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The dorsal spinal cord synthesizes a variety of neuropeptides that modulate the transmission of nociceptive sensory information. Here we use genetic fate mapping to show that Tlx3+ spinal cord neurons and their derivatives represent a heterogeneous population of neurons, marked by partially overlapping expression of a set of neuropeptide genes, including those encoding the anti-opioid peptide CCK, pronociceptive Substance P (SP) and Neurokinin B, and a late wave of somatostatin (SOM). Mutations of Tlx3 and Tlx1 result in a loss of expression of these peptide genes. Brn3a, a homeobox transcription factor whose expression is partly dependent on Tlx3, is required specifically for the early wave of SP expression. These studies suggest that Tlx1 and Tlx3 operate high in the regulatory hierarchy that coordinates specification of dorsal horn pain-modulatory peptidergic neurons.
The dorsal horn of the spinal cord is an integrative center that processes and transmits somatic sensory information. Morphological and functional studies have revealed a tremendous diversity of dorsal horn neurons (Christensen and Perl, 1970; Lima and Coimbra, 1986; Todd and Spike, 1993 ; Han et al., 1998 ; Grudt and Perl, 2002; Todd and Koerber, 2006). Diversity of the dorsal horn neurons is further suggested by the expression of neuropeptides, including the opioid-like peptides Dynorphin (DYN) and enkephalin (ENK), the anti-opioid peptide cholecystokinin (CCK), the tachykinin peptides Substance P (SP) and Neurokinin B (NKB), somatostatin (SOM), and others (Marti et al., 1987 ; Todd and Spike, 1993 ; Todd and Koerber, 2006; Polgar et al., 2006 ). Functionally, neuropetides modulate the transmission of somatic sensory information, particularly those involved with pain perception (Kajander et al., 1990; Xu et al., 1993 ; Wang et al., 2001; Wiesenfeld-Hallin et al., 2002).
The past decade has seen important progress in understanding dorsal horn neuron development (Caspary and Anderson, 2003; Helms and Johnson, 2003; Fitzgerald, 2005 ; Ma, 2006). Signals derived from the roof plate pattern the dorsal neural tube, such that precursors are divided into distinct compartments along the dorsoventral axis (Caspary and Anderson, 2003; Helms and Johnson, 2003). Early born neurons (DI1–DI6) migrate ventrally and settle in deep dorsal horn laminae, while late born neurons (DILA and DILB) settle in superficial dorsal horn laminae (Caspary and Anderson, 2003; Helms and Johnson, 2003). With the exception of DI1–DI3 neurons, most dorsal horn neurons express Lbx1 at embryonic stages E11.5–E13.5 (Gross et al., 2002; Müller et al., 2002). Lbx1+ neurons are divided into two populations, based on their non-overlapping expression of the homeobox proteins Pax2 (DI4, DI6 and DILA) versus Tlx3 plus Lmx1b (DI5 and DILB) (Gross et al., 2002; Müller et al., 2002; Cheng et al., 2004). A set of transcription factors acts to specify the excitatory versus the inhibitory neuron cell fates (Cheng et al., 2004; Cheng et al., 2005; Glasgow et al., 2005; Mizuguchi et al., 2006; Hori et al., 2008). Lbx1 determines a basal GABAergic inhibitory neuron cell fate (Cheng et al., 2005). GSH1 and GSH2 control the expression of Tlx3, which in turn antagonizes Lbx1 to determine the glutamatergic excitatory neuron cell fate (Cheng et al., 2004; Cheng et al., 2005; Mizuguchi et al., 2006). Ptf1a acts in combination with RBPjk to suppress Tlx3 expression and to promote GABAergic differentiation (Glasgow et al., 2005; Mizuguchi et al., 2006; Hori et al., 2008).
Despite this progress, transcriptional regulation of neuropeptides in the developing spinal cord is poorly understood. In this study, we used genetic fate mapping to show that Tlx3+ neurons or their derivatives express a set of neuropeptides, including SP, CCK, NKB, and a late wave of SOM. Accordingly, expression of these peptide genes is eliminated in mice that lack Tlx3 and Tlx1. Mechanistically, Tlx1 and Tlx3 activate a variety of downstream transcription factors, including the homeobox protein Brn3a that controls the expression of an early wave of SP expression. Tlx1/3 therefore act as master regulators that coordinate the development of dorsal horn excitatory peptidergic neurons.
