Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus
Aurore Thélie1*, Simon Desiderio1*, Julie Hanotel1, Ian Quigley2, Benoit Van Driessche6,Anthony Rodari6, Mark D Borromeo3, Sadia Kricha1, François Lahaye4, Jenifer Croce4, Gustavo Cerda-Moya5, Jesús Ordoño Fernandez1, Barbara Bolle1, Katharine Lewis7, Maike Sander8, Alessandra Pierani9, Michael Schubert4, Jane E.
Johnson3, Christopher R. Kintner2, Tomas Pieler10, Carine Van Lint6, Kristine A.
Henningfeld10, Eric J. Bellefroid1◊, Claude Van Campenhout1◊
1Laboratory of Developmental Genetics, Université Libre de Bruxelles (ULB), Institute of Molecular Biology and Medecine (IBMM) and ULB Neuroscience Institute, B-6041 Gosselies, Belgium; 2The Salk Institute for Biological Studies, La Jolla 92037, California, USA; 3Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390, USA; 4Sorbonne Universités, UPMC Université Paris 06, CNRS UMR 7009, Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR 7009), Observatoire Océanologique de Villefranche-sur- Mer, 06230 Villefranche-sur-Mer, France; 5Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK; 6Laboratory of Molecular Virology, ULB, IBMM, B-6041, Gosselies, Belgium; 7Department of Biology, Syracuse University, 107 College Place, Syracuse, NY 13244, USA;
8Departments of Pediatrics and Cellular and Molecular Medicine, Pediatric Diabetes Research Center, University of California, San Diego, La Jolla, CA 92093-0695, USA; 9Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, 75205 Paris Cedex 13, France ; 10Institute of Developmental Biochemistry, Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University of Goettingen, 37077 Goettingen, Germany. *AT and SD contributed equally to the work.
◊Corresponding authors: Eric J. Bellefroid and Claude Van Campenhout, Email: ebellefr@ulb.ac.be; cvcampen@ulb.ac.be.
ABSTRACT
V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain however largely undefined. Here we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, but also chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors due to the repressive action of Dbx1 and Nkx6.1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part by directly repressing Dbx1 and Nkx6 genes, likely by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.
INTRODUCTION
In the ventral neural tube, distinct classes of neurons (V0, V1, V2 and V3 interneurons, INs) and motor neurons are generated at specific dorsoventral levels in response to a gradient of sonic hedgehog activity. Shh functions by regulating the expression of genes encoding homeodomain (HD) (i.e Pax6, Irx3 and Dbx1/2 repressed and Nkx6 and Nkx2 induced by Shh) and basic helix-loop-helix (bHLH) transcription factors (TFs). These TFs produce a combinatorial transcriptional code that delineates spatially the distinct ventral progenitor domains (Briscoe and Novitch, 2008; Dessaud et al., 2008). Cross-repressive interactions between complementary pairs of these TFs further refine and sharpen the boundaries of the progenitor domains. Perturbation of this code of HD and bHLH TFs leads to aberrant dorsoventral patterning and cell type misspecification (Briscoe and Novitch, 2008;
Dessaud et al., 2008).
In the adult mammalian spinal cord, likely as the result of evolutionary pressures toward functional specialisation during transition from swimming to terrestrial locomotion, there is a large diversity of spinal INs modulating motor output that arise from this limited number of progenitor domains (Goulding, 2009; Grillner and Jessell, 2009). As an example, one can cite the V1 class of inhibitory premotor INs that, at the onset of post-mitotic differentiation, is characterized in all vertebrates studied so far by the expression of Engrailed-1 (En1) (Saueressig et al., 1999; Wenner et al., 2000;
Li et al., 2004; Higashijima et al., 2004; Alvarez et al., 2005). In aquatic vertebrates, this class of INs comprises only one type of multifunctional INs, known as circumferential ipsilateral ascending neurons (CiA) in fish and ascending interneurons (aINs) in frogs. In mammals, the V1 class of INs that plays a crucial role in regulating the rhythm of locomotor outputs and flexor-extensor inhibition (Gosgnach et al., 2006; Zhang et al., 2014) comprises a large variety of derivatives, including Renshaw cells (RCs) and putative reciprocal Ia inhibitory INs (Ia INs) (Alvarez et al., 2005;
Siembab et al., 2010; Benito-Gonzalez and Alvarez, 2012; Francius et al., 2013). In the chick, the population of En1-expressing neurons in the embryonic spinal cord has equally been shown to be heterogeneous (Wenner et al., 2000). However, how these V1 INs and their subtypes are generated remains largely unknown.
Prdm proteins are a family of epigenetic zinc finger transcriptional regulators with important roles in cell differentiation and diseases (Fog et al., 2012; Hohenauer
and Moore, 2012). In mouse and zebrafish embryos, Prdm12 is expressed in specific regions of the developing brain, in spinal cord p1 progenitors and in dorsal root and trigeminal ganglia (Kinameri et al., 2008). In P19 embryonic carcinoma cells, Prdm12 is induced by retinoic acid (RA) and inhibits cell proliferation (Yang and Shinkai, 2013). Recently, it has been shown that Prdm12 is required for V1 interneurons in zebrafish (Zannino et al., 2014). However, the mechanisms that are responsible for its selective expression in p1 progenitors and its mode of action remain elusive. Here, we approached these mechanisms and dissected its mode of action in Xenopus V1 cell fate specification.
MATERIALS AND METHODS
Prdm12 cDNAs and expression constructs
Xenopus laevis (EST BM179581), zebrafish (Danio rerio) (EST BC085382) and chicken (Gallus gallus) (EST BU233582) Prdm12 cDNA clones were identified by BLAST searches of EST databases (NCBI), using the mouse Prdm12 cDNA sequence as a bait. An amphioxus (Branchiostoma lanceolatum) Prdm12 cDNA was amplified by PCR from cDNA generated from total RNA from adults, using primers forward 5’-
ATGAAGCCGACCCTGTTTGATC-3’ and reverse 5’-
ACATGGCGACCGAACGAACGTC-3’ (Oulion et al., 2012).
Prdm12 and previously described expression vectors used in frog microinjections and in ovo electroporations are described (supplementary material).
Xenopus embryos micro-injections and animal caps
Synthetic mRNAs were prepared using the SP6 mMessage mMachine kit (Ambion). The pCS2-Flag-dnG9a construct was obtained by PCR from pCDNA3- human dnG9a (Gyory et al., 2004) using primers 5’-GACGA TGACAAGAA
TTCAAGTGA TGA TGTCCACTCACTGGGAAAG-3’ and reverse 5’-
GTTCTAGAGGCTCGAGTCATGTGTTGACAGGGGGCAGGGA-3’ and cloned into the EcoRI and XhoI sites of pCS2-FLAG. The Prdm12 antisense morpholino oligonucleotides (MO) (5’-CCGGCAGCACCGAGCCCATCATTAA-3’ and 5’- CATTAATTCTGCCTGCGAGTCTGAC-3’) and the control Prdm12 antisense mismatch MO (5’-CCCGCACCACGGAGCGCATGATTAA-3’) were injected at 20 ng/blastomere. Pax6, Dbx1, Nkx6.1 and Nkx6.2 MOs were as described (Dichmann and Harland, 2011; Ma et al., 2013; Ma et al., 2011; Rungger-Brändle et al., 2010).
For in situ hybridization (ISH) analyses, embryos were injected in one cell of two to four-cell stage embryos. In all experiments, embryos were coinjected with LacZ mRNA (50 pg/blastomere) and LacZ activity was revealed by X-gal staining (in light blue or red). For animal cap assays, mRNA was microinjected into the animal region of each blastomere of four-cell stage embryos. Dissected animal caps were cultured in 1X Steinberg medium, 0.1% BSA with or without RA (10 uM) until sibling embryos reach stage 28, unless indicated. All microinjections were repeated independently at least two times.
In situ hybridization and immunohistochemistry
ISH and immunohistochemistry (IHC) procedures used in amphioxus, frog, chick, zebrafish and mouse embryos are detailed in supplementary material.
