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
fear/anxiety.
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.
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
fear/anxiety.
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)
Research authored by Dr. Yamaguchi
Click the tab and a window will appear.
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" along with 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 |
Questions to the Scientific & Medical Advisory Board,
Please use the contact Scientific & Medical Advisory Board Tab or you may email directly
AdvisoryBoard@mheresearchfoundation.org
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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
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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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: yyamaguchi@burnham.org.
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.
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: yyamaguchi@burnham.org.
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.
e-mail: kmatsu@burnham.org.
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.
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.
e-mail: kmatsu@burnham.org.
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.
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Intute: health & life sciences a free online service providing access to the very best Web resources for education and research located in the UK
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