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Tissue-specific action of Msx genes in the regulation of skull vault development

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Tissue-specific action of Msx genes in the regulation of skull vault development

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Content Tissue-specific action of Msx genes in the regulation of skull vault
development

by

Paul G. Roybal

_____________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2009

Copyright 2009 Paul G. Roybal
i
Table of Contents

List of Figures………………………………………………………..................... ii
Abstract…………………………………………………………………………...1

Introduction………………………………………………………………….…....2

Materials and Methods……………………………………………………………6

Results……………………………………………………………………….…... 9
An unexpected regulative response in the frontal bones of Wnt1-Cre;
Msx1/2flox/flox mutant embryos……………….............................................. 9
Regulative frontal bone in Wnt1-Cre; Msx1/2 mutants is derived from a
population of neural crest whose normal fate is non-osteogenic……............. 13
Timing of action of Msx genes……………………………………………… 20
Dysregulation of Bmp signaling in Msx1-2 mutants…………………………22

Discussion…………………………………………………………………….…. 24
Bibliography……………………………………………………………………... 30

ii
List of Figures

Figure 1: Dual functions of Msx1/2 in promoting frontal bone development and
suppressing heterotopic bone formation in early migrating cranial
mesenchyme …………………………………………………………………….. 10

Figure 2: Prospective heterotopic bone is detectible at E13.5…………............. 12

Figure 3: Neural crest origin of heterotopic bone.……………........................... 14

Figure 4: EphA4 expression was dramatically altered in Twist mutants………. 16

Figure 5: Inactivation of Msx1/2 in neural crest causes a change in the fate map
of early migrating cranial mesenchyme.…………………………......................... 18

Figure 6: Osteogenic precursor cells can migrate apically from the supraorbital
ridge in Msx1/2 flox/flox; Wnt1-Cre mutant embryos.…………………………... 19

Figure 7: Increased number of osteogenic cells within the coronal sutures of
compound heterozygotes compared with individual heterozygotes……………... 21

Figure 8: Dysregulated Bmp signaling correlated with heterotopic bone in
Msx1/2 mutants.………………………………………………………………….. 23
1
Abstract

In an effort to understand the morphogenetic forces that shape the bones of the skull,
we inactivated Msx1 and Msx2 conditionally in neural crest and other cell
populations potentially relevant to skull development. We show that Wnt1-Cre
inactivation of up to three Msx1/2 alleles results in a progressively larger defect in
the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx
alleles, the large defect is filled in with mispatterned bone. This bone also partially
obliterates the coronal sutures, thus producing craniosynostosis. The bone is derived
from neural crest, not mesoderm, and originates in a normally non-osteogenic layer
of cells through with the rudiment grows. Associated with the heterotopic
osteogeneis is an upregulation of Bmp signaling in this cell layer. These results
suggest that Msx genes have a dual role in calvarial development. They are required
for the differentiation and proliferation of osteogenic cells within rudiments, and they
are also required to suppress an osteogenic program in a cell layer within which the
rudiments grow. We suggest that the inactivation of this repressive activity may be
one cause of Wormian bones, ectopic bones that are a feature of a variety of
pathological conditions in which calvarial bone development is compromised.

2
Introduction

The vertebrate skull vault consists of several component bones of mixed lineage
origin. In the mouse, these are the paired frontal and parietal bones and a single
interparietal bone. The frontal bones are derived from neural crest, the parietal bones
from mesoderm (Jiang et al., 2002). The interparietal bone is a composite, its central
portion derived from neural crest and its lateral portions from mesoderm. The
patterned growth of the vertebrate skull is a complex and as yet poorly understood
process that involves an exquisitely controlled series of migratory and proliferative
events, as well as the specification and differentiation of osteogenic and
chondrogenic cells among others (Chai and Maxson, 2006; Morriss-Kay and Wilkie,
2005). During the first stages of skull vault development, precursor neural crest and
mesodermal cells migrate to positions on the lateral aspects of the cerebral
hemispheres. The frontal and parietal bone rudiments become evident at E12.5 as
condensations within these mesenchymal cell populations. They express early
osteoblast markers, and then expand apically through the remainder of prenatal
development and several days of postnatal development, ultimately coming into
apposition with their paired counterparts at the midline (Ishii et al., 2003; Jiang et al.,
2002).

