University of Southern California Dissertations and Theses

Genetic interaction between androgen receptor and Lef1 in bone mass control

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Genetic interaction between androgen receptor and Lef1 in bone mass control

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

August 2007
Copyright 2007 Archana V. Tank

GENETIC INTERACTION BETWEEN ANDROGEN RECEPTOR AND LEF1 IN
BONE MASS CONTROL

by

Archana V. Tank

ii

Dedication

Lead me from the unreal to the real, from ignorance like darkness to knowledge
like light. Lead me from death to eternal life. May there be peace everywhere.
-Rig Veda

iii

Acknowledgments

USC’s master’s degree has given me a new perception of how I look towards
science and what real world science is. Performing science and understanding what
science is what I learned in my mentor Dr. Baruch Frenkel. I am thankful to him for the
opportunity he gave me to work in his lab. He encouraged me as a mentor when many
time my own mistakes left me agitated with myself to learn from them and move ahead.
Various experiences of these two years including cloning experiments and micro
CT usage have helped me understand that science is about resilience, patience and
precision. I am thankful to fellow lab members Nathalie Leclerc and Unnati Jariwala
who trained me with lab techniques that I was totally inexperienced with. Using micro
CT machine was truly a nice experience, which is added as an asset to my experience. I
am thankful to Jon Cogan for teaching me the micro CT usage very well. And project
mate Tommy Noh for explaining me the intricacies of the project and helping in thesis
writing. I am thankful to other lab members Artem, Steve, Paul and Sanjeev for various
technical helps and making my overall laboratory working experience memorable.
At the end I would like to express my gratitude towards my committee members
Dr. Zoltan Tokes and Dr. Joseph Hacia for their guidance and time.
Lastly I am thankful to my family and friends who have been a constant support
during my academic endeavors.

iv
Table of Contents
Dedication i
Acknowledgments ii
List of Tables vi
List of Figures vii
Abstract viii

Chapter 1: Introduction
1.1 Wnt Signaling 1
1.1.1 Wnt proteins and receptors 3
1.1.2 Mechanism of Wnt signaling 4
1.2 Wnt Signaling and bone mass 7
1.2.1 Mechanism of Action 9
1.3 Wnt signaling and sex steroids 12
1.4 Sex steroid actions in skeletal development 13
1.4.1 Effects of estrogen and progesterone 14
1.4.2 Effects of androgens 15
1.5 Role of androgen receptor in bone biology 17
1.6 Tfm- Testicular feminization mutation 19
1.7 Lef1 and low bone mass phenotype in female mice 20
1.8 Hypothesis 21
1.9 Aim 21

Chapter 2: Methods and Materials
2.1 Genetic setup and breeding scheme of mice 22
2.1.2 Specimen preparation 23
2.2 Micro computed tomography scanner 24
2.3 Data acquisition and scanning procedure 26
2.3.1 Scanner geometry calibration 26
2.3.2 Center offset calibration 27
2.3.3 Camera setup 27
2.3.4 Radiograph 28
2.3.5 CT Scan 29
2.4 MicroCAT file reconstruction 29
2.5 MicroCAT data analysis 30

v
2.5.1 Loading data 32
2.5.2 Cropping 32
2.6 Cortical Analysis 33
2.6.1 Cortical measurements 36
2.7 Trabecular Analysis 36
2.7.1 Trabecular measurements 37
2.8 Bone Length measurement 39
2.9 Statistical test 39

Chapter 3: Results
3.1 XY Lef1
+/+
vs. XY Lef1
+/-
40
3.2 X
T
Y Lef1
+/+
vs. X
T
Y Lef1
+/-
42
3.3 XX Lef1
+/+
vs. XX Lef1
+/-
44
3.4 X
T
X Lef1
+/+
vs. X
T
X Lef1
+/-
46
3.5 Measurement characteristics and quality control 49

Chapter 4: Discussion
4.1 Lef1 haploinsufficiency effects on bone mass 51
4.2 Lef1 haploinsufficiency effect being sex specific 52
4.3 Bone loss in female mice due to Lef1 53
4.4 Bone loss in male mice due to Lef1 53
4.5 Possible factors causing variations in bone mass 54
4.5.1 Genetic background of strains 54
4.5.2 Effects on other parts 55
4.6 Future directions 55

References 57

vi

List of Tables

Table 1 XX Lef1
+/+
vs. XX Lef1
+/-
41

Table 2 X
T
X Lef1
+/+
vs. X
T
X Lef1
+/-
43

Table 3 XY Lef1
+/+
vs. XY Lef1
+/-
45

Table 4 X
T
Y Lef1
+/+
vs. X
T
YLef1
+/-
47

Table 5 Variation in repeated analysis of specimen 50

vii
List of Figures

Fig. 1: Canonical Wnt signaling pathway 2

Fig. 2: Nuclear factors in Wnt signaling 6

Fig. 3: A- Role Wnt pathway molecules in bone development 8
B- Scheme of skeletal actions of sex hormones 18

Fig. 4: Genetic breeding scheme of the mice with Tfm mutation 23

Fig. 5: A- Screenshot images of data acquisition software and 25
camera and radiograph setup panel.
B- Radiograph image of specimen femur 25

Fig 6: A- Screenshot image of CT setup panel 31
B- Raw data parameter window screenshot 31
C- Reconstruction software COBRA 32

Fig 7: A, B: Voltex reconstruction of cortical region and highlighting. 35

C, D: Isosurface reconstruction of trabecular and highlighting of
slices 35

viii
Abstract

Canonical Wnt signaling plays a major role in bone development. The
transcription factors TCF/LEF are activated in the nucleus in response to Wnt signaling.
Preliminary results from our lab have shown that Lef1 heterozygous Lef1 female mice
have low bone volume w as compared to wild type females, whereas Lef1 heterozygous
males have normal bone volume. Because sex steroids have significant role in bone
development, we were interested to know if androgens protected the male mice from
bone loss that otherwise occurs as a result of Lef1 haploinsufficiency.
To understand if the androgen signaling actually played a role in protecting Lef1
haploinsufficient males we used Tfm mutant male mice, which have aberrant androgen
receptor. Lef1 haploinsufficient male mice were bred with female mice having the Tfm
mutation. Bone volume density was assessed in F1 generation male and female mice
with Tfm mutation and Lef1 heterozygosity. Using micro computed tomography (µCT),
we imaged the distal femurs of 9-20 bones for each of the eight genotypes, and
calculated both cortical and trabecular bone parameters.
We observed that our results were consistent with the preliminary data that the
bone loss was specific and to female mice. Male mice with Tfm mutation showed bone
loss compared to male mice that don’t have Tfm mutation. Most importantly,
comparison of Lef1+/- with Lef1+/+ male mice on a Tfm mutant background showed
bone loss as a result of Lef1 haploinsufficiency, although the p value was only
marginally significant (p<0.059). Hence we conclude that there is an interaction
between Lef1 and androgen signaling in bone development.

1
Chapter 1: Introduction
1.1 Wnt Signaling:

Cells respond to their microenvironment. Cell signaling being a part of
intercellular communication, regulates the cell activities and coordinates various
physiological processes. Various molecules that act as signaling molecule families are
the Wnts, the bone morphogenetic proteins (BMPs), the Hedgehogs, the fibroblast
growth factors (FGFs). The general pattern of this mechanism is receptor activation
upon ligand binding(Logan and Nusse 2004).
The Wnt family consists of glycoproteins of 19 kinds, which act on specific
locations in the cell and have singular physiological implications. The usual mechanism
of Wnt signaling, which is through its receptors Fzd (Frizzled) and β-catenin is known
as canonical signaling pathway. Unlike this, mechanisms wherein the Frizzled
receptors and β-catenin are not involved are called noncannonical signaling pathway
(Wodarz and Nusse 1998).
Fig.1 shows a scheme of the Wnt signaling pathway. As shown in the figure
Wnt proteins bind to specific cell surface receptor complexes. The receptors are of two
kinds one belongs to Frizzled (Fzd) family and the other to low density lipoprotein
receptor –related protein (LRP) family(Songyang, Fanning et al. 1997). These receptor
molecules further act upon various molecules like Dishevelled (Dsh) (Klingensmith,
Nusse et al. 1994), glycogen synthase kinase-3β (GSK-3β) (Dominguez, Itoh et al.

2
1995), Axin, Adenomatous Polyposis Coli and β-catenin. β-catenin further regulates
transcription factors such as Lymphoid enhancer factor (LEF), T-cell factor to affect
gene transcription.(Behrens, von Kries et al. 1996)

Fig 1.
Canonical Wnt signaling pathway: In cells devoid of Wnt signal (left panel), β-
catenin is degraded through interactions with Axin, APC, and the protein kinase GSK-
3. Wnt proteins (right panel) bind to the Frizzled/LRP receptor complex at the cell
surface. These receptors transduce a signal to Dishevelled (Dsh) and to Axin, which
may directly interact (dashed lines). As a consequence, the degradation of β-catenin is
inhibited, and this protein accumulates in the cytoplasm and nucleus. β-catenin then
interacts with TCF to control transcription. (Logan 2004)

3

Evolutionary origins of Wnts are traced with the help of genome work show a
catalogue of 19 genes in humans & mice, 7 in Drosophila and 5 in C. elegans. Between
Drosophila and mammals, there is a fairly extensive conservation of Wnt genes.
(Danielian and McMahon 1996)
These signaling pathways play a significant role in diseases and developmental
defects. Altered progress in the signaling pathway (LRP-5 defect) causes errors in
somite formation and chondrogenesis (Boyden, Mao et al. 2002). Rare human genetic
disease Tetra-amelia has been proposed to develop due to loss-of-function mutation in
WNT3(Niemann, Zhao et al. 2004). FZD4 mutation is responsible for causing retinal
angiogenesis defects (Robitaille, MacDonald et al. 2002). Similarly, Axin2 and APC
are responsible for causing colorectal and colon cancer respectively (Kinzler, Nilbert et
al. 1991; Lammi, Arte et al. 2004).
1.1.1 Wnt Proteins & Receptors:
The Wnt proteins consist of a highly conserved cysteine distribution. The Wnt
proteins tend to get palmitoylated and are hydrophobic. The process of palmitoylation
is found on the cysteine domains, which makes them hydrophobic. Mutation analysis
shows that the cysteines are essential for the function. Treating the Wnt proteins with
the enzyme acyl protein thioesterase results in a form that is no longer hydrophobic or
active, providing further evidence that palmitoylation is critical for signaling (Willert,
Brown et al. 2003).