Real-time RT-PCR
Real-time RT-PCR was performed as reported (Hanotel et al., 2013). Gene expression levels were normalized with GAPDH and, unless indicated, compared to the level observed in naïve RA-treated neuralized caps (defined as 1). Primers are listed in supplemental Table S1. All assays were carried out in duplicates or triplicates. Error bars represents standard deviations. The values indicated in the figures are derived from one representative experiment.
Chick embryo in ovo electroporation
Stage HH12-14 embryos were electroporated in the neural tube as described (Hanotel et al., 2013). 48 hours (HH24-25) or 72 hours (HH27-28) after electroporation, embryos were harvested, fixed and processed for IHC or ISH.
Embryos electroporated with the 5.7 kb mouse Dbx1 promoter fragment were collected 24 hours later (HH18) and processed for β-gal activity as described (Lu et al., 1996).
Mouse embryo preparation
Pax6Sey/Sey, Dbx1LacZ/LacZ, Nkx6.1-/- and Nkx6.2Cre/Cre mutant mice were as described (Baudet et al., 2008; Ericson et al., 1997; Pierani et al., 2001; Sander et al., 2000).
Histone methyltransferase activity
The HMTase assays were conducted as described (Hanotel et al., 2013).
RNA sequencing and Chromatin immunoprecipitation
RNAseq from Xenopus animal caps was performed essentially as described (Ma et al., 2014). ChIP was essentially conducted in mouse (Borromeo et al., 2014) and in Xenopus as described (Ma et al., 2014; Colin et al., 2011) (see supplementary material).
RESULTS
Cloning and expression of Xenopus, chicken and amphioxus Prdm12 during development
Xenopus Prdm12 encodes a nuclear protein with a N-terminal PR domain followed by three zinc finger motifs (Hanotel et al., 2013). As previously reported for the mouse protein (Yang and Shinkai, 2013), Xenopus Prdm12 exhibits histone methyltransferase activity. Prdm12 is highly conserved during evolution (Nagy et al., 2015) and we identified orthologous sequences in the chicken and the cephalochordate amphioxus (Branchiostoma lanceolatum), a basal chordate (supplementary material Fig. S1).
Prdm12b is selectively expressed in p1 progenitors of the zebrafish and mouse neural tube (Kinameri et al., 2008; Zannino et al., 2014 and supplementary material Fig. S2). To determine Prdm12 expression during Xenopus neural development, we used ISH. Prdm12 is first detected at stage 12.5-13 in two bilateral longitudinal stripes of cells running along the posterior neural plate as well as in cranial placodes (Fig. 1A-C). At this stage, these stripes have an anterior limit in rhombomere 7. After neural tube closure, Prdm12 is also detected in specific brain regions and its expression in neural tube ventral progenitors has a rostral limit corresponding to the anterior hindbrain (Fig. 1D, F and supplementary material Fig. S3). Within the neural plate, Prdm12 expressing cells are included in the Pax6 domain. They are located between the medial and intermediate stripes of the primary neuronal marker N- tubulin, at the medial boundary of Dbx1 that in the mouse spinal cord marks p0 progenitors (Fig. 1H-J) and at the lateral boundary of Nkx6.1 and Nkx6.2 (Fig. 1K, L).
At this stage, the medial limit of Prdm12 appears to correspond to the lateral limit of Nkx6.2, while Nkx6.1 is separated from Prdm12 by a gap. At later tailbud and tadpole stages, the dorsal limits of Nkx6.1 and Nkx6.2 appear to be similar and correspond to the ventral border of Prdm12. Thus, in contrast to the mouse (Vallstedt et al., 2001), Nkx6.1 and Nkx6.2 are broadly coexpressed, from neurula to tadpole stages, and appear excluded from p1 progenitors. Dbx2 is very weak at neurula stage. In tadpoles, it is more broadly expressed than Dbx1, suggesting that, like in the mouse (Pierani et al., 2001), Dbx2 extends ventrally into the p1 progenitor territory (supplementary material Fig. S4, 5). In tadpoles, Prdm12 staining in the ventricular zone of the neural
tube appears in the marginal zone at the level of En1, a selective marker of Xenopus aINs and mouse V1 INs (Matise and Joyner, 1997; Li et al., 2004; Fig. 1M). Thus, as in zebrafish and mouse, Prdm12 is selectively expressed in the spinal cord in p1 progenitors (Fig. 1N). A similar restricted expression was also observed in the neural tube of chick embryos (Fig. 1O) and in the ventral nerve cord of the larvae of the basal chordate amphioxus (Fig. 1P, Q).
Xenopus Prdm12 expression in the spinal cord is dependent on RA signaling and Pax6
RA plays a role in spinal cord neurogenesis and is known to posteriorize induced neural tissue (Briscoe and Novitch, 2008; Durston et al., 1989). To examine whether RA regulates Prdm12 during Xenopus neural development, we analyzed its expression in animal cap ectodermal explants. These explants are specified to become epidermal tissue, but can be converted to derivatives of all three germ layers.
Embryos were injected with noggin mRNA to neuralize the cells, and dissected animal caps were cultured to the equivalent of stage 28 in the presence or absence of RA. Total RNA was isolated and Prdm12 expression was analyzed by RT-qPCR. We also monitored En1 and, as controls, the epidermal marker Ep. Keratin, the anterior neural marker Otx2, the hindbrain markers Krox20 and Mafb and the posterior marker Hoxb9 (Hb9). As expected, in neuralized caps, Ep.keratin is inhibited and Otx2 is strongly expressed. Krox-20, MafB and Hoxb9 are only weakly upregulated, demonstrating that the explants have adopted an anterior neural fate. In neuralized caps treated with RA, Otx2, Krox20 and MafB were downregulated and HoxB9 was induced, consistent with their differentiation into spinal cord. Prdm12 and En1 were not efficiently induced in neuralized caps, but were strongly upregulated in neuralized explants treated with RA (Fig. 2A). The requirement of RA for Prdm12 expression during spinal cord development was next evaluated in both frog and chick embryos.
In Xenopus, RA signaling was blocked by overexpressing the RA-catabolizing enzyme Cyp26. In the chick, we interfered with RA signaling using a dominant negative form of the human RA receptor α (dnRAR403). As shown in supplementary material Fig. S6, Prdm12 is reduced in both Xenopus and chick embryos in response to the inhibition of RA. Thus, RA appears important for Prdm12 expression in the ventral spinal cord. However, we cannot exclude that Prdm12 up- and downregulation
observed in our manipulations of RA is indirect, related to the change of A/P identity of the cells.
Downstream of RA signaling, Pax6 constitutes one of the major stimuli for IN development in the intermediate spinal cord (Ericson et al., 1997; Pierani et al., 1999).
Therefore, we investigated the regulation of Prdm12 by Pax6. As a first step, we assessed the effects of Pax6 overexpression on Prdm12 expression in the frog and chick embryos. Fig. 2B shows that Pax6 overexpression in Xenopus neurula-stage embryos is not sufficient to upregulate Prdm12 (71% unaffected, 29 % decreased, n=24), and similar results were obtained in the chick (supplementary material Fig.
S7). We next assessed in both frog and mice, whether the loss of Pax6 affects Prdm12 expression. To block Pax6 activity in Xenopus, embryos were injected with a Pax6 MO. Fig. 2C shows that injection of the Pax6 MO inhibits Prdm12. The penetrance of this phenotype, however, was weak (58% inhibited, 42% unaffected, n=45), which may be due to incomplete depletion of the protein (Rungger et al., 2010). We therefore examined Prdm12 expression in Pax6Sey/Sey mutants. In Pax6Sey/Sey embryos, in which En1 is lost (Burrill et al., 1997), Prdm12 was strongly reduced (Fig. 2D, E).
In silico, we identified several potential Pax6 binding sites in a conserved 600- bp sequence stretch located within the first intron of the Prdm12 locus with enhancer activity in the neural tube of E11.5 mouse embryos (Visel et al. 2007). To determine if Pax6 binds this enhancer, ChIP-qPCR was performed on caudal neural tube tissues from E11.5 embryos using an anti-Pax6 antibody (Coutinho et al., 2011) and primers that span the identified potential Pax6 binding sites. This region was found to be significantly enriched compared to a negative control region (Kirrel2) (Borromeo et al., 2014) (supplementary material Fig. S8). Thus, Prdm12 is activated, may be directly, by Pax6. However, this activation could be also indirect as the absence of Pax6 results in the dysregulation of other TFs expressed in ventral progenitors (Takahashi et al., 2002).
