scRNA-Seq Analysis Reveals Human Cortical tRG Lineage at GW21-GW26
Our recent study has demonstrated that the presence of EGFR+ progenitors in the E16.5 mouse cortex is a strong signal for the onset of cortical gliogenesis (and OBiN genesis) . To investigate whether a similar process may be occurring in humans, we studied the molecular identity of human cortical EGFR+ cells using published scRNA-Seq data. First, we analyzed data from 3,355 EGFR-immunopanned cells obtained from GW21-GW26 human frontal cortex (Fig. 1A) . 13 clusters were recognized (C0-C12) using t-distributed stochastic neighbor embedding (tSNE) and Seurat clustering  (Fig. 1B). We identified the identity of the cell types in these clusters based on gene expression. To our surprise, we found that cluster 6 contained cortical tRGs (see below). Furthermore, we obtained evidence that these tRGs (cluster 6) may be lineally related to the PyN-IPC clusters (C4, C2 and C0, 1,531 cells, 46% of EGFR+ cells) and bMIPC clusters (C10, C3 and C1) (Fig. 1B), as they all expressed EGFR and ASCL1 (Fig. 1G). Other major cell types were APCs, OPCs and OBiN-IPCs. Consistent with previous analysis, immature ependymal cells (EP), pericytes and endothelial (Endo) cells also expressed EGFR (Fig. 1B), but the authors did not recognize EGFR expression in the cortical tRG . Below, we focus on the molecular features of the tRG, PyN-IPC, bMIPC, APC, OPC and OBiN-IPC cell types.
Cortical RGCs in late embryogenesis express molecules that are typical of the astrocyte lineage cells (including APCs, immature astrocytes and mature astrocytes) [2, 6]. Indeed, our analysis revealed that human cortical tRGs expressed more than 50 genes that are astrocyte-lineage markers (Fig. 1C, Fig. S1), including RGC markers FABP7, HES1, SLC1A3, SOX2 and VIM (Fig. 1C), and astrocyte markers, ALDOC, AQP4, DBI, GFAP, GLUL, S100B, SLC1A2 and SOX9 etc. (Fig. S1A–G). Notably, HOPX, FAM107A, LIFR and TNC, markers of vRG and oRG [27, 44, 45], were also expressed by tRGs and APCs (Fig. S1). In addition, FGF and BMP signaling pathways were activated in tRGs and APCs (Fig. S2A, B). tRGs had distinctive properties based on their expression of ANXA1, ANXA2, APOE, CD38, CRYAB, CXCL12, GLI3, GPX3, MT1E, PDGFD, PDGFRB and TMEM47; these genes were not expressed by APCs at GW21-GW26 (Fig. S2C, D). On the other hand, CXCL14, DIO2, DUSP6, SDC3, SPARCL1, SPRY1, OLIG2, OLIG1, and ID1 were mainly expressed in APCs but not in tRGs (Fig. 1G; Fig. S2B, E). At GW21-GW26, tRGs did not express ACSBG1, ALDH1L1, FBN2, HS6ST1, HS6ST2 and NOG (Fig. S2F); some of these genes are markers of oRG .
In general, EGFR was weakly expressed in tRGs and PyN-IPCs (PAX6+, EOMES+ and NEUROG2+), and its expression was downregulated in immature PyNs because very few NEUROD6+TBR1+ PyNs expressed EGFR (Fig. 1D–G). Based on the expression of specific marker genes across major cell types, we deduced likely continuities between specific cell types and probable developmental lineages. For example, while oRGs never expressed EGFR and ASCL1, tRGs expressed EGFR, ASCL1 and PAX6, and PyN-IPCs continued to express them (Fig. 1D–G), providing evidence that EGFR+ PyN-IPCs are derived from tRGs. Following similar logic, PAX6+EGFR+ASCL1+BCAN+ tRGs appear to give rise to EGFR+ASCL1+BCAN+ bMIPCs (Fig. 1D–G; Fig. S1A). bMIPCs, expressing higher levels of EGFR and ASCL1 than tRGs, also expressed OLIG2, OLIG1, DLL3 and PDGFRA (Fig. 1F–H); these genes are typical markers for OPCs. This might be the reason why previous studies termed bMIPC as “Pre-OPCs” or “Pri-OPCs” (IPC types preceding committed OPC) [35, 46,47,48,49]. Slingshot analysis , predicting a developmental trajectory and pseudo-timeline progression of IPCs, suggest that bMIPCs give rise to OPCs (ASCL1+OLIG2+OLIG1+PDGFRA+SOX10+NKX2-2+PCDH15+) (Fig. 1H), APCs (EGFR+ OLIG2+OLIG1+HOPX+ID1+HES1+) (Fig. 1G; Fig. S1C) and OBiN-IPCs (EGFR+ASCL1+GSX2+DLX2+DLX1+GAD2+) (Fig. 1I). Early born OPCs and OBiN-IPCs expressed weak EGFR and downregulated its expression very soon, whereas APCs expressed higher level of EGFR (Fig. 1G).
