BMP SIGNALING MEDIATES THE TIMING OF INTRAMEMBRANOUS OSSIFICATION
*Merrill, AE; *Eames, BF; *Weston, SJ; *Heath, T; +*Schneider, RA
+* Orthopaedic Surgery, University of California, San Francisco, CA
+ ras@itsa.ucsf.edu
Introduction: In order to develop novel strategies for regenerating
skeletal tissues affected by disease and trauma, endogenous molecular
mechanisms that drive the differentiation of mesenchymal stem cells
into bone need to be identified. Toward this goal, we investigated in vivo
signaling interactions between mesenchyme and epithelia, which
establish the precise timing of intramembranous ossification. We employ
an avian chimeric system that exploits the divergent maturation rates of
quail and duck embryos and allows us to manipulate temporal
information being conveyed between embryonic populations of
mesenchyme and epithelium. We transplant pre-osteogenic
mesenchyme, from quail to duck and produce chimeric embryos that
have an accelerated population of quail donor mesenchyme alongside
relatively slower duck host epithelium. We find that quail donor
mesenchyme maintains its faster timetable for osteogenesis within the
slower environment of duck hosts based on molecular and histological
markers for bone. From these observations and in light of our published
work where donor mesenchyme regulated gene expression in adjacent
host epithelia [1,2], we hypothesize that pre-osteogenic mesenchyme
controls the timing of intramembranous ossification by regulating
expression of molecules known to function during bone formation such
as members and targets of the Bone Morphogenetic Protein (BMP)
pathway. To test our hypothesis we first defined the precise stages
during which tissue interactions are required for intramembranous
ossification of the mandible and determined the extent to which preosteogenic mesenchyme governs these interactions. Second, we
identified candidate molecules that may mediate this tissue interaction
based on donor-induced changes to spatiotemporal expression patterns.
Third, we ascertained the potential of exogenous BMP to regulate the
timing of skeletal differentiation. Together, our results identify BMPs as
candidate mediators in the timing of intramembranous ossification.
Results: To determine the precise stage during which tissue interactions
between epithelium and mesenchyme occur, we cultured control duck
mandibular mesenchyme in the presence and absence of mandibular
epithelium for eight days. We found that HH25 mandibles formed bone
with or without overlying epithelium. In contrast, control HH23
mandibles were only able to form dermal bone in the presence of
overlying epithelium. The presence of cartilage in all samples served as
an internal control, since cartilage can form in the absence of an
epithelium and ensures that the cultures were viable. To determine the
extent to which neural crest-derived mesenchyme governs this
interaction, mandibles from chimeric embryos were cultured with and
without mandibular epithelium. Chimeric HH23 mandibles, where older
quail neural crest-derived mesenchyme lies adjacent to relatively
younger duck epithelium, formed bone in the absence of epithelium. To
identify candidate molecules that may temporally regulate the epithelialmesenchymal interactions required for bone formation, we examined
expression patterns of members of the BMP family. In situ analysis
revealed expression patterns that correlated spatiotemporally with the
osteo-inductive tissue interaction. Bmp4 and Bmp7 transcripts were
restricted to mandibular epithelium at HH23. By HH25, Bmp4 and
Bmp7 transcripts were localized to only the mesenchyme. B m p 5
transcripts were undetectable in either mandibular epithelium or
mesenchyme at HH23. By HH25, Bmp5 transcripts were expressed in
the mesenchyme. To test the ability of exogenous BMP to regulate the
timing of skeletal differentiation, surgically extracted HH23 quail
mandibles were treated with beads soaked in BMP4. Mandibles cultured
for three days did do not show any histological evidence of bone on
either the BMP4 or BSA treated side. Those mandibles cultured for five
days showed an average seven-fold increase in bone volume on the side
treated with BMP4 compared to the contralateral side treated with BSA.
Methods: Fertilized eggs of Japanese quail and white Pekin duck were
incubated until stage-matched at embryonic stage (HH) 9.5 [3]. All
embryos were handled in accordance with University and NIH
guidelines. Neural crest cells from the rostral hindbrain and midbrain
were excised from quail donors and transplanted into duck hosts. For
controls, orthotopic grafts were made within each species. Chimeric and
control embryos were collected at embryonic stages when epithelialmesenchymal signaling interactions required for bone formation are
believed to occur (HH19-HH23). We surgically extracted mandibles at
successive embryonic stages from control and chimeric embryos,
removed the epithelia, cultured the mesenchyme in vitro, and assayed
for molecular and histological evidence of bone. As a control, mandibles
were similarly processed without removing the epithelium.
Mesenchymal condensations are first detectable at HH25, and begin to
deposit bone matrix by HH32. Based on this timetable, tissue explants
were grown in organ culture with differentiation media for eight days,
allowing time for even the earliest stages (HH19) to form bone. Tissues
were fixed, paraffin embedded, and cut into sections. To detect donor
cells in chimeric mandibles, representative sections were immunostained
with the quail nuclei-specific Q¢PN antibody. We used Milligan’s
Trichrome as a histological stain to assay for bone and cartilage in
sections containing labeled donor neural crest-derived quail cells.
Mandibles from successive embryonic stages were assayed for temporal
changes in the expression of BMP pathway members using in situ
hybridization. In situ hybridization was performed with 35S-labeled
antisense riboprobes to chick Bmp4, Bmp5, and Bmp7. Mandibles from
HH23 quail embryos were surgically extracted, placed on transwell
membranes, and treated contralaterally with Affigel agarose beads
equilibrated in BMP4 or with .1% bovine serum albumin (BSA) for
controls. After 24 hours, mandibles were cultured in differentiation
medium for three and five days and assayed for the histological presence
of bone. In order to estimate the amount of bone in cultured mandibles,
representative histological sections were digitized in Adobe Photoshop.
Bone volume (BV) was estimated as a function of the total number of
pixels comprising domains (A) of stained bone matrix using the equation
for a conical frustum BV=(1/3h)[(Ai + Aii +1)(√AiAii+1)].
Discussion: Understanding molecular mechanisms that impart precise
temporal control of mesenchymal differentiation into bone is essential
for generating effective therapies to treat skeletal tissues affected by
disease and trauma. In this context, we have developed an in vivo avian
embryonic stem cell transplantation system, which can be exploited to
identify molecular and cellular interactions that regulate the timing of
osteogenesis. While time-dependent epithelial-mesenchymal interactions
are known to regulate intramembranous ossification, our results reveal
BMP family members as candidate mediators of this critical process.
Our in situ analyses demonstrate that spatiotemporal changes in Bmp
expression correlate with the ability of mandibular mesenchyme to form
bone in the absence of epithelium. These findings, coupled with the
ability of exogenous BMP4 to induce premature bone formation in
cultured mandibles, indicates that molecular-based therapies have strong
potential to promote regeneration of skeletal tissues in clinical situations
such as following trauma or in cases of degenerative skeletal diseases.
HH23
HH25
Bmp5
A
HH23
B
HH25
Figure: In situ hybridization of frontal sections through quail mandibles
shows that Bmp5 transcripts are not present at HH23 (A). At HH25
Bmp5 transcripts (white signal) are localized to mandibular epithelium.
Acknowledgements: Supported by R03 DE014795-01 and R01
DE016402-01 from the NIDCR, Research Grant 5-FY04-26 from the
March of Dimes Birth Defects Foundation, and UCSF Academic Senate
and REAC grants to R.A.S.
1. Schneider and Helms (2003) Science 299: 565-8.
2. Eames and Schneider (2005) Development 132(7): 1499-509.
3. Hamburger and Hamilton (1951) Journal of Morphology 88: 49-92.
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 1657