The generation of Tlx1 and Tlx3 mutant mice, Lbx1 mutant mice and Brn3a mutant mice has been described previously (Roberts et al., 1994; Shirasawa et al., 2000 ; Gross et al., 2002; Quina et al., 2005 ). The generation of Tlx3Cre knock-in mice is described in Supplementary Figure 1. In order to fate-map Tlx3-expressing neurons, the Tlx3Cre knock-in mice were then crossed with Cre-dependent Rosa26-lacZ reporter mice (Soriano, 1999), as described in Supplementary Figure 2. Tlx3Cre mice were also crossed with another Cre-dependent reporter line, Tau-nLacZ (Hippenmeyer et al., 2005 ), as described in Figure 1. In all timed matings, the morning that vaginal plugs were observed was considered to be E0.5. All animal procedures are contained in protocols reviewed and approved by the Animal Care Committees at the Dana-Faber Cancer Institute (DFCI), Harvard Medical School.
Detailed methods for single and double color in situ hybridization (see Supplementary Figure 4) have been described previously (Qian et al., 2001 ). The following mouse in situ probes were amplified with gene-specific sets of PCR primers and cDNA templates prepared from P0 or P7 mouse brain/spinal cords, including Sst (NM_009215, 0.5 kb), Tac1 (NM_009311, 0.85 kb), CCK (NM_031161, 0.34 kb), Pdyn (NM_018863, 0.7 kb), Penk1 (NM_001002927, 0.63 kb), and Tac2 (D14423, 0.44 kb). Chick Tac1 (BI395005, 0.4 kb) and chick CCK (NM_001001741, 0.4kb) was amplified from cDNA from E10 chick spinal cord. In situ probes for dorsal horn functional genes (Supplementary Figure 7) were described previously (Qian et al., 2002; Cheng et al., 2004). To produce double-color in situ hybridization (Supplementary Figure 4), the first in situ hybridization signal (purple, with NBT/BCIP substrates) was photographed, followed by the development of the second signal (brown, with INT/BCIP as substrates). This sequential photographic process is helpful in determining whether a cell expresses a single gene or two genes.
The following antibodies were used for single or double immunostaining, rabbit anti-Pax2 antibody (Zymed Laboratories Inc.), rabbit anti-Brn3a antibody (E. Turner, University of California, San Diego), and guinea pig anti-Lbx1, rabbit anti-Tlx3, and guinea pig anti-Tlx3 antibodies (T. Müller and C. Birchmeier, Max-Delbrück-Centrum for Molecular Medicine).
For in situ hybridization combined with fluorescent immunostaining, in situ hybridization was first performed without proteinase K treatment. After post-hybridization washing, Tlx3, Pax2, Lbx1 or Brn3a proteins were detected by incubation with appropriate antibodies and then with Alexa-488 conjugated secondary antibody (1:200, Molecular Probes) in PBT solution. After the fluorescent signals were photographed, sections were incubated with alkaline phosphotase-conjugated anti-digoxigenin antibody, followed by development of the in situ hybridisation signal with NBT/BCIP substrates. The bright field views of the in situ hybridization images were inverted, and then merged with the fluorescent images. This process avoids the masking of low-level fluorescent signals by non-fluorescent in situ signals.
Immunostaining combined with X-gal staining was also performed sequentially, with the immunostaining done first. The bright field images of X-gal staining were inverted, and then merged with the immunofluorescence images, thus avoiding the masking of low-level fluorescent signals by lacZ staining signals. In situ hybridization combined with X-gal staining was performed by a similar sequential process, with the X-gal staining performed and photographed first.
