Setting up an experimental strategy to identify NeuroG2 early response genes.
To identify overall NEUROG2 early response genes, we combined a gain of function (GOF) strategy using in ovo
electroporation in the chick neural tube, allowing for the targeting of a thousand spinal neural cells per embryo at a precise time, with a global measure of transcript levels using chicken microarrays. We first determined which time window would allow identification of early response genes in our experimental system. Based on our previous experiments, showing that DLL1
is upregulated and PAX6
is downregulated 6 h following NEUROG2 electroporation in the neural tube (3
), we performed a time course of PAX6
extinction between 4 h and 6 h following NEUROG2 electroporation. PAX6
transcripts start to be notably diminished 5 h after electroporation and are strongly repressed 6 h after electroporation (see Fig. S1 in the supplemental material). We then checked the differentiation status of NEUROG2 electroporated cells 6 h after electroporation: NEUROG2 cells have activated markers of young neurons, such as NEUROD4
), but we do not detect the neuronal class III β-tubulin protein (TUBB3 or Tuj1) whose expression starts in differentiating neurons (see Fig. S1). This suggests that NEUROG2 cells are engaged in the neuronal program but are not yet differentiated. Therefore, a 6-h time window appears to be quite appropriate to identify NEUROG2 early response genes.
To be able to distinguish between targets for NEUROG2 transcriptional activity (i.e., E-box binding) versus those that are independent of DNA binding, we generated a chick version of the NEUROG2 mutant form NEUROG2AQ. This mutant form cannot bind DNA and is unable to activate the neurogenic cascade in the cortex (17
). However, it retains the ability to interact with protein partners such as CBP/p300. We verified that as in the cortex, NEUROG2AQ is unable to promote neurogenesis in the neural tube (see Fig. S1 in the supplemental material). The use of this NEUROG2 mutant form thus allows selecting specifically NEUROG2 targets involved with transcriptional activity.
Identification of NEUROG2 early response genes in the developing spinal cord.
To compare the transcriptome of neural precursors overexpressing (i) only GFP, (ii) NEUROG2, or (iii) NEUROG2AQ, we transfected the neural tube of E1.5 chick embryos by electroporation with expression vectors coding for GFP, NEUROG2-GFP, or NEUROG2AQ-GFP. GFP-positive cells were collected 6 h later using FACS and processed for RNA probe preparation and hybridization on Affymetrix microarrays (; see also Materials and Methods). Expression profiling of neural cells electroporated with control, NEUROG2, or NEUROG2AQ identified 1,038 probe sets corresponding to 942 unique genes, revealing significantly modified expression with a 0.01 false-discovery rate (FDR) (false-positive rate accepted, ≤1%) (; see also Table S1 in the supplemental material). As expected for a transcription factor acting mainly as a transcriptional activator, 670 probe sets were upregulated by NEUROG2. However, we also detected 368 probe sets downregulated by NEUROG2. This suggests that an important fraction of NEUROG2 activity also goes to the repression of transcriptional targets. Of these 1,038 probe sets, only 75 have their expression similarly modified by NEUROG2 and NEUROG2AQ, indicating that in the neural tube, NEUROG2 function requires mainly DNA binding.
Fig 1 Identification of NEUROG2 early target genes in the neural tube. (A) Schematic representation of the step-by-step protocol. Neural tubes were electroporated at stage E1.5 with NEUROG2-GFP, NEUROG2AQ-GFP, or CTL-GFP and dissected 6 h later. GFP-positive (more ...)
