Author response: Cell type composition and circuit organization of clonally related excitatory neurons in the juvenile mouse neocortex
2020-02-21

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Clones of excitatory neurons derived from a common progenitor have been proposed to serve as elementary information processing modules in the neocortex. To characterize the cell types and circuit diagram of clonally related excitatory neurons, we performed multi-cell patch clamp recordings and Patch-seq on neurons derived from Nestin-positive progenitors labeled by tamoxifen induction at embryonic day 10.5. The resulting clones are derived from two radial glia on average, span cortical layers 2–6, and are composed of a random sampling of transcriptomic cell types. We find an interaction between shared lineage and connection type: related neurons are more likely to be connected vertically across cortical layers, but not laterally within the same layer. These findings challenge the view that related neurons show uniformly increased connectivity and suggest that integration of vertical intra-clonal input with lateral inter-clonal input may represent a developmentally programmed connectivity motif supporting the emergence of functional circuits. Introduction The mammalian neocortex carries out complex mental processes such as cognition and perception through the interaction of billions of neurons connected by trillions of synapses. We are just beginning to understand how networks of neurons become wired together during development to give rise to cortical computations (Polleux et al., 2007; Cadwell et al., 2019). During cortical neurogenesis, which lasts from approximately embryonic day 10.5 (E10.5) through E17.5 in the mouse (Caviness et al., 1995; Takahashi et al., 1996), radial glial cells undergo asymmetric division to generate postmitotic excitatory neurons that migrate radially to populate the cortical plate. Neurogenesis occurs in an inside-out gradient, such that early born neurons occupy the deep cortical layers and later-born neurons reside in progressively more superficial layers (Angevine and Sidman, 1961; Rakic, 1974; Caviness et al., 1995). The ventricular zone is subdivided by glial septa into well-defined columns of precursor or stem cells referred to as 'proliferative units' that give rise to a radial unit of clonally related excitatory neurons, sometimes referred to as an ontogenetic column (Torii et al., 2009; Kriegstein and Noctor, 2004; Noctor et al., 2001; Noctor et al., 2007; Rakic, 1988). However, these radial units of clonally related neurons are only loosely clustered and are heavily intermixed with numerous nearby unrelated neurons (Walsh and Cepko, 1988; Tan et al., 1995) and there is substantial tangential migration of clonally related neurons as they traverse the subventricular zone and intermediate zone en route to the developing cortical plate (Torii et al., 2009). In contrast to excitatory neurons, inhibitory interneurons are generated in the ganglionic eminences and migrate tangentially to disperse throughout the developing cortical mantle (Letinic et al., 2002; Kriegstein and Noctor, 2004; Tan et al., 1998; Mayer et al., 2015). Recent advances in single-cell RNA-sequencing technology (Tang et al., 2009; Picelli et al., 2013; Picelli et al., 2014a) have enabled unbiased cell type classification in heterogeneous tissues including the cerebral cortex (Zeisel et al., 2015; Tasic et al., 2016; Tasic et al., 2018). In contrast to inhibitory interneurons, excitatory neurons in the adult mouse (Tasic et al., 2018) and developing human (Nowakowski et al., 2017) cortex are largely region-specific at the level of transcriptomic cell types, with several dozens of excitatory cell types per area (Tasic et al., 2018; Hodge et al., 2019). While it is well-established that the vast majority of cells within radial clones are excitatory neurons (Tan et al., 1998), it remains controversial whether individual progenitors give rise to the full diversity of excitatory neuron cell types within a given cortical area, or only to a restricted subset of transcriptomic cell types (Franco et al., 2012; Gil-Sanz et al., 2015; Eckler et al., 2015; Kaplan et al., 2017; Llorca et al., 2019). A series of studies using a retroviral lineage tracing method has suggested that clonally related excitatory neurons are more likely to be synaptically