- Email: [email protected]
Investigation of the neurodegenerative impact of CAG expansion in TBP gene through transgenic mouse model
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
response depends mostly on OPL-derived signals and not brain signals. We cross-bred WBC mice with mice knock-out for the Dlx5 homeogene, a model of lack of olfactory innervation. Grafting of Dlx5 mutant OPL onto WT slice cultures indicates that the connectivity defect is primarily axonal. Conversely, grafting of a Dlx5 mutant OB in WT newborn animals resulted in OB innervation, suggests that the mutant OB is able to accept incoming neurites. We show that in Dlx5 mutant embryos WBC responsive cells are nearly absent, as the expression of several Wnt mRNAs is reduced in the adjacent territory. At the same time, the basement membrane of the forebrain fails to be fenestrated and the formation of the OB nerve layer is prevented. The role of Wnt signalling in olfactory connectivity has been assayed by the use of purified DKK-1 or sFRP2, blockers of the WBC pathway, in slice cultures. Results will be presented. These data highlight the existence of a complex interplay between cell populations of different embryonic origins for the establishment of olfactory connections, in which Wnt signals play a key role.
585
ronal population is complex and not yet fully understood. This makes it important to investigate additional vertebrate models in terms of conserved developmental mechanisms. We performed for the first time a precise mapping of the expression patterns of the chicken orthologs for the mouse mesDA marker genes Aldh1a1, Nr4a2/Nurr1, Pitx3, Lmx1b, En1 and Th during early chicken development. With the in situ hybridisation technique on whole mount embryos or consecutive paraffin sections we investigated the mesDA marker genes at different developmental stages of the chicken embyro. We found striking differences in the spatiotemporal expression of some of these marker genes between the two species mouse and chicken, for example cAldh1a1 was not expressed at all in the developing ventral midbrain in chicken embryos. These differences may reflect partly different functions of the corresponding genes in birds and mammals that arose during evolution, whereas the similarities might show conserved pathways in the development of mesDA neurons. Keywords: mesDA neurons; Chicken; Aldh1a1; Expression pattern doi:10.1016/j.ijdevneu.2006.09.268 [P209] Investigation of the neurodegenerative impact of CAG expansion in TBP gene through transgenic mouse model H.M. Hsieh-Li 1,∗ , C.Y. Lin 1 , L.S. RO 2 , C.M. Chen 2 , G.J. Lee-Chen 1 1 National
Keywords: Wnt; Olfactory; Dlx; Axon growth doi:10.1016/j.ijdevneu.2006.09.267 [P208] Mapping of the midbrain dopaminergic system during development of the chicken embryo reveals evolutionary differences between birds and mammals R. Klafke 1,∗ , A. Wizenmann 2 , W. Wurst 1 , N. Prakash 1 1 Technical University Munich, Germany; 2 GSF-Research Cen-
ter for Environment and Health, Institute of Stem Cell Research, Germany Dopamine-synthesizing (DA) neurons constitute a prominent neuronal population in the vertebrate brain as they are involved in the control and modulation of key cerebral functions. The mesencephalic dopaminergic (mesDA) neurons of the mouse arise from the ventral midbrain during embryonic development. In mouse there are several key factors identified which control their induction, specification, proliferation and differentiation, like Fgf8, Shh and Wnt1. Even though the mouse mesDA system is the best studied, the spatiotemporal development of this neu-
Taiwan Normal University, Taiwan; Memorial Hospital, Taiwan
2 Chang
Gung
Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disorder disease belonging to the autosomal dominant cerebellar ataxia (ADCA). SCA17 is caused by expansion of CAA/CAG trinucleotide repeats in the TATA box-binding protein (TBP) gene. It was proposed that the length of polyglutamine tract encoded by the CAA/CAG repeats related to the disease progression. To investigate the TBP trinucleotide expansion effect on neurodegeneration, we generate transgenic mice expressing the human TBP gene with either normal or expanded CAA/CAG tracts. The transgene was confirmed by Southern blot analysis. RNA and protein expressions of transgenes were detected by RT-PCR ad Western blot analyses, respectively. Behavior analyses were conducted by Rota-rod and grasp strength test. Three lines of these mice have shown behavioral abnormalities. Line-16 mice were observed to have a hind-limb clasping phenotype, which was reported by Huntington’s disease transgenic mice. Line-54 homozygous (tg/tg) mice showed obvious ataxia phenotype at 6-month of age. Line-69 transgenic mice showed the most severe phenotypes with a significant reduction in the Rota-rod latency identified since 2-month-old of age, and ataxia phenotype was observed at 9-month-old of heterozygous (tg/+) mice and 1-month-old homozygous (tg/tg) mice,
586
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
respectively. These results suggest that abnormal TBP with trinucleotide repeat expansion may interfere the normal cerebellar development, which in turn cause the ataxia phenotype of the transgenic mice.
