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A shared transcriptional code orchestrates temporal patterning of the central nervous system

['Andreas Sagner', 'The Francis Crick Institute', 'London', 'United Kingdom', 'Faculty Of Biology', 'Medicine', 'Health', 'The University Of Manchester', 'Manchester', 'Institut Für Biochemie']

Date: 2021-11

The molecular mechanisms that produce the full array of neuronal subtypes in the vertebrate nervous system are incompletely understood. Here, we provide evidence of a global temporal patterning program comprising sets of transcription factors that stratifies neurons based on the developmental time at which they are generated. This transcriptional code acts throughout the central nervous system, in parallel to spatial patterning, thereby increasing the diversity of neurons generated along the neuraxis. We further demonstrate that this temporal program operates in stem cell−derived neurons and is under the control of the TGFβ signaling pathway. Targeted perturbation of components of the temporal program, Nfia and Nfib, reveals their functional requirement for the generation of late-born neuronal subtypes. Together, our results provide evidence for the existence of a previously unappreciated global temporal transcriptional program of neuronal subtype identity and suggest that the integration of spatial and temporal patterning mechanisms diversifies and organizes neuronal subtypes in the vertebrate nervous system.

Funding: This work was funded by the Francis Crick Institute (to JB), which receives its core funding from Cancer Research UK, the UK Medical Research Council and Wellcome Trust (all under FC001051); and by the European Research Council under European Union (EU) Horizon 2020 research and innovation program grant 742138 (to JB). AS was supported by the Human Frontier Science Program (LTF000401/2014-L), the University of Manchester Presidential Fellowship and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 455354162. Cancer Research UK (CRUK) supported IZ (C157/A23459). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Sequencing data has been obtained from sources indicated in the Experimental Procedures section provided in the manuscript. Custom computer code is provided as Supplemental Experimental Procedures and available via the Github repository as stated in the manuscript. All other relevant data are within the paper and its Supporting Information files.

Here, we demonstrate by EdU birthdating that a set of TFs comprise a temporal TF code that identifies neurons based on their time point of birth. We find that the same sequence of TF expression applies throughout the brain, including the forebrain, midbrain, hindbrain, and retina, and for stem cell–derived in vitro generated neurons with defined dorsal–ventral and axial identities. We also document a temporal patterning code for progenitors throughout the nervous system and provide evidence that TGFβ signaling controls the pace of the temporal program. Finally, to characterize the genetic programs that control the temporal specification of neurons, we perturb the function of the TFs Nfia and Nfib and show that their activity is required for the generation of late neuronal subtypes. Taken together, our data reveal conserved temporal patterning of neurons and progenitors in large parts of the nervous system that is under the control of the TGFβ signaling pathway and suggest a close link between the developmental programs that control the switch from neuro- to gliogenesis and the specification of neuronal diversity.

TGFβ signaling has been implicated in the timing of developmental temporal switches in the nervous system [ 48 , 49 ]. The transition from MN to serotonergic neurons and from ocular MNs to red nucleus neurons is accelerated by TGFβ signaling [ 48 ]. TGFβ signaling also promotes the expression of the late progenitor marker Nfia in neurogenic neural stem cells [ 50 ]. Furthermore, Growth differentiation factor 11 (Gdf11), a ligand of the TGFβ family that signals via Activin receptors [ 51 , 52 ], has been implicated in the timing of MN subtype generation and onset of gliogenesis in the spinal cord [ 53 ]. TGFβ signaling is also important for controlling the timing of fate switches in the Drosophila nervous system [ 49 ], raising the possibility that it may serve as a general timer for the sequential generation of cellular subtypes.

