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Reduced Β-Catenin Expression Affects Patterning of Bone Primordia, but Not Bone Maturation Tobias Pflug, Uyen Huynh-Do and Stefan Rudloff*

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Reduced Β-Catenin Expression Affects Patterning of Bone Primordia, but Not Bone Maturation Tobias Pflug, Uyen Huynh-Do and Stefan Rudloff* © 2017. Published by The Company of Biologists Ltd | Biology Open (2017) 6, 582-588 doi:10.1242/bio.023572 RESEARCH ARTICLE Reduced β-catenin expression affects patterning of bone primordia, but not bone maturation Tobias Pflug, Uyen Huynh-Do and Stefan Rudloff* ABSTRACT disassembled and β-catenin accumulates in the cell and is Wnt/β-catenin signaling is involved in patterning of bone primordia, subsequently translocated to the nucleus, where it transactivates but also plays an important role in the differentiation of chondrocytes canonical Wnt target genes (Cadigan and Peifer, 2009; Heuberger and osteoblasts. During these processes the level of β-catenin must be and Birchmeier, 2010; Roose et al., 1998). Additional players that β tightly regulated. Excess β-catenin leads to conditions with increased affect the level of -catenin during bone development and repair bone mass, whereas loss of β-catenin is associated with osteoporosis include the extracellular Wnt inhibitors Dickkopf and Sclerostin or, in extreme cases, the absence of limbs. In this study, we examined (Florio et al., 2016; Ke et al., 2012; Qiang et al., 2008). β skeletogenesis in mice, which retain only 25% of β-catenin. These Early during embryonic development Wnt/ -catenin signaling embryos showed severe morphological abnormalities of which the plays crucial roles in determining the future body axes, and lack of hindlimbs and misshaped front paws were the most striking. maintaining gastrulation and mesoderm production (Hikasa and Surprisingly however, calcification of bone primordia occurred Sokol, 2013; Holstein, 2012). During later stages, Wnt signals are normally. Moreover, the Wnt-dependent regulatory network of important for induction, growth and organization of the future limbs transcription factors driving the differentiation of cartilage and bone, (Kengaku et al., 1998). Hereby, reciprocal signals between as well as the expression of extracellular matrix components, were mesenchymal and ectodermal cells promote the formation of the preserved. These findings show that 25% β-catenin is insufficient for limb organizer, called the apical ectodermal ridge (AER) (Barrow the correct patterning of bone primordia, but sufficient for their et al., 2003; McQueeney et al., 2002; Narita et al., 2007). The AER mineralization. Our approach helps to identify bone morphogenetic promotes the proliferation of the underlying mesenchymal cells via processes that can proceed normally even at low β-catenin levels, in Fgf signaling and induces the zone of polarizing activity (ZPA) at contrast to those that require high β-catenin dosages. This information the posterior side of the growing limb bud. Limb patterning involves could be exploited to improve the treatment of bone diseases by fine- multiple signaling protein gradients (Wnt, Fgf and Shh), originating tuning the individual β-catenin dosage requirements. from either AER or ZPA, that in turn affect the expression of different Hox genes (Wellik and Capecchi, 2003; Capdevila and KEY WORDS: β-catenin dosage, Canonical Wnt signaling, Bone Izpisúa Belmonte, 2001; Chiang et al., 2001). The individual bones development, Limb patterning of stylopodium (humerus or femur), zeugopodium (radius/ulna or tibia/fibula) and autopodium (hand or foot) are initially set up as INTRODUCTION aggregates of tightly packed mesenchymal cells (Hall and Miyake, Canonical Wnt signaling activates the transcription of β-catenin- 2000). These mesenchymal condensations differentiate into dependent target genes during many stages of bone development cartilage and are later replaced by bone via endochondral and maintenance (Duan and Bonewald, 2016), whereby a osteogenesis. The orderly progression of these processes again cytoplasmic multi-protein complex composed of the scaffolding crucially depends on the timely activation of Wnt/β-catenin proteins Axin and APC (Heuberger and Birchmeier, 2010; signaling in the respective cell types (Lu et al., 2013; Tamamura Munemitsu et al., 1995), and the kinases CK1α and GSK3β et al., 2005). The molecular mechanisms underlying bone formation (Behrens et al., 1998; Munemitsu et al., 1995), tightly regulates the are highly complex and involve the activation of mutually exclusive level of β-catenin. In the absence of canonical Wnt signaling this transcription factors and extracellular matrix genes in chondrocytes degradation machinery continuously phosphorylates β-catenin at its and osteoblasts. As the master regulator of chondrogenesis, Sox9 N-terminal region. This marks the