Our genetic fate mapping study demonstrated that mouse cortical Egfr+Ascl1+Olig2+Olig1+ bMIPC population give rise to most of the OPCs and APCs in the mouse cortex and a subpopulation of OBiN-IPCs . Thus, our re-analysis of scRNA-Seq from human cortical EGFR+ cells provides evidence for that mouse and human share the same developmental origins of cortical oligodendrocytes and astrocytes, and cortex-derived OBiNs.
The Coexistence of vRGs, tRGs and oRGs in the Human Cortex at GW17-GW18
To gain a deeper understanding of the molecular features and the relationships between vRGs, tRGs and oRGs, we next re-analyzed published scRNA-Seq data from the mid-gestation human neocortex (GW17-GW18), which provided a high-resolution transcriptome map of 33,976 cortical cells (Fig. 2A) . vRGs and tRGs expressed PDGFD (Fig. S2D) whereas oRGs did not [44, 51]. oRGs expressed higher levels of HOPX, FAM107A and LIFR than tRGs [27, 44]. Thus, based on expression patterns of PDGFD, HOPX, FAM107A and LIFR, we show evidence that vRGs, tRGs and oRGs coexist in the human cortex at GW17-GW18 (Fig. 2B).
Here, we propose that vRGs might directly give rise to oRGs and tRGs simultaneously at the end of their final mitosis because vRGs, tRGs and oRGs expressed many shared glial genes, such as HOPX, FAM107A, LIFR, ALDOC, AQP4, BCAN, GFAP and TNC (Fig. 2B, C). Perhaps because tRGs contact the lateral ventricle, they may gradually acquire their unique properties, including expression of EGFR, ASCL1, ANXA1, CRYAB and CXCL12 (Fig. 2D); these genes were not expressed in vRGs or oRGs. On the other hand, oRGs, contacting the pial surface, expressed ACSBG1 and FBN2, which were not expressed in tRGs (Fig. 2E). In the cortex at GW17-GW18, IPCs mainly consisted of PyN-IPCs that expressed PAX6, NEUROG2 and EOMES (Fig. 2D, F), suggesting that PyN genesis is the primary cellular product. Indeed, except for the medial ganglionic eminence (MGE)-derived OPCs (Fig. 2G), we did not observe cortex-derived OPC or APC clusters, suggesting that gliogenesis had not yet commenced at GW17-GW18. Notably, while EGFR was expressed in tRGs and a subpopulation of PyN-IPCs (progeny of tRG), EGFR expression was absent in the OPCs and cortical interneurons that were derived from the MGE and caudal ganglionic eminence (CGE) (Fig. 2D). This demonstrated that EGFR+ cells in the cortex were derived from the cortex itself. ASCL1, in contrast, was continuously expressed in a subpopulation of immature PyNs, MGE- and CGE-derived cortical interneurons, and MGE-derived OPCs in the cortex (arrows in Fig. 2D). Consistent with previous report , we did not find IPC clusters in the human cortex for generating cortical interneurons, further providing evidence that most if not all human cortical interneurons are derived from the ventral telencephalon [29, 37].
Taken together, by a re-analysis of published scRNA-Seq datasets from human fetal brains at GW17-GW18 and GW21-GW26, we have found that: 1) EGFR is expressed in tRGs but not in vRGs or oRGs; 2) EGFR+ tRGs appear to generate EGFR+ PyN-IPCs and EGFR+ bMIPCs; these bMIPCs then give rise to cortical OPCs, APCs, and OBiN-IPCs; 3) HOPX, FAM107A, TNC and LIFR are expressed in tRGs, in addition to vRGs and oRGs; 4) tRGs express numerous hallmarks of cells in the astrocyte lineage.