4
It has been proven that Fzd proteins are the primary receptors for the Wnts. Fzd
is a seven-transmembrane receptor with a long N-terminal extension called cysteine-
rich domain (CRD). Wnt proteins fit into the Fzd CRD (Bhanot, Brink et al. 1996).
Moreover, it is proven that Wnt ligand determines the activation of the Fzd receptor in
canonical signaling (Bhanot, Fish et al. 1999).
LRP(low density lipoprotein receptor-related protein), which acts as a co-
receptor to Wnt proteins like Fzd; gets phosphorylated following the Wnt signals
(Tamai, Semenov et al. 2000). On phosphorylation due to Wnt binding, LRP interacts
with Axin in the cytoplasm, as Axin docks on the LRP cytoplasmic tail to further down
signal (Mao, Wang et al. 2001; Tolwinski, Wehrli et al. 2003).
A protein, Norrin has been reported to be binding to Fzd receptor although it is
not structurally similar to Wnt proteins(Xu, Wang et al. 2004). Similarly, Derailed has
been recently reported to act as a Wnt receptor. It is a transmembrane tyrosine kinase
belonging to RYK subfamily (Yoshikawa, McKinnon et al. 2003).
1.1.2 Mechanism of Wnt Signaling:
Upon binding, Wnt proteins induce structural changes in transmembrane
domains of Fzd. Consequently Dishevelled (Dsh) protein is activated as shown in Fig1.
Dsh interacts with Fzd at C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif
(Umbhauer, Djiane et al. 2000). These receptors transduce signal to several
intracellular proteins Axin, APC (Adenomatous Polyposis coli).
Secreted frizzled related proteins (sFRPs) are negative modulators of Wnt
pathway. sFRPs contain a cysteine rich domain, which interacts with Wnt proteins,

5
preventing it from binding to Frizzled and/or co-receptor LRP5/6 (Hoang, Moos et al.
1996).
In the cytoplasm as shown in Fig1, the degradation pathway of β-catenin is
inhibited and the β-catenin level is regulated by the down-signaling due to Wnt
binding. β-catenin phosphorylation is activated by the absence of Wnt signaling, which
causes β-catenin degradation. β-catenin on further accumulation plays role in nuclear
transcription regulation .
In the nucleus, β-catenin is known to interact with Tcf1 (T-cell factor-1) and
Lef1 (lymphoid enhancer-binding factor1) in the nucleus (Molenaar, van de Wetering
et al. 1996). In the nucleus β-catenin is thought to convert the TCF repressor complex
into a transcriptional activator complex. This may occur through displacement of
Groucho from Tcf1 /Lef1 (Cavallo, Rubenstein et al. 1997).
As shown in the figure 2., inside the nucleus β-catenin drives interferes with
the TCF groucho complex. The separation of groucho from the TCF complex activates
it for the transcription. Further the ability of Tcf1/Lef1 to interact with DNA is
regulated by β-catenin and plays a critical role in modulation of Wnt target gene
expression (Lo, Gay et al. 2004).

6

Fig 2.
Nuclear factors in Wnt signaling: The interaction between Groucho and TCF is
thought to down-regulate transcriptional activation (left panel). β-catenin is also
negatively regulated on binding to Chibby. TCF activity in the nucleus can be
modulated by phosphorylation by Nemo-like kinase (NLK), and in C. elegans, a 14-3-
3-like protein has been shown to facilitate nuclear export of TCF (thinarrow). β-catenin
interferes with the interaction between TCF and Groucho,, and together with TCF,
activates gene expression. β-catenin also binds to other components such as
Legless(Lgs), Pygopus (Pygo), CREB-binding protein (CBP), and Brg1. Negative
regulators areshown in black. Positively acting components are outlined in gray color.

7
1.2 Wnt Signaling & Bone Mass:
The two types of cells that play a role in the bone physiology are osteoblasts,
which are bone forming cells and osteoclasts, which are bone resorbing cells. The
osteoblasts arise from mesenchymal cells of the bone marrow, and osteoclasts from the
hematopoietic precursors. Together they work in the process of bone remodeling
wherein there are structural and functional modifications like replacing old bones with
new and repairing the damaged ones. In the remodeling process the osteoclasts are
employed which remove the mineralized matrix. The complete physiological process
takes about 6 months(Owen 1980; Owen and Friedenstein 1988).
The Wnt signaling pathway is proven to play a significant role in bone mass in
vivo (Karsenty and Wagner 2002; Logan and Nusse 2004). Various experimental
evidences of null mutations of the proteins required for canonical Wnt Signaling have
shown significant alterations in bone development, which we shall discuss further in
section1.2.1. Simultaneously the dysfunction of Wnt pathway antagonists have shown
increased bone mass (Bodine, Billiard et al. 2005). However, the pathway mechanisms
affecting the bone development and regulation functions are not well understood.
The Wnt signaling pathway is linked with the transcription factors
Runx2/Cbfa1/AML3, which are essential components of osteoblast differentiation
(Kobayashi and Kronenberg 2005; Komori 2005). Embryonic lethality of the β-catenin
null mice exhibit lack of skeletal structures derived from cranial neural crest and arrest
of osteoblast differentiation (Haegel, Larue et al. 1995). Thus demonstrating the

8
importance of canonical Wnt pathway during early developmental stages and bone
formation.

Fig 3-A: Role of Wnt pathway molecules in bone development:
Wnt 10b plays significant role in driving mesenchymal precursor, and β-catenin plays
significant role in pre-osteoblast formation

9
1.2.1 Mechanism of action:
Various molecular components of the pathway have different roles to play in
the bone development. And their roles are better understood with the help of mutation
studies in the mice.
Wnts: The pathway ligand Wnt, is important for the signaling to progress,
however the actual characteristics of the Wnts that are involved in the bone related
signaling is not understood. Wnts are expressed in osteoblasts and the level is up
regulated during differentiation process (Hu, Hilton et al. 2005). As shown in Fig.3,
Wnt1 and Wnt10 play a role in the formation of precursors like osteoblasts,
chondrocytes, monocytes and adipocytes from mesenchymal cells, and suppress
adipogenesis in preadipocyte cells(Ross, Hemati et al. 2000). Mice with mutated
Wnt10b show altered bone mass and decreased level of trabeculae(Bennett, Longo et
al. 2005).
LRP: LRP5 acts as co receptor for Wnt proteins, similar to Fzd. A mutation in
the Wnt receptor LRP5 results in a high bone mass trait in humans (Boyden, Mao et al.
2002; Little, Carulli et al. 2002). LRP-5 loss of function mutant in the mice model
exhibits osteopenia. In the mouse model with loss of Lrp5 there is osteoporosis and
blindness syndrome. In situ hybridization has revealed the presence of Lrp5 in
osteoblast lineage cells.(Gong, Slee et al. 2001; Kato, Patel et al. 2002; Little, Carulli et
al. 2002) In the case of gain of function of LRP-5 there is higher bone mass phenotype
in humans, change in single amino acid glycine to valine causes this phenotype.
However, with the mutation in LRP-5 there is no change in the osteoclast parameters.

10
LRP5-/- mice show decreased number of osteoblasts and bone formation rate and
osteoblast proliferation rate without any signs of apoptosis. (Kato, Patel et al. 2002)
Contrary to LRP-5, LRP-6 mutations have not shown apparent effects in
humans. However, LRP-6 -/- generated by mutation, die at birth (Pinson, Brennan et al.
2000). The LRP-6 hypomorphic mutants exhibit functional defects in adult mice. The
mice show digit and neural tube defects and axial skeletal defects, with delays in
ossification centers during embryogenesis and decreased bone mass, which suggests
that LRP6 could be more vital receptor of Wnt than LRP5(Kato, Patel et al. 2002;
Kokubu, Heinzmann et al. 2004).
β β β β-catenin: β-catenin is an adhesive molecule in cells as it interacts with E-
cadherin and α-catenin (Hirohashi and Kanai 2003). β-catenin plays a role in Wnt
signaling by entering in the nucleus and heterodimerizing with Lef/Tcf transcription
factors and regulating Wnt target genes. Inactivation mutation of β-catenin drives
osteoblast differentiation pathway to ectopic chondrocytes formation in mice. In mature
bones, β-catenin reduces osteoclast differentiation and/or function, whereas the
osteoblast function and number remains unchanged. This results with the increase in
osteoprotegerin (Opg) level, which inhibits osteoclast differentiation (Simonet, Lacey
et al. 1997; Glass, Bialek et al. 2005). Contrary to this when β-catenin was inactivated
in osteoblasts, the mice showed decrease in bone mass due to increase in osteoclasts
resulting in bone resorption.
A mouse18.5 days old with a conditional knockout of β-catenin, exhibits lack
of bones, though has cartilage. Likewise, the role of β-catenin was studied during

11
postnatal murine bone acquisition by conditionally deleting β-catenin and observations
show that trabecular and cortical bone volume was reduced. Osteopenia is correlated with
a decrease in osteoblast differentiation and matrix mineralization, as well as an increase
in osteoclast differentiation that resulted from down-regulation of osteoblastic
osteoprotegerin (OPG) expression and up-regulation of receptor activated by nuclear
factor-κB ligand (RANKL) expression (Holmen, Zylstra et al. 2005).
Similarly, GSK3-β deficient mice are embryonically lethal at E13.5-14.5 due to
defective NF- κB signaling and hepatocyte apoptosis (Hoeflich, Luo et al. 2000). With
the help of inhibitor of GSK3-β, which is lithium, osteopenia due to LRP5 -/- is
rescued(Clement-Lacroix, Ai et al. 2005).
Lef/Tcf: Wnt signaling regulates the functioning of the Lef/Tcf transcription
factor action. Expression pattern shows that Tcf1 and Tcf4 are expressed in adult
primary osteoblast cultures (Glass, Bialek et al. 2005). Mice deficient of this
transcription factors were shown to have increase in osteoclast numbers without
rendering any changes to osteoblast number or functioning (Glass and Karsenty 2006).
Tcf1 and β-catenin mutant genotyped mice have a low bone mass not seen in either of
the singly heterozygous with an increased number of osteoclasts and decreased
expression of Opg (Glass, Bialek et al. 2005).
Wnt pathway antagonists: Axin-2, is an intracellular inhibitor of canonical
Wnt signaling. In mice targeted disruption of Axin2, is shown to cause increased
osteoblast differentiation and matrix mineralization. The Axin-2 -/- in mice shows

12
increase in the β-catenin level and its loss causes increase in cell proliferation and
differentiation (Yu, Jerchow et al. 2005).
Dkk-2 is an extracellular antagonist of LRP-5 & LRP-6, with the deletion of
Dkk-2 it contrary to expectations of increased bone formation caused, osteopenia in
mice. Although canonical Wnt signaling is elevated, the loss of Dkk-2 causes delays in
matrix mineralization (Li, Liu et al. 2005).
Sfrps- secreted frizzled recptor proteins are a class of Wnt antagonists. Sfrp1 is
highly expressed during the transition from preosteoblast to osteoblast (Bodine, Billiard
et al. 2005). Sfrps -/- mice show increase in trabecular bone level and cortical bone
volume. Moreover, the knockout mice have shortened growth plates and increased
calcification of the hypertrophic zone of chondrocytes, indicating increased
chondrocytes differentiation and endochondral ossification. Hence molecule serves as
the negative regulator of osteogenesis and chondrogenesis (Bodine, Billiard et al. 2005;
Gaur, Lengner et al. 2005; Gaur, Rich et al. 2006).
Similar to the above-described molecules, there are other molecules like Ror2,
SOST, OG2 play a role in bone development and functioning.

