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Asynchronous transcription and translation of neurotransmitter-related genes characterize the initial stages of neuronal maturation in Drosophila [1]

['Graça S. Marques', 'Nova Medical School', 'Faculdade De Ciências Médicas', 'Nms', 'Fcm', 'Universidade Nova De Lisboa', 'Lisboa', 'José Teles-Reis', 'Nikolaos Konstantinides', 'Department Of Biology']

Date: 2023-05

Neuron specification and maturation are essential for proper central nervous system development. However, the precise mechanisms that govern neuronal maturation, essential to shape and maintain neuronal circuitry, remain poorly understood. Here, we analyse early-born secondary neurons in the Drosophila larval brain, revealing that the early maturation of secondary neurons goes through 3 consecutive phases: (1) Immediately after birth, neurons express pan-neuronal markers but do not transcribe terminal differentiation genes; (2) Transcription of terminal differentiation genes, such as neurotransmitter-related genes VGlut, ChAT, or Gad1, starts shortly after neuron birth, but these transcripts are, however, not translated; (3) Translation of neurotransmitter-related genes only begins several hours later in mid-pupa stages in a coordinated manner with animal developmental stage, albeit in an ecdysone-independent manner. These results support a model where temporal regulation of transcription and translation of neurotransmitter-related genes is an important mechanism to coordinate neuron maturation with brain development.

Funding: This project has received funding from the European Research Council (ERC; https://erc.europa.eu/ ) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2017-STG-GA 759853-StemCellHabitat to C.C.F.H.); by Wellcome Trust ( https://wellcome.org/grant-funding ) and Howard Hughes Medical Institute ( https://www.hhmi.org/ ; HHMI-208581/Z/17/Z-Metabolic Reg SC fate to C.C.F.H.); European Molecular Biology Organization (EMBO) Installation grant ( https://www.embo.org/ ; H2020-EMBO-3311/2017/G2017 to C.C.F.H.); by Fundação para a Ciência e Tecnologia ( https://www.fct.pt/ ; PTDC/BIA-BID/0681/2021; IF/01265/2014/CP1252/CT0004 to C.C.F.H. and PD/BD/114253/2016 to G.S.M.). N.K. was supported by the National Eye Institute ( https://www.nei.nih.gov/ ; K99 EY029356-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We show that neuron maturation can be divided in 3 phases: a first phase, immediately after neuron birth, when neurons are specified but do not transcribe terminal differentiation genes; a second phase, which starts shortly after birth (<12 h), when neurons start transcribing, but not translating terminal differentiation genes such as the NT-related genes vesicular glutamate transporter (VGlut), choline acetyltransferase (ChAT), and glutamic acid decarboxylase 1 (Gad1); and a third phase when these NT-related genes are translated in coordination with the animal developmental stage. We additionally show that translation inhibition or onset of NT-related gene translation occurs in an ecdysone-independent manner.

Several advances have been made in understanding how axon guidance occurs and how neurons choose and connect to their specific partners to form circuits [ 25 ]. However, to date, very little is known about the mechanisms that trigger neuronal maturation [ 26 ]. Interestingly, it was shown that the age-related transcriptomic diversity of Drosophila neurons is partially lost as early as 15 h after neuron birth, resulting in transcriptomic convergence in mature neurons [ 27 ]. This highlights the need to study young neurons as the transcriptomic profiles involved in the initial phases of neuron maturation can be quickly lost and may no longer be detectable in adult neurons. However, the transcriptomic datasets originated so far did not focus on young secondary neurons, not allowing for their clear distinction from mature neurons [ 18 , 27 – 35 ]. In this study, we characterized the transcriptional changes that lead to mature neurons in Drosophila larval central brain (CB) and ventral nerve cord (VNC) lineages. We devised a conditional genetic strategy to label, select, and sequence single-cell transcriptomes of secondary neuronal lineages, including only 0-h- to 12.5-h-old neurons (time relates to neuron birth, i.e., when the neuron is generated by a ganglion mother cell (GMC), it is considered to be 0 h old; 12.5 h later, this same neuron will be 12.5 h old). This time window was chosen as it is prior to neuron transcriptomic convergence [ 27 ]. The analysis of these young neurons allowed us, for the first time, to transcriptionally characterize the initial phases of neuron maturation.

