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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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Enlarged hepatocytes and brain and spinal cord neurons in tsc2vu242/vu242 mutant zebrafish and suppression of hepatocyte size by inhibition of TORC1 activity. (A–F) Transverse section of liver (A,B), brain (C,D) and spinal cord (E,F). (G) Hepatocyte size differences in control siblings and tsc2vu242/vu242 mutants are shown. (H) The relative size of neuronal cells in the brain and spinal cord were compared. (I) Homozygous mutant zebrafish have increased size of their spinal cord. (J) Cell number within the spinal cord in control and tsc2vu242/vu242 mutants. (K) Transverse sections through the trunk of 4.5-day-old embryos treated with different concentrations of rapamycin from 3.5 dpf to 4.5 dpf were stained with antibody to phospho-S6 (red). (L) Transverse section of livers from wild-type and tsc2vu242/vu242 mutants without rapamycin treatment, or with 5 nM or 50 nM rapamycin treatment. Cells outlined in yellow were measured and soma size compared. (M) Graph of measurements of relative sizes of cells from embryo shown in L. Numbers on the each bar represent relative cell size. Statistical significance of each analysis is indicated at the bottom of the graph. Scale bars: 50 μm.
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f4-0040255: Enlarged hepatocytes and brain and spinal cord neurons in tsc2vu242/vu242 mutant zebrafish and suppression of hepatocyte size by inhibition of TORC1 activity. (A–F) Transverse section of liver (A,B), brain (C,D) and spinal cord (E,F). (G) Hepatocyte size differences in control siblings and tsc2vu242/vu242 mutants are shown. (H) The relative size of neuronal cells in the brain and spinal cord were compared. (I) Homozygous mutant zebrafish have increased size of their spinal cord. (J) Cell number within the spinal cord in control and tsc2vu242/vu242 mutants. (K) Transverse sections through the trunk of 4.5-day-old embryos treated with different concentrations of rapamycin from 3.5 dpf to 4.5 dpf were stained with antibody to phospho-S6 (red). (L) Transverse section of livers from wild-type and tsc2vu242/vu242 mutants without rapamycin treatment, or with 5 nM or 50 nM rapamycin treatment. Cells outlined in yellow were measured and soma size compared. (M) Graph of measurements of relative sizes of cells from embryo shown in L. Numbers on the each bar represent relative cell size. Statistical significance of each analysis is indicated at the bottom of the graph. Scale bars: 50 μm.

Mentions: The enlarged liver seen in tsc2vu242/vu242 mutant larvae (Fig. 2C,D) could result from either increased numbers or size of hepatocytes. Using BrdU-incorporation experiments, we did not detect increased cell proliferation in tsc2vu242/vu242 mutants compared to wild-type embryos by 7 dpf (data not shown). To measure cell size, we crossed tsc2vu242/+ carriers with fish harboring the Tg(β-actin:mGFP) transgene, which expresses green fluorescent protein (GFP) at the cell membrane in all tissues under the control of the β-actin promoter (Cooper et al., 2005). We crossed the resulting tsc2vu242/+;Tg(β-actin:mGFP) fish and determined cell size in tsc2vu242/vu242 homozygous mutants in various organs at 9 dpf. These analyses revealed increased size of hepatocytes by approximately twofold in tsc2vu242/vu242 homozygous mutants as compared with wild-type siblings (Fig. 4A,B,G).


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)

Enlarged hepatocytes and brain and spinal cord neurons in tsc2vu242/vu242 mutant zebrafish and suppression of hepatocyte size by inhibition of TORC1 activity. (A–F) Transverse section of liver (A,B), brain (C,D) and spinal cord (E,F). (G) Hepatocyte size differences in control siblings and tsc2vu242/vu242 mutants are shown. (H) The relative size of neuronal cells in the brain and spinal cord were compared. (I) Homozygous mutant zebrafish have increased size of their spinal cord. (J) Cell number within the spinal cord in control and tsc2vu242/vu242 mutants. (K) Transverse sections through the trunk of 4.5-day-old embryos treated with different concentrations of rapamycin from 3.5 dpf to 4.5 dpf were stained with antibody to phospho-S6 (red). (L) Transverse section of livers from wild-type and tsc2vu242/vu242 mutants without rapamycin treatment, or with 5 nM or 50 nM rapamycin treatment. Cells outlined in yellow were measured and soma size compared. (M) Graph of measurements of relative sizes of cells from embryo shown in L. Numbers on the each bar represent relative cell size. Statistical significance of each analysis is indicated at the bottom of the graph. Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4-0040255: Enlarged hepatocytes and brain and spinal cord neurons in tsc2vu242/vu242 mutant zebrafish and suppression of hepatocyte size by inhibition of TORC1 activity. (A–F) Transverse section of liver (A,B), brain (C,D) and spinal cord (E,F). (G) Hepatocyte size differences in control siblings and tsc2vu242/vu242 mutants are shown. (H) The relative size of neuronal cells in the brain and spinal cord were compared. (I) Homozygous mutant zebrafish have increased size of their spinal cord. (J) Cell number within the spinal cord in control and tsc2vu242/vu242 mutants. (K) Transverse sections through the trunk of 4.5-day-old embryos treated with different concentrations of rapamycin from 3.5 dpf to 4.5 dpf were stained with antibody to phospho-S6 (red). (L) Transverse section of livers from wild-type and tsc2vu242/vu242 mutants without rapamycin treatment, or with 5 nM or 50 nM rapamycin treatment. Cells outlined in yellow were measured and soma size compared. (M) Graph of measurements of relative sizes of cells from embryo shown in L. Numbers on the each bar represent relative cell size. Statistical significance of each analysis is indicated at the bottom of the graph. Scale bars: 50 μm.
Mentions: The enlarged liver seen in tsc2vu242/vu242 mutant larvae (Fig. 2C,D) could result from either increased numbers or size of hepatocytes. Using BrdU-incorporation experiments, we did not detect increased cell proliferation in tsc2vu242/vu242 mutants compared to wild-type embryos by 7 dpf (data not shown). To measure cell size, we crossed tsc2vu242/+ carriers with fish harboring the Tg(β-actin:mGFP) transgene, which expresses green fluorescent protein (GFP) at the cell membrane in all tissues under the control of the β-actin promoter (Cooper et al., 2005). We crossed the resulting tsc2vu242/+;Tg(β-actin:mGFP) fish and determined cell size in tsc2vu242/vu242 homozygous mutants in various organs at 9 dpf. These analyses revealed increased size of hepatocytes by approximately twofold in tsc2vu242/vu242 homozygous mutants as compared with wild-type siblings (Fig. 4A,B,G).

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