For electroporation studies in chick embryos, a cDNA fragment encoding a Myc-tagged mouse Brn3a fusion protein was cloned to the RCASBP chick viral expression vector (Morgan and Fekete, 1996), to produce the construct RCAS-Brn3a. The purified plasmid DNA was re-suspended at concentrations of 5 μg/μl. RCAS-Brn3a plus a GFP expression vector, pCAX-IRES-GFP or pCAX-GFP (Gross et al., 2002), were co-injected into the spinal neural tubes of E2 chick embryos. After electroporation, the embryos were incubated at 39 °C for a further 72–120 h (E5–E7). Embryos with a high level of GFP fluorescence were fixed, and changes in the expression of genes of interest in the spinal cord were analyzed.
To count neurons that express Tac1, Sst, Tac2, and Pemk1 per thoracic spinal section of E14.5 or E18.75 wild-type and Tlx1/3 double mutants, 3 sets of thoracic transverse sections from 3 pairs of wild-type and mutant embryos (14-μm thickness) were hybridized with probes derived from the cDNAs for each peptide. Positive cells with clear nuclear morphology in the dorsal spinal cord were counted. Values were presented as mean ± s. d.. The differences in values were considered to be significant at P < 0.05 by Student’s t-test. To determine the percentage of Tlx3+, Pax2+ or Lbx1+ neurons that express a peptide gene, we again only counted those isolated cells with clear nuclear morphology.
Tlx3 exhibits dynamic expression in the developing spinal cord (Qian et al., 2002). To follow the fate of those neurons derived from Tlx3+ cells, we generated a Tlx3Cre knock-in mouse line, in which the Cre recombinase gene was inserted into the first coding exon of the Tlx3 locus (Supplementary Figure 1). To determine whether Tlx3Cre expression faithfully reflects in vivo Tlx3 expression, we crossed Tlx3Cre mice with a Cre-dependent lacZ reporter line, ROSA26-LacZ (Supplementary Figure 2) (Soriano, 1999). In ROSA26-LacZ (Tlx3Cre) mice, Cre-mediated removal of a transcriptional termination cassette allows a constitutive expression of the lacZ protein product, beta-galactosidase (Soriano, 1999). Consequently, all derivatives that undergo successful Cre-mediated DNA recombination are labeled by X-gal staining (also called lacZ staining). Examined at E11.5, ROSA26-LacZ (Tlx3Cre) embryos exhibited a LacZ staining pattern that matched endogenous Tlx3 expression revealed by whole mount in situ hybridization (Supplementary Figure 2), demonstrating that Tlx3Cre mice are an effective tool for fate mapping experiments.
To facilitate fate-mapping experiments, we next crossed Tlx3Cre mice with another Cre-dependent reporter mouse line, Tau-nlacZ, with the resulting double heterozygous mice referred to as Tau-nlacZ(Tlx3Cre) mice. Upon Cre-mediated removal of a transcriptional termination cassette, this reporter gene encodes beta-galactosidase linked to a nuclear localization signal (nLacZ) and is driven from the pan-neuronal Tau promoter (Figure 1A) (Hippenmeyer et al., 2005 ). In Tau-nlacZ(Tlx3Cre) mice at postnatal day 7 (P7), X-gal staining showed that nlacZ+ neurons were enriched in the dorsal spinal cord, but also present in small numbers in the ventral spinal cord (Figure 1B). Tlx3+ cells normally give rise to glutamatergic neurons that are intermingled with inhibitory interneurons marked by the expression of Pax2 (Cheng et al., 2004). Consistent with this, virtually no nlacZ+ neurons coexpressed Pax2 (Figure 1C), providing a key validation of the fidelity of nlacZ expression in Tau-nlacZ(Tlx3Cre) mice. Double staining of Tlx3 protein and nLacZ showed that neurons with persistent Tlx3 expression (nlacZ+;Tlx3+) were enriched in the superficial dorsal horn, whereas neurons with transient Tlx3 expression (nlacZ+;Tlx3−) were distributed throughout the spinal cord but are enriched in areas from deep dorsal horn laminae to the ventral spinal cord (Figure 1D). Transient Tlx3 expression in a subset of dorsal horn neurons is consistent with the previous finding that Tlx3 expression is switched off in DI3 and a portion of DI5 neurons that settle in deep dorsal horn laminae (Qian et al., 2002).
