrocyte, Osteoblast, Zebrafish
3992 RESEARCH ARTICLE
MATERIALS AND METHODS In situ hybridisation
In situ labelling was performed as previously described (Schulte-Merker, 2002). The markers used were osterix, osteocalcin, RANKL and collagen1alpha2 (for primers, see Table S1 in the supplementary material). In all cases in situs were carried out with mutant and siblings, analysed blind and subsequently genotyped by DNA isolation and sequencing, using the primers shown in Table S1 in the supplementary material. Mutant lines
ptc1, ptc2 and dre mutant lines are ptc1hu1602, lep(ptc2)tj222 and dretm146d, respectively (Koudijs et al., 2008; Koudijs et al., 2005). Ihhahu2131 stocks were obtained from the Sanger Centre (Cambridge, UK). Lines were crossed to the transgenic lines Tg(osterix:nuGFP) line (Spoorendonk et al., 2008) or Tg(kdr-l:gfp)s843, originally referred to as Tg(flk1:EGFP)s843 (Jin et al., 2005). Drug treatment
Cyclopamine (Sigma-Aldrich) was used at a concentration of 75 M, added directly to the E3 medium in which the larvae were grown. Purmorphamine (Calbiochem) was used at a concentration of 20 M. For both drugs, treatment began at 2 days post-fertilisation (dpf) to prevent gross effects on patterning or heart formation, and solutions were replaced approximately every 12 hours. Controls were incubated in E3, to which appropriate amounts of ethanol (cyclopamine) or DMSO (purmorphamine) were added. BrdU labelling
BrdU labelling was performed as previously described (Kimmel et al., 1998). In short, BrdU was diluted to a working concentration of 3 mM in E3 medium. Embryos were incubated in this solution overnight and fixed subsequently. Immunohistochemistry
Embryos were briefly fixed in 4% PFA and stored in MeOH. Embryos were rehydrated, blocked in PBS with 5% lamb serum and incubated with 1/100 anti-BrdU primary antibody (DAKO), anti-GFP (1/500 Torrey Pines Biolabs) or anti-Collagen II (1/500 DSHB) overnight at 4°C. Embryos were washed extensively then incubated in Alexa-Fluor secondary antibodies (Molecular probes) diluted 1/500 in blocking solution for 3 hours at room temperature. Embryos were washed extensively in the dark, with DAPI (Sigma-Aldrich) added at 1/1000 to one wash, then mounted for analysis. For DAB staining the secondary antibody was anti-mouse IgG-biotin, followed by incubation in ABC reagent (DAKO) and development of the DAB stain. BAC transgenesis
mCherry was recombined directly after the ATG site of the Collagen 2a1 on a bacterial artificial chromosome (BAC) clone, using similar principles to those previously (Kimura et al., 2006). The BAC was CH73-184B14, containing around 39.5 kb upstream of Col2 and around 11 kb downstream. Primer sequences for cloning available on request.
Alcian Blue and Alizarin Red staining
Bone and cartilage labelling was performed as described previously (Spoorendonk et al., 2008; Walker and Kimmel, 2007). TRAP staining
Tartrate-resistant acidic phosphatase (TRAP) staining was performed as described (Albertson and Yelick, 2005), with the following modifications: zebrafish were fixed in cold methanol (–20°C) overnight, rehydrated with PBS, and incubated in freshly made TRAP medium, for 1.5 hours at 37°C. Fish were subsequently bleached in 10% H2O2 for 4 hours and post-fixed in 4% PFA. Microscopy
In situ hybridisations were analysed and photographed with a Leica 480C camera on a Zeiss Axioplan microscope. For cell number analyses in transgenic lines, images were captured on a Leica TCS-SPE confocal system, and the stacks were analysed and cells counted using Velocity software. In the cell-counting experiments, a minimum of four different individuals for each genotype were counted. Results are presented as mean±1 standard deviation (s.d.), significance was ascertained by performing two-tailed paired Student’s t-tests of each data set to the wildtype situation.
