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• W4386422771 abstract "Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Peer review Author response Article and author information Metrics Abstract The telencephalon and eye in mammals are originated from adjacent fields at the anterior neural plate. Morphogenesis of these fields generates telencephalon, optic-stalk, optic-disc, and neuroretina along a spatial axis. How these telencephalic and ocular tissues are specified coordinately to ensure directional retinal ganglion cell (RGC) axon growth is unclear. Here, we report self-formation of human telencephalon-eye organoids comprising concentric zones of telencephalic, optic-stalk, optic-disc, and neuroretinal tissues along the center-periphery axis. Initially-differentiated RGCs grew axons towards and then along a path defined by adjacent PAX2+ VSX2+ optic-disc cells. Single-cell RNA sequencing of these organoids not only confirmed telencephalic and ocular identities but also identified expression signatures of early optic-disc, optic-stalk, and RGCs. These signatures were similar to those in human fetal retinas. Optic-disc cells in these organoids differentially expressed FGF8 and FGF9; FGFR inhibitions drastically decreased early RGC differentiation and directional axon growth. Through the RGC-specific cell-surface marker CNTN2 identified here, electrophysiologically excitable RGCs were isolated under a native condition. Our findings provide insight into the coordinated specification of early telencephalic and ocular tissues in humans and establish resources for studying RGC-related diseases such as glaucoma. eLife assessment In this important study, the authors present a human telencephalon-eye organoid model that exhibits remarkable pathfinding and growth of retinal ganglion cell (RGC) axons. The identification of cell-surface markers for RGCs could have value for understanding the molecular mechanisms involved in RGC axon development and regeneration. The strength of evidence is compelling for future studies to investigate RGC neurite outgrowth and brain-eye connectivity in humans. https://doi.org/10.7554/eLife.87306.3.sa0 About eLife assessments Introduction Our understanding of eye and brain development in humans is mostly deduced from animal studies. In mice, fate mapping of the anterior neural plate reveals that the eye field is in rostral regions surrounded anteriorly and laterally by the telencephalic field and caudally and medially by the hypothalamic field, indicating the proximity of their embryonic origins (Inoue et al., 2000). Subsequent evagination of the eye field generates bilateral optic vesicles and optic stalks, and the optic stalk connects the optic vesicle to the forebrain. The optic vesicle then invaginates ventrally, resulting in the formation of the double-layered optic cup in which the inner and outer layers develop into the neuroretina and retinal pigment epithelium (RPE), respectively. The posterior pole of the optic cup forms the optic disc (also known as optic nerve head). In the central neuroretina close to the nascent optic disc, retinal ganglion cells (RGCs) start to appear as the optic fissure nearly closes. Early RGC axons find their path toward the optic disc and then enter the optic stalk to reach the brain. Concentrically organized growth-promoting and growth-inhibitory guidance cues around the optic disc regulate RGC axon growth and pathfinding through multiple mechanisms (Erskine and Herrera, 2007; Erskine and Herrera, 2014; Oster et al., 2004). Therefore, early eye morphogenesis leads to coordinated specification of the telencephalic, optic stalk, optic disc, and retinal tissues along a spatial axis. Early telencephalic and eye development is marked and regulated by a group of tissue-specific transcription factors and signal transduction molecules (Wilson and Rubenstein, 2000). In mice, Foxg1 is specifically expressed in the presumptive telencephalon and is essential for the development of the cerebral hemispheres (Xuan et al., 1995). Pax6 is specifically expressed in the eye field and is essential for the development of multiple ocular tissues, such as the neuroretina, lens, and RPE (Ashery-Padan et al., 2000; Bäumer et al., 2003; Marquardt et al., 2001; Zuber et al., 