connected to each other (Yu et al., 2009; Yu et al., 2012; He et al., 2015) and have similar preferred orientations in primary visual cortex (V1) compared to unrelated neurons (Li et al., 2012), providing support for the long-standing hypothesis that radial clones may constitute elementary circuit modules for information processing in the cortex (Rakic, 1988; Mountcastle, 1997; Buxhoeveden and Casanova, 2002). The projection pattern of vertical, across-layer connections between related neurons was qualitatively similar to the canonical circuit of layer-specific connections in adult cortex (Yu et al., 2009); however, a direct comparison of related and unrelated pairs for each layer-specific connection type was not done, and lateral connections between clonally related cells within the same cortical layer were not examined. Therefore, it remains unclear whether all local connections are uniformly increased between clonally related excitatory neurons, although this assumption has become dogma in the field (Li et al., 2018). Given the complexity of the local cortical circuit and the different functional roles of layer-defined connections (Lefort et al., 2009; Feldmeyer, 2012; Lübke et al., 2000; Lübke et al., 2003), clarifying the effect of cell lineage on the underlying layer-specific connectivity matrix may have important implications regarding the mechanism and functional purpose of lineage-associated connectivity. The difficulty of multi-patching experiments combined with the relatively low connectivity rates between excitatory neurons (Jiang et al., 2015; Markram et al., 1997; Barth et al., 2016; Jiang et al., 2016) necessitate testing a very large number of connections and pose an enormous technical challenge to fully answer this question. Using an enhancer trap Cre line to label neural progenitors at an earlier developmental stage, yielding much larger clones (approximately 670–800 neurons per clone compared to 4–6 neurons per clone in Yu et al., 2009), a separate group has also reported an association between shared lineage and orientation tuning in V1 (Ohtsuki et al., 2012), although with a much smaller effect size than reported in Li et al. (2012) for small clones. Another study examined lateral connections within layer 4 (L4) of large clones labeled in chimeric mice at approximately E3.5 (Tarusawa et al., 2016) and found transient increases in connectivity between clonally related cells. These studies raise the possibility that shared lineage may serve as an important predictor of large-scale functional circuits even in more distantly related neurons derived from symmetrically dividing neural stem cells (Smith and Fitzpatrick, 2012). However, the relationship between lineage and the layer-specific connectivity matrix has not been systematically studied in large clones, and there remains a major disconnect in scale between studies of lineage-associated connectivity and the development of larger, functional cortical units that are thought to implement computations. Here we use a tamoxifen-inducible Cre-lox system to label progenitors at approximately the onset of neurogenesis, resulting in intermediate-sized translaminar clones that span cortical layers 2–6 and ask: (a) what is the cell type composition of individual translaminar clones and (b) does the layer-specific connectivity matrix among clonally related excitatory neurons differ from unrelated neurons. We find that clones of excitatory neurons labeled at embryonic day 10.5 (E10.5) are derived from two radial glia on average, and are composed of diverse transcriptomic cell types. Vertical connections linking cells across cortical layers, and in particular connections from upper cortical layers onto layer 5 (L5) excitatory neurons, are selectively increased among clonally related neurons compared to unrelated neurons. In contrast, clonally related excitatory neurons are not preferentially connected laterally, within the same cortical layer, despite being located in close proximity to one another. These findings argue against the prevailing view that preferential connectivity between clonally related excitatory neurons mirrors that of the canonical cortical circuit, and suggest that integration of vertical input from related neurons with lateral inputs from unrelated neurons may represent a developmentally programmed motif for assembling functional cortical circuits. Results