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Zebra fish as a model for studies on the hereditary multiple exostosis.
Abstract 2005 MHE Conference
Malgorzata Wiweger, Aurélie Clément and Henry Roehl
Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Firth Court, Sheffield
S10 2TN, UK.
Zebra fish is an easy to maintain, small tropical fish with transparent embryos that develop outside the mother’s body. Their short
generation time (3-4 months), high fertility rate (hundreds of eggs per week), rapid development (most organs develop within first
48 hours post fertilization) and advanced genetic techniques (transgenics, forward and reverse genetics) make them an
outstanding model for biomedical research. Furthermore, development of their cartilaginous skeleton occurs by similar mechanisms
to that of humans, which means zebra fish are suitable as a model for studies on human skeletal diseases.
Using forward genetic screens, hundreds of zebra fish mutations that affect cartilage morphogenesis and/or differentiation have
been identified. We positionally cloned two mutations in exostosin genes: dackel (dak/ext2) and boxer (box/exlt3) (Lee et al.,
2004, Neuron. 44: 947-960) and are currently using these to study the development of exostoses. Homozygous dak/ext2
mutants show a similar disorganization of cartilaginous skeleton to that seen in HME tumors i.e. chondrocytes, instead of forming
long stacks of flattened cells, form non-polarized clusters of rounded cells.
Cartilage formation and differentiation remains unaffected in dak/ext2 mutants, which suggest that the dak/ext2 phenotype
probably results from changes in cell division planes or/and cell movements. In support, electron microscope observations verified
the presence of abnormalities in the cytoskeleton of dak/ext2 mutant chondrocytes. Furthermore, malformations of cartilage similar
to those seen in dak/ext2 are also present in another zebrafish mutant called pipetail (ppt/wnt5a) that is involved in the non-
canonical Wnt/Ca2+ planar polarity pathway.
Interestingly, both dak and ppt mutants show a significant delay in endochondrial ossification whereas membranous bones are
formed normally. The similarities between these two mutants suggest Ext2 may act through non-canonical Wnt signaling.
In addition to the exostoses-like phenotype, dak/ext2 and box/exlt3 also share other developmental defects (missorted
retinotectal projections and malformed pectoral fins) and both of these mutants have significantly reduced level of heparan sulfate
proteoglycans (HSPGs). A third mutant, called pinscher (pic), also has the mentioned above defects, suggesting that pic is in the
same genetic pathway.
We have recently positionally cloned pic and shown it does not belong the to exostosin gene family.
This strengthens the possibility that non-Ext1/Ext2-related cases of HME might be due to mutations in other genes involved in the
synthesis of HSPGs.
This work is supported by Wellcome Trust and Cancer Research UK.
Abstract 2005 MHE Conference
Malgorzata Wiweger, Aurélie Clément and Henry Roehl
Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Firth Court, Sheffield
S10 2TN, UK.
Zebra fish is an easy to maintain, small tropical fish with transparent embryos that develop outside the mother’s body. Their short
generation time (3-4 months), high fertility rate (hundreds of eggs per week), rapid development (most organs develop within first
48 hours post fertilization) and advanced genetic techniques (transgenics, forward and reverse genetics) make them an
outstanding model for biomedical research. Furthermore, development of their cartilaginous skeleton occurs by similar mechanisms
to that of humans, which means zebra fish are suitable as a model for studies on human skeletal diseases.
Using forward genetic screens, hundreds of zebra fish mutations that affect cartilage morphogenesis and/or differentiation have
been identified. We positionally cloned two mutations in exostosin genes: dackel (dak/ext2) and boxer (box/exlt3) (Lee et al.,
2004, Neuron. 44: 947-960) and are currently using these to study the development of exostoses. Homozygous dak/ext2
mutants show a similar disorganization of cartilaginous skeleton to that seen in HME tumors i.e. chondrocytes, instead of forming
long stacks of flattened cells, form non-polarized clusters of rounded cells.
Cartilage formation and differentiation remains unaffected in dak/ext2 mutants, which suggest that the dak/ext2 phenotype
probably results from changes in cell division planes or/and cell movements. In support, electron microscope observations verified
the presence of abnormalities in the cytoskeleton of dak/ext2 mutant chondrocytes. Furthermore, malformations of cartilage similar
to those seen in dak/ext2 are also present in another zebrafish mutant called pipetail (ppt/wnt5a) that is involved in the non-
canonical Wnt/Ca2+ planar polarity pathway.
