In silico analysis identifies a putative cell-of-origin for BRAF fusion-positive cerebellar pilocytic astrocytoma.

Childhood cancers are increasingly recognized as disorders of cellular development. This study sought to identify the cellular and developmental origins of cerebellar pilocytic astrocytoma, the most common brain tumor of childhood. Using publicly available gene expression data from pilocytic astrocytoma tumors and controlling for driver mutation, a set of developmental-related genes which were overexpressed in cerebellar pilocytic astrocytoma was identified. These genes were then mapped onto several developmental atlases in order to identify normal cells with similar gene expression patterns and the developmental trajectory of those cells was interrogated. Eight known neuro-developmental genes were identified as being expressed in cerebellar pilocytic astrocytoma. Mapping those genes or their orthologs onto mouse neuro-developmental atlases identified overlap in their expression within the ventricular zone of the cerebellar anlage. Further analysis with a single cell RNA-sequencing atlas of the developing mouse cerebellum defined this overlap as occurring in ventricular zone progenitor cells at the division point between GABA-ergic neuronal and glial lineages, a developmental trajectory which closely mirrors that previously described to occur within pilocytic astrocytoma cells. Furthermore, ventricular zone progenitor cells and their progeny exhibited evidence of MAPK pathway activation, the paradigmatic oncogenic cascade known to be active in cerebellar pilocytic astrocytoma. Gene expression from developing human brain atlases recapitulated the same anatomic localizations and developmental trajectories as those found in mice. Taken together, these data suggest this population of ventricular zone progenitor cells as the cell-of-origin for BRAF fusion-positive cerebellar pilocytic astrocytoma.

Introduction

A developmental origin of childhood cancer is well recognized [1]. For example, the neoplastic cells which give rise to pediatric leukemia are often present at birth, years before manifestation of disease [2-6]. Moreover, the mutations occurring within childhood cancers often inhibit cellular differentiation and treating the neoplastic cells with agents which induce differentiation has proven to be a highly effective therapeutic approach [7, 8]. Thus, understanding the developmental processes which have gone awry during tumorigenesis is crucial to understanding the biology of pediatric tumors and may inform therapeutic approaches.

Tumors of the central nervous system (CNS) are the most common solid malignancy of childhood and are the leading cause of cancer-related deaths in children and adolescents [9, 10]. Moreover, many of those children who are cured must confront and manage treatment-related morbidity due to toxicity associated with contemporary radiation and chemotherapy treatment regimens [11-13].

Spatiotemporal restriction of driver mutations in pediatric CNS tumors suggests that these mutations are only oncogenic within certain cellular contexts [14, 15]. As such, pediatric CNS cancer is widely recognized to be a disorder of neural development, whereby oncogenic mutations hijack normal developmental pathways within the cell-of-origin to drive tumor initiation, growth, and progression [16]. For example, medulloblastoma is now understood to represent a heterogenous disease with distinct cellular and developmental origins [15, 17, 18]. Treatment approaches are now stratified based upon these distinctions [19], underscoring the importance of understanding the developmental biology and cell-of-origin of pediatric CNS tumors.

Cerebellar pilocytic astrocytoma is the most common CNS tumor in children, with over 500 being diagnosed in the United States each year [10]. Though complete resection is curative, surgery is often associated with significant morbidity, and not all tumors are amenable to surgery, necessitating the use of adjuvant radiation or chemotherapy with resultant increased risk of aforementioned treatment-related morbidity [12, 13]. Further study of the underlying biology of cerebellar pilocytic astrocytoma is needed in order to inform improved therapeutic approaches.

By far, the most common driver mutation in cerebellar pilocytic astrocytoma is fusion of the BRAF locus with a variety of fusion partners, the most frequent being KIAA1549 [20-23]. The common effect of these fusions is constitutive activation of the MAPK pathway with resultant driving of cellular growth and division [24]. Whereas this molecular signaling has been well studied, the cellular and developmental origins of cerebellar pilocytic astrocytoma remain shrouded in mystery. The objective of this study was to elucidate and define the cellular and developmental origins of pediatric cerebellar pilocytic astrocytoma. The central hypothesis of this study is that cerebellar pilocytic astrocytoma arises from a distinct cell-of-origin within the developing brain and that the developmental gene expression program thereof is retained within the tumor cells.

Materials and methods

Microarray gene expression analysis

Gene expression data was obtained from the Gene Expression Omnibus (GEO) in the form of a GEO dataset using the accession number GSE44971 [25] and the R package GEOquery [26]. The rest of the data analysis was performed using a variety of R packages [27-34]. Samples without a documented BRAF fusion were removed from the dataset. Two infratentorial tumors with a BRAF fusion were removed since their location was listed as “Brainstem” and “fourth ventricle” rather than “cerebellum.” Gene expression contrast was run using a linear model as implemented in the R package limma [35] for infratentorial vs. supratentorial tumors. The resulting p-value was adjusted for multiple testing via the Benjamini and Hochberg method. A P value of < 0.001 was considered to be statistically significant.