Prdm12 is confined to p1 progenitors due to the repressive action of Dbx1 and Nkx6
Progenitor identity in the spinal cord often relies on cross-repressive interactions between specific TFs (Briscoe and Novitch, 2008; Dessaud et al., 2008).
We therefore asked whether Prdm12 is regulated by Dbx1, Nkx6.1 and Nkx6.2. In the frog, we found that their overexpression inhibits Prdm12 in neurula-stage embryos
(71% reduced, n=28 for Dbx1; 78% reduced, n=18 for Nkx6.1; 71% reduced, n= 17 for Nkx6.2) (Fig. 2F, G and data not shown). As in the frog embryo, misexpression of Dbx1 or Nkx6.1 in the chick neural tube resulted in a decrease of Prdm12 (supplementary material Fig. S9). The relationship between Prdm12 and these genes was further analyzed in the mouse by examining Prdm12 expression in Dbx1LacZ/LacZ and Nkx6.1-/- knockout mice (Pierani et al., 2001; Sander et al., 2000; Vallstedt et al., 2001). Compared to control mice, Prdm12 is expanded in Dbx1LacZ/LacZ mutants (Fig.
2H, I). Comparing Prdm12 and Prdm13 expressed in dp2-dp6 (Chang et al., 2013), this expansion appears limited to the p0 domain, which is defective in Dbx1LacZ/LacZ embryos (supplementary material Fig. S10). In Nkx6.1-/- knockout mice, the Prdm12 domain was also expanded (Fig. 2J, K). This expansion is however also limited, likely due to a switch of identity of some more ventral progenitors to p1 (Sander et al., 2000). Together, these results reveal that Prdm12 expression in the Xenopus ventral spinal cord is excluded from the p0 and p2 domains due to the repressive action of Dbx1 and Nkx6.1/2 and suggest that other TFs may also participate in its repression.
In the mouse, Nkx6.1 alone is likely responsible for the ventral restriction of Prdm12, as Nkx6.2 is expressed in p1 and no change in Prdm12 could be detected in Nkx6.2Cre/Cre null mice (data not shown).
Xenopus and mouse, but not amphioxus Prdm12, induces a V1 cell fate
Loss of Prdm12 in zebrafish inhibits the V1-specific marker En1 and affects swimming movements (Zannino et al., 2014). To determine whether Prdm12 has a similar function in Xenopus, we designed a Prdm12 translation blocking MO and inject it bilaterally at the 2-cell stage. As in zebrafish, Prdm12 morphants show impaired swimming movements and reduced En1 (99% reduced, n=78) (Fig. 3A, supplementary material Fig. S11 and movie online). Similar results were obtained with a second independent MO (data not shown). Injection of a Prdm12 mismatch MO had no effect on En1 (none affected, n=21) and the phenotype could be rescued by coinjection of mRNA encoding mouse Flag-Prdm12 (150 pg mPrdm12/blastomere) which is insensitive to the Prdm12 MO (94% increased, n=33) (Fig. 3B, C).
We next determined whether the postmitotic V0 and V2 markers Evx1 and Vsx1 are affected in Prdm12 morphants. While Vsx1 is unaffected in most injected
embryos (87% unaffected, n=47) (data not shown), Evx1 is increased (92%
upregulated, n=78) (Fig. 3D).
Zannino et al. reported that the dorsal limit of Nkx6.1 is expanded in ventral neural tube progenitor cells of zebrafish prdm12b morphants (Zannino et al., 2014).
We thus analyzed Nkx6.1 as well as Nkx6.2, Pax6 and Dbx1. In most Prdm12 MO- injected embryos, Pax6 and Dbx1 were unaffected (Pax6, none affected, n = 7; Dbx1, 89% unaffected and 11% increased, n=28). In the case of Nkx6.1, a slight dorsal expansion was detectable in 66% of the injected embryos (n=44). This expansion was most noticeable in embryos stained for both Nkx6.1 and Dbx1, as the region of expansion corresponds to a gap normally present between the expression domains of the two markers (Fig. 3E,F). The Nkx6.2 signal also appears to be extended dorsally in some Prdm12 MO-injected embryos (37% expanded, n=8) (data not shown). Thus, as in zebrafish, Prdm12 is required for the specification of V1 INs and these neurons are important for swimming movements
To further characterize the function of Prdm12, we next performed gain-of- function assays, in the frog and chick embryos. While frog embryos overexpressing a Flag version of mouse or Xenopus Prdm12 appear morphologically normal at the early tadpole stage, they did not respond to touch (supplementary material Fig. S11 and movie online), suggesting that Prdm12 affects neuronal specification. In these Prdm12 mRNA injected embryos, we first examined En1 expression. In embryos overexpressing Xenopus or mouse Prdm12, En1 was induced (all induced, n=35, for Xenopus and n=23 for mouse Prdm12). The induction observed was, however, stronger with mouse Prdm12 (Fig. 4A and data not shown). We therefore performed all our subsequent Prdm12 overexpression experiments using the Flag version of mouse Prdm12. We next looked at the effects of Prdm12 overexpression on the postmitotic V0 and V2 markers Evx1 and Vsx1. While Vsx1 is mainly unaffected (92% unchanged, n=75) (data not shown), Evx1 was often reduced (89% decreased, n=90) (Fig. 4B). We next investigated the effects of Prdm12 overexpression on Pax6 and Xiro3, coexpressed with Prdm12 in p1 progenitors, Dbx1, Nkx6.1 and Nkx6.2 expressed in adjacent progenitor domains and on Pax3, Pax7 and Ptf1a expressed in neural progenitors of the dorsal neural tube. Prdm12 overexpression had no effect on Pax6 (89% unaffected, n=19), Xiro3 (100%, unaffected, n= 14), Pax3, Pax7 and Ptf1a (supplementary material Fig. S12). In contrast, Dbx1, Nkx6.1 and Nkx6.2 were decreased (97% decreased, n=33 for Dbx1; 71% decreased, n=42, for Nkx6.1; 76%
decreased, n=42, for Nkx6.2) (Fig. 4C-F and supplementary material Fig. S12). Thus, in Xenopus, Prdm12 activity appears mainly limited to the repression of neighbouring genes.
The effects of Prdm12 overexpression were further examined in animal caps.
We found that Prdm12 induces En1 in neuralized explants but not in naïve caps and that Prdm12 is much more active in RA-treated neuralized caps than in untreated ones (Fig. 4G). We also observed that in RA-treated neuralized caps, Prdm12 overexpression efficiently increases En1 level only starting from tailbud stages (st.
20), thus at around its normal onset of expression during development (Fig. 4H). We next further evaluated Prdm12 activity by analyzing the expression of Pax6, Dbx1/2, Nkx6.1/2 (and Prdm12 itself) as well as of the postmitotic markers Evx1 and Vsx1.
Fig. 4I shows that, as observed in embryos, Dbx1, Nkx6.1, Nkx6.2 that are excluded from p1 progenitors and Evx1 were all down-regulated by Prdm12 overexpression.
Vsx1, that appears unaffected in embryos, was also downregulated. In contrast, Pax6 was not affected and Dbx2, which is coexpressed with Prdm12 in p1 progenitors, was only slightly reduced. Interestingly, Prdm12 was upregulated by its own overexpression. Finally, we also tested the activity of the amphioxus Prdm12 protein.
Fig. 4J shows that in contrast to the mouse protein, amphioxus Prdm12 is unable both to increase En1 and to efficiently downregulate Dbx1 and Nkx6.1. Thus, amphioxus and vertebrate Prdm12 proteins appear to differ functionally.
In the chick neural tube, Prdm12 increases En1 and induces markers of the different subtypes of V1 INs (supplementary material Fig. S13). Together, these results indicate that Prdm12 is involved in patterning ventral neuronal progenitors and acts as a general determinant of V1 cell fate, a property that appears specific to the vertebrate lineage.