Keywords: Chromatin remodeling; ISWI; Brain development; SNF2L
Keywords: TBP; SCA17; Transgenic mice
[P211]
doi:10.1016/j.ijdevneu.2006.09.269
Mammalian ISWI gene knockdown modulates growth and differentiation properties of neuronal progenitor cells
[P210] SNF2L-mediated control of cell number in the developing brain D.J. Yip ∗ , A. de Maria, J. Coulombe, M. Rudnicki, D.J. Picketts Molecular Medicine Program, Ottawa Health Research Institute, Canada Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however knowledge of the role of these proteins in normal CNS development is limited. In Drosophila, ISWI is essential for development and the protein functions as the ATPhydrolysing component in several chromatin-remodeling complexes. The two mammalian ISWI orthologs, SNF2H and SNF2L are differentially expressed, suggesting that they possess distinct developmental roles. Prevalent expression of SNF2H occurs during neuroprogenitor proliferation while SNF2L transcripts have increased levels in maturing neurons. Here we use conditional Cre-loxP gene-targeting to inactivate the murine Snf2l gene in order to assess its role in neurodevelopment. Heterozygous females (Snf2lf/x ) were crossed to mice expressing Crerecombinase under the control of the GATA-1 promoter. SNF2Lnull male mice were viable and born at classic Mendelian ratios. These mice displayed no overt developmental or behavioral abnormalities; however, the loss of Snf2l resulted in a 2-fold increase in the brain weight to body weight ratio. This was accompanied by a concomitant increase in cell number in the hippocampus and cerebral cortex ranging from 0.5- to 2-fold in the six distinct cortical layers and a 2-fold increase in all hippocampal strata. Moreover we observed a proportionate increase in BrdU staining of the ventricular and intermediate zones of day 15.5 embryonic cortices, coupled with an approximately 3fold increase in cells undergoing mitosis. These results suggest that the increased cell number within the adult brain arose from enhanced embryonic neuroprogenitor proliferation. Finally, loss of Snf2l protein was accompanied by an increase in Snf2h protein levels, suggesting that Snf2h can functionally compensate for the loss of Snf2l in the mutant mice. Taken together, our results indicate that a proper balance of Snf2h to Snf2l protein ratios is an important regulator of brain size and/or cell number.
doi:10.1016/j.ijdevneu.2006.09.270
S. Rennick 1,∗ , D. Picketts 2 1 Ottawa
Health Research Institute, Molecular Medicine Program, Canada; 2 University of Ottawa, Canada Epigenetic modifications during cellular differentiation are critical for establishing the expression of tissue specific genes characterizing particular cell types. There are two groups of chromatin modifying enzymes, the ATP-dependent complexes, and the histone modifying complexes. In ATP-dependent remodelling complexes, the imitation switch (ISWI) group has been studied extensively in the past. Initially ISWI members were discovered in Drosophila and mammals contain two orthologs called SNF2H and SNF2L. Past experiments demonstrated that these remodelers have distinct roles in cellular differentiation and proliferation. SNF2H expression is greatest in proliferating neuroprogenitor populations, whereas SNF2L predominates in mature neurons. Additionally, SNF2H null mice are periimplantation lethal due to defective proliferation. Other studies showed that neuronal cell cultures ectopically expressing increased levels of SNF2L display a dramatic increase in neurite extension and spontaneous differentiation. These findings point to requirements for SNF2L during neuronal differentiation but the distinct manner in which ISWI proteins oversee this process remains unknown. To elucidate ISWIs roles for regulation of neuronal proliferation and differentiation we used shRNA transfection to stably knockdown the genes of interest in established neuronal cell lines. Concurrently, we examined the growth and differentiation properties of primary neuronal cultures derived from mice functionally ablated for Snf2L. Initial results in neuroblastoma cultures show that upon Snf2L knockdown proliferation is maintained in 30% of the population under differentiation conditions. In contrast, Snf2H knockdown cells proliferate normally but undergo extensive apoptosis when induced to differentiate. Aberrant cell replication was also observed in E14 neurosphere cultures and resulted in a 2-fold decrease in the number of neurons generated and a 4-fold reduction in astrocyte production. Moreover, both astrocytes and neurons expressing differentiation markers had reduced neurite extensions and branching patterns suggesting a distinct developmental delay. Taken together these studies define distinct roles for ISWI in growth and differentiation of CNS cells. Keywords: ISWI; Chromatin remodeling; Snf2L doi:10.1016/j.ijdevneu.2006.09.271
response depends mostly on OPL-derived signals and not brain signals. We cross-bred WBC mice with mice knock-out for the Dlx5 homeogene, a model of lack of olfactory innervation. Grafting of Dlx5 mutant OPL onto WT slice cultures indicates that the connectivity defect is primarily axonal. Conversely, grafting of a Dlx5 mutant OB in WT newborn animals resulted in OB innervation, suggests that the mutant OB is able to accept incoming neurites. We show that in Dlx5 mutant embryos WBC responsive cells are nearly absent, as the expression of several Wnt mRNAs is reduced in the adjacent territory. At the same time, the basement membrane of the forebrain fails to be fenestrated and the formation of the OB nerve layer is prevented. The role of Wnt signalling in olfactory connectivity has been assayed by the use of purified DKK-1 or sFRP2, blockers of the WBC pathway, in slice cultures. Results will be presented. These data highlight the existence of a complex interplay between cell populations of different embryonic origins for the establishment of olfactory connections, in which Wnt signals play a key role.
