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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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The neuroblastoma-associated 676delG and K155X PHOX2B variants cause decreased terminal differentiation in the SCG.Whole-mount ISH for th (A–F) and dbh (G–L) expression in 3-dpf embryos in which human neuroblastoma-derived mutations were overexpressed (lateral views are shown). The area encompassing the SCG (boxed) is shown enlarged to the right of each panel. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B and the R100L, 676delG, K155X mutations were injected into one-cell embryos. CT, control water-injected. Relative intensity levels of th (M) and dbh (N) expression in the embryos depicted in panels A–F and G–L respectively. Data are presented as means ± SD (*P<0.01 vs. control-injected embryos; n = 6 per group). Whole-mount ISH for th (O) and dbh (P) in 3-dpf embryos expressing the phox2b MO and PHOX2B K155X mutant mRNA (P2BMO+K155X). Arrow indicates the region of the SCG.
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pgen-1003533-g003: The neuroblastoma-associated 676delG and K155X PHOX2B variants cause decreased terminal differentiation in the SCG.Whole-mount ISH for th (A–F) and dbh (G–L) expression in 3-dpf embryos in which human neuroblastoma-derived mutations were overexpressed (lateral views are shown). The area encompassing the SCG (boxed) is shown enlarged to the right of each panel. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B and the R100L, 676delG, K155X mutations were injected into one-cell embryos. CT, control water-injected. Relative intensity levels of th (M) and dbh (N) expression in the embryos depicted in panels A–F and G–L respectively. Data are presented as means ± SD (*P<0.01 vs. control-injected embryos; n = 6 per group). Whole-mount ISH for th (O) and dbh (P) in 3-dpf embryos expressing the phox2b MO and PHOX2B K155X mutant mRNA (P2BMO+K155X). Arrow indicates the region of the SCG.

Mentions: We next examined the effects of three distinct neuroblastoma-associated PHOX2B mutations on PSNS development (Figure S4, Figure 3). Overexpression of the 676delG frameshift [5] and K155X truncation [8] variants led to a significant decrease in the expression of th (Figure 3D, 3E, 3M) and dbh in the SCG (Figure 3J, 3K, 3N) as compared to that in control (Figure 3A, 3G) and WT (Figure 3B, 3H) phox2b RNA-injected animals, but had a similar effect to MO knockdown (Figure 3F, 3L). By contrast, overexpression of the R100L homeodomain missense mutation [7] did not lead to a discernible change in the expression of either th or dbh in the SCG (Figure 3C, 3I). To mimic the heterozygous situation seen in patients with PHOX2B mutations, we repeated these experiments in the setting of phox2b MO knockdown. In this context, overexpression of K155X and 676delG led to an even more striking reduction and, in some embryos, to an almost complete absence of th and dbh expression in the SCG (Figure 3O, 3P). These results suggest that the block in differentiation imposed by the 676delG and K155X mutations cannot be rescued by the expression of endogenous wild-type phox2b; rather, these variants appear to function dominant-negatively.


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)

The neuroblastoma-associated 676delG and K155X PHOX2B variants cause decreased terminal differentiation in the SCG.Whole-mount ISH for th (A–F) and dbh (G–L) expression in 3-dpf embryos in which human neuroblastoma-derived mutations were overexpressed (lateral views are shown). The area encompassing the SCG (boxed) is shown enlarged to the right of each panel. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B and the R100L, 676delG, K155X mutations were injected into one-cell embryos. CT, control water-injected. Relative intensity levels of th (M) and dbh (N) expression in the embryos depicted in panels A–F and G–L respectively. Data are presented as means ± SD (*P<0.01 vs. control-injected embryos; n = 6 per group). Whole-mount ISH for th (O) and dbh (P) in 3-dpf embryos expressing the phox2b MO and PHOX2B K155X mutant mRNA (P2BMO+K155X). Arrow indicates the region of the SCG.
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Related In: Results  -  Collection

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pgen-1003533-g003: The neuroblastoma-associated 676delG and K155X PHOX2B variants cause decreased terminal differentiation in the SCG.Whole-mount ISH for th (A–F) and dbh (G–L) expression in 3-dpf embryos in which human neuroblastoma-derived mutations were overexpressed (lateral views are shown). The area encompassing the SCG (boxed) is shown enlarged to the right of each panel. Capped mRNA (100 ng/µl) for wild-type (WT) human PHOX2B and the R100L, 676delG, K155X mutations were injected into one-cell embryos. CT, control water-injected. Relative intensity levels of th (M) and dbh (N) expression in the embryos depicted in panels A–F and G–L respectively. Data are presented as means ± SD (*P<0.01 vs. control-injected embryos; n = 6 per group). Whole-mount ISH for th (O) and dbh (P) in 3-dpf embryos expressing the phox2b MO and PHOX2B K155X mutant mRNA (P2BMO+K155X). Arrow indicates the region of the SCG.
Mentions: We next examined the effects of three distinct neuroblastoma-associated PHOX2B mutations on PSNS development (Figure S4, Figure 3). Overexpression of the 676delG frameshift [5] and K155X truncation [8] variants led to a significant decrease in the expression of th (Figure 3D, 3E, 3M) and dbh in the SCG (Figure 3J, 3K, 3N) as compared to that in control (Figure 3A, 3G) and WT (Figure 3B, 3H) phox2b RNA-injected animals, but had a similar effect to MO knockdown (Figure 3F, 3L). By contrast, overexpression of the R100L homeodomain missense mutation [7] did not lead to a discernible change in the expression of either th or dbh in the SCG (Figure 3C, 3I). To mimic the heterozygous situation seen in patients with PHOX2B mutations, we repeated these experiments in the setting of phox2b MO knockdown. In this context, overexpression of K155X and 676delG led to an even more striking reduction and, in some embryos, to an almost complete absence of th and dbh expression in the SCG (Figure 3O, 3P). These results suggest that the block in differentiation imposed by the 676delG and K155X mutations cannot be rescued by the expression of endogenous wild-type phox2b; rather, these variants appear to function dominant-negatively.

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