The process of progenitors undergoing cell fate decisions to determine specific cellular identity and tissue patterning is fundamental in the field of developmental biology. Neural development is an excellent model system to study cell fate processes to understand how neural restricted progenitors differentiate into diverse types of neurons through complex interactions of transcriptional activators and repressors. Multiple regulatory design principles involving combinations of transcription factors have emerged through studies from vertebrate and invertebrate model systems that have significant repercussions in determining and maintaining cell identity (Deneris and Hobert, 2014; Dessaud et al., 2008; Lai et al., 2016; Matise, 2013). During progenitor fate specification in the developing nervous system, extensive cross-repressive activities between TFs expressed in neighboring domains in the vertebrate neural tube provide bistable switches needed for generating precise cellular identities (Briscoe et al., 2000; Gowan et al., 2001; Muhr et al., 2001). An additional cell fate design principle was recently demonstrated in the generation of progenitor diversity and in terminal differentiation genes that involves the intersection of a broadly expressed transcriptional activator with progenitor-specific repressor proteins (Kerk et al., 2017; Kutejova et al., 2016; Nishi et al., 2015). In contrast, cell fate specification in the dorsal neural tube may represent a modification of these principles where the activity of progenitor-specific transcriptional activator proteins must be suppressed to limit expression of inappropriate genes. Here we identify the transcriptional repressor, PRDM13, as a critical factor in keeping ventral cell-type specification genes silenced in the dorsal neural tube, highlighting the importance of a balanced network of transcriptional activators and repressors for generating precision in neuronal identities throughout populations in the dorsal ventral axis of the neural tube.

The dorsal spinal cord functions as an essential center for integration of somatosensory information (Liu and Ma, 2011; Ross, 2011). Appropriate processing of this information requires the correct composition of neuronal subtypes, and regulated generation of this neuronal diversity relies on discrete patterns of expression of basic helix-loop-helix (bHLH) and homeodomain (HD) TFs (Lai et al., 2016) (Figure 1A). In the dorsal neural tube, genetic studies have identified the bHLH factors ASCL1 and PTF1A as promoters of excitatory and inhibitory neuronal fate, respectively (Glasgow et al., 2005; Gowan et al., 2001; Helms et al., 2005; Mizuguchi et al., 2006; Nakada et al., 2004). The bHLH TFs execute their function by directly promoting transcription of genes encoding HD TFs, as well as other genes for neuronal function (Borromeo et al., 2014). ASCL1 induces the excitatory neuron specification HD factors TLX1 and TLX3, while PTF1A induces the inhibitory neuron specification HD factor PAX2 (Batista and Lewis, 2008; Borromeo et al., 2014; Chang et al., 2013; Cheng et al., 2004; Cheng et al., 2005; Glasgow et al., 2005; Mizuguchi et al., 2006) (Figure 1B). These studies highlight the essential role bHLH TFs play in determining neuronal diversity in the dorsal spinal cord and serve to demonstrate some basic principles of cell fate specification. In order to drive a multipotent progenitor to a specific lineage, the bHLH TFs must be capable of promoting the fate-specific program while simultaneously suppressing the opposing transcriptional programs for alternative fates (Gowan et al., 2001; Helms et al., 2005). The all-or-nothing nature of this genetic switch is apparent in that gain or loss of function of either factor does not give rise to neurons of mixed identity. Since both ASCL1 and PTF1A function as transcriptional activators (Beres et al., 2006; Nakada et al., 2004), the mechanism for repression was discovered to be indirect, involving PTF1A increasing levels of the transcriptional repressor PRDM13 (Chang et al., 2013). Thus, a network of TFs including bHLH and PRDM factors control levels of HD factors that generate the necessary balance of excitatory and inhibitory neurons in the dorsal spinal cord that modulate somatosensory information processing (Figure 1A,B).