In Drosophila, neurons are formed in 2 waves. The first wave occurs in embryos to form the central nervous system of the embryo itself and of the larva (primary neurogenesis), while the second wave occurs in larva and early pupa and is responsible for the generation of the majority of the neurons that will populate the adult brain (secondary neurogenesis) [ 14 ]. Although secondary neurons are formed during a large developmental time window, neurons are described to remain immature until mid-pupal stages when synaptogenesis synchronously starts [ 15 – 19 ]. During this maturation period, neurons establish their morphological and electrophysiological properties and axon guidance occurs, ultimately allowing them to reach their target regions and connect with other neurons [ 20 – 23 ]. This process requires the coordinated expression of a combination of effector genes, responsible for the terminal differentiation of neurons, such as cell surface molecules, ion channels, and neurotransmitter (NT) receptors [ 18 , 24 ].

During brain development, a small number of neural stem cells (NSCs) gives rise to the large neuronal diversity found in the adult brain (reviewed in, for instance, [ 1 , 2 ]). Studies in Drosophila have been fundamental to show how each neuronal fate is determined by the combination of several layers of transcription factors (TFs) and signaling pathways acting at the level of the NSC, intermediate progenitors, or even at the level of the differentiating neuron [ 3 – 9 ]. The core mechanisms of neural lineage progression and neuronal fate specification have been shown to be well conserved in mammals [ 8 , 10 – 13 ].

Brain development is a complex process that requires the coordinated generation and maturation of thousands of neurons and glia cells. Ultimately, the formation of the right number and type of neurons and their synaptic connections results in the complex neuronal circuitry that will allow proper brain functioning.

Results

Neurons in Phase 2 of maturation transcribe VGlut and ChAT, but no protein can be detected The expression of NT pathway genes, ion channels, and other terminal differentiation genes in secondary neurons in larval stages was surprising as these neurons will only initiate synaptogenesis days later in pupal stages [17]. Hence, to further validate our observations done by scRNA-Seq, we assessed the presence of mRNA molecules in vivo by single-molecule fluorescence in situ hybridization (smFISH). We analysed the mRNA expression pattern for the most abundant NT pathway markers VGlut, ChAT, and Gad1. We labeled cells using a similar strategy as to previously described, raising the animals at 18°C and shifting them to 25°C 18 h before dissection, at an equivalent developmental time of 105 h ALH (S4A Fig). In this set of experiments, we used a permanent GFP labeling strategy to allow us to label the secondary neurons formed and to track these neurons until later time points. A clonal strategy is required to identify secondary neurons in the larval and pupal CB, as in the CB secondary neurons coexist with primary neurons, which were made during embryogenesis and are already mature at these stages. This strategy to label secondary neurons uses a ubiquitin promotor to control Stinger expression (a nuclear-localized EGFP) upon removal of an FRT cassette by a UAS-FLP [64]. The described smFISH analysis detected VGlut, ChAT, and Gad1 mRNA molecules in the progeny generated in this 18-h period, showing that indeed early-born secondary neurons (<12.5 h old) can already transcribe these terminal differentiation genes (Figs 3A, S4A, and S4B). Based on our transcriptomic analysis, these 18-h clones should also contain Phase 1 neurons that have not yet started NT gene transcription. To quantitatively determine the amount of mRNA in young versus older neurons, and as antibodies for lineage fate markers do not function well with the smFISH protocol used, we devised a quantification strategy based on the distance from the cell to the mother NB. NB lineages are stereotypical and as cells do not migrate, cells closer to the mother NB are younger than cells further away, which are pushed inward the brain with the birth of new cells. Consistently, in the cells closest to the NB (Region 1/green; S4C Fig), the number of smFISH spots representing the abundance of mRNA of ChAT or Gad1 was significantly lower than the number of spots present in the cells further away from NBs (Region 2/magenta; S4C and S4D Fig, S1 Data). The quantification of the number of smFISH spots of VGlut in younger versus older progeny was difficulted as independent lineages were hard to separate and thus only a small number of cells could be accurately counted. Nonetheless, this quantification shows that secondary neurons express VGlut mRNA. Additionally, the cells further away from the NB, predicted to be older/Phase 2 neurons, tend to have higher number of spots when compared to cells closer to the NB, although this difference was not statistically significant (S4D Fig, S1 Data). Next, we wanted to determine if the presence of mRNA in the Phase 2 neurons is already accompanied by the expression of the respective protein. Therefore, we again generated 18-h clones but this time tested for the presence of VGlut or ChAT proteins with available antibodies. Interestingly, none of the secondary neurons (GFP+) generated in the 18-h window is stained with either VGlut or ChAT antibody, indicating that these proteins are still not expressed (Fig 3B, outlines). These results indicate that even though the oldest neurons in our dataset (Phase 2) already express VGlut and ChAT mRNA, these molecules are either not yet being translated into protein or that the levels of the proteins made are somehow kept bellow detection limit in these maturing neurons. PPT PowerPoint slide

