Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics
© Seredick et al.; licensee BioMed Central Ltd. 2012
Received: 31 August 2012
Accepted: 16 October 2012
Published: 5 November 2012
Precise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons.
Zebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology.
Our results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.
KeywordsZebrafish Mnx Motoneuron Interneuron
Caudal primary motoneuron
Middle primary motoneuron
Motor neuron and pancreas homeobox
Progenitor of motoneuron
Rostral primary motoneuron
Ventral lateral descending.
The ability of an animal to carry out behavior depends on precise innervation of each muscle by the appropriate motoneuron subtype. Motoneuron subtype identity is specified by the combination of transcription factors expressed by a cell during its differentiation, and recognized by characteristic features, such as soma position, axon trajectory and muscle innervation pattern. Although specification of motoneuron subtype identity has been well-studied [1, 2], we still have an incomplete picture of the molecular mechanisms regulating this process. Here we take advantage of the ability to recognize individual primary motoneurons (PMNs) in the spinal cord of embryonic zebrafish to explore the roles of Mnx family transcription factors in motoneuron subtype specification.
Spinal cord neurons develop from distinct progenitor domains defined by expression of specific transcription factors [2, 3]. Zebrafish PMNs are derived from the progenitor of motoneuron (pMN) domain  and comprise three subtypes: CaP, MiP and RoP, each of which can be distinguished based on soma position, axon trajectory and muscle innervation . A fourth PMN, VaP, is variably present, initially equivalent to CaP, and later typically dies [6, 7]. Here we focus on CaP, which innervates ventral myotome and MiP, which innervates dorsal myotome. Initially both CaP and MiP express Islet1, a transcription factor required for PMN development. In the absence of Islet1, PMNs develop axon trajectories and express the neurotransmitter characteristic of VeLD interneurons , which are also derived from the pMN domain . Later in development, CaP down-regulates Islet1 and expresses a related protein, Islet2a. MiP also down-regulates Islet1, but then re-expresses it about an hour later [9, 10]. The second phase of Islet1 expression is regulated by Nkx6 transcription factors. In the absence of Nkx6 proteins, MiP axon formation begins normally with the extension of a ventral axon to the muscle pioneers, an identified set of muscle fibers that separate dorsal and ventral muscle . However, MiP then fails to extend its normal axon collateral to dorsal muscle, and instead develops an interneuron-like axon within the spinal cord . This interneuron-like axon often resembles axons of V2a interneurons [12, 13] that are derived from the p2 domain situated just dorsal to the pMN domain . The p2 domain, which generates excitatory V2a and inhibitory V2b neurons, has been shown to be closely related to the pMN domain based on shared expression of a number of transcription factors .
The vertebrate Mnx family comprises homeodomain transcription factors originally isolated in human and subsequently isolated in chick and mouse . Mnx2 (previously called MNR2 and Hlxb9l ) was isolated from a single chick cell induced to become a motoneuron . Mnx2 is expressed in motoneuron progenitors and in post-mitotic motoneurons. Ectopic expression of Mnx2 is sufficient to induce motoneuron differentiation in Islet1-positive spinal cord neurons; whether Mnx2 is necessary for motoneuron differentiation has not been tested. Mnx1 (previously called Hb9 and Hlxb9 ) was isolated in mice and shown to be necessary for normal differentiation of many motoneurons [16, 17]. In its absence, motoneurons still project axons to the periphery, but the axon projections are abnormal and the cells inappropriately express a marker of V2a interneurons . In both chick and mouse, Mnx proteins are exclusive to motoneurons at early stages of spinal cord development [15–17]; however, later in development Mnx1 is expressed in a small set of interneurons .