For the heatmap, a distance matrix was constructed using the non-centered Pearson method [(1—sum(x_i y_i) / sqrt [sum(x_i^2) sum(y_i^2)] as implemented in the R package amap [36]. Unsupervised hierarchical clustering using the complete linkage method was then performed on the distance matrix. Gene expression data was viewed globally using the Barnes-Hut implementation of t-weighted stochastic neighbor embedding (t-SNE) wrapped in the package Rtsne [34] with the following non-default parameters: perplexity = 12, theta = 0, max_iter = 2500.

Gene ontology analysis

Gene ontology analysis was conducted as described in the Bioconductor workflow “maEndtoEnd” [37]. Briefly, genes with similar absolute expression intensities to genes which were differentially expressed between infratentorial and supratentorial tumors were selected as a background set. Thus, the gene universe consisted of differentially expressed genes and a background set of genes with similar expression intensities. The package topGO [31] was then applied to this dataset using the “Biological Processes” ontology mode and Fisher exact testing for statistical significance. Gene annotation data was imported from the Affymetrix Human Genome U133 Plus 2.0 Array annotation data [28]. The top five gene ontology terms were reported.

Selection of genes for analysis

Similar to the method described by Gibson, et al. [15], genes which were overexpressed in BRAF fusion-positive infratentorial tumors were selected as candidate marker genes for the cell-of-origin. Then, the Allen Developing Mouse Brain Atlas (Allen Institute; 2008) and the Cell Seek single cell RNA sequencing atlas [38] were interrogated for these genes or their mouse orthologs. Several genes, though present in the one or both atlases, exhibited no expression (Ppargc1A, Ntrk3, Camk4, Gdf11) or nonspecific expression (i.e. all cell types) across the entire brain and/or developing cerebellum (Cntn1, Cntn3, Tsc22D1, Gria4). As a result, these genes were excluded from subsequent analysis. The remaining genes (Ascl1, Irx2, Irx5, Klf15, Meis1, Msx2, Pax3, and Pbx3) were validated in an independent pilocytic astrocytoma gene expression dataset and used for downstream analysis. These genes are hereafter referred to as pilocytic astrocytoma-developmentally related (PA-DR) genes.

Gene expression mapping using Allen Developing Mouse Brain Atlas

Because data on gene expression in the developing human cerebellum is sparse while mouse gene expression atlases are more robust, analysis was first performed using data from mice. Brain Explorer 2 is a desktop software application which can visualize the Allen Developing Mouse Brain Atlas gene expression data in three dimensions. The software also allows highlighting of particular anatomic structures. The methodology used to generate this and map this data is provided by the Allen Institute for Brain Science at http://developingmouse.brain-map.org/content/explorer. Data for all eight PA-DR genes at embryonic day 13.5 and 15.5 were interrogated as shown in S2 Fig. Images were taken in the sagittal and coronal anatomic planes. Rhombomere 1 was highlighted in each captured image as was the cerebellar anlage. For the summary data shown in Fig 2A and 2B, high resolution ISH images from the Developing Mouse Brain Atlas were accessed for each PA-DR gene. Using the associated reference atlas, each gene was manually called to be expressed or not in the ventricular zone and the external granule cell layer. Individual microscopic images used for this analysis are available in S1 Data while the results are available in S2 Table.

Fig 2

Neurodevelopmental-related genes overexpressed in infratentorial pilocytic astrocytoma are co-expressed in the ventricular zone of the developing cerebellum.

(A-B) Summary data indicating percentage of PA-DR genes expressed within the indicated anatomic zone on gestation day 13.5 (A) and 15.5 (B) demonstrating that PA-DR genes are enriched within the ventricular zone. Images represent sagittal sections of the developing mouse cerebellum from the Allen Brain Reference Atlas. Cell nuclei are stained yellow. The regions outlined by black lines represent different developmental structures. The external granule cell layer and ventricular zone layer are labeled and colored based on the percentage of PA-DR genes expressed in that region. See also S2 Table and S1 Data.

Cell seek analysis

The Cell Seek database was accessed at https://cellseek.stjude.org/cerebellum. Data for each of the PA-DR genes was accessed and an image of the resulting expression data was taken. The overlap image shown in Fig 3A was generated using the GNU Image Manipulation Program (https://www.gimp.org) in the following manner. The images for the top four or all PA-DR genes was loaded into GIMP as a layer. Each layer was made transparent to 25% and merged with the layer mode set to “Merge.” Selection of cells for subsequent lineage analysis was guided to incorporate each of the major cell types seen within the cluster enriched for PA-DR genes (see Fig 3A). Monocle, BEAM, and transcription factor correlation were performed using the built in functions of Cell Seek [38]. For details of the implementation therein, see reference [38]. The q-value threshold for BEAM was set at 0.001.

Fig 3

PA-DR genes are co-expressed in early ventricular zone progenitors and these cells differentiate along GABA-ergic neuron, astrocyte, and glial progenitor lineages.