Interestingly, both dak and ppt mutants show a significant delay in endochondrial ossification whereas membranous bones are
formed normally. The similarities between these two mutants suggest Ext2 may act through non-canonical Wnt signaling.
In addition to the exostoses-like phenotype, dak/ext2 and box/exlt3 also share other developmental defects (missorted
retinotectal projections and malformed pectoral fins) and both of these mutants have significantly reduced level of heparan sulfate
proteoglycans (HSPGs). A third mutant, called pinscher (pic), also has the mentioned above defects, suggesting that pic is in the
same genetic pathway.
We have recently positionally cloned pic and shown it does not belong the to exostosin gene family.
This strengthens the possibility that non-Ext1/Ext2-related cases of HME might be due to mutations in other genes involved in the
synthesis of HSPGs.
This work is supported by Wellcome Trust and Cancer Research UK.
The MHE Research Foundation would like to acknowledge and thank Dr. Karlstrom, Biology Department, University of Massachusetts
and Dr. Kane, Dept of Biology, University of Rochester for the use of the zebrafish video.
and Dr. Kane, Dept of Biology, University of Rochester for the use of the zebrafish video.
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Research authored by Dr. Roehl
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List of Publications via PubMed
(NIH National Library of Medicine)
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List of Publications via PubMed
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| Henry Roehl, Ph.D., research |
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Dr. Roehl serves on the Scientific and Medical Advisory Board of the MHE Research Foundation
Dr. Roehl presented his latest research during the 2007 Society For Glycobiology Conference held in Boston.
Regulation of skeletal morphogenesis by sulphated proteoglycans in zebrafish
To read the abstract from this conference presentation Click Here
Regulation of skeletal morphogenesis by sulphated proteoglycans in zebrafish
To read the abstract from this conference presentation Click Here
Press Release 8/02/08
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
Abstract
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.
To read full text publication Click Here
The MHE Research Foundation would like to extend its appreciation to Dr. Henry Roehl who is a member of our foundation's
Scientific and Medical Advisory Board and to Dr. Malgorzata Wiweger for all of their continuing, dedicated research efforts on
behalf of all people affected by MHE/MO/HME
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
Abstract
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.
To read full text publication Click Here
The MHE Research Foundation would like to extend its appreciation to Dr. Henry Roehl who is a member of our foundation's
Scientific and Medical Advisory Board and to Dr. Malgorzata Wiweger for all of their continuing, dedicated research efforts on
behalf of all people affected by MHE/MO/HME
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2009 Conference abatract
Zebrafish As a Model for Multiple Hereditary Exostoses
Aurelie Clement, Malgorzata Wiweger and Henry H. Roehl
Department of Biomedical Sciences, University of Sheffield.
e-mail: h.roehl@sheffield.ac.uk
As part of a large scale forward genetic screen for zebrafish developmental mutants, more then twenty alleles (representing >6
genes) were isolated that confer a "cartilage-tumour" phenotype. In collaboration with the Chein Laboratory (University of Utah,
Salt Lake City), we have positionally cloned alleles for three of these genes and found that they encode null mutations in Ext2, Extl3
and Papt1. We have shown that all three genes are required for synthesis of heparin sulphate (HS). Surprisingly, homozygous
mutant embryos develop normally and have only have defects in four processes: pectoral fin development, axon sorting, osteoblast
differentiation and cartilage morphogenesis. This is explained in part by the presence of maternally provided transcripts for these
three genes in the early embryo.
In order to elucidate the roles of HS during skeletal development, we have analysed the skeletal defects of ext2-/- fish. During early
development, nascent chondrocytes flatten and intercalate to give rise to a morphology that resembles a 'stack of pennies'. In
ext2-/- embryos this process does not occur and chondrocytes remain round and disorganized. Early chondrogenesis proceeds
normally indicating that HS is required during morphogenesis and not differentiation of the cartilage skeleton. Mutations in zebrafish
wnt5a confer a similar phenotype, suggesting that HS acts with the non-cannonical Wnt pathway to establish cell polarity within
pre-cartilage condensations. In addition, both perichondral bone formation as well as chondrocyte hypertrophy are lost in ext2-/-
mutants indicating that chondral ossification also requires HS. Finally, dermal bone ossification and expression of markers of
osteoblast differentiation are reduced in ext2-/- larvae.