585
ronal population is complex and not yet fully understood. This makes it important to investigate additional vertebrate models in terms of conserved developmental mechanisms. We performed for the first time a precise mapping of the expression patterns of the chicken orthologs for the mouse mesDA marker genes Aldh1a1, Nr4a2/Nurr1, Pitx3, Lmx1b, En1 and Th during early chicken development. With the in situ hybridisation technique on whole mount embryos or consecutive paraffin sections we investigated the mesDA marker genes at different developmental stages of the chicken embyro. We found striking differences in the spatiotemporal expression of some of these marker genes between the two species mouse and chicken, for example cAldh1a1 was not expressed at all in the developing ventral midbrain in chicken embryos. These differences may reflect partly different functions of the corresponding genes in birds and mammals that arose during evolution, whereas the similarities might show conserved pathways in the development of mesDA neurons. Keywords: mesDA neurons; Chicken; Aldh1a1; Expression pattern doi:10.1016/j.ijdevneu.2006.09.268 [P209] Investigation of the neurodegenerative impact of CAG expansion in TBP gene through transgenic mouse model H.M. Hsieh-Li 1,∗ , C.Y. Lin 1 , L.S. RO 2 , C.M. Chen 2 , G.J. Lee-Chen 1 1 National
Keywords: Wnt; Olfactory; Dlx; Axon growth doi:10.1016/j.ijdevneu.2006.09.267 [P208] Mapping of the midbrain dopaminergic system during development of the chicken embryo reveals evolutionary differences between birds and mammals R. Klafke 1,∗ , A. Wizenmann 2 , W. Wurst 1 , N. Prakash 1 1 Technical University Munich, Germany; 2 GSF-Research Cen-
ter for Environment and Health, Institute of Stem Cell Research, Germany Dopamine-synthesizing (DA) neurons constitute a prominent neuronal population in the vertebrate brain as they are involved in the control and modulation of key cerebral functions. The mesencephalic dopaminergic (mesDA) neurons of the mouse arise from the ventral midbrain during embryonic development. In mouse there are several key factors identified which control their induction, specification, proliferation and differentiation, like Fgf8, Shh and Wnt1. Even though the mouse mesDA system is the best studied, the spatiotemporal development of this neu-
Taiwan Normal University, Taiwan; Memorial Hospital, Taiwan
2 Chang
Gung
Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disorder disease belonging to the autosomal dominant cerebellar ataxia (ADCA). SCA17 is caused by expansion of CAA/CAG trinucleotide repeats in the TATA box-binding protein (TBP) gene. It was proposed that the length of polyglutamine tract encoded by the CAA/CAG repeats related to the disease progression. To investigate the TBP trinucleotide expansion effect on neurodegeneration, we generate transgenic mice expressing the human TBP gene with either normal or expanded CAA/CAG tracts. The transgene was confirmed by Southern blot analysis. RNA and protein expressions of transgenes were detected by RT-PCR ad Western blot analyses, respectively. Behavior analyses were conducted by Rota-rod and grasp strength test. Three lines of these mice have shown behavioral abnormalities. Line-16 mice were observed to have a hind-limb clasping phenotype, which was reported by Huntington’s disease transgenic mice. Line-54 homozygous (tg/tg) mice showed obvious ataxia phenotype at 6-month of age. Line-69 transgenic mice showed the most severe phenotypes with a significant reduction in the Rota-rod latency identified since 2-month-old of age, and ataxia phenotype was observed at 9-month-old of heterozygous (tg/+) mice and 1-month-old homozygous (tg/tg) mice,
586
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
respectively. These results suggest that abnormal TBP with trinucleotide repeat expansion may interfere the normal cerebellar development, which in turn cause the ataxia phenotype of the transgenic mice.