(A) Overlap of top four PA-DR genes in a single cell RNA-sequencing atlas of the developing mouse cerebellum. Each hexagon denotes a single cell or closely related group of cells plotted via t-SNE dimensionality reduction of the RNA sequencing data. Overlap was identified by stacking images from each of the four top differentially expressed PA-DR genes, revealing those cells with co-expression. The box denotes the region of strongest overlap. Expression scale is a heatmap depicting the log2 of the expression value. (B) Lineage analysis of early ventricular zone progenitor cells which express PA-DR genes reveals that these cells differentiate along three distinct lineages. Labels are added after manual curation of respective lineage gene expression. The blue color gradient refers to the position of each respective cell in “pseudotime,”–that is, degree of calculated differentiation (see reference for a description of the Monocle software and the concept of pseudotime [47]). The gradient of white to blue represents less and more differentiated cells, respectively. (C) Expression of genes which differentiate between the two main lineages marked by point 1 in (B), revealing one major lineage as GABA-ergic neurons. The middle of the figure represents the cell fate decision–each direction extending from the middle white line denotes one branch along the two different cell fate lineages. The blue-red gradient represents genes which are down- or up-regulated, respectively, as one travels along that lineage. (D) Expression of genes which differentiate between lineages marked by point 2 in (B). The trajectory toward glial progenitor cells is marked by expression of stem cell-like markers (e.g. Pax6) while the other lineage is enriched for astrocyte markers. Colors and interpretation as in (C).

Expression in developing human brain

The BrainSpan atlas of the developing human brain [39] (http://www.brainspan.org) was used to interrogate the expression of PA-DR genes therein. The resulting heatmap was sorted by structure. For organoid expression data, the scApeX portal from the accompanying publication [40] was accessed (https://bioinf.eva.mpg.de/shiny/sample-apps/scApeX/). The expression of each PA-DR gene was interrogated and reported as it resulted in the portal (i.e. no further analysis or alteration was done).

Results

Gene expression contrast of BRAF fusion-positive infratentorial pilocytic astrocytoma identifies neural development related genes

In order to identify the developmental origins of cerebellar pilocytic astrocytoma, a publicly available gene expression dataset was used which contains information from 49 pilocytic astrocytomas and 9 normal cerebellar tissues [25]. Two principles drove subsequent analysis. First, presumably the most common driver mutation in pilocytic astrocytoma (i.e. BRAF fusion) produces a similar set of gene expression changes regardless of tumor location/cell of origin. Second, contrasting gene expression between tumors of differing location/cell of origin but with identical driver mutation should mask those changes induced by the BRAF fusion, leaving only those genes expressed in the presumptive cell of origin (i.e. those genes which are unique to that tumor location/cell of origin). Therefore, the tumor dataset was filtered to include only those tumors with a documented BRAF fusion, regardless of fusion partner. This left a set of 37 tumors, 5 supratentorial and 32 infratentorial. All subsequent analyses were performed on this set.

Consistent with the overarching hypothesis, dimensionality reduction failed to distinguish between supra- and infra-tentorial tumors (Fig 1A and 1B), suggesting that BRAF fusion drives similar gene expression changes in both groups of tumors [41, 42]. Despite this similarity, gene expression contrast between infra- and supra-tentorial tumors identified multiple differentially expressed genes. This data suggests that these differentially expressed genes represent those genes expressed in the cell of origin for each respective tumor location. Indeed, consistent with prior reports [25] (albeit ones which included non-BRAF translocated tumors), several neuro-developmental related genes were found to be overexpressed in infratentorial tumors (Fig 1C), and top gene ontology terms for differentially expressed genes were largely related to cellular/neural development (Fig 1D). This result demonstrates the potential utility of contrasting tumors with identical driver mutations but differing anatomic locations in order to reveal the underlying developmental program operative in the cell of origin.

Fig 1

Gene expression of BRAF fusion-positive pilocytic astrocytoma identifies several neural developmental-related genes in infratentorial tumors.

(A) Heatmap of global gene expression data from all BRAF fusion-positive tumors in the cohort. (B) T-weighted stochastic neighbor embedding (t-SNE) plot of gene expression from BRAF fusion-positive tumors. (C) Volcano plot of gene expression data from infratentorial vs. supratentorial BRAF fusion-positive pilocytic astrocytomas. Known developmental genes which were found to be differentially expressed are labeled (see text). (D) Gene ontology terms for differentially expressed genes in infratentorial vs. supratentorial BRAF fusion-positive pilocytic astrocytomas. P-value represents that derived from a Fisher’s exact test.

Of note, very few genes were differentially expressed, with only 97 being significantly upregulated and 26 significantly downregulated (S1 Table). This is in keeping with the relatively quiescent genomic perturbances which characterize pilocytic astrocytoma [25, 43, 44].

