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. Author manuscript; available in PMC: 2012 Jan 15.
Published in final edited form as: Dev Biol. 2010 Oct 28;349(2):378–386. doi: 10.1016/j.ydbio.2010.10.030

Nkx6 genes pattern the frog neural plate and Nkx6.1 is necessary for motoneuron axon projection

Darwin S Dichmann 1, Richard M Harland 1,1
PMCID: PMC3018535  NIHMSID: NIHMS256239  PMID: 21035438

Abstract

Neuronal subtypes originate from an undifferentiated neural epithelium that is progressively divided into progenitor domains by homeodomain transcription factors such as members of the Nkx family. Here we report the functional analysis of Nkx6.1 and Nkx6.2 in Xenopus. While Nkx6.2 is expressed early in a large region of the medial neural plate , Nkx6.1 is restricted to a region overlapping with the region of motor neuron formation. By mRNA injection we show that both can inhibit primary neurogenesis as well as expression of intermediate neural plate markers. However, they do not form auto-regulatory loops and fail to induce ectopic motor neurons as they do in the chick. Using morpholino-mediated knockdown in X. laevis and X. tropicalis we show that Nkx6.1 knockdown results in paralyzed tadpoles. Using DiI labeling and immunohistochemistry we show that the underlying mechanism is a failure of spinal motor neurons to extend axons to their targets. Analysis of neural pattern reveals that ventral Lhx3+ and Pax2+ interneurons are dependent on Nkx6.1 function, but overall neural patterning is not. This study illustrates that while important aspects of Nkx6 gene function are conserved in vertebrate neural patterning, others are not.

Keywords: Xenopus laevis, Xenopus tropicalis, Nkx6, neural plate, patterning, axon guidance, differentiation, motor neuron, interneuron, motility, paralysis

INTRODUCTION

During embryogenesis interactions of the neural plate with the surrounding mesoderm and ectoderm specify cell fates along the mediolateral (M–L) and anterior-posterior (A–P) axes (Lee and Jessell, 1999; Sasai and De Robertis, 1997). In Xenopus, mediolateral patterning of the neural plate is evident in the open neural plate from the expression of differentiated markers such as neural-specific beta-tubulin (Chitnis et al., 1995), and this differentiation can be traced back to gastrulation when neuronal progenitor domains express neurogenin. During neurulation, medial progenitor cells become the ventral motor neurons (MN), intermediate cells become interneurons (IN), and lateral cells become the dorsal sensory neurons (SN; also called Rohon-Beard cells). As the neurons differentiate and form functional circuits, they extend precise axonal projections to their target tissues.

Significant progress has been made in understanding the molecular mechanisms specifying the neuronal progenitor cells in the spinal cord from studies in chick and mouse embryos. Two signaling centers, the ventral floor plate and the dorsal roof plate, secrete signaling molecules that set up distinct neural progenitor domains defined by specific transcription factor codes. In the amniote neural tube, Sonic hedgehog (Shh) induces the homeodomain (HD) transcription factors Nkx2.2, Nkx6.1, and Nkx6.2 while repressing Pax3, Pax6, Irx3, and Dbx1/2 (Briscoe et al., 2000). These HD proteins act as transcriptional repressors that confer progenitor identity by repressing alternative cell fates (Briscoe et al., 2000; Muhr et al., 2001; Vallstedt et al., 2001). Similarly, in the dorsal neural tube members of the TGF-β family govern a transcription factor code consisting of HD and basic helix-loop-helix (bHLH) proteins, including Olig3, Msx1/2, and Pax3 (Liem et al., 1997; Liem et al., 1995; Scardigli et al., 2001; Timmer et al., 2002).

In the ventral neural tube of chick and mouse embryos the regional transcription factor code depends on Nkx6 gene function (reviewed in (Briscoe and Ericson, 2001; Lee and Pfaff, 2001; Marquardt and Pfaff, 2001)). Nkx6.1 and Nkx6.2 are highly similar proteins that confer transcriptional repression by interaction of their engrailed-homology domain with Groucho/TLE transcriptional co-factors (Muhr et al., 2001). They determine ventral cell fates by repressing expression of members of the Dbx family of HD proteins that are expressed in more intermediate positions of the neural tube (Briscoe et al., 2000; Vallstedt et al., 2001). Nkx6 function is necessary for MN formation and misexpression of Nkx6.1 can induce ectopic MNs (Briscoe et al., 2000). In addition, Nkx6 mouse mutants have been shown to have defects in cranial nerve projections (Pattyn et al., 2003). Nkx6 genes from Drosophila, Xenopus and zebrafish have also been described (Broihier et al., 2004:Zhao, 2007 #258; Cheesman et al., 2004).

Although the principles that govern patterning of the neural plate are similar in the chick and mouse, differences in the deployment of particular genes have been found; for example, in the mouse Nkx6.1 and 6.2 show cross repressive interactions, while in the chick they do not (Vallstedt et al., 2001). To determine whether the general principles of neural plate patterning are shared among the vertebrates, and to address the origins of patterning mechanisms, it is important to examine mechanisms in other vertebrate groups. Cheesman et al. (2004) found that Nkx6 gene function in zebrafish is also able to induce ectopic motor neurons, but loss of Nkx6 function, while affecting secondary motoneurons, did not affect the initial population of motoneurons. In this paper, we have addressed whether common mechanisms are used to determine the pattern of the spinal cord by examining the function of Nkx6 genes in Xenopus. We show that both can repress intermediate neural plate markers but are unable to induce MNs. In contrast to what has been reported in mouse, we show that frog Nkx6.1 only plays a minor role in neural plate patterning but is critical in controlling MN axonal projections. This study confirms some conserved functions of this important class of genes but also provides new insight into the variation in mechanisms of neural plate patterning in vertebrates.