2003). Vsx2 and Mitf are specifically expressed in the neuroretina and RPE, respectively, and are essential for retinal development (Bharti et al., 2012; Horsford et al., 2005; Liu et al., 2010; Rowan et al., 2004). Pax2 is expressed in the optic stalk, optic vesicles, central neuroretina, and optic disc; Pax2 is essential for optic stalk and nerve development (Macdonald et al., 1997; Martinez-Morales et al., 2001; Schwarz et al., 2000; Torres et al., 1996). Fgf8 is specifically expressed at the rostral forebrain at early stages, induces Foxg1 expression (Shimamura and Rubenstein, 1997), and regulates telencephalic patterning in a dose-dependent manner (Storm et al., 2006). Fgf8 also maintains Pax2 expression in the optic stalk (Soukkarieh et al., 2007), and Fgf8 and Fgf3 coordinate the initiation of retinal differentiation in chicks (Martinez-Morales et al., 2005; Vogel-Höpker et al., 2000). Despite these findings in vertebrates, it is unclear how telencephalic and ocular tissues are specified in humans. Human organoids are transformative since they enable us to study the mechanisms of cell specification and differentiation directly in human tissues (Lancaster and Knoblich, 2014). Self-organized three-dimensional (3-D) retinal organoids are originally reported by Sasai’s group and further improved in follow-up studies (Cowan et al., 2020; Eiraku et al., 2011; Fligor et al., 2021; Kim et al., 2019; Lowe et al., 2016; Meyer et al., 2011; Nakano et al., 2012; Reichman et al., 2014; Zhong et al., 2014). We and others have demonstrated that 3-D retinal organoids derived from human embryonic stem cells (hESCs) display a stratified structure containing all major types of retinal cells. Although RGCs are differentiated in 3-D retinal organoids, there is no proper RGC axon outgrowth toward the optic disc as seen in vivo since the optic disc-like tissue is missing in 3-D retinal organoids. When these organoids are dissociated into single cells or cut into pieces for adherent culture, RGCs generate neurites (Fligor et al., 2018; Langer et al., 2018; Teotia et al., 2017). A variety of brain organoids have been described (Kadoshima et al., 2013; Lancaster et al., 2013; Mariani et al., 2012; Qian et al., 2017; Velasco et al., 2019). Although rudimentary ocular tissues are occasionally found in some brain organoids (Gabriel et al., 2021), optic disc- and stalk-like tissues are absent. Collectively, RGC axon outgrowth and pathfinding directed by optic disc- and stalk-like tissues in organoids have not been reported. Tissue patterning and coordinated specification are fundamental for body plan formation in vivo. Remarkably, concentric zones of trophectoderm, endoderm, mesoderm, and ectoderm (Etoc et al., 2016; Minn et al., 2020), neural plate and neural plate border (Xue et al., 2018), and ectodermal cells (Hayashi et al., 2016) are self-organized from single pluripotent stem cells, indicating patterning and coordinated specification of these tissues in vitro. Nevertheless, optic disc and stalk tissues are not reported in any of these structures. We hypothesize that coordinated specification from the anterior ectodermal epithelium via morphogen gradients leads to self-organization of telencephalic and ocular tissues, including optic disc- and stalk-like tissues that provide guidance cues for RGC axon growth and pathfinding. In support of this hypothesis, we generated self-formed human telencephalon-eye organoids that comprise concentric zones of anterior ectodermal progenitors (CONCEPT), including FOXG1+ telencephalic, PAX2+ VSX2- optic stalk, PAX2+ VSX2+ optic disc, and VSX2+ neuroretinal cells along the center-periphery axis. We call this system as an organoid since it displays a spatially organized structure with cell identities mimicking those tissues in vivo. In CONCEPT organoids, early differentiated RGCs grew their axons toward and then along a path defined by adjacent PAX2+ VSX2+ optic disc cells. Single-cell RNA sequencing of CONCEPT organoids not only confirmed telencephalic and ocular identities but also discovered expression signatures of cell clusters. Additionally, CONCEPT organoids and human fetal retinas had similar expression signatures. Furthermore, PAX2+ VSX2+ optic disc cells differentially expressed FGF8 and FGF9; inhibition