We provide evidence for a novel role of Mnx proteins in zebrafish motoneuron subtype specification. Zebrafish have three Mnx proteins, Mnx1 and two co-orthologs of Mnx2, Mnx2a and Mnx2b , all of which are expressed primarily in post-mitotic neurons. We show that each Mnx family member is expressed in a distinct pattern in each PMN subtype, and that this pattern is dynamic during PMN differentiation. In contrast to early developmental stages in chicks and mice when Mnx expression within the spinal cord is exclusive to motoneurons [15–17], two zebrafish Mnx family members are expressed in VeLD interneurons. We used morpholino antisense oligonucleotides (MOs ) to knock down Mnx function and found, to our surprise, that CaPs and VeLDs developed normally. In contrast, Mnx proteins are required for normal MiP development. In their absence, the second phase of MiP Islet1 expression is initiated at the appropriate time, but is lost a few hours later. MiPs in mnx MO-injected embryos express markers of V2a interneurons, similar to what has been reported in mouse mutants [16, 17]. However, in contrast to mouse mutants, these MiPs also developed V2a-like axons in addition to peripheral axons projecting to muscle. Surprisingly, the peripheral axons of these MiPs did not extend to their normal dorsal muscle targets, but instead projected ventrally alongside normal CaP axons. In some cases, MiPs appeared to be entirely transformed to a CaP morphology in the absence of Mnx proteins. These studies identify Mnx proteins as essential in preventing MiPs from expressing characteristics of V2a interneurons. They also reveal an unexpected role for Mnx proteins in specifying MiP subtype identity by preventing MiPs from developing as CaPs.
mnx family genes are dynamically expressed in primary motoneurons and VeLD interneurons
We characterized expression of mnx1, mnx2a and mnx2b within the zebrafish spinal cord using RNA in situ hybridization. To determine the specific PMNs that express each mnx gene, we simultaneously labeled islet1 mRNA, which is initially expressed in all PMNs  and each of the mnx family members. At later stages we included either islet1, which is expressed in RoP and MiP, or islet2a, which is expressed in CaP and VaP . After 20 hpf, islet1 and islet2a are also expressed by smaller, more ventrally-located secondary motoneurons [9, 22, 23] that we excluded from our analyses.
In addition to expression in PMNs, mnx1 and mnx2b are also expressed in a slightly more dorsal cell first visible at about 14 hpf (Figure 1A, I). mnx1 expression persisted in this cell through 24 hpf, but mnx2b expression was extinguished around 20 hpf. The position and early appearance of these mnx1 + mnx2b + cells suggested that they were VeLD interneurons, which can be uniquely identified based on their lateral position, soma shape, and axon trajectory [4, 24–26]. To test this, we labeled VeLDs by injecting the UAS:GFP plasmid into Tg(mnx1:GAL4) embryos and verified that they expressed Mnx1 by immunohistochemistry (Figure 1N). We also showed that mnx1 is co-expressed with the VeLD marker gad (glutamic acid decarboxylase, an enzyme in the synthetic pathway for the neurotransmitter GABA; [8, 27–29]), but not with vglut (vesicular glutamate transporter), a marker of excitatory, glutamatergic V2a interneurons  (Additional file 1: Figure S1).
To explore the possibility that mnx genes are expressed by other interneurons with descending axons we looked for co-expression of mnx1 and vsx2 or gata3, markers of V2a and V2b fate, respectively [13, 29]. Expression of mnx1 and these markers was always mutually exclusive (Additional file 1: Figure S1 and data not shown), ruling out expression of mnx family genes in V2a and V2b interneurons. Based on its descending axon, early appearance and gad expression, the interneuron positive for expression of both mnx1 and mnx2b is VeLD.
In mice and chicks, members of the mnx gene family are expressed in motoneuron progenitors prior to exit from the cell cycle. In zebrafish, PMNs and VeLDs adjacent to somites 5 to 15 emerge from olig2:GFP + progenitors in the pMN domain , exit the cell cycle between 9 and 16 hpf , and then down-regulate olig2, although GFP persists for a short time. To determine if zebrafish Mnx proteins are expressed in PMN progenitors, we examined expression in Tg(olig2:GFP) embryos (Additional file 2: Figure S2). We found Mnx1+ cells that were either GFP- or expressed low levels of GFP; these cells often co-expressed Elavl3, a marker of post-mitotic neurons but did not co-express phosphohistone H3 (PH3), a marker of mitotic cells. Similarly, Mnx2a was often co-expressed with Elavl3 and never co-expressed with PH3, even though Mnx2a was expressed in some cells with high levels of GFP. Although mnx2b RNA was present as early as 14 hpf, we could not detect Mnx2b protein until at least 20 hpf, and then it was present only in MiPs (Figure 1O). Together these data are most consistent with the idea that all three Mnx proteins are first expressed in post-mitotic neurons and that expression of Mnx2a precedes expression of Mnx1.