Most dorsal horn neurons, including Tlx3+ excitatory neurons and Pax2+ inhibitory neurons, develop from Lbx1+ cells (Gross et al., 2002; Müller et al., 2002). In P7 spinal cord of Tau-nlacZ(Tlx3Cre) mice, Lbx1 protein, however, was virtually absent in a majority of nlacZ+ neurons (Figure 1E), as well as in most Pax2+ neurons (data not shown), implying a transient Lbx1 expression in most dorsal horn neurons. Residual Lbx1+ neurons were located in an intermediate dorsal horn lamina, most of which derive from Tlx3+ neurons, as indicated by the coexpression of Lbx1 and nlacZ (Figure 1E, arrows). In summary, both Tlx3 and Lbx1 exhibit dynamic expression in the developing spinal cord.
We next examined the expression of the following six peptide genes: Tachykinin 1 or Tac1 encoding the precursor for the Substance P (SP) and Neurokinin A (NKA), Tachykinin 2 or Tac2 encoding the precursor for the Neurokinin B (NKB), Cholecystokinin or CCK encoding the precursor for CCK peptides, Somatostatin or Sst encoding the precursor for Somatostatin (SOM), Prodynorphin or Pdyn encoding the precursor for dynorphin (DYN), and Preproenkephalin 1 or Penk1 encoding the precursor for enkephalin (ENK). In the remaining text, we will refer to these genes as Tac1, Tac2, CCK, Sst, Pdyn, and Penk1.
Figure 2 shows the spatial and temporal expression patterns of these peptide genes. Several features are noteworthy. First, expression of different peptide genes is established at distinct developmental stages. In the dorsal spinal cord, Sst expression starts at E11.5, followed by Tac1 expression at E12.5, CCK expression at E14.5, Pdyn and Penk1 expression at E16.5–P0, and finally Tac2 expression at P5–P7 (Figure 2, data not shown). Second, as previously reported (Todd and Spike, 1993 ), each peptide gene exhibits a unique lamina-specific expression pattern (Figure 2). Specifically, in the P7 spinal cord, Sst expression is enriched in superficial laminae but is also widely distributed, Tac2 and CCK expression is confined to the intermediate laminae, Tac1 expression is enriched in the deep laminae, Pdyn expression is enriched in superficial laminae, and Penk1 expression is widely distributed (Figure 2).
To better understand the relationship between transcriptional regulators and neuropeptide phenotype in the dorsal spinal cord, we undertook a series of double staining experiments that combined in situ hybridization with peptide cDNAs as the probes and immunostaining with Tlx3 or Pax2 antibodies (Figures 3 and and4).4). Tac2 expression was confined to a subset of Tlx3+ neurons in intermediate laminae of P7 dorsal spinal cord (Figure 3A). Only a portion of Tac1-expressing neurons expressed Tlx3 at E13.5 (Figure 3B). At P0 or P7, about 22.5% (124/551) of Tac1-expressing neurons and 21.2% (95/442) of CCK-expressing neurons coexpressed Tlx3 (Figure 3C and 3D). However, in P7 Tau-nlacZ(Tlx3Cre) fate mapping mice, Tac1 and CCK expression was confined exclusively to nLacZ+ neurons (Figure 3E and 3F), implying that all Tac1-expressing and CCK-expressing neurons are derived from Tlx3+ neurons, but Tlx3 expression is transient in most of these peptidergic neurons.
Sst exhibited a more complex expression pattern. At E13.5, about 65.3% (68/104) of Sst-expressing cells in the dorsal spinal cord expressed Pax2 (Figure 4B), but none of them expressed Tlx3 (Figure 4C). At P7, cells coexpressing Pax2 and Sst were confined to deep laminae (Figure 4E). At this stage, a new population of Sst-expressing neurons was detected in superficial laminae that coexpressed Tlx3 (Figure 4F). Further examination of Sst expression in Tau-nlacZ(Tlx3Cre) mice showed that a majority of Sst-expressing neurons in superficial laminae were nlacZ+ (Supplementary Figure 3), and were thus derived from Tlx3+ neurons. Therefore, early and late waves of Sst-expressing neurons are primarily associated with Pax2+ and Tlx3+ neurons (and their derivatives), respectively, although some Sst-expressing neurons may develop from cells that do not express Pax2 or Tlx3. Pdyn and Penk1 were expressed exclusively in neurons that coexpressed Pax2 (Figure 4H and 4K), but not Tlx3 (Figure 4I and 4L).