Figure 1

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PRDM13 is a transcriptional repressor present in progenitor domains of the dorsal but not the ventral neural tube from hindbrain regions to the spinal cord (Chang et al., 2013) (Figure 1C). It is a member of the PRDM family of proteins defined by an N-terminal PR domain and a varying number of C-terminal zinc-finger (ZF) domains (Hohenauer and Moore, 2012). PRDM factors function in a variety of cell lineages to promote cell proliferation and cell type specification (Bikoff et al., 2009; Eom et al., 2009; Huang et al., 1998; Ross et al., 2012). Mechanistically they rely on protein-protein and protein-DNA interactions through the PR and ZF domains to repress or activate transcription. The role of PRDM13 as a cell type specification factor has been demonstrated in neural tube (Chang et al., 2013) and retina (Watanabe et al., 2015). In retina, PRDM13 functions to specify subsets of retinal amacrine interneurons. Within the dorsal spinal cord, PRDM13 functions as a downstream effector of PTF1A to suppress the excitatory neuronal program within the inhibitory neuronal lineage. Several PRDM family members have been shown to have intrinsic histone methyltransferase activity mediated by the PR domain (Di Zazzo et al., 2013; Fog et al., 2012; Fumasoni et al., 2007; Hohenauer and Moore, 2012). PRDM13 has been suggested to have intrinsic histone methyltransferase activity, although the relevance of this in vivo remains unclear (Hanotel et al., 2014) since the overexpressed PRDM13 ZF domain is sufficient to specify inhibitory neurons in retina and neural tube (Chang et al., 2013; Watanabe et al., 2015).

To further probe the function of PRDM13 in spinal cord development, and to uncover molecular mechanisms that determine neuronal diversity, we generated multiple Prdm13 mutant mouse strains. In contrast to the previously reported Prdm13 mutant deleting exons 2 and 3 (Watanabe et al., 2015), Prdm13 mutant mice generated here are neonatal lethal and appear to reflect a null phenotype. Analysis of the neural tube phenotype confirms the role of PRDM13 in regulation of neuronal specification factors PAX2 for inhibitory neuronal lineages and TLX1/3 for excitatory neuronal lineages during early neurogenesis (Chang et al., 2013). However, unbiased approaches probing gene expression changes in mutant neural tubes reveal much broader roles for PRDM13 in neuronal specification. These include a negative feedback loop between PRDM13 and its activator, PTF1A, and a requirement for PRDM13 in direct suppression of multiple ventral neuronal-subtype specification transcription factors. Mechanistically, PRDM13 is bound to genomic regions overlapping those bound by neural bHLH factors, and PRDM13 represses the ability of these bHLH factors to activate enhancer driven reporters. Together, a model is presented for recruitment of PRDM13 to bHLH bound enhancers to ensure silencing of lineage inappropriate factors, a function that promotes precision in generating neuronal subtype specific fates.

To understand the role PRDM13 plays in spinal cord development in vivo, and to uncover mechanistic insights into PRDM13 activity, we generated multiple mutant alleles of Prdm13 in mice. These include a GFP knockin (Prdm13^GFP) that disrupts generation of PRDM13, and a deletion within exon 4, the genomic region that encodes the terminal three zinc fingers (ZFs) of PRDM13 (Prdm13^ΔZF) (Figure 1D). This ZF domain is predicted to be essential for PRDM13 function because in overexpression assays this domain is sufficient to mimic the activity of the full-length protein, and overexpression of mutant PRDM13 variants that disrupt each ZF lose activity relative to wild type (Chang et al., 2013). The phenotypes observed in both Prdm13^GFP and Prdm13^ΔZF mutants are identical. Heterozygous animals are indistinguishable from wild-type littermates, but when homozygous for the mutant allele they die at birth, similar to the neonatal lethality seen in other mutants null for TFs that regulate neuronal differentiation and specification such as the bHLH factors ASCL1 and PTF1A (Glasgow et al., 2005; Guillemot and Joyner, 1993). PRDM13-specific antibodies generated to the C-terminal domain (Watanabe et al., 2015) do not detect protein in these mutants at E11.5 by Western blot or immunofluorescence (Figure 1F–H,J). A third mutant was generated with a 115 bp deletion near the N-terminus that causes a frame shift (Prdm13^∆115) (Figure 1E). Surprisingly, Prdm13^{∆115/∆115} survives into adulthood, similar to a previously reported Prdm13 mutant that deleted exons 2 and 3 that encodes much of the PR domain of the protein (Watanabe et al., 2015). Western analysis and immunohistochemistry reveal that a protein containing at least the C-terminal ZF domain is translated in Prdm13^∆115 (Figure 1I,J). This mutant PRDM13 has sufficient function for normal survival (see Discussion). To uncover the requirement for PRDM13 in spinal cord development, we focused the remaining experiments on the Prdm13^GFP and Prdm13^ΔZF mutants.