of FGF signaling with an FGFR inhibitor during early RGC differentiation drastically decreased the number of RGCs somas and nearly ablated directional axon growth. Using the RGC-specific cell-surface marker CNTN2 identified in our single-cell RNA-sequencing, we developed a one-step method for isolating electrophysiologically-excitable RGCs under a native condition. Our studies provide deeper insight into the coordinated specification of telencephalic and ocular tissues in humans and establish resources for studying neurodegenerative diseases such as glaucoma. Results Generation of telencephalon-eye organoids that are composed of concentric zones of anterior ectodermal progenitors (CONCEPT) The telencephalon and eye in mammals are originated from adjacent fields at the anterior neuroepithelium (Inoue et al., 2000). Morphogenesis of these embryonic fields leads to the formation of telencephalon, optic stalk, optic disc, and neuroretina along a spatial axis. Early differentiated retinal ganglion cells (RGCs) in the neuroretina grow axons toward the optic disc and then along the optic stalk to reach the brain. How these telencephalic and ocular tissues are specified coordinately to ensure directional RGC axon growth is unclear. Cysts are hollow spheres of a columnar epithelium that is induced from human pluripotent stem cells via embedding hESC sheets in Matrigel; they are used to generate retinal cells (Kim et al., 2019; Lowe et al., 2016; Zhu et al., 2013). Nevertheless, developmental potentials of cysts have not been characterized. Using the cysts, here we generate CONCEPT telencephalon-eye organoids (Figure 1A and B). Cysts were generated as previously described (Kim et al., 2019; Lowe et al., 2016). The epithelial structure of cysts was demonstrated by apical localization of the TJP1::GFP reporter at the lumen (Figure 1C), consistent with previous findings (Lowe et al., 2016; Zhu et al., 2013). To assess developmental potentials of cysts, individual cysts were manually picked and then seeded onto the Matrigel-coated surface at low densities (Figure 1A). After attaching to the culture surface, cysts grew as dome-shaped individual colonies. Subsequent culture of the colonies in a KSR medium (see Materials and methods) led to the self-formation of concentric zones of anterior ectodermal progenitors: an elevated central zone surrounded by multiple zones. This morphology was identifiable under a stereomicroscope (Figure 1D) or an inverted microscope. If multiple cysts were fused together or cysts for seeding were too big or small, CONCEPT structures were affected or incomplete. Overall, the concentric structure was abundant. Figure 1 with 3 supplements see all Download asset Open asset Generation of telencephalon-eye organoids comprising concentric zones of anterior ectodermal progenitors (CONCEPT). (A) A scheme of the procedure. (B) Diagrams of developing CONCEPT organoids showing concentric zones of the anterior ectodermal progenitors. A summary of Figures 1, 2 and 7, Figure 1—figure supplements 1–3. (C) Morphology of cysts at day 2 showing the epithelial structure indicated by apical localization of the reporter TJ::GFP at the lumen. (D) Morphology of CONCEPT organoids at day 26. (E–G) Expression of telencephalon (Tel) marker Foxg1, neuroretinal (NR) markers Vsx2 and Pax6 in mouse eyes at E10-10.5. Rostral optic stalk (OS) connected the telencephalic vesicle to the optic cup. (H–O) FOXG1+ telencephalic progenitors, VSX2+ and/or PAX6+ retinal progenitors formed concentric zones in CONCEPT organoids. N>5 experiments. (P–W) In CONCEPT organoids, morphogens FGF8, BMP4, and BMP7 mRNA expression started at early stages and subsequently formed circular gradients. N>5 experiments. Scale bars, 100 µm (C, E, M, O, P, S, T), 200 µm (I, K), 500 µm (Q, U), 1 mm (D, H, J, L, N, R, V, W). CONCEPT structures expressed gene markers for telencephalon and eye in concentric patterns. In mice, Foxg1 was specifically expressed in the E10 telencephalon at high levels; it was also expressed in the optic stalk, invaginating optic cup, and lens at lower levels, with the rostral optic stalk connecting the telencephalic vesicle to the invaginating optic cup (Figure 1E; Xuan et al., 1995); Vsx2 was specifically expressed in the E10 neuroretina (Figure 