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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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Impaired differentiation in the SCG due to phox2b deficiency is not rescued by retinoic acid.(A–F) Dorsal views of 3-dpf embryos injected with phox2b MO (D–F) or mismatched control MO (A–C) and treated with increasing concentrations of 13–cis retinoic acid (RA). (G) Relative intensity measurements of th expression in the SCG of embryos injected with various MOs and treated with different concentrations of RA. ATGK1, translation-blocking phox2b MO; P2BT2, splice blocking phox2b MO. (H–O) Whole-mount ISH of 3-dpf embryos in which the specified RNAs were overexpressed and analyzed for th expression following exposure to RA. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B, and the R100L, 676delG, K155X mutations were injected into embryos at the one-cell stage. (P) Quantification of the relative intensity of th staining in the embryos depicted in H–O. Data are presented as means ± SD (*P<0.05; n = 10 per group).
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pgen-1003533-g004: Impaired differentiation in the SCG due to phox2b deficiency is not rescued by retinoic acid.(A–F) Dorsal views of 3-dpf embryos injected with phox2b MO (D–F) or mismatched control MO (A–C) and treated with increasing concentrations of 13–cis retinoic acid (RA). (G) Relative intensity measurements of th expression in the SCG of embryos injected with various MOs and treated with different concentrations of RA. ATGK1, translation-blocking phox2b MO; P2BT2, splice blocking phox2b MO. (H–O) Whole-mount ISH of 3-dpf embryos in which the specified RNAs were overexpressed and analyzed for th expression following exposure to RA. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B, and the R100L, 676delG, K155X mutations were injected into embryos at the one-cell stage. (P) Quantification of the relative intensity of th staining in the embryos depicted in H–O. Data are presented as means ± SD (*P<0.05; n = 10 per group).

Mentions: PHOX2B complements exogenous differentiating agents such as retinoic acid (RA) by promoting cellular differentiation in vitro [36]. To determine whether phox2b loss might obviate the induction of differentiation by RA, we treated control and phox2b MO embryos with various concentrations of 13-cis-retinoic acid, which is commonly used in the treatment of patients with neuroblastoma (Figure 4A–4F). Control-injected embryos showed an increase in th expression in the SCG after RA treatment (Figure 4A–4C, 4G), which was not apparent in the SCG of the phox2b morphant embryos (Figure 4D–4F, 4G). A similar impairment in RA-induced differentiation was observed after expression of the 676delG variant and, to a lesser extent, the K155X variant (Figure 4H–4P). Similar effects in dbh expression were also seen (data not shown). Together, these findings reinforce the strict requirement for Phox2b in the differentiation of sympathetic neuronal progenitors and suggest that loss of this transcription factor cannot be overcome with RA treatment.


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)

Impaired differentiation in the SCG due to phox2b deficiency is not rescued by retinoic acid.(A–F) Dorsal views of 3-dpf embryos injected with phox2b MO (D–F) or mismatched control MO (A–C) and treated with increasing concentrations of 13–cis retinoic acid (RA). (G) Relative intensity measurements of th expression in the SCG of embryos injected with various MOs and treated with different concentrations of RA. ATGK1, translation-blocking phox2b MO; P2BT2, splice blocking phox2b MO. (H–O) Whole-mount ISH of 3-dpf embryos in which the specified RNAs were overexpressed and analyzed for th expression following exposure to RA. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B, and the R100L, 676delG, K155X mutations were injected into embryos at the one-cell stage. (P) Quantification of the relative intensity of th staining in the embryos depicted in H–O. Data are presented as means ± SD (*P<0.05; n = 10 per group).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675015&req=5

pgen-1003533-g004: Impaired differentiation in the SCG due to phox2b deficiency is not rescued by retinoic acid.(A–F) Dorsal views of 3-dpf embryos injected with phox2b MO (D–F) or mismatched control MO (A–C) and treated with increasing concentrations of 13–cis retinoic acid (RA). (G) Relative intensity measurements of th expression in the SCG of embryos injected with various MOs and treated with different concentrations of RA. ATGK1, translation-blocking phox2b MO; P2BT2, splice blocking phox2b MO. (H–O) Whole-mount ISH of 3-dpf embryos in which the specified RNAs were overexpressed and analyzed for th expression following exposure to RA. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B, and the R100L, 676delG, K155X mutations were injected into embryos at the one-cell stage. (P) Quantification of the relative intensity of th staining in the embryos depicted in H–O. Data are presented as means ± SD (*P<0.05; n = 10 per group).
Mentions: PHOX2B complements exogenous differentiating agents such as retinoic acid (RA) by promoting cellular differentiation in vitro [36]. To determine whether phox2b loss might obviate the induction of differentiation by RA, we treated control and phox2b MO embryos with various concentrations of 13-cis-retinoic acid, which is commonly used in the treatment of patients with neuroblastoma (Figure 4A–4F). Control-injected embryos showed an increase in th expression in the SCG after RA treatment (Figure 4A–4C, 4G), which was not apparent in the SCG of the phox2b morphant embryos (Figure 4D–4F, 4G). A similar impairment in RA-induced differentiation was observed after expression of the 676delG variant and, to a lesser extent, the K155X variant (Figure 4H–4P). Similar effects in dbh expression were also seen (data not shown). Together, these findings reinforce the strict requirement for Phox2b in the differentiation of sympathetic neuronal progenitors and suggest that loss of this transcription factor cannot be overcome with RA treatment.

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