There are currently two models for the genetic mechanism that gives rise to osteochondromas in MHE patients: reduced gene
dosage and loss-of-heterozygosity(LOH). One caveat in the LOH model is that HS is secreted and thus a homozygous mutant cell
that arises may be rescued by neighbouring cells. To test this directly, we transplanted ext2-/- cells into wildtype fish and assayed
chondrocyte polarity. In most cases, ext2-/- cells were rescued by neighbouring wildtype chondrocytes and stacked normally.
However, in several cases the mutant chondrocytes behaved autonomously and formed an apolar cluster of cells on the edge of the
cartilage. This data argues that mutant cells can loose polarity and that cells that lie on the edge of the cartilage may become
autonomous because of the lack of contact with neighbouring wildtype cells.
Zebrafish As a Model for Multiple Hereditary Exostoses
Aurelie Clement, Malgorzata Wiweger and Henry H. Roehl
Department of Biomedical Sciences, University of Sheffield.
e-mail: h.roehl@sheffield.ac.uk
As part of a large scale forward genetic screen for zebrafish developmental mutants, more then twenty alleles (representing >6
genes) were isolated that confer a "cartilage-tumour" phenotype. In collaboration with the Chein Laboratory (University of Utah,
Salt Lake City), we have positionally cloned alleles for three of these genes and found that they encode null mutations in Ext2, Extl3
and Papt1. We have shown that all three genes are required for synthesis of heparin sulphate (HS). Surprisingly, homozygous
mutant embryos develop normally and have only have defects in four processes: pectoral fin development, axon sorting, osteoblast
differentiation and cartilage morphogenesis. This is explained in part by the presence of maternally provided transcripts for these
three genes in the early embryo.
In order to elucidate the roles of HS during skeletal development, we have analysed the skeletal defects of ext2-/- fish. During early
development, nascent chondrocytes flatten and intercalate to give rise to a morphology that resembles a 'stack of pennies'. In
ext2-/- embryos this process does not occur and chondrocytes remain round and disorganized. Early chondrogenesis proceeds
normally indicating that HS is required during morphogenesis and not differentiation of the cartilage skeleton. Mutations in zebrafish
wnt5a confer a similar phenotype, suggesting that HS acts with the non-cannonical Wnt pathway to establish cell polarity within
pre-cartilage condensations. In addition, both perichondral bone formation as well as chondrocyte hypertrophy are lost in ext2-/-
mutants indicating that chondral ossification also requires HS. Finally, dermal bone ossification and expression of markers of
osteoblast differentiation are reduced in ext2-/- larvae.
There are currently two models for the genetic mechanism that gives rise to osteochondromas in MHE patients: reduced gene
dosage and loss-of-heterozygosity(LOH). One caveat in the LOH model is that HS is secreted and thus a homozygous mutant cell
that arises may be rescued by neighbouring cells. To test this directly, we transplanted ext2-/- cells into wildtype fish and assayed
chondrocyte polarity. In most cases, ext2-/- cells were rescued by neighbouring wildtype chondrocytes and stacked normally.
However, in several cases the mutant chondrocytes behaved autonomously and formed an apolar cluster of cells on the edge of the
cartilage. This data argues that mutant cells can loose polarity and that cells that lie on the edge of the cartilage may become
autonomous because of the lack of contact with neighbouring wildtype cells.
| Photo's taken during the Third International MHE Research Conference |
2012 publication
HSPG-Deficient Zebrafish Uncovers Dental Aspect of Multiple Osteochondromas.
Wiweger MI, Zhao Z, van Merkesteyn RJ, Roehl HH, Hogendoorn PC.
PLoS One. 2012;7(1):e29734. Epub 2012 Jan 11.
To read this full text publication Click here
HSPG-Deficient Zebrafish Uncovers Dental Aspect of Multiple Osteochondromas.
Wiweger MI, Zhao Z, van Merkesteyn RJ, Roehl HH, Hogendoorn PC.
PLoS One. 2012;7(1):e29734. Epub 2012 Jan 11.
To read this full text publication Click here
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