1.3 Wnt signaling and sex steroids:
There exists correlation between sex steroids and Wnt signaling. β-catenin
which is the mediator of the pathway interacts with the androgen receptor. It has been
shown that β-catenin tends to localize in the nucleus with the help of
immunolocalization techniques. The β-catenin levels are well regulated because of the

13
phosphorylation of GSK-3β and proteosomal degradation. Further it translocates in the
nucleus and via Tcf/Lef regulates transcription of the targeted genes. Ligand bound
androgen receptor enhances the transcriptional activity related to β-catenin /TCF. It has
been shown using truncated AR and mutant with nuclear translocation defect
(R617K618, 632, 633M) that neither receptors nor catenin was translocated to the
nucleus. The carboxyl terminus of the AR is required for the translocation of β-catenin
to the nucleus. It is also shown that unregulated nuclear accumulation of β-catenin
leads to transformed phenotype by up regulating the transcription of responsive genes.
Complete nuclear translocation of β-catenin occurs only when the AR is ligand bound
and in a correct conformation.(Pawlowski, Ertel et al. 2002) Further impacts due to AR
binding on TCF/LEF transcription factors are not understood.

1.4 Sex steroid action in skeletal development:
Bone is metabolically, a highly active tissue, a loss of bone homoeostasis may
result in a decrease in bone mass and deterioration of the microarchitecture of the
skeleton (osteoporosis), or a defect in the mineralization of bone, as seen in
osteomalacia. Clinical studies reveal, in vitro and in vivo suggest that steroid hormones
regulate normal bone development and the maintenance of intact bone. Sex steroids,
estrogen and androgens play a significant role in bone modeling. Mutations in estrogen
receptors, aromatase, and androgen receptor have provided knowledge about the in vivo
mechanism of bone development (Bland 2000).

14
The actions of the steroid hormones, retinoids, vitamin D and thyroid hormones
are mediated by the ligand binding to its homologous nuclear receptors, which act on
ligand-dependent transcription factors to either activate or repress target gene
expression (Evans 1988).
It was a traditional view that in male and female bodies the testosterone and the
estrogen play a parallel role in the bone formation, contrary to this it was shown that in
male the deletion of ER-α had unfused epiphyses regardless of the testosterone level.
Clinical studies suggest that combined therapy of androgens and estrogens may
enhance bone mineral density and bone mass to a more significant therapy than
estrogen therapy in women in postmenopausal women (Christiansen, Christensen et al.
1980).Both estrogens and androgens act on the bone marrow stromal / osteoblast cells
to prevent the production of local factors that induce osteoclast development. The
mechanism by which the sex steroids act is through the receptor mediated regulation of
interleukin-6 (IL-6), which is an osteoclastogenic cytokine (Syed F 2005 Mar 18).

1.4.1 Effects of estrogen and progesterone:
Estrogens play a significant role in bone remodeling. Women in
postmenopausal state that have loss of estrogen tend to suffer from osteoporosis.
Estrogens are thought to be diffusing passively in the cells, and are
preferentially retained in the target cells by forming complexes with the intracellular
receptors. The lower number of the hormone specific receptor in the osteoblast cells

15
indicates more indirect effect in the physiological process (Higgins, Rousseau et al.
1973).
Estrogens are reported to have stimulatory effects on both osteoblasts and
osteoclasts. 17-β-estradiol is reported to have effects of bone formation and resorption
(Ernst 1989; Qu 1998). Estrogen stimulates the mRNA expression and/or activity of a
number of osteoblast marker genes, including collagen type I, osteocalcin, osteopontin,
osteonectin and AP. (Ernst, Schmid et al. 1988; Robinson, Harris et al. 1997; Qu,
Perala-Heape et al. 1998). This change in osteoblast marker causes differentiation in
osteoblast cells, increase in deposition and mineralisation of matrix.
The bone remodeling is regulated by basic multicellular units (BMUs) which
are temporary anatomic units comprising of osteoclasts in front and osteoblasts in the
rear. Loss of estrogen triggers the osteoclastic precursors in the marrow.
The osteoblasts have progesterone receptors present in two receptor forms A
and B, which have various impacts on bone mass (Eriksen, Colvard et al. 1988).

1.4.2 Effects of androgens:
Androgens play important role in bone development and homeostasis. Although
testosterone is the major circulating androgen, there is evidence that its skeletal effects
are at least partially

mediated by metabolites produced by enzymes present in bone.
Effects of androgens on osteoblastic cells have been demonstrated in animals
and humans. Stimulation of proliferation

and differentiation has

been reported
(Kasperk, Wergedal et al. 1989). In vivo animal studies have shown that androgens

16
promote chondrocyte maturation, metaphysial ossification, and the growth

of long
bones; this contrasts with the effect of estrogens that

promote epiphysial closure and
hence reduce longitudinal growth (ES. 1996).In growing male rats and mice, castration
is associated

with a reduction in cortical and cancellous bone mass(Hock, Gera et al.
1988; Ornoy, Giron et al. 1994; Vanderschueren, Jans et al. 1994), probably due to an
increase in bone turnover and in osteoclastic

activity.
The mechanisms by which androgen depletion and repletion affect the human
skeleton have been less understood. Studies in men

undergoing orchidectomy or
rendered hypogonadal by administration

of gonadotrophin releasing hormone analogs
have shown rapid

bone loss.
There is also evidence that androgens play an important role in the female
skeleton (Slemenda, Hui et al. 1987; Vanderschueren, Jans et al. 1994). Thus in
females affected

by the androgen insensitivity syndrome, there is resistance to

androgens, and endogenous estrogen production is also reduced. Markers of

bone
resorption and formation, indicate increased bone turnover (Goldray, Weisman et al.
1993).
The presence of aromatase(Nawata, Tanaka et al. 1995), which converts

testosterone to estradiol and androstenedione and dehydroepiandrosterone

(DHEA) to
estrone, and 5 -reductase (Vittek, Altman et al. 1974; Schweikert, Rulf et al. 1980),
which reduces

testosterone to androstenedione and DHT, has been reported in

bone.
The female mice with aromatase knockout have been shown to have higher
bone turnover. Both male and female mice were shown to have reduced trabecular

17
thickness and trabecular bone volume. Also some results shown in male and female
mice have shown reduced bone volume and increased which is in consistency with
some human findings in aromatase deficient males.
1.4 Role of androgen receptor in bone physiology:
Androgen receptor (AR) is a member of nuclear steroid/thyroid hormone
receptor gene superfamily. It is a transcription factor that is stimulated by
ligand(Mangelsdorf, Thummel et al. 1995; Takeyama, Ito et al. 2002). Like all other
tissues bone cells; osteoblasts and osteoclasts possess AR, and the impacts of
androgens tend to act on genes to activate or suppress the expression of target genes in
bones (Compston 2001).
In the cartilage and bone, AR is expressed in chondrocytes, osteoblasts and
osteocytes and osteoclasts. (Ogata, Chikazu et al. 2000)
To understand better the implications of the AR in the bone physiology the AR
-/- mice show important implications on the bone (Sato, Matsumoto et al. 2003). As
shown in Fig 4; AR knockout (ARKO) mice suggested that the males developed
osteopenia and retarded growth curves, with a normal shape of the bone. The clear
impact of the AR role is still not understood as the androgen is aromatized to the
estrogen (Simpson and Davis 2000). In the case of female ARKO mice the bone loss is
not detected hence it is believed that AR is the determining factor for the male bones.
As the major impact of sex steroid on bone remodeling is due to estrogen and estrogen
receptor mediated signaling.

18
AR located on the X chromosome, male mice with AR-/- exhibit reduced
trabecular volume and cortical volume with increase in the bone turnover, and are
hypogonadal, and have low testosterone levels. In the case of female mice the AR-/-
does not render any abnormalities on the skeleton due to the double copies of the gene.

Fig 3-B: Schema of skeletal sex hormone action: In male WT mice, skeletal sex
hormone activities are mediated by both AR and ER. In female WT mice, skeletal
function of ER is likely to dominate over that of AR as serum levels of AR ligands in
females are quite low. In male ARKO mice, testicular testosterone production is
severely impaired by hypoplasia of the testes, leading to a lack of skeletal sex hormone
actions. In contrast, female ARKO mice may not be greatly affected by disruption of
AR signaling. (Hirotaka Kawano July 18, 2003)

19
1.6 TFM (Testicular feminized mutation):
The X-linked syndrome of androgen insensitivity (also known as testicular
feminization (Tfm)‘) results from a failure of tissue response to androgen. In humans,
phenotypic expression ranges from partial forms, in which 46,XY phenotypic males are
infertile, to the complete syndrome, in which the phenotype is female (J. B. 1983).In
affected genetic males, Mullerian ducts are absent because of the effect of testicular
Mullerian-inhibiting substance, but androgen-dependent male accessory sex glands fail
to develop(J. B. 1983).The arginine 734 to glutamine in the Tfm mutation prevents
phosphorylation at one or more critical sites in the steroid-binding domain. Other
possible consequences of the single base mutation include distortion of protein folding
and/or loss of dimer formation or interactions with other proteins(Pratt, Jolly et al.
1988; Bresnick, Dalman et al. 1989). A single base mutation greatly reduces androgen-
binding capacity. Arginine at position 734 is involved in one or more mechanisms
required for normal functioning of the androgen binding domain. Naturally occurring
AR mutations associated with androgen insensitivity provide experimental models that
serve in correlating structural, physiological, developmental, and behavioral aspects of
receptor function.
Testosterone effects on sexual behavior in the Tfm rat have been attributed to
the conversion of testosterone to estrogen in the brain; male type sexual behavior
occurs to a limited extent in response to estrogen or testosterone, but not to
dihydrotestosterone, a non aromatizable androgen (Yarbrough, Quarmby et al. 1990).

20
The Tfm serve as a good parameter to employ to understand the effect of
androgen in bone physiology. To determine androgen effect in Tfm male mice would
explain important details.

1.7 LEF1 and low bone mass phenotype in heterozygous female mice:
Graduate student of the lab Tommy Noh’s work done on Lef-1 heterozygous
mice shows that they have low bone mass phenotype; the homozygous knockout mice
die within 1-2 days of birth (unpublished observation). Along with low bone mass it
has been observed that it is seen specifically in female mice. On comparison of 17-
week-old female Lef-1 +/- and +/- it is observed that they have similar bone length,
diameter and trabecular thickness. However, statistically significant differences are
observed in cortical thickness and trabecular connectivity (work in progress).