Two models have been put forward to explain the apical expansion of the frontal and
parietal bones (Lana-Elola et al., 2007; Ting et al., 2009; Yoshida et al., 2008);. One
posits proliferation and migration of precursor cells located at base, the second,
3
differentiation of preexisting precursors. In favor of the first model, DiI injections
into the rudiment at E13.5, followed by exo utero culture of embryos revealed that
during apical expansion cells migrate from the base of the rudiments to the leading
edge of the growing bone. Cells within the rudiment are also proliferating during this
period of expansion. Thus migration and proliferation are probably important
morphogenetic forces contributing to the growth of the calvarial bones. Arguing
against the second model are results of diI injections into mesenchymal cells located
apical to the rudiment. Labeled cells do not have an ostoegenic fate, but are located
in a non-osteogenic layer flanking the bone (Yoshida et al., 2008). We note, however,
that when diI injections are performed at late stages in the presumptive sagittal suture,
a small number of labeled cells are found in bone (Lana-Elola et al., 2007). Thus at
least some sutural cells are capable of differentiating into osteoblasts. The emerging
view of at least the early stages of calvarial bone growth is that the bone rudiments
grow by end-addition of migratory cells through a layer of non-osteogenic
mesenchyme.

Twist, Msx1, and Msx2 have been shown to have major influences on calvarial
growth and patterning (Chai and Maxson, 2006). Twist, a basic helix-loop-helix
transcription factor, is mutated in Saethre-Chozen Syndrome, characterized by
craniosynostosis as well as other craniofacial and limb defects (el Ghouzzi et al.,
1997). Twist is required for proper targeting of migratory osteogenic cells to the
leading edges of growing bones. This activity requires EphA4, which functions in a
layer of cells in which osteogenic precursors migrate, flanking the prospective bone
4
(Merrill et al., 2006; Ting et al., 2009). Twist, EphA4 and Twist-EphA4 mutants
exhibit inappropriate migration of osteogenic cells into the coronal suture and
consequent differentiation of normally non osteogenic suture cells. The result is
synostosis of the frontal and parietal bones (Ting et al., 2009).

Msx genes function in the apical expansion of the frontal and parietal bone rudiments
(Han et al., 2007; Ishii et al., 2003). In Msx2 conventional mutants, the growth of the
rudiments is retarded and cells within the rudiments proliferate at a reduced rate
(Ishii et al., 2003). In combination Msx1-2 mutants, the frontal and parietal bones do
not form and many embryos exhibit exencephaly (Han et al., 2007). The severity of
this set of phenotypes precluded a detailed analysis of the role of Msx genes in
calvarial bone growth. This limitation, together with the critical role of Msx genes in
the apical expansion of the rudiments, prompted us to undertake a more detailed
analysis of the activities of Msx1-2.

We produced floxed alleles of Msx1 and Msx2 (Fu et al., 2007). In the present study,
as part of an effort to understand more fully the morphogenetic forces shaping
calvarial bones, we inactivated Msx1 and 2 conditionally in neural crest and other
cell populations relevant to skull vault development. We show that Wnt1-Cre-
mediated inactivation of up to three Msx1/2 alleles results in a progressively larger
frontal bone defect. Unexpectedly, in embryos lacking all four Msx alleles, the large
defect is largely filled with bone, which is mispatterned and present in sutures. This
bone is derived from neural crest, not mesoderm, and originates in the normally non-
5
osteogenic layer of cells through with the rudiment grows. Associated with the
heterotopic osteogeneis is an upregulation of Bmp signaling in this cell layer. These
results suggest that Msx genes have a dual role in calvarial development. They are
required for the differentiation and proliferation of osteogenic cells within rudiments,
and they are also required to suppress an osteogenic program in a normally non-
osteogenic cell layer within which the rudiments grow.

6
Materials and Methods

Mouse mutants and genotyping
Mutant lines were maintained in a C57Bl/6 mixed background. The R26R (Soriano,
1999), Wnt1-cre (Danielian et al., 1998) and Msx1 and 2 Floxed (Fu et al., 2007)
alleles have been described. We genotyped Msx1 and 2 Floxed, R26R, and Wnt1-cre
alleles by PCR as described (Fu et al., 2007; Jiang et al., 2002).