In addition to examining Mnx expression in early-developing PMNs and VeLD interneurons, we also characterized Mnx expression in later-developing secondary motoneurons (SMNs). By 26 hpf, Mnx1 is expressed in a subset of mostly dorsally-located SMNs (Additional file 3: Figure S3), Mnx2a is expressed in a subset of mostly ventrally-located SMNs (Additional file 3: Figure S3), and Mnx2b is rarely expressed in SMNs (data not shown).
mnx expression is independent of Islet1
Islet1 and Lhx3 cooperate to regulate Mnx1 expression in chicks [32, 33]. To learn whether this relationship is conserved in zebrafish, we injected MOs to knock down either Islet1 , or Lhx3 and Lhx4 (Hutchinson SA, Seredick S, Van Ryswyk L, Talbot JC, Eisen JS: Lhx3 and Lhx4 regulate interneuron fate and prevent motoneurons from co-expressing interneuron characteristics, unpublished), and examined mnx gene expression. Surprisingly, expression of all three mnx genes was unaffected by Islet1 knockdown (Additional file 4: Figure S4). Moreover, at 24 hpf only mnx2b expression was eliminated in the absence of Lhx3 and Lhx4, revealing that Islet1 and Lhx3 do not cooperate to regulate mnx expression in zebrafish (Additional file 4: Figure S4).
Overexpression of Mnx proteins in chicks induces formation of ectopic motoneurons expressing Islet, Lhx3 and other Mnx paralogs [15, 34]. To test the hypothesis that Mnx proteins regulate expression of lhx genes in zebrafish, we injected mnx MOs and examined expression of lhx3 and lhx4. We found that neither lhx3 nor lhx4 expression was affected by the absence of Mnx proteins. We also eliminated expression of Mnx1, Mnx2a and Mnx2b individually and examined expression of each mnx gene. In the absence of any one mnx gene, expression of other paralogs was unaffected, revealing that each member of the gene family is regulated independently of the others (Additional file 4: Figure S4).
Mnx proteins are unnecessary for formation of primary motoneurons and VeLD interneurons
To assess whether VeLD development was compromised in the absence of Mnx proteins, we examined expression of Gad65/67, the biosynthetic enzyme for GABA, in Tg(vsx1:GFP) embryos. VeLDs express Gad65/67 but not vsx1:GFP, and can be uniquely identified by their lateral position and soma shape. At 20 hpf, the number of VeLDs in the absence of all three Mnx proteins was indistinguishable from controls (Figure 2E, F). Moreover, at 28 hpf VeLDs were morphologically normal in the absence of Mnx proteins (data not shown). Together these data provide evidence that Mnx proteins are not required for VeLD or PMN generation, and that both cell types acquire aspects of their mature identity in the absence of Mnx proteins.
Mnx proteins promote formation of normal MiP axons
Mnx proteins are required for MiP formation
CaP axons Normal
MiP axons Truncated
n = 30
n = 85
n = 85
n = 85
n = 85
n = 85
n = 85
n = 85
n = 87
n = 87
n = 87
n = 87
Mnx proteins prevent MiPs from acquiring molecular characteristics of V2a interneurons
Although V2a interneurons are the only cells in the ventral spinal cord that express glutamatergic markers before 32 hpf , we also examined expression of vsx2, a definitive V2a marker . In 22 hpf control Tg(nrp1a:GFP) embryos, we found 1.2 vsx2 + cells per spinal hemisegment. In controls, expression of vsx2 and the motoneuron markers islet1 and nrp1a:GFP was mutually exclusive (Figure 5C). In the absence of Mnx proteins, we found 2.3 vsx2 + cells per spinal hemisegment. Often, the extra vsx2 + cell weakly expressed GFP and was located near the somite boundary (Figure 5D), in the position occupied by the MiP soma, suggesting the hybrid cells expressing both PMN and interneuron markers were MiPs. By comparison, vsx2 was never expressed in CaPs, which continued to express islet2a in the absence of Mnx proteins (Figures 3B and 5D). These results suggest that Mnx proteins act to block expression of V2a interneuron markers specifically within MiPs.
