Author response: Activity-dependent modulation of synapse-regulating genes in astrocytes (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders. Introduction Synapses are points of contact where electrochemical signals are transferred between neurons in a given circuit (Petzoldt and Sigrist, 2014; Südhof, 2018). Synapse development occurs in several molecularly and functionally defined stages, which include initiation, maturation, and pruning steps prior to stabilization and establishment of mature circuits. In the rodent cortex, this occurs over the period of the first postnatal month, initiating at around postnatal day (P) 7, peaking at P14, and stabilizing towards P28 (Blue and Parnavelas, 1983a; Blue and Parnavelas, 1983b; Farhy-Tselnicker and Allen, 2018; Li et al., 2010). Synaptic deficits, for example, caused by mutations in synapse-related genes, are associated with developmental disorders such as autism spectrum disorder and epilepsy (Lepeta et al., 2016). Therefore, understanding how synaptic development is regulated will provide important insights into how circuits form and function in health and are altered in disease. The majority of synapses in the mammalian cortex are contacted by astrocytes, a type of glia and key regulators of circuit development and function (Allen, 2013; Batool et al., 2019; Bernardinelli et al., 2014a; Genoud et al., 2006). Astrocytes express many genes encoding proteins that regulate distinct stages of synapse formation and maturation (Baldwin and Eroglu, 2017). For example, thrombospondin family members induce formation of structurally normal but functionally silent synapses (Christopherson et al., 2005; Eroglu et al., 2009). Glypicans induce the formation of active synapses by recruiting GLUA1 to the postsynaptic side (Allen et al., 2012; Farhy-Tselnicker et al., 2017) and chordin-like 1 induces synapse maturation by recruiting GLUA2 to the postsynaptic side (Blanco-Suarez et al., 2018). These and other signals have been identified using in vitro cell culture approaches and analyzed individually across ages and brain regions in vivo. Yet, a systematic analysis of their expression patterns in a complete circuit in vivo, which would reveal distinct regulatory roles for specific synaptic connections, has not been attempted. The mouse visual circuit is a well-characterized and powerful model to investigate the role of astrocytes in regulating different stages of synaptogenesis. Visual information perceived by the retina is relayed to the visual cortex (VC) via the neurons of the thalamic lateral geniculate nucleus (LGN). Before eye opening (from birth to ~P12 in mice), spontaneous retinal activity evokes correlated cortical responses (Gribizis et al., 2019; Hanganu et al., 2006) that are important for the correct establishment of thalamocortical synapses (Cang et al., 2005). Eye opening marks a step towards synapse maturation in the VC, with the appearance of visually evoked neuronal responses across the retinal-LGN-VC circuit (Espinosa and Stryker, 2012; Hooks and Chen, 2006; Hooks and Chen, 2020). Perturbing this process by methods of visual deprivation, such as dark rearing, delays both synaptic (Albanese et al., 1983; Desai et al., 2002; Freire, 1978; Funahashi et al., 2013; Hsu et al., 2018; Ishikawa et al., 2014; Ko et al., 2014; Majdan and Shatz, 2006; Tropea et al., 2006) and astrocyte maturation (Müller, 1990; Stogsdill et al., 2017). The VC’s glutamatergic neurons are arranged in spatially defined layers, with distinct transcriptomic, functional, and connectivity profiles (Bannister, 2005; Douglas and Martin, 2004). For example, neurons in layers 1 and 4 receive input from the LGN, while layer 5 neurons, the main output to subcortical regions, send their dendrites to layer 1, where they receive input from both local and subcortical projections. Recent work has shown that cortical astrocytes are also spatially arranged in diverse populations (Batiuk et al., 2020; Bayraktar et al., 2020; John Lin et al., 2017; Lanjakornsiripan et al., 2018), in line with evidence from other brain regions showing astrocyte heterogeneity (Chaboub and Deneen, 2012; Chai et al., 2017; Khakh and Deneen, 2019; Oberheim et al., 2012; Rusnakova et al., 2013; Schitine