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TIFF original image Download: Fig 3. The timing of VGlut and ChAT protein detection depends on animal age. (A, B) 18-h clones induced at 87 h ALH with the VT201094-Gal4 driver using G-TRACE; VT201094-Gal4;tubGal80ts was used to permanently label with GFP NB-derived lineages using G-TRACE; clones were analysed at 105 h ALH (wandering third instar larvae); oldest neurons in the clone are approximately 12.5 h old. (A) smFISH against VGlut or ChAT. Individual mRNA molecules are displayed as magenta dots; neural cells are labeled with nuclear GFP (green). Dashed line delimitates a neural clone; pink arrowhead indicates the closest cell to the neuroblast where mRNA is visible; yellow asterisk identifies neuroblast. Z-projection from 21 slices with 0.25 μm interval, resulting in a total z range of approximately 5 μm (roughly the size of a neuron); scale bar, 5 μm. (B) Immunofluorescence images for VGlut or ChAT antibody staining (magenta). Neural cells in clones are labeled with nuclear GFP (green); outlines indicate examples of lineages with GFP-positive cells; yellow asterisk identifies neuroblast. Scale bar, 20 μm. (C, D) 72-h clones induced at 87 h ALH and analysed at 159 h ALH (approximately 48 h APF) using G-TRACE; oldest neurons in the clone are approximately 66.5h. (C) Schematic representation of the temporal strategy used to induce 72-h clones in neural lineages represented in green. Timings used for the scRNA-Seq dataset (18-h clones induced at 87 h ALH and analysed at 105 h ALH) are represented in grey. (D) Immunofluorescence images for Vglut or ChAT antibody staining (magenta). VT201094-Gal4 drives nuclear GFP (green) expression in NBs, daughter cells generated by NBs during clone period inherit GFP expression. Outlines indicate clones; scale bar, 20 μm. (E, F) 72-h clones induced at 50 h ALH and analysed at 122 h ALH (approximately 12 h APF) using G-TRACE; oldest neurons in the clone are approximately 66.5 h. (E) Schematic representation of temporal strategy used to induce clones in neural lineages. (F) Immunofluorescence images for VGlut or ChAT antibody staining (magenta). VT201094-Gal4 was used to drive nuclear GFP (green) expression in NBs; outlines indicate neurons clones; scale bar, 20 μm. See also S4 Fig. ALH, after larval hatching; APF, after puparium formation; ChAT, choline acetyltransferase; NB, neuroblast; scRNA-Seq, single-cell RNA sequencing; smFISH, single-molecule fluorescence in situ hybridization; VGlut, vesicular glutamate transporter. https://doi.org/10.1371/journal.pbio.3002115.g003