Prdm12 requires both its PR and zinc finger domains and acts as a repressor Prdm family members contain two major motifs, the PR domain similar to the SET domain found in histone methyltransferases and zinc fingers. To determine their functional contribution, we tested the activity of a series of mutants (Yang and Shinkai, 2013) in the chick neural tube (Fig. 5A). Fig. 5B shows that mutations that disrupt either the PR or the zinc fingers impair its ability to induce En1.
To determine whether the activity of Prdm12 to induce V1 INs reflects a transcriptional activator or repressor function, we created fusion constructs of
Xenopus Prdm12 with the engrailed repressor (EnR) (Smith and Jaynes, 1996) or with the VP16 activator domain (Triezenberg et al., 1988) and tested the En1-inducing activity of the chimeric proteins in the frog embryo. We found that EnR-Prdm12 functions as the wild type protein in that it induces En1 (97%, n=78). In contrast, VP16-Prdm12 functions as an antimorphic mutant as its overexpression inhibits En1 (98%, n=125). Interestingly, Nkx6.1 that is downregulated by Prdm12 is upregulated by VP16-Prdm12 (64%, n=22). In animal caps, like in the embryos, EnR-Prdm12 induces En1. It also downregulates Nkx6.1, Nkx6.2 and Dbx1 while VP16-Prdm12 upregulates their expression (Fig. 5C).
Unlike other Prdm proteins, Prdm12 lacks direct histone methyltransferase activity and repress target gene expression by recruiting the methyltransferase G9a to dimethylate histone H3 at lysine 9 (H3K9me2) (Yang and Shinkai, 2013). Therefore, we asked whether G9a is necessary for Prdm12 function in the spinal cord. We found that a dominant negative G9a overexpressed together with Prdm12 in RA-treated neuralized caps diminishes its ability to induce En1 and at high dose slightly attenuates Nkx6.1 and Dbx1 repression (Fig. 5D).
The amphioxus Prdm12 protein appears functionally distinct to the vertebrate Prdm12 proteins. To identify the regions responsible for this differential activity, we generated two mouse-amphioxus Prdm12 chimeric proteins, substituting their PR or zinc finger domains, and tested their activity using our animal cap assay. The chimeric protein retaining the mouse zinc finger domain (aPR-mZF) was still able to induce En1 and to repress Dbx1 and Nkx6.1. In contrast, the other chimeric protein with the amphioxus zinc finger domain (mPR-aZF) was inactive (Fig. 5E). Together, these data indicate that Prdm12 acts in V1 cell specification as a G9a-dependent transcriptional repressor, whose activity requires both the PR and zinc finger domains.
They also suggest that differences in the C-terminal part of the protein, including the zinc fingers, are responsible for the differential functions of the amphioxus and vertebrate Prdm12 proteins.
Dbx1 and Nkx6 genes overexpression blocks Prdm12 activity and their knockdown upregulates En1
The above results suggest that Prdm12 may induce V1 expressing cells by repressing Dbx1 and Nkx6 genes. To test this idea, we coexpressed them with Prdm12 in RA-treated neuralized caps. Fig. 6A shows that the induction of En1 by Prdm12 is
abrogated by the overexpression of Dbx1, Nkx6.1 or Nkx6.2. Coexpression of Dbx2 or LacZ had no such repressive effect. We next tested whether the knockdown of Dbx1 and/or Nkx6.1 and Nkx6.2 in RA-treated neuralized caps could be sufficient to increase En1. Fig. 6B shows that En1 is upregulated when Dbx1, Nkx6.1 or Nkx6.2 were knocked down, and that En1 level was increased even further when combinations of these MOs were used. As expected, Prdm12 was also upregulated when Dbx1, Nkx6.1 or Nkx6.2 were knocked-down, Thus, Prdm12 promotes V1 cell fate in Xenopus mainly by repressing Dbx1 and Nkx6 genes.
RNA-Seq and ChIP-Seq analyses support a role for Prdm12 in promoting V1 cell fate through direct repression of Dbx1 and Nkx6 genes
To gain further insights into the transcription network regulated by Prdm12, RNAseq analyses were carried out on RA-treated neuralized caps derived from wild type or Prdm12-overexpressing embryos (Fig. 7A). Applying a minimal cutoff of 2 fold-change and p < 0.05, a total of 299 differential expressed genes were identified.
Of these, 62 were downregulated and 237 were upregulated by Prdm12 (Table S2).
Gene ontology (GO) analyses revealed that genes positively regulated by Prdm12 are enriched in GO categories relevant to neuronal function such as transmission of nerve impulses, neurotransmitter transport, secretion, synapse and channel activity (Fig 7B).
Enriched in the both up- and down-regulated categories were genes involved in sequence-specific DNA binding and neuron differentiation. Fig. 7C shows a curated list of the most highly upregulated and downregulated genes (together with their fold change). The list of upregulated genes contains as expected many genes know to be expressed in the postmitotic V1 domain, including TFs En1, Pax2 and FoxD3.
Consistent with the inhibitory neurotransmitter phenotype of V1 neurons, markers such as Gad1 and Slc32a1 were also found upregulated. In the list of downregulated genes were TFs expressed in both the progenitor and postmitotic cells of the adjacent domains including Olig2, Nkx6.1, Lhx3, Dbx1 and Vsx1/Chx10.
To obtain additional evidence that those genes are indeed regulated by Prdm12, the transcriptome of RA-treated neuralized caps overexpressing the EnR- Prdm12 or VP16-Prdm12 constructs was determined. Consistent with our results suggesting that Prdm12 functions as a repressor, 76% of the genes downregulated by Prdm12 were present in the list of the 379 genes downregulated by EnR-Prdm12 and 92% of the genes upregulated by Prdm12 were present in the 862 genes induced by
EnR-Prdm12. The overlap of common differentially expressed genes between Prdm12 and Prdm12-VP16 was limited, with the highest overlap represented by the 30 genes upregulated by Prdm12 and downregulated by VP16-Prdm12-VP16 (Fig.7D and supplemental Table S2-3).
For some of these genes, we have confirmed their disregulation by Prdm12 using RT-qPCR. Fig. 7E shows that two of the most upregulated genes in our RNAseq list, the gastrin-releasing peptide receptor (GRPR) that is involved in neurogenesis (Walton et al., 2014) and neurexophilin2 (Nxph2), a member of a family of secreted glycoprotein that function as neuropeptides (Missler et al., 1998), are indeed strongly increased by Prdm12. The upregulation of Pax2, Lhx5 (induced over 2 fold by EnR-Prdm12 but not by Prdm12) and Lhx1 (induced by both Prdm12 and EnR-Prdm1, but below 2-fold), which are expressed in spinal cord V1 INs, as well as of the inhibitory markers Gad1, Slc32a1 and Slc6a5 was also validated. We were further able to verify that Lhx3/Lim3 and cyclin Dx, which are expressed more ventrally, as well as Neurog2 and Sox3, which are expressed in undifferentiated neural cells, are indeed downregulated by Prdm12. Finally, we also tested p27Xic1 encoding a cyclin-dependent kinase inhibitory protein and the pan-neuronal differentiation marker N-tubulin as those genes were found upregulated by Prdm12 in P19 cells (Yang and Shinkai, 2013). Both of them were also upregulated by Prdm12 in our neuralized RA-treated caps. The deregulation of two of these targets, Pax2 and Gad1, has further been validated by ISH in Prdm12 overexpressing and morphant embryos (Fig. 7F). Their up- and dowregulation by Prdm12-EnR and Prdm12-VP16 has been equally confirmed by RT-qPCR (Fig 7G).
To identify Prdm12 direct downstream targets, chromatin immunoprecipitation sequencing (ChIP-Seq) experiments were performed using neuralized RA-treated animal caps overexpressing mouse Flag-Prdm12 and anti Flag antibodies. Naïve animal caps overexpressing the mouse Flag-Prdm12 construct were used as a control, as Prdm12 is unable to promote En1 in epidermal cells (Fig 4G). In these conditions, Prdm12 binds to several hundreds of sites within the genome (supplemental Table S4).