Dorsal neuronal populations are defined largely by the combination of HD factors they express, and in E10.5 neural tube they are designated as dorsal interneurons 1–6 (dI1-dI6) (for review see [Lai et al., 2016]). Prdm13 was identified as an important specification gene in the dorsal neural tube required for the balance of inhibitory (PAX2⁺;LHX1/5⁺, dI4) interneurons and excitatory (TLX1/3⁺, dI5) interneurons in the chick neural tube (Chang et al., 2013). Overexpression of PRDM13 by electroporation into the chick neural tube led to a suppression of TLX1/3 and a subsequent increase in the number of PAX2⁺ cells. The latter is thought to be an indirect consequence of TLX1/3 function in repressing PAX2 (Chang et al., 2013; Cheng et al., 2004; Cheng et al., 2005). Support for these functions of PRDM13 is seen in Prdm13^GFP and Prdm13^ΔZF mutant embryos with the loss of PAX2⁺;LHX1/5⁺ dI4 neurons and an increase in TLX1/3⁺ dI5 neurons in the dorsal neural tube. At E10.5, antibodies specific for these factors were used to examine changes in the dorsal interneuron populations (Figure 2). A complete loss of dI4 (PAX2⁺;LHX1/5⁺) and an increase in dI3/5 (TLX1/3⁺) in both the Prdm13^GFP/GFP and Prdm13^ΔZF/ΔZF neural tubes was seen at this stage. These phenotypes are identical to the Ptf1a null mouse (Figure 2G) (Glasgow et al., 2005), and are opposite to the Prdm13 overexpression phenotypes in the chick (Chang et al., 2013). Thus, these findings confirm PRDM13 functions as a repressor of the excitatory neuronal gene program within the inhibitory neuronal lineage in the dorsal neural tube.

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PRDM13 is required for specification of dorsal interneurons in the spinal cord.

Associated with Figure 2G. Quantification of the number of dI2 (Tab LHX1/5), dI4 (Tab PAX2 LHX1/5) and dI3/dI5 (Tab TLX1/3) cells per neural tube section in WT, Prdm13 mutants, and for comparison, Ptf1a^Cre nulls. Cell numbers from transverse section at forelimb level were counted for each half of the neural tube and average was considered for the analysis. The numbers of embryos for each genotype are indicated. SEM was used for error bars. Student’s t-test was used to determine significant differences relative to WT.

https://doi.org/10.7554/eLife.25787.004

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The Prdm13 mutant phenotype, however, does not completely mimic that seen for Ptf1a mutants in the dorsal neural tube. One important distinction is the marked reduction of the dI2 population (LHX1/5⁺; PAX2^-) in the Prdm13 mutants that is not seen with the loss of Ptf1a (Figure 2). This is not unexpected given that there is Ptf1a-independent expression of Prdm13 that places it in a broader domain than that of Ptf1a (Chang et al., 2013) (Figure 1A). Another notable difference between the Prdm13 and Ptf1a mutants is detected at later stages of neural tube development. The Ptf1a mutants are striking in that there is essentially a complete loss of PAX2⁺ neurons in the dorsal spinal cord with a subsequent absence of GABAergic and glycinergic neuronal lineages (Glasgow et al., 2005) (Figure 3). Both Prdm13^GFP/GFP and Prdm13^ΔZF/ΔZF neural tubes show only partial loss of PAX2⁺ cells dorsally at E12.5 and E16.5, markedly different from the absolute loss of PAX2 observed in the Ptf1a^CRE/CRE null (Figure 3A–G), and at earlier stages of neurogenesis (Figure 2). This partial loss of PAX2⁺ cells was accompanied by a modest increase in the number of TLX1/3⁺ cells in the Prdm13 mutants, which was not as dramatic as the increase seen in the Ptf1a null (Figure 3H). Consistent with only a partial loss of PAX2+ cells in the Prdm13 mutants, Gad1, required for synthesis of the inhibitory neurotransmitter GABA, is also only partially lost dorsally as seen by in situ hybridization at E16.5 (Figure 3I–K). Again this contrasts to the complete loss of this marker in the Ptf1a null animals at these later stages (Glasgow et al., 2005). The partial loss of Gad1 dorsally in the absence of PRDM13 presumably reflects loss of the inhibitory neurons derived from the E10.5 dI4 PAX2⁺ population (Figure 2). Similarly, only a modest increase in Vglut2 (Slc17a6), a marker of excitatory neurons, is detected in the lateral region of the domain lacking Gad1 (Figure 3L–N). These results highlight overlapping but distinct consequences to dorsal spinal cord development with the loss of PRDM13 versus its upstream regulator PTF1A.