protein for ubiquitination and drives not only the expression of cartilage-specific collagen type II proteasomal degradation (Aberle et al., 1997) and the transcription and Aggrecan, but also maintains the proliferation of chondrocytes of canonical Wnt target genes is repressed. Upon binding of a and prohibits their differentiation into osteoblasts and thus canonical Wnt ligand to the co-receptor pair Frizzled and Lrp5/6 ossification (Lefebvre and de Crombrugghe, 1998; Dy et al., (Heuberger and Birchmeier, 2010), the degradation machinery is 2012; Hattori et al., 2010). The activity of Sox9 is itself regulated by several upstream signals, of which canonical Wnt signaling exerts Department of Clinical Research, Department of Nephrology and Hypertension, an inhibiting effect. Lastly, Sox9 inhibits the expression of Runx2, a Bern University Hospital, University of Bern, Freiburgstrasse 15, Bern CH-3010, key transcription factor for bone development (Liu and Lee, 2013). Switzerland. Conversely, activation of canonical Wnt signaling in pre-osteoblasts *Author for correspondence ([email protected]) triggers the expression of Runx2 and other bone-promoting factors such as Sp7 or Atf4 (Huang et al., 2007; Yang and Karsenty, 2004). S.R., 0000-0001-6333-0830 Their importance for osteoblast differentiation has been shown in This is an Open Access article distributed under the terms of the Creative Commons Attribution knock-out mice or overexpression studies, which result in loss of License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. bone formation or ectopic ossification, respectively (Komori et al., 1997; Ueta et al., 2001). Furthermore, these transcription factors Received 13 December 2016; Accepted 20 March 2017 activate genes like type I collagen alpha 1, osteocalcin or bone Biology Open 582 RESEARCH ARTICLE Biology Open (2017) 6, 582-588 doi:10.1242/bio.023572 sialoprotein that are characteristic for the extracellular matrix of Phenotypically, mutant embryos showed major developmental bone (Ducy et al., 1997; Kern et al., 2001; Ganss et al., 1999). defects with increasing severity in the caudal half of the embryo It is therefore not surprising that many human genetic diseases are (hindlimbs and tail), as well as in the distal parts of the forelimbs associated with mutations that cause a loss of function (LOF) or a (Fig. 1; Fig. S2). In more detail, most mutant embryos did not have gain of function (GOF) of different genes of the Wnt signaling any hindlimb structures (Fig. 1A,C and G), displayed a spina bifida pathway. GOF mutations in both mice and humans lead to unusually (white arrowhead in Fig. 1A,B), an unusually curled tail (asterisk in high bone mass (Regard et al., 2012), while LOF mutations are Fig. 1A,C and Fig. S2A) and only rudimentary pelvic bones (black usually associated with low bone mass. One of the most extreme arrowhead in Fig. 1C). In very few embryos, truncated hindlimbs LOF mutations is the Tetra-amelia syndrome. It is caused by were present (black arrowhead in Fig. S2A). Forelimbs were always mutations in WNT3, which leads to the complete loss of the developed; however, their appearance was abnormal, and the formation of all four limbs (Niemann et al., 2004). In this study, we digits showed severe malformations (Fig. 1A). To gain more restricted β-catenin expression during bone development without insight into the skeletal changes, we performed Alcian Blue disturbing the machinery of the Wnt signaling pathway. To this end, (cartilage) and Alizarin Red (calcified bone) staining on whole we conditionally expressed β-catenin only from the ROSA26 locus mount embryos (Fig. 1C,D; Fig. S2A) and isolated forelimbs (Rudloff and Kemler, 2012) in all tissues caudal to the heart (Fig. 1E,F; Fig. S2B-D). Shape and size of the skeletal elements of (Hierholzer and Kemler, 2009). We found that the morphogenesis forelimb zeugopodia and stylopodia seemed not to be altered of the distal forelimbs, hindlimbs and the tail was disturbed. We between control (Fig. 1F) and β-catenin knockdown embryos further showed that the expression of genes underlying the patterning process in the forelimb autopodia was severely disturbed. Yet, the formation of mineralized matrix in these bone anlagen appeared to proceed normally. More surprisingly, we could show that the underlying molecular machinery that controls the differentiation of chondrocytes and osteoblasts was preserved in β- catenin knockdown mice. Thus, low β-catenin expression levels are insufficient for correct patterning of the skeleton, but adequate for the maturation of skeletal primordia. RESULTS Knockdown of β-catenin leads to developmental defects in the caudal embryo Expression of Cdx1::Cre mediates recombination in the three germ layers of the primitive streak region throughout the posterior embryo, caudal to the heart at mid-gastrulation (Hierholzer
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