et al., 2015). However, whether this astrocyte diversity is reflected in their regulation of synapses across the distinct layers of the VC is unknown. To understand how the diverse astrocytic signals act together to regulate formation of a complete circuit, it is important to determine both temporal and spatial expression patterns of astrocytic synapse-regulating genes in vivo as well as identify the mechanisms that control their level of expression and the subsequent effect on synapse formation. Here, we use RNA sequencing and in situ hybridization to obtain the developmental transcriptome of astrocytes in the mouse VC in vivo. We find that astrocyte synapse-regulating genes display differential temporal and spatial expression patterns, which correspond to stages of synapse initiation and maturation. Furthermore, we find that developmental regulation of these genes, namely glypican 4 and chordin-like 1, depends on thalamocortical neuronal activity, with additional regulation by astrocyte IP3R2-dependent Ca2+ activity. Manipulating either neuronal or astrocytic activity leads to shifts in synaptic development and maturation. Finally, single-nucleus RNA sequencing analysis reveals diverse populations of astrocytes in the developing VC and identifies novel groups of genes that are regulated by neuronal and astrocyte activity. These findings demonstrate how astrocyte expression of synapse-regulating genes is controlled during development, and how synapse maturation is dependent on neuron-astrocyte communication. These data further provide an important resource for future studies of astrocyte development and astrocyte regulation of synapse formation. Results To address key questions regarding astrocyte regulation of synapse development, this study has three major goals. First, to characterize the spatio-temporal profile of astrocyte development during distinct stages of cortical synaptogenesis at the transcriptomic level. Second, to investigate the mechanisms that influence spatio-temporal expression levels of select astrocytic synapse-regulating genes, focusing on neuronal and astrocyte activity. Third, to determine the global dependence of the astrocyte transcriptome on neuronal and astrocyte activity using an unbiased RNA sequencing approach. All analyses were performed in the mouse VC to enable comparison of these metrics within a defined circuit, providing a blueprint for future analysis of the functional roles of astrocytes in synaptic development. Developmental profiling of the astrocyte transcriptome in the postnatal VC Astrocytes appear in the cortex at birth, and migrate, proliferate, and mature throughout the first postnatal month (Ge et al., 2012), coincidently with the stages of synapse development. To determine the transcriptomic profile of astrocytes at these stages (P7, P14, P28, and adult, P120), we used the astrocyte Ribotag mouse model to isolate mRNA for bulk RNA sequencing (B6N.129-Rpl22tm1.1Psam/J crossed to B6.Cg-Tg(Gfap-cre)73.12Mvs/J – labeled Astrocyte-RiboTag) (Figure 1, Figure 1—figure supplement 1, Figure 1—source data 1; Boisvert et al., 2018; Chai et al., 2017). In this mouse line, cre recombinase drives the expression of an HA-tagged ribosomal subunit, enabling purification of cell-type-specific ribosomes and associated mRNA for analysis by RNA sequencing (Figure 1A and B). We found a significant enrichment in astrocytic genes over other cell types in the mRNA isolated by HA immunopurification (IP; labeled Astro) compared to total VC mRNA (input; IN; Figure 1—figure supplement 1A). Furthermore, immunostaining analysis of VC sections at P28 shows a high overlap between the HA ribosome tag and the astrocyte marker S100β, but not with other cell-type markers (Figure 1—figure supplement 1B-D), with more than 95% of S100β-positive cells also positive for the HA tag, suggesting high astrocyte coverage of our mRNA isolation, consistent with our previous analysis in the adult (Boisvert et al., 2018). The complete RNA sequencing dataset is available in a searchable format online (http://igc1.salk.edu:3838/astrocyte_transcriptome/). Figure 1 with 1 supplement see all Download asset Open asset Developmental profiling of the astrocyte transcriptome in the postnatal visual