MATERIALS AND METHODS

Plasmids

Cloning of Xenopus laevis Nkx6.1 and Nkx6.2

Cloning of Nkx6 genes has been described by Zhao et al., (2007). cDNAs used in this work were isolated by screening a X. laevis whole tadpole cDNA library (Grammer et al., 2000). One clone, #111-M17, contained the entire CDS as well as 5’- and 3’-UTR sequence. The CDS of Nkx6.1 was cloned by PCR using primers designed based on sequence information from the X. tropicalis genome (http://www.jgi.org) from X. laevis stage 14 oligo-dT primed cDNA. The PCR fragment was cloned into the XbaI and XhoI sites in pCS108 to give pCS108-Nkx6.1. The sequences determined for the coding regions are identical to those in Zhao et al. (2007). For in situ hybridization the CDS for X. laevis Irx3 was cloned by PCR and subcloned into bluescript SK(−) vector to give pBS-Irx3. X. laevis Isl1 was obtained from IMAGE clone #4058863 and subcloned into bluescript SK(−) using EcoRI and NotI to give pBS-Isl1. X. laevis Olig3 was obtained from NIBB clone #056o10 and subcloned into pCS108 using EcoRI and XhoI to give pCS108-Olig3.

Whole mount in situ hybridization

Whole mount in situ hybridization on X. laevis embryos employed digoxygenin-labeled RNA probes as described previously (Sive et al., 2000). To examine potential overlap, double in situ hybridizations used two digoxygenin-labeled probes simultaneously. Antisense probes for Nkx6.1 and Nkx6.2 were synthesized from pCS108-Nkx6.1 and clone 111-M17, respectively, linearized with Sal1 and transcribed with T7 polymerase. Antisense probe for Irx3 was synthesized from pBS-Irx3 linearized with HindIII and transcribed with T7; for Isl1 pBS-Isl1 was linearized with HindIII and transcribed with T7; for Olig3 pCS108-Olig3 was linearized with BamHI and transcribed with T7.The following probes have been described previously: Pax2 (Heller and Brandli, 1997), Slug (Richter et al., 1988), Nrp1, N-tubulin both (Richter et al., 1990), Krox20 (Nieto et al., 1991), Dbx1(Xdbx) (Gershon et al., 2000), Shh (Ruiz i Altaba et al., 1995), Pax6 (Hirsch and Harris, 1997), Cytokeratin-81 (Jamrich et al., 1987), Nkx2.2 (Saha et al., 1993), Hb9 (Saha et al., 1997), Lhx3 (Xlim3) (Taira et al., 1993) , Msx1 (Suzuki et al., 1997), Pax3 (Mariani et al., 2001) and Sox2 (Grammer et al., 2000)

Embryo culture and microinjection

Embryos of X. laevis and X. tropicalis was obtained by in vitro fertilization and manipulated as described in (Khokha et al., 2002; Sive et al., 2000). Capped X. laevis Nkx6.1 and Nkx6.2 mRNA were synthesized using SP6 mMessage Machine kit (Ambion) with pCS106-Nkx6.1 or clone 111-M17 (Nkx6.2) linearized with AscI as template. Embryos were injected in one blastomere at the 2-cell stage with 100–200 pg Nkx6.1 mRNA or 500–1000 pg of Nkx6.2 mRNA. Injected mRNA was traced by coinjecting 100 pg nuclear beta-galactosidase and staining with Red-Gal substrate (Research Organics). All phenotypes described in this study were observed in at least 80% of the injected embryos (n≥40 in at least 3 independent experiments).

Morpholino design and injections

Translation blocking MOs were obtained from Genetools. For Nkx6.1 knockdown in both X. laevis and X. tropicalis, the morpholino Xl-6.1MO1 (MO1) (5’-ATCCATCTGCCCTAAGGTTAACAT-3’) was used (translation start codon underlined). A second MO (MO2) specific for the 5’UTR of Nkx6.1 in X. laevis had the sequence (5’-CACTGAGCCACCTGCACTGCAGCCC-3’). For knockdown of Nkx6.1 in X. laevis, unless indicated otherwise, 37.5 ng MO was injected per blastomere at the 2-cell stage and 5 ng of fluoresceinated control morpholino (GeneTools) was co-injected as a tracer. MO injections in X. tropicalis used one-fifth of the amount and volume compared to X. laevis. Injected embryos were sorted according to which side was injected based on control MO fluorescence prior to further analysis. A polyclonal rabbit antibody recognizing Nkx6.1 (a gift from Dr. Palle Serup) was used to confirm knockdown of the protein in Nkx6.1MO injected X. laevis embryos.

Movies of Nkx6.1MO injected embryos

Control embryos and embryos injected with Nkx6.1MO in both blastomeres at the 2-cell stage were cultured until stage 37. Escape reflex responses of embryos in an agarose dish were recorded using ImagePro software set at the fastest shutter speed using a DFC480 camera (Leica) mounted on a MZ FLIII microscope (Leica).

Whole mount immunohistochemistry

Whole mount immunohistochemistry on X. laevis embryos was performed as described previously (Sive et al., 2000). Mouse antibody 6F11 (Harris and Hartenstein, 1991), recognizing N-CAM, was diluted 1:5. Mouse antibody 12/101 staining skeletal muscle (Kintner and Brockes, 1984) was used undiluted. HRP-conjugated secondary antimouse antibody (Jackson) was diluted 1:500.