21
1.8 Hypothesis: Androgen Signaling prevents bone loss that occurs in Lef1
haploinsufficient mice.
The female mice had a bone loss due to the lack of androgen signaling and the
protection in male mice would be due to anabolic effects of the androgens. And as
discussed in the section 1.5 androgen receptor plays a role in bone physiology.

1.9 Aim: To understand the interaction between Lef1 haploinsufficiency and
androgen receptor.
As discussed in section 1.7 the observations have shown the effects are sex
specific, we became curious in understanding the reason of the male skeleton being
protected from the bone loss due to haploinsufficiency of the Lef-1. Male mice that
would have Tfm mutation would have androgen insensitivity and thus female like
bones in the absence of androgen effect.
Our aim was to determine the possible effect of androgen signaling in the male
mice and its relation with bone mass. The mice with mutated androgen receptor were
bred with mice having Lef-1 heterozygosity, which is described in the methods and
materials section.

22
Chapter 2: Methods and Materials

2.1 Genetic setup and breeding scheme of mice:
The Tfm mutation being on the X chromosome makes it difficult to breed a
male with Tfm mutation, as the male mice with this mutation have under developed
testis and cannot effectively breed (Olsen 1990). Thus a female mouse that has Tfm
mutation on the X chromosome was bred with a mouse that has LEF1 heterozygous
phenotype.
As discussed in the introduction chapter we were interested to know if there
was any impact of androgen signaling that prevented bone loss in Lef1
haploinsufficient males. Fig 4. describes a breeding scheme of mice done by Tommy
Noh. The F1 generation off springs had wild type female mice group without Lef1
mutation (XX Lef1
+/+
) with a sample size of 20 and mice with Lef1 mutation
(XXLef1
+/-
) with a sample size of 20. Similarly, there was a comparison of wild type
male mice with homozygous Lef1 (XY Lef1
+/+
) with a sample size of 10 and male mice
with heterozygous Lef1 (XY Lef1
+/-
) with a sample size of 18. A group of female mice
with Tfm mutation and homozygous Lef1 allele (X
T
X Lef1
+/+
) had a sample size of 15
were compared with heterozygous Tfm mutant female mice group of 9 samples.
Likewise the male Lef1 homozygous mice group with Tfm mutation (X
T
Y Lef1
+/+
) had
a sample size 15 and the Tfm mutant Lef1 heterozygous group (X
T
Y Lef1
+/-
) had 13
samples. In total the scanning procedure was done on 111 mice. The wild type male
and female groups and the Tfm mutant female groups served as the control for the test

23
groups of Tfm mutant male groups. The details about the scanner and the scanning
procedure done are as follows.
Genotypes:
• X
T
- Tfm mutation
• X - Normal chromosome
• Y- Normal chromosome
• LEF-1
+/-
genotype

2.1.2 Specimen preparation:
The mice femurs used for scanning were immersed in 70% ethanol and
mounted in a 0.6 ml microcentrifuge tubes.

Fig-4: Genetic scheme describing the Tfm mutant mice breeding with
Lef1 haploinsufficient mice.

24
The tube containing the specimen was taped of the bed of the scanning machine
with the distal end towards the end of the bed.

2.2 Micro Computed Tomography Scanner:
The experiment was designed to understand the variation in bone mass due to
Lef1 gene dose combined with androgen signaling. To understand the resultant
phenotype, we studied femurs dissected from these mice. To understand the bone
structure we used the microCAT
TM
II X-ray computed tomography system (Siemens
Preclinical solutions Inc., Knoxville, TN.), which facilitates acquisition and 3-D
manipulation of tomographic data from small animals. The scanner has two modes rat
mode and mouse mode. The mode used for high resolution and detailed imaging is rat
mode. The mode changing involves variation in the relative position of key
components like X-ray source and X-ray detector. These components positions are
manipulated during the calibration of the scanner as described in section 2.3.1 and
2.3.2.
X-ray source: The position of the source is modulated for either mode of
scanning. The angular position facilitates exposing the specimen to X-ray at different
angles. The X-ray source is also provided with aluminum filters to prevent X-ray
deflection and improved quality of imaging. The X-ray voltage was 80.0 kVp and the
anode current 250μAmp. Since the specimen material was dense, a higher voltage
range was chosen.

25

Fig 5-A. A screenshot image of the
data acquisition the microCAT
TM
II software

Fig 5-B X-ray camera setup menu
screenshot
Fig 5-C: Radiograph setup menu screenshot

Fig 5-D Radiograph image of the
specimen femur

26
X-ray detector: The X-ray detector is enabled with modulation in position with
reference to the X-ray source. The X-ray detector is placed perpendicular to the animal
bed in order to capture the X-rays in a transaxial manner. On calibrating the machine,
the position of the detector also varies.

2.3 Data acquisition and scanning procedure:
The microCAT
TM
II software executes the data acquisition of the X-ray imaging
work. It operates on a Windows XP
TM
workstation platform. The software also
regulates the power, calibration of the geometry, and other hardware modifications.
The hardware is initialized with the help of the software. The working screenshot of the
software on start up is as shown in Fig 5-A. Various features are X-ray stop button for
immediate termination of X-ray, scan status window showing the progress, X-ray
activity indicator bulb on right. As described in section 2.2 the positioning of the source
and the detector are done on the basis of calibration procedure.
2.3.1 Scanner geometry calibration:
Selecting ‘geometric calibration’ from the tools menu of the software does the
procedure. A metallic rod that substitutes a tissue for scanner calibration is called a
phantom. The X-ray beams are shown as two blue gridlines. The blue gridlines are
aligned with the phantom axis by dragging with the help of the computer mouse. On
the basis of the aligned gridlines the computer automatically calculates the relative
position of the X-ray source and the detector.

27
On the basis of high-resolution mode the source and the detector positions and
distances are calculated. The calculations are like source to detector distance
(~350.13mm); source to center distance (~105.5mm); detector pitch (~0.03 mm);
transaxial length; axial length; horizontal & vertical rot-axis bed angle.
The set parameters are saved as a log file. The observed geometry is recorded
and further repeated to verify consistency with consecutive reading.
2.3.2 Center offset calibration:
Center offset is the distance between the CCD (charged couple device) detector
and the scanner isocenter. In other words it is midpoint calibration of the subject to get
precise resolution. Like geometric calibration the computer performs an automated
calibration at high-resolution mode; by selecting the center offset calibration option in
the tools menu as shown in Fig.5-A. The beams represented by the wires are aligned
with the phantom. The calibration is done for BinX1 (~43.5μ); BinX2 (~20.5μ); BinX4
(~10.5μ) modes, the processes are repeated approximately ten times for consistency.
For accuracy of the resolution a variation in the range of only 0.1μ can be allowed. The
calibration phantom is replaced with the animal bed after calibration process is done.
Scanner is then ready for the setup of other features necessary for scanning, like
camera, radiograph and CT scan.
2.3.3 Camera setup:
The camera captures every angle at which the specimen is exposed to X-ray
source. Its setup enables to modulate the width and the length of the image size to best

28
fit the specimen. The camera setup option is selected from the tools menu. The
screenshot of the setup menu is as per Fig 5-B.
Transaxial length is the length across the X-axis. (Values range from 128 pixels
to the full CCD, length 2048) The pixel range chosen in this particular set of scanning
was 2048x2048. By varying the transaxial length, the data set was cropped to cover the
region of interest. Transaxial Binning Factor stands for the individual slice width.
BinX2 option was selected for this set of experiment.
In axial length the BinX2 option was selected as well. Axial length is the length
across the Y-axis. (Values range from 128 to 2048). The pixel range chosen for this
study is 2048 X 2048. By varying the transaxial length, the data set may be cropped to
a region of interest vertically. BinX2 option was selected for this parameter as well for
scanning.
CCD (Charged couple device) Exposure Time is the duration to which the
material is exposed to X-ray shot. Selecting a particular value at this level causes
variation in the brightness and contrast level of the image. It is measured in
milliseconds and the value selected in here was of 750.
The software has built in data verification and correction options, warp
correction is applied to prevent any kind of distortion.
2.3.4 Radiograph:
Radiograph feature is meant to have the pilot image of the CT scan. It enables
to view the image at various angles to determine the accuracy of the scanning.

29
Fig. 5-C describes the menu page of the radiograph setup panel. The position
and the height of the bed is 10 mm. The machine determines these parameters set
automatically during geometric calibration as described in section 2.3.1. The degree
values of 0 and 90 are chosen to get a view of the positioning and the image to be
scanned. The X-ray source is not turned off after the radiograph is performed.
The “Set at Center” option is selected from the tools menu, to position the
image frame properly with reference to the subject to be scanned.
2.3.5 CT Scan:
Finally, the scanning procedure is started by selecting the CT scan option from
acquire menu. The data is saved on computer as CAT file. For this procedure filling the
details in menu option does the setup. Various important parameters of the setup are
shown in Fig 5-A. Rotating start position is 90 degrees and the detector total rotation
value is set to 210 degrees. The bed position and height values are the same as those
measured for the radiograph setup. The data is written to a raw file and hence the
option is selected. The real time reconstruction is not selected as the reconstruction
process is independently performed latter using different software. For consistency in
repeated scans the data is saved as a protocol file on the hard disk. Every time these
specimens were scanned the set protocol file was loaded before performing the scan.
The total scan time is about 20-25 minutes at the end of which along with the
main cat file. The image of the specimen CT scanned is the same as the radiograph
image.

30
2.4 Micro CAT file Reconstruction:

The software used for the reconstruction of the data is Exxim’s Cone Beam
Reconstruction Software package, COBRA version 5. The process being there are 4
different processing terminals used for the reconstruction. The .cat file is reconstructed
into 1024 slices by four terminals and finally .CT files are formed. The processing
machine is attached to the acquisition computer by the LAN mechanism.
The RVA software at the same terminal as the x-ray scanning software
was used to initiate the x-ray reconstruction process. The basic data input file is cat file.
The reconstruction parameters are the same as those set in microCat file. On the basis
of 512 steps input from the cat file, 1024 CT slices are created. The slices eventually
after the reconstruction are transferred back to the data computer with the cat file.

2.5 Micro CAT data analysis:

The software used for the analysis is Amira 4.1.0 Windows 64 bit. Amira is
adapted to image processing functions and analysis of micro CAT data. It also enables
various image formats.

31

Fig 6-B: Raw data parameters menu
to load the .ct files of in Amira.