Only sites differentially bound at 4-fold were retained, which numbered approximately 20,000. To determine where Prdm12 binds relative to other genomic features, we annotated its positions and found that Prdm12 strongly prefers enhancers to promoters (data not shown). We then asked how many Prdm12 peaks were
flanking genes regulated by Prdm12, as shown by our RNA-seq. We found Prdm12 peaks flanking many of these targets, such as Dbx1, Nkx6.1, Nkx6.2, Vsx1, Lhx3, Prdm14, Sox3 and Neurog2 (Fig. 8A). In some cases, genes were surrounded by a large number of Prdm12 peaks, hinting at complex regulation. For example, Dbx1 has some 18 Prdm12 peaks flanking its promoter in distances ranging from 2-70 kb.
Among the genes upregulated by Prdm12, En1 and genes such as Slc32a1, Slc6a5 and Nxph2 had no or very low peaks, which is consistent with the idea that they are indirectly regulated by Prdm12.
Overexpression of Prdm12 increases H3K9me2 and H3K9me3 levels in Xenopus embryos (Chen et al., 2015; Matsukawa et al., 2015). To investigate whether Prdm12 methylates histone H3 at lysine 9 at the Dbx1, Nkx6.1 and Nkx6.2 promoters, ChIP-qPCR experiments were performed with anti-H3K9me2 and H3K9me3 antibodies on RA-treated neuralized animal caps overexpressing or not Flag- mPrdm12. Fig. 8B shows an enrichment of H3K9me2 on the Dbx1, Nkx6.1 and Nkx6.2 promoter regions bound by Prdm12 in caps overexpressing Prdm12 compared to controls. An enrichment of H3K9me3 marks was also observed, but only in the Nkx6.1 and Nkx6.2 upstream sequences.
A 5.7 kb DNA fragment of the mouse Dbx1 promoter is sufficient to direct the expression of a LacZ transgene in the intermediate region of the developing spinal cord where Dbx1 is expressed (Lu et al., 1996). This fragment includes a conserved non coding sequence (CNS) present in the human, chicken, Xenopus tropicalis and zebrafish that corresponds to the distal Prdm12 peak identified by ChIP-Seq (data not shown).
As previously reported, when a construct containing this mouse 5.7 kb Dbx1 promoter fragment cloned upstream of a LacZ reporter was electroporated into the chick neural tube, staining was mainly restricted to the intermediate spinal cord where Dbx1 is expressed (Fig. 8C). Upon co-electroporation with Prdm12, LacZ staining was strongly reduced. Thus Prdm12 bound region in the Dbx1 promoter appears functional. Together, these data suggest Prdm12 promotes V1 cell fate through, at least in part, direct repression of Dbx1 and Nkx6 genes.
DISCUSSION
Prdm12 function in vertebrate V1 IN specification
In the mouse, V1 INs play an important role in locomotion (Gosgnach et al., 2006; Zhang et al., 2014). Our Prdm12 knockdown experiments indicate that the aINs, the homologues of V1 INs, are required for swimming movement in Xenopus, as observed for CiA INs in zebrafish (Zannino et al., 2014). Thus, V1 INs have a conserved role in controlling locomotor outputs in vertebrates. Whether changes in these V1 INs occur in the metamorphing frog during the transition from swimming to walking behavior remains to be determined.
As in zebrafish (Zannino et al., 2014), loss of Prdm12 in Xenopus affects En1- expressing V1 INs. En1 is also lost in the spinal cord of Prdm12 mutant mice (unpublished data), indicating that Prdm12 has an evolutionarily conserved role in V1 IN specification in vertebrates. In zebrafish, Prdm12 overexpression does not lead to ectopic V1 neurons (Zannino et al., 2014). In contrast, our data indicate that Xenopus or mouse Prdm12 promotes En1-positive V1 INs when overexpressed in the frog or chick embryo. Human Prdm12 also induces En1 when overexpressed in the frog (Nagy et al., 2015). The reasons for this functional difference between zebrafish and tetrapode vertebrate Prdm12 are unclear and remain to be determined. We also observed that the amphioxus Prdm12 ortholog when overexpressed in the frog does not induce En1. This suggests that Prdm12 function in V1 IN specification has specifically evolved in the vertebrate lineage after the split of the amphioxus and vertebrate lineages. This function of Prdm12 might have however already been present in the last common ancestor of all chordates and then specifically been lost in the lineage leading to extant cephalochordates. We favor the first scenario, given that vertebrate diversification was marked by a significant increase of the diversity of neuronal cell types, which required the elaboration of novel regulatory circuits, mediated, for example, by novel protein functions, as we report here for Prdm12 and as has previously been reported for other Prdm proteins (Vincent et al., 2012).
Prdm12 mechanism of action in V1 INs
Our results suggest that Prdm12 acts in p1 progenitors as a repressor by suppressing Dbx1, Nkx61 and Nkx6.2. Whether Prdm12 can in addition also act as an activator is unclear. Prdm12 ChIP-Seq peaks around genes such as Pax2, FoxD3,
Lhx5 and Lhx1, all expressed in postmitotic V1 INs, have been observed. As 1) Prdm12 is not maintained in postmitotic cells and 2) these ChIP-Seq data have been obtained in conditions of Prdm12 overexpression, whether they correspond to true binding sites is questionable and remains to be investigated. It is not clear why Dbx1, which is increased by VP16-Prdm12 in noggin-injected RA-treated caps, is not upregulated in most Prdm12 morphants and in VP16-Prdm12 overexpressing embryos (data not shown). Dbx1 is also unaffected by the loss of Prdm12 in zebrafish embryos (Zannino et al., 2014). It is possible that Dbx1 is not upregulated in the Prdm12 morphants and in VP16-Prdm12 overexpressing embryos because other TFs cooperate with Prdm12 to exclude Dbx1 from p1 progenitors. The fact that Vsx1 appears unaffected in embryos but is however downregulated in the noggin mRNA injected RA-treated explants is also puzzling.
Our results also indicate that the proneural gene Neurog2 is downregulated by Prdm12 while the negative regulators of the cell cycle p27Xic and the pan-neuronal differentiated marker N-tubulin are upregulated. These findings which are consistent with the observation that Prdm12 has anti-proliferative activity in P19 cells (Yang and Shinkai, 2013) suggest that Prdm12 may play a role in the coordination of the proliferation and differentiation of p1 progenitors.
Our data indicate that Prdm12 acts as a G9a dependent transcriptional repressor in V1 cell specification, as in P19 cells (Yang and Shinkai, 2013). Prdm12 recruits G9a via its second zinc finger (Yang and Shinkai, 2013). While confirming the importance of the zinc fingers for Prdm12 activity, our results suggest a specific role for the PR domain. We speculate that Prdm12 may recruit, via its PR domain, other chromatin modifying proteins to create a repressive state. This has previously been shown for Prdm6 that associates with HDAC1-3 via its PR domain (Davis et al., 2006).
The key differences responsible for the differential function of the amphioxus and vertebrate Prdm12 proteins appear to be located in the C-terminal part of the protein, including the zinc fingers. In a recent study, different homozygous mutations have been identified throughout Prdm12 that all abrogates its histone-modifying activity (Chen et al., 2015). Some of these mutated forms of Prdm12, when overexpressed in Xenopus, exhibit an impaired activity to induce En1 (Nagy et al., 2015). Whether the amphioxus Prdm12 is inactive because it has impaired histone- modifying potential remains to be tested.
Our results indicate that Prdm12 occupies regions in the Dbx1, Nkx61 and Nkx6.2 loci, increases in these regions H3K9 methylation and represses the transcriptional activity of a Dbx1 promoter construct. These observations strongly suggest that Prdm12 acts as a patterning gene in V1 cell fate determination by directly repressing Dbx1 and Nkx6. The high number of postmitotic genes that are upregulated upon Prdm12 overexpression is likely an indirect consequence of its role in progenitors in the induction of V1 and sensory cell lineages. Whether Prdm12 binds directly to DNA or indirectly, via other TFs, is unknown. De novo motif analysis of the Prdm12 peaks revealed a strong enrichment for Sox and homeobox binding motifs (data not shown). Whether Prdm12 binds DNA via these TFs or whether it cooperates with them at these sites remains to be tested.
ACKNOWLEDGEMENTS
The authors acknowledge M. Andreazzoli, F. Clotman, J. Ericson, M. Götz, R.