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PRDM13 is not required for specification of dorsal interneurons during the late wave of neurogenesis.

Associated with Figure 3D. Quantification of the number of dI4 (Tab E12.5 PAX2) cells per neural tube section in WT, Prdm13 mutants, and for comparison, Ptf1a^Cre nulls at E12.5. Associated with Figure 3H. Quantification of the number of dI4 (Tab E16.5 PAX2) cells and dI3/5 (Tab E16.5 TLX1/3) at E16.5. Cell numbers from transverse section at forelimb level were counted for each half of the embryo and average was considered for the analysis. The numbers of embryos for each genotype are indicated. SEM was used for error bars. Student’s t-test was used to determine significant differences relative to WT.

https://doi.org/10.7554/eLife.25787.006

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To discover additional PRDM13 functions in neural development, we identified differentially expressed genes (DEGs) in dorsal neural tube cells in the presence and absence of PRDM13. RNA-seq was performed on GFP⁺ cells isolated by fluorescence-activated cell sorting from E11.5 Prdm13^GFP/+ versus Prdm13^GFP/GFP neural tubes (Figure 4A, Table 1, and Supplementary file 1). 837 genes had greater than a 1.5-fold difference in expression and an FPKM >1 in at least one of the conditions. Gene ontology (GO) analysis determined that a large number of the DEGs in the Prdm13 mutants play a role in cell proliferation, neuronal specification and cell cycle regulation (Supplementary file 2).

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We also performed PRDM13 ChIP-Seq to identify candidate direct gene targets of PRDM13 to advance our mechanistic understanding of its function. To increase confidence in PRDM13 bound sites identified, we performed ChIP-seq experiments using three different antibodies (see Materials and methods), and used only those sites identified in at least two experiments. We identified 220 PRDM13 binding sites largely located at distal sites relative to the transcription start sites of genes. These targets included a known target of PRDM13, Tlx3 (Chang et al., 2013). The genome tracks for the Tlx3 locus showing RNA-Seq and ChIP-Seq data illustrate the increase in Tlx3 transcripts in the dorsal neural tube of Prdm13 mutants relative to wild type, and the binding of PRDM13 to a region >20 kb 5’ of Tlx3 (Figure 4B). This region was previously shown to drive expression of a reporter gene to the dI3/dI5 domains in the chick neural tube, identifying it as a Tlx3 enhancer (Chang et al., 2013).

De novo motif analysis failed to identify a novel binding motif unique to PRDM13, but PRDM13 bound sites were enriched for motifs for known neural TFs such as E-box (bHLH) (40%), Rfx (17%), Pdx (HD) (25%) and Sox (30%) motifs (Figure 4C). Indeed, almost half of the PRDM13 bound sites overlap with the E-box binding factors ASCL1 (15), PTF1A (48), or both (39) (Figure 4D). The lack of a unique binding motif is consistent with a model where PRDM13 is recruited to sites by other site-specific TFs.