Neural development related genes overexpressed in infratentorial pilocytic astrocytoma are co-expressed in the ventricular zone of the developing cerebellum

Similar to the methodology described by Gibson, et al. [15], genes overexpressed in infratentorial tumors or their mouse orthologs were cross-referenced with the Allen Developing Mouse Brain Atlas (www.brain-map.org, Allen Institute) in order to identify the anatomic origins of cerebellar pilocytic astrocytoma. Using in situ hybridization, this resource catalogs expression of over 2000 genes within the developing mouse brain across 7 developmental stages. Of 97 upregulated genes in infratentorial tumors (i.e. those genes expressed in infratentorial but not supratentorial pilocytic astrocytoma), 16 were found to be present in the atlas. Of these, four genes exhibited no expression (Ppargc1A, Ntrk3, Camk4, Gdf11) across the developing brain and four (Cntn1, Cntn3, Tsc22D1, Gria4) exhibited diffuse expression across the entire brain and/or developing cerebellum. As a result, these genes were excluded from subsequent analysis. This left eight genes to be analyzed (Ascl1, Irx2, Irx5, Klf15, Meis1, Msx2, Pax3, and Pbx3). In order to validate these genes, an independent dataset of gene expression from pilocytic astrocytoma tumors was interrogated [45]. All eight of these genes were reported to be overexpressed in infratentorial as compared to supratentorial tumors in this independent dataset, validating these findings. Hereafter, this group of genes is referred to as pilocytic astrocytoma development-related (PA-DR) genes.

First, the temporal expression pattern of each of these eight genes was interrogated. The temporal patterns of PA-DR gene expression differed depending on the particular gene in question (S1 Fig). However, embryonic days 13.5 and 15.5 emerged as being the time point in development with the most significant overlap in expression amongst all PA-DR genes. Therefore, these time points were selected for further analysis.

To further annotate anatomic localization, this data was visualized using Brain Explorer 2, an application which plots the in situ hybridization data of the Allen Developing Mouse Brain Atlas in three dimensions. Six out of eight PA-DR genes exhibited expression within rhombomere 1, the embryological structure which gives rise to the cerebellum, on both embryonic day 13.5 and 15.5 (S2A to S2B Fig). Furthermore, many PA-DR genes exhibited overlap in their localization thereof, suggesting cellular and/or regional overlap in expression. This suggests that cerebellar pilocytic astrocytoma arises from a cell within the cerebellar anlage, unlike some cerebellar tumors which have origins outside the cerebellum proper (e.g. Wnt-subtype medulloblastoma) [15].

The many different cell types of the adult cerebellum can be conceived of as arising from two distinct developmental pathways. Glutamatergic neurons arise from the external granule cell layer, while GABA-ergic neurons as well as cerebellar glial cells arise from the ventricular zone (see excellent review by Martinez et al., 2013) [46]. Note that the neurons of the deep cerebellar nuclei also arise from the ventricular zone with a stereotypic migration pattern from the ventricular zone to a structure known as the nuclear transitory zone. Thus, further analysis was conducted to identify which of these embryologic structures are responsible for the gene expression overlap described above.

As shown in Fig 2A and 2B, the ventricular zone of the cerebellar anlage exhibited significantly more overlap in expression of PA-DR genes than the external granule cell layer (see S2 Table and S1 Data), suggesting a ventricular zone origin for BRAF fusion-positive cerebellar pilocytic astrocytoma.

PA-DR genes are co-expressed in early ventricular zone progenitors and straddle the differentiation point between GABA-ergic neuron and glial progenitor cells

Since multiple cell types are present within the ventricular zone, further definition of the individual cells which display co-expression of PA-DR genes was needed. To accomplish this, Cell Seek, a single cell RNA sequencing dataset of the developing mouse cerebellum [38] was used. As shown in Fig 3A, a small population of cells exhibited overlap in expression of PA-DR genes. In particular, Pax3 and Ascl1 exhibited highly specific expression in the Cell Seek database (S3A Fig). These cells are isolated from the embryologic day 13, 14, and 15 (S3B Fig) and are classified by the Cell Seek software as “progenitor,” “GABA progenitor,” “glia,” and “astrocytes,” based upon their expression of known cellular markers (S3C Fig) [38].

In order to further define the cellular and developmental trajectory of this cell population, 2,262 cells compromising the aforementioned four major lineages were selected to perform further analysis upon (S4A Fig). Lineage analysis with Monocle identified a cluster of early precursor cells with three different developmental trajectories (Fig 3B). The first lineage decision was between cells with markers of the early GABA-ergic lineage (i.e. Ptf1a) and a lineage characterized by stem cell/neuronal progenitor markers (e.g. Pax6 and Otx2) (Fig 3C). This latter cell lineage was further divided into astrocytes and a cryptic group of cells which express some markers of glutamatergic cell lineage (e.g. Pax6, Id1, Ybx1), yet lack expression of canonical glutamatergic neuronal progenitors (Atoh1) (Fig 3D). The Cell Seek software identifies these cells as early glial cells (Fig 3D and S4B Fig).

PA-DR genes exhibited enrichment along specific cell lineages. Pax3, the most significantly overexpressed gene in BRAF fusion-positive infratentorial tumors, together with Irx5 and Irx2, exhibited expression across both early progenitors, GABA progenitors, and astrocytes (S5A to S5C Fig), with little expression in cells from earlier or later cell types, suggesting that Pax3 plays an important role at this key developmental stage. Similarly, Ascl1 and Pbx3 were expressed in earlier and later progenitors, respectively, while the remaining PA-DR genes were expressed in cells of the early astrocytic lineage (Meis1, Klf15, Msx2) (S5A to S5C Fig).