Immunohistochemical Identification of tRGs and oRGs in the Human Developing Cerebral Cortex
To validate the molecular signatures of tRG and oRG identified by the scRNA-Seq analysis, we examined expression of cell type specific marker proteins in fixed cortical sections at GW18 (n = 1) and GW23 (n = 2) (Fig. 1A). HOPX and GFAP were strongly expressed in somas and/or basal processes of oRGs in the GW18 cortex (Fig. 3A, D, E). At GW23, we observed similar expression patterns of these proteins in the cortex (Fig. S3A–D). On the other hand, double- or triple-immunofluorescence analysis of GW18 cortical sections showed that EGFR, CRYAB, HOPX and GFAP were expressed in tRGs (Fig. 3A–C, E). Consistent with previous observations , CRYAB+ tRG basal processes terminated in the cortical OSVZ, and thus did not contact the pia (Fig. 3C, Fig. S3A).
CRYAB is a marker for tRGs at GW18 as it is mainly expressed in tRGs. However, at GW23, we observed CRYAB+ astrocyte lineage cells that also expressed HOPX and GFAP in the cortical IFL and MZ (Fig. S3B, C) suggesting that CRYAB is more widely expressed in cortical cells at this later fetal stage (Fig. S2C). Taken together, HOPX is expressed in tRGs, in addition to expression in vRGs and oRGs, consistent with our scRNA-Seq analysis (Fig. S1C). This is also consistent with the expression pattern of HOPX in the macaque monkey cortical VZ and OSVZ at E70 and E125  and in the ferret cortical VZ and OSVZ at E36 .
A Proposed Model of the Generation of tRGs and oRGs from vRGs
It has been proposed that human cortical oRGs are generated from vRGs by a process that resembles epithelial-mesenchymal transitions, because oRGs express genes that promote extracellular matrix production, such as TNC, ITGB5, PTN and PTPRZ1 [27, 44]. However, these genes were also expressed in tRGs; furthermore, TNC, PTN and PTPRZ1 were also expressed in vRGs (Fig. 2C, Fig. S1) [36, 44]. Thus, our scRNA-Seq analysis demonstrated that oRGs expressed most of the genes that were also expressed in tRGs (Fig. 1, Figs. S1, S2).
HOPX expression marked multiple progenitor subtypes at GW18. Immunohistochemistry of fixed cortical sections showed HOPX expression in tRGs in the VZ, oRGs in the OSVZ, and in cells in the ISVZ and IFL with long basal processes that appear to be oRGs migrating into the OSVZ (Fig. 4A, B). Previous study using time-lapse imaging have observed that oRGs and tRGs emerge as the daughter cells of horizontally dividing vRGs . Thus, based on HOPX immunostaining results (Fig. 4A, B), combined with scRNA-Seq data (Fig. 1, 2; Figs. S1, S2) and time-lapse imaging analysis , we propose that the final mitosis of a HOPX+ vRG concomitantly generates a HOPX+ oRG and a HOPX+ tRG (Fig. 4C). oRGs inherit the long basal fiber of vRGs while tRGs inherit the apical domain of vRGs. Like cortical vRGs, both oRGs and tRGs can self-renew (Fig. 4C).
HOPX Is also Expressed in Cortical Cells in the Astrocyte Lineage
scRNA-Seq analysis showed that human cortical APCs expressed EGFR, HOPX, GFAP, OLIG2 and OLIG1 (Fig. 1G, Fig. S2B, C). We thus examined the expression pattern of EGFR in the GW18 cortex (Fig. 5A). Because endothelial cells and pericytes expressed EGFR (scRNA-Seq data, Fig. 1B), cortical blood vessels were EGFR+ (Fig. 5A). EGFR was expressed in the cortical VZ and co-labeled with HOPX, further confirming that tRGs expressed EGFR and HOPX (Fig. 5C). However, HOPX+ oRGs did not express EGFR (Fig. 5B). From the cortical ISVZ to CP, scattered EGFR+HOPX+ cells with small somas were observed (Fig. 5B). These cells were APCs, as most of them also expressed GFAP (Fig. 5D, E), consistent with scRNA-Seq analysis (Fig. 1G, Fig. S1B, C). There were ~10-fold more EGFR+ cells in the cortical VZ, ISVZ and IFL than that in the OSVZ, IZ, SP and CP (Fig. 5F), suggesting that cortical EGFR+ cells are generated in the VZ, and migrate toward the CP. This observation is consistent with previous report . In contrast, there were 4-fold more HOPX+ cells in the OSVZ than VZ (Fig. 5F). In the cortical IZ, SP and CP, 94% of EGFR+ cells expressed HOPX, and 87% of HOPX+ cells expressed EGFR (Fig. 5G).
