Phenotypes of conditional Ext1 knockout mice:
Insights into non-skeletal symptoms of MHE
Abstract 2005 MHE Conference
Yu Yamaguchi, M.D., Ph.D.
Developmental Neurobiology Program The Burnham Institute, La Jolla, CA 92037
Heparan sulfate proteoglycans have been implicated in various cell biological and developmental processes, such as growth
factor and morphogen signaling, cell adhesion and migration, and extracellular matrix assembly.
To study the physiological roles of heparan sulfate proteoglycans in the mammalian development, we created conditional allele of
Ext1, the gene encoding a glycosyltransferase required for heparan sulfate biosynthesis.
Mice carrying this allele have been crossed with several Cre transgenic mice to determine the function of heparan sulfate in
different embryonic and adult tissues. I will present our recent findings on the phenotypes of these conditional knockout mice,
and discuss molecular mechanisms underlying such phenotypes and potential implications into non-skeletal symptoms of MHE.
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.
Our conditional knockout mice, which allow knocking out EXT1 at the site and time of researchers' desire, they are very useful
for diverse studies on the function of EXT1/heparan sulfate. Our mice have already been distributed to more than a dozen
laboratories in the world (US, Europe, and Japan) to help studies by other MHE investigators. Meanwhile, the main focus of my
laboratory is the brain, nerves, and muscles.
Through an informal survey conducted by Sarah Ziegler, we have realized that, 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, and muscle weakness (easy to get tired) and pain.
We believe these symptoms can be explained by the deficiency of heparan sulfate in nerve and muscle cells. Indeed, our recent
analysis of knockout mouse behavior has suggested that these mice have deficits in social interaction and reduced levels of
In addition, we have recently discovered that knockout of EXT1 in the “neural crest cell” (which is a cell type that develops into
various bones as well as nerves), causes skeletal defects. This finding has provided us with a new insight into the reason why
“exostosis” develops in MHE patient and into potential MHE treatment paradigm.
Dr. Yamaguchi serves on the Scientific and Medical Advisory Board of the MHE Research Foundation
Research authored by Dr. Yamaguchi
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List of Publications via PubMed
(NIH National Library of Medicine)
|Yu Yamaguchi, M.D., Ph.D., research
|On September 30, 2007 during the FUNTASIA Research banquet Dr. Yamaguchi was presented with the
"The Humanitarian Scientific Achievement Award"
CITATION from Borough of Brooklyn City of New York, Office of the President, Presented
by the President Marty Markowitz.
PROCLAMATION from New York State Senate, Presented by Senator Martin J. Golden.
CERTIFICATE OF RECOGNITION in honor of their Commitment to the MHE Research
Foundation from United States Congress U.S. House of Representatives, Presented by
Congressman Vito J. Fossella.
To read more about this event Click Here
Press Release 5/ 09 / 08
We are pleased to announce Yu Yamaguchi, M.D., PH.D. has been named as one of the three senor investigators at the new
Sanford Children’s Health Research Center located at the Burnham Institute for Medical Research in San Diego CA, where he will
continue his research efforts to read this Press Release Click Here
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Multiple Hereditary Exostoses / Multiple
2009 Conference abstract
Deficiency of Heparan Sulfate in Excitatory Neurons Causes Autism-like Behaviors in Mice
Fumitoshi Irie and Yu Yamaguchi
Sanford Children's Health Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA
92037, USA. e-mail: email@example.com.
The role of heparan sulfate (HS) in mammalian brain development is well established. The function of HS in the brain, however, is
not limited to development. HS is highly concentrated in synapses in the adult brain and the HS proteoglycan syndecan-2
controls the formation of dendritic spines, the key postsynaptic structure of excitatory neurons. Electrophysiologically,
enzymatic elimination of HS has been shown to impair the expression of hippocampal synaptic plasticity. AMPA-class glutamate
receptors have been shown to bind heparin. Considering these biological observations suggesting a synaptic function for HS, it
is of interest that there are reports, both scientific and anecdotal, that MHE is sometimes associated with neurological (such as
generalized pain) and mental (such as autistic traits) conditions.
To study the role of HS in adult brain function and behavior, we generated conditional Ext1 knockout mice specifically targeted to
postnatal excitatory neurons using a CaMKII-Cre transgene. This particular CaMKII-Cre transgene drives recombination
specifically in forebrain excitatory neurons starting only after P14. This property allows us to essentially rule out developmental
defects of the brain as the cause of possible physiological or behavioral phenotypes. Indeed, extensive histological analyses
revealed no detectable abnormalities in the cytoarchitecture of the brain of CaMKII-Cre;Ext1flox/flox mice. Moreover,
CaMKII-Cre;Ext1flox/flox mice have normal visual, olfactory, and motor functions.