PRDM13 peak-to-gene associations were identified using GREAT (McLean et al., 2010), which identified 384 genes. GO analysis of these genes also showed enrichment for those that play a role in cell proliferation, neuronal specification and cell cycle regulation (Supplementary file 2). 60 putative direct targets of PRDM13 were identified by intersecting these genes with the DEGs identified in Prdm13^GFP neural tubes (Figure 4A, Supplemental file 3). Consistent with the reported function of PRDM13 as a transcriptional repressor (Chang et al., 2013), mRNA levels for 54 of the putative targets are increased in Prdm13 mutants relative to controls (p-value=1.26e-27) whereas only six are decreased (p-value=0.16) (Figure 4A).

The genes controlled by PRDM13 are notable in multiple respects. First, the function for PRDM13 in repressing the gene expression program for the excitatory neuronal lineage dI5 as was shown in the chick neural tube (Chang et al., 2013) is evident in the Prdm13 mutants. For example, PRDM13 binds the Tlx3 enhancer and restricts its expression at E10.5/E11.5 (Figure 4B, eTlx3). Consistently, Tlx3 is elevated 17-fold, while Pax2 is reduced 6.3-fold in the absence of Prdm13 (Table 1). However, RNA-seq in the mutants also uncovered new functions for PRDM13, revealing a much broader function in restricting essential ventral neuronal subtype specification genes from being expressed in the dorsal neural tube (Table 1). The following experiments support these conclusions and probe mechanisms of PRDM13 activity in spinal cord development.

PRDM13 directly represses its upstream regulator Ptf1a through the Ptf1a auto-regulatory enhancer

Ptf1a was upregulated 3.5-fold by RNA-seq in the Prdm13 mutants relative to control (Table 1). To determine the pattern of this increase in expression we performed mRNA in situ hybridization and immunofluorescence for PTF1A. The increase in PTF1A is localized within its normal dorsal progenitor domain 4 (dP4) (Figure 5A–F). Because PTF1A is a transcriptional activator of Prdm13 transcription (Chang et al., 2013) these data reveal a negative feedback loop between PRDM13 and PTF1A (Figure 5G) where the product of the activated gene (PRDM13) feeds back to repress transcription of its activator (Ptf1a) (Alon, 2007).

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PRDM13 represses transcription of its upstream regulator Ptf1a through the Ptf1a auto-regulatory enhancer.

Associated with Figure 5M. Quantification of GFP intensity from transverse sections of chick neural tubes co-electroporated with a GFP reporter driven by the 2.3kb-Ptf1a autoregulatory enhancer (Tab Ptf1a enhancer). Electroporation of the enhancer with ectopic expression of PTF1A or PRDM13 are labelled accordingly. GFP intensity was calculated using ImageJ software for both electroporated and control side of the neural tube, and the difference between these was analyzed. The numbers of embryos for each electroporation condition are indicated. SEM was used for error bars. Student’s t-test was used to determine significant differences relative to control enhancer expression.

https://doi.org/10.7554/eLife.25787.011

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Ptf1a transcription is regulated through a 2.3 kb autoregulatory enhancer (e2.3Ptf1a) (Masui et al., 2008; Meredith et al., 2009) (Figure 5H). PTF1A binds and activates transcription through this enhancer in a trimeric transcription activator complex called PTF1 that comprises PTF1A, an E-protein, and RBPJ. Perturbations in the PTF1 trimer binding motifs (E-box plus a TC-box) cause a loss of enhancer activity in reporter assays in chick and in transgenic mice (Masui et al., 2008; Meredith et al., 2009). One possible mechanism for the negative feedback by PRDM13 is recruitment of PRDM13 to this Ptf1a autoregulatory enhancer resulting in blocking activity through this site. Indeed, ChIP-seq experiments demonstrate PRDM13 is bound to this enhancer region (Figure 5H and Figure 5—figure supplement 1).