As expected, many of the already-known NEUROG2 transcriptional targets have their expression consistently modified in our screen. For example, PAX6
(see introduction), and HES6
), known to be regulated by NEUROG2 in the neural tube, are found significantly upregulated (DLL1
, and HES6
) or downregulated (PAX6
) in our experimental context ( and ). Moreover, we compared our data with those of screens already performed to identify NEUROG2 targets in mouse embryonic cortex (18
) or in the Xenopus
). We retrieved by our approach 25 out of the 183 genes that were identified in the cortex and 9 out of the 59 that were identified in the Xenopus
neurectoderm. Among them are the NHLH1
, or CXCR4
genes that are known to be expressed in the neural tube. Furthermore, we analyzed randomly selected putative targets, and all of them are expressed in the neural tube and have their expression modified 6 h after NEUROG2 electroporation ( and not shown). As our experimental set up was efficient in retrieving classical NEUROG2 targets, it is likely that a large proportion of the novel genes identified are bona fide spinal progenitor NEUROG2 targets.
Examples of some NEUROG2 targets according to gene ontology analysis
NEUROG2 rapidly modulates the expression of major players of neural cell proliferation.
To extract from our data set all the genes related to cell cycle control, we performed a global analysis of the main biological processes modified after NEUROG2 expression using the Genecodis software (9
) ( and ). Among the 942 NEUROG2 targets, 669 annotated genes were processed further. A total of 624 were assigned one or more GO terms: biological process (BP; ) or KEGGS pathways (Kyoto Encyclopedia of Genes and Genomes; KP; ). A large set of targets is involved in biological processes such as cell differentiation (P
= 3.87e−08, 34 genes) or nervous system development (P
= 3.08e−07, 30 genes, e.g., PAX6
, or LHX1
). As expected, numerous members of the NOTCH signaling pathway are significantly regulated (P
= 4.30e−06, 10 genes, e.g., JAG1
, or HES1
). Several genes related to neuronal function are also enriched in the NEUROG2 transcriptome, such as genes associated with axon guidance (SEMA6B
), synaptic transmission (KCNMB4
), chemotaxis (CCL19
), or glutamate receptor activity (GRIN3A
). Thus, this global analysis allows identifying gene groups in coherence with known NEUROG2 functions.
Fig 2 Ontological classification of genes modified by NEUROG2. Graphs showing the number of genes per singular ontological annotation determined by GeneCodis 2.0. (A) Biological processes; (B) KEGG pathways (B) (Kyoto Encyclopedia of Genes and Genomes). The (more ...)
Importantly, NEUROG2 also regulates a high proportion of genes involved in the cell cycle, allowing us to examine the molecular mechanisms by which NEUROG2 induces cell cycle arrest (, Cell cycle). Indeed, cell cycle is one of the most enriched biological processes in KEGGS pathways (BP, P = 1.63e−09, 38 genes; KP, P = 4.06e−09, 19 genes). This subset of NEUROG2 targets includes transcription factors influencing cell proliferation, such as MYCN or FOXM1, as well as genes involved in the core cell cycle machinery, such as WEE1, AURKA, and BUB1, or direct cell cycle regulators, such as CCND1 or CCNE1/2. All these putative targets involved in promoting proliferation are downregulated by NEUROG2. Thus, this global analysis indicates that one of the first functions of NEUROG2 is to repress the expression of positive players of cell proliferation.
Core regulators of the G1 and S phases of the cell cycle are rapidly downregulated by NEUROG2.
Many cell cycle-related genes are rapidly downregulated after NEUROG2 overexpression, and we analyzed in detail the expression of core cell cycle regulators present in the array (). First, we found that p27kip1
expression is not modified by NEUROG2 overexpression (). We confirmed this result by immunohistochemistry showing that p27kip1
protein is not activated 6 h postelectroporation (), whereas, as previously reported, it clearly accumulated in NEUROG2-transfected cells 48 h hours postelectroporation (see Fig. S2 in the supplemental material) (20
). We made similar observations for other inhibitors belonging to the CIP/Kip or the INK families (). Thus, upregulation of CKIs is not an early response to NEUROG2.
Fig 3 G1 and S cyclins are rapidly downregulated by NEUROG2. (A) Schematic drawing of the relationship between core regulators of the different phases of the cell cycle. Specific combinations of CDK/cyclin heterodimers allow progression in the different phases (more ...)