Fig 6-A: Screenshot image of CT setup
panel

32

2.5.1 Loading Data:
The raw data, which is reconstructed as CT files is loaded to create an Amira
file. The software compiles 1024 CT slices as raw data. Selecting the series of the .ct
files does the loading of the data files. The slow data reading option is selected.
Thereafter as per Fig. 6-C the values of the parameters are selected. The machine on
the basis of the .ct files loaded selects the data dimensions. The requested and the file
size should match in the menu while loading the files. The voxel size in the data
window should be equivalent to the micron size of the binning factor selected while
scanning. The selected option in this experiment was 0.02 mm as the BinX2 factor. To
further modify the data by reducing it to required length and size it is cropped.
2.5.2 Cropping and Sections:
Fig 6-C: Reconstruction software COBRA

33
In order to crop, after loading the file, voltex function is applied. It generates a
3D view of the object. Options selected in this function are constant color and a data
window with the range of 100-1000, with a down sample value 4 in all the three
dimensions. Voltex function also allows transformation.
Thereafter transformation tool is used, for the vertical or horizontal
orientation of the object to rectify any misplacement in the object position. This
application is not necessary for all the specimens.
Cropping step is necessary for all the image objects. Selecting the crop tag in
the menu shows grid lines around the object frame. These gridlines in all the
dimensions are dragged with the help of the mouse and placed to reduce the non-object
portion of the frame. However, care is taken that any region of the bone is not cropped.
This is made sure by looking at the object from all the dimensions. The cropped section
is finally saved as a full Amira mesh file with extension .am.
To verify the cropped object the orthoslice feature located in the right panel is
used and the slices are viewed by scrolling. Specific regions of the bones were studied
in detail and the regions were cropped out of the whole bone on the same basis.

2.6 Cortical Analysis:
The cortical analysis of the bone is done to determine its cortical wall thickness
in the center of the bone. The region of interest of the bone was selected from the
center slice to 1mm distal length. Considering the individual slice length to be 20μ,
1mm equivalent of it would be 50 slices from the midpoint of the bone. Hence a value

34
of 50 in the z dimension of the crop menu is entered. Inclusive of the initial slice 49
slices are cropped. The file is renamed and saved as a cortical section in Amira mesh
file format. Fig 7-A shows a voltex reconstruction of the cortical section of the bone
after cropping. Fig 7-B shows highlighting of the cortical wall.
To enhance the image resolution median filter with 3D option is selected. It
also adds accuracy to the result.
The main step of the analysis procedure is segmentation procedure.
Segmentation assigns a label to each pixel. This helps assigning individual pixels to the
parts of bones they belong to. This specific assigning of the pixels does the volumetric
measurements of the specific regions. The segmentation done with the help of separate
data object is known as Labelfield. Labelfield is applied by right click on the file and
selecting label field from display menu.
Applying labelfield opens a new window, which has four different dimension views.
Single xz view is selected out of the 4 by default views of the Labelfield. The zooming
is adjusted with 3:1 ratio. Thereafter, selecting the data window option from
segmentation menu is selected to set the brightness contrast level. The values are in the
range of minimum and maximum range. The region of interest of the cortical section is
then highlighted by the coloring function of the magic wand. In the magic wand tool
the values are set for the min/max level to cover the whole bone region including the
wall and the area inclusive of the wall. All the slices are highlighted. In the materials
menu located in the upper half right panel a new material named ‘TV-BV’ (Total

35
volume-Bone volume) is created. The highlighted slices are added to the material with
the help of adding tool.
Select threshold option from selection menu. Select a threshold value that
highlights only the bone material and not the including region. Create a new material
named BV. Finally, add the highlighted region to BV. The data is saved as an Amira
file.

Fig 7-A: Bone cortical region voltex B: Slice highlighting of the cortical region
C: Isosurface reconstruction of trabecular regio D: Trabecular highlighting of
the slice

7-A 7-B

7-C 7-D

36

2.6.1 Cortical Measurements:
The saved Amira file is then used to determine tissue statistics. It is the voxel
based counting of the highlighted values in the analysis process. The tissue statistics
function is selected by right clicking on the measure tab on the right panel. Applying
the measure function a text file is generated, which shows calculated the cortical region
volume, the parameters determined are TV-BV and BV. From these values other
measurements like TV, and the ratio BV/TV is calculated.
TV: The total highlighted region determines the TV, which is total volume. The
total region consists of the wall and the cortical cavity. Output data gives the values
TV-BV and BV; hence TV is determined by adding the two available values.
BV: The bone volume is determined by subtracting the cavity volume from the
total volume. The bone volume consists of the cortex wall of the bone region.
BV/TV: The BV was also normalized with TV to obtain the relative bone
volume. The BV/TV ratio helps to determine the volume of the cortical wall with
reference to the total bone volume.

2.7 Trabecular Analysis:
The trabecular region is analyzed to determine the trabecular micro-
architecture of the particular bone. The region above the cartilage plate to the length of
3.2 mm towards the proximal end has been selected as the trabecular region of interest.
Again considering the individual width to be 20μ, 160 slices would measure 3.2 mm of

37
the bone length. The values of the initial and final slices are set in the z-dimension of
the crop menu.
Median filter with 3D option is applied to the cropped object file. Creating
Labelfield continues the segmentation process further. The data window value is
adjusted with brightness of contrast. And the zoom ratio set to 3:1 value.
Every 5
th
slice of the section is highlighted using the paintbrush feature in the
tools menu. In the case of trabecular bone highlighting the peripheral wall is not
highlighted with the paintbrush and only the inner cavity is highlighted, which contains
trabeculae. Followed by the painting is the interpolation, which enables the
highlighting of the whole section of interest without painting individual slices. The
total highlighted bone is shrunk with the help of shrinking tool that would separate the
trabecular region from the peripheral wall region. Volume shrinking is done 5 times
and the data is added to the newly formed material labeled ‘TV-BV’. Further selecting
the threshold option, the trabeculae, which are visually apparent, are highlighted.
Making a new material and labeling it as BV thereafter the highlighted region is added
to it. The Labelfield is saved as an Amira mesh file. Fig.7-C shows the iso-surface
reconstruction of the trabeculae and the separation from the peripheral bone. And Fig
7-D shows trabeculae highlighting of the bone.
2.7.1 Trabecular measurements:
For the statistical measurement of the tissue, the measure tissue function is
selected. This generates the text file, which gives the primary values TV-BV and BV.
And value like TV and BV/TV ratios thus are calculated from the primary tissues.

38
Similarly the surface area measurement is determined by selecting the option
compute and surfacegen in that option. After applying the surfacegen calculations, the
surface area is determined by applying to surfacegen-generated file. The raw values
obtained from it are inner shaft and exterior – triangle, volume, and area; trabecular -
triangle, volume and area.
TV: The total volume here consists of the trabeculae and the cavity enclosed in
the peripheral wall, excluding the wall. The total volume that is determined by the
region highlighted.
BV: The bone volume for the trabecular section consists of the trabeculae alone,
which is excluded from the total volume region.
BV/TV: The volume of the trabeculae (BV) is the internal trabecular volume;
BV is also normalized with TV to obtain the relative bone volume (BV/TV).
Bone/Tissue Volume: The tissue volume consists of dividing the trabecular
bone volume by the inner shaft volume of the trabecular segment. The formula for
calculating the specific parameter is
Bone/Tissue Volume = Trabecular bone/Total internal volume
Bone Surface/Volume (mm
-1
): The bone surface (BS) area was calculated
using trabecular bone area (mm
2
) and the trabecular volume (mm
3
). The BS value was
obtained by normalizing trabecular area with the trabecular volume, to obtain the
relative bone surface. The formulae for determining it is:
Bone surface/volume = Trabecular Area / Trabecular Volume

39
Trabecular Thickness (mm): Mean trabecular thickness (Tb.Th) is determined
by dividing 2 with tissue volume.
Trabecular thickness = 2 / Bone/Tissue volume.
Trabecular Spacing (mm): Trabecular spacing (Tb.Sp) is calculated by inverse
of subtraction of trabecular number from trabecular thickness.
Trabecular spacing = 1/ Trabecular thickness – Trabecular number.
Trabecular Number (mm
-1
): The trabecular number (Tb.N) is calculated by
taking the ratio of tissue volume by the trabecular thickness.
Trabecular number = Bone/Tissue Volume / Trabecular thickness

2.8 Bone length measurement:
The bone length was measured by subtracting the initial slice number from the
terminal slice number. The numbers of slices were multiplied by 0.02 mm as each slice
width is 0.02 mm.

2.9 Statistical test:
The MS Office Excel T-test is performed to determine the statistical
significance of the experimental results. The mean of one group was entered as array1
and the other as array2. It is 1 tailed and with equal variance of sample chosen to
perform the t-test. The significance level of p-value is set to 0.05.

40
Chapter 3: Results

To understand the bone loss effect due to different genetic alleles groups were
compared. The first comparison group is the wild type female with and without Lef1
mutation (XX Lef1
+/+
and XX Lef1
+/-
). The second group consists of the Tfm mutant
female mice with homozygous and heterozygous alleles (X
T
X Lef1
+/+
and X
T
X Lef1
+/-
). Similarly in the male mice the two groups were wild type Lef1 homozygous male
mice and heterozygous Lef1 (XY Lef1
+/+
and XY Lef1
+/-
). The three groups above
mentioned served as the control for the male Tfm mice group with homozygous and the
heterozygous alleles (X
T
Y Lef1
+/+
and X
T
Y
+/-
). The results of the comparison studies
are as follows:

3.1 XY Lef1
+/-
vs. XY Lef1
+/+
:
Trabecular: The trabecular parameters calculated, as shown in Fig 10 A and B,
the mean trabecular BV/TV of XY Lef1
+/+
is 0.313 with standard deviation value 0.149,
and the mean of XY Lef1
+/-
is 0.267 and standard deviation 0.056.
The p-value determined from the t-test that is performed is 0.126, which shows
that the difference between the mean of the two groups is not statistically significant.
Cortical: As shown in Fig 10-C and D, the cortical value of XY Lef1
+/+
mean is
0.556 with a standard deviation 0.034 and the mean of XY Lef1
+/-
is 0.554 with a
standard deviation 0.032. The p-value for the different data sets is determined by the t-
test performed on the mean value of the data sets, which gives 0.444.

41
Bone length: The bone length value as shown in table 10-A for the XY Lef1
+/+

had

a value of 15.751 mm and XY Lef1
+/-
a value of 15.865 mm. The standard
deviation values are 0.383 and 0.377 respectively. The p-value estimated by t-test had a
value of 0.231.

Table 1 B. Table 1 C.

Cortical BV/TV
0
0.1
0.2
0.3
0.4
0.5
0.6
BV/TV (m.m.)
XY Lef1 +/+
XY Lef1 +/-

Trabecular BV/TV
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
BV/TV (m.m.)
XY Lef1+/+
XY Lef1 +/-
Trabecular (BV/TV) Mean SD P-value
XY Lef1
+/+
(n = 10) 0.313 0.150
XY Lef1
+/-
(n = 18) 0.267 0.055
0.125
Cortical (BV/TV)

XY Lef1
+/+
0.556 0.034
XY Lef1
+/-
0.555 0.032
0.444
Bone Length

XY Lef1
+/+

15.751
0.383
XY Lef1
+/-
15.865 0.377
0.231

Table1 A.