DiI labeling

Orthograde labeling of axonal tracts was peformed using using CellTracker CM-DiI (Molecular Probes) dissolved in 70% ethanol to 0.25% and then in 0.3M sucrose to 0.1% for injection. DiI solution was backloaded into a pulled glass needle (World Precision Instruments, Inc.) and injected into the neural tube of stage 39/40 embryos using a Picospritzer II (General Valve Corp.). After injection the embryos were allowed to recover for 30 minutes followed by fixation for 2–4 weeks at 37°C in methanol-free 4% paraformaldehyde (Electron Microscopy Services).

RESULTS

Expression of Nkx6 genes during frog development

We examined Nkx6 gene expression during neural patterning by whole mount in situ hybridization on X. laevis embryos. As described by Zhao et al. (2007) Nkx6.1 expression was first detected at stage 13 in two stripes in the neural plate (Fig.1A,B) and later more robustly extending from the midbrain-hindbrain boundary (MHB) through the spinal neural plate (Fig.1C,D). We found that Nkx6.1 expression overlapped with the region where MNs form as marked by Hb9 expression at stage 17 (compare Fig.1D,M). Following neural tube closure Nkx6.1 expression persisted in the ventral spinal cord and hindbrain, and could be detected in the foregut from stage 34 (Fig.1E).

Fig.1. Expression of Nkx6 genes in X. laevis.

Fig.1

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(A–E) Whole mount in situ hybridization showing Nkx6.1 expression. (A) Nkx6.1 is not expressed during gastrulation. (B) Expression begins at stage 13 in two lateral stripes (red arrowhead) and intensifies at stage 14 (C). At stage 17 (D) Nkx6.1 expression marks where Hb9-positive MNs will form (compare to (M). (E) At stage 34, Nkx6.1 is expressed in the ventral spinal cord and hindbrain as well as the foregut (red arrowhead). (F–J) Whole mount in situ hybridization showing Nkx6.2 expression. (F) Nkx6.2 is expressed during gastrulation in the dorsal ectoderm. Red line indicate the dorsal blastopore lip. (G) At stage 13, Nkx6.2 expression is detected in a broad part of the medial neural plate. At stage 14 (H) and stage 17 (I) Nkx6.2 expression extends through the medial developing hindbrain and spinal neural plate. (J) At stage 34, Nkx6.2 expression is detected in the ventral spinal cord and hindbrain as well as the foregut (red arrowhead). Nkx6.1 and Nkx6.2 expression is confined to the medial part of the neural plate when compared to the pan-neural marker Sox2 expression (compare (C, H) to (K); and (D, I) to, (L)). (N,O) Neither Nkx6.1 (N; red arrowhead) or Nkx6.2 (O; red arrowhead) expression overlaps with Dbx1 expression (N,O; black arrows) at stage 16.

Nkx6.2 is expressed during gastrulation from stage 10.5 in a broad region of the neurogenic ectoderm (Fig.1F) and later in the medial part of the neural plate extending anteriorly into the hindbrain (compare Fig.1H,I to 1K,L). This expression pattern persisted after neural tube closure in the ventral part of the spinal cord and hindbrain. At stage 34, Nkx6.2 expression could be detected in the foregut in a broader domain than Nkx6.1 (Fig.1J).

Nkx6 genes form cross-repressive pairs with members of the Dbx family of HD transcription factors in amniotes. To determine if expression of Nkx6 and Dbx1 gene expression overlap in frog embryos we performed double in situ hybridization at neural plate stages. In these in situ experiments, Nkx6.1 and Dbx1, or Nkx6.2 and Dbx1, showed distinct and non-overlapping expression patterns (Fig.1N,O). The expression patterns suggest that Nkx6 genes in X. laevis are likely to be involved in neural development, but does not support a direct cross-repressive role, since the domains of expression are separated. The spatiotemporal expression of Nkx6.2 suggests a role in early neural patterning and for Nkx6.1 in MN development.

Misexpression of Nkx6 genes inhibits neurogenesis and expression of intermediate progenitor domain markers

To determine the function of Nkx6 genes, we injected synthetic mRNA into one blastomere of 2-cell frog embryos and investigated the effect on neural plate markers. In the following experiments we did not detect any qualitative difference in the activity of Nkx6.1 or Nkx6.2, suggesting that, at least in their ability to change neural plate markers when misexpressed, their functions are similar.

First, we investigated if Nkx6 misexpression could induce ectopic nervous tissue. When mRNA was targeted to the non-neural ectoderm the effect was a robust increase in the pan-neuronal marker Nrp1 (Fig.2A–C; Nkx6.1: 15/17 embryos with proper targeting, Nkx6.2: 13/15). This increase in nervous tissue was accompanied by a reduction in epidermal cytokeratin (CK81) (Fig.2D–F; Nkx6.1: 16/18, Nkx6.2: 15/17).

Fig.2. Effect of Nkx6 mRNA injection on neural development.

