Expression of the Neuroblastoma-Associated ALK-F1174L Activating Mutation During Embryogenesis Impairs the Differentiation of Neural Crest Progenitors in Sympathetic Ganglia

Abstract

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.

Keywords: ALK; PHOX2B; SOX10; differentiation; mouse embryos; neural crest cells; neuroblastoma; sympathetic ganglia.

Figure 1

ALK-F1174L expression in Sox10⁺ cells NCCs mediates an embryonic lethality. Representative images of both WT and Sox10-Cre;LSL-ALK-F1174L embryo phenotypes for stages from E9.5 to E12.5. Embryos were genotyped by PCR as described in the Material and Method section.

Figure 2

ALK mRNA is expressed from E10.5 in SG. Analysis of endogenous ALK mRNA expression in WT embryos. (A) Representative pictures of ALK ISH (red) staining of WT embryos at E9.5 (magnifications: x20 for top panel and x40 for lower panels). (B) Representative pictures of ALK ISH (red) staining in WT SG from E10.5 to E12.5 (×40 magnification). (A,B) Phox2b IF (green) staining was applied after ISH to highlight migrating sympathoblasts and SG. Specific ALK ISH signals (red, small dots) are identified by white arrows, and non-specific signals (diffuse cytoplasmic or membranous staining) by yellow arrowheads. Numbers of embryos analyzed: E9.5 n = 3, E10.5 n = 4, E11.5 n = 3, and E12.5 n = 2. Representative images of ISH with the positive control probe Mm-Ppib (white), showing mRNA integrity of the embryo sections, and the negative control DapB (pink), showing absence of the background due to molecule trapping in the tissue, are also displayed for each embryonic stages with Dapi staining (blue).

Figure 3

Increased SG size in Sox10-Cre;LSL-ALK-F1174L embryos. (A) Representative images of WT and Sox10-Cre;LSL-ALK-F1174L embryo sections stained for Sox10 (red) and Phox2b (green), with DAPI (blue) at E9.5 and E10.5 (x10 magnification). SG are surrounded by white circle. (B) Representative images of SG IF staining for Sox10 (red) and Phox2b (green) (x40 magnification). (C) Box-plot of SG section areas in arbitrary units (AU) (upper panel) and of the total numbers of cells per SG section (lower panel) at E10.5 and E11.5 in WT and Sox10-Cre;LSL-ALK-F1174L embryos (One-way Anova multiple comparison, ^****p < 0.0001, ^*p = 0.0306, not significant (ns) comparisons are not shown). Numbers of SG sections analyzed at E10.5 and E11.5, respectively: WT n = 22 and n = 23, Sox10-Cre;LSL-ALK-F1174L n = 14 and n = 16.

Figure 4

ALK-F1174L affects the differentiation of sympathetic progenitors. (A) The fractions of Sox10⁺/Phox2b⁻, Sox10⁺/Phox2b⁺, and Sox10⁻/Phox2b⁺ cell populations in WT and Sox10-Cre;LSL-ALK-F1174L embryos at E10.5 (top) and E11.5 (bottom) are shown in Box-plots (Mann Whitney test or unpaired t-test depending on data distributions, ^****p < 0.0001, ^**p < 0.01). (B) The evolution from E10.5 to E11.5 of the three cell populations in WT (top) and Sox10-Cre;LSL-ALK-F1174L (bottom) embryos are illustrated in Box-plots (Krukal-Wallis and One-way Anova respectively, paired comparisons, ^****p < 0.0001, ^**p = 0.0043, ns comparisons are not shown). Numbers of SG sections analyzed at E10.5 and E11.5, respectively: WT n = 22 and n = 23, Sox10-Cre;LSL-ALK-F1174L n = 14 and n = 16. (C) The mean percentages of each cell population are plotted in grouped representations at E10.5 and E11.5 for WT (top) and Sox10-Cre;LSL-ALK-F1174L (bottom) embryos.

