what-when-how
In Depth Tutorials and Information
In
Runx2/Cbfa1
null mice, osteoblast differentiation is
arrested in both the endochondral and intramembra-
nous skeleton.
16,17
In addition,
Runx2/Cbfa1
also has a
positive role in the differentiation of hypertrophic chon-
drocytes, a property that should prime the process by
which the cartilaginous skeleton is replaced by bone.
18-20
Another polypeptide, Indian hedgehog (Ihh), a member
of the Hedgehog family of secreted proteins, is required
for endochondral but not for intramembranous ossifica-
tion.
21
It has been demonstrated that Ihh is needed for
the establishment of the osteogenic portion of the peri-
chondrium/periosteum acting upstream of
Runx2/Cbfa1
.
This and additional roles of Ihh coordinate several criti-
cal events in endochondral bone formation.
10
A zinc finger-containing transcription factor called
Osterix (Osx) is expressed in osteoblasts of all endochon-
dral and membranous bones. In
Osx
null mutant mice,
no endochondral and no intramembranous bone forma-
tion occur. In addition, arrest in osteoblast differentiation
occurs at a later step than in
Runx2/Cbfa1
null mice.
In the
Osx
null mice, Nakashima et al.
10
demon-
strated that Osx is essential for osteoblast differentiation.
This transcription factor is required for osteoblast differ-
entiation and for bone formation. In the absence of Osx,
no cortical bone and no bone trabeculae were formed
through either intramembranous or endochondral ossi-
fication. In endochondral skeletal elements of
Osx
null
mutants, mesenchymal cells from the perichondrium/
periosteum, together with blood vessels, invaded the
mineralized matrix of the zone of hypertrophic chon-
drocytes. Moreover, the presence of multinucleated
osteoclasts in the mineralized cartilage matrix of
Osx
null mutants and degradation of this matrix indicated
that osteoclast differentiation and active cartilage matrix
degradation were taking place. However, mesenchymal
cells migrating together with osteoclasts and blood ves-
sels could not deposit bone matrix, so no ossification
could occur in the matrix of this mesenchyme. Similarly,
no ossification took place in the condensed mesenchyme
of membranous skeletal elements. The degraded miner-
alized cartilage matrix and the absence of bone matrix
accounted for the severe bending of long bones. In con-
trast to the absence of bone matrix, the cellular organiza-
tion of the cartilage growth plate was normal in
Osx
null
mutants except for the disordered organization of the
deep layers of the zone of hypertrophic chondrocytes.
Furthermore, Osx appeared to have no role in pattern-
ing of the skeleton. Indeed, at E12.5, a time when many
of the patterning decisions of the embryonic skeleton
have been made,
Osx
null embryos were indistinguish-
able from wild-type embryos. In addition, in newborn
mutants, densely packed mesenchymal cells in mem-
branous skeletal elements, unable to differentiate into
osteoblasts, occupied the same space as that occupied by
osteoblasts in wild-type embryos.
10
THE S
P7 PROTEIN AND THE
SP7
GENE
OSX is a 431 amino acid protein that belongs to the
specificity protein (Sp) subgroup of the Krüppel-like fam-
ily of transcription factors characterized by the invariable
presence of three tandem Cys2-His2 zinc-finger DNA-
binding domains at their carboxy-terminus.
22-24
Based on
crystallography and nuclear magnetic resonance studies
performed in some members of the family, it has been
established that each protein inger recognizes a consecu-
tive trinucleotide sequence, with the three zinc-fingers
contributing to the strength and specificity of the DNA-
protein interaction.
25-27
SP1, which is the best character-
ized member of the Sp group and closely related to Sp7,
binds GC-rich DNA boxes of nine base pairs. Previous
experimental studies including DNA-binding assays
using Sp1 peptides (530-623 amino acids) in which either
the N-terminal (1) or the C-terminal (3) zinc-fingers were
deleted demonstrated a greater contribution of inger 3
to the DNA-binding affinity than the other two remain-
ing ingers.
28,29
The p.E351Gfs19X change in the patient
we recently described
9
(see below) leads to a truncated
SP7 protein that lacks the last 81 amino acids including
the third zinc-finger domain and carries 18 new residues
downstream of codon 351 instead (
Figure 18.1
) Thus, in
the case that the OSX mutant variant is not degraded by
the proteasome, its DNA-binding properties are expected
to be altered and consequently the SP7/Osterix medi-
ated transcription regulation is impaired. Other genetic
anomalies in zinc-fingers have been reported in human
diseases. Thus, mutations in the third zinc-finger of EGR2,
a very similar Cys2-His2 transcription factor, have been
described in patients with autosomal recessive congeni-
tal hypomyelinating neuropathy (MIM 605253) proving
that mutations in the third zinc-finger can clearly lead to
disease.
30
OSX
MOUSE MODEL
The murine Osx protein is comprised of 428 amino
acids yielding a molecular weight of 46 kDa and con-
tains a zinc-finger motif of 85 amino acids. This motif
serves as the DNA-binding motif, which recognizes
GC and GT boxes. This DNA interacting motif displays
high sequence homology with other members of the
SP
family of transcription factors. Analysis of
Osx
mouse
models has revealed that Osx plays an essential role in
regulating the maturation of osteoblasts in a step down-
stream of
Runx2
,
10
another transcription factor that is a
master regulator for osteoblast differentiation, and that
when mutated is responsible for cleidocranial dyspla-
sia in humans (MIM: 119600).
31
While
Runx2
null mice
do not express
Osx
,
Runx2
expression was shown to be
unaffected in the developing bones of
Osx
−/−
mutants.
10