1F); Pax6 was specifically expressed in the E10.5 neuroretina, RPE, lens vesicles, and surface ectoderm (Figure 1G; Liu et al., 2010). In CONCEPT structures, the expression of FOXG1, VSX2, and PAX6 generally exhibited concentric patterns spanning from the center to the periphery at days 15–17 (Figure 1H–K, Figure 1—figure supplement 1; a scheme in the left panel in B). At this stage, PAX6 was expressed in multiple concentric zones at distinct levels, with a higher level at a circular zone that also expressed VSX2 (Figure 1J, Figure 1—figure supplement 1). Concentric zones of FOXG1, VSX2, and PAX6 expression were also found at later stages. At day 26, VSX2 expression expanded peripherally compared to its expression at day 17 (Figure 1—figure supplement 1C, Figure 1—figure supplement 2C) and largely overlapped with PAX6 expression (Figure 1—figure supplement 2A–C). Additionally, VSX2 expression was higher in a zone close to the center and relatively lower in more peripheral zones (Figure 1—figure supplement 2C). Therefore, the CONCEPT structures comprise spatially organized telencephalic and ocular tissues and thus are named as telencephalon-eye organoids. Morphogens FGFs and BMPs play crucial roles in patterning the forebrain and eye in vivo. In mice, Fgf8 is specifically expressed at the rostral forebrain at early stages, induces Foxg1 expression (Shimamura and Rubenstein, 1997), and regulates telencephalic patterning in a dose-dependent manner (Storm et al., 2006). Bmp4 and Bmp7 are expressed in the dorsomedial telencephalon, optic vesicles, and presumptive lens placodes (Danesh et al., 2009; Dudley and Robertson, 1997; Furuta and Hogan, 1998; Solloway et al., 1998). Bmp4 is required for lens induction (Furuta and Hogan, 1998), and Bmp7 is required for proper patterning of the optic fissure (Morcillo et al., 2006). In CONCEPT telencephalon-eye organoids, FGF8, BMP4, and BMP7 were expressed in attached cell colonies starting from early stages (Figure 1P, S and T) and subsequently exhibited circular patterns. At day 10, the expression of BMP4 and BMP7 delineated multiple rings, with a big ring mostly at the center surrounded by numerous small rings (Figure 1P and S). These observations suggest that the attachment of a single-lumen cyst to the culture surface caused differences in cell behaviors: some cells separated from the original cyst and migrated peripherally; some of the separated cells formed small ring-like structures; the cells that remained at the center formed a big ring-like structure. At day 17, circular expression patterns of BMP4 and FGF8 emerged (Figure 1Q and U). At day 25, BMP4, FGF8, and BMP7 expression clearly marked circular zones (Figure 1R, V, W). Expression profiles of FGFs and BMPs were highly reproducible (Figure 1—figure supplement 3A–D). Hence, the expression of FGFs and BMPs spontaneously form circular gradients, which likely dictate coordinated cell specification in CONCEPT telencephalon-eye organoids. Early differentiated RGCs grow directional long axons toward and then along a path defined by PAX2+ VSX2+ cells in CONCEPT telencephalon-eye organoids To assess cell differentiation in CONCEPT telencephalon-eye organoids, we examined marker expression for RGCs, the first type of cells that differentiate in the neuroretina. In mice, transcription factor Pou4f2 is expressed in the early differentiated RGCs and required for the development of a large set of RGCs (Gan et al., 1996). Tubb3 is expressed in the somas and axons of differentiating RGCs. In CONCEPT organoids, POU4F2 and TUBB3 were detectable as early as day 17 and increased to higher levels at day 22. RGC somas were marked by POU4F2 and TUBB3 co-expression; RGC axons were marked by TUBB3 expression. Interestingly, RGCs grew directional long axons that followed a circular path (Figure 2A and B). The circular path of RGC axon outgrowth became more evident in organoids at day 26 (Figure 2C and D). In mice, the initially differentiated RGCs are adjacent to the optic disc; their axons grow towards the optic disc to exit the eye and then navigate within the optic stalk to reach their targets in the brain. The optic disc and stalk specifically expressed Pax2 at distinct levels (Figure 2E–G), consistent with previous findings (Bassett et al., 