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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, but not ascl1, is indispensable for sympathetic neuronal differentiation.(A–F) Whole-mount ISH of 3-dpf embryos for th and dbh following ascl1 MO knockdown. Lateral views depict a minimal decrease in expression of th (A,B) and dbh (D,E) in ascl1 MO-injected embryos compared to control MO-injected (A,D) embryos. Simultaneous knockdown of both ascl1 and phox2b led to a significant decrease in expression of th and dbh in the SCG (C,F). (G) Relative intensity levels of dbh-staining in the SCG of embryos expressing ascl1 MO, phox2b MO or the combination of the two. Data are presented as means ± SEM (***P<0.001; n = 15 per group). (H–M) Whole-mount ISH for expression of ascl (H–J) and phox2b (K–M) in embryos in which ascl1 expression was abrogated by MO knockdown, either singly (I, L) or in combination with phox2b (J, M). (N,O) Whole-mount ISH for phox2a expression in 4-dpf embryos in which ascl1 expression was abrogated by MO knockdown. Arrows point to region of SCG.
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pgen-1003533-g007: phox2b, but not ascl1, is indispensable for sympathetic neuronal differentiation.(A–F) Whole-mount ISH of 3-dpf embryos for th and dbh following ascl1 MO knockdown. Lateral views depict a minimal decrease in expression of th (A,B) and dbh (D,E) in ascl1 MO-injected embryos compared to control MO-injected (A,D) embryos. Simultaneous knockdown of both ascl1 and phox2b led to a significant decrease in expression of th and dbh in the SCG (C,F). (G) Relative intensity levels of dbh-staining in the SCG of embryos expressing ascl1 MO, phox2b MO or the combination of the two. Data are presented as means ± SEM (***P<0.001; n = 15 per group). (H–M) Whole-mount ISH for expression of ascl (H–J) and phox2b (K–M) in embryos in which ascl1 expression was abrogated by MO knockdown, either singly (I, L) or in combination with phox2b (J, M). (N,O) Whole-mount ISH for phox2a expression in 4-dpf embryos in which ascl1 expression was abrogated by MO knockdown. Arrows point to region of SCG.

Mentions: During noradrenergic development in the chick embryo, Phox2b and Ascl1 are expressed together in response to BMP signaling [44], [48]; in fact, elimination of Ascl1 has also been reported to cause impaired sympathetic differentiation [14], [16]. To determine whether ascl1 is as critical as phox2b to PSNS differentiation in the zebrafish, we studied the effects of ascl1 knockdown using a translation-blocking MO (Figure S6A; Figure 7A, 7B, 7C, 7E). Abrogation of ascl1 alone led to only a marginal decrease in th and dbh expression in the SCG (Figure 7B, 7E, 7G), while a striking decrease in both th and dbh expression occurred with simultaneous knockdown of both ascl1 and phox2b, similar to the result with phox2b knockdown alone (Figure 7C, 7F, 7G). Moreover, in contrast to the outcome of phox2b knockdown, expression of hand2, gata3 and tfap2a was not affected in the ascl1 morphants (Figure S6B–G). These findings identify Phox2b as the central driver of terminal differentiation of sympathetic neurons in the zebrafish model.


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, but not ascl1, is indispensable for sympathetic neuronal differentiation.(A–F) Whole-mount ISH of 3-dpf embryos for th and dbh following ascl1 MO knockdown. Lateral views depict a minimal decrease in expression of th (A,B) and dbh (D,E) in ascl1 MO-injected embryos compared to control MO-injected (A,D) embryos. Simultaneous knockdown of both ascl1 and phox2b led to a significant decrease in expression of th and dbh in the SCG (C,F). (G) Relative intensity levels of dbh-staining in the SCG of embryos expressing ascl1 MO, phox2b MO or the combination of the two. Data are presented as means ± SEM (***P<0.001; n = 15 per group). (H–M) Whole-mount ISH for expression of ascl (H–J) and phox2b (K–M) in embryos in which ascl1 expression was abrogated by MO knockdown, either singly (I, L) or in combination with phox2b (J, M). (N,O) Whole-mount ISH for phox2a expression in 4-dpf embryos in which ascl1 expression was abrogated by MO knockdown. Arrows point to region of SCG.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003533-g007: phox2b, but not ascl1, is indispensable for sympathetic neuronal differentiation.(A–F) Whole-mount ISH of 3-dpf embryos for th and dbh following ascl1 MO knockdown. Lateral views depict a minimal decrease in expression of th (A,B) and dbh (D,E) in ascl1 MO-injected embryos compared to control MO-injected (A,D) embryos. Simultaneous knockdown of both ascl1 and phox2b led to a significant decrease in expression of th and dbh in the SCG (C,F). (G) Relative intensity levels of dbh-staining in the SCG of embryos expressing ascl1 MO, phox2b MO or the combination of the two. Data are presented as means ± SEM (***P<0.001; n = 15 per group). (H–M) Whole-mount ISH for expression of ascl (H–J) and phox2b (K–M) in embryos in which ascl1 expression was abrogated by MO knockdown, either singly (I, L) or in combination with phox2b (J, M). (N,O) Whole-mount ISH for phox2a expression in 4-dpf embryos in which ascl1 expression was abrogated by MO knockdown. Arrows point to region of SCG.
Mentions: During noradrenergic development in the chick embryo, Phox2b and Ascl1 are expressed together in response to BMP signaling [44], [48]; in fact, elimination of Ascl1 has also been reported to cause impaired sympathetic differentiation [14], [16]. To determine whether ascl1 is as critical as phox2b to PSNS differentiation in the zebrafish, we studied the effects of ascl1 knockdown using a translation-blocking MO (Figure S6A; Figure 7A, 7B, 7C, 7E). Abrogation of ascl1 alone led to only a marginal decrease in th and dbh expression in the SCG (Figure 7B, 7E, 7G), while a striking decrease in both th and dbh expression occurred with simultaneous knockdown of both ascl1 and phox2b, similar to the result with phox2b knockdown alone (Figure 7C, 7F, 7G). Moreover, in contrast to the outcome of phox2b knockdown, expression of hand2, gata3 and tfap2a was not affected in the ascl1 morphants (Figure S6B–G). These findings identify Phox2b as the central driver of terminal differentiation of sympathetic neurons in the zebrafish model.

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