Tamoxifen induction at E10.5 generates translaminar clones spanning cortical layers 2–6 To label radial clones, we induced sparse recombination in progenitors at approximately the onset of neurogenesis using a tamoxifen-inducible Cre-lox transgenic system driven by the Nes promoter (Figure 1A,B). In contrast to viral lineage tracing methods, which are routinely performed at E12.5 or later (Yu et al., 2009; Yu et al., 2012; Li et al., 2012), and enhancer trap methods (Ohtsuki et al., 2012) that do not precisely control the timing and density of labeling, our tamoxifen-inducible system can reproducibly and sparsely label progenitor cells in the developing forebrain across a range of timepoints (Figure 1C). We first compared clone size and layer distribution at postnatal day 10 (P10) following tamoxifen administration at E9.5, E10.5, and E11.5. Tamoxifen administration at E9.5 resulted in clones containing on average 196 neurons (median, interquartile range [IQR] 75–346 neurons) and spanning 347 μm in width (median, IQR 223–402 μm; Figure 1D,E). Tamoxifen administration at E10.5 resulted in clones containing on average 60 neurons (median, IQR 26–117) and spanning 224 μm in width (median, IQR 140–298 μm; Figure 1D,E). Tamoxifen administration at E11.5 resulted in clones containing on average 39 neurons (median, IQR 24–63) and spanning 220 μm in width (median, IQR 162–276 μm; Figure 1D,E), similar to the width of clones labeled at E10.5. However, a substantial fraction of clones labeled at E11.5 did not contain neurons in the deep cortical layers 5–6 (10/50 clones, 20%), which was only rarely seen in clones labeled by induction at E10.5 (1/39 clones, 3%), and never following induction at E9.5 (0/35 clones, 0%; (Figure 1F and Figure 1—figure supplement 1). These data suggest that progenitors labeled at E11.5 or later may be diverse, with a subset of these no longer generating deep layer neurons. Given the variability in clones seen with induction at E11.5, we focused our study on clones generated by tamoxifen induction at E10.5. Figure 1 with 2 supplements see all Download asset Open asset Tamoxifen induction at E10.5 generates translaminar clones spanning cortical layers 2–6. (A) Schematic of tamoxifen-inducible Cre-loxP system for lineage tracing. (B) Manual reconstruction of a clone across multiple slices. In this example, larger red spots are morphologically consistent with glial cells at high magnification. Scale bar: 100 μm. (C) Examples of reconstructed clones labeled at E9.5, E10.5 or E11.5. Scale bars: 100 μm. Differences in thickness are due to tangential sectioning of the cortex rostrally (approximate rostrocaudal position of each clone is shown in gray beneath the reconstruction). (D and E) Number of neurons (D) and clone width (E) at postnatal day 10 following tamoxifen induction at E9.5, E10.5, or E11.5 (n = 35, 39, and 50 clones; n = 3, 4 and 3 mice per condition; p-values computed using Wilcoxon rank sum). (F) Percent of clones that are do not contain neurons in L5 or L6 following tamoxifen induction at E9.5, E10.5, or E11.5 (n = 0/35, 1/39, and 10/50 clones; n = 3, 4 and 3 mice per condition; p-values computed using Fisher's exact test). Error bars show 95% Clopper-Pearson confidence intervals. (G) Developing cortical plate with immunohistochemical staining for Pax6 (green), Tbr2 (magenta), and tdTomato (red) to characterize progenitors within individual clones at E12.5 following tamoxifen induction at E10.5. Scale bar, 50 μm. (H) High magnification of clone shown in G, demonstrating two PAX6+/Tbr2- radial glia within one proliferative unit. Scale bar, 5 μm. In panels (G) and (H), the images are oriented with the ventricular zone on the left and the pial surface on the right. (I and J) Summary of the number of radial glia and the number of proliferative units per clone for all clones analyzed at E12.5 (n = 30 clones from three mice). Shades of gray in panels (I and J) denote data obtained from different animals for each treatment condition. See also Figure 1—figure supplements 1 and 2 and Figure 1—source datas 1 and 2. Figure 1—source data 1 Clone quantification data, related to Figure 1. Number of cells and clone width for each clone analyzed at postnatal day 10 (P10). https://cdn.elifesciences.org/articles/52951/elife-52951-fig1-data1-v3.xlsx Download elife-52951-fig1-data1-v3.xlsx Figure 1—source data 2 Number and type