2010; Martinez-Morales et al., 2001; Mui et al., 2005). Additionally, presumptive optic disc cells expressed VSX2, whereas optic stalk cells did not (Figure 1F, Figure 2E; Liu et al., 2010). In CONCEPT organoids, there were two PAX2+ cell populations that formed two adjacent rings (Figure 2H–L). POU4F2+ RGCs grew TUBB3+ axons toward and then navigated along the adjacent PAX2+ VSX2+ cell population (Figure 2H–L), mimicking axon growth from the nascent RGCs toward the optic disc in vivo. Meanwhile, the PAX2+ VSX2- cell population at the inner zone set up an inner boundary of the path for RGC axon growth, mimicking the optic stalk that spatially confines RGC axon growth in vivo. Based on these findings, we designate PAX2+ VSX2+ cells and PAX2+ VSX2- cells in CONCEPT organoids as optic disc and optic stalk cells, respectively. Taken together, our findings demonstrate that RGCs grow directional axons toward and then along a path defined by PAX2+ VSX2+ cells in CONCEPT telencephalon-eye organoids. Figure 2 Download asset Open asset Retinal ganglion cells (RGCs) grow axons toward and then along a path defined by PAX2+ VSX2+ cells in CONCEPT telencephalon-eye organoids. N>5 experiments. (A–D) POU4F2+ RGCs grew TUBB3+ axons toward and then along a path with a circular or a portion of circular shape. (E, F) In mice, Pax2 was expressed in central regions of the retina and optic stalk at E10.5 (E) and in the optic disc and optic stalk at E13.5 (F). Tubb3+ axons from the initial RGCs grew toward the optic disc, exited the eye, and navigated within the optic stalk (G). (H–L) In CONCEPT organoids at day 26, TUBB3+ RGC axons grew toward and then along a path defined by an adjacent PAX2+ VSX2+ cell population (arrowhead in H, brackets in I, J); the PAX2+ VSX2- cell population set up an inner boundary of RGC axon growth. (L) A diagram summarizing RGC axon growth, PAX2+ VSX2+ optic disc (OD), and PAX2+ VSX2- optic stalk (OS) in CONCEPT organoids. The area labeled by the asterisk may appear as false signals in a low-resolution printout but it is clearly a background in digital display. (M) A count of CONCEPT organoids showing directional retinal ganglion cell axons. Scale bars, 50 µm (E, F, G), 100 µm (B), 200 µm (A, H, I). CONCEPT telencephalon-eye organoids also contain lens cells that undergo terminal differentiation Besides anterior neuroectodermal cells, we wondered whether CONCEPT organoids also contain non-neural ectodermal cells. CONCEPT telencephalon-eye organoids at around day 25 contained transparent structures reminiscent of the ocular lens. To determine their cell identity, we performed immunostaining. Starting at day 22, lens markers CRYAA and beta crystallin (CRY B) were found (Figure 3A and B) and continuously expressed in these transparent cell structures (Figure 3C, D and K). Interestingly, these transparent structures were not stained by DAPI (Figure 3C–F), indicating denucleation in these lens cells. When CONCEPT organoids were detached using Dispase at around day 28 and continuously grown as suspension cultures, crystal-like clusters with fused transparent spheres—lentoid bodies—were found, and they continuously survived for months (Figure 3I and J). Lentoid bodies highly expressed gamma crystallin (CRY G; Figure 3L), confirming their lens identity. In mice, terminally differentiated lens fiber cells are free of organelles and featured by specialized interlocking cell membrane domains shown as ball-and-sockets and protrusions (Biswas et al., 2010). Using transmission electron microscopy, we found that our lentoid bodies were free of organelles and exhibited ball-and-socket structures (Figure 3M and N). Taken together, our findings demonstrate that CONCEPT telencephalon-eye organoids also contain lens cells that undergo terminal differentiation; FGFs in CONCEPT organoids likely promote terminal lens differentiation as seen in other settings (Lovicu and McAvoy, 2001). Figure 3 Download asset Open asset CONCEPT telencephalon-eye organoids contain lens cells that undergo terminal differentiation. N>5 experiments. (A–L) In CONCEPT organoids, lens markers CRYAA and beta crystallin (CRY B) were expressed at day 22 (A, B) and day 39 (C, D, K; a count in H). Lens cells were not stained by DAPI (E, F); they exhibited a crystal-like shape (G). When CONCEPT organoids were detached using Dispase at around day 28 and grown in suspension, crystal-like clusters, named as lentoid bodies, were found (I) and survived for months (J). These lentoid bodies highly expressed gamma crystallin (CRY G), as revealed by Western blot (L). (M–N) These lentoid bodies were free of organelles and exhibited ball-and-socket structures (K, L), as revealed by electron microscopy. Scale bars, 100 µm (A, B, C, D, I), 200 µm (J), 5 µm (K), 200 nm (L). Single-cell RNA sequencing analysis identifies telencephalic and ocular cell populations in CONCEPT telencephalon-eye organoids To fully characterize cell populations in CONCEPT organoids and the mechanisms underlying RGC axon pathfinding, we performed single-cell RNA sequencing (10 x Genomics) of the organoids at day 24, around the stage when RGCs grew long axons toward and along the PAX2+ VSX2+ cell population. Cell Ranger mapping showed that 11158 single cells were sequenced at a depth of 27,842 reads and 2967 genes per cell, and the dataset were further analyzed using Seurat (v3.2.0) (Stuart et al., 2019). Cell filtration (nFeature_RNA >200 & nFeature_RNA <6000 & percent.mt <20) resulted in 10,218 cells. Cell clustering grouped cells into 14 clusters (Figure 4A; Table 1), and cell cycle phases were identified based on cell cycle scores using an established method (Tirosh et al., 2016; Figure 4B). Several cell clusters, for example, clusters 4, 8, 9, were separated along cell cycle phases (Figure 4A and B). Figure 4 with 11 supplements see all Download asset Open asset scRNA-seq of CONCEPT organoids identifies telencephalic and ocular cells, including PAX2+ VSX2+ optic disc cells, PAX2+ VSX2- optic stalk cells, and CNTN2+ RGCs. CONCEPT organoids at day 24 were used for profiling. (A) Identification of 14 cell clusters. (B) Cell cycle phases revealed by cell cycle scores. (C) FOXG1 expression marked telencephalic cells. (D, E) The expression of PAX6 and/or VSX2 marked retinal cells. (F) PAX2+ cells were found in two major cell populations: PAX2+ VSX2+ cells were assigned as the optic disc (OD), whereas PAX2+ VSX2- FOXG1+ cells were assigned as the optic stalk (OS). (G–L) The expression of major DEGs in cluster 2, the major cell population that mimics the optic disc. (M–P) The expression of major gene markers for PAX2+ VSX2- optic stalk cells. (Q–T) Identification of CNTN2 as a specific marker for early human RGCs. A large portion of cluster 11 differentially expressed neurogenic retinal progenitor marker ATOH7 and RGC markers POU4F2 and SNCG. The expression of CNTN2 and POU4F2 largely overlapped. (U–X) Two small portions of cluster 11 differentially expressed early photoreceptor cell markers (OTX2 and CRX, U, V) and amacrine/horizontal cell markers (TFAP2C and PTF1A, W, X), respectively. Table 1 Cell counts for clusters in the scRNA-seq dataset of CONCEPT organoids at day 24. Clusters012345678910111213IdentsMixed(lower counts)dTelODvTeldTel/OSNRRPENRdTel/ OSdTel/ OSvTelRGC/PR/AC/HCvTelUD# Cells1295115896493192187174774372960345039333380percent0.1270.1130.0940.0910.090.0850.0730.0730.0710.0590.0440.0380.0330.008 Abbreviations: Idents, assigned cell identities; # Cells, cell number; Tel, telencephalon; NR, neural retina; OD, optic disc; OS, optic stalk; RPE, retinal pigment epithelial cells; RGC, retinal ganglion cells; PR, photoreceptor cells; AC, amacrine cells; HC, horizontal cells; UD, undetermined. We next assessed cell identities using established markers. Mesoderm, endoderm, and neural crest are identified by a group of gene markers (Poh et al., 2014; Simões-Costa and Bronner, 2015; Varga et al., 2021; Yao et al., 2017). In CONCEPT organoids, gene markers for mesoderm (TBXT, GATA2, HAND1), endoderm (GATA1, GATA4, SOX17), and neural crest (SNAI1, SOX10, FOXD3) were not expressed (Figure 4—figure supplement 1). In contrast, gene markers for the anterior neuroectoderm were widely expressed. CONCEPT organoids at day 24 were mostly composed of FOXG1+ telencephalic cells (clusters 10, 3, 12, 1, 4, 8, 9, 13) and PAX6+ and/or VSX2+ retinal cells (clusters 2, 7, 5, 6, 11; Figure 4A–E). Cluster 0, which comprised both telencephalic and retinal cells (Figure 4C–E), was marked by negative gene markers (Figure 4—figure supplement 2A–D). Cluster 