Computation of transcription factor networks (i.e. transcription factors whose expression are correlated) for these cell types recapitulated these two major lineages (GABA-ergic neuronal progenitors and glial cells). Seven out of eight PA-DR genes were computed to be in the same transcription factor network as these established markers of cell lineage (S6 Fig). Taken together, these data support a developmental paradigm operative within BRAF fusion-positive cerebellar pilocytic astrocytoma [42] which recapitulates this early developmental process whereby ventricular zone progenitors differentiate into GABA-ergic and glial lineages.

Glial progenitors and astrocytes of the ventricular zone display evidence of MAPK activation

Given the central role of the canonical mitogen-activated protein kinase cascade in the pathogenesis of pilocytic astrocytoma [24], the expression of key markers of MAPK activity was interrogated within the developing cerebellum, namely, the transcription factors Fos and Jun. Early astrocytic progenitor cells, but not ventricular zone progenitors or GABA-ergic progenitors, exhibited strong expression of both Fos and Jun (Fig 4A to 4B), suggesting MAPK activation within these cells.

Fig 4

Glial progenitors and differentiating astrocytes express markers of MAPK activation.

Expression of MAPK cascade transcriptional mediators Fos (A) and Jun (B) along Monocle lineages. Note the intense expression in intermediate progenitors (see also Fig 3D).

Data from the developing human brain recapitulates expression of PA-DR genes within the developing cerebellum

Since all of the aforementioned developmental data is from mouse studies, validation of these findings in human data is needed. First the anatomic sites of PA-DR gene expression were identified within the developing human brain using BrainSpan (www.brainspan.org, Allen Institute). All PA-DR genes were expressed in the developing cerebellum, including many with exclusive expression therein (Fig 5A). Additionally, expression of PA-DR genes was sparse in postnatal brain samples, further underscoring the developmental nature of pilocytic astrocytoma. Second, in an organoid based model of brain development [40], PA-DR genes were expressed along the hindbrain neuron and astrocytic developmental lineages (Fig 5B). Taken together, these data support similar developmental trajectories and spatiotemporal localization of human cells expressing PA-DR genes as that found in the developing mice brain.

Fig 5

Expression of PA-DR genes in the developing human brain recapitulates those patterns found in mice.

(A) Expression of PA-DR genes within the Developing Human Brain Atlas (Allen Institute). Image is ordered first by structure. Within each structure, each sample/donor is ordered by age (i.e. earliest samples are first within each structure subunit). The red box indicates cerebellar tissue. Expression values are in reads per kilobase per million (RPKM) as indicated in the heatmap. Note the exclusivity of PA-DR overlap within the early, developing cerebellum. (B) Expression of PA-DR genes within a pluripotent stem cell-derived developing brain organoid [40]. Data represent a t-SNE plot of single-cell RNA sequencing data from the developing brain organoid whereby each cell is a single point in the plot. Cell identities are indicated. For each PA-DR gene, the log2 expression value in each cell is shown by the color gradient. Note the trajectory of cells expressing PA-DR genes–most notably, Pax3 and Irx5, the most highly upregulated PA-DR genes in cerebellar pilocytic astrocytoma–are chiefly along the astrocyte and hindbrain neuronal lineages, recapitulating an identical developmental trajectory of PA-DR gene expressing cells in mice.

Thus, in conclusion, these data support early ventricular zone progenitor cells on the cusp of their GABA-ergic/astrocytic differentiation point as the cell of origin for BRAF fusion-positive cerebellar pilocytic astrocytoma.

Discussion

Many pediatric cancers are characterized by perturbations and co-opting of normal developmental pathways that drive tumor development and growth. Understanding those pathways helps reveal the underlying tumor biology, shed light on predisposing factors, and suggests therapeutic approaches. The data presented herein map developmental genes overexpressed in BRAF fusion-positive cerebellar pilocytic astrocytoma to the normal developmental pathway of ventricular zone progenitor cells, suggesting that these cells represent the cell-of-origin for this tumor.

The unique temporal profiles of PA-DR genes within the Cell Seek atlas is of particular interest, given the documented developmental paradigm which has been described in pilocytic astrocytoma. Reitman and colleagues describe a developmental program operative within pilocytic astrocytoma tumors whereby neoplastic cells differentiate along three separate lineages [42]. The data presented here synergize therewith, mapping a nearly identical developmental paradigm occurring in normal cerebellar development (Fig 6). Thus, it is likely that the developmental program described in pilocytic astrocytoma tumors is a recapitulation of the normal pathway of cerebellar ventricular zone development described in this paper. Based upon this, it is tempting to speculate that BRAF fusion within a ventricular zone progenitor cells blocks normal differentiation “locking” the cell into a permanent embryonic state, similar to that process described in pediatric leukemia.

Fig 6

Pilocytic astrocytoma cells recapitulate the developmental trajectory of ventricular zone progenitor cells.

Data from Reitman, et al. 2019 [42] shows that the neoplastic cells of pilocytic astrocytoma differentiate along three distinct lineages. Early ventricular zone progenitor cells show very similar developmental architecture, differentiating along three distinct lineages, suggesting that pilocytic astrocytoma recapitulates this normal developmental pathway. The colored arrows are designed to draw attention to the very similar architecture of developmental trajectories between the cerebellar ventricular zone and pilocytic astrocytoma cells: namely, two cell fate decision points ultimately resulting in three different cell types.