cortex (VC). See also Figure 1—figure supplement 1, Figure 1—source data 1. (A) Outline of experiment: VCs from Rpl22-HAf/+; Gfap-cre 73.12 mice were collected at different time points corresponding to synapse development and maturation, and subjected to Ribotag pulldown protocol, followed by RNA purification, library preparation and sequencing. (B) Example images of VC at postnatal day (P)7 and P28 as labeled, showing colocalization between Ribotag (green, HA) and astrocyte marker S100β (red). Scale bars = 20 μm. (C) Principal component analysis of RNAseq data shows P7 and P14 samples clustering separately from other ages, while P28 and P120 samples cluster together, suggesting similar gene expression profiles (N = 3 at P7, 4 at P14, 5 at P28, 3 at P120; for statistical comparisons, 3xP120 samples published in Boisvert et al., 2018 were added to increase the power of the analysis, giving an N = 6 P120). (D) Pairwise comparison of differentially expressed genes (DEGs; red: upregulated; blue: downregulated) between each time point showing total genes (light hue, all DEGs identified with FPKM >1), astrocyte expressed genes (darker hue, expression level in pulldown sample/input >0.75), and astrocyte-enriched genes (dark hue, expression level in pulldown sample; astro/input >3). (E) Percent of all astrocyte-enriched genes that are differentially expressed between each age. Percent DEGs is highest between P7 and P14. (F) Heatmaps of top 20 astrocyte-enriched genes at each age, sorted by expression level, along with 13 genes common to all time points (left), and top 5 most enriched genes at each age (right). Colors represent log2 FPKM of expression level. (G, H) Gene Ontology (GO) terms analysis with String db of Biological Process (BP) in astrocyte-enriched genes at each time point. (G) Plot of GO terms common to all time points with a gene ratio >0.5. (H) Plot of GO terms unique to each age with a gene ratio >0.5. Bar length is gene ratio, fill color is false discovery rate (FDR). See also Figure 1—source data 1B. (I, J) Heatmaps of select genes related to astrocyte function (I) and synaptic regulation (J) plotted as log2 fold change (FC) at each age relative to P120. * Adjusted p value (FDR) < 0.05 by DESeq2 with Benjamini–Hochberg’s correction when comparing P120 to each age. Figure 1—source data 1 Temporal profiling of astrocyte transcriptome in the postnatal visual cortex (VC). (A) Complete list of genes (expression levels shown as FPKM) at each developmental stage as indicated. Expression levels for each sample, as well as average, are shown, as well as pairwise analysis and false discovery rate (FDR) values. (B) Complete list of Gene Ontology (GO) terms (Biological Process) identified for astrocyte-enriched genes at each developmental time point as indicated. https://cdn.elifesciences.org/articles/70514/elife-70514-fig1-data1-v2.xlsx Download elife-70514-fig1-data1-v2.xlsx To assess if there are broad changes in the transcriptomic profiles of astrocytes across development, we performed principal component analysis (PCA; Figure 1C). This showed that P7 and P14 astrocytes form distinct clusters, while P28 and P120 astrocytes cluster together. To investigate this further, we analyzed the number of differentially expressed genes (DEGs; fragments per kilobase of exon per million mapped fragments [FPKM] > 1; false discovery rate [FDR] < 0.05) between each age group. DEGs are classified into total genes (all changes detected), genes that are expressed by astrocytes (IP/input > 0.75), and genes that are enriched in astrocytes (IP/input > 3; Figure 1D, for definitions, see also Boisvert et al., 2018). The largest number of DEGs is between P7 and P28 (~6000 total genes), and smallest numbers between P14 and P28 (~1000 total genes), and P28 and P120 (~2000 total genes). Analysis of astrocyte-enriched genes (IP/input > 3) showed that ~60% of all astrocyte-enriched genes are significantly changed from P7 to P14, while only ~20% are changing between P28 and P120 (Figure 1E). This shows that most changes in astrocyte gene expression are occurring between the first and second postnatal weeks, a time of transition from synapse formation to synapse maturation, and in the VC, from spontaneous to visually