Intriguingly, CaMKII-Cre;Ext1flox/flox mice displayed an array of behavioral deficits that are relevant to human autism, namely:
(i) impairment in social activities, such as reduced social interaction with littermates of the same genotype and the avoidance of
unfamiliar wild-type mice; (ii) reduced fear of physical danger; (iii) stereotyped behavior; (iv) hyperlocomotion; and (v)
hypersensitivity to certain types of sensory stimuli. Neuronal activation following social or fear stimulation, as assayed by
immunodetection of rapid induction of c-Fos, was attenuated in the amygdala of CaMKII-Cre;Ext1flox/flox mice.
Electrophysiological analysis revealed that AMPA glutamate receptor-mediated excitatory postsynaptic activity is reduced in the
basal amygdala pyramidal neurons of CaMKII-Cre;Ext1flox/flox mice. Surface expression of AMPA receptors is decreased in the
Ext1-null primary neurons, which is restored by reintroduction of Ext1 by transfection. Mice carrying heterozygous inactivation
of Ext1 in excitatory neurons (CaMKII-Cre;Ext1flox/+) or in the entire brain (Nestin-Cre;Ext1flox/+) displayed a partial
behavioral phenotype. Our results demonstrate that HS plays a physiological role in the regulation of synaptic transmission, and
that its elimination from synapses results in electrophysiological and behavioral deficits. These results also suggest that the
anecdotal information known among families with MHE patients regarding the frequent association of autistic and Asperger-like
traits may be true, and that the mental aspect of MHE would require a systematic scientific study.
2009 Conference abstract
Stochastic Conditional Knockout of Ext1 Reveals an Unexpected Relationship between Biallelic Inactivation of the
Gene and the Development of Multiple Exostoses
Kazu Matsumoto1, Fumitoshi Irie1, Susan Mackem2, and Yu Yamaguchi1
1Sanford Children's Health Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla,
CA 92037, USA. 2Laboratory of Pathology, National Cancer Institute, Bethesda, MD, 20892, USA.
Individuals with MHE carry heterozygous loss-of-function mutations of Ext1 or Ext2, which together encode an enzyme
essential for heparan sulfate synthesis. Despite the unambiguous identification of causative genes, there are a number of
enigmatic issues and unanswered questions surrounding MHE. Among them, three questions are of particular interest: (i)
whether osteochondroma in MHE is a true neoplasm or a developmental defect; (ii) whether loss of heterozygosity is the
underlying genetic mechanism of MHE; and (iii) why Ext1+/– mutant mice, which faithfully mimic the genotype of human MHE,
are resistant to osteochondroma formation, especially in long bones.
To test the hypothesis that biallelic inactivation of Ext1 occurring in a small fraction of chondrocytes is the pathogenic
mechanism of MHE, we employed a method of stochastic inactivation of loxP-flanked Ext1 alleles (Ext1flox) using a tamoxifen-
dependent Cre transgene driven by the Col2a1 promoter (Col2-CreERT). We originally intended to control the level of
recombination using different doses of tamoxifen. Unexpectedly, Col2-CreERT;Ext1flox/flox mice developed multiple
osteochondromas and other MHE-like bone deformities without tamoxifen treatment. We found that the non-induced Col2-
CreERT transgene drives stochastic recombination in a small fraction of chondrocytes (~5% in long bones). (Col2-CreERT;
Ext1flox/flox mice that are raised without tamoxifen treatment are designated as Ext1-SKO [stochastic knockout] mice.) The
penetrance of the long bone exostosis phenotype in Ext1-SKO mice was 100%, whereas bowing deformity and subluxation of
the radius and scoliosis were observed in 92% and 58% of Ext1-SKO mice, respectively. In contrast, neither heterozygous
Ext1-SKO mice (i.e., Col2-CreERT;Ext1flox/+) or Prx1-Cre;Ext1flox/+ mice developed these phenotypes at all, supporting the
requirement for biallelic inactivation. Surprisingly, osteochondromas (cartilage cap region) developed in Ext1-SKO mice are not
clonal growths of Ext1-null chondrocytes, but mixtures of Ext1-null and wild-type chondrocytes at highly variable ratios. This
heterogeneous nature of osteochondroma might be a part of the reason why previous studies on loss of heterozygosity have
not generated an unequivocal conclusion. Our results indicate that, although biallelic inactivation of Ext1 is required for its
initiation, chondrocytes comprising osteochondroma are not clonal, and therefore osteochondroma is not considered to be a
neoplasm in its strictest sense. Our results also suggest that Ext1-null chondrocytes exert unexpectedly potent cell non-
autonomous effects on the behavior of wild-type chondrocytes. This mouse model provides novel insight not only into the
genetic mechanism of MHE but also how heparan sulfate controls tissue development.