Initiation of VGlut and ChAT protein detection is coordinated with animal developmental stage rather than neuron age The fact that VGlut and ChAT transcripts are present, but their proteins are absent, suggests that there is a mechanism delaying the expression or accumulation of these proteins in maturing neurons. To understand what determines the time point when VGlut/ChAT protein is first detected in maturing neurons, we tested whether (1) the presence of VGlut/ChAT protein is dependent of neuron age and requires neurons to be older/of a certain age; or (2) VGlut/ChAT protein levels are coordinated with animal development starting to be translated or accumulated only at a certain stage of brain development. In order to test these hypotheses, we again used a permanent labelling strategy to fluorescently label neural lineages, thus labelling neurons from their birth, and follow them until a certain age. We have previously shown that in third instar larvae, neurons younger than 12.5 h VGlut/ChAT protein cannot be detected (Fig 3B). To test if in older neurons there is already VGlut/ChAT protein, we have allowed these neurons to age for a longer time. To do this, we induced clone formation at the same time as previously (87 h ALH), but instead, we let them develop for 72 h. Hence, the older neurons would be up to 66.5 h old, and the animal would be 159 h ALH (approximately 48 h APF; Fig 3C). In these clones, coexpression of GFP and either VGlut or ChAT protein is detected in several neurons cell bodies (Fig 3D, outline). However, these results still fit both hypotheses, since neurons may have VGlut/ChAT protein because they are older than in the previous clones, or simply because the animal itself is older and closer to the onset of synaptogenesis [17]. In order to uncouple neuronal age from the animal’s age, and tease apart these 2 options, we generated a clone for the same duration of 72 h but starting at an earlier time in animal development. We have thus induced clone formation at 50 h ALH (Fig 3E). This new timeline still allows neurons to age up to 66.5 h old, but the animal itself, although still a pupa, would be only approximately 12 h APF (122 h ALH). Interestingly, in these clones, there is no codetection of GFP and VGlut or ChAT (Fig 3F). Hence, the presence of VGlut and ChAT protein is not necessarily triggered by neuronal age itself, being rather dependent on the age of the animal. Overall, our results indicate that the detection of protein for NT pathway genes in maturing neurons depends on the animal developmental stage, representing what we propose to be a third phase of neuronal maturation.

