Deltoid tuberosity develops through endochondral bone formation
Very little is known about the formation of bone ridges. As a model for studying the development of these structures we chose the deltoid tuberosity, a bone ridge located on the shaft of the humerus that exhibits a well-defined morphology.
To document DT development, we stained skeletons of E14.5–E18.5 wild type (WT) embryos with Alizarin red and Alcian blue. At E14.5, we observed the emergence of the tuberosity from the lateral aspect of the proximal third of the humerus (). As DT development proceeded, it maintained its growth; concomitantly, it underwent ossification, except for a cartilaginous part at its tip ().
Developmental analysis of the deltoid tuberosity
To study the cellular mechanism by which the DT forms, we next analyzed histologically sagittal and transverse sections of E13.5–E18.5 WT limbs. At E13.5, a group of cells with a distinctive appearance emerge from the proximal epiphysis of the humerus, indicating the initiation of DT formation (). At E14.5, these cells appeared similar to chondrocytes in the proliferative zone of the humerus primary growth plate (). At later stages (E16.5), we observed pre-hypertrophic and hypertrophic chondrocytes (). Their emergence was followed by blood vessel invasion and by E18.5 () bone formation was clearly observed. This sequence of events suggests that the DT develops through endochondral ossification.
The hallmark of endochondral ossification is the formation of a growth plate. To validate our hypothesis that the tuberosity is formed by endochondral ossification, we examined the expression of growth plate markers in E16.5 WT embryos (). The markers we analyzed included collagen type II alpha 1 (Col2a1
), a marker for resting and proliferating chondrocytes, Indian hedgehog (Ihh
) and parathyroid hormone-related peptide receptor (PTHrPR
), markers for pre-hypertrophic chondrocytes and collagen type X alpha 1 (Col10a1
), a marker for hypertrophic chondrocytes (Kronenberg, 2003
). The differential expression of theses markers in the developing DT () strongly supports our conclusion that the tuberosity develops by endochondral ossification.
To determine the growth direction of the tuberosity growth plate, we examined cell proliferation by analyzing BrdU incorporation into cells of the DT. In E16.5 WT embryos, we detected a group of proliferating chondrocytes at the tip of the DT (). This result indicates that the direction of DT growth is different from that of the humerus primary growth plate.
Tuberosity initiation is muscle independent
Studies performed over the last century have established the contribution of mechanical load created by muscle contraction in the formation of bone ridges (Hamburger, 1938
; Hamburger, 1940
)Hall and Herring, 1990; Hosseini and Hogg, 1991; (Pai, 1965a
; Rot-Nikcevic et al., 2006
; Tremblay et al., 1998
). However, all those studies document bone ridge development in ossified bones at relatively advanced stages of skeleton development. Our finding that the DT emerges as a cartilaginous template at an earlier stage of skeletogenesis prompted us to study the role of the musculature in regulating the initial stages of DT development.
We examined the skeletons of two strains of mutant mice with muscular defects. The splotch delayed mutant (Spd
) mice harbor a naturally occurring point mutation in the Pax3
gene. Homozygous Spd
embryos lack all limb musculature due to a defect in the migration of muscle progenitor cells to the developing limb (Dickie, 1964
; Franz et al., 1993
). The second mouse strain, which carries the naturally occurring autosomal recessive mutation muscular dysgenesis (mdg
), lack excitation-contraction coupling, leading to the absence of skeletal muscle contractility (Pai, 1965a
First we analyzed skeletal preparations of forelimbs from E18.5 homozygous Spd
embryos. Consistently with previous findings, these skeletons lacked the DT () (Pai, 1965a
; Rot-Nikcevic et al., 2006
; Tremblay et al., 1998
). While bone ridge development was dramatically impaired, a mild effect on the humerus longitudinal growth and ossification was also observed at E18.5 (data not shown).
Bone ridge development is disrupted in mice with defective musculature
To determine the influence of the musculature on tuberosity initiation, we examined embryonic skeletons at E14.5, one day after DT development initiates. Surprisingly, the DT was present in homozygous Spd
embryos (). Additionally, chondrocytes in the DT of the mdg
mutant mice continued their differentiation to hypertrophy (); however, they failed to maintain normal proliferation rate (Sup. 2
These results demonstrate that DT development is a biphasic process, consisting of initiation and growth phases. The initiation phase is muscle-independent, whereas the subsequent growth phase depends on muscle contraction.
Tendons are necessary for bone ridge formation
Intrigued by the biphasic nature of DT development, we sought to identify the trigger of the initiation phase. Recent reports have demonstrated the lack of DT in embryos where the expression of Tgf-β
receptor II (Tgf-βRII
) was conditionally targeted in limb mesenchyme, using the Prx1-Cre
as a deletor mouse (Prx1-Tgf-βRII
) (Seo and Serra, 2007
; Spagnoli et al., 2007
)(Baffi et al., 2004
; Logan et al., 2002
). Recently, we showed that TGF-β signaling was essential for tendon development, as in the Prx1-Tgf-βRII
embryos tendons and ligaments were lost (Pryce et al., 2009). In light of this evidence and given the biphasic nature of tuberosity formation, we decided to use the Prx1-Tgf-βRII
embryos to study the role of tendon cells in DT initiation.