Histology, immunostaining and in situ hybridization
Heads of embryos were embedded in OCT medium (Histoprep, Fisher Scientific)
before sectioning. Frozen sections were cut at 10 μm. Analysis of β-galactosidase
activity of Wnt1-Cre/R26R reporter gene expression was carried out as described
(Ishii et al., 2003). Immunostaining of frozen sections was largely carried out as
previously reported (Ishii et al., 2003). Immunohistochemistry was performed using
rabbit anti-P-Smad1/5/8 (pSmad1/5/8) (Cell Signaling) or rabbit anti-phosphorylated
Histone3 (pH3) (Cell Signaling) diluted in 1%BSA/PBS and incubated overnight at
4°C. Detection of anti-p-Smad1/5/8 or anit-pH3 was performed by incubating
rhodamine-labeled goat anti-rabbit IgG (1:100 for anti-pSmad1/5/8) or (1:50 for anti-
pH3) for1 hour at room temperature followed by DAPI counterstaining and
examination by epifluorescence microscopy. Non-radioactive section in situ
hybridization using the tyramide signal amplification (TSA) method was performed
as described (Adams, 1992; Paratore et al., 1999; Yang et al., 1999). Briefly, to
analyze mRNA expression by TSA, DIG-labeled or FLlabeled riboprobes were
7
allowed to hybridize with the section and were detected with anti-DIG or anti-FL
antibodies conjugated to horseradish peroxidase (POD). Indirect TSA fluorescence
system (TSA-biotin/avidin-FITC) was used to detect the POD-conjugated antibody
(Perkin Elmer). RNA probes were generated as reported: DIG-labeled Bmp4 probe
(courtesy Malcolm Snead) and FL-labeled Bmp2(courtesy Malcolm Snead).

Whole-mount skull Alizarin Red S staining
Skulls from P0-day-old postnatal mice were stained for bone with 2% Alizarin Red S
in 1% KOH for 1 to 2 days. The specimens were then cleared and stored in 100%
glycerol.

Whole-mount alkaline phosphatase (ALP) staining
Whole-mount staining for alkaline phosphatase was carried out as described (Ishii et
al., 2003). Embryonic day 12.5-14.5 (E12.5-E14.5) embryo heads were fixed in 4%
paraformaldehyde in PBS, and were bisected midsagitally after fixation. Presumptive
calvarial bones were stained with NBT and BCIP (Roche).

Exo utero DiI labeling of migratory osteogenic precursor cells
Details of the exo utero manipulation have been described (Muneoka et al., 1986;
Serbedzija et al., 1992). Briefly, E13.5 embryos with embryonic membranes were
carefully exposed by incising the uterine wall. Two embryos from each side of the
uterine horns were designated as the experimental group, and all others were
removed. DiI (Molecular Probes, 1:10 dilution from 0.5% stock solution) was
8
injected into the area of the frontal bone rudiments under a dissecting microscope
with a microelectrode (tip diameter, 20 μm) attached to a mouth pipette (Yoshida,
2005). After injection, the embryos were returned to the peritoneal cavity of dams
and allowed to continue development exo utero. After 2-3 days of additional
development, the embryos were removed and examined by epifluorescence
microscopy. The survival rate of the embryos after DiI injection was greater than
70%.

Exo utero DiI labeling of Cranial Mesenchyme
The surgery for this was carried out exactly as with the labeling of migratory
osteogenic precursors. However, instead of labeling the frontal bone rudiment, the
cranial mesenchyme of E13.5 embryos was labeled at a height of 2 ½ - 3 eye
diameter above the mid point of the eye. Survival rate was greater than 70%.
9
Results

An unexpected regulative response in the frontal bones of Wnt1-Cre;
Msx1/2flox/flox mutant embryos

We first assessed the effect of a neural crest-specific knockout of Msx1 and Msx2 on
the development of the neural crest-derived frontal bone and the mesoderm-derived
parietal bone. We produced an allelic series of floxed Msx1 and Msx2 alleles
together with Wnt1-Cre, and examined the morphology of skulls at the newborn
stage (Figure 1). We showed previously that Wnt1-Cre caused a highly efficient
knockout of Msx1 and Msx2 (Fu et al., 2007). As is evident in Figure 1, the skulls
exhibited a defect in the frontal bone which became progressively larger as the
number of inactivated Msx alleles increased. Different allelic combinations of Msx1
and Msx2 revealed that the two genes were equivalent in their effects on the frontal
bone defect (Figure 1; data not shown).