Similar to the current study, data from Vladoiu and colleagues implicates the ventricular zone as the origin for cerebellar pilocytic astrocytoma [48]. The current study supports and extends those findings, further defining the developmental trajectory of the cerebellar ventricular zone, demonstrating MAPK pathway activation within ventricular zone-derived glial progenitor cells, and, uniquely, mapping these transcriptional and developmental data to the developing human brain.

Though de-differentiation of a mature cell type induced by BRAF fusion is not excluded, these data raise the intriguing possibility that BRAF fusion-positive cerebellar pilocytic astrocytoma has a prenatal origin, similar to other pediatric cancers [1]. For example, using newborn blood spots, several studies have shown that for children who develop acute lymphoblastic leukemia, the leukemic clone is present at birth [2-5]. Notably, however, not all neonates who have a leukemic clone at birth will go on to develop childhood leukemia, indicating that other genetic and environmental factors influence disease risk [6, 49]. It is tempting to speculate that a similar paradigm is true of BRAF fusion-positive cerebellar pilocytic astrocytoma. Given the lack of tissue for examining such a question and the obvious unethical nature of obtaining such samples, future studies examining this question will have to rely on other markers (e.g. circulating tumor cells).

This study has several limitations. First, it is possible that PA-DR genes simply represent gene expression of hindbrain neurons trapped within the tumor tissue rather than the tumor cells themselves. However, since most PA-DR genes are not expressed postnatally (see Fig 5A), this is unlikely.

Second, being in silico, while these data show consistency of PA-DR gene localization within the developing cerebellum across a variety of murine and human datasets, it cannot causally identify cerebellar ventricular zone progenitor cells as the cell-of-origin for cerebellar pilocytic astrocytoma. One approach to validating these findings in vivo would be to identify whether BRAF fusion alone within ventricular zone progenitor cells can lead to tumorigenesis. Given the relative impotence of BRAF fusion to cause neoplasia in other cell types [50], it would be fascinating to determine whether this genetic alteration is more oncogenic in the cellular context of ventricular zone progenitor cells.

Third, admittedly, the number of genes used in the analyses described herein is small; several factors contribute to this limitation. First, very few genes were differentially expressed between supra- and infra-tentorial tumors. Moreover, given the nature of the analyses, specific expression within the developing brain was required, further restricting the number of genes to analyze. Nevertheless, the consistency of spatio-temporal localization of even this small set of genes across multiple datasets partially mitigates this limitation. Moreover, several PA-DR genes, most notably Pax3 and the members of the Iroquois family (Irx5 and Irx2), have consistently been reported to be differentially methylated [25, 51, 52] and expressed at both the mRNA [25, 45, 51, 53] and protein levels [53] in infratentorial tumors, strengthening the selection of genes used in this study.

Fourth, this analysis is restricted to a single subtype of pilocytic astrocytoma; namely, those occurring in the cerebellum with a BRAF fusion. It is unlikely that this data can be extended to pilocytic astrocytomas with different drivers or those occurring in different locations, as those tumor entities likely have a distinct cell-of-origin. However, given that the cerebellum is the most common location for pediatric pilocytic astrocytoma, and that BRAF-fusion is the most common driver, this data is highly relevant. In addition, the approach described in this paper, when coupled with appropriate developmental data, could be applied to pilocytic astrocytomas with different driver mutations, further defining the cellular and developmental origins of pilocytic astrocytoma.

In conclusion, with the above limitations in mind, these data provide compelling evidence for cerebellar ventricular zone progenitor cells as the cell-of-origin for BRAF fusion-positive cerebellar pilocytic astrocytoma. The current findings provide a key first step toward future validation research that may ultimately guide improved and targeted treatment for BRAF fusion-positive cerebellar pilocytic astrocytoma.

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List of genes differentially expressed between BRAF fusion-positive infratentorial vs. supratentorial tumors with accompanying statistical data.

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Table detailing the expression or lack thereof of PA-DR genes in three regions of the developing cerebellum.

(XLSX)

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PA-DR genes are expressed early in development of the mouse brain.

Expression for each PA-DR gene in the Allen Developing Mouse Brain Atlas. Note the particular enrichment on embryonic days 13.5 and 15.5 for all genes.

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Neurodevelopmental-related genes overexpressed in infratentorial pilocytic astrocytoma are expressed in rhombomere 1.

(A-D) Saggital and coronal images of the developing mouse brain with individual gene expression as marked. The purple highlight marks rhombomere 1. The white asterisk marks the cerebellar anlage. Many PA-DR genes exhibit a morphogenic gradient. Six out of eight PA-DR genes are expressed within rhombomere 1. Four are also expressed rostrally and five are also expressed caudally, suggesting rhombomere 1 as the region of overlap for these morphogenic gradients.

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PA-DR genes are enriched in early ventricular zone progenitors isolated from embryonic day 13–15.