Although the role of these TFs had not been conceptualized as part of a globally coordinated temporal code, some have been implicated in the specification of neuronal subtypes. Onecut TFs, for example, are required in early-born V1 and MNs for the specification of Renshaw cells and medial lateral motor column (LMCm) neurons, respectively [ 31 , 36 ]. Onecut TFs and Pou2f2 also control the distribution of neurons from multiple dorsal-–ventral domains [ 37 – 39 ]. Neurod2/6 control neuropeptide expression in inhibitory neurons in the dorsal horns of the spinal cord [ 40 ], and characterization of V2a neuron heterogeneity revealed that Zfhx3 and Neurod2/Nfib divide this neuronal class into a lateral and medial population [ 29 ]. Recent evidence further suggests that Zfhx3 and Nfib/Neurod2 partition neurons in the spinal cord into long-range projection and local interneurons [ 41 ]. Similar to the spinal cord, Onecut, Pou2f2, and Nfi-TFs label early and late-born neuronal subtypes in the retina and are required for their generation [ 42 – 44 ]. Zfhx3 and Nfi TFs also define distinct subpopulations of neurons generated by the midbrain floor plate, including dopaminergic neurons [ 45 ], and in the cerebral cortex [ 46 ]. Furthermore, Nfi TFs have also previously been identified as core components of a neurogenic transcriptional network in adult neural stem cells [ 47 ]. These observations raise the possibility that this temporal TF code is conserved in large parts of the central nervous system.

(A) Distinct cohorts of TFs are induced at different developmental stages in neurons from all dorsal–ventral domains in the spinal cord. (B) Scheme depicting EdU birthdating of neurons. (C) Dams were injected with EdU at e9.5, e10.5, e11.5, or e12.5 and embryos collected at e13.5. Colocalization between EdU and temporal TFs was then assessed in spinal cord cryosections. (D) Zfhx3-positive neurons are labeled by EdU, when EdU is administered at e9.5, but not at e12.5. (E) EdU labels Nfib-positive neurons when administered at e12.5, but not at e9.5. (F) Neurod2-postive neurons are labeled when EdU is administered at e11.5, but not when EdU is administered at e9.5. (G) Percentage of EdU-positive neurons labeled by Zfhx3, Nfib, and Neurod2 in the spinal cord. Underlying data are provided in S1 Data . Scale bars in D, E, and F = 200 μm. TF, transcription factor.

The vertebrate spinal cord is an experimentally tractable system to address the basis of neuronal diversity. In this region of the nervous system, neurons process sensory inputs from the periphery relaying the information to the brain or to motor circuits that control and coordinate muscle activity. The temporally stratified generation of some of these neuronal subtypes has been documented, including inhibitory and excitatory neurons located in the dorsal horn as well as ventral motor and interneurons [ 25 – 32 ]. Nevertheless, a comprehensive picture is lacking, and the genetic programs that orchestrate the temporal patterning of the spinal cord are largely unclear. To this end, we recently characterized the emergence of neuronal diversity in the embryonic spinal cord [ 33 ]. This revealed sets of transcription factors (TFs), expressed at characteristic time points during the neurogenic period of spinal cord development, which further partition all major neuronal subtypes ( Fig 1A ). In all domains, the earliest neurons express Onecut family TFs, intermediate neurons express Pou2f2 and Zfhx2-4, while at late stages, subsets of neurons start to express Nfia/b/x, Neurod2/6, and Tcf4 [ 33 – 35 ]. This suggested the existence of a previously unappreciated temporal dimension to neuronal subtype generation in the spinal cord.

Temporal mechanisms—the sequential production of different cell types at the same location—have been proposed to contribute to the generation of cell type diversity [ 10 , 11 ]. In the Drosophila nervous system, individual neuroblasts produce a characteristic temporal series of distinct neuronal subtypes [ 12 – 15 ]. Similar mechanisms have been documented in various regions of the vertebrate nervous system [ 11 , 16 , 17 ]. In the cortex, distinct subtypes of glutamatergic neurons are sequentially generated [ 18 – 20 ], in the hindbrain, first motor neurons (MNs) and later serotonergic neurons are generated from the same set of progenitors [ 21 ], while in the midbrain, the production of ocular MNs is followed by red nucleus neurons [ 22 ]. Moreover, progenitors throughout the nervous system typically produce neurons first and later generate glial cells such as astrocytes and oligodendrocytes [ 23 , 24 ]. However, whether temporal programs are a universal feature of neuronal subtype specification in the vertebrate nervous system and whether these are implemented by common or location specific gene expression programs is unclear.