Table 1:

A- XY Lef1
+/+
vs. XY Lef1
+/-
trabecular BV/TV, cortical and bone length values
comparison. B, C - Graphical description of XY Lef1
+/+
vs. XY Lef1
+/-
trabecular,
cortical comparison.

42

3.2 X
T
Y (+/-) vs. X
T
Y (+/+):

Trabecular: The trabecular parameters calculated, as shown in Fig 11 A and B,
the mean trabecular BV/TV of X
T
Y Lef1
+/+
is 0.059 with standard deviation value
0.0350, and the mean of X
T
Y Lef1
+/-
is 0.041 and standard deviation 0.02.
The p-value determined from the t-test that is performed is 0.059, which shows
that the difference between the mean of the two groups is statistically significant.
Cortical: As shown in Fig 11-C and D, the cortical value of X
T
Y Lef1
+/+
mean
is 0.532 with a standard deviation 0.036 and the mean of X
T
Y Lef1
+/-
is 0.545 with a
standard deviation 0.045. The p-value for the different data sets is determined by the t-
test performed on the mean value of the data sets, which gives 0.182.
Bone Length
0
2
4
6
8
10
12
14
16
18
20
XY Lef1+/+
XY Lef1 +/-

Table 1: D - Graphical description of XY Lef1
+/+
vs. XY Lef1
+/-
bone
length values comparison

43
Bone length: The bone length value as shown in table 11-A for the XY Lef1
+/+

had

a value of 15.670 mm and XY Lef1
+/-
a value of 15.726 mm. The standard
deviation values are 0.434 and 0.583 respectively. The p-value estimated by t-test had a
value of 0.384.
Table 2 A.

B. C.

Table 2:

A- X
T
Y Lef1
+/+
vs. X
T
Y Lef1
+/-
trabecular BV/TV, cortical and bone length values
comparison.
B, C, - Graphical description of X
T
Y Lef1
+/+
vs. X
T
Y Lef1
+/-
trabecular, cortical
and bone length values comparison.

Trabecular (BV/TV) Mean SD P-value
X
T
Y Lef1
+/+
(n = 15) 0.059 0.035
X
T
Y Lef1
+/-
(n = 13) 0.041 0.021
0.059
Cortical (BV/TV)

X
T
Y Lef1
+/+
0.532 0.036
X
T
Y Lef1
+/-
0.546 0.045
0.183
Bone Length

X
T
Y Lef1
+/+
15.670
0.434
X
T
Y Lef1
+/-
15.726 0.583
0.384

C o rtic al B V /T V
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
BV/TV (mm)
T fm X Y L e f1 + /+
T fm X Y L e f1 + /-
T rab ecu lar B V/T V
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Tfm XY L e f1 + /+
Tfm XY L e f1 + /-

44

3.3 XX Lef1
+/-
vs. XX Lef1
+/-
:

Trabecular: The trabecular parameters calculated, as shown in Fig 8 A and B,
the mean trabecular BV/TV of XX Lef1
+/+
is 0.103 with standard deviation value
0.034284, and the mean of XX Lef1
+/-
is 0.085 and standard deviation 0.034.
The p-value determined from the t-test that is performed is 0.054, which shows
that the difference between the mean of the two groups is statistically significant.
Cortical: As shown in Fig 8-A, C; the cortical value of XX Lef1
+/+
mean is
0.56608 with a standard deviation 0.034 and the mean of XX Lef1
+/-
is 0.574 with a
Bone Length
0
2
4
6
8
10
12
14
16
18
20
Tfm XY Lef1 +/+
Tfm XY Lef1 +/-
Table 2: D - Graphical comparison between the two group X
T
Y Lef1
+/+
vs.
X
T
Y Lef1
+/-
trabecular, cortical and bone length values comparison.

45
standard deviation 0.034844. The p-value for the different data sets is determined by
the t-test performed on the mean value of the data sets, which gives 0.217.
Bone length: The bone length value as shown in fig 8-A for the XX Lef1
+/+ had
a value of 15.546 mm and XX Lef1
+/-
a value of 14.54 mm. The standard deviation
values are 0.392 and 0.556 respectively. The p-value estimated by t-test had a value of
0.109.

Trabecular (BV/TV) Mean SD P-value
XX Lef1
+/+
(n = 20) 0.103 0.034
XX Lef1
+/-
(n =20 ) 0.085 0.034
0.055
Cortical (BV/TV)

XX Lef1
+/+
0.566 0.034
XX Lef1
+/-
0.574 0.034
0.217
Bone Length

XX Lef1
+/+

15.546
0.392
XX Lef1
+/-
14.54 3.556
0.109

Trabecular BV/TV
0
0.02
0.04
0.06
0.08
0.1
0.12
XX Lef1+/+
XX Lef1 +/-

Table 3 B. Table 3 C.
Table 3A.

Cortical BV/TV
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
XX Lef 1 +/ +
XX Lef 1 +/ -

46

3.4 X
T
X Lef1
+/-
vs. X
T
X Lef1
+/+
:

Trabecular: The trabecular parameters calculated, as shown in Fig 9 A and B,
the mean trabecular BV/TV of X
T
X Lef1
+/+
is 0.121 with standard deviation value
0.027, and the mean of X
T
X Lef1
+/-
is 0.092 and standard deviation 0.037.
The p-value determined from the t-test that is performed is 0.020, which shows
that the difference between the mean of the two groups is statistically significant.
Cortical: As shown in Fig 9-A and D, the cortical value of X
T
X Lef1
+/+
mean
is 0.549 with a standard deviation 0.039 and the mean of X
T
X Lef1
+/-
is 0.547 with a
Bone Length
0
2
4
6
8
10
12
14
16
18
20
X X Lef1 +/+
X X Lef1 +/-

D.
Table 3: D - Graphical description of XX Lef1
+/+
vs. XX Lef1
+/-
bone length values
comparison.

Table 3: A, B C- Table of contents with Graphical comparison between the two group
XX Lef1
+/+
vs. XX Lef1
+/-
trabecular, cortical and bone length values comparison.

47
standard deviation 0.022. The p-value for the different data sets is determined by the t-
test performed on the mean value of the data sets, which gives 0.425.
Bone length: The bone length value as shown in table 9-A for the X
T
X Lef1
+/+

had

a value of 15.504 mm and X
T
X Lef1
+/-
a value of 15.466 mm. The standard
deviation values are 0.376 and 0.479 respectively. The p-value estimated by t-test had a
value of 0.109.
Table 4: A.

Table 4 B. Table4 C.

Cortical BV/TV
0
0.1
0.2
0.3
0.4
0.5
0.6
Tfm XX (+/+) & (+/-)
BV/TV (m.m.)
Tfm XX
Lef1+/+
Tfm XX Lef1+/-
Trabecular BV/TV
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
BV/TV (m.m.)
Tfm XX +/+
Tfm XX +/-
Trabecular (BV/TV) Mean SD P-value
X
T
X Lef1
+/+
(n = 15) 0.122 0.027
X
T
X Lef1
+/-
(n = 9) 0.092 0.037
0.020
Cortical (BV/TV)

X
T
X Lef1
+/+
0.550 0.040
X
T
X Lef1
+/-
0.547 0.022
0.425
Bone Length

X
T
X Lef1
+/+

15.504 0.376
X
T
X Lef1
+/-
15.466 0.479
0.413

48

3.5 Measurement characteristics and quality control:

The brightness and the contrast setting levels play a significant role these
settings further help in defining the regions of the bones to be highlighted. To prevent
any kind of variation the particular contrast and the brightness levels was set for the
whole batch, which was the same for the control and the test bones this helped in
preventing any sort of bias.
The cortical region highlighting was done using the fixed values of the magic
wand tool, which were absolute numbers. Similarly the threshold values were set
Bone Length
0
2
4
6
8
10
12
14
16
18
20
Tfm XX Lef1 +/+
Tfm XX Lef1 +/-

D.
Table 4:

A- X
T
X Lef1
+/+
vs. X
T
X Lef1
+/-
trabecular BV/TV, cortical and bone length values comparison.
B, C, - : Graphical description of X
T
X Lef1
+/+
vs. X
T
X Lef1
+/-
trabecular, cortical comparison.

Table 4: D- Graphical description of X
T
X Lef1
+/+
vs. X
T
X Lef1
+/-
and bone
length values comparison.

49
common for the whole batch to maintain consistency of the procedure and highlighting.
As a result the cortical values on repeating the analysis of the same bone give the same
results without any percent of variation.
The trabecular analysis as shown in the methods section was done using
highlighting of paintbrush which could lead to some variation on repeating the same
procedure. The measurement accuracy of the analysis was determined by repeating the
measurements and the variations on repeated measurements. As shown in figure 16 the
measurement results of specimens TL- 50, in the initial analysis procedure on repeating
gave a result of BV value 0.757 and a TV value of 4.877, final ratio of BV/TV resulted
to be 0.155. The repetition of the particular value gave a result of BV value of 0.772
and TV value 4.898 and finally the BV/TV ratio resulted in 0.1577. Hence the
resultant variation on repeating the analysis is 1.5%.
Customized highlighting of the individual slices and the precise interpolation of
the trabecular region maintain the accuracy of the measurements and final ratios of
BV/TV, which are devoid of any sort of large variations.

50

Specimens: BV TV BV/TV Variation
TL-50 0.75730401 4.87676411 0.155288218
TL-50 Repeat 0.772424 4.897983 0.157702467
1.5%
TL-161 2.9923 6.66663 0.448847469
TL-161 Repeat 3.02422 6.71344 0.450472485
0.3%

Table 5: Variation results showing repetition of specimens TL-50 and TL-161

51

Chapter 4: Discussion:

4.1 Lef1 haploinsufficiency effects on bone mass:
Canonical Wnt signaling plays important role in bone formation as discussed in
section 1.2. And the case of mice that have mutant Lef1 action the canonical Wnt
signaling pathway would be affected. This can affect normal bone formation process.
Effects on the bone length: The bone length determined as described in the
methods section has shown results that there is no significant difference in the bone
length and there is no statistically significant difference with the p-value being greater
than 0.05 in both control and the test group. Hence the bone length is not affected due to
Lef1 haploinsufficiency.
Effects on the cortical region of the bone: The comparison of control groups
as stated in the methods and materials section, has the p-value of XY Lef1
+/+
vs. XY
Lef1
+/+
is 0.444 and that of XX Lef1
+/+
vs. XX Lef1
+/-
is 0.297. Similarly, the X
T
X
Lef1
+/+
vs. X
T
X Lef1
+/-
p-value is 0.425 and male group X
T
Y Lef1
+/+
vs. X
T
Y Lef1
+/-
has
p-value 0.182.
The above data suggests that there is no impact on the cortical bone mass. The
cortical region BV/TV values do not vary in the groups with Lef1 haploinsufficiency.
Cortical bone mass in males or females is not affected.
Effects on the trabecular region of the bone: The trabecular BV/TV region
comparison of bone mass between XX Lef1
+/+
vs. XX Lef1
+/-
shows a bone mass loss of
about 17.4% with a p-value 0.054. XY Lef1
+/+
vs. XY Lef1
+/-
p-value is 0.125. The p-

52
value of difference between the mean of the X
T
X Lef1
+/+
vs. X
T
X Lef1
+/-
group bone
loss is 24% with p-value 0.020 and that of X
T
Y Lef1
+/+
vs. X
T
Y Lef1
+/-
bone loss of
30.3%, p-value 0.059 suggest that Lef1 haploinsufficiency causes problem in the
normal trabeculae formation.
The trabeculae BV/TV in the case of female +/- as oppose to that of the female
Lef1 +/+ shows that the trabecular mass is reduced to greater extent which is in
confirmation with the previous data that female mice have greater bone loss. The
canonical Wnt signaling in the heterozygous mice is affected and that resulted in the
hampered growth of the trabecular region. The female mice show loss of bone more
compared to that of the male counter parts. This suggests that the bone loss prevention
in male mice could be due to the androgen signaling.