Instead, we found that CCND1, CCNE1, CCNE2, and CCNA2, acting at the G1 and S phases of the cell cycle, are downregulated within 6 h (). The expression of their associated CDKs is not modified under these conditions (). Moreover, the expression of CCNB2 involved in the G2/M transition is not changed (). From the three D-type cyclins (CCND) involved in G1 progression, only CCND1 is downregulated, with CCND2 and CCND3 being unaffected (). Analysis of our data set therefore suggests that among direct cell cycle regulators, only a subtype of cyclins has its expression modified by NEUROG2, but not by NEUROG2AQ, indicating that this regulation is DNA binding dependent. We validated these data obtained with our transcriptomic approach by analyzing in more detail the regulation of two of these cyclins: CCND1 and CCNE2. Using in situ hybridization, we confirmed that CCND1 and CCNE2 are rapidly downregulated following NEUROG2 gain of function (). To ascertain the specificity of NEUROG2 action on these cell cycle regulators, we analyzed their expression in loss-of-function conditions, using two different siRNAs to target endogenous NEUROG2 (see Materials and Methods). We started verifying that our siRNAs extinguish specifically NEUROG2 transcript and lead to a reduction of neuronal differentiation (see Fig. S3 in the supplemental material). We then tested the expression of CCND1 and CCNE2 24 h after siRNA electroporation. As seen on , the acute reduction of endogenous NEUROG2 provokes an accumulation of CCND1 and CCNE2 transcripts without affecting CCND2 expression as expected from our transcriptomic data. This upregulation doesn't occur when the siRNA is coelectroporated with a vector expressing the mouse NeuroG2, confirming the specificity of the siRNAs (). NEUROG2 is thus necessary for CCND1 and CCNE2 downregulation in spinal progenitors. Hence, among the core regulators controlling the cell cycle, only a subset of cyclins governing the G1 and S phases are rapidly and specifically downregulated in response to NEUROG2.
Although NEUROG2 has not been shown to act as a transcriptional repressor, we nevertheless tested whether this repression could be direct. We used two NEUROG2 modified forms: a NEUROG2VP16 form acting as a constitutive activator and a NEUROG2EnR form acting as a constitutive repressor in the cerebral cortex (C. Schuurmans, personal communication). We assumed that if NEUROG2 acts as a transcriptional activator, NEUROG2VP16 would mimic the effect of NEUROG2, NEUROG2EnR having no or the opposite effect. We validated these constructs by looking at PAX6 expression that we know to be indirectly repressed by NEUROG2 in the neural tube (M. Lacomme, unpublished observation). As expected, PAX6 expression is strongly repressed by NEUROG2VP16 but not significantly affected by NEUROG2EnR in the neural tube (see Fig. S4 in the supplemental material). We tested the expression of CCND1 and CCNE2 in the same experimental conditions. We found that CCND1 is strongly repressed by NEUROG2VP16, NEUROG2EnR having little or no effect (), indicating that CCND1 is repressed indirectly by NEUROG2. However, the regulation of CCNE2 appears to be more complex, as we observed a reduction of CCNE2 transcripts with both NEUROG2VP16 and NEUROG2EnR (). Hence, these experiments indicate that NEUROG2 represses CCND1 and CCNE2 in an indirect manner but open the possibility that CCNE2 is also repressed by a direct mechanism (see Discussion).
NEUROG2 rapidly impedes cell cycle reentry of spinal progenitors.
