Yu Yamaguchi, Ph.D. (Please click this link to read more about the new mouse model research on the brain and bone)
Burnham Institute in La Jolla, California:
My laboratory has been studying the role of EXT1/heparan sulfate in mouse embryonic development. We have created a
conditional EXT1 knockout mouse model. These conditional EXT1 knockout mice are being used for genetic studies to figure
out how the deficiency of EXT1/heparan sulfate causes MHE.
These conditional knockout mice, which allow knocking out EXT1 at the site and time of researchers' desire, are very useful for
diverse studies on the function of EXT1/heparan sulfate.
Dr. Yamaguchi and his lab have been able to distribute these mice to more than 20 laboratories around the world (US, Europe,
and Japan) to help studies by other MHE investigators. Using this model system, Dr. Yamaguchi has demonstrated that
mutations of EXT1 influence not only bones but also the nervous system. Through an informal survey conducted by Sarah
Ziegler and Dr. Yamaguchi, although frequently ignored in the clinical front, MHE patients tend to have some mental,
neurological, and muscular symptoms. Such symptoms include: mild social interaction deficits (excessive shyness, adherence to
routines), heightened sensitivities to sensory stimulation (sounds, touch, taste), difficulties to concentrate, sleep issues and
muscle weakness(easy to get tired) and pain. Dr. Yamaguchi believes these neurological symptoms can be explained by the
deficiency of heparan sulfate in nerve cells. Indeed, recent analysis of knockout mouse behavior has revealed that these mice
have deficits in certain aspects of learning and the levels of fear/anxiety, as well as alterations in nerve cell wiring.
In addition, Dr. Yamaguchi has recently discovered that knockout of EXT1 in stem cells that destined to become bones and
cartilage causes severe bone abnormalities. These findings have provided us with a new insight into the reason why MHE
patients frequently associate a variety of symptoms in addition to exostosis /osteochondroma formation, and suggests
potential novel MHE treatment paradigms.
Jeffrey D. Esko, Ph.D.
University of California, San Diego:
“Hereditary multiple exostoses (HME) is a dominant genetic disorder resulting in the formation of generally benign cartilage-
capped tumors in various bones.
Recent work from a number of laboratories indicates that the disease results from mutations in EXT genes involved in making a
complex sugar, or polysaccharide, called heparan sulfate.
Since heparan sulfate interacts with many factors involved in cell growth, this discovery may shed light on the cause of the
disease, which in turn may suggest new treatments.
Studies of HME have relied on analysis of human exostosis samples made available when patients have surgery to remove
Progress understanding the cause of the disease has been frustrated by the paucity of material available for study and only
rare opportunities to compare the exostosis to normal tissue.
Recently, we discovered that mice bearing mutations in EXT genes also develop exostoses, which mimic many key features of
the human tumors.
One finding that emerged from studies of the HME mice is that the frequency of exostoses is highly variable and depends on
other genetic traits in the mice.
Since we can breed mice rapidly, we are now in a position to identify these other genes that may contribute to the severity of
Additionally, we need to work out methods to detect exostoses in live animals, assess treatment strategies for reducing the
frequency and growth of exostoses, and develop systems to look at exostosis development in isolated bones."
Dan Wells, Ph.D.
University of Houston:
Multiple Hereditary Exostoses (MHE) is an autosomal dominant skeletal disorder most frequently caused by mutations in the
MHE affects proper development of endochondral bones, such that all affected individuals present with exostoses adjacent to
the growth plate of long bones, while some individuals exhibit additional bone deformities. EXT1 functions as a heparan sulfate
(HS) co-polymerase, and when defective causes improper elongation of glycosaminoglycan side chains on core proteins of HS
Although analysis of heterozygous EXT1-deficient mice has failed to reveal any significant gross morphological variations in
skeletal development, significant alterations in molecular signaling occur in the developing long bones.
