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Distinct neuroblastoma-associated alterations of PHOX2B impair sympathetic neuronal differentiation in zebrafish models.

Pei D, Luther W, Wang W, Paw BH, Stewart RA, George RE - PLoS Genet. (2013)

Bottom Line: The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects.This effect on terminal differentiation is associated with an increased number of phox2b(+), ascl1(+), elavl3(-) cells that respond poorly to retinoic acid.These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b(+), ascl1(+), elavl3(-) cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

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phox2b-deficient embryos show impaired differentiation of sympathetic neurons in the SCG.(A–F) Lateral/oblique views of 4-dpf embryos after whole-mount ISH for th (A–C) and dbh (D–F) in control and phox2b–deficient embryos. Arrows indicate the SCG. Knockdown of phox2b by injection of a splice MO (4 ng) (MOsplice) inhibits the expression of th and dbh (B, E) which is rescued by coexpression of human PHOX2B mRNA (10 ng/µl) (C, F). Relative intensity levels of th (G) and dbh (H) expression in embryos injected with phox2b MOs that inhibit translation (MOATG) or splicing (MOsplice). Mismatched control MO (MOmm) and PHOX2B mRNA-rescue (MOsplice/PHOX2B) are also shown. Data are presented as means ± SD (***P<0.001; **P<0.01; n = 15 for each group).
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pgen-1003533-g002: phox2b-deficient embryos show impaired differentiation of sympathetic neurons in the SCG.(A–F) Lateral/oblique views of 4-dpf embryos after whole-mount ISH for th (A–C) and dbh (D–F) in control and phox2b–deficient embryos. Arrows indicate the SCG. Knockdown of phox2b by injection of a splice MO (4 ng) (MOsplice) inhibits the expression of th and dbh (B, E) which is rescued by coexpression of human PHOX2B mRNA (10 ng/µl) (C, F). Relative intensity levels of th (G) and dbh (H) expression in embryos injected with phox2b MOs that inhibit translation (MOATG) or splicing (MOsplice). Mismatched control MO (MOmm) and PHOX2B mRNA-rescue (MOsplice/PHOX2B) are also shown. Data are presented as means ± SD (***P<0.001; **P<0.01; n = 15 for each group).

Mentions: To study the consequences of allelic PHOX2B deletion in patients with neuroblastoma [35], we performed morpholino (MO) knockdown of zebrafish phox2b, using two non-overlapping antisense oligonucleotide sequences targeted to the phox2b gene: a translation-blocking MO (MOATG) and a splice-blocking MO (MOsplice) directed to the second exon/intron splice junction (Figure S2A). phox2b knockdown was confirmed by immunoblotting in the case of the ATG MO (which showed ∼70–80% knockdown) and RT-PCR for the splice MO (Figure S2B). Experiments were performed in both wild-type and p53 mutant embryos (tp53M214K/M214K) [42] to account for potential nonspecific effects associated with some MO injections, with similar results obtained in both backgrounds (Figure S2C). MO knockdown of phox2b led to a marked reduction in th and dbh expression in the SCG at 3 dpf, as compared with mismatched control MO-injected (MOMM) or uninjected wild-type (WT) siblings (Figures S3A, S3B, S3D, S3E, S3G, S3H). This phenotype was consistent with both the ATG and the splice MOs. To ensure that the decrease in th and dbh was not secondary to a general delay in development due to MO injection, we examined the SCG at 4 dpf (Figures 2A, 2B, 2D, 2E, 2G, 2H) and later (5 dpf; data not shown), again noting a decrease in the expression of these genes. To confirm that the phenotype was specific to phox2b, we rescued the th- and dbh-expressing cells by coexpressing human PHOX2B mRNA with both MOs, which led to an increase in th and dbh expression in the SCG (Figures 2C, 2E; 2G, 2H; Figures S3C, S3F; 3G, 3H). These results indicate that PHOX2B is necessary and sufficient for the terminal differentiation of sympathetic neuronal precursors.


