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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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Ectopic expression of truncated forms of Tsc2 increases TORC1 activity and causes morphological defects in the brain. (A–D) Lateral view of uninjected wild-type control (A), tsc2 wild-type RNA-injected (B), tsc2vu242 RNA-injected (C) and tsc2/NruI-truncated RNA-injected (D) embryos at the one-somite stage (10.3 hpf). Embryos were labeled with an antibody to phospho-S6 (A–H). (E–H) Dorsal view of A–D. (I–L) Lateral views (anterior to the left) of control (I), tsc2 RNA-injected (J), tsc2vu242 RNA-injected (K) and tsc2/NruI-truncated RNA-injected (L) embryos at 27 hpf. Solid lines mark the hindbrain boundary (I–L). (M) Schematic of constructs used for the production of synthetic RNAs for injection experiments. Scale bars: 100 μm.
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f3-0040255: Ectopic expression of truncated forms of Tsc2 increases TORC1 activity and causes morphological defects in the brain. (A–D) Lateral view of uninjected wild-type control (A), tsc2 wild-type RNA-injected (B), tsc2vu242 RNA-injected (C) and tsc2/NruI-truncated RNA-injected (D) embryos at the one-somite stage (10.3 hpf). Embryos were labeled with an antibody to phospho-S6 (A–H). (E–H) Dorsal view of A–D. (I–L) Lateral views (anterior to the left) of control (I), tsc2 RNA-injected (J), tsc2vu242 RNA-injected (K) and tsc2/NruI-truncated RNA-injected (L) embryos at 27 hpf. Solid lines mark the hindbrain boundary (I–L). (M) Schematic of constructs used for the production of synthetic RNAs for injection experiments. Scale bars: 100 μm.

Mentions: To test whether the tsc2vu242 nonsense mutation can interfere with TORC1 signaling, we overexpressed full-length or truncated Tuberin by injecting synthetic wild-type tsc2 or tsc2vu242 RNA, or an additional truncated-Tuberin-encoding RNA, into wild-type embryos at the one-cell stage. We observed enlarged cells in the superficial enveloping layer in embryos injected with 400 pg of tsc2 RNA containing the tsc2vu242 nonsense mutation, and these cells had increased levels of phospho-S6 during early development (10–11 hpf) (Fig. 3C,G) compared with wild type. A similar phenotype was also generated by overexpressing a similar truncated form of Tuberin, made from a NruI deletion construct of wild-type tsc2 (Fig. 3D,H). By contrast, embryos injected with 300 pg of wild-type tsc2 RNA did not show such abnormalities (Fig. 3B,F). These results suggested that truncated Tuberin generated by the tsc2vu242 nonsense mutation or a deletion construct might interfere with the function of endogenous Tuberin. By 27 hpf, abnormal brain morphology with a dorsally expanded hindbrain was observed in embryos injected with RNA encoding the truncated forms of Tuberin (Fig. 3K,L) but not in those injected with wild-type tsc2 RNA (Fig. 3J). In addition, we observed slightly decreased levels of tsc2 transcripts in heterozygous fish and a marked decrease in homozygous mutants at 26 hpf (supplementary material Fig. S5E,F). Given the position of the premature stop codon, this is probably caused by nonsense-mediated mRNA decay (NMD) (Silva and Romao, 2009). Although levels of mutant Tuberin could then decrease over time compared with wild-type Tuberin, the amount of phospho-Tuberin (Ser939) protein (supplementary material Fig. S3) was relatively stable in mutant larvae at 26 hpf.


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)

Ectopic expression of truncated forms of Tsc2 increases TORC1 activity and causes morphological defects in the brain. (A–D) Lateral view of uninjected wild-type control (A), tsc2 wild-type RNA-injected (B), tsc2vu242 RNA-injected (C) and tsc2/NruI-truncated RNA-injected (D) embryos at the one-somite stage (10.3 hpf). Embryos were labeled with an antibody to phospho-S6 (A–H). (E–H) Dorsal view of A–D. (I–L) Lateral views (anterior to the left) of control (I), tsc2 RNA-injected (J), tsc2vu242 RNA-injected (K) and tsc2/NruI-truncated RNA-injected (L) embryos at 27 hpf. Solid lines mark the hindbrain boundary (I–L). (M) Schematic of constructs used for the production of synthetic RNAs for injection experiments. Scale bars: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046101&req=5

f3-0040255: Ectopic expression of truncated forms of Tsc2 increases TORC1 activity and causes morphological defects in the brain. (A–D) Lateral view of uninjected wild-type control (A), tsc2 wild-type RNA-injected (B), tsc2vu242 RNA-injected (C) and tsc2/NruI-truncated RNA-injected (D) embryos at the one-somite stage (10.3 hpf). Embryos were labeled with an antibody to phospho-S6 (A–H). (E–H) Dorsal view of A–D. (I–L) Lateral views (anterior to the left) of control (I), tsc2 RNA-injected (J), tsc2vu242 RNA-injected (K) and tsc2/NruI-truncated RNA-injected (L) embryos at 27 hpf. Solid lines mark the hindbrain boundary (I–L). (M) Schematic of constructs used for the production of synthetic RNAs for injection experiments. Scale bars: 100 μm.
Mentions: To test whether the tsc2vu242 nonsense mutation can interfere with TORC1 signaling, we overexpressed full-length or truncated Tuberin by injecting synthetic wild-type tsc2 or tsc2vu242 RNA, or an additional truncated-Tuberin-encoding RNA, into wild-type embryos at the one-cell stage. We observed enlarged cells in the superficial enveloping layer in embryos injected with 400 pg of tsc2 RNA containing the tsc2vu242 nonsense mutation, and these cells had increased levels of phospho-S6 during early development (10–11 hpf) (Fig. 3C,G) compared with wild type. A similar phenotype was also generated by overexpressing a similar truncated form of Tuberin, made from a NruI deletion construct of wild-type tsc2 (Fig. 3D,H). By contrast, embryos injected with 300 pg of wild-type tsc2 RNA did not show such abnormalities (Fig. 3B,F). These results suggested that truncated Tuberin generated by the tsc2vu242 nonsense mutation or a deletion construct might interfere with the function of endogenous Tuberin. By 27 hpf, abnormal brain morphology with a dorsally expanded hindbrain was observed in embryos injected with RNA encoding the truncated forms of Tuberin (Fig. 3K,L) but not in those injected with wild-type tsc2 RNA (Fig. 3J). In addition, we observed slightly decreased levels of tsc2 transcripts in heterozygous fish and a marked decrease in homozygous mutants at 26 hpf (supplementary material Fig. S5E,F). Given the position of the premature stop codon, this is probably caused by nonsense-mediated mRNA decay (NMD) (Silva and Romao, 2009). Although levels of mutant Tuberin could then decrease over time compared with wild-type Tuberin, the amount of phospho-Tuberin (Ser939) protein (supplementary material Fig. S3) was relatively stable in mutant larvae at 26 hpf.

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