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Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small-cell lung cancer (SCLC), choroid plexus tumors, and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional redundancy between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain insight into this in vivo redundancy. We show that while rb1 inactivation is sufficient to induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking tp53 mutant X. tropicalis line, we demonstrate increased malignancy of rb1/rbl1-mutant glioma towards glioblastoma upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, the tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental setup, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1, generating new insights in the functional redundancy within the retinoblastoma protein family in suppressing neuroendocrine pancreatic cancer and glioma/glioblastoma.

Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small cell lung cancer (SCLC), choroid plexus tumors and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional compensation between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation ofrb1andrbl1by multiplex CRISPR/Cas9 genome editing in the true diploidXenopus tropicalisto gain insight into thesein vivocompensatory mechanisms. We show that whilerb1inactivation is sufficient to induce choroid plexus papilloma, combinedrb1andrbl1inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimickingtp53mutantX. tropicalisline, we demonstrate increased malignancy of retinoblastoma-mutant neural malignancies upon concomitant inactivation oftp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, thetp53status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental set-up, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1 and generate new insights in functional compensation within the retinoblastoma protein family in suppressing pancreatic and specific neural cancers.

Many developmental signals are associated with changes in proliferative response. Also, growing organs and tissues can contain different cellular subpopulations with a defined status in the cell cycle, e.g., quiescent in stem cells, high proliferation in progenitors, cell cycle exit in differentiating cells. This chapter describes a method for isolation of individual cell populations from the Xenopus tadpole brain and determination of their cell cycle status using flow cytometry.

Many developmental signals are associated with changes in proliferative response. Also, growing organs and tissues can contain different cellular subpopulations with a defined status in the cell cycle, e.g., quiescent in stem cells, high proliferation in progenitors, cell cycle exit in differentiating cells. This chapter describes a method for isolation of individual cell populations from the Xenopus tadpole brain and determination of their cell cycle status using flow cytometry.

The four R-spondin secreted ligands (RSPO1–RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1–3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers. Independently of the LGR4/5/6 receptors, RSPO2 acts as a direct antagonistic ligand to RNF43 and ZNRF3 during embryogenesis, and specifies the position and number of limbs that an embryo should form.

Wnt/β-catenin signaling plays a crucial role throughout all stages of brain development and remains important in the adult brain. Accordingly, many neurological disorders have been linked to Wnt signaling. Defects in Wnt signaling during neural development can give rise to birth defects or lead to neurological dysfunction later in life. Developmental signaling events can also be hijacked in the adult and result in disease. Moreover, knowledge about the physiological role of Wnt signaling in the brain might lead to new therapeutic strategies for neurological diseases. Especially, the important role for Wnt signaling in neural differentiation of pluripotent stem cells has received much attention as this might provide a cure for neurodegenerative disorders. In this review, we summarize the versatile role of Wnt/β-catenin signaling during neural development and discuss some recent studies linking Wnt signaling to neurological disorders.

In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.