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Zebrafish as a model for studies on 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.
Zebrafish 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 zebrafish are suitable as a model for studies on human skeletal diseases. Using
forward genetic screens, hundreds of zebrafish 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.
Zebrafish 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 zebrafish are suitable as a model for studies on human skeletal diseases. Using
forward genetic screens, hundreds of zebrafish 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.
| Zebrafish Model Research |
Dr. Yost's research lab
Neuron. 2004 Dec 16;44(6):947-60.
Lee JS, von der Hardt S, Rusch MA, Stringer SE, Stickney HL, Talbot WS, Geisler R, Nusslein-Volhard C, Selleck SB, Chien CB,
Roehl H.
Department of Neurobiology and Anatomy, University of Utah, 20 North 1900 East, Salt Lake City, Utah 84103, USA.
Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish
dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum
topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan
sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical
analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued
by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants,
suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in
robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides
clues to begin understanding the underlying molecular mechanisms.
Lee JS, von der Hardt S, Rusch MA, Stringer SE, Stickney HL, Talbot WS, Geisler R, Nusslein-Volhard C, Selleck SB, Chien CB,
Roehl H.
Department of Neurobiology and Anatomy, University of Utah, 20 North 1900 East, Salt Lake City, Utah 84103, USA.
Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish
dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum
topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan
sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical
analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued
by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants,
suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in
robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides
clues to begin understanding the underlying molecular mechanisms.
Dev Dyn. 2005 Feb;232(2):498-505.
http://www3.interscience.wiley.com/cgi-bin/abstract/109859362/ABSTRACT?CRETRY=1&SRETRY=0
Siekmann AF, Brand M.
Max Planck Institute for Molecular Cell Biology and Genetics, and Department of Genetics,
Dresden University of Technology, Pfotenhauerstr. 108, 01307 Dresden, Germany.
Proteins of the EXT (Exostosin) 1 family are known for their role in human disease. Mutations in EXT1 cause hereditary multiple
exostoses (HME), benign outgrowths of the bones, and therefore were classed as tumor suppressors. More recently, their role
during embryonic development of Drosophila and mouse was addressed, revealing important functions of EXT1 genes in major
signaling pathways. Here, we report the isolation of three zebrafish members of the EXT1 family, which we named ext1a, ext1b,
and ext1c, respectively. They are expressed in restricted temporal and spatial domains during development.
Both ext1a and ext1b are provided maternally and expressed during gastrulation: ext1a in the neurectoderm and ext1b in the
embryonic midline and in the involuting mesendoderm of the germ ring.
During somitogenesis stages, transcripts of all three ext genes can be found in the somitic mesoderm.
Furthermore, ext1a is expressed in the dorsal neural tube. These expression domains become more pronounced at 24 hr
postfertilization (hpf). At 48 hpf, ext1 genes are present in the brain, while somitic expression ceases. Zebrafish have three
members of the EXT1 family, in contrast to only one EXT1 gene in mammals or Xenopus, consistent with the occurrence of
partial genome duplications in the teleost lineage.
Our expression analysis reveals that the three ext genes have distinct expression patterns, reflecting functional divergence
after duplication. In addition, expression of ext1a and ext1c responds to elevated and reduced levels of Sonic hedgehog (shh)
signaling in the somites, whereas expression of ext1b does not.
This suggests a differential relationship between the shh pathway and individual ext gene function in zebrafish. Copyright 2004
Wiley-Liss, Inc.
http://www3.interscience.wiley.com/cgi-bin/abstract/109859362/ABSTRACT?CRETRY=1&SRETRY=0
Siekmann AF, Brand M.
Max Planck Institute for Molecular Cell Biology and Genetics, and Department of Genetics,
Dresden University of Technology, Pfotenhauerstr. 108, 01307 Dresden, Germany.
Proteins of the EXT (Exostosin) 1 family are known for their role in human disease. Mutations in EXT1 cause hereditary multiple
exostoses (HME), benign outgrowths of the bones, and therefore were classed as tumor suppressors. More recently, their role
during embryonic development of Drosophila and mouse was addressed, revealing important functions of EXT1 genes in major
signaling pathways. Here, we report the isolation of three zebrafish members of the EXT1 family, which we named ext1a, ext1b,
and ext1c, respectively. They are expressed in restricted temporal and spatial domains during development.
Both ext1a and ext1b are provided maternally and expressed during gastrulation: ext1a in the neurectoderm and ext1b in the
embryonic midline and in the involuting mesendoderm of the germ ring.
During somitogenesis stages, transcripts of all three ext genes can be found in the somitic mesoderm.
Furthermore, ext1a is expressed in the dorsal neural tube. These expression domains become more pronounced at 24 hr
postfertilization (hpf). At 48 hpf, ext1 genes are present in the brain, while somitic expression ceases. Zebrafish have three
members of the EXT1 family, in contrast to only one EXT1 gene in mammals or Xenopus, consistent with the occurrence of
partial genome duplications in the teleost lineage.
Our expression analysis reveals that the three ext genes have distinct expression patterns, reflecting functional divergence
after duplication. In addition, expression of ext1a and ext1c responds to elevated and reduced levels of Sonic hedgehog (shh)
signaling in the somites, whereas expression of ext1b does not.
This suggests a differential relationship between the shh pathway and individual ext gene function in zebrafish. Copyright 2004
Wiley-Liss, Inc.
Development. 2005 Nov;132(22):4963-73. Epub 2005 Oct 12.
Norton WH, Ledin J, Grandel H, Neumann CJ.
European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf
proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in
vertebrates. Here, we report the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as
the gene disrupted in daedalus.
We find that fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required
for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development.
Consistent with this proposal, we observe a strong genetic interaction between fgf10 and extl3 mutants. Furthermore,
application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast,
application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are
differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of
HSPGs in regulating distinct vertebrate Fgfs.
Norton WH, Ledin J, Grandel H, Neumann CJ.
European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf
proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in
vertebrates. Here, we report the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as
the gene disrupted in daedalus.
We find that fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required
for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development.
Consistent with this proposal, we observe a strong genetic interaction between fgf10 and extl3 mutants. Furthermore,
application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast,
application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are
differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of
HSPGs in regulating distinct vertebrate Fgfs.
For more detailed information please read Dr. Yost's foundation's website page 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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here to verify.# HON Conduct 282463 and is linked on the NIH National Library of Medicine, Directory of Health Organizations (SIS) website, as well as
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online service providing access to the very best Web resources for education and research located in the UK
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