In summary, Tlx3+ neurons or their derivatives express Tac1, Tac2, CCK, and a late wave of Sst, whereas Pax2+ neurons express a different set of peptide genes, including Pdyn, Penk1, and an early wave of Sst (summarized in Figure 4M). Double-color in situ hybridizations further showed that Tac1, Tac2, CCK, and Sst exhibited partially overlapping expression patterns (Supplementary Figure 4), thereby revealing a tremendous diversity of dorsal horn peptidergic neurons.
We next analyzed peptide gene expression in mice that lacked both Tlx3 and its related gene Tlx1 because Tlx3 and Tlx1 exhibit a partial redundancy in cervical and thoracic spinal cord (Cheng et al., 2004). Expression of Tac1 and CCK in the dorsal spinal cord was virtually eliminated in Tlx1/3−/− mice, from E13.5 to E18.75 (Figure 5A–D, also see Figure 6). Because increased cell death has not been observed in Tlx1/3−/− spinal cords during embryonic development (Qian et al., 2002), the simplest interpretation of these results is that Tlx1/3 are required to establish these peptidergic transmitter phenotypes.
Tlx1/3, however, exerted both negative and positive effects on Sst expression. At E14.5, the number of Sst-expressing neurons in dorsal thoracic spinal cord increased by 5 fold in Tlx1/3−/− embryos compared with wild-type embryos (Figure 5I, 5J and 5M), and most of these Sst-expressing neurons were confined to the intermediate and deep dorsal laminae (Figure 5J). We previously reported that there is a marked increase of Pax2+ neurons in Tlx1/3−/− spinal cord (Cheng et al., 2004). Surprisingly, a double staining of Sst and Pax2 showed that only 28.2% of Sst-expressing neurons in E14.5 Tlx1/3−/− dorsal horn coexpressed Pax2 (Supplementary Figure 5), implying that most of these ectopic Sst-expressing neurons were derived from Tlx1/3−/− cells that are incapable of switching on Pax2 expression. A potential source could be DI3 interneurons that express Tlx3, but not Lbx1, which is required for Pax2 expression (Helms and Johnson, 2003; Fitzgerald, 2005).
By E18.75, Sst expression in the superficial dorsal horn, which is largely derived from Tlx3+ neurons, was eliminated in Tlx1/3−/− mice (Figure 5K and 5L), whereas Sst expression in deep laminae was not affected (Figure 5L). As a result of this, there is a 5-fold reduction in the number of Sst-expressing neurons in the dorsal spinal cord of E18.75 Tlx1/3−/− mice compared with wild-type mice (Figure 5M). These data suggest a dual function of Tlx1/3: activating and repressing Sst expression in superficial and deep dorsal horn laminae, respectively.
Expression of Pdyn and Penk1, which are confined to Pax2+ cells in wild-type embryos, did not exhibit obvious changes in E18.75 Tlx1/3−/− mice (Figure 5E–H). The number of Penk1-expressing cells per dorsal horn section at thoracic axial levels was 115 ± 12 in wild-type mice and 123 ± 17 in Tlx1/3−/− mice (P > 0.5). The numbers of Pdyn-expressing cells in E18.75 dorsal spinal cord were also comparable, 65 ± 6 in wild-type mice versus 69 ± 4 in Tlx1/3−/− mice. However, the distribution of Pdyn-expressing cells may have been slightly affected, with an apparent increase of the density of Pdyn-expressing cells in the superficial laminae (Figure 5H vs. 5G). We previously showed that mutations of Tlx1 and Tlx3 result in a transformation of glutamatergic neurons into Pax2+ GABAergic neurons (Cheng et al., 2004). The lack of a significant increase of Pdyn-expressing and Penk1-expressing neurons suggests an incomplete switch in cell fate.
In summary, Tlx1/3 are required to establish the expression of a set of peptide genes, including Tac1, CCK and Sst.