evoked neuronal activity. To determine the different astrocyte functions at each age, we focused on the astrocyte-enriched gene lists (~1000 total genes FPKM >1, astro IP/input > 3). To identify common genes expressed at all ages, as well as astrocyte-enriched genes that are most highly expressed at each age, we performed Gene Ontology (GO) analysis, focusing on Biological Process (BP) terms (Figure 1F–H, Figure 1—figure supplement 1F and G). This showed that genes common to all ages are enriched in GO terms related to cholesterol processing and serine synthesis, confirming the previously established important role of astrocytes in regulating brain cholesterol (Orth and Bellosta, 2012; Figure 1G). Analysis of GO terms unique to each age showed that at P7 astrocyte genes are enriched in GO terms related to cortical development, while at P14 astrocyte genes are enriched in GO terms related to Wnt and BMP signaling pathways. Conversely, adult astrocytes (P120) are enriched in terms related to regulation of extracellular matrix assembly and contact inhibition (Figure 1H). In all we found both the number of genes and the GO terms common to all ages constitute about 50% of all terms identified for each age, while genes and GO terms unique to each age consist less than 10% of all terms (Figure 1—figure supplement 1F and G). These results demonstrate that astrocytes perform their core functions across all developmental stages similarly, with several age-specific functions that turn on and off depending on the developmental stage and the environment in the VC. An important analysis to be performed with this dataset is determining the temporal expression changes of known astrocyte genes, for example, to identify potential astrocyte markers that are either global or age specific. We found that Apoe and Cst3 are the most highly expressed genes in astrocytes at all ages (peak FPKM ~10,000; Figure 1F, Figure 1—source data 1), while amongst highly expressed astrocyte genes (FPKM >100) Lars2 is the most astrocyte-enriched gene at all ages (astro IP/ input ~40; Figure 1—figure supplement 1E). These genes are highly expressed in adult astrocytes across multiple brain regions (Morel et al., 2017), thus can be potentially utilized to mark or target astrocytes globally. The expression of another well-known astrocytic gene Aldh1l1 (Morel et al., 2017; Figure 1I) is stable across all ages, making it an optimal marker for astrocytes during both development and in the adult (Srinivasan et al., 2016). On the other hand, S100b expression is upregulated later in development, making it more suitable to mark adult astrocytes (Figure 1I). For genes that encode proteins important for astrocyte function, we found that the metabotropic glutamate receptor Grm5 (mGluR5) is most highly expressed at P7 and then declines with maturation, confirming previous reports (Catania et al., 1994; Sun et al., 2013), while the glutamate transporter Slc1a2 (Glt1) and the connexins (Gja1, Gjb6) are significantly upregulated from P14 onwards (Figure 1I). Astrocytic synapse-regulating genes show differential spatio-temporal expression patterns Our goal is to characterize how astrocytes regulate synapses, so we next used the astrocyte Ribotag dataset to investigate the developmental expression changes of key synapse-regulating genes (Figure 1J). These include astrocyte-secreted thrombospondins (Thbs), which induce silent synapse formation. The family members expressed by astrocytes in the CNS show divergent expression, with Thbs1 being significantly higher at P7 than later ages, whereas Thbs4 is significantly higher at P120 than P7 (Figure 1J). This temporal expression profile fits with previous studies that have demonstrated important roles for Thbs1 in initial synapse formation at P7 (Christopherson et al., 2005), and suggested roles for Thbs4 in the adult brain (Benner et al., 2013). Similarly, glypican (Gpc) family members have a divergent expression. Though redundant in culture for their ability to regulate immature synapse formation (Allen et al., 2012), Gpc4 and Gpc6 show different temporal expression in vivo. While Gpc4 is most highly expressed at P7 and gradually declines with maturation, Gpc6 peaks at P14–P28. Gpc5, a glypican family