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

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.

SUBMITTER: Pei D

PROVIDER: S-EPMC3675015 | biostudies-other | 2013 Jun

REPOSITORIES: biostudies-other

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

Pei Desheng D Luther William W Wang Wenchao W Paw Barry H BH Stewart Rodney A RA George Rani E RE

PLoS genetics 20130606 6

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 ter ...[more]

PMID: 23754957

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Project description:BackgroundChromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines.MethodsNB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 μM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies.ResultsTreatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor.ConclusionsTreatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

Project description:Neuroblastoma (NB) is an embryonal malignancy derived from the abnormal differentiation of the sympathetic nervous system. The Anaplastic Lymphoma Kinase (ALK) gene is frequently altered in NB, through copy number alterations and activating mutations, and represents a predisposition in NB-genesis when mutated. Our previously published data suggested that ALK activating mutations may impair the differentiation potential of neural crest (NC) progenitor cells. Here, we demonstrated that the expression of the endogenous ALK gene starts at E10.5 in the developing sympathetic ganglia (SG). To decipher the impact of deregulated ALK signaling during embryogenesis on the formation and differentiation of sympathetic neuroblasts, Sox10-Cre;LSL-ALK-F1174L embryos were produced to restrict the expression of the human ALK-F1174L transgene to migrating NC cells (NCCs). First, ALK-F1174L mediated an embryonic lethality at mid-gestation and an enlargement of SG with a disorganized architecture in Sox10-Cre;LSL-ALK-F1174L embryos at E10.5 and E11.5. Second, early sympathetic differentiation was severely impaired in Sox10-Cre;LSL-ALK-F1174L embryos. Indeed, their SG displayed a marked increase in the proportion of NCCs and a decrease of sympathetic neuroblasts at both embryonic stages. Third, neuronal and noradrenergic differentiations were blocked in Sox10-Cre;LSL-ALK-F1174L SG, as a reduced proportion of Phox2b+ sympathoblasts expressed βIII-tubulin and almost none were Tyrosine Hydroxylase (TH) positive. Finally, at E10.5, ALK-F1174L mediated an important increase in the proliferation of Phox2b+ progenitors, affecting the transient cell cycle exit observed in normal SG at this embryonic stage. Altogether, we report for the first time that the expression of the human ALK-F1174L mutation in NCCs during embryonic development profoundly disturbs early sympathetic progenitor differentiation, in addition to increasing their proliferation, both mechanisms being potential crucial events in NB oncogenesis.

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

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

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