(A) t-SNE plots of single cell gene expression data as in Fig 3A for each PA-DR gene. (B) Developmental day of isolation for single cells shown in Fig 3A. The box denotes the region of overlap of top four PA-DR genes, showing these cells are isolated from embryonic days 13–15. (C) Cell Seek derived cell type for single cells shown in Fig 3A. Based on expression of known cellular markers, cells co-expressing PA-DR genes are identified as early ventricular zone progenitor cells, GABA-ergic neurons, glia, and astrocytes.

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Lineage analysis of single cells from the developing mouse cerebellum which co-express PA-DR genes.

(A) Selection of cells used for subsequent lineage analysis. Bolded hexagons indicate cells which were selected while grayed out hexagons indicate cells which were excluded. (B) Cell seek derived cell types plotted along Monocle derived lineages revealing three main cell types are derived from early ventricular zone progenitor cells: GABA-ergic neuronal progenitors, glial precursor cells, and astrocytes.

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PA-DR genes are individually enriched along certain lineages derived from ventricular zone progenitor cells.

(A) Zoomed region of interest from Fig 3A showing cell type for those cells with strongest overlap in expression of PA-DR genes. (B) Individual PA-DR gene expression for region of interest. Note the temporal relationship and lineage-specific expression of each PA-DR gene (C) Expression data for each PA-DR gene is shown along the Monocle-derived lineages. Note the enrichment of Pax3, Irx5, and Irx2 along all lineages. Ascl1 is enriched for early ventricular zone progenitor cells. Meis1, Klf15, and Msx2 are enriched along the glial progenitor and astrocytic lineages. Pbx3 is expressed chiefly in GABA-ergic neuron progenitor cells.

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Transcription factor correlation network reinforces cell developmental trajectories and places PA-DR genes within the same functional network as known regulators of cellular development.

Note that seven out of eight PA-DR genes are represented within the transcription factor network and localization therein recapitulates expression patterns/cell lineage restriction shown in S5 Fig.

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29 Jul 2020

PONE-D-20-17252

In Silico Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma

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Please submit your revised manuscript by Sep 12 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Marta M. Alonso, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study, Younes investigates the cell or origin for pediatric cerebellar juvenile pilocytic astrocytoma through an in silico approach. This is an ambitious study and carries relevance for the study of pediatric low-grade glioma. However, the study and especially the figures are often confusing and challenging to interpret. The lack of in vitro or in vivo validation of the findings is also a major issue. Additional issues are raised below.

Introduction:

- Line 73 would say radiation or chemotherapy, since both would rarely be necessary in these tumors.

- Would add a section on the known molecular alterations common to cerebellar pilocytic astrocytomas

Methods:

- Line 87, translocation -- does this refer to BRAF fusions?

- Need to define PA-DR at first mention in this section, not in the middle of Results

- There needs to be more justification of the use of a mouse atlas.

Results

- Line 162: Again, would change to BRAF fusion (throughout manuscript)

- The legend for Figure 2 is unclear -- what do the yellow dots represent?

- Figure 3 is also unclear -- color gradients need to be better defined, and images need to be labeled; screen captures from analysis programs generally are not good manuscript figure panels without editing to clarify and remove some unnecessary portions (e.g. a "Refresh" button)

- Wouldn't the lack of transcriptional upregulation of MAPK markers within the developmental cerebellum be because the fusion protein deregulating the pathway in tumors is not present?

- Again, Figure 5 is not clear and needs to be better explained in both the figure (via labels) and legend.

- These findings could likely be validated on a protein level through IHC/IF in human tumor samples, which would substantially strengthen the conclusions.

Discussion

- Figure 6: While usually figures should be associated with Results, this may be acceptable if allowed by editors; figure again needs to be better explained, however -- what do the colored arrows represent?

- More discussion is needed regarding the use of investigating markers from human tumor samples in a mouse brain atlas and then revalidating with a human atlas -- why not just use the human brain atlas throughout? This may be justifiable in terms of these developmental atlases not being available for humans, but this merits discussion.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

21 Oct 2020

Response to Reviewers

We would like to thank the reviewer and the editor for their thoughtful review and suggestions for improvement. In response, the manuscript has been extensively revised to incorporate the aforementioned reviewer and editor comments.

Editor Comments

Comment: The paper could use further review by a biostatistician, as the methodology used is complex.

Response: The methodology has been reviewed by Dr. Kurt Showmaker, a bioinformatician with expertise in differential gene expression analyses. His comments have been used to revise the description of the methodology used.

Comment: I would also advise you to ask a further review by a senior colleague that could give further input.

Response: Dr. Betty Herrington, a pediatric neuro-oncologist, has carefully reviewed the manuscript and analyses. Based on her contributions, she has been added as an author on the manuscript.

Reviewer Comments

Comment: In this study, Younes investigates the cell or origin for pediatric cerebellar juvenile pilocytic astrocytoma through an in silico approach. This is an ambitious study and carries relevance for the study of pediatric low-grade glioma. However, the study and especially the figures are often confusing and challenging to interpret. The lack of in vitro or in vivo validation of the findings is also a major issue.

Response: The figures and accompanying text have been extensively revised in order to make their interpretation clearer. In particular, the figure legends have been expanded to more carefully walk the reader through the data presented. Regarding the lack of in vitro or in vivo validation, the discussion has been updated to address these potential shortcomings. Most notably, potential avenues of in vivo validation have been proposed.