member with yet unknown function, has low expression at P7, and is significantly increased at all later ages. Astrocyte-secreted chordin-like 1 (Chrdl1) regulates synapse maturation and its expression peaks at P14, confirming previous analysis (Blanco-Suarez et al., 2018; Figure 1J). These temporal expression profiles are not limited to factors that promote synapse formation. Astrocyte phagocytic receptors involved in synapse elimination, Megf10 and Mertk (Chung et al., 2013), significantly increase in expression between P7 and P14, coincident with the initiation of synapse elimination. Our RNA sequencing analysis demonstrates that in the developing VC synapse-regulating genes show differential temporal expression patterns. However, whether these levels are equal across astrocytes in all cortical layers throughout development is unknown. During the first postnatal weeks, when astrocytes regulate synapse development, they are still migrating and dividing (Ge et al., 2012). Yet, how developing astrocytes populate each of the cortical layers, and their ratio relative to other cortical cells across development has not been quantified. To address this, we utilized the well-established mouse line where astrocytes express GFP under the Aldh1l1 promoter (Cahoy et al., 2008; Dougherty et al., 2010; John Lin et al., 2017; Stogsdill et al., 2017; Tien et al., 2012). Immunostaining of brain sections from Aldh1l1-GFP mice at P7 and P28 with antibodies against known astrocyte markers ALDH1L1, S100β, and SOX9 showed high overlap between GFP and marker-positive cells, suggesting the majority of astrocytes in the VC express GFP in this mouse line (Figure 2—figure supplement 1A–C), further validating its usage. We quantified astrocyte numbers within each of the six neuronal layers at the developmental time points, which correspond to astrocyte and synapse development throughout the first postnatal month (P1, P4, P7, P14, P28; Figure 2A–C). At birth (P1), very few astrocytes are present, comprising 0.5–2% of the total cell number in the VC (represented as GFP-positive cells as a percentage of all cells marked by the nuclear dye DAPI), with a significantly higher percentage of astrocytes in deeper layers than upper layers (Figure 2C and D, Figure 2—source data 1A). The astrocyte percentage increases with development in all cortical layers, peaking at P21–P28. At this time, astrocytes are ~10% of total cell number in layers (L) 2–6, and ~50% in L1 (Figure 2C and D, Figure 2—source data 1A). As the brain develops, the distance between cells grows to accommodate the increase in cell size and complexity, as evident by a significant decrease in DAPI-positive nuclei per mm2 of VC that occurs from P1 to P14–P28 (Figure 2—figure supplement 1D, Figure 2—source data 1A). Despite this decrease in total cell density, the proportion of astrocytes remains constant in all cortical layers and across ages, with the exception of L1–2/3, where it is significantly lower at P1 (Figure 2C and E). This stability in astrocyte density is likely explained by new astrocytes still being generated in the weeks after birth (Ge et al., 2012). Figure 2 with 2 supplements see all Download asset Open asset Astrocytic synapse-regulating genes show differential spatio-temporal expression patterns. See also Figure 2—figure supplements 1 and 2, Figure 2—source data 1. (A) Schematic of experiments described in Figures visual cortex (VC) sections from Aldh1l1-GFP mice analyzed for astrocyte gene expression, and synaptic proteins at developmental time points as indicated. (B) of VC neuronal and connectivity Astrocytes are in all Astrocyte number increases in the VC across development. Example images of the VC from Aldh1l1-GFP mice at time points GFP marks astrocytes (L) labeled by numbers on the in each (D) of astrocytes as a percentage of total cells within each cortical (E) of number of astrocytes per mm2 of VC within each Scale in N = 4 mice for postnatal day N = 3 mice for show are of by comparing expression between time points within each see also Figure 2—source data genes show differential spatio-temporal expression patterns. Example images showing Gpc5, or mRNA in astrocytes at each age and layer as on the on the (G, of (F) Gpc4 expression is