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Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin.

Kim SH, Speirs CK, Solnica-Krezel L, Ess KC - Dis Model Mech (2010)

Bottom Line: However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner.These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC.The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

View Article: PubMed Central - PubMed

Affiliation: Vanderbilt University, Department of Biological Sciences, Nashville, TN 37232, USA.

ABSTRACT
Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2(vu242) allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2(vu242) is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2(vu242/vu242) homozygous zebrafish, and is moderately increased in tsc2(vu242/+) heterozygotes. Forebrain neurons are poorly organized in tsc2(vu242/vu242) homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

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Adult brain abnormalities after tsc2vu242/vu242 mutant cell transplantation. (A–L) Coronal sections of adult brain at 1 year of age. (A) Green (GFP) indicates transplanted wild-type cells, blue (DAPI) shows nuclei and red shows phospho-S6 staining. (B) DAPI channel of A. (C–L) Cell clusters were found 1 year after transplantation in wild-type host zebrafish of tsc2vu242/vu242;Tg(β-actin:mGFP) donor cells. (C,E,G,I,L) Green cells indicate transplanted tsc2vu242/vu242;Tg(β-actin:mGFP) cells. (D,H,K) DAPI staining showing disruption of gray and white matter in wild-type host brain. (C,G) Distance between the two cortical sections shown is 50 μm. (F,J) Red shows phospho-S6 staining. (D,H) Selected areas surrounded by yellow line indicate a disruption of gray and white matter. (C) Merged image of D–F; (L) merged image of I–K. (M) Schematic of brain structure (rectangle) for I–L. TeO, tectum opticum; HaV, ventral habenular nucleus; AT, anterior thalamic nucleus; VL, ventrolateral thalam. Scale bars: 50 μm (A–H); 200 μm (I–L).
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f7-0040255: Adult brain abnormalities after tsc2vu242/vu242 mutant cell transplantation. (A–L) Coronal sections of adult brain at 1 year of age. (A) Green (GFP) indicates transplanted wild-type cells, blue (DAPI) shows nuclei and red shows phospho-S6 staining. (B) DAPI channel of A. (C–L) Cell clusters were found 1 year after transplantation in wild-type host zebrafish of tsc2vu242/vu242;Tg(β-actin:mGFP) donor cells. (C,E,G,I,L) Green cells indicate transplanted tsc2vu242/vu242;Tg(β-actin:mGFP) cells. (D,H,K) DAPI staining showing disruption of gray and white matter in wild-type host brain. (C,G) Distance between the two cortical sections shown is 50 μm. (F,J) Red shows phospho-S6 staining. (D,H) Selected areas surrounded by yellow line indicate a disruption of gray and white matter. (C) Merged image of D–F; (L) merged image of I–K. (M) Schematic of brain structure (rectangle) for I–L. TeO, tectum opticum; HaV, ventral habenular nucleus; AT, anterior thalamic nucleus; VL, ventrolateral thalam. Scale bars: 50 μm (A–H); 200 μm (I–L).

Mentions: Given the predominance of rodent models of human disease and the marked difficulty in performing transplantation experiments in these models, it remains unknown whether Tsc1 or Tsc2 mutant cells can produce the focal brain hamartomas that are almost universally seen in patients with TSC. To address whether tsc2vu242/vu242 mutant cells can produce ‘tuber-like’ abnormalities, we transplanted membrane GFP-tagged tsc2+/+;Tg(β-actin:mGFP) and tsc2vu242/vu242;Tg(β-actin:mGFP) donor cells into wild-type host embryos at the blastula stage. The resulting chimeras were raised to adulthood and sacrificed 1 year after transplantation. We analyzed transverse sections of the chimeric brains with respect to TORC1 activation and tissue architecture. These analyses revealed that the transplanted wild-type Tg(β-actin:mGFP) cells exhibited normal levels of TORC1 pathway activity and did not cause any obvious defects in the host brain (Fig. 7A,B). By contrast, transplanted tsc2vu242/vu242;Tg(β-actin:mGFP) cells showed elevated levels of TORC1 activity compared with the surrounding wild-type cells (Fig. 7C,F). Moreover, we noted clusters of transplanted mutant cells in the gray-white matter boundary (Fig. 7G,H) and within the gray matter in a series of sections (Fig. 7C,D). These GFP-positive cell clusters were detected through 100 μm of contiguous sections (data not shown), suggesting that they formed an extensive cluster. There seemed to be at least two types of transplanted cells in each cell cluster: relatively small and rounded cells were present (Fig. 7E) but there were also elongated and enlarged cells with dendrites, suggestive of astrocytes and/or glial cells (Fig. 7G). We were unable to confirm the lineage of these intriguing cells because there are few antibodies currently available in zebrafish that are specific to glial cell populations. These brain lesions with mixed cell types and increased TORC1 signaling suggest the formation of brain hamartomas. Further examples of these lesions in the thalamic region are notable for evidence of further disruption of brain architecture (Fig. 7I-M). These mosaic experiments indicate that tsc2vu242/vu242 mutant cells can produce local lesions with increased TORC1 signaling as well as more-extensive malformations in the adult wild-type brain.


Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin.