Fig.2

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Gene expression patterns analyzed by whole mount in situ hybridization. Top row: uninjected control embryos. Middle row: embryos injected with Nkx6.1 mRNA. Bottom row: embryos injected with Nkx6.2 mRNA. Injected side is to the right and red stain indicate beta-galactosidase lineage tracer. All embryos are analyzed at stage 16, except embryos stained for Pax3 (V–X), which were analyzed at stage 17. (A–C) Pan-neural marker Nrp1 is expanded in Nkx6 injected embryos. (D–F) Epidermal marker cytokeratin-81 (CK81) is reduced in Nkx6 injected embryos. (G–I) Primary neurogenesis is inhibited by Nkx6 mRNA injected as visualized by loss of N-tubulin (N-tub) staining. (J–L) Dbx1 expression is inhibited in the spinal neural plate when embryos are injected with Nkx6 mRNA. (M–O) Spinal neural plate expression of Pax2 is repressed by Nkx6 mRNA, whereas the MHB is intact. (P–R) Spinal, but not rhombomeric, expression of Irx3 is repressed by Nkx6 misexpression. (S–U) Pax6 expression in the neural plate is repressed by ectopic Nkx6 misexpression. (V–X) Pax3 expression and Msx1 (Y–Æ) expression is unchanged in response to Nkx6 injection.

During primary neurogenesis in frog embryos, three lateral stripes of neurons on each side of the embryo give rise to MNs, INs, and SNs at progressively more lateral positions. Upon Nkx6 misexpression, primary neurogenesis was inhibited resulting in little or no formation of primary neurons in the injected region (Fig.2G–I; Nkx6.1: 20/27, Nkx6.2: 28/40). Depending on the targeting of the mRNA, all three classes of primary neurons could be inhibited (data not shown).

During mid-neurula stages the neural plate is divided into distinct progenitor domains that can be distinguished by the expression of different homeobox genes. To determine if these were changed by Nkx6 misexpression, we first examined the effect on Dbx1 (Xdbx; (Gershon et al., 2000)), which is expressed in the two longitudinal stripes along the spinal neural plate and ventral telencephalon. Injection of mRNA encoding either Nkx6 protein completely inhibited Dbx1 expression in the spinal cord whereas the forebrain expression remained (Fig.2J–K; Nkx6.1: 32/34, Nkx6.2: 26/31 and not shown). Similarly, Pax2, which is expressed in several parts of the developing nervous system including the spinal neural plate, was repressed in response to Nkx6 injection. Again, the effect was confined to the spinal domain whereas the MHB expression was intact, though usually shifted slightly posteriorly on the injected side (Fig.2M–O; Nkx6.1: 40/40, Nkx6.2: 36/37).

In mouse and chick embryos, Nkx6 proteins act specifically to inhibit expression of Dbx family transcription factors (Briscoe et al., 2000; Vallstedt et al., 2001). To test if this is also the case in frogs, or if they had broader effects, we investigated the effect of Nkx6 misexpression on Irx3 and Pax6. Surprisingly, both Irx3 (Fig.2P–R; Nkx6.1: 23/28, Nkx6.2: 24/30) and Pax6 (Fig.2S–U; Nkx6.1: 14/17, Nkx6.2: 13/16) were repressed in the neural plate in response to Nkx6 misexpression. Interestingly, the repression of Irx3 was restricted to the spinal region whereas the hindbrain expression was resistant to Nkx6 repression (Fig.2P–R). Pax6 repression was restricted to the hindbrain and spinal region of the neural plate while the forebrain expression was normal (Fig.2S–U).

To examine if even more lateral neural markers could be repressed by ectopic Nkx6 expression we tested the response of Pax3 and Msx1 to Nkx6 mRNA injection. In contrast to the more medial markers, Pax3 expression was not inhibited by Nkx6 expression (Fig.2V–X; Nkx6.1: 25/25 retain expression, Nkx6.2: 19/19). Similarly, Msx1 expression, which marks the boundary of the neural plate, was unchanged (Fig.2Y–Æ; Nkx6.1: 13/15 normal, Nkx6.2: 12/13). Together, our misexpression experiments show that Nkx6 proteins have more pleiotropic effects on medial neural gene expression than do their amniote counterparts, and can potently repress intermediate neural plate marker genes and primary neurogenesis, though they show considerable specificity, in that they do not inhibit expression of lateral neural plate markers, nor the brain domain of other markers. In addition, the Nkx proteins can expand the neural territory of the ectoderm at the cost of epidermal gene expression.

Frog Nkx6 genes do not repress each other or induce ectopic MNs

In mouse embryos Nkx6.1 and Nkx6.2 are expressed in mutually exclusive domains and repress each others’ expression whereas in chicken Nkx6 genes do not (Vallstedt et al., 2001). To determine if frog Nkx6 genes are mutually repressive, we injected mRNA encoding each protein into one cell of 2-cell embryos and monitored changes in expression of the other Nkx6 gene by in situ hybridization. Nkx6.2 mRNA injection did not repress Nkx6.1 expression. In fact, we frequently observed a modest broadening of the Nkx6.1 domain (Fig.3A,B; 10/26). Similarly, Nkx6.1 injection did not repress Nkx6.2, but instead caused ectopic expression in the spinal neural plate (Fig.3C,D; 8/18). The expanded expression of Nkx6.2 in response to Nkx6.1 mRNA injection was always confined to the spinal region of the neural plate. These results show that, in contrast to the mouse, Nkx6 genes in frogs are not cross-repressive. These results also show that the inhibitory effects in the overexpression experiments show considerable specificity, with no inhibition of Nkx genes, while other medial genes such as Dbx1 and Pax2 are inhibited.

Fig.3. Effect of Nkx6 mRNA injection on Nkx6 gene expression and MN formation.

Fig.3

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Nkx6 mRNA injected embryos analyzed by whole mount in situ hybridization at stage 17. Injected side is to the right and red stain indicate the beta-galactosidase lineage tracer. (A,C,E) Uninjected control embryos, (B,G) embryos injected with Nkx6.2 mRNA, (D,F) embryos injected with Nkx6.1 mRNA. (A,B) Nkx6.1 expression is not repressed in response to Nkx6.2 mRNA injection. (C,D) Nkx6.2 is not repressed in embryos injected with Nkx6.1 mRNA. (E–G) Embryos injected with Nkx6 mRNA do not form ectopic Hb9-positive MNs.