Mnx proteins regulate MiP axon formation by maintaining Islet1 expression
Previously we found that in the absence of Nkx6.1 and Nkx6.2, most MiPs failed to form dorsal axons, and instead projected both their normal short ventral axon to the muscle pioneers and an interneuron-like axon within the spinal cord . The similarity of the Nkx6 and Mnx knockdown phenotypes suggested that the genes might be part of the same pathway. To test this hypothesis, we injected nkx6 MOs and examined Mnx expression, and we injected mnx MOs and examined Nkx6 expression. We found that mnx expression was unaffected by the absence of Nkx6 proteins (Additional file 4: Figure S4). Similarly, nkx6 expression was unaffected by the absence of all three Mnx proteins (Figure 5). This indicates that Mnx proteins influence MiP development independently of Nkx6.
Mnx expression in interneurons
Spinal cord expression of mnx genes was originally thought to be restricted to motoneurons [15–17]. However, the Drosophila melanogaster mnx paralog, hb9, is expressed in both motoneurons and interneurons [38, 39]. More recently, a small population of Mnx1-expressing interneurons was identified in the mouse spinal cord . Here we identify a class of ventral spinal interneurons in zebrafish, VeLDs, that express two mnx genes, mnx1 and mnx2b. The Mnx1-expressing interneurons in mouse are active components in the locomotor central pattern generator [19, 40, 41]. Although these cells have been extensively characterized during fictive locomotion in isolated spinal cord preparations [42–47], their exact role in the locomotor network in intact animals is unknown. Indeed, mouse Mnx1+ interneurons have proven resistant to genetic analysis, in part because their developmental provenance is entirely unclear.
It seems unlikely that VeLDs are the zebrafish equivalent of mouse Mnx1+ spinal interneurons. VeLDs are born early, GABA+ and have ipsilateral axons that descend many segments within the spinal cord [4, 8, 27]. In contrast, although the Mnx1+ mouse interneurons may arise from the same domain as motoneurons , they are likely to be born later than motoneurons as they have not been described in lineage studies. Mouse Mnx1+ interneurons are glutamatergic and likely make strictly local projections to motoneuron pools within the same segment [42, 46]. We have also noticed some ventromedially-located Mnx1+ interneurons that appear at about three days post-fertilization and do not seem to make projections to adjacent segments. Given the striking parallels between well-characterized components of the locomotor network in zebrafish and mouse [12, 48], it will be important to follow development of these Mnx1+ interneurons in vivo to learn their origins. Assessing their role in zebrafish motor behavior should have implications for understanding the contribution of Mnx1+ interneurons to locomotion in other vertebrate species.
Despite expression in VeLDs, Mnx proteins appear unnecessary for VeLD development. However, we only assayed axon projection and neurotransmitter phenotype, thus our results do not rule out a role for Mnx proteins in regulating some other aspect of VeLD differentiation.
Mnx proteins promote MiP subtype identity
The acquisition of MiP and CaP subtype identities are differentially affected by the absence of Mnx1, Mnx2a and Mnx2b. In the absence of all three Mnx proteins, CaPs fail to down-regulate islet1 expression. However, CaP subtype identity appears unaffected as both axon projection and neurotransmitter expression are normal. This finding is consistent with our previous studies showing that islet1 and islet2a can play equivalent roles in CaP specification . In contrast, a late, MiP-specific phase of islet1 expression is misregulated in the absence of Mnx proteins. In the absence of high levels of Islet1, MiPs fail to form their characteristic dorsal axons. This is reminiscent of the phenotype observed in the absence of Nkx6 , but whereas Nkx6 proteins are required to initiate the late phase of islet1 expression in MiP, Mnx proteins seem to be required to maintain high levels of Islet1 in MiP, similar to what has been reported in a mouse Mnx1 knock out [16, 17].
Variability in the amount of Islet1 protein or the precise time at which it is cleared might account for variability in MiP morphologies in the absence of Mnx proteins. One possibility is that MiPs that maintain Islet1 expression relatively late retain sufficient motoneuron character to project an axon out of the spinal cord. In contrast, those that down-regulate Islet1 relatively early might fail to express factors necessary to guide growth cones out of the spinal cord and into the periphery. This is consistent with the finding that early expression of Islet1 in CaP is sufficient to permit axon growth into the periphery , and could be tested in future experiments with photo-activatable morpholinos  to block islet1 translation at different times and assess the frequency with which MiP axons exit the spinal cord.