Kim SH, Speirs CK, Solnica-Krezel L, Ess KC - Dis Model Mech (2010)

Adult brain abnormalities after tsc2vu242/vu242 mutant cell transplantation. (A–L) Coronal sections of adult brain at 1 year of age. (A) Green (GFP) indicates transplanted wild-type cells, blue (DAPI) shows nuclei and red shows phospho-S6 staining. (B) DAPI channel of A. (C–L) Cell clusters were found 1 year after transplantation in wild-type host zebrafish of tsc2vu242/vu242;Tg(β-actin:mGFP) donor cells. (C,E,G,I,L) Green cells indicate transplanted tsc2vu242/vu242;Tg(β-actin:mGFP) cells. (D,H,K) DAPI staining showing disruption of gray and white matter in wild-type host brain. (C,G) Distance between the two cortical sections shown is 50 μm. (F,J) Red shows phospho-S6 staining. (D,H) Selected areas surrounded by yellow line indicate a disruption of gray and white matter. (C) Merged image of D–F; (L) merged image of I–K. (M) Schematic of brain structure (rectangle) for I–L. TeO, tectum opticum; HaV, ventral habenular nucleus; AT, anterior thalamic nucleus; VL, ventrolateral thalam. Scale bars: 50 μm (A–H); 200 μm (I–L).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f7-0040255: Adult brain abnormalities after tsc2vu242/vu242 mutant cell transplantation. (A–L) Coronal sections of adult brain at 1 year of age. (A) Green (GFP) indicates transplanted wild-type cells, blue (DAPI) shows nuclei and red shows phospho-S6 staining. (B) DAPI channel of A. (C–L) Cell clusters were found 1 year after transplantation in wild-type host zebrafish of tsc2vu242/vu242;Tg(β-actin:mGFP) donor cells. (C,E,G,I,L) Green cells indicate transplanted tsc2vu242/vu242;Tg(β-actin:mGFP) cells. (D,H,K) DAPI staining showing disruption of gray and white matter in wild-type host brain. (C,G) Distance between the two cortical sections shown is 50 μm. (F,J) Red shows phospho-S6 staining. (D,H) Selected areas surrounded by yellow line indicate a disruption of gray and white matter. (C) Merged image of D–F; (L) merged image of I–K. (M) Schematic of brain structure (rectangle) for I–L. TeO, tectum opticum; HaV, ventral habenular nucleus; AT, anterior thalamic nucleus; VL, ventrolateral thalam. Scale bars: 50 μm (A–H); 200 μm (I–L).
Mentions: Given the predominance of rodent models of human disease and the marked difficulty in performing transplantation experiments in these models, it remains unknown whether Tsc1 or Tsc2 mutant cells can produce the focal brain hamartomas that are almost universally seen in patients with TSC. To address whether tsc2vu242/vu242 mutant cells can produce ‘tuber-like’ abnormalities, we transplanted membrane GFP-tagged tsc2+/+;Tg(β-actin:mGFP) and tsc2vu242/vu242;Tg(β-actin:mGFP) donor cells into wild-type host embryos at the blastula stage. The resulting chimeras were raised to adulthood and sacrificed 1 year after transplantation. We analyzed transverse sections of the chimeric brains with respect to TORC1 activation and tissue architecture. These analyses revealed that the transplanted wild-type Tg(β-actin:mGFP) cells exhibited normal levels of TORC1 pathway activity and did not cause any obvious defects in the host brain (Fig. 7A,B). By contrast, transplanted tsc2vu242/vu242;Tg(β-actin:mGFP) cells showed elevated levels of TORC1 activity compared with the surrounding wild-type cells (Fig. 7C,F). Moreover, we noted clusters of transplanted mutant cells in the gray-white matter boundary (Fig. 7G,H) and within the gray matter in a series of sections (Fig. 7C,D). These GFP-positive cell clusters were detected through 100 μm of contiguous sections (data not shown), suggesting that they formed an extensive cluster. There seemed to be at least two types of transplanted cells in each cell cluster: relatively small and rounded cells were present (Fig. 7E) but there were also elongated and enlarged cells with dendrites, suggestive of astrocytes and/or glial cells (Fig. 7G). We were unable to confirm the lineage of these intriguing cells because there are few antibodies currently available in zebrafish that are specific to glial cell populations. These brain lesions with mixed cell types and increased TORC1 signaling suggest the formation of brain hamartomas. Further examples of these lesions in the thalamic region are notable for evidence of further disruption of brain architecture (Fig. 7I-M). These mosaic experiments indicate that tsc2vu242/vu242 mutant cells can produce local lesions with increased TORC1 signaling as well as more-extensive malformations in the adult wild-type brain.

Bottom Line: However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner.These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC.The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

View Article: PubMed Central - PubMed

Affiliation: Vanderbilt University, Department of Biological Sciences, Nashville, TN 37232, USA.

ABSTRACT
Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2(vu242) allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2(vu242) is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2(vu242/vu242) homozygous zebrafish, and is moderately increased in tsc2(vu242/+) heterozygotes. Forebrain neurons are poorly organized in tsc2(vu242/vu242) homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

Show MeSH
Related in: MedlinePlus