In mammals, the function of the nervous system depends on hundreds of molecularly and functionally distinct cell types [ 1 ]. This diversity requires the generation of different neuronal subtypes at the right place, time, and quantity during development. In turn, this guides the wiring of functioning neural circuits. The molecular mechanisms that direct the specification of distinct neuronal classes at characteristic positions, by subdividing the developing nervous system into topographical territories, have received considerable attention [ 2 , 3 ]. However, spatial patterning programs are not sufficient to account for the diversity of neuronal subtypes observed in the nervous system. Even within the same region of the nervous system, most neuronal classes can be further partitioned into distinct subtypes based on molecular and functional properties [ 4 – 9 ].

Results

EdU birthdating reveals a temporal TF code in spinal cord neurons We previously identified sets of TFs that are expressed in multiple subsets of neurons in the spinal cord. As the onset of expression of these TFs occurred at different times during development, we speculated that they subdivide neurons in the spinal cord based on their time point of birth [33] (Fig 1A). We and others have demonstrated before that Onecut TFs are expressed in early-born neurons and that their expression is rapidly extinguished as neurons mature [27,31,33,36,37]. We therefore focused on the TFs Zfhx3, Nfib, and Neurod2, which start to be expressed in neurons at intermediate or late stages during the neurogenic period, respectively, and analyzed the birthdate of neurons expressing these TFs by EdU incorporation (Fig 1B). Pregnant dams were injected with EdU at embryonic day (e)9.5, e10.5, e11.5, or e12.5 (Fig 1C). Embryos were collected at e13.5 and forelimb-level spinal cord cryosections assayed for colocalization between EdU and Zfhx3, Nfib, and Neurod2 in neurons (Figs 1D–1F and S1). Consistent with the hypothesis of a temporal TF code, a high proportion of EdU-labeled neurons expressed Zfhx3, when EdU was administered at e9.5 and e10.5, while there was little, if any, colocalization between EdU and Zfhx3 when EdU was given at later time points (Figs 1G and S1A). By contrast, few EdU-positive neurons expressed Nfib when EdU was administered before e11.5, but more than 80% of EdU+ neurons were positive for Nfib when it was given at e12.5 (Figs 1G and S1B). Neurod2 followed a similar trend to Nfib until e11.5 (Figs 1G and SS1C), consistent with the high degree of coexpression between these genes [33]. However, the proportion of Neurod2-positive neurons decreased when EdU was given at e12.5 (Figs 1G and S1C). This may be due to the relatively late onset of Neurod2 expression after neuronal differentiation. Furthermore, Neurod2 is not expressed in late-born dorsal excitatory neurons [40], which are generated at high frequency during late neurogenic stages in the spinal cord [54]. The mutually exclusive birthdates of Zfhx3 and Nfib/Neurod2-positive neurons indicate that these TFs label largely nonoverlapping subsets of neurons. To test this prediction directly, we stained e13.5 spinal cord sections for either Zfhx3 and Nfib or Zfhx3 and Neurod2 (S2A and S2B Fig). Although each of these markers labeled a large number of neurons, the expression of Zfhx3 and Nfib or Zfhx3 and Neurod2 was mutually exclusive. These results are consistent with a model in which Zfhx3 is specifically expressed and maintained in neurons born before e11.5 but not in later-born neurons, which instead express Neurod2/6 and Nfi-family TFs. Together, the data argue against sequential expression of these TFs during neuronal maturation because, in such a model, TFs with an early onset of expression would be specific for early maturation stages and would thus, contrary to our observations, expected to be labeled by EdU given at late developmental time points. Consistent with this interpretation, a recent study found similar birthdates for Zfhx3- and Neurod2-positive neurons in the perinatal spinal cord [41]. We therefore conclude that these TFs comprise a temporal code and label distinct subsets of neurons based on their time point of birth in the spinal cord.