Our results indicate that defects in EXT1 and the resulting reduction in HS lead to enhanced Indian Hedgehog diffusion causing
an increase in chondrocyte proliferation and delayed hypertrophic differentiation.
Andrea Vortkamp, P.h.D.
Propagation of Ihh signaling:
One important question to understand the IhhP/THrP feedback loop is how the Ihh signal is transported through the growth
plate. In Drosophila the glycosyltransferase ‘tout velu’ (ttv) is necessary to transport the hedgehog signal in the developing
Mutations in the human homologues, Ext-1 and Ext-2, result in, Heritable multiple exostoses’ (HME), disease characterized by
benign bone tumors and short stature.In the developing bone we found Ext-1 and Ext-2 both expressed in domains flanking
the Ihh expression domain.
Using transgenic mice and a gene trap line targeting the Ext1 locus we aim to analyze the role of Ext1 during bone devolopment
and to identify a potential function in Ihh transport.
Marion Kuche-Gullberg, Ph.D.
Characterization of enzymes involved in heparan sulfate biosynthesis:
Our area of interest is the structure and function of heparan sulfate (HS). HSs play dynamic functional roles in a diverse number
of biological events related to intracellular signaling, cell-cell interactions and tissue morphogenesis.
HS execute its function by the binding to a variety of molecules including growth factors, serine protease inhibitors and
extracellular matrix proteins.
The biological activities of HS largely depend on the amount and distribution of its sulfate groups that provide specific binding
sites for proteins.
Our overall goal is to understand the mechanisms generating specific saccharide structures and to provide insight into the link
between cell type specific expression of HS modifying enzymes and the biological function of the polysaccharide.
Our research focus on
1. UDP-glucose dehydrogenase, which converts UDP-glucose to UDP-glucuronic acid providing one of the building blocks for
2. heparan sulfate polymerases (EXT1 and EXT2) giving rise to the polysaccharide backbone(mice with a gene trap mutation in
3. 2-O- and 6-O-sulfotransferases, incorporating sulfate groups in specific positions, generating biological active heparan
4. Sulfs, cell associated HS 6-O endosulfatases, that remove sulfate groups in specific positions, thus modulating HS
dependent growth factor signaling.
Howard Hughes Medical Institute Holiday Lectures on Science Programs. This four part lecture series held in 2002 will give you a
better insight and understanding of research that is now being conducted in MHE now. Once you have viewed these 4 lectures
you can view other illustrations on this web-page. Click this link
Chondrocytes (from Greek chondros cartilage + kytos cell) are the only cells found in cartilage. They produce and maintain the
cartilaginous matrix, which consists mainly of collagen and proteoglycans.
To view a short video of what is chondrocyte is please click the tab
Slide from video presentation " What is MHE Research?" by Jeffrey D. Esko, Phd
This presentation will open in a new browser window
Heparan Sulfates - Regulators of Cell Functions
Heparan sulfates (HS): are glycans (complex sugars) found on all cell surfaces
which act by binding selectively to a variety of proteins and pathogens and
are critically relevant to many disease processes (eg., inflammation,
neurodegeneration, angiogenesis, wound healing, cancer, cardiovascular
disorders and infectious diseases). Many of these activities have been detected
using heparin, which is a subclass of the HS family of glycans.
Heparin and heparan sulphates act by binding to proteins and regulating
their biological activities.
The picture shows the interaction of a small heparin hexasaccharide (6 sugar
units) with the growth factor called basic FGF that controls the growth and
differentiation of many cell types.
The HS family of sugars are composed of long chains of repeating disaccharide
units of uronic acid and glucosamine residues, decorated by variable patterns of
sulphate and carboxyl groups, giving them very strong negative charge.
They are produced in living cells by a complex multi-step enzymatic biosynthetic
Heparin is a highly sulphated and relatively structurally homogenous molecule
compared to cellular heparan sulphates, which have increased sequence
diversity and fulfil many complex biological functions by interacting with proteins
and influencing their biological activities.