By GW23, the density of EGFR+ cells and HOPX+ cells were greatly increased in the cortex compared with GW18 (Figs. S4A and 5A). Again, tRGs expressed EGFR and oRGs did not (Fig. S4B). EGFR+HOPX+ APCs were widely distributed from the cortical ISVZ to SP, but EGFR was not expressed in most of the HOPX+ cells in the cortical CP (Fig. S4B), indicating that EGFR expression is downregulated in HOPX+ immature astrocytes. GFAP and HOPX double immunostaining revealed that nearly all HOPX+ GW23 cortical cells were GFAP+ tRGs, oRGs and astrocyte lineage cells (Fig. 6A–C). The high density of astrocyte lineage cells (HOPX+GFAP+ cells) in the GW23 cortex suggests a high level of gliogenesis.
To further confirm that HOPX expression marks astrocyte lineage cells, we performed HOPX, SOX10 and OLIG2 triple immunostaining on GW18 cortical sections (Fig. S5A). All SOX10+ cells (OPCs) in the cortex expressed OLIG2, but none of them expressed HOPX (Fig. S5B–F), confirming that HOPX is not expressed in OPCs. On the other hand, At GW18, most HOPX+ APCs expressed OLIG2 (Fig. S5B–F), consistent with the scRNA-Seq data (Fig. 1G). The ratio of OPCs (SOX10+OLIG2+ cells) to APCs (HOPX+OLIG2+ cells) was 2:1 (Fig. S5G), demonstrating that there were more OPCs than APCs in the GW18 cortex. We propose that in the GW18 cortex, most OPCs were derived from the ventral telencephalon , whereas most APCs were derived from tRGs, because both APCs and tRGs expressed EGFR and HOPX, and because only a small number of cortical bMIPCs were observed at this stage (Figs. 7, 8, 9). Thus, the earliest macroglial cells in the human cortex are MGE-derived OPCs, followed by cortical tRG-derived EGFR+HOPX+ APCs.
We also observed that there were more GFAP+HOPX+ APCs in the ventral cortex than in the dorsal cortex at GW18 (Fig. S6A–C). Thus, in both mouse and human brains, PyNs and glial cells are first generated in the ventral cortex, followed by dorsal and medial cortex , suggesting this is a common rule in mammalian brain. Taken together, our immunohistochemistry results demonstrated that in addition to its expression in vRGs, tRGs and oRGs, HOPX marks astrocyte, but not oligodendrocyte, lineage cells in the human cortex.
Human Cortical tRGs Generate PyN-IPCs and bMIPCs
Our scRNA-Seq analysis provides evidence that human cortical tRGs give rise to PyN-IPCs and bMIPCs (Fig. 1). We thus examined the progeny of tRGs in the GW18 and GW23 human cortex. EOMES and EGFR double immunostaining showed that while EGFR was expressed in both the ganglionic eminences and cortex, EOMES was only expressed in the cortex (Fig. 7A, B). Furthermore, we never observed EOMES+ cells (PyN-IPCs) expressing OLIG2 (Fig. 7C, G). Thus, we propose that EGFR+ tRGs generate both EGFR+EOMES+ PyN-IPCs and EGFR+OLIG2+ bMIPCs (Fig. 7H).
