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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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Cell-autonomous activation of TORC1 in tsc2vu242/vu242 mutant cells and non-cell-autonomous disruption of white matter. (A) Schematic of mosaic analyses: cells from wild-type tsc2;Tg(h2afv:GFP) or tsc2vu242/vu242;Tg(h2afv:GFP) donors were transplanted into wild-type host blastulae at 4 hpf, and host embryos were analyzed at 7.5 dpf. (B–L) Coronal brain sections from wild-type embryos receiving either Tg(h2afv:GFP) wild-type donor cells (B–D) or tsc2vu242/vu242; Tg(h2afv:GFP) mutant donor cells (E–L). (C,G) Green (GFP); (D,H) red (phospho-S6); (E,I) green (GFP), red (phospho-S6), blue (DAPI) merged images. (B) Merged image of C and D; (F) merged image of G and H to delineate transplanted cells and those with increased mTORC1 signaling. (J–N) Magnified views of rectangle in I. Asterisks point to wild-type host cells (GFP negative and phospho-S6 negative), which seem to be ectopically positioned within the white matter (M,N). (J–N) The area outlined in yellow marks the normal gray-white matter limits. lfb, lateral forebrain bundle. Scale bars: 50 μm.
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f6-0040255: Cell-autonomous activation of TORC1 in tsc2vu242/vu242 mutant cells and non-cell-autonomous disruption of white matter. (A) Schematic of mosaic analyses: cells from wild-type tsc2;Tg(h2afv:GFP) or tsc2vu242/vu242;Tg(h2afv:GFP) donors were transplanted into wild-type host blastulae at 4 hpf, and host embryos were analyzed at 7.5 dpf. (B–L) Coronal brain sections from wild-type embryos receiving either Tg(h2afv:GFP) wild-type donor cells (B–D) or tsc2vu242/vu242; Tg(h2afv:GFP) mutant donor cells (E–L). (C,G) Green (GFP); (D,H) red (phospho-S6); (E,I) green (GFP), red (phospho-S6), blue (DAPI) merged images. (B) Merged image of C and D; (F) merged image of G and H to delineate transplanted cells and those with increased mTORC1 signaling. (J–N) Magnified views of rectangle in I. Asterisks point to wild-type host cells (GFP negative and phospho-S6 negative), which seem to be ectopically positioned within the white matter (M,N). (J–N) The area outlined in yellow marks the normal gray-white matter limits. lfb, lateral forebrain bundle. Scale bars: 50 μm.

Mentions: The disruption of gray and white matter organization that we found in the pallium, subpallium and thalamic regions could be caused by intrinsic loss of Tsc2, but the heterogeneity of human tubers suggests that additional mechanisms might be possible. To address this issue, we made mosaic zebrafish by transplanting tsc2vu242/vu242 cells into wild-type host embryos (schematic in Fig. 6A). Donor cells were obtained from a tsc2vu242/+;Tg(h2afv:GFP) transgenic line, in which all cell nuclei express GFP (Pauls et al., 2001). Tsc2vu242/+;Tg(h2afv:GFP) fish were intercrossed and their progenies were used as donor embryos in the transplantation experiment (Fig. 6B-N). During transplantations at the late blastula stage (4 hpf), donor cells were taken from the animal pole region and transplanted to the same region because most of the cells in this region normally give rise to forebrain and retina (Woo and Fraser, 1995). Analyses of chimeric embryos at 7.5 dpf revealed that tsc2vu242/vu242;Tg(h2afv:GFP) mutant cells, marked by GFP expression in the nuclei, showed a strongly increased level of TORC1 activity, as indicated by increased levels of phospho-S6 (Fig. 6F-L; n=20/20). We also observed a marked disruption of gray-white matter borders in a subset of the chimeric embryos (Fig. 6I, rectangle, enlarged in J–N; n=3/18), similar to the phenotype observed in tsc2vu242/vu242 mutants (Fig. 5E-H). Strikingly, we also noted several wild-type host cell bodies (GFP negative), which, as expected, showed no increase in phospho-S6 staining, that were surrounded by mutant cells within the lateral forebrain bundle (Fig. 6J-N). By contrast, wild-type and tsc2vu242/+ cells transplanted into wild-type hosts did not have increased TORC1 signaling and did not cause any brain abnormalities (Fig. 6B-E; n=6/6). These results suggest that tsc2vu242/vu242 homozygous mutant cells activate TORC1 in a cell-autonomous manner but also in a non-cell-autonomous manner, which can cause wild-type cells to be abnormally positioned within the white matter. Because such abnormalities are seen in the brains from patients with TSC (Park et al., 1997; Shepherd et al., 1995), this observation further supports the intriguing possibility that LOH might not be absolutely required in all cells for the formation of brain hamartomas (tubers) in TSC (Talos et al., 2008).