Since the calvarial bones do not develop in conventional Msx1/2 knockouts (Han et
al., 2007), we expected that complete Wnt-Cre-mediated inactivation of Msx1/2
would result in a more severe defect than in homozygous-heterozgyous combinations.
Strikingly, however, upon inactivation of the final Msx allele, a new phenotype
became evident: Alizarin stained bone was present over much of the area where we
expected to see an unossified persistent foramen. Thus Msx1/2 mutant embryos
“regulated” and partially repaired the frontal bone defect. This “regulative” bone
10
Figure 1. Dual functions of Msx1/2 in promoting frontal bone development
and suppressing heterotopic bone formation in early migrating cranial
mesenchyme. Skulls of Wnt1-Cre; Msx1flox/flox; Msx2flox/flox mutants at the
newborn stage were stained with Alizarin Red S to reveal bone. A, wild type; B,
Msx1flox/+;Msx2flox/+; C, Msx1flox/+;Msx2flox/flox; D, Msx1flox/flox;
Msx2flox/+; E, Msx1flox/flox; Msx2flox/flox. Note increasing size of frontal
foramen with decreasing Msx gene dosage up to homozygote-heterozytgote
combination (arrows). Note unpatterned, heterotopic bone in area of posterior
frontal bone in E (bracket).
11
obliterated part of the frontal suture and was irregular in shape, suggesting that it was
not subject to normal patterning mechanisms. In addition, the extent of apical growth
of the parietal bones was reduced in Msx1/2 homozygous mutant embryos. This
effect is non-autonomous since the parietal bones are derived from mesoderm.

Finally, defects were apparent in the interparietal bone. Double heterozygous Msx1/2
mutants had a cleft in the posterior portion of the bone. In double homozygous
mutants, an additional defect was evident in the anterior of the interparietal bone.
Both defects occurred in the central portion of the bone, which is derived from neural
crest (Jiang et al., 2002).

We examined a developmental series of embryos to determine when frontal bone
regulation was first detectible. We stained embryos in whole mount for the early
osteoblast marker, alkaline phosphatase (ALP) (Figure 2). In control embryos at
E12.5, the frontal bone rudiment is evident as a crescent of ALP-stained cells in the
supraorbital ridge. Immediately posterior to the rudiment is an ALP-free area
corresponding to the presumptive coronal suture, and posterior to that is the parietal
bone rudiment. In Msx1/2 flox/flox mutants, no staining was apparent in the area of
the rudiment, nor was there staining on the apical portion of the head in the area
where the heterotopic bone will form.

At E13.5, as revealed by ALP staining the frontal and parietal bone rudiments of
control embryos were larger (Figure 2). No ALP activity was present apical to the
12
Figure 2. Prospective heterotopic bone is detectible at E13.5. Embryonic
heads were stained in whole mount for alkaline phosphatase expression at E12.5
(A, B) and E13.5 (C, D). Note that in the Wnt1-Cre; Msx1/2 flox/flox mutant at
E12.5, the frontal bone rudiment is not detectible (B, boxed area). At E13.5, the
rudiment is visible, and an area of ALP stain is evident apical to the rudiment,
extending approximately 2/3 of the distance to the dorsum of the head (D,
arrows).
13
rudiments. In contrast, in Msx1/2 Wnt1-Cre mutants, the rudiments were irregular in
shape, and areas of apical ALP staining were evident.

Regulative frontal bone in Wnt1-Cre; Msx1/2 mutants is derived from a
population of neural crest whose normal fate is non-osteogenic

To understand the processes that gave rise to the regulative bone in Msx1/2 mutants,
we sought to determine its tissue of origin. We considered two possibilities. The first
was that the bone arose from mesoderm-derived cells that normally form the parietal
bone. The close proximity of the heterotopic bone to the parietal bone made this an
attractive possibility. Also consistent with this possibility is the finding that
regulative bone does not occur in the frontal bone territory of conventional Msx1/2
knockouts, suggesting that its development may depend on Msx gene function in a
non-neural crest cell type.

We crossed the R26R marker allele into Wnt1-Cre; Msx1/2 mutants and examined
the distribution of lacZ positive cells in double homozygous floxed mutant and
control embryos at E13.5 (Figure 3). In control embryos a lacZ positive layer of
loose mesenchyme was evident between the epidermis and the meninges. This layer
is composed of neural crest cells that migrate apically prior to the growth of the
frontal bone rudiment; thus we refer to them as “early migrating” neural crest cells.
Msx1 and Msx2 are coexpressed in this layer, commencing when its component cells
first migrate apically, between E9.5 and E10.5 (Ishii et al., 2003; unpublished
14
Figure 3. Neural crest origin of heterotopic bone. To visualize neural crest-
derived cells, we produced mice carrying the R26R marker allele along with
Wnt1-Cre; Msx1flox/flox; Msx2flox/flox. Embryos were taken at E13.5, and
heads were sectioned in the indicated plane. Adjacent sections were stained
either for lacZ (A, C, E, G) or ALP (B, D, F, H). Boxed areas in A-D correspond
to areas of heterotopic ALP activity (see Figure 2). Arrows in H point to ALP
stain of heterotopic prospective bone. Note that these ALP-positive cells are in a
lacZ positive cell layer and are therefore derived from neural crest.
Abbreviations, fb, frontal bone, b, brain.
15
observations). Figure 4 shows the distribution of Msx1 and Msx2 transcripts in this
layer at E11.5, E12.5 and E13.5.