Animated picture shows an extended helical
heparin sequence with sulphate groups
(yellow/red) decorating the backbone
(image courtesy of Dr Barbara Mulloy,
National Institiute of Biological Standards,
HS and heparin are produced on cells by a complex process involving
the sequential action of multiple enzymes which knit together the repeating
disaccharide units (polymerases) and then modify them with exquisitely
complex patterns of sulphate groups (sulfotransferases). The resulting
structural motifs bind to specific proteins and influence their biological
Heparan sulphate binds proteins
Heparin and heparan sulphates act by binding to proteins and regulating their
The picture shows the interaction of a small heparin hexasaccharide (6 sugar
units) with the growth factor called basic FGF that controls the growth and
differentiation of many cell types.
This information was provided by intellthep
We are grateful for the use of this information
heparan sulfate proteoglycans (HSPGs), are ubiquitous glycoproteins present at the cell surface and in the extracellular matrix,
and have their roles in neuron migration, process outgrowth and guidance and in synapse formation. HSPGs contain a protein
core substituted with heparan sulphate (HS) polysaccharide chains, which encode complex sugar sequences with variant
sulfation patterns that confer biological functions as protein regulators. HS/HSPGs play essential roles in controlling cell
differentiation, tissue morphogenesis and homeostasis. In the nervous system, HS and HSPGs have been implicated in neuron
migration, axon guidance, synapse formation and maturation and control of physiological responses such as feeding, learning
HS and heparin are long, linear chains of sugars, composed of repeating
disaccharide units made up of alternating uronic acid (glucuronic or iduronic
acid) and glucosamine residues. The backbone structure is then decorated
with complex patterns of sulphate groups at various positions.
For more detailed information concerning the Perichondrium, chondrocytes, PTHrP, Ihh and other signaling
pathways affected by the defect in the EXT genes please view the video link below.
To view this video presentation given by Dr. Henry Kronenberg during this conference please click the link tab
The MHE Research would like to thank all for the use of the presentation of The Perichondrium in Bone Development
on the MHE Research Foundation website. This presentation was from the April 25–28, 2007, the 2nd Conference
on Skeletal Biology and Medicine held in NYC.
This meeting, was jointly hosted at the New York Academy of Sciences and Mount Sinai School of Medicine, was organized and
chaired by Mone Zaidi, professor of endocrinology, geriatrics and adult development, and structural and chemical biology at
Mount Sinai. Cochairs were Gerard Karsenty of Columbia University and Steven Teitelbaum of the Washington University School
Henry H. Roehl, Ph.D.
Department of Biomedical Science, The University of Sheffield, United Kingdom
The Roehl laboratory focuses on the role of heparin sulphate proteoglycans (HSPGs) during development of the zebrafish.
Although HSPGs are ubiquitous structural components of the extracellular matrix, they are also thought to play very specific
roles in cell-cell signalling during development. The disaccharide repeats that make up the heparan chains come in 32 different
varieties making heparan sulphate the most information-dense biopolymer found in nature. Binding studies and X-ray
crystallography have identified many specific interactions between oligosaccharides and secreted proteins. Mutational analysis of
genes involved with proteoglycan synthesis has shown that Wingless, Decapentaplegic, Fibroblast Growth Factor and
Hedgehog signalling pathways all depend on proteoglycans at different times during Drosophila development. These data
together have led to the hypothesis that different HSPGs have highly specialized roles including limiting or facilitating signal
diffusion, blocking signal degradation and modulating signal/receptor complex formation.
Our work in this field began with the positional cloning of a small family of zebrafish mutants that all have similar phenotypes
suggesting that their gene products interact or are in the same pathway. These genes, pinscher (pic/papst1), boxer
(box/extl3) and dackel(dak/ext2), are all required for development of the pectoral fins, sorting of the retinotectal projections
and morphogenesis of the skeleton. This cloning project has been a collaboration between three groups (Chi-Bin Chien, U. of
Utah; Robert Geisler, M.P.I. Tuebingen; Henry Roehl, U. of Sheffield). Together, we have found that box and dak encode
glycosyltransferases responsible for synthesis of the heparin sugar chain (EXTL3 and EXT2 respectively), and pic encodes a
sulphur transporter that is involved with the sulphation of all proteogycans (PAPST1). These finding have allowed us to begin
to address the functional requirements of HSPGs during zebrafish development.