Direct link to Yu Yamaguchi's Sanford-Burnham Institute for Medical Research webpage Click Here
Compound heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs and
Zak BM, Schuksz M, Koyama E, Mundy C, Wells DE, Yamaguchi Y, Pacifici M, Esko JD.
Bone. 2011 May 1;48(5):979-87. Epub 2011 Feb 15.
To read this full text publication Click Here
Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases
EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent
mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting
that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain
greater insights into pathogenic mechanisms, we created heterozygous Ext1(+/-) and compound Ext1(+/-)/Ext2(+/-) mice.
Like Ext2(+/-) mice described previously (Stickens et al. Development 132:5055), Ext1(+/-) mice displayed rib-associated
exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more
importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1(+/-)Ext2(+/-) exostoses contained
very low levels of immuno-detectable heparan sulfate, and Ext1(+/-)Ext2(+/-) chondrocytes, endothelial cells and fibroblasts in
vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors
such as FGF-18. We also found that rib outgrowths formed in Ext1(f/+)Col2Cre and Ext1(f/+)Dermo1Cre mice, suggesting
that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study
indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that
exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate
that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.
A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary
Kazu Matsumotoa, Fumitoshi Iriea, Susan Mackemb, and Yu Yamaguchia
To read this full text publication Click Here
Multiple hereditary exostoses (MHE) is one of the most common skeletal dysplasias, exhibiting the formation of multiple
cartilage-capped bony protrusions (osteochondroma) and characteristic bone deformities. Individuals with MHE carry
heterozygous loss-of-function mutations in Ext1 or Ext2, genes which together encode an enzyme essential for heparan
sulfate synthesis. Despite the identification of causative genes, the pathogenesis of MHE remains unclear, especially with regard
to whether osteochondroma results from loss of heterozygosity of the Ext genes. Hampering elucidation of the pathogenic
mechanism of MHE, both Ext1+/− and Ext2+/− heterozygous mutant mice, which mimic the genetic status of human MHE, are
highly resistant to osteochondroma formation, especially in long bones. To address these issues, we created a mouse model in
which Ext1 is stochastically inactivated in a chondrocyte-specific manner. We show that these mice develop multiple
osteochondromas and characteristic bone deformities in a pattern and a frequency that are almost identical to those of human
MHE, suggesting a role for Ext1 LOH in MHE. Surprisingly, however, genotyping and fate mapping analyses reveal that
chondrocytes constituting osteochondromas are mixtures of mutant and wild-type cells. Moreover, osteochondromas do not
possess many typical neoplastic properties. Together, our results suggest that inactivation of Ext1 in a small fraction of
chondrocytes is sufficient for the development of osteochondromas and other skeletal defects associated with MHE. Because
the observed osteochondromas in our mouse model do not arise from clonal growth of chondrocytes, they cannot be
considered true neoplasms.
Roles of Heparan Sulfate in Mammalian Brain Development: Current Views Based on the Findings from Ext1
Conditional Knockout Studies
Yu Yamaguchi, Masaru Inatani, Yoshihiro Matsumoto, Junko Ogawa and Fumitoshi Irie
Development of the mammalian central nervous system proceeds roughly in four major steps, namely the patterning of the
neural tube, generation of neurons from neural stem cells and their migration to genetically predetermined destinations,
extension of axons and dendrites toward target neurons to form neural circuits, and formation of synaptic contacts. Earlier
studies on spatiotemporal expression patterns and in vitro function of heparan sulfate (HS) suggested that HS is functionally
involved in various aspects of neural development. Recent studies using knockout of genes involved in HS biosynthesis have
provided more physiologically relevant information as to the role of HS in mammalian neural development. This chapter reviews
the current understanding of the in vivo function of HS deduced from the phenotypes of conditional Ext1 knockout mice.
Link to abstract
Prog Mol Biol Transl Sci. 2010;93:133-52.
To view the two video presentations of this research Video 1 Click Here Video 2 Click Here
|The 3rd Annual SBMRI Rare Disease Symposium held Feb 24, 2011.
Once again supported by our foundation, to view the video presentations and during this symposium
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Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate.
Irie F, Badie-Mahdavi H, Yamaguchi Y.
Proc Natl Acad Sci U S A. 2012 Mar 12.
Genetic Disease Program, Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA
Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently
have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its
synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly
characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue,
we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential
for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the
cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including
impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as
well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal
activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal
neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of
synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal
functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies
characteristic for autism.