Surprisingly, when MiPs in mnx MO-injected embryos project axons into the periphery, most of them aberrantly extend to ventral muscle along a pathway normally reserved for CaP axons. Notably, MiP ventral axons stop normally at the muscle pioneers in the absence of Nkx6 proteins and late-phase Islet1 expression . Thus, while Mnx proteins promote formation of dorsal MiP axons by maintaining Islet1 expression, they exclude MiP axons from ventral muscle independently of Islet1.
PMN subtype identity, as revealed by axon trajectory, is influenced by positional signals that normally act during a specific window of developmental time [25, 50]. These signals affect motoneuron subtype, at least in part, by regulating expression of factors involved in axon pathfinding. Motor axons navigate toward their appropriate muscle targets by following subtype-specific guidance cues. The cues that are differentially recognized by CaP axons and MiP axons are unknown. Our results suggest that Mnx proteins regulate expression of receptors that recognize cues that prevent the MiP growth cone from progressing ventral of the muscle pioneers, and thus prevent MiPs from becoming CaP-like. To our knowledge, this is the first report of genes that can cause MiPs to transform to a CaP morphology.
Mnx proteins prevent MiP from acquiring V2a interneuron characteristics
In the absence of Mnx proteins, MiPs, but not CaPs, often form hybrids that have features of both motoneurons and interneurons. Based on expression of vsx2 and vglut and axon morphology, MiPs appear to have acquired features of zebrafish V2a interneurons . As this phenotype is only observed in the combined absence of Mnx1, Mnx2a and Mnx2b, the three zebrafish paralogs act redundantly to suppress the formation of MiP-V2a hybrids. The acquisition of V2a features is reminiscent of the phenotype of Mnx1 knockout mice in which Vsx2 is inappropriately expressed in a subset of Islet1+ motoneurons [16, 17]. However, MN-V2a hybrids in Mnx1 knockout mice fail to project interneuron-like axons within the spinal cord and whether they express glutamatergic markers has not been assessed. Our results suggest that zebrafish MiP-interneuron hybrids acquire a more complete set of V2a features. Regardless, our results reveal a conserved role for the mnx gene family in segregating motoneuron from V2a interneuron cell fate in specific motoneuron subtypes.
We previously reported that knocking down Islet1 resulted in PMNs developing as interneurons and that knocking down the Met receptor tyrosine kinase resulted in PMNs co-expressing motoneuron and interneuron characteristics [8, 35]. In both of these cases, PMNs expressed the neurotransmitter, GABA. Clonal analysis in zebrafish has revealed that PMNs can be siblings with either KA’ or VeLD interneurons , both of which express GABA [4, 27]. These observations supported a model whereby many factors expressed by PMNs cooperate to suppress acquisition of characteristics of closely-related interneurons derived from the pMN domain. Here we show that in the absence of Mnx proteins, PMN-interneuron hybrids inappropriately express V2a interneuron characteristics. These data support a model whereby post-mitotic Mnx expression in PMNs suppresses acquisition of characteristics of more distantly-related interneurons from the adjacent p2 domain. A striking aspect of the PMN-V2a phenotype is that it is limited to MiP. This suggests that MiPs are more similar to V2a interneurons than are CaPs. This is consistent with the observation that in the absence of Nkx6 proteins, many MiP-interneuron hybrids have axons with a V2a morphology . However, it is important to note that in our previous Nkx6 knockdown studies, MiPs did not express vsx2. As Mnx proteins suppress vsx2 expression in MiPs (Figure 5), we suspect their continued expression in the absence of Nkx6 (Additional file 4: Figure S4) accounts for our inability to detect vsx2 expression in MiP in our previous Nkx6 knockdown studies. These combined results imply that other genes in addition to vsx2, genes not regulated by Mnx proteins, contribute to acquisition of V2a interneuron axonal projections.
V2as, like PMNs, originate from a domain that expresses Nkx6.1 , and continue to express Lhx3 after they exit the cell cycle . Moreover, recent lineage-tracing in mouse has shown that many V2a neurons have expressed Olig2 during their developmental history [51, 52], revealing that they may be even more similar to motoneurons than had been previously appreciated. A more detailed lineage analysis in zebrafish of the relationship between PMNs, VeLD interneurons and V2a neurons could help resolve the relationships among these neurons.