Figure 5

ALK-F1174L expression in sympathoblasts impairs noradrenergic differentiation. (A) Representative IF images of TH (red) and Phox2b (green) staining in WT and Sox10-Cre;LSL-ALK-F1174L embryos at E10.5 and E11.5 (x40 magnification). (B) The quantifications of the proportions of Phox2b⁺/TH⁺ cells over total Phox2b⁺ cells in WT and Sox10-Cre;LSL-ALK-F1174L embryos are illustrated in Box-plot for E10.5 and E11.5 (Kruskal-Wallis test, multiple comparisons, ^****p < 0.0001, ^***p = 0.0003, ns comparisons are not shown). Numbers of SG sections analyzed at E10.5 and E11.5, respectively: WT n = 30 and n = 30; Sox10-Cre;LSL-ALK-F1174L n = 18 and n = 16. (C) Mean percentages of TH⁻/Phox2b⁺ and TH⁺/Phox2b⁺ cell populations are plotted in grouped representations at E10.5 and E11.5 for WT (left panel) and Sox10-Cre/LSL-ALK-F1174L SG (right panel) embryos. (D) Representative IF images of double staining for human ALK (h-ALK) (yellow) and TH (red) in two Sox10-Cre/LSL-ALK-F1174L embryos at E10.5. Numbers of SG sections analyzed: n = 10.

Figure 6

Sox10-Cre;LSL-ALK-F1174L SG displayed reduced neuronal differentiation. (A) Representative IF images of Phox2b (green) and βIII-tubulin (red) staining (x40 magnification). (B) Box-plot illustration of the fractions of βIII-tubulin⁺ cells over total Phox2b⁺ cells per section at E10.5 and E11.5 in WT and Sox10-Cre;LSL-ALK-F1174L embryos (Kruskal-Wallis test, paired comparisons, ^****p < 0.0001, ^**p = 0.0034, ^*p = 0.023, ns comparisons are not shown). Numbers of SG sections analyzed at E10.5 and E11.5, respectively: WT n = 20 and n = 28, Sox10-Cre;LSL-ALK-F1174L n = 12 and n = 13. (C) The mean percentages of βIII-tubulin⁻/Phox2b⁺ and βIII-tubulin⁺/Phox2b⁺ cell populations are plotted in grouped representations at E10.5 and E11.5 for WT (left panel) and Sox10-Cre;LSL-ALK-F1174L (right panel) embryos.

Figure 7

Increased proliferation of Phox2b⁺ cells in Sox10-Cre;LSL-ALK-F1174L SG. (A) Representative IF images of Phox2b (green) and Ki67 (red) staining at E10.5 and E11.5 in WT and Sox10-Cre;LSL-ALK-F1174L SG (x40 magnification). SG are surrounded by white circles. (B) Blox-plot representation of the fractions of Phox2b⁺/Ki67⁺ over total Phox2b⁺ cells in WT and Sox10-Cre;LSL-ALK-F1174L SG at E10.5 and E11.5 (Kruskal-Wallis test, multiple comparisons, ^****p < 0.0001, ns comparisons are not shown). Numbers of SG sections analyzed at E10.5 and E11.5, respectively: WT n = 24 and n = 22, Sox10-Cre;LSL-ALK-F1174L n = 16 and n = 16.

Figure 8

Schematic model proposed for the impact of the ALK-F1174L mutation on sympathetic differentiation. The expression of the ALK-F1174L mutation in NCCs affects various steps of the sympathetic differentiation during SA lineage development. ALK-F1174L increases the number of NCCs at early embryonic stages (marked by red halo). The transition from NCCs to Sox10⁺/Phox2b⁺ progenitors, and further to sympathetic progenitors is incomplete in Sox10-Cre;LSL-ALK-F1174L embryos (red dashed arrows). The expression of Sox10 is maintained in a large proportion of cells, while the cell population upregulating Phox2b is reduced, and no variation occur between embryonic stages. The Sox10⁻/Phox2b⁺ sympathetic progenitors do not acquire noradrenergic properties highlighting that ALK-F1174L hinders their maturation into noradrenergic neurons. The proportion of proliferating Phox2b⁺ cells is increased in Sox10-Cre;LSL-ALK-F1174L SG at E10.5 (red circular arrows). Thus, ALK deregulation leading to reduced SA differentiation and proliferative excess may be an essential path for NB initiation. NCC, neural crest cell; DP, differentiating progenitor; SP, sympathetic progenitor; NP, noradrenergic progenitor.