We next tested the ability of PRDM13 to regulate transcription through the Ptf1a autoregulatory enhancer using the chick electroporation assay and a GFP reporter under the control of the 2.3 kb Ptf1a autoregulatory enhancer (e2.3kb-Ptf1a::GFP) (Meredith et al., 2009). As was previously reported, this enhancer directs GFP expression to the Ptf1a domain in the dorsal neural tube and co-electroporation with a Ptf1a expression vector dramatically increases enhancer activity (Figure 5I–J,M). In contrast, co-electroporation with a Prdm13 expression vector alone or along with that for Ptf1a blocks reporter activity completely (Figure 5K–M). Finally, we show that PRDM13 and PTF1A interact, possibly forming a repressor complex, using co-IP assays with lysates from HEK293 cells transfected with FLAG-PRDM13 and MYC-PTF1A (Figure 5N). Because PRDM13 also interacts by co-IP with ASCL1 (Chang et al., 2013) (Figure 5O), we tested whether these interactions are mediated through an E-protein, the common heterodimeric partner for these bHLH factors. We detected no interaction between PRDM13 and the E-proteins E47 and E12 (2 isoforms of Tcf3), and HEB (Tcf12) (Figure 5O). We propose a model whereby PRDM13 directly inhibits Ptf1a transcription by interrupting PTF1A auto-regulation through binding with PTF1A to the Ptf1a autoregulatory enhancer (Figure 5P).

The increased PTF1A expression may be responsible for a difference detected between the Prdm13 and Ptf1a loss of function phenotypes in the second wave of neurogenesis in the dorsal neural tube (Figure 3).

PRDM13 blocks PTF1A activation of Olig2 in the dorsal neural tube

OLIG2 is restricted to the motor neuron progenitor domain (pMN) in the ventral spinal cord at these early stages of neurogenesis, and it is required for specification of motor neurons (Mizuguchi et al., 2001; Novitch et al., 2001; Zhou et al., 2000) (Figure 1A). Upon loss of PRDM13, the linked genes Olig1 and Olig2 show striking ectopic dorsal expression within Prdm13^GFP/GFP neural tubes (Table 1, 217-fold and 1114-fold, respectively). To determine the pattern of this increase in expression we performed immunofluorescence for OLIG2. The ectopic OLIG2 is localized just lateral to the progenitor domain in the dorsal neural tube (Figure 6A–C). This aberrant expression of OLIG2 is not detected in the Ptf1a null even though there is a partial loss of PRDM13 in these mutants (Figure 6D). Co-labeling of OLIG2 with TLX1/3, PTF1A, and OLIG3 demonstrate the ectopic OLIG2 is restricted to the dI4 domain (Figure 6E–H and Figure 6—figure supplement 1). Notably, the ectopic OLIG2 expressing cells co-express TLX1/3 and PTF1A (Figure 6E–H), factors that are never co-expressed during normal development, thus indicating a severe disruption of neuronal subtype identity in the dorsal neural tube.

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PRDM13 is required to block Olig2 expression in the dorsal neural tube via inhibition of PTF1A.

Associated with Figure 6O,T,U,V. Quantification of GFP intensity from transverse sections of chick neural tubes co-electroporated with a GFP reporter driven by the e2Olig enhancer, e2Olig-PTF1Mut and e2Olig-allEboxMut (Tab e2Olig), e1Olig enhancer (Tab e1Olig) and e3Olig enhancer (Tab e3Olig). Electroporation of the enhancer with ectopic expression of PTF1A, ASCL1 or PRDM13 are labelled accordingly. GFP intensity was calculated using ImageJ software for both electroporated and control side of the neural tube, and the difference between these was analyzed. The numbers of embryos for each electroporation condition are indicated. SEM was used for error bars. Student’s t-test was used to determine significant differences relative to control enhancer expression.