The three zebrafish Mnx transcription factors have distinct expression patterns in each of the zebrafish PMN subtypes and in VeLD interneurons. These expression patterns are dynamic during the period when these cells are extending axons and initiating neurotransmitter expression. Despite their expression in CaP motoneurons and VeLD interneurons, Mnx proteins appear dispensable for development of these two cell types. In contrast, Mnx proteins are required for development of MiP motoneurons. In the absence of Mnx proteins, MiPs extend aberrant axons and express an interneuron-specific neurotransmitter (Figure 7).
Wild-type (AB), Tg(olig2:GFP) vu12 , Tg(nrp1a:GFP) js12  and Tg(vsx1:GFP) nns5  and Tg(mnx1:GFP) ml2  zebrafish were maintained in a laboratory breeding colony according to established protocols . Embryos collected from natural crosses were allowed to develop at 28.5°C, and staged by hours post-fertilization (hpf) according to morphological criteria .
Generation of transgenic fish lines
A 3-kb fragment of the mnx1 promoter  was subcloned into p5E MCS. Multi-site Gateway® technology (Life Technologies; Eugene, OR, USA) was used to assemble an mnx1:GAL4VP16:pA construct flanked by Tol2 terminal inverted repeats. Tg(mnx1:GAL4VP16) lines were generated by co-injecting plasmid DNA and Tol2 transposase RNA  into the yolk of one-cell stage embryos. Multiple founders were recovered and characterized; Tg(mnx1:GAL4VP16) b1222 was chosen for this study because transgene expression faithfully mirrored endogenous Mnx1 protein expression (data not shown).
Approximately 2.5 nl of 100 μM translation-blocking morpholinos (Gene Tools, LLC; Philomath, OR, USA) against mnx1 (5’-ACCTCACAAACAGATTAACGCCTCG-3’), mnx2a (5’-ACCTCACAAACAGATTAACGCCTCG-3’) and mnx2b (5’-GACTTTTCCATTGCAACACTTTTGT-3’) were injected into one- to two-cell stage embryos; this was sufficient to suppress translation as assayed by whole-mount immunohistochemistry (Additional file 5: Figure S5) without elevated cell death as assayed by acridine orange staining (data not shown). These morpholinos have been previously validated .
Other previously validated morpholinos used in this study include: random control oligonucleotide (5’-N25-3’), 2.5 nl of 100 μM islet1 E2 (5’-TTAATCTGCGTTACCTGATGTAGTC-3’) plus 100 μM islet1 E3 (5’-GAATGCAATGCCTACCTGCCATTTG-3’)  to knockdown islet1; 2.5 nl of 400 μM nkx6.1 (5’-CGCAAGAAGAAGGACAGTGACCCG-3’)  plus 400 μM nkx6.2 (5’-CGCGCAAAACTCACCCGCACAGGGA-3’)  to knockdown nkx6.1 and nkx6.2; and 2.5 nl of 280 μM lhx3 (5’-GTTCTAACAACATTCTGGCGATAAA-3’) plus 280 μM lhx4 (5’-GCAGCACAGCCGCACTTTGCATCAT-3’) to knockdown lhx3 plus lhx4 (Hutchinson SA, Seredick S, Van Ryswyk L, Talbot JC, Eisen JS: Lhx3 and Lhx4 regulate interneuron fate and prevent motoneurons from co-expressing interneuron characteristics, unpublished). Morpholino effectiveness was verified by whole mount immunohistochemisry.
Fluorescent RNA in situ hybridization
RNA in situ hybridization was performed according to standard protocols  with the following modifications. For two-color fluorescent in situ hybridization anti-sense probes were labeled with digoxigenin-UTP (Roche Applied Sciences, Indianapolis, IN, USA) and dinitrophenol-UTP (Perkin-Elmer, Waltham, MA, USA). Following overnight hybridization, unbound probe was removed with three 30-minute washes at 68°C in 50% formamide, 5x SSC and 0.1% SDS, followed by stringent washes in 50% formamide, 2x SSC and 0.1% Tween-20. Labeled probes were detected with HRP-conjugated anti-DIG (1:2,000; Jackson ImmunoResearch, West Grove, PA, USA) or HRP-conjugated anti-DNP (1:2,000; Perkin-Elmer) and stained with fluorescein, Cy3- or Cy5-tyramide (1:100; Perkin-Elmer) for 1 to 10 minutes.