of progenitors per clone, related to Figure 1. Number of radial glia, intermediate progenitors and proliferative units for each clone analyzed at embryonic day 12.5 (E12.5). https://cdn.elifesciences.org/articles/52951/elife-52951-fig1-data2-v3.xlsx Download elife-52951-fig1-data2-v3.xlsx Given that Nestin is expressed by neuroepithelial stem cells and ventricular radial glia (Hockfield and McKay, 1985; Misson et al., 1988) and that the transition between neuroepithelial stem cells and radial glia occurs between E10.25 and E11.5 (Misson et al., 1988; Anthony et al., 2004; Nowakowski et al., 2011), it is possible that a subset of our clones are derived from neuroepithelial stem cells. However, there is a substantial delay in onset and prolonged pharmacokinetic activity of tamoxifen following oral administration (Robinson et al., 1991), and an inherent imprecision in the timing of pregnancies. Therefore, to empirically determine the type of progenitors labeled in our experimental model, embryos were sacrificed at E12.5 following tamoxifen administration at E10.5 and immunohistochemistry for Pax6 (expressed in radial glial and intermediate progenitors, Götz et al., 1998) and Tbr2 (also known as Eomes, a marker of intermediate progenitors, Englund et al., 2005; Mihalas et al., 2016) were performed to characterize the type and number of progenitors within individual clones (Figure 1G,H and Figure 1—figure supplement 2). On average, individual clones contained two radial glia (median, range 1–4, Figure 1I and Figure 1—figure supplement 2). As described by Rakic (1988), proliferative units can be identified as well-defined columns of precursor or stem cells within the ventricular zone; it has been proposed that the polyclonal precursors within a proliferative unit collectively give rise to a population of postmitotic neurons that share a common radial glial scaffold and form a morphologically identifiable stack of neurons in the cortex termed an 'ontogenetic' or 'embryonic' column (Rakic, 1988). Although no well-accepted criteria are available for defining the boundaries of a proliferative unit, we found that the vast majority of our clones labeled at E10.5 were arranged in a single vertical track at E12.5 consistent with their belonging to a single proliferative unit (24 out of 30 clones, 80%, Figure 1J and Figure 1—figure supplement 2). These findings show that tamoxifen administration at E10.5 labels predominantly individual radial glia and neuroepithelial stem cells in their final 1–2 cycles of symmetric cell division. Region-specific differences in gene expression are present in juvenile mouse neocortex Recent transcriptomic cell atlases of adult mouse neocortex showed that different cortical areas are composed of distinct transcriptomic excitatory neuron cell types (Tasic et al., 2018; Saunders et al., 2018). Given that our lineage tracing strategy labels translaminar clones across many cortical regions, we next asked whether region-specific excitatory neurons are present also in juvenile mouse neocortex. To test this, we cut acute parasagittal slices spanning primary visual (V1) and primary somatosensory (SS1) cortices from juvenile (P15-P20) mice. Translaminar clones were identified by their intrinsic fluorescence (Figure 2A,B), and the contents of individual labeled neurons as well as nearby unlabeled control neurons were aspirated through a patch pipette following brief electrophysiological recording using our recently described Patch-seq protocol (Cadwell et al., 2017; Cadwell et al., 2016; Scala et al., 2019). We analyzed 206 neurons (after quality control, see Figure 2—figure supplement 1 and Methods) which had approximately 0.36 million uniquely mapping reads (median; IQR 0.17–0.69 million reads; Figure 2C) and approximately 7000 genes detected (median: 7007; IQR 6152–7920 genes; Figure 2D) on average per cell. For all downstream analyses, we used 12,841 genes that had on average >1 count/cell (see Methods). Alternatively filtering genes based on the number of cells expressing each gene had no substantial effect on our results (data not shown). There were modest differences in the average library size (Figure 2—figure supplement 1E,F) and number of genes expressed (Figure 2—figure supplement 1I,J) between different cortical areas and different layers. However, there were no significant differences between tdTomato-positive (clonally related) and tdTomato-negative (nearby unrelated) neurons in either of these two measures (Figure 2—figure supplement 1G,K). Count data were normalized using a pool-based strategy developed specifically for single-cell RNA sequencing analysis (Lun et al., 2016). Size factors largely correlated with library size (Figure 2—figure supplement 1H), suggesting that our cell population is relatively homogenous and that systematic differences in gene counts in our dataset are driven primarily by technical factors such as capture efficiency and sequencing depth. The normalized counts (Figure 2—source data 1) were used in all subsequent analyses. Figure 2 with 2 supplements see all Download asset Open asset Region-specific differences in gene expression are present in juvenile mouse neocortex. (A) Overview of experimental approach using Patch-seq. (B) Example tdTomato-positive translaminar clone spanning cortical layers 2–6 in an acute cortical slice used for Patch-seq experiments. Overlay of bright field and fluorescence image was performed in Adobe Photoshop. Scale bar: 100 μm. (C and D) Box plots showing library size (C) and number of genes detected (D) for all cells passing quality control criteria (n = 206). (E) Density plot of the percent of variance in normalized log-expression values explained by different experimental factors. Each curve corresponds to the variance in gene expression across all genes (n = 12,841 genes) that can be explained by a single variable, with right-shifted curves reflecting variables that explain a higher fraction of the variance. (F) T-distributed stochastic neighbor embedding (t-SNE) plots using the top highly variable and correlated genes across all cells (n = 91 genes; n = 87, 22, 84, and 13 cells in layers 2/3, 4, 5, and 6, respectively), colored by layer position. (G) Performance of a generalized linear model (GLM) trained to predict region from gene expression data of L2/3 neurons (n = 12,841 genes and 85 cells) with model performance (black dot) compared to the chance-level performance estimated using shuffled data (gray, mean and 95% coverage interval; one-tailed p-value computed from shuffled data, shuffling region). (H) Performance of a GLM trained to predict region from gene expression data of L5 neurons (n = 12,841 genes and 77 cells) as described in (G). See also Figure 2—figure supplements 1 and 2 and Figure 2—source data 1. Figure 2—source data 1 Gene expression data, related to Figure 2. Normalized counts, normalized log counts, and metadata for all Patch-seq neurons included in our analysis. https://cdn.elifesciences.org/articles/52951/elife-52951-fig2-data1-v3.xls Download elife-52951-fig2-data1-v3.xls Consistent with recent studies (Zeisel et al., 2015; Tasic et al., 2016; Tasic et al., 2018; Nowakowski et al., we found that layer position and cortical region were the of transcriptomic variability among excitatory neurons in our dataset (Figure and Figure 2—figure supplement 2). using stochastic neighbor embedding (t-SNE) that neurons clustered primarily by layer position (Figure and a generalized linear model (GLM) predict layer position from the gene expression data with approximately (data not shown). L2/3 and neurons from V1 and in gene (data not but a generalized linear model (GLM) predict position than (Figure suggesting that L2/3 and L5 excitatory neurons in juvenile mouse neocortex have region-specific transcriptomic we that in to determine whether individual progenitors are to generate a restricted subset of transcriptomic cell types, it be to the cell type composition of clonally related neurons to nearby unlabeled neurons with a similar position. Translaminar clones labeled at E10.5 are composed of diverse transcriptomic of excitatory neurons While a model of excitatory neurogenesis, individual progenitors give rise to many different excitatory neuron cell types through (Tan and et al., 2013; et al., studies suggest that a subset of progenitors are early on to give rise to excitatory neurons (Franco and 2013; et al., 2012; Gil-Sanz et al., 2015). In one recent study that a large fraction of clones spanning the full thickness of the cortex are composed of projection neurons et al., 2019). To characterize the diversity of cell types within our translaminar clones, we our single-cell to a recently cell type of adult mouse cortex (Tasic et al., 2018). neurons in L2/3 to L2/3 types