Harland, C. Logan, B. Mao, A. Moore, A.H. Monsoro-Burq, T. Müller, B. Novitch, Y. Shinkai and E. Winterbottom and K. Wright for reagents, N. Bessodes for experimental help, L. Delhaye for technical assistance and G. Salinas-Riester and T.
Linger for the RNAseq analysis. This work was supported by grants from the FNRS (to E.B. and C.V.L.), from the French ANR (11-JSV2-002-01) to M.S, from the MRC to K.E.L. and from the NIH/NIDDK to M.S. A.T. and C.V.C. are FNRS postdoctoral fellow. S.D. and J. H. are FRIA doctoral fellows.
AUTHOR CONTRIBUTIONS
A.T., S.D., E.B. and C.V.C. designed the experiments and analyzed the data, with contributions from J.H., I.Q., A.R., M.D.B., B.V.D., C.K., T.P. and K.A.H. A.T, S.D., J.H., S.K., E.B. and C.V.C. carried out all the experiments with contributions from I.Q, A.R., M.D.B., F.L., J.C., B.V.D., G.C., J.O.F. and B.B. S.D, E.B. and C.V.C prepare the manuscript incorporating comments from K.L., M.Sa., A.P., C.V.L., M.Sc., J.E.J. and K.A.H.
REFERENCES
Alvarez, F. J., Jonas, P. C., Sapir, T., Hartley, R., Berrocal, M. C., Geiman, E. J., Todd, A. J. and Goulding, M. (2005). Postnatal phenotype and localization of spinal cord V1 derived interneurons. J Comp Neurol 493, 177- 192.
Anders, S., Huber, W. (2010). Differential expression analysis for sequence count data. Genome Biol. 2010;11 : R106.
Anders, S., Pyl, P.T., Huber, W. (2014). HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics. pii: btu638. [Epub ahead of print]
Bang, A. G., Papalopulu, N., Kintner, C. and Goulding, M. D. (1997).
Expression of Pax-3 is initiated in the early neural plate by posteriorizing signals produced by the organizer and by posterior non-axial mesoderm.
Development 124:2075-85.
Baudet, C., Pozas, E., Adameyko, I., Andersson, E., Ericson, J. and Ernfors, P. (2008). Retrograde signaling onto Ret during motor nerve terminal maturation. J Neurosci 28, 963-975.
Bellefroid, E. J., Kobbe, A., Gruss, P., Pieler, T., Gurdon, J. B. and Papalopulu, N. (1998). Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification. EMBO J 17:191-203.
Bel-Vialar, S., Itasaki, N. and Krumlauf, R. (2002). Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development 129, 5103-5115.
Benito-Gonzalez, A. and Alvarez, F. J. (2012). Renshaw cells and Ia inhibitory interneurons are generated at different times from p1 progenitors and differentiate shortly after exiting the cell cycle. J Neurosci 32, 1156-1170.
Borromeo, M. D., Meredith, D. M., Castro, D. S., Chang, J. C., Tung, K. C., Guillemot, F. and Johnson, J. E. (2014). A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord.
Development 141, 2803-2812.
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435-445.
Briscoe, J. and Novitch, B. G. (2008). Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube.
Philos Trans R Soc Lond B Biol Sci 363, 57-70.
Burrill, J. D., Moran, L., Goulding, M. D. and Saueressig, H. (1997). PAX2 is expressed in multiple spinal cord interneurons, including a population of EN1+ interneurons that require PAX6 for their development. Development 124, 4493-4503.
Cerda, G.A., Hargrave, M., Lewis, K.E. (2009). RNA profiling of FAC-sorted neurons from the developing zebrafish spinal cord. Dev Dyn. 238:150-61.
Chang, J. C., Meredith, D. M., Mayer, P. R., Borromeo, M. D., Lai, H. C., Ou, Y. H. and Johnson, J. E. (2013). Prdm13 mediates the balance of inhibitory and excitatory neurons in somatosensory circuits. Dev Cell 25, 182- 195.
Cheesman, S. E., Layden, M. J., Von Ohlen T., Doe C. Q., Eisen J. S.
(2004). Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development. 131:5221-32.
Chen, Y. C., Auer-Grumbach, M., Matsukawa, S., Zitzelsberger, M., Themistocleous, A. C., Strom, T. M., Samara, C., Moore, A. W., Cho, L. T., Young, G. T., Weiss, C., Schabhüttl, M., Stucka, R., Schmid, A. B., Parman, Y., Graul-Neumann, L., Heinritz, W., Passarge, E., Watson, R. M., Hertz, J. M., Moog, U., Baumgartner, M., Valente, E. M., Pereira, D., Restrepo, C. M., Katona, I., Dusl, M., Stendel, C., Wieland, T., Stafford, F., Reimann, F., Von Au, K., Finke C., Willems P.J., Nahorski M.S., Shaikh S.S., Carvalho O.P., Nicholas, A. K., Karbani, G., McAleer, M. A., Cilio, M.
R., McHugh, J. C., Murphy, S. M., Irvine, A. D., Jensen, U. B., Windhager, R., Weis, J., Bergmann, C., Rautenstrauss, B., Baets, J., De Jonghe, P., Reilly, M. M., Kropatsch, R., Kurth, I., Chrast, R., Michiue, T., Bennett, D.
L., Woods, C. G. and Senderek, J. (2015). Transcriptional regulator PRDM12 is essential for human pain perception. Nat Genet. doi:
10.1038/ng.3308.
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C.
(1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375, 761-766.
Chow, R. L., Altmann, C. R., Lang, R. A. and Hemmati-Brivanlou, A.
(1999). Pax6 induces ectopic eyes in a vertebrate. Development 126, 4213- 4222.
Colin, L., Dekoninck, A., Reichert, M., Calao, M., Merimi, M., Van den Broeke, A., Vierendeel, V., Cleuter, Y., Burny, A., Rohr, O. and Van Lint, C. (2011). Chromatin disruption in the promoter of Bovine Leukemia Virus during transcriptional activation. Nucleic Acids Res. 39:9559-73.
Coutinho, P., Pavlou, S., Bhatia, S., Chalmers, K. J., Kleinjan, D. A. and van Heyningen, V. (2011). Discovery and assessment of conserved Pax6 target genes and enhancers. Genome Res 21, 1349-1359.
D'Autilia, S., Decembrini, S., Casarosa, S., He, R. Q., Barsacchi, G., Cremisi, F. and Andreazzoli, M. (2006). Cloning and developmental expression of the Xenopus homeobox gene Xvsx1. Dev Genes Evol 216, 829- 834.
Davis, C. A. and Joyner, A. L. (1988). Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. Genes Dev 2, 1736-1744.
Davis, C. A., Haberland, M., Arnold, M. A., Sutherland, L. B., McDonald, O. G., Richardson, J. A., Childs, G., Harris, S., Owens, G. K. and Olson, E. N. (2006). PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells. Mol Cell Biol 26, 2626- 2636.
Dennis G. Jr., Sherman B.T., Hosack D.A., Yang J., Gao W., Lane H.C., Lempicki R.A. (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 4: P3.
Dessaud, E., McMahon, A. P. and Briscoe, J. (2008). Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network. Development 135, 2489-2503.
Dichmann, D. S. and Harland, R. M. (2011). Nkx6 genes pattern the frog neural plate and Nkx6.1 is necessary for motoneuron axon projection. Dev Biol 349, 378-386.
Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29,15-21.
Dullin, J. P., Locker, M., Robach, M., Henningfled, K. A., Parain, K., Afelik, S., Pieler, T. and Perron, M. (2007). Ptf1a triggers GABAergic neuronal cell fates in the retina. BMC Dev Biol 7:110.
Durinck, S., Spellman, P. T., Birney, E., Huber, W. (2009). Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc. 4,1184-91.
Durston, A. J., Timmermans, J. P., Hage, W. J., Hendriks, H. F., de Vries, N. J., Heideveld, M. and Nieuwkoop, P. D. (1989). Retinoic acid causes an
anteroposterior transformation in the developing central nervous system.
Nature 340, 140-144.
Eizema, K., Koster, J. G., Stegeman, B. I., Baarends, W. M., Lanser, P. H.
and Destrée, O. H. (1994). Comparative analysis of Engrailed-1 and Wnt-1 expression in the developing central nervous system of Xenopus laevis. Int J Dev Biol 38, 623-632.