Nkx6.1 misexpression is sufficient to induce ectopic MNs in chicken and zebrafish embryonic spinal cord (Cheesman et al., 2004; Vallstedt et al., 2001) . To test if frog Nkx6 genes had a similar function, we injected Nkx6.1 or Nkx6.2 mRNA and examined embryos for changes in expression of the MN markers. However, neither mRNA induced changes in the MN markers Hb9 (Fig.3E–G), Lhx3, or Isl1 (data not shown), even over a range of mRNA doses. In addition, we examined embryos at stage 34 and also tested if simultaneous injection of both Nkx6 transcripts could induce MNs. However, in neither case did we observe ectopic MNs (data not shown).

Morpholino mediated knockdown of Nkx6 genes in frogs

To determine the in vivo function of Nkx6.1 in frog neural development we designed an antisense morpholino oligonucleotides (MO) that targeted the translation start site to inhibit translation of the endogenous transcript. To address effects caused by unintended hybridization to other transcripts, we performed the knockdown experiments in X. laevis as well as in the closely related species X. tropicalis. Because the CDS of Nkx6.1 genes in X. laevis and X. tropicalis are highly conserved in the 5’ region we designed a morpholino that would inhibit translation in both species (Xl-6.1MO), although with a single base mismatch in the 3’ terminus of the MO in X. tropicalis (Fig.4A). When we injected this MO into frog embryos of either X. laevis (Fig.4B,C) or X. tropicalis (Fig.4D,E) both usually appeared normal until stage 40.

Fig.4. Morpholino mediated knockdown of Nkx6 genes in X. laevis and X. tropicalis.

Fig.4

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MO design and morphology of MO injected tadpoles. (A) Alignment of Nkx6.1 sequences in X. laevis (Xl; top) and X. tropicalis (Xt; bottom) used for MO design. Vertical lines indicate identical residues and translation start codon is in bold type. Sequence targeted by Xl-Nkx6.1MO is shown in blue and is identical to the corresponding X. tropicalis sequence in 24/25 positions. (B) Uninjected stage 40 X. laevis tadpole. (C) Tadpole injected with 80 ng Xl-6.1MO at the 1-cell stage are morphologically normal but paralyzed. (D) Uninjected stage 40 X. tropicalis tadpole. (E) X. tropicalis tadpole injected with 8 ng Xl-6.1MO in both blastomeres at the 2-cell stage is morphologically normal but paralyzed, phenocopying the X. laevis injection in (C).

Nkx6.1 knockdown results in reduction of Pax2+ interneurons, but does not prevent MN formation

To determine the function of Nkx6.1 during neural plate patterning we performed whole mount in situ hybridization for genes expressed in different cell populations of the neural plate. First, we investigated N-tubulin expression in differentiated primary neurons. The embryos were prescreened for presence of the fluoresceinated control morpholino in half of the spinal cord region prior to in situ hybridization. Unilateral Injection of Nkx6.1MO did not change primary neurogenesis (Fig.5A,B). To test if MN differentiation was normal we analyzed the expression of Hb9, which marks differentiated MNs, and Lhx3, which in other vertebrates is expressed early during ventral interneuron and MN specification (Thaler et al., 1999; Thaler et al., 2002). In Nkx6.1MO injected embryos Hb9 expression at stage 17 was unaffected (Fig.5C,D). In contrast, Nkx6.1MO injected embryos frequently showed a reduction of Lhx3 positive cells on the injected side at stage 16 (Fig.5E,F; 14/33).

Fig.5. Knockdown of Nkx6.1 has minor effects on neural plate patterning.

Fig.5

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Gene expression pattern in the neural plate of Nkx6.1MO injected X. laevis embryos. Top row shows uninjected control embryos, bottom row shows embryos injected with 37.5 ng Nkx6.1MO in one half of the embryos. The injected side is to the right. (A,B) N-tubulin expression pattern marking primary neurons is normal at stage 16. (C,D) Hb9-positive MNs form normally in Nkx6.1 knockdowns (stage 18). (E,F) Lhx3-positive cells are reduced in Nkx6.1MO injected embryos at stage 16 (black arrowhead). (G,H) Pax2-expressing interneurons are reduced at stage 15. Red brackets indicate medial and lateral Pax2-positive stripes in the spinal neural plate. Only one stripe is present on the injected side (H and insert). (I,J) At stage 18 the spinal Pax2+ interneurons form one stripe that is reduced in response to Nkx6.1 knockdown (J, red arrowhead). Interneuron progenitor markers such as Pax6 (K,L), Dbx1 (M,N), and Nkx2.2 (O,P) are not affected by Nkx6.1 knockdown. Nkx6.1 is not required for its own (Q,R) or for Nkx6.2 expression (S,T).

We proceeded to investigate more subtle changes in neural plate patterning. During development Pax2 is expressed in multiple domains of the nervous system including several classes of postmitotic spinal interneurons (Burrill et al., 1997). We investigated Pax2 expression at mid- and late neural plate stages. Early spinal Pax2+ interneurons normally form in two lines on each side that later appear to form a single line on each side of the midline (compare Fig.5G,I). Embryos injected with Nkx6.1MO and analyzed at mid neurula stages had reduction of early forming Pax2 expressing neurons on the injected side (Fig.5H; 25/32). The affected population seemed primarily to be the medial-most stripe that also extend more posteriorly along the spinal neural plate. Later, at stage 18, this showed as a thinner and shorter Pax2 domain in the spinal cord (Fig.5J; 17/24).