4.2 Lef1 haploinsufficiency effect being sex specific:

As discussed in section 1.6 with the alteration in the androgen receptor of the cell
the mutation causes, the ligand binding and the down signaling due to the androgen
receptor binding and ligand is affected. This further affects the physiological processes
that are androgen dependent in the cell. Bone development is one of the processes,
which is dependent on the androgen signaling in the osteoblast cells.
The alteration of Tfm shows that since there is problem with the androgen
receptor in the mutated mice, there seems problem with the bone physiology too, which
is driven by the mechanism of androgen.

53
4.3 Bone loss in female mice due to Lef1 haploinsufficiency:

With the comparison of the female mice groups between the wild type (the mice
that do not have Tfm mutation), which exhibit that the Lef1 haploinsufficiency causes
trabecular bone mass difference (17.4%) in the case of the female with and without
mutation the p-value of the trabecular bone volume (BV/TV) ratio is 0.054.
Similarly, the comparison of female mice between that of the tfm mutated mice
with Lef1 heterozygous allele shows that there is significant difference as the p-value is
0.020.The androgen receptor and the lack of androgen effect in the females may cause
altered bone development compared to that in male mice. The results it has been
observed that in the case of Tfm female mice with and without LEF1 mutation tend to
have 30% low bone mass.
However, in the female mice there is no greater impact of androgen in bone
development. The bone development is more dependent on estrogen hence mutation in
Tfm would not affect the bone physiology to a greater extent.

4.4 Bone loss in Tfm male mice due to Lef1 haploinsufficiency:

The androgens bind to the androgen receptor and further cause down signaling
via the ligand receptor binding mechanism the causes further signaling and would affect
bone development. The males having the androgen playing a role in the bone
development tend to have protecting function against the bone loss due to Lef1. In Tfm
mice the androgen receptor that would be affected by the Tfm mutation would result in
the altered bone formation.

54
Comparing the Tfm male mutant mice with the homozygous Tfm male mice
there is a bone loss as the androgen receptor mediated signaling is not normal. However
the comparison between the mean and the p-value determined by the t-test is 0.059. The
male Tfm mutants tend to have 30.3% low bone mass.
The results show a trend of bone loss as compared to that of the male mice that
do not have Tfm mutation and haploinsufficiency. The haploinsufficiency causes a
marginally significant bone loss. But being an in vivo study the bone physiology is
dependant on various other factors, which we will discuss further in the following
sections.

4.5 Possible factors causing variations in bone loss:

4.5.1 Genetic make up and the background of strains:

The genetic cross setup primarily used breeding with the males that have
heterozygous Lef1 and the female mice that have Tfm mutation on one of the X
chromosome. Since the mutation is on X chromosome and the effects of it are under
development of gonads it makes it very difficult to breed and get off springs that have
exactly the same genetic make up as expected. The genetic background of the strains
used for breeding plays important role in bone volume density.
Moreover, apart from the Lef1 haploinsufficient alleles and Tfm genes which
are tested by genotyping methods all the other genes are randomly inherited, which may
play a role in the regulation of bone physiology. That could be a possible factor of
variation in bone mass as expected.

55
4.5.2 Effect on other parts of the skeleton:

As observed in the results section the cortical values of the determined data
does not show any difference between the mutant and the wild type mice. The
trabecular region exhibits the normal bone mass being affected. This also implies that
the effects of the mutation are location specific in the body of the mice. Hence one can
believe that if the bone mass is determined of the regions like the vertebrae, one could
see apparent bone loss in the case of the mutants.

4.6 Future Directions:

As we observed in the results section that the statistical significance was
marginal we plan to increase the number of samples to study and determine group mean
again to see the consistency of results in a more significant manner.
In the analysis of the bone structure we saw that the cortical region did not show
any apparent bone loss as oppose to the trabecular region.
Trabecular region was observed to have variation in the pattern of micro
architecture in the bone images; hence we further intend to determine the bone
architecture values like trabecular spacing, trabecular thickness, and trabecular number.
These will help understand the detailed impact of the haploinsufficiency.
The formation of the trabecular micro-architecture is observed to get more
detailed at the distal end near the primary spongiosa, hence to determine the specific
bone volume density we intend to determine the reduced trabecular length.

56
Further the region of interest would be other skeleton parts like the vertebrae,
which would help understand the impact of the Lef1 haploinsufficiency. So we intend to
determine the vertebrae bone volume density.

57
References:
Behrens, J., J. P. von Kries, et al. (1996). "Functional interaction of beta-catenin with
the transcription factor LEF-1." Nature 382(6592): 638-42.

Bennett, C. N., K. A. Longo, et al. (2005). "Regulation of osteoblastogenesis and bone
mass by Wnt10b." Proc Natl Acad Sci U S A 102(9): 3324-9.

Bhanot, P., M. Brink, et al. (1996). "A new member of the frizzled family from
Drosophila functions as a Wingless receptor." Nature 382(6588): 225-30.

Bhanot, P., M. Fish, et al. (1999). "Frizzled and Dfrizzled-2 function as redundant
receptors for Wingless during Drosophila embryonic development."
Development 126(18): 4175-86.

Bland, R. (2000). "Steroid hormone receptor expression and action in bone." Clin Sci
(Lond) 98(2): 217-40.

Bodine, P. V., J. Billiard, et al. (2005). "The Wnt antagonist secreted frizzled-related
protein-1 controls osteoblast and osteocyte apoptosis." J Cell Biochem 96(6):
1212-30.

Boyden, L. M., J. Mao, et al. (2002). "High bone density due to a mutation in LDL-
receptor-related protein 5." N Engl J Med 346(20): 1513-21.

Bresnick, E. H., F. C. Dalman, et al. (1989). "Evidence that the 90-kDa heat shock
protein is necessary for the steroid binding conformation of the L cell
glucocorticoid receptor." J Biol Chem 264(9): 4992-7.

Cavallo, R., D. Rubenstein, et al. (1997). "Armadillo and dTCF: a marriage made in the
nucleus." Curr Opin Genet Dev 7(4): 459-66.

Christiansen, C., M. S. Christensen, et al. (1980). "Prevention of early postmenopausal
bone loss: controlled 2-year study in 315 normal females." Eur J Clin Invest
10(4): 273-9.

Clement-Lacroix, P., M. Ai, et al. (2005). "Lrp5-independent activation of Wnt
signaling by lithium chloride increases bone formation and bone mass in mice."
Proc Natl Acad Sci U S A 102(48): 17406-11.

Compston, J. E. (2001). "Sex steroids and bone." Physiol Rev 81(1): 419-447.

58
Danielian, P. S. and A. P. McMahon (1996). "Engrailed-1 as a target of the Wnt-1
signalling pathway in vertebrate midbrain development." Nature 383(6598):
332-4.

Dominguez, I., K. Itoh, et al. (1995). "Role of glycogen synthase kinase 3 beta as a
negative regulator of dorsoventral axis formation in Xenopus embryos." Proc
Natl Acad Sci U S A 92(18): 8498-502.
Eriksen, E. F., D. S. Colvard, et al. (1988). "Evidence of estrogen receptors in normal
human osteoblast-like cells." Science 241(4861): 84-6.

Ernst, M., Heath, J. K. and Rodan, G. A. (1989). "Estradiol effects on proliferation,
messenger ribonucleic acid for collagen and insulin-like growth factor-I, and
parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells
from calvariae and long bones." Endocrinology 125, 825–833.

Ernst, M., C. Schmid, et al. (1988). "Enhanced osteoblast proliferation and collagen
gene expression by estradiol." Proc Natl Acad Sci U S A 85(7): 2307-10.

ES., O. (1996). Androgens. In: Principles of Bone Biology.

Evans, R. M. (1988). "The steroid and thyroid hormone receptor superfamily." Science
240(4854): 889-95.

Gaur, T., C. J. Lengner, et al. (2005). "Canonical WNT signaling promotes osteogenesis
by directly stimulating Runx2 gene expression." J Biol Chem 280(39): 33132-
40.

Gaur, T., L. Rich, et al. (2006). "Secreted frizzled related protein 1 regulates Wnt
signaling for BMP2 induced chondrocyte differentiation." J Cell Physiol 208(1):
87-96.

Glass, D. A., 2nd, P. Bialek, et al. (2005). "Canonical Wnt signaling in differentiated
osteoblasts controls osteoclast differentiation." Dev Cell 8(5): 751-64.

Glass, D. A., 2nd and G. Karsenty (2006). "Molecular bases of the regulation of bone
remodeling by the canonical Wnt signaling pathway." Curr Top Dev Biol 73:
43-84.

Goldray, D., Y. Weisman, et al. (1993). "Decreased bone density in elderly men treated
with the gonadotropin-releasing hormone agonist decapeptyl (D-Trp6-GnRH)."
J Clin Endocrinol Metab 76(2): 288-90.

Gong, Y., R. B. Slee, et al. (2001). "LDL receptor-related protein 5 (LRP5) affects bone
accrual and eye development." Cell 107(4): 513-23.

59

Haegel, H., L. Larue, et al. (1995). "Lack of beta-catenin affects mouse development at
gastrulation." Development 121(11): 3529-37.

Higgins, S. J., G. G. Rousseau, et al. (1973). "Nature of nuclear acceptor sites for
glucocorticoid- and estrogen-receptor complexes." J Biol Chem 248(16): 5873-
9.

Hirohashi, S. and Y. Kanai (2003). "Cell adhesion system and human cancer
morphogenesis." Cancer Sci 94(7): 575-81.

Hoang, B., M. Moos, Jr., et al. (1996). "Primary structure and tissue distribution of
FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal
morphogenesis." J Biol Chem 271(42): 26131-7.

Hock, J. M., I. Gera, et al. (1988). "Human parathyroid hormone-(1-34) increases bone
mass in ovariectomized and orchidectomized rats." Endocrinology 122(6): 2899-
904.