Tlx1/3 acts to antagonize Lbx1 to specify the glutamatergic transmitter phenotype in dorsal horn excitatory neurons (Cheng et al., 2005). Accordingly, a loss of the expression of Slc17a6, which encodes the vesicular glutamate transporter VGLUT2 and the specific marker for dorsal horn glutamatergic neurons, in Tlx3−/− embryos is restored in Tlx3−/−;Lbx1−/− embryos (Cheng et al., 2005). To determine if peptide transmitter phenotypes are established in a similar way, we analyzed peptide gene expression in Tlx3−/− and Lbx1−/− single knockout mice and Tlx3−/−;Lbx1−/− double knockout mice at E14.5. E14.5 was chosen because cell death occurs after E14.5 in the caudal spinal cord of Lbx1 mutant mice (Gross et al., 2002; Cheng et al., 2005). In addition, lumbar spinal cords were analyzed because Tlx3, but not Tlx1, operates at this axial level (Cheng et al., 2005).
Expression of Tac1 was eliminated in E14.5 Tlx3−/− mice (Figure 6A vs. 6B), but not affected in Lbx1−/− mice (Figure 6A vs. 6C). Furthermore, unlike a restoration of VGLUT2 expression (Cheng et al., 2005), Tac1 expression was not recovered in Tlx3−/−;Lbx1−/− mice (Figure 6D), suggesting that Tlx3 controls Tac1 expression through an Lbx1-independent pathway. More surprisingly, CCK expression was eliminated in Tlx3−/− and Lbx1−/− single knockout mice and in Tlx3−/−;Lbx1−/− double knockout mice (Figure 6E–6H ), suggesting that both Lbx1 and Tlx3 are required for the expression of CCK. Tlx3 therefore uses distinct pathways to specify glutamate and peptide transmitters.
Tlx1/3 are required for the expression of a set of transcription factors in the dorsal spinal cord (Qian et al., 2002). We hypothesized that Tlx1/3 might use these downstream transcription factors to control the expression of peptide genes. To test this hypothesis, we examined the expression of neuropeptide genes in mice with a null mutation of the Pou4f1 gene, encoding the Brn3a homeobox transcription factor (Quina et al., 2005 ).
Brn3a was expressed primarily in deep dorsal horn laminae and to a lesser extent in the most superficial laminae in P0 spinal cord (Figure 7A and 7B). Double immunostaining showed that only a portion of Brn3a+ neurons coexpressed Tlx3 (Figure 7A, arrow). This is consistent with previously demonstrated Brn3a expression in early-born DI1 and DI2 interneurons that lack Tlx3 expression (Gowan et al., 2001; Qian et al., 2002; Helms and Johnson, 2003). Accordingly, Brn3a expression was largely, but not completely, eliminated in Tlx1/3−/− mice (Figure 7B).
Since Tac1 is also expressed predominantly in deep dorsal horn laminae, we performed a double staining of Brn3a protein and Tac1 mRNA in the developing spinal cord. We found that at E12.5, virtually all Tac1-expressing neurons in the intermediate level of the spinal cord coexpressed Brn3a, but only a fraction of Brn3a+ neurons exhibited Tac1 expression (Figure 7C). From E12.5 to P0, a new population of Tac1-expressing neurons that did not express Brn3a emerged (Figure 7C).
Consistent with this expression pattern, Tac1 expression was virtually eliminated in the caudal spinal cord of E12.5 Brn3a−/− embryos (Figure 7D), but only reduced in E14.5 Brn3a−/− spinal cord (Figure 7D), suggesting a specific role of Brn3a in controlling the early wave of Tac1 expression.
Because of incomplete loss of Brn3a expression in Tlx1/3−/− mice (Figure 7B), two distinct models may explain a loss of Tac1 expression in both Tlx1/3−/− and Brn3a−/− mice. First, Tlx1/3 and Brn3a act in a cascade to control Tac1 expression (in other words, Tac1 is established in cells in which Brn3a expression is dependent on Tlx1/3). Second, Tlx1/3 and Brn3a act in combination, meaning that Tac1 expression is established in Tlx3+;Brn3a+ neurons in which Brn3a expression is independent of Tlx3. To help to distinguish these models, we analyzed Tac1 and Brn3a expression in E12.5 wild-type and Tlx1/3−/− embryos. At this stage, Tac1 expression was confined to a lateral region in the middle of the wild-type spinal cord (Supplementary Figure 6). In Tlx1/3−/− embryos, expression of both Tac1 and Brn3a was eliminated from this lateral region, whereas Tlx3-independent Brn3a expression was located in a dorso-medial area (Supplementary Figure 6). This data is more consistent with the first model that Tlx3 and Brn3a may act sequentially to control Tac1 expression.
