Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival

Abstract

Activating anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system, we employed CRIPSR/Cas9, incorporating orthologs of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibited enhanced Alk signaling phenotypes, but unexpectedly depended on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains displayed hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We showed that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing, we identified perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of Alk-activating mutations to perturb specification and promote survival in neuronal lineages. This article has an associated First Person interview with the first author of the paper.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in theDrosophilaCNS

“…To further explore the observed enrichment of Alk-regulated TaDa loci in neuroendocrine cells, we used a Dimmed (Dimm) transcription factor reporter ( Dimm-Gal4>UAS-GFPcaax ), as a neuroendocrine marker (Park et al , 2008), to confirm Alk protein expression in a subset of neuroendocrine cells in the larval central brain, ventral nerve cord and neuroendocrine corpora cardiaca cells (Figure 2g, Figure 2 – figure supplement 1) . This could not be confirmed at the RNA level, due to low expression of dimm in both our and publicly available single cell RNASeq datasets (Brunet Avalos et al , 2019; Michki et al , 2021; Pfeifer et al ., 2022).…”

“…We compared CNS TaDa Alk targets with our previously published embryonic visceral mesoderm TaDa datasets that were derived under similar experimental conditions (Mendoza-Garcia et al ., 2021) and found 775 common genes (Figure 1e , Table S1) . Gene ontology (GO) analysis identified GO terms in agreement with previously reported Alk functions in the CNS (Bai & Sehgal, 2015; Bazigou et al ., 2007; Cheng et al ., 2011; Gouzi et al ., 2011; Orthofer et al ., 2020; Pfeifer et al ., 2022; Rohrbough & Broadie, 2010; Rohrbough et al ., 2013; Woodling et al , 2020) such as axon guidance, determination of adult lifespan, nervous system development, regulation of gene expression, mushroom body development, behavioral response to ethanol and locomotor rhythm ( Figure 1f ). Many of the differentially regulated identified loci have not previously been associated with Alk signaling and represent candidates for future characterisation.…”

“…To further characterize Alk-regulated TaDa loci, we set out to examine their expression in scRNA-seq data from wild-type third instar larval CNS (Pfeifer et al ., 2022). Enrichment of TaDa loci were identified by using AUCell, an area-under-the-curve based enrichment score method, employing the top 500 TaDa hits (Aibar et al , 2017) (Figure 2a-b , Table S1) .…”

“…Using a newly generated antibody we characterized the expression of Spar in the Drosophila CNS, showing that its expression overlaps with the Dimm transcription factor that is expressed in the fly neuroendocrine system (Hewes et al ., 2003), suggesting that Spar is expressed along with multiple other neuropeptides in pro-secretory cells of the CNS (Park et al ., 2008). Spar is also expressed in well-established structures such as the mushroom bodies (Crocker et al , 2016) (Table 1), which are known to be important in learning and memory and regulate food attraction and sleep (Joiner et al , 2006; Pitman et al , 2006), and where Alk is also known to function (Bai & Sehgal, 2015; Gouzi et al ., 2011; Pfeifer et al ., 2022). Interestingly , Spar is expressed in a subset of peptidergic neurons which emerge from the ventral nerve cord (VNC) and innervate larval body wall muscle number 8.…”

First person – Kathrin Pfeifer

“… First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Kathrin Pfeifer is first author on ‘ Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival ’, published in DMM. Kathrin is a postdoc in the lab of Ruth Palmer at the University of Gothenburg, Gothenburg, Sweden, investigating aberrant Alk signalling in Drosophila .…”