Keywords: Chromatin remodeling; ISWI; Brain development; SNF2L
Keywords: TBP; SCA17; Transgenic mice
[P211]
doi:10.1016/j.ijdevneu.2006.09.269
Mammalian ISWI gene knockdown modulates growth and differentiation properties of neuronal progenitor cells
[P210] SNF2L-mediated control of cell number in the developing brain D.J. Yip ∗ , A. de Maria, J. Coulombe, M. Rudnicki, D.J. Picketts Molecular Medicine Program, Ottawa Health Research Institute, Canada Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however knowledge of the role of these proteins in normal CNS development is limited. In Drosophila, ISWI is essential for development and the protein functions as the ATPhydrolysing component in several chromatin-remodeling complexes. The two mammalian ISWI orthologs, SNF2H and SNF2L are differentially expressed, suggesting that they possess distinct developmental roles. Prevalent expression of SNF2H occurs during neuroprogenitor proliferation while SNF2L transcripts have increased levels in maturing neurons. Here we use conditional Cre-loxP gene-targeting to inactivate the murine Snf2l gene in order to assess its role in neurodevelopment. Heterozygous females (Snf2lf/x ) were crossed to mice expressing Crerecombinase under the control of the GATA-1 promoter. SNF2Lnull male mice were viable and born at classic Mendelian ratios. These mice displayed no overt developmental or behavioral abnormalities; however, the loss of Snf2l resulted in a 2-fold increase in the brain weight to body weight ratio. This was accompanied by a concomitant increase in cell number in the hippocampus and cerebral cortex ranging from 0.5- to 2-fold in the six distinct cortical layers and a 2-fold increase in all hippocampal strata. Moreover we observed a proportionate increase in BrdU staining of the ventricular and intermediate zones of day 15.5 embryonic cortices, coupled with an approximately 3fold increase in cells undergoing mitosis. These results suggest that the increased cell number within the adult brain arose from enhanced embryonic neuroprogenitor proliferation. Finally, loss of Snf2l protein was accompanied by an increase in Snf2h protein levels, suggesting that Snf2h can functionally compensate for the loss of Snf2l in the mutant mice. Taken together, our results indicate that a proper balance of Snf2h to Snf2l protein ratios is an important regulator of brain size and/or cell number.
doi:10.1016/j.ijdevneu.2006.09.270
S. Rennick 1,∗ , D. Picketts 2 1 Ottawa
Health Research Institute, Molecular Medicine Program, Canada; 2 University of Ottawa, Canada Epigenetic modifications during cellular differentiation are critical for establishing the expression of tissue specific genes characterizing particular cell types. There are two groups of chromatin modifying enzymes, the ATP-dependent complexes, and the histone modifying complexes. In ATP-dependent remodelling complexes, the imitation switch (ISWI) group has been studied extensively in the past. Initially ISWI members were discovered in Drosophila and mammals contain two orthologs called SNF2H and SNF2L. Past experiments demonstrated that these remodelers have distinct roles in cellular differentiation and proliferation. SNF2H expression is greatest in proliferating neuroprogenitor populations, whereas SNF2L predominates in mature neurons. Additionally, SNF2H null mice are periimplantation lethal due to defective proliferation. Other studies showed that neuronal cell cultures ectopically expressing increased levels of SNF2L display a dramatic increase in neurite extension and spontaneous differentiation. These findings point to requirements for SNF2L during neuronal differentiation but the distinct manner in which ISWI proteins oversee this process remains unknown. To elucidate ISWIs roles for regulation of neuronal proliferation and differentiation we used shRNA transfection to stably knockdown the genes of interest in established neuronal cell lines. Concurrently, we examined the growth and differentiation properties of primary neuronal cultures derived from mice functionally ablated for Snf2L. Initial results in neuroblastoma cultures show that upon Snf2L knockdown proliferation is maintained in 30% of the population under differentiation conditions. In contrast, Snf2H knockdown cells proliferate normally but undergo extensive apoptosis when induced to differentiate. Aberrant cell replication was also observed in E14 neurosphere cultures and resulted in a 2-fold decrease in the number of neurons generated and a 4-fold reduction in astrocyte production. Moreover, both astrocytes and neurons expressing differentiation markers had reduced neurite extensions and branching patterns suggesting a distinct developmental delay. Taken together these studies define distinct roles for ISWI in growth and differentiation of CNS cells. Keywords: ISWI; Chromatin remodeling; Snf2L doi:10.1016/j.ijdevneu.2006.09.271