at P14 in (H) Gpc6 expression is increased at P14 in (J) expression is increased at P14 in all (L) expression is increased at P14 in Scale bars in = 20 μm. in mark astrocyte cells on the N = 3 for each Data as with are mRNA in to age are N = 3 = age and statistical analysis are on N = per by comparing expression between time points within each See also Figure 2—source data and Figure 2—source data 1 Astrocytic synapse-regulating genes show differential spatio-temporal expression patterns. (A) statistical analysis of astrocyte number changes during development. comparison astrocyte comparison between ages within each is labeled (B) statistical analysis of developmental changes in mRNA expression of synapse-regulating genes by in situ hybridization and analysis for N = that per (C) statistical analysis of developmental changes in mRNA expression of synapse-regulating genes by and analysis for = that total number of astrocytes per the three Download established the developmental profile of astrocytes, we next determined if there are layer-specific developmental changes in mRNA expression of synapse-regulating genes by in situ hybridization on brain sections of Aldh1l1-GFP We for genes that regulate distinct of active synapse-regulating – (Gpc) synapse maturation regulating – chordin-like 1 and silent synapse-regulating – thrombospondins 2, (Figure Figure 2—figure supplement 2, Figure 2—source data and Expression of each gene (represented as total of in labeled as analyzed within the of GFP-positive astrocytes in each cortical layer at similar time points as P4, P7, P14, and P28, when most in astrocyte transcriptome (Figure and synapse development and Allen, 2018), and when the numbers of astrocytes in the cortex are very control used to determine the of (Figure 2—figure supplement and We first analyzed factors that promote active synapse formation. RNAseq showed that Gpc4 expression by astrocytes is highest at P7 and gradually declines with maturation (Figure Figure 1—source data 1A). analysis, however, shows that these are by astrocytes in Gpc4 expression between P7 and P14 only in L1 astrocytes, stable across development in all other layers P7 P14 P28 Figure and Figure 2—source data and on the other hand, peaks at P14–P28 in bulk sequencing. analysis showed that this increase occurs in astrocytes between P7 and P14 in with significant in and high at P28 P7 P14 P28 Figure and Figure 2—source data and is upregulated at P14 and remains high at P28 in astrocytes in all layers, the bulk RNAseq data P7 P14 P28 Figure and Figure 2—source data and The expression of the synapse maturation peaks at P14 in the bulk RNAseq data (Figure Figure 1—source data 1A). analysis that this increase from P7 to P14 is with the largest increase in occurring in upper layer astrocytes with significant in P7 1; P14 P28 1; Figure and Figure 2—source data and at its of expression, is highest in astrocytes compared to a expression across layers, as previously et al., 2020; et al., 2018). Finally, we analyzed astrocyte factors that induce silent synapse formation (Figure 2—figure supplement mRNA levels in the VC are lower at their expression than or consistent with our bulk RNAseq results (Figure Figure 1—source data and previous studies showing low thrombospondin expression in the and an by or et al., 2019; et al., we an increase in expression in at P28 P7 P14 1 P28 Figure 2—figure supplement and Figure 2—source data and an increase in Thbs4 at P28 in all layers P7 P14 P28 Figure 2—figure supplement and Figure 2—source data The astrocyte synapse-regulating genes we analyzed using all regulate formation and function of glutamatergic In the of and they regulate glutamatergic synapse development by of glutamate receptor (Allen et al., 2012; et al., 2018). studies in the cortex have identified developmental in expression and 2008; et al., however, their spatio-temporal expression patterns in the developing mouse VC, and whether these with the expression of astrocytic genes that regulate have not been analyzed (Figure and B). To postsynaptic glutamate we for GLUA1 and GLUA2 (Figure Figure data To the corresponding glutamatergic we for to identify local (Figure and Figure supplement Figure data and to identify thalamocortical