Expression of other Tlx3-dependent genes, including CCK, Sst, the Gria2 glutamate receptor gene (Cheng et al., 2004) and the TRPC3 transient receptor potential channel gene (Li et al., 2006 ), was not grossly affected in Brn3a mutants (Supplementary Figure 7), suggesting a specific role of Brn3a in controlling the early wave of Tac1 expression.
To determine if Brn3a is sufficient to promote Tac1 expression, we performed gain-of-function analyses by using chick electroporation technique (Itasaki et al., 1999). Electroporation of a Brn3a expression vector, RCAS-Brn3a, in E2 chick neural tubes resulted in an induction of Tac1 expression at E5 (Figure 8B) and even more at E7 (Figure 8D). Electroporation with control vectors did not affect Tac1 expression (Figure 8E and 8F), suggesting that Tac1 induction by RCAS-Brn3a electroporation was not caused by side effects associated with proviral vector electroporation (Hermann and Logan, 2003 ). As the case in wild-type mouse spinal cord (Figure 7C), only a portion of Brn3a+ neurons coexpressed Tac1 (data not shown), suggesting that Brn3a needs a specific cellular context to activate Tac1.
This study suggests that dorsal horn peptidergic neurons emerge from distinct neuronal populations. Expression of Tac1, Tac2, CCK, and the late wave of Sst is confined to Tlx3+ neurons or their derivatives, and the development of these peptidergic neurons is compromised in mice that lack Tlx3 and Tlx1. Expression of Pdyn, Penk1, and a portion of early wave Sst is restricted to Pax2+ neurons, and their development is independent of Tlx1 or Tlx3. Our data also suggest that some Sst-expressing neurons may develop from cells that do not express Tlx3 or Pax2. Tlx3 and Pax2 are associated with excitatory and inhibitory neurons, respectively, at least at embryonic stages (Cheng et al., 2004). Consistently, neurons that produce Neurokinin B (the product of Tac2), Substance P (the product of Tac1) and a late wave of SOM (the product of Sst) belong to glutamatergic excitatory neurons (Proudlock et al., 1993; Todd et al., 2003; Todd and Koerber, 2006; Polgar et al., 2006 ). Also consistent with an association with Pax2+ neurons, a small number of SOM+ neurons located in the deep dorsal horn are inhibitory neurons (Proudlock et al., 1993).
Tlx1/3 are known to antagonize Lbx1 to control the expression of VGLUT2, the vesicular glutamate transporter and the specific marker for dorsal horn glutamatergic neurons (Todd et al., 2003; Cheng et al., 2004; Fremeau et al., 2004). Loss of VGLUT2 expression in Tlx3 mutant mice is restored in Tlx3−/−;Lbx1−/− double mutants (Cheng et al., 2005). However, expression of the Tlx1/3-dependent peptide genes is not restored in Tlx3−/−;Lbx1−/− double mutants, implying that Tlx1/3 use distinct pathways to coordinate glutamate and peptide transmitters. A separate control of these transmitters is supported by the fact that all dorsal horn excitatory neurons use glutamate as a fast transmitter, whereas individual peptide transmitters are confined to a small subset of dorsal horn neurons.