Conservation of the temporal TF code in the retina Similar to the spinal cord, Pou2f2, Neurod2/6, and TFs of the Onecut and Nfi families are required for the generation of early and late-born neurons in the retina [42–44,55,56]. We therefore speculated that the temporal TF code is preserved in the retina. To test this hypothesis, we analyzed a published single-cell RNA sequencing (scRNAseq) time course of mouse retina development [43] (S3 Fig). Performing dimensionality reduction by Uniform Manifold Approximation and Projection (UMAP) from prenatal and perinatal stages (e14, e16, e18, P0) resulted in clear trajectories from retinal progenitors to horizontal cells, amacrine cells, retinal ganglion cells, and cone and rod photoreceptors (S3A and S3B Fig). Examining Onecut2, Pou2f2, Zfhx3, and Nfib revealed different expression of these genes along these differentiation trajectories (S3C Fig). As expected, Onecut2 was strongly enriched in horizontal cells, an early-born cell type in the retina, although some expression was also observed in retinal ganglion cells, amacrine cells, and cones. Nfib expression was largely restricted to late progenitors and rods (S3C Fig). By contrast, Pou2f2 and Zfhx3 were enriched in amacrine and retinal ganglion cells. Furthermore, both genes were expressed in subsets of retinal progenitors. To further characterize the expression of Onecut2, Pou2f2, Zfhx3, and Nfib genes in retinal neurons, we plotted their expression levels in the individual classes of neurons stratified by developmental stage (S3D Fig). This analysis revealed a clear link between the expression of these TFs and developmental stage. Onecut2 was enriched in amacrine cells, retinal ganglion cells, and cones at e14 (S3D Fig). Zfhx3 was absent at this stage but was enriched in amacrine and retinal ganglion cells at e18 and P0 (S3D Fig). Pou2f2 and Zfhx3 were not detected in cone and rod photoreceptors at any stage, suggesting that not all aspects of this temporal program apply to all neuronal subtypes (S3C and S3D Fig). These data support the hypothesis that the temporal TFs are expressed in different retinal cell types born at distinct time points and raise the possibility that the expression of these genes further subdivide distinct classes of retinal neurons based on their birthdates.

EdU birthdating confirms sequential generation of Zfhx3 and Nfib-positive neurons We next sought to confirm experimentally that Zfhx3 and Nfib-positive neurons are generated at distinct time points in the midbrain and hindbrain using EdU birthdating. To this end, we injected pregnant dams at e10.5 or e12.5 with EdU and assayed the proportion of EdU-positive neurons expressing Zfhx3 or Nfib at e13.5 in the midbrain or hindbrain. As for the spinal cord (Fig 1), Zfhx3-positive neurons in both tissues were labeled by EdU at e10.5 but not at e12.5 (Figs 2I and 2J and S7A–S7D). Moreover, in the hindbrain, a high proportion of EdU-positive neurons expressed Nfib when EdU was given at e12.5 (Figs 2I, S7E, and S7F). By contrast, the proportion of Nfib-positive neurons in the midbrain did not increase markedly (Fig 2J). This was because most neurons labeled by EdU at e12.5 resided in the dorsal part of the midbrain where Nfib is not expressed in neurons at this stage (S7G and S7H Fig). These neurons did not express Zfhx3 (S7D Fig), suggesting that the lack of Nfib-positive neurons in this area was not due to prolonged generation of Zfhx3 neurons. Restricting the analysis to the intermediate and ventral midbrain resulted in the expected increase of EdU-positive neurons expressing Nfib (Fig 2K). Taken together, these observations provide further experimental evidence that Zfhx3 and Nfib label sequentially generated neurons in large regions of the midbrain and hindbrain.