0, together with cluster 10, had much lower counts of captured RNA and genes compared to other clusters (Figure 4—figure supplement 2E–G) and was therefore not dissected further. In contrast to telencephalon and retinal markers, diencephalon markers (GBX2, WNT3, SOX14; Chatterjee and Li, 2012; Martinez-Ferre and Martinez, 2012) and midbrain/hindbrain markers (EN2, PAX7, TFAP2B; Yao et al., 2017) were rarely expressed (Figure 4—figure supplement 3). Lens markers CRYAA and FOXE3 were expressed in a very small cell population. Those denucleated lens cells were not captured in the scRNA-seq since the absence of nucleus prevented active transcription. Therefore, lens cells did not form a separate cluster probably due to their very low abundance. Taken together, these findings indicate that CONCEPT telencephalon-eye organoids at day 24 are mostly composed of FOXG1+ telencephalic cells and PAX6+ and/or VSX2+ ocular (mostly retinal) cells. We next characterized FOXG1+ telencephalic cells via assessing differentially expressed genes (DEGs) of clusters. Top DEGs in clusters 4, 8, 9, 1 included SOX3, FGFR2, PRRX1, and EDNRB (Figure 4—figure supplement 4A–D), which mouse orthologs are specifically expressed in the dorsal telencephalon (Figure 4—figure supplement 5B–E). Top DEGs in clusters 12, 3, 10 included DLX2, DLX6-AS1, DLX1, and RGS16 (Figure 4—figure supplement 4E–H), which mouse orthologs are specifically expressed in the ventral telencephalon (Figure 4—figure supplement 5F–I). Consistently, Foxg1 is expressed in both dorsal and ventral telencephalon in E14.5 mouse embryos (Figure 4—figure supplement 5A). Therefore, telencephalic cells in CONCEPT organoids at day 24 comprise both dorsal and ventral telencephalic cells. Cluster 13 also expressed FOXG1, but it was separated from other FOXG1+ cells. Top DEGs in cluster 13 included TRIB3, DWORF, SLC7A11, GDF15, and UNC5B; cell identities of cluster 13 were undetermined. PAX6+ and/or VSX2+ retinal cells were grouped into several clusters. VSX2 was expressed in clusters 2, 7, 5, and 0. Clusters 2 was at G1 phase, cluster 7 was at G1 and S phases, and cluster 5 was at S and G2M phases (Figure 4A, B and E). Cluster 6 did not express VSX2 but expressed PAX6 and was at the G1 phase (Figure 4A, B, D and E). Cluster 6 differentially expressed RPE markers, e.g., PMEL, HSD17B2, DCT, and MITF (Figure 4—figure supplement 6), indicating they were mostly differentiating RPE cells. Taken together, our single-cell RNA sequencing analysis of CONCEPT telencephalon-eye organoids at day 24 confirms their telencephalic and ocular identities, establishing a valuable transcriptomic dataset for mechanistic studies. Identification of two PAX2+ cell populations that mimic to the optic disc and optic stalk, respectively, in the scRNA-seq dataset of CONCEPT telencephalon-eye organoids To identify PAX2+ cell populations that defined the path for RGC axon outgrowth, we examined PAX2 expression in the dataset. PAX2 was mostly expressed in cluster 2 and subsets of clusters 7, 5, 0, 4, 8, and 9 (Figure 4A and F). Since clusters 2, 7, 5, and 0 expressed VSX2 (Figure 4E and F), we deduced that PAX2+ VSX2+ cells in cluster 2 and subsets of clusters 7, 5, and 0 corresponded to those PAX2+ VSX2+ cells that defined the path for RGC axon growth (Figure 2H–L) and therefore were assigned as optic disc (OD) cells (Figure 4F). PAX2+ VSX2+ cells also expressed optic disc and optic stalk marker SEMA5A (Oster et al., 2003; Figure 4—figure supplements 7A and 9A, E). We particularly focused on cluster 2 since PAX2+ VSX2+ cells were mostly found in this cluster. Interestingly, COL9A3 and COL13A1 were differentially expressed in cluster 2 (Figure 4G and H; Figure 4—figure supplement 7A). Previous in situ hybridization results indicate that COL13A1 is prominently expressed in the optic disc of human fetal retinas (Sandberg-Lall et al., 2000). These findings support that PAX2+ VSX2+ ce" @default.
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• W4386422771 title "eLife assessment: Self-formation of concentric zones of telencephalic and ocular tissues and directional retinal ganglion cell axons" @default.
• W4386422771 doi "https://doi.org/10.7554/elife.87306.3.sa0" @default.
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