The early migrating neural crest layer did not stain appreciably for ALP. In contrast,
in mutant embryos, the regulative prospective bone was detectible as a patchy ALP
stained layer apical to the frontal bone rudiment. Cells of this layer appeared more
tightly condensed than their wild type counterparts, as well as more numerous.
Staining for lacZ revealed that these ALP-positive cells were entirely or almost
entirely of neural crest origin. Thus, although the regulative response may require
Msx gene function in a non-neural crest cell type, it does not entail recruitment of
mesoderm-derived cells to the frontal bone defect. Neither is the response likely a
result of selective proliferation of local cells. Brdu labeling revealed a general
increase in the number of proliferative cells in tissues of the skull and brain, but not a
selective increase in the early migrating cranial mesenchyme (data not shown).

We next addressed the source of the ALP-positive, neural crest-derived cells that
form the regulative bone of Msx1/2 mutants. The simplest possibility was that the
cells originate from the early migrating neural crest. It was also possible that they
were the result of an aberrant migration, one potential source being a population of
osteogenic precursor cells that migrate from the supraorbital ridge to the leading
edge of the growing frontal bone (Yoshida et al., 2008; Ting et al., 2009). To test
these possibilities, we carried out a series of diI cell marking experiments.

16
Figure 4. Expression of Msx1/2 in early migrating cranial mesenchyme.
Embryos at E12.5 were sectioned in the coronal plane and sections were stained
for ALP activity (A, E) and incubated with Msx1 (B, F) and Msx2 (F, G) probes.
Hybridization signals were visualized by immunofluorescence. Msx1 is in red,
Msx2 green. Note largely overlapping expression of Msx1 and Msx2 in layer of
neural crest-derived mesenchyme surrounding the brain. Apical to the frontal
bone rudiment (fb) this mesenchyme is normally non-osteogenic. Abbreviations:
frontal bone, fb; b, brain; m, meninges; nom, neural crest-derived, non-
osteogenic mesenchyme.
17
We first labeled cells of the early migrating neural crest layer prior to the formation
of heterotopic bone, and asked whether they become incorporated into such bone.
DiI was injected into embryonic heads at E13.5, during the early stages of the
formation of heterotopic bone. The dye was placed apically, beyond the dorsal
margin of bone at this stage (see Figure 2). Embryos were allowed to develop exo
utero and examined for the distribution of the dye (Figure 5). At E16.5, the dye was
located almost exclusively in ALP-expressing cells of heterotopic bone. We obtained
substantially similar results in three repetitions of this experiment. In wild type
embryos, in contrast, dye placed in this area became localized in a cell layer flanking
the bone (Figure 5). These results suggest that in Wnt1-Cre; Msx1/2 flox/flox mutants
at E13.5, cells of the early migrating layer of neural crest are allocated to form
regulative bone.

To determine whether neural crest specific inactivation of Msx1/2 caused changes in
osteogenic precursor cell migration from the area of the frontal bone rudiment, we
carried out DiI labeling of the frontal bone rudiment at E13.5 and assessed the
distribution of dye at E15.5 (Figure 6). In control embryos, labeled cells were found
in both the ectocranial layer in which the cells migrate, as well as in the prospective
bone, which is their ultimate fate. No difference in the distribution of labeled cells
was apparent in Wnt1-Cre; Msx1-2 mutants. The results of diI labeling of the early
migrating neural crest and the frontal bone rudiment together suggest that the
regulative frontal bone cells arise from a heterotopic location—the early migrating
neural crest layer.
18

Figure 5. Inactivation of Msx1/2 in neural crest causes a change in the fate
map of early migrating cranial mesenchyme. We injected DiI into heads of
E13.5 control and Msx1/2 flox/flox; Wnt1-Cre embryos exo utero and assessed
the distribution of dye after exo utero development until E16.5. Dye was placed
near the apex, in the area in which the frontal bone will develop in control
embryos and heterotopic bone will develop in Msx1/2flox/flox; Wnt1-Cre
mutants. The placement of dye is illustrated in A and B. Embryos were allowed
to develop to E16.5, and were then sectioned in the indicated plane and
photographed (C-H). In control embryos, dye was distributed in a layer of cells
flanking the prospective bone. Few if any labeled cells were found in the
prospective bone. In mutant embryos, dye was located largely in the developing
bone.
19
Figure 6. Osteogenic precursor cells can migrate apically from the
supraorbital ridge in Msx1/2 flox/flox; Wnt1-Cre mutant embryos. To
assess the apical migration of osteogenic precursor cells, DiI was placed in the
supraorbital ridge of control and mutant embryos as illustrated in A-C. Embryos
were allowed to develop exo utero until E16.5, and were then sectioned in the
indicated plane and photographed. Note labeled precursor cells in the ectocranial
layer as previously described (arrowheads) (Ting et al., 2009; Yoshida et al.,
2008). These cells add to the leading edge of the growing bone in both control
and mutant embryos, suggesting that growth of the frontal bone rudiment is
normal in Msx1/2flox/flox; Wnt1-Cre mutants.