https://doi.org/10.7554/eLife.25787.014

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We showed that PRDM13 localizes to a PTF1A bound enhancer and represses enhancer activity (Figure 5). To explore whether a similar mechanism is involved in regulating the linked genes Olig1 and Olig2, we identified three genomic regions surrounding the Olig genes bound by PRDM13 from ChIP-seq (Figure 6I and Figure 6—figure supplement 1). All three of these sites, designated as e1Olig, e2Olig and e3Olig, are also bound by PTF1A and/or ASCL1. Of these three sites, only e2Olig contains the compound E-box/TC-box motifs that represent the site bound by the PTF1 trimeric complex (Figure 6J). We utilized the chick electroporation system to test the ability of each region to drive expression of a GFP reporter gene. All three regions directed expression of GFP in the neural tube, activity that was suppressed by PRDM13 (Figure 6K–l,O,U,V and Figure 6—figure supplement 1). We tested whether PTF1A or ASCL1 were sufficient to activate the Olig enhancers by co-electroporating them individually with each enhancer-reporter. e2Olig::GFP but not e1Olig::GFP or e3Olig::GFP was activated by PTF1A, and this activity was suppressed by PRDM13 (Figure 6M–O and Figure 6—figure supplement 1E,J). ASCL1 did not increase activity through these enhancers. Although these enhancers do not direct expression restricted to the endogenous Olig2 domain, reporters require E-boxes within the enhancers for activity (Figure 6P–T and Figure 6—figure supplement 1). Indeed, e2Olig::GFP enhancer activity and its induction by PTF1A is lost when E-boxes that conform to a PTF1 motif are mutated (Figure 6P,R).

Our data suggest a model for the requirement for PRDM13 to restrict Olig1 and Olig2 expression to the ventral neural tube (Figure 6W). Transcriptional activators such as PTF1A can bind and activate transcription through an enhancer located between Olig1 and Olig2. However, the Olig genes are not expressed in the PTF1A domain in the dorsal neural tube because PRDM13 is also recruited to these sites to silence transcription. In this way, the Olig genes are restricted to the ventral neural tube.

PRDM13 represses Prdm12 in the dorsal neural tube via inhibition of NEUROG1 and NEUROG2

Prdm12 is normally restricted to ventral progenitor domain 1 (p1) and is required for generation of V1 interneurons (Thélie et al., 2015) (Figure 1A). Like Olig1 and Olig2, Prdm12 mRNA is strikingly upregulated in the Prdm13^GFP/GFP mutants (Table 1, 24-fold). We verified the ectopic Prdm12 expression in Prdm13^GFP/GFP and Prdm13^ΔZF/ΔZF neural tubes by mRNA in situ hybridization (Figure 7A–C). This misregulation of Prdm12 is not detected in Ptf1a mutants (Figure 7D). Similar to the conclusions with regulation of the Olig genes, these findings demonstrate a role for PRDM13 in restricting Prdm12 to the ventral neural tube.

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PRDM13 is required to restrict Prdm12 from expression in the dorsal neural tube via inhibition of NEUROG1/2.

Associated with Figure 7—figure supplement 7S. Quantification of GFP intensity from transverse sections of chick neural tubes co-electroporated with a GFP reporter driven by the e1Prdm12 (Tab e1Prdm12) and e2Prdm12 enhancer (Tab e2Prdm12). Electroporation of the enhancer with ectopic expression of PTF1A, NEUROG1, NEUROG2 or PRDM13 are labelled accordingly. GFP intensity was calculated using ImageJ software for both electroporated and control side of the neural tube, and the difference between these was analyzed. The numbers of embryos for each electroporation condition are indicated. SEM was used for error bars. Student’s t-test was used to determine significant differences relative to control enhancer expression.

https://doi.org/10.7554/eLife.25787.017

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To determine if PRDM13 and PTF1A are directly regulating Prdm12, we analyzed the Prdm12 genomic region for sites bound by these factors. PRDM13 ChIP-seq identified a site, e1Prdm12, overlapping a binding site for PTF1A (Figure 7E and Figure 7—figure supplement 1). e1Prdm12 contains an E-box, but no compound E-box/TC-box for the PTF1 trimer complex binding (Figure 7F). e1Prdm12::GFP directs reporter expression localized to the endogenous Prdm12 domain and is repressed by PRDM13 (Figure 7G,H,J). However, co-electroporation with Ptf1a had no effect (Figure 7I,J). This latter result is consistent with the lack of PTF1 trimer binding motif, which is the motif for the PTF1A activator complex (Hori et al., 2008; Meredith et al., 2009).