England S., Batista M.F., Mich J.K., Chen J.K. and Lewis K.E. (2011).
Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons. Development 138, 5121- 5134.
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.
Cell 90, 169-180.
Fog, C. K., Galli, G. G. and Lund, A. H. (2012). PRDM proteins: important players in differentiation and disease. Bioessays 34, 50-60.
Francius, C., Harris, A., Rucchin, V., Hendricks, T. J., Stam, F. J., Barber, M., Kurek, D., Grosveld, F. G., Pierani, A., Goulding, M., Clotman F.
(2013). Identification of multiple subsets of ventral interneurons and differential distribution along the rostrocaudal axis of the developing spinal cord. PLoS One 8, e70325.
Gosgnach, S., Lanuza, G. M., Butt, S. J., Saueressig, H., Zhang, Y., Velasquez, T., Riethmacher, D., Callaway, E. M., Kiehn, O. and Goulding, M. (2006). V1 spinal neurons regulate the speed of vertebrate locomotor outputs. Nature 440, 215-219.
Goulding, M. (2009). Circuits controlling vertebrate locomotion: moving in a new direction. Nat Rev Neurosci 10, 507-518.
Grillner, S. and Jessell, T. M. (2009). Measured motion: searching for simplicity in spinal locomotor networks. Curr Opin Neurobiol 19, 572-586.
Gribble, S. L., Nikolaus, O. B. and Dorsky, R. I. (2007). Regulation and function of Dbx genes in the zebrafish spinal cord. Dev. Dyn. 236, 3472-3483.
Gyory, I., Wu, J., Fejér, G., Seto, E. and Wright, K. L. (2004). PRDI-BF1 recruits the histone H3 methyltransferase G9a in transcriptional silencing. Nat Immunol. 5:299-308.
Hack, M. A., Sugimori, M., Lundberg, C., Nakafuku, M. and Götz, M.
(2004). Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol Cell Neurosci 25, 664-678.
Hanotel, J., Bessodes, N., Thélie, A., Hedderich, M., Parain, K., Driessche, B. V., Brandão, K. D., Kricha, S., Jorgensen, M. C., Grapin- Botton, A., Serup P., Van Lint C., Perron M., Pieler T., Henningfeld K.A., Bellefroid E.J. (2014). The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube. Dev Biol. 386, 340-347.
Heller, N., Brändli, A.W. (1997). Xenopus Pax-2 displays multiple splice forms during embryogenesis and pronephric kidney development. Mech. Dev.
69, 83-104.
Higashijima, S., Masino, M. A., Mandel, G. and Fetcho, J. R. (2004).
Engrailed-1 expression marks a primitive class of inhibitory spinal interneuron.
J Neurosci 24, 5827-5839.
Hohenauer, T. and Moore, A. W. (2012). The Prdm family: expanding roles in stem cells and development. Development 139, 2267-2282.
Holland, L. Z., Holland, P. W. H. and Holland, N. D. (1996). Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. In Molecular Zoology: Advances, Strategies and Protocols (ed. J. D. Ferraris and S. R. Palumbi), pp. 267-282; 473-483. New York, NY: Wiley-Liss.
Hollemann, T., Chen, Y., Grunz, H. and Pieler, T. (1998). Regionalized metabolic activity establishes boundaries of retinoic acid signalling. EMBO J 17, 7361-7372.
Hutchinson, S. A., Cheesman, S. E., Hale, L. A., Boone, J. Q. and Eisen, J. S. (2007). Nkx6 proteins specify one zebrafish primary motoneuron subtype by regulating late islet1 expression. Development. 134, 1671-7.
Ishibashi, S. and Yasuda, K. (2001). Distinct roles of maf genes during Xenopus lens development. Mech Dev. 101:155-66.
Kessler, D. S. (1997). Siamois is required for formation of Spemann's organizer. Proc Natl Acad Sci U S A 94, 13017-13022.
Kinameri, E., Inoue, T., Aruga, J., Imayoshi, I., Kageyama, R., Shimogori, T. and Moore, A. W. (2008). Prdm proto-oncogene transcription factor family expression and interaction with the Notch-Hes pathway in mouse neurogenesis. PLoS One 3, e3859.
Li, W. C., Higashijima, S., Parry, D. M., Roberts, A. and Soffe, S. R.
(2004). Primitive roles for inhibitory interneurons in developing frog spinal cord. J Neurosci 24, 5840-5848.
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R.; 1000 Genome Project Data Processing Subgroup. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics. 25, 2078-2079.
Logan C., Wizenmann A., Drescher U., Monschau B., Bonhoeffer F., Lumsden A. (1996). Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression. Curr Biol. 6(8):1006-14.
Lu, S., Shashikant, C. S. and Ruddle, F. H. (1996). Separate cis-acting elements determine the expression of mouse DBX gene in multiple spatial domains of the central nervous system. Mech Dev. 58:193-202.
Ma, P., Zhao, S., Zeng, W., Yang, Q., Li, C., Lv, X., Zhou, Q. and Mao, B.
(2011). Xenopus Dbx2 is involved in primary neurogenesis and early neural plate patterning. Biochem Biophys Res Commun 412, 170-174.
Ma, P., Xia, Y., Ma, L., Zhao, S. and Mao, B. (2013). Xenopus Nkx6.1 and Nkx6.2 are required for mid-hindbrain boundary development. Dev Genes Evol 223, 253-259.
Ma, L., Quigley, I., Omran, H. and Kintner, C. (2014). Multicilin drives centriole biogenesis via E2f proteins. Genes Dev. 28:1461-71.
Matise, M. P. and Joyner, A. L. (1997). Expression patterns of developmental control genes in normal and Engrailed-1 mutant mouse spinal cord reveal early diversity in developing interneurons. J Neurosci 17, 7805- 7816.
Matsukawa, S., Miwata, K., Asashima, M. and Michiue, T. (2015). The requirement of histone modification by PRDM12 and Kdm4a for the development of pre-placodal ectoderm and neural crest in Xenopus. Dev Biol.
399:164-76.
Missler M. and Südhof T.C. (1998). Neurexophilins form a conserved family of neuropeptide-like glycoproteins. J Neurosci. 18, 3630-3638.
Nagy, V., Cole, T., Van Campenhout, C., Khoung, T.M., Leung, C., Vermeiren, S., Novatchkova, M., Wenzel, D., Cikes, D., Polyansky, A.A., Kozieradzki, I., Meixner, A., Bellefroid, E.J., Neely, G.G., Penninger, J.M.
2015. The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception. Cell Cycle 14, 1799-1808.
Nieto, M. A., Bradley, L. C. and Wilkinson D. G. (1991). Conserved segmental expression of Krox-20 in the vertebrate hindbrain and its relationship to lineage restriction. Development suppl 2:59-62.
Novitch, B. G., Wichterle, H., Jessell, T. M. and Sockanathan, S. (2003). A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron 40, 81-95.
Oulion, S., Bertrand, S., Belgacem, M. R., Le Petillon, Y. and Escriva, H.
(2012) Sequencing and analysis of the Mediterranean amphioxus (Branchiostoma lanceolatum) transcriptome. PLoS One 7, e36554.
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. M. (1999). A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97, 903-915.
Pierani, A., Moran-Rivard, L., Sunshine, M. J., Littman, D. R., Goulding, M. and Jessell, T. M. (2001). Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1. Neuron 29, 367- 384.
Ruiz i Altaba A. (1990). Neural expression of the Xenopus homeobox gene Xhox3: evidence for a patterning neural signal that spreads through the ectoderm. Development 108:595-604.
Rungger-Brändle, E., Ripperger, J. A., Steiner, K., Conti, A., Stieger, A., Soltanieh, S. and Rungger, D. (2010). Retinal patterning by Pax6-dependent cell adhesion molecules. Dev Neurobiol 70, 764-780.
Sander, M., Paydar, S., Ericson, J., Briscoe, J., Berber, E., German, M., Jessell, T. M. and Rubenstein, J. L. (2000). Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates. Genes Dev 14, 2134-2139.
Saueressig, H., Burrill, J. and Goulding, M. (1999). Engrailed-1 and netrin- 1 regulate axon pathfinding by association interneurons that project to motor neurons. Development 126, 4201-4212.