Most cortical excitatory neurons express late temporal TFs Molecularly and functionally distinct excitatory neurons in the mammalian cortex are arranged in distinct layers based on their time point of generation [18,20,61]. As this is one of the best-established models for temporal neuronal subtype generation, we wondered how the temporal patterning program we describe relates to the temporal patterning of the cortical layers. To this end, we analyzed scRNAseq of cortical excitatory neurons from e10 to e14 [9] (S8A and S8B Fig). Consistent with the extended period of neurogenesis in the cortex compared to other regions of the nervous system, we found that most cortical excitatory neurons express TFs characteristic of the late temporal identity, including Nfia, Nfib, Neurod2, Neurod6, and Tcf4 (S8B and S8C Fig). However, a small cluster (cluster 7; S8D Fig) of excitatory neurons lacked expression of these markers. Instead, these neurons expressed Pbx3, Meis1, Meis2, Tshz2, Barhl2, and Zfhx3 (S8C and S8E Fig). Consistent with the timing of generation of Zfhx3 neurons in the rest of the nervous system, the cortical Zfhx3-positive neurons appear to be generated earlier during development than Nfia/b Neurod2/6-positive neurons (S8F Fig). Taken together, these data suggest that the temporal patterning program we describe applies to cortical neurons and that most excitatory cortical neurons express TFs characteristic of a late temporal identity. These findings match recent observations by Moreau and colleagues, demonstrating a subdivision of early cortical excitatory neurons into Pbx3/Zfhx3 and Nfi/Neurod2/Neurod6-positive subtypes.

Conservation of temporal TF expression at later developmental stages Zfhx3 and Neurod2/Nfib also partition spinal cord neurons in the perinatal and adult spinal cord [41]. We therefore investigated if the subdivision into Zfhx3 and Nfib-positive neurons is maintained in other regions of the nervous system. To address this, we analyzed scRNAseq data from late embryonic (e16 to e18) forebrain and midbrain [9]. Characterization of the gene expression patterns of intermediate (Pou2f2, Zfhx3, Zfhx4) and late (Nfia, Nfib, Neurod2, Neurod6, Tcf4) TFs indicated that these TFs continue to be expressed in largely nonoverlapping populations of neurons (S9A, S9B, S9F, and S9G Fig). Nfia/Nfib-positive cells expressed the neuronal markers Elavl3 and Tubb3 but did not express the glial markers S100b and Slc1a3 (also known as Glast), confirming their neuronal identity (S9C and S9H Fig). Moreover, hierarchical clustering of the gene expression patterns and correlation rank plots for Zfhx3 and Nfib revealed similar coexpression patterns as at earlier developmental stages (S9D, S9E, S9I, and S9J Fig). Collectively, these results suggest that the anticorrelated expression of intermediate and late TFs are broadly retained until late embryonic stages in the forebrain and midbrain.