In contrast to the dI4 restricted ectopic expression seen with OLIG2, the ectopic expression of Prdm12 in the dorsal neural tube of the Prdm13 mutants was in a pattern reminiscent of two Ebox binding bHLH factors, NEUROG1 and NEUROG2, that are normally present in progenitors to the dI2 and dI6 domains as well as in the ventral neural tube (Gowan et al., 2001) (Figure 7K,M). Support for these bHLH factors being involved in the ectopic dorsal expression of Prdm12 was suggested by the RNA-seq data that showed both Neurog1 and Neurog2 as being increased in Prdm13^GFP/GFP mutants (Table 1, 3.4- and 1.7-fold respectively). Indeed, mRNA in situ hybridization shows both of these factors increased in the dorsal neural tube of the Prdm13 mutants (Figure 7L,N). We co-electroporated expression constructs for Neurog1 and Neurog2 with the Prdm12 enhancer reporter e1Prdm12::GFP and found that both drive activity of the reporter, activity that is suppressed when Prdm13 is added (Figure 7O–S). And finally, we show that PRDM13 interacts with NEUROG1 and NEUROG2 using co-IP assays with lysates from HEK293 cells transfected with these factors (Figure 7T).

These data demonstrate the requirement for PRDM13 to restrict Prdm12 expression to the ventral neural tube (Figure 7U). Transcriptional activators such as NEUROG1 and NEUROG2 can activate transcription through an enhancer located more than 25 kb upstream of Prdm12. In the absence of PRDM13, there is ectopic expression of Prdm12 in the dorsal neural tube, expression that may be a consequence of increased NEUROG1 and NEUROG2 combined with the loss of PRDM13.

The ability to generate precise cell identities within an organ is a fundamental process in developmental biology. Central to this process in the developing nervous system are mechanisms that include combinatorial expression of TFs, and TF networks that rely on extensive feedforward and feedback mechanisms to direct cell-type specific gene transcription. To specify the neuronal subtypes in the dorsal spinal cord involved in somatosensory information processing, the transcriptional repressor PRDM13 plays a critical role. We demonstrate its importance in restricting expression of inappropriate regulatory TFs within the lineage that generates the inhibitory neurons of the dorsal spinal cord (summary Figure 8). Within the TF network, PRDM13 is a component of a negative feedback loop where the PTF1 trimeric complex activates transcription of Prdm13 then PRDM13 feeds back to repress transcription of Ptf1a, ensuring PTF1A is present only transiently during neural development. Furthermore, a primary mechanism for PRDM13 repression appears to be recruitment to chromatin sites bound by neural bHLH transcription activators, thus providing a molecular explanation for why localization of the bHLH activator does not always result in active gene transcription. Finally, this study demonstrates PRDM13 is required to restrict expression of multiple neuronal specification factors to the ventral neural tube to ensure precision in cell-fate identity in the dorsal spinal cord (Figure 8 and Figure 8—figure supplement 1).

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Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Jane E. Johnson (jane.johnson@utsouthwestern.edu).

1. 1. Mona B
   2. Uruena A
   3. Kollipara RK
   4. Ma Z
   5. Borromeo MD

   (2016) Repression by PRDM13 is critical for generating precise neuronal identity

   Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE90938).

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE90938

1. 1. Borromeo MD
   2. Meredith DM
   3. Castro DS
   4. Chang JC
   5. Tung K
   6. Guillemot F
   7. Johnson JE

   (2014) Transcription Factor Network Specifying Inhibitory versus Excitatory Neurons in the Dorsal Spinal Cord [ChIP-Seq]

   Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE55840).

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55840

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Author details

1. Bishakha Mona

   Department of Neuroscience, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology, Writing—original draft

   Contributed equally with

   Ana Uruena

   Competing interests

   No competing interests declared

2. Ana Uruena

   Department of Neuroscience, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Investigation, Methodology, Writing—original draft

   Contributed equally with

   Bishakha Mona

   Competing interests

   No competing interests declared

3. Rahul K Kollipara

   McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Software, Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

4. Zhenzhong Ma

   Department of Neuroscience, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

5. Mark D Borromeo

   Department of Neuroscience, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Joshua C Chang

   Department of Neuroscience, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Jane E Johnson

   1. Department of Neuroscience, UT Southwestern Medical Center, Dallas, United States
   2. Department of Pharmacology, UT Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing—original draft

   For correspondence

   Jane.Johnson@UTSouthwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8605-2746

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