Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation

Abstract

BackgroundUnderstanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults.ResultsUsing a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages.ConclusionsWe conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2215-8) contains supplementary material, which is available to authorized users.

Single cell analyses of the effects of Amyloid-beta42 and Interleukin-4 on neural stem/progenitor cell plasticity in adult zebrafish brain

“…Additionally, NBs are highly regulated by Notch signaling (Figure 3M). These results are consistent with published literature as Fgf signaling is important for NSPC proliferation (Ganz et al, 2010; Kaslin et al, 2009), Wnt signaling is active in adult zebrafish brain (Shimizu et al, 2018; Tallafuss et al, 2015), chemokine signaling is a prominent interaction mechanism for maintenance and regenerative ability of NSPCs (Belmadani et al, 2006; Diotel et al, 2010; Kizil et al, 2012a), and Notch signaling determines the neurogenic competency in neuronal progenitors (Alunni et al, 2013; Than-Trong et al, 2018). We also identified other potential interactions with less studied ligands and receptors such as agrn , appa , ctgfa , gnai2 , penkb , ptn , serpine , edil3a and hbegf (Dataset S1L), which might provide novel candidates for regulation of NSPC plasticity.…”

“…Since these genes are associated with a more progenitor state of the radial glia, we can speculate that s100a10+/cx43+ clusters could represent a radial glial state that is more progenitor-like. This is consistent with the absence of immature neuronal fate marker elavl3 ( Kizil et al, 2012b ; Tallafuss et al, 2015 ) in dRGC1 and mRGC1 but its presence in dRGC2 and mRGC2 (Dataset S1B, Figure S3). Given that all RGC clusters express widely-used glial markers s100b (Raponi et al, 2007) , her4 , fabp7a (Ito et al, 2010) , gfap (Lam et al, 2009) , msi1 (Kaneko et al, 2000) , slc1a3b (Untiet et al, 2017) , glula (Grupp et al, 2010) , sox2 (Ellis et al, 2004) , vim (Doetsch et al, 1997) , hopx (Li et al, 2015) and notch3 (Alunni et al, 2013) (Figure 2; Figure S3; Dataset S1B), our unbiased separation of RGCs into six clusters with differential expression of regional markers in important in terms of delineating the heterogeneity of different glial progenitors.…”

“…Unique MF GO-terms included various signaling pathways and receptor activity such as Wnt/Frizzled binding (dRGC1), epinephrine binding (mRGC1), collagen binding (vRGC1), retinoic acid binding (vRGC2), chromatin binding (NB), insulin binding (NE), and glutamate receptor binding (GIs+) (Figure 3G, Dataset S1H). Many of these signaling pathways were shown to be required for normal functioning and plasticity of the zebrafish brain (Bhattarai et al, 2016b; Diotel et al, 2013; Jiao et al, 2011; Shimizu et al, 2018; Sloin et al, 2018; Tallafuss et al, 2015; Than-Trong et al, 2018); however, their specific involvement for certain subtypes of NSPCs was not shown before. Therefore, our results provide a detailed resource on specific molecular regulatory networks, constitute a starting point for underlying the heterogeneity of the NSPC populations, and serves as a comprehensive repository for researchers interested in certain molecular programs.…”

Investigation of Islet2a function in zebrafish embryos: Mutants and morphants differ in morphologic phenotypes and gene expression

“…In addition, a splice blocking MO, Sp-MO (10 mg/ml) was designed to a sequence overlapping the splice junction between intron 1–2 and exon 2 ( ; S1 Fig ). Comparisons of the MO and islet gene sequences supports specificity of the MOs targeting islet2a ( Table 1 and S1 Table ) [ 28 , 29 ]. The T-MO and Sp-MO produced similar motor neuron morphological results and we report results obtained with T-MO.…”

EC-tagging allows cell type-specific RNA analysis

“…TU-tagging relies on cell type-specific expression of uracil phosphoribosyltransferase (UPRT) to convert a modified uracil, 4-thiouracil (TU), into 4-thiouridine (4sUd) monophosphate that is subsequently incorporated into nascent RNAs. TU-tagging has been used to study cell type-specific gene expression in Drosophila ( 10 , 12 ), zebrafish ( 13 , 14 ), mammalian tissue culture cells ( 15 ) and mice ( 16 , 17 ). TU-tagging has also been used to measure cell type-specific mRNA decay in Drosophila embryos ( 18 ).…”