Temporal TF expression correlates with the acquisition of distinct neuronal identities in the ventral midbrain We next investigated whether the temporal TF code is responsible for the establishment of neuronal populations with specific functions. To test this, we first focused on the sequential generation of oculomotor and red nucleus neurons in the ventral midbrain. The switch from oculomotor to red nucleus neurons occurs between e10.5 and e11.5 [22,48]. We therefore speculated that this switch may coincide with the switch from early (Onecut2) to intermediate (Zfhx3) TF expression. Consistent with this hypothesis, expression of the red nucleus neuron marker Pou4f1 is mutually exclusive with the expression of Onecut2 in the ventral midbrain (S10A Fig), while most Pou4f1 neurons express Zfhx3 (S10B Fig). These data support the hypothesis that the temporal TFs are involved in the sequential generation of neurons with distinct functions. Of note, Pou4f1 and Onecut2 are coexpressed in subsets of neurons in the dorsal midbrain, suggesting that these TFs do not always mutually cross-repress each other. Dopaminergic neurons are a neuronal population of medical interest because their degeneration causes Parkinson disease. During development, these neurons are born from the midbrain floor plate and can be discriminated based on the expression of the TFs Lmx1a, Lmx1b, and Pitx3 as well as the enzymes tyrosine hydroxylase (TH) and the dopamine transporter Slc6a3 (also known as Dat). Strikingly, previous characterization of neurons generated from the midbrain floor plate suggested that these neurons can be broadly subdivided into Nfia/b and Zfhx3 expressing subsets. The Zfhx3-positive population expresses dopaminergic neuron markers such as Slc6a3 and high levels of TH [45]. By contrast, the Nfi-positive population lacked the molecular machinery for the synthesis of dopamine and expressed markers characteristic for excitatory neurons such as Slc17a6 [45]. These findings, in combination with our observation that Zfhx3 and Nfi TFs define temporal neuronal populations in the midbrain, suggest that midbrain dopaminergic neurons may constitute a temporal neuronal subtype born from the midbrain floor plate. We therefore examined if Zfhx3-positive neurons are generated before Nfia/b-positive neurons from the midbrain floor plate. Assays at e11.5 revealed widespread expression of Zfhx3 in floor plate–derived Lmx1b-positive neurons (Fig 3A). At this stage, Nfib expression just commenced in Sox2-positive neural progenitors (Fig 3B). In contrast, at e13.5, numerous Nfib-positive neurons expressing Lmx1b were found in the vicinity of the midbrain floor plate (Fig 3C and 3D), likely corresponding to the N-Datlow population [45]. Zfhx3-positive neurons at this stage had migrated to a more lateral position (Fig 3C). These neurons coexpressed the Zfhx TFs, Zfhx3, and Zfhx4 and also increased levels of TH (Fig 3E and 3F), suggesting that these populations correspond to the AT-Dathigh, T-Dathigh, and VT-Dathigh neurons described by Tiklová and colleagues. These conclusions are also consistent with previous birthdating experiments that concluded that the majority of TH-positive dopaminergic neurons are born before and around e12.5 [62,63]. Taken together, these data suggest that the sequence of temporal TF expression is preserved in neurons derived from the midbrain floor plate, that the expression of different temporal TFs correlates with the acquisition of different neuronal subtype identities in these neurons, and that dopaminergic neurons correspond to the Zfhx3-positive temporal neuronal population. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Midbrain dopaminergic neurons are a temporal population of neurons derived from the midbrain floor plate (see also Midbrain dopaminergic neurons are a temporal population of neurons derived from the midbrain floor plate (see also S10 Fig ). (A) Coexpression of Zfhx3 and Lmx1b in neurons derived from the midbrain floor plate at e11.5. (B) Nfib is restricted to Sox2-positive neural progenitors in the ventral midbrain at e11.5. (C) Mutually exclusive expression of Zfhx3 and Nfib in Lmx1b-positive neurons at e13.5. (D) Nfib labels Lmx1b-positive neurons directly adjacent to Sox2-positive progenitors at e13.5. (E) Colocalization between Zfhx3 and Zfhx4 in Lmx1b-positive neurons at e13.5. (F) Zfhx4 labels Lmx1b-positive neurons expressing high levels of TH at e13.5. Scale bars = 100 μm. https://doi.org/10.1371/journal.pbio.3001450.g003