The development of Tlx1/3-dependent peptidergic neurons is subject to complex genetic control. The early, but not late, Tac1 expression is dependent on Brn3a. Moreover, Tlx1/3 can exert both positive and negative effects on Sst expression in different dorsal horn lamina. A surprising result is that CCK expression is dependent on both Lbx1 and Tlx3, despite that Tlx3 antagonizes Lbx1 to control VGLUT2 expression. One potential solution for these seemly conflicting Tlx3 activities is that glutamate and CCK transmitter phenotypes are established at distinct stages. VGLUT2 expression is established soon after cells exit from the cell cycle, and at this stage Tlx3 acts to remove an inhibitory effect of Lbx1 on VGLUT2 expression (Cheng et al., 2005). CCK expression, however, is established at E14.5 (Figure 2), when Tlx3 protein has been extinguished in most CCK-expressing neurons (Supplementary Figure 8). Our hypothesis is that Tlx3 extinguishment allows Lbx1 to escape a Tlx3-mediated inhibition, and Lbx1 might in turn act together with an unknown Tlx3-dependent event (established at earlier stages) to control CCK expression.
Our findings argue that Tlx1/3 act as “master regulators” in coordinating dorsal horn excitatory neuron development. Virtually all known functional genes that are preferentially expressed in glutamatergic neurons within the dorsal spinal cord are eliminated in Tlx1/3−/− mice, including VGLUT2 (Cheng et al., 2004), the glutamate receptor gene Gria2 (Kerr RC, 1998; Cheng et al., 2004), the channel gene TRPC3 (Li et al., 2006), and a set of peptide genes described in this study. Tlx1/3 activate a set of downstream transcription factors, some of which appears to control a portion of Tlx1/3-dependent differentiation programs (Figure 9). For example, Brn3a is required for the early wave of Tac1 expression, but is dispensable for the expression of other Tlx1/3-dependent genes (Supplementary Figure 6). DRG11, encoded by the homeobox gene Prrxl1 (Saito et al., 1995 ; Chen et al., 2001), is required for the expression of TRPC3 (Li et al., 2006), but not Gria2 or any category I peptide genes (Lopes et al., unpublished data). Finally, other Tlx1/3-dependent transcription factors, such as Islet1, Phox2a, and EBF2 are all expressed in a fraction of dorsal horn neurons (Tiveron et al., 1996; Qian et al., 2002; Li et al., 2006), and they therefore likely contribute to specification of other specialized dorsal horn neuron subtypes. One challenging unsolved question is to understand how Tlx1/3 are able to activate distinct downstream differentiation programs in distinct dorsal horn neuron contexts.
One important concept in developmental biology is that specification of individual neuronal cell types is controlled by a unique combination of transcription factors (TFs), or combinatorial TF codes (Shirasaki and Pfaff, 2002; Thor and Thomas, 2002 ). However, after late neuronal phenotypes are analyzed, it becomes increasingly evident that a TF code established in newly born neurons specifies more than one neuronal cell type. As aforementioned, a set of peptidergic neurons and other excitatory neurons in the superficial dorsal horn develop from DI5/DILB neurons that share the same TF code, by coexpressing Tlx3 and Lmx1b (Gross et al., 2002; Müller et al., 2002; Cheng et al., 2004), and Tlx3 coordinates the development of these neurons. In the ventral spinal cord, Engrailed1+ V1 interneurons are composed of multiple neuron subtypes involved with locomotion controls (Sapir et al., 2004 ; Alvarez et al., 2005). In dorsal root ganglia, the Runx1 runt domain transcription factor is required for the development of a variety of nociceptive sensory neurons (Chen et al., 2006; Ibanez and Ernfors, 2007 ; Marmigere and Ernfors, 2007 ; Woolf and Ma, 2007 ). The emerging theme is that a TF code established in newly formed neurons coordinates specification of a heterogeneous group of neurons that carry out related physiological functions, thereby implying a modular control of the development of the mammalian nervous system.
We thank Drs. Senji Shirasawa and Stan Korsmeyer for Tlx1 and Tlx3 null mice, Silvia Arber for Tau-nlacZ reporter mice, Philippe Soriano for ROSA26-lacZ reporter mice, Mengqing Xiang for early phase analysis of Brn3a mutant phenotype, and Thomas Muller and Carmen Birchmeier for Lbx1 and Tlx3 antibodies. We also thank Drs. Jane Johnson and Chuck Stiles for critical reading of this manuscript. This work is supported by NIH grants from NINDS (R01NS47710 and P01NS047572). CL is supported by a visiting student scholarship from Portugal and QM is a Claudia Adams Barr Scholar.