Siembab, V. C., Smith, C. A., Zagoraiou, L., Berrocal, M. C., Mentis, G. Z.
and Alvarez, F. J. (2010). Target selection of proprioceptive and motor axon synapses on neonatal V1-derived Ia inhibitory interneurons and Renshaw cells. J Comp Neurol 518, 4675-4701.
Sive, H. L., Grainger, R. M. and Harland, R. M. (2000). Early development of Xenopus laevis : a laboratory manual. Cold Spring Harbor: CSHL Press.
Smith, S. T. and Jaynes, J. B. (1996). A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141- 3150.
Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos.
Cell 70, 829-840.
Takahashi, M., Osumi, N. (2002). Pax6 regulates specification of ventral neuron subtypes in the hindbrain by establishing progenitor domains.
Development 129, 1327-1338.
Triezenberg, S. J., Kingsbury, R. C. and McKnight, S. L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev 2, 718-729.
Vallstedt, A., Muhr, J., Pattyn, A., Pierani, A., Mendelsohn, M., Sander, M., Jessell, T. M. and Ericson, J. (2001). Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31, 743-755.
Vincent, S. D., Mayeuf, A., Niro, C., Saitou, M., Buckingham, M. (2012).
Non conservation of function for the evolutionarily conserved prdm1 protein in the control of the slow twitch myogenic program in the mouse embryo. Mol Biol Evol. 29, 3181-3191.
Visel, A., Minovitsky, S., Dubchak, I., Pennacchio, L. A. (2007). VISTA Enhancer Browser - a database of tissue-specific human enhancers. Nucleic Acids Res. 35(Database issue):D88-92.
Walton, N. M., De Koning, A., Xie, X., Shin, R., Chen, Q., Miyake, S., Tajinda, K., Gross, A. K., Kogan, J. H., Heusner, C. L., Tamura, K., Matsumoto, M. (2014). Gastrin-releasing peptide contributes to the regulation of adult hippocampal neurogenesis and neuronal development. Stem Cells 32 , 2454-2466.
Wenner, P., O'Donovan, M. J. and Matise, M. P. (2000). Topographical and physiological characterization of interneurons that express engrailed-1 in the embryonic chick spinal cord. J Neurophysiol 84, 2651-2657.
Wilkinson, D. G. and Nieto, M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol 225, 361-373.
Yang, C. M. and Shinkai, Y. (2013). Prdm12 is induced by retinoic acid and exhibits anti-proliferative properties through the cell cycle modulation of P19 embryonic carcinoma cells. Cell Struct Funct 38, 195-204.
Zannino, D. A., Downes, G. B. and Sagerström, C. G. (2014). prdm12b specifies the p1 progenitor domain and reveals a role for V1 interneurons in swim movements. Dev Biol 390, 247-260.
Zhang, J., Lanuza, G. M., Britz, O., Wang, Z., Siembab, V. C., Zhang, Y., Velasquez, T., Alvarez, F. J., Frank, E. and Goulding, M. (2014). V1 and v2b interneurons secure the alternating flexor-extensor motor activity mice require for limbed locomotion. Neuron 82, 138-150.
FIGURE LEGENDS
Fig. 1. Prdm12 expression in Xenopus, chicken and amphioxus embryos. (A-G) (A-G) Whole-mount ISH analysis of Prdm12 expression in Xenopus. In C, E, F transverse sections at the levels indicated in B,D are shown. (H-M) Double ISH comparing Prdm12 with the indicated genes. In panels H, L and M, both Prdm12 and the second probe are revealed in dark blue. In panels I, J and K, Prdm12 is in dark blue and the second probe is in light blue. (N) Diagram summarizing the expression domains of Prdm12 and of HD TFs in the ventricular zone of the Xenopus ventral caudal neural tube. (O) A transverse section of the neural tube of a chick embryo hybridized for Prdm12 and immunostained for Dbx1 (red). (P, Q) Prdm12 and Engrailed are coexpressed in restricted cells of the ventral nerve cord (arrows) of amphioxus larvae (48 hpf). Shown are lateral and dorsal views as well as a transverse section.
Fig. 2. Prdm12 is dependent on RA signaling and Pax6 and is repressed by Dbx1 and Nkx6. (A) RT-PCR analysis of the expression of the indicated genes in animal caps isolated from wild-type or noggin-injected (100 pg/blastomere) embryos, cultivated in the presence or absence of RA. Expression levels were compared to the expression level of uninjected caps, which was defined as 1. (B, C, F, G) Prdm12 expression in Xenopus neurula stage embryos injected unilaterally at the 2 cell-stage with the indicated mRNA (500 pg/blastomere). (D, E, H, K). Prdm12 is downregulated in the spinal cord of E12.5 Pax6Sey/Sey mutants and is expanded in Dbx1LacZ/LacZ and Nkx6.1-/- embryos (brackets) (for all mutant lines, n ≥ 2). IS: injected side.
Fig. 3. Prdm12 knockdown in Xenopus dramatically reduces the V1 marker En1 and affects gene expression in ventral spinal cord progenitors. (A-F) Transverse sections at the neural tube of stage 32 embryos unilaterally injected with 20 ng of Prdm12 MO and hybridized as indicated. The disappearance of the gap between Nkx6.1 and Dbx1 expression in Prdm12 morphants is indicated by an arrow. IS:
injected side.
Fig. 4. Xenopus and mouse Prdm12, but not amphioxus Prdm12, downregulates Dbx1, Nkx6.1 and Nkx6.2 and induces En1 in Xenopus. (A-F) Transverse sections at the neural tube of stage 32 embryos injected unilaterally with mPrdm12 mRNA (200 pg/blastomere) and hybridized with indicated probes. The decrease of Dbx1, Nkx6.1 Nkx6.2 and Evx1 is indicated by arrows. IS: injected side. (G-J) RT-PCR analysis of the expression of indicated genes in RA-treated neuralized animal caps isolated from embryos injected with mouse Prdm12 mRNA (50 or 250pg/blastomere), amphioxus Prdm12 and chimeric constructs mRNA (250 pg/blastomere) as indicated.
In G and H, expression levels were compared to the expression level of uninjected caps, defined as 1.
Fig. 5. Prdm12 function requires both the PR and zinc finger domains and acts as a G9a dependent repressor to induce En1. (A) Diagram of the Flag-mPrdm12 wild-type and mutants. (B) Average number of En1-positive cells detected on the electroporated side of embryos overexpressing the indicated constructs. P < 0.05: *, P
< 0.01: **, P < 0.001: ***; (n ≥ 3). (C) Top panels: scheme of the VP16-Prdm12 and EnR-Prdm12 constructs and transverse sections of the neural tube of embryos overexpressing the indicated constructs (250pg/blastomere for EnR-Prdm12 and 50pg/blastomere for VP16-Prdm12) hybridized with the indicated probes. The upregulation of En1 by EnR-Prdm12, the downregulation of En1 and the upregulation of Nkx6.1 by VP16-Prdm12 are indicated (arrows). Bottom panels: RT-PCR analysis of the expression of the indicated genes in RA-treated neuralized animal caps overexpressing mouse Prdm12 (250pg/blastomere), EnR-Prdm12 (250pg/blastomere) or VP16-Prdm12 (50pg/blastomere) mRNA as indicated. (D) A dominant negative form of G9a (25, 500 and 100 pg mRNA injected) reduces the ability of Prdm12 to induce En1 and slightly attenuates the repression of Dbx1 and Nkx6.1 in RA-treated neuralized animal caps. (E) The mouse-amphioxus Prdm12 chimeric protein aPR- mZF but not the complementary chimeric protein mPR-aZF induces En1 and represses Dbx1 and Nkx6.1 in RA treated neuralized animal caps.
Fig. 6. Dbx1 and Nkx6 overexpression blocks Prdm12 activity and their knockdown upregulates En1. (A) RT-PCR analysis of En1 in neuralized animal caps overexpressing mouse Prdm12 mRNA (250 pg/blastomere), Dbx1, Dbx2, Nkx6.1 or Nkx6.2 mRNA (50 and 250 pg/blastomere) and treated with RA as indicated. (B)