RESULTS Increased Hh signalling leads to premature chondral mineralisation Recently two papers have implicated the Hedgehog membrane receptor patched 1 (Ptch1) in mouse bone development, but came to somewhat contradictory conclusions. The first, studying heterozygous deficiency for Ptch1, concluded that decreased Ptch1 leads to increased bone deposition (Ohba et al., 2008), whereas the other found that conditional knockout of Ptch1 in osteocalcinexpressing cells leads to decreased bone density and increased osteoclast activity (Mak et al., 2008a). Owing to embryonic lethality before the onset of bone mineralisation, it is impossible to resolve these issues in homozygous Ptch1 mutant mice. In zebrafish, however, ptc1 mutants can typically survive to around 12 dpf, and ptc2 mutants are even sub-viable, with small numbers reaching 3 months of age; as ptc1, ptc2, dre and ihha are expressed in positions where cartilage later forms (see Fig. S1A in the supplementary material) (Avaron et al., 2006; Thisse and Thisse, 2005), and as bone development in zebrafish is first apparent from 3 dpf, this gives us a window of opportunity in which to study bone development in a variety of Hh mutants. In wild-type zebrafish, the first evidence of mineralisation detected by Alizarin Red staining is at 3 dpf in the cleithrum (data not shown). By 4 dpf the dermal bones, cleithrum, operculum and notochord tip are mineralised, and at this stage ptc1, ptc2 and dre mutants were indistinguishable from their wild-type siblings (Fig. 1A). However, by 7 dpf all three mutants could be distinguished by premature mineralisation of the ceratohyal and, in the case of ptc2 and dre, the hyosimplectic – both bones of chondral origin (Fig. 1A, black arrowheads). Osterix expression is increased in endochondral bones In order to better understand why an increase in Hh signalling leads to increased bone deposition in chondral bones, we performed in situ hybridisations for osterix. Osterix is a marker of early osteoblast development and is thought to be the master regulator of osteoblast differentiation (Nakashima et al., 2002). In ptc1, ptc2 and dre mutants at 3.5 dpf, expression of osterix mRNA expression was
DEVELOPMENT
smoothened on osteoblast precursors. We therefore sought to investigate the effect of manipulating smoothened activity in vivo in the zebrafish. Together the study of these mutants and titration of smoothened activity allows us to build up a comprehensive picture of the effects both of an increase or depletion of Hh signalling on early bone formation in vivo. We show that, at least in the early stages of development, alteration of Hh signalling has no effect on dermal bone formation. However, in chondral bone elements Hh signalling critically controls both the differentiation of osteoblasts and the onset of osteoclast activity. In addition we demonstrate that many cells that are located within the cartilage element retain a level of plasticity that allows them, on receipt of high levels of Hh signalling, to differentiate as osteoblasts.
Development 136 (23)
Hedgehog signalling and osteogenesis
RESEARCH ARTICLE 3993 Fig. 1. Levels of Hh signalling are critical for endochondral ossification and proliferation of chondrocytes. (A)Alizarin Red staining of representative Hh mutants reveals no differences at 4 dpf. At 7 dpf, however, premature mineralisation of endochondral bone elements can be seen (black arrowheads mark the ceratohyal, purple arrowheads mark the hyosymplectic), while the extent of ossification in the cleithrum is unchanged (red arrowheads). (B)In situ analysis of mutants at 4 dpf reveals increased expression of osterix in the endochondral bone, where premature mineralisation is later seen (black arrowheads point to ceratohyal, purple to hyosymplectic), while osterix expression is unchanged or slightly reduced in dermal bone elements such as the cleithrum (red arrowheads). Insets show ventral views of osterix expression in the ceratohyal. (C)Alcian Blue/Alazarin Red double staining of 17 dpf ihha and wild-type sibling fish. The second panel is an enlargement of the boxed area for each genotype. (D)Whole-mount antibody staining for BrdU incorporation between 4.5 and 5 dpf in wild type and ptc2 mutants. Excessive proliferation can be seen in the ptc2 mutant in the tectum (black arrowhead) and jaw cartilages (blue arrowhead). (E)Representative single confocal images of the Meckel’s cartilage in 5 dpf zebrafish. Proliferating chondrocytes (blue arrowheads) are labelled with anti-BrdU (green), the dashed red line shows the shape of a single chondrocyte. (F)Quantitation of BrdU-positive cells in the Meckel’s cartilage at 5 dpf. Data are mean±s.d. taken of at least five fish per genotype. *P