The rapid downregulation of G1/S cyclins suggests that within 6 h, neural cells overexpressing NEUROG2 no longer enter S phase. To test this hypothesis, we measured the percentage of cells in S phase in transfected populations overexpressing NEUROG2, NEUROG2AQ, or CTL 6 h after electroporation, by applying a 30-min BrdU pulse just before collecting the embryos (A). As expected from the array data, NEUROG2AQ does not modify the BrdU incorporation rate (38.2% ± 8% of the NEUROG2AQ cells incorporated BrdU compared with 41.8% ± 14% in the CTL condition). In contrast, we observed a dramatic fall of this percentage upon NEUROG2 overexpression (2.1% ± 2%) (), indicating that cells overexpressing NEUROG2 are retained in some cell cycle phases. If NEUROG2 controls the G1/S transition, neural cells overexpressing NEUROG2 should be kept in G1. We thus performed FACS analysis to determine the distribution of transfected cells in the different phases of the cell cycle 6 h after NEUROG2 overexpression. We observed that NEUROG2-positive cells clearly start to accumulate at the G1/G0 phase of the cell cycle () (74.6% ± 6.4% of the NEUROG2-positive cells versus 60.6% ± 2.9% in CTL cells; P < 0.05). As these cells do not yet express p27kip1 or the β tubulin protein (see Fig. S1 in the supplemental material), our results indicate that NEUROG2 upregulation causes a rapid arrest in G1 preceding the upregulation of CKIs.
Fig 4 NEUROG2 blocks S phase reentry via the repression of CCND1 and CCNE. (Aa to Ac and Ca to Cd) Close-ups on transversal sections of embryos electroporated with the mentioned constructs and incubated 30 min with BrdU just before harvesting, 6 h following (more ...) Shutting down CCND1 and CCNE is needed to hold cells in G1 downstream of NEUROG2.
If the cell accumulation in G1 phase that we observed following NEUROG2 electroporation is directly linked to the repression of G1/S cyclin expression, maintaining one of these cyclins in otherwise NEUROG2-expressing cells should restore cell cycle progression. To test this, we electroporated NEUROG2 alone or in conjunction with CCND1 or CCNE and 6 h after performed a 30-min BrdU pulse just before collecting the embryos (). Cotransfection of either CCND1 or CCNE with NEUROG2 fully rescues the lack of BrdU incorporation observed with NEUROG2 alone, to a level similar to that observed with CCND1 or CCNE alone () (63.8% ± 2% of GFP+ BrdU+ cells with NEUROG2 and CCND1 and 87% ± 8% with NEUROG2 and CCNE versus 2.1% ± 2% with NEUROG2 alone). In addition, FACS analysis shows that when coelectroporated with NEUROG2, CCND1 prevents cell accumulation in the G1/G0 phases observed with NEUROG2 alone () (74.6% ± 6.4% of the NEUROG2+ cells versus 57% ± 6.5% in NEUROG2+ CCND1+ cells; P < 0.05). This finding links the early NEUROG2-driven G1 accumulation to the repression of G1 and S cyclins.
NEUROG2 coordinates cell cycle exit with neuronal differentiation.
The results presented so far show that NEUROG2 represses specifically a subset of cell cycle regulators that drive cell cycle arrest in G1
. Given that NEUROG2 also represses patterning genes and initiates differentiation genes, we wondered if these different events were dependent on each other or could be controlled independently by NEUROG2. It has been proposed that in the developing cortex, CCND1 downregulation is sufficient to trigger neuronal differentiation, suggesting that neuronal differentiation could be a consequence of cell cycle exit. We checked to see whether in our experimental conditions CCND1 downregulation was sufficient to induce precocious neuronal differentiation. The downregulation of CCND1, using a short hairpin RNA (shRNA) construct (RNAiD1) (34
), leads to a significant reduction of BrdU incorporation as seen 24 h postelectroporation (see Fig. S5 in the supplemental material) (27% ± 4.6% of RNAiD1+
cells and 37.5% ± 7.4% of GFP+
cells in a control). However, it is not accompanied by an increase of TUJ1+
cells, indicating that reducing the CCND1 level is not sufficient to promote neuronal differentiation (see Fig. S5) (5.9% ± 1.5% of RNAiD1+
cells and 6.1% ± 1.3% of GFP+
cells in a control).