Probes used include mnx1, mnx2a and mnx2b; chat; islet1, islet2a and lhx3; lhx4 (Hutchinson SA, Seredick S, Van Ryswyk L, Talbot JC, Eisen JS: Lhx3 and Lhx4 regulate interneuron fate and prevent motoneurons from co-expressing interneuron characteristics, unpublished).; gad1b and gad2 (collectively referred to as gad), slc17a6a, slc17a6b and slc17a7 (collectively referred to as vglut) and slc6a9 and slc6a5 (collectively referred to as glyt) ; vsx2; and gata3.
To prepare Mnx1 and Mnx2b antisera, cDNAs corresponding to amino acids 245 to 311 of Mnx1 or amino acids 224 to 301 of Mnx2b were His-tagged, over-expressed in E. coli and purified by nickel column chromatography under native conditions. These regions are C-terminal to the homeodomain, and are the most divergent regions of the gene family. Purified recombinant proteins were used to immunize rabbits, and the resulting antisera screened by whole mount immunohistochemistry. Attempts to generate antisera against Mnx2a were not successful.
Embryos were fixed for 2 hours in 4% paraformaldehyde and 1x Fix Buffer  at 4°C, and then treated with 0.5% Triton X-100 in 1x PBS for 15 minutes at room temperature. Embryos were blocked in 5% normal goat serum, 2.5% DMSO and 0.1% Tween-20 in 1x PBS before overnight incubation in diluted primary antibody at 4°C. Unbound primary antibodies were removed by washing for two hours in 1x PBS plus 0.1% Tween-20, followed by overnight incubation in diluted secondary antibody at 4°C. Anti-Mnx1, anti-Mnx2a and anti-Mnx2b were detected with HRP-conjugated goat anti-rabbit and stained with fluorescein-, Cy3- or Cy5-tyramide (1:100; Perkin-Elmer) for one minute; all other primary antibodies were detected with dye-labeled secondary antibodies.
Antibodies used include rabbit polyclonal anti-Mnx1 (1:1,000) and anti-Mnx2b (1:1,000), anti-Mnx2a (1:1,000; AnaSpec, Fremont, CA, USA), anti-Lhx3 and anti-Lhx4 (Hutchinson SA, Seredick S, Van Ryswyk L, Talbot JC, Eisen JS: Lhx3 and Lhx4 regulate interneuron fate and prevent motoneurons from co-expressing interneuron characteristics, unpublished), mouse monoclonal anti-Elavl3/4 (1:10,000; A21271, Life Technologies), anti-Gad (1:500; ab11070, Abcam, Cambridge, MA, USA), anti-GFP (JL-8; Clontech, Mountain View, CA, USA; or A-11120, Life Technologies), anti-Histone H3 (phospho S10; 1:1000; ab14955, Abcam), anti-Islet (39.4D5; DSHB, Iowa City, IA, USA), and anti-Nkx6.1 (F55A10; DSHB).
Subtype-specific cell labeling
To correlate cell morphology with gene expression, we injected UAS:EGFP plasmid with Tol2 transposase RNA and selected embryos with GFP-expressing cells for immunohistochemistry. Since our mnx1 morpholino also suppressed expression from our Tg(mnx1:GAL4VP16) transgene, individual neurons in mnx morpholino-injected fish were dye-labeled with 5% tetramethylrhodamine-dextran (D-3308; Life Technologies) in 0.2 M KCl .
All images were acquired on a Zeiss Pascal confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, New York, USA) using a 40x water immersion objective. The brightness and contrast of images was adjusted using Photoshop CS5 (Version 12.0, Adobe Systems, Inc., San Jose, CA, USA).
All observations of PMNs were made in the mid-trunk region of the spinal cord adjacent to somites 8 to 12. We examined at least 30 segments from 10 embryos for each condition, unless otherwise noted in the figure legends.
We thank Marnie Preston and Chris Doe for comments on the manuscript, the Chien lab for Gateway vectors, Dirk Meyer for mnx plasmids, Mike Marusich for help generating Mnx antibodies, and Amanda Lewis, Jacob Lewis, Dayna Lamb, and the UO Zebrafish Facility staff for animal husbandry. Supported by NIH grants NS23915 and HD22486 (JSE), HD007348 (SAH and LVR), and an NSERC fellowship (SS).
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