The temporal TF code applies to in vitro generated midbrain, hindbrain, and spinal cord neurons We next sought to investigate whether the temporal code was preserved in vitro during the directed differentiation of embryonic stem (ES) cells to neurons with specific axial and dorsal–ventral identities [64–66]. We reasoned that in vitro putative global signaling cues, originating from distant signaling centers, should be absent. We examined if the same sequence of temporal TF factor expression can be observed in stem cell–derived neurons with midbrain and hindbrain and spinal cord identities. ES cells were differentiated to appropriate identities using established protocols [64] (Fig 4A), as confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) for Foxg1, Otx2, Hoxa4, Hoxb9, and Hoxc8 (S11A Fig). As expected, cells differentiated to midbrain identity induced Otx2, but not the forebrain marker Foxg1 or the hindbrain marker Hoxa4, which was induced in hindbrain conditions. By contrast, the posterior Hox genes Hoxb9 and Hoxc8 were only induced when cells were differentiated to a spinal cord identity. We next assayed the expression of the temporal TFs Onecut2, Zfhx3, Nfia, and Neurod2 under these differentiation conditions by flow cytometry from days 6 to 13 (Figs 4B and S11B). The overall expression dynamics of these markers observed in vivo were preserved under the different conditions. Most neurons expressed Onecut2 at days 6 and 7, while the proportion of Zfhx3-positive neurons increased between days 7 and 9, and Nfia and Neurod2-positive neurons were typically not detected before day 11. These results closely resemble our previous observations of the temporal patterning of neurons in the developing nervous system. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Conservation of the temporal TF code in stem cell–derived neurons with different axial and dorsal–ventral identities (see also Conservation of the temporal TF code in stem cell–derived neurons with different axial and dorsal–ventral identities (see also S11 Fig ). (A) Schematics of the differentiation protocols for the generation of progenitors and neurons with different axial and dorsal–ventral identities. (B) Flow cytometry analysis of temporal TF expression indicates that neurons with different axial and dorsal–ventral identities display the same temporal progression in vitro as in vivo. (C) Flow cytometry analysis of Nkx2.2 and Pax3 expression in neural progenitors in dorsal and ventral spinal cord differentiations. (D) Percentage of neural progenitors expressing Pax3 and Nkx2.2 in ventral and dorsal spinal cord differentiations between days 7–11. Underlying data are provided in S3 Data. FGF, fibroblast growth factor; RA, retinoic acid; SAG, Shh pathway agonist; TF, transcription factor. https://doi.org/10.1371/journal.pbio.3001450.g004 We next investigated if the progression of the temporal TF code is preserved in neurons with different dorsal–ventral identities. We have previously demonstrated that exposure of spinal cord progenitors to appropriate concentrations of the Sonic Hedgehog (Shh) pathway agonist (SAG) promotes the generation of progenitors and neurons with different dorsal–ventral identities [66]. We therefore focused on the spinal cord condition and either ventralized cells by exposing them from day 3 to day 9 to 500 nM SAG or dorsalised them in the absence of SAG. Samples for flow cytometry were collected at days 7, 9, and 11 (Fig 4C). Consistent with our previous observations [66], in the absence of Shh pathway activation, most progenitors expressed the dorsal progenitor marker Pax3, while prolonged high-level Shh pathway activation leads to the majority of progenitors acquiring an Nkx2.2-positive ventral p3 identity (Fig 4C and 4D). Consistent with this, most neurons generated in the absence of Shh pathway activation expressed the intermediate dorsal marker Lbx1, while Shh pathway activation led to the generation of Sim1-positive V3 neurons (S11C Fig). We therefore refer to these conditions as dorsal and ventral, respectively. Assaying the expression of the temporal TFs in neurons in the ventral differentiation condition revealed similar expression dynamics for these markers as previously observed under dorsal spinal cord conditions, although notably a higher proportion of neurons expressed Nfia and Neurod2 at later stages of the differentiations (Fig 4B). Based on these oberservations, we conclude that the temporal TF code is preserved in in vitro generated neurons with different axial (midbrain, hindbrain, and spinal cord) and dorsal–ventral identities. Furthermore, the time scale over which the temporal patterning unfolds is similar in vivo and in vitro, corresponding in both cases to approximately 4 to 5 days (in vivo approximately e9.5 to e13.5; in vitro approximately day 7 to day 11). These results argue against a model in which global signaling cues orchestrate the temporal patterning program. We note, however, that this analysis also uncovered reproducible differences in the proportions of neurons expressing the respective markers between the different axial identities. Cells differentiated under hindbrain conditions induced late temporal TFs at a faster pace, while cells under midbrain conditions seemed to progress slowest to a later temporal identity. These differences may be indicative of cell-intrinsic programs that allow progenitors and/or neurons to progress through the temporal TF code at a speed characteristic for their axial identity.

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