Distinct neuroblastoma-associated alterations of PHOX2B impair sympathetic neuronal differentiation in zebrafish models.

Pei D, Luther W, Wang W, Paw BH, Stewart RA, George RE - PLoS Genet. (2013)

phox2b-deficient embryos show impaired differentiation of sympathetic neurons in the SCG.(A–F) Lateral/oblique views of 4-dpf embryos after whole-mount ISH for th (A–C) and dbh (D–F) in control and phox2b–deficient embryos. Arrows indicate the SCG. Knockdown of phox2b by injection of a splice MO (4 ng) (MOsplice) inhibits the expression of th and dbh (B, E) which is rescued by coexpression of human PHOX2B mRNA (10 ng/µl) (C, F). Relative intensity levels of th (G) and dbh (H) expression in embryos injected with phox2b MOs that inhibit translation (MOATG) or splicing (MOsplice). Mismatched control MO (MOmm) and PHOX2B mRNA-rescue (MOsplice/PHOX2B) are also shown. Data are presented as means ± SD (***P<0.001; **P<0.01; n = 15 for each group).
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pgen-1003533-g002: phox2b-deficient embryos show impaired differentiation of sympathetic neurons in the SCG.(A–F) Lateral/oblique views of 4-dpf embryos after whole-mount ISH for th (A–C) and dbh (D–F) in control and phox2b–deficient embryos. Arrows indicate the SCG. Knockdown of phox2b by injection of a splice MO (4 ng) (MOsplice) inhibits the expression of th and dbh (B, E) which is rescued by coexpression of human PHOX2B mRNA (10 ng/µl) (C, F). Relative intensity levels of th (G) and dbh (H) expression in embryos injected with phox2b MOs that inhibit translation (MOATG) or splicing (MOsplice). Mismatched control MO (MOmm) and PHOX2B mRNA-rescue (MOsplice/PHOX2B) are also shown. Data are presented as means ± SD (***P<0.001; **P<0.01; n = 15 for each group).
Mentions: To study the consequences of allelic PHOX2B deletion in patients with neuroblastoma [35], we performed morpholino (MO) knockdown of zebrafish phox2b, using two non-overlapping antisense oligonucleotide sequences targeted to the phox2b gene: a translation-blocking MO (MOATG) and a splice-blocking MO (MOsplice) directed to the second exon/intron splice junction (Figure S2A). phox2b knockdown was confirmed by immunoblotting in the case of the ATG MO (which showed ∼70–80% knockdown) and RT-PCR for the splice MO (Figure S2B). Experiments were performed in both wild-type and p53 mutant embryos (tp53M214K/M214K) [42] to account for potential nonspecific effects associated with some MO injections, with similar results obtained in both backgrounds (Figure S2C). MO knockdown of phox2b led to a marked reduction in th and dbh expression in the SCG at 3 dpf, as compared with mismatched control MO-injected (MOMM) or uninjected wild-type (WT) siblings (Figures S3A, S3B, S3D, S3E, S3G, S3H). This phenotype was consistent with both the ATG and the splice MOs. To ensure that the decrease in th and dbh was not secondary to a general delay in development due to MO injection, we examined the SCG at 4 dpf (Figures 2A, 2B, 2D, 2E, 2G, 2H) and later (5 dpf; data not shown), again noting a decrease in the expression of these genes. To confirm that the phenotype was specific to phox2b, we rescued the th- and dbh-expressing cells by coexpressing human PHOX2B mRNA with both MOs, which led to an increase in th and dbh expression in the SCG (Figures 2C, 2E; 2G, 2H; Figures S3C, S3F; 3G, 3H). These results indicate that PHOX2B is necessary and sufficient for the terminal differentiation of sympathetic neuronal precursors.

Bottom Line: The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects.This effect on terminal differentiation is associated with an increased number of phox2b(+), ascl1(+), elavl3(-) cells that respond poorly to retinoic acid.These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b(+), ascl1(+), elavl3(-) cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

Show MeSH
Related in: MedlinePlus