Hoeflich, K. P., J. Luo, et al. (2000). "Requirement for glycogen synthase kinase-3beta
in cell survival and NF-kappaB activation." Nature 406(6791): 86-90.

Holmen, S. L., C. R. Zylstra, et al. (2005). "Essential role of beta-catenin in postnatal
bone acquisition." J Biol Chem 280(22): 21162-8.

Hu, H., M. J. Hilton, et al. (2005). "Sequential roles of Hedgehog and Wnt signaling in
osteoblast development." Development 132(1): 49-60.

J. B., W., J. B., Fredrickson, D. S., Goldstein, J. L., (1983). "The metabolic basis of
inherited disease."

Karsenty, G. and E. F. Wagner (2002). "Reaching a genetic and molecular
understanding of skeletal development." Dev Cell 2(4): 389-406.

Kasperk, C. H., J. E. Wergedal, et al. (1989). "Androgens directly stimulate
proliferation of bone cells in vitro." Endocrinology 124(3): 1576-8.

Kato, M., M. S. Patel, et al. (2002). "Cbfa1-independent decrease in osteoblast
proliferation, osteopenia, and persistent embryonic eye vascularization in mice
deficient in Lrp5, a Wnt coreceptor." J Cell Biol 157(2): 303-14.

Kinzler, K. W., M. C. Nilbert, et al. (1991). "Identification of FAP locus genes from
chromosome 5q21." Science 253(5020): 661-5.

60
Klingensmith, J., R. Nusse, et al. (1994). "The Drosophila segment polarity gene
dishevelled encodes a novel protein required for response to the wingless
signal." Genes Dev 8(1): 118-30.

Kobayashi, T. and H. Kronenberg (2005). "Minireview: transcriptional regulation in
development of bone." Endocrinology 146(3): 1012-7.

Kokubu, C., U. Heinzmann, et al. (2004). "Skeletal defects in ringelschwanz mutant
mice reveal that Lrp6 is required for proper somitogenesis and osteogenesis."
Development 131(21): 5469-80.

Komori, T. (2005). "Regulation of skeletal development by the Runx family of
transcription factors." J Cell Biochem 95(3): 445-53.
Lammi, L., S. Arte, et al. (2004). "Mutations in AXIN2 cause familial tooth agenesis
and predispose to colorectal cancer." Am J Hum Genet 74(5): 1043-50.

Li, X., P. Liu, et al. (2005). "Dkk2 has a role in terminal osteoblast differentiation and
mineralized matrix formation." Nat Genet 37(9): 945-52.

Little, R. D., J. P. Carulli, et al. (2002). "A mutation in the LDL receptor-related protein
5 gene results in the autosomal dominant high-bone-mass trait." Am J Hum
Genet 70(1): 11-9.

Lo, M. C., F. Gay, et al. (2004). "Phosphorylation by the beta-catenin/MAPK complex
promotes 14-3-3-mediated nuclear export of TCF/POP-1 in signal-responsive
cells in C. elegans." Cell 117(1): 95-106.

Logan, C. Y. and R. Nusse (2004). "The Wnt signaling pathway in development and
disease." Annu Rev Cell Dev Biol 20: 781-810.

Logan, C. Y. a. R. N. (2004). "The Wnt signaling pathway in development and disease."
Annu Rev Cell Dev Biol 20: 781-810.

Mangelsdorf, D. J., C. Thummel, et al. (1995). "The nuclear receptor superfamily: the
second decade." Cell 83(6): 835-9.

Mao, J., J. Wang, et al. (2001). "Low-density lipoprotein receptor-related protein-5
binds to Axin and regulates the canonical Wnt signaling pathway." Mol Cell
7(4): 801-9.

Molenaar, M., M. van de Wetering, et al. (1996). "XTcf-3 transcription factor mediates
beta-catenin-induced axis formation in Xenopus embryos." Cell 86(3): 391-9.

61
Nawata, H., S. Tanaka, et al. (1995). "Aromatase in bone cell: association with
osteoporosis in postmenopausal women." J Steroid Biochem Mol Biol 53(1-6):
165-74.

Niemann, S., C. Zhao, et al. (2004). "Homozygous WNT3 mutation causes tetra-amelia
in a large consanguineous family." Am J Hum Genet 74(3): 558-63.

Ogata, N., D. Chikazu, et al. (2000). "Insulin receptor substrate-1 in osteoblast is
indispensable for maintaining bone turnover." J Clin Invest 105(7): 935-43.

Olsen, K. L. (1990). Handbook of Behavioral Neurobiology.

Ornoy, A., S. Giron, et al. (1994). "Gender dependent effects of testosterone and 17
beta-estradiol on bone growth and modelling in young mice." Bone Miner 24(1):
43-58.

Owen, M. (1980). "The origin of bone cells in the postnatal organism." Arthritis Rheum
23(10): 1073-80.

Owen, M. and A. J. Friedenstein (1988). "Stromal stem cells: marrow-derived
osteogenic precursors." Ciba Found Symp 136: 42-60.

Pawlowski, J. E., J. R. Ertel, et al. (2002). "Liganded androgen receptor interaction with
beta-catenin: nuclear co-localization and modulation of transcriptional activity
in neuronal cells." J Biol Chem 277(23): 20702-10.

Pinson, K. I., J. Brennan, et al. (2000). "An LDL-receptor-related protein mediates Wnt
signalling in mice." Nature 407(6803): 535-8.

Pratt, W. B., D. J. Jolly, et al. (1988). "A region in the steroid binding domain
determines formation of the non-DNA-binding, 9 S glucocorticoid receptor
complex." J Biol Chem 263(1): 267-73.

Qu, Q., M. Perala-Heape, et al. (1998). "Estrogen enhances differentiation of osteoblasts
in mouse bone marrow culture." Bone 22(3): 201-9.

Qu, Q., Perala-Heape, M., Kapanen, A. et al (1998). "Estrogen enhances differentiation
of osteoblasts in mouse bone marrow culture." Bone 22(3):201-9.

Robinson, J. A., S. A. Harris, et al. (1997). "Estrogen regulation of human osteoblastic
cell proliferation and differentiation." Endocrinology 138(7): 2919-27.

Robitaille, J., M. L. MacDonald, et al. (2002). "Mutant frizzled-4 disrupts retinal
angiogenesis in familial exudative vitreoretinopathy." Nat Genet 32(2): 326-30.

62

Ross, S. E., N. Hemati, et al. (2000). "Inhibition of adipogenesis by Wnt signaling."
Science 289(5481): 950-3.

Sato, T., T. Matsumoto, et al. (2003). "Late onset of obesity in male androgen receptor-
deficient (AR KO) mice." Biochem Biophys Res Commun 300(1): 167-71.

Schweikert, H. U., W. Rulf, et al. (1980). "Testosterone metabolism in human bone."
Acta Endocrinol (Copenh) 95(2): 258-64.

Simonet, W. S., D. L. Lacey, et al. (1997). "Osteoprotegerin: a novel secreted protein
involved in the regulation of bone density." Cell 89(2): 309-19.

Simpson, E. R. and S. R. Davis (2000). "Another role highlighted for estrogens in the
male: sexual behavior." Proc Natl Acad Sci U S A 97(26): 14038-40.

Slemenda, C., S. L. Hui, et al. (1987). "Sex steroids and bone mass. A study of changes
about the time of menopause." J Clin Invest 80(5): 1261-9.

Songyang, Z., A. S. Fanning, et al. (1997). "Recognition of unique carboxyl-terminal
motifs by distinct PDZ domains." Science 275(5296): 73-7.
Syed F, K. S. (2005 Mar 18). "Mechanisms of sex steroid effects on bone." Biochem
Biophys Res Commun. 328(3): 688-96.

Takeyama, K., S. Ito, et al. (2002). "Androgen-dependent neurodegeneration by
polyglutamine-expanded human androgen receptor in Drosophila." Neuron
35(5): 855-64.

Tamai, K., M. Semenov, et al. (2000). "LDL-receptor-related proteins in Wnt signal
transduction." Nature 407(6803): 530-5.

Tolwinski, N. S., M. Wehrli, et al. (2003). "Wg/Wnt signal can be transmitted through
arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity." Dev Cell
4(3): 407-18.

Umbhauer, M., A. Djiane, et al. (2000). "The C-terminal cytoplasmic Lys-thr-X-X-X-
Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling." Embo J
19(18): 4944-54.

Vanderschueren, D., I. Jans, et al. (1994). "Time-related increase of biochemical
markers of bone turnover in androgen-deficient male rats." Bone Miner 26(2):
123-31.

63
Vittek, J., K. Altman, et al. (1974). "The metabolism of 7alpha-3H-testosterone by rat
mandibular bone." Endocrinology 94(2): 325-9.

Willert, K., J. D. Brown, et al. (2003). "Wnt proteins are lipid-modified and can act as
stem cell growth factors." Nature 423(6938): 448-52.

Wodarz, A. and R. Nusse (1998). "Mechanisms of Wnt signaling in development."
Annu Rev Cell Dev Biol 14: 59-88.

Xu, Q., Y. Wang, et al. (2004). "Vascular development in the retina and inner ear:
control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair." Cell
116(6): 883-95.

Yarbrough, W. G., V. E. Quarmby, et al. (1990). "A single base mutation in the
androgen receptor gene causes androgen insensitivity in the testicular feminized
rat." J Biol Chem 265(15): 8893-900.

Yoshikawa, S., R. D. McKinnon, et al. (2003). "Wnt-mediated axon guidance via the
Drosophila Derailed receptor." Nature 422(6932): 583-8.

Yu, H. M., B. Jerchow, et al. (2005). "The role of Axin2 in calvarial morphogenesis and
craniosynostosis." Development 132(8): 1995-2005.

Abstract (if available)

Abstract Canonical Wnt signaling plays a major role in bone development. The transcription factors TCF/LEF are activated in the nucleus in response to Wnt signaling. Preliminary results from our lab have shown that Lef1 heterozygous Lef1 female mice have low bone volume w as compared to wild type females, whereas Lef1 heterozygous males have normal bone volume. Because sex steroids have significant role in bone development, we were interested to know if androgens protected the male mice from bone loss that otherwise occurs as a result of Lef1 haploinsufficiency.

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Asset Metadata

Creator Tank, Archana V. (author)

Core Title Genetic interaction between androgen receptor and Lef1 in bone mass control

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2007-08

Publication Date 08/07/2007

Defense Date 06/18/2007

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

Tag Bone uCT imaging,OAI-PMH Harvest,Wnt signaling

Language English

Advisor Frenkel, Baruch (committee chair), Hacia, Joseph G. (committee member), Tokes, Zoltan A. (committee member)

Creator Email tank@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m774

Unique identifier UC1490544

Identifier etd-Tank-20070807 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-535758 (legacy record id),usctheses-m774 (legacy record id)

Legacy Identifier etd-Tank-20070807.pdf

Dmrecord 535758

Document Type Thesis

Rights Tank, Archana V.

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