20
Timing of action of Msx genes

DiI labeling suggested that in Wnt1-Cre/Msx1-2 mutants, the early migrating neural
crest cells are mis-allocated to form bone as early as E13.5. Thus Msx genes must be
required for the proper allocation of this cell layer at or before E13.5. To determine
when Msx genes are required to suppress heterotopic bone formation, we used a
Tamoxifen-inducible Cre mouse line (Hayashi and McMahon, 2002). We produced
mice carrying the Cagg-ER transgene together with floxed alleles of Msx1 and Msx2.
We used a dosage of tamoxifen that we determined to be sufficient to cause efficient
recombination within 24 hours of injection (data not shown; see also Hayashi and
McMahon, 2002). We injected tamoxifen into pregnant females at 24 hour intervals
from 9.5 days pc to 12.5 days pc and examined newborns for heterotopic bone in the
area that the frontal bone would normally occupy. We found that ER-Cre; Msx1f/f;
Msx2f/f embryos injected at E9.5 had small areas of heterotopic bone; embryos
injected at E10.5 had substantially larger areas (Fig7B,C). Embryos injected at E11.5
or E12.5 did not have heterotopic bone. Given that complete Cre-mediated
recombination occurs approximately 24 hours after injection of tamoxifen, the
interval during which Msx1/2 function to suppress heterotopic bone formation is
between E10.5 and E11.5. This is the interval during which the early migrating
neural crest migrates apically (Ishii et al., 2003).

21

Figure 7. Timing of Msx1/2 gene action in heterotopic bone formation. We
used a Tamoxifen-inducible (Cagg-Er-Cre) to inactivate Msx1/2 at different
times during development. Tamoxifen was injected IP into pregnant females at
the indicated times post coitum. Pups were taken at the newborn stage and bones
of the skull vault visualized by staining with Alizarin Red S. Note that
Tamoxifen injected at E10.5 caused heterotopic bone (arrows). Tamoxifen
injected at E11.5 and E12.5 resulted in retarded growth of the frontal and parietal
bones, but not heterotopic bone. Thus Msx1/2 are required between E10.5 and
E11.5 to suppress heterotopic bone formation.
22
Dysregulation of Bmp signaling in Msx1-2 mutants

The well-documented role of Bmp signaling in osteogenesis and the known
relationship between Msx genes and the Bmp pathway (Brugger et al., 2004; Chai
and Maxson, 2006; Maxson and Ishii, 2008) prompted us to examine the status of
Bmp signaling during heterotopic bone formation in Wnt1-Cre/Msx1/2 mutants. We
assessed the expression of Bmp2 and Bmp4, both known to be expressed in calvarial
tissues (Kim et al., 1998). In addition we examined the distribution of P-Smad1/5/8,
as an indicator of the sum of canonical Bmp signaling activity (Figure 8). In wild
type embryos at E12.5, Bmp2 was expressed in the meningeal layer and at a low
level in the early migrating mesenchyme. Bmp4 was also expressed in the meningeal
layer and at a much higher level in the early migrating mesenchyme. In Wnt1-Cre;
Msx1/2 flox/flox mutants Bmp2 expression in the early migrating mesenchyme
increased and Bmp4 expression decreased. A similar result was evident at E13.5.
Immunostaining for p-smad1/5/8 at E13.5 showed elevated levels in the early
migrating mesenchyme layer, indicating that the net change in Bmp signaling was
positive. The upregulation of Bmp signaling in Msx mutants in association with the
development of heterotopic bone suggests that the Bmp pathway has a role in this
process.

23
Figure 8. Dysregulated Bmp signaling correlated with heterotopic bone in
Msx1/2 mutants. We examined the expression of ALP (A, B, G, H), Bmp2 (C
and D), Bmp4 (E and F), and P-smad1/5/8 (I, J) in the apical cranial
mesenchyme. ALP was detected by histochemistry, Bmps2 and 4 by in situ
hybridization, P-smad1/5/8 by immunostaining. Note reduced expression of
Bmp2 in the miningeal layer and increased expression of Bmp4 in the
mesenchymal layer in Wnt1-Cre; Msx1/2flox/flox mutants. Note the net increase
in Bmp signaling in the ALP-positive mesenchymal layer as indicated by an
increase in the number of P-smad1/5/8 positive cells. Abbreviations, cm, cranial
mesenchyme, b, brain.