To determine if other ventral or intermediate progenitor domains in the spinal neural plate was affected by Nkx6.1 knockdown we performed in situ hybridization for Nkx2.2 (Fig.5K,L), Pax6 (Fig.5M,N), and Dbx1 (Fig.5O,P) but found no changes. Lastly, we tested if Nkx6.1 is necessary for either its own expression or that of Nkx6.2. However, Nkx6.1 and Nkx6.2 expression was normal in Nkx6.1MO injected embryos at stage 17 (Fig.5Q–T) as well as stage 34 (data not shown). The largely normal expression of most genes in the neural plate indicate that the patterning effect of Nkx6.1 knockdown is limited to a restricted population of ventral interneurons in the neural plate. However, considering the partial overlapping expression of Nkx6.1 and Nkx6.2 it is possible that knockdown of both Nkx6 genes is necessary to reveal their function fully.

Nkx6.1 knockdown results in paralysis and impaired axonal outgrowth

Although Nkx6.1MO injected embryos were morphologically normal, even at late stages, they exhibited complete loss of mobility. Tadpole escape reflexes are an adaptation to stimulus that integrates sensory input into a coordinated motor response leading to flight away from a potential danger (Roberts, 1990). When poked with a pipette tip uninjected embryos at stage 37 initiated an escape reflex (Fig.6A, top panel). In contrast, embryos injected bilaterally with Nkx6.1MO did not move (Fig.6A lower panel). Unilateral injection of morpholino resulted in tadpoles that were unable to coordinate swimming movements but did respond to stimuli by flexing the uninjected side (data not shown). These experiments show that although the Nkx6.1MO injected embryos are morphologically normal their behavior is not. The paralysis could be caused by defects in the neural circuit involved in the escape reflex or in the muscle effectors. Visual inspection of embryos and immunostaining for skeletal muscle using 12/101 antibody (Kintner and Brockes, 1984) showed that paraxial muscle formation was normal (data not shown).

Fig.6. Nkx6.1 knockdown results in paralysis and axon misguidance.

Fig.6

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(A) Narrative frames from a time lapse series showing uninjected controls (top panel) and Nkx6.1MO injected embryos (bottom). Uninjected embryos initiate escape reflex when poked with a pipette tip (frames 12 and 17; pipette tip painted black). Poking of Nkx6.1MO injected embryos does not trigger escape reflex (bottom). Nkx6.1MO injected embryos were injected in both blastomeres at the 2-cell stage. (B,C,F,H) Orthograde DiI-labeling of stage 39/40 embryos injected unilaterally with Nkx6.1MO. (B) Control side showing labeled nerve fibers emanating from the neural tube. (C) Nkx6.1MO injected side show labeled nerves. (D,E,G,I) Immunostaining for N-CAM using antibody 6F11 showing axonal extensions. (D) Uninjected side of stage 39/40 tadpole showing nerve fibers protruding from the spinal cord and hindbrain. (E) Nkx6.1MO injected side of same tadpole does have greatly reduced and disorganized axons and cranial nerves are severely affected (blue arrowhead). (F, H) High magnification of boxes in (B) and (D), respectively. White arrow heads in (F) indicate DiI labeled axons. (H,I) High magnification of boxes show in (C) and (E), respectively. Asterisks in (H) indicate fluorescence from the uninjected side out of the plane of focus. (J) Effect of different MO1 and MO2 doses, individually and in combination, on tadpole mobility. Embryos were injected in all blastomeres at the one or two cell stage. Total MO amount injected is listed under bar. Number in parenthesis indicate number of embryos analyzed. MO1+2 injections were performed with equal amounts of each. The two last bars show effect on MO1 when injected into X. tropicalis. Embryos were scored at stage 32–39.

To determine if impaired axon guidance could explain the paralysis in Nkx6.1MO injected embryos we visualized the nerve fiber pattern by two different techniques. First, we performed orthograde DiI-labeling of tadpoles that had been injected with Nkx6.1MO on one side of the embryo. On the control side, the DiI labeled the axonal tracts extending from the trunk neural tube towards the axial muscle (Fig.6B,F). In contrast, DiI completely failed to label any axonal tracts on the Nkx6.1MO injected side (Fig.6C,H; 11/14 embryos).

Next, we stained neural extensions in unilaterally injected Nkx6.1MO tadpoles with the mouse monoclonal antibody 6F11, that recognizes N-CAM (Lamb et al., 1993). On the uninjected side we observed nerve fibers extending from the neural tube in a highly regular pattern marking the trunk motor columns and cranial nerves (Fig.6D,G). In contrast, the nerve fibers on the Nkx6.1MO injected side were abnormal (Fig.6E,I). Nerve fibers projecting from the spinal cord of the MO injected side were significantly thinner and diffuse compared to controls (Fig.6D,E; 31/35 embryos). In addition, the cranial nerves emanating from the hindbrain were notably affected and their projections stunted (Fig.6D,E). The defects appeared confined to the MN axonal nerve fibers as the overall morphology of the neural tube and brain seemed normal as well as the ridge of sensory Rohon-Beard cells dorsal to the neural tube (Fig.6E). At high magnification the Nkx6.1-deficient motor nerves were irregular in shape in contrast to defined pattern of the normal spinal nerves (Fig.6G,I). To further demonstrate the relation between Nkx6.1 knockdown with the observed mobility defects, we designed a second antisense morpholino (MO2) that hybridize to the 5’UTR of X. laevis Nkx6.1 directly upstream of, but not overlapping with the MO1 target sequence. When injected into X. laevis embryos MO2 phenocopied MO1, demonstrating the specific relation between Nkx6.1 knockdown and the mobility defects although it was less effective (Fig.6J) and caused some morphology defects that we did not observe when injecting MO1. Co-injecting MO1 and MO2 at various doses had additive effects further demonstrating their specific action (Fig.6J). Together, these experiments show that Nkx6.1 is essential for movement in Xenopus tadpoles and that absence of Nkx6.1 function results in aberrant axon extensions of the trunk and cranial motor nerves.

