Neuroblastoma Phox2b Variants Stimulate Proliferation and Dedifferentiation of Immature Sympathetic Neurons

Reiff, Tobias; Tsarovina, Konstantina; Majdazari, Afsaneh; Schmidt, Matthias; Pino, Isabel del; Rohrer, Hermann

doi:10.1523/jneurosci.5368-09.2010

Cited by 54 publications

(66 citation statements)

References 54 publications

(94 reference statements)

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“…Rather, sympathetic ganglion progenitors were less proliferative in these mutants. These results appear contrary to the previous observation that overexpression of NPARM PHOX2B mutants in chick sympathetic ganglion cells leads to increased proliferation of those cells (42). This may be because different types of NPARM PHOX2B mutants were used in that study.…”

Section: Discussioncontrasting

confidence: 99%

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

Nagashimada¹,

Ohta²,

Li³

et al. 2012

J. Clin. Invest.

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The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine β-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

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Section: Discussioncontrasting

confidence: 99%

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

Nagashimada¹,

Ohta²,

Li³

et al. 2012

J. Clin. Invest.

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“…6A). This finding, together with the previous demonstration that Hand2 controls proliferation of sympathoadrenergic precursors and immature neurons in vitro and in vivo (Hendershot et al, 2008;Reiff et al, 2010;Schmidt et al, 2009) and the presence of Hand2 expression in sympathetic ganglia (Fig. 6B), implicated Hand2 in the regulation of Alk expression.…”

Section: Autoregulatory Control Of Alk Expression In Sympathetic Neursupporting

confidence: 83%

“…, and pCAGGS expression plasmids for Hand2, Phox2b wt and Phox2b K155X have been described previously (Janoueix-Lerosey et al, 2008;Reiff et al, 2010).…”

Section: R1275qmentioning

confidence: 99%

“…Mutations in the PHOX2B gene were identified in familial cases of NB and result in tumor predisposition (McConville et al, 2006;Mosse et al, 2004;Trochet et al, 2004;van Limpt et al, 2004). NB PHOX2B mutations affect proliferation and differentiation of embryonic sympathetic neurons and the proliferation of NB cell lines (Raabe et al, 2008;Reiff et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…By contrast, cell cycle genes Cdk6 and Ccnblip identified as Hand2 target genes in developing heart tissue (Holler et al, 2010) were not affected in sympathetic neurons (not shown). As Phox2b has been implicated, together with Hand2, in the control of sympathetic neuron proliferation (Bachetti et al, 2010; Coppola et al, 2010;Reiff et al, 2010) we investigated whether wild-type Phox2b (Phox2b wt ) or a NBrelated Phox2b variant (Phox2b K155X ), ectopically expressed in cultured immature sympathetic neurons, would affect Alk expression . However, we were unable to detect effects of Phox2b wt and Phox2b K155X on Alk expression levels by qPCR (data not shown).…”

mentioning

confidence: 99%

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Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition

et al. 2011

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SUMMARYNeuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression. Activation of Alk signaling, in particular overexpression of ALK wt and NB ALK mutants resulted in the upregulation of NMyc and trkB, which may contribute to continued proliferation and NB predisposition. MATERIALS AND METHODS Expression plasmidsPcDNA3.1 expression plasmids for human ALK wt , ALK F1174L and ALK R1275Q, and pCAGGS expression plasmids for Hand2, Phox2b wt and Phox2b K155X have been described previously (Janoueix-Lerosey et al., 2008;Reiff et al., 2010). shRNA constructsThe shRNA against Gallus gallus Alk and Mk were designed using BLOCK-iT RNAi Designer (Invitrogen, Karlsruhe, Germany) and target the following sequences: shAlk, 5Ј-AAU GGU UUC UCU CUA UGU CCA ACU C-3Ј; shMk, 5Ј-GAG CUG ACU GCA AGU ACA AGU UUG A-3Ј. Scrambled shRNAs (sc-shRNA, Invitrogen, Karlsruhe, Germany) served as controls. In situ hybridizationNon-radioactive in situ hybridization on cryosections and preparation of digoxigenin labeled probes for chick Phox2b was carried out as described previously (Ernsberger et al., 1997;Stanke et al., 1999). qPCR analysisEqual amounts of RNA were used to synthesize cDNA with Oligo(dT)-primers and Superscript-III-reverse-transcriptase according to manufacturer's instructions (Invitrogen, Karlsruhe, Germany). The PCR was carried out using Abgene's Absolute Blue SYBR-Green qPCR Mix (Abgene, Epsom, UK) in a Stratagene Mx3000p Light Cycler (Stratagene, Waldbronn, Germany). All primers (MWG Biotech AG, Ebersberg, Germany) were designed to anneal optimally at 58°C with PerlPrimer (Marshall, 2004) (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, repeated for 50 cycles and a subsequent dissociation curve). The primer pairs (see Table S1 in the supplementary material) were ...

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Prox1 identifies proliferating neuroblasts and nascent neurons during neurogenesis in sympathetic ganglia

Holzmann

Hennchen

Rohrer

2015

Developmental Neurobiology

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Neurogenesis in embryonic sympathetic ganglia involves neuroblasts that resume proliferation following neuronal differentiation. As cell cycle exit is not associated with neuronal differentiation, the identity of proliferating neuroblasts is incompletely understood. Here, we use sympathetic ganglia of chick embryos to define the timing of neurogenesis and neuroblast identity focusing on the expression and function of the transcription factor Prox1. We show that a large fraction of neuroblasts has initially withdrawn from the cell cycle at embryonic day 3 (E3), which is reflected by a high proportion of p27(+)/Islet1(+) neuroblasts (63%) and low numbers of EdU(+)/Islet1(+) cells (12%). The proportion of proliferating Islet1(+) neuroblasts, identified by EdU pulse labeling and by the absence of the postmitotic marker p27 increases to reach maximal levels at E5, when virtually all neuroblasts are in the cell cycle (95%). Subsequently, the proportion of EdU-labeled and p27(-) neuroblasts is reduced to reach low levels at E11. Interestingly, the expression of the transcription factor Prox1 is restricted to the neuronal lineage, that is, Sox10(+)/Phox2b(+) neuron progenitors, proliferating p27(-)/Islet1(+) neuroblasts and nascent neurons but is rapidly lost in postmitotic neurons. In vitro and in vivo knockdown and overexpression experiments demonstrate effects of Prox1 in the support of neuroblast proliferation and survival. Taken together, these results define the neurogenesis period in the chick paravertebral sympathetic ganglia including an initial cell cycle withdrawal and identify Prox1 as a marker and regulator of proliferating sympathetic neuroblasts.

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Neuroblastoma Phox2b Variants Stimulate Proliferation and Dedifferentiation of Immature Sympathetic Neurons

Cited by 54 publications

References 54 publications

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition

Prox1 identifies proliferating neuroblasts and nascent neurons during neurogenesis in sympathetic ganglia

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