VGlut, ChAT, and Gad1 are transcribed days before protein is present also in optic lobe neurons To determine if the observed pattern of expression of NT pathway genes is also conserved in other brain regions and neuron types, we analysed maturing neurons in the optic lobe. The neurons that compose the adult visual system are all generated in post-embryonic stages, i.e., they are all secondary neurons [65], and establish synapses in mid-pupal stages at approximately 55 h APF [15,19,66]. We have thus stained larval and pupal optic lobe regions with antibodies against VGlut or ChAT. Since antibodies anti-Gad1 are not available, we have assayed Gad1 expression utilizing an allele of Gad1 endogenously tagged with EGFP [67,68]. This analysis revealed that during third instar larval stages, no VGlut, ChAT, or Gad1 are detected in the entire region of the optic lobe (Figs 4A, wL3 and 0 h APF; S4E, wL3). VGlut, ChAT, and Gad1 proteins were only detected, albeit at still low levels, at approximately 24 h APF (Figs 4A, 24 h APF; S4E Fig, 24 h APF). As a large fraction of optic lobe neurons are interneurons, whose cell bodies and projections remain within the optic lobe [65], the absence of NT proteins in the entire optic lobe region additionally shows that there is no protein in neither neuron cell bodies or in their extensions. As Gad1 is also normally expressed in cell bodies [69–71], this experiment also supports that NT protein absence in young stages is not related to the status of vesicle formation or maturation. To determine if NT pathway genes are also transcribed in the optic lobe during the stages when no protein is detected, we have reanalysed the single-cell transcriptome dataset generated in [18] where the single-cell transcriptome of the optic lobe was sequenced from 0 to 96 h APF in 12-h intervals. This analysis showed that optic lobe neurons transcribe NT pathway genes at 0 h APF (normalized average expression ranging from 0.21 to 5.74; S2 Data), several hours before protein starts being detected in mid-pupal stages. One possibility is that protein only accumulates to detectable levels when the levels of transcripts reach a certain threshold, and this is why protein is not visible up to 24 h APF. To test this hypothesis, we selected a subset of neurons that represent glutamatergic, cholinergic, or GABAergic neurons and for which counts for the corresponding NT transcript can be detected in all time points. We have thus selected 2 glutamatergic neurons (VGlut+; L1, Mi9), 2 cholinergic neurons (ChAT+; L5, Mi1), and 2 GABAergic neurons (Gad1+; Dm10, Mi4) (S4F Fig, S2 Data). This analysis revealed that NT genes are expressed at constant levels from early pupal stages (0 h APF) to approximately 48 h APF (S4F Fig, S2 Data). Although the expression levels of VGlut were found to be more variable, the constant levels of Gad1, ChAT (S4F Fig, S2 Data) show that there is not a consistent increase in the levels of transcripts preceding the start of protein detection in optic lobe neurons (approximately 24 h APF; Fig 4A). So, an increase in transcription levels cannot by itself justify the detection of protein at 24 h APF. The observed increase in NT gene transcription levels from approximately 48 h APF onward has been previously reported, and it is thought to be related to neuronal activity [18]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. VGlut and ChAT proteins are not detected in wL3 OL. (A) insc-Gal4,UAS-CD8::GFP was used to label NB lineages in the OL. GFP(cyan), VGlut (yellow), and ChAT (magenta) protein expression in the OL at wL3 stages, 0 h APF, and 24 h APF. Insc-Gal4 is expressed in both OL and CB regions; OL is delimitated by a dashed line. Arrows in 24 h APF panel refer to VGlut protein (yellow) or ChAT protein (magenta) expression. Scale bar, 50 μm. (B, C) Mean fluorescence intensity measured in wL3 raised in food supplemented with MG132 diluted in DMSO, or in DMSO alone (control). (B) Arm mean fluorescence intensity was measured in OL neuroepithelia (control, n = 6; MG132, n = 5). (C) VGlut and ChAT mean fluorescence intensity was measured in medulla neurons (control, n = 7; MG132, n = 5). Plotted data were normalized to the mean of the control. Data shown as mean ± SEM; statistical analysis was done using unpaired two-tailed t test; **P value < 0.01, ns = nonsignificant. The data underlying this figure are contained within S3 Data. (D) UAS-Stinger::GFP (green) expression driven by VGlut-T2A-Gal4 or ChAT-T2A-Gal4 in wL3 stages. DAPI (magenta). OL delimited by dashed line. CB located to the left of the OL. Scale bar, 50 μm. (F) Model for the asynchronous onset of VGlut and ChAT transcription and translation during the first phases of neuron maturation. See also S4 Fig. APF, after pupa formation; Arm, Armadillo; CB, central brain; ChAT, choline acetyltransferase; NB, neuroblast; OL, optic lobe; VGlut, vesicular glutamate transporter; wL3, wandering L3. https://doi.org/10.1371/journal.pbio.3002115.g004 Overall, these results show that NT pathway genes are also transcribed in larval optic lobe neurons days before protein is detected, showing that this is a conserved mechanism occurring in maturing neurons in multiple brain regions. These results further show that the beginning of VGlut, ChAT, or Gad1 protein detection does not occur as a consequence of transcript accumulation.