24
Discussion

Here we show that Msx genes, shown previously to be required for calvarial bone
development (Han et al., 2007; Ishii et al., 2003), also suppress bone formation in a
tissue that is normally non-osteogenic. Whereas inactivation of up to three Msx1/2
alleles by means of Wnt1-Cre results in a progressive increase in size of a defect in
the frontal bone, inactivation of the final Msx1/2 allele results in the filling in of the
defect with disorganized bone. Thus the suppression of “ectopic” regulative bone
formation requires a single Msx allele. This bone is derived from neural crest, not
mesoderm, and develops in a population of neural crest cells that migrate prior to the
cells that normally compose the frontal bone. We suggest that this activity may be
one cause of Wormian bones, ectopic bones that are a feature of a variety of
pathological conditions in which calvarial bone development is compromised.
Finally, we demonstrate that increased expression of Bmp2 is associated with the
development of ectopic bone.

Given the well documented function of Msx genes in skull vault development (Chai
and Maxson, 2006; Maxson and Ishii, 2008), the bone suppressive activity was a
surprise. In conventional Msx1/2 knockout mice, the calvarial bones do not form
(Han et al., 2007). Thus Msx1/2 have a positive role in bone formation. Detailed
analysis showed roles in proliferation and differentiation of osteogenic cells. Since
the mispatterned bone does not occur in the frontal bone territory of conventional
Msx1/2 knockouts, its development in Wnt1-Cre Msx1/2 mutants appears to depend
25
on Msx gene function in a non-neural crest cell type. The appearance of the bone in
the posterior of the frontal bone led to the strong prediction that cells normally
allocated to the parietal bone were migrating into the defect, filling it in. However,
Wnt1-Cre mapping showed that the bone is entirely neural crest in origin. Thus
ectopic bone appears to depend on the autonomous function of Msx genes in the
neural crest and a non-autonomous function in an as yet unidentified cell type.

What specifically is the cell of origin of the ectopic bone? Clues came from recent
work on the mechanism of growth of the skull vault. The frontal and parietal bones
grow by end-addition of migratory precursors (Ting et al., 2009; Yoshida et al.,
2008). The bone rudiment grows through a pre-existing layer of mesenchyme. DiI
labeling of cells in this layer showed that they do not become located in bone in
appreciable numbers (Yoshida et al., 2008). Instead, they are found in a layer that
flanks bone on the outside. Intriguingly, DiI labeling of Msx1/2 mutants showed that
cells of this layer are incorporated into bone. Thus cells in the “early migrating”
mesenchyme form ectopic bone. These cells are not likely to come from the frontal
bone rudiments, since diI labeling of rudiments at E13.5 showed no difference
between mutant and wild type in terms of numbers of migratory cells, or migratory
properties of cells. The simplest interpretation of our results is that cells of the early
migrating layer convert to an osteogenic fate, although we stress that definitive proof
of this would require a demonstration of a fate change at the level of individual cells.
We emphasize that our analysis of the fate of the early migrating mesenchymal layer
26
does not preclude a role in the adult. These cells could be, for example, be a source
of adult stem cells for replenishment or regeneration of calvarial bones in the adult.

Mesoderm-derived mesenchyme of the supraorbital ridge is perhaps the most likely
tissue in which Msx genes could function to influence regulative bone growth in the
neural crest. At the onset of their apical migration, early migrating neural crest cells
are flanked by this tissue, providing an opportunity for a signaling interaction.
Moreover, this tissue expresses Bmp2 and Bmp4 which are strong candidates for
growth factors that influence the fate of the crest population. Experiments with the
Cagg-ER tx-inducible Cre provided results consistent with this hypothesis.
Inactivation of Msx1/2 during the interval between E10.5 and E11.5 resulted in
ectopic bone. Mesoderm and crest are in close juxtaposition in the supraorbital ridge
during this interval.

Our data strongly suggest that the Bmp pathway is at least part of the cause of the
ectopic bone. Analysis of Bmp2 and Bmp4 expression showed an upregulation of
Bmp2 in the early migrating neural crest at E12.5, prior to the detection of ectopic
ALP. An increase in the number of P-smad 1/5/8 positive cells became evident by
E13.5. These findings suggest that Bmp signaling may have a role in causing the
differentiation of early migrating mesenchyme into osteoblasts.