Some EGFR+EOMES+ cells had an apical process extended to the ventricular surface (Fig. 7C, G), exhibiting morphologies of cortical SNPs. At GW18 and GW23, EGFR+EOMES+ PyN-IPCs were mainly in the cortical ISVZ, whereas EGFR+OLIG2+ bMIPCs were mainly in the IFL (Fig. 7D, E). At GW18, only a few EGFR+OLIG2+ bMIPCs were observed in the cortical IFL (Fig. 7C, D), suggesting that at this stage tRGs mainly produce PyN-IPCs. Thus, there were two germinal zones (niches) for PyN genesis in the GW18 cortex: one in the VZ and the other in the OSVZ. By GW23, there was a high density of EGFR+OLIG2+ bMIPCs in the IFL (Fig. 7E, G), a high density of astrocyte lineage cells (Fig. 6) and a high density of OPCs (see below, Fig. S10) in the cortex, indicating a high level of gliogenesis. Thus, at GW23 there were two germinal zones: one was the VZ for tRG PyN genesis and gliogenesis, and the other was the OSVZ mainly for PyN genesis from oRGs (see below, Fig. S12, S13). We propose that tRGs and oRGs both generate upper layer PyNs (Fig. 7H, see discussion).
Recently, we have shown that mouse E16.5 cortical RGCs (did not express EGFR) begin to generate EGFR+ aMIPCs that differentiate into bMIPCs . To investigate whether mouse EGFR+ IPCs also produce PyN-IPCs, like we show in the human cortex, we performed EGFR, EOMES and ASCL1 triple immunostaining on E17.5 and E18.5 mouse cortical sections. We observed EGFR+EOMES+ PyN-IPCs in the mouse cortical SVZ (Fig. S7A, B), whereas most of the EGFR+ASCL1+ (also OLIG2+) bMIPCs  were in the SVZ/IZ border and IZ (Fig. S7A, B). This provides evidence that both human cortical tRGs and mouse cortical RGCs generate EGFR+ PyN-IPCs and EGFR+ bMIPCs (Fig. 7H and Fig. S7C).
bMIPCs Are Mainly Distributed in the Human Cortical IFL at GW18-GW23
To further confirm bMIPCs were mainly distributed in the human cortical IFL, we examined cell types in the cortical ISVZ. Immunostaining showed that there were many EOMES+ IPCs in the cortical ISVZ (Fig. 8A), but there were many more cortical interneurons, forming a migratory stream, in the cortical ISVZ at GW18 (Fig. 8B). These cortical interneurons expressed GABA, NR2F2 (COUP-TFII) and SP8 (Fig. 8B) suggesting that they are mainly CGE-derived cortical interneurons [29, 37]. Again, very few EGFR+ASCL1+OLIG2+ bMIPCs were in the ISVZ; they were mainly in the IFL (Fig. 8C, D). In the mouse cortex, chemokine CXCL12 is mainly expressed in PyN-IPCs [56, 57], whereas in the human cortex, our scRNA-Seq analysis showed that CXCL12 is mainly expressed in tRGs (Fig. S2C). CXCR4, a CXCL12 receptor, is expressed in nearly all human cortical interneurons . Because CXCL12 is mainly expressed by tRGs in the human cortical VZ, but not by oRGs and their progeny in the OSVZ, cortical ISVZ becomes the main corridor for tangentially migrating cortical interneurons. This may be a key reason for the distribution of bMIPCs mainly in the cortical IFL at GW18-GW23 (Fig. 8D).
bMIPCs Give Rise to OPCs, APCs and OBiN-IPCs in the Human Cortex
We next examined the lineage of bMIPCs in the human cortex at GW18 and GW23 in vivo. First, EGFR+ASCL1+ tRGs in the cortical VZ were identified (Fig. 9C, F), consistent with scRNA-Seq analysis. Based on proximity, tRGs then generated EGFR+ASCL1+OLIG2+ bMIPCs (Fig. 9B, E). There were only a few bMIPCs in the GW18 cortical IFL, whereas a 10-fold higher density of bMIPCs were observed in the GW23 cortical IFL (Fig. 7C–G, Fig. 9B, E), consistent with the evidence for increased gliogenesis in the GW23 cortex. We suggest that EGFR+ASCL1+OLIG2+ bMIPCs in the cortical IFL give rise to both ASCL1+OLIG2+ OPCs (Fig. 9A, D), and to EGFR+OLIG2+ APCs (Fig. 9A, D).