First, to verify the loss of tendons at the attachment site we examined the expression of scleraxis (SCX), a marker for tendons, in embryos homozygous for floxed-Tgf-βRII and heterozygous for Prx1-Cre alleles (Prx1-Tgf-βRII) and embryos heterozygous for floxed-Tgf-βRII and Prx1-Cre alleles (control). As expected, no Scx expression could be observed in Prx1-Tgf-βRII forelimbs (). Next, we studied DT initiation in the Tgf-βRII-ablated limbs. Skeletal preparations of E14.5 Prx1-Tgf-βRII forelimbs lacked DT initiation (). These results strongly imply that tendons are necessary for the initial signal that induces the formation of the DT.
Tendons regulate deltoid tuberosity initiation through scleraxis
SCX is necessary for deltoid tuberosity initiation
Our finding that tendons are necessary for DT initiation prompted us to screen for molecules that mediate this interaction. We previously showed that Scx−/−
newborn mice lacked the tuberosity and suggested that the phenotype might be attributed to a failure in the transmission of mechanical load from muscles to the skeleton, due to tendon abnormality (Murchison et al., 2007
). Having found an involvement of tendons in the induction of DT formation, we postulated that SCX might regulate tuberosity initiation. SCX is essential for tendon differentiation and in Scx−/−
mice, some of the tendons appeared rudimentary and others were completely missing (Murchison et al., 2007
). Therefore, we first had to verify that tendons formed at the Scx−/−
humerus attachment site, prior to DT initiation. To this end, we crossed the ScxGFP transgenic tendon reporter mouse into the Scx−/−
background (Pryce et al., 2007
). In sagittal sections through the humeri of E13.5 embryos, tendons attaching at the initiation site of the DT, visualized as ScxGFP positive cells, were observed in both Scx+/−
(control) and Scx−/−
mutants (). Thus, despite the presence of tendon cells at the humerus insertion site of Scx−/−
mutant embryos, these embryos lacked DT initiation ().
If Scx is required for DT initiation, it should be expressed in the Spd and mdg mutant mice, which display normal initiation. As expected, Scx was expressed at the initiation site in both strains, similarly to control mice (). These results implicate SCX as a key molecule in the regulation of DT initiation by tendons.
Bmp4 expression in tendons is regulated by SCX
The regulation of DT initiation by SCX must operate through a cell-nonautonomous mechanism, since Scx expression is restricted to tendons. Because SCX is a transcription factor, we performed a mini screen of candidate molecules to identify secreted factors that act downstream to SCX and mediate the nonautonomous initiation signal from the tendons to the skeleton. We detected bone morphogenetic protein 4 (Bmp4) expression in tendon cells at the attachment site at E13.5, making it a likely candidate to initiate tuberosity formation (). Moreover, Bmp4 expression in tendons was co-localized with Scx expression, mainly at the attachment site (). The co-expression of Bmp4 and Scx was also observed in other tendon-cartilage intersections, such as the elbow joint ().
BMP4 acts downstream of scleraxis
The co-expression of Scx and Bmp4 in tendon-cartilage intersections prompted us to examine whether SCX is necessary for Bmp4 expression in tendons. The specific loss of Bmp4 expression at the tendon insertion site of Scx−/− embryos implied that SCX acts upstream to BMP4 in tendon cells ().
SCX regulates the transcription of Bmp4
The co-expression of Scx and Bmp4 and the reduction in Bmp4 expression in tendon-cartilage intersection point of Scx−/− mice led us to determine whether or not SCX regulates the transcription of Bmp4. First, we examined the ability of Scx overexpression to induce an increment in Bmp4 expression in cell culture, by either transiently or stably transfecting Scx into C3H10T1/2 cells. In both cases, quantitative RT-PCR revealed an elevation in Bmp4 expression in Scx-transfected cells (). To determine whether or not the increased steady-state level of Bmp4 mRNA was a consequence of increased transcription, we transiently cotransfected C3H10T1/2 and HeLa cell lines with a construct that contained a 0.9 kb of Bmp4 promoter region fused to luciferase cDNA (pGL3 BMP4 full) and Scx-expressing vector. These experiments revealed approximately three-fold induction of luciferase activity relative to the control vector (; for C3H10T1/2 cells, data not shown).
Next, we searched the mouse Bmp4
promoter for SCX consensus binding sites (also referred to as E-box). We identified two putative binding sites in positions −242 (CACGTG) and −230 (CAGGTG) upstream to the transcription initiation site (); these consensus sequences were previously shown to bind scleraxis (Lejard et al., 2007
; Liu et al., 1997
). In order to examine whether these two E-boxes were necessary for SCX induction of Bmp4
expression, we made two constructs: one with mutated E-boxes (pGL3 B4mut: CACGTG -> CACAAA, CAGGTG -> CAGAAA) and another that contained a region downstream of the E-Boxes (pGL3 del1). We compared the ability of SCX to induce luciferase activity in the mutated promoter and in the intact promoter. As can be seen in , as well as indicating a deletion of the Bmp4
promoter, the two mutations of the E-boxes blocked the ability of SCX to induce luciferase activity.