Comment: Line 37 should say radiation or chemotherapy, since both would rarely be necessary in these tumors.

Response: The line in question has been updated to correctly state that radiation or chemotherapy would be used for the treatment of pilocytic astrocytoma.

Comment: Would add a section on the known molecular alterations common to cerebellar pilocytic astrocytoma.

Response: A paragraph commenting on the known molecular alterations in cerebellar pilocytic astrocytoma has been added to the introduction.

Comment: Line 87, “translocation” – does this refer to BRAF fusions?

Response: This line did indeed seek to refer to BRAF fusion. The entire manuscript has been updated to properly state, “BRAF fusion.”

Comment: Need to define “PA-DR” at first mention in this section, not in the middle of Results.

Response: A line defining the designation of PA-DR has been added to the methods section

Comment: There needs to be more justification of the use of a mouse atlas.

Response: Several lines have been added explaining why we chose to utilize mouse atlases first. Briefly, as the reviewer suggests in a later comment, gene expression atlases of the developing human brain are sparse. On the other hand, mouse developmental atlases are more readily available with more robust data. Additionally, a section has been added to the discussion to more fully justify the use of a mouse gene expression atlas.

Comment: Line 162: Again, would change to BRAF fusion (throughout manuscript)

Response: All references throughout the manuscript have been changed to read, “BRAF fusion.”

Comment: The legend for Figure 2 is unclear – what do the yellow dots represent?

Response: The legend for Figure 2 has been revised to explain what is depicted.

Comment: Figure 3 is also unclear – color gradients need to be better defined, and images need to be labeled; screen captures from analysis programs generally are not good manuscript figure panels without editing to clarify and remove some unnecessary portions (e.g. a “refresh” button).

Response: Figure 3 has been extensively revised. The legend has been expanded and the figures themselves have been annotated to better direct the reader to the key data points.

Comment: Wouldn’t the lack of transcriptional upregulation of MAPK markers within the developmental cerebellum be because the fusion protein deregulating the pathway in tumors is not present?

Response: The results section in question has been revised to address a comment by the editor. Namely, the editor requested that the comment, “data not shown,” be removed from the manuscript. With the removal of that sentence, the reviewer’s comment has been relieved.

Comment: Again, figure 5 is not clear and needs to be better explained in both the figure (via labels) and legend.

Response: Figure 5 has been extensively revised and annotated to better convey the key data points.

Comment: These findings could likely be validated on a protein level through IHC/IF in human tumor samples, which would substantially strengthen the conclusions.

Response: A review of the literature was performed. Protein-level expression data could only be identified for a single PA-DR gene, namely, Pax3, confirming that it is overexpressed in cerebellar pilocytic astrocytoma. Unfortunately, a lack of funding precludes the ability to interrogate other markers.

Comment: Figure 6: While usually figures should be associated with Results, this may be acceptable if allowed by editors; figure again needs to be better explained, however -- what do the colored arrows represent?

Response: The figure legend has been revised.

Comment: More discussion is needed regarding the use of investigating markers from human tumor samples in a mouse brain atlas and then revalidating with a human atlas -- why not just use the human brain atlas throughout? This may be justifiable in terms of these developmental atlases not being available for humans, but this merits discussion.

Response: The discussion has been revised to more clearly state our rationale for having used mice atlases.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

3 Nov 2020

PONE-D-20-17252R1

In Silico Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma

PLOS ONE

Dear Dr. Younes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please, address the following minor concerns: Improve the color keys to the color gradients in panels 3C, 3D, and 5A on the figures themselves to assist readers in interpreting these figures; 3C-D could likely also use text and/or arrows on the panels themselves to help with interpretation

Please submit your revised manuscript by Dec 18 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Marta M. Alonso, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have done well in revising the manuscript and have addressed the majority of my concerns. While the figure captions are improved, I would still like to see better keys to the color gradients in panels 3C, 3D, and 5A on the figures themselves to assist readers in interpreting these figures; 3C-D could likely also use text and/or arrows on the panels themselves to help with interpretation.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Nov 2020

Response to Reviewers

We would like to thank the reviewer for their thoughtful review and suggestions for improvement. In response, the manuscript has been revised to incorporate the aforementioned reviewer comments.

Reviewer Comments

Comment: The authors have done well in revising the manuscript and have addressed the majority of my concerns. While the figure captions are improved, I would still like to see better keys to the color gradients in panels 3C, 3D, and 5A on the figures themselves to assist readers in interpreting these figures; 3C-D could likely also use text and/or arrows on the panels themselves to help with interpretation.

Response: Figures 3C, 3D, and 5A have each been updated to include a color gradient key. In addition, arrows and text descriptions have been added to figures 3C and 3D to aid the reader in interpreting which direction represents which cell lineage.

Submitted filename: Response to Reviewers R2.docx

Click here for additional data file.

4 Nov 2020

In Silico Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma

PONE-D-20-17252R2

Dear Dr Younes,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Marta M. Alonso, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

6 Nov 2020

PONE-D-20-17252R2

In

Dear Dr. Younes:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Marta M. Alonso

Academic Editor

PLOS ONE