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Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

Ríos Y, Melmed S, Lin S, Liu NA - PLoS Genet. (2011)

Bottom Line: Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression.Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon.These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

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usp39 mutation lead to expansion of all pituitary cell lineages at 48 hpf as indicated by pituitary hormone markers.A–L: Whole-mount double in situ hybridization with probes indicated on the side. usp39 mutant embryos exhibit higher expression of all pituitary hormone markers compared to wild-type (wt) embryos. Spatial distribution of prl, tsh, pomc and cga are normal in the usp39 mutant. (A, B, E, F, I, J, M, and N) ventral view and (C, D, G, H, K, L, O, and P) lateral view, with anterior to the left. Columns 1 (A, E, I, and M) and 3 (C, G, K and O) show wt siblings; columns 2 (B, F, J, and N) and 4 (D, H, L, and P) show usp39 mutant embryos. A–D: gh (purple) and prl (red) transcripts. (C) The spatial distribution of gh is normally found in the proximal pars distalis (white arrow) and prl is found in the rostral pars distalis (black arrow). (D) Note the spatial distribution of gh in the usp39 mutant; gh is abnormally expressed in the rostral pars distalis (black arrow). E–H: tsh (purple) and pomc (red) transcripts. I–L: prl (purple) and pomc (red) transcripts. M–P: Whole-mount in situ hybridization with cga transcript.
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pgen-1001271-g003: usp39 mutation lead to expansion of all pituitary cell lineages at 48 hpf as indicated by pituitary hormone markers.A–L: Whole-mount double in situ hybridization with probes indicated on the side. usp39 mutant embryos exhibit higher expression of all pituitary hormone markers compared to wild-type (wt) embryos. Spatial distribution of prl, tsh, pomc and cga are normal in the usp39 mutant. (A, B, E, F, I, J, M, and N) ventral view and (C, D, G, H, K, L, O, and P) lateral view, with anterior to the left. Columns 1 (A, E, I, and M) and 3 (C, G, K and O) show wt siblings; columns 2 (B, F, J, and N) and 4 (D, H, L, and P) show usp39 mutant embryos. A–D: gh (purple) and prl (red) transcripts. (C) The spatial distribution of gh is normally found in the proximal pars distalis (white arrow) and prl is found in the rostral pars distalis (black arrow). (D) Note the spatial distribution of gh in the usp39 mutant; gh is abnormally expressed in the rostral pars distalis (black arrow). E–H: tsh (purple) and pomc (red) transcripts. I–L: prl (purple) and pomc (red) transcripts. M–P: Whole-mount in situ hybridization with cga transcript.

Mentions: The zebrafish adenohypophysis consists of six different hormone-secreting cell types distributed along the anterior-posterior axis: lactotropes and corticotropes are located anteriorly in the rostral pars distalis, thyrotropes, gonadotropes and somatotropes are found medially in the proximal pars distalis whereas melanotropes are situated posteriorly in the pars intermedia (Figure 3). To determine if additional pituitary lineages are affected by the usp39 mutation, we performed double color RNA in situ hybridization analysis with combinatory pituitary lineage specific marker genes pomc, gh, prl, tsh, and with cga that encodes the glycoprotein α-subunit heterodimerizing with TSHβ, LHβ, or FSHβ subunit [2], [16]. This analysis revealed expansion of all the analyzed cell lineages without apparent cell fate transformation in the usp39 mutant pituitary at 48 hpf (Figure 3A–3P). Cell expansion was most marked in corticotropes and lactotropes, indicating that usp39 is important for regulating embryonic pituitary cell populations (Figure 3B, 3D, 3F, 3H, 3J, and 3L).


Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

Ríos Y, Melmed S, Lin S, Liu NA - PLoS Genet. (2011)

usp39 mutation lead to expansion of all pituitary cell lineages at 48 hpf as indicated by pituitary hormone markers.A–L: Whole-mount double in situ hybridization with probes indicated on the side. usp39 mutant embryos exhibit higher expression of all pituitary hormone markers compared to wild-type (wt) embryos. Spatial distribution of prl, tsh, pomc and cga are normal in the usp39 mutant. (A, B, E, F, I, J, M, and N) ventral view and (C, D, G, H, K, L, O, and P) lateral view, with anterior to the left. Columns 1 (A, E, I, and M) and 3 (C, G, K and O) show wt siblings; columns 2 (B, F, J, and N) and 4 (D, H, L, and P) show usp39 mutant embryos. A–D: gh (purple) and prl (red) transcripts. (C) The spatial distribution of gh is normally found in the proximal pars distalis (white arrow) and prl is found in the rostral pars distalis (black arrow). (D) Note the spatial distribution of gh in the usp39 mutant; gh is abnormally expressed in the rostral pars distalis (black arrow). E–H: tsh (purple) and pomc (red) transcripts. I–L: prl (purple) and pomc (red) transcripts. M–P: Whole-mount in situ hybridization with cga transcript.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020934&req=5

pgen-1001271-g003: usp39 mutation lead to expansion of all pituitary cell lineages at 48 hpf as indicated by pituitary hormone markers.A–L: Whole-mount double in situ hybridization with probes indicated on the side. usp39 mutant embryos exhibit higher expression of all pituitary hormone markers compared to wild-type (wt) embryos. Spatial distribution of prl, tsh, pomc and cga are normal in the usp39 mutant. (A, B, E, F, I, J, M, and N) ventral view and (C, D, G, H, K, L, O, and P) lateral view, with anterior to the left. Columns 1 (A, E, I, and M) and 3 (C, G, K and O) show wt siblings; columns 2 (B, F, J, and N) and 4 (D, H, L, and P) show usp39 mutant embryos. A–D: gh (purple) and prl (red) transcripts. (C) The spatial distribution of gh is normally found in the proximal pars distalis (white arrow) and prl is found in the rostral pars distalis (black arrow). (D) Note the spatial distribution of gh in the usp39 mutant; gh is abnormally expressed in the rostral pars distalis (black arrow). E–H: tsh (purple) and pomc (red) transcripts. I–L: prl (purple) and pomc (red) transcripts. M–P: Whole-mount in situ hybridization with cga transcript.
Mentions: The zebrafish adenohypophysis consists of six different hormone-secreting cell types distributed along the anterior-posterior axis: lactotropes and corticotropes are located anteriorly in the rostral pars distalis, thyrotropes, gonadotropes and somatotropes are found medially in the proximal pars distalis whereas melanotropes are situated posteriorly in the pars intermedia (Figure 3). To determine if additional pituitary lineages are affected by the usp39 mutation, we performed double color RNA in situ hybridization analysis with combinatory pituitary lineage specific marker genes pomc, gh, prl, tsh, and with cga that encodes the glycoprotein α-subunit heterodimerizing with TSHβ, LHβ, or FSHβ subunit [2], [16]. This analysis revealed expansion of all the analyzed cell lineages without apparent cell fate transformation in the usp39 mutant pituitary at 48 hpf (Figure 3A–3P). Cell expansion was most marked in corticotropes and lactotropes, indicating that usp39 is important for regulating embryonic pituitary cell populations (Figure 3B, 3D, 3F, 3H, 3J, and 3L).

Bottom Line: Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression.Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon.These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

Show MeSH
Related in: MedlinePlus