In order to demonstrate a direct binding of scleraxis protein to the Bmp4 promoter we performed a chromatin immunoprecipitation (ChIP) experiment. Chromatin was prepared from C3H10T1/2 cells lysate stably expressing Scx tagged with Xpress epitope. The lysate was incubated with either an anti-Xpress-tag antibody or anti-GFP antibody (as control). qRT-PCR analysis using specific primers to the E-boxes region of Bmp4 promoter revealed binding of SCX to the indicated promoter region ().
To further validate the specificity of the two identified E-box sequences for SCX binding, we utilized a plasmid IP experiment. To that end, HeLa cells where cotransfected with Scx-expressing plasmid together with either the intact Bmp4 promoter (pGL3 B4 full) or the Bmp4 promoter with the mutated E-boxes (pGL3 B4mut). As can be seen in , qRT-PCR analysis revealed that the mutations in the E-boxes completely ablated the binding of SCX to the Bmp4 promoter. Therefore, we conclude that scleraxis binds the Bmp4 promoter and regulates its transcription.
Bmp4 expression in tendons regulates deltoid tuberosity initiation
To establish that BMP4 mediates signaling from tendons to cartilage in the regulation of bone ridge initiation, we wanted to demonstrate that BMP signaling was activated in the initiating DT and was required for its formation. To detect an activation of BMP signaling in chondrocytes of the forming DT we examined the expression of Alk3
, a type IA receptor for BMP4 (BmprIA
) (Yamaji et al., 1994
). In E14.5 control mice, Alk3
was expressed within muscles and cartilage, but not in tendons (). Next, we directly tested the activation of the BMP pathway by examining the activation of Smad proteins, the downstream mediators of the BMP signaling cascade (Shi and Massague, 2003
), using a specific antibody that detects the phosphorylated form of Smad 1, 5 and 8 (P-Smad). P-Smad 1 5 8 staining in E14.5 WT forelimb was observed in DT chondrocytes (). Taken together, these results support the notion that SCX-dependent expression of Bmp4
in tendons activates BMP signaling in tuberosity-forming chondrocytes.
BMP4 mediates deltoid tuberosity initiation
To establish that BMP signaling to chondrocytes was imperative for DT initiation, we blocked the expression of Bmp4
in limb mesenchyme using the Prx1-Cre
as a deletor (Prx1-Bmp4
). Examination of skeletal preparation and histological sections of E14.5 Prx1-Bmp4
limbs revealed the lack of DT initiation (). To further demonstrate that this phenotype was specifically related to Bmp4
abolishment in tendon cells, we blocked the expression of Bmp4
-expressing cells using the Scx-Cre
as a deletor (Scx-Bmp4;
mice will be reported elsewhere). To evaluate the effectiveness and specificity of Scx-Cre
as a deletor in tendon cells, we crossed the Scx-Cre
mice with R26R-lacZ
reporter mice (Rountree et al., 2004
; Soriano, 1999
). Examination of Scx-Cre,
heterozygous embryos at E14.5 revealed lacZ
expression in the forming tendons ().
Next, we examined skeletal preparations and histological sections of limbs from E14.5 Scx-Bmp4
embryos and detected no DT initiation (). To exclude the possibility that tuberosity loss resulted from the lack of tendons in these mutants’ limbs, or that Bmp4
expression was necessary for the expression of Scx
in these limbs, we examined histological section and the expression of Scx
in limbs of Prx1-Bmp4
expression was not affected in the mutated limbs () and tendons did form in their presumed attachment site to the humerus (Sup. 3B,C
); moreover, these tendons apparently attached directly to the perichondrium instead of to the DT, as in the control limbs. Further analysis of tendon integrity was conducted on the Scx-Bmp4
embryos by examining the expression of Scx,
collagen type I alpha 1 (Col1a1)
, tenomodulin (Tnmd
) and tenascin C (Tnc
), markers of tendon differentiation. The expression of Scx, Col1a1, Tnmd
in mutants’ tendons () indicated normal differentiation.
Finally, to evaluate the contribution of Bmp4 expression in tendons to bone ridge formation throughout the skeleton we examined the development of a wide variety of bone ridges in 2-week-old Scx-Bmp4 mice by micro-CT scanning. As can be seen in , the loss of Bmp4 in tendons led to aberrant formation of numerous ridges in the axial and appendicular skeleton; however, not all the ridges were affected.
The loss of Bmp4 in tendons has a wide effect on bone ridge development throughout the skeleton
These results strongly support our hypothesis that SCX regulation of Bmp4 expression in tendons regulates bone ridge initiation. Moreover, these results implicate BMP4 as a key molecule in the mechanism that mediates signaling from tendon cells to chondrocytes at their attachment site.