We next investigated whether cortical bMIPCs also give rise to OBiN-IPCs as suggested by the scRNA-Seq analysis (Fig. 1F, I). We first examined GSX2 expression in the GW23 cortex; the GSX2 homeodomain is at the top of the hierarchical gene regulatory network that governs OBiN development in the mouse cortex and dorsal lateral ganglionic eminence (LGE) [39, 58,59,60,61]. As expected, GSX2+ cells were observed in the GW23 cortex; these cells were also mainly located in the cortical IFL (Fig. S8A-D). We next performed GSX2, EGFR and OLIG2 triple immunostaining, and found some GSX2+ cells that expressed EGFR and OLIG2 (Fig. 10A, B). This provided strong evidence that bMIPCs also produced cortical GSX2+ cells. We also observed a subset of EGFR+GSX2+ cells that already downregulated OLIG2 expression (Fig. 10B). GSX2 promotes DLX gene expression in the cortex [58,59,60, 62]. Indeed, some EGFR+ASCL1+DLX2+ cells were identified in the cortical IFL (Fig. 10C), further suggesting that EGFR+ASCL1+OLIG2+ bMIPCs give rise to EGFR+ASCL1+GSX2+DLX2+ OBiN-IPCs.
Human Cortical oRGs Do not Generate OPCs
Two recent studies suggested that cortical oRGs were an additional source of OPCs in the developing human  and macaque monkey  cortex. The authors found some HOPX+ cells expressing EGFR in the cortical OSVZ, therefore suggesting that oRGs generated cortical Pre-OPCs and OPCs [49, 52]. In the human cerebral cortex, Huang et al., also found a small number of HOPX+ cells expressing PDGFRA, further proposed that oRGs generated Pre-OPCs . However, our results do not support these conclusions. We have identified that HOPX+EGFR+ cells were cortical tRGs and APCs, but not oRGs (Fig. 5, 6 and Fig. S4).
Next, we examined the expression of PDGFRA in the human cortex at GW18 and GW23. The vast majority of PDGFRA+ cells were OPCs in the cortex based on their co-expression with OLIG2 and SOX10 (Fig. S9A–F, Fig. S10). In addition, we found that about 16% of PDGFRA+ cells between the GW18 cortical ISVZ and SP expressed HOPX (Fig. S9E, G); they were cortical APCs. We also found that about 16% of PDGFRA+ cells in the GW18 cortical IFL and OSVZ expressed neither HOPX nor SOX10 (Fig. S9E, G); they were bMIPCs. These observations were consistent with the scRNA-Seq analysis results (Fig. 1H). Notably, in the GW23 human fetal brain, a large number of OPCs (Fig. S10) and APCs (Fig. 6) were already generated in the cortex, but oRGs were still making PyN-IPCs (see below, Fig. S12, S13), further suggesting that oRGs do not produce OPCs.
Thus, based on the expression patterns of EGFR, ASCL1, PDGFRA, OLIG2, HOPX and EOMES in the cortex, as well as the scRNA-Seq analysis results, we concluded that EGFR+ tRG-derived bMIPCs are the major source of cortical OPCs (and APCs) in the human brain. There are a small number of OPCs in the human cortex that are derived from the MGE, but we did not find any evidences supporting the claim that oRGs are an additional source of cortical OPCs.
PyN Genesis Continues at GW23
There were two germinal zones, VZ and OSVZ, in the GW18 and GW23 cortex. HOPX and EOMES double immunostaining revealed a large number of EOMES+ cells in both the cortical ISVZ and OSVZ at GW18; some of them expressed ASCL1 (Fig. S11A–D) [28, 37]. In the GW23 cortex, a large number of EOMES+ cells in the OSVZ and ISVZ were observed (Fig. S12A–C), further confirming that the GW23 cortical OSVZ is still in the peak of PyN genesis, whereas cortical VZ is making both PyNs and glia. A recent study reported that the number of EOMES+ cells was reduced in the GW19-GW20 cortex and proposed that the production of PyNs ceases in the human fetal cortex after GW20 . However, as indicated above, we observed a large number of EOMES+ cells in the GW23 cortex (Fig. S12); all of these EOMES+ cells expressed PAX6 (Fig. S13) and none of them expressed OLIG2 (Fig. 7G), suggesting that human cortical PyN genesis indeed occurs at GW23. Our observation was again supported by the scRNA-Seq data: among 3,355 tRG-derived EGFR-immunopanned cells obtained from GW21-GW26 human frontal cortex, there were 1,531 cells belonged to PyN-IPCs (46% of EGFR+ cells) (Fig. 1A) . Thus, during GW21-GW26, human cortical tRGs and oRGs are still making PyN-IPCs.