We then examined if, on the contrary, the maintenance of one G1/S cyclin would impede the capacity of NEUROG2 to push cells out of the progenitor state and trigger neuronal differentiation. We started analyzing the expression pattern of PAX6 6 h following NEUROG2 and CCND1 electroporation (). PAX6 transcripts expression is repressed by NEUROG2 even in the presence of CCND1. We also observed that DLL1 is upregulated in NEUROG2 and CCND1 transfected cells (). These data show that NEUROG2 cells, despite maintaining CCND1 expression, are still able to extinguish progenitor genes and to commit to the neuronal fate. We asked if these NEUROG2 and CCND1 transfected cells were able to initiate neuronal differentiation by analyzing the differentiation status of cells overexpressing NEUROG2, CCND1, CCNE, or combinations of them 24 h postelectroporation. As already described, electroporation of NEUROG2 alone leads to precocious neuronal differentiation () (64.3% ± 6.7% of NEUROG2+ cells express TUJ1 instead of 6.2% ± 1.9% in a control). Interestingly, we also detect many GFP+ TUJ1+ cells in the ventricular zone of the neural tube electroporated either with NEUROG2 and CCND1 or NEUROG2 and CCNE () (60.4% ± 3.7% or 31.7% ± 7.5%, respectively), whereas neither of these cyclins alone is able to induce an early expression of TUJ1 (1.2% ± 0.9% of GFP+ TUJ1+ cells for CCND1 and 1% ± 0.5% for CCNE). This indicates that NEUROG2 can promote expression of differentiation markers independently of G1/S cyclin repression.
Fig 5 NEUROG2 can control differentiation independently of cell cycle exit. (A) Detection of PAX6 (a′, c′, and e′) and DLL1 (b′, d′, and f′) transcripts on transversal sections by in situ hybridization 6 h following (more ...)
This could be due to the fact that cells coelectroporated with NEUROG2 and CCND1 or CCNE manage to escape the cell cycle to differentiate. To check if these cells are still cycling, we performed a 30-min BrdU pulse 24 h postelectroporation. Cells misexpressing NEUROG2 alone do not incorporate BrdU at 24 h as expected from data obtained at 6 h () (0.93% ± 2.3% of NEUROG2+ BrdU+ cells). However, cells transfected with NEUROG2 and CCND1 or NEUROG2 and CCNE are still proliferating 24 h postelectroporation () (37.5% ± 7.3% of GFP+ BrdU+ control cells, 40% ± 4.6% of NEUROG2+ CCND1+ BrdU+ cells, and 41.5% ± 4.4% of NEUROG2+ CCNE+ BrdU+ cells). This suggests that a large proportion of cells being kept in the cell cycle are able to initiate their differentiation.
We further analyzed in these different conditions the number of cells both cycling and expressing TUJ1. To circumvent incompatibility between antibodies, we used EdU instead of BrdU to mark proliferating cells. We applied a 30-min pulse of EdU before harvesting the embryos and measured differentiating neurons that incorporated EdU (). When electroporating a control vector, 0.77% ± 0.48% transfected cells are EdU+ TUJ1+, indicating that few cells start to express neuronal markers before they accomplish their last division. Similarly when electroporating NEUROG2 alone, only 1.1% ± 1.1% of transfected cells are EdU+ TUJ1+, consistent with the fact that NEUROG2 rapidly triggers cell cycle exit. In contrast, the proportion of EdU+ TUJ1+ cells is increased by 20-fold in the GFP+ population when NEUROG2 is coelectroporated either with CCND1 or CCNE (20.7% ± 8.8% and 20.2% ± 5.2% of EDU+ TUJ1+ cells, respectively). This indicates that a large proportion of NEUROG2 and CCND1 or NEUROG2 and CCNE cells start to differentiate despite being still in the cell cycle.
All together, these observations show that in the developing neural tube NEUROG2 can trigger expression of markers of neuronal differentiation independently of cell cycle arrest. This indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but rather an event which is controlled in parallel by NEUROG2 (). Hence, in the developing spinal cord, cell cycle exit can be uncoupled from neuronal differentiation, and a main function of NEUROG2, in the course of normal development, is to coordinate these two separable events.