and for L5 and L6 (Figure and Figure data 1). The were due to neurons mapping to a transcriptomic type from a neurons from L2/3 mapping to types neurons from L5 mapping to types and neurons from L5 mapping to L6 types These results are not given that transcriptomic cell types are also present in cortical layers (Tasic et al., 2018). cells to types tdTomato-positive and two and one cell to an type which may mapping of our dataset to the dataset or of an cell. was not specifically in the cell we found that cells from V1 and in our dataset predominantly to V1 excitatory neuron types of V1 cells and of Figure data 1) with only a mapping to excitatory neuron types of V1 cells and of the quality of the mapping was for V1 and cells to for for V1 mean suggesting that the adult cell type is an for excitatory neurons in juvenile V1 and Figure 3 with 2 supplements see all Download asset Open asset Translaminar clones labeled at E10.5 are composed of diverse transcriptomic of excitatory neurons. (A) T-distributed stochastic neighbor embedding (t-SNE) plot showing of our Patch-seq data (data with n = 87, 22, 84, and 13 cells in layers 2/3, 4, 5, and with a recently mouse cell type (data with no n = from Tasic et al. denote transcriptomic types and are from the The of the dataset and the of Patch-seq cells were performed as described in and see The size of the Patch-seq data the of the mapping (see Materials and small high (B) of labeled (n = and unlabeled (n = cells mapping to transcriptomic cell types types with than three neurons are not test). (C and D) of related and unrelated cell pairs mapping to the same transcriptomic cell type either n = related n = unrelated or on layer position n = related n = unrelated test). For bars are 95% Clopper-Pearson confidence and p-values are computed using test we used for each of the 13 See also Figure supplements 1 and 2 and Figure data 1. Figure data 1 to transcriptomic cell types, related to Figure for each of our cells onto transcriptomic cell types, for the and for projection of our data onto the with a of Download the labeled neurons within our clones to all of the excitatory cell and in similar to unlabeled control neurons Figure supplement 1A,B). We had at three cells to each of transcriptomic cell types in Tasic et al. (Figure data 1). There was a in the distribution of labeled and unlabeled neurons among these cell types Figure and that the L2/3 transcriptomic cell type was in our labeled clones compared to unlabeled control neurons test with for 13 Figure for the other transcriptomic cell These findings that the progenitors labeled in our study collectively give rise to the majority of transcriptomic cell types present in the areas of cortex but suggest that the L2/3 transcriptomic cell type may from a distinct progenitor individual clones, we found that pairs of related neurons were no more likely to to the same transcriptomic Figure supplement and Figure supplement or cell type Figure and Figure supplement compared to pairs of nearby unrelated neurons from the same cortical possibility is that our clones to contain different cell types they multiple layers, but within a layer they may be restricted to give rise to a particular cell To test this, we also compared pairs of clonally related neurons within the same cortical layer to pairs of unrelated neurons in the same layer and found no among clonally related neurons to to the same Figure supplement or Figure transcriptomic cell While these findings the possibility of of individual radial glia or intermediate progenitors labeled at later they suggest that individual progenitors labeled at E10.5 give rise to a range of excitatory cell types. across-layer connections are selectively increased between excitatory neurons in translaminar clones To determine whether clonally related neurons within our translaminar clones were preferentially we performed a separate series of experiments using multiple recordings as described (Jiang et al., to neurons including clonally related cells and nearby unlabeled control cells (Figure In we neurons labeled and from clones in mice. The layer position of each cell was using contrast at the of recording and later using staining as described (Jiang et al., 2015; Cadwell et al., 2017; Scala et al., 2019). The cells were throughout L2/3 (n = (n = cells) and L5 (n = To test we brief into each neuron to and the of all