Recently we have turned our attention to the role of HSPGs play in a disease called Hereditary Multiple Exostoses (HME). HME is
an autosomal dominant disorder that affects 1 in 50,000 among the general population. Patients with HME have a short stature
and develop numerous cartilage-capped tumours (called exostoses or osteochondromas) from the growth plates of their
longbones. Mutations in human EXT2 account for a large percentage of the cases of this disease. While osteochondromas are
normally benign, they can lead to complications and patients have a 1-2% risk of developing chondrosarcoma or osteosarcoma.
The dominant and sporadic nature of tumour formation in HME patients has led to the proposal of two genetic models.
Osteochondromas may arise from a loss of heterozygosity (LOH) at one of the EXT loci in a developing chondrocyte resulting in
unregulated growth and clonal expansion. In support of this model, somatic mutations or aneuploidy have been found in 3 out
of 46 osteochondromas analysed. The alternative model is that reduced EXT gene dose results in a structural change that
allows chondrocytes to occasionally escape normal developmental constraints to give rise to an osteochondroma.
|Names of researchers listed on this
Regulation of Zebrafish Skeletogenesis by ext2/dackel and papst1/pinscher
Aurélie Clément1,2, Malgorzata Wiweger1,2, Sophia von der Hardt3, Melissa A. Rusch4,5, Scott B. Selleck4,5, Chi-Bin Chien6,7,
Henry H. Roehl1,2*
1 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom2 Department of
Biomedical Science, University of Sheffield, Sheffield, United Kingdom3 Abteilung Genetik, MPI für Entwicklungsbiologie,
Tuebingen, Germany4 Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, United States of America5
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, United States of
America6 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, United States of America7 Brain
Institute, University of Utah, Salt Lake City, Utah, United States of America
Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in
50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood.
Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those
seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it
encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are
required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are
also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that
dak−/− cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic−/− chondrocytes always act
autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new
model to explain the aetiology of HME.
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||Wings of HOPE as we REACH
for the CURE to
Multiple Hereditary Exostoses / Multiple
Maurizio Pacifici, Ph.D.
Department of Orthopaedic Surgery, Thomas Jefferson University, School of Medicine, Philadelphia, PA.
The mechanisms by which exostoses form along the growth plates of long bones and other skeletal elements remain largely
unclear. Since the majority of HME patients carry loss-of-function mutations in Ext1 or Ext2, other groups previously created
heterozygous null Ext1 (Ext1+/-) mice that were expected to display traits of HME patients.
Surprisingly, the mutant mice did not completely mimic the human phenotype. Exostosis-like masses were observed in 10-20%
of the mice only, and the ectopic masses were rather small and atypical in organization and were limited to the ribs. Based on
immunohistochemical evidence that human exostoses contain far less heparan sulfate (HS) than would be expected of a
heterozygous Ext mutation (about 50% of control levels), we reasoned that mice producing lower amounts of HS chains may be
able to mimic the human condition more closely. Thus, we created and examined double heterozygous Ext1+/-/ Ext2+/- mice.
Indeed, the double hets mice did display stereotypic exostoses along their long bones that were characterized by a distal
cartilaginous cap followed by a pseudo growth plate and were oriented at a 90 degree angle with respect to the long axis of the
We even observed osteochondromas masses at other locations. The data strongly indicate that exostosis formation and
organization are intimately sensitive to, and dependent on, HS production and/or content and that frequency of exostosis
formation can be increased by progressive decreases in Ext expression. Data from an additional mouse model of HME and data
from mesenchymal cell cultures that provide important insights into the mechanisms of exostosis induction and formation.
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