Absence of VGlut and ChAT proteins in young maturing neurons in larval stages cannot be explained by low levels of translation or proteasomal protein degradation To try to understand the mechanism that temporally regulates the levels of VGlut or ChAT protein in the different phases of maturing neurons/animal stages, we next tested several hypotheses: One interesting hypothesis is that there is translation of VGlut and ChAT during larval and young pupal stages but that the protein formed during these early stages is quickly degraded. Later in mid-pupal stages, these proteins would no longer be degraded and would therefore accumulate. This would be interesting on its own as it would indicate that there is an active mechanism to degrade these proteins and ensure that NT protein levels are maintained at low levels until mid-pupal stages potentially coordinated with synaptogenesis. To test this hypothesis, we blocked protein degradation by inhibiting the proteasome with the well-described proteasome inhibitor MG132 [72]. We treated larvae with MG132 as previously described [73] to determine if proteasome inhibition would lead to accumulation of VGlut or ChAT protein in larval optic lobes (when and where normally no protein is detected). To ensure that the treatment with MG132 was effective, we quantified the levels of Armadillo (Arm) in optic lobe neuroepithelia as a positive control. Drosophila Arm is targeted for destruction by the proteasome unless stabilized by extracellular Wnt signals [74]. Confirming that inhibition of the proteasome by MG132 was effective, Arm was significantly accumulated in the neuroepithelia (Fig 4B, S3 Data). However, proteasome inhibition with MG132 did not lead to the accumulation of VGlut or ChAT in optic lobe (Fig 4C, S3 Data; analysis done in medulla neuron region that contains both glutamatergic and cholinergic neurons) [65]. In addition, we have inhibited the proteasome by knocking down several subunits of the proteasome using previously validated RNAi lines [75]. Consistent with the results obtained with MG132, knockdown of these proteasome subunits in optic lobe neurons did not cause accumulation of VGlut of ChAT (S4G Fig, S4 Data). These results indicate that NT proteasomal protein degradation is not the mechanism responsible for the undetectable levels of protein in young neurons in larval brains. To further test if there are low levels of translation of NT proteins in larval stages that might be below the detection limit of antibodies, we have used a transgenic line in which a T2A-Gal4 is knocked-in downstream of endogenous VGlut or ChAT (VGlut-T2A-Gal4 and ChAT-T2A-Gal4; [76]). 2A peptide-linked polycistronic vectors can be used to express multiple proteins from a single open reading frame; a cleavage event at the 2A sequence then separates the individual proteins. Thus, if there is translation of VGlut or ChAT proteins, then Gal4 would also be translated. Gal4, a TF originally cloned from yeast, contains a DNA-binding domain (DBD) and a transcription activation domain (AD), and binds to a specific sequence, UAS (upstream activation sequence) [77]. The Gal4/UAS system results in the expression of downstream genes at much higher levels than endogenous tissue-specific promoters [78]. Therefore, this amplification process generates high levels of gene expression. We have thus crossed these NT-T2A-Gal4 to UAS-Stinger-GFP (nuclear fast-maturing, insulated, stable enhanced-GFP; [79]) to determine if there are low levels of translation occurring. The brightness and stability of this Stinger-GFP protein potentiates for even a low number of molecules to be detected. We have performed these analyses in the optic lobe in larval stages, when normally no NT protein can be detected with antibodies. However, we have observed no GFP expression in VGlut-T2A-Gal4; UAS-Stinger-GFP or ChAT-T2A-Gal4; UAS-Stinger-GFP animals (Fig 4D, outline). As a confirmation that these lines are functioning, we can detect GFP signal in the mature primary neurons in the larval central brain (Fig 4D, to the left of the dashed lines). Combined, all these results provide strong evidence supporting that VGlut and ChAT are not translated in secondary neurons in larval stages. Interestingly, this suggests that there is a delay in translation initiation of these NT genes and that translation then starts in mid-pupa stages in a coordinated manner with animal development.

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