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)

Cell-autonomous activation of TORC1 in tsc2vu242/vu242 mutant cells and non-cell-autonomous disruption of white matter. (A) Schematic of mosaic analyses: cells from wild-type tsc2;Tg(h2afv:GFP) or tsc2vu242/vu242;Tg(h2afv:GFP) donors were transplanted into wild-type host blastulae at 4 hpf, and host embryos were analyzed at 7.5 dpf. (B–L) Coronal brain sections from wild-type embryos receiving either Tg(h2afv:GFP) wild-type donor cells (B–D) or tsc2vu242/vu242; Tg(h2afv:GFP) mutant donor cells (E–L). (C,G) Green (GFP); (D,H) red (phospho-S6); (E,I) green (GFP), red (phospho-S6), blue (DAPI) merged images. (B) Merged image of C and D; (F) merged image of G and H to delineate transplanted cells and those with increased mTORC1 signaling. (J–N) Magnified views of rectangle in I. Asterisks point to wild-type host cells (GFP negative and phospho-S6 negative), which seem to be ectopically positioned within the white matter (M,N). (J–N) The area outlined in yellow marks the normal gray-white matter limits. lfb, lateral forebrain bundle. Scale bars: 50 μm.
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Related In: Results  -  Collection

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f6-0040255: Cell-autonomous activation of TORC1 in tsc2vu242/vu242 mutant cells and non-cell-autonomous disruption of white matter. (A) Schematic of mosaic analyses: cells from wild-type tsc2;Tg(h2afv:GFP) or tsc2vu242/vu242;Tg(h2afv:GFP) donors were transplanted into wild-type host blastulae at 4 hpf, and host embryos were analyzed at 7.5 dpf. (B–L) Coronal brain sections from wild-type embryos receiving either Tg(h2afv:GFP) wild-type donor cells (B–D) or tsc2vu242/vu242; Tg(h2afv:GFP) mutant donor cells (E–L). (C,G) Green (GFP); (D,H) red (phospho-S6); (E,I) green (GFP), red (phospho-S6), blue (DAPI) merged images. (B) Merged image of C and D; (F) merged image of G and H to delineate transplanted cells and those with increased mTORC1 signaling. (J–N) Magnified views of rectangle in I. Asterisks point to wild-type host cells (GFP negative and phospho-S6 negative), which seem to be ectopically positioned within the white matter (M,N). (J–N) The area outlined in yellow marks the normal gray-white matter limits. lfb, lateral forebrain bundle. Scale bars: 50 μm.
Mentions: The disruption of gray and white matter organization that we found in the pallium, subpallium and thalamic regions could be caused by intrinsic loss of Tsc2, but the heterogeneity of human tubers suggests that additional mechanisms might be possible. To address this issue, we made mosaic zebrafish by transplanting tsc2vu242/vu242 cells into wild-type host embryos (schematic in Fig. 6A). Donor cells were obtained from a tsc2vu242/+;Tg(h2afv:GFP) transgenic line, in which all cell nuclei express GFP (Pauls et al., 2001). Tsc2vu242/+;Tg(h2afv:GFP) fish were intercrossed and their progenies were used as donor embryos in the transplantation experiment (Fig. 6B-N). During transplantations at the late blastula stage (4 hpf), donor cells were taken from the animal pole region and transplanted to the same region because most of the cells in this region normally give rise to forebrain and retina (Woo and Fraser, 1995). Analyses of chimeric embryos at 7.5 dpf revealed that tsc2vu242/vu242;Tg(h2afv:GFP) mutant cells, marked by GFP expression in the nuclei, showed a strongly increased level of TORC1 activity, as indicated by increased levels of phospho-S6 (Fig. 6F-L; n=20/20). We also observed a marked disruption of gray-white matter borders in a subset of the chimeric embryos (Fig. 6I, rectangle, enlarged in J–N; n=3/18), similar to the phenotype observed in tsc2vu242/vu242 mutants (Fig. 5E-H). Strikingly, we also noted several wild-type host cell bodies (GFP negative), which, as expected, showed no increase in phospho-S6 staining, that were surrounded by mutant cells within the lateral forebrain bundle (Fig. 6J-N). By contrast, wild-type and tsc2vu242/+ cells transplanted into wild-type hosts did not have increased TORC1 signaling and did not cause any brain abnormalities (Fig. 6B-E; n=6/6). These results suggest that tsc2vu242/vu242 homozygous mutant cells activate TORC1 in a cell-autonomous manner but also in a non-cell-autonomous manner, which can cause wild-type cells to be abnormally positioned within the white matter. Because such abnormalities are seen in the brains from patients with TSC (Park et al., 1997; Shepherd et al., 1995), this observation further supports the intriguing possibility that LOH might not be absolutely required in all cells for the formation of brain hamartomas (tubers) in TSC (Talos et al., 2008).

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