Ectopic bone formation driven by Bmp signaling is also observed in fibrodysplasia
ossificans progressiva, a disorder characterized by congenital great toe
27
malformations and progressive heterotopic ossification in which skeletal muscles and
connective tissues are transformed into bone (Billings et al., 2008). This disorder is
associated with a specific mutation in ACVR1, which encodes a bone morphogenetic
protein type I receptor. The ectopic skeleton is derived largely from cells of vascular
origin (Shore et al., 2006.

The ectopic bone in Msx1/2 mutants has the appearance of Wormian bone, which is a
disorganized bone that appears in the area of prospective sutures, particularly when
bone growth is abnormally slow (Sanchez-Lara et al., 2007). Such slow growth can
be the result of an unusually large head, or of genetic defects that result in
compromised bone growth. Among these defects are osteogenesis imperfecta and
cleidocranial dysplasia (Rauch and Glorieux, 2004; Ziros et al., 2008). Wormian
bone is thus in a sense a regulative response to a deficiency in bone growth, a
circumstance similar to the one caused by neural crest-specific inactivation of Msx1
and Msx2. The tissue of origin of Wormian bone has not been investigated.

The relationship between the Wormian bone and deficiencies in cranial bone growth
implies communication between the growing bone and the tissue from which the
Wormian bone arises, presumably early migrating cranial mesenchyme. For example,
in the absence of a signal indicating normal bone growth, the mesenchyme would
convert to bone. In the presence of the signal, the mesenchyme would remain
undifferentiated. The Msx-dependent signaling that maintains the early migrating
mesenchyme in an undifferentiated state could be part of such a mechanism. We
28
suggest that a deficiency in bone growth suppresses Msx expression, and thus causes
the early migrating mesenchyme to differentiate along an osteogenic pathway. It will
be interesting to assess Msx gene expression and function in genetic models in which
Wormian bones are a feature.

Finally, we are intrigued by the potential evolutionary implications of the Msx-
dependent program we have uncovered. Our results, together with recent DiI cell
marking experiments; (Ting et al., 2009; Yoshida et al., 2008), document two distinct
osteogenic programs in skull vault development. One consists of the patterned
growth of the calvarial rudiments by end addition of migratory osteogenic precursor
cells. The other consists of the differentiation of the early migrating mesenchyme
along an osteogenic pathway. The former appears to be the primary mechanism by
which the frontal and parietal bones grow. The latter mechanism, we suggest, may be
an evolutionary remnant of a program present in basal vertebrates with a mode of
calvarial bone growth distinct from that of mammals. Skulls of fish typically have
many loosely connected elements; those of mammals are more tightly connected and
have fewer elements (de Beer, 1985; Romer, 1997). These changes in skull structure
must be a result of changes in the developmental program underlying skull growth.
One scenario is that the osteogenic program in the early migrating mesenchyme was
the primitive condition, accounting for the multiplicity of loosely patterned skull
bones in early vertebrates. The migratory program then arose secondarily. The loss
of bones would then be a result of suppression of the primitive program—e.g., by
acquisition of Msx-dependent repression of osteogenesis—in specific regions,
29
accompanied by an expansion of the growth by end addition. Analysis of cranial
development in extant agnathans may shed light on this scenario.

30
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Abstract (if available)

Abstract In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest and other cell populations potentially relevant to skull development. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx alleles, the large defect is filled in with mispatterned bone. This bone also partially obliterates the coronal sutures, thus producing craniosynostosis. The bone is derived from neural crest, not mesoderm, and originates in a normally non-osteogenic layer of cells through with the rudiment grows. Associated with the heterotopic osteogeneis is an upregulation of Bmp signaling in this cell layer. These results suggest that Msx genes have a dual role in calvarial development. They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised.

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Asset Metadata

Creator Roybal, Paul G. (author)

Core Title Tissue-specific action of Msx genes in the regulation of skull vault development

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 12/08/2009

Defense Date 07/01/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag conditional knockout,embryo,mouse,Msx1,Msx2,OAI-PMH Harvest,Skull,Wnt1-Cre

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Maxson, Robert E. (committee chair), Chai, Yang (committee member), Chuong, Cheng-Ming (committee member)

Creator Email maxson@usc.edu,Robert.maxson@med.usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2790

Unique identifier UC1154335

Identifier etd-Roybal-3119 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-301071 (legacy record id),usctheses-m2790 (legacy record id)

Legacy Identifier etd-Roybal-3119-0.pdf

Dmrecord 301071

Document Type Dissertation

Rights Roybal, Paul G.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu