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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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Zebrafish tsc2 is maternally and zygotically expressed during development. (A–D) Ubiquitous expression of wild-type tsc2 at the eight-cell (A), sphere (B), 75% epiboly (C) and five-somite (D) stage. (E–H) At 25 hpf, tsc2 expression is detected in discrete regions of the developing eye and forebrain and midbrain (E), lateral part of the hindbrain (F), otic vesicle (F,G), pharyngeal arch (G), and blood vessels (H). (I) At 32 hpf, tsc2 expression is maintained in the eyes and brain. (J) Transverse section of the hindbrain region shows that tsc2 expression is limited to the ventrolateral region in wild-type embryos. (K,L) At 3 dpf, tsc2 expression is detected in the eye (K), heart and intestine (L). (M) Structure of the predicted protein domains of wild-type human TSC2 and zebrafish tsc2. The vu242 mutation causes a premature stop codon within exon 26. Blue indicates the hamartin-interacting domain, green the tuberin domain and red the GAP domain. fb, forebrain; mib, midbrain; hib, hindbrain; bv, blood vessel; h, heart; i, intestine; ov, otic vesicle. Scale bars: 250 μm (bar in K is for J, K; bar in L is for A–I, L). (A–D,H,I,L) Lateral views; (E,F) dorsal views; anterior to the left.
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f1-0040255: Zebrafish tsc2 is maternally and zygotically expressed during development. (A–D) Ubiquitous expression of wild-type tsc2 at the eight-cell (A), sphere (B), 75% epiboly (C) and five-somite (D) stage. (E–H) At 25 hpf, tsc2 expression is detected in discrete regions of the developing eye and forebrain and midbrain (E), lateral part of the hindbrain (F), otic vesicle (F,G), pharyngeal arch (G), and blood vessels (H). (I) At 32 hpf, tsc2 expression is maintained in the eyes and brain. (J) Transverse section of the hindbrain region shows that tsc2 expression is limited to the ventrolateral region in wild-type embryos. (K,L) At 3 dpf, tsc2 expression is detected in the eye (K), heart and intestine (L). (M) Structure of the predicted protein domains of wild-type human TSC2 and zebrafish tsc2. The vu242 mutation causes a premature stop codon within exon 26. Blue indicates the hamartin-interacting domain, green the tuberin domain and red the GAP domain. fb, forebrain; mib, midbrain; hib, hindbrain; bv, blood vessel; h, heart; i, intestine; ov, otic vesicle. Scale bars: 250 μm (bar in K is for J, K; bar in L is for A–I, L). (A–D,H,I,L) Lateral views; (E,F) dorsal views; anterior to the left.

Mentions: To better understand the role of TSC2 in normal development and disease, we first cloned full-length zebrafish tsc2 cDNA. The predicted protein overall shows 60% identity and 73% similarity to human tuberin (supplementary material Fig. S2). The hamartin-interacting, tuberin and GAP domains are all highly conserved, with 71%, 70% and 75% identical amino acids between the two species, respectively (Fig. 1M). Analysis of the spatiotemporal expression pattern by whole-mount in situ hybridization revealed that tsc2 transcripts are maternally deposited (Fig. 1A) and ubiquitously expressed during blastula, gastrula and segmentation stages (Fig. 1B-D). By 25 hours post-fertilization (hpf), tsc2 expression is highly enriched in the developing eye, forebrain, midbrain, lateral hindbrain region and otic vesicles (Fig. 1E-G), as well as in the blood vessels (Fig. 1H). At 36 hpf, strong expression persists in the brain (Fig. 1I) and transverse sections show that tsc2 is also expressed in the ventrolateral region of the hindbrain (Fig. 1J). At 3 days post-fertilization (dpf), we detected low levels of tsc2 expression in the eye (Fig. 1K), heart and intestine, in addition to the continued higher levels in the brain (Fig. 1L).


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)

Zebrafish tsc2 is maternally and zygotically expressed during development. (A–D) Ubiquitous expression of wild-type tsc2 at the eight-cell (A), sphere (B), 75% epiboly (C) and five-somite (D) stage. (E–H) At 25 hpf, tsc2 expression is detected in discrete regions of the developing eye and forebrain and midbrain (E), lateral part of the hindbrain (F), otic vesicle (F,G), pharyngeal arch (G), and blood vessels (H). (I) At 32 hpf, tsc2 expression is maintained in the eyes and brain. (J) Transverse section of the hindbrain region shows that tsc2 expression is limited to the ventrolateral region in wild-type embryos. (K,L) At 3 dpf, tsc2 expression is detected in the eye (K), heart and intestine (L). (M) Structure of the predicted protein domains of wild-type human TSC2 and zebrafish tsc2. The vu242 mutation causes a premature stop codon within exon 26. Blue indicates the hamartin-interacting domain, green the tuberin domain and red the GAP domain. fb, forebrain; mib, midbrain; hib, hindbrain; bv, blood vessel; h, heart; i, intestine; ov, otic vesicle. Scale bars: 250 μm (bar in K is for J, K; bar in L is for A–I, L). (A–D,H,I,L) Lateral views; (E,F) dorsal views; anterior to the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-0040255: Zebrafish tsc2 is maternally and zygotically expressed during development. (A–D) Ubiquitous expression of wild-type tsc2 at the eight-cell (A), sphere (B), 75% epiboly (C) and five-somite (D) stage. (E–H) At 25 hpf, tsc2 expression is detected in discrete regions of the developing eye and forebrain and midbrain (E), lateral part of the hindbrain (F), otic vesicle (F,G), pharyngeal arch (G), and blood vessels (H). (I) At 32 hpf, tsc2 expression is maintained in the eyes and brain. (J) Transverse section of the hindbrain region shows that tsc2 expression is limited to the ventrolateral region in wild-type embryos. (K,L) At 3 dpf, tsc2 expression is detected in the eye (K), heart and intestine (L). (M) Structure of the predicted protein domains of wild-type human TSC2 and zebrafish tsc2. The vu242 mutation causes a premature stop codon within exon 26. Blue indicates the hamartin-interacting domain, green the tuberin domain and red the GAP domain. fb, forebrain; mib, midbrain; hib, hindbrain; bv, blood vessel; h, heart; i, intestine; ov, otic vesicle. Scale bars: 250 μm (bar in K is for J, K; bar in L is for A–I, L). (A–D,H,I,L) Lateral views; (E,F) dorsal views; anterior to the left.
Mentions: To better understand the role of TSC2 in normal development and disease, we first cloned full-length zebrafish tsc2 cDNA. The predicted protein overall shows 60% identity and 73% similarity to human tuberin (supplementary material Fig. S2). The hamartin-interacting, tuberin and GAP domains are all highly conserved, with 71%, 70% and 75% identical amino acids between the two species, respectively (Fig. 1M). Analysis of the spatiotemporal expression pattern by whole-mount in situ hybridization revealed that tsc2 transcripts are maternally deposited (Fig. 1A) and ubiquitously expressed during blastula, gastrula and segmentation stages (Fig. 1B-D). By 25 hours post-fertilization (hpf), tsc2 expression is highly enriched in the developing eye, forebrain, midbrain, lateral hindbrain region and otic vesicles (Fig. 1E-G), as well as in the blood vessels (Fig. 1H). At 36 hpf, strong expression persists in the brain (Fig. 1I) and transverse sections show that tsc2 is also expressed in the ventrolateral region of the hindbrain (Fig. 1J). At 3 days post-fertilization (dpf), we detected low levels of tsc2 expression in the eye (Fig. 1K), heart and intestine, in addition to the continued higher levels in the brain (Fig. 1L).

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