Attempts to knock down Nkx6.2 function

During our studies we attempted MO knockdown of Nkx6.2 in both X. laevis and X. tropicalis. However, despite testing two different MO in X.laevis and an additional MO specific to the X.tropicalis allele we failed to achieve specific Nkx6.2 knockdown. In fact, both MOs tested in X.laevis caused an unspecific inhibition of all primary neurons (motor-, inter-, and sensory neuron), as well as Dbx1 expression. The unspecific nature of the MO effect was demonstrated by injection of the MO in the lateral part of the prospective neural plate at the 32-cell stage (i.e.. outside of the Nkx6.2 expression domain). Injection in this part resulted in repression of Dbx1 and N-tubulin expression. When the MOs was targeted to the medial part of the neural plate we could occasionally observe an expansion of Dbx1 expression towards the midline, suggesting that indeed Nkx6.2 acts to repress intermediate neural plate gene expression in frogs as well. We are currently examining other strategies for removing Nkx6.2 function that are not dependent upon MO injection.

DISCUSSION

In this report we show that the two Nkx6 genes in Xenopus have similar functions during neural development when misexpressed and that their functions differ in important aspects from their orthologues in other vertebrates. Confirming work of Zhao et al., 2007, Nkx6.2 is broadly expressed in the medial neural plate, whereas Nkx6.1 is highly restricted in its expression (Fig.1). In addition, we show that the domains of gene expression do not abut the domains of Dbx1 expression, as they do in amniotes, suggesting that there is no simple cross repressive activity. Furthermore, we find that Nkx6.1 is mainly required for controlling MN axon growth (Fig.6) and ventral interneuron specification (Fig.5). This differs from the reported roles of Nkx6.1 in mouse, chicken and zebrafish, where it is expressed broadly in the ventral neural tube and acts a critical patterning gene (Vallstedt et al., 2001). In frogs, the expression pattern of Nkx6.2 suggests that it could have a central function in neural patterning since it is expressed early during gastrulation and in a broad region of the neurogenic ectoderm. This is consistent with the earlier and more general expression of Nkx6.2, when compared to Nkx6.1. This broader expression and overexpression phenotype of Nkx6.2 argues for a critical and central role of Nkx6.2 in patterning and a more restricted role for Nkx6.1 in MN and interneuron differentiation. The ability of ectopic Nkx6 expression to repress Dbx1 and Pax2 expression suggests a evolutionary conserved role of these genes in specifying ventromedial cell fates by repressing more intermediate ones.

Nkx6.1 knockdown function in MN and interneuron development

The restricted effect of Nkx6.1 knockdown to the MNs of the trunk and cranial nerves (that in most cases are composites of sensory and MNs) is consistent with a role of in MN axon outgrowth or guidance (Fig.6). The similar paralysis in both X.laevis and its sister species X.tropicalis, supports the argument that the effect is specific and attributable to Nkx6.1 knockdown. The Nkx6.1MO injected embryos form Hb9 positive MNs which are present in seemingly normal numbers (Fig.5). Hence, our results argue that the biological cause of the paralysis is a defect in MN axons reaching their muscle targets. Indeed, detailed tracing of MN trajectory in Nkx6.1 mouse mutants shows that these have defects in connecting to their proper muscular targets in the limbs (De Marco Garcia and Jessell, 2008) and face (Prakash et al., 2009). In addition, studies of mouse Nkx6.1/2 double mutant embryoshave shown that hindbrains of these embryos also have defective motor nerve outgrowth(Pattyn et al., 2003). It has also been shown that the fly Nkx6 ortholog is involved in MN axon guidance, underlining the evolutionary importance of this transcription factor family in MN development (Broihier et al., 2004).

The neurobiological basis of the escape reflex in Xenopus tadpoles has been delineated (reviewed in (Roberts et al., 1998)). Apart from MNs and SNs, the neural circuit also involves spinal INs. The SNs (Rohon-Beard neurons) involved in the escape reflex are present in the Nkx6.1MO injected tadpoles, suggesting that the defect is downstream of the sensory input. As Pax2+ or Lhx3+ interneuron development was also affected in Nkx6.1MO injected embryos, it cannot be excluded that these neurons are necessary for escape reflexes (Fig.5). In fact, both Pax2+ V1 and Lhx3+ V2 interneurons are involved in locomotion in mice although the phenotypic effects of ablating these neurons are relatively mild (Gosgnach et al., 2006)(Crone et al., 2009). In principle, both the axon guidance defect and failure to form interneurons could independently cause loss of escape reflexes. Nonetheless, the striking absence of axonal extensions from the ventral neural tube strongly suggests this as the primary cause of the paralysis (Fig.6). At present it is not known what function, if any, Pax2+ and Lhx3+ interneurons have in coordinated tadpole movement, and further study of these interneurons should clarify the issue.

Frog Nkx6 genes are not cross-repressive

Studies of Nkx6 mouse mutants have clearly shown that Nkx6 genes repress the expression of each other and are expressed in mutually exclusive domains (Vallstedt et al., 2001). Our study shows that this is not the case in frogs as misexpression of either gene leads to a small expansion of the expression domain of the other (Fig.3). Indeed, Nkx6.1 is expressed in a subset of the Nkx6.2 domain making cross-repression unlikely (Fig.1).

Ectopic Nkx6 expression represses intermediate neural gene expression

While misexpression experiments should be interpreted cautiously, we believe that the ectopic expression data provide useful insight to Nkx6 function. The dose of Nkx6 mRNA used for misexpression was the lowest that would cause robust Dbx1 repression and far lower than the maximum tolerated by the embryos. Additional evidence for specificity comes from the absence of cross repression of Nkx6.1 and Nkx6.2, and the absence of repression of several genes in the brain. Although difficult to verify, we consider it likely that Dbx1 repression in the spinal cord is a primary endogenous function of Nkx6.

Nkx6 gene expression partly overlaps with Irx3 and Pax6 in the medial-most part of the Nkx6 domain, as has also been shown to be the case in mouse and chick neural tube (Briscoe et al., 2000). It is therefore puzzling that Nkx6 mRNA represses these genes when mis-expressed in the neural plate (Fig.2). Briscoe et al. (2000) found that early misexpression of Nkx6 could specify ventral identity in most of the intermediate neural tube, whereas delayed misexpression (after the onset of Irx3 expression) had a minor effect. This suggests that the function of Nkx6 depends on the prepatterning of the neural tissue and that Irx3 expression could close this window of competence. In frogs, we observe strong expression of Nkx6.2 as early as the formation of the neural plate whereas spinal expression of Pax6 and Irx3 starts later. Consistent with this model of Irx3 mediated loss-of-competence, and supporting specific action of the mRNA, early rhombomeric expression of Irx3, which is refractory to Nkx6 repression, appears to overlap Nkx6.2 expression. While it is unclear how the intermediate region of the Nkx6 domain acquires Irx3 and Pax6 expression at midneurula stages we speculate that Nkx6 mediated repression of Irx3 and Pax6 might be antagonized by auxiliary factors that are not present in the early neural plate but are expressed at later stages of neural development, and possibly in the hindbrain region that coexpresses nkx6.2 and irx3.

In our ectopic expression experiments we also show that Nkx6 mRNAs are able to repress markers of differentiated neurons most notably N-tub, but also Pax2. Again this appears counter intuitive since Nkx6 genes are normally expressed in differentiated motor neurons that express N-tub. In vertebrates Nkx6 genes are initially expressed in neural progenitor cells, but are extinguished as they exit the cell cycle (Vallstedt et al., 2001). Interestingly, Nkx6 gene expression then reappears as MN axonal projections emerge (De Marco Garcia and Jessell, 2008). Perhaps this temporary absence of Nkx6 expression is necessary for neural cells to move from progenitors to differentiating neurons; thus, forced Nkx6 expression throughout their development would disrupt the normal differentiation program and, possibly, arrest the neurons in a progenitor state. This would also explain the failure to form pax2+ cells.

Nkx6 expression in frogs is not sufficient to induce MNs

Ectopic expression of Nkx6.1 in chick or fish neural tube causes ectopic MN induction (Briscoe et al., 2000; Cheesman et al., 2004). In frog embryos we tested a range of doses, as well as both Nkx6 genes in combination, but were unsuccessful in generating ectopic MNs, indicating that Nkx6 function is not sufficient to initiate MN development (Fig.3). Ectopic MN induction in chick is inhibited by Irx3 expression in the neural tube, which closes the window of competence (Briscoe et al., 2000). A similar mechanism for the lack of ectopic MN induction in our study is unlikely since the mRNA is injected at the 2-cell stage and furthermore prevents Irx3 expression in the spinal neural plate. It is possible that other members of the Irx-family prevent ectopic MN formation in our experiments. One candidate is Irx2, which is expressed in the neural plate (Gomez-Skarmeta et al., 1998), but is not repressed by Nkx6 misexpression (data not shown). In conclusion, our results show that Nkx6.1 function is necessary but not sufficient for proper MN differentiation in frogs.

Redundant Nkx6 functions

Mouse mutant studies show that Nkx6.1 and Nkx6.2 have highly redundant functions, and consequently, inactivation of both genes is necessary to reveal their function in ventral neural tube patterning (Vallstedt et al., 2001). Despite testing three different MOs, we were unable to specifically inhibit Nkx6.2 function. Nonetheless, it is likely that Nkx6.2 has important functions, based on its early and prominent expression and overexpression phenotype. Specific inhibition of the function of this gene in combination with the results presented here would provide a more comprehensive understanding of conserved, and non-conserved features of M-L patterning of the frog neural plate.

Research Highlights

Nkx6.1 and Nkx6.2 mRNA injections inhibit primary neurogenesis and intermediate neural plate markers. Nkx6.1 antisense morpholino oligonucleotide injected tadpoles are paralyzed. Nkx6.1 MO injected tadpoles fail to extend motor axons.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. Jen-Yi Lee for critical reading of the manuscript and members of the Harland lab for helpful suggestions and comments. We thank Drs. Johan Ericsson, Maike Sander, Palle Serup, and Kathryn Zimmerman for reagents. This work was supported by the Alfred Benzon